CHARACTERIZATION OF SUCROSE METABOLIZING
ENZYMES IN SUGARCANE UNDER HEAT STRESS
FAISAL MEHDI
DEPARTMENT OF AGRICULTURE AND AGRIBUSINESS
MANAGEMENT, UNIVERSITY OF KARACHI,
KARACHI, PAKISTAN
2019
ii
CHARACTERIZATION OF SUCROSE METABOLIZING
ENZYMES IN SUGARCANE UNDER HEAT STRESS
By
FAISAL MEHDI
A thesis submitted in
fulfilment of the requirements for the degree of
Doctor of Philosophy (Ph.D.) in Agriculture
DEPARTMENT OF AGRICULTURE AND AGRIBUSINESS
MANAGEMENT, UNIVERSITY OF KARACHI,
KARACHI, PAKISTAN
2019
iii
Dedication
I dedicate this dissertation to my beloved parents
Ghulam Mehdi Masroor and Amina Masroor
for their love, endless support, encouragement and sacrifices
without whom none of my success would be possible.
iv
CHARACTERIZATION OF SUCROSE METABOLIZING
ENZYMES IN SUGARCANE UNDER HEAT STRESS
Thesis Approved
Supervisor
Dr. Saddia Galani Associate Prof.
Dr. A.Q Khan Institute of Biotechnology and Genetic Engineering
University of Karachi, Karachi, Pakistan
Supervisor’s Signature:
Examiner’s Signature:
v
Acknowledgements
All Praise to ALLAH, for giving me the blessing, the strength, the chance and endurance
to complete this dissertation. My special gratitude and appreciation to my parents, my
brothers and sisters for encouraging, supporting and always believing me to follow my
dreams throughout the study. I gratefully acknowledge to My Supervisor Dr. Saddia
Galani Associate Professor in the Karachi Institute of Biotechnology and Genetic
Engineering (KIBGE), University of Karachi she has been a tremendous mentor for me.
Without her continuous guidance, advice, effort, support, suggestions and constant
feedback throughout the research, this Ph.D. would not have been achievable. My
utmost gratitude to Prof. Dr. Abid Azhar Director General Meritorious Professor in the
Karachi Institute of Biotechnology and Genetic Engineering (KIBGE), University of Karachi
for providing me the lab facilities to carry out my research successfully. My deepest
gratitude goes to Prof. Dr. Saleem Shahzad Registrar University of Karachi and Ex-
chairperson department of agriculture and agribusiness management university of
Karachi. I am indebted to Dr. Saboohe Raza chairperson department of agriculture and
agribusiness management university of Karachi. I also acknowledge to Mr. Touqeer Mirza
Cane Procurement and Development Officer Mehran Sugar Mills Ltd and Mr. Sharif Khan
Deputy General Manager Mirpurkhas Sugar Mills Ltd. Last but not least, my particular
appreciation goes to Dr. Afsheen Aman, Dr. Urooj Javed, Dr. Kazim Ali, Dr. Shagufta,
Sahar, Dr. Ishrat Jameel, Riaz Ahmed, Sohail Ahmed and Abid Hussian as well as my
fellow lab mates Neshaman Huma, Zubia Rashid, Nabeela Afzal, Zunaira Riaz, Lubna
Faraz, Fatima Haider, Maleeha Akbar, Iffat Imran, Ali Muntazir Naqvi and lab assistant
Aijaz bhai and Zaheer bhai for their support and encourage during all experiments.
Faisal Mehdi
vi
TABLE OF CONTENTS
Description Page no.
Acknowledgments v
Table of Contents vi-xii
List of Tables xi-xii
List of Figures xii-xvi
List of Abbreviations xvi-xviii
Abstract xix-xx
Abstract in Urdu xxi-xxii
Section # 1 Introduction 1
1.1. Introduction 2-6
1.2. Objectives 7
Section # 2 Literature Review 8
2.1. Importance of Sugarcane 9
2.2. Phenology of Sugarcane 10
2.3. Heat Stress 11
2.4. Effect of Heat Stress on Sugarcane 11-12
2.5. Effect of High Temperature on Growth and Development 13
2.6. Physiological Indicators of Tolerance to Increased Temperatures
2.6.1 Cell Membrane Thermosability
2.6.2 Accumulation of Compatible Solutes (Proline)
2.6.3 Malondialdehyde (MDA)
2.6.4 Hydrogen peroxide (H2O2)
13-16
3.1. Molecular Indicators of Tolerance to Increased Temperatures
3.1.1. Reactive Oxygen Species (ROS)
3.1.2. Heat Shock Protein (HSPs)
17-18
4.1. Antioxidant Defense Response to Heat Induced Oxidative Stress
4.1.1. Enzymatic Antioxidants
4.1.2. Non-Enzymatic Antioxidants
19
5.1. Plant Adaptation to Heat Stress 20
vii
6.1. Mechanism of plant Adaptation to Heat stress
6.1.1. Avoidance Mechanism
6.1.2. Tolerant Mechanism
20-21
7.1. Sucrose Metabolism and Regulation in Sugarcane
7.1.1. Biosynthesis of Sucrose in Sugarcane Plant
7.1.2. Source-sink Regulation of Sucrose Accumulation in
Sugarcane
21-23
8.1. Sucrose Metalizing Enzymes Response Under Heat Stress
8.1.1. Invertases
8.1.2. Soluble Acids
8.1.2.1. Soluble Acid Invertase (Vacuolar Invertase)
8.1.2.2. Insoluble Acid Invertase (Cell Wall Invertase)
24-26
8.2. Cytoplasmic Invertase 26
8.3. Sucrose Synthase 26-28
8.4. Sucrose Phosphate Synthase 28
8.5. Sugar Recovery Rate 29
9.1. The Mitigation of Heat Stress Strategies
9.1.1. Cultural Method 30-31
9.2. Genetics and Genomics Strategies
9.2.1. Omics-Led Breeding and Marker-Assisted Selection
9.2.2. Genome Wide Association Studies for Stress Tolerance
9.2.3. Genetic Engineered Plants for Stress Tolerance
31-33
9.3. Genome Editing Strategies 33-34
Section # 3 Methodology 35
3. General Experimental Details 36
3.1. Physiochemical Properties of Soil and Water
3.1.1. Soil and Water Analysis 37
3.2. Crop Husbandry
3.2.1. Cultivation of Sugarcane 37
3.3. Heat Stress Treatments 38
3.4. Sample Collection 39
viii
3.5. Morphological Analysis 39
3.6. Physiological Analysis
3.6.1. Cell Membrane Thermostability (CMT)
3.6.2. Proline Estimation (Osmolyte accumulation)
3.6.3. H2O2 Quantification
3.6.4. Determination of Lipid Peroxidation
40-43
3.7. Biochemical Analysis
3.7.1. Sugar Extraction
3.7.2. Total Sugar Estimation
3.7.3. Reducing Sugar Estimation
3.7.4. Non-Reducing Sugar Estimation
3.7.5. Protein and Enzymes Extraction
3.7.5.1. Extraction
3.7.5.2. Total Soluble Protein Quantification
43-47
3.8. Quantification of Sugar Metabolizing Enzymes
3.8.1. Vacuolar Acid Invertase (VAI)
3.8.2. Quantitative Analysis of Cell Wall Invertase (CWI)
3.8.3. Quantification of Cytoplasmic Invertase (CyIN)
3.8.4. Quantification of Sucrose Phosphate Synthase (SPS)
3.8.5. Sucrose Synthase (SS)
47-52
3.9. Qualitative Analysis of Isozymes through Native PAGE
3.9.1. Sample Extraction
3.9.2. Native Polyacrylamide Gel Electrophoresis
3.9.2.1. Preparation of Reagents
3.9.2.2. Preparations of Buffers
3.9.2.3. Preparation of Resolving Gel (lower gel) (pH-8.8)
3.9.2.4. Preparation of Stacking Gel (Upper gel) (pH-6.8)
3.9.2.5. Preparation of Running Buffer (1X)
52-56
3.9.3. Invertase Zymography 56-57
3.9.4. SDS-PAGE Protein Profiling 57
3.9.5. Quality Parameters Analysis 57-59
ix
3.9.5.1. Pol (%) Estimation
3.9.5.2. °Brix Estimation
3.9.5.3. Moisture Content
3.9.5.4. Fiber Content (%)
3.9.5.5. Sugar Recovery Rate Estimation
3.9.6. Statistical Analysis 59
Section # 4 Results 60
4.1. Morphological Analysis
4.1.1. Shoot Length (cm)
4.1.2. Root Length (cm)
4.1.3. Number of Tillers (plant-1)
4.1.4 Number of Leaf (plant-1)
4.1.5. Leaf Length (cm)
4.1.6. Leaf Width (cm)
4.1.7. Fresh to Dry Weight Ratio (%)
4.1.8. Stem Diameter (cm)
4.1.9. Number of Nodes (plant-1)
4.2.0 Number of Internodes (plant-1)
4.2.1. Internode Distance (plant-1)
61-71
4.3. Stress Damage Indicators Quantification
4.3.1 Malondialdehyde (MDA)
4.3.2 Proline Estimation
4.3.3 Hydrogen-peroxide
4.3.4 Relative Membrane Permeability (RMP)
72-79
4.4. Biochemical Analysis
4.4.1. Total Sugar Quantification
4.4.2. Reducing Sugar Quantification
4.4.3. Non-reducing Sugar Quantification
4.4.4. Total Soluble Protein Analysis
80-89
4.5. Sugar Metabolizing Enzymes
4.5.1. Quantitative Analysis 90-100
x
4.5.1.1. Sucrose Synthase (SS)
4.5.1.2. Sucrose Phosphate Synthase (SPS)
4.5.1.3. Cytoplasmic Invertase (CyINV)
4.5.1.4. Cell Wall Invertase (CWIN)
4.5.1.5. Vacuolar Invertase (VIN)
4.5.2. Qualitative Analysis
4.5.2.1. Cytoplasmic Invertase
4.5.2.2. Vacuolar Invertase
4.5.2.3. Cell wall Invertase
101-110
4.6. Quality Parameters Analysis
4.6.1. °Brix Estimation
4.6.2. Fiber Content
4.6.3. Pol Estimation
4.6.4. Sugar Recovery Estimation
111-114
4.7. Protein Profiling at Different Growth Stages
4.7.1. Protein Profiling at Vegetative Stage
4.7.2. Protein Profiling at Grand Growth Stage
4.7.3. Protein Profiling at Maturity Stage
115-120
4.8. Correlation 121-122
4.9. Heat Map 123
Section # 5 Discussion 124
5.1. Morphological Analysis 125-127
5.2. Thermotolerant Indicators Analysis 127-131
5.3. Sugar Analysis 131-132
5.4. Sugar Recovery Rate Analysis 132-134
5.5. Sugar Metabolizing Enzymes Analysis
5.5.1. Qualitative Analysis
5.5.2. Quantitative Analysis of Invertase Isozymes
134-140
5.6. SDS-PAGE Protein Profiling 141-142
5.7. Correlation 142-143
Key Findings 144-145
xi
Future Directions 146
Conclusion 147
References 148-188
Oral Presentations 189
Publications 189
Poster Presentations 189
LIST OF TABLES
Table no. Title Legends Page no.
1. Five year statistics of area, can production and yield 9
2. Five year statistics of sugar crushing, production and sugar
recovery rate. 9
3. Heat stress treatment used for the estimation of plant heat
tolerant based on the common ion leakage measurement (EC). 14
4. Details of physiochemical properties of soil and water used in
experiment 37
5. Morphological analysis of both cultivars S2003-US-633 and
SPF-238 under control (30±2°C), heat shock (45±2°C) and
recovery (30±2°C) for 24, 48 and 72 h at vegetative stage.
Cultivar (C), Treatments (T) and Cultivar × Treatments (C×T) at
p level p<0.05.
69
6. Morphological analysis of both cultivars S2003-US-633 and
SPF-238 under control (30±2°C), heat shock (45±2°C) and
recovery (30±2°C) for 24, 48 and 72 h at grand growth stage.
Cultivar (C), Treatments (T) and Cultivar × Treatments (C×T) at
p level p<0.05.
70
7. Morphological analysis of both cultivars S2003-US-633 and
SPF-238 under control (30±2°C), heat shock (45±2°C) and
recovery (30±2°C) for 24, 48 and 72 h at maturity stage. Cultivar
(C), Treatments (T) and Cultivar × Treatments (C×T) at p level
p<0.05.
71
xii
8. Quality parameters estimation of both cultivars S2003-US-633
and SPF-238 under control (30±2°C), heat shock (45±2°C) and
recovery (30±2°C) for 24, 48 and 72 h at vegetative, grand
growth and maturity stages. Cultivar (C), Treatments (T) and
Cultivar × Treatments (C×T) at p level p<0.05.
114
9. Correlation among sugar profile, sugar metabolizing enzymes
and quality parameters at all growth stages. 122
10. Status of thermotolerant sugarcane cultivars. 123
LIST OF FIGURES Figure no. Description Page no.
1. ROS accumulation in sugarcane plant cells as consequence of
heat shock. These ROS generated from different organelles
such as mitochondria, chloroplast, endoplasmic reticulum,
peroxisome and extracellular side of cell membrane.
12
2. Free radicals are known to exist in crops. The Lewis diagram of
these free radicals presented in black, with unpaired electrons
highlighted in red. The half-life time (t1/2) is given for each type
of radicals. Colour coded with highest value for H2O2 (red) and
lowest value for OH• (green). Abbreviations: (ms=milli second,
µs=micro second, and ns= Nano second).
17
3. Sucrose biosynthesis: Sucrose is synthesized from uridine
diphosphate synthase (UDP) glucose and fructose 6-
phosphate, which are synthesized from triose phosphates in
the plant cell cytosol. The sucrose 6-phosphate synthase of
most plant species is allosterically regulated by glucose 6-
phosphate and Pi.
22
4. Schematic presentation of sucrose metabolism and
transportation from source (leave) to stem (sink). 23
5. Annual mean temperature and relative humidity of sugarcane
field for the year 2016-2018. 38
xiii
6. MDA quantified of sugarcane cultivars S2003-US-633 and SPF-
238 under control (30±2°C), heat shock (45±2°C) and recovery
(30±2°C) for 24, 48 and 72 h at vegetative, grand growth and
maturity stages.
73
7. Free proline estimated of sugarcane cultivars S2003-US-633
and SPF-238 under control (30±2°C), heat shock (45±2°C) and
recovery (30±2°C) for 24, 48 and 72 h at vegetative, grand
growth and maturity stages.
75
8. H2O2 estimated of sugarcane cultivars S2003-US-633 and SPF-
238 under control (30±2°C), heat shock (45±2°C) and recovery
(30±2°C) for 24, 48 and 72 h at vegetative, grand growth and
maturity stages.
77
9. Electrolytes leakage quantified of sugarcane cultivars S2003-
US-633 and SPF-238 under control (30±2°C), heat shock
(45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at
vegetative, grand growth and maturity stages.
79
10. Total sugar estimated of sugarcane cultivars S2003-US-633 and
SPF-238 under control (30±2°C), heat shock (45±2°C) and
recovery (30±2°C) for 24, 48 and 72 h at vegetative, grand
growth and maturity stages.
82
11. Reducing sugar estimated of sugarcane cultivars S2003-US-633
and SPF-238 under control (30±2°C), heat shock (45±2°C) and
recovery (30±2°C) for 24, 48 and 72 h at vegetative, grand
growth and maturity stages.
85
12. Nonreducing sugar estimated of sugarcane cultivars S2003-US-
633 and SPF-238 under control (30±2°C), heat shock (45±2°C)
and recovery (30±2°C) for 24, 48 and 72 h at vegetative, grand
growth and maturity stages.
87
13. Total soluble protein estimated of sugarcane cultivars S2003-
US-633 and SPF-238 under control (30±2°C), heat shock 89
xiv
(45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at
vegetative, grand growth and maturity stages.
14. Specific activity quantified of sucrose synthase (SS) of
sugarcane cultivars S2003-US-633 and SPF-238 under control
(30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48
and 72 h at vegetative, grand growth and maturity stages.
92
15. Specific activity quantified of sucrose phosphate synthase (SPS)
of sugarcane cultivars S2003-US-633 and SPF-238 under
control (30±2°C), heat shock (45±2°C) and recovery (30±2°C)
for 24, 48 and 72 h at vegetative, grand growth and maturity
stages.
94
16. Specific activity quantified of cytoplasmic invertase (CyIN) of
sugarcane cultivars S2003-US-633 and SPF-238 under control
(30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48
and 72 h at vegetative, grand growth and maturity stages.
96
17. Specific activity quantified of cell wall invertase (CWIN) of
sugarcane cultivars S2003-US-633 and SPF-238 under control
(30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48
and 72 h at vegetative, grand growth and maturity stages.
98
18. Specific activity quantified of vacuolar invertase (VIN) of
sugarcane cultivars S2003-US-633 and SPF-238 under control
(30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48
and 72 h at vegetative, grand growth and maturity stages.
100
19. Native–PAGE analysis of cytoplasmic invertase (CyIN) of
cultivar S2003-US-633 subjected to control (C) heat shock
(45±2°C) and recovery treatments for 24, 48 and 72 h at all
growth stages. (Markers used ovalbumin (45 kDa), albumin
bovine (monomer 67 kDa and dimer 134 kDa) and gama
globulin human (160 kDa).
103
20. Native–PAGE analysis of cytoplasmic invertase (CyIN) of
cultivar SPF-238 subjected to control (C) heat shock (45±2°C) 104
xv
and recovery treatments for 24, 48 and 72 h at all growth
stages. (Markers used ovalbumin (45 kDa), albumin bovine
(monomer 67 kDa and dimer 134 kDa) and gama globulin
human (160 kDa).
21. Native–PAGE analysis of vacuolar invertase (VIN) of cultivar
S2003-US-633 subjected to control (C) heat shock (45±2°C) and
recovery treatments for 24, 48 and 72 h at all growth stages.
(Markers used ovalbumin (45 kDa), albumin bovine (monomer
67 kDa and dimer 134 kDa) and gama globulin human (160
kDa).
106
22. Native–PAGE analysis of vacuolar invertase (VIN) of cultivar
SPF-238 subjected to control (C) heat shock (45±2°C) and
recovery treatments for 24, 48 and 72 h at all growth stages.
(Markers used ovalbumin (45 kDa), albumin bovine (monomer
67 kDa and dimer 134 kDa) and gama globulin human (160
kDa).
107
23. Native–PAGE analysis of cell wall invertase (CWIN) of cultivar
S2003-US-633 subjected to control (C) heat shock (45±2°C) and
recovery treatments for 24, 48 and 72 h at all growth stages.
(Markers used ovabumin (45 kDa), albumin bovine (monomer
67 kDa and dimer 134 kDa) and gama glubulin uman (160 kDa).
109
24. Native–PAGE analysis of cell wall invertase (CWIN) of cultivar
SPF-238 subjected to control (C) heat shock (45±2°C) and
recovery treatmnets for 24, 48 and 72 h at all growth stages.
(Markers used ovabumin (45 kDa), albumin bovine (monomer
67 kDa and dimer 134 kDa) and gama glubulin uman (160 kDa).
110
25. SDS-PAGE protein profiling of sugacane cultivars (A) S2003-US-
633 (B) SPF-238 at formative stage under control at (30±2°C),
heat shock (45±2°C) and recovery treatments (30±2°C) for 24,
48 and 72 h.
116
xvi
26. SDS-PAGE protein profiling of sugacane cultivars (A) S2003-US-
633 (B) SPF-238 at grand growth stage under control at
(30±2°C), Heat shock (45±2°C) and recovery treatments
(30±2°C) for 24, 48 and 72 h.
118
27. SDS-PAGE protein profiling of sugacane cultivars (A) S2003-US-
633 (B) SPF-238 at maturity stage under control at (30±2°C),
heat shock (45±2°C) and recovery treatments (30±2°C) for 24,
48 and 72 h.
120
LIST OF ABBREVIATIONS / ACRONYMS ANOVA Analysis of Variance
AOS Active Oxygen Species
APS Ammonium Per Sulfate
AS Amount of Sample
°Bx °Brix
BSA Bovine Serum Albumin
C Control Treatment
°C Degree(s) Celsius
CBB Commassie Brilliant Blue
CMT Membrane Thermostability
Conc Std Concentration of Standard
CWIN Cell Wall Invertase
CyIN Cytoplasmic Invertase
DNA Deoxyribonucleic Acid
DNSA Dinitro Salicylic Acid
EC Electrical Conductivity
E.C Enzyme Commission
Ext pol Extraction of pol
EDTA Ethylene Diamine Tetra Acetic Acid
FAO Food and Agriculture Organization
FW Fresh Weight
FYM Farm Yard Manure
xvii
G Gram
GDP Gross Domestic Product
H Hour
HCl Hydrochloric Acid
H2O2 Hydrogen-peroxide
HSPs Heat Shock Proteins
HST Heat Shock Treatment
IPCC Intergovernmental Panel on Climate Change
KDa Kilo Dalton
KI Potassium Iodide
KOH Potassium Hydroxide
M Mole
Min Minute
MY Marketing Year
MNFSR Ministry of National Food Security and Research
MDA Malondialdehyde
MOPS Morpholino Propane Sulfonic Acid
mg ml-1 Milligram Per Milli Liter
MgCl2 Magnesium Chloride
MW Molecular Weight
N Normality
Nm Nano Meter
NaCl Sodium Chloride
NASA The National Aeronautics and Space Administration
NPK Nitrogen, Phosphorous and Potassium
OD Std Standard of Optical Density
ODT Optical Density of Test Sample
1O2 Singlet Oxygen
O.-2 Super Oxide
OH Hydroxyl Radical
PAGE Polyacryl Amide Gel Electrophoresis
xviii
PAR Photo Synthetically Active Radiation
Pb Plumbum (Lead)
PCD Programmed Cell Death
pH Power of Hydrogen Ions
PSMA Pakistan Sugar Mills Association
R Recovery
ROS Reactive Oxygen Species
RMP Relative Membrane Permeability
RPM Revolution Per Minute
RT Reaction Time
SR Sugar Recovery
SPS Sucrose Phosphate Synthase
SPSS Statistical Package for the Social Sciences
SS Sucrose Synthase
SSA Sulfosalicylic Acid
T Temperature
TBA Thiobarbituric Acid
TCA Trichloroacetic Acid
TEMED Tetramethylethylenediamine
U Unites
UDPG Uridine Diphosphate Glucose
USDA The United States Department of Agriculture
µg ml-1 Microgram Per Milli Liter
VIN Vacuolar Invertase
xix
Abstract Sugarcane is a valuable cash crop in Pakistan. It is cultivated in tropical and
subtropical areas worldwide and produced 80 % sugar globally. It requires optimum
temperature 30°C-32°C for economic production but shift in temperature declined
sugarcane production. Heat stress causes serious yield reduction in sugarcane by
affecting its morphology, physiology, biochemical, molecular levels which leading to
poor yield production and low sugar recovery rate. In this study, two local sugarcane
cultivars, high sucrose accumulation (S2003-US-633) and low sucrose accumulation
(SPF-238) were analyzed for morphological, physiological and biochemical analysis as
well as sugar quality parameters to heat stress. With particular reference to sucrose
metabolizing enzymes such as sucrose phosphate synthase (SPS), sucrose synthase
(SS) and invertase isozymes (cytoplasmic CyIN, vacuolar (VIN) and cell wall bound
(CWIN) were investigated under heat stress and recovery treatments. For this both
sugarcane cultivars subjected to extreme heat shocked treatments at (45±2°C) for
different episodes (T24, T48 and T72 h) and recovery treatments at (30±2°C) for
different episodes (R24, R48 and R72 h). The samples were collected at different
growth stages such as vegetative, grand growth and maturity stages. Results revealed
that after exposure of heat stress for different episodes 24 (T24), 48 (T48) and 72
(T72) h altered most of the morphological attributes due to the differences in the
thermotolerant potential. Both the cultivars indicated the differential expression of
proteins and sucrose metabolizing enzymes at all growth stages. Efficient activity of
sucrose metabolizing enzymes and maximum proline content, highest sucrose
accumulation, maximum sugar recovery rate, reduced electrolytes leakage (EC),
xx
declined malondialdehyde (MDA) and hydrogen peroxide (H2O2) improved
thermotolerance in cultivar S2003-US-633. While lower content of free proline,
sugars, poor performance of ROS scavenger, lower or irregular expression of enzymes
and proteins failed to protect cellular damages caused by extreme temperature,
subsequently hampered growth and development along with reduced sugar recovery
rate in cultivar SPF-238. Present study revealed that high and low molecular mass
protein bands ranging from 15 kDa to 150 kDa proteins in cultivar S2003-US-633. It is
expected that heat shock proteins (HSPs) might be play significant role in
development of thermotolerant without inhibiting the expression or activity of
sucrose metabolizing enzymes. In additions, maximum accumulation of free proline
that performance as a computable solute or osmolytes and oxidative markers,
responsible to stabilize the activity of sucrose metabolizing enzymes activity under
heat stress conditions. It can be concluded that cultivars S2003-US-633 showed more
thermotolerant cultivar with great sucrose accumulation as well as high recovery rate
under different regimes of heat shock treatment conditions. Consequently, these
biochemical attributes can index the degree of thermotolerant of sugarcane crop to
exhibit under heat stress conditions given that insight to molecular breeders to
recognize the thermotolerant sugarcane cultivars with improved recovery rate of
sugarcane.
xxi
خلاصہ
گن
پاکستتتتماںایکقیمی نم کاشتتتتر دنی اک سق فصتتتت۔ قدا دں مگرمعلایقونگرمک
ق عالن ستتتپر ر سق -حاصتتت۔ک جات چین گن ستتت ف صتتتد(08ستتت ایکک جات قون
قچ شتتتتتتتتتتتترقون کا بمیسستتتتتتتتتتتتت ن گ ک ستتتتتتتتتتتتتیس دقوقنک لئ نجہحرقنتشتتتتتتتتتتتتت اعاک
گل کن نجہحرقنتایک-ضتتتتتتتتتتترونت دت ک تبدیل ک وجہستتتتتتتتتتتت کاف کن ن ایک دقوقن
کا-آئ لدج ،اانفدلدج ،ک د ےتناؤگرا کدںستتتتتتتتتتتتپ دستتتتتتتتتتتتالنات قونبائیدک ن ک۔فز ا
ایکک کرماثرا ک دقوقن ستتتتتتابےما کن شتتتتتدیدگن دقوقنکگن جسک وجہستتتتت -کا
چ ک ن قون ین جتتات کدنی کتتاف کن آ ک -ک شتتتتتتتتتتتتتتریایک گن قساپتتالع ایک، واقتتاا
کر وقل ی قک ا کر قون322-یدق س3882-ستتتتمق سقیستتتتام،د ا کستتتتدکرود قک ا کمستتتتتدکرود
گ ا320- قیفستتتمق سی وقل ک ا ک و دںقیستتتام-کدقستتتمعنای حرقنت نجہکد،د ا کگن
ییر نجتہ قون لدج ،ک بتاؤ فدلدج ،فز ا اان وقل ایکنکتککر قونک ن ک۔بائیدحرقنتک بتاؤ
ت ا چین ک گ ا ک ا تجز ہ دگرکساتساتکق ک اع انک یرقای ردکا جیساکہخاارےوقل بنا ش
زقیندنٹیسآسددقئنقونق س( ق سےم یسسدسدکرو ق س(، ق سسےم یسفاستی ستدکرود
قونستتتتتتتتائ لاستتتتتتتتنم، یعن گ ا(ستتتتتتتت ۔وقیو کیدلر ک ا ب تجز ہ ک -کا گن و دں قسک لئ
ک د وںکد،قی نجہک ڈگریستتتتتتتت ن گ (54±3 نمالیس یعن شتتتتتتتتدیدگرا ایکستتتتتتتتام
ک قڑتتتتتتالیسقون،حرقنت رچ بیس گ ن د ک ییرہجبکتتتتویی بہمر بتتتتتاؤوقل نجتتتتتہحرقنت
ک لئ نجہ چ بیسڈگری(28±3حرقنتتیس د وں بہمرگ ن دںقڑتالیسقون،س ن گ ر
-ک ویی رنک گئ ک ارقحتت کی ند اخملفنشتتتتتتتتتتتتتتدو نتتا جنع ونقںقست قیقک-گئ ۔ ر
گ ا ک ا اخملفویی ر نجہحرقنت رک (ڈگریستتت ن گ 54±3کہ نمالیس یہاشتتتا دک
گئ چ بیس،قڑتالیسقون نک ثرخصتتتتتتتتتتتتتدصتتتتتتتتتتتتتت ات رق د وںک ظا ریبہمرگ ن دںک ویی ر
xxii
ک صتتتتتتتتلاح ر و دںقیستتتتتتتتامایکقل قں-ق دقد دئ اخملف- تقل حرقنتبر قشتتتتتتتترکر
بنا وقل خااروں دگر شتت ک ارحل ایک و دںقیستتتامایکل ن اتقون ک قیکستتتشر شتتتننشتتدو نا
د ا ک د ا،ل ن ات- دئ کاف فرقظا رب ایک کا بنا وقل خااروںک قیکستتشر شتتن دگر شتت
قک اد ا ک د ا،د ا کک قجزق دگر د ا،ستتتتتدکرود
قیستتتتت ،قون د ا،د ا کن کدنیک شتتتتتریشتتتتت
ایک322-ق سید3882-ق سکہرتا کثابریہ د اکماکقےڈیقیمآکستتتتتتتتتتتتتائ ،ر ای نوجن
ک صتتتتتتتتتتلاح ربر قشتتتتتتتتتترحرقنت دگرکا د ا،کماقدقنل ن اتک جبکہ-ت بہمرکر
کمشتتتتتتتتتت
قو خااروںک ظا رک ایصکانکر گ ،ب یائدکل ن اتق سقستتتتتتتتتتتتکدقینجرد د ا،آن د ا،قون
ن دگر
ک ستتتتتتتتتتتتاتکستتتتتتتتتتتتتاتکشتتتتتتتتتتتتت قس-کاوٹ دئ ب ن کدنیایکخلیدںک ت یظایک اکامنہن
یمستتد ندنکستت قایکبہرستتانےستتالنات ودںوقل 322-یدق س-3882ق س ونقںت قیقک
ستتکم -ل ن اتق کشتتاہ دئ ک ک ڈیقےپچاس تدیعک جا قیچ-قسستت یہ یشتتن دئ یا
بنا وقل خااروںک دکہق س یس(ج دگر
کاوٹکئ بغیر د وںایکد ا ککدن ستتتتتتتترگرایدںشتتتتتتت
ک صتتتلاح رکدبہمرکر ایکق کرگرا بر قشتتترکر ق ق ا کعلاوکقد ند- یکستتتکم مکر قن
قک ا د ا،جد رولین ایتک باؤک حگرا جدکہداانکرقونآکست ی دستندییسسبپدنآکہکا
کاااہ قن ایک ک خصتتتتتدصتتتتت ات ن ائ قسک تیج ایکح ات ات ک-قستتتتتم کام کد۔فصتتتتتگن
ک گرا گرا بر قشتترد-ستتکم کراشتتتا دکحایتایکک باؤ ک صتتتلاح روقلکر ا ک
دگرک شتتتتتتتتناخرک یستتتتتتتتمک گن
-بہمریت شتتتتتتتتریایکب ک ن کدنیستتتتتتتتاتکستتتتتتتتاتکشتتتتتتتت
1
SECTION # 1 INTRODUCTION
2
1.1 Introduction
Sugarcane (Saccharum Officinarum) is a crop of immense agronomical value and due
to high sucrose content in it, that is cultivated in more than hundred countries and
it contributes almost 65 % of world sugar production (Carson and Botha, 2002).
