RESULTS

RESULTS

Rubber content

Fig. 1 shows the rubber contents in two-year-old guayule cultivars: 11591,

Cal-1 and USSZX. The stem portions had the highest rubber content on a dry weight

basis in all the cultivars. The roots had lower rubber content and the leaves contained

the lowest amount of rubber. Of the three cultivars studied, 11591 possessed the

highest amount of rubber in stem (8.5 %) followed by Cal-1 (5.7 %) and USS2X

(4.2 %).

The effect of water stress on rubber accumulation in guayule was determined

(Fig. 3). All the thrte guayule cultivars showed a significant increase in the rubber

content under water s a s s . As the leaf water potential (Y) dropped from 0.5 -MPa to

2.5 -MPa, the rubber content increased. Significantly, rubber accumulation was high

In the plants possessing leaf water potentials of 2.5 -MPa. Water stressed (2.5 -MPa)

stem tissues of 11591 also exhibited maximum rubber accumulation (Fig.4). The

effect of water stress on rubber accumulation per plant was determined (Fig. 5). With

the decrease in leaf water potential from 0.5 -MPa to 2.5 -MPa, the total rubber yield

per individual plant i n d from 38.3 g to 62.6 g in 11591.

The data in Table 1 indicate the reiationship betwen rubber content (% d.w.

and g and the biomass yields in different guayule cultivars. Rubber content

was mars in 1 1591 while its biomass was significantly low compared with Cal-1 and

USSZX. Rubber aaxmulation and the biomass content in relation to watm status in

Fi(r. 1 : Rubber Content in the stan tbsuer of ZCmonth-old auavule dants. Rubber horn atem portions wem mhncbd into hexane fmdions and L i e d & desaibed in matodds and methods. Vertical bars indicate slandard m r ( ~ 3 ) .

Stem Rwts Leaves

Po& of th. plant

Fig. 2 : Rubber content in different plant portions of 11591. Veltlcai ban ~ndicate standard rrrw (n-3)

10,

Flo. 4 : Rubber content In different plant pa- of 11591 in well watered (-1 0 MPa) and wnter stressed (-2.5 MPa) guayule. Vertical bars indicate standard error (n=3)

Fig. 6 : EffW of water sSrsss on rubber content per lndivldual plant of differem guayuls wllvars (llSO1, C I C 1 a d USS2X). Vectlcrl bars indicate mdard arm (n=3)

Leaf mtn p0t.nti8l (MPa)

T a m 1 : Rubber content and Blomass yieW in different guayule wltivan

Biomass

g plat"

293.2 . 456.5

400.5 -

Guayuk

UMS

11591

-1

U r n

Rubber content

H

13.6

8

7.1

g @ant''

85.2

42.6

42.9

11591 are depicted in Fig. 6. There was a slight decrease in biomass content with

increasing water shw.

The effect of low night temperature cycles (30115 O C ) on rubber accumulation

was shown in Fig. 7. Low night temperature significantly enhanced the rubber

content in all the thne cultivars. Guayule plants (11591) exposed to low night

temperature possessed significantly high rubber content (14 % d.w.) aftn 60 cycles of

15 OC night temperature compared to that in control (34/28 "C) plants (8 % d.w.).

Rubber particle proteins

Rubber particles and rubber particle proteins (Rep) were isolated and purified

from stem bark tissue of guayule. The extracted rubber particle proteins fmm these

fractions were purified by a two step procedure: (NH4hSOd precipitation and

repetitive centrifugation/ flotation. Equal volumes of saturated @&t4)2SO4 was used

to precipitate RPP as it tends to give better recoveries than precipitation with solid

(NH.(hSO4 (Scopes, 1994). The precipitate was desalted by using Sephadex G-25

column. Mu extensive washing aod centrifugation, the proteins with 17.64-fold

purification and 17.3 % recovery was mrdcd from the guayule ~ b b e r particle

fraction with a specific activity of 1.5 nkat mg prote~' 0:abie 2). Our d y t i d

centrifugation technique to isolate the rubber particles gave us enough rubber

particles to investigate tk protein profile of the w d d rubbar pmtidcs (WRP) from

gunyule. Tho nibbu parPicls jmth protile was d y m d tluuugb SDS-PAGE (Plate

1). SDS.PA()E myski irdicltsd fht the rubber putides on -4011 showed a

Fig. 6 : Blomass yields md rubber wntenl in relotion to water status in 11591. VerUul ban Indicate 8tondrrtl enw (n-3)

800

500

$ 400

z. 300 - -

fi i

100 - - --C- - - --

20 f -m-Rubbr-

10

0 . r 0 -1 .O -1.5 -2.0 -2.5 -3.0 -3.5

Lort water potential (-IMP.)

Fig. 7 : Effect of low night temperature on ~ b b e r content in U l m c~lthran (1 1581. CaC1 and USsZ)O of ausyule. Day tempedure was 30 .C and growth i w t intensity was 900 pE mJ s-'. \ / A 4 1 barn &i$e standad emr (n=3)

Table 2 : Purification of rubber particle probins from Partheniurn argentaturn Grey

(1 1591)

* Protein estimation was done by the method of Bradford (1976) using BSA as the

standard

Preparation

Crude extract

NH4SO4 Precipitate

* * Rubba -faape (RUT) activity was m e a s d as inwrporation of radiolabelled

isoprene from ("c) isopentenyl pymphosphate (IPP) into guayule lubber particla.

One unit of the cnyme is d e h d as the incorporation of one pmol of radiolabelled

isopnae ("c) br~pentmyl pymphosphatc (IPP) into rubbcr particle Wions pa

hour at 16T.

1 Repetitive 1.2 1.8 1 1.5 17.64 ( 17.3 1 centrifugation1

flotation

Total

Proloin*

(mg)

122.02

6.23

Told

Activity**

(nk4

10.4

2.12

Spec@

Activiry

(nwmgmg')

0.085

0.34

P w & d o n

(-fold)

1

4.1

YiiU

(96)

100

20.38

plat, 1 : Analysis of proteins in m h e d rubber particle8 by 12 % SDS-PAGE. Thnxr- yearold 9uPyule plants (11581) were subjected to law nigM temperatwe trePtmsnt ( a1 5 OC).

