The tomato mutant ars1 (altered response to salt stress 1) identifies an R1-type MYB transcription factor involved in stomatal closure under salt acclimation

Abstract

A screening under salt stress conditions of a T-DNA mutant collection of tomato (Solanum lycopersicum L.) led to the identification of the altered response to salt stress 1 (ars1) mutant, which showed a salt-sensitive phenotype. Genetic analysis of the ars1 mutation revealed that a single T-DNA insertion in the ARS1 gene was responsible of the mutant phenotype. ARS1 coded for an R1-MYB type transcription factor and its expression was induced by salinity in leaves. The mutant reduced fruit yield under salt acclimation while in the absence of stress the disruption of ARS1 did not affect this agronomic trait. The stomatal behaviour of ars1 mutant leaves induced higher Na(+) accumulation via the transpiration stream, as the decreases of stomatal conductance and transpiration rate induced by salt stress were markedly lower in the mutant plants. Moreover, the mutation affected stomatal closure in a response mediated by abscisic acid (ABA). The characterization of tomato transgenic lines silencing and overexpressing ARS1 corroborates the role of the gene in regulating the water loss via transpiration under salinity. Together, our results show that ARS1 tomato gene contributes to reduce transpirational water loss under salt stress. Finally, this gene could be interesting for tomato molecular breeding, because its manipulation could lead to improved stress tolerance without yield penalty under optimal culture conditions.

Keywords: MYB transcription factor; Solanum lycopersicum; insertional mutagenesis; salt stress; stomatal aperture; transpiration.

Figure 1

The dominant salt‐sensitive ars1 mutant identifies an R1‐MYB gene of tomato. (a) Phenotype of wild type and T₁ ars1 mutant plants in control condition without NaCl and after 20 days of 200 mm NaCl treatment, in shoot and root. (b) DNA‐blot analysis to determine number of T‐DNA insertions in the T₁ ars1 mutant plant using the coding region of the nptII gene as probe. Single restriction fragments observed in genomic DNA digested with BamHI (12 kb) and EcoRI (1 kb) indicate the presence of a single T‑DNA insertion in the ars1 genome. (c) Identification of ARS1, a R1‐MYB type gene tagged by the T‐DNA and characterization of the insertional event. The presence in the 5′‐untranslated region (5′UTR) of an ABA‐responsive element is indicated by vertical black straight line. Exons and UTRs are represented by black and grey boxes, respectively, whereas introns are represented by horizontal lines. Start and stop codons for translation are indicated, as well as the SANT domain characteristic of this family of transcription factors. Positioning of primers designed for detecting presence of T‐DNA insertion and for genotyping is showed in the ARS1 genomic sequence as well as in the T‐DNA insert.

Figure 2

Sequence analysis of ARS1. (a) Phylogenic tree constructed with MEGA5 software based on neighbour‐joining method after sequences alignment with Clustal‐X. Sequences of Arabidopsis CCA1‐like single MYB‐like domain proteins had previously been described (Yanhui et al., 2006). The single MYB‐like domain proteins from potato (StMYB1R1, Shin et al., 2011) and rice (OsMYB48‐1, Xiong et al., 2014) implicated in salt stress resistance were also included (bold letters). Proteins integrated in the CCA1‐like clade are indicated with a bracket including ARS1 protein (bold letters). Scale indicates percentage of substitutions. (b) Multiple sequence alignment of the conserved MYB‐like and adjacent P‐rich domains of the CCA1‐like proteins showed in (a). An arrow indicates the residue where the T‐DNA insertion changes ARS1 reading frame including three amino acids (VVC) before a stop codon.

Figure 3

The null ars1 mutant shows salt sensitivity to long term. (a) Plants of wild type (WT) and ars1 mutant were grown in greenhouse. Salt stress (100 mm NaCl) was applied when the plants had ten true leaves. Pictures are representatives of the eight plants per treatment after 0, 30 and 60 days of salt treatment (DST). 0 DST means just before the start of the salt treatment. (b) Fruit yield of WT and ars1 mutant without NaCl (control) and salt stress condition at the end of the assay. (c) Evolution of the Na⁺ concentration in leaves of WT and ars1 during 50 DST. (d) Stomatal conductance in leaves of WT and ars1 plants without NaCl (control) and after 30 DST (salt). Measurements were taken at dawn and after 2 h of light. (e) Evolution of the stomatal conductance between 2 and 5 h of light in leaves of WT and ars1 plants grown during 50 DST. Values are means ± SE of eight individual plants per line and condition. Asterisks indicate significant differences by Student's t‐test between WT and mutant plants (P < 0.05).

Figure 4

The ars1 mutant shows increased stomatal aperture and Na⁺ accumulation under salt stress. Wild type (WT) and ars1 mutant plants were grown in hydroponic culture adding 200 mm NaCl to the Hoagland solution for 10 days when plants had developed ten true leaves. Measurements were taken in 3rd and 4th developed leaves. (a) Stomatal conductance and transpiration rate in leaves of WT and mutant without NaCl (control) and after 1 day of salt treatment (DST). (b) Stomatal aperture and percentage of open stomata in leaves of WT and ars1 after three and seven DST, and representative images of stomatal aperture in both genotypes and conditions without NaCl (control) and salt stress. (c) Shoot Na⁺ partitioning in WT and mutant plants, in stem (left hand side graphic) and leaves (right hand side graphic) after ten DST. Values are means ± SE of six individual plants per line. Asterisks indicate significant differences between WT and mutant plants by Student's t‐test (P < 0.05).

Figure 5

ars1 mutant responses to dehydration and ABA. (a) Plants of wild type (WT) and ars1 were submitted to two successive cycles of withholding irrigation followed by 1 day of rewatering at the eight‐leaf developmental stage, and stomatal conductance and transpiration rate were measured throughout the second dehydration cycle. (b) Water loss rate measured in detached leaf. The leaves were detached from light‐grown plants with eight fully developed leaves. Measures were taken during 8 h of incubation at room temperature. (c) Stomatal aperture of WT and ars1 mutant leaves treated with increasing ABA concentrations. Values are means ± SE of six individual plants per line. Asterisks indicate significant differences by Student's t‐test between WT and mutant plants (P < 0.05).

Figure 6

The overexpressing (OE) and silencing (RNAi) ARS1 lines corroborate that ARS1 gene is required in regulating stomatal conductance (g _s) and transpiration rate (E) under salt stress. (a) At the end of the salt stress assay (10 days at 200 mm NaCl), the leaves of OE lines did not show chlorosis, while ars1 mutant and RNAi lines showed a high level of leaf chlorosis, with WT leaves showing an intermediate response. (b) Relative values with respect to WT of g _s and E in the 3rd developed leaf of ars1, OE and RNAi lines after 3 days of salt treatment. The measurements were taken as indicated in Table S3. Asterisks indicate significant differences between WT and each one of the other lines by Student's t‐test (P < 0.05).

Figure 7

The relative expression of SlSOS1 and SLHKT1;2 increases with salinity in roots of the ars1 mutant and RNAi lines and decreases in roots of OE lines, compared with WT. Results of expression prior to salt treatment (no NaCl), after 12 h at 100 mm NaCl and other 36 h at 200 mm NaCl. The expression of WT prior to salt stress was set to 1. Values are means ± SE of six individual plants per line.