[01] Seshadri Shivashankar, Division of Plant Physiology and Biochemistry, ICAR-Indian Institute of Horticultural Research, Bengaluru, India. [02] Manoharan Sumathi, Division of Plant Physiology and Biochemistry, ICAR-Indian Institute of Horticultural Research, Bengaluru, India.

Sapota fruit, Manilkara achras (Mill) Fosberg cv. Cricket ball shows high incidence of the physiological disorder known as Corky tissue under rainfed cultivation due to poor seed viability. Interestingly, however, fruits naturally infested by sapota seed borer (SSB), Trymalitis margaritas Meyrick (Tortricidae: Lepidoptera), remain free from the disorder. Hence, this study was carried out to elucidate the mechanism by which SSB infestation prevents corky tissue development in sapota. Comparative biochemical analyses of the uninfested seeds of SSB affected fruits and the seed of H and CT fruits showed that the activities of dehydrogenases, PEP carboxylase, concentrations of key metabolites such as, ATP, pyruvate, NAD and NADH and the contents of the growth hormones, ABA, IAA and GA were significantly higher in SSB seed showing that it was metabolically more active compared to seed from H and CT. The significantly higher rate and percentage of germination of uninfested seeds of SSB affected fruits confirmed higher seed viability and the markedly higher ratio of seed dry weight to fruit dry weight indicated higher sink strength and sink capacity compared to H and CT affected fruits. The study thus, confirmed that seeds from SSB affected fruit were more vigorous and viable compared to seed from CT infested and healthy fruits. The relatively lower levels of ROS and MDA coupled with lower electrical conductivity and higher pH reflected a higher level of tissue integrity of the SSB seed. Taken together, the study unequivocally established that infestation of a single seed by SSB led to enhanced seed vigour and viability in the neighbouring seeds and thus ensured that the infested sapota fruits remained free from corky tissue disorder. This novel finding could pave the way for prevention of incidence of corky tissue disorder in sapota by enhancement of seed viability during fruit growth.

Corky Tissue, Dehydrogenases, Free Radicals, Sapota cv. Cricket Ball, Sapota Seed Borer, Seed Viability, TCA Cycle Intermediates

[01] Shivashankar S., Jaya Joshi., Sumathi M. 2013. The role of seed viability in development of corky tissue in sapota fruit. J. Hort. Sci. Biotech. 88: 671-677. [02] Shukla A. 2009. Seasonal incidence and biology of sapota seed borer, Trymalitis margaritas Meyrick. Pakistan. J. Entomol. 2: 31. [03] Patel Z. P. 2001. Record of seed borer in sapota (Manilkara achras (Mill) Fosberg). Insect Environ. 6: 149. [04] Nakai Z., Kondo T., Akimoto S. 2011. Parasitoid attack of the seed-feeding beetle Bruchus loti enhances the germination success of Lathyrus japonicas seeds. Arth. Plant Int. 5: 227-234. [05] Karban R., Lowenberg G. 1992. Feeding by seed bugs and weevils enhances germination of wild Gossypium species. Oecologia 92: 196-200. [06] Harms K. E., Dalling J. W. 2000. A bruchid beetle and a viable seedling from a single diaspore of Attalea butyracea. J. Trop. Ecol. 16: 319-325. [07] International Rules for Seed Testing. 1985. Seed Sci. Tech 1. 3: 299-335. [08] International Rules for Seed Testing. 1993. Seed Sci. Tech. 21: 1-298. [09] Bernfeld P. 1955. Amylases. In: Methods in Enzymology. (Colowick, S. P. and Kaplan, N. O., Eds.). Academic Press, New York, NY, USA. 149–158. [10] Lowry O. H., Rosenbrough N. J., Farr A. L., Randall R. J. 1952. Protein measurement with Folin- phenol reagent. J. Biol. Chem. 193: 265–267. [11] Jayaraman J. 1981. Laboratory manual in biochemistry. New Delhi, Wiley Eastern Ltd. [12] Lee Y. P., Takahashi T. 1966. An improved colorimetric determination of amino acid with the use of ninhydrin. Anal. Biochem. 14: 71–77. [13] Burrell M., Mooney P., Blundy M., Carter D., Wilson F., Green J. 1994. Genetic manipulation of 6-phosphofructokinase in potato tubers. Planta 194: 95–101. [14] Selvaraj Y., Pal D. K., Singh R., Roy T. K. 1995. Biochemistry of uneven ripening in Gulabi grape. J. Food Biochem. 18: 325–340. [15] Sadasivam S., Gowri G. 1981. Activities of phosphoenolpyruvate carboxylase, NAD+-malate dehydrogenase and aspartate aminotransferase and chlorophyll a/b ratio in leaves of sorghum cultivars during plant growth. Photosyn. 