Lane A- Molecular weight marker

Lane B- Crude extract of WRPs

Lane C - M e d ~bbe r particle protein (RPP) in control plants

Lane D- RPP a h 60 cycles of cold tanpmtm treatment (3011 5 OC)

single abundant protein of 50 kDa (Plate 1, Lane C). Interestingly, quantitative RPP

analysis from stem barks of guayule plants treated with 60 cycles of low night

temperature (30115 OC) revealed greater accumulation of the 50 kDa RPP as noticed

by a heavy band (Plate 1. Lane D).

Rubber bansfemre activity

Fig. 8 shows thc analysis of three-year-old guayule cultivars (11591, Cal-1

and USS2X) for rubber transferax activity, which indicates that 11591 had the

highest rubber bansferase activity (40 nmol mg prot'l K') compared to other two

cultivars Cal-l (35 ntnol mg prot" K1) and USS2X (31 nmol mg K').

The activity of rubber transferase was increased 100% in water stressed plants

(Fig. 9). The results clearly indicate that the highest activity of rubber transferase was

evident in young stem portions subjected to water stress with leaf water potential of

2.5 -MPa As the leaf water potential dropped, the rubber transferase activity

increased about 2-fold in all water stressed plants (-2.5 MPa) compared to controls

(Fig. 10).

Tbe effect of low night temperature treatment on rubber transferase activity in

guayule cultivars followad a pattan similar to that seen in the case of rubber contat.

Under low ni@t taqmtwc (15 OC) conditions, highest activity was o b d at

medium didon (900 pEi ma dl) with maximum ectivity after 50 cycles of cold

trratmsm (Fig. 11). Ihe enzyma actreds fmm f j O - d ~ ~ y M guayule plants showed

Fig. I : Rubbar lmnsfer~se odivlty horn the stem slicer of 3-year-dd guayule plants. Vbrtiosl ban Indicate standard m ' ( n - 3 ) .

USSW

Fig. 8 : Effect of waler dresr on rubber transferase activity in differant plant p o h m of 11591. Vertical bm Indicate standard error ( ~ 3 )

FIg.10 : Effed of water streas on arbber trsnrfellure adhrlty In different puayule wlivam (11591. Cab1 and USS2X). Vartlul bars lndlcate standard e m ( ~ 3 ) .

Fip. 1 I : Effed of low night temperature (30/15 OC) on ~ b b e r transferase activity in thrw-year& guayule plants (radiation - 900 pE m" s-'). Vertical bars indicate

approximately two-fold increase $ the activity which were well comlatcd with the

rubber lrcoumulation under low night tempmature treatmmts (Fig. 7 & Fig. 11).

However percentage increase was more in stem bark tissues of 11591 (60%)

compared to Cal-1 (51%) and USS2X (48%). Guayule plants subjected to 30115 "C

treatment were also exposed to different radiation conditions. The plants showed

variation in the rubber transferax activities (Fig. 12). At a growth temperature of

30130 "C and medium growth radiation (900 pE ma s'l), the rubber tmnsferase

activity was 50.8 nmol rng h" in guayule cultivar (I 1591) which also showed a

significantly higher rubber transferax activity (74.1 nmol mg h") at 30115 OC

temperatures. When guayule plants were exposed to a range of combinations of

temperatun and radiation conditions, rubber transferase activity was found to be

maximum at 30115 OC and at a radiation of 900 pE m'2 i' in the controlled phytotron

environment (Fig. 13).

" ~ - i a c o ~ r a t i o n rates into newly formed rubber molecules were followed

using different allylic diphosphatc molecules as initiators with WRPs obtained from

the low night tunpcrature (30115 OC) brtated guayule plants. This study revealed that

the highest rubber d u a s e activity could be obtained with geranyl gaanyl

d i p h o s p k (OOPP) as initiator molecule (Fig. 14). Incubations of guayle stem-bark

WRPs with EDTA completely inhibited the '4~-incorporntion into PP. The activity

of rubbar transfmee as affected by the addition of exogmous h@ was shown in

Fig. 15.

Fig. 12 : Effect of growth mdistion on Nbber transferaso adlvity in byear-old gwyub (11 501) treated wlth low nbht temperature (3W15 q. Varying growth radlstions are lndlutod as low (450 fl m" C), medium (OW pE m.' a') and hbh (1500 )rE mas-'). Vertical ban lndhte standard error ( ~ 3 ) .

Fig. 13 : Effect of temperature and radiation ambinations on ~ b b e r transferaso adMty in byear-old guayule (1 1591) plants. The light mimes are same as lndiceted in Fig. 12

Flg.14 : E f f d Of different ~ l l y l l ~ ~mph~sphste initiators (DMAPP. QPP, FPP & GGPP) at 20 VM retuntlng c u n c o ~ on rubber transfoma adlvlty in guayuie (11591) treated wlVl00 cycles of night temperature (3W15 OC) under varying gmth conditions. Vartlcsl bnrs indhto atandad omr ( ~ 3 ) .

MMPP QPP FPP GQPP

Fig.16 : Effed of Mg2' concentration and EDTA (25 mM) on rubber transferase acllvity in guayulo (11591).

ELISA of Rubber Paltitle Proteins

To establish that the purified 50 kDa is the major protein, the reactivity of 50

kDa protein with polyclonal entisera raised in albino rabbits agaiost rubber particle

antigens was tested by ELISA. Varying dilutions of serum was used and good

reactivity was observed for 50 kDa upto 1:3200 dilution of serum (Fig. 16).

The levels of 1gG antibodies specific to the 50 kDa protein fraction in the

serum of plants treated to diffmnt environmental regimes which include the plants

treated with water stress, low night temperature and growth radiations was evaluated

by ELISA. A range of dilutions (1:100 to 1 :12800) was used for each serum and the

IgG titre levels for each antigen were determined (Fig. 17). The results showed that

the antibody levels in plants treated with different environmental regimes wcre

comparable. A higher 1gG titre value was observed with the serum h m stressed

plants treated with water stress, low night temperature and growth radiations

compared to the control.

