TY - JOUR
AU - Leng, Ping
AB - Abstract Abscisic acid (ABA) plays a vital role in coordinating physiological processes during fresh fruit ripening. Binding of ABA to receptors facilitates the interaction and inhibition of type 2C phosphatase (PP2C) co-receptors. However, the exact mechanism of PP2C during fruit ripening is unclear. In this study, we determined the role of the tomato ABA co-receptor type 2C phosphatase SlPP2C3, a negative regulator of ABA signaling and fruit ripening. SlPP2C3 selectively interacted with monomeric ABA receptors and SlSnRK2.8 kinase in both yeast and tobacco epidermal cells. Expression of SlPP2C3 was ABA-inducible, which was negatively correlated with fruit ripening. Tomato plants with suppressed SlPP2C3 expression exhibited enhanced sensitivity to ABA, while plants overexpressing SlPP2C3 were less sensitive to ABA. Importantly, lack of SlPP2C3 expression accelerated the onset of fruit ripening and affected fruit glossiness by altering the outer epidermis structure. There was a significant difference in the expression of cuticle-related genes in the pericarp between wild-type and SlPP2C3-suppressed lines based on RNA sequencing (RNA-seq) analysis. Taken together, our findings demonstrate that SlPP2C3 plays an important role in the regulation of fruit ripening and fruit glossiness in tomato. ABA signaling, cuticle, fruit glossiness, fruit ripening, negative regulator, protein phosphatase 2C, RNA-seq, SlPP2C3, tomato Introduction The phytohormone abscisic acid (ABA) functions in various physiological processes during the plant lifecycle, including seed dormancy and germination, root growth, stomatal guard cell expansion, leaf senescence, and fruit ripening (Schroeder et al., 2001; Finkelstein et al., 2002; Zhang et al., 2009). Plant ABA responses rely on signal transduction pathways (Cutler et al., 2010). The dominant ABA signaling pathway is composed of the PYRABACTIN RESISTANCE 1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTOR (PYR/PYL/RCAR) family of receptor proteins, group A type 2C protein phosphatases (PP2Cs), and sub-family 2 of the sucrose-nonfermenting 1-related kinases (SnRK2s; Fujii et al., 2009; Ma et al., 2009; Park et al., 2009; Umezawa et al., 2009). In the canonical ABA signaling model, binding of ABA to the PYR/PYL/RCAR receptor induces a conformational change in the receptor, resulting in competition between SnRK2 kinase and PP2C inhibition. The released SnRK2s can be activated by Raf-like kinases, and downstream substrates can be phosphorylated by the activated SnRK2s. These substrates include ABF (ABA-responsive element-binding factors) transcription factors or the SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1) ion channel, to elicit ABA responses (Cutler et al., 2010; Weiner et al., 2010; Hauser et al., 2011). PP2C was the first key component of the ABA signaling pathway to be identified (Koornneef et al., 1984). Arabidopsis group A PP2C members ABI1 (ABA INSENSITIVE 1) and ABI2 were cloned from abi1-1 and abi1-2 mutants with ABA-insensitive phenotypes, and confirmed to play a negative role in ABA signaling regulation with overlapping functions (Leung et al., 1994; Meyer et al., 1994; Merlot et al., 2001). To date, nine group A PP2C members have been identified as ABA signaling pathway components in Arabidopsis; ABI1, ABI2, HAB1 (HYPERSENSITIVE TO ABA1), HAB2, AHG1 (ABA-HYPERSENSITIVE GERMINATION 1), AHG3/AtPP2CA, and three HAIs (HIGHLY ABA-INDUCED PP2C GENE; (Saez et al., 2004; Nishimura et al., 2007; Bhaskara et al., 2012). PP2Cs interact with, and phosphorylate SnRK2s, and the activity of PP2Cs is inhibited by ABA receptors via both ABA-dependent and ABA-independent mechanisms (Hao et al., 2011; Nemoto et al., 2018). Structural studies indicate that Ser residues of the PYR/PYL/RCAR receptor insert into the PP2C active site and competitively occlude the access of substrates (Melcher et al., 2009; Miyazono et al., 2009). In this model, two residues are important for the interaction, one of which, Gly-180 of ABI1, is next to the PP2C active site; the Gly-to-Asp mutation (G180D in ABI1, G168D in ABI2 and G246D in HAB1) results in reduced PP2C-PYL binding affinity (Sheen, 1998; Robert et al., 2006; Umezawa et al., 2009). Trp-300 of ABI1, and the corresponding Trp residue of related PP2Cs, is another important residue, since it is the only residue of