Lipoxygenase activity in fresh, stored, and germinating pearl millet ...

Lipoxygenase activity in fresh, stored, and germinating pearl millet seeds resistant and susceptible to downy mildew disease K. C. NAGARATHNA, SUDHEER A . SHETTY,A N D H. S. PRAKASH Department of Studies in Applied Botany, University of Mysore, Mar~asagangotri,Mysore 570 006, India

S. G. BHAT Biochemistry Section, Food Chemistry Department, Central Food Technological Research Institute, Mysore 570 013, India AND

H. SHEKARA SHETTY'

Can. J. Bot. Downloaded from www.nrcresearchpress.com by Peking University on 05/28/13 For personal use only.

Department of Studies in Applied Botany, University of Mysore, Manasagangotri, Mysore 570 006, India Received January 7, 1992 NAGARATHNA, K. C., SHETTY, S. A,, PRAKASH, H. S., BHAT,S. G., and SHETTY, H. S. 1992. Lipoxygenase activity in fresh, stored, and germinating pearl millet seeds resistant and susceptible to downy mildew disease. Can. J . Bot. 70: 2028 -2031. Lipoxygenase activity was studied in fresh, stored, and germinating pearl millet seeds of downy mildew resistant and susceptible genotypes. In both fresh and stored seeds, the enzyme activity was greater in resistant genotypes compared with that of the susceptible ones. In seeds stored for a period of 6 months, a significant reduction in the lipoxygenase activity was recorded, the reduction being greater in resistant seeds. Lipoxygenase activity decreased significantly in germinating seedlings of resistant genotypes, whereas it increased significantly in the susceptible seedlings. The high vigour in the resistant seeds of downy mildew resistant genotypes is attributed to their high enzyme activity. Key words: lipoxygenase, pearl millet, downy mildew, resistant, susceptible. K. C., SHETTY, S. A,, PRAKASH, H. S., BHAT,S. G., et SHETTY, H. S. 1992. Lipoxygenase activity in fresh, NAGARATHNA, stored, and germinating pearl millet seeds resistant and susceptible to downy mildew disease. Can. J. Bot. 7 0 : 2028 -2031. Les auteurs ont CtudiC l'activitt de la lipoxygenasse chez des graines de millet per16 en rtserve et en germination appartenant ?I des gtnotypes rtsistants ou susceptibles au mildiou. Dans les graines en rtserve aussi bien que dans les graines en germination, l'activitt enzymatique est plus forte chez les gtnotypes rtsistants que chez Les gtnotypes susceptibles. Dans les graines mises en rtserve pendant 6 mois, on observe une reduction significative de l'activitt lipoxygtnaseique; cette reduction est plus marqute chez les graines des gtnotypes rtsistants. Pour les graines en germination, l'activitt de la lipoxygenase diminue significativement chez les plantules de gtnotypes rtsistants alors qu'elle augmente de f a ~ o nsignificative dans les plantules de genotypes susceptibles. La grande vigeur des graines de gtnotypes rtsistants au mildiou serait attribuable ?I leur forte activitt enzymatique. Mots cles : lipoxygtnase, millet perlt, mildiou, rtsistant, susceptible. [Traduit par la redaction]

Introduction The enzyme lipoxygenase (LOX, EC 1.13.1 1.12) catalyzes the oxidation of free fatty acids such as linoleic and linolenic acids containing the cis,cis-methylene interrupted diene system forming the conjugated cis,trans diene hydroperoxide. This enzyme is well studied in legume seeds, notably in soybean (Glycine m m L.) and oil seeds (Galliard and Chan 1980). In cereals, the LOX activity has been studied in wheat (Triticum aestivum L.) (Hsieh and McDonale 1984) and maize (Zea mays L.) (Gardner and Weisleder 1970). Although the LOX activity has been well studied in plant systems, its physiological role in plants is still ambiguous. The enzyme is known as a wound hormone (Galliard and Chan 1980) and its role in seed germination and disease resistance has also been described. Vick and Zimmerman (1976) suggested that LOX could function in the conversion of linoleic acid to a suspected plant hormone. Several of the hydroperoxide decomposition products of LOX activity have antimicrobial activity, and some volatile products are similar to a range of fungicides (Galliard and Chan 1980). Siedow (1991) has provided more information on LOX in his recent review. Downy mildew disease, incited by Sclerospora graminicola (Sacc.) Schroet, is a severe problem in pearl millet (Pennisetum 'Author to whom correspondence should be addressed. Prinled in Canndn I Imprime au Canada

