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

Tsutomu Ishimaru,Naoko K Nishizawa,Sakiko Hirose,Masashi Ida,Mikio Nakazono,Satoshi Shimamura,Motohiko Kondo,Takehiro Masumura

doi:10.1186/s12284-015-0057-2

Abstract

BackgroundRice 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.ResultsTarget 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12284-015-0057-2) contains supplementary material, which is available to authorized users.

Highlights

Rice endosperm is composed of aleurone cells in the outermost layers and starchy endosperm cells in the inner part
Lipid is accumulated in aleurone cells, whereas starch is accumulated in starchy endosperm, starting at 5 days after flowering (DAF) (Hoshikawa 1967)
The Laser microdissection (LM)-based expression analysis conducted in this study provided the novel finding that the clear gradient of transcripts of OsAGPL2 and OsAGPS2b may be responsible for the large difference in starch accumulation among at the dorsal side (AL), starchy endosperm in the lateral regions (SEL), and starchy endosperm in the central region (SEC) in the early storage phase

Summary

Introduction

Rice endosperm is composed of aleurone cells in the outermost layers and starchy endosperm cells in the inner part. The rice sucrose transporter gene family comprises five genes, OsSUT1-5 (Aoki et al 2003) Among these five OsSUT genes, OsSUT1 is expressed after 5 DAF (Hirose et al 1997; Hirose et al 2002) in aleurone cells in developing endosperm (Furbank et al 2001; Ishimaru et al 2007) and plays a critical role in starch accumulation in endosperm (Scofield et al 2002). All of the genes associated with starch biosynthesis, including ADP-glucose pyrophosphorylase (Ohdan et al 2005), plastid translocator (Toyota et al 2006), starch synthase (Hirose and Terao 2004), branching enzyme, starch debranching enzyme, phosphorylase, and disproportionating enzyme (Ohdan et al 2005) have been cloned, and some of them are expressed concomitantly with the onset of endosperm starch accumulation at 5 DAF (Hirose and Terao 2004; Ohdan et al 2005; Toyota et al 2006). Genetic analyses using mutants and gene manipulation of starch biosynthesis-related genes have revealed the critical role(s) of some genes in grain phenotypes and starch properties in rice endosperm (Lee et al 2007; Fujita et al 2006; Fujita 2014; Umemoto et al 2004; Fujita et al 2007; Ryoo et al 2007; Itoh et al 2003; Satoh et al 2003; Satoh et al 2008; Nishi et al 2001)

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