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Laser microdissection-based gene expression analysis in the aleurone layer and starchy endosperm of developing rice caryopses in the early storage phase

Ishimaru, Tsutomu, Ida, Masashi, Hirose, Sakiko, Shimamura, Satoshi, Masumura, Takehiro, Nishizawa, Naoko K., Nakazono, Mikio, Kondo, Motohiko
Rice 2015 v.8 no.1 pp. 22
adenosine triphosphate, aerobiosis, aleurone cells, carbohydrate metabolism, carbon dioxide fixation, endosperm, fruits, gene expression, genes, glucose-1-phosphate adenylyltransferase, lipids, messenger RNA, mitochondria, oxidative phosphorylation, oxygen, quantitative polymerase chain reaction, reverse transcriptase polymerase chain reaction, rice, ripening, starch granules, sucrose, tissues, tricarboxylic acid cycle
BACKGROUND: Rice endosperm is composed of aleurone cells in the outermost layers and starchy endosperm cells in the inner part. The aleurone layer accumulates lipids, whereas starchy endosperm mainly accumulates starch. During the ripening stage, the starch accumulation rate is known to be asynchronous, depending on the position of the starchy endosperm. Different physiological and molecular mechanisms are hypothesized to underlie the qualitative and quantitative differences in storage products among developing rice endosperm tissues. RESULTS: Target cells in aleurone layers and starchy endosperm were isolated by laser microdissection (LM), and RNAs were extracted from each endosperm tissue in the early storage phase. Genes important for carbohydrate metabolism in developing endosperm were analyzed using qRT-PCR, and some of the genes showed specific localization in either tissue of the endosperm. Aleurone layer-specific gene expression of a sucrose transporter, OsSUT1, suggested that the gene functions in sucrose uptake into aleurone cells. The expression levels of ADP-glucose pyrophosphorylase (AGPL2 and AGPS2b) in each endosperm tissue spatially corresponded to the distribution of starch granules differentially observed among endosperm tissues. By contrast, expressions of genes for sucrose cleavage—hexokinase, UDP-glucose pyrophosphorylase, and phosphoglucomutase—were observed in all endosperm tissues tested. Aleurone cells predominantly expressed mRNAs for the TCA cycle and oxidative phosphorylation. This finding was supported by the presence of oxygen (8 % concentration) and large numbers of mitochondria in the aleurone layers. In contrast, oxygen was absent and only a few mitochondria were observed in the starchy endosperm. Genes for carbon fixation and the GS/GOGAT cycle were expressed highly in aleurone cells compared to starchy endosperm. CONCLUSIONS: The transcript level of AGPL2 and AGPS2b encoding ADP-glucose pyrophosphorylase appears to regulate the asynchronous development of starch granules in developing caryopses. Aleurone cells appear to generate, at least partially, ATP via aerobic respiration as observed from specific expression of identified genes and large numbers of mitochondria. The LM-based expression analysis and physiological experiments provide insight into the molecular basis of the spatial and nutritional differences between rice aleurone cells and starchy endosperm cells.