Western blot of Rubber Particle Proteins

Rabbit antibodies wen raised successfully against RPP from the stressed and

control guayule plants (11591) that had been bound to nitrocellulose and

subsequently solublized in DMSO. An immunoblot of washed mbba particles

(WRPs) md the WRPs fiam plants treated to 60 cycles of low night tcmpcratrrrr

shorn that the serum reacts with the 50 kDa RPP in both samples (Phte 2).

Fig. 16 : Readlvlty of 50 kDp prolein fmlon with pdydonrl a W r a raised agrlnst rubber partlde antlgens In guayuie (1 1591)

Fig. 17 : Lev& of IgG antibodies s p M c to RPP antlgens (1 1591) subjected to dlfferenl envlmnmenlal mgimes

Plat. 2 : Wsstorn Blot ealysis of rubber particle prodem (50 kDa)

Lane A- RPP from guayule line Cal-l

Lane B- RPP from guayule line 1 1 591

Lane C- RPP from 11591 after 60 cycles of cold temperatwe treatment (3011 5 "C)

Analysis of Flcur rubber parkicles

Rubber particles from F h s latex were isolated and rubber particle proteins

(RPP) were purified. Rubber particles fiom Ficus latex were purified which yielded

two rubber particle fractions that were designated as the light buoyant fraction and the

heavy fraction in the pellet. The extracted rubber particle proteins from these

fractions were purified by a two-step procedure as described earlier for guayule

rubber particles (Table 3). Proteins with 20.18-fold purification and 18.61 %

recovery were recorded from the buoyant rubber particle fraction with a specific

activity of 1.675 nkat mg p r o t e ~ ' (Table 3). Ihe heavy fraction recorded 15.80 fold

purification and 22.58% recovery with a specific activity of 0.98 nkat mg-' protein.

SDS-PAGE of purified rubber particle proteins in F i w latex showed

the presence of a 20 Ld)a polypeptide in the heavy rubber particle fraction and two

protein bands of 20 kDa and 30 kDa in the buoyant rubber particle preparations

(Plate 3). Studies on the role of different dlylic pyrophosphate initiators for rubber

biosynthesis were performed using the purified rubber particles. These in vilro kinetic

characteristics indicate that geranyl geranyl pyrophosphate (GGPP) acts as a potential

initiator during the polymerization of IPP into rubber in both buoyant and heavy

fractions (Fig. 18).

Purification of pbosphoribosylpyrophosphate synthetase (PRS) was carried

out using the &entable fraction of the Ficw latex. The pcrcmt m v a y of PRS

activity ww 18.42 with a purification of 83.3 fold (Table 4).

Table 3 : P~riRcrti~n of r ~ b b r parklo pmbIn8 from Ficus elasth latex

* Prokin estimation was done by the mahod of B r a d

standard

' f i t m a

Crodeexeact

NKSQ Precipitate

+tivc

centrifugation/

Flotation

* * R u b k t r a r u f e ~ e (RUT) activity was m d as incorporation of radiolabcllad

isopnne from ("(7 C)tmyI pyrophospk (IPP) into rubber particle h d o p s .

One unit of the enymc is defined as the incorporation of one p o l of

radiolabelled isopmc ("c) iqcntenyl pyrophosphate (IPP) into rubber particle

fractions pa hour It 16*C.

~~n

Buoyant

Heavy

Buoyant

Heavy

Buoyant

Heavy

TdrJ A.olrin*

f

86.74

85.1

5.31

6.04

0.8

1.22

TOM

AaWv**

flw)

7.21

5.27

1.5

1.6

1.34

1.19

SpCcF

Adhriry

fr*lllm8-3

0.083

0.062

0.282

0.265

1.675

0.98

Pur~fiation

6/o&J

1

1

3.4

4.27

20.18

15.8

Ywld

(%)

100

100

20.83

30.36

18.61

22.58

Pkb 3 : SDWAQE proRle of purW rubber partides from Ficus elestica latex.

Lane A- Molecular weight marker

Lane B- Proteins from heavy rubber particle fraction

Lane C- Proteins from buoyant rubber particle fraction

(The most abundant rubber pmtein has a MI of 20 kDa in both the buoyant and heavy rubber particle fractions)

Fb. 16 : Effect of pyrophosphate Initiators on rubber biosynthesis initiation In Fkus measured as rumr transferass activity. Vertkai bars indicate standard ermr ( ~ 3 ) .

Buoyant Heavy

35 s

-- 300- k -

OFPP UGPP

f UGQPP

Table 4 : Purification of phosphoribosylpyrophosphate synthetase (PRS) from the

latex cytosol of Ficus elatdice

--

Preparation

Crude extract

W S O a

Precipitate

Blue

s e p h = eluate

Total

Protein

(mg)

225.3

26.25

0.612

Total

Activity

(nkal)

9.3

3.3

1.75

Specifc

Activity

mg.9

0.042

0.126

3.5

Purifcation

(-fold)

1

3.1

83.3

Yirld

(77)

100

34.7

18.42

Photooynthetlc Rator

The iixation of "COZ by leaves was determined in all the control and treated

plants. The rates of photosynthesis in the leaves of guayule cultivars were in the range

of 28-36 mg C@ dm" If' ( F i 19). Differences in photosynthetic rates among the

cultivars were highly significant. Cultivar 11591 exhibited highest photosyntkt~c

rate (35.75 mg C01 dm'' h") while USS2X exhibited the lowest rates (28.32

mg C@ dm" K').

Photosynthetic rates were also measured in the leaves of cultivar 11591 under

different water regimes (Pig. 20). Inhibition in the rates of "CO~ uptake was noticed

under water stress in all the three guayule cultivars studied.

Fig. 21 shows the effect of low night temperature txeatment on photosynthetic

rates in guayule. The leaves showed a decline in the rates of photosynthesis in the

initial stages and later the C@ assimilation rates gradually increased. Guayule plants

(1 1591) exposed to 60 nights of low temperature exhibited higher photosynthetic

rates (44 mg C& d d h") compared to the control plants (36 mg C@ dm" K').

