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| Status |
Public on Dec 10, 2018 |
| Title |
Laser microdissection-based tissue specific transcriptome analyses reveals novel regulatory network of genes involved in heat-induced grain chalk in rice endosperm. |
| Organism |
Oryza sativa |
| Experiment type |
Expression profiling by array
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| Summary |
Heat stress occurrence during endosperm development and seed filling forms chalky portion in the limited zone of starchy endosperm of rice grains. In this study, isolation of aleurone, dorsal, central and lateral tissues of developing endosperm by laser-microdissection (LM) coupled with gene expression analysis of 44K microarray was performed to identify key regulatory genes involved in the formation of milky-white (MW) and white-back (WB) chalky grains during heat stress. Gene regulatory network analysis classified the genes changed under heat stress into five modules. the modules of genes changed in developing starchy endosperm corresponding to MW and WB portion under heat stress suggested different regulatory genes involved in each type of grain chalk. The most distinct expression pattern was observed in a M1 and M2 modules where most of the small heat shock proteins and cellular organisation genes being upregulated under heat stress in dorsal aleurone cells and dorsal starchy endosperm zones The histological observation supported the significant increase in cell number and size of dorsal aleurone cells in WB grains. With regard to the central zone of starchy endosperm, preferential downregulation of high molecular weight heat shock proteins (HMW HSPs) including a prominent member encoding for endoplasmic reticulum (ER) chaperones by heat stress were observed, while expression of starch-biosynthesis genes remained unaffected. Characterization of transgenic plants supressing endosperm lumenal binding protein (BiP1), an ER chaperone preferentially downregulated at MW portion under heat stress, showed an evidence of forming the chalky grains without disturbing the expression of starch-biosynthesis genes. The present LM-based comprehensive expression analyses provides novel inferences that HMW HSPs play important role in controlling the redox, nitrogen and amino aicd metabolism in endosperm leading to the formation of MW and WB chalky grains.Keywords ; developing endosperm, gene expression, grain chalkiness, heat stress, laser-microdissection, Oryza sativa.
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| Overall design |
Microarray experiment was conducted based on the method of Takehisa et al. (2012). Total RNA (5.0 ng) was amplified to obtain complementary RNA (cRNA) using two-color Quick Amp Labeling kit (Agilent technologies), according to the modified manufacturer’s instruction. RNA extracted from control and high-temperature condition was labeled with cyanine-3 (Cy3)-CTP and cyanine-5 (Cy5)-CTP, respectively. Three biological replicates were prepared from high temperature conditions as well under control conditions. 1250 ng of labeled cRNAs were fragmented and hydridized on a slide glass of rice 4 × 44K microarray RAP-DB (G2519F#1524 ; Agilent Technologies). Slides were scanned on an Agilent G2505B DNA microarray scanner, and background of the Cy3 and Cy5 raw signals was corrected using the Feature Exraction (ver. 10.5.1.1, Agilent Technologies). The two channels Agilent microarray expression data of 12 microdissected samples of dorsal aluerone cells (AL), dorsal starchy endosperm (DSE), central starchy endosperm (CSE) and lateral starchy endosperm (LSE) at 7DAF from control and at 6DAF from heat stressed tissues consisting of three replicates was further processed (ratio computation, log transformation and baseline transformation) using the software GeneSpring GX version 12 (Agilent, Santa Carla, CA).
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| Contributor(s) |
Ishimaru T, Parween S, Saito Y, Shigemitsu T, Yamakawa H, Nakazono M, Masumura T, Nishizawa NK, Kondo M, Sreenivasulu N |
| Citation(s) |
30517758 |
| Submission date |
Nov 02, 2018 |
| Last update date |
Dec 10, 2018 |
| Contact name |
Tsutomu Ishimaru |
| E-mail(s) |
cropman@affrc.go.jp
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| Organization name |
Central Region Agricultural Research Center, National Agriculture and Food Research Organization (CARC/NARO)
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| Street address |
1-2-1 Inada, Joetsu, Niigata
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| City |
Niigata |
| ZIP/Postal code |
941-0193 |
| Country |
Japan |
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| Platforms (1) |
| GPL8852 |
Agilent-015241 Rice Gene Expression 4x44K Microarray (Probe name version) |
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| Samples (24)
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| GSM3455520 |
C-ALR1-7DAF: Control Aleurone Cell Replicate 1 |
| GSM3455521 |
C-ALR2-7DAF: Control Aleurone Cell Replicate 2 |
| GSM3455522 |
C-ALR3-7DAF: Control Aleurone Cell Replicate 3 |
| GSM3455523 |
C-DSER1-7DAF: Control Dorsal Starchy Endosperm Replicate 1 |
| GSM3455524 |
C-DSER2-7DAF: Control Dorsal Starchy Endosperm Replicate 2 |
| GSM3455525 |
C-DSER3-7DAF: Control Dorsal Starchy Endosperm Replicate 3 |
| GSM3455526 |
C-CSER1-7DAF: Control Central Starchy Endosperm Replicate 1 |
| GSM3455527 |
C-CSER2-7DAF: Control Central Starchy Endosperm Replicate 2 |
| GSM3455528 |
C-CSER3-7DAF: Control Central Starchy Endosperm Replicate 3 |
| GSM3455529 |
C-LSER1-7DAF: Control Lateral Starchy Endosperm Replicate 1 |
| GSM3455530 |
C-LSER2-7DAF: Control Lateral Starchy Endosperm Replicate 2 |
| GSM3455531 |
C-LSER3-7DAF: Control Lateral Starchy Endosperm Replicate 3 |
| GSM3455532 |
S-ALR1-6DAF: Stress Aleurone Cell Replicate 1 |
| GSM3455533 |
S-ALR2-6DAF: Stress Aleurone Cell Replicate 2 |
| GSM3455534 |
S-ALR3-6DAF: Stress Aleurone Cell Replicate 3 |
| GSM3455535 |
S-DSER1-6DAF: Stress Dorsal Starchy Endosperm Replicate 1 |
| GSM3455536 |
S-DSER2-6DAF: Stress Dorsal Starchy Endosperm Replicate 2 |
| GSM3455537 |
S-DSER3-6DAF: Stress Dorsal Starchy Endosperm Replicate 3 |
| GSM3455538 |
S-CSER1-6DAF: Stress Central Starchy Endosperm Replicate 1 |
| GSM3455539 |
S-CSER2-6DAF: Stress Central Starchy Endosperm Replicate 2 |
| GSM3455540 |
S-CSER3-6DAF: Stress Central Starchy Endosperm Replicate 3 |
| GSM3455541 |
S-LSER1-6DAF: Stress Lateral Starchy Endosperm Replicate 1 |
| GSM3455542 |
S-LSER2-6DAF: Stress Lateral Starchy Endosperm Replicate 2 |
| GSM3455543 |
S-LSER3-6DAF: Stress Lateral Starchy Endosperm Replicate 3 |
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| Relations |
| BioProject |
PRJNA503622 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE122115_raw_data_files.tar.gz |
162.0 Mb |
(ftp)(http) |
TAR |
| Processed data included within Sample table |
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