Changes in Proteins and Enzymes during Grain Development in Pearl Millet

Biochem. Physiol. Pflanzen 175, 15 - 22 (1980)

Changes in Proteins and Enzymes during Grain Development in Pearl Millet D. N. GUPTA, M. K. Roy, J. N. SINGH, G. S. SINGHAL and S. L. MEHTA Nuclear Research Laboratory, Indian Agricultural Research Institute, New Delhi, India Key Term Index: protein, enzymes, grain development; Pennisetum typhoides.

Summary Changes in protein, peroxidase, GDHl) and glutamate oxalo-acetate transaminase were studied in Pennisetum hybrids HB-3, PHB-14 and their parents during grain development. Dry weight and protein content of grain increased during development but the rate of accumulation varied. Protein percentage showed only slight variations. HB-3 maintained higher dry weight and protein compared to either parent while PHB-14 had lower dry weight as well as protein as compared to female parent at maturity. Both the hybrids had lesser protein percentage than their female parent but were almost similar to their male parent. HB-3 was superior to PHB-14 in respect of dry weight and protein in grain. Peroxidase activity of HB-3 was similar to Tift-23 A while for PHB-14 it was considerably higher than either of the parents at maturity. GDH activity was found to be higher in HB-3 as compared to its parents at dough and maturity stages while in case of PHB-14 the activity was lower than either of the parents at these stages. GOT activity in HB-3 was almost twice that of Tift-23A and 50 % higher compared to J-l04 at maturity while in PHB-14 the activity at maturity was lower than either of the parents. These results lend support to the hypothesis that the heterosis-effects in different hybrids are manifested differently and activities in hybrids are higher than their parents.

Introduction

In recent years plant breeders have devoted their attention on increasing crop yields. In pearl millet several new hybrids have been released. One of the important aspects of plant breeding programme is the improvement in protein quality among cereal grains. The protein quality of pearl millet is better than sorghum (SAWHNEY and NArK 1969). In addition, higher content of lipid compared to other cereal grains, makes it energy rich food (SA WHNEY and NAIK 1969). The physiological analysis of many hybrids have been undertaken but information about the changes occurring in protein and various enzymes relating to growth and development is lacking. Therefore, in present study two hybrids have been compared with their parents with respect to morphological characters, dry weight, protein, peroxidase, glutamate dehydrogenase and glutamate oxaloacetate transaminase. Peroxidase has been selected mainly due to its role in growth and development (SIEGEL and GALSTON 1967; FRICK 1971). GDH and GOT are the key enzymes involved in the final assimilation of ammonia for the synthesis of various amino acids. 1) Abbreviations: GDH, glutamate dehydrogenase; GOT, glutamate oxaloacetate transaminase.

16

D. N. GUPTA et al.

Materials and Methods Pel1niselum typhoides Two hybrids HB-3 and PHB-14 along with their parents, i. e., Tift-23A, J 104 and L 111 A, PIB-228 and maintainer i. e. Tift-23B and L 111 B were grown on IARI farm. Morphological observations of the plants were taken from time to time. Ears were harvested at milky (10 days after pollination), dough (15 days after pollination), and mature stages, grains separated and analysed for peroxidase, GDH and GOT activity, dry weight and protein, or stored in liquid nitrogen until further use.

Enzyme extraction Grains were extracted by hand grinding in a chilled pestle and mortar with 50 mM Tris-HCl buffer (pH 7.6) containing 5 mM 2-mercaptoethanol for GDH and GOT. Mercaptoethanol was omitted for peroxidase. All the operations were carried out at 4°C. Homogenate was centrifuged at 10,000· g for 20 min and supernatant used for enzyme activity.