glaucum (L.) R. Br.) growing areas. Several genotypes of pearl millet, with different degrees of resistance to downy mildew disease, are available. The present study describes the differential pattern of LOX activity in fresh, stored, and germinating seeds of pearl millet genotypes with different degrees of resistance to downy mildew disease.

Materials and methods Pearl millet seeds that are resistant (P-310-17, 841 B and 700651) and susceptible (23 B, HB3 and 843 B) to downy mildew disease were obtained from the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, India, and the project co-ordinator, All India Co-ordinated Pearl Millet Improvement Programme (AICPMIP), Pune, India. All six genotypes were tested to confirm their downy mildew reactions in the downy mildew disease plots of the Department of Applied Botany, University of Mysore, where a well-established, highly susceptible genotype HB3 scores above 90% for downy mildew disease. The seeds of different genotypes were sown in the disease plot by randomized block design, using HB3 as indicator plants. Disease incidence in the plants was recorded on the 30th and 60th days after sowing by adopting the standard procedure for downy mildew screening (Williams et al. 1981). Determination of LOX activity In fresh seeds Pearl millet seeds belonging to the Qx different genotypes, harvested in October 1989, were collected and stored in brown paper bags. The

2029

NAGARATHNA ET AL.

TABLE1. Lipoxygenase activity in fresh seeds and seeds stored for 6 months of downy mildew resistant (R) and susceptible (S) genotypes of pearl millet

21 0

Can. J. Bot. Downloaded from www.nrcresearchpress.com by Peking University on 05/28/13 For personal use only.

18

Genotypes

Fresh seeds

P-310-17 (R) 841 B (R) 700651 (R) 843 B (S) HB3 (S) 23 B (S)

18.61k2.011 (7) 18.44k2.146 (10) 15.36k1.725 (10) 9.60k0.910 (10) 8.76k0.996 (13) 7.04k1.426 (9)

NOTE: LOX activity is given as A/(mg protein. rnin) parentheses are the number of replicates. *Significant at 5 % level.

X

lo-'.

-

X

Student's t-value

Stored seeds 8.35f0.200 7.65k0.219 7.26k0.550 5.14k0.396 6.29k0.252 4.77k0.139

0

(3) (4) (5) (3) (3) (3)

P-310-17 (Resistant) HE3 (Susceptible)

7.08* 9.79* 10.07* 8.06* 4.85* 3.19*

Values within the

moisture content of the seed samples were determined by the oven method (International Seed Testing Assn. 1985). The seeds were used for enzyme study within 1 month of harvest.

In stored seeds A portion of the above seed materials was stored in brown paper bags for 6 months under laboratory conditions at 45-50% RH and 25k2"C. These stored seeds were used for testing the LOX activity.

Seedling age (days) FIG. 1. Lipoxygenase activity in two different genotypes of pearl millet during seed germination. *, significant at 5 % level. shoot lengths of all the normal seedlings were measured. Since the seed germination was above 70% in the first count, the second count was not taken. The vigour index (VI) was calculated using the formula

During seedling development Healthy seeds of 841 B, P-310-17 (downy mildew resistant), 23 B, and HB3 (downy mildew susceptible) were germinated aseptically on moist blotters in Petri dishes in an incubator under darkness at 25f 1°C for 7 days. The seedlings (0.5 g) were harvested at 1-day regular intervals for 7 days and subjected to enzyme study.

Comparison of root and shoot lengths between resistant and susceptible genotypes was based on the Student's t-test at P = 0.005 (Zar 1984).

