Transcriptome analysis reveals specific modulation of abscisic acid signaling by ROP10 small GTPase in Arabidopsis

Abstract

Abscisic acid (ABA) is a hormone that modulates a variety of agronomically important growth and developmental processes and various stresses responses, but its signal transduction pathways remain poorly understood. ROP10, a member of ROP small GTPases in Arabidopsis (Arabidopsis thaliana), is a plasma membrane-associated protein specifically involved in negative regulation of ABA responses. To dissect the ROP10-mediated ABA signaling, we carried out transcriptome analysis using the Arabidopsis full-genome chip. Our analysis revealed a total of 262 and 125 genes that were, respectively, up- and down-regulated (> or =2-fold cutoff) by 1 mum ABA in wild type (Wassilewskija [Ws]); 42 up-regulated and 38 down-regulated genes have not been identified in other studies. Consistent with the nonpleiotropic phenotypes of rop10-1, only three genes were altered in rop10-1 in the absence of ABA treatment. In response to 1 microm ABA, 341 and 127 genes were, respectively, activated and repressed in rop10-1. Interestingly, a particular subset of 21 genes that were not altered by 1 microm ABA in Ws but only activated in rop10-1 was identified. Reverse transcription-polymerase chain reaction analysis revealed the existence of three distinct categories of ABA dose-response patterns. One novel category is characterized by their ABA unresponsiveness in Ws and activation in rop10-1 at 1 microm but not 10 and 100 microm of ABA. This indicates that ROP10 gates the expression of genes that are specific to low concentrations of ABA. Furthermore, almost all of these 21 genes are known to be highly induced by various biotic and abiotic stresses. Consequently, we found that rop10-1 enhanced the sensitivity of seed germination inhibition to mannitol and sodium chloride. Our results suggest that ROP10 negatively regulates ABA responses by specifically and differentially modulating the ABA sensitivity of a subset of genes including protein kinases and zinc-finger family proteins.

Figure 1.

RT-PCR analysis of gene expression in response to ABA doses. A total of 19 genes were investigated using 7-d-old wild-type (Ws) and rop10-1 (r10) seedlings treated with 0, 1, 10, and 100 μm of ABA for 4 h. A, Five genes that show similar ABA activation in Ws and rop10-1. B, One gene that exhibits similar ABA repression in Ws and rop10-1. C, One gene that shows ≥2-fold activation by 1 μm ABA in rop10-1 but weakly in Ws. D to F, Nine genes from the particular subset of 21 genes that show ABA activation in rop10-1. D, Four genes in the first category that is not responsive to all concentrations of ABA in Ws but induced in rop10-1 only by 1 μm ABA. E, Three genes in the second category that is not responsive to 1 μm ABA but only very weakly induced by higher doses of ABA in Ws. These genes are induced in rop10-1 only by 1 μm ABA. F, Two genes in the third category that is significantly activated by 1 μm ABA in rop10-1 but not greatly induced by 1 μm ABA in Ws. However, it is induced by higher ABA concentrations in both Ws and rop10-1. G, MYB2 and two MYB2 closely related MYB genes that exhibit differential expression patterns. H, ACT2, an internal control. Three biological replicates showed the similar patterns, and one of the representative RT-PCR gel pictures was shown for each gene.

Figure 2.

Real-time PCR analysis of transcript changes in response to ABA doses. Samples were essentially the same as in Figure 1. Three genes in the first category (A–C) and one gene each for the second (D) and third (E) categories were selected for quantitative analysis. Relative mRNA levels were determined by first normalizing their PCR threshold cycle numbers with those of the reference (ACT2) and setting the relative mRNA levels for each gene in nontreated Ws at 1. The bar indicates the sd (n = 3).

Figure 3.

rop10-1 enhances the sensitivity of seed germination inhibition to mannitol and NaCl. Both wild-type (Ws) and rop10-1 seeds were sown on the half-strength MS medium supplemented with ABA, mannitol, or NaCl. Germination was scored 2 d after the cold treatment. Each data point represents the average and sd of germination percentage for three replicates, each with 60 to 90 seeds.