Rlbulore bisphorphat. carboxylase activity (RuBPcase)

The activity of RuBPcax was assayed in the leaf extracts of guayule. The

levels of the enzyme in d i h t culti~us are depicted in Fi 22. Large variation in

the enzyme activity was noticed among the cultivars. RuBPcase from 11591 exhibited

the highest activity (302.15 p o l (3% mg cN' K') while the lowest activity was in

Rg. 18 : Photosynthetic rates In the leaves of 24-months-old guayule cultivan. Vertlml bars indicate standard error (1123).

11591 CaC1 USSW

QUayu* culvar8

Flg. 20 : Effed of wrtw stms on photosynthetic rates In the leaves of 2 C m o ~ l d guayub w#ivPm. V e r W bua Indicate a!andard m r ( ~ 3 ) .

Fig. 21 . Effect of low night temperature on photosynthesis in the leaves of lyear- old guayule cultivam. Day tempsrature was 30 % end growth light intensity was 800 VE ma s". Vettical ban Indime aandard emr ( ~ 3 ) .

Fig. 22 : Rlkrloro Msphwphlte cscboxyhse (RuBPcase) activity In leaf exwaus of 2CmonUlcdd wyub Warn (1 1591, Cab1 and USSW. Vertical bars hdiorte rtrndard e m (n-3).

Fig. 23 : Effed of water dress on ribulote bisphobphate cahcuylase (RuBPcase) adlvity In leaf ex!rads ZCmonths-old guayule wHivan (1 1591, CaCl and USSZX). V M bars indicate standard error (n-3).

USS2X was the lowest (250 pmol C02 mg chl-' K'). Tbm was a significant positive

oomlation between the pho~synthctic rates and RuBPcase activity among the three

guayule cultivars.

The activity was RuBPcase in all the guayule cultivars under watm stress

revealed that with the drop in leaf water potential fiom 1.0 - m a to 3.0 -ma, thm

was a decrease in the activity of the enzyme (Fig. 23). Marked changes in the

activities of RuBPcase wen also noticed in guayule leaf extracts subjected to wld

night temperature (Fig. 24). The activity of RuBPcase showed an initial dec l i i up to

20 days of wld night temperature treatment and later rapidly i n d up to 50 days

of treatment. The activity of this enzyme dwng the low temperature treatment was

positively wmlated with the rates of leaf photosynthetic rates (Fig. 21 & Fig. 24).

However, RuBPcase activity in plants grown under low and high radiation wnditions

were lower when wmpared to the activity in plants grown under medium radiation

(Fig. 25).

The properties of the purified RuBPcase were determined for its kinetic

charactaistics (Table 5). Among the three guayule cultivars, the enyme fiom 11591

exhibited lower K,,,(HCO.>) value (0.3 mM) and high V, (284.5 w o l mg IN' v').

However, then were no significant differencts in the K, (RuBP) values m o n g the

lhnt guayule cultivars.

Fig. 24 : Effect of low nbht temperature on ribulose Msphosphste carboxylase (RuBPcMo) sdhrity In leaf e m u s of guayule wlthrars (11591, Cab1 and USS2X). Day t8mpemtum waa 30 F and growth UgM Intensity was 900 pE ma s". Vertical bm Indicate standard e m (n-3).

Fig. YS . Ritu lwe bisphosphate cadmxylase (RuBPcase) adivlty in guayule WHivars wbjsded to varying gmwVl ligM regimes. Growth temperature was 30125 OC. Vertical bars Indicate slandard error ( ~ 3 ) .

Low medium ~ d l l k n w mS sd)

Tablr 6 : Kinetic characteristics of purified RuBPcase from guayule leaf extracts

(Values are mean ?: SE of three deten~nations)

Fmctou bbphorph9b.6 activity (FBPaso)

F 4 26 shows the analysis of two-year-old guayule cultivars 11591, Cal-1 and

USS2X for FBPase activity. 11591 had the highest fructose bisphosphatase activity

(3 15 pmol mg chl" Y1) compared to the other two cultivars Cal- 1 (285 pmol mg chi-'

K') and USSW (281 pmol mg chl.' K1). FBPase activity of all the three cultivm was

also detenninul in the leaves at different leaf water potentials. The enzyme activity

deed in all the three cultivars with increasing wate~ stress (Fig. 27).

Marked changes in the activities of h s e bisphosphatase were noticod in

guayule leaf extracts subjected to cold night temperature treatment (Fig. 28). The

activity of FBPase followed a similar pattern to that seen in RuBPcase activity. The

activity showed an initial decline up to 20 days of cold night temperature treatment

and later rapidly increased up to 50 days of treatment (Fig. 24 & Pig. 28). Highest

fructose bisphosphatase activity was observed in plants grown at medium growth

radintion conditions compared to plants grown at low or high growth radiations. At

medium growth radiation (9C3 pE rn-' i') after 60 cycles, hctose bisphosphatase

activity was 320 pmol mg chl.' h" in 11591 followed by 280 p o l mg chl" h-' in

Cal-1 and 270 pnol mg cW1 K1 in USS2X (Fig. 29).

Sucrou phorphatr synthrw activity (SPS)

~ h e ~ p h o s p h a t c s ~ t h ~ s e ~ v i t y w a s m o n i n 1 1 5 9 1 ( 1 7 ~ o l m g c h l . '

K') comgP#i to Cal-1 (15 p o l mg chl.' Y') and USSW (12 pnol mg dC1 Y')

(FiL.30): In11591,withthcdropinloafwattrpotentialhml.O-MPato3.O-hdPa,

Plg. t a : FrcMtow bbptwsphabm QBPw) adMty in leaf exhUn of dllfemnl guayule OUWR (11591. Cab1 ud USS2X). VeNul barn Indicate standard emr ( ~ 3 ) .

11591 CaCl USSW

Ouryule cultivars

Fig. 27 : Effed of water stress on fructose bisphosphatase (FBPase) activity in leaf axtmts of diffemnt guayuie cullivarn (1 1591. Cab1 end USSW. Vertical bars indicate stendud e m (n-3).

Ftg. 28 : Efhd of low nbM temperature on fivct080 bisphosphatase (FBPoss) adlvity In leaf mlndr of different guayub arltivan (11591, CaCl and US-. Day temperature was 30 F and growth lbM IntOnSIty was 900 uE ma s-'. Vettical ban indicate standard ermr (1153).

