physio-biochemical analysis and transcript profiling of...
TRANSCRIPT
ORIGINAL PAPER
Physio-biochemical analysis and transcript profilingof Saccharum officinarum L. submitted to salt stress
Madhuri Chandrakant Pagariya • M. Harikrishnan •
Pranali Arun Kulkarni • Rachayya Mallikarjun Devarumath •
Prashant Govindrao Kawar
Received: 13 July 2010 / Revised: 16 November 2010 / Accepted: 7 December 2010
� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2010
Abstract In an attempt to understand the molecular basis
of salt-stress response in sugarcane, physio-biochemical
assays and cDNA-RAPD-based gene expression studies
under high salt (2% NaCl) supply regimes were initiated.
The comparative rates of total protein, proline content and
lipid peroxidation were found steadily increased, while
total chlorophyll content was decreased in leaves of salt-
treated over untreated sugarcane plants at corresponding
increase in soil electrical conductivity. The comparative
transcript responses to salt stress were monitored by ribo-
typing of both treated and untreated sugarcane plants at
early growth stage. Among 335 differentially expressed
transcript-derived fragments, 156 up- and 85 down-regu-
lated were reamplified and sequenced. They were
functionally categorized as metabolism, DNA/RNA/cellular
processes, signal transduction/cell rescue/defense, cell wall
modifications, transcriptional regulation, transport/traffick-
ing, retroelements and unknown/hypothetical proteins.
Keywords Salt stress � cDNA-RAPD � Lipid
peroxidation � Proline content � Chlorophyll content �Ribotyping
Abbreviations
ABA Abscisic acid
DAP Days after planting (stress)
EC Electrical conductivity
HSPs Heat shock proteins
MDA Malondialdehyde
RAPD Randomly amplified polymorphic DNA
ROS Reactive oxygen species
TDFs Transcript-derived fragments
Introduction
Abiotic stresses adversely affect the crop productivity and
quality worldwide. Among the various stresses, salinity is a
major threat to agriculture. FAO estimates suggest that
around 6% of the world’s total land area and 20% of irri-
gated land are affected by high salinity (Verma et al. 2007).
Plants have developed elaborate mechanisms to perceive
external signals and to manifest adaptive responses with
proper physiological and morphological changes (Xiong
and Yang 2003). Salinity affects numerous physiological or
biochemical processes, imposing ionic, osmotic and sec-
ondary stress such as nutritional disorders and oxidative
Communicated by T. Moriguchi.
M. C. Pagariya � M. Harikrishnan � P. A. Kulkarni �R. M. Devarumath � P. G. Kawar (&)
Molecular Biology and Genetic Engineering Division,
Vasantdada Sugar Institute, Manjari (Bk), Tal. Haveli,
Pune 412307, Maharashtra, India
e-mail: [email protected]; [email protected]
M. C. Pagariya � R. M. Devarumath
Department of Botany, Shivaji University,
Vidyanagari, Kolhapur 416004, Maharashtra, India
Present Address:R. M. Devarumath
Centre for Plant Conservation Genetics,
Southern Cross University, Lismore, NSW, Australia
Present Address:M. Harikrishnan
Plant Molecular Biology Lab, M S Swaminathan Research
Foundation, Chennai 600 113, Tamil Nadu, India
123
Acta Physiol Plant
DOI 10.1007/s11738-010-0676-6
stress leading to membrane disorganization, metabolic
toxicity and inhibition of photosynthesis, many of which
are seen at the cellular level. However, the underlying
molecular mechanisms for adaptation to salt stress in plants
remain unclear (Sakamoto et al. 2008; Lee et al. 2009).
Sugarcane (Saccharum officinarum L.) is an important
industrial crop of tropical and subtropical region; it serves
as a source of sugar and is one of the important bioenergy-
generating crops (Han and Wu 2004). In glycophytes,
sodium toxicity represents the major ionic stress associated
with high salinity enforcing ion imbalance or disequilib-
rium, and hyperionic and hyper-osmotic stress, thus dis-
rupting the overall metabolic activities and causing plant
demise (Zhu 2001). Sugarcane being a glycophyte shows
high sensitivity to salinity at various growth stages, like
majority of other crop species, and cannot tolerate high
salinity at early stages of growth.
Many features associated with salt tolerance are reflec-
ted at the cellular level and glycophytes are believed to
have special cellular mechanisms for salt tolerance
(Hasegawa et al. 2000). Improved complex mechanisms for
adaptation are generally associated with osmoregulation
adjustment by using some osmotic regulators such as
proline, change in cell membrane permeability, soluble
protein content, percent soluble sugar, etc., ultimately
affecting photosynthesis and growth rate (Parvaiz and
Satyawati 2008). Thus, plants have developed a wide array
of strategies either to avoid or cope with the stress condi-
tions. The salt stress-mediated tolerance in plants is a
complex quantitative trait regulated by a large number of
up- and down- regulated genes inducing multiple signaling
pathways. Currently, several molecular techniques such as
differential display reverse transcription-polymerase chain
reaction (DD-RTPCR), representational difference analysis
(RDA), serial analysis of gene expression (SAGE), sup-
pression subtractive hybridization (SSH), ribotyping by
random decamer primers and cDNA microarray are avail-
able for transcriptome analysis. By using these techniques,
efforts were made to decipher the nature of pathways
operational under salt stress and assignment of putative
identities to a subset of the genic fragments through elec-
tronic database homology searches. However, the studies
toward assessing the effects of high salinity in sugarcane
are limited to morphological, physiological and few of the
salt-responsive genes (Pagariya et al. 2010; Patade et al.
2009; Hussain et al. 2004; Joshi and Naik 1981), and hence
efforts are being made to explore the molecular responses
of sugarcane in this regard.