Preparation of cell-free extract Seeds, either fresh or stored (0.5 g dry weight), were soaked in 10 mL distilled water and incubated in darkness for 12 h in a seed germinator at 2 5 k 1°C. The soaked seeds were blotted with filter paper to remove excess water. The seeds were macerated to a fine paste with 0.5 g acid-washed sand in a mortar at 4°C with 5 mL of cold 0.1 M sodium phosphate buffer (pH 6.5). The homogenate was centrifuged at 9000 x g for 20 min at 4°C. The supernatant was used as enzyme source. Similar enzyme extracts were prepared from seedlings of 841 B, P-310-17, 23 B, and HB3.

Results In the field-screening trial, carried out in the downy mildew disease plot, the disease incidence ranged between 1.4 and 97 % . The genotypes P-3 10- 17, 841 B, and 70065 1 that had 0, 2.0, and 10% disease incidence, respectively, were categorized as resistant. The genotypes 843 B, HB3, and 23 B that had 25, 95 and 97 % disease incidence, respectively, were considered to be susceptible.

Measurement of LOX activity LOX activity was measured following the procedure of Borthakur et al. (1987). Activity was determined spectrophotometrically by monitoring the appearance of conjugated diene hydroperoxide, absorbing at 234 nm. The reaction mixture contained 2.7 mL of 0.2 M sodium phosphate buffer (pH 6.5), 0.3 mL of 10 mM linoleic acid (Hi-Media, Bombay, India) in Tween 20 (Hi-Media, Bombay, India), and 15 pL of the enzyme extract. The protein content of the extracts was determined by the procedure of ~0wr-yet al. (1951). The enzyme activity was expressed as a change in the absorbance (A) per milligram protein per minute. Each of the above experiments was repeated twice and statistical analyses of the data were carried out. LOX activity in fresh and stored seeds was compared by using the Student's 1-test. Since the correlation coefficient was significant, simple linear regression analysis was carried out by the method of least squares for LOX activity during germination of both resistant and susceptible seeds (Zar 1984). Vigour analysis The vigour of germinating, fresh seeds of P-310-17 (resistant) and HB3 (susceptible) was evaluated by adopting the standard procedure of International Seed Testing Assn. (1985). One hundred seeds were subjected to the germination test by the top of paper method. Twentyfive seeds were plated on top of a moist blotter in a 9-cm diameter Petri dish and incubated under a 12 h light : 12 h dark cycle at 25 1 1"C. Four replicates were maintained. In the first count, made on the 3rd day, percent seed germination of each sample and root and

[I] VI

=

(mean shoot length

+ mean root length) x % germination

Determination of LOX activity LOX activity in fresh and stored seeds The LOX activity was higher in the seeds of downy mildew resistant genotypes and lower in susceptible genotypes in both fresh and stored seeds (Table 1). The seeds of P-310-17 (resistant) had a maximum enzyme activity of 0.1861/(mg protein . min) while a minimum activity of 0.0764/(mg protein . min) was observed in 23 B (susceptible). The LOX activity was low in stored seeds compared with that of fresh seeds of all the six genotypes (Table 1). This decrease in the LOX activity was statistically significant. In downy mildew resistant genotypes, reduction in the LOX activity was greater in stored seeds ( > 5 0 % ) compared with that of susceptible seeds ( < 50 %). LOX activity during seedling development The LOX activity decreased in germinating seedlings of resistant genotypes but increased in susceptible genotypes. In P-310-17 (resistant), the LOX activity started declining from the 1st day and continued to do so until the end of the test period of 7 days. Up to the 3rd day of germination the enzyme activity was greater in P-310-17 than in HB3 (susceptible) seedlings, but from the 4th day onwards, the level of LOX started declining in P-310-17 seedling compared with that of HB3. On the 7th day of germination the activity was as low

2030

CAN. J. BOT. VOL. 70, 1992

TABLE 2. Vigour analysis of downy mildew resistant (R) and susceptible (S) genotypes of pearl millet Genotypes

Root length (cm)

Shoot length (cm)

Vigour index

P-310-17 (R) HB3 (S) t-value*

1.71+0.885 0.61 +0.327 22.23

0.44k0.213 0.39+0.181 26.53

189.2 76

Can. J. Bot. Downloaded from www.nrcresearchpress.com by Peking University on 05/28/13 For personal use only.

