cadmium elevates level of protein, amino acids and alters activity of proteolytic enzymes in...

ACTA PHYSIOLOGIAE

PLANTARUM Vol. 20. No. 2. 1998:189-196

Cadmium elevates level of protein, amino acids and alters activity of proteolytic enzymes in germinating rice seeds

Kavita Shah and R, S. Dubey*

Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi--221 005, India, Fax + 91-542-317074 *Author for correspondance: e-mail " [email protected]

Key words: amino acids, c a d m i u m , ca rboxypep t i -

dase, germina t ion , leucine aminopept idase , prote-

ase, protein, Oryza sativa

Abstract

When seeds of two rice cvs. Ratna and Jaya were germinated under increasing levels of cadmium nitrate (0, 100 and 500 ~tM) in the medium, a marked decrease in germination percentage was observed with Cd treatments, as compared to controls. There was more absorbed Cd in embryo axes than in endosperms. More uptake resulted with increasing Cd levels in the growth medium in embryo axes. In both rice cultivars, during a germination period of 0 - 120 h, an increased level of protein as well as tree amino acids was noted in Cd treatments. Protease activity in general decreased in both embryo axes as well as endosperms due to Cd treatment. In vitro studies showed an enhancement in protease activity in Cd treatments at low Cd levels (50 - 100 laM), whereas concentrations above this caused inhibition in enzyme activity. Under 500 ~tM Cd treatments in vivo there was about 30 to 50 percent decline in leucine aminopeptidase (LAP) activity in endosperms, however, carboxypeptidase activity showed a marked increase in endosperms beyond 24 h under Cd treatments. In embryo axes of germinating seeds there was always a decline in peptidase activities, under the influence of cadmium. The leucine amino peptidase and protease activity were always greater in embryo axes in cv. Ratna than cv. Jaya. However, the carboxypeptidase activity was higher in Jaya when compared to Ratna in endosperms under Cd treatments. The results suggest possible suppression ofprotease and peptidase activities due to Cd treat-

ments in germinating rice seeds leading to altered levels of protein and amino acids.

Introduction

E n v i r o n m e n t a l stresses c o m e in m a n y forms, yet

the m o s t p reva len t s tresses lead to cer ta in c o m m o n

adapta t ions in plants. (Bohner t et al. 1995). It m a y

lead to the a c c u m u l a t i o n or deple t ion o f certain me-

taboli tes, al terations in e n z y m a t i c activities and de

novo synthesis o f stress specif ic prote ins so as to

surv ive under stressful condi t ions (Dubey 1994,

Shah and D u b e y 1995). H e a v y meta ls in the soil en-

v i ronmen t are a ma jo r r isk for the g rowth of plants.

C a d m i u m , a potent e n v i r o n m e n t a l pol lutant , is in- c reas ing in the soil e n v i r o n m e n t with t ime, because

its add i t i on b e c o m e s g rea t e r t han its r e m o v a l

th rough leakage and plant harves t ing (Greger and

Johansson , 1992, Na idu et aI. 1994).

T h e use o f Cd and C d - c o n t a m i n a t e d goods like

ores, metal concent ra te , etc. result in e n h a n c e d soil

C d levels. Also the use o f fert i l isers and s ewage

s ludge result in e leva ted levels o f Cd in the soil. ( F l y h a m m a r 1995).

189

KA VITA SHAH AND R. S. DUBEY

Under many stressful conditions the solutes that accumulate include sugars, amino acids, organic acids, proline and glycine-betaine (Jones and Turner 1980, Good and Zaplachinski 1994, Shah and Dubey 1997, Shah and Dubey 1998).

The protein of a seed, either enzymatic or storage reserves are synthesized during seed development and maturation and are deposited within membrane bound protein bodies. At the onset of germination protein becomes degraded and the products trans- ported to different parts of the growing plant for use in biosynthesis (Duarte et al. 1996). The break- down of proteins in germinating seeds and in various parts of the plant is accomplished by the activities of proteases and peptidases (Dubey and Rani 1989, Yamaguchi el al. 1995). Cellular protein pat- terns change both qualitatively and quantitatively with changes in environmental conditions. Limited proteolysis and complete polypetide degradation are a closely interacting process (Muntz 1996).

