IC4R004-Microarray-2012-22827941

Project Title

• Microarray analysis reveals overlapping and specific transcriptional responses to different plant hormones in rice

The Background of This Project

• Hormones govern almost every aspect of plant life cycle from germination to seed development. Several molecular genetics studies have demonstrated the crucial role of various hormones in plant growth and development processes. The analyses of mutants with altered hormone biosynthesis and signaling have revealed the role of auxin, brassinosteroid, cytokinin and gibberellin in cell expansion along the longitudinal axes and ethylene along the transverse axes. Abscisic acid acts antagonistically on plant growth and is majorly involved in abiotic stress responses. Jasmonic acid and salicylic acid are involved in plant defense responses against pathogens.
• Most of our knowledge in hormone biology is based on the work done in Arabidopsis. Only a few studies have been performed to show the effect of different hormones at whole genome level in crop plant rice. 8,17-19 Further, the comparison of transcriptional responses of different plant hormones is also not available in rice. In this study, we performed microarray analy- sis in rice to study the effect of different hormones, including auxin, cytokinin, abscisic acid, ethylene, salicylic acid and jas- monic acid, on gene expression at whole genome level. We further examined the overlap in the transcriptional responses to different hormones and identified several hormone-specific marker genes.

Plant Materials & Treatment

• Plant material and hormone treatments. Rice (Oryza sativa subspecies indica variety IR64) seeds were treated and grown as described previously. 2 Different hormone treatments were given to the 7-d-old light-grown rice seedlings as described. 8,10 The seedlings were transferred to the beakers containing 50 μM solution of indole-3-acetic acid (auxin) and benzyl aminopurine (cytokinin) and 100 μM solution of abscisic acid, 1-aminocyclopropane-1-carboxylic acid (ethylene derivative), salicylic acid and jasmonic acid for hormone treatments. The seedlings were kept in respective solutions for 3 h, at 28 ± 1°C before harvesting. The seedlings kept in water for 3 h, at 28 ± 1°C served as control. At least three independent biological replicates of each tissue sample were harvested.

Figure 1. Differential expression of rice genes in the presence of different plant hormones. (A) Number of genes significantly (at least 2-fold with P-value ≤ 0.01) up- and downregulated in the presence of different plant hormones is shown. (B) The representative significantly enriched gene ontology (GO) categories in the genes showing differential expression are given. Only the data of significantly enriched (P-value < 0.001) GO categories are shown. IAA, auxin; BAP, cytokinin; ABA, abscisic acid; ACC, ethylene derivative; SA, salicylic acid; JA, jasmonic acid.

Research Findings

• Plant hormones control various cellular processes by regulating the expression of several genes. To reveal the transcriptional responses of different hormones in rice, microarray analysis was performed using total RNA isolated from seedlings treated exogenously with various plant hormones involved in development and stress responses as per interest of our laboratory, including auxin (IAA), cytokinin (BAP), abscisic acid (ABA), ethylene derivative (ACC), salicylic acid (SA) and jasmonic acid (JA), using Affymetrix rice whole genome arrays. The microarray data are available at Gene Expression Omnibus database at NCBI under the series accession number GSE37557.
• The microarray data analysis identified a total of 4171 genes differentially expressed significantly (≥ 2-fold with at least 0.01 P-value) under at least one hormone condition tested. The list of all these genes with fold-change and P-value under different hormone conditions is given in Supplementary Table S1. Among these, 428, 279, 3635, 183, 798 and 615 genes were differentially expressed on IAA, BAP, ABA, ACC, SA and JA application, respectively (Fig. 1A). The results indicate that ABA altered the expression of largest number of genes, while ACC least number of genes under our experimental conditions. In each case, the number of upregulated genes was higher than the number of downregulated genes.
• To gain insights into the putative functions of hormone-responsive genes, their annotation was explored from the Rice Genome Annotation Project database (http://rice.plantbiology. msu.edu/). Several genes well-known to be induced by different hormones were represented in our list of hormone-responsive genes. For example, Aux/IAA and GH3 gene family members, type-A response regulators and several stress-responsive genes were included in the IAA-, BAP- and ABA-induced gene lists, respectively. These results confirmed the reliability of our micro-array experiments. The analysis revealed that genes involved in various cellular processes and pathways were regulated by different plant hormones. Several genes encoding for transcription factors belonging to different families were also represented in the hormone-responsive genes, indicating the regulatory role of plant hormones in transcription.
• Further, we analyzed various gene ontology (GO) categories enriched in the hormone- responsive genes (Fig. 1B). Some GO categories were enriched in all/most of the hormone-responsive genes and others were enriched under two or more hormone conditions. For example, the molecular function category, oxidoreductase activity, was highly enriched in all the hormone-responsive genes. Likewise, genes involved in metabolic processes and localized in nucleus were also enriched in most of the hormone-responsive gene lists. The genes related to stress response were found to be enriched on ABA, IAA and SA application as expected. Overall, these results suggest that different hormones largely regulate similar cellular processes in rice. Similar results have been obtained based on microarray analysis in Arabidopsis.

Labs working on this Project

• National Institute of Plant Genome Research (NIPGR); Aruna Asaf Ali Marg; New Delhi, India

Corresponding Author

Mukesh Jain (email: mjain@nipgr.res.in )