0 10 20 30 40 50 60

Night temperatun t m b m n t at 15 'C (cycles)

Fig. 29 : Effect of varylw growVl light rqime on frudose bisphosphatase (FBPese) adMty In leaf eximcts of Quayule cultlvan. Growth temperature was 30/25 F. Vettical ban Micats standard srmr ( ~ 3 ) .

L w medium

bdwon (yz ma 8-7

the SPS activity decreased h m 21 pmol mg c ~ ' li' to 15 pmol mg chl.' h-' (Fig.

31). In the othn two cul t iva SPS activity was significantly affected by water stress.

SPS activity was also assayed in plants treated to 60 cycles low night

temperatures (30/15 OC). It was noticed that the enzyme activity initially dropped up

to 20 cycles and then increased upto 50 cycles (Fig. 32). Similar to the pattern in

RuBPcase and FBPasc activities, higher activities of SPS (21 pmol mg chi“ li') was

observed in 1 1591 under medium growth radiation (Fig. 33).

Photochemical Activities

The activities of photosystem I1 were measured in isolated chloroplasts by

following the photoreduction of 2,CDichlorophenol Indophenol (DCPIP) and

Fmicyanide (FeCN). Among the control guayule cultivars studied, 11591 is

characterized by the higher rates of DCPIP and FeCN reduction followed by Cal-1

and USS2X (Fig. 34 & Fi 38).

Water stress resulted in the d d activity of photosystem II as evidenced

by the decrease in the photoreduction of DCPIP and Femcyanide (Fi 35 & Fig. 39).

Low night temper- treatment caused an initial decrease in the activity of

photosystcm I1 ad lrtcr increased (Fig. 36 & Fig. 40). AAa 30 cycles of low night

tempemme treatment, the photonduction of the FeCN and DCPIP wm reduced to

the tune of 44% and 4 1 % respectively in 1 159 1. 'be leaves recowred thereefta and

the photondudion of DCPIP and FcCN incnased in the leaves of gusyule plants.

Pb. 52 : Effa d low nlgM tempetaturn on sucrose phosphDte synth~rre (SPQ activity In leaf axhcb of m m m gwyub wltivan (11591, CaCl and USSW). Day temperature wu 30 F and growth light intensity was 900 pE m-' 6'. Vertical bars indicate rtMdsld cnrw (n-3).

Fig. 33 : Sucroos phosphate synthase (SPS) adivny in baf wlmds of different guayule arlthran subjeded to varylng ~rowth light intensity. Growlh temperature was 3M5 F. VerUcd ban Indlcata standard emr (n-3).

Fig. U: Photowitem II BSII) adivity (FeCN redudon) in isolated ch~om@ssto of dlffemnt g u a w culUvan (11591. Cab1 and USSW). Venial bars Indime otandeFd m r (n-3).

11591 CaCl USSW

Ouayu* CUfUvur

Fig. 3S : Effed of waler strsss on photosystem II (PSII) adivity (FeCN redudion) in isolntd chloroplasts of different guayule cultivan. Vertical ban indicate standard error (n=3).

Fig. 36 : Effed of low nbM temperature on photosystem II (PSII) activity (FQeCN rsdudlon) In bmlatecl chlomplasts of different guayule cultlvars (1 1591, Cab1 & USSW). Dey temperatun mci 30 F end growth llght lntendty was 900 ME m" s-'. Vertical bars lndlutr standard error (n=3).

Fig. 37 : Photosystem I1 (PSII) adMty (FeCN reduction) in isolated chloroplasts of different guayule cutllvars subjected to varying gmvth llght Intensity. Growth temperature was 30125 F. Vettical bars indicate standard error (n=3).

Low n\.dun

ml.tlon ma 1")

no. $8 : Ptwwsttlm II P811) sdlvity W P I P redudlon) In Isolated chlomplpstcr of &ffmnl WYule cuRIvata (11501, Cab1 a d USSZX). V-1 ban indkute standard

CaCl USSW

~ u a y u k cumvan

Fig. 3s : Effect of water Jtrsss on pholosystern II (PSII) activity (DCPIP reduction) In Weted chloroplesls of dinersnl guayule wlvan. Vertical ban indicate standard m r (n=3).

fig. 40 : Effect of low nighl temperalure on photosystem II (PSII) adlvlty @CPIP raduction) in balated ohloroplasta of dmerenl guayule culthran (1 1591. Cab1 and U S W . Day temperatun, was 30 C and orvwth lipM Intensity was 900 900 ma 5 ' .

V m l M bsn Indicate dswrd e m (n=3).

Fig. 41 : Phdosystem I1 (PSII) edivrty (DCPIP reduuion) In isolated chlomplasts of dmerrmt guayule artlivan subjected to varytng growth light intensity. Growth temperature wu 3W25 %-. VVertlul bars indicate standard ermr (n=3).

When compand to plants grown under low and medium radiation conditions, the

plants grown under medium growth radiation (900 pE mS2 sit) exhibited higher

capacity for the photoreduction of FeCN and DCPIP (Fig. 37 & Fig. 41).

Compared to PSI1 activity, the PSI activity in isolated chloroplasts was not

much affected. The NADP reduction capacity (Photosystem 1 activity) of chloroplasts

among the three guayule cultivars was not much different (Fig. 42). The photosystem

I activity in isolated chloroplasts of guayule cultivars was also not much affected by

low night temperature treatment (Fig. 43) and different growth radiation conditions

(Fig. 44).

Leaf Pigments

The contents of total chlorophyll and carotenoids were estimated in leaves of

control and treated plants. Fig. 45 shows the chlorophyll content in different guayule

cultivars. Tnen was not much difference in the chlorophyll content among the three

guayule cult~vars. Similarly. there was also not much difference in the carotenoid

contents in the leaves of three guayule cultivars (Fig. 46). The total leaf protein

content in 11591. Cal-1 and USS2X was shown in Fig. 47. 11591 registered

maximum foliar protein content (5800 mg m-'). Cal-1 showed 5650 mg m-2 protein

and USS2X p o d 5478 mg mm2.

