| The booting stage, flowering stage and filling stage of rice plant growth and development were most vulnerable to the change environmental temperatures, leading to the reduction in spikelet fertility together with inferior palatability property and nutritional quality of cooking rice cultivars. In present study, on the basis of heat resistance screening in rice cultivars in our lab, the rice genotypes differing in susceptible to temperature were used to investigate effects of high temperature (HT) on rice floral injury, seed production characteristics and grain quality in relation to carton and nitrogen mechanism. Furthermore, protein disulfide isomerase (PDI) RNAi and over-expression vectors were constructed to reveal the relationship between PDI expression and pollen viability affected by HT. The main results as followed:1. Spikelet fertility and pollen viability significantly decreased exposure to HT at meiosis and microspore development stage, with the greater negative effect exposure to HT at meiosis stage than at microspore development stage. Sugar profiles showed significant increase in glucose and fructose levels and remarkable decrease in sucrose and starch levels in rice anthers responsive to HT at two stages. DNA microarray and qRT-PCR data indicated that the HT occurring at meiosis stage accelerated sucrose transport and cleavage processes in rice anthers, sucrose invertase genes (CIN1) were more sensitive to HT than sucrose synthase genes (SUS4, SUS5, SUS6) in sucrose cleavage in rice anthers. SUT3and CIN1were mainly responsible for sucrose signal metabolism of rice floral in response to HT due to their significant increase in the transcript expressions under HT. The impairment of MST8-mediated hexose transport probably contributes to hexose accumulation and starch deficiency in rice anthers under the elevated temperature conditions.2. Significant increase in apparent amylose content (AAC) and hot-water-soluble starch content (HWS) in mutant9311eha were genetically caused by a substitution from AGTTATA to AGGTATA at the leader intron5’splice site in Wx gene. This mutation resulted in different mRNA transcript level, mRNA splicing efficiency and protein levels of Wx between the two rice genotypes, which also lead to the genotype-dependent alteration in the temporal pattern of Wx transcription and GBSS activity in response to HT. However, changes in the activities of other starch synthesizing enzymes and their expressions of distinct isoform genes were insignificant with the Wx gene mutation. The temporal-specific expression of multiple isoform genes responsive to different temperature regiments indicated that the reduction of GBSS transcript expression under HT was generally accompanied by the decreased expressions of SSSIIa, SSSIIIa and SBEIIb. The temperature-dependent alteration of amylose content was not only attributed to the reduced expression of GBSS, but also associated with the complimentary effect of SSSIIa and SBEIIb. Meanwhile, HT remarkably increase proportion of very large size starch granules, gelatinization temperature (GT), and proportion of intermediate and long B chains amylopectin for two genotypes. The higher GT in response to HT was attributed to combination of enhancement in intermediate and long B chains amylopectin and proportion of large starch granules size.3. HT significantly decreased rice grain weight, but remarkably increased total protein content (TPC) in brown, milled grains and aleurone fraction at the filling stage irrespective of rice genotypes. The ratios of glutelin to prolamin in brown and milled grains were significantly increased at HT due to enhancing percentage of glutelin to TPC and reduced percentage of prolamin to TPC. SDS-PAGE analysis revealed that HT increased the deposited accumulation of13-kD prolamin in brown and milled grains at the early filling stage, but decreased the accumulation of13-kD prolamin at the late filling stage; whereas accumulation of pro-glutein, a-glutelin and β-glutelin subunit in brown and milled grains was increased during the whole filling period. The semi-quantitative PCR data revealed that HT had a remarkable influence on the temporal patterns of GluA and GluB subfamily genes, with highly expressed level and also significantly rising transcripts of GluAl, GluA2, GluA3, GluBl, GluB2, GluB3, and GluB5in response to HT at the early filling stage. In contrast, the expressions of Pro13, Prol4, Prol7encoding13-KD prolamin were significantly decreased during the whole filling period. Transcription factor genes RISBZ1and RPBF, as well as PDI and BiP exhibited similar temporal patterns with glutelin and prolamin subfamily genes responsive to HT. Meanwhile, the semi-quantitative PCR data also indicated greatly altered temporal expression profiles of various genes involved in grain nitrogen assimilation (GS1, GDH1, GOT), distribution of carbon and nitrogen (SnRKla), and starch biosynthesis (GBSS1) in heat-stressed plants. These data suggest that increased grain protein induced by HT was not only attributed to altered temporal expression profiles of genes related to storage protein synthesis, but also due to altered temporal expression profiles of genes involved in transcriptional regulation, protein folding and processing, grain nitrogen assimilation and starch biosynthesis.4. We constructed a binary-vector pTCK303-RiOsPDI containing intron hpRNA (ih-pRNA) and over-expression vector pTCK303-OsPDI, then transformed them into the callus of wild type Nipponbare mediated by EHA105. The T-DNA region for PDI RNA interference and over-expression in regenerating rice plants were integrated with rice genome via single copy in TO generation transgenic plants, and showed a3:1genetic mode in T1trans genic population, which could be conformed by PCR amplifying analysis. The qRT-PCR for PDI gene expression in different organs showed that there were much higher levels of PDI expression in stem, sheath, leaf, grain and pollen for PDI over-expression transgenic rice compared with those for wild type Nipponbare, with much lower level of PDI expression in different organs for PDI silence transgenic rice. Moreover, the variation of PDI expressions in PDI transgenic rices was greater in pollen than in other organs. The influence of HT on seed setting traits, panicle agronomic traits and grain quality were further examined with T2generation of transgenic plants, the results indicated that no significant differences were observed in grain total protein and amylose content between PDI transgenic plants and its wild type. However, PDI silence transgenic plants had a remarkable lower seed setting rate under HT, whereas there was no significant difference in seed setting rate between PDI over-expression transgenic rice and its wild type under HT, suggesting that PDI gene should be probably responsible for rice tolerance to high temperature stress. Notably, anther size and pollen viability were significantly decreased in PDI silence transgenic plants, and the more reduction in that were observed in HT than in NT for PDI silence transgenic plants; while no significant differences were observed between PDI over-expression transgenic rice and its wild type under HT, suggesting that PDI gene probably play an important role in development of anther and tolerance of pollen to high temperature stress. |
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