PROTEIN HYDROLYSIS AND NITROGEN TRANSFER DURING GERMINATION OF MAIZE SEEDS AT VARIOUS TEMPERATURES

PROTEIN HYDROLYSIS AND NITROGEN TRANSFER DURING GERMINATION OF MAIZE SEEDS AT VARIOUS

TEMPERATURES

BANDANA BosE AND H.S. SRIVASTAVAl Department of Botany, University ofGorakhpur

Gorakhpur-273aOl

Revised March 10, L981

SUMMARY

Changes in ethanol-soluble and insoluble nitlogen contents of maiz~ endospetm from seeds germinated in darkness at 10 to 50°C for 50 h were measured to Sludythe effect of temperature on nitrogen solubilization. Nitrogen content of the embryo was also measured to determine the extent of translocation of organic nitrogen during germination. The parameters examined showed maxima at 30 to 35°C which corre­ lated well with the effect of temperature on the germination of seeds and on the pro­ tcare activity in the endosperm. At higher temperatures, (40-50°C), a major portion of the nitrogen lost from the endosperm wa s leached out to the medium. When the seeds were soaked at different temperatures before sowing, soaking at optimum tem­ perature (30-35°C) supported maximum growth of the seedling and ethanol soluble and insoluble nitrogen content and nitrate reductase activity in the'primary leaves. These experiments demonstrate a correlated effect of temperature on seed germination, protease activity and nitrogen solubiliza.tion in the endosperm and its translocation to the embryo.

INTRODDCTION

The effects of temperature on seed germination and seedling growth are well docUJB8Dted (Went 1953, Sen and Chawan 1970,Sylvia 1973, Takeba and Matsubara 1976, Simon et al., 1976). The process of germination is initiated by the uptake flf water (Wassink and Stolwijk 1956). During this period the seed rapidly changes from a quiscent state to a dynamic state and metabolic activities increase. During germina­ tion, the storage products of cotyledons or endosperm are hydrolysed and transferred

Received on Oct. 10, 1980.

142 BANDANA BOSE AND B.S. SlUVAETAVA

to the embryonic axis involving activation and synthesis of several macromolecules (Beevers 1968, Beevers and Spliitstoesser 1968, Ching 1972, Stoddart et al., 1973). Temperature as an effector of seed germination may modify anyone of the biochemical reactions during germination.

To examine whether the effects of temperature on these biochemical processesr

_t

are correlated with the effect of temperature on seed germination and seedling growth, i

the present study was undertaken.

MATERIALS AND METHODS

Seeds of Zea mays L. Cv. Sankar makka Gang-2 were purchased from a local dealer. After sterilising them with a dilute solution of bleaching powder (l%) for about 5 min, they were washed thoroughly and then imbibed in distilled water for 6 h at desired temperature in darkness. After that they were placed on filter papers mois­

tened~ith 10 mM CaCIlI.2HIIO, contained in large petri dishes, and incubated for the

I

desired period at the desired temperature. Endosperm and embryo were dissected

I

out aseptically for analysis. Scutellum was included in the embryo portion.

For studying the effect of pre-sowing treatment at different temperatures, the I

seeds were imbibed at various temperatures for 50 h and then planted in the pots. , i I i

,Seedlings were raised in identical environments for 8 days, before the leaves were used

I

,for various' analyses. Dry weight of leaves was measured as an index of growth.

Leaves from uniformly grown seedlings were collected and dried in 'an ovenat 60°C for

i

24 h.. Ethanol soluble and insoluble nitrogen in oven dried samples of endosperm,

embryo and leaves were measured according to the modified micro-Kjeldahl method

(Lang 1958).

I

ProteaSe activity in the endosperm w~s measured according to a slightly modified

I

procedure of Harvey and Oaks (1974). Bovine Serum albumin (BSA) and Haemo­

globin (Bovine type U,.Hb), purchased from Sigma Chemical Co., U.S.A. were used as substrates. The details of the procedure have been given earlier (Bose and Srivastava 1980).

In vivo activity of nitrate reductase in the leaves wasineasured as described earlier (Srivastava 1974).

Data presented are mean values of three observations.

RESULTS

Nitrogtm content of the seeds imbibed for 50 h at various temperatures: In the

143

tnlpeTature~ raap'sfudicd (UHo 5()°O). the insolubcl.nitrogeuof- -the-endosperm <Wacased:with

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iDel'ease in temper~ture upto 30~C and with.a correlated -incrcasein tho

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:lfitrogen (Fig.

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Further jncreaseilr temperature uptoSo.~C caused an;

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TEMPERATURE ,-., 1

Fig. 1 Nitrogen content of the maize seeds imbibed at different temperatures in the dark for SO h.

Circ1es-embryo; trianglcs-cndoseperm. Continuous lines-ethanol-insoluble nitrogen; Dashed lines-ethanol-soluble nirogen.

144 BANDANA BOSE AND H.S. SRIVASTAVA

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increase in insoluble nitrogen and decrease in soluble nitrogen. Optimum temperature: for. the solubilization of endosperm protein, appeared to be 30-35°C.. Gain in the· embryo nitrogen was also maximum in this temperature range. Temperature lower or

higher than this range caused decline in both soluble and insoluble nitrogen of the embryo. (Fig. 1).