According to Food and Agriculture Organization and United state department of
Agriculture stated that Asian countries contribute 37 % of the world’s total sugar
production while around 46 % of sugar consumption, confirming its role as a key
player in the global sugar market (FAO 2015; USDA 2015). In Pakistan, cane is a
valuable cash crop and plays an imperative role in the economy of the country and
it provides raw material to 81 sugarcane mills and thus employment to over 4 million
people with sugarcane. It is also reported that Pakistan Sugar Mills Association
(PSMA) and Ministry of National Food Security and Research (MNFSR), Marketing
year (MY 2017-2018), sugarcane produced 82 million tons with 9 % increase as
compared to 2016/2017.
Sugarcane yield and sugar recovery are the primary objectives of famers and sugar
industrialists. Unfortunately, Pakistan has low sugar yield and recovery rate as
compared to the international market. The sugar production depends mainly on
cane yield and percentage of recovery. Despite extension in sugarcane production
in Pakistan, average sugar recovery rate is reported 8.1 % only which is far beyond
from other developed countries (PSMA Report, 2019). On the other hand, altering
climate conditions along with low sugar recovery potential of sugarcane poses a
challenge to sugarcane industry.
3
The increasing threat of climate shift is already having an extensive effect on
agricultural production all over the world as heat wave causes substantial yield
decline with great risk for upcoming global food security (Christensen and
Christensen, 2007). However, there are so many biotic and abiotic factors such as
pests, diseases and high temperature are the major constrain to achieve high cane
production and sugar yield (Khalid et al., 2005). NASA, 2017 reported that increase in
temperature up to 0.99°C makes agricultural crops most susceptible and projected
to have negative affect on plant growth and development, resulting in wide spread
famine or food security will encounter in near future. It is projected that increase in
temperature due to climate change will have effect production (Neumeister, 2010)
negatively affect growth and development of sugarcane (Rasheed et al., 2011)
because Pakistan having wide range of temperature range and varied environmental
condition (Hameed et al 2013).
Heat stress effects the crop physiology, biochemical and morphological alterations
such as decrease in internode distance, shoot and root growth inhibition
(Hasanuzzaman et al., 2013) which disturb plant growth and development and may
lead to drop the crop yield, while temperature threshold for sugarcane growth and
development is 32-33°C (Wahid et al., 2007). Heat stress also affects the sugarcane
crop by reduced concentration of sucrose content, decline in shoot dry mass,
increased number of tillers and smaller internodes at greater than 40°C (Azevedo et
al., 2011). While, cultivars were usually indexed on basis of these agronomically
important traits for improvement of sugarcane yield and sugar content and
susceptible to environmental constrains (Abbas et al., 2013).
4
This poses a serious problem for achieving high yield and sugar recovery for industrial
sector and there is more demand for more potential for sucrose accumulation in cane
tissues (Datir and Joshi, 2016; Batta et al., 2011). It is reported that limited number
of cultivars with the low capability to store high sucrose contents are major cause for
low sugar recovery and it also depends on the ratio of sucrose synthesis to sucrose
cleavage (Office of the Cane and Sugar Board, 2013) regulated by sugar metabolizing
enzymes. Sucrose metabolism enzymes such as acid invertase, cytoplasmic invertase,
sucrose synthase and sucrose phosphate synthase play important role in primary
metabolism and plant development in parenchyma cells of sugarcane plant (Schaffer
and Quick, 2017; Batta et al., 2008; Botha and Black, 2000; Batta et al., 1995).
Invertase (E.C 3.2.1.26) involves in sucrose hydrolyses (Zhu et al., 1997), while
sucrose-phosphate synthase (E.C 2.4.1.14) synthesizes sucrose and sucrose synthase
(E.C 2.4.1.13) can either degrade or synthesize sucrose (Geigenberger and Stitt,
1993). Regarding localization, invertases are divided in to three subgroups, viz cell
wall, cytoplasmic and vacuolar (Ruan et al., 2010). Sucrose and its hydrolysis product
glucose and fructose not only provide energy to growing tissues but also function as
signals in regulation of gene expression and thus invertase activity at wrong time and
place can drastically affects plant viability and development (Xu et al., 1996). Various
physiological functions have been suggested for invertases, that is, to provide
growing tissue with reducing sugar (glucose and fructose ) as a source of energy, to
produce a sucrose concentration gradient and to partition sucrose between source
(leaf) and sink tissues (cane stalk), as well as to aid sucrose transport (Chandra et al.,
2012). In sugarcane, sugar (sucrose) is translocated through the phloem to the sinks,
where it is used for cell growth, metabolism, respiration or storage (Ayre, 2011; Wang
5
et al., 2013). These sucrose metabolizing enzymes in sugarcane genotypes are
diversely affected during different growth stages (Tana et al., 2014). It has been
investigated that accumulation of sucrose in sugarcane tissue is mainly dependent on
soluble acid invertase and sucrose phosphate synthase activities (Ansari et al., 2013),
which are adversely affected by climatic alterations. It has been reported that there
are substantial yield reductions observed at temperature more than 45°C, also
affecting the sugar recovery rate in sugarcane leading to huge economic losses (Mali
et al., 2014; Bonnett et al., 2006). This sugar reduction is due to the down regulation
of specific genes in carbohydrate metabolism which may lead to the altered activities
of sugar enzymes with compromised sucrose accumulation and sucrose synthesis
under heat stress (Ruan et al., 2010). There is contradictory opinion regarding the
association between the sucrose level and the activities of enzymes contributing to
sucrose accumulation in the culm (Botta et al., 2011). The growth of internodes
decrease along with reduced sucrose in sugarcane due to significant rise in
temperature (Bohnert et al., 2006). Heat stress inhibits the processes of
photosynthesis and respiration mainly by limiting electron transport and rubisco
activase activity (Sage and Kubien, 2007). Due to small difference in the temperature
requirement for cyclic reactions may result in large changes in the net amount of
sucrose, the temperature dependence of the overall process of sucrose accumulation
is unpredictable (Ebrahim et al., 1998). On the other hand, heat stress results in the
production of reactive oxygen species (ROS) such as, hydrogen peroxide (H2O2)
superoxide anion (O-2) and hydroxyl radical (OH)-, which are highly reactive and can
change the metabolism of plant through oxidative damage to membrane, denaturing
of protein and nucleic acids leading to cell death (Pastore et al., 2007). Moreover,
6
H2O2 is involved in disruption of various metabolic activities like calvin cycle (Akram
et al., 2012).
Although it is known that sugarcane is severely affected by heat stress, cellular,
biochemical, physiological and molecular mechanisms of the response of sugarcane
to heat stress is needed to be elucidate. In present scenario of global warming, the
major challenges for crop scientists is to produce new thermotolerant varieties to
fetch the growing population with improved sugarcane productivity and sugar
recovery. These sugarcane varieties development programmes are dependent on
biochemical, molecular and biotechnological strategies in order to develop new
varieties having important economic quality characteristics (Khanum et al., 2006;
Moore, 2005).
Due to present scenario, development of thermotolerant cultivars are the significant
approach in adaptation of climate change. In this regard, biochemical
characterization is fundamental step to identify cultivars with desirable agronomic
characters or traits to meet sugar industry requirements. The current study was
designed to determine the biochemical, physiological, molecular and quality
parameters analysis as well as expression of sucrose metabolizing enzymes in
sugarcane at formative, grand growth and maturity stages under heat stress
conditions. Thus, these findings can be helpful in providing information for
engineering sugar improvement along with thermotolerance in sugarcane varieties
and providing new avenues towards the economic development of the country.
7
1.2 Objectives This study was planned with the following objectives:
• To study the temporal expression of sucrose metabolizing enzymes and their
characteristic role in sucrose accumulation in different varieties of sugarcane under
heat stress.
• To elucidate the biochemical mechanism involved in sucrose production
underlying heat stress.
• To find out a genotype that produces maximum sucrose under heat stress.
• To evaluate the characteristic role of sucrose metabolizing enzymes isoforms
responsible for sugar metabolism under heat stress.
8
SECTION # 2 LITERATURE REVIEW
9
Literature Review
2.1 Importance of Sugarcane
Globally sugarcane production for the marketing year 2019-2020 is forecast 181
million metric tons (USDA, 2019). Among sugarcane producing countries, Australia,
India, China, Brazil and Pakistan are the top sugarcane producing countries. In
Pakistan, the sugar industry is the 2nd largest agro-base industry after textiles and it
is being cultivated on 1343 m hectors with production 83,333 tons and its production
accounts for 2.9 % value addition in agriculture sector and 0.5 % in overall Gross
Domestic Production (GDP) (Pakistan Bureau of Statistics 2018-2019). Sugar industry
plays a vital role in the national economy of the country. Five year statistic of
sugarcane production, sugar recovery rate, and yield presented in Table 1-2 (PSMA
annual report, 2018-2019).
Table 1. Five Year Statistics of Area, Can Production and Yield.
Year Area Hectares Production Tons Yield Tons/Hectare
2013-2014 1,171,687 67,427,975 57.55
2014-2015 1,113,161 62,794,827 56.41
2015-2016 1,130,820 65,450,704 57.88
2016-2017 1,216,894 75,450,620 62.00
2017-2018 1,340,926 83,289,340 62.11
Table 2. Five Year Statistics of Sugar Crushing, Production and Sugar Recovery Rate.
Year Cane crushing Tons Sugar Production Tons Recovery %
2013-2014 56,460,524 5,587,568 9.90
2014-2015 50,795,218 5,139,566 10.12
2015-2016 50,024,249 5,082,110 10.16
2016-2017 70,989,946 7,005,678 9.87
2017-2018 65,615,550 6,576,534 10.02
10
2.2. Phenology of Sugarcane
Plant development phases of sugarcane are germination (seedling), vegetative
(tillering), grand growth (elongation) and maturity (ripening) stages (Silva et al.,
2008). Optimum temperature for germination of sugarcane is about 28°C to 30°C
which begins from 10 to 35 days after planting in field conditions. The germination of
bud is influenced by biotic and abiotic factors especially high temperature (Farooq et
al., 2009) and affected early seedling growth in sugarcane and other plants (Wahid et
al., 2007). Vegetative stage (tillering formation) is a physiological process of repeated
branches of cane and it provides maximum stalk for higher yield commencing from
around 40 days and may last up to 180 days after planting. The optimum temperature
for this stage is 26.2 °C which is the most important for crop growth and
development. Grand growth stage is considered from 120 to 250 days after planting
of sugarcane, where cane formation and elongation take place. Under favorable
conditions stem grow swiftly almost 5 to 6 internodes per month. Its optimal
temperature from (26.2°C to 35.5°C) also plays imperative role during the active
growth stage (Samui et al., 2003). At maturity or ripening stage starts from 9 to 12
months with declined vegetative growth with maximum sugar accumulation in
sugarcane stem. Sugarcane ripening is very important in improving quality of
sugarcane but evaluated temperature due to climate change affect the sugarcane
natural ripening and quality (Gawander, 2007).
11
2.3. Heat Stress
Heat stress is defined as the increase in temperature away from a threshold level for a
period of time sufficient to cause irreparable loss to plant growth and development.
According to NASA the surface temperature increased by 0.8 °C worldwide (NASA'S
Goddard Institute for Space Studies, 2018). While the world metrological organization
reported that global mean temperature has increased by 1.1°C (WMO, 2019). Climate
changes partially and completely damaged regional crop production (Abdelrahman et al.,
2017; Lobell et al., 2011). A single degree increase from the threshold level is consider
heat stress (Hasanuzzaman et al., 2013) and may cause 2.5 % and 10 % in crop yield
reduction (Hatfield et al., 2011). As manifested by massive yield decline in many crops,
the increasing extreme impact of heat stress are putting global food security at high risk.
Global food productions must rise by 70 % to meet the demand of a projected increasing
in population growth to nine billion by 2050 (Stratonovitch and Semenov, 2015). High
population growth rate countries are working more aggressively to improving crop yields.
To mitigate high temperature stress and for the development of thermotolerant crop
varieties, it is very important to understand heat stress mechanisms in plant.
2.4. Effect of Heat Stress on Sugarcane
Climate related event such as carbon dioxide, temperature, precipitation are key factors
for sugarcane production (D. Zhao and Li, 2015). Heat stress may cause alteration in
physiology, morphology, as well as other biomolecules during the various growth stage
of crop. Mostly sugarcane propagated by setts, germination of can sett is badly affected
by high temperature. It is reported that reduced growth and water relation were evident
in sugarcane at temperature greater than 36°C (Wahid et al., 2007).
12
Optimum temperature of the sugarcane is between 8°C to 34°C but low temperature
declined the photosynthesis and leaf growth rate ultimately reducing yield (Gawander,
2007). High temperature results increased number of nodes, short internodes, higher
fiber content in stem and lower sucrose content (Bonnett et al., 2006). High temperature
also triggers the production of reactive oxygen species (ROS) which are strong oxidizers
and react with a large variety of biological molecules in plant cells. When these ROS and
antioxidants are in equilibrium, plant shows normal activities. However, any
environmental imbalance between ROS and antioxidants that causes oxidative stress to
plant cells. This oxidative stress may lead to molecular damages, such as improper
functioning of membranes and eventually plant cell death (Fig 1).
Fig 1: ROS accumulation in sugarcane plant cells as consequence of heat shock: These ROS
generated from different organelles such as mitochondria, chloroplast, endoplasmic reticulum,
peroxisome and extracellular side of cell membrane.
13
2.5. Effect of High Temperature on Growth and Development
Temperature plays a vital role in dry matter, transpiration, partitioning (Crawford et
al., 2012; Zhao et al., 2013) respiration and photosynthetic activity (Sage and
Kocacinar, 2012; Atkin and Tjoelker, 2003). Plants survive at optimal temperature
while high and low temperatures decline the growth rate (Sanchez et al., 2015; Ciais
et al., 2005). It has been investigated that the rise in temperature to optimal
thresholds effect biochemical mechanisms, causing altered slow growth and
development rates (Thornton et al., 2014; Cleland et al., 2007) imposing devastating
impact on crop yield (Chmielewski et al., 2004). The reproductive stage is crucial,
mostly effected by fluctuation of high temperature such as plant reproductive male
and female organs and seeds subsequently declining the pollen viability and yield
(Yang et al., 2018; Chao et al., 2017; Tashiro and Wardlaw, 1990). Crop yield is
negatively affected by high temperature due to biochemical changes, physical
damages and physiological distribution. For instance, yield some major crops are
declined such as, wheat (6.0 %), rice (3.2 %), maize (7.4 %), and soybean (3.1 %) (Zhao
et al., 2017), sorghum (44 %) (Tack et al., 2017) and sunflower (10 %) (Debaeke et al.,
2017), tobacco plants (Yang et al., 2018), oil palm, rapeseeds, barely, cassava and
sugarcane (Ray et al., 2019).
2.6. Physiological Indicators of Tolerance to Increased Temperatures
2.6.1. Cell Membrane Thermostability
Electrolyte leakage (EC) is a stress induce marker of membrane injury, has been used
successfully to quantity cell membrane thermostability due to various environmental
stresses (Demidchik et al., 2014; J. Lui et al., 2006). Cytoplasmic membranes are
14
considered the most sensitive components of all plant cells as they are the primary
sites for damage (Abraham Blum, 2018). During heat stress, the plants undergo
transition phase from solid-gel structure to flexible liquid-crystalline structure. This
denaturization of protein and rise in unsaturated fatty acids consequence in
enhanced fluidity of the cell membrane (Savchenko et al., 2002). The unsaturated
fatty acids are less rigidly packed with membrane due to non-linearity of fatty acid
chains (Horvath et al., 2012; Cyril et al., 2002) along with initiation of lipids base
signaling cascades, calcium2+influx and cytoskeleton reorganization (Bita and Gerats,
2013). Under stress conditions, organic and inorganic ion leakage from cells occurred
due to membrane damage. Overexpression of Ppexp1 gene in tobacco observed a
minimum EC and membrane lipid peroxidation damage than wild type plant (Xu et
al., 2014). Cell membrane thermo-stability has been successfully employed to
measure heat resistance in various crops (ElBasyoni et al., 2017; Khaushal et al., 2016)
(Table 3).
Table 3. Heat stress treatment used for the estimation of plant heat tolerant based
on the common ion leakage measurement (EC).
Plant Samples Heat Shock Treatments References
Cicer arietinum
Wheat Triticum aestivum
Zea mays
Arabidopsis thaliana
Oryza sativa
Nicotiana Tabacum
Agrostis capillaris
Cynodon transvaallensis
Festuca arundinacea
40°C /30°C d/n
30°C /40°C d/n
42°C /26°C d/n
44°C /22°C d/n for 2d
42°C for 2d
42°C for 10d
38°C /33°C d/n for 28d
42°C for 6h
35°C /30°C d/n for 1d
Kumar et al., 2013
Kumar et al., 2016
Naveed et al., 2016
Lin et al., 2015
Feng et al., 2015
Liu et al., 2016
Jespersen et al., 2016
Wang et al., 2016
Bi et al., 2016
SECTION 2 LITERATURE REVIEW
15
2.6.2. Accumulation of Compatible Solutes (Proline)
Proline is compatible solute that maintain the cell’s water environment and helps the
organism to sustain under severe environmental stresses during its life cycle (Singh
et al., 2015) and is correlated with degree of stress tolerance (Carline and Santos,
2009). Plants defense through various osmolytes such as trehalose, free proline and
glycine betaine and that contribute to maintain equilibrium in cellular structures like
protein, enzymes and cell membrane via hydrophilic interaction and hydrogen
bonding (Ahanger et al., 2014). Under stress conditions plants accumulate proline
content hundred times more than favorable conditions (Verbruggen and Hermans,
2008). There are various studies on proline accumulation in plants under different
stresses (Das et al., 2014) such as in drought (Anjum et al., 2017; Ajithkumar et al.,
2014; Anjum et al., 2017), salinity (Wang et al., 2004), osmotic (Conde et al., 2011),
heavy metal (Sharma and Dietz, 2006), temperature (Munns and Tester, 2008), light
and pesticides stresses (Ningthoujam et al., 2013). It has many functions such as it
acts as stabilizer for subcellular structures energy, signaling molecules and play role
in homeostasis of metabolic pathways under various stress conditions (Sharma et al.,
2011; Szabados and Savoure, 2010).
2.6.3. Malondialdehyde (MDA)
When ROS level increases due to heat stress, may lead to lipid peroxidation in
biological membranes, altering the fundamental properties of the membrane, such
as fluidity, loss of activity of enzymes and denaturing consequently cell death (Zafar
et al., 2018; Sharma et al., 2012; Gill et al., 2010). Among the oxidative damaging
indicators malondialdehyde (MDA) is one of them and it is a by-product of lipid
16
peroxidation under biotic and abiotic stresses (Hameed and Iqbal 2014; Aly et al.,
2012; Dallagnol et al., 2011). Maximum malondialdehyde (MDA) content was
exhibited in rice and wheat under high temperature stress respectively (Sanchez-
Reinoso et al., 2014; Savicka and Skute, 2010), also reported in rape seed oil (Kong et
al., 2016), Algerian Plants (Bentahar et al., 2016) and sugarcane (Abbas et al., 2014).
2.6.4. Hydrogen peroxide (H2O2)
Hydrogen peroxide (H2O2) is a chemical compound discovered in 1818 by Louis
Jacques and also a by-product of aerobic metabolism in plants (Mittler, 2002).
Reactive oxygen species (ROS) produced in plants and animal cells due to various
biotic and abiotic stresses (Sieas et al., 2017). In plants it produced in different
organelles such as peroxisome, chloroplast and mitochondria. Hydrogen peroxide
play imperative roles in plant physiological and developmental processes including
programmed cell death (Cheng et al., 2015), root development (Hernandez et al.,
2015), stomatal aperture regulation (Ge et al., 2015). H2O2 can be produced either by
enzymatically or non-enzymatically. There are different routes of hydrogen peroxide
production in plant cell, such as redox reaction and photorespiration. H2O2 is a
signaling molecules in normal condition in the signaling pathway which relates to
abiotic stress response. Many studies revealed that hydrogen peroxide could respond
to abiotic stresses such as cold (Orabi et al., 2015), high temperatures (Wu et al.,
2015). Heat stress which cause oxidative stress in both plants and animals (Kotak et
al., 2007).
17
3.1. Molecular Indicators of Tolerance to Increased Temperatures
3.1.1. Reactive Oxygen Species (ROS)
Reactive Oxygen Species (ROS) commenced in 20th century, initially it was defined as
intermediate organic and inorganic compounds. ROS are derivatives of oxygen, more
reactive than oxygen molecules under stress conditions (Mittler et al., 2017) and
mainly produced in different compartments of plant cell such as chloroplast,
mitochondria, peroxisomes, apoplast, cell wall, cell membrane and cytosol (Noctor
and Foyer, 2016). Among various forms of reactive oxygen species, singlet oxygen
(half life time 1 to 4 µ second) can oxidize lipids and proteins while superoxide with
same half life time of 1O2 and react with Fe-S proteins and hydroxyl radicals (half life
time 1 nano second) are extremely reactive and unstable (Waszczak et al., 2018) (Fig
2). While hydrogen peroxide are fair stable signaling molecules and treated by various
enzymes like catalases (CATs) and ascorbate peroxidases (APXs) in plant cell.
Fig 2: Free radicals are known to exist in crops: The Lewis diagram of these free radicals
presented in black, with unpaired electrons highlighted in red. The half-life time (t1/2) is
given for each type of radicals. Colour coded with highest value for H2O2 (red) and lowest
value for OH· (green). Abbreviations: (ms=milli second, µs=micro second, and ns= Nano
second).
18
3.1.2. Heat Shock Proteins (HSPs)
Heat shock protein was discovered by Italian scientist R. Ritossa in Drosophila
melanogaster and named as heat shock proteins (Tissieres et al., 1974). Under stress
conditions, plants produce cluster of proteins called heat shock or heat induced
proteins. Overall, many heat inducible genes are expressed that is called as heat
shock genes which encode heat shock proteins, essential for existence plant in heat
stress conditions (Charng et al., 2007). Several kinds of HSPs have been recognized
in many organisms (Bharti and Nover, 2002). In plants, according to their molecular
mass and their activities, five classes of heat shock proteins were characterized as:
HSPs60, HSPs70, HSPs90, HSPs100 and small heat shock proteins (Kotak et al., 2007).
The HSPs60 and HSPs70 normally conserved protein in nature and play vital role in
heat stress conditions (Kultz, 2003). HSPs70 present in different organelles such as
nuclear, chloroplast, cytosol, mitochondria and endoplasmic reticulum (Usman et
al., 2017) in barley crop (Landi et al., 2019) while HSPs90 in Arabidopsis thaliana
(Toumi et al., 2019). Heat shock protein play important role in the stabilization of
protein, avoiding the aggregation of polypeptide and facilitate the protein
maturation (Hartl et al., 2011). It is reported that HSP90 play vital role in plant
defense mechanisms (Bao et al., 2014), cellular homeostasis, growth and
reproductive and flowering development (Margaritopoulou et al., 2016).
19
4.1. Antioxidant Defense Response to Heat Induced Oxidative Stress
Thermotolerant plants have ability to protect against the damaging effects of reactive
oxygen species during stress conditions, which produces different enzymatic and non-
enzymatic biomolecules for detoxification of ROS (Apel and Hirt, 2004). These enzymatic
and non-enzymatic antioxidant defense system are as follows:
4.1.1. Enzymatic Antioxidants
In higher plants, ROS scavenging enzymes are catalase (CAT), ascorbate peroxidase
(APX), glutathione peroxidase (GPX), peroxiredoxins (Prx) and thioredoxins (Trx).
