Salt-Related MYB1 Coordinates Abscisic Acid Biosynthesis and Signaling during Salt Stress in Arabidopsis

Abstract

Abiotic stresses, such as salinity, cause global yield loss of all major crop plants. Factors and mechanisms that can aid in plant breeding for salt stress tolerance are therefore of great importance for food and feed production. Here, we identified a MYB-like transcription factor, Salt-Related MYB1 (SRM1), that negatively affects Arabidopsis (Arabidopsis thaliana) seed germination under saline conditions by regulating the levels of the stress hormone abscisic acid (ABA). Accordingly, several ABA biosynthesis and signaling genes act directly downstream of SRM1, including SALT TOLERANT1/NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3, RESPONSIVE TO DESICCATION26, and Arabidopsis NAC DOMAIN CONTAINING PROTEIN19. Furthermore, SRM1 impacts vegetative growth and leaf shape. We show that SRM1 is an important transcriptional regulator that directly targets ABA biosynthesis and signaling-related genes and therefore may be regarded as an important regulator of ABA-mediated salt stress tolerance.

Figure 1.

SRM1 impacts germination on medium with high salt levels. A, T-DNA insertion site in the SRM1 gene. Black boxes indicate untranslated regions, white boxes indicate exons, the black thick line denotes intron, and ATG and TAA indicate start and stop codons, respectively. FP (Forward Primer) and RP (Reverse Primer) indicate primers used for SRM1 RT-PCR. B, RT-PCR of SRM1 transcript in the wild type and srm1 (3-week-old soil-grown seedlings). Glyceraldehyde 3-phosphate dehydrogenase (GADPH) was used as mRNA control. C, Quantification of seedling greening on NaCl and KCl plates. D, Cotyledon greening of wild-type and srm1 seedlings on MS medium or MS medium supplemented with 150 mm NaCl. E, Germination time courses of srm1, Col-0, and T3 homozygous transgenic plants carrying the SRM1 genomic fragment (gSRM1) in the srm1 mutant background in the presence of 125 mm NaCl. All germination and cotyledon greening experiments were performed in triplicate, and more than 100 seeds and seedlings were used for each replicate. F, A functional 35S-driven SRM1-GFP in srm1 mutant background (35S:SRM1-GFP srm1) localizes to the nucleus in Arabidopsis seedling root cells. Error bars indicate sd. *, P < 0.05; and **, P < 0.01 (Student’s t test). Bars = 5 mm (D) and 5 µm (F).

Figure 2.

SRM1 overexpressing plants display opposing phenotypes to srm1 mutant. A, qRT-PCR of SRM1 in the wild type, srm1, complementation line 35S:SRM1-GFP in srm1 mutant background (35S:SRM1-GFP srm1), and two SRM1 overexpression lines (OX6-4 and OX4-3). Three-week-old soil-grown plants were used for mRNA samples. Data are from one out of three experiments with similar profiles. B, Cotyledon greening of the wild type, srm1, and the two SRM1 overexpressing lines (OX6-4 and OX4-3) on MS medium or MS medium supplemented with 150 mm NaCl. C, Quantification of seedling greening on plates such as those in B. Three independent experiments were performed. D, Four-week-old rosettes of soil-grown wild type, srm1, and the two SRM1 overexpressing lines under long-day conditions. Error bars indicate sd. *, P < 0.05; and **, P < 0.01 (Student’s t test). Bars = 5 mm (B) and 2 cm (D).

Figure 3.

Microarray and qRT-PCR analysis of altered gene expression in srm1 mutant. A, Pie chart of overrepresented PageMan ontology terms associated with genes up- (log₂FC > 1, P < 0.05) or down-regulated (log₂FC < –1, P < 0.05) in the srm1 compared with the wild type. B, Confirmation of microarray data using qRT-PCR. Blue and yellow colors indicate decrease and increase of gene expression, respectively. Scale bar shows log₂ fold changes. C, qRT-PCR of three genes (NCED3/STO1, RD26, and ANAC019) differentially expressed from the microarray experiments in the wild type, srm1, and two SRM1 overexpression lines. Data represent three biological replicates. Error bars indicate sd. *, P < 0.05; and **, P < 0.01 (Student’s t test). CHO, CARBOHYDRATE; CYP83A1, CYTOCHROME P450 FAMILY83-SUBFAMILY A-POLYPEPTIDE1; FLN2, FRUCTOKINASE-LIKE PROTEIN2; FSD1, FE-SUPEROXIDE DISMUTASE1; GRF11, GENERAL REGULATORY FACTOR11; PP2C, PROTEIN PHOSPHATASE 2C; UNE15, UNFERTILIZED EMBRYO SAC15.