15: 453-456. [16] Kelen M., Demiralay E. C., Ozakan S. G. 2004. Separation of abscisic acid, indole-3-acetic acid, gibberellic acid in 99 R (Vitisberlandieri × Vitisrupestris) and rose oil (Rosa damascene Mill.) by reversed-phase liquid chromatography. Turk. J. Chem. 28: 603– 610. [17] Chiwocha S. D. S., Abrams S. R., Ambrose S. J., Cutler A. J., Loewen, M., Ross A. R., Kermode A. R. 2003. A method for profiling classes of plant hormones and their metabolites using liquid chromatography-electrospray ionization tandem mass spectrometry: an analysis of hormone regulation of thermo-dormancy of lettuce (Lactuca sativa L.) seeds. The Plant J. 35: 405–417. [18] Segarra G., Jauregui O., Casanova E., Trillas I. 2006. Simultaneous quantitative LC–ESI- MS/MS analyses of salicylic acid and jasmonic acid in crude extracts of Cucumis sativus under biotic stress. Phytochem. 67: 395–401. [19] Serrano M., Romojaro F., Casas J. L., Acosta M. 1991. Ethylene and polyamine metabolism in climacteric and nonclimacteric carnation flowers. HortSci. 26: 894–896. [20] Galdón B. R., Rodríguez C. T., Rodríguez E. M. R., Romero C. D. 2009. “Fructans and Major Compounds in Onion Cultivars (Allium cepa).” J. Food Com. Anal. 22: 25-32. [21] Folch J., Lees M., Stanley G. S. H. 1957. A simple method for the isolation and purification of lipids in animal tissues. J. Biol. Chem. 226: 497–509. [22] Liu K. 1994. Preparation of fatty acid methyl esters for gas chromatographic analysis of lipids in biological materials. J. American Oil. Chem. Soci. 71: 1179–1187. [23] Doke N. 1983. Involvement of superoxide anion generation in the hypersensitive response of potato tuber tissues to infection with an incompatible race of Phytophthorainfestans and to the hyphal wall components. Physiol. Plant Pathol. 23: 345–357. [24] Von Tiedemann A. 1997. Evidence for a primary role of active oxygen species in induction of host cell death during infection of bean leaves with Botrytis cinerea. Physiol. Mol. Plant Pathol. 50: 151–166. [25] Schopfer P., Plachy C., Frahry G. 2001. Release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide and hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light, gibberellin, and abscisic acid. Plant Physiol. 125: 1591–1602. [26] Draper H. H., Hadley M. 1990. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 186: 421–431. [27] Tezuka T., Yamaguchi F., Ando Y. 1994. Physiological activation in radish plants by UV-A radiation. J. Photochem. Photobiol. B: Biology 24: 33–40. [28] Stitt M., Lilley R., Gerhardt R., Heldt H. 1989. Metabolite levels in specific cells and subcellular compartments of plant leaves. Methods in Enzymol. 174: 518–553. [29] Chen L., Lin Q., Nose A. 2002. A comparative study on diurnal changes in metabolite levels in the leaves of three crassulacean acid metabolism (CAM) species, Ananas comosus, Kalanchoe diagremontiana and K. pinnata. J. Exp. Bot. 53: 341–350. [30] Panse V. G., Sukhatme P. V. 1978. Statistical Methods for Agricultural Workers, ICAR, New Delhi, India, pp. 108. [31] Seifert M., Wermelinger B., Schneider D. 2000. The effect of spruce cone insects on seed production in Switzerland. J. App. Entomol. 124: 269-278. [32] Marcelis L. F. M., Hofman-Eijer L. R. 1997. Effects of seed number on competition and dominance among fruits in Capsicum annuum L. Ann. Bot. 79: 687-693. [33] Richard J. P., Fabiana C., Carmen C. 2014. Mechanisms regulating auxin action during fruit development. Physiol. Plant. 151: 62–72. [34] Sjut V., Bangerth F. 1984. Induced parthenocarpy–a way of manipulating levels of endogenous hormones in tomato fruits (Lycopersicon esculentum Mill.) 2. Diffusible hormones. Plant Growth Reg. 2: 49-56. [35] Hoekstra F. A., Haigh A. M., Tetteroo F. A. A., Van Roekel T. 1994. Changes in soluble sugars in relation to desiccation tolerance in cauliflower seeds. Seed Sci. Res. 4: 143-147. [36] Ooms J. J. J., Léon-Kloosterziel K. M., Bartels D., Koornneef M., Karssen C. M. 1993. Acquisition of desiccation tolerance and longevity in seeds of Arabidopsis thaliana: a comparative study using abscisic acid-insensitive abi₃ mutants. Plant Physiol. 