These antioxidant enzymes have various temperature ranges but the activity of
these enzymes increases at high temperature. It is reported that the activities of CAT,
APX and SOD were increased in high temperature while POX and GR declined their
activities in temperature ranges from 20°C to 50°C (Chakraborty and Pradhan, 2011).
This thermotolerance and susceptibility depend on crop varieties, growth phase and
growing season (Almeselmani et al., 2006).
4.1.2. Non-Enzymatic Antioxidants
Non-enzymatic antioxidants including ascorbic acid, tocopherol glutathione and
carotenes work with antioxidant enzymes against intracellular ROS, which may help
plant growth and development as well as strengthen the responses against harsh
environmental circumstances. Non-enzymatic antioxidants such as carotene,
tocopherol, ascorbic acid and glutathione defends against oxidative stress (Sairam
et al., 2000). Heat stress increased the glutathione level that enhanced the tolerance
of wheat crop under high temperature (Chauhan, 2005).
20
5.1. Plant Adaptation to Heat Stress
On the basis of temperature tolerance plant can be divided into fallowing groups:
(i) Psychrophilic: Plant grow at very low temperature range from 0°C to 10°C.
(ii) Mesophylls: Its moderate temperature between 10°C to 30°C for growing plant.
(iii) Thermophiles: Those plant can be cultivated between 30°C to 65°C (Zrobek, 2012).
Different plant species have different responses and resistance to heat stress. Another
scientist grouped pant species into three categories: (i) heat sensitive species (ii) relative
heat resistant species and (iii) heat tolerant species (Larcher, 1995).
6.1. Mechanism of Plant Adaptation to Heat Stress
6.1.1 Avoidance Mechanism
Plant as sessile organism which cannot move from one place to another, under high
temperature. Plants have developed different mechanisms to avoid any environmental
stress such as short term and long-term avoidance mechanisms. First long-term
mechanism which includes evolutionary phonological and morphological adaptations
and secondly, short term avoidance mechanism in which plants change leaf direction,
change lipid compositions and close stomata to prevent water losses (Srivastava et al.,
2012). Plants can also reduce heat intensity by protective waxy covering, leaf rolling.
Under heat stress physiological leaf rolling was evident in wheat plant (Sarieva et al.,
2010). During grand growth stage crops are extremely sensitive to heat stress. Good
agriculture practices like selecting proper sowing method (late or early sowing), crop
varieties can also be avoided against high temperature stress (Hall, 2011).
21
6.1.2. Tolerant Mechanism
Plants have various responses against different environmental stresses, which depend on
stress types, intensity and duration (Queitsch et al., 2000). Plants fight against any biotic
and abiotic stresses by through many tolerance mechanisms that includes expression of
heat shock proteins, accumulation of osmolytes, ion transporters and antioxidant
defense systems (Rodriguez and Borras, 2005; Wang et al., 2004). This stress responsive
mechanisms maintain the homeostasis and protect enzymes, protein and membrane
damages (Vinocur and Altman, 2005).
7.1. Sucrose Metabolism and Regulation in Sugarcane
The word sucrose was coined by William Miller in 1857. It is non-reducing sugar
(disaccharide) which synthesized by hexose sugar (glucose and fructose) that naturally
occurs in many plants such as sugarcane and sugar beets.
7.1.1. Biosynthesis of Sucrose in Sugarcane Plant
During photosynthesis, sugarcane leaves produce more carbohydrate (as
triphosphate) then it is converted to sucrose and transported to other compartment
of plant cells such as stalk or stem. Sucrose is the primary form of stored sugar in
sugarcane and sugar beet. Sucrose is synthesized at different compartment such as
plastid and cytosol. After condensation of two triphosphates to form fructose 1, 6
biphosphate), hydrolysis by fructose 1, 6 bisphosphates yields fructose 6-phosphate.
Sucrose 6-phosphate synthase then catalysis the reaction of fructose 6-phosphate
with UDP-glucose to form sucrose 6-phosphate. Mostly, the triose phosphate
generated by carbon dioxide fixation process is converted to sucrose as following
reaction:
22
Fig 3: Sucrose biosynthesis: Sucrose is synthesized from uridine diphosphate synthase
(UDP) glucose and fructose 6-phosphate, which are synthesized from triose phosphates in
the plant cell cytosol. The sucrose 6-phosphate synthase of most plant species is
allosterically regulated by glucose 6-phosphate and Pi.
7.1.2. Source-sink Regulation of Sucrose Accumulation in Sugarcane
Sucrose is accumulated in stem according to their capacity, supply of sources (leaves)
and sink demand. When source supply is not fulfilled for sink demand that is called
source limited plant. In contrast, when source supply exceeds sink demand, it is
called sink limited plant. Most of the sugar plants are in last category (sink limited)
(W. Patrick et al., 2013). Furthermore, high sucrose accumulating cultivars of
sugarcane had lower photosynthesis activity than low sucrose accumulating cultivars
(McCormick et al., 2008). Sugarcane has a typical source to sink system, sucrose
transported parenchyma and apoplastic pathway with concentration ranged from
400 to 700 nm (Welbaum and Meinzer, 1990; Moore and Cosgrove, 1991). Suberized
cell walls barrier prevent the apoplastic sucrose to reverse into phloem (Welbaum
et al., 1992). So, sucrose transporting from vacuole to apoplast is a controlling step
in sucrose accumulation. In juvenile internodes, 66 % of carbon utilizes for
respiration and synthesis of protein and fibers while 34 % of carbon stores as
sucrose, but reverse the case in mature internodes (Bindon and Botha, 2002). The
23
sucrose content depends on many enzymatic activities which play important role in
sucrose metabolism in sugarcane. In sugarcane, sucrose metabolizing enzymes are
sucrose phosphate synthase (SPS), sucrose synthase (SS) and invertases are
expressed in leaves and stem (Kalwade and Devarumath, 2014). Sucrose synthase
(SS) is key enzyme for sucrose synthesis playing dual role of hydrolysis or synthesis
but invertases only cleaves the sucrose into hexose sugar (glucose and fructose). The
sucrose phosphate synthase activity is higher in high sucrose content cultivars and
mature internode than low sucrose cultivars and immature internodes (Verma et al.,
2011). Sucrose synthesized in photosynthetic leave (source) is translocated through
phloem to stalk (Fig 4).
Fig 4: Schematic presentation of sucrose metabolism and transportation from source
(leave) to stem (sink).
24
8.1. Sucrose Metalizing Enzymes Response Under Heat Stress
In the past thirty years, quick development in understanding the dynamic of sucrose
metabolizing enzymes during leaf growth (Ruan, 2014). Sucrose metabolizing enzymes
are divided into two groups first group is hydrolyzed enzymes including sucrose synthase
and invertases which hydrolyzed sucrose irreversibly into glucose and fructose while
second is synthesis enzyme group such as sucrose phosphate synthase which sucrose
reversibly in the presence of uridine diphosphate (UDP) into uridine diphosphate-glucose
and fructose (UDPG-F), SPS is also responsible for sucrose synthesis in leaves (Wang,
2013).
8.1.1. Invertases
Invertase also called beta-fructofuranosidase (E.C.3.2.1.26). It is found commonly in
plants such as in pear, pea, grapes, oats and microorganisms like S.cerevisia, Candida
utilus and niger ect. In sugarcane, invertases are important for growth and
development (Moore, 1995; Zhu et al., 1997). There are different types of invertases
terms as isoform or isozymes codded by the different gene or one enzyme more than
one locus gene duplication. Sucrose transported from source (Leaf) to stem (sink)
through different cellular compartments cell wall, cytoplasm and vacuole (Ma et al.,
2000). Different invertase isozymes present in plant with different functions,
properties and beneficial role to the crop (Lahiri et al., 2012; Kim et al., 2011). Based
on subcellular localization, solubility, isoelectric point and pH plant invertase can be
divided into following groups.
8.1.2. Soluble Acids
In sugarcane two types of acid invertase have been reported based on solubility and
cellular localization.
25
8.1.2.1. Soluble Acid Invertase (Vacuolar Invertase)
Vacuolar acid invertase present on vacuole, has an acidic pH-4.5 to 5.0. It is believed
to be important in the regulation of hexose level in certain tissues, rate of return
sugar from storage, sucrose import, sugar signaling and remobilization of stored
sucrose from the vacuole (Sturm et al., 1999). High accumulation of sucrose directly
proportional to soluble acid invertase (SAI) in sugarcane plant during rapid growth
phase. The substrate of this enzyme is sucrose while metal ions such as mercury and
silver are inhibitors having molecular weight of is about 70 kDa, 80 kDa and 86 kDa
respectively (Hashizume et al., 2003). Vacuolar invertase also have more than two
isoforms, these isozymes can be characterized and purified from many plants such
as barley, pear, tobacco etc.
8.1.2.2. Insoluble Invertase (Cell Wall Invertase)
Acid invertase, glycosylated protein belongs to family GH32 and play significant role
in sucrose partitioning, growth and development (Roitsch and Gozale, 2004). It is
true member of β-fructofuranosidase, sucrose and raffinose as substrate (Belcarz et
al., 2002). Cell wall invertase is confined to the cell wall (ionically bond) and
exhibiting optimum activity at pH-3.2 to 3.6 and temperature 45°C with different
molecular weight ranges from 28 kDa to 64 kDa. The first cloned acid invertase was
found in carrot and tobacco (63 kDa), in tomato (68 kDa) (Klann et al., 1992; Konno
et al., 1993), Potato 58 kDa (Bracho and Whitaker, 1990) (Weil and Rausch, 1990). In
mung bean 70 kDa protein and subunit heterodimer 30 kDa and 38 kDa were
exhibited. Acid invertase dimer in barley 64 kDa (Avigad and Dey, 1997), rice 46 kDa
(Isla et al., 1995) proteins were observed. Cell wall invertase also act as gateway for
26
the entry of sucrose into the cell in juvenile tissues. It is found in maize seed kernel
and also found in seed coat of parenchyma cell.
8.2. Cytoplasmic Invertase (Alkaline/Neutral)
Cytoplasmic invertase is a non-glycosylated polypeptide but belong to the GH100
family (lammens et al., 2009) expressed at low level ranging from 54 kDa to 65 kDa
molecular weight at different growth and development stages. Cytoplasmic
invertase was localized in cytosol, mitochondria chloroplast and nucleus (Vargas and
Salerno, 2010) and involved in flowering, seed germination, growth and
development (Jia et al., 2008; Barratt et al., 2009). It is reported that cytoplasmic
invertase activities negatively correlates with sucrose content (Chandra and
Solomon, 2012). While, in inter nodal tissues it exhibited positive correlation with
sucrose concentration and isozymes of molecular weight (60, 120 and 240 kDa) were
reported (Vorster and Botha, 1998). It is antioxidant against reactive oxygen species
homeostasis (Xiang et al., 2011).
8.3. Sucrose Synthase (pH 7.5)
Sucrose synthase (SS) (EC 2.4.1.13) is very important sucrose metabolizing enzyme
belongs to subfamily glycosyltransferases and its protein are characteristically
considered homotetramers (Schmolzer et al., 2016). Sucrose synthase (SS)
expressed in vasculature of many plant species in phloem (Goren et al., 2017). SS
plays important role in high temperature in plants and may play signaling role in the
development of flowers (Cho et al., 2018). It is reported that SUS3 allele that is highly
expressed during seed maturity stage may confer resistance to chalky grain in brown
rice caused by heat stress (Takehara et al., 2018). Additionally, heat resistant sucrose
27
synthase was purified in wheat line (WH-1Q21). It catalyses the reversible hydrolyse
of sucrose into fructose in the presence of NDP-glucose (Kolman et al., 2015) as
following:
NDP-glucose + D-fructose ⇌ NDP + sucrose
Both NDP-glucose and D-fructose are substrate of this enzyme while D-fructose and
sucrose are products. The product of sucrose hydrolyzed by sucrose synthesis
enzymes are important for energy production, primary metabolites as well as starch
synthesis. SS is present in cytosol, cell wall, vacuole and mitochondria. The optimal
SS activity is pH 5.5-7.5 and with molecular mass of approximately 90 kDa. It is also
found in other plants such as., banana with 110 kDa (Yang and Su, 1980) Arabidopsis
with 107 kDa (Baud et al., 2004) wheat with 63 kDa (Verma et al., 2018), bean with
78 kDa (Fujii et al., 2010). When SS activity was reduced, growth and development
is affected with low tolerance against stress conditions. Whereas overexpression of
SS activity had shown increased starch content and growth, making SS high possible
candidate gene for the development of economical important crops. Activity of this
enzyme is regulated by two phosphorylation positions such as, serine
phosphorylation site at position 11 to 15 which is assumed to play important role in
membrane association and other site also serine, at about position 170 which is
supposed to regulate protein degradation (Hardin et al., 2003). In rice this
phosphorylation protein Rsus1-3 could promote sucrose synthases activity (Takeda
et al., 2017). Sucrose synthase isozymes have also been noticed in cell wall in various
plants for example, rice, carrot, tomato, sugarcane and cotton (Salnikov et al., 2003)
in tobacco pollen tubes (Persia et al., 2008). However, it is demonstrated that
sucrose synthase localized to the cell wall remains unclear (Brill et al., 2011). Under
28
stress conditions, sucrose synthase play important role in metabolism. Sucrose
synthase genes were found in various plants species, in rice 6 genes (Hirose et al.,
2008) in grapes and sugarcane 5,5 genes are characterized (Zhang et al., 2013; Zhu
et al., 2017). Sucrose synthase genes exhibited in apples (Tong et al., 2018) while 30
in Chinese-pear (Abdullah et al., 2018). It activity was noticed in in delayed ripening
in straw berry fruits (Zhao et al., 2017) and in immature internodes in sugarcane stalk
(Schafer et al., 2004).
8.4. Sucrose Phosphate Synthase (pH 7.5)
Sucrose phosphate synthase (SPS) (EC 2.4.1.14) is the most important enzyme
among sucrose metabolizing enzymes. Sucrose phosphate synthase (SPS) and
Sucrose synthase (SS) activity declines at increasing temperature of 42°C in
sugarcane (Gomathi et al., 2013). Sucrose phosphate synthase found in both
photosynthetic and heterotrophic tissues such as leave, stem, roots and nodules
(Aleman et al., 2010; Haigler et al., 2007). Sucrose phosphate synthase has very
important role in growth and development for many plants such as maize plant
(Causse et al., 1995a) and its activity associated with dry mass yield (Causse et al.,
1995b). In transgenic rice its activity is directly proportional to growth rate (Ishimaru
et al., 2004), in sugarcane non reducing sugar (sucrose) accumulation in the stalk
depends on sucrose phosphate synthase (Zhu et al., 1997). SPS also present in alfalfa,
pea (Aleman et al., 2010), it is also reported that Arabidopsis has 4 genes encoding
SPS enzymes (Lunn and MacRae, 2003; Langenkämper et al., 2002). SPS crucial role
in water stress condition and low temperature in potato crop (Krause et al., 1998;
Geigenberger et al., 1999). However, it activity gradually decreased at 28°C but it
inactivated when temperature at 60°C by heat stress (Neliana et al., 2019).
29
8.6. Sugar Recovery Rate
High population growth rate countries are working more aggressively to improving
crop yields. Sugar recovery rate is the percentage (%) of sugar production in metric
ton to the sugarcane crushed in metric ton. Sugar recovery is a significant parameter
for both farmers and industrialist (Costa et al., 2014). Quality cane depends on some
characteristics such as sugar recovery rate, high °brix, pol and high sucrose content
in stem while minimum non-sugar and optimum fibers. But the other factors like
climate change adversely affects on sugar recovery rate. Recovery rate of Pakistan is
9.87 to 10.02 % (PSMA report, 2019). Which is less than other sugarcane producing
countries. Average sugar recovery rate countries are Brazil (14.6 %), Australia (13.8
%, European Union (13 %), USA (11.7 %) Mexico (11.6 %), Egypt (11.5 %), Thailand
(11.3 %) and India (10 %) (Roy et al., 2018; PSMA report, 2007). Furthermore, there
are many other factors which also contribute to decline sugar recovery rate. For
instance, late harvesting, transportation, storage, processing and improper hygienic
conditions etc. Soon after harvesting sugar recovery rate start to decline, after 24 h
is consider reduction in cane weight due to moisture loss, reduction in sucrose
content due to sucrose hydrolyse (Roy et al., 2018). This is one of the most
challenging factors for sugarcane producing countries (Suma et al., 2000). It is
suggested that due to staling of canes for 4 days, there is reduction in sugarcane
mass (7.4 % to 17.0 %) (Datir and Joshi, 2015), recovery rate 2 % (Rakkiyappan et al.,
2009). So, improvement in sugar recovery rate is very important with high sugarcane
yield against biotic and abiotic factors.
30
9.1. The Mitigation of Heat Stress Strategies
Variation in environment has long lasting impact on agriculture and food security
worldwide. Food security is threatened by global warming and severe climate conditions.
This changing global temperature is challenging for crop scientists to combat this
problem. Therefore, to cope theses changing climate, development of thermotolerance
crop varieties against high temperature stress are required.
9.1.1. Cultural Method
There are so many agronomical practices to combat the climate change such as changing
sowing and harvesting time, crop rotation, irrigation techniques and variation in cropping
scheme. Under heat stress condition these approaches are useful for crop adaptability
Deligios et al., 2019; Duku et al., 2018). So the choice of suitable sowing and harvesting
time, planting density, best irrigation practices are essential techniques to tackle any
environmental stress conditions (Battisti et al., 2018). Fertilizer also play very important
role under stress conditions, it provides to support better adoptability, to provide energy
to maintain the soil fertility and increase the crop productivity (Henderson et al., 2018).
Under high temperature stress, macronutrients (k, Ca) and micronutrients (B, Se and Mn)
regulate stomata functions and also activate the physiological and metabolic activities
contributing to maintain high water potential in tissues and reduce toxicity to ROS by
enhancing the activity of antioxidant enzymes in plant cell ( Waraich et al., 2012).
Under any environmental stress conditions, plant breeding is the best techniques for
enhancement of crop productivity. It provides potential to guarantee food security,
escape from stress through a crucial growth and development phases by developing
stress resistant cultivars (Abraham Blum, 2018). For this, genetic divergent analysis is
31
considered a significant method for the improvement of new varieties based on genetic
distance and similarities (Raza et al., 2019; Raza et al., 2018).
9.2. Genetics and Genomics Strategies
9.2.1. Omics-Led Breeding and Marker-Assisted Selection (MAS)
Omic approaches provide valuable resources to elucidate biological functions of any
genetic information for crop progress and growth (Stinchcombe and Hoekstra, 2008). The
breeding program is attached with genomic approaches to succeed great heights in
molecular breeding and to screen elite germ plasms with multiple trait assembly (Bevan
and Waugh, 2007). Genomic also allows exploration of the molecular mechanism
underlining the abiotic stresses resistance. These approaches assistance in the
improvement of climate smart crops for high yield and production under high
temperature stress (Roy et al., 2011). With the initiation of high quantity of sequencing
and genomic led breeding paved the way for recognizing various stresses that are
projected to badly affect on crop production. In addition, the data available on many
environmental stresses, DNA fingerprinting and qualitative trait loci (QTL) mapping
permits the screening of best germplasms under heat stress (Kotle et al., 2015). QTL
dissention of yield related characteristic under high temperature stress allows the
development of new varieties with better adaptability in abiotic stress (Collins et al.,
2008). Molecular plant breeding is very important approach for improving crop yield
(Gosal and Wani, 2018). For the immediate breeding progression marker assisted
selection (MAS) presents a fundamental part in the improvement of crop trait and yield.
With the development in crop genomics, DNA markers have been recognized which are
valuable for marker-assisted breeding (Da Silva, 2014).
32
9.2.2. Genome Wide Association Studies (GWAS) for Stress Tolerance
Genome wide association studies is a powerful tool for understanding the whole set of
genetic variants in different crop varieties to identify allelic variants connected with any
particular characteristic (Manolio, 2010). Genome Wide Association Studies (GWAS)
mostly highlight association among SNPs and characteristic and based on GWAS design,
genotyping tools, statistical models for examination and results interpretation (Bush and
Moore, 2012). In many crop GWAS has been carried out to exploit the genetic process
responsible for genetic resistant under heat high temperature stress conditions
(Mousavi-Derazmahalleh et al., 2019). In plants, GWAS has extensive applications
associated to environmental stresses. GWAS have been applied to describe salt tolerance
(Wan et al., 2017), drought tolerance (Thoen et al., 2017) and thermo tolerance (Lafarge
et al., 2017).
9.2.3. Genetic Engineered Plants for Stress Tolerance
The genetic alteration via biotechnology is an important tools and powerful strategy for
the improvement of plant against biotic and abiotic stresses. Encouraging data collected
from genetics which can be exploited significantly to numerous biotic and abiotic stresses
including heat stress. Identification of stress response transcription factors are influential
discoveries to improve thermotolerant crop varieties. These transcription factors can
control the phenotypes of genes in genetic engineered crops associated with different
environmental stresses (Reynolds et al., 2015). There are many transgenic plants which
have been recognized by genetic engineering to tackle the various environmental
stresses. These transgenic plants revealed important resistance under heat stress
conditions as compared to normal plants (Nejat and Mantri, 2017; Shah et al., 2016).
33
Various plant specific TFs are identified such as AP2/ERFBP group (Riechman and
Meyerowits, 1998). This family or groups of TFs is responsible for plant growth and
development pathway and has functions in different ecological stresses (Licausi et al.,
2010). The subfamilies of TFs including DREB (dehydration-responsive element-binding
protein) and ERF play vital role under biotic and abiotic tresses conditions (Phukan et al.,
2017).
9.3. Genome Editing Strategies
Genome editing (GE) is very important tool to manipulate the plant genome by means of
sequence-specific nucleases. This GE tools use for crop development has the
extraordinary capability to tackle food insecurity and advances in climate-smart
agriculture system (Liu et al., 2013). It has been using for many years due to fast and
precise manipulation in crop genomes to defend them against various environmental
stresses including heat stress (Taranto et al., 2018). This genome editing tools including
Crisper-case9, zinc-finger nucleases (ZFNs) and transcription activator like effector
nucleases (TALENs) (Zhu et al., 2017). This crisper-case9 has been used in many plant
genome editing to survive against different stresses (Manolio, 2010). Many plant
manipulated by crisper-case9 gene editing tools against different stresses, 21 KUP genes
identified against stress conditions in cassava (Ou et al., 2018), herbicides resistant was
developed in rice (Shen et al., 2017) and enhance the seed size in wheat (Wang et al.,
2018). To develop new thermotolerant crop varieties regarding global climate change,
there is urgent need to study about thermotolerance mechanisms at physiological,
biochemical and molecular levels. In present scenario, we must understand heat stress
mechanism for avoiding heat shock induced detrimental changes that play a vital role in
34
crop survival. In this situation of global warming, the major concern for plant scientist is
to develop new cultivars or varieties resistant to biotic and abiotic stresses (Zhang et al.,
2005). In the coming decade, due to high temperature, crop yield will decline but
constant growing population, will increase food demand, that create a gap between the
current crop yield achievement and yield potential (Koevoets et al., 2016). There are
many molecular techniques that have been used to develop high yielding and
thermotolerant crop varieties. Present study analysis effect of heat shock for sucrose
metabolizing enzymes that might reveal ways to develop thermotolerance cultivars that
vital in adverse environmental conditions.
35
SECTION # 3 METHODOLOGY
36
Methodology
3. General Experimental Details
This study was performed using two local sugarcane cultivars S2003-US-633 (high
sucrose accumulation) and SPF-238 (low sucrose accumulation) provided by “Cane
Testing Lab” Mehran Sugar Mills Limited. Tando Allahyar, Pakistan. All the
experiments were conducted Plant Care Unit, Agricultural Biotechnology Section, The
Karachi Institute of Biotechnology and Genetic Engineering (KIBGE), University of
Karachi, Karachi, Pakistan. Experiment was conducted in Completely Randomized
Design (CRD) with three replicates per treatment group at vegetative (30 to 50 days),
grand growth (150 days) and maturity stages (250 days). All chemicals and reagents
of either analytical or optical grades were purchased from Aldrich, Bio-Rad, BDH,
Fluka, Merck, Scharlau and Sigma. Crop was cultivated in February, 2016 and
morphological analysis was measured along with determination of thermo tolerance
indicators such as proline, electrolytes leakage, lipid per oxidation malondialdehyde
(MDA), hydrogen peroxide (H2O2). For second year 2017, sugar metabolism in terms
of total sugar, reducing sugar, non-reducing sugar, total soluble protein, sucrose
metabolizing enzymes sucrose phosphate synthase (SPS), sucrose synthase (SS) and
Invertase isozymes such as cytoplasmic invertase (CyIN) cell wall invertase (CWIN)
and vacuolar invertase (VIN) as well as sugar recovery was quantified. During last year
2018 invertase isozymes were analyzed through Native-PAGE and differential staining
for proteins SDS-PAGE electrophoresis was done, in addition to confirmation of
previous year’s results. The experiments were conducted consecutively for three
years 2016 to 2018.
37
3.1. Physiochemical Properties of Soil and Water
3.1.1. Soil and Water Analysis
Table 4: Detail Physiochemical Properties of Soil and Water Used in Experiment.
Year 2016 2017 2018
Soil texture Clay (%) Silt (%) Sand (%) Soil textural class EC (dSm
-1)
pH Fertilizer Value NO
3-N
P K Water properties EC (dSm
-1)
pH
11.6 12.4 74
Sandy loamy 0.36 7.92
1.23 2.91 192
5.9 7.6
10.5 13.8 75
Sandy loamy 0.38 7.81
1.20 2.82 190
6.2 7.9
12.2 11.3 78
Sandy loamy 0.40 7.75
1.30 3.10 193
6.6 8.0
3.2. Crop Husbandry
3.2.1. Cultivation of Sugarcane
Before sowing, the sugarcane sets were kept in hot water at 50 C temperature for
two hours (h) to control red-rot, grassy shoots and other virus diseases and to
improve germination. Sugarcane were cultivated in pots filled with 20 kg loamy soil
and 5 kg farm yard manure (FYM). All agronomic practices were done such as
application of fertilizers (NPK), weeding and regularly application of supplement with
half strength nutrients (Hoagland and Arnon, 1950). During experiment, temperature
and relative humidity were recorded on daily bases, at vegetative (50 days), grand
growth (150 days) and maturity stages (250 days) (Fig 5).
38
Figure 5: Annual mean temperature and relative humidity of sugarcane field for the year
2016-2018.
3.3. Heat Stress Treatments
All sugarcane plants were shifted to growth room for heat shock treatments. Where,
white fluorescent tube lights/mercury lamps were used for maintaining
photosynthesis active radiation (PAR) ranging from 650 to 700 µmol m-2 s-1 in long
day conditions (16 h light/8 h dark). Temperature was set at 45±2°C and 34±2°C for
day and night respectively while humidity was maintained at 60% to 70 %. For air
circulation, fans were adjusted. Heat shock treatments at 45±2°C for 24 h (T24), 48h
39
(T48) and 72 h (T72) along with control (30±2°C) at different time intervals were
imposed at vegetative (50 days) grand growth (150 days) and maturity stage (250
days) after planting. The plants were then allowed to recover. For recovery
experiment, pots were again shifted from growth room to field at 30±2°C for 24 h
(R24), 48 h (R48) and 72 h (R72).
3.4. Sample Collection
Samples were collected for control, heat stress and recovery treatments at all
growth phases (vegetative, grand growth and maturity) and stored at -80°C for
further analysis.