Needs storage proteins in rice are essentially globu- lins which are formed in the specific storage tissue, the endosperm, during seed development. While these proteins accumulate the respective organs and storage tissues become nitrogen and carbon sinks and act as nitrogen and carbon source when the protein reserves are reactivated. Within this changing sink/source relationship, storage protein metabolism represents a unique example for the develop- mentally and environmentally controlled fc'-ma- tionldegradation of proteins (Muntz 1996). This neccessitates studies on cadmium which causes pertinent imbalances in the levels of biomolecules including proteins in growing plant parts due to its effect on various synthetic and hydrolytic enzymes.

Herein, we have examined the influence of increasing levels of Cd in situ on the metabolic status of proteins, amino acids and the behaviour of proteolytic enzyms in germinating rice seeds.

Materials and Methods

Plant material

Seeds of two rice (Oryza sativa L.) cvs. Ratna and Jaya were used. After surface sterilization and im- bibition in water for 24 h, seeds were germinated in

Petri dishes for 0 - 120 h at 28+1 °C in a growth chamber with 80 % relative humidity and 12 h light followed by dark periods. Cadmium nitrate solutions of concentrations 100 IJM and 500 tJM served as treatment solutions and water was used as the control. Seeds were taken at 24 h intervals and endosperms and embyro axes were separated. All analyses and enzymatic determinations were done in triplicate.

Evaluation o f germination percentage

Germination percentage was evaluated using 50 healthy seeds from each cultivar in triplicate. Ger- minated seeds were counted till 120 hours at 24 h intervals.

Extraction and estimation of cadmium, soluble

proteins and amino acids

Fresh endosperms/embryo axes from germinating rice seeds, were washed with 1 mM EDTA at 24 h mtc~vm~ to remove excess butiacc t)uunu t,u, and then dried in an oven at 70 °C for 5 days. Dried samples were weighed and acid digested. Cd was then estimated using a Perkin Elmer-337 Atomic Ab- sorption spectrophotometer and expressed in terms of mg.g -1 dry wt. sample. For protein estimation seed parts were homogenized in 5 ml of 0.2 M Na- phosphate buffer (pH 6.5), containing 0.5 M NaC1. After centrifugation, the soluble proteins were pre- cipitated from the supernatant with lml 60 % pcr- chloric acid and dissolved in hnl 0.1 N NaOH. Pro- tein was estimated according to the method of Lowry et. al. (1951) using B SA (Sigma) as standard and amino acids according to Roscn (1959), using glycine as standard.

Assay o f proteolytic enzymes

Protease was assayed in endosperms/embryo axes according to the method of Dubey (1982). Plant samples weighing 200 mg were homogenized at 4 °C in 5 ml 0.1 M Na-phosphate buffer (pH 6.5) and centrifuged. The supernatant after dialysis in cello- phane membrane tubings in cold for 24 h, served as the source of the enzyme. The assay mixture contained lml of 20 mg.m1-1 purified casein (Merck) in the same buffer and 1 ml enzyme. After incubatioa at 30 °C for 2 h, the reaction was stopped by adding

190

C A D M I U M ZN G E R M I N A T I N G R I C E S E E D S

3 ml of 5 % TCA. After centrifugation, TCA soluble peptide fragments were estimated according to Lowry et al. (1951). Enzyme specific activity was expressed as pg peptide fragments released.min-l.mg -1 protein.