Enzyme activities Peroxidase activity in the supernatant was assayed according to the method used by SHANNON et al. (1966). GDH activity in the supernatant was assayed by measuring NADH oxidation at 340 nm in the reaction mixture which contained in 3 ml: Tris (pH 8) 0.125 mMole; iX-ketoglutarate 20 ,uMole; (NH4)2S04 15 mMole; NADH 0.2,uMole; enzyme 0.1 m!. Blank contained no NADH. The'reaction was initiated by the addition of NADH as the last component. GOT was assayed in the supernatant at 37°C. The reaction mixture contained in 3 ml: Tris-HCL buffer (pH 8) 0.15 mMole; aspartate 20 ,uMole; pyridoxal phosphate 25 ,ug; NaCN 0.1 ,uMole; enzyme extract 0.1 ml, iX-ketoglutarate 20,uMole. iX-Ketoglutarate was omitted in blank and 2.7 ml of buffer instead of 2.6 ml taken. The reaction was initiated by the addition of iX- Ketoglutarate as the last component and enzyme activity measured at 280 nm. The enzyme activities have been reported as the change in optical density per g fresh weight or per grain per min. Protein determination

Protein content of grains was estimated by micro-Kjeldahl method (A.O.A.C. 1965).

Dry u'eight After taking fresh weight the grains were oven dried at 110°C for 24 h and cooled in dessicator and weighed. This was repeated till constant weight.

Results and Discussion

Morphological characters Morphological characters of the hybrids along with their parents shown in Table 1 indicate different patterns for hybrids HB-3 and PHB-14. HB-3 inherited characters like length of ear head and girth of ear head from its male parent J-I04 while PHB-14 inherited length of ear head, length of leaves, and number of leaves per plant from its female parent L 111 A and girth of ear head and plant height from its male parents PIB-228. Heterotic effects are seen with respect to plant height and width of leaves in PHB-14, and plant height, width and length of leaves in HB-3. These results show that the two hybrids show differences in relation to their similarity to either of their parents.

to

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Table 1. Morphological characters of pearl millet varieties

170.0 117.0 124.2 159.2

6.38 2.28

3.91

± ±

±

11 ± 0.59 12 ± 1.51 11 9 ± 0.31

13 ± 0.4 10 ± 0.3 9 9 ± 0.3

132.0 ± 3.9 104.6 ± 2.45 116.0 11.4 ± 2.3

Date of sowing - 20.7. 1976 (a) observation taken at maturity (28-30/10/1976) (b), (c) & (d) observation taken at ear emergence (25. 9.1976) (e) & (f) observation taken at matured earhead.

PHB-14 J,)l1 A L 111 B PIB-228

J 104

Tift-23 B

Hyb-3 Tift-23 A

Number of leaves per plant

Height of plants (ems)

2.33 2.20

1.14

1.78 2.85

± 2.58

± ±

±

± ±

67 64 66 61

51 39 47 38

Length of leaves (em)

± 0.11

± 0.11 ± 0.29

4.2 ± 0.25 3.4 ± 0.11 3.9 3.2 ± 0.25

3.1 2.2 2.9 2.1

Width of leaves

±

± ±

0.20

0.15 0.12

8.0 ± 0.31 6.1 ± 0.36 7.2 9.0 ± 0.72

6.5 5.3 5.9 6.3

Girth of ear head (em)

2.7

±

2.85

± 0.80 ± 1.48

±

± 1.1 ± 1.48

31.2 29.8 28.8 35.2

19.6 17.0 20.8 18.8

Length of earhead (em)

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D. N.

GUPTA

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For explaining biological and physiological basis of heterosis it has been suggested that the hybrid follows one of the better parents (COYNE 1965; KHANNA 1974). In other words, it means that the superior characters from either of the parents are inherited in the hybrids. While this is true of some characters which follow the quantitative behaviour, it may not be true of many other characters relating to enzyme activity (HAGEMAN et al. 1967). On examining dry weight accumulation during grain development (Table 2), it is seen that HB-3 is superior to either of its parents and at maturity it had substantially higher dry weight compared to its parents, whereas another hybrid PHB-14 had an intermediate pattern of dry weight accumulation compared to its parents upto dough stage. At maturity it had lower dry weight than its parents. The pattern of dry weight accumulation in PHB-14 resembled its male parent PIB-228. The female parent L 111 A accumulated much more dry weight during later stages of development and this character has not been inherited in the new hybrid. Therefore, the dry weight accumulation in HB-3 and PHB-14 is quite distinct.