3.5. Morphological Analysis
The morphological observations were recorded in replicates from each treatment as
shoot length (cm), root length (cm), leaf length (cm), leaf width (cm), stem diameter
(cm), number of leaves, number of tillers, number of nodes, number of internodes
and internode distance (cm).
Fresh weight of shoot was determined on electronic weighing balance immediately
after harvesting while dry weight was taken after drying shoot from oven at 60 °C
for a week. Measurement for shoot, root, leaf, stem diameters and internode
distances were taken using meter scales.
40
3.6. Physiological Analysis
Following physiological parameters were analyzed for assessing heat stress imposed
damages in sugarcane cultivars at different growth stages.
3.6.1. Cell Membrane Thermosability (CMT)
Relative membrane permeability (RMP) in terms of % measured by assessing
electrolytes leakage (EC) using the method by Yang et al., (1996), with the help of
electrical conductivity meter. Fresh leaves (500 mg) cut into small pieces and soaked
in 25 ml distilled water then vortexed for 20 to 30 seconds. Initial electrical
conductivity (EC₀) was measured within 10 min while (EC1) measured after
incubation of tubes for overnight at 4°C then tubes were autoclaved for 20 min, all
tubs were placed at room temperature till it cooled down and (EC2) was measured,
same steps were repeated three times.
Calculation
The electrolyte leakage was calculated as following formulae;
RMP(%) =EC1 − EC₀
EC2 − EC₀ × 100
3.6.2. Proline Quantification
Reagents
Ninhydrin: 1.25 g ninhydrin was dissolved in 30 ml glacial acetic acid and 20 ml 6 M
ortho phosphoric acid.
3 % Aqueous Sulphosalicylic Acid (SSA): 3 g of sulphosalicylic acid was dissolved in
100 ml deionized water.
Proline (Osmolyte accumulation) was determined in sugarcane using method by Bate
41
et al., (1973). Leaf tissues (100 mg) were extracted in 2 ml sulphosalicylic acid with
mortar and pestle. The residue was removed by centrifugation at 12000 rpm for 10
min. Supernatant was used for estimation of proline. In 1 ml aliquot, 1 ml ninhydrin
reagent, 1 ml acetic acid were added, the mixture then heated at 100 °C for 1 h and
transferred on ice bath for termination of reaction. The reaction mixture was
extracted with 4 ml toluene then vortexed for 20 to 30 seconds and kept for 30 min
at room temperature, upper phase (toluene layer) was separated in a dry glass tube
then the absorbance or intensity at 520 nm using spectrophotometer was measured,
while toluene was used as a blank. Standard curve of proline ranging from 10 µg to
50 µg / 2 ml was constructed and slope value was used in formula for calculation.
Calculation
The Proline was calculated as following formulae;
Proline mg ml −1 =Slope × Absorbance
Extract Used (ml)
Proline (uMg−1FW) =µg Proline × 115.5 × Volume of Extract
Toluene (ml) × Sample(g)
Note: (Where 115.5 is the molecular weight of proline)
3.6.3. H2O2 Quantification
Reagents
0.1 % w/v TCA: 0.01 g trichloroacetic acid (TCA) dissolved in 10ml water.
10 mM phosphate buffer (pH 7.0): Disodium phosphate (0.141g) and monosodium
phosphate (0.119 g) dissolved in 100ml distilled water. Mixed both solution and
adjusted pH at 7.0.
42
1 M Potassium iodide (KI): 16.6 g Potassium iodide dissolved in 100ml distilled water.
Hydrogen peroxide (H2O2) content was assayed by the method of (Jessup et al., 1994).
For this 0.1 g sugarcane leaf was homogenized in 2 ml 0.1 % trichloro acetic acid in
mortar and pestle. The extract was centrifuged at 12000 rpm for 15 min. From
supernatant, an aliquot of 0.5 ml is added to 1 ml of phosphate buffer and 1.5 ml of
potassium iodide. The mixture was vortexed for 15 to 20 seconds then its absorbance
was measured at 390 nm. 2ml of potassium iodide and 1ml potassium buffer were
used as blanks in the absence of leaf extract. Standard curve of H2O2 was constructed
using different concentration of H2O2 standards. Results were expressed as μmol
H2O2 g-1 fresh weight.
3.6.4. Determination of lipid peroxidation
Reagents
5 % Tetracholoroacitic acid (TCA): 0.5 g of TCA was dissolved in 10 ml distilled water.
0.5 % Thiobarbituric acid (TBA): 0.05 g of TBA was dissolved in 10 ml distilled water.
To estimate the malondialdehyde (MDA) content, byproduct of membrane lipid
peroxidation was quantified as described by (Heath and Packer, 1968). Fresh leaf of
sugarcane (100 mg) was homogenized in pestle and mortar in 2 ml tetracholoroacitic
acid solution. After centrifugation at 12000 rpm for 15 min, 1 ml supernatant was
mixed with 1 ml thiobarbituric acid then kept in water bath for 30 min at 95 C. The
mixture was allowed to cool rapidly on an ice bath and again centrifuged at 10,000
rpm for 10 min then absorbance of MDA was measured at 532 nm. The value of non-
specific absorbance at 600 nm were subtracted by using spectrophotometer, 5 % TCA
used as blank.
43
Calculation
Lipid peroxidation was expressed as nmole g-1 FW using the formula;
MDA =A532 − A600
15500 × 106
3.7. Biochemical Analysis
Sugar analysis in terms of total, reducing and non-reducing sugars were carried out
from both sugarcane varieties from heat shock, recovery along with control
treatments at all growth stages.
3.7.1. Sugar Extraction
For extraction of reducing and total sugar, 100 mg of sugarcane leaves were
homogenized in 5 ml of 80 % ethanol.
3.7.2. Total Sugar Estimation
Reagents
Standard sucrose stock—10 mg sucrose dissolved in 10 ml distilled water.
Working standard: 1 ml stock solution was added into 3 ml water (1:3) for working
standard and diluted as (25 to 250 µg ml-1).
Anthrone reagent: 100 mg anthrone was dissolved in 50 ml of ice-cold 95 %
hydrochloric acid (H2SO4).
Total sugars were determined by using Anthrone reagent method (Hedge and
Hofreiter, 1962). The homogenate was centrifuged at 1000 rpm for 10 min and used
for the estimation of total sugars. Volume was made up 975 µl distilled water added
in 25 µl supernatant of ethanol extraction. 5ml of anthrone reagent was added and
the reaction mixture was heated for 15 min in a boiling water bath, cooled rapidly
44
and vortexed. The absorbance of the green colour solution was measured at 620 nm
by using spectrophotometer. Sucrose was used as standard. The total sugar content
was expressed in terms of percentage on fresh weight basis.
Calculation
Total sugar was calculated as µg ml-1 using the formula:
Total sugar (µg ml−1 ) =ODT × Conc Std (µg)
OD Std × AS
whereas,
ODT= Optical Density of Test (Sample)
Conc std= Concentration of Standard
OD std= Optical Density of Standard
AS= Amount of Sample
3.7.3. Reducing Sugar Estimation
Reagents
0.5 gm DNS: dissolved in 25 ml distill water on stirring. After solubilizing 15 gm
potassium sodium tartrate was added after completed solubalization milky yellow
solution was obtained.
0.8 gm NaOH: dissolved in 10 ml of distill water and added NaOH solution in to DNS
solution it was turned into clear orange solution made volume up to 50 ml then kept
in refrigerator in dark bottle.
Standard Stock: 0.05 gm glucose dissolved in 50 ml distilled water and diluted (0.1
to 1.0 mg ml-1).
Reducing sugar was estimated by using DNSA (dinitro salicylic acid) reagent method
45
(Miller, 1959). The homogenate was centrifuged at 1000 rpm for 10 min and used
for the estimation of reducing sugar. 1 ml DNS was added with 1ml reaction mixture
and kept in a boiling water bath for 5 min. After cooling 9 ml distilled water was
added and then vortexed. The absorbance of the orange colored solution was
measured at 546 nm using spectrophotometer. Glucose was used as standard. The
reducing sugar content was expressed in terms of percentage on fresh weight basis.
Calculation
Reducing sugars was calculated a mg ml-1 using the formula:
Reducing sugar (mg ml−1 ) =ODT × Conc Std (mg)
OD Std × AS
3.7.4. Non-Reducing Sugar Estimation:
Non-reducing sugar was estimated by following formula;
Non reducing sugar ( mg ml−1) = Total sugar − Reducing sugar
3.7.5. Total Soluble Proteins and Sugar Metabolizing Enzymes
Extraction:
Reagents
500 mM MOPS: 10.465 gm MOPS dissolved in 100 ml deionized water.
15 mM MgCl2: 0.914 gm MgCl2 dissolved in 300 ml deionized water.
10 mM EDTA: 0.371 gm EDTA dissolved in 100 ml deionized water.
10 mM DTT: 0.462 gm DTT dissolved in 300 ml deionized water.
Bovine serum albumin (BSA): 0.25 gm BSA dissolved in 50 ml deionized water (freshly
prepared).
0.05 % (v/v) Triton X-100
46
Working solution:
50 ml MOPS, 16 ml MgCl2, 50 ml EDTA, 125 ml DTT, 50 ml BSA and 0.25 ml Triton X-
100 were added and adjusted pH through NaOH and stored at 4°C.
3.7.5.1. Extraction
Sucrose metabolizing enzymes were extracted by buffer containing, MOPS-NaOH
(pH -7.5), MgCl2, EDTA, DTT, Triton X-100 and Bovine serum albumin (BSA). For this
100 mg tissues were homogenized in 2 ml buffer and then centrifuged at 12000 rpm
for 30 min. Supernatant were separated into other tubes and then stored at -20 for
further biochemical analysis.
3.7.5.2. Total Soluble Protein Quantification
Reagents
Coomassie Brilliant Blue (CBB) G-250 dye: 0.01 g CBB (G-250) was dissolved it in 5
ml 95 % ethanol. 10 ml of orthophosphoric acid was added to make the volume up
to 20 ml with deionized water. Reagent was filtered with Whatman paper # 01 and
stored in dark bottle at 4°C.
0.15N NaCl: 0.87 g NaCl was dissolved in 100 ml deionized water.
Bovine Serum Albumin (BSA): 0.01 g of bovine serum albumin was dissolved in 10
ml water.
Total soluble proteins were calculated through Bradford Assay (Bradford, 1976). The
3 ml reaction mixture containing, 50 µl protein sample, Bradford dye 150 µl and 0.15
N NaCl 2800 µl vortexed for 5 to 10 seconds then incubated at room temperature
15-20 min and read at 595 nm by spectrophotometer. Protein standard curve was
constructed by using known concentration of BSA (10 to 100 µg ml-1).
47
Calculation
Total soluble protein was calculated as mg ml-1 using the formula;
Protein (mg ml−1 ) =ODT × Conc Std (mg)
OD Std × AS
3.8. Quantification of Sugar Metabolizing Enzymes
3.8.1. Vacuolar Acid Invertase (VAI) pH 5.0
Unit Definition:
One unit is defined as that quantity of vacuolar invertase that will convert or
generate sucrose to 1.0 µmol of glucose per minute at pH 5.0 at 37 °C under assay
conditions.
Principle
Sucrose + water → Glucose + Fructose
Reagents
0.05 M potassium-acetate buffer (pH 5.0): 4 g sucrose was dissolved in 100 ml
acetate buffer (freshly prepared).
DNSA reagent (1.6 % NaOH): (As mentioned in reducing sugar protocols).
Soluble acid invertase activity was assayed using modified method of (Hatch et al.,
1963) and (Voster and Botha, 1999). The assay medium consisted of 0.05 M
potassium-acetate buffer (pH 5.0), 250 µl 4 % sucrose, 500 µl plant extraction and
250 µl deionized water. All control reactions tubes were stopped at 0 minute with 1
ml of (1.6 % NaOH, DNSA reagent) before incubation. The assay mixture was
incubated at 37 °C for 1 h. After that reaction mixture was neutralized with 1 ml of
(1.6 % NaOH, DNSA reagent) and heated at 100 °C for 30 min in a boiling water bath.
After cooling, 9 ml of water added in each tub then absorbance was measured at
48
540 nm using spectrophotometer. The blank tube contained all mixture excluding
enzyme. The enzyme activity was measured as glucose mg ml-1 protein mint-1 .
Calculation
Vacuolar acid invertase was calculated µmole ml−1 using the formula;
Vacuolar invertase (units ml−1min−1) =ODT × Conc Std (mg)
OD Std × AS × MW × RT × TSP
whereas,
ODT=Optical Density of Test (Sample)
Conc std=Concentration of Standard
OD std=Optical Density of Standard
AS=Amount of Sample
MW=Molecular Weight
RT=Reaction Time
TSP=Total Soluble Protein
3.8.2. Quantitative Analysis of Cell Wall Invertase (CWI) pH 3.5
Reagents
0.05 M potassium-acetate buffer (pH 3.0): 4 g sucrose was dissolved in 100 ml
acetate buffer (freshly prepared).
Unit Definition:
One unit is defined as that quantity of cell wall invertase that will convert or generate
sucrose to 1.0 µmol of glucose per min at pH 3.0 at 37 °C under assay conditions.
Principle
Sucrose + water → Glucose + Fructose
49
The activities of cell wall bond invertase using with modified procedure by (Hatch et
al., 1963). The assay medium consisted of (250 μl) 0.05 M potassium-acetate buffer
(pH 5.0) with 4 % sucrose, (250 μl) supernatants and (500 μl) deionized water. The
pellet was washed with extraction buffer and re-suspended in 2000 μl of MOPS-
NaOH (pH-7.5), kept for overnight at 4°C. Then aliquot was used for activity assay in
1 ml containing acetate buffer (pH-3.5) with 4 % sucrose as a substrate (250 µl), for
control and blank (excluding 4 % sucrose buffer) were used. The reaction mixture
was incubated at 37 °C for one hour. All reactions were terminated by added 1 ml of
(1.6 % NaOH, DNSA reagent) after incubation, boiling at 100°C in water bath for 5
min. After cooling at room temperature, samples were re-centrifuged at 14000 rpm
for 5 min and absorbance was taken at 540 nm using known glucose standard curve.
Calculation
Cell wall invertase was calculated µmole ml-1 using the formula;
Cell wall invertase (units ml−1min−1) =ODT × Conc Std (mg)
OD Std × AS × MW × RT × TSP
3.8.3. Quantification of Cytoplasmic Invertase (CyIN) pH- 7.0
Unit Definition:
One unit is defined as that quantity of neutral invertase that will convert or generate
sucrose to 1.0 µmol of glucose per minute at pH 7.0 at 37 °C under assay conditions.
Principle
Sucrose + water → Glucose + Fructose
Reagents
0.1 M Potassium-phosphate buffer (pH 5.0): Solution # A: 1.74 g K2HPO4 was
dissolved in 100 ml deionized water and solution # B: 1.36 g KH2PO4 sucrose was
50
dissolved in 100 ml deionized water and both solution mixed and adjusted the pH 7.0.
DNSA reagent (1.6 % NaOH): (As mentioned in reducing sugar protocols)
Procedure
The assay medium consisted of 0.1 M potassium-phosphate buffer (pH 7.0), 250 μl
4 % sucrose, 500 μl plant extraction and 250 μl deionized water. All control reactions
tubes were stopped at 0 min with 1 ml of (1.6 % NaOH, DNSA reagent) before
incubation. The assay mixture was incubated at 37 °C for 60 min. After that reaction
mixture was neutralized with 1 ml of (1.6 % NaOH, DNSA reagent) and heated at 100
°C for 30 min in a boiling water bath. After cooling, 9 ml of water added in each tub
then absorbance was measured at 540 nm using spectrophotometer. The blank tub
contained all mixture excluding enzyme.
Calculation
Cytoplasmic was calculated µmole ml-1 using the formula;
Cytoplasmic invertase (units ml−1min−1) =ODT × Conc Std (mg)
OD Std × AS × MW × RT × TSP
3.8.4. Quantification of Sucrose Phosphate Synthase (SPS) pH-7.5
Principle
Higher plants synthesize sucrose through two processes. Suc 6'-phosphate is then
dephosphorylated by Suc-P phosphatase (E.C 2.3.1.14; SPS) to produce sucrose as
the final product. The sucrose phosphate synthase a soluble enzyme located in the
cytoplasm that catalysis the reaction:
UDPGlc + Fru6-phosphate ⇌ UDP + Suc6'-phosphate
51
Reagents
50 mM MOPS-NaOH (pH-7.5)
2 mM UDGP (Substrate)
5 mM MgCl2
1 mM EDTA
4 mM Frutose-6-p
20 mM Glucose-6-p
30 % KOH
0.14 % (w/v) anthrone reagent in 95 % sulphuric acid (H2SO4)
Procedure
Sucrose phosphate synthase activities were quantified according to the method
described by (Huber et al., 1989). The reaction mixture containing 10 μl enzymes, 25
μl (UDPG) substrate, 70 μl KoH, MOPS-NaOH (pH -7.5) buffer consisted of MgCl2 and
EDTA, Fru 6-P, Glu 6-P. The mixture was incubated at 37°C for 20 min before adding
70 µl 30 % (w/v) KOH and heating for 10 min at 100°C. To this 5 ml anthrone reagent
was then added. The mixture was incubated at 100°C for 20 min and A620 was
measured.
Calculation
Sucrose phosphate synthase (SPS) was calculated µmole ml-1 using the formula;
SPS (units ml−1min−1) =ODT × Conc Std (mg)
OD Std × AS × MW × RT × TSP
52
3.8.5. Sucrose Synthase (SS) pH-7.5
Principle
Sucrose synthase (E.C 2.4.1.13) which catalysis a reversible reaction:
UDP - glucose + fructose ⇌ sucrose + UDP
Reagents
50 mM MOPS-NaOH (pH-7.5)
2 mM UDGP (Substrate)
5 mM MgCl2
1 mM EDTA
4 mM fructose
30 % KOH
0.14 % (w/v) anthrone reagent in 95 % sulphuric acid (H2SO4)
Procedure
The procedure followed for the assay of sucrose synthase (SS) was similar to that of
sucrose phosphate synthase (SPS), except that 4 mM fructose was used instead of
fructose 6-p and glucose 6p in the reaction mixture.
Calculation
Calculation also mentioned in sucrose phosphate synthase (SPS) protocol.
3.9. Qualitative Analysis of Isozymes through Native PAGE
Native or non-denaturing gel electrophoresis is technique for analysis and
separation of macromolecules such as nucleic acids and proteins in their native state
separation of proteins in the absence of SDS.
53
3.9.1. Sample Extraction
Invertase isozymes (CWIN, VIN and CyIN) were extracted with MOPS-NaOH (pH-7.0).
For this, 10 g of leaf tissues were crushed into liquid nitrogen and homogenized with
buffer and left for 30 min, then centrifuged at 12000 rpm for 30 min. Supernatant
was filtered by syringe filter. To concentrate the sample, centricon device was used.
For this 3 ml supernatant was collected in centricon centrifugal filter device and
centrifuged at 2000 rpm till concentrated and stored -20°C for gel electrophoreses.
3.9.2. Native Polyacrylamide Gel Electrophoresis
3.9.2.1. Prepration of Reagents
a. Acrylamde bisacrylamide (solution A)
Acryle amide 30 g, bisacryle amide 0.8 g, both polymer and cross linkers were
dissolved in 80ml double deionized filrtrate water and the volume was filtred via
whatman filter paper # 01 and stored at 4°C for further use.
b. 1.5 M Tris-Hcl (pH-8.8)( solution B)
36.3 g trizma base was dissolved in 200 ml double deionized water to achive 1.5 M
concerntrantion and pH-8.8 was maintained by gradually incorporated 1 N HCL.
c. 0.5 M Tris-Hcl (pH-6.8)( solution C)
6.05 g trizma base was dissoved in 100 ml double deionized water to get 0.5 M
concerntration and the pH was adjusted upto 6.8 by gradually incorporated in HCl.
The buffer was stored at 4°C.
d. Ammonium Per Sulphate (APS 10 % solution D)
Fresh solution of ammonium per sulphate was prepared. For this, 10 g APS salt was
dissolved in 100 ml double deionized water and placed on ice bath before use.
54
e. N,N,N,N-Tetramethyleethylenediamine (TEMED)
TEMED was stored at 4°C.
f. 0.025 M Tris-glycine Buffer pH 8.4-8.6 (Reservior buffer)
First 14.4 g of glycine was dissolved in 1000 ml double deionized water. After
complete dissolving then 3.025 g of trizma base was added and pH was adjusted at
8.4 and stored at 4°C.
g. Sample Diluting Buffer (SDB):
For this, glycerol (2 ml) was mixed with 0.5 M Tris-HCL ( 2.5 ml) and few crsytals of
bromophenole blue were added. The volume was made upto 10 ml using double
deionized water. Aliquotes (1 ml) were prepared and stored at 4°C.
3.9.2.2 Preprations of Buffers:
a. 0.05 M Sodium Acetate Buffer pH-3.5 and pH-5.0
Solution # A: 0.4 g sodium acetate dissolved in 100 ml double deinized water.
Solution # B: 0.14 ml acetic acid was added in 50 ml double deionized water.
Gradually acid (solution # A) incorporated into base(solution # B) to achieve upto
pH-3.5 and pH-5.0.
b. 0.1 M Potasium Phosphate Buffer pH-7.0
Solution # A: 1.36 g of KH2PO4 dissolved 100 ml double deinized water
Solution # B: 1.74 g of K2HPO4 dissolved 100 ml double deinized water.
Both solutions (A and B) were mixed to get pH-7.0
c. 0.3 %- 2,3,5 Triphenyle Tetrazolium Chloride (TTC) in 4 %- NaOH
Tetrazolium chloride (TTZ) solution was prepared fresh.
For this, 2, 3, 5 triphenyle tetrazolium chliride (TTZ) (0.1 g) was dissolved in 4 %
NaOH.
55
The solution was stored at 4°C.
d. Prepration of Staining Solution
Coomassie Briliant Blue G-250
Coomassie briliant blue G-250 ( 0.05 g ) was mixed with 50 ml 2-propanol and 30 ml
gacail acitic acid. The final volume was made upto 200 ml with double deionized
water. The prepared dye was filtered through whatman filter paper # 01 and stored
at 4°C.
e. Prepration of Destaining Solution
For this, 25 ml ethanol and 75 ml glacail acitic acid were mixed. The final volume was
raised upto 1000 ml with double deionized water. The prepared solution was stored
at 4°C.
3.9.2.3. Preparation of Resolving Gel (pH-8.8)
3.9.2.4. Preparation of Stacking Gel (pH-6.8)
56
3.9.2.5. Preparation of Running Buffer (1X)
Extracted proteins were resolved on NATIVE-PAGE gel (Laemmli, 1970). Spacer
plates (1.0 mm) were used to cast the gel. Resolving (12 %) and stacking (4 %) gels
were prepared by combination of acrylamide and bis-acrylamide solution, resolving
(pH 8.8), stacking buffers (pH 6.8) fresh APS (10 %), TEMED and double distilled
water. For invertase isozymes sample preparation, ultra-filtration or centricon (10
kda) were used. Concentrated protein samples (100 µg to 140 µg) mixed with sample
diluting buffer SDB (20 µl): Sample (80 µl) ratio, placed in ice prior to loading. Sample
(50 µl) was loaded in each well and gel was run at 120 voltage for 2 h. The gel was
incubated with substrate solution at 37°C for overnight with slightly shaking. Next
day the gel was stained by 2,3,5,-tripbenyltetrazolium chloride in NaOH solution.
3.9.3. Invertase Zymography
Principle
Alkaline 2,3,5,-tripbenyltetrazolium chloride (TTC) forms a red insoluble formazan in
reaction with glucose and fructose. For Invertase isozymes gel was stained with
some modification by the method discribed by (Cairns and Ashton, 1991). Samples
were prepared 1:4 (Dye : sample) mixed 4 part of samples with 1 part of craking
solution or (SDB). Sample (60 ul) was loaded in each well. Two gels were run parallel;
one for commassie staining while second for (TTC) satining. The gel was run at 120
voltage for 2 h.
57
At the end of gel electrophoresis, the gel was washed thrice with double distilled
deinized water followed by K-acetate buffer pH-5.0 for vacoular invertase while K-
Phospahte buffer pH-7.0 for alkaline or cytoplasmic invertase at 37°C for 2 h. And
the buffer was replaced by substrate buffer (20 % sucrose) and further incubated at
37°C for overnight with slight shaking. Next day substrate was changed and again
incubated at 37°C for 2 h. Then the gel was washed tree time with buffer. The hexose
released was visualized by soaking the gel in 50 ml of 0.1 % (TTC) in 4 %-NaOH
solution and heated in microvave oven for 30 seconds. Alkaline TTC formed a red
insoluble formazan is reacted with glucose and fructose and the red band was
appeared with light pick background. The second gel was stained with commasie G-
250 solution for overnight shaking. Then the gel was destained for visualizaton of
bands. Both gels were scaned and documented.
3.9.4. SDS-PAGE
Protein qualitative analysis was carried out by Laemmli method (Laemmli, 1970).
Method of gel preparation and buffer composition detailed already given in Native-
PAGE.
All procedures are same as in Native-PAGE but the differences are 10 % SDS (250 µl)
was added in resolving, stacking and sample diluting buffers. Blue protein marker pre-
stained (16 to 270 kDa, Cat No PM-PM-002-500, size 500 µl) was used for each gel
electrophoresis.
3.9.5. Quality Parameters Analysis
3.9.5.1. Pol (%) Estimation
Pol % was estimation the protocol used (Tahir et al., 2014a). For this, 4000 ml water
58
was mixed with 400 g crushed sample then disintegrated for 30 min. Lead (pb)
powder 1-2 g was added in 150 ml of sieved cane extraction and filtered, 20 ml of
filtered sample was measured using polari meter, this procedure was repeated three
times. And pol cane % was calculated by following formula;
𝐏𝐨𝐥 𝐜𝐚𝐧𝐞 (%) =Ext Pol × 0.26 (W + C − (0.0125F × C)
Specific Gravity × C
whereas,
Ext pol=Extraction of Pol
W=water and C=Sugarcane
3.9.5.2. °Brix Estimation
Degree brix (symbol °Bx) is the sugar concentration in water solution. One degree
brix is one gram of sugar in hundred gram of solution and represents the strength of
the solution as percentage by mass.
Brix was measured using brix meter and calculated by this formula:
°𝐁𝐫𝐢𝐱 𝐜𝐚𝐧𝐞 =Ext B × W − (0.25C) − (0.0125C × M)
C × 1 − (0.0125 × ExtB)
whereas,
ExtB=Extraction °Brix
W=Water
C=Cane
M=Moister
3.9.5.3. Moisture Content
For moisture calculation. 50 g of crushed sample was placed in oven at 40-60 min at
150°C then dry weight was measured following formula;
59
Moister Content = Fresh Weight-Dry Weight-Tare weight
3.9.5.4. Fiber Content (%)
Fiber content was calculated using this formula;
Fiber Content (%) = 100-moisture-calculated °brix
3.9.5.5. Recovery Estimation
Sugar recovery was estimated according to Foster (Foster, 1955). For this, 1 kg
sugarcane stems were crushed in crusher machine and weighted with the weighing
balance. Sample was divided into two portions one (400 g) for pol % and other (50
g) for moisture.
Recovery (%) =Pol %-2.5 %
Note: (2.5 % is operational loss in sugar mills during processing which is differ
according to different mills).
3.9.6. Statistical Analysis:
Data was statistically analyzed for analysis of variance (ANOVA), Pearson Correlation
and statistical significance was determined at p < 0.05 level using the LSD test. All
calculation and data analysis were done using the SPSS package program, version
17.