Figure 4.

SRM1 affects plant responses to ABA and impacts ABA production. A, Germination assay of the wild type, srm1, and two SRM1 overexpressing lines on MS medium or on medium supplemented with different concentrations of ABA. B, Root growth assay of the wild type, srm1, and two SRM1 overexpressing lines. Seeds for the different genotypes were germinated on MS medium and grown for 7 d under 16-h-light/8-h-dark conditions. Seedlings were then transferred to MS medium supplemented with 50 µm ABA or to plates with control medium. Root growth was measured after an additional 10 d on the new medium. C, LDA plot of leaf shape analysis of srm1 and the wild type with or without ABA treatment. Seven-day-old MS-grown seedlings were transferred to DMSO-containing and ABA-containing (2 µm) MS medium for another 2 weeks for growing. D and E, ABA levels in germinating seedlings (3-d-old seedlings and seeds grown in control plates and 5-d-old seedlings and seeds in 125 mm NaCl-containing plates) and in 3-week-old rosette leaves (without or with 2-h 200 mm NaCl treatment). All experiments were performed independently three times. Error bars indicate sd. *, P < 0.05; and **, P < 0.01 (Student’s t test). F.W., fresh weight.

Figure 5.

SRM1 is induced by stress but not by ABA and is necessary for induction of NCED3/STO1, RD26, and ANAC019 during salt stress. A, qRT-PCR of an estradiol-inducible SRM1 line after 1, 2, and 4 h of induction. B, Expression of NCED3/STO1, RD26, and ANAC019 after induction of SRM1 using estradiol. ACTIN2 was used for normalization of expression. Seven-day-old light-grown seedlings were used for mRNA extraction. C, Induction of SRM1 after treatment with 50 µm ABA, dehydration, or 200 mm NaCl in light-grown 7-d-old seedlings. Expression was normalized against ACTIN2. D to F, Differential mRNA accumulation of NCED3/STO1, RD26, or ANAC019 in srm1 mutant under stress condition. Light-grown 7-d-old wild-type and srm1 seedlings were treated with 200 mm NaCl and 50 µm ABA for 2 h, respectively. All experiments were performed independently three times. Error bars indicate sd. *, P < 0.05; and **, P < 0.01 (Student’s t test).

Figure 6.

SRM1 binds to specific elements in the promoters of NCED3/STO1, RD26, and ANAC019 and activates them in vitro and in vivo. A and B, PLACE and MEME analyses identified an AACCAT motif in the promoters of NCED3/STO1, RD26, and ANAC019 (A) at sites indicated in B. Numbers indicate base pairs from the ATG start site of the respective genes. C, Probes used for EMSAs in D. Sequence P was the probe designed from the NCED3/STO1 promoter (N3R2), C1 was the competitor with the same sequence as P, and C2, C3, and C4 were the competitors with a mutated binding motif in their sequences. D, EMSA analyses to test binding of SRM1 to the DNA fragment harboring the AACCAT motif. A segment corresponding to the second AACCAT motif in the NCED3/STO1 promoter (segment P in B) was used for the analysis. SRM1 interacted with this segment probe as indicated by the band shift in D (left section), which could be reversed if an excess of unlabeled probe was included in the assay. The right section shows competitive EMSA analyses with mutated AACCAT motifs in the probe segments. Note that only the nonmutated probe can completely outcompete the labeled probe. CK indicates the probe alone without protein or competitor addition. E, ChIP-qPCR analyses of immune precipitates of a functional SRM1-GFP. Different regions of the promoters that contained AACCAT related motifs were used as templates for the qPCR analysis (see outline in B). GFP control plants were used as normalization for the qPCR. Experiments were performed independently three times. F, Transactivation assays for the NCED3/STO1, RD26, and ANAC019 promoters driving luciferase genes by the SRM1. Three-week-old rosette leaves from the srm1 mutant were used for protoplast isolation, and the relative luciferase activity was normalized by Renilla luciferase activity and represented as fold change. Error bars indicate sd. *, P < 0.05; and **, P < 0.01 (Student’s t test).

Figure 7.

Schematic model for SRM1 function during salt stress. SRM1 is induced in response to salt and dehydration but not to ABA. SRM1 controls expression of NCED3/STO1, RD26, and ANAC019 both during normal growth conditions and under salt stress conditions. However, ABA can, by a currently unknown mechanism, induce RD26 and ANAC019 in the absence of SRM1, indicating a complex regulatory principle for RD26 and ANAC019. The activation of the ABA synthesis and signaling components by SRM1 impacts the growth and development of the plant and its ability to withstand external stress.