PP2C that approaches the ABA molecule in the interaction complex, and plays an important role in complex stabilization (Melcher et al., 2009; Dupeux et al., 2011). Several studies show selective interaction among PP2C and PYR/PYL/RCAR receptors (Antoni et al., 2012; Fuchs et al., 2014), but the underlying mechanism remains unclear. Most PP2Cs have redundant functions and display additive effects in ABA signaling. Single PP2C loss-of-function mutants exhibit mild phenotypic effects, while double or triple mutants show constitutive ABA responses (Rubio et al., 2009). Although the physiological functions of PP2C have been described in the model plants Arabidopsis and rice (Saez et al., 2004; Nishimura et al., 2007; Bhaskara et al., 2012), the functions of PP2C in fresh fruit development and ripening are unknown. Recent studies suggest that ABA plays a role in metabolism in tomato fruit ripening regulation (Galpaz et al., 2008; Zhang et al., 2009; Sun et al., 2012a, b, 2017). However, little is known about the function of ABA signaling components during fresh fruit ripening. Overexpression of the tomato ABA receptor gene SlPYL9 leads to early fruit ripening, while silencing using SlPYL9-RNAi affects mesocarp thickness and petal abscission (Kai et al., 2019). The function of SlPP2C1 has also been elucidated in tomato. Suppression of SlPP2C1 expression significantly accelerated fruit ripening, increased endogenous ABA accumulation, advanced the release of ethylene, and resulted in hypersensitivity to ABA, but it led to abnormal flower development, which may reduce fruit yield (Zhang et al., 2018). To further understand ABA signaling during fruit ripening, we explored the roles of SlPP2C3 in tomato fruits in the present work. We found that SlPP2C3 may play a crucial role in fruit maturation and quality by affecting the expression of genes related to ethylene and cuticle metabolism. Materials and methods Plant materials All tomato plants (Solanum lycopersicum L. ‘Micro Tom’), including wild type (WT) and transgenic lines, were grown in mixture of peat and roseite (2:1, v/v), under standard greenhouse conditions (25−30 °C day, 15−20 °C night, 70% humidity, natural lighting). For gene expression analysis, fruits were sampled at various developmental stages, including mature green (MG), three days after breaker (B+3), and 10 days after breaker (B+10). Generation of SlPP2C3 transgenic tomato lines For SlPP2C3-OE construction, the SlPP2C3 (Solyc06g076400.2.1) coding sequence (CDS) was cloned into the pRI101-AN vector (Takara Bio Inc, Shiga, Japan) under the control of the 35S promoter. For SlPP2C3-RNAi construction, a fragment of the N-terminus-specific sequence was amplified from the SlPP2C3 cDNA, and cloned in both sense and antisense orientations into the pCambia1305.1 plasmid (Cambia, Canberra, Australia) driven by the 35S promoter, to form a hairpin structure for RNA interference (RNAi). Primers are listed in SupplementaryTable S2, and schematic diagrams of SlPP2C3-OE and SlPP2C3-RNAi constructs are shown in Supplementary Fig. S1. Plasmids were introduced into Agrobacterium tumefaciens LBA4404 using the freeze-thaw transformation method (Chen et al., 1994). Briefly, 500 ng plasmid DNA was mixed with 50 μl LBA4404 competent cells and incubated on ice for 10 min. Cells were frozen in liquid nitrogen for 1 min, then incubated at 37 °C for 5 min. A 1 ml sample of Luria-Bertani (LB) medium was added to cells and incubated at 28 °C for 4 h. Cells were spread on an LB agar plate containing appropriate antibiotics for selection of transformants. The SlPP2C3-OE and SlPP2C3-RNAi constructs were introduced into tomato (‘Micro-Tom’) via Agrobacterium tumefaciens-mediated transformation, according to a previous report (Sun et al., 2006). Regenerated plants with corresponding antibiotic (kanamycin for SlPP2C3-OE, hygromycin B for SlPP2C3-RNAi) resistance were selected. Overexpressing (OE) lines were identified through kanamycin resistance (50 mg l-1) and PCR detection. Positive SlPP2C3-RNAi transgenic plants were identified through hygromycin B resistance (5 mg l-1) and β-glucuronidase (GUS) staining. T0 plants were self-pollinated to generate T1 and T2 plants. Phenotypic and molecular analyses of plants and fruits were performed on T2 plants after selfing of a single homozygous T1 plant, while drought resistance, seed germination and primary