Tomato PYR/PYL/RCAR abscisic acid receptors show high expression in root, differential sensitivity to the abscisic acid agonist quinabactin, and the capability to enhance plant drought resistance

Abstract

Abscisic acid (ABA) plays a crucial role in the plant's response to both biotic and abiotic stress. Sustainable production of food faces several key challenges, particularly the generation of new varieties with improved water use efficiency and drought tolerance. Different studies have shown the potential applications of Arabidopsis PYR/PYL/RCAR ABA receptors to enhance plant drought resistance. Consequently the functional characterization of orthologous genes in crops holds promise for agriculture. The full set of tomato (Solanum lycopersicum) PYR/PYL/RCAR ABA receptors have been identified here. From the 15 putative tomato ABA receptors, 14 of them could be grouped in three subfamilies that correlated well with corresponding Arabidopsis subfamilies. High levels of expression of PYR/PYL/RCAR genes was found in tomato root, and some genes showed predominant expression in leaf and fruit tissues. Functional characterization of tomato receptors was performed through interaction assays with Arabidopsis and tomato clade A protein phosphatase type 2Cs (PP2Cs) as well as phosphatase inhibition studies. Tomato receptors were able to inhibit the activity of clade A PP2Cs differentially in an ABA-dependent manner, and at least three receptors were sensitive to the ABA agonist quinabactin, which inhibited tomato seed germination. Indeed, the chemical activation of ABA signalling induced by quinabactin was able to activate stress-responsive genes. Both dimeric and monomeric tomato receptors were functional in Arabidopsis plant cells, but only overexpression of monomeric-type receptors conferred enhanced drought resistance. In summary, gene expression analyses, and chemical and transgenic approaches revealed distinct properties of tomato PYR/PYL/RCAR ABA receptors that might have biotechnological implications.

Keywords: Abscisic acid (ABA); drought resistance; tomato ABA receptor; tomato clade A PP2C..

Fig. 1.

Cladogram and amino acid sequence alignment of tomato PYR/PYL ABA receptors. (A) Cladogram of the multiple sequence alignment of tomato and Arabidopsis PYR/PYL receptors, indicating three major subfamilies and the ungrouped 2g076770. Those tomato receptors further described in the text are in bold face. (B) Sequence and secondary structure alignment of tomato PYR/PYL ABA receptors and Arabidopsis PYR1 protein. The predicted secondary structure of the tomato proteins was indicated, taking as a model the crystallographic structure of PYR1 (Protein DataBank Code 3K90) and using the Espript interface (http://espript.ibcp.fr/). Boxes indicate the position of the gate and latch loops. Black asterisks mark residues K59, A89, E94, R116, Y120, S122, and E141 of PYR1 involved in ABA binding. Grey asterisks mark conserved residues of the tomato receptor family that differ in tomato 2g076770 protein. (This figure is available in colour at JXB online.)

Fig. 2.

Relative gene expression of tomato ABA receptors in leaf, root, and fruit was determined by RNA-Seq and microarray analysis. (A, B) The transcriptome of the inbred tomato cultivar Heinz 1706 was analysed using Illumina RNA-Seq technology (Tomato genome Consortium, 2012). Data show gene transcription of tomato receptors grouped in three subfamilies and the ungrouped 2g076770 in leaf and root (A) and during fruit development and ripening (B). Significant expression of 2g076770 and 12g095970 was not detected in these tissues. RPKM, reads per kilobase of exon model per million mapped reads; MG, mature green stage; B, breaker stage. (C, D) Relative RNA abundance based on GC-RMA values (GC content–robust multiarray analysis) obtained from the Affymetrix exon tomato microarray (EUTOM3) hybridized with fruit pericarp RNA of Moneymaker and TO-937 (C) and fruit epidermis RNA of the Microtom background (D) at the breaker stage.

Fig. 3.

