GSE119109
Transcriptome analysis of flooded rice spikelets in response to high temperature at flowering
Purpose: understanding the complex genetic control of heat tolerance can be reached using whole transcriptome sequencing (RNA-seq) that is currently the most powerful tool to identify genes differentially regulated. Heat responses differ greatly between sensitive and tolerant genotypes, mainly in terms of the amount of genes and pathways involved in the response and differences in the expression level of constitutive genes in many pathways. The present study aims to compare the molecular response of Brazilian flooded rice cultivars with variable sensitivity to heat stress at anthesis stage and to discuss the possible processes involved in heat tolerance. Methods: Panicle mRNA profiles of cultivars IRGA 428 (sensitive) and BR-IRGA 409 (tolerant) were generated by deep sequencing, in triplicate (each replicate is composed of two plants), under three days of heat stress and control condition at anthesis stage, using Illumina Hiseq 2500. The sequence reads from each sample that passed quality filters were mapped independently, generating contigs that were aligned with the reference genome sequences in rice (cv. MH63-indica). The transcripts were analyzed at the expression level with statistic test (EdgeR – Rstudio) to identify the significant changes in the expression level of the genes for each treatment condition and cultivar follow by identification of metabolic pathways (MapMan and Cytoscape) based on different programs. Results: Using an optimized data analysis workflow, we identified 16,871 transcripts in the rice spikelet of sensitive and tolerant cultivars with HISAT workflow. Using filtered and aligned reads, transcript levels were used to find differentially expressed genes (DEGs) at different thermal condition and cultivars. We identified 2,064 genes for BR-IRGA 409 and 1,078 genes for IRGA 428 that showed a significant change in expression in response to heat stress (LogFC =0.5 and adjusted p-value <0.001). When it was examined the multi-factor responsive genes, that is genes with an interaction between cultivar and heat stress (multi-factor analysis - cultivars vs control and high temperature), it was identified 65 DEGs in response to heat stress (LogFC =0.5 and adjusted p-value <0.001). Comparison of our data set with other RNA-seq studies shows a significant overlap in heat responsive genes (hypergeometric enrichment test). This core set of genes common among RNA-seq studies indicates that it may be associated with basal response to heat. In the present study, genes related to light reaction pathway were induced in the panicle of the cultivar IRGA 428, while these genes in cultivar BR-IRGA 409 were already expressed highly in control conditions. The basal level of these genes in BR-IRGA 409 was as high as the heat-induced level in the sensitive IRGA 428. Photosynthesis is known to occur in rice panicles, but little has been reported about the photosynthetic characteristics of such panicles. To cope with heat stress, plants have developed a sophisticated mechanism to repair photosystem damage. Therefore, in IRGA 428 the induction of these genes suggests the activation of a repair mechanism to reverse damage on the photosynthetic apparatus caused by the heat stress. The difference in basal levels of the photosynthetic genes may be a contributing factor to the differences in spikelet fertility between the cultivars under heat stress. Conclusion: This is the first study to evaluate the heat tolerance of Brazilian rice cultivars. In panicle tissue, most canonical heat responsive genes, such as HSF family, small HSP family and FKBP family, show a similar response in both Brazilian cultivars, despite the fact that these cultivars have not been specifically selected for heat tolerance. The response of photosynthetic genes to heat stress is a major difference between the cultivars BR-IRGA 409 and IRGA 428. This study indicates that the higher basal expression of photosynthetic genes in the panicle provides improved spikelet fertility under heat stress. This may provide potential markers that can be used to identify heat tolerant genotypes among rice breeding programs. This study emphasizes that the characterization of heat stress responses in individual cultivars provides insights into both the basic mechanisms of heat response and assists with understanding the complex phenomenon of responses to high temperature stress. Overall design: Panicle mRNA profiles of cultivars IRGA 428 (sensitive) and BR-IRGA 409 (tolerant) were generated by deep sequencing, in triplicate (each replicate is composed of two plants), under three days of heat stress and control condition at anthesis stage, using Illumina Hiseq 2500 150bp single end reads.