Microarray Analysis of Rice d1 (RGA1) Mutant Reveals the Potential Role of G-Protein Alpha Subunit in Regulating Multiple Abiotic Stresses Such as Drought, Salinity, Heat, and Cold

Abstract

The genome-wide role of heterotrimeric G-proteins in abiotic stress response in rice has not been examined from a functional genomics perspective, despite the availability of mutants and evidences involving individual genes/processes/stresses. Our rice whole transcriptome microarray analysis (GSE 20925 at NCBI GEO) using the G-alpha subunit (RGA1) null mutant (Daikoku 1 or d1) and its corresponding wild type (Oryza sativa Japonica Nipponbare) identified 2270 unique differentially expressed genes (DEGs). Out of them, we mined for all the potentially abiotic stress-responsive genes using Gene Ontology terms, STIFDB2.0 and Rice DB. The first two approaches produced smaller subsets of the 1886 genes found at Rice DB. The GO approach revealed similar regulation of several families of stress-responsive genes in RGA1 mutant. The Genevestigator analysis of the stress-responsive subset of the RGA1-regulated genes from STIFDB revealed cold and drought-responsive clusters. Meta data analysis at Rice DB revealed large stress-response categories such as cold (878 up/810 down), drought (882 up/837 down), heat (913 up/777 down), and salt stress (889 up/841 down). One thousand four hundred ninety-eight of them are common to all the four abiotic stresses, followed by fewer genes common to smaller groups of stresses. The RGA1-regulated genes that uniquely respond to individual stresses include 111 in heat stress, eight each in cold only and drought only stresses, and two genes in salt stress only. The common DEGs (1498) belong to pathways such as the synthesis of polyamine, glycine-betaine, proline, and trehalose. Some of the common DEGs belong to abiotic stress signaling pathways such as calcium-dependent pathway, ABA independent and dependent pathway, and MAP kinase pathway in the RGA1 mutant. Gene ontology of the common stress responsive DEGs revealed 62 unique molecular functions such as transporters, enzyme regulators, transferases, hydrolases, carbon and protein metabolism, binding to nucleotides, carbohydrates, receptors and lipids, morphogenesis, flower development, and cell homeostasis. We also mined 63 miRNAs that bind to the stress responsive transcripts identified in this study, indicating their post-transcriptional regulation. Overall, these results indicate the potentially extensive role of RGA1 in the regulation of multiple abiotic stresses in rice for further validation.

Keywords: G-protein; RGA1; cold; drought; heat; rice; salt; stress.

Figure 1

Stress responsive genes among RGA1-regulated genes in rice. The left panel shows Venn selections between the subsets of all rice abiotic stress-responsive genes listed at STIFDB2.0. The inset shows Venn selection between all 626 abiotic stress-responsive genes listed at STIFDB2.0 and 2270 RGA-1-regulated DEGs identified on our microarray. The left panel shows the break-up of the 106 RGA-regulated stress-responsive genes identified in the inset in terms of individual stresses viz., heat (94), drought (13), salt (6), and cold (4).

Figure 2

Hierarchical clustering of the transcripts of 106 RGA1-regulated stress-responsive genes in various abiotic stress studies in rice (118 perturbations). The red and green colors indicate up-regulation (log2 [2.5]) and down-regulation (log2 [−2.5]), respectively as shown in the color bar. Hierarchical clustering was done using average linkage based on Euclidean distance subsets of individual stress conditions such as heat, cold, drought/dehydration, salt, submergence, and shift from aerobic to anaerobic germination cold and drought. The expression data were obtained using Genevestigator (Zimmermann et al., 2004).

Figure 3

Expression profiles of 106 RGA1-regulated stress-responsive genes in cold stress (39 perturbations from literature). The red and green colors indicate up-regulation (log2 [2.5]) and down-regulation (log2 [−2.5]), respectively, as shown in the color bar. The expression data in the left panel were obtained using Genevestigator. The table compares their regulation in normal plants under stress in literature with actual fold-change values in the RGA1 mutant.

Figure 4

Expression profiles of 106 RGA1-regulated stress- responsive genes in drought stress (30 perturbations from literature). The red and green colors indicate up-regulation (log2 [2.5]) and down-regulation (log2 [−2.5]), respectively, as shown in the color bar. The expression data in the left panel were obtained using Genevestigator. The table compares their regulation in normal plants under stress in literature with actual fold-change values in the RGA1 mutant.

Figure 5

Venn selection of RGA1 regulated stress responsive genes mined from RiceDB, STIFDB, and GO term. The overlap among the three sets revealed that the genes mined using GO term stress and stress responsive genes from STIFDB are largely subsets of the 1886 DEGs identified using Rice DB.

Figure 6

Meta-data analysis of RGA1-regulated genes regulated under various abiotic stresses. The 2270 RGA1-regulated genes (1242 up and 1028 down) were used as input query at Rice DB to generate genes responsive to cold (878 up/810 down), drought (882 up/837 down), heat (913 up/777 down), and salt stress (889 up/841 down) with 1498 genes common to all four stresses and totaling 1886 unique genes. Their Venn selections are depicted as total (A), up-regulated (B) and down-regulated (C) sets, using the online tool Venny (Oliveros, 2007–2015).

Figure 7

qRT-PCR validation of RGA1-regulated genes identified as common to various abiotic stresses. Out of the 1498 RGA1-regulated genes identified as common to multiple abiotic stresses on the microarray, 12 of the most up/down-regulated genes were validated by qRT-PCR. Their fold change data are shown based on averages of two biological replicates and two technical replicates of total RNA, along with microarray results. The left panel in red shows the up-regulated genes and the right panel in green shows the down-regulated genes.

Figure 8

Gene Ontology enrichment of RGA1-regulated, stress responsive genes from Rice DB. The 1498 genes common to all four major abiotic stresses were subjected to GO enrichment using AgriGO with default settings. (A) Biological process categorization of the RGA1-regulated genes shared by salt, heat, cold, and drought stresses. (B) Molecular function categorization and (C) Subcellular localization of the RGA1-regulated genes shared by all four abiotic-stresses.