102: 1185–1191. [37] Lu W., John W. P., Yong-Ling R. 2018. Live Long and Prosper: Roles of Sugar and Sugar Polymers in Seed Vigor. Molecular Plant 11: 1–3. [38] Alhadi Fatima A., AL-Asbahi A. A. S., Alhammadi A. S. A., Abdullah Q. A. A. 2012. The effects of free amino acids profiles on seeds germination/dormancy and seedlings development of two genetically different cultivars of Yemeni Pomegranates. J. Stress Physiol. Biochem. 8: 114-137. [39] Cantisan S., Force E. M., Ortega R. A., Garces R. 1999. Lipid characterization in vegetable tissues of high-saturated fatty acid sunflower mutants. J. Agri. Food Chem. 47: 78-82. [40] Ghodratollah S. 1998. The effect of seed colour and linolenic acid concentration on germination, seed vigour, seed quality and agronomic characteristics of flax. Doctor of Philosophy in the Department of Crop Science and Plant Ecology University of Saskatchewan Saskatoon, Canada. [41] Munshi S. K., Sandhu S., Sharma S. 2007. Lipid composition in fast and slow germination sunflower (Helianthus annuus L.) seeds. Gen. Appl. Plant Physiol. 33: 235-246. [42] Marambe B., Nagaoka T., Ando T. 1993. Identification and biological activity of germination- inhibiting long-chain fatty acids in animal-waste composts. Plant Cell Physiol. 34: 605-612. [43] Huang Y., Lin C., He F., Li Z., Guan Y., Hu Q., Hu J. 2017. Exogenous spermidine improves seed germination of sweet corn via involvement in phytohormone interactions, H₂O₂ and relevant gene expression. BMC Plant Biol. 17: DOI 10.1186/s12870-016-0951-9. [44] Mukhopadhyay A., Choudhuri M. M., Sen K., Ghosh B. 1983. Changes in polyamines and related enzymes with loss of viability in rice seeds. Phytochem. 22: 1547-1551. [45] Handa A. K., Mattoo A. K. 2010. Differential and functional interactions emphasize the multiple roles of polyamines in plants. Plant Physiol. Biochem. 48: 540–546. [46] Rodríguez-Gacio M. C., Matilla-Vázquez M. A., Matilla A. J. 2009. Seed dormancy and ABA signaling: the breakthrough goes on. Plant Sig. Behav. 4: 1035–1048. [47] Liu Y., Ye N., Liu R., Chen M., Zhang J. 2010. H₂O₂ mediates the regulation of ABA catabolism and GA biosynthesis in Arabidopsis seed dormancy and germination. J. Exp. Bot. 61: 2979–2990. [48] Finch-Savage W. E., Leubner-Metzger G. 2006. Seed dormancy and the control of germination. New Phytol. 171: 501–523. [49] Shoeb F., Yadav J. S., Bajaj S., Rajam M. V. 2001. Polyamines as biomarkers for plant regeneration capacity: improvement of regeneration by modulation of polyamine metabolism indifferent genotypes of Indica rice. Plant Sci. 160: 1229-1235. [50] Zeng X. Y., Chen R. Z., Fu J. R. 1998. The effects of water content during storage on physiological activity of cucumber seeds. Seed Sci. Res. 8: 65-68. [51] Giri A. P., Winsche H., Mitra S., Zavalal J. A., Muck A., Svatos A., Baldwin I. T. 2006. Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuate, VII. Changes in the plant’s proteome. Plant Physiol. 142: 1621-1641. [52] Plaxton W. C. 1996. The organization and regulation of plant glycolysis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 185–214. [53] Kletzien R. F., Harris P. K. W., Foellmi L. A. 1994. Glucose 6 phosphate dehydrogenase: a “housekeeping” enzyme subject to tissue-specific regulation by hormones, nutrients, and oxidart stress. FASEB J. 8: 174–181. [54] Yang Y., Fu Z., Su Y., Zhang X., Li G., Guo J., Que Y., Xu L. 2014. A cytosolic glucose-6-phosphate dehydrogenase gene, ScG6PDH, plays a positive role in response to various abiotic stresses in sugarcane Sci. Rep. 4: 7090: DOI: 10.1038/srep07090. [55] Schuller K. A., Plaxton W. C., Turpin D. H. 1990. Regulation of C3 phosphoenolpyruvate carboxylase from the green alga Selenastrum minutum. Properties associated with replenishment of TCA cycle intermediates during amino acid biosynthesis. Plant Physiol. 93: 1303-1311. [56] Latzko E., Kelly G. J. 1983. The many-faceted function of phosphoenolpyruvate carboxylase in C₃ plants. Physiol. Veg. 21: 805-815. [57] Briggs A. G., Bent A. F. 2011. Poly (ADP-ribosyl) ation in plants. Trends in Plant Sci. 16: 372-380. [58] Kibinza S., Vinel D., Coˆme D., Bailly C., Corbineau F. 2006. Sunflower seed deterioration as related to moisture content during ageing, energy metabolism and active oxygen species scavenging. Physiol. Planta. 128: 496–506. [59] Oenel A., Fekete A., Krischke M., Faul S. C., Gresser G., Havaux M., Mueller M. J., Berger S. 2017. Enzymatic and Non-Enzymatic Mechanisms Contribute to Lipid Oxidation During Seed Aging. Plant Cell Physiol. 58: 925-933.