The chlorophyll and protein contents in the leaves of guayule were nduced by

W a t u stress as depicted in Fig. 48. The reduction in chlorophyll and protein contents

Fig. 42 : Photosystem I (PSI) actlv~ty (NADP reduction) in Isolated chlomplasts of different ~uoyule cultivars (11591. Cal-1 and USSZX) Vertical bars indicate standard error (~3).

11591 CaCl USSW

Ouryule cultivrn

Flg. 46 : Eff- of low night temperature on photosystem I (PSI) adMty (NADP mdudlon) In is~iolated chlomplasts of different guayule cultivars (1 1591, Cal-I and US82)0. Day kmpereture was 30 PC and gmwth ligM intensity was WO ME ma d. VeltlCd bPn ifldicale Stand~rd emr (11x3).

275

*- a0 e - t 5 225

t ::: (E! 125

100 0 10 20 30 40 50 80

NbM temperature treatment at 15 t (cycles)

Fig. 44: Photasystem I (PSI) adlvity (NADP redudton) in isolated chloroplasts of dlfhmnt gwyuk &an subjected to varying growth ligM intensity.Gmwth temperature WM 30/W 'C. V ~ I I W bars indicale standard ermr (n=3).

300 m119D1 m u 9

Fig. Total chlomphyll content in the leaves of different guayule cultivars (1 1591, Cab1 and U6S2X). Vertical ban indicate standard error (n=3)

11581 Cal-1 USSW

Guayule cultivars

Fig. 46 : Total camtenoid wnlent m the leaves of d~fferent guayule cult~vars (11591, CoCl and USSZX). Vertical bars indicate standard error (n=3)

Fb. 47: Soluble protein content in the leaf extracts of different guayule culthrars (1 1591. Cab1 and US-. Vsltical bars indicate standard error (n=3).

Fig. 48. Effect of water stress on chlorophyll and protein content in the leaves of 11591. Vefilcal bars indicate standard error (n=3)

was significant at the leaf water potential of 3.0 -MPa. Total chlorophyll content

estimated from guayule d t ivar 11591 treated with 60 cycles of low night

temperature (30115 O C ) showed an initial decline in the pigment content upto 30

cycles aftm which there was an increase upto 60 cycles (Fig. 49). Compared to

chlorophyll content, the total protein has markedly increased (15%) with increase in

cycles of low night temperature treatment (Fig. 49).

Fig. 50 shows the effect of different growth radiations on the chlorophyll and

protein contents in guayule leaves. Higher contents of chlorophyll (450 mg m-2) and

protein (5800 mg m'2) were recorded in plants grown at 900 pE m-2 s" growth

radiation.

Starch and Sucrose contents

All lhe three guayule cultivars have shown a drop in the sucrose content under

water stress (Fig. 51). In 11591 leaves, sucrose content dropped from 12.3 pmol mg-'

chl" at 1.0 -MPa to 9.9 pmol mg'l chrl at 3.0 M a . The starch content steadily

increased from 56.3 pmol mg.' chl.' at 1.0 - M a to 78.8 pmol mg" CW' at 3.0 -MPa

The results clearly indicate that the water stressed guayule plants accumulated

uctnmely high amounts of starch compared to control plaats.

Low night temperature initially caused a substantial increase in the foliar

st& content in guuyule (li591) while the content of sucrose showed an initial

dtclii followed by jvogressive increase from 20 days of treatment pig. 52). It was

Fig. 48: E M oflw nlgM temperature on total ohlomphyll and pmteln content In the leaves of 11591. Day tempemtutu was 30 F and growth ligM Intensity was OM) vE m-'~". Vertical bars indicate standard error ( ~ 3 ) .

360 4 ! 5200 0 10 20 30 40 50 60

NlgM tempwature treatment st I S 'C (cycles)

Fig. SO. Chlomphyll and Plotem contents In the leaves of 11 591 subledad to varying gfwwth IlgM Intensky Growth temperature was 30/25 "C Vertical ban indicate standard error (17-3).

Flg. 81: Effect of water stress on Starch and Sucrose contents in leaf exlrads of 11 691. Vmlbal ban indicate standard e m (m3).

Fig. 62: Effed of low nigM temperature on Starch and Sucrose contents in leaf extracts of 11 591. Day temperature was 30 "C end gmwlh intensity was 900 VE m" s" . Vertical ban indicmte standard error (1113).

inttmsting to notice that with increase in cycles of low night temperatwe tmtment,

there was an increase in sucrose content (Fig. 52).

Nitrogen content

' l l~c results on foliar nitrogen content in 11591 show that the basal portions

contain more nitrogen t . the apical ones (Fig. 53). The nitrogen content in the

leaves of guayule increased water stress upto 2.5 -MPa leaf water potential as

depicted in Fig. 54.

Nitrate reductase activity (NR)

The activity of nitrate reductase was assayed in both control and treated

guayule cultivars. Among the guayule cultivars, 11591 exhibited higher activity (4.3

pmol N& mg prot"h''), while lower activity was in USSZX (3.7 pmol NO*

mg prot" h") (Fig. 55). It was also observed that NR activity was higher in basal

portions of guayule when compared to the apical and middle portions (Fig. 56).

Fig. 57 shows the effect of wate~ stress on nitrate reductase activity in guayule

cultivars. It was obscrved that with the drop in leaf water potential h m 1.0 to 2.5

-MPa, the enzyme activity decmsed. Nitrate reductase activity increased under low

night temperature treatment upto 40 cycles after which the activity red& (Fig. 58).

Highst activity of nitrate reductase was observed in plants grown under mcdium

radiation conditions (Fig. 59).

Fig. 63: Follrr nilwen content in different parts of guayule culvar. 11581. Vertlcel ban Indicate standard error (n=3).

1 .o Base Middle Apical

PorHon of the plant

Fig. S4. Effed of water stress on follar nitrogen content in different guayule cultivan (11591. Cab1 and USSZX) Vertical ban Indicate standard error ( ~ 3 ) .

Fig. 66 : Nitrate reducab adivity in the leaves of different guayule cultivars (1 1591, CaCl and USSZX). Veltical ban Indicate standard emr (11x3).

