Comprehensive expression profiling of rice grain filling-related genes under high temperature using DNA microarray

Abstract

To elucidate the effect of high temperature on grain-filling metabolism, developing rice (Oryza sativa) "Nipponbare" caryopses were exposed to high temperature (33 degrees C/28 degrees C) or control temperature (25 degrees C/20 degrees C) during the milky stage. Comprehensive gene screening by a 22-K DNA microarray and differential hybridization, followed by expression analysis by semiquantitative reverse transcription-PCR, revealed that several starch synthesis-related genes, such as granule-bound starch synthase I (GBSSI) and branching enzymes, especially BEIIb, and a cytosolic pyruvate orthophosphate dikinase gene were down-regulated by high temperature, whereas those for starch-consuming alpha-amylases and heat shock proteins were up-regulated. Biochemical analyses of starch showed that the high temperature-ripened grains contained decreased levels of amylose and long chain-enriched amylopectin, which might be attributed to the repressed expression of GBSSI and BEIIb, respectively. SDS-PAGE and immunoblot analysis of storage proteins revealed decreased accumulation of 13-kD prolamin, which is consistent with the diminished expression of prolamin genes under elevated temperature. Ripening under high temperature resulted in the occurrence of grains with various degrees of chalky appearance and decreased weight. Among them, severely chalky grains contained amylopectin enriched particularly with long chains compared to slightly chalky grains, suggesting that such alterations of amylopectin structure might be involved in grain chalkiness. However, among high temperature-tolerant and sensitive cultivars, alterations of neither amylopectin chain-length distribution nor amylose content were correlated to the degree of grain chalkiness, but rather seemed to be correlated to grain weight decrease, implying different underlying mechanisms for the varietal difference in grain chalkiness. The possible metabolic pathways affected by high temperature and their relevance to grain chalkiness are discussed.

Figure 1.

Grain filling using plant growth incubators. A, Change in the fresh weight of rice caryopses developing under 33°C/28°C (black circles) or 25°C/20°C (white circles). Values are the mean of at least 50 grains. B, Appearance and weight of dehulled grains. The ratio of perfect (translucent), immature (mainly chalky), damaged, abortive, and colored grains was determined by a grain-grading machine, ES-1000 (Shizuoka Seiki), and is indicated from left to right of the bars in order. Because colored gains were few, the symbol is inside lines. Black and white arrowheads indicate almost translucent and severely chalky grains ripened under 33°C/28°C, respectively. C, Scanning electron micrographs of transverse sections of 25°C/20°C-ripened translucent (left) and 33°C/28°C-ripened chalky grains (right). Top and bottom, Light microscope and scanning electron microscope images, respectively. The areas indicated by boxes were analyzed by scanning electromicroscopy. Bars = 10 μm.

Figure 2.

Expression of genes related to starch metabolism, storage protein synthesis, and heat stress response in developing caryopses, as revealed by semiquantitative RT-PCR analysis. The time-course profiles for ripening under 25°C/20°C and 33°C/28°C are shown in the top and bottom rows for each gene, respectively. The developmental stage of the caryopsis is indicated by DAF. The expression levels were quantified densitometrically, and the ratio of accumulation of the transcript levels for 8 to 30 DAF of 33°C/28°C to that of 25°C/20°C (H/L) is indicated for each gene.

Figure 3.

Comparison of expression of genes for SSs, BEs, and α-amylases, and cyPPDKB. The expression levels were determined by semiquantitative RT-PCR analysis and densitometry, and the value of 25°C/20°C, 11 DAF was set to “1” for each gene. Results from four independent PCRs are shown with error bars (sd).

Figure 4.

Comparison of the chain-length profile of amylopectin in ‘Nipponbare’ grain ripened under different temperatures. A, Chain-length profile. Debranched amylopectin extracted from 25°C/20°C-ripened (white bars) or 33°C/28°C-ripened (black bars) grains were analyzed by HPAEC-PAD, and the relative peak area of the chromatogram is shown for the individual DP. The data is the mean ± sd of five independent measurements. B, Difference in the chain-length distribution of amylopectin. The difference in the relative peak area in A between 33°C/28°C-ripened and 25°C/20°C-ripened grains is shown in the DP range of 6 to 53.

Figure 5.

SDS-PAGE and immunoblot analyses of seed storage proteins. Total protein was extracted from mature grains ripened under 25°C/20°C or 33°C/28°C, and five independent extracts for each were separated on a SDS-polyacrylamide (18%) gel, followed by Coomassie Brilliant Blue staining or immunoblot detection with a polyclonal antibody raised against purified 13-kD prolamin. Large and small subunits of glutelin and 13-kD prolamin are indicated on the right side, and their relative amount was quantified densitometrically. For the SDS-PAGE, the ratio of 13-kD prolamin amount to the sum of glutelin large and small subunits is shown at the bottom, and, for the immunoblot, the relative amount of 13-kD prolamin is shown.

Figure 6.

Comparison of the chain-length profile of amylopectin in perfect (translucent) and milky-white (severely chalky) grains ripened under 33°C/28°C. A, Chain-length profile. Debranched amylopectin extracted from ‘Nipponbare’ perfect (white bars) or milky-white (black bars) grains was analyzed by HPAEC-PAD, and the relative peak area of the chromatogram is shown for the individual DP. The data is the mean ± sd of five independent measurements. B, Difference in the chain-length distribution of amylopectin. The difference in the relative peak area in A between milky-white and perfect grains is shown in the DP range of 6 to 63.

Figure 7.

Comparison of high temperature-tolerant (‘Koshiibuki’ and ‘Tentakaku’) and -sensitive (‘Sasanishiki’ and ‘Hatsuboshi’) cultivars. A, Appearance grade of grains matured under 25°C/20°C (white bar) and 33°C/28°C (black bar). The ratio of perfect (translucent) grain was determined by a grain-grading machine, ES-1000 (Shizuoka Seiki). Appearance of grains ripened under 33°C/28°C is shown in the photograph with the ratio of reduction of grain weight to that of the 25°C/20°C-treated control. B, Difference in the chain-length distribution of amylopectin. For the respective cultivars, the difference in the relative peak area on the HPAEC-PAD chromatogram between 33°C/28°C-ripened and 25°C/20°C-ripened grains is shown in the DP range of 6 to 59.

Figure 8.

Ratio of cumulative expression level during 8 to 30 DAF of starch metabolism-related genes. The cumulative expression levels during 8 to 30 DAF were determined by semiquantitative RT-PCR and densitometry for grain filling under 25°C/20°C and 33°C/28°C, as described in the legend of Figure 2, and the ratio of cumulative transcript level for 33°C/28°C to 25°C/20°C is shown for the respective genes encoding enzymes/translocators on the starch-metabolizing pathway. The genes induced >1.5-fold and the corresponding reaction step are indicated in bold and with a thick arrow, while the genes repressed to <0.70-fold and the corresponding steps are indicated in bold and with thin arrows. The products of AGPL1 and AGPS1 genes have been estimated to be localized in amyloplasts, while those of AGPL2 and AGPS2b in the cytosol (Akihiro et al., 2005; Ohdan et al., 2005). The pathways described in the text are solely depicted. MOS, Maltooligosaccharide; PKc, cytosolic pyruvate kinase.