Protein content The differences observed in dry weight of the grain during development were also reflected in the differences in the amount of protein per grain. Patterns for protein accumulation were also identical to the patterns for dry weight accumulation for both the hybrids (Table 2). The hybrid PHB-14 contained only 50% of the protein per grain Table 2. Dry weight and protein in grains at different stages of development in Pearl millet Variety

Dry weight (g/100 grain)

Percent Protein

Protein (mg/100 grain)

Milky

Milky

STAGES Milky

Dough Mature

HB-3

0.294

0.598

Tift-23A

0.282

0.380

0.434

17.32

18.18

17.67

48.8

69.1

76.7

Tift-23 B

0.215

0.287

0.330

15.53

18.10

18.42

33.4

51.9

60.8

0.474

0.550

15.03

16.78

71.2

92.3

J 104

0.831

Dough Mature 15.48

15.38

Dough Mature 92.6

127.8

PHB-14

0.246

0.522

0.600

13.06

12.84

12.71

32.1

67.0

76.3

1111 A

0.224

0.390

0.799

17.04

17.51

17.69

38.2

68.3

141.3

1111 B

0.384

0.410

0.587

16.67

13.11

14.88

64.0

53.7

87.3

PIB-228

0.380

0.703

0.674

11.09

12.42

11.93

42.1

87.3

80.4

as compared to its female L 111 A while HB-3 had 70% higher protein compared to its female parent. HB-3 has 70% more protein per grain compared to PHB-14 at maturity. Protein percentage in each variety did not show much variation during development. Both the hybrids resembled their respective male parents with respect to protein percentage. Similarity in protein percent during development indicates parallel synthesis of starch and protein.

Protein Changes in Penniselum Grain Development

19

Enzyme activities Because of the differences in the rate of protein accumulation in the two hybrids as well as their parents further studies on the two key enzymes involved in the amino acid metabolism were undertaken. The activity of GDH and GOT was studied during grain development in hybrids along with their parents. The results presented in Table 3 Table 3. Glutamate dehydrogenase activity in developing grains of Pearl millet Variety

G.D.H. activity (,1 O.D. 340/1000 grains/min) STAGE Milky

Dough

Mature

HB-3

1.02

2.70

4.51

Tift-23 A

1.38

2.12

2.80

Tift-23 B

1.27

1.49

2.77

J 104

1.33

1.85

3.61

PHB-14

0.60

1.07

3.12

1111 A

0.58

0.95

3.77

L 111 B

0.97

0.89

2.97

PIB-228

0.97

2.04

4.55

showed differences in the GDH activity as well as GOT activity of hybrids and parents. GDH activity per grain of HB-3 was higher as compared to either of the parents at dough and mature stages while its activity in PHB-14 was lower as compared to male parent PIB-228 upto dough stage. However, the GDH activity per g fresh weight showed different trend (Table 4). During development GDH activity on fresh weight basis increased in HB-3, 23A and PIB-228. While in case of PHB-14, L 111 A and J 104 there was slight decrease in GDH activity at dough stage and substantial increase at maturity. Both the hybrids had slightly lower GDH activity compared to their parents during grain development. Table 4. Glutamate dehydrogenase activity in developing grains of Pearl millet Variety

Activity (,1 O.D./g. fr.wt./min) STAGE Milky

Dough

Mature

HB-3

0.30

0.38

0.46

Tift-23 A

0.40

0.42

0.56

Tift-23 B

0.49

0.42

0.69

J 104

0.38

0.30

0.56

PHB-14

0.17

0.15

0.44

L 111 A

0.21

0.18

0.40

L 111 B

0.20

0.18

0.42

PIB-228

0.21

0.24

0.57

2*

20

D. N.

GUPTA

et al.