Leucine aminopeptidase (LAP) activity was assayed by homogenizing 200 mg endosperms/embryo axes in 5 ml of 25 mM citrate phosphate buffer (pH 5.5), containing 10 mM ~3-mercaptoethanol at 4 °C. After centrifugation LAP was assayed, in the dialyzed supernatant according to the method of Chrispeels and Boulter (1975). The assay mixture contained 0.1 ml enzyme and 2 ml of 2 mM L- leucine p-nitroanilide (Sigma) in 50 mM Na- phosphate buffer (pH 6.8). After lh incubation at 30 °C the reaction was stopped by adding lml of 1M perchloric acid. After centrifugation, total amino acid was estimated in the supernatant according to the method of Rosen (1959). Enzyme specific activity was expressed as gmoles amino acids released.min -1 .mg -1 protein.

Enzyme extraction for carboxypeptidase was simi- lar to that used for LAR It was assayed according to Chrispeels and Boulter (1975). The assay mixture contained 0.1 ml enzyme and 2 ml of 2 mM N-

carbobenzoxy 1-phenylalanyl-l-alanine (Sigma) and 0.5 mM EDTA in 25 mM citrate phosphate buffer (pH 5.0). After incubation at 30 °C for 1 h, the reaction was stopped by adding 1 ml of 15 % (w/v) TCA. Total amino acids level was determined in the supernatant according to Rosen (1959). Enzyme activity was expressed as gmole amino acid released.rain -1 .mg -1 protein. In all enzyme perpara- tions, protein was estimated according to Lowry et at. (1959) using BSA (Sigma) as standard.

T h e e f fec t of increas ing concen t ra t ions of Cd(NO3) 2 in vitro on protease activity was studied using dialyzed enzyme preparations from endosperms/embryo axes of 72 h germinated seeds grown without Cd in the medium. The assay mixture, in addition to the normal constituents of the medium, contained increasing concentrations (0 - 2000 pM) of Cd(NO3) 2.

Results

Effect o f Cd in vivo on seed germination

Figure 1 shows the effect of increasing levels of Cd(NO3)2 in the medium on germination percent of

I - - Z L~I C )

0-

100

80

6 0

RAINA

.L 2'4 4'8 7'2 9'6 1:20

JA ¥A Ioo ~~ 80

60

~0

L ~ - L I I I I

0 24 48 72 g6 120-- HOURS OF GERMINATION

Fig. 1. Germination percent of seeds of rice cvs. Ratna and Jaya 0 - 120 h after germination, with increasing concentration of Cd(NO3)2 in the medium : (o), control; (.), 100~tM Cd(NO3)2; (V), 500ktM Cd(NO3)2. Values are means based on three independent determinations.

f f I

'T o~ IE 0

~=8 o.z tM

t- 0,3 W

o 0.2 U

~r ~ 0.1

=i 0 Q

u 0.1 O

0.2 Ill

R A T N A

~[ll lO0/JMCd2: 500/J M Cd 2

- [ ] 500/JM Cd 2.

J A Y A

--J---~--L_.__~ 24 48 "/2 96 120

HOURS OF GERMINATION

Fig. 2. Cadmium content of endosperm and embryoaxes of rice cvs. Ratna and Jaya 0 - 120 h germination, with increasing concentrations of Cd(NO3)2 in the medium. Values are mean based on three independent determinations. Bars indicate standard deviations.

191


] R A T N A io cowrRo~ ~. Ioo ~ Cd

v 5 0 0 #~ Cd +

f i i : ........................... * . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................................... ::::::::::::::::::::::::::::::::::::: 2 ....... ..................................... ~ '

Z 0 • - - - - - - - - - - - T

= JAYA

,~ .......................... • ::::::::::: .....................

O ,,:;::::.. .... .

2 ......... i " ............... • ................

0 24 48 72 96 120

H O U R S OF GERMINATION

10

8

6

4-

Fig. 3. Extractable protein of endosperm ( ) and embryoaxes ( ............ ) of rice cvs. Ratna and ]aya 0 - 120 h after germinations, with increasing concentrations of Cd(NO3)2 in the medium. Values are means _+ s.d. based on three replicates and bars indicate standard deviations.

