DEVELOPMENT OF MITOCHONDRIA IN HORSE GRAM COTYLEDONS DURING GERMINA nON

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DEVELOPMENT OF MITOCHONDRIA IN HORSE GRAM COTYLEDONS DURING GERMINAnON

D.KARUNAGARAN·AND~RAM~SHNARAO

Department of Biochemistry, Sri Krishnadevaraya University Anantapur-SIS 003

Received on 18 June, 1990

SUMMARY

Time-course of changes in activities of mitochondrial enzymes in cotyledons of germinating horse gram seeds showed an upward trend during the initial stages and a decline during the later stages of germination. While succinate dehydrogenase artd malate dehydrogenase activities enhanced upto day I, activities of cytochrome-c oxidase, 2­ oxog)utarate dehydrogenase, isocitrate dehydrogenase and glutamate dehydrogenase increased upto day 3. Treatment with cycloheximide or chloramphenicol had no inhibitory effect on appearance of the enzyme activity, Mitochondrial maturation in horse gram is probably m=diated by activation of pre-existing enzymes rather than by their de novo synthesis. Influence of axis was indicated by the restricted development of enzyme activity in the absence of axis.

INTRODUCTION

Mitochondria in the cotyledons of dry seeds of legumes are structurally and enzymatically underdeveloped. During germination mitochondria become well developed to meet the energy-requirement for utilization of the mobiJized food reserves. Mitochondrial development takes place by the repair and activation of pre-existing immature mitochondria or biogenesis of new mitochondria (Bewley and Black, 1978; Morohashi, 1986). As the cotyledonary reserves get depleted, mito­

chondria become disorganized and gradually Ios~ their respiratory efficiency, enzyme

complement and activity (Bewley and Black, 1978). Earlier studies on mitochon­ drial development have been restricted to a few leguminous seeds with no detailed information on the biochemical processes. Therefore, the present study is aimed at understanding the mode of development of mitochondria in cotyledons of germinat­ ing horse gram see.ds in terms of enzymatic changes. Time-course of changes in

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activities of a few mitochondrial enzymes were followed for a period of 4 days. Effects of cytosolic and mitochondrial protein synthesis inhibitors on activities of succinate dehydrogenase and cytochrome-c oxidase were also investigated. Since embryonic axis is suggested to influence the development of mitochondria (Morohashi and Bewley, 1980) and hydrolases (Morohashi. 1982; Sharma, 1988) during germination, possible role of axis was examined as well.

MATERIALS AND METHODS

Uniform-sized seeds of Co 1 variety of horse gram (Macrotyloma uniflorum Lam.) used in this study were procured from Tamil Nadu Agricultural University,

Coimbatore. Seeds were surface sterilized with 0.1

%

mercuric chloride for 5 min

and rinsed thoroughly with distilled water. Seeds were then soaked in water for 4 hrs and a bafch of 20 seeds was spread on two layers of filter paper, in a 10 cm Petri dish, moistened with 8 ml of water. These were placoo in a wooden tray, covered tightly with polythene sheets and left for germination in dark at 28±2°C. Sterile conditions were maintained by including 20 ppm of streptomycin sulfate in water and test solutions. To evaluate the influence of axis, seed coats were removed and cotyledons separated from each other, so that the axis remained attached to one of them. The cotyledons with (attached) or without (detached) axis were incubated under the conditions described, for the specified length of time. The time, when the

plant materials were transferred to the Petri dishes was considered as day O.

Preparation of mitochondrial fraction : Mitochondria were isolated according to the method of Bonner (1967). Cotyledons were ground in a mortar and pestle

at 0-4°C using a solution containing 0.3 M mannitol, 0.1

%

BSA, 0.05~1,. cysteine

and 1 mM EDTA (tissue; medium=l : 2). The pH was maintained at 7.2 by adding a few drops of 1 N KOH and the homogenate was filtered through cheese cloth, centrifuged at 1000 X g for 15 min and the supernatant was centrifuged at 10,000 X g. for 15 min in a refrigerated centrifuge Remi K24A model. The mitochondrial pellet was washed. resedimented and suspended in K-Pi buffer (0.1 M. j>H 7.2) for use.