60
SECTION # 4 RESULTS
61
Results
4.1. Morphological Analysis
Morphological characterization is very important role in identification of a cultivar
and source of variability. Morphological analysis was carried out on two sugarcane
cultivars S2003-US-633 (high sucrose accumulation) and SPF-238 (low sucrose
accumulation) under different temperature regimes (45±2°C) for 24, 48 and 72 h at
different growth stages.
4.1.1. Shoot Length (cm)
Statistical analysis revealed that there were significant differences (p<0.05) between
cultivars (C), treatments (T) and their interactions (C×T) at formative stage for shoot
length. Under control conditions, maximum shoot length was observed in cultivar
S2003-US-633 (90 cm).While, under heat stress conditions, both cultivars declined in
shoot length but the drastic reduction in shoot length was exhibited in cultivar SPF-
238. Upon recovery, swift improvement was observed in cultivar S2003-US-633 while
slow progress in shoot length was observed in cultivars SPF-238 at vegetative stage
(Table 2). At grand growth stage, only the cultivars were significantly influenced
(p<0.05) while heat stress treatments and interactions were not significantly different
(p>0.05). After 48 and 72 h of heat exposure, both cultivars exhibited similar shoot
lengths. However, highest shoot length was depicted in cultivar S2003-US-633 under
recovery treatments (Table 3). At maturity stage, only significant changes in shoot
length was noted upon heat stress treatments, overall (Table 4). With consistently
decline it is evident from the results that varietal differences were also observed
among cultivars.
62
4.1.2. Root Length (cm)
Root length is an important morphological parameter, result revealed that root
length was statistically significant (p<0.05) affected by heat stress treatments while
no significant differences (p<0.05) were observed for their intersections (C×T) at
formative and grand growth stages. During formative stage, under control conditions,
root length were showed in both cultivars S2003-US-633 (19.7cm) and SFP-238 (18.3
cm) respectively. Moreover, both cultivars manifested the same trend for root length
under heat stress but the cultivars SPF-238 (17) was decreased root length after
exposure of heat stress as compared to S2003-US-633 (18) while recovery treatments
cultivar SPF-238 slowly improvement was observed upon recovery conditions (Table
2). At grand growth stage, when heat stress applied for different episodes root length
was gradually decreased in both varieties, maximum root length declined was
showed in cultivar S2003-US-633. Upon recovery treatments root length was
increased, comparatively, quick improvement was observed in cultivar SPF-238 as
compared to S2003-US-633 (Table 3).At maturity stage, results revealed that S2003-
US-633 (82 cm) and SFP-238 (78 cm) under control conditions. However, root length
was constantly declined under high temperature stress conditions. While
improvement of root length was found under recovery treatments in both cultivars
(Table 4).
4.1.3. Number of Tillers (plant-1)
It is manifest from results that number of tillers per sugarcane plant was not
significantly affected (p>0.05) by heat stress treatments at both formative and
maturity stages. At formative stage means of number of tillers exhibited that under
63
control conditions, maximum number of tillers were found in cultivar S2003-US-633
(5) as compared to cultivar SPF-238 (3). Inconsistent trend was observed for number
of tillers for heat shock and recovery treatments for both varieties but no drastic
reduction in number of tiller was observed (Table 2).At grand growth stage, no
remarkable differences were observed at heat stress and recovery treatments for the
number of tillers per plant (Table 3). Number of tillers were significantly affected by
heat stress for cultivars (C) and their interactions (C×T) but not significantly (p>0.05)
affected for treatments (T) at maturity stage. Although among all stages, highest
number of tillers were evident at maturity stage in both cultivars (Table 4).
4.1.4. Number of Leaf (plant-1)
It is evident that no significant difference (p>0.05) for number of leaf count per plant
at formative and grand growth stages were noted. At formative stage, results
indicated that maximum number of leaves (18) were found in cultivar S2003-US-633
as compared to SPF-238. Upon heat stress conditions, number of leaves were reduced
in both cultivars ranging from 18 to 15 showing reduction from control (Table 2).At
grand growth stage, similar number of leaves were recorded in both cultivars under
control, heat stress and recovery treatments on both cultivars (Table 3).While at
maturity stage, maximum number of leaves were observed under control conditions.
Significant differences (p<0.05) were observed between treatments (T) but not for
their interactions (C×T) and cultivars (C). Although heat stress treatments should
reduce the number of leaves from control in both varieties (Table 4).
64
4.1.5. Leaf Length (cm)
Leaf length was measured to explore the effect of high temperature treatment.
Statistical analysis for treatments, cultivars and their intersections showed non-
significant differences (p>0.05) at vegetative, grand growth and maturity stages.
Under control condition leaf length exhibited in S2003-US6-33 (65 cm) and SPF-238
(63 cm) respectively at formative stage. Under heat stress, no differences were
observed in leaf length while upon recovery conditions in both cultivar SPF-238 (Table
2). At grand growth stage, similar pattern was found in both cultivars (Table 3). At
maturity stage, highest leave length (127 cm) was recorded in cultivar S2003-US-633,
although there is a varietal difference but no notable difference was recorded
between both cultivars at all treatment (Table 4).
4.1.6. Leaf Width (cm)
Leaf width was measured of sugarcane at all growth stages. No significant difference
(p>0.05) was found in the leaf width among treatments (T), cultivars (C) and their
interactions (C×T) at vegetative stages. Under control conditions, the leaf width was
observed in both cultivars S2003-US-633 (1.67 cm) and SFP-238 (1.5 cm).While it was
reduced after 48 and 72 h heat treatments in both cultivars. However, upon recovery
treatments, leaf width was continuously reduced in cultivar SFP-3238 (Table 2). Only
significant differences were observed between cultivars at grand growth stage. Under
control conditions, both cultivars S2003-US-633 (2.67cm) and SFP-238 (2.5 cm) were
exhibited respectively. Under high temperature stress and recovery treatments, the
leaf width did not consistently increased or decreased in both cultivars (Table 3).
While at maturity stage, leaf width was significantly different (p<0.05) between
65
treatments (T) but no difference was found in cultivars and interactions. Leaf width
showing declined after heat stress compared to control but the reduction was
remained consistent from 24 to 72 h (T24 to T72) in cultivar S2003-US-633 (Table 4).
4.1.7. Fresh to Dry Weight Ratio (%)
At vegetative stage analysis of data for fresh to dry weight ratio (%) revealed,
significant differences (p<0.05) between treatments (T) and non-significant
differences (p>0.05) among cultivars (C) and their interactions (C×T). Under control
conditions, fresh to dry weight ratio was observed 27.9 % in cultivar S2003-US-633
and 26.49 % in cultivar SPF-238. During the heat treatments, initially the fresh weight
to dry weight ratio was slightly declined at 24 h (T24) but drastic reduction were
observed at after 72 h (T72) in both cultivars. Upon recovery, rapid improvement was
observed in cultivar SPF-238 (Table 2). While at grand growth stage, there were
significant differences for treatments and their interactions except cultivars (C). At
control conditions fresh to dry weight ratio was 43 % in cultivar SPF-238. But under
heat shock conditions, fresh to dry weight ratio was consistently reduced in cultivar
SPF-238.However, after 72h of recovery conditions, similar results were found in
both cultivars (Table 3). At maturity, there were significantly affected (p<0.05) for
cultivars and treatments but non-significant (p>0.05) for their interactions (C×T).
Fresh to dry weight ratio were found in both cultivars, S2003-US-633 (44 %) and SPF-
238 (40 %) at control conditions. Fresh to dry weight ratio of sugarcane significantly
reduced due to heat shock in both cultivars. Initially both cultivars slowly declined
fresh to dry weight ratio but at T72 h drastic reduction was observed in both cultivars.
Upon recovery conditions, similar pattern of improvement was exhibited in both
66
cultivars. Overall, cultivar S2003-US-633 had better performance under high
temperature and earlier recovery than other cultivar.
4.1.8. Stem Diameter (cm)
At grand growth stage, significant differences (p<0.05) were evident for stem
diameter among cultivars, treatments but not for their interactions (cultivars ×
treatments). Under control conditions, maximum stem diameter was observed in
cultivar S2003-US-633 (2.2 cm) and SPF-238 (1.8 cm) respectively. Under heat stress
treatments, minimum stem diameter was showed in both cultivars. However, upon
recovery treatments, stem diameter was found in both cultivars S2003-US-633 (1.9
cm) and SPF-238 (1.7 cm) respectively (Table 3). While at maturity phase, there were
non-significant (p>0.05) for all parameters. Stem diameter were increased as
compared previous stages in both varieties both varieties S2003-US-633 and SPF-238.
Although, there varietal differences but maximum stem diameter was found in
cultivar S2003-US-633 (Table 4). This result indicated that cultivar S2003-US-633
showed higher growth rate and better performance at various environmental
conditions than cultivar SPF-238.
4.1.9. Number of Nodes (plant-1)
There was no significant (p>0.05) difference between cultivars (C) and treatments
(C×T) at grand growth and maturity stages. At grand growth stage, number of nodes
per plant was noted in cultivars S2003-US-633 and SPF-238 in all treatments. Same
number of nodes of sugarcane were found in both cultivars S2003-US-633 (23) and
SPF-238 (23) under control conditions. No changes were found of number of nodes
under stress and recovery conditions (Table 3). The above mentioned results were
67
also noticed at maturity stage (Table 4). Although there were no differences (p>0.05)
between cultivars, treatments and their interactions at maturity stage but the
number of nodes were increased as compared to grand growth stage.
4.2.0. Number of Internodes (plant-1)
At grand growth, number of internodes per plant was also counted after each
treatment. Data revealed that there was no significant difference (p>0.05) among
cultivars, treatments and their interactions (C×T). At control conditions same number
of internodes (22, 22) were observed in both cultivars. Upon the heat stress
treatments, number of internodes reduced at T48 h after heat exposure in both
cultivars. However, upon recovery conditions, no changes occurred in number of
internodes in both cultivars (Table 6). Again no difference was exhibited at maturity
stage for all parameters (Table 7). Similar trend of number of internodes was shown
in both cultivars at all treatments, control and recovery conditions. At both stages,
the study revealed that no remarkable change was observed upon heat stress and
recovery treatments in both cultivars.
4.2.1. Internode Distance (plant-1)
Internode distance is very important morphological parameters because sucrose
content depends on internode distance. Internode distance is directly proportional
to sucrose content in sugarcane stalk. Analysis of data for internode distance per
plant revealed non-significant differences (p>0.05) between treatments (T), cultivars
(C) and their interactions (C×T) at grand growth stage. In control conditions,
internode distance was recorded in both cultivars S2003-US-633 (8.3 cm) and SPF-
238 (8 cm) respectively. Upon heat shock and recovery treatments internode distance
68
were at T48, R24 and R72 in cultivar S2003-US-633 (7.7 cm) while equal internode
distance (7.7 cm) were observed at R48 in recovery conditions (Table 6).
At maturity stage, no statistically significant affect was observed by heat shock
treatment for internode distance. Mostly the highest internode distance (9 cm) was
recorded in cultivar S2003-US-633 as against SPF-238 (Table 7). However, the length
of internode were slightly increased in cultivar S2003-US-633 at grand growth stage
as compared to maturity stage. This results indicated that higher length of internodes
may increase sucrose content in sugarcane stalk.
69
Table 5: Morphological parameters of both cultivars S2003-US-633 and SPF-238 under control at (30±2°C), heat shock (45±2°C) and recovery (30±2°C)
for 24, 48 and 48 h at vegetative stage. Cultivar (C), Treatments (T) and Cultivar × Treatments (C×T) at p level p<0.05.
Vegetative Stage
Parameters Varieties
Mean ± SEM P Value
Control Heat shock Recovery Cultivar Treatment Interaction
C T24 T48 T72 R24 R48 R72 C T C×T
Shoot length
(cm)
S2003-US-633 90.00±0.57 89.66±0.33 89.33±0.88 88.33±0.88 88.00±0.66 87.66±0.33 88.66±0.33 * * *
SPF-238 88.00±0.88 87.00±0.57 84.00±0.57 84.00±0.57 84.00±0.57 84.33±0.33 84.33±0.66
Root length
(cm)
S2003-US-633 19.0±0.30 19.3±0.30 19.3±0.70 18.3±0.90 18.7±0.30 19.0±0.06 19.3±0.30 * * ns
SPF-238 18.3±0.30 17.3±0.30 17.0±0.00 16.0±0.00 16.3±0.30 16.3±0.30 17.7±0.30
Number of
tiller
S2003-US-633 5.00±0.58 2.67±0.67 3.67±0.67 4.00±0.58 3.67±0.67 5.00±0.58 4.00±0.58 ns ns ns
SPF-238 3.67±0.67 4.00±0.58 4.33±0.88 6.00±0.58 4.00±0.58 5.00±0.58 4.33±0.88
Number of
leaf
S2003-US-633 18.00±0.58 18.00±0.58 17.00±0.88 16.00±0.33 16.00±0.58 17.00±0.33 18.00±0.33 ns ns ns
SPF-238 17.00±0.58 16.00±0.33 16.00±0.33 15.00±0.33 15.00±0.33 16.00±0.33 16.00±0.58
Leaf length
(cm)
S2003-US-633 65.00±1.0 66.00±0.6 66.00±1.5 65.00±2.61 64.00±1.20 64.00±0.00 63.00±1.3 ns ns ns
SPF-238 63.00±0.6 63.00±1.8 63.00±1.5 63.00±2.6 63.00±2.4 65.00±0.6 64.00±2.00
Leaf width
(cm)
S2003-US-633 1.67±0.29 1.83±0.29 1.17±0.17 1.50±0.29 1.50±0.29 1.83±0.33 1.67±0.33 ns ns ns
SPF-238 1.50±0.33 1.50±0.17 1.67±0.17 1.50±0.29 1.50±0.29 1.33±0.17 1.33±0.33
Fresh to dry
wt ratio (%)
S2003-US-633 27.94±0.32 27.30±0.02 26.01±0.72 24.89±0.45 25.27±0.22 25.32±0.71 25.79±0.48 ns * ns
SPF-238 26.49±0.59 25.12±1.11 25.02±1.35 24.79±0.33 25.45±0.13 25.97±0.60 26.03±0.43
Significant difference p<0.05 (*) and non-significant differences p<0.05 (ns)
70
Table 6: Morphological parameters of both cultivars S2003-US-633 and SPF-238 under control at (30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at grand growth stage. Cultivar (C), Treatments (T) and Cultivar × Treatments (C×T) at p level p<0.05.
Significant difference p<0.05 (*) and non-significant differences p<0.05 (ns)
Grand Growth Stage
Parameters Varieties
Mean ± SEM P Value
Control Heat shock Recovery Cultivar Treatment Interaction
C T24 T48 T72 R24 R48 R72 C T C×T
Shoot length (cm)
S2003-US-633 167.3±0.33 166.7±0.30 167.3±0.47 167.3±0.33 166.3±0.33 166.3±0.33 166.6±0.88 * ns ns
SPF-238 165.6±0.51 166.3±1.06 165.3±0.33 165.3±0.33 165.6±1.35 166.1±0.88 164.5±0.88
Root length (cm)
S2003-US-633 41.0±0.58 36.5±1.80 35.67±1.77 34.33±0.33 35.0±0.58 33.67±0.88 31.33±0.67 * * ns
SPF-238 38.18±1.09 38.0±0.58 36.33±0.33 36.00±0.58 36.67±0.88 37.0±1.00 37.33±0.33
Number of tiller
S2003-US-633 5.0±0.58 5.0±1.00 5.0±0.58 4.0±0.58 5.0±0.00 5.0±1.00 5.0±1.16 ns ns ns
SPF-238 4.0±1.00 4.0±0.58 5.0±0.58 5.0±0.58 5.0±0.58 6.0±0.58 5.0±1.16
Number of leaf S2003-US-633 24.0±0.58 24.0±0.58 25.0±0.67 25.0±0.00 24.0±0.33 26.0±0.33 24.0±0.58
ns ns ns SPF-238 24.0±1.16 25.0±0.00 25.0±0.67 26.0±1.00 26.0±0.33 26.0±0.67 25.0±1.33
Leaf length (cm)
S2003-US-633 125±0.29 125±0.88 125±0.88 124±0.99 124±0.80 125±0.99 126±1.31 ns ns ns
SPF-238 125±0.59 124±0.59 124±0.40 123±0.68 126±0.00 125±0.00 125±0.33
Leaf width (cm)
S2003-US-633 2.67±0.17 2.63±0.13 2.50±0.29 2.70±0.47 2.57±0.07 2.47±0.03 2.80±0.06 * ns ns
SPF-238 2.50±0.29 2.37±0.37 2.27±0.15 2.50±0.06 2.13±0.13 2.33±0.09 2.44±0.06
Fresh to dry wt ratio (%)
S2003-US-633 39.47±0.48 38.0±0.54 37.6±1.19 38.8±0.75 39.8±0.85 41.8±2.21 34.2±0.94 ns * *
SPF-238 43.0±1.44 42.3±1.13 39.6±0.81 38.9±0.69 30.6±2.55 34.0±1.57 34.2±3.77
Stem diameter (cm)
S2003-US-633 2.23±0.10 1.77±0.12 1.67±0.17 1.57±0.03 1.70±0.06 1.67±0.09 1.90±0.06 * * ns
SPF-238 1.83±0.09 1.60±0.06 1.53±0.03 1.53±0.03 1.60±0.06 1.60±0.15 1.70±0.06
Number of nodes
S2003-US-633 23.0±0.00 22.7±0.33 22.7±0.33 23.0±0.00 22.7±0.33 22.7±0.33 22.7±0.33 ns ns ns
SPF-238 23.0±0.00 23.0±0.00 22.7±0.33 22.7±0.33 23.0±0.00 23.0±0.00 23.0±0.00
Number of internodes
S2003-US-633 22.0±0.00 21.7±0.33 21.7±0.33 22.0±0.00 21.7±0.33 21.7±0.33 21.7±0.33 ns ns ns
SPF-238 22.0±0.00 22.0±0.00 21.7±0.33 21.7±0.33 22.0±0.00 22.0±0.00 22.0±0.00
Internodes distance (cm)
S2003-US-633 8.33±0.33 8.00±0.00 7.70±0.33 8.00±0.00 7.70±0.33 8.3±0.33 7.70±0.33 ns ns ns
SPF-238 8.00±0.00 8.33±0.33 8.33±0.33 8.33±0.33 8.00±0.00 7.7±0.33 8.33±0.33
71
Table 7: Morphological parameters of both cultivars S2003-US-633 and SPF-238 under control at (30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at maturity stage. Cultivar (C), Treatments (T) and Cultivar × Treatments (C×T) at p level p<0.05.
Significant difference p<0.05 (*) and non-significant differences p<0.05
Maturity Stage
Parameters Varieties
Mean ± SEM P Value
Control Heat shock Recovery Cultivar Treatment Interaction
C T24 T48 T72 R24 R48 R72 C T C×T
Shoot length (cm)
S2003-US-633 317.3±1.33 316.0±0.58 315.6±0.88 314±1.58 314±0.33 315.0±0.58 315.33±0.88 ns * ns
SPF-238 316.33±0.33 315.33±0.33 314.33±0.33 313.6±0.88 314.6±0.33 315.3±0.33 315.67±0.67
Root length (cm)
S2003-US-633 82.00±1.70 81.00±0.60 78.00±0.90 76.00±0.90 77.00±0.90 78.00±0.90 79.00±1.50 * * *
SPF-238 78.00±0.30 77.00±0.90 77.00±0.60 76.00±0.30 76.00±.0.90 77.00±0.70 77.00±0.30
Number of tiller
S2003-US-633 6.67±0.33 6.33±0.33 6.33±0.17 6.17±0.17 6.00±0.00 6.23±0.23 6.17±0.17 * ns *
SPF-238 6.33±0.33 6.00±0.00 5.83±0.17 5.67±0.33 5.83±0.17 5.67±0.17 5.87±0.13
Number of leaf
S2003-US-633 30.00±0.58 27.67±0.33 27.00±0.58 27.33±0.33 27.33±0.33 27.67±0.33 28.67±0.33 ns * ns
SPF-238 29.33±0.33 28.00±0.58 27.67±0.33 27.33±0.33 27.67±0.33 26.33±0.33 27.33±0.33
Leaf length (cm)
S2003-US-633 127.7±1.20 127.3±2.91 127.3±2.85 126.3±0.33 127.7±1.20 127.7±1.86 127.3±0.67 ns ns ns
SPF-238 123.7±0.88 127.7±1.20 127.7±0.88 127.7±1.67 126.0±0.58 126.3±2.41 126.3±0.88
Leaf width (cm)
S2003-US-633 2.93±0.07 2.77±0.03 2.70±0.06 2.70±0.00 2.57±0.03 2.63±0.09 2.77±0.03 ns * ns
SPF-238 2.70±0.06 2.80±0.09 2.70±0.10 2.70±0.10 2.73±0.09 2.57±0.07 2.83±0.07
Fresh to dry wt
ratio (%)
S2003-US-633 44.0± 1.29 40.99±0.37 39.29±1.73 37.91±0.08 38.16±0.35 38.73±0.50 40.29±0.55 * * ns
SPF-238 40.47±0.33 39.84±0.57 38.57±0.49 37.06±0.42 37.57±0.39 37.87±2.22 38.25±0.21
Stem diameter
S2003-US-633 2.50±0.06 2.53±0.03 2.47±0.03 2.33±0.18 2.43±0.07 2.57±0.03 2.53±0.03 ns ns ns
SPF-238 2.33±0.17 2.30±0.15 2.33±0.17 2.17±0.17 2.23±0.15 2.33±0.17 2.37±0.19
Number of nodes
S2003-US-633 26.00±0.00 26.00±0.58 25.00±0.33 26.00±0.58 25.26±0.00 26.00±0.33 26.00±0.58 ns ns ns
SPF-238 25.00±0.33 26.00±0.33 25.00±0.33 26.00±0.33 26.00±0.33 25.00±0.00 26.00±0.00
Number of internodes
S2003-US-633 25.00±0.00 25.00±0.58 24.00±0.33 25.00±0.58 24.26±0.00 25.00±0.33 25.00±0.58 ns ns ns
SPF-238 24.00±0.33 25.00±0.33 24.00±0.33 25.00±0.33 25.00±0.33 24.00±0.00 25.00±0.00
Internodes distance
(cm)
S2003-US-633 9.00±0.00 9.00±0.00 9.00±0.00 8.83±0.17 8.33±0.33 8.67±0.33 9.00±0.00 ns ns ns
SPF-238 8.33±0.58 8.67±0.33 8.33±0.33 8.77±0.33 8.33±0.33 8.83±0.20 9.00±0.00
72
4.3. Stress Damage Indicators Quantification
Stress damage indicators such as malondialdehyde, hydrogen per oxide and relative
membrane permeability and proline were quantified as following.
4.3.1. Malondialdehyde (MDA)
MDA is the indicator of lipid peroxidation, it was quantified in sugarcane plant and
is expressed in terms of nano-mole per ml. It is a vital resistant physiological index
of plant in any environmental stress conditions. As shown in the below (Figure 6).
MDA content was statistically different (p<0.05) for treatments (T) and cultivars (C)
and their interactions (C×T) at all stages. Under control conditions, the amount of
MDA content was observed for cultivar, SPF-238 (12.74 n mole ml-1) and S2003-US-
633 (11.78 n mole ml-1) at vegetative stage. Exposure of heat stress for 72 h trigger
the highest MDA content in cultivar SPF-238 (42.4 n mole ml-1) as compared to
S2003-US-633 (38.36 n mole ml-1). While under recovery treatments, both cultivars
depicted similar pattern of MDA accumulation. However, at grand growth stage,
maximum MDA content was also exhibited under heat stress but upon recovery
conditions both cultivars slowly declined the MDA content. At maturity stage, the
amount of MDA content was significantly lower than the MDA quantifies under
control, heat stress and recovery conditions at grand growth stage. However, under
control conditions, cultivar S2003-US-633 indicated lowest MDA accumulation than
SPF-238 at maturity stage. Heat stressed plants slightly increased MDA content over
no heat stressed plants. But between cultivar S2003-US-633 was observed quick and
better recovery of MDA than cultivar SPF-238.
73
Fig 6: MDA quantified of sugarcane cultivars S2003-US-633 and SPF-238 under control
(30±2oC), heat shock (45±2oC) and recovery (30±2oC) for 24, 48 and 72 h at vegetative,
grand growth and maturity stages.
74
4.3.2. Proline Estimation
Higher accumulation of osmolyte (proline) is an important physiological response
in sugarcane plant exposed to different environmental stresses (biotic and abiotic).
Free proline was quantified in terms of µmole gm-1 FW. The amount of proline was
significantly different (p<0.05) between cultivars (C) and treatments at all stages
except their interactions (C×T) at grand growth stage. While proline content was not
significantly different (p>0.05) for their interactions (C×T) at vegetative and maturity
stages. Under control conditions, both cultivars did not show any differences for the
accumulation of free proline at vegetative stage (Fig 7). Under heat shock conditions,
the amount of free proline was significantly higher than control and recovery
conditions in both cultivars. However, during recovery conditions, proline content
was slightly declined in both cultivars S2003-US-633 and SPF-238 but SPF-238
showed a drastic reduction than S2003-US-633. While at grand growth stage,
significant differences for the free proline content among all factors. Under heat
shock conditions, both cultivars indicated an enhancement in the free proline
contents however proline accumulation was noticeably greater in cultivar S2003-US-
633 than SPF-238. Upon recovery, proline content dropped sharply in both cultivars.
At maturity stage, proline content was accumulated in cultivar S2003-US-633 (61.4
µM g-1 FW) and SPF-238 (58.3 µM g-1 FW). After exposure to heat stress for 24 h
(T24) the amount of proline content slightly increased and after 48 h (T48) much
more increased. While, after 72 h (T72) it increased many folds in both cultivars
S2003-US-633 (190 µM g-1 FW and SPF-238 (173.8 µM g-1 FW) respectively.
75
Fig 7: Free proline estimated of sugarcane cultivars S2003-US-633 and SPF-238 under
control (30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at
vegetative, grand growth and maturity stages.
76
4.3.3. Hydrogen peroxide
Hydrogen peroxide is produced in plant cell and it plays vital role as signaling
molecules in different physiological processes. In plant cell hydrogen peroxide
content increases under biotic and abiotic stresses. Hydrogen peroxide was
estimated in terms of µmole g-1 FW. The amount of hydrogen peroxide exhibited
significant differences (p<0.05) between treatments (T) and cultivars (C). While non-
significant differences (p>0.05) were observed between their interactions (C×T) at
all stages. At formative stage, the amount of hydrogen peroxide was (64.39 µM g-1
FW) S2003-US-633 and (92.42 µM g-1 FW) SPF-238 under control conditions. Under
heat stress treatments, its content gradually increased with the increasing
temperature. Maximum hydrogen peroxide content was noted in SPF-238 (208.2 µM
g-1 FW) than S2003-US-633 (187.8 µM g-1 FW). Under recovery conditions, rapid
improvement was depicted in cultivar S2003-US-633. At grand growth phase, the
amount of hydrogen peroxide exhibited in S2003-US-633 (66 µM g-1 FW) and SPF-
238 (86 µM g-1 FW) respectively. But after 72 h (T72) exposure to heat stress, the
content of hydrogen peroxide was increased in both cultivars S2003-US-633 (154.9
µM g-1 FW) and cultivar SPF-238 (173.35 µM g-1 FW), respectively. However, upon
recovery conditions, lower accumulation of hydrogen peroxide was depicted in both
cultivars. Same results were also found at maturity stage in both cultivars. Figure 8
indicates cultivar S2003-US-633 showing tolerance under heat stress conditions.
77
Fig 8: H2O2 estimated of sugarcane cultivars S2003-US-633 and SPF-238 under control
(30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at vegetative,
grand growth and maturity stages.