The cDNA-RAPD, a cost-efficient technique that pro-
vides complex phenotype, reflecting changes in the abun-
dance of hundreds of RNAs under various conditions, does
not require specialized expertise to handle as in other
highly technical activities. Recently, this technique has
been successfully used for identifying and isolating sug-
arcane grassy shoot (SCGS) phytoplasma TDFs and
differentially expressed genes in sugarcane under phy-
toplasma infection condition (Kawar et al. 2010). In
another attempt toward isolating chickpea TDFs expressed
during root infection by Fusarium oxysporum (Nimbalkar
et al. 2006) and transcriptomic analysis of tissue culture-
derived peloric mutants of Phalaenopsis orchids (Chen
et al. 2005), this method was found to be highly fruitful.
Accordingly, the objective of this study was to assess
the biochemical changes associated with the sugarcane
plants under salinity, and to identify the key genes that
were differentially expressed in sugarcane leaves at early
growth stage in response to salinity stress using cDNA-
RAPD technique.
Materials and methods
Plant material, growth and stress treatments
A pot (L:1200, W:5�00, H:5�00) experiment was executed
for salt stress induction in Saccharum sp. hybrid cv. Co
62175, a moderately salt-tolerant variety (Patade et al.
2009; http://sugarcane-breeding.tn.nic.in/physiology.htm)
at Vasantdada Sugar Institute, Pune, India. The entire
transverse nodal culm sections, bearing a single intact
axillary bud, were planted in ten rectangular plastic pots
filled with soil mix (soil:green manure, 10:1 [w/w]) in
replicates of three and maintained in greenhouse conditions
(16/8 h photoperiod; 25�C). Sodium chloride (2%) was
added after every 3 days interval and leaf samples from
both stressed and their respective controls were harvested
at 15 and 30 DAP, frozen in liquid nitrogen and stored at
-80�C before being scheduled for physiological tests and
RNA isolation.
Physio-biochemical attributes
Soil salinity and electrical conductivity
Soil salinity was estimated measuring the EC of a soil-
saturated paste before and after adding 2% salt solution in
soil using Rayment and Higginson (1992) methodology.
Estimation of chlorophyll content
Total chlorophyll (chlorophyll a ? b) content of unstressed
and salt-stressed sugarcane leaves of 15 and 30 DAP
intervals were extracted in 80% (v/v) aqueous acetone and
absorption measured in a Shimadzu UV-1700 model
spectrophotometer at 645 and 663 nm following the
method of Arnon (1949).
Acta Physiol Plant
123
Protein determinations
Protein concentration in the extracts was quantified at
595 nm by the dye-binding assay of Bradford (1976) using
bovine serum albumin as a standard (Merck, fraction V).
Estimation of proline
Free proline content for control and salt-stressed plant
samples was determined at 520 nm by ninhydrin method
according to the procedure of Bates (1973).
Determination of malondialdehyde content
For the measurements of lipid peroxidation in leaves, the
thiobarbituric acid (TBA) test, which determines mal-
ondialdehyde (MDA) as an end product of lipid peroxi-
dation (Heath and Parker 1968), was used. Absorbance of
the supernatant was measured at 532 nm, subtracting the
value for non-specific absorption at 600 nm. The amount of
MDA–TBA complex (red pigment) was calculated from
the extinction coefficient 155 mm-1 cm-1.
Statistical analysis
All the experimental data values were mean of three rep-
licates of ten plants each of control and test, and the data
were expressed as mean ± SE. Statistical significance was
evaluated with the Student’s t test, and differences were
considered significant at 1% level (Gao et al. 2008).
Molecular attributes
RNA isolation and cDNA synthesis
Total RNA was extracted from the frozen sugarcane leaf of
control and stressed plant samples using the RNeasy Plant
kit (Qiagen Inc., Valencia, CA) and stored in 100 ll of
elution buffer (10 mM Tris–Cl, pH 8.5). Reverse tran-
scription of transcripts and second-strand synthesis from
1 lg total RNAs was carried out using SMARTTM PCR
cDNA Synthesis Kit (Clontech, USA) and quantified by
measuring OD at 260 nm.
RAPD primer screening with cDNA templates
PCR amplification of quantified second-strand cDNA from
both stressed and their respective control plant samples (15
and 30 DAP) were achieved using 10-mer RAPD primers
(OPA, AB, G and K, Operon Technologies, Inc., Alameda,
CA). As much as 25 ll of PCR reaction mixture contained
10 ng of cDNA, 2.5 ll of 109 PCR buffer, 2.5 mM
MgCl2, 2 mM dNTPs, 15 ng of primer and 1 unit of Taq
Polymerase (Banglore Genei, India). Amplifications were
performed for 45 cycles: 4.30 min at 92�C, 1 min at 35�C,
2 min at 72�C; followed by 44 cycles each of 1 min at
92�C, 1 min at 35�C, and 2 min at 72�C followed by a final
extension for 15 min at 72�C. The PCR products were
resolved on 1.5% agarose in 19 TAE. All the reactions
were repeated thrice, and consistently reproducible bands
were scored.
Profile scoring and data analysis
Profile of the amplicons was scored and data analyzed
based on the consensus results of three independent runs.
Clearly resolved bands (TDFs) of both stressed samples (15
and 30 DAP) were scored manually on the basis of their
presence/absence or intensity of the bands and were
assigned up- and down-regulated in comparison with their
respective controls.
Cloning, colony PCR screening and sequencing
of differentially expressed TDFs
The individual differentially expressed TDF was cut from
the gel with a sharp surgical blade, avoiding any contam-
inating fragment(s) and eluted in 30 ll of sterile double
distilled water using the QIAEX1 II gel extraction kit
(Qiagen Inc., Valencia, CA). Aliquot of 1 ll was used for
reamplification of individual TDF using the same set of
corresponding primer with slight modification in amplifi-
cation cycles and final extension time period. The PCR
products were analyzed on 1.5% agarose in 19 TAE,
ligated into the pGEM�-T Easy vector (Promega Corpo-
ration, Madison, USA) according to the manufacturer’s
instructions and transformed into E. coli strain DH5a.
