The reproductive stage of rice is the most sensitive to heat stress, which can lead to spikelet sterility. Thus, heat-tolerant and heat-susceptible genotypes were used to investigate their differences in terms of phenotypic responses and expression changes of Hsf genes at the pre-flowering stage under heat stress. Results clearly showed that panicles had the highest temperature compared with other plant parts under both natural and heated conditions. However, the temperatures of tolerant and susceptible genotypes were not significantly different. In terms of spikelet fertility, the tolerant lines M9962 and M7988 had high seed set because their anther dehiscence, pollen viability and pollen germination were only slightly affected. In contrast, the susceptible line Sinlek showed severe effects at all steps of fertilization, and the pollen viability of M7766 was slightly affected under heat stress but was more affected in terms of anther dehiscence and pollen germination. Both susceptible lines showed dramatically decreased seed set. In addition, the expression of six HsfA genes in the flag leaves and spikelets at the R2 stage of plants under heat stress showed different responses. Notably, expression of the HsfA2a gene was predominantly upregulated in the flag leaf and spikelets under heat stress in M9962. Therefore, it can be concluded that heat stress has severe effects on the stamen, and that different genotypes have different susceptibilities to heat stress.

To understand the regulated expression of stage-specific genes of schistosomes, it is necessary to identify regulatory DNA elements and DNA-binding proteins that control the level of gene transcription. Here we describe the preparation of Schistosoma mansoni extracts with active transcription factors detected by the electrophoretic mobility shift assay (EMSA). We analysed the hsp70 system of S. mansoni because the promoters of the hsp70 gene contain two heat shock elements (HSE) that differ from the consensus sequence (CnnGAAnnTTCnnG) at one (HSEI) or three (HSEII) positions, and it is known that transcriptional activation of hsp70 genes is mediated by interaction of HSE with the heat shock factor (HSF). Analyses of parasite extracts from different developmental stages demonstrate the presence of putative HSF that correlates with the pattern of hsp70 mRNA expression (cercariae-, schistosomula+, adult worms+). Cercarial extracts did not show binding of 32P-labelled HSEI or HSEII. Extracts of schistosomula and of adult worms kept at 37 or 42 °C showed binding of HSEI but not HSEII. The specificity of HSEI-HSF complex formation was ascertained by inhibition experiments. The EMSA experiments and structural features of the hsp70 promoter indicate that HSEI is the major DNA element responsible for transcriptional activation of the hsp70 gene, while the HSEII may be a redundant sequence with minor (if any) regulatory function. The extracts reported here can be used to study transcriptional control of other schistosome genes.

The DNA-binding domain of the yeast heat shock transcription factor (HSF) contains a strictly conserved proline that is at the center of a kink. To define the role of this conserved proline-centered kink, we replaced the proline with a number of other residues. These substitutions did not diminish the ability of the full-length protein to support growth of yeast or to activate transcription, suggesting that the proline at the center of the kink is not conserved for function. The stability of the isolated mutant DNA-binding domains was unaltered from the wild-type, so the proline is not conserved to maintain the stability of the protein. The crystal structures of two of the mutant DNA-binding domains revealed that the helices in the mutant proteins were still kinked after substitution of the proline, suggesting that the proline does not cause the α-helical kink. So why are prolines conserved in this and the majority of other kinked α-helices if not for structure, function, or stability? The mutant DNA-binding domains are less soluble than wild-type when overexpressed. In addition, the folding kinetics, as measured by stopped-flow fluorescence, is faster for the mutant proteins. These two results support the premise that the presence of the proline is critical for the folding pathway of HSF's DNA-binding domain. The finding may also be more general and explain why kinked helices maintain their prolines.