Changes in Soluble Carbohydrates during Seed Storage1

The soluble sugars present in the maize (Zea mays L.) embryo may serve as important components of protection or may contnbute to the deteriorative changes occurring during seed storage. Examination of the changes in sugars during accelerated aging of maize seeds indicates that the decline in vigor is associated with a marked decline in monosaccharides and in raffinose. Sucrose content remains relatively stable. The depletion of raffinose may have special relevance to the decline in seed vigor. shown by a-galactosidase. These facts reinforce the expectation that changes in sugars may occur during seed storage. We have carried out experiments on the sugar contents of maize seeds as a function of accelerated aging, with the intent to examine the possible relation of carbohydrate changes to the deterioration of vigor, and then to the loss of germinability. MATERIALS AND METHODS The decline of seed quality during storage is expressed first as a decrease in the growth rate of the germinating axis (vigor) and subsequently as a loss of actual germinability. Changes in the soluble carbohydrate contents could contribute to both the declines of vigor and of germinability of the seeds. It is known that soluble carbohydrates generally decline with seed aging (15), and this decline might result in limited availability of respiratory substrates for germination (9). Another possibility is that depletions of disaccharides may lessen the protective effects of sugars on structural integrity of membranes (7) or may limit the ability of the seeds to maintain the vitrified state, a noncrystalline liquid state of high viscosity (3, 20). Finally, the presence of reducing sugars may lead to deterioration of protein components through Amadori and Maillard reactions. These are nonenzymatic carbonyl-amine reactions that take place preferentially in dry systems (11). They may contribute to seed deterioration (19). In quiescent seeds, the main soluble embryonic carbohydrate reserves are sucrose, usually associated with lesser amounts of the oligosaccharides, raffinose, stachyose, and/or verbascose (1). Sucrose is exceptionally effective in protecting membrane integrity in dry systems (6), as well as being one of the best vitrifying sugars (12). Raffinose is known to enhance the protective effects of sucrose by limiting crystalization (5). Interconversion of sugars may occur in dry seeds. It has been found that a-galactosidase activity was detectable in dry seeds of cotton (16). Invertase activity is also present in dry seeds, but its activity is at least 5 to 10 times less than that 'Research supported in part by the International Board for Plant Genetic Resources and in part by the Institute for International Education. Plant Material Maize seeds (Zea mays L.) NYCO 11 3XNYPA33 (C2) were obtained from the New York Seed Improvement Cooperative, Ithaca, NY. Before storage, the seeds were surface-sterilized with 3.5% sodium hypochlorite twice, for 3 min, and each time, they were rinsed with sterilized deionized water and blotted dry. All manipulations were done aseptically. The seeds were then placed in storage at 30°C and 75% RH, using a saturated NaCl solution to buffer the humidity. The term water activity is used to refer to a material that has been equilibrated in an atmosphere at a given proportion of saturation. Thus, these seeds were at a water activity of 0.75 during the storage treatment. Germination and Vigor Seeds were germinated in wet paper rolls (Anchor Paper Company, St. Paul, MN) in the dark at 25C. Vigor was expressed as the length of the axis (coleoptile plus radicle) measured after 5 d of germination. Sugar Determinations After various periods of accelerated aging, maize embryos were excised, and three replicate samples of 75 to 100 mg were homogenized with a Polytron in 75% ethanol containing melezitose as internal standard. The homonogenate was allowed to steep for 30 min, centrifuged at 5000g for 5 min at room temperature, and the pellet was washed with 75% ethanol and centrifuged again. The supernatants were pooled and dried under reduced pressure. The dry residue was resuspended in water and filtered through a column consisting of PVPP and a mixed bed of ion-exchange resins (2). The eluent was divided into two aliquots before lyophilization. One dried aliquot was dissolved in a mixture of 75% acetonitrile and 25% water, filtered through a 45 gm pore size nylon filter, and analyzed by HPLC according to Koster and Leopold (13). 1207 www.plantphysiol.org on January 16, 2018 Published by Downloaded from Copyright © 1992 American Society of Plant Biologists. All rights reserved. BERNAL-LUGO AND LEOPOLD The other dry aliquot was dissolved in 0.5 mL of N-trimethylsilylimidazole (Supelco) to form the trimethylsilyl-derivatives of sugars, and analyzed by GC. Derivatized samples were injected into a Hewlett-Packard 5890A gas chromatograph equipped with an Ailtech SE-54 capillary column and flame-ionization detector. Chromatographic conditions were temperature programmed from 100 to 200°C at 8°C/min. Injection port and detector temperatures were set at 150 and 100°C, respectively. Response factors were determined utilizing a Hewlett-Packard model 3308S integrator. Standards of D-fructose, D-galactose, and D-glucose were derivatized as described above and analyzed to determine response factors, which were linear over the concentration ranges evaluated.