Recombinants were selected on LB agar medium contain-
ing ampicillin (50 lg ml-1), X-Gal (20 lg ml-1) and
IPTG (0.1 mM) (Sambrook et al. 1989). The presence of
insert DNA was confirmed by colony PCR and sequenced
using automated DNA sequencer (Model 3100, Applied
Biosystems, USA) at SaiRaj Biotech, Pune.
Homology analysis
The sequences of the TDF (with vector sequences trimmed
off, where plasmid was used as the template) were ana-
lyzed for their homology against the publicly available
nonredundant genes/ESTs/transcripts in the NCBI Gen-
Bank nucleotide and protein database using BLAST algo-
rithms (http://www.ncbi.nlm.nih.gov/Blast.cgi) (Altschul
et al. 1997).
Acta Physiol Plant
123
Results and discussion
The effects of salinity on physio-biochemical
parameters in sugarcane cv. Co 62175
Salinity represents one of the most important environ-
mental stresses since it limits crop plant production dis-
turbing the normal physiology and entire metabolic
balance. Salinity measurements provide information on the
ability of a site to support plant growth. EC measured using
conductivity meter is a gross measure of dissolved salts in
soil solution as it correlates with texture, nutrients and crop
yield (Moore and Wolcott 2001). In our experiment, 2%
NaCl was added after every 3-day interval and EC was
checked before and after adding the salt solution to soil.
The soil EC increased considerably from 0.55 to
5.81 mS cm-1 (Fig. 1a; Table 1) with a remarkable dif-
ference between control and NaCl treated, inhibiting the
plant growth in 30 DAP plant as compared to control by
affecting the total chlorophyll content of sugarcane leaf.
Chlorophyll a, b and total chlorophyll content
The results showed that there was clear effect of soil salination
on the leaf pigment contents, normal physiology and entire
metabolic balance due to stress. It was observed that chloro-
phyll a/b content increased along the period of time in control
plants, while remarkably reduced in salt stress-induced plants
thus proving that the salinization (NaCl) induced a significant
decrease in the contents of pigment fractions (chlorophyll a
and b) and consequently the total chlorophyll content
(Fig. 1b–d; Table 1). The reduced level of total chlorophyll
content under salt stress condition may be due to chloroplastid
membrane deterioration, leading toward lesser accumulation
of chlorophyll and decrease in photosynthetic efficiency as
reported earlier by several researchers (Turan et al. 2009;
Dhanapackiam and Ilyas 2010).
Total soluble proteins
The total soluble proteins of the salt-stressed plants was
increased drastically in 30 DAP plants with respect to
15 DAP, but with a slight noticeable increase in untreated
controls as illustrated in Fig. 1e and Table 1, indicating
that proteins accumulate during stress. As soluble proteins
are localized in the cytoplasm (cytoplasmic proteins) dur-
ing salt- or water stress, it may play a role in osmotic
adjustment (Loponen et al. 2004). Similar results have been
observed in salt-tolerant cultivars of barley, sunflower,
finger millet and rice (Ashraf and Harris 2004) implying
that plants under stress have powerful protein turnover by
compensating for the increased oxidation or loss of
antioxidants, at the same time providing cells the ability to
withstand a high degree of salt stress (Hasegawa et al.
2000; Abdel et al. 2003).
Proline accumulation
Proline accumulation in stressed plants is a primary
defense response to maintain osmotic pressure in a cell.
There was significant increase in proline concentration in
stressed plants as compared to its control; however, proline
level was noticeably increased in 30 DAP compared to
15 DAP salt-treated plants (Fig. 1f; Table 1). Proline is
known to accrue widely in higher plants in response to
salinity, playing an adaptive role in mediating osmotic
adjustment and protecting the sub-cellular structures. In
many studies, a positive correlation between the accumu-
lation of proline and stress tolerance in plants has been
noted (Lutts et al. 1996; Kumar et al. 2003).
MDA content (lipid peroxidation)
MDA is regarded as a marker for evaluation of lipid per-
oxidation or damage to plasmalemma and organelle
membranes that increases with environmental stresses. The
NaCl treatment influenced the level of MDA content (lipid
peroxidation) and was higher in stressed sugarcane plants
as compared to control; furthermore, it was significantly
high in 30 DAP stressed plants (Fig. 1g; Table 1). Perox-
idation of membrane lipids is an indication of membrane
damage and leakage under salt stress conditions. Growth
inhibition under salinity has a proven correlation with
increased lipid peroxidation levels (Khan and Panda 2008;
Azooz 2009; Reezi et al. 2009) and the results of our
studies also mimic the similar correlation with the increase
in MDA content under salinity.
cDNA synthesis and profiling for differentially
expressed TDFs
The quantified cDNA of both stressed plants (15 and 30
DAP) with their respective controls was profiled using 80
decamer primers. Altogether, 27 primers produced repro-
ducible and scorable profiles, yielding 354 scorable bands.
No PCR products were observed among 39 primers, while
14 primers showed no variation in amplified DNA frag-
ments in all samples. A maximum of 131 bands were
produced by the OPG series followed by 87, 78 and 58
bands by OPAB, OPK and OPA series, respectively. The
transcript size ranged between 200 and 1.8 kb in up- and
250 bp to 1.7 kb in down-regulated TDFs. Out of 354
bands, 19 bands were monomorphic, 179 bands were up-
Acta Physiol Plant
123
regulated in which 29 bands were common in both, and 75
bands each were from 15 and 30 DAP stressed plants. A
total of 156 bands were found to be down-regulated, of
which 69 were in 15 DAP, 60 in 30 DAP and 27 were
common in both stress-treated plants (Fig. 2).