78
4.3.4. Relative Membrane Permeability (RMP)
Membrane thermo stability is a significant stress damage indicator in various
physiological processes. It is quantified by measuring electrical conductivity (EC) of
sugarcane plant. It is evident from the results EC was significantly different (p<0.05)
between treatments, cultivars and their interactions but non-significant difference
(p>0.05) was observed among their interactions (C×T) at vegetative and grand
growth stages. At formative stage, electrolyte leakage (EC) was exhibited in S2003-
US-633 (14.7 %) and SPF-238 (16.6 %) respectively. After 24 h heat stress treatment
(T24) initially maximum EC content was observed in both cultivars S2003-US-633 (20
%) and SPF-238 (27.7 %) and at 72 h (T72), EC content was continuously increased in
both cultivars S2003-US-633 (24.3 %) and SPF-238 (28.71 %) respectively. Recovery
treatments for 72 h (R72) showed maximum EC in cultivar S2003-US-633 (19.2 %)
while minimum RMP manifested by SPF-238 (18.5 %). At grand growth stage, data
stated that EC was observed in both cultivars S2003-US-633 (15.24 %) and SPF-238
(18.71 %) of untreated plants. But after heat treatments EC was increased in both
cultivars with increasing heat for different episodes (T24, T48 and T72). Upon
recovery treatments, EC was declined in both cultivars with same pattern (Fig 9). At
maturity stage, data revealed that significant difference between cultivars and
treatments except their interactions (C×T). Minimum RMP was observed in
untreated plants in both cultivars. Under heat stress conditions, EC increased
gradually after 72 h heat treatments in both cultivars. Upon recovery both cultivars
recovered EC content after 72 h (R72). However, minimum EC was displayed in
cultivar S2003-US-633 that shows tolerance under heat stress conditions.
79
Fig 9: Electrolytes lekeage quantified of sugarcane cultivars S2003-US-633 and SPF-238
under control (30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at
vegetative, grand growth and maturity stages.
80
4.4. Biochemical Analysis
The biochemical analysis of two sugarcane cultivars S2003-US-633 (high sucrose
accumulation) and SPF-238 (low sucrose accumulation) under different temperature
regimes (45±2°C) for 24, 48 and 72 h at vegetative, grand growth and maturity stages.
4.4.1. Total Sugar Estimation
Total sugar was quantified in terms of (µg ml-1). Results revealed that total sugar
showed statistically significant differences (p<0.05) between cultivars (C) and
treatments (T) at all stages. While non-significant differences (p>0.05) were
exhibited between their interactions (C×T) at both vegetative and maturity stages
but only significant differences (p<0.05) were found between their interactions (C×T)
at grand growth stage. Under control conditions, maximum total sugar content was
exhibited in cultivar S2003-US-633 (712.6) as compared to cultivar SPF-238 (573).
Upon the application of thermal stress, total sugar content gradually decreased at
24, 48 h (T24-T48) and reached maximum reduction at 72 h (T72) in both cultivars
S2003-US-633 (263.15) and SPF-238 (228). However, upon recovery treatments after
24, 48 and 72 h (R24, R48 and R72) both cultivars recovered sugar loss with same
pattern at vegetative stage. At grand growth stage, maximum total sugar content
was showed in S2003-US-633 (1641). Incontrast minimum sugar content was found
SPF-238 (1581.1) under control conditions. But during exposure to heat stress both
cultivars showed gradual declined from 24 to 72 h (T24, T48 and T72) in cultivar
S2003-US-633 (1623.9), (1367.37) and (0.1213.67) while in cultivar SPF-238
(1581.19), (1402.2) and (1241.37) respectively. However, during recovery
treatments, total sugar content progressively increased from 24 to 72 h (R24, R48
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and R72) in cultivar S2003-US-633 (1230.76), (1316.23) and (1461.53) and SPF-238
(1213.675), (1307.69) and (1410.25) against heat stress conditions. At maturity
stage, the amount of total sugar was found in cultivar S2003-US-633 (10818.3) and
SPF-238 (8573.31) under control conditions. But under high temperature stress
conditions, total sugar content was declined in cultivar S2003-US-633 at 24
(9471.84), at 48 (9044.49) and at 72 h (8663.16) and SPF-238 at 24 (8573.31), at 48
(7020.93) and at 72 h (7027.62) respectively. However, during recovery treatments
from (24, 48 and 72 h) regained total sugar loss in cultivar S2003-US-633 at 24
(9020.38) at 48 (9298.78) and at 72 h (9714.22) and SPF-238 at 24 (7392.92), at 48
(7131.78) and at 72 h (7388.57) respectively. Although, there is varietal difference,
but it was revealed that the amount of total sugar gradually increased from
vegetative stage to grand growth stage and maturity stage (Fig 10).
82
Fig 10: Total sugar estimated of sugarcane cultivars S2003-US-633 and SPF-238 under
control (30±2°C),heat shock (45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at
vegetative, grand growth and maturity stages.
83
4.4.2. Reducing Sugar Estimation
Reducing sugar of both varieties during this study estimated in terms of (mg ml-1) at
all three growth stages. Data revealed significant difference between cultivars and
treatments except their interactions at vegetative and grand growth stages. Under
control conditions, reducing sugar content was observed in S2003-US-633 (0.25) and
in SPF-238 (0.19) respectively (Fig 11). Under high temperature stress treatments
the amount of reducing sugar was significantly reduced in cultivar S2003-US-633 at
24, 48 and 72 h (0.15), (0.13) and (0.12) while SPF-238 (0.15), (0.12) and (0.11)
respectively. During recovery conditions, both varieties showed positive adaptation
with regaining of reducing sugar loss. In addition, the content of reducing sugar was
recovered in cultivar S2003-US-633 after 24, 48 and 72 h (0.19), (0.20) and (0.21) and
cultivar SPF-238 (0.18 ), (0.182) and (0.187) at vegetative stage respectively. At
grand growth stage, under control conditions, the amount of reducing sugars were
in both cultivars S2003-US-633 (0.52) and SPF-238 (0.61) but significant variation was
noticed depending upon the duration of high temperature and recovery treatment.
Reducing sugars were declined in SPF-238 after 24, 48 and 72 h (0.58), (0.58) and
(0.44) and S2003-US-633 (0.48), (0.38) and (0.31) during stress treatments. The
amount of reducing sugar was maximum in SPF-238 as compared to S2003-US-633.
However, both cultivars improved sugar loss in recovery treatment with same
pattern. At maturity stage, only significant differences (p<0.05) were observed
between cultivars (C) but non-significant (p>0.05) differences were found among
their interactions (C×T). Plant under control conditions, reducing sugars content
found in S2003-US-633 (0.23) and SPF-238 (0.35) but under thermal stress
84
conditions, the amount of sugar slightly declined in both cultivars while
improvements were exhibited during recovery treatments. These responses were
noticed in both varieties. Collectively, heat stress caused reduction in sugar profile.
The amount of reducing sugar was maximum in SPF-238 as compared to S2003-US-
633 in all three stages but highest level of reducing sugar was noticed at grand
growth stage than the other two stages. In addition, the amount of reducing sugar
was declined in formative stage and maturity stage but increased in grand growth
stage.
85
Fig 11: Reducing estimated sugar of sugarcane cultivars S2003-US-633 and SPF-238 under
control (30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at
vegetative, grand growth and maturity stages.
86
4.4.3. Non-reducing Sugar Estimation
Non-reducing sugar also quantified in terms of (mg ml-1). Statistical analysis results
were found same as total sugar at all growth stages.
At formative stage, the amount of non-reducing sugar was observed in S2003-US-
633 (0.45) while in cultivar SPF-238 (0.37) under control condition. While after 72 h
of heat exposure the amount of non-reducing sugar was declined in cultivar S2003-
US-633 (0.14) and in SPF-238 (0.11). However, after72 h recovery treatments both
varieties showed same pattern of improvement in both cultivars S2003-US-633
(0.39) and SPF-238 (0.39) respectively. At grand growth stage, reduction of non-
reducing sugars content was noticed from 24, 48 and 72 h after heat shock
treatments as compared to under control condition S2003-US-633 (0.30) and SPF-
238 (0.14) and similarly recovered sugar losses in both varieties. At maturity stage,
under control conditions, SPF-238 had less amount of non-reducing sugars (8.23)
than S2003-US-633 had (10.59 ) while during different episode of heat stress at 24,
48 and 72 h (9.24), (8.8 ) and (8.44) in S2003-US-633 but in SPF-238 (7.34), (6.6) and
(6.71) showed substantial reduction respectively. Cultivar S2003-US-633 got better
recovery than SPF-238. Despite of different cultivars, maximum amount of non-
reducing sugar was revealed at vegetative stage and maturity stage while minimum
contents of non-reducing sugars were found in grand growth stage in both cultivars.
(Fig 12).
87
Fig 12: Non reducing sugar estimated of sugarcane cultivars S2003-US-633 and SPF-238
under control (30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at
vegetative, grand growth and maturity stages.
88
4.4.4. Total Soluble Protein Analysis
Total soluble protein (TSP) was quantified in terms of (mg ml-1) in both cultivars.
Significant differences (p<0.05) were observed among all cultivars (C), treatments
(T) and their interactions (C×T) at all stages. At formative stage, TSP content was
noted in cultivar S2003-US-633 (1.187) and cultivar SPF-238 (1) under control
conditions. While TSP concentration declined under heat stress conditions in both
cultivars. Comparatively, minimum accumulation of TSP content was exhibited in
SPF-238. After exposure of heat shock treatments at different time intervals (24, 48
and 72 h), TSP content showed in cultivar S2003-US-633 (1.11), (0.938) and (0.819)
while in SPF-238 (0.879), (0.652) and (0.634) respectively. Upon recovery
treatments, the amount of protein in cultivar SPF-238 minimum recovered than
cultivar S2003-US 633. At grand growth stage, under normal conditions, protein
concentration was exhibited in both cultivars, S2003-US-633 (1.8) and cultivar, SPF-
238 (1.7) respectively. But during the heat shock treatments both cultivar was
exhibited less amount of TSP. In addition under stress conditions, the amount of
protein observed in S2003-US-633 (1.6) (1.5) and (1.4) and in SPF-238 (1.5), (1.4) and
(1.2) respectively. However, after 24 and 48 h of recovery treatments both cultivars
had same amount of protein contents, except 72 h (1.6) and (1.5). At maturity stage,
cultivar S2003-US-633 (3) while other variety, SPF-238 (2.8) were noted under
control conditions. However, similar results were exhibited under high temperature
stress conditions as at both vegetative and grand growth stages. Comparatively,
cultivar S2003-US-633 showed best resistance in heat stress conditions as well as
swift recovery in recovery treatments than SPF-238 (Fig 13).
89
Fig 13: Total soluble protein estimated of sugarcane cultivars S2003-US-633 and SPF-238
under control (30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48 and 72 h at
vegetative, grand growth and maturity stages.
90
4.5. Sugar Metabolizing Enzymes
Sugar metabolizing enzymes such as sucrose synthase (SS), sucrose phosphate
synthase (SPS) and invertase isozymes such as cytoplasmic invertase (neutral), cell
wall invertase (acidic) and vacuolar invertase (acidic) of two sugarcane cultivars
S2003-US-633 and SPF-238 were quantified at vegetative, grand growth and
maturity stages. Characterization of enzymes activities were carried out through
quantitative and qualitative analysis as follows:
4.5.1. Quantitative Analysis
4.5.1.1. Sucrose Synthase (SS)
Sucrose synthase (SS) activities exhibited significant differences (p<0.05) among
cultivars (C), treatments (T) and their interactions (C x T) at maturity stage. However,
non-significant differences (p>0.05) variation were observed for cultivars (C) and
their interactions (C x T) at vegetative and grand growth stage. The exposure of heat
stress declined the enzymes activities in both varieties as compared to the control
(Fig 14). Drastic reduction in SS activity was noted after 72 h of heat stress in S2003-
US-633 (92.14 U ml-1min-1) and SPF-238 (69.68 U ml-1min-1) while increased in
recovery treatments after (139 and 142 U ml-1min-1) respectively. While, at grand
growth stage, sucrose synthase (SS) activity exhibited significant differences (p>0.05)
only for treatments (T). The activity of sucrose synthase was found in both cultivars
S2003-US-633 (386.5 U ml-1min-1) and SPF-238 (388.3 U ml-1min-1) at normal growth
condition. But after heat shock treatments for 24, 48 and 72 h the SS activities were
observed in S2003-US-633 (382.251 and 259.2 U ml-1min-1) and SPF-238 (382.8,
91
254.7 and 245.4 U ml-1min-1) respectively. Furthermore, cultivar S2003-US-633 was
found to quickly recover the activity of SS after heat shock. At maturity stage, it is
evident from the results that SS activity was sequentially decreased as episodes of
heat stress gradually increased, maximum reduction (189.4 U ml-1min-1) was
observed at heat shock treatment (T72) in SPF-238. While both cultivars recovered
the maximum SS activity after 72 h of recovery treatment (R72).
92
Fig 14: Specific activity quantified of sucrose synthase (SS) of sugarcane cultivars S2003-
US-633 and SPF-238 under control (30±2°C), heat shock (45±2°C) and recovery (30±2°C)
for 24, 48 and 72 h at vegetative, grand growth and maturity stages.
93
4.5.1.2. Sucrose Phosphate Synthase (SPS)
For sucrose phosphate synthase (SPS) activity, significant differences (p<0.05) were
found for cultivars (C) and treatments (T) while their interactions (C x T) at all growth
stages but non-significant (p>0.05) difference between cultivars at grand growth
stage. At vegetative stage, heat shock treatments for 24 and 48 h slightly decreased
the SPS activity in both cultivars but depicted maximum declined in activity at T72 in
S2003-US-633 (2257.95 U ml-1min-1). While for recovery treatment after 72 h both
varieties recorded the enzymes activity. At grand growth stage, SPS activity exhibited
the same pattern in heat shock and recovery conditions as at vegetative stage. At
maturity stage, SPS activity under control condition was slightly high in S2003-US-
633 than SPF-238 (Fig 15). However, under heat stress conditions (T24, T48 and T72)
S2003-US-633 exhibited reduction in this attribute but rapid recovery from heat
stress was observed through improved SPS activity as compared to heat shock
conditions. Comparatively among different growth phases, maximum SPS activity
was exhibited at maturity stage as compared with previous two stages (vegetative
and grand growth) in both cultivars but S2003-US-633 had better performance under
heat stress conditions. This results indicated that cultivar S2003-US-633 has better
sink strength to accumulate sucrose in stem.
94
Fig 15: Specific activity quantified of sucrose phosphate synthase (SPS) of sugarcane
cultivars S2003-US-633 and SPF-238 under control (30±2°C), heat shock (45±2°C) and
recovery (30±2°C) for 24, 48 and 72 h at vegetative, grand growth and maturity stages.
95
4.5.1.3. Cytoplasmic Invertase (CyINV)
In plant different invertase isoform are presented at different cellular localization
which are subcategorized based on pH which play various different important roles.
These invertase isozymes are cell wall invertase (acidic), vacuolar invertase (acidic)
and cytoplasmic invertase (neutral). Data suggest significant difference (p<0.05) for
temperature treatments, cultivars and their interactions (C x T) both vegetative and
grand growth phase while non-significant differences (p>0.05) were observed
among all treatments at maturity stage. At vegetative stage, the exposure of heat
stress at 24 and 48 h slightly declined the enzymes activities but after 72 h drastic
reduction was noted in both varieties as compared to the control. Under recovery
treatments both cultivars depicted varietal differences for cytoplasmic invertase
activity. As SPF-238 showed fast recovery of enzyme activity S2003-US-633. At grand
growth stage, maximum enzyme activity was noted in SPF-238 than S2003-US-633
under normal growth conditions. Although SPF-238 showed maximum activity but
heat shock exposure drastically affected the enzyme activity in both cultivars.
However, in maturity stage, there were no significant differences (p>0.05) for
cultivars (C), treatments (T) and their interactions (C x T). At maturity stage,
furthermore, maximum cytoplasmic invertase specific activity was noted in cultivar
SPF-238 than S2003-US-633 at all growth stages (Fig 16).
96
Fig 16: Specific activity quantified of cytoplasmic invertase (CyIN) of sugarcane cultivars
S2003-US-633 and SPF-238 under control (30±2°C), heat shock (45±2°C) and recovery
(30±2°C) for 24, 48 and 72 h at vegetative, grand growth and maturity stages.
97
4.5.1.4. Cell Wall Invertase (CWIN)
For cell wall invertase activity, significant differences (p<0.05) were found for
treatments (T) while cultivar and their interaction (C x T) were non-significant (p>
0.05) at formative and grand growth stages. However, at maturity stage significant
differences (p<0.05) were noted for cultivar and their interaction (C x T). At
vegetative stage, with increasing episodes of heat shock for 24, 48 and 72 h
decreased activity of cell wall invertase activity was observed. After 72 h of heat
stress, cell wall invertase activity declined drastically in both cultivars S2003-US-633
(0.233 U ml-1 min-1) and SPF-238 (0.211 U ml-1 min-1). Moreover, quick recovery was
observed only in SPF-238 at 72 h (0.30 U ml-1 min-1) as compared to S2003-US-633
(0.33 U ml-1 min-1). Whereas, at grand growth stage, no significant differences
(p>0.05) were evident. Only temperature treatment manifested the significant
difference (p<0.05) for cell wall invertase activity. However, maximum cell wall
enzyme activity was observed in cultivar SPF-238 (0.371 U ml-1 min-1) than cultivar
S2003-US-633 (0.301 U ml-1 min-1) control condition at grand growth stage. At
maturity stage, under heat stress conditions, both cultivars indicated reduction in
the activity of cell wall invertase enzyme, but this activity was substantially greater
in SPF-238 than S2003-US-633. During the recovery enzyme activity improved same
way in both cultivars. Collectively, SPF-238 had more invertase than S2003-US-633
in all treatments at all stages (Fig 17).
98
Fig 17: Specific activity quantified of cell wall invertase (CWIN) of sugarcane cultivars S2003-
US-633 and SPF-238 under control (30±2°C), heat shock (45±2°C) and recovery (30±2°C) for
24, 48 and 72 h at vegetative, grand growth and maturity stages.
99
4.5.1.5. Vacuolar Invertase (VIN)
At vegetative stage, data revealed significant differences (p<0.05) for treatments (T)
and their interaction (C×T) while non-significant differences (p>0.05) were observed
for cultivars (C) (Fig 18). Under control conditions, vacuolar invertase activity was
(10.1 U ml-1 min-1) in S2003-US-633 and (8.2 U ml-1 min-1) in SPF-238 but heat stress
significantly reduced the activity of vacuolar invertase in both cultivars while S2003-
US-633 exhibited slow recovery of the activity of this enzyme under recovery
conditions. Furthermore, at grand growth stage, vacuolar invertase activity was
declined in all treatments as compared to control but not significant differences
(p>0.05) were found for cultivar (C), treatment (T) and their interaction (C×T).
Maximum vacuolar enzyme activity was observed in SPF-238 under control, heat
shock and recovery treatments at maturity stage. There was decline in enzyme
activity with increasing episode of temperature in both cultivars. Statistical analysis
revealed significant differences (p<0.05) for cultivar and treatment but non-
significant differences (p>0.05) were noted for their interaction (C×T). Moreover, the
activity of vacuolar enzyme activity was more in vegetative stage than grand growth
and maturity stage in both cultivars.
100
Fig 18: Specific activity quantified of vacuolar invertase (VIN) of sugarcane cultivars S2003-
US-633 and SPF-238 under control (30±2°C), heat shock (45±2°C) and recovery (30±2°C) for
24, 48 and 72 h at vegetative, grand growth and maturity stages.
101
4.5.2. Qualitative Analysis
Invertase isozymes are present in plant at different location, based on location plant
invertase divided into three classes cytoplasmic, cell wall and vacuolar invertases.
Among sucrose metabolizing enzymes invertase is the most important sucrose
cleavage enzymes which was studied through Native-PAGE electrophoresis in both
cultivars S2003-US-633 and SPF-238 at different growth stages. Qualitative analysis
or differential staining of invertase isozymes (cell wall, vacuolar and cytoplasmic)
were carried out on NATIVE-PAGE at all growth stages.
4.5.2.1. Cytoplasmic Invertase (CyINV)
Results revealed that at formative stage, cultivar S2003-US-633 expressed
cytoplasmic or neutral invertase of 160 kDa. During heat stress treatment for T24 low
expression of cytoplasmic invertase while upon recovery conditions at (R24)
maximum intensity was exhibited. At grand growth stage, three types of cytoplasmic
invertases molecular weight (134, 150 and 160 kDa) were exhibited. Band intensity
of 134 kDa was decreased at T72 but expression was observed under recovery
treatments. Intense banding pattern of cytoplasmic invertases (134 and 160 kDa) was
observed than (150 kDa) at all treatments. Bands of cytoplasmic invertase (150 kDa)
were visible under control condition but its intensity decreased at (T72). However, its
expression was increased at R24, R48 and R72 upon recovery. At maturity stage,
expression of cytoplasmic invertase different at all treatments as compared to other
growths stages. Invertase type one (160 kDa) expressed only under recovery
condition after R48 and R72 h. Cultivar S2003-US-633 explored multiple type of
invertases at grand growth but only one type of invertase was exhibited at vegetative
102
and maturity stages (Fig 19). Qualitative analysis of cytoplasmic invertase of cultivar
SPF-238 at different growth stages (formative, grand growth and maturity) is
presented in (Fig 20). At vegetative stage, in SPF-238, one types of invertases
molecular weight (160 kDa) was observed. Under control conditions, band intensity
was high but diminished as temperature increased while presence of cytoplasmic
invertase was evident under recovery conditions. At grand growth stage, cultivar SPF-
238, multiple bands (160, 150 and 134 kDa) were detected. Expression pattern of this
invertase isoform 160 kDa was visible at control, T24 h and T48 h, but it was
diminished at T72 h than again reappeared at all recovery conditions. However, other
invertase isoforms (150 and 134 kDa) exhibited weak signal only but the sharp
intensity of band was evident under recovery condition. At maturity stage, cultivar
SPF-238 revealed the presence of invertase isoform molecular weight (160 kDa) at all
heat shock and recovery treatments with varying band intensity. Sharp bands were
observed only in control and heat shock condition (T72).
103
Fig 19: Native–PAGE analysis of cytoplasmic invertase (CyIN) of cultivar S2003-US-633
subjected to control (C) heat shock (45±2°C) and recovery treatments for 24, 48 and 72 h at
all growth stages. (Markers used ovalbumin (45 kDa), albumin bovine (monomer 67 kDa
and dimer 134 kDa) and gama globulin human (160 kDa).
104
Fig 20: Native–PAGE analysis of cytoplasmic invertase (CyIN) of cultivar SPF-238 subjected
to control (C) heat shock (45±2°C) and recovery treatments for 24, 48 and 72 h at all growth
stages. (Markers used ovalbumin (45 kDa), albumin bovine (monomer 67 kDa and dimer
134 kDa) and gama globulin human (160 kDa).
105
4.5.2.2. Vacuolar Invertase (VIN)
Quantitative analysis of vacuolar invertase at formative stage revealed that explored
one type of isozyme (160 kDa) with uniform but at heat shock treatments band
intensity was declined at T72 h. After recovery treatment maximum enzymes
expression was exhibited at R24, R48 and R72 h. At grand growth stage, only 134 kDa
molecular weight isozyme was observed, their expression was confirmed at heat
shock treatments T24, T48 and T72 h, band intensity of vacuolar invertase was
reduced. Furthermore, two types of vacuolar invertases (150 and 160 kDa) were
observed in S2003-US-633 variety, at control condition the band intensity was low
but then exposure to high temperature its expression was increased at maturity stage
while during recovery conditions only, isoforms (150 kDa) was expressed under heat
shock treatment (Fig 21). In cultivar SPF-238, only molecular weight of 160 kDa
isozyme was exhibited, the expression pattern initially increased, but declined with
increased in heat stress application (T72) and recovery conditions (R24) at formative
stage (Fig 22). At grand growth stage, band at 134 kDa was evident at control
conditions and recovery treatment (R72) but band not visible at any heat stress
treatment. While under control condition vacuolar invertase band intensity (160 kDa)
was maximum but the band intensity gradually declined at T24, T48 and T72 h after
heat exposure. However, under recovery treatments, the band intensity gradually
increased at R24 and R72 h at maturity stage.
106
Fig 21: Native–PAGE analysis of vacuolar invertase (VIN) of cultivar S2003-US-633 subjected
to control (C) heat shock (45±2°C) and recovery treatments for 24, 48 and 72 h at all growth
stages. (Markers used ovalbumin (45 kDa), albumin bovine (monomer 67 kDa and dimer
134 kDa) and gama globulin human (160 kDa).
107
Fig 22: Native–PAGE analysis of vacuolar invertase (VIN) of cultivar SPF-238 subjected to
control (C) heat shock (45±2°C) and recovery treatmnents for 24, 48 and 72 h at all growth
stages. (Markers used ovalbumin (45 kDa), albumin bovine (monomer 67 kDa and dimer
134 kDa) and gama globulin human (160 kDa).
108
4.5.2.3. Cell Wall Invertase (CWIN)
Multiple bands of molecular weight (140,150 and16 kDa) were expressed in cultivar
US-633 under recovery treatments at vegetative stage. Heat stress severely affected
on enzymes activity, especially after 72 h of heat exposure (T72). On the other hand,
upon recovery, maximum enzymes expression was observed at R24 h. Sharp bands
of 150 kDa and 140 kDa molecular weight isozymes were exhibited after heat shock
treatment after 48 h (T48) at grand growth stage maximum involvement of cell wall
invertase (Fig 23). At maturity stage, only 160 kDa isozyme was evident among all the
heat shock and recovery treatments except R72 (recovery treatment after 72 h). Only
band intensity varied. In SPF-238 cultivar, only 160 kDa molecular weight band was
displayed at vegetative stage. Band of cell wall invertase was sharp under control
conditions but at heat stress treatments (T24, T48 and T72 h) expression of the cell
wall invertase was affected (Fig 24). Upon recovery treatment, only its expression was
confirmed at R24 but at R48 and R72 h the band intensity was diminished. While at
grand growth stage, their expression was not visible at T24 and T48 h after heat shock
treatment. At maturity phase, only 160 kDa isozyme band was displayed same
pattern of band intensity as at vegetative stage.
109
Fig 23: Native–PAGE analysis of cell wall invertase (CWIN) of cultivar S2003-US-633
subjected to control (C) heat shock (45±2°C) and recovery treatments for 24, 48 and 72 h at
all growth stages. (Markers used ovalbumin (45 kDa), albumin bovine (monomer 67 kDa
and dimer 134 kDa) and gama globulin human (160 kDa).
110
Fig 24: Native–PAGE analysis of cell wall invertase (CWIN) of cultivar SPF-238 subjected to
control (C) heat shock (45±2°C) and recovery treatments for 24, 48 and 72 h at all growth
stages. (Markers used ovalbumin (45 kDa), albumin bovine (monomer 67 kDa and dimer
134 kDa) and gama globulin human (160 kDa).
111
4.6. Quality Parameters Analysis
Quality traits or parameters such as °brix, polarity, fiber content and sugar recovery
rates are the quality parameters of sugarcane (Table 8).
