| Summary: | 博士 === 國立臺灣大學 === 農藝學研究所 === 101 === The chalkiness of rice grain is a primary defect under high temperature stress. The building of chalky endosperm was a result of low energy status, high oxidative stress, high endosperm acidification, and high ethylene evolution within caryopsis developing under high temperature. To gain insight into the mechanisms caused the physiological and biochemical changes, the expression profiles of miRNAs under high temperature in the developing caryopsis of TK9 were studied by microarray analyses. TK9 (Oryza sativa L. Japonica cv. Taiken 9) is a heat sensitive rice variety having a higher ratio of chalky grain under high temperature. After verified by qRT-PCR (real-time quantitative reverse transcriptase-polymerase chain reaction), thirty six miRNAs were confirmed to respond to high temperature. Compared the chromosomal locations of potential miRNA targets with that of QTLs (quantitative trait loci) related to the buildup of chalky endosperm, thirty out of 81 potential target genes were confirmed to be separately down-regulated by 17 miRNAs. Under high temperature, at 6- to 9-day caryopsis, two miRNAs, miR812 and miR818, were induced, while their predicted target genes, citrate carrier and pex14, showed expected inverse patterns. The decreased expressions of citrate export (citrate carrier) from mitochondria and pex14 may result in improved TCA cycle and reduced function of peroxisome, respectively.
Moreover, the transcriptomes of a high temperature resistant mutant, SA1737, and its corresponding wild type, TNG67 (Oryza sativa L. Japonica cv. Tainung 67) was profiled by DNA microarray analyses. In total, 6,009 genes having significantly different expression levels were detected in compare with high temperature. 7 of them are the putative target genes of 5 miRNAs. These miRNAs in TK9 involving in responsiveness to high temperature indicated that miRNAs may have a regulatory role in the development of chalky grain upon high temperature. LON protease was a predicted target of siR806, suppression of LON protease in SA1737 suggested inhibition of proteolysis within mitochondria was crucial for the resistance to high temperature in developing caryopsis. LPP3 (lipid phosphate phosphatase 3), the enzyme catalyzed the dephosphorylation of PA (phosphatidic acid) was predicted to be a regulatory target of miR319. Suppression of LPP3 and accumulation of PA in TNG67 could activate ethylene signaling pathway under high temperature.
In addition, two genes (pyruvate kinase and alcohol dehydrogenase) involved in energy metabolism were examined in TK9. Both genes were co-regulated under high temperature, indicating that the energy production in developing caryopsis has been switched to the anaerobic pathway. Furthermore, to test whether ethylene accumulation in the developing caryopsis is related to the endosperm acidification and chalky occurrence under high temperature, an inhibitor of ethylene production (AgNO3) was applied to the developing caryopses of TK9 plants. As expected, the level of endosperm acidification and the ratio of chalky occurrence were reduced under high temperature. In this study, we identified miRNA-mediated regulations that responded to high temperature in developing rice grain. The data shed light on transcriptional gene regulations in chalky grain development under high temperature. The information obtained here could further be used by breeders to develop potential strategies for breeding heat-tolerant rice cultivars.
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