Screening for differential expression and sequence
analysis
Of 335 differentially expressed TDFs, 156 up- and 85
down-regulated ones were reamplifed, cloned and
a
0
1
2
3
4
5
6
(15DAP) (30DAP)
Control Induced Control Induced
Control Induced
Control Induced
Control Induced
Control Induced
Control Induced
b
0
1
2
3
4
5
6
(15DAP) (30DAP)
c
0
1
2
3
4
5
6
(15DAP) (30DAP)
d
0
0.5
1
1.5
2
2.5
3
3.5
4
(15DAP) (30DAP)
e
0
2
4
6
8
10
12
(15DAP) (30DAP)
f
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
(15DAP) (30DAP)
EC
mS
/cm
of
soil
Ch
loro
ph
yll b
mg
mg
-1 F
W
Ch
loro
ph
yll a
mg
mg
-1 F
W
To
tal C
hlo
rop
hyl
l mg
mg
-1 F
W
Pro
line
µM
mg
-1 F
W o
f le
af
To
tal P
rote
in U
nit
s 50
mg
-1 F
W o
f le
af
g
0
0.1
0.2
0.3
0.4
0.5
0.6
(15DAP) (30DAP)
MD
A µ
M m
g-1 F
W o
f le
af
Fig. 1 Effect of NaCl stress on
a electrical conductivity,
b chlorophyll a pigment,
c chlorophyll b pigment. d Total
chlorophyll pigment. e Total
protein, f proline, g MDA (lipid
peroxidation) in Saccharumofficinarum L. plants of cv. Co
62175 submitted to 15 and
30 days of salt stress. The data
are mean value ± SE for ten
plants in three replicates (t test
significant at 1% level)
Acta Physiol Plant
123
subjected to nested PCR amplification. Over 80% of
recombinants revealed amplicons ranging from 150 to
1,500 bp that accounted for 268 positive clones. One
hundred differentially expressed TDFs were sequenced, of
which 68 inserts that showed unambiguous sequence reads
were submitted to the NCBI database and are presented in
Table 2 with the GenBank accession numbers, while 13
sequences showed no reads.
Table 1 Effect of NaCl on electrical conductivity, chlorophyll a, chlorophyll b, total chlorophyll, MDA content, total protein and proline
content in sugarcane
Physio-biochemical test 15 (DAP) 30 (DAP)
Control Test Control Test
EC (mS cm-1 of soil) 0.547 ± 0.149 3.504 ± 0.210 1.229 ± 0.163 5.778 ± 0.088
chl a (mg mg-1 FW) 2.317 ± 0.23 1.428 ± 0.079 5.612 ± 0.092 3.072 ± 0.408
chl b (mg mg-1 FW) 2.749 ± 0.203 1.934 ± 0.080 4.035 ± 0.322 2.211 ± 0.244
Total chl (mg mg-1 FW) 2.419 ± 0.193 1.727 ± 0.045 3.579 ± 0.074 2.071 ± 0.118
MDA (lM mg-1 FW) 0.109 ± 0.006 0.239 ± 0.031 0.139 ± 0.012 0.504 ± 0.072
Total proteins units (50 mg-1 FW) 1.646 ± 0.012 3.266 ± 0.0412 4.819 ± 0.0733 11.721 ± 0.235
Proline (lM mg-1 FW) 0.068 ± 0.004 0.111 ± 0.010 0.036 ± 0.004 0.726 ± 0.040
The data are mean value ± SE for ten plants in three replicates (t test significant at 1% level)
M 1 2 3 4 5
OPK19
K31
L 1 2 3 4 5
OPG06
G02
M 1 2 3 4 5
A03
OPA11
A09
M 1 2 3 4 5
OPA15
AB07
AB06
G19
M 1 2 3 4 5
OPAB07
M 1 2 3 4
OPG09
Fig. 2 Representative amplification profiles generated by RAPD
primers using cDNA templates, gel showing the differentially
expressed TDFs under different stages of salt stress in sugarcane
cv. 62175. M: 100 bp ladder (Fermentas), L: 1 kb ladder (Fermentas),
1 control 15 DAP, 2 stress-induced 15 DAP, 3 control 30 DAP,
4 stress-induced 30 DAP, 5 water control. Arrows indicate TDFs up-
regulated ( ), down-regulated ( ), monomorphic ( ) and ( )
indicate the TDF ID represented in Table 2
Acta Physiol Plant
123
Table 2 Salt-stressed sugarcane TDFs listed according to functional classification
TDF
ID
GenBank
depositories
Length
(bp)
Best hits (BLASTN/X)
analyses to database
Organism E value Percent identity Functional
category
Metabolism
G01 FF281697 784 Hydroxymethylglutaryl-CoA lyase
(ACG37628)aZea mays 1.00E-
46
64/67 (95%) Secondary
metabolism/
leucine
degradation
G02 FF281698 603 ADP-glucose pyrophosphorylase subunit SH2,
transcriptional regulator, NADPH-dependent
reductase A1-a and NADPH-dependent reductase
A1-b genes (AF010283)
Sorghumbicolor
3.00E-
56
302/404 (74%) Carbohydrate
metabolism
G20 HO112150 501 Sesquiterpene synthase (TA40885)a Solanumlycopersicum
2 194/328 (59%) Secondary
metabolism
K33 FF281729 467 Putative prephenate dehydratase (BAD46234)a Oryza sativa 2.3 14/26 (53%) Secondary
metabolism
A14 FF281745 871 SU1 isoamylase (sugary1) gene (AF030882)a Zea mays 6.00E-
10
85/109 (77%) Starch
biosynthesis
AB02 FF281746 783 Aminoalcoholphosphotransferase (AF466199)a Sorghumbicolor
0.021 47/58 (81%) Metabolic
enzymes
AB06 FF281750 913 Sucrose phosphate synthase III (SPSIII)
(EU278618)
Saccharumofficinarum
0.00 788/828 (95%) Starch and
sucrose
metabolism/
metabolic
pathways
B07 FF281751 530 Putative chain A, crystal structure of
phosphopantothenoylcysteine synthetase
(CA299327)
Oryza sativa 0.031 111/174 (63%) Coenzyme A
