IC4R004-RNA-Seq-2015-26032497

Project Title

Five pectinase gene expressions highly responding to heat stress in rice floral organs revealed by RNA-seq analysis

The Background of This Projec

• High temperature stress or heat stress is defined as the temperature beyond a cretical threshhold for a peroid of time. In this paper, we performed Digital Gene Expression Profiling via Illumina/Solexa sequencing technology to parallelally sequence the floral transcriptomes of a rice line that was heat stressed at the booting stage with the normal check.

Plant Culture & Treatment

• Oryza sativa L. ssp. japonica heat-sensitive cultivar 'Ningjing 4' was used in this study. Ningjing 4 has been widely grown from the middle to lower districts of the Yangz River. Rice plants of Ningjing 4 were grown in 2013 at the experiemntal farm of Anhui Agricultural University.
• At 10 d before the heat stress at 38 ℃ was applied, individual plants at similar developmental stage were selected and transplanted to plastic pots sized 40×40×30 cm 3 . At meiophase of the pollen mother cell (pulvinus flat), the plants were moved to an artificial climate chamber for optimal and high temperature treatments for 3d. The optimal chamber (control) was set as day 08:00-18:00, 32 ℃ and night 18:00-08:00, 25 ℃ , and the high temperature chamber was set as day 08:00-18:00, 2 ℃and night 18:00-08:00, 30 ℃ .

Illumina Sequencing

• The RNAprep Pure Plant Total RNA extraction kit purchased from TIANGEN company was used to extract total RNA of floral organs for both control (A) and heat stress treatment (B). Flower organs from five sampled panicles were pooled as a technical replicate for RNA extraction, two replicates for each temperature treatment.
• cDNA libraries for RNA-Seq sequencing were prepared with the mRNA-Seq Sample Preparation Kit (Illumina) according to the protocol of the manufacturer, and sequencing of the RNA-Seq li- braries was performed by Beijing Genomics Institute (BGI) using the 10G Illumina Genome Analyzer. The Illumina Genome Analyzer system was used to statisticaly analyze the transcriptome sequencing results of RNA isolated from floral organs under normal (A) and heat stress (B) treatments comparatively.

Research Findings

• After low-quality sequences including impurities and 3 0 linker sequences were removed from the raw sequence reads, the normal library (A) contained 660,800 or 4.2% more clean reads than the heat stress library (B). However, the data size was smaller in A library than it in B library, in which A had 14,541,433 or 0.8% less nucleotides than B. The average read length was ~130 bp, and the Q20 ratio (sequencing error rate <1%) of sequences was 100% in the two libraries. The data size and Q20 ratio demonstrated a high quality of sequencing data in both A and B libraries with a completion transcriptome sequencing.
• The 34 GO categories belonged to three fuctional domains, biological process domain with 16 including 2906 DEGs, cellular component domain with 9 including 3368 DEGs and molecular function domain with 9 including 904 DEGs (Fig. 3). In the biological process domain, a great majority of DEGs (44.6%) were involved in metabolic processes category, followed by cellular processes (32.2%) and stress process in response to stimulus (10.9%).
• In the cellular component domain, those DEGs were mainly involved in functions of the cell (45.6%), cell part (33.7%), and organelles (15.3%). In the molecular function domain, the DEGs were mainly involved in functions of the binding (42.2%) and catalytic activity (39%). These GO mapping results indicated that the ma- jority of DEGs responding to heat stress were involved in metabolic processes, cells and catalytic activity, and enzymatic activity. These affected activities suggest that heat stress treatment mainly brought about functional changes in physiological metabolism and cell differentiation.

• Pathway enrichment analysis determines the main biochemical metabolic pathways and signal transduction pathways in which the DEGS are involved. 14 pathways were found to be significantly enriched in response to heat stress treatment. In the 14 significant pathways, a total of 1753 DEGs were pathway-annotated.

• The ko04626 for Plant-pathogen interaction had the most DEGs as 492, followed by ko04075 for Plant honene signal transduction as 257, ko00500 for Starch and sucrose metabolism as 170, ko00230 for Purine metabolism as 162, and ko00940 for Phenylpropanoid biosynthesis as 124. The numbers of DEGs were less than 100 for the rest of metabolic pathways.
• Using DESeq software [26] to inspect the scatter plots of all the genes expressed in the rice floral organs of A and B identified a total of 7178 DEGs. The differential expressions due to heat stress treatment between A and B for the identified genes were highly significant at a probability of p_value ≤ 0.001, FDR (false discovery rate) ≤ 0.001, and corrected value (Q_value)≤0.01, jLog2 (A/B)j ≤ 1. Among those significant DEGs, 4355 or 61% of DEGs showed up-regulated expressions, while other 2823 or 39% of DEGs showed down-regulated expression.

Labs working on this Project

• Center of Cooperative Innovation for Crop, Agronomy College, Anhui Agricultural University, Hefei 230036, Anhui, China
• Jiangsu Collaboraive Innovation Center for Modern Crop Production, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China