4.6.1. °Brix Estimation
°Brix is the sucrose content of aqueous solution. It indicates the percentage of cane
sugar (sucrose) by weight (1 g / 100 ml of water). ° Brix is conventionally used in wine
and sugarcane and fruit industries. Results indicates that significant differences
(p<0.05) were observed between treatments (T) and their interactions (C×T) except
cultivar (C) at grand growth stage. Under control conditions, greater °brix content
was depicted in cultivar S2003-US-633. Although heat shock treatment maximum
reduction affected the °brix content by declining in both cultivars. After 72 h of heat
stress in °brix content was evident in cultivar SPF-238 as compare to S2003-US-633.
Upon recovery conditions, both cultivars gradually improved after 48 h and 72 h of
recovery. Comparatively, rapid recovery was exhibited in cultivar S2003-US-633 than
SPF-238. However, at maturity stage, non-significant differences (p>0.05) were
exhibited for °brix content between treatments and their interactions (C×T) except
cultivar (C). Moreover, increased °brix content was observed in both cultivars at
maturity stage than grand growth stage. Temperature episodes also affected °brix
contents in both cultivars, after 72 h (T72) °brix content was drastically declined in
both cultivars. While cultivar SPF-238 showed a greater reduction in °brix content
than S2003-US-633. However, during recovery conditions, both cultivars gradually
improved in °brix contents while quick recovery was observed in S2003-US-633 after
72 h of recovery.
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4.6.2. Fiber Content
Regarding fiber content, it is defined as the dry fibrous insoluble materials of
sugarcane plant. Significant differences (p<0.05) were observed among cultivars (C),
treatments (T) and their interactions at grand growth stage. Under control
conditions, maximum fiber contents were noted in SPF-238 than S2003-US-633.
When temperature episodes were increased gradually, fiber content was decreased
in both cultivars while minimum fiber content was observed in cultivar S2003-US-
633 than SPF-238. But, the percentage of fiber content was progressively increased
after recovery treatments in both cultivars S2003-US-633 and SPF-238 respectively.
At maturity stage, significant differences (p<0.05) for cultivars (C), treatments (T) but
not their interactions (C×T) were observed. Overall, fiber content was slightly
increased at all treatments at maturity stage as compared to grand growth stage.
4.6.3. Pol Estimation
Pol % was investigated in both sugarcane cultivars at both growth and maturity
stages. Statistical analysis revealed that significant differences (p<0.05) were
exhibited for pol among cultivars (C) and their interactions (C×T) at growth stage.
Pol percentage at grand growth stage was gradually declined after applying heat
shock for 7 h than control conditions. In addition, in cultivar S2003-US-633 higher
pol content was noted under heat shock treatments after at (24 h T24) and (72 h
T72). Exhibiting that high temperature affected the pol of sugarcane at grand growth
stage. Moreover, upon recovery pol rate was improved in both cultivars with similar
pattern. While maximum pol percentage was found in cultivar S2003-US-633 than
SPF-238. At maturity, pol content of sugarcane reduced significantly due to heat
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stress. There were non-significant differences (p>0.05) between cultivars (C) and
treatments (T) together with a non-significant (p>0.05) interactions (C×T) of these
factors. Under heat shock conditions, pol % was gradually declined as compared to
control conditions. But both varieties exhibited same pattern under recovery
treatments. Comparatively, current results designated cultivar S2003-US-633 had
higher percentage of pol than other at both stages.
4.6.4. Sugar Recovery Rate Estimation
Data revealed that statistically significant differences were evident among all factors
for sugar recovery rate (%) at grand growth stage. High sugar recovery rate was
depicted in cultivar S2003-US-633 under control conditions. However, under heat
stress conditions both cultivars exhibited a relatively lesser reduction in this
attribute. In addition, rapid recovery was showed from stress in both cultivars. This
trend also was observed in heat stress and recovery treatments in cultivars SPF-238
(Table 8). At maturity, both cultivars and treatments indicated significant (p<0.05)
differences but not their interactions (C×T). In control conditions, S2003-US-633 (13
%) showed greater sugar recovery rate than SPF-238. Under the stress conditions,
SPF-238 depicted more declined in sugar recovery rate while under recovery cultivar
S2003-US-633 recovered readily more than SPF-238. Above mentioned results
showed that both cultivars losses sugar recovery at harsh environmental conditions
and after recovery treatments for different time durations (24-72 h) both cultivars
observed improvement in sugar recovery rate. At maturity stage both varieties
showed higher percentage of sugar recovery rate than previous both vegetative and
grand growth stage.
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Table 8: Quality parameters of both cultivars S2003-US-633 and SPF-238 under control (30±2°C), heat shock (45±2°C) and recovery (30±2°C) for 24, 48
and 48 h at vegetative, grand growth and maturity stages. Cultivar (C), Treatments (T) and Cultivar ×Treatments (C×T) at p level p<0.05.
Grand Growth Stage
Parameters Varieties
Mean ± SEM P Value
Control Heat shock Recovery Cultivar Treatment Interaction
C T24 T48 T72 R24 R48 R72 C T C×T
°Brix
S2003-US-633 13.78±0.12 12.85±0.37 12.82±0.06 12.41±0.13 12.14±0.11 12.52±0.13 12.74±0.05
ns * * SPF-238 13.44±0.19 13.12±0.06 12.25±0.10 11.88±0.07 12.44±0.15 12.46±0.12 12.56±0.12
Fiber content
(%)
S2003-US-633 17.55±0.40 15.48±0.70 14.51±0.38 13.92±0.29 15.19±0.39 16.81±0.33 16.92±0.33
* * * SPF-238 18.89±0.35 17.22±0.29 17.08±0.58 16.45±0.39 14.56±0.15 17.87±0.98 18.44±0.60
Polarization
S2003-US-633 12.13±0.66 10.76±0.25 10.31±0.24 9.47±0.245 9.36±0.250 10.28±0.18 9.81±0.325
* * * SPF-238 10.25±0.19 10.05±0.27 9.31±0.130 9.00±0.329 9.36±0.004 9.39±0.011 10.35±0.01
Recovery (%)
S2003-US-633 9.63±0.66 8.26±0.253 7.81±0.245 6.97±0.246 6.86±0.251 7.78±0.187 7.31±0.325
* * * SPF-238 7.75±0.18 7.55±0.272 6.81±0.130 6.50±0.329 6.86±0.001 6.89±0.015 7.58±0.007
Maturity Stage
°Brix
S2003-US-633 18.13±0.48 17.41±0.28 17.40±0.35 16.65±0.62 16.40±0.65 17.20±0.66 17.64±0.58
* ns ns SPF-238 16.69±0.48 16.41±0.67 16.16±0.59 15.48±0.71 15.54±0.33 16.18±1.03 16.14±1.03
Fiber content
(%)
S2003-US-633 14.40±1.71 13.39±0.28 11.60±0.36 11.35±0.62 11.60±0.66 11.39±0.67 11.86±0.59
* * ns SPF-238 18.68±0.73 17.71±0.67 15.17±0.31 14.52±0.13 14.79±0.58 15.82±1.00 15.59±1.03
Polarization
S2003-US-633 16.99±0.25 16.14±0.06 15.60±0.06 15.43±0.20 15.53±0.36 15.99±0.50 16.08±0.40
* * ns SPF-238 16.18±0.08 15.70±0.18 14.83±0.15 14.68±0.08 15.18±0.30 15.70±0.76 16.00±0.88
Recovery (%)
S2003-US-633 14.49±0.25 13.64±0.06 13.10±0.87 12.93±0.25 13.03±0.36 13.49±0.49 13.58±0.39
* * ns SPF-238 13.68±0.08 13.20±0.18 12.33±0.15 12.18±0.08 12.37±0.24 31.21±0.75 13.50±0.87
Significant difference p<0.05 (*) and non-significant differences p>0.05 (ns)
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4.7. Protein Profiling at Different Growth Stages
4.7.1. Protein Profiling at Vegetative Stage
At vegetative stage, protein expression was analyzed through SDS-PAGE for both
varieties. Proteins of different molecular weights were differentially expressed
during heat stress conditions. The highest molecular weight protein approximately
150 kDa (black) was clearly expressed upon heat stress and recovery treatments. The
band intensity of the protein remained stable during all the episodes of heat stress
24, 48 and 72 h (T24, T48 and T72) but during the recovery phase it was diminished
after 24 and 48 h (R24, R48) and then reappeared at 72 h (R72). Similarly, among
high molecular weight proteins 90 kDa (white), 70 kDa (yellow) and 60 kDa (red)
protein bands were consistently expressed during the heat shock treatments but at
the initial stages of recovery (R24-R48) these were not visible (Fig 25A). Thus, in both
varieties same pattern of protein expression was observed in high molecular weight
protein but in case of low molecular weight proteins there was a sharp high intensity
band observed at approx. 15 kDa (green) in cultivar S2003-US-633 which was not
present in cultivar SPF-238. During the SDS PAGE analysis 15 kDa protein might be
differentially expressed in S2003-US-633 as compared to SPF-238 during the heat
shock and recovery phases. However, protein band nearly 35 kDa (blue) evident at
heat shock as well as after 72 h recovery treatments (R72) while it was absent in
control conditions in cultivar SPF-238 (Fig 25B).
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Fig 25: SDS-PAGE protein profiling of sugacane cultivars (A) S2003-US-633 (B) SPF-238 at
formative stage under control at (30±2°C), heat shock (45±2°C ) and recovery treatments
(30±2°C) for 24, 48 and 72 h.
117
4.7.2. Protein Profiling at Grand Growth Stage
At grand growth stage, analysis of protein profiling observed induction of different
proteins molecular mass bands approximately 100 kDa, 66 kDa, 30 kDa and 35 kDa.
The 70 kDa protein band (Red) was prominent after 72 h exposure to heat shock
(T72) while under control conditions, the band intensity was slightly observed but
not prominently. Upon recovery, 70 kDa protein band was disappeared at 24 h (R24)
then after 48 and 72 h recovery treatment the bands, again reappeared at R48 and
R72. Same pattern of results was exhibited of 100 kDa protein (white). However, in
cultivar S20003-US633, the expression of 30 kDa and 35 kDa protein bands were not
higher at all treatments (Fig 26A). In cultivar SPF-238 both 66 kDa and 100 kDa
showed expression after 72 h (T72) heat treatments as compared to other
treatments. Another new protein 52 kDa (green) also observed under control
conditions and after 24 h (T24) heat shock conditions (Fig 26B). And also 30 kDa band
(yellow) molecular mass protein was expressed only under control and under heat
stress conditions at 24 h (T24) but rest of the samples showed no bands.
118
Fig 26: SDS-PAGE protein profiling of sugacane cultivars (A) S2003-US-633 (B) SPF-238 at
grand growth stage under control at (30±2°C), heat shock (45±2°C) and recovery treatments
(30±2°C) for 24, 48 and 72 h.
119
4.7.3. Protein Profiling at Maturity Stage
At maturity stage, in both cultivars (S2003-US-633 and SPF-238) the expression
pattern was quite different than vegetative stage. In cultivar S2003-US-633, protein
band approximately 66 kDa molecular mass (red), was observed very clearly with
higher intensity under control conditions, heat shock after 24-48 h (T24 and T48) as
well as after 48 h (R48) recovery conditions while 30 kDa protein band intensity was
very low after 72 h (T72) exposure of heat treatments. However, induction of 100
kDa molecular weight protein was not visible under heat shock T72, recovery R24 and
R72 but the band was slightly visible in other treatments (Fig 27A). In cultivar SPF-
238, 66 kDa (red) and 100 kDa (white) proteins bands were dominantly expressed in
all treatment except after heat exposure at 48 h (T48). Both proteins (66 kDa and 100
kDa) were not expressed at T72 h after heat shock treatments. However, another
differential expression exhibited as 30 kDa band molecular mass (yellow) protein was
under heat shock treatment and recovery but the expression level was very low in
control conditions (Fig 27B).
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Fig 27: SDS-PAGE protein profiling of sugacane cultivars (A) S2003-US-633 (B) SPF-238 at
maturity stage under control at (30±2°C), heat shock (45±2°C ) and recovery treatments
(30±2°C) for 24, 48 and 72 h.
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4.8. Correlation
Sugar metabolizing enzymes play vital role in sugarcane plant for synthesis and
hydrolyze sugar at different growth stages. Furthermore, theses enzymes also help
sugar loading and unloading at different compartment or location in plant cells to
balance the cellular mechanisms of different biomolecules. Present study was
performed to investigate correlation among sugar profile, sugar metabolizing
enzymes such as sucrose synthase, sucrose phosphate synthase, cell wall invertase,
cytoplasmic or neutral invertase and vacuolar invertase and quality parameters like
pol, fiber content, °brix and sugar recovery rate at vegetative, grand growth and
maturity stages. The correlation results of the all parameters at different growth
phases were presented in (Table 9). Data was tested at a significant p<0.01 (**) and
p<0.05 (*). Results revealed that the relationship among total sugar with sucrose
synthase (SS) and sucrose phosphate synthase (SPS) were strongly positively
correlated at all growth stages. While its relationship with reducing sugar at maturity
stage was negative at maturity stage but positive to relation was exhibited at
vegetative and maturity stage. The association of quality parameters such as °brix,
recovery rate and pol with total sugar was positive but negative association was
observed with reducing sugar at maturity stage. Sugar recovery relationship among
cell wall invertase (CWIN) and vacuolar invertase (VIN) were negatively correlated at
both vegetative and maturity stages. This result indicates that the °brix, pol and total
sugar were inversely proportional to reducing sugar in both cultivars. However, the
association of invertase isozymes such as vacuolar and cell wall invertases with total
sugar and non-reducing sugar as well as sugar recovery rate, pol and °brix were
negatively correlated at maturity stage.
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**Significant correlation at p<0.01 and *Significant correlation at p<0.01
Table 9: Correlation among sugar profile, sugar metabolizing enzymes and quality parameters at all growth stages.
Parameters Stages TS RS NRS SS SPS CyIN VIN CWIN °Bx Fb Pol RC
Total Sugar (TS)
V 1 .732** .972** .694** .707** .501** .114 .315* - - - -
G 1 .448** .390* .771** .755** .540** .378* .532** .746** .378* .739** .739**
M 1 -.701** 1.000** .629** .667** .037 -.250 -.310* .473** -.430** .451** .451**
Reducing Sugar (RS)
V .732** 1 .551** .572** .579** .245 -.150 .221 - - - -
G .448** 1 -.648** .464** .514** .627** .372* .383* .387* .721** .200 .200
M -.701** 1 -.723** -.294 -.298 .273 .513** .573** -.425** .668** -.296 -.296
Non-reducing Sugar (NRS)
V .972** .551** 1 .654** .671** .534** .193 .308* - - - -
G .390* -.648** 1 .180 .115 -.183 -.059 .065 .240 -.417** .424** .424**
M 1.000** -.723** 1 .622** .659** .023 -.264 -.326* .476** -.446** .449** .449**
Sucrose Synthase (SS)
V .694** .572** .654** 1 .715** .532** .325* .511** - - - -
G .771** .464** .180 1 .658** .534** .439** .494** .734** .414** .553** .553**
M .629** -.294 .622** 1 .822** .328* .152 .094 .503** -.009 .588** .588**
Sucrose Phosphate Synthase (SPS)
V .707** .579** .671** .715** 1 .480** .086 .326* - - - - G .755** .514** .115 .658** 1 .522** .370* .626** .750** .342* .647** .647**
M .667** -.298 .659** .822** 1 .375* .170 .130 .459** .073 .597** .597**
Cytoplasmic Invertase (CyIN)
V .501** .245 .534** .532** .480** 1 .155 .360* - - - -
G .540** .627** -.183 .534** .522** 1 .207 .408** .579** .562** .325* .325*
M .037 .273 .023 .328* .375* 1 .415** .362* .098 .385* .077 .077
Vacuolar Invertase (VIN)
V .114 -.150 .193 .325* .086 .155 1 .230 - - - -
G .378* .372* -.059 .439** .370* .207 1 .450** .443** .245 .431** .431**
M -.250 .513** -.264 .152 .170 .415** 1 .659** .024 .526** -.074 -.074
Cell Wall Invertase (CWIN)
V .315* .221 .308* .511** .326* .360* .230 1 - - - -
G .532** .383* .065 .494** .626** .408** .450** 1 .579** .293 .504** .504**
M -.310* .573** -.326* .094 .130 .362* .659** 1 -.140 .640** -.010 -.010
°Brix (°BX) G .746** .387* .240 .734** .750** .579** .443** .579** 1 .249 .703** .703**
M .473** -.425** .476** .503** .459** .098 .024 -.140 1 -.474** .308* .308*
Fiber Content (FC) G .378* .721** -.417** .414** .342* .562** .245 .293 .249 1 .204 .204
M -.430** .668** -.446** -.009 .073 .385* .526** .640** -.474** 1 .050 .050
Polarization (Pol) G .739** .200 .424** .553** .647** .325* .431** .504** .703** .204 1 1.000**
M .451** -.296 .449** .588** .597** .077 -.074 -.010 .308* .050 1 1.000**
Sugar Recovery (SR) G .739** .200 .424** .553** .647** .325* .431** .504** .703** .204 1.000** 1
M .451** -.296 .449** .588** .597** .077 -.074 -.010 .308* .050 1.000** 1
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4.9-Heat Map
Table 9: Status of Thermotolerant Sugarcane Cultivars
Growth Stages
Cultivars
Sugar Analysis Sucrose Metabolizing Enzyme Analysis
Sugar Recovery Analysis Thermotolerance Indicators Analysis
Remarks TS
RS
NRS
SS
SPS
CyIN
CWIN
VIN
°Bx
Pol
Fb
SR
EC
MDA
H2O2
Pr
Vegetative
S2003-US-633
Nil Nil Nil Nil
Least Susceptible
SPF-238
Nil Nil Nil Nil
Most Susceptible
Grand
Growth
S2003-US-633
Moderately Susceptible
SPF-238
Moderately Tolerant
Maturity
S2003-US-633
Most Tolerant
SPF-238
Least Tolerant
Most Susceptible Moderately Susceptible Least Susceptible Least Tolerant Moderately Tolerant Most Tolerant
Susceptible (SPF-238 ) Tolerant (S2003-US-633)
Total Sugar (TS), Reducing Sugar (RS), Non-reducing Sugar (NRS), Sucrose Synthase (SS), Sucrose Phosphate Synthase (SPS), Cytoplasmic Invertase (CyIN), Cell Wall Invertase (CWIN), Vacuolar Invertase (VIN),°Brix (°Bx), Sugar Recovery (SR), Polarization (Pol), Fiber (Fb), Proline (Pr), Hydrogen Peroxide (H2O2 ), Electrolytes leakage (EC) and Malondialdehyde (MDA).
124
SECTION # 5 DISCUSSION
125
Discussion
5.1. Morphological Analysis
Yield parameters are potential and reliable morphological indicators to select and
differentiate the thermotolerant sugarcane cultivars. In the present study, heat stress
conditions decreased the plant height and root length, fresh to dry weight ratio, stem
diameter and biomass accumulation that eventually reduced yield and related
attributes of both sugarcane cultivars at vegetative, grand growth and maturity
stages (Table 5-7).
Among all stages, vegetative stage is considered more critical, severely affecting the
shoot length by application of heat stress for different episodes but slowly
improvement was shown upon recovery conditions in SPF-238. While possible reason
for taller shoot length of sugarcane cultivar S2003-US-633 may be due to high yield
characteristics than cultivars SPF-238 exhibiting its potential for stress tolerance
(Abubakar et al., 2013; Gravois and Legendre, 2011). Varietal differences were
evident with respect to root length (Table 6) effecting root length of both sugarcane
cultivars upon heat stress. While longest root length (82 cm) was observed in S2003-
US-633 compared to SPF-238 (78 cm) indicating its deep root branching for more
water absorption to combat water loss due to evaporation (Madhav et al., 2017).
Roots play a vital role for supply of nutrients and water for growth and development
of crop and tendency to develop deep root system observed may be used as selection
criteria for drought and thermotolerant as well (Wasaya et al., 2018).
126
Another important agronomic trait, number of tillering influenced by biotic and
abiotic factors because the optimal temperature for tillering formation is 30°C.
Maximum number of tillers in SPF-238 depicts its low sugar recovery potential in
stalk, delayed accumulation of sucrose in culm and stunted growth due to maximum
energy utilization during tiller formation (Misheck, 2013; Inman-Bamber et al., 2009).
No external changes were observed in sugarcane leaf length, leaf width, due to short
episode of temperature however, leaf colors were slightly changed when the plants
exposed at different heat shock episodes. High temperature resulted pre and post-
harvest damages such as, scrolling of leaf, sunburn of leaf, leaf senescence, shoot and
root growth inhibition, stem diameter and declined the yield for long exposure
(Vollenweider and Gunthardt-Goerg, 2005). Leaf structure also affected by heat
stress frequently causing of thinner leaf with higher leaf area (Poorter et al., 2009).
These morphological alterations are supported with changes in leaf anatomy. Leaf
under high temperature and drought usually have smaller cells and higher stomatal
density (Shahinnia et al., 2016) but limited data available regarding to leaf anatomy
alterations due to heat stress (Wahid et al., 2007). In this study, no changes were
observed in number of nodes and internodes per plant in both cultivars at all stages
due to heat stress. However, number of nodes was increased in maturity stage as
compared to grand growth stage in S2003-US-633. Number of nodes is important
criteria for sucrose accumulation and there is inverse relationship between number
of nodes and sucrose content in sugarcane stalk (Bonnett et al., 2006). Number of
nodes and internodal distances are also controlled by genetic factors and may also be
127
affected by plantation date or other environmental conditions (Shahzad et al., 2002;
Shahzad et al., 2016).
Under high temperature stress drastic reduction was exhibited in fresh to dry weight
ratio in both cultivars. Highest fresh to dry matter ratio was found in cultivar, S2003-
US-633 upon heat stress only at vegetative stage and attributed due to increased
photosynthetic rate. Heat stress at 40 / 24 °C (day/night) significantly declined the
shoot dry weight in quinoa (Hinojosa et al., 2019), sugarcane, corn and pearl millet
(Wahid et al., 2007; Zhao et al., 2006). In this study, cultivar S2003-US-633 could be
ranked as thermotolerant on morphological basis due to high yielding characteristics,
deep rooting system and high fresh to dry matter ratio (Table 10).
5.2. Thermotolerant Indicators Analysis
Malondialdehyde (MDA) content as by-product of lipid per oxidation, was quantified
in both cultivars of sugarcane plant under heat stress. Increase in lipid peroxidation
also a marker of oxidative stress (Goel and Sheoran, 2003) for abiotic and biotic
stresses (Hameed and Iqbal, 2014) and it could be used as an important
thermotolerant indicator of physiological damages during crop growth and
development. In current study, maximum accumulation of MDA content was
revealed in both cultivars S2003-US-633 and SPF-238 under high temperature
conditions displaying substantial lipid peroxidation of biological membrane leading to
the reactive oxygen production along with losing membrane integrity (Boaretto et al.,
2014). The S2003-US-633 cultivar had minimum MDA content accumulation than SPF-
238 cultivar (Fig 6). Electrolyte leakage also closely related with the degree of cell
membrane
128
injury and relative membrane thermosability under heat stress. Plasma membrane is
very sensitive to abiotic stress (high temperature) in plant cell, it is primary site for
injury (Blum, 2018). This injury can be measured by the loss of membrane integrity
reflected in ion leakage from plant cells (Liu and Huang, 2000; Jiang and Huang,
2001a; Salvucci et al., 2004) and has been used as stress induced markers in under
stress conditions. Under heat shock conditions, plant cells extremely damage and
even cell death may occur within minutes. This phenomena is extensively used as a
test for the stress induced damage of plant tissue and measure of plant. Recent study
revealed significant damage to the membrane thermal stability of SPF-238
(susceptible cultivar) under heat stress conditions, whereas S2003-US-633 (tolerant
cultivar) maintained a membrane thermal stability with minimal electrolytes leakage
(Fig 9). This may be attributed due to cell membrane damages by losing membrane
integrity with increase in unsaturated fatty acids leading to increased fluidity of the
membrane (Horvath et al., 2012) which further increase Ca2+ influx (Bita and Gerats,
2013). These outcomes are supported by previous study (Zhang et al., 2005) where
excessive permeability of membrane severely damaged the mesophyll cells and led
to the increased electrolytes leakage under heat stress conditions (Savchenko et al.,
2002; ElBasyoni et al., 2017 ; Kumar et al., 2013). It is also reported that EC content
was associated with grain yield reduction (Khan et al., 2019). Among all growth
stages, both cultivars significantly influenced under heat shock treatments at
vegetative and grand growth stages. Comparatively maximum EC content was
observed in vegetative stage than other stages. Cell membrane thermostability
depends on species, tissue, cell types and can be triggered by biotic and abiotic
129
factors including pathogen attack (Maffei et al., 2007), salinity (Demidchik et al.,
2010), oxidative stress (Demidchik et al., 2003, 2010), heat (Liu and Huang, 2000) and
others. Heat stress could lead to over production of reactive oxygen species (ROS)
which deteriorate photosynthetic machineries in plants (Wang, 2004). High levels of
ROS accumulation significantly effect on physiological and biochemical functions such
as destruction of plasma membrane, lipid per oxidation, protein denaturation,
destruction of enzymes, DNA, RNA and figments (Bose et al., 2013; S Li et al., 2018)
resulted reduced crop yield and quality. (Sharma et al., 2017; You and Chan, 2015;
Gaschler and Stockwell, 2017). Among the different types of ROS hydrogen peroxide
is a lethal reactive oxygen species, which has harmful effect on plant cells (R. Sairma
and Srivasta, 2002). It is also most important signaling molecule playing vital role in
the photosynthesis (Rodrigues et al., 2017), cell wall cross linkage (Li et al., 2017)
stress acclimation (Lv et al., 2018) and antioxidant defense system (Liu et al., 2016).
In this study, increased hydrogen peroxide contents in both sugarcane cultivars were
exhibited under heat stress conditions which indicated the manifestation of oxidative
stress. But maximum hydrogen peroxide content was found in SPF-238 exhibiting
damages and low integrity of cell membrane. The level of hydrogen peroxide was
higher at vegetative stage than other growth stages (Fig 8). In general, when the level
of hydrogen peroxide level exceeds then caused damage to essential cellular
components (Mittler, 2006). Similar results were also observed in wheat (Hamurcu et
al., 2014) and canola (Akram et al., 2018) plants under stress conditions. Studies at
physiological, molecular, biochemical levels suggest that osmolytes perform
important role in mitigating heat stress in plant by decreasing reactive oxygen species
130
(ROS) level by protecting cell membrane and protein (Hayat et al., 2012). Free proline
and other compatible solutes such as glycine and soluble sugar is essential to regulate
osmotic activities and protect cellular structures, membrane stability by maintaining
the cell water balance, improve cytoplasmic acidosis and by buffering redox potential
(Khan et al., 2019; Farooq et al., 2008; Chen and Murata, 2002). As the synthesis of
proline depend on plant to plant, species to species, plant tissue under investigation
past memory and genetic factors (Ashraf and Foolad, 2007; Qaun et al., 2004).
Therefore, both cultivars S2003-US-633 and SPF-238) had ability to accumulation of
thermotolerant osmolyte (proline) under heat stress conditions at different growth
phases. Cultivar S2003-US-633 had more potential to accumulate free proline as
compared to other cultivar SPF-238 under heat stress conditions at all growth stages
(Fig 7). This higher accumulation of osmolytes such as proline maintained water
environment in the cells (Gupta et al., 2013; Giri et al., 2011; Sakamoto and Murata,
2000) and providing thermotolerance (Tanveer et al., 2019; Sharma et al., 2019) to
S2003-US-633. On the other hand, proline also serve as energy for respiration,
ammonia sources during stress conditions and directly participating in plant
metabolism after stress relief (Hussain et al., 2019) and act as electron acceptor,
osmolyte and protect the cell membrane (Bartels and Sunkar 2005; Gupta et al.,
2013). Accumulation of free proline in cultivar S2003-US-633 might contribute
minimum ROS production such as hydrogen peroxide with compromised MDA and
electrolytes leakage than cultivar SPF-238 under heat stress conditions. These
biochemical attributes can index the degree of tolerance of sugarcane to exhibit
adaptability under stressful conditions providing the insights to molecular breeders
131
to identify the thermotolerant varieties with improved recovery of sugar. In this
regard, cultivar S2003-US-633 is ranked as thermotolerant variety.
