| Summary: | Orthodox and recalcitrant seeds exhibit differential tolerance to water loss. Recalcitrant seeds are not able to tolerate desiccation and die when dried, while the orthodox seeds can be stored dry without losing viability for years. Spartina is a good model to study recalcitrance, because unlike most other recalcitrance studies, which contain only a recalcitrant species, this system has both recalcitrant S. alterniflora and orthodox species, S. pectinata and S. spartinae, as close-related physiological comparators. Lack of protective proteins, e.g. late embryogenesis abundant proteins (LEAs), has been proposed to be the cause of recalcitrant seed death. A common feature of these protective proteins is their heat stability. In order to identify any heat-stable proteins that may be associated with a lack of desiccation tolerance in S. alterniflora, it is necessary to optimize the protocol of heat-stable protein extraction first. Heating the protein extracts at 95C for 40 minutes and centrifuging the heated protein extracts at 20,000g and 4C for 40 minutes yield a constant protein concentration of heat-stable fractions both in the S. alterniflora and S. pectinata. Comparisons of one-dimensional SDS-PAGE gels or total protein concentration provide little information about the minimum amount of protease inhibitor needed to stop the protease activity in Spartina seed protein extracts. Results of the Protease Determine Quick testTM protease assay indicated that 50 ul of protease inhibitor were sufficient to totally quench the protease activity in protein extracts in both S. pectinata and S. alterniflora. To investigate an association between heat-stable fraction percentage and desiccation tolerance, heat-stable fractions of S. alterniflora and S. pectinata were compared. There was no association between heat-stable fraction percentage and desiccation tolerance in Spartina. Comparative 1-DE profiles between dry S. alterniflora and dry S. pectinata did not reveal any differences. Therefore, two-dimensional gel electrophoresis, which has a much higher capability to resolve proteins, was used to investigate the differences in protein patterns between recalcitrant S. alterniflora and orthodox S. pectinata.
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