biosynthesis
pathway
G27 FF281716 846 Putative 2-oxoglutarate dehydrogenase,
E1 subunit (CA163992)
Oryza sativa 5.00E-
09
123/162 (75%) Citric acid cycle/
cellular
functions
G29 HO112149 381 Chain A, rice ketolacid reductoisomerase in
complex with Mg2?-NADPH, chain B, rice
ketolacid reductoisomerase in complex with
Mg2?-NADPH (EEE64772.1)a
Oryza sativa 3.00E-
49
91/94 (96%) Secondary
metabolism/
amino acid
metabolism
G30 FF281719 792 Putative ketol-acid reductoisomerase
(TA267544547)aOryza sativa 3.00E-
114
518/538 (96%) Secondary
metabolism/
amino acid
metabolism
DNA/RNA/cellular processes
G06 FF281700 356 Putative nucleic acid-binding protein
(AAM74443.1)aOryza sativa 0.003 17/49 (34%) RNA processing
G08 FF281701 603 Eukaryotic translation initiation factor 3
subunit A (AAD39834.1)aZea mays 9.00E-
40
138/184 (75%) Cellular functions
A01 FF281735 933 Small nuclear ribonucleoprotein F
(ACG39375.1)aZea mays 0.077 19/20 (95%) Post-translational
modification/
A08 FF281740 907 HAT family dimerization domain-containing
protein (ABB47649.2)aOryza sativa 8.1 17/48 (35%) DNA binding/
protein
dimerization
activity
A10 FF281742 616 Soluble inorganic pyrophosphatase
(ACG34367.1)aZea mays 2.00E-
92
172/188 (91%) Nucleotide
metabolism
AB05 FF281749 639 RNA-directed DNA polymerase;
HMG-I and HMG-Y, DNA-binding,
putative (ABN08144.1)a
Medicagotruncatula
4.00E-
42
83/134 (61%) Transcription
factor/
chromatin
structural factor
AB18 FF281760 765 Chloroplast ribonuclease III domain
protein (ABR53724.1)aZea mays 1.00E-
127
221/243 (90%) Cellular processes
Acta Physiol Plant
123
Table 2 continued
TDF
ID
GenBank
depositories
Length
(bp)
Best hits (BLASTN/X)
analyses to database
Organism E value Percent identity Functional
category
K27 FF281724 783 Intermediate filament-like
protein (BU634593)
Aspergillusoryzae
5.3 79/123 (64%) Cellular
organization
and homeostasis
AB04 FF281748 845 Topoisomerase II-like protein
(ADB28927.1)aZea mays 2.00E-
08
25/30 (83%) DNA
modification/
cellular
processes
G12 FF281705 313 Putative WD-repeat protein (AAT38033)a Oryza sativa 0.071 17/21 (80%) Cellular functions
Signal transduction/cell rescue/defense
G10 FF281703 366 ABA-induced RAB17-like gene (AY177889) Sorghumbicolor
9.00E-
16
78/89 (87%) Regulators
G23 FF281712 190 Glutaryl-CoA dehydrogenase (ACG31721)a Zea mays 6.1 12/31 (38%) Stress related
G28 FF281717 192 Putative receptor protein kinase,
aminoalcoholphosphotransferase, putative
growth-regulating factor 1(AF466199)
Sorghumbicolor
0.65 38/45 (84%) Signaling/
phosphatases/
kinases
K30 FF281726 794 Metallothionein-like protein mRNA
(EF120467)
Saccharumofficinarum
1.00E-
06
64/74 (86%) Redox
K31 FF281727 556 Putative zinc finger protein (Z438D03.1),
putative kinase (Z438D03.7) (AY530950)
Zea mays 2.00E-
06
109/157 (69%) Phosphatases/
kinases
K36 FF281730 859 MAPKKK14/ATP binding/kinase/protein kinase/
protein serine/threonine kinase (NP180565)aZea mays 0.9 12/32 (37%) Phosphatases/
kinases
K39 FF281731 865 Nodulation receptor kinase (EU972265) Zea mays 8.00E-
116
358/437 (81%) Phosphatases/
kinases
K46 FF281733 882 Lipoxygenase (CX110685)a Oryza sativa 0.76 128/209 (61%) Cell rescue/signal
transduction
K23 FF281722 823 Protein kinase family protein (NP680446.1)a Arabidopsisthaliana
3.2 20/65 (30%) Phosphatases/
kinases
K32 FF281728 626 Hsp70 interacting protein/thioredoxin chimera
(DW087330)
Vitis labrusca 0.37 101/149 (67%) Regulators/stress
response
K40 FF281732 855 Peroxidase precursor (TA394814558) Cenchrusciliaris
0.03 82/114 (71%) Oxidase
K21 FF281720 266 Putative 12-oxophytodienoate reductase
(BAD09954)aOryza sativa 6.1 16/46 (34%) Redox
homeostasis
A09 FF281741 921 Alcohol dehydrogenase superfamily,
zinc-containing (TA245924558)
Sorghumbicolor|
9.00E-
06
88/112 (78%) Zinc ion binding/
oxidoreductase
activity
AB10 FF281753 764 Kinesin motor domain-containing protein
(ABF99106.1)aOryza sativa 2.2 17/36 (47%) Signal
transduction
AB14 FF281756 835 Early drought-induced protein
(AAM46895.1)aOryza sativa 3.00E-
19
31/43 (72%) Cell rescue/
defense
Cell wall modifications
G11 FF281704 211 Remorin-like protein (AAS07383.1)a Oryza sativa 10 14/40 (35%) Cell wall
G15 FF281707 537 Catalytic/glucuronoxylan
glucuronosyltransferase (83 9635)aArabidopsis
thaliana7.3 16/36 (44%) Cell wall
A11 FF281743 853 Heavy meromyosin-like protein
(AAO72662)aOryza sativa 7.5 29/85 (34%) Cell wall
modification/
lignification
AB04 FF281748 845 Proline-rich protein (PRP) gene
(AF331851)
Saccharumhybrid