Interactions between PYR/PYL ABA receptors and clade A PP2Cs. Interaction was determined by growth assay on media lacking histidine and adenine (–H, –A), which were supplemented or not with 50 μM ABA (+ABA). Dilutions (10^–1, 10^–2, and 10^–3) of saturated cultures were spotted onto the plates. (A) Interaction of tomato receptors with Arabidopsis PP2Cs. (B) Interaction of a tomato PP2CA-like phosphatase (5g052980) and Arabidopsis (left) or tomato (right) PYR/PYL ABA receptors. (C) Interaction of a tomato ΔN HAB1-like phosphatase (12g096020) and Arabidopsis (left) or tomato (right) PYR/PYL ABA receptors. (D) Elution profiles after size-exclusion chromatography of four tomato ABA receptors in the absence of ABA. The lines show the absorbance recorded at 280nm. Molecular mass markers are indicated in kDa. (This figure is available in colour at JXB online.)

Fig. 4.

ABA-dependent PP2C inhibition mediated by tomato ABA receptors. PP2C activity was measured in vitro using a phosphopeptide substrate in the absence or presence of ABA at a 1:4 ratio of phosphatase:receptor (0.5:2 μM stoichiometry). Data are averages ±SD for three independent experiments. (A) ABA-dependent inhibition of AtABI2 by tomato receptors. Values represent percentage activity compared with 100% in the absence of receptor and ABA. (B) Phosphatase activity of tomato 5g052980 in the presence of tomato receptors. (C) Phosphatase activity of tomato ΔN 12g096020 PP2C in the presence of tomato receptors. PP2C activity was measured in the absence or presence of 0, 0.1, 0.5, 1, or 10 μM ABA or QB. The column labelled as buffer contained an equivalent volume of HIS elution buffer and 0.5% DMSO. (D) Inhibition of tomato seed germination is more sensitive to ABA than QB. Seed germination was scored 72h after sowing. * indicates P<0.05 (Student’s t-test) when comparing data of plates supplemented with ABA or QB with plates lacking these chemicals. (E) QB treatment induces expression of ABA- and stress-responsive genes. Ten-day-old tomato seedlings were either mock treated or treated with 10 μM ABA or QB for 3h. The histograms indicate the relative induction by ABA or QB treatment of the indicated tomato genes with respect to mock conditions (value 1). (This figure is available in colour at JXB online.)

Fig. 5.

Overexpression of monomeric-type tomato receptors in Arabidopsis confers enhanced response to ABA and drought resistance. (A) Immunoblot analysis using antibody against the haemagglutinin (HA) tag shows expression of tomato ABA receptors in 21-day-old seedlings (two independent Arabidopsis T₃ transgenic lines for each tomato receptor). Ponceau staining is shown below. RBC indicates ribulose-1,5-bisphosphate carboxylase. (B) ABA-mediated inhibition of seedling establishment in transgenic lines compared with non-transformed Col plants and the hab1-1abi1-2 double mutant. * indicates P<0.05 (Student’s t-test) when comparing data of transgenic lines and the hab1-1abi1-2 mutant with non-transformed Col plants in the same assay conditions. Approximately 100 seeds of each genotype (three independent experiments) were sown on MS plates lacking or supplemented with 0.25 μM ABA. Seedlings were scored for the presence of both green cotyledons and the first pair of true leaves after 8 d. Values are averages ±SE. (C) Enhanced sensitivity to ABA-mediated inhibition of root growth of transgenic lines and the hab1-1abi1-2 mutant compared with non-transformed Col plants. Photographs show representative seedlings 10 d after the transfer of 4-day-old seedlings to MS plates lacking or supplemented with 5 μM ABA. Right panel: quantification of ABA-mediated root growth inhibition (values are means ±SE; growth of Col wild type on MS medium was taken as 100%). (D–F) Transgenic lines overexpressing monomeric-type receptors show enhanced drought resistance and survival, and higher RWC compared with non-transformed plants. (D) Two-week-old plants were deprived of water for 20 d and then re-watered. Photographs were taken at the start of the experiment (0-d), after 16 d and 20 d of drought, and 3 d after re-watering. (E) Percentage survival of non-transformed Col, hab1-1abi1-2, and transgenic lines 3 d after re-watering. (F) RWC of non-transformed Col, hab1-1abi1-2, and transgenic lines after 11, 14, and 17 d of water withdrawal. (This figure is available in colour at JXB online.)