We have conducted a detailed investigation into the effects of temperature stress on gene expression in maize. Young (3-5 day old) plumules of Oh43, subjected to a brief shift in incubation temperature, exhibit a striking change in polypeptide synthetic patterns--from the production of a broad spectrum of different proteins to the new and/or enhanced synthesis of a small group of "heat-shock" proteins (HSPs). One-dimensional (1D) SDS-PAGE and fluorographic analysis of the newly synthesized polypeptides reveals the enhanced synthesis of polypeptides with molecular masses (Mr) of 108, 89, 84, 76, 73 and 18 kilodaltons (kd), and the apparent depressed synthesis of a major polypeptide with Mr = 93 kd (actively synthesized in the controls) following a one-hour shift from 27 C to 41 C. The six Mr classes of HSPs resolve into at least 18 spots by 2D IEF-SDS PAGE; some spots clearly show new or enhanced synthesis relative to the control while others show no apparent differences in intensity between control and heat-shocked samples.
Seedlings germinated and grown at 27 C require temperatures at or exceeding 35 C for detectable synthesis of these HSPs. The response is rapid; by 15 minutes following a shift to 41 C, enhanced synthesis of some of the HSPs is noted. When heat shocked seedlings are returned to 27 C, the polypeptide synthetic patterns recover to the control pattern after six to eight hours.
Although recovery of polypeptide synthetic patterns occurs following heat shock, we were interested in knowing if other cellular processes in the seedlings had been affected to prevent continued development of the plants. Five-day-old seedlings were heat shocked for one hour at 35 C, 41 C, 44 C, or 50 C, returned to 27 C for at least six hours and then transplanted to our nursery. While emergence was less than 100% in all cases due to stress from transplanting, there was a clear reduction in the number of emergent seedlings treated at 44 C and a complete absence of emergent seedlings from a treatment at 50 C. Emergence for each heat shock temperature as a percentage of the controls was as follows: 35 C (94%), 41 C (82%), 44 C (28%), 50 C (0%). Casual examination revealed no phenotypic differences among emergent seedlings. They were monitored to maturity. Our results show no differences in the heat-shock response between the various treatments. Some minor changes in band position in other regions of the gels suggest that we may be detecting some genetic variability in the sampling population.
We have also isolated total RNA from individual shoots of both control (27 C) and heat-shocked (41 C) seedlings, translated these RNAs in vitro in a rabbit reticulocyte lysate system, and analyzed the translated products by 1D and 2D PAGE. The results clearly demonstrate that 5 of the 6 Mr classes of HSPs noted in vivo are also translated in vitro and that differences in polypeptide number and intensity can be detected by 2D IEF-SDS PAGE between the in vivo and in vitro translated products. We are exploiting these apparent differences to examine the level of regulation of HSP induction.
The characterization of the heat shock response as described here provides a system for investigating: a) the induction of gene activity by environmental stress, b) the effect of tissue source or genotype on this response, and c) the influence of transcriptional and/or translational control on the regulation of gene expression in maize.
C. L. Baszczynski, D. B. Walden and B. G. Atkinson
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