root growth assays were performed on T3 seeds obtained from selfing of homozygous T2 plants. Sub-cellular localization and bimolecular fluorescence complementation (BiFC) assay To investigate the sub-cellular localization of SlPP2C3, the open reading frame (ORF) sequence of SlPP2C3 tagged with green fluorescence protein (GFP) at the C-terminus was cloned into the pCambia1300 vector between restriction sites KpnI and SalI. The specific primers are listed in Supplementary Table S3. A. tumefaciens GV3101 cells carrying constructs were grown overnight at 28 °C, cells collected by centrifugation at 3220 × g, then resuspended in MES buffer (10 mM MES, 10 mM MgCl2, pH 6.5, with 100 μM acetosyringone added after sterilization) to an optical density at 600 nm (OD600) of ~1. After incubation at 25 °C for 4 h, cells were injected into the lower epidermis of five to six-week-old Nicotiana benthamiana leaves using a sterile 1 ml syringe without a needle. After 2 d culturing at 25 °C with 50% relative humidity in the dark, the infiltrated leaves were examined, and the GFP signal was monitored by an Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan) at an optical wavelength of 470 nm and 200× magnification (Grefen et al., 2008). For bimolecular fluorescence complementation, the cDNA of SlPP2C3 was cloned into pSPYCE(M), while SlPYLs and SlSnRK2s were cloned into pSPYNE173 (Waadt et al., 2008). Primers are listed in Supplementary Table S3. A. tumefaciens GV3101 carrying pSPYCE(M) or pSPYNE173 fusion constructs were co-infiltrated into five to six-week-old N. benthamiana leaves. After 2 d, the infiltrated leaves were examined using an Olympus BX51 fluorescence microscope (Olympus). Yeast two-hybrid (Y2H) assay The Y2H assay was performed using a Matchmaker Gold Yeast Two-Hybrid System (Takara, Cat. No. 630489) according to the user manual. Briefly, full-length, site-directed mutagenic, and catalytic core (residues 105−411) SlPP2C3 constructs were separately cloned into the Y2H ‘prey’ vector pGADT7. Full-length SlPYLs and SlSnRK2s were cloned into the ‘bait’ vector pGBKT7. Prey and bait vectors were co-transformed into the Y2HGold yeast strain and grown on SD-Leu/-Trp plates at 30 °C for 3 d. Interactions were examined on SD-Leu/-Trp control media and SD-Leu/-Trp/-His/-Ade selective media in the presence or absence of 10 μM ABA. Yeast cells containing proteins of interest in combination with empty pGADT7 or pGBKT7 vectors were used as controls. The primers used for Y2H assay are listed in Supplementary Table S4. Site-directed mutagenesis Mutants were created using a Fast Mutagenesis System (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. Mutagenesis was performed using pGADT7-SlPP2C3 as template. All mutagenesis primer sequences are provided in Supplementary Table S5. Seed germination and primary root growth assays Wild-type and transgenic T3 generation seeds obtained from T2 homozygous plants were harvested, air-dried, and stored at 4 °C for three weeks. After surface sterilization, seeds (~50 seeds for each replicate) were sown onto half-strength Murashige and Skoog (MS) medium containing 0 or 3 µM ABA, and germinated in the dark at 25 °C for 4 d or 10 d. Seed germination rates were investigated daily. Radicle emergence >1 mm indicated successful germination. Three replicate plates were included in each treatment. For the primary root growth assay, WT, SlPP2C3-OE and SlPP2C3-RNAi seedlings were grown on half-strength MS medium for 3 d. Seedlings with roots of the same length were transferred onto fresh half-strength MS medium supplemented with 0 or 10 μM ABA and grown for 5 d. Root length was measured from the base of the hypocotyl to the root tip. Experiments were performed three times. Analysis of drought stress, relative water content (RWC), and detached leaf water loss Sixteen day-old WT, OE4 and RNAi13 plants were subjected to a 22 d drought stress treatment by stopping watering, and plants were subsequently rewatered for 5 d. The survival rate of plants was then measured, and photographs were taken at 0, 11, 15, 19, and 22 d after drought treatment and 5 d after rewatering. RWC in leaves was measured at 0, 11, 15, 19, and 22 d after the treatment. RWC was calculated using the formula [(fresh weight − dry weight) / (turgid weight − dry weight)]. To measure water loss from detached leaves, healthy leaves were detached