MITOCHONDRIA IN GERMINATING HORSE GRAM 221

RESULTS AND DISCUSSION

Time-course of changes in mitochondrial enzyme activity: Mitochondria are important centres of energy production and changes in metabolic state of cotyledons

may be reflected through the pattern of mitochondrial enzyme activity. Mitochondri~l

enzyme activities assayed in the cotyledons of horse gram during germination exhibited two discernible patterns of development over a 4-day period. The activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) showed (Fig. 1) an initial increase from day 0 to day 1 with a progressive decline thereafter. How­ ever, activity of the other set of enzymes (Table I) continued to increase upto day 3 (except for the initial decline in glutamate dehydrogenase) and then declined on day 4. The observed pattern of development of mitochondrial enzymes in horse gram is

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D. ICARUNAGARAN AND P. RAMAKlUSHNA RAO

Table I. Changes in activity of some mitochondrial enzymes in the cotyledons of horse gram during germination

Time (days)

2.()xoglutarate

dehydrogenasea dehydrogenaseIsocitrate b dehydrogenasec Glutamate

Cytocbrome-c oxidased

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4.26 ±0.08 26 ±0.73 2.84 ±0.06 1.69 ±0.04

1 4.57 ±0.07 96*+1.93 1.12*+0.04 3.11*+0.09

2 8.33*+0.20 153*+3.67 3.41*+0.11 3.85*+0.08

3 11.79*+0.22 177 +4.27 5.22*+0.18 4.68*+0.07

4 2.44*+0.04 38*+0.83 1.39*+0.04 2.76*+0.03

Each value is the mean ± SE of six values.

&. Activity expressed as nmoles of ferrocyanide Hberated/hr/l0 cotyledons.

b. Activity expressed as nmoles 'of products (2, 4-DNP hydrazones of 2-oxoglutarate and NADPH) formed X lo-'/min/l0 cotyledons.

c. Activity expressed as nmoles of oxoglutarate formed/hr /10 cotyledons. d. Activity expreSSed as 6. A X 10.../min/10 cotyledons.

*Significant difference at each day interval (p

<

0.001).

Table

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Effects of cyclohe,ximide and chloramphenicol on mitochondrial succinate

dehydrogenase and cytochrome-c oxidase activities

Succinate dehydrogenase activity (nmoles of Cytochrome-c oxidase activity (6. A x 10-a/

dichlorophenol indophenol reduced/min/lO min/lO cotyledons) cotyledons)

Time Control 0.1 mMCH 2mMCP Control 0.1 mMCH 2mMCP

(Days) treated treated treated treated

89Cl+29 900+31 910+32 3.11+0.09 3.20+0.10 3.19+0.13

2 745+23 750+25 740+26 3.85+0,08 3.90+0.12 3.87+0.11

3 669+21 680+23 670+24 4.68+0.07 4.71+0.16 .4.70+0.12

4 433+16 440+15 450+18 2.76+0.03 2.63+0.08 2.68+0.06

Each value is the mean±SE of six values.

No significant difference between control and treated.

consistent with that in starch-storing legumes (Morohashi, 1986). Nawa et al. (1973), reported a decrease in mitochondrial respiratory activity in pea cotyledons at the later stages of germination accompanied by a loss of mitochondrial enzymes.

Effect ofprotein synthesis inhibitors: Seed germination was completely inhibit­ ed by cycloheximide (CH) at a cone. of 0.1 mM (no protrusion of radicle occurred

J

MITOCHONDRIA IN GERMINATING HORSE GRAM

223

radicle on day 1 as compared to 1.2 cm in control). However, both CH and CP

failed to inhibit the development of SDH and cytochrome oxidase (Table II) in

horse gram suggesting tbat the de novo syntbesis of proteins may not be involved in

the development of mitochondrial enzymes. These results indicate that tbe develop­ ment of mitochondria is achieved by incorporation of pre-existing cytoplasmic pro­ teins into tbe immature mitochondria as is the case with peas and soybeans (Malhotra et al., 1973; Nawa and Asahi, 1973; Singh et al., 1987).

Effect ofaxis excision on enzyme activity: The changes in the activities of two representative enzymes, SDH and cytochrome-c oxidase, were followed in the pre­ sence and absence of axis and are shown in Fig. 2 and 3, respectively. Detached cotyledons possessed lower activity of tbe enzymes. The pattern of enzyme activities

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Fig. 2. Effect of axis excision on mitO<?hondri~1 succi~ate dehydrogen~ activity in the cotyledoQs

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D. KARrJNAOARAN AND P. RAMAKRISHNA RAO

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was similar in attached and detached cotyledons for both enzymes upto day 3. From day 3 to day 4 no perceptible changes were noticed in detached cotyledons. In agreement with the present work, excision of axis resulted in a relatively lesser development of mitochondrial enzyme activities in peanut cotyledons (Morohashi

et 01., 1981) and cucumber (Morohashi and Matsushima, 1988). However, in peas lack of embryonic axis control over mitochondrial activity has been reported

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