NOTE: Data are means f SD from four replicates. each with 25 seedlings. Vigour analysis made on the 3rd day of germination. *Significant at 5% level.

as 0.0786/(mg protein . min) in P-310-17. Regression analysis showed that the reduction in the LOX activity (Y) with age (X) was significant (Y = 15.79 - 1.674X) (Fig. 1). In the case of HB3, a gradual, significant increase in the LOX activity was recorded during germination (Y = 11.24 0.47X). A similar trend for enzyme activity was recorded in another set of resistant (841 B) and susceptible (23 B) genotypes.

+

Vigour analysis Seed germination on the 3rd day of incubation was 88% in P-3 10-17 (resistant) while that of HB3 (susceptible) was 7 6 % . The mean root and shoot lengths were also significantly higher in P-310-17. This was further reflected by the vigour index, which was greater in the resistant genotype than in the susceptible genotype (Table 2).

Discussion It is clear from the present study that the LOX activity is higher in fresh seeds of different genotypes of pearl millet than in seeds stored for 6 months. It is also evident that seeds of downy mildew resistant genotypes have a higher LOX activity compared with their susceptible counterparts. Wang et al. (1990) observed a decrease in LOX activity in aged soybean seeds. The current work on LOX in pearl millet also showed a decline in the enzyme activity in seeds stored for 6 months. However, it is not known whether the decrease in the enzyme activity is the cause or the consequence of seed aging. The reason for the greater decrease in LOX activity in resistant genotypes than in susceptible ones during storage is unclear at this time. Lynch et al. (1985) suggested that LOX and its hydroperoxide products might directly participate in senescence, whereas Mack et al. (1987) argued against such a role for LOX. In pearl millet, as seen in the present study, the pattern of LOX activity differs in developing seedlings of downy mildew resistant and susceptible genotypes. Increases in the enzyme activity in developing seedlings could be related to the disease susceptibility of the genotype and decreases with resistance. Thus, enzyme activity during seed germination either increased, as in 23 B and HB3 (downy mildew susceptible), or decreased, as in P-310-17 and 841 B (downy mildew resistant); the pattern of initial increase in LOX activity followed by a decrease (or vice versa) that has been reported in other crops was not observed in pearl millet. An increase in LOX activity in the early stages of germination, followed by a subsequent decrease, was observed in wheat (Guss et al. 1968), mustard (Oelze-Karow et al. 1970), rice (Ohta et al. 1986), cucumber (Matsui et al. 1988), and lupin (Beneytout et al. 1988). In pea seeds, Anstis and Friend (1974) showed that an early decrease in LOX activity in pea seeds is followed by an increase at later stages of germination. There appears to be a correlation

between the amount of LOX activity found in a given plant tissue and its rate of elongation, the highest levels of LOX being found in rapidly growing tissues. What role LOX might play in such tissues is not presently understood (Siedow 199 1). It is interesting to note that the downy mildew resistant genotype (P-310-17) that had high LOX activity in the seeds had high seedling vigour, and the susceptible genotype (HB3) that showed a comparatively low LOX activity had low seedling vigour. According to Vick and Zimmerman (1976), the product of LOX might have a role during seed germination and in high vigour. The enzyme may be indirectly involved in cell differentiation or in cell division and cell enlargement. which is an indication of vigour. The initial higher LOX activity in the seeds of resistant genotypes might result in the vigorous growth of the seedlings. It is well known that in seeds, stored lipid is mobilized during germination (Slack et al. 1977; Bewley and Black 1985). A higher LOX activity in the seeds of resistant genotypes, in the eirly days of germination, and a drastic reduction later could be due to greater depletion of stored lipid in the resistant seeds than in susceptible seeds during germination. Thus, the behaviour of LOX in germinating seeds might be used as a biochemical parameter to screen different genotypes of pearl millet for their resistance or susceptibility t o downy mildew disease.