5.3. Sugar Analysis
In current study, biochemical parameters such as (total sugar, reducing sugar and
nonreducing sugar) were quantified upon heat stress in both sugarcane varieties.
These biochemicals not only play imprative role in growth and development of crop
but also act as osmolytes in any environmental stress conditions such as biotic and
abiotic for protect the plant cells (Zhou et al., 2017). It is evident from results that
heat stress declined the content of total sugar in both sugarcane varieties but upon
the recovery treatment, sugar content improved only at maturity stage. These results
are in agreement with Datir et al., 2015 finding that total sugar was low at 300 day
after planting and then gradually increased at 360 day after planting (Datir et al.,
2015). There is variability in sugar content in different sugarcane varieties at different
growth stages, sucrose content was predominant at maturity stage (Tana et al.,
2014). However, higher total and non-reducing sugar content was observed in
cultivar S2003-US-633 at all stages (Fig 10 and 12). This accumulation of sucrose may
be due to the decreased invertase activity and delayed ripening processes in S2003-
US-633 (Sachdeva et al., 2003). However, reducing and nonreduing sugar analysis
depicted same pattern of decrease under high temperature in both varieties at
formative, grand growth and maturity stages. This reduction in total sugar content is
attributed due to less carbon assimilation and subsequent partitioning of carbon
derived energy products including sucrose, from source (leaves) to sink (stem),
tissues or increased respiratory demand
132
(Huntingford et al., 2017; O sullivan et al., 2017; Amthor et al., 2019). Results
revealed that maximum reducing sugar was observed in cultivar SPF-238 only under
heat stress conditions at grand growth and maturity stage. But upon recovery not
much recovered reducing sugar content (Fig 11). It is assumed that enhanced
invertase activity may be responsible for increased reducing sugar (Tana et al., 2014).
High temprature stress declined the activity of key sucrose metabolizing enzymes
sucah as adenosine diphosphate glucose pyrophophorylase, SPS and invertase
affecting the sucrose accumulation (Vu et al., 2001). These alteration in sucrose
content under heat stress is fundamental for understanding the biochemical
pathways associated in the molecular response of plant (Rodziewicz et al., 2014).
5.4. Sugar Recovery Rate Analysis
Sugar industrialists and farmers required more sucrose contents, cane yield as well
as high sugar recovery rate. Quality parameters play vital role in marker assisted
selection for high cane yield and sugar recovery rate. Sucrose recovery rate analysis
comprised of different quality characteristics like sugarcane pol, brix, moisture, and
fiber content and sugar recovery rate. Present study demonstrated that S2003-US-
633 had highest sugar recovery (14.4 %) while in cultivar SPF-238 (13.6 %) were
recorded under control conditions. It is reported that pol % is directly proportional to
sugar recovery (Khan et al., 2018). It is suggested that maximum pol (18.38 %) was
exhibited in cultivar MS-92-CP-99 with highest sugar recovery (12.44 %) (Ali et al.,
2019) These high and low cane yield and sugar recovery potential are associated with
various morphological and genetical characters (Ali et al., 2019; Khan et al., 2018).
133
However, it is challenging to get simultaneously cane yield and sugar recovery rate in
the same cultivar (Khan et al., 2018). Due to the highest CCS %, yield and sugar
recovery potential, cultivar S2003-US-633 was recommended for commercial cultivar
in Punjab Pakistan (Tahir et al., 2014). It is reported previously that cultivar SPF-238,
sugarcane produced 115 ton per hector and sugar recovery rate 6.02 % while cultivar
S2003-US-633 can produced 130 ton per hector and recovery rate 12.90 %
(Anonymous 2009-2010). Whereas, the cultivar S2003-US-633 was produced average
15 % commercial cane sugar (CCS) also produced excellent recovery, this variety is
fast growth rate, salt tolerant as well as highest sugar recovery potential while
cultivar, SPF-238 was banned because low yield with 43.48 to 53.40 t / hec and
recovery rate 7.96 % to 9.48 % respectively (Afzal et al., 2011). Maximum sugar
recovery was exhibited at temperature from 22°C to 26°C at maturity stage or
ripening (Srivastava et al., 1995).
However, results revealed that exposure of heat treatments declined the sugar
recovery rate in both cultivars at both grand growth and maturity stages. Upon the
recovery conditions, both cultivars recovered with same pattern. Fluctuation in
temperature and humidity adversely effect on sugar recovery and sugar
accumulation in sugarcane crop (Pathak et al., 2019) by affecting metabolizing
enzymes activities. High temperature denatures enzymes and protein ultimately
declining the sugar recovery rate (Kohila and Gomathi, 2018) and these sugar
recovery and sucrose metabolizing enzymes such as SPS and SS positively associated
with sucrose content. Cultivar SPF-238 exhibited declined SPS and SS activities at high
temperature (45 ± 2°C) but thermotolerant cultivar S2003-US-633 had highest
134
sucrose metabolizing enzymes activity. Low sugar recovery rate is also attributed due
to high fiber content in sugarcane stalk (Rakkiyappan et al., 2003). Present
experiment revealed that among cultivars SPF-238 had maximum fiber content
comparison with cultivar S2003-US-633 at grand growth and maturity stages (Table
8). Regarding pol %, it content also declined due to improper harvesting season (early
or late) (Rakkiyappan et al., 2009; Kulkarni et al., 2010). Early mature cultivar of
sugarcane and less fibrous percentage are tolerant cultivar to postharvest sucrose
loss (Siddhant et al., 2009). Recent data showed that cultivar S2003-US-633 had less
fibrous percentage but high pol % than cultivar SPF-238. Sowing date also most
important factor for high sugar recovery rate, it is suggested that sugarcane crop
cultivated in September achieved high sugar recovery rate and sugarcane yield as
compared to March (Fazal-ur-Rehman, 2018). So cultivar S2003-US-633 may be the
high yielding, resistant in extreme temperature conditions and potential to high
recovery rate as compared to cultivar SPF-238.
5.5. Sucrose Metabolizing Enzymes Analysis
5.5.1. Quantitative Analysis
Sucrose metabolizing enzymes such as sucrose phosphate synthase (SPS), sucrose
synthase (SS), cell wall (CWIN), cytoplasmic (CyIN) and vacuolar (VIN) invertases, are very
important enzymes for sugar metabolism in sugarcane. There is need to understand the
characteristics of sucrose metabolizing enzymes for improvement of sugar recovery rate
against abiotic stress especially high temperature. In sugarcane, sucrose synthesis in
135
leaves is carried out by photosynthesis and transported to stems which is fundamental
requirement for plant growth and development (Bihmidine, 2013; Yadav, 2015).
Moreover, this sugar synthesis and transports are affected under biotic and abiotic stress
conditions (Lemoine, 2013) and regulated by sucrose phosphate synthase (SPS). SPS
activity was higher in high sucrose accumulation cultivars in mature nodes than lower
sucrose accumulation cultivars (Verma et al., 2011). While, SPS synthesized sucrose -6-
phosphate by protein phosphorylation with glucose 6-p and resynthesize sucrose after
apoplastic hydrolysis in sugarcane (Oparka et al., 1992; Gayler and Glasziou ,1972). In this
study, sucrose phosphate synthase (SPS) activity was evaluated at different heat shock
conditions in both sugarcane cultivars (Fig 15). Quantitative analysis revealed that
significant decline in SPS enzyme was exhibited in both cultivars but cultivar S2003-US-
633 had higher SPS enzymatic activity than SPF-238 under heat stress conditions (45 ± 2
°C for 24, 48 and 72 h) at maturity stage (Gomathi et al., 2017). This swiftly inactivation
of SPS activity under heat stress was attributed due to denaturation of enzyme structure
under heat stress (Neliana et al., 2019). Another enzyme sucrose synthase, catalyzes the
reversible hydrolysis of sucrose using UDP to yield fructose and UDP-G (Granot and Stein,
2019). At vegetative stage, minimum SS activity was found in both cultivars as compared
to grand growth and maturity stages under heat stress exhibiting enzyme may be
hydrolyzed into fructose and glucose for as fuel for growth and developments at
vegetative stage (Ruan, 2014). But at maturity stage, the SS activity was declined due to
limited sink strength especially in low sucrose accumulation cultivar (SPF-238) under heat
stress (Fig 14). Temperature is very import factor for enzyme activity, sucrose synthase
showed optimal activity at 37°C (Verma et al., 2018) and at 30 °C (Schmolzer et al., 2016).
136
Other studies also confirmed that SS activity can either decline or enhance at maturity or
ripening stage (Botha and Black 2000; Joshi et al., 2013). It has been reported that sucrose
metabolizing enzymes activities were found dissimilar in different cultivars at different
growth stages (Tana et al., 2014). As sucrose accumulation depends on SS and invertases
isoforms in sugarcane and sucrose synthase was negatively associated with invertases
while positive correlation with SPS enzymes activities (Gutierrez-Miceli et al., 2002;
Siswoyo et al., 2007). Now-a-days, efforts have been made for the increased sucrose
contents or sugarcane production in sugarcane crop through manipulating genes
associated with sucrose (Conradie, 2011). During ripening of rice seed, maximum SS
activity was exhibited under heat stress (Takehara et al., 2018). In addition, potential
thermotolerant SS was observed in wheat crop (Wh-1021). Another significant enzyme
involved in sucrose metabolism is invertase, hydrolyzes sucrose to hexose as a fuel for
cell growth, elongation and other metabolic processes (Roitsch and Gonzalez, 2004).
Invertase plays vital role in sucrose accumulation and have been well documented in
sugarcane plant since five decades (Gayler and Glasziou, 1972; L. Wang et al., 2017).
Invertases isoforms are classified according to subcellular location and optimum pH and
designated cell wall invertase at apoplectic space, vacuolar invertase at in vacuoles and
cytoplasmic invertase in cytoplasm (Ma et al., 2000). These enzymes declined the
expression under heat stress, some increase upon recovery. In this study the enzyme
activity of invertase isoforms (CyIN, CWIN and VIN) of cultivar S2003-US-633 and SPF-238
were carried out at various growth phases under heat stress conditions. Results showed
that when heat stress was applied for different episodes (T24, T48 and T72) both cultivars
declined the activity of cytoplasmic invertase at all stages. Comparatively, highest CyIN
137
activity exhibited at maturity stage in SPF-238. Similar results reported that the
cytoplasmic invertase activity or their expression was increased while maturation process
as this play important role is in sugars accumulating at maturity stage (Sachdeva et al.,
2011). In sugarcane, invertase activity is higher in juvenile tissue where fast cleavage
happens to provide fuel for cell growth or biosynthesis and other metabolic activities (A
Chandra et al., 2015). Lower CyIN activity was observed in cultivar S2003-US-633 at this
maturity stage (Fig 16). In contrast, results were suggested that minimum CyIN activity
was showed in mature tissues in early maturity sugarcane cultivar (Rossouw et al., 2010).
Cell wall invertase that is believed to be present in sugarcane plant (Vorster and Botha.,
1998) have variable functions on the bases of specificity and enzymatic characterization
(Wan et al., 2018). CWIN plays imperative role for plant growth and development,
reproduction, germination and ovary activity in plants (Goetz et al., 2017; Wan, Wu, Yang,
Zhou, and Ruan, 2018; Lv et al., 2018). This study reported the significant decrease in the
activity of CWIN from 24-72 h after heat stress treatments in both cultivars (S2003-US-
633 and SPF-238). In tomato crop, CWIN activity increased under heat stress conditions,
which suppresses reactive oxygen species independent in cell death (Liu et al., 2016).
Among cultivars, the activity of CWIN was substantially greater in SPF-238 than cultivar
S2003-US-633 at vegetative and maturity stages with low sucrose accumulation (Fig 17).
It is proposed that the highest level of CWIN associated with low level of sucrose
accumulating cultivar in juvenile tissues while lowest level of activity was associated with
higher level of sucrose accumulating cultivar in mature sugarcane stem or stalk (Zhu et
al., 1997). CWIN activity is consider as the most significant enzyme regulating the sucrose
content in sugarcane stalk as compared to other sucrose metabolizing enzymes (SPS, SS
138
and CyIN). Maximum activity of soluble acid invertase (VIN) and insoluble acid invertase
(CWIN) in immature tissues of S2003-US-633 were observed (Batta et al., 2008). It is
assumed that CWIN play important role in this variety in sugar signaling and transporting,
supplying nutritional for elongation of pollen tubs (Goetz et al., 2017). Comparatively
CWIN activity, acid invertase and cytoplasmic invertase were declined in mature tissues
S2003-US-633 due to least growth and development processes (Verma et al., 2011). This
low invertase activity may govern active and maximum sucrose accumulation in the
vacuoles and in storage sinks (Chandra et al., 2012). For example, knock out of cell wall
invertase genes declined grain mass in corn (Miller and Chourey, 1992), inhibited
expression of cell wall invertase genes led to declined root growth in carrot (Tang et al.,
1999). Regarding vacuolar invertase activity, present study revealed that both cultivars
(S2003-US-633 and SPF-238) declined VIN activity but during control, heat shock and
recovery treatments, maximum reduction was observed in cultivar S2003-US-633 at
maturity stage. As the short-term response of heat stress might differ from long term
responses but 72h might be a significant time point for initiating long term heat stress
response in sugarcane crop. Acid invertase cleavage sucrose during cane maturity and
post-harvest (Chandra et al., 2015), may be due to this reason minimum VIN activity was
exhibited in cultivar S2003-US-633 at maturity stage. It is assumed that storage of sucrose
in vacuoles leads to high sucrose accumulation or high sink strength in sugarcane stalk
(Fig 18). On the other hand, sugar metabolizing gene inhibition, disturbed biochemical
activity and deactivated regulatory network eventually result decline growth rate, sugar
production or yield in sugarcane due to heat shock. The activity of VIN depended on
multitude of signals, sugars hormones and other environmental stresses and exhibited
139
maximum activity when rapid growth observed (Lontom et al., 2008; Koch, 2004). In case
of down regulation of VIN genes in sugarcane at maturity stage may enhance sucrose
content or sugar recovery percentage along with regulation of source to sink strength of
sucrose by physiological and biochemical alteration (Tauzin and Giardina, 2014). It is also
reported other crop that the VIN invertase gene suppression can be minimize or could
control sugar-end defect formation in potato (Zhu, 2014). Cleavage of sucrose in the
intracellular space and vacuole affect the sucrose content in sugarcane stalk (Wang et al.,
2013).
5.5.2. Qualitative Analysis of Invertase Isozymes
In the present study cytoplasmic invertase (CyIN) of multiple molecular weights were
identified (134,150 and 160 kDa) in both sugarcane cultivars (S2003-US-633 and SPF-
238) at different growth phases. Although, increasing temperature declined the
expression of invertase activity but minimum CyIN expression was exhibited in
cultivar S2003-US-633 as compared to SPF-238 at vegetative and maturity stages due
to presence of invertase inhibitors (Fig 19). It is suggested that invertase inhibitor
gene homologs (18.17 kDa ShINH1) and (19.97 kDa ShINH2) were identified in
sugarcane (Shivalingamurthy et al., 2018). Regarding cell wall invertase that multiple
CWIN molecular mass band proteins (140, 150 and 160 kDa) were expressed in
cultivar S2003-US-633 at vegetative and grand growth stages suggesting is
involvement in thermotolerance (Fig 20). Only 160 kDa molecular mass CWIN found
in SPF-238 at all growth stages (Pressman et al., 2006). Among VIN, three types of
molecular weight (134, 155 and 160 kDa) were exhibited in both cultivars at different
140
growth phases. Only 160 kDa molecular mass was observed at vegetative and
maturity stages in both cultivars. While only 134 kDa molecular mass VIN band was
observed at grand growth stage in both cultivars. Previous study also reported that
60, 120, 240 kDa (Vorster and Botha 1999), 150 kDa was found in yeast Candida utilis
(Chavez et al., 1997), 218 kDa in sugarcane (Rahman et al., 2004), 52.94 kDa
molecular weight of VIN was observed in sugarcane (L. Wang et al., 2017). It is also
reported that 55 to 70 kDa molecular weight VIN was observed which is N-
glycosylated at multiple sites (Tymowska et al., 1998). First, genotypic evidence that
maximum VIN activity are likely to play key role in heat resistant in sugarcane.
Moreover, VIN activity was significantly higher in juvenile tissue in sugarcane. This
result suggested that VIN activity declined under heat stress as well maturity stages
(Fig 21 and 22) in high sucrose accumulating cultivar while at maturity stage VIN
activity increased in low sucrose accumulating variety (Gayler and Glasziou., 1972).
However, complete information about invertase enzymes is unclear, so further study
is required to explore thermotolerant mechanism in sugarcane crop under heat stress
conditions.
141
5.6. SDS-PAGE Protein Profiling
Proteins play vital role in energy production, growth and development by prevention
of severe damage to the photosynthetic machinery in plant cells (Ford et al., 2011).
In this study, quantitative analysis of total soluble protein contents was declined in
both cultivars under heat stress, but drastic reduction was exhibited in cultivar SPF-
238 as compared to S2003-US-633. After SDS-PAGE analysis differential protein
expression (high molecular weight proteins) was observed during heat stress (Fig 25-
27) in both cultivars at all stages but higher expression were found at vegetative
stage. It is assumed that these expressed protein may belong to class of stress
proteins such as HSPs, dehydrins and osmotins etc. Heat shock protein (HSPs)
involved in transcription and translation might be important in response to heat
stress (Kültz, 2003; Hasanuzzaman et al., 2003). Multiples bands protein molecular
mass such as 60, 90 100 kDa these proteins were higher expression under stress
conditions in both cultivars S2003-US-633 and SPF-238 but the level of expression
was higher in cultivar S2003-US-633 under heat stress conditions. This result
indicating that these molecular mass protein might also be significant in the response
of sugarcane crop to heat stress conditions. These differential expression protein
might be different types of heat shock proteins which expression was higher in heat
stress conditions. Under heat shock treatments, expression of heat shock or stress
proteins are significant adaptation to cope with abiotic stresses. Most of the heat
shock protein water soluble so contribution to stress resistance apparently through
hydration of cellular structure (Wahid and Close, 2007). Molecular weight of heat
shock proteins ranging from 10 kDa to 250 kDa, have chaperon like role and under
142
heat stress it is involved in signal transduction (Shinozaki, 1999), protect cellular
structure (Sanmiya et al., 2004) and protein folding (Cho and Choi, 2009) and act as
molecular chaperones (Li et al., 2013). Heat shock protein play important role in
conferring heat, drought and light stresses resistant in plants (Montero-Barrientos et
al., 2010). For example, in sugarcane overexpression of HSP70 heat stress protein
confers abiotic stress (Augustine et al., 2015). These HSPs support other large number
of proteins in stress condition to protect other protein and maintain the functions of
biomolecules in plant cell (Cho and Choi, 2009). These epigenetic alteration can
regulate the expression of important genes related to thermotolerance (Grativol et
al., 2012) and can activate the transcription of genes that respond to environmental
stress (Probst and Scheid, 2015); Rausell et al., 2003). Thermotolerant sugarcane
cultivars present a more abundance of protein involved in protein biosynthesis,
proposed that these proteins are significant in the response of sugarcane crop under
heat stress conditions (Pacheco et al., 2013).
5.7. Correlation
Results revealed that total sugar negatively associated with VIN and CWIN activities
at maturity stage this results agreement witw previous study a negative and
significant correlation between CyIN and SAI with sucrose accumulating (Siswoyo et
al., 2016) and maximum activity of acid invertase was correlated with low levels of
sucrose (Lontom et al., 2008; Pan et al., 2009). Present results indicating that
invertase activity was lowest in cultivar S2003-US-633 with high sucrose
accumulation and invertase was highest in cultivar SPF-238 with low sucrose
accumulation (Table 9). Invertase my play important role in sucrose accumulation in
143
sugarcane crop and also stated that invertase activity had negative association with
and significant in the accumulation of sucrose. Cell wall invertase increased at
maturity stage. Our results lined with (Botha et al., 1996). However, it is suggested
hydrolyze and resynthesize model for sucrose unloading and storage in sugarcane
stem (parenchyma) where sucrose is hydrolyzed in to hexose (glucose and fructose).
Incontrast, it is demonstrated that sucrose could be transported in sugarcane but did
not hydrolyzed and resynthesize played no role in sugarcane storage (Lingle, 1989).
CWIN was negatively correlated with sugar recovery and sucrose content in both
cultivars. Regarding sugar recovery rate, pol, °brix were showed strongly positive
association with sugar recovery at both growth stages. Same results also reported (Ali
et al., 2019). So, CWIN may be a good candidates for high sugar recovery rate and
sugar accumulation in sugarcane crop.
144
Key Findings
1. Morphological study showed that maximum nodes, tillers while minimum internodal
distance, stem diameter, fresh to dry weight ratio, shoot and root length were
observed in cultivar SPF-238 with compared to S2003-US-633. This proposed that
SPF-238 was susceptible cultivar.
2. Thermotolerant damage indicators studies revealed that maximum cell membrane
thermostability, hydrogen peroxide, MDA contents were accumulated under heat
stress treatments in SPF-238. However, maximum proline content was accumulated
in cultivar S2003-US-633. This higher accumulation of proline play a protective role
under heat stress suggested that cultivar S2003-US-633 has ability to cope any harsh
environmental stress conditions. But this character or ability was lack in SPF-238.
3. Sugar quantification revealed that higher total sugar and nonreducing sugar contents
were exhibited in cultivar S2003-US-633 while maximum reducing sugar content
found in SPF-238 under heat shock treatments. This higher concentration of reducing
sugar in cultivar SPF-238 may be sucrose cleavages into hexose sugar due to higher
expression of invertase in sugarcane culm at maturity stage resulting sugar recovery
rate loss.
4. Sugar recovery rate is positively associated with attributes with SPS, SS and total
sugar content while negatively associated with invertase isozymes and reducing
sugar.
5. Sugar recovery parameters study revealed that exposure of heat stress severely
effected on sugar recovery rate, pol and brix in both cultivars. Both the cultivars had
different thermotolerant potential. Maximum sugar recovery rate was observed in
145
cultivar S2003-US-633 at all treatments. This indicated that cultivar S2003-US-633 has
higher sink strength (less sucrose hydrolysis in stem at maturity stage) and less fiber
content in stem.
6. Different molecular mass protein bands (70, 90 and 100 kDa) may be heat shock
protein which involved under heat stress conditions in both cultivars. This heat shock
protein (HSPs) help to folding denatured proteins and provide cellular protection
against heat induced damages. This indicated that cultivar S2003-US-633 had better
performance under heat stress and upon recovery treatments, quick recovery rate
was exhibited as compared to SPF-238.
7. Native PAGE and zymography studies revealed that the differential expression of
invertase isozymes at various growth phases in both cultivars. Their expression or
activity to be growth stage specific. The vegetative stage of plant growth phase
appears to be more affected by heat shock as compared to other stages. Both
cultivars indicated three types of cytoplasmic invertase (134, 150 and 160 kDa) at
grand growth stage. Cultivar S2003-US-633 was better performance under heat stress
as compared to SFP-238.
8. Regarding cell wall invertase, multiple bands (140, 150 and 160 kDa) were observed
at vegetative and grand growth stages due to higher demand of sugars, juvenile cells
use this sugars as fuel for growth and development during growth phase. While
vacuolar invertase expression pattern was same in both cultivars at all growth stages.
146
Future Directions
Due to complex genome structure of sugarcane, marker assisted breeding, genetic
transformation and genome editing approaches require the complete information of
all heat stress tolerant mechanisms as well as sucrose metabolism enzymes
characterization particularly invertase isozymes for improved sugar recovery rate
and thermotolerance. Many commercial agriculture biotechnological companies
have been heavily invested in developing high yielding as well thermotolerant
commercial sugarcane cultivars. So there is urgent need to grow thermotolerant crop,
unfortunately there is limited thermotolerant varieties have been developed. For the
development of high yielding thermotolerant varieties, enzymes controls points
involved in sucrose metabolic pathway, degree of phloem loading and unloading and
rate of sucrose assimilation needs to be explore for increasing sucrose accumulation.
Engineering of sugar metabolizing enzymes through genetic transformation may lead
to the increased sucrose accumulation and sugar recovery rate. At this point
fundamental research plays vital role by providing molecular physiology of the plant
heat stress response and can speed up biotechnological modification of heat tolerant
traits.
Biotechnology intervention for vacuolar targeted expression of sucrose metabolizing
enzymes may not only depict enhanced sucrose transport but sink strength.
Post-transcriptional gene silencing can be done to target suppress the expression of
invertase and its isozymes in maturity stage for more accumulation of sucrose.
147
Conclusion
Environmental fluctuations are the major limitations for growth, yield and production
of sugarcane plant. The cultivar S2003-US-633 revealed to show thermotolerant
under heat stress than SPF-238. More osmolytes, sugars and total soluble protein
content were evident in S2003-US-633. While less amount of hydrogen peroxide,
MDA and EC content in cultivar S2003-US-633 suggesting that directly associated with
osmotic homeostasis in sugarcane during the exposure of heat stress. Heat shock
proteins (HSPs) and their differential expression in cultivar S2003-US-633 might be
directly play in maintenance of pant growth and development under heat stress. The
multiple molecular bands proteins and different enzymes (multiple types of invertase
isozymes) identified during heat stress and their linked biochemical pathways provide
new avenue regarding sugarcane improvement programme with respect to high
temperature. Cultivar S2003-US-633 is ranked in high yielding and high sugar
recovery rate variety due to high °brix, pol and total sugar content despite severe
environmental condition.
148
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A-Oral Presentations
1. Faisal Mehdi*, Kazim Ali, Nesheman Huma, Abid Azhar and Saddia Galani. Biochemical
analysis of high and low sucrose accumulation sugarcane varieties at formative stage
under heat stress. Dynamic Trends in Plant Sciences: Fostering Environment and Food
Security, May 9-11, 2017. Organized by Pakistan Botanical Society (PBS) at Sardar
Bahadur Khan Women's University (SBK), Quetta, Pakistan.
2. Faisal Mehdi*, Kazim Ali, Nesheman Huma, Abid Azhar and Saddia Galani.
Quantitative and qualitative analysis of invertase isozymes regulating sucrose mechanism
in heat shock sugarcane. “Molecular biosciences: research and innovations” Fourteenth
Biennial Conference of Pakistan Society for Biochemistry and Molecular Biology (PSBMB),
December 9-12, 2018. Organized by Pakistan Society for Biochemistry and Molecular
Biology (PSBMB) at KIBGE University of Karachi.
B-Publication
1. Faisal Mehdi*, Kazim Ali, Nesheman Huma, Iqbal Hussain, Abid Azhar and Saddia
Galani (2019). Comparative biochemical analysis of high and low sucrose accumulating
sugarcane varieties at formative stage under heat stress. Accepted to be published in the
Journal of Agricultural Sciences for issue 2020/Vol 26/Issue 1.
C-Poster Presentation
1. Faisal Mehdi1*, Shaghufta Sahar, Maleeha Akbar, Abid Azhar and Saddia Galani.
“Recent Innovations in Molecular Sciences”. The Conference organized by the
University of the Punjab (Quaid-e-Azam Campus), Lahore during November 06-08,
2019.