Fig. 4. Free amino acids level in endosperm ( _ _ ) and embryoaxes ( ............ ) of rice cvs. Ratna and Jaya 0 - 120 h after germination, with increasing concentrations of Cd(NO3)2 in the medium. Values are means _+ s.d. based on three replicates. Bars indicate standard deviations.

b

i b~

2~ u

o Z

rM rM

r .

! 2

10

8

~[

2

0

~2

10

8

6

4

2

0

RATNA

J

JAYA

24 4 8 72 9 5

HOURS OF GERMINATI0N

CONTROL 2. I 0 0 , am C d

~7 50O # ~ Cd 2÷

1117:::

120

rice seeds ofcvs. Ratna and Jaya at different times. In controls (without Cd) upto 96 h, i 0 0 % germination was observed. Inhibition in germination o f seeds was noted due to the presence o f Cd in the medium. Al though 100 #M Cd treatment stimulated germination yet the values were lower when compared with controls. With 500 phi Cd(NO3)2 in the medium a 10 to 20 percent decrease in germination o f the seeds could be observed.

Uptake and localization of cad-

mium in germinating rice seeds

The absorption o f cadmium and its distribution in the endosperms and embryo axes o f germinating rice seeds is s h o w n in Figure 2. In both cuitivars, with increase in time, there is a gradual increase in the level o f Cd absorbed. The uptake o f Cd increased with an increase in Cd concentration in the medium. In both the rice cultivars, with the 500 ~tM Cd treatment Cd levels o f 16.8 to 76.0 ppm were noted in embryo axes and 11.20 to 16.68 ppm in endosperms, after 120 hours o f germination.

Cadmium effect on protein level

Figure 3 s h o w s a gradual rise in the Icvcl o f extractable proteins in embryo axes o f rice cvs. Ratna (0 - 72h) and Jaya (0 - 120h) with an increase in germination time. An increase in Cd levels in the germination m e d i u m led to a marked increase in protein level. The in- c reased e f f e c t w a s m o r e pro- nounced during 48 - 96h in Jaya but not in Ratna where it is at 0 - 48 h. With a moderate Cd level o f 100 pM in cv. Jaya, about 1 to 2 t imes more increase in protein

192

CADMIUM IN GERMINATING RICE SEEDS

CONTROL 800 RATNA , o . . . . . . . ::

, . . ~7 500 ~ Cd .~;> .< ...........

! 400

~c~ 200

>~

['~ 8OO

r,.)

6{30

c,.

r...)

[.=3 400 Pu

f~

200 F~ 0

0

, r , ,

JAYA

i r ± I i

24 48 72 96 120 HOURS OF G E R M I N A T I O N

Fig. 5. Protease specific activity in endosperm ( _ _ ) and embryoaxes ( ............ ). of rice cvs. Ratna and Jaya at increasing honrs of germination under increasindg levels of Cd(NO3h in the medium. Vanles are mean -+ s.d. based on three observations and bars indicate standard deviations.

Fig. 6. Effect of increasing concentrations of Cd(NO3h (0 - 2000 # M ) in vitro on protease specific activity of dialyzed enzyme preparations obtained from control grown endosperms and embryoaxes of rice cvs. Ratna and Jaya 72 h after germination. Values are means _+ s.d. based on three observations Bars indicate standard deviations.

800

600

'7' 400

7

200 @

°

8 0 0

5O0

4 0 0

o 200

2 5 5 0 IOC 20O

C d ( N O a ) ~ CONCENTRATION (/zM)

5 0 0 1000 ZOO0

was observed in the embryo axes during 48 - 72h of germination, whereas with 500 uM Cd this level increased to 2 - 3 times, compared to controls. With the 500 }aM Cd treatment a higher protein level was a lso o b s e r v e d in the endosperms at 24 and 72 h in ca,. Jaya as compared to controls.