3.00E-
57
171/197 (86%) Cell wall
modification/
lignification
Transcriptional regulation
G19 FF281710 891 Putative wuschel homeobox protein
(CAM32351)aZea mays 2.1 18/41 (43%) Transcriptional
regulation
Acta Physiol Plant
123
Table 2 continued
TDF
ID
GenBank
depositories
Length
(bp)
Best hits (BLASTN/X)
analyses to database
Organism E value Percent identity Functional
category
G29 HO112151 659 Helix–loop–helix DNA-binding domain,
putative (TA771524530)aOryza sativa 6.00E-
08
113/155 (72%) Transcriptional
regulation
G30 HO112153 630 WRKY25-superfamily of TFs having WRKY
and zinc finger domains (ACG44142.1)aZea mays 4.5 18/59 (30%) Transcriptional
regulation
A03 FF281737 956 m19 gene for putative MADS-domain
transcription factor (AJ850302)
Zea mays 2.00E-
54
261/332 (78%) Transcriptional
regulation
A12 FF281744 891 Double WRKY type transfactor (AI960217) Solanumtuberosum
9.7 73/111 (65%) Transcriptional
regulation
AB17 FF281759 752 Myb family transcription factor-like
(CA266446)
Oryza sativa 3.30E-
03
65/74 (87%) Transcriptional
regulation
Transport/trafficking
G03 FF281699 728 ABC transporter-like protein Arabidopsisthaliana
2.3 15/36 (41%) Sugar and protein
transport
AB16 FF281758 843 Putative vesicle transport-related protein
(TA332934)
Oryza sativa 0.0041 342/603 (56%) Vesicular
trafficking
Retroelements/transposition
G24 FF281713 381 Putative polyprotein (AAU10819)a Oryza sativa 3.00E-
27
54/82 (65%) Retroelements
G25 FF281714 715 Retrotransposon protein, putative,
Ty3-gypsy subclass (ABA97082)aOryza sativa 4.3 13/27 (48%) Retroelements
G26 FF281715 625 Putative TNP2-like protein (FJ627006) Zea mays 0 528/612 (86%) Transposition
K22 FF281721 616 Retrotransposon protein (ABA94477)a Oryza sativa 9.2 23/72 (31%) Retroelements
A05 FF281738 682 Retrotransposon-like element Levithan,
50 LTR sequence (U07815.1)
Sorghumbicolor
3.00E-
18
208/297 (70%) Retroelements
AB09 FF281752 663 Retrotransposon protein, putative,
unclassified (AAX96066)aOryza sativa 7.00E-
09
36/75 (48%) Retroelements
AB13 FF281755 772 Retrotransposon protein, putative,
Ty3-gypsy subclass (ABA93999)aOryza sativa 0.12 37/90 (41%) Retroelements
Others
G30 HO112152 689 22 kDa kafirin cluster (AF061282) Sorghumbicolor
2.00E-
47
164/200 (82%) Storage proteins
AB11 FF281754 617 Serine esterase family protein (NP001152051)a Zea mays 4.00E-
06
24/24 (100%) Others
Unclear classification
G17 FF281708 323 Hypothetical protein (CAI73731)a Theileriaannulata
6.6 12/25 (48%) Unknown protein
G18 FF281709 508 No significant similarity found – – – No hits
G20 FF281711 98 No significant similarity found – – – No hits
G09 FF281702 156 No significant similarity found – – – No hits
K25 FF281723 466 No significant similarity found – – – No hits
K28 FF281725 817 Hypothetical protein (NP001056399)a Oryza sativa 3.4 16/26 (61%) Unknown protein
AK47 FF281734 383 Unknown protein (BU763719) Glycine max 4.00E-
00
129/220 (58%) Unknown protein
A02 FF281736 514 No significant similarity found – – – No hits
A06 FF281739 636 Hypothetical protein SORBIDRAFT
(XP002449503)aSorghum
bicolor2.00E-
44
87/108 (80%) Unknown protein
AB03 FF281747 455 Hypothetical protein (NP001059118)a Oryza sativa 0.12 17/37 (45%) Unknown protein
AB15 FF281757 567 Hypothetical protein SORBIDRAFT
(XP002465565)aSorghum
bicolor2.00E-
49
51/73 (69%) Unknown protein
a BLASTX result
Acta Physiol Plant
123
Functional classification and comparative analysis
of TDFs with abiotic stress-regulated transcriptome
The sequence comparison of 68 cloned TDFs against the
database toward identifying the cellular processes affected
by salt stress revealed homology to genes with known
functions and majority of the TDFs were involved in cell
rescue/defense and signal transduction. However, 17%
TDFs showed none or only poor sequence similarity to any
database entries, hence no function could be assigned to
them. These may represent potential transcripts involved in
stress resistance and plant defense reaction in sugarcane
and need to be further studied (Fig. 3). Comparative
analysis was carried out across species for correlating the
existence of salt stress-regulated transcriptome and to
provide an insight into the salt stress-regulation mechanism
in sugarcane at the molecular level, as less work has
been done in this area. The possible role of important
salt-responsive genes of sugarcane in comparison with
stress-regulatory transcriptomes from other plant species is
discussed in relation to stress adaptation.
