Our objective was to identify amylases that may participate in starch degradation in alfalfa (Medicago sativa L.) taproots during winter hardening and subsequent spring regrowth. Taproots from field-grown plants were sampled at intervals throughout fall, winter, and early spring. In experiment 1, taproots were separated into bark and wood tissues. Concentrations of soluble sugars, starch, and buffer-soluble proteins and activities of endo-and exoamylase were determined. Starch concentrations declined in late fall, whereas concentrations of sucrose increased. Total amylolytic activity (primarily exoamylase) was not consistently associated with starch degradation but followed trends in soluble protein concentration of taproots. This was especially evident in spring when both declined as starch degradation increased and shoot growth resumed. Activity of endoamylase increased during periods of starch degradation, especially in bark tissues. In experiment 2, a low starch line had higher specific activity of taproot amylases. This line depleted its taproot starch by late winter, after which taproot sugar concentrations declined. As in experiment 1, total amylolytic activity declined in spring in both lines, whereas that of endoamylase increased in both lines even though little starch remained in taproots of the low starch line. Several isoforms of both amylases were distinguished using native polyacrylamide electrophoresis, with isoforms being similar in bark and wood tissues. The slowest migrating isoform of endoamylase was most prominent at each sampling. Activity of all endoamylase isoforms increased during winter adaptation and in spring when shoot growth resumed. Endoamylase activity consistently increased at times of starch utilization in alfalfa taproots (hardening, spring regrowth, after defoliation), indicating that it may serve an important role in starch degradation.concentrations of soluble sugars, and freezing tolerance, increase (6, 16). The enzymes participating in starch degradation in alfalfa taproots during winter hardening have not been identified.Alfalfa taproots contain high amylolytic activity. Duke and Doehlert (10) reported a large range in total amylolytic activity in taproots of hardy and nonhardy cultivars in early fall, but these activities were not associated with sucrose accumulation. Tysdal (28) found that, although no association between total diastatic (amylolytic) activity and winter hardiness was apparent, hardy cultivars produced a heat-stable (70°C) diastase during hardening. This suggested that forms of amylase present in fall and winter may differ from those found in taproots in summer and spring. Multiple isoforms of amylase have been reported in alfalfa taproots (18,19), with specific isoforms being more heat stable in hardened cultivars. Exoamylase is the predominant amylase found in alfalfa taproots, and it exists in multiple isoforms that may differ in mol. wt. (8). Procedures are now available that enable us to determine accurately endoamylase activity in tissues c...
The occurrence of heat-shock proteins (HSPs) in response to high temperature stress is a universal phenomenon in higher plants and has been well documented. However, in agriculturally important species, less is known about the expression of HSPs under natural environments. A review of the heat-shock response in wheat (Triticum aestivum L.) is presented and recent results on the expression of wheat HSPs under diurnal stress and field conditions are reported. In the field experiment, flag leaf blade temperatures were obtained and leaf blades collected for northern blot analysis using HSP 16.9 cDNA as a probe. Temperatures of leaf blades ranged from 32 to 35°C under the tested field conditions at New Deal near Lubbock, Texas. Messenger RNAs encoding a major class of low molecular weight HSPs, HSP 16.9, were detected in all wheat genotypes examined. The results suggested that HSPs are synthesised in response to heat stress under agricultural production, and furthermore, that HSPs are produced in wheats differing in geographic background. In the controlled growth chamber experiment, HSP expression in two wheat cultivars, Mustang (heat tolerant) andSturdy (heat susceptible) were analysed to determine if wheat genotypes differing in heat tolerance differ in in vitro HSP synthesis (translatable HSP mRNAs) under a chronic, diurnal heat-stress regime. Leaf tissues were collected from seedlings over a time-course and poly (A)+RNAs were isolated for in vitro translation and 2-D gel electrophoresis. The protein profiles shown in the 2-D gel analysis revealed that there were not only quantitative differences of individual HSPs between these two wheat lines, but also some unique HSPs which were only found in the heat tolerant line. This data provides evidence of a correlation between HSP synthesis and heat tolerance in wheat under a simulated field environment and suggests that further genetic analysis of HSPs in a segregating population is worthy of investigation. In conclusion, the results of this study provide an impetus for the investigation of the roles of HSP genes in heat tolerance in wheat.
The adverse effects of heat stress are a major limitation to realizing the genetic potential for productivity of wheat (Triticum aestivum L.). This study was undertaken to characterize the temperature‐dependent induction response of heat‐shock protein (HSP) synthesis in wheat seedlings, and to determine if HSPs could be detected in flag leaves of flowering wheat plants at nonlethal temperatures similar to those encountered during agricultural production. The HSP profile was characterized by isoelectric focusing and polyacrylamide gel electrophoresis of 35S‐methionine labeled products of an in vitro translation of poly (A)+ RNA obtained from seedling leaf blades exposed to temperatures of 22, 25, 28, 31, 34, and 37 °C for 2 h and in flag leaf blades exposed to 32 °C for 2 h. Synthesis of HSPs was detected in seedling leaf tissue during heat shock between 28 and 31 °C and in flag leaves at 32 °C. This observation in seedlings was confirmed by northern blot analysis utilizing HSP 16.9 and HSP 70 32P‐labeled probes. These results indicate that both low and high molecular weight HSPs are synthesized in wheat as a response to heat stress when leaf temperatures increase ≈ 10 °C above the optimal growth temperature of 18 to 23 °C. Detection of low and high molecular weight HSPs synthesized in seedlings and flag leaves of flowering plants suggests that HSPs are synthesized before leaf temperatures reach levels that are considered injurious to growth and development.
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