Cadmium effect on level o f free amino acids

In the embryo axes of both cultivars, with an increase in the Cd level in the medium, a concomi- tant increase in amino acid content was noticed during 24 - 96 h of germinat ion (Fig. 4). At 96h abou t 1 fo ld i nc rease in f ree amino acids levels was noticed in e m b r y o axes o f 100 pM Cd treated seeds whereas at 72 h with the 500 }aM Cd treatment the increase was up to 2 to 3 fold in both the cultivars. In endosperms an increase in free amino acids was observed with 100 }aM Cd levels during 48 -72 h, compared to the controls, whereas with 500 }aM Cd there was a decrease in amino acid levels beyond 72 h of germination. As with extractable proteins, the level of amino acids was always h igher in embryo axes than in endosperms, in both control and Cd treatments.

Effect o f cadmium in vivo on protease activity

Figu re 5 shows the specific activity of protease in endosperms and embryo axes of rice seeds as a function of time with increasing concentrat ions of cadmium in the medium. A decrease in protease activity was observed at 24 h due to the Cd treatments in both endosperms and embryo axes in cv.

193

K A V I T A S H A H A N D R. S. D U B E Y

"Z

0 , 4 I 0 CONTROL 2+

7 = I . RATNA { v~ ~ ~+

I O 0 1 l I

~- 0 .4 - - -

(2,

0.3

< 0.2

0 0.;

0,0

r r I I

.;.~::::. JAYA

, ,,.

i i . . . . i t I

24 48 72 96 120

HOURS OF GERMINATION

Fig. 7. Specific activity of Leucine aminopept idase in endospe rm ( _ _ ) and embryoaxes ( ............ ). o f rice c v s . Ratna and Jaya 0 - 1 20 h after germinat ion with increasing levels o f C d ( N O 3 h in the medium. Values are mean __ s.d. based on three observations. Bars indicate standard deviations.

Fig. 8. Specific activity of carboxypept idase in endosperm ( . _ _ ) and embryoaxes ( ............ ). o f rice c v s . Ratna and Jaya 0 - 1 20 h after germinat ion growing with increasing levels o f Cd(NO3)2 in the medinm. Values are mean _+ s.d. based on three observations. Bars indicate standard deviations.

0 . 8

o

'7 0 6 E

T

0 4

-~ 0 .2

oo

N

o.6 U 03

0 . 4

N

13. 0 " 2 t > ,

o~

0 . 0 0

0 CONTROL a,

X7 600 t,.~ Cd ~4

~ ;~i+ i121121: :: ::::: : ....... . . . . . . . . . . . . . . . . . . . . . . . . .

p r - - - - , - - - -

JAYA z

1

2 4

]72::: .:::::.:;::::::112121111111 ............. :::::: ....................

....... :: !!!!!!!!!!!!i!!iiii~ t q r

48 72 96 120

HOURS 0P GERMINATION

Jaya. In seeds germinating without cadmium, during 0 to 48 h, there was an increase in protease activity followed.by a gradual decline in the embryo axes. In endosperms, however, during 0 - 72 h an increase in activity was observed, which declined thereafter. In all cases the activity of protease was higher in endosperms, compared to the embryo axes.

Cd(N03)2 in vitro and protease

activity

In vitro studies indicated a slight increase in protease activity at low Cd concentration of 50 - 100 laM in cv. Jaya, whereas higher concentrations beyond this level caused a marked inhibition in enzyme activity in enzyme preparations from both endosperms and embryo axes (Fig. 6). Initially, a Cd concertration of 1 - 50 #M in vitro led to a gradual decline in protease activity, but a sharp rise in the activity was found at 100 laM Cd level followed by a gradual decline thereafter. A Cd concentration of 1000 to 2000 gM ap- peared to be highly inhibitory to the protease activity.

Effect o f Cd in vivo on LAP ac-

tivity

In cndospcrms of seeds germinating without Cd, an increase in LAP activity was observed at 0 - 48 h, followed by a gradual decline thereafter (Fig. 7). In vivo Cd treatments of both 100 and 500 pM were inhibitory to the enzyme beyond 24 h of germination.. A 500 pM Cd caused about 50 percent decline in LAP activity in endosperms. An increase in LAP activity was observed in embryo axes in the two cultivars.

