Transcriptome analysis of grain-filling caryopses reveals involvement of multiple regulatory pathways in chalky grain formation in rice

Abstract

Background: Grain endosperm chalkiness of rice is a varietal characteristic that negatively affects not only the appearance and milling properties but also the cooking texture and palatability of cooked rice. However, grain chalkiness is a complex quantitative genetic trait and the molecular mechanisms underlying its formation are poorly understood.

Results: A near-isogenic line CSSL50-1 with high chalkiness was compared with its normal parental line Asominori for grain endosperm chalkiness. Physico-biochemical analyses of ripened grains showed that, compared with Asominori, CSSL50-1 contains higher levels of amylose and 8 DP (degree of polymerization) short-chain amylopectin, but lower medium length 12 DP amylopectin. Transcriptome analysis of 15 DAF (day after flowering) caryopses of the isogenic lines identified 623 differential expressed genes (P < 0.01), among which 324 genes are up-regulated and 299 down-regulated. These genes were classified into 18 major categories, with 65.3% of them belong to six major functional groups: signal transduction, cell rescue/defense, transcription, protein degradation, carbohydrate metabolism and redox homeostasis. Detailed pathway dissection demonstrated that genes involved in sucrose and starch synthesis are up-regulated, whereas those involved in non-starch polysaccharides are down regulated. Several genes involved in oxidoreductive homeostasis were found to have higher expression levels in CSSL50-1 as well, suggesting potential roles of ROS in grain chalkiness formation.

Conclusion: Extensive gene expression changes were detected during rice grain chalkiness formation. Over half of these differentially expressed genes are implicated in several important categories of genes, including signal transduction, transcription, carbohydrate metabolism and redox homeostasis, suggesting that chalkiness formation involves multiple metabolic and regulatory pathways.

Figure 1

Starch properties of Asominori and CSSL50-1. (A) Rice grains of Asominori (left) and CSSL50-1 (right); (B) Scanning electron microscopy of starch granule transverse sections of Asominori (left) and CSSL50-1 (right); (C) Chain-length profile of Asominori and CSSL50-1 and differences in the chain-length distribution of amylopectin; (D) RVA profile, Rapid Visco Analyzer profile (E) PGWC, Percentage of grain with chalkiness; (F) DEC, Degree of endosperm chalkiness; (G) StC, Starch content; (H) AC, Amylose content; (I) SuC, sucrose content; (J) PC, protein content. Bars = 3.0 μm.

Figure 2

Comparison of grain-filling rates and activities of starch synthesis enzymes between CSSL50-1 and Asominori. (A) Fresh weight of rice grains; (B) Dry weight of rice grains; (C) Sucrose synthase (SuSy); (D) ADP-glucose pyrophosphorylase (AGPase); (E) Starch branching enzyme (SBE); (F) Starch debranching enzyme (DBE).

Figure 3

Functional classification and distribution of 623 differentially expressed genes. (A) Total differentially expressed genes (623); (B) Genes up-regulated (324); (C) Genes down-regulated (299).

Figure 4

Overlays of differentially expressed genes onto starch and non-starch polysaccharide metabolism pathways. Up arrows designate up-regulation of the genes and down arrows down-regulated genes. The number of arrows indicates the number of genes. AC, amylose content; AGPase, ADP-glucose pyrophosphorylase; DBE, starch debranching enzyme; GPI, glucose-6-phosphate isomerase; GT5, glycosyltransferase family 5 protein; SBE, starch branching enzyme; SPP, sucrose phosphatase; SPS, sucrose phosphate synthase.

Figure 5

Differentially expressed genes associated with redox homeostasis networks in plants. Up arrows designate up-regulation of the genes and down arrows down-regulated genes. The number of arrows indicates the number of genes. APX, ascorbate peroxidase; DHA, dehydroascrobate; GLR, glutaredoxin; Glx, glyoxalase; GPX, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; GST, glutathione-S-transferase; LOX5, lipoxygenase-5; MDA, monodehydroascorbate; MDAR, MDA reductase; PrxR, peroxiredoxin; ROS, reactive oxygen species; SOD, superoxide dismutase; Trx, thioredoxin.

Figure 6

An overview of the major gene networks closely associated with rice grain endosperm chalkiness. AC, amylose content; a-D-Xyase, Alpha-D-xylosidase; AGPase, ADP-glucose pyrophosphorylase; a-L-AFase, Alpha-L-arabinofuranosidase; AP-2, APETALA2 domain-containing protein; APX, ascorbate peroxidase; ARR, arabidopsis thaliana response regulator; BES1/BZR1, BES1/BZR1 homolog protein; BR, brassinosteroid; BTB/POZ, BTB/POZ domain protein; C2H2/C3HC4 Zn RING, C2H2/C3HC4-type zinc finger proteins; CesA, cellulose synthase; DBE, starch debranching enzyme; GA, gibberellin; GH28, Glycosyl hydrolase family 28 protein; GLR, glutaredoxin; GPX, glutathione peroxidase; GST, glutathione-S-transferase; GT5, glycosyltransferase family 5 protein; Hpt, Hpt domain protein; HSP, heat shock protein; LIM, LIM domain protein; Maf/bZIP, Maf family protein; MAPK, mitogen-activated protein kinase; MDAR, monodehydroascorbate reductase; Myb, Myb transcription factor; OXI1, serine/threonine protein phosphatase; PA, phosphatidic acid; PDK, phosphoinositide-dependent kinase; PHD, PHD finger transcription factor; PI(3/4)P, phosphatidylinositol-4-phosphate; PLD, phospholipase D; PrxR, peroxiredoxin; ROS, reactive oxygen species; SBE, starch branching enzyme; SOD, superoxide dismutase; SPP, sucrose phosphatase; SPS, sucrose phosphate synthase; TF, transcription factor; Trx, thioredoxin; XIP, Xylanase inhibitor protein.

Figure 7

RT-PCR confirmation of the microarray data. Twenty-one differentially expressed genes were randomly selected for RT-PCR analysis. (A) 15 up-regulated genes; (B) 6 down-regulated genes. Actin was used as the control.