In general, HB-3 and its parents had higher GDH activity compared to PHB-14 and its parents both on the grain and fresh weight basis. The increase in GDH activity was substantially higher towards later part of grain development. GOT activity per grain (Table 5) of HB-3 was almost two fold compared to its female parent Tift-23A and 50% higher compared to male parent J 104 at maturity. On the Table 5. Glutamate-oxaloacetate transaminase activity in developing grains of Pearl millet Variety

HB-3 Tift-23 A Tift-23 B J 104 PHB-14 L1llA L 111 B PIB-228

Activity (LJ O.D./1000 grain/min) STAGE Milky

Dough

Mature

6.12 4.36 4.29 8.54 4.44 2.89 5.69 4.14

8.76 6.20 4.26 6.47 6.86 5.41 3.57 9.18

12.54 6.40 4.98 8.24 7.24 10.64 7.99 12.69

other hand the activity in PHB-14 was substantially lower compared to either of its parents at maturity. HB-3 maintained higher GOT activity per grain as well as per g fresh weight (Table 6) compared to PHB-14 during grain development. GOT activity in hybrids PHB-14 and HB-3 at dough stage and maturity was similar to the activity of their female parent. These results suggest that the higher protein content in HB-3 could be mainly due to the higher GDH and GOT activity per grain while the lower Table 6. Glutarnate-oxaloacetaie transaminase activity in Pearl millet Variety

HB-3 Tift-23 A Tift-23 B J 104 PHB-14 L 111 A L 111 B PIB-228

Activity (.1 O.D./g. fr.wt./min) STAGE Milky

Dough

Mature

1.80 1.26 1.65 2.44 1.25 1.05 1.17 0.90

1.23 1.23 1.20 1.05 0.96 1.02 0.72 1.08

1.28 1.28 1.24 1.28 1.02 1.13 1.13 1.59

protein content of PHB-14 could be ascribed to the decreased activity of these enzymes. The relationship of GDH, GOT with protein accumulation has also been observed in Sorghum and maize (MEHTA et al. 1978; OAKS 1978). It is now well established that glutamine synthetase, glutamate synthetase, GDH and GOT play an important role in nitrogen assimilation in plants. Some of the workers have shown that glutamate

Protein Changes in Pennisetum Grain Development

21

synthetase is the key enzyme as far as ammonia assimilation is concerned. However, for the synthesis of other amino acids from glutamic acid, transaminases play an important role. L 111 A and HB-3 which have substantially higher protein per grain also have much higher GDH and GOT activity at maturity. PIB-228 also had considerably higher GDH and GOT activity, yet the total protein accumulated in it towards the later stages of grain development was much less. This could perhaps be due to limitation of free amino acids. Peroxidase is known to play an important role in growth and development of the plants. Therefore, in the present study, peroxidase activity was determined during grain development in hybrids along with their parents. Peroxidase activity on fresh weight basis (Table 7) in HB-3 grains wa, lower a<; compared to its parents at Table 7. Peroxidase activity in developing grains of Pearl millet Variety

Activity (Ll O.D./g. fr.wt./min) STAGE Milky

HB-3 Tift-23 A Tift-23 B J 104

Dough

Mature

548 664 1,160 580

488 688 648 368

508 586 800 920

PHB-14

596

328

756

L 111 A

520

432

566

L 111 B

568

504

744

PIB-228

488

412

586

maturity. In PHB-14, the peroxidase activity was higher than its parents. Higher peroxidase activity has been reported to be associated with low grain weight in triticale (RAO et al. 1976). Highly significant and negative association between plant height and peroxidase activity has also been observed (SINGHAL et al. 1979). While the exact function of the peroxidase is not known, morphological roles are suggested by its action in producing (RIDDLE and MAZELIS 1964) and in activating auxin (GALSTON et al. 1953), in converting hydroxyphenylpropanes such as coniferyl alcohol to lignin like materials, and in oxidizing metabolic compounds such as reduced NAD and its phosphate (WILLIAMS et al. 1959). Further peroxidase and IAA oxidase activities have been shown to be due to identical enzymes (SHIN and NAKAMURA 1962). In present investigation also, a negative correlation seem to exist between the grain weight and peroxidase activity. Similar negative relationship between peroxidase activity and grain weight in wheat has also been observed (SINGHAL et al. 1979). Seeds of HB-3 and L 111 A which had higher grain weight had lower peroxidase activity while the seeds of J-104 and Tift-23B having lower grain weight possessed substantially higher peroxidase activity. Tift-23A deviated from such relationship. Variation in peroxidase activity during grain development was also observed.

