The Arabidopsis RNA-binding protein AtRGGA regulates tolerance to salt and drought stress

Abstract

Salt and drought stress severely reduce plant growth and crop productivity worldwide. The identification of genes underlying stress response and tolerance is the subject of intense research in plant biology. Through microarray analyses, we previously identified in potato (Solanum tuberosum) StRGGA, coding for an Arginine Glycine Glycine (RGG) box-containing RNA-binding protein, whose expression was specifically induced in potato cell cultures gradually exposed to osmotic stress. Here, we show that the Arabidopsis (Arabidopsis thaliana) ortholog, AtRGGA, is a functional RNA-binding protein required for a proper response to osmotic stress. AtRGGA gene expression was up-regulated in seedlings after long-term exposure to abscisic acid (ABA) and polyethylene glycol, while treatments with NaCl resulted in AtRGGA down-regulation. AtRGGA promoter analysis showed activity in several tissues, including stomata, the organs controlling transpiration. Fusion of AtRGGA with yellow fluorescent protein indicated that AtRGGA is localized in the cytoplasm and the cytoplasmic perinuclear region. In addition, the rgga knockout mutant was hypersensitive to ABA in root growth and survival tests and to salt stress during germination and at the vegetative stage. AtRGGA-overexpressing plants showed higher tolerance to ABA and salt stress on plates and in soil, accumulating lower levels of proline when exposed to drought stress. Finally, a global analysis of gene expression revealed extensive alterations in the transcriptome under salt stress, including several genes such as ASCORBATE PEROXIDASE2, GLUTATHIONE S-TRANSFERASE TAU9, and several SMALL AUXIN UPREGULATED RNA-like genes showing opposite expression behavior in transgenic and knockout plants. Taken together, our results reveal an important role of AtRGGA in the mechanisms of plant response and adaptation to stress.

Figure 1.

Expression analysis of RGGA in potato and Arabidopsis. A, Expression of RGGA in cells of potato in control conditions and after gradual (PEG-adapted) or abrupt (PEG-shocked) exposure to PEG. B, Gene expression of RGGA in Arabidopsis MM2D cells exposed for 24 h to NaCl (150 mm), ABA (50 μm), or 10% (w/v) PEG. C, AtRGGA expression in 14-d-old seedlings of Arabidopsis treated for 24 h with different concentrations of NaCl as indicated. D, AtRGGA expression in Arabidopsis seedlings after 48 h of exposure to 35% (w/v) PEG, NaCl (120 mm), or ABA (10 μm). Gene expression analyses were conducted by quantitative reverse transcription (qRT)-PCR.

Figure 2.

A, Schematic representation of Arabidopsis AtRGGA protein domain organization. Gray boxes indicate the locations of the Stm1 N-terminal domain (Stm1; InterPro no. IPR019084) and the hyaluronan/mRNA-binding domain (HABP4_PAI1_RBP1; InterPro no. IPR006861). B, EMSA of Arabidopsis RNA incubated with recombinant AtRGGA (His-RGGA). RNA was extracted from NaCl-treated (Salt Stress RNA) or untreated (Control RNA) plants and labeled with biotin. Unlabeled RNA (160-fold) was used as a competitor. Recombinant PYR1 (His-PYR1) served as a negative control. C, EMSA of Arabidopsis total, poly(A⁺), and poly(A⁻) RNA incubated without or with recombinant AtRGGA (His-RGGA). The brackets indicate labeled RNA, and the arrows indicate RGGA-bound RNA.

Figure 3.

AtRGGA promoter activity in tissues of Arabidopsis. GUS staining was performed in vegetative and reproductive tissues of transgenic Arabidopsis plants expressing the GUS reporter gene under the control of the AtRGGA promoter. Five-day-old seedling (A), root (B), leaves (C), inflorescences (E), and siliques (L–N) were stained. Closeup views of stomata (D), anther (F), stigma (G), ovary (H), and ovule (I) are also shown.

Figure 4.

AtRGGA protein localization in Arabidopsis. Confocal microscopy visualization was performed for transgenic Arabidopsis plants expressing a YFP-RGGA or RGGA-YFP (D) fusion protein. Propidium iodide staining, YFP fluorescence, and merged images of root apex (A), root elongation zone (B and D), and leaf epidermal cells (C) are shown.

Figure 5.

Characterization of plants with modified expression of AtRGGA. A, Identification of an RGGA knockout mutant. The top shows a representative model of the At4g16830 locus encoding RGGA in Arabidopsis, showing the location of the T-DNA insertion in SALK_143514 (rgga). The bottom shows semiquantitative reverse transcription-PCR analysis to confirm that the expression of At4g16830 is abolished in the rgga mutant. β-Actin was amplified as an internal standard. B, Immunoblot using α-FLAG antibody of total proteins extracted from Arabidopsis plants transformed to overexpress the fusion protein FLAG-RGGA (35S::FLAG-RGGA). Different transgenic lines (#10, #15, #18, and #20), along with the wild type (Col-0) and controls transformed with the empty binary plasmid (empty vector), are shown. Ponceau staining of Rubisco small subunit (RbcS) served as a loading control. C, Germination analysis of AtRGGA knockout and transgenic plants in the presence of NaCl (120 mm). Germination was scored in terms of fully expanded cotyledons 7 d after stratification. Data reported are means ± sd from three independent experiments. The asterisk denotes a significant difference between Col-0 and rgga (P < 0.05) according to Student’s t test. D, Survival test of 18-d-old seedlings germinated on germination medium (GM; 4.3 g L⁻¹ MS salts, 30% [w/v] Suc, pH 5.7) and transferred to NaCl (180 mm) medium. Survival was scored daily in terms of absence of necrotic or bleached leaves. Data are means ± sd of three independent experiments (n = 30). Asterisks denote statistically significant differences versus Col-0 assessed by χ² test (*P < 0.05, **P < 0.01).

Figure 6.

Phenotypes of RGGA knockout and overexpressing plants. A, Phenotypes of Col-0, rgga, and 35S::FLAG-RGGA plants grown on GM for 14 d and exposed for 7 d to NaCl (180 or 200 mm). B, Water loss of leaves detached from Col-0, rgga, and 35S::FLAG-RGGA plants. Data are presented as percentages of initial weight lost at different time points (1, 2, and 3 h). Each point consists of average values ± sd (n = 5 for each line). Data relative to 35S::FLAG-RGGA represent means of three independent transgenic lines. C, Quantification of primary root length of 14-d-old seedlings germinated for 4 d on GM and transferred to control GM medium or medium containing 20 µm ABA. Values are means ± sd (n = 25). The asterisk indicates a statistically significant difference assessed by Student’s t test (P < 0.001). D, Photograph of seedlings grown as described in C. E, Survival test of 18-d-old seedlings germinated on GM and transferred to ABA (50 µm) medium. Survival was scored daily in terms of absence of necrotic or bleached leaves. Data are means ± sd of three independent experiments (n = 30). Asterisks denote statistically significant differences versus Col-0 assessed by χ² test (*P < 0.05, **P < 0.01).

Figure 7.

Phenotypes of AtRGGA knockout and overexpressing plants. A, Representative 4-week-old plants of Col-0, rgga, and 35S::FLAG-RGGA genotypes grown in control conditions or after 7-d NaCl (300 mm) or drought treatment. B, Pro concentrations in Col-0, rgga, and 35S::FLAG-RGGA plants treated as described in A. Asterisks indicate statistically significant differences assessed by Student’s t test (P < 0.01).