TDFs involved in metabolism and energy production
Salinity stress up-regulated key enzyme TDFs found in our
analysis included carbohydrate (FF281698, FF281750)
starch mobilization (FF281745), energy production
(FF281706, FF281716) and secondary metabolite hydro-
xymethylglutaryl-CoA lyase (FF281697). These were
reported to be triggered by jasmonates during stress leading
to ketogenesis on exhaustion of cellular carbohydrate
stores, making energy available to cell by breakdown of
stored fatty acids (Creelman and Mullet 1995; Walia et al.
2006). Induction of free amino acids (HO112149,
FF281719) as osmolytes in response to abiotic stress is
thought to play a role in plant stress tolerance (Joshi et al.
2010) toward adaption to the changing environment. Dif-
ferent secondary metabolites such as terpenes (HO112150)
are defense chemicals found in wide groups of plants. The
terpene synthase family gene prephenate dehydratase
(FF281729) contributes significantly to the terpenoids’
production (Zwenger and Basu 2007). During stress, it
enhances the shikimate pathway activity, accumulates
more phenylalanine and produces aromatic secondary
compounds, which act as tools to overcome stress
constraints in plants (Edreva et al. 2008).
TDFs involved in DNA/RNA metabolism and cellular
processes
Cell division and cell cycle progression in plants are often
altered in response to various environmental stresses such
as wounding, low temperature and salt (Potters et al. 2007).
TDFs involved in DNA methylation, chromatin remodeling
and post-transcriptional regulation, such as small nuclear
ribonucleoprotein F (FF281735), topoisomerase II-like
protein (FF281748), putative nucleic acid-binding protein
(FF281700) and eukaryotic translation initiation factor 4GI
(eIF4GI) (FF281701), were upregulated in both 15 and
30 days stressed plants. HAT enzyme (FF281740) was
found up-regulated in 30 DAP plant, a catalytic subunit of
protein complexes that acetylate specific lysine residues on
the N-terminal regions of the chromatin histone compo-
nents, and promoted gene activation. This might be more
responsive to osmotic stress, which is created a bit later
than the ionic stress experienced immediately after impo-
sition of salt stress (Stockinger et al. 2001). Generally up-
regulation of WD-repeat proteins (FF281705) was reported
during salt stress, playing an important role in intracellular
signal transduction and apoptosis (Lee et al. 2010), but
astonishingly in our case it was down-regulated in 15 days
stressed plant and absent in 30 days stressed plant. This
may be either because these proteins are intimately
involved in cellular functions, such as cell growth, prolif-
eration by regulating various developmental processes
hampered due to stress at early stages of induction
(van-Nocker and Ludwig 2003; Chantha et al. 2007), or
due to limited efficiency of the present method by the
preferential amplification, a well-known drawback of PCR-
based ribotyping methods (Kawar et al. 2010).
TDFs involved in signal transduction/cell rescue/
defense
In the present study, a number of genes that are classified in
the signal transduction, cell rescue or defense category
were up-regulated in response to salt stress. Plants develop
a sophisticated signaling pathway to deal with dramatic
environmental changes; perception of extracellular stimuli
and the subsequent activation of defense responses require
a complex interplay of signaling cascades, in which
reversible protein phosphorylation plays a central role (Li
et al. 2008). Several plant responses to environmental
stress are mediated by phytohormones, with a well-known
Functional Distribution
Metabolism
DNA/RNA/Cellular processes
Cell rescue/Defense/ Signal TransductionCell wall modifications
Transcriptional regulation
Transport/ Trafficing
Others
Retroelements/ Transposition
Unknown and Hypothetical
Fig. 3 Functional distribution of the salt-stressed sugarcane TDFs
derived from the cDNA-RAPD on the basis of their homology
Acta Physiol Plant
123
cross-talk between them (Gazzarrini and Mccourt 2003).
The plant hormone ABA (FF281703) is an important
intermediate in transducing signals of stresses, since the
ABA levels are often elevated upon stress and are involved
in many aspects of plant responses to salt stress, besides
inducing the expression of many salt-responsive genes
(Leung and Giraudat 1998).
Many TDFs up-regulated in stress-induced plants have
been identified having a potential role in regulation of
cellular processes in response to salt stress that includes
MAP kinase (FF281730) and different protein kinases
(FF281717) (FF281722) (FF281727) (FF281731). These
are positive regulators controlling stress signal transduction
in plants, and their homologous genes have been reported
to be induced by water deficit, low temperature, dehydra-
tion, pathogen and other stresses in previous studies (Seong
et al. 2007; Prabu et al. 2010).
Among genes in the category ‘cell rescue, defense, cell
death and aging’, up-regulation of genes encoding HSPs
(FF281728) has been reported in a variety of stresses such
as oxidative and heat stress (Wang et al. 2004). Lipoxy-
genase (FF281733) involved in jasmonate biosynthesis has
been studied by a few researches in salt-stressed plants. An
increase in lipoxygenase and hydroperoxide degradation
activities was observed and seems to indicate that several
enzymes of the lipoxygenase pathway are involved in the
plant response to salt stress (Roychoudhury et al. 2008).
Recently, a pro-apoptotic effect of lipoxygenase, and of the
hydroperoxides produced thereof, has been reported in
different cells and tissues, leading to cell death (Maccar-
rone et al. 2001; Delaplace et al. 2009). Zinc finger proteins
(FF281727), which were observed to be up-regulated in
stressed plant, are among the most abundant stress-
responsive proteins in eukaryotes having involvement in
various cellular functions such as transcriptional activation,
regulation of apoptosis, protein folding and assembly
(Laity et al. 2001).
A recent study suggested a new model of redox
homeostasis during stress, predicting that the metallothio-
nein-like protein (FF281726) chelates heavy metals
through its large number of Cys residues and removes ROS
to avoid the oxidative damage (Li et al. 2010) and was also
seen up-regulated in 30-day stressed plant in our studies.