22

Protein Changes in Pennisetum Grain Development

The results reported in this study, therefore, indicate that the hybrids differ markedly from their parents, in respect of some enzymes, dry weight accumulation and protein accumulation. Comparison of the enzyme activity, protein and dry weight accumulation in hybrids HB-3 and PHB-14 indicate lack of similarity between these two hybrids. Improvement in the accumulation rates of dry weight and protein compared to either of parents indicates clear heterosis effects. These results further indicate that in some of the characters, hybrids may follow one of the better parents, while in others it may have improvement over either of the parents due to true heterotic behaviour. References A.

o. A. C.,

Official methods of analysis of Association of Official Agricultural Chemists. MicroKjeldt\hl method, 774 (1965). COYNE, D. P.: Component interaction in relation to heterosis for plant height in Phaseolus vulgaris L. variety crosses. Crop Science 5, 17 (1965). FRICK, F.: Enzymes of indole acetic acid degradation in barley leaves. Biologia (Bratislava) 26, 677 (1971). GALSTON, H. W., BORNER, J., and EAKEH, R. S.: Flavoprotein and peroxidase as components of the indole acetic acid and oxidase system of peas. Arch. Biochem. Biophys. 42, 456 - 470 (1953). HAGEMAN, R. H., LENG, E. R., and Dl'DLEY, J. W.: A biochemical approach to crop breeding. Advanc. Agron. 19, 45 (1967). KHANNA, R.: Physiological and biochemical analysis of heterosis in sorghum. Ph. D. Thesis. !ARI. Kew Delhi 1974. :MEIITA, S. 1., DONGHE, A. B., J OH"\HI, R. P., LODIIA, lVI. 1., and NArK, 1\1. S.: Biochemical constraints that determine protein quality and grain yield in cereal grains. Proe. IAEA Symp. 1, 241 (1979). Rxo, N. G. P.: Genetic analysis of some exotic and Indian crosses in sorghum. 1. Heterosis and its interaction with seasons. Ind. J. Genetic & Plant Breeding 30, 347 (1970a). - Genotype & environment interaction in grain sorghum hybrids. Indian J. Genetics and Plant breeding, 39, 75 (1970 b). RAo, V. R., MEHn, S. L., and JOSIIl, 1\I. G.: Peroxidase and amylase activity in developing grains of triticale, wheat and rye. Phytochemistry 5, 893 (1976). RIDDLE, W. M., and MAZELIS, M.: A role of peroxidase in biosynthesis of auxin. N atnre 202, 391 (1964). SAWHNEY, S. K., and NAIK, M. S.: Amino acid composition of protein fractions of pearl millet and the effects of nitrogen fertilization on its proteins. Indian J. Genetics 29, 395 (1969). SHANNON, L. 1\1., KAY, E., and LEW, J. Y.: Peroxidase isoenzymes from horse radish roots - isolation and physical properties. J. BioI. Chern. 241, 1266 (1966). SHIN, M., and NAKAMUlU, W.: Indole acetic acid oxidase activity of wheat peroxidase J. Biochem. 52, 444 (1962). SIEGEL, B. Z., and GUSTON, A. W.: The isoperoxidases of Pisum sativu1Il. Plant Physiology 42, 221 (1967). SINGHAL, N. C., MEHTA, S. L., and SINGH, M. P.: Peroxidase activities in relation to plant height and grain weight in bread wheat Triticum aestivum 1. Theoretical and Applied Genetics 55, 87 (1979). WILLIAMS, A., CeHMAN, H. G., and KLAVINS, H.: Two enzymic mechanisms for hydrogen transport by phenolic oestrogens. Nature 184, 427 (1959).

Received July 18, 1979. Authors' address: D. N. GpPTA, Nuclear Research Laboratory, Indian Agricultural Research Institlltc, New Delhi - 110012, India.