194

CADMIUM IN GERMINATING RICE SEEDS

From 48 - 72 h from germination in c~: Jaya LAP activity increased in controls followed by a subse- quent decline in both embryo axes and endosperm. Cadmium treatments of 100 as well as 500 gM in situ reduced the LAP activity in embryoaxes.

Effect o f Cd in vivo on carboxypeptidase activity

The activity of carboxypeptidase declined in embryo axes of germinating seeds of both rice cultivars due to Cd treatments. However , in endosperms, especially in the 500 ~tM Cd treatment and during 48 to 96 h of germination, a marked increase in carboxypeptidase activity was noticed (Fig. 8). Contrary to protease and LAP activities, results indicated enhancement in carboxypeptidase activity in endosperms and a decline in embryo axes due to cadmium.

Discussion

Our results indicate a decrease in germination percentage, an increase in levels of soluble proteins and amino acids and altered behaviour of proteolytic enzymes in rice seeds germinating in the presence of Cd in the medium. Differential absorption of cadmium in germinating seed parts i.e., its greater localisation in embryo axes than in endosperms was observed, which suggests that, distribution of absorbed Cd in rice is organ specific. Cadmium uptake and its distribution inside the plant depends upon species, soil type, pH and on the presence of competing ions (Shah and Dubey 1995).

Many workers have observed that, under various environmental stresses like heat, water, salinity and heavy metals, certain new proteins are synthesised (Kumar and Singh 1991, Alia and Saradhi 1991, Esaka and Hayakawa 1995, Shah and Dubey 1995). The increased protein levels, under Cd stress, as observed in our study might possibly be due to increased synthesis of new stress specific proteins (Shah and Dubey 1995, Reddy and Prasad 1995), as well as to inhibition in the activity of proteolytic enzymes. A considerable increase in the level of amino acids was reported in various plant parts under salinity stress (Dubey 1982, Dubey and Rani 1989). Accumulation of amino acids was suggested mainly due to induction of protein hydrolysis by the

enzyme proteases, under stressful conditions (Reddy and Vora 1985). However, this was not the case with our experiments, as we noted decreased proteolytic activity due to Cd. Cadmium was shown to be the strongest inducer of protine accumulation in plants (Alia and Saradhi 1991 Chen and Kao 1995). It has been suggested that free amino acids may be produced in stressed plants at the cost of materials needed for developmental processes (Alia and Saradhi 1991).

Peptidases and proteases hydrolyse endosperm storage proteins during the germination of cereals (Mikola 1983) and play an important role during seed germination. In our experiments an inhibition in protease, LAP and carboxypeptidase activity was observed in embryo axes due to Cd treatment. De- creased activity of endospermic protease and LAR especially 72 to 120 h from the start of germination, under Cd stress, suggests a loss in degradation of peptides, leading to an imbalance in the level of proteins and amino acids. Carboxypeptidase activity increased to a large extent in endosperms 72-96 h from germination.

Different levels of protein and amino acids and especially a higher level of proline, in germinating seed parts, is directly correlated with the degree of metal tolerance (Alia and Saradhi 1991). Cadmium in the present investigations, elicited transient changes in the activities of protease, aminopeptidase and carboxypeptidase in endosperms. This suggests that the presence of Cd in the medium is likely to cause marked perturbations in protein metabolism of germinating rice seeds and growing plants. This might contribute towards decreased germination and impared growth of rice seedlings in a Cd polluted soil environment.

Acknowledgements

This work was supported by the grants from Ministry of Envi- ronment and Forests, Govt of India in form of a research proj- ect. One of us (KS) is grateful to CSIR, New Delhi for provid- ing a research associateship.

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Received May 16, 1997; accepted September 22, 1997

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