Seed gennination and protease activity: Optmum temtJCrature range for germi~ nation of seeds and for development of protease activity in the endosperm was also found to be 30~35oe (Table I). Seeds did not germinate at all upto 50 h at 10,20 or 500_{e temperature and there was little germination at 25 or 4Ooe. Similarly. no prote~}

ase activity was detected at extreme temperatures of 10 or 5~e and the activity at 20. 25 or 400

e was also low.

Table I. Germination of seeds and protease activity in the endosperm of seeds germinated at different temperatures in dark

Temperature ·C Germination percent Protease activity in the endosperm (mg protein after SO h. hydrolysed/hI endosperm) after 96 h.

Substrate - . BSA Hb

10

20

25

30

35

40

SO

0

0

20

89

82

IS

0

1.242

1.870

4.047

4.022

2.010

2.012

2.903

6.139

6.030

3.193

Critical

_±

36.27 1.549 2.329

difference

Temperature and nitrogen balance: The nitrogen distribution in the maize seeds germinated at various temperatures is shown in Table II. The transt:er of nitrogen from endosperm to the embryo increased between 10 and 300e with QUJ

value of about 1. 2.. At temperatures above 350

e

_{a major portion of nitrogen}

PROTEIN HYDltOL YSIS AND NITROGEN TRANSFER IN MAIZE SIIED 145

Table 11. Effect of temperature on transfer of total nitrogen from endosperm to the embryo between 6 and 50 h of germination

Temperature °C Nitrogen, mg/part

Nitrogen lost Nitrogen gained LeachingiD

from eooosperm in embryo the medium

(difference)

0.150 0.151 0

0.220 0.221 0

0.295 0.294 0.001

0.368 0.354 0.014

'0.365 0.342 0.023

0.290 0.174 0.116

0.319 0.115 0.214

10 20 25 30 3S

40

50

Growth. nitrllte reductase activity and nitrogen content of t he primary leaves:

It was observed that in leaves obtained from pretreated seeds at 30 and 3SoC. all the parameters studied viz. fresh-weight. dry weight. nitrate reductase activity. soluble and insoluble nitrogen were higher i.n comparison to ·other temperature treatments (Table III).

Table III. Growth. nitrate reductase activity and nitrogen content of primary leaves as affected by pretreatment of seeds at various temperature in dark

Pretreatment

temperature Leaf parameters

°C

Fresh wt. mglleaf

Dry wt. ms/leaf

Nitrate reduct­ ase activity.

nmoles No./h/g fresh wt.

Ethanol-soluble

nitrogen mslg dry wt.

Insoluble nitrogen

mslg dry wt.

10 30.70 3.90 91.2 4.46 8.57

20 63.75 6.f5 350.0 5.63 11.29

25 85.65 7.60 468.0 7.02 12.85

30 110.50 11.25 680.0 10.20 18.50

3S 103.00 10.60 560.0 8.45 15.l2

40 50

73·40 6.91 448.0 6.67 12.00

-­

Critical

_±

dilference 36.64 3.58 227.78 3.015 5.40

Seeds were imbibed in distilled water for 50 h at various temperature. They were planted in

pots and seedlings raised for 8 days in the garden at an average day and night temperature of 30 and 26·C respectively. photoperiod was about 11 h.

146 BANDANA BOSE AND B.S. SRIVASTAVA

DISCUSSION

The results demonstrate'that at 30-350_{C the solubilization of nitrogen in embryo}

and endosperm are maximum. But at lower and at higher temperatures this process is inhibited. At 30-35°C the synthesis of new protein in the embryo and the transfer of soluble nitrogen from endosperm to embryonic axis are also maximum. Although the effect of temperature on germination has been studied by several workers(PoUock and Toole 1966. Sen and Chawan 1970, H~ndricks and Tayl~rS'&n 197{), Simon et al.. 1976, Hard Castle 1978), the process of nitrogen solubilization in the endosperm and its transfer to the embryo has received little attention. The optimum temperatnre recorded for germination in the present investigation is similar as observed by Went (1953). It was also observed that germination is the manifestation of a number of biochemical reactions in relation to nitrogen metabolism (Dute 1960, Beevers 1968, AbduJ-Baki 1969). An increase in germination and related changes in the nitrogen of embryonic axis with increase in temperature upto 35°C indicated that there is a posi­ tive cori'el~tion between the percent germination and nitrogen solubilization in the endosperm. Inhibition of these processes at higher temperatures appears to be due to leaching of metabolites. Similar observation at higher temperatures has also been reported by Hendricks and Taylorson (1976). The activity of protease in the endos­ perm was also correlated with the germination and nitrogen changes. However, the procedul1eemployed for extraction and assay of proteases in the present investigation (Harvey and Oaks 1974) accounts only for the acidic proteases whick have a narrow specificity cleaving the peptide linkage involving the a carboxyl group of either aspartic acid or glutamic aeid (Garg and Virupaksha 1970). Besides, several other proteases. have,. also. been" cha,/:aa.el'ised<tn the st.omge' tissues (Penner and AshtoJ1 1967), which may be equally active in the hydrolysis of storag~ proteins and thus may contribute in the germination and growth of young seedlings.

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PROTEIN HYDROLYSlS AND NITROGEN TRANSFI!ll IN MAIZE SEED 141­

ACKNOWLEDGEMENT

The- senior author (BB) is greatful to the C. S. I. R., N@w Delbi for the award of a S. R. F. and to Professor S·. N. Mathur, Head of the Botany Department for laboratory faeilities and encouragement •

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14~ BANDANA BOSE AND B.S. SlUVATAVA

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