The enzyme oxophytodienoate reductase (FF281720)
activates antioxidative systems in cells possessing a
stronger capability for removing H2O2 by reducing its
intracellular levels to attenuate the oxidative damage ulti-
mately establishing a new redox homeostasis. Such anti-
oxidative systems playing an important role in maintaining
the survival and growth of rice seedlings under strong and
sustained oxidative stress have been reported by Wan and
Liu (2008). Most importantly, the status of antioxidants in
the cell (FF281741) provides essential information on
cellular redox state, and influence the expression of stress-
responsive genes to maximize defense. Some earlier
evidences suggested a model for redox homeostasis in
which the ROS–antioxidant interaction acts as a metabolic
interface for signals from metabolism and environment.
This interface modulates the appropriate induction of
acclimation processes or, alternatively, execution of cell
death programs (Foyer 2005).
TDFs involved in transcription activation
Transcription factors function as transcriptional activators
in the expression of stress-inducible genes (Chen et al.
2005). Transcription factor orthologs of the MYB
(FF281759), WRKY (FF281744, HO112153) regulated by
the SOS (salt overly sensitive) signaling pathway under salt
stress conditions (Kamei et al. 2003), MADS (FF281737)
and helix–loop–helix DNA-binding domain (HO112151)
were evidenced as up-regulated in both 15- and 30-day
stressed plants. These are known to be role players in the
stress responses, and their induction during drought, cold
and freezing stress has been reported earlier (Heim et al.
2003; Ramamoorthy et al. 2008; Prabu et al. 2010).
Wuschel homeobox protein (FF281710) was upregulated
only in 15-day stressed plant, while absent in 30-day
stressed plants. Perhaps, these genes are either involved in
diversification of cell function, specification of cell fate and
may be silenced due to prolonged stress as reported by Zhu
et al. (2004) in Arabidopsis, or the efficiency of the present
method is limited by the preferential amplification, a
well-known drawback of PCR-based ribotyping methods
(Kawar et al. 2010).
TDFs involved in cell wall modifications
Cell wall reinforcement being a well-known defense
mechanism of plants, the gene expression data by Raffaele
et al. (2007) suggested that some remorins (FF281704)
have key functions during responses to biotic and abiotic
stimuli, predicting their involvement in hormone-mediated
responses and primary signal transduction. In this study, a
remorin TDF was found up-regulated in 15-day and was
absent in 30-day stressed plant, implying that hormone
signaling is active at stress initiation. Studies have dem-
onstrated that heavy meromyosin enzymes (FF281743) are
differentially expressed in response to environmental
stresses and have a role in the early stages of wound
healing in plants (Krawczyk 1977).
TDFs involved in transport/trafficking
Vesicular transport is increased in stressed cells either to
facilitate membrane turnover, or to decrease unnecessary
Acta Physiol Plant
123
secretion (Brands and Ho 2002). ABC transporters evolved
to have important plant-specific functions in plant devel-
opmental and physiological regulations. It is predicted that
they may function in the excretion of a cytotoxic and
secondary metabolites that are accumulated under condi-
tions of salinity stress (Smart and Fleming 1996; Yazaki
2006). In the present study, two TDFs (FF281758 and
FF281699) up-regulated in 15-day stressed plant showed
high homology with protein trafficking genes.
TDFs involved in transposition (retroelements)
Activation of retrotransposons like TNP2-like protein,
Ty3-gypsy subclass and Levithan, 50 LTR (FF281715,
FF281721, FF281738, FF281752, FF281755) by stresses
and external change is common in all eukaryotes,
including plants. The transcriptional activation of several
well-characterized plant retrotransposons seems to be
tightly linked to molecular pathways activated by stress,
and most of the time activation is under the control of
cis-regulatory sequences strikingly similar to those of
plant defense genes (Zhao et al. 2009). These regulatory
sequences are highly variable, suggesting that retrotrans-
posons could evolve through modification of their regu-
latory features and are reported to be induced during
biotic and abiotic stress from different plants (Wessler
1996; Grandbastien et al. 2005).
Unclear classification
Among the TDFs characterized, 17% TDFs could not be
associated with any genes described in the GenBank,
thus encoding for unknown/hypothetical proteins. Such
unknown sequences may contribute to the salt stress
tolerance phenotype and further studies with these genes
will help to decipher the molecular mechanism associated
with salt stress in sugarcane.
Conclusion
The present study presented the physio-biochemical and
transcriptional changes in sugarcane under salinity stress.
These findings on biochemical and physiological indicators
at the cellular level may serve as in vitro selection criteria
for salt tolerance in sugarcane. Moreover, a fast, simple
and inexpensive cDNA-RAPD approach having abilities to
compare multiple experimental samples simultaneously
have been fruitfully used to produce a reliable cDNA
library from sugarcane under salt stress condition. Our
library represented most of the up-regulated sugarcane
genes’ comparative expression in 15- and 30-day stressed
plants and their respective controls. The 17% TDFs, which
are new to database, may represent potential transcripts
involved in stress resistance and plant defense in sugarcane
and needs to be further studied. Moreover, this information
provides a foundation for future studies toward determi-
nation of functional importance of these salt-responsive
genes for developing stress-tolerant plants. TDF-based
isolation of full-length genes, their expression and knock-
out studies will provide insights for managing salt toler-
ance trait in sugarcane. These candidate genes can be
further utilized as molecular markers for early identifica-
tion of salt-tolerant progenies in sugarcane hybridization
programs.
Acknowledgments The authors gratefully acknowledge the Direc-
tor General Vasantdada Sugar Institute, India, for financial and
research support, and Prof VS Ghule, Dr KH Babu, Mr. MR Shinde,
VSI and Prof DT Prasad, GKVK, Bangaluru, India for their valuable
suggestions during the research work.
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