Acknowledgements We thank Dr. G. Harinarayana, Project Co-ordinator, All India Co-ordinated Pearl Millet Improvement Programme, Pune, India and Dr. S. D. Singh, International Crops Research Institute for the Semi-Arid Tropics, Hyderabad, India for providing the seed samples. The senior author is grateful to the Council of Scientific and Industrial Research, New Delhi for the financial assistance. This work was supported by the Indian Council of Agricultural Research, New Delhi. Anstis, P. J. P., and Friend, J . 1974. The isozyme distribution of etiolated pea seedling lipoxygenase. Planta, 115: 329-335. Beneytout, J. L., Najid, A,, and Tixier, M. 1988. Changes in lipoxygenase activity during seedling development of Lupinus albus. Plant Sci. 58: 35 -41. Bewley, J. D., and Black, M. 1985. Seeds: physiology of development and germination. Plenum Press, New York. Borthakur, A. B., Bhat, B. G., and Ramadoss, C. S. 1987. The positional specificities of the oxygenation of linoleic acid catalysed by two forms of lipoxygenase isolated from Bengal gram (Cicer arietinum). J. Biosci. 11: 257-263. Galliard, T., and Chan, H. W. S. 1980. Lipoxygenases: hz The biochemistry of plants. Vol. 4. Edited by P. K. Stumpf. Academic Press, New York. pp. 131- 161. Gardner, H. W., and Weisleder, D. 1970. Lipoxygenase from Zea mays: 9-D-hydroperoxy-trans-10, cis-12-octadecadienoic acid from linoleic acid. Lipids, 5: 678 -683. Guss, P. L., Macko, V., Richardson, T., and Stahmann, M . A. 1968. Lipoxidase in early growth of wheat. Plant Cell Physiol. 9: 415-422. Hsieh, C. C., and McDonale, C. E. 1984. Isolation of lipoxygenase isoenzymes from flour of durum wheat endosperm. Cereal Chem. 61: 392-398. International Seed Testing Assn. 1985. Seed Sci. Technol. 13: 299-573. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.

Can. J. Bot. Downloaded from www.nrcresearchpress.com by Peking University on 05/28/13 For personal use only.

NAGARATHNA ET AL.

Lynch, D. V., Sridhara, S., and Thompson, J. E. 1985. Lipoxygenasegenerated hydroperoxides account for the non-physiological features of ethylene formation from 1-aminocyclopropane-1-carboxylic acid by microsomal membranes of carnations. Planta, 164: 121 - 125. Mack, A. J., Peterman, T. K., and Siedow, J. N. 1987. Lipoxygenase isozymes in higher plants: biochemical properties and physiological role. Isozymes Curr. Top. Biol. Med. Res. 13: 127- 154. Matsui, K., Kajiwara, T., Hayashi, K., and Hatanaka, A. 1988. Tissue specific heterogeneity of lipoxygenase in cucumber seedlings. Agric. Biol. Chem. 52: 3219-3221. Oelze-Karow, H., Schopfer, P., and Mohr, H. 1970. Phytochromemediated repression of enzyme synthesis (lipoxygenase a threshold phenomenon). Proc. Natl. Acad. Sci. U.S.A. 65: 51-57. Ohta, H., Ida, S., Mikarni, B., and Morita, Y. 1986. Changes in lipoxygenase components of rice seedlings during germination. Plant Cell Physiol. 27: 91 1 -918.

203 1

Siedow, J. N. 199 1. Plant lipoxygenase: structure and function. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42: 145- 188. Slack, P. T., Black, M., and Chapman, J. M. 1977. Testa and lipid mobilization in cucumber. J. Exp. Bot. 28: 569-577. Vick, B. A , , and Zimmerman, D. C. 1976. Lipoxygenase and hydroperoxide lyase in germinating watermelon seedlings. Plant Physiol. 57: 780-788. Wang, J., Fujumoto, K., Miyazawa, T . , Endo, Y., and Kitamura, K. 1990. Sensitivities of lipoxygenase-lacking soybean seeds to accelerated aging and their chemiluminescence levels. Phytochemistry, 29: 3739 -3742. Williams, R. J., Singh, S. D., and Pawar, M. N. 1981. An improved field screening technique for downy mildew resistance in pearl millet. Plant Dis. 65: 239-241. Zar, J. H. 1984. Biostatistical analysis. Prentice-Hall International Ltd., London.