Fig. 66 : Nltratr rsducatse adlvity in different parts of guayule cultivar, 11591. Vectlcsl bars lndlcste standard error (nz3).

Base Middle Aprcal

Portion of the plant

Fig. 67 . Effed of water stress on Nitrate reducatse activity ~n the leaves of guayule culllvan (1 1591, Cal-1 and USSZX). Vertical ban indicate standard error (1113).

Flg. 68 : Effed of low night temperature on nitrate reducetse a M y in the leaves of guayule (11501, Cab1 and USSW). Day temperature was 30 F and growth liiM intensky was WO pE mQ s". Vertical ban indicate standard error (n=3).

Nighttsmperaturn trnatment at 15 'C (cycles)

Fig. 59 : Effect of valying growth light lntenslty on N~trate reducatse activity In guayule leaves. Growth temperature was 30125 OC. Vertical ban indicate standard error (n=3).

Low medium

R.dl.tion (WE ma s')

Prolino content

The study on proliie content in different guayule cultivars showed that 11591

exhibited higher contents of proliie (3 mg g'l d.w.) compared to the other two

cultivars (Fig. 60). The effect of water stress on the amount of proliie was depicted

in Fig. 61. The data clearly show that the water stressed plants exhibited greater

enhancement in the proliie content. At 3.0 -MPa, the proline content was 4.5 mg gl

d.w. in 1 1591 compared to control plants (3 mg g-l d.w.).

Low temperature treatment of guayule cultivars also resulted in an increase in

the proline content in all the guayule cultivars (Fig. 62). Proline content in plants

grown under low and high radiation conditions were lower when compared to the

proline contents in plants grown under medium radiation (Fig. 63).

Proline dehydrogenase activity (PDH)

The PDH activity was estimated from the leaves of both control and stressed

plants. Among untreated guayule cultivars, highest activities of proliie

dehydrogenase were observed in 11591 (Fig. 64). Water stress enhanced the enzyme

activity in all the three cultivars (Fig. 65). The low night temperature keatment also

resulted in an enhancement in the activity of proline dehydrogenase (Fig. 66). Highest

activity of PDH was observed under 900 pE m'2 i' medium radiation (Fig. 67).

Suporoxide dismutase activity (SOD)

Among the three guayule cultivars, high activity of superoxide dismutnse was

noticed in 11591 (213 Units mg chl-I ~ ' ) followed by Cal-1 (185 Units mg chl"

PIg.60 : P d n e content in the leaves of different guayule wltivan ( I 1591. Cab1 and U S m . Vertkal ban indicate standard error (n=3).

Fig. 61 : Effect of water stress on Pmline content in guayule leaves (1 1581, CaCl and USSZX). Veltlcel ban Indicate standard ermr (1113).

Nlght temperature treatment at 15 OC (cycles)

Fig. 02 : Emct of low nlght temperature on fdiar Proline content of guayule (I 1501, Cab1 and USSZX). Day temperature was 30% and gmwth light Intensity was OW pE ma s-'. Vertlcal bars indicate standard error (nn3).

6 - - 5.- d 0 - 0

i : E 0

2 2 *I1591

Fig. 63 : Effed of valylng growth light intensly on Pmline content in guayule leaves. Growth temperature was 30125 OC. Vertical bars indicate standard ermr (n=3).

I,

Low medium

~n l~a t i on (5 m' s4)

*caCl +USSW

'L

0 10 20 30 40 50 80

Fia. 64 : Proline dehydrogensse adivily in Quayule leaves (11591, Cab1 and USS2)o. Vertlcal bars Indicate standard error (n.3).

11591 CaCl USSW

Ouayule cultivam

Fig. 66 . Effect of water stress on Pmline dehydmgenase acttvity ~n the leaf extracts of guayule (llSB1, Cal-1 and USS2X). Vettical ban ind~cate standard e m r (n=3).

800

1 m-- I x

I I x Z

1 T 7-

t t

"00 H -- -0-11s1 -0-CT1 --+--uBB1X

400 i -1 .O -1.5 -2.0 -2.5 -3.0

Leaf watw potential I-MPa)

Fig. 88 : Effed of low night temperature on Pmline dehydmgenese activity in guayuie leaf extracts. Day temperature was 30 OC and growth light intensity was 900 pE ma s". Vertlcsl bers indicate standard error (n.3).

10 20 30 40 50

NigM temperature treatment at I5 OC (cycler)

Fig. 67 . Effect of varylng growth light ~ntensity on Pmline dehydrogenase activity in guayule leaf extracts. Growth temperature was 30125 'C. Vertical baa indicate standard emr (n=3).

run.

mid') and USSZX (154 Units mg chl.' mine') (Fig. 68). Fig. 69 shows the effect of

water stress on the superoxide dismutase activity in different guyule cultivars. There

was a significant increase in the enzyme activity with decrease in leaf water potential

from 1.0 -MPa to 3.0 -MPa. The maximum extractable activity of superoxide

dismutase measured in guayule leaves on chlorophyll basis increased significantly

with 60 cycles of low night temperature treatment (Fig. 70). On the other hand,

maximum activity of superoxide disrnutase was also observed in plants grown under

medium growth radiation (Fig. 71)

Cahlase activity (CAT)

The data in Fig. 72 indicate catalase activity in different guayule cultivars.

The enzyme activity was high in 11591 (76 mmol mg chl" mid') followed by Cal-1

(66 mrnol mg chl" mid') and USSZX (58 mmol rng chl" min-I). Fig. 73 shows the

effect of water stress on the activity of catalase in guayule plants. There was a

uniform increase upto three-fold in the enzyme activity with the drop in the leaf water

potential. Under low night temperature treatment also, an increase in the activity of

catalase was observed with 60 cycles of treatment (Fig. 74). In 11591, at 0 cycles the

catalase activity was (76 mmol mg chl.' *-I), while after 60 cycles of low night

temperature treatment the catalase activity was enhanced to 195 mrnol mg chl.' min".

Catalase activity in plants grown under low and high radiation conditions were lower

when compared to the activity in plants grown under medium radiation (Fig. 75).

fIg. 68 : Sup~nxkb dismutasa (SOD) adivky in ieaf extracts of different ~ueyule oufflvan (11501, CICI and USS2X). Vectical brs indicate standard ermr (n=3).

11591 Cal-1 USSW Guayule cultlvar8

Fig. 69 : Effed of water stress supemxide dismutase (SOD) activity in guayule ieaf extra&. Vertical ban ~ndicate standard emr (n-3).

Fig. TO : Superoxide disrnutw (SOD) activity after 60 cydes of low nlgM lemperatwe at 15 .C. Day tmpetntum was 30 OC and gmwth ligM intensity was 900 pE m" 8. Velticsl ban indicate standard emr (ne3)

11591 Cab1 USSW

Ouayul* cultivars

Fig. 71 : Effect of varying gmwth ltght intensity on superoxide dismutase (SOD) activity in guayule leaf extracts. Gmwth temperature was 30125 OC. Vertical ban indicate standard e m r (n=3).

Low medlurn

~d~.tlon (16 ma 8-'1

Fig. 72 : Cstelase (CAT) adMty in leaf extrads of guayule wlUvan (1 1591, Cab1 and USSZX). Vertical ban indlcste standard emr (n=3).

11501 Cal-1 USSW

Guayule cultlvan

Fig. 73 : Effect of water stress on cetalase (CAT) actlvity In guayule leaf extracts. Vertical ban indicate standard error (1113)

Fig. 74 : Cataiase (CAT) edlvity afler 60 cycles of low night temperatun at 15 OC. Day temperature was 30 "C and growth IigM intensity was eW pE mv2 s". Vertlcai ban indicate standard enur ( ~ 3 ) .

Fig. 75 : EffeU of varying growth iigM intensity on cataiase (CAT) activity in guayuie leaf extracts. Growth temperature was 30125 OC. Vertical ban indicate standard e m r (11x3).

Low medium Rdi.Hon (LC m a s ' )

Poroxidasr activity (POD)

Figurer 76 - 80 show the activities of the key peroxidases, namely, the

guaiacol peroxidase and ascorbate peroxidase in guayule cultivars. 11591 exhibited

the highest activities of both the peroxidases (Fig. 76 & Fig. 80). Progressive

stimulation in the activity of peroxidases (guaiacol pemxidase and axorbate

peroxidase) from the leaves of guayule 11591, Cal-1 and USS2X were recorded

under water stress (Fig. 77 & Fig. 81). A consistent increase in peroxidase activity

during the water stress period fiom 1.0 -MPa to 3.0 -MPa leaf water potential was

noticed. An increase in the peroxidases activity was also observed in all the three

guayule cultivars treated to low night temperatures (Fig. 78 & Fig. 82) and medium

growth radiation conditions (Fig. 79 & Fig. 83).

Glutathione reductase activity (GR)

The GR activity was higher in 11591 (1055 pmol mg chl" K') when

compared to Cal-1 (889 pmol mg chl-' K') and USS2X (755 pmol mg W' K') (Fig.

84). The leaf GR activity increased from 1056 pmol mg chl" K' to 1522 p o l mg

CM' h" in leaf extracts of 11591 with increase in water stress (Fig. 85). GR activity in

guayule cultivars also increased significantly under low night tempemtun: treatment

(Fig. 86). All the cultivars exhibited maximum GR activity under medium p w t h

radiation conditions (Fig. 87). Highest GR activity of 1056 pnol mg W' K' was

observed in 1 1591 under 900 pE mq2 i' growth radiation.

Flg. 78 : Pemxldase (POD) activity In leaf exiracts of guayule cukivan (1 1591, Cal-1 and U582X). Vertical ban lndlcate standard emr (n.3).

Fig. 77 : Effect of water stress on pemxidase (POD) actlv~ty in guayule leaf extracts. Vertical ban indicate standard ermr (n13).

Fte 7a : P8oxidam BOD) adlvity after 60 cydss of low night tempamtun, at 15 t. Day Zanpentura WM 30 OC and gfwwth IQM intensity wos 900 UE ma 6'. Vertiwl barn indlcete standard m r ( ~ 3 ) .

Fig. 79 : Effect of varying gr0Wth light intensly on pemxidae (POD) activity in guayule leaf extracts. Growth temperature was 30125 t. Vertical ban indicate standard error ( ~ 3 ) .

Low medium

Radlatlon (pe ma 8'1

Fig. M : Asmbab p ~ ~ x i d s s e (APX) adhrlty in leaf extracts of guayule culivars (1 1591. CIII-1 and U S W . Vettical bars indicate standard error (n=3).

11581 Cat-I USSW

Ouayuk cumvan

Fig. 81 : Effect of water stress on ascorbate pemxidase (APX) activity in guayule leaf extracts. VecticPl bars indicate standard ermr (n=3)

Fig. 82 : Ascorb.1e pemddase (APX) adlvlty after 60 cycles of low nlgM temperature et 15 t. Day tanpentun, was 30 t and gmwth light intensity was 900 pE mQ s". Vertical bars Indicale siandard error (n13).

Fig. 83 : Effect of varying growth light intensity on ascorbate pemxldase (APX) activity In guayule leaf extracts. Growth temperature was 30125 OC Vertical ban indicate standard error (nn3).

Flg. M : GM.thlone rsductase (OR) adlvky in leaf extracts of guayule culthrars (1 1501, Cab1 and USSZX). Verticel bars indicate standad emr (1153).

Fig. 86 : E W of water stress on glutathtone redudase (GR) activity In guayule leaf exifads. Vertical bars indicate standard error (n=3).

-1.5 -2.0 -2.5 -3.0 Led mt.r wtmnw (-MP.)

Fig. 116 : Glutathione rsductase (OR) adivity afier 80 cycles of low night temperaturn at 15 "C. Day temperaturn was 30 OC and gmwth lQht intensity was 800 pE ma s". Vertlcal ban indicste standard error (n-3).

115B1 CaC1 USSW

Fig. 67 : Effect of varying gmwth light intensly on Glutathione reductae (GR) activity in auawle leaf extracts. Growth temperature was 30/25 OC Vert~cal ban indicate sI&dard error ( ~ 3 ) .

Low medium