from each line and placed abaxial side up in an open plate at 25 °C with 35% relative humidity. Water loss was measured over a period of 8 h. All experiments were repeated three times. Quantitative real-time PCR (qRT–PCR) Total RNA was extracted from tomato tissues according to the improved hot borate method (Wan and Wilkins, 1994), followed by DNase digestion using recombinant DNase I (RNase-free, Takara) according to the manufacturer’s instructions. DNase-treated RNA was used for cDNA synthesis with a PrimeScript II 1st Strand cDNA Synthesis Kit (Takara) according to the manufacturer’s instructions. qRT–PCR was performed using SYBR Premix Ex Taq (Takara) and a Rotor-Gene 3000 system (QIAGEN, Hilden, Germany). Samples were denatured at 95 °C for 30 s, followed by 40 cycles at 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 15 s. qRT–PCR was conducted in three biological replicates and data were normalized against SlSAND (Solyc03g115810), SlEXP (Expressed, Solyc07g025390) and SlCAC (Clathrin adaptor complexes medium subunit, Solyc08g006960) as reference genes (Expósito-Rodríguez et al., 2008; González-Aguilera et al., 2016; Choi et al., 2018). Relative expression for each gene was calculated by Rotor-Gene Q software using the 2-ΔΔCT method (Livak and Schmittgen, 2001). Primers used for qRT–PCR analysis are listed in Supplementary Table S6. Agarose gel electrophoresis was used to assess the quality of the extracted RNA (Supplementary Fig. S2), and the RNA concentration was measured by a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) instrument. Measurement of abscisic acid and ethylene Extraction and measurement of ABA were performed as described previously (Sun et al., 2017). Briefly, fruit (3 g) was ground and extracted with 80% (v/v) methanol at –20 °C for 18 h. After centrifugation, the pellet was extracted twice with 80% methanol. The supernatant was dried under vacuum, and the residue was dissolved in 0.02 M phosphate buffer (pH 8.0). Petroleum ether was used to remove pigments, and insoluble polyvinylpolypyrrolidone was used to remove polyphenols. ABA was extracted three times by ethyl acetate. The ethyl acetate phase was dried under vacuum and dissolved in 50% methanol (v/v). ABA content was determined via HPLC using an Agilent LC 1200 instrument (Agilent Technologies, Santa Clara, CA, USA) equipped with a 4.8 x 150 mm C18 column (Agilent Technologies), and (±)-abscisic acid (Sigma-Aldrich, St. Louis, MO, USA) as a standard. Three replicates were conducted for each sample. Ethylene production was measured by enclosing two to four fruits in a 50 ml airtight container for 2 h at 25 °C, and 1 ml of headspace gas was withdrawn and injected into an Agilent GC 6890N gas chromatography instrument (Agilent Technologies) equipped with a flame ionization detector and an activated alumina column (Zhang et al., 2009). Determination of carotenoids, total sugars, and titratable acid Total carotenoids of fruit were determined by spectroscopy, as described previously (Lichtenthaler and Wellburn, 1983). The lycopene and β-carotene content of fruit was determined as described by Nagata et al. (2007). Determination of total sugar content was performed using the 3,5-dinitrosalicylic acid colorimetric method, according to Sumner and Graham (1921). The content of total titratable acid was determined by acid-base titration. The fruit (5 g) was ground in liquid nitrogen and dissolved in 40 ml water, then incubated at 75−80 °C in a water bath for 30 min. After cooling, water was added to 50 ml, the mixture was filtered through a filter paper, and 20 ml of the sample was used for titration. A drop of phenolphthalein was added into the sample and titrated with 0.1 M NaOH until a light red color was observed. The volume (V) of NaOH used was recorded, and total titratable acid was calculated according to the equation: titratable acid content (%) = V×0.1×0.067×50/20/5×100%. RNA–seq Total RNA was extracted from the skin of WT and SlPP2C3-RNAi4 fruits at the mature green stage. RNA degradation and contamination were monitored on 1% agarose gels. RNA integrity was assessed by an RNA 6000 Pico Assay Kit and a Bioanalyzer 2100 system (Agilent Technologies). The results are shown in Supplementary Fig. S3. RNA (3 μg per sample) was used for mRNA purification and library construction with a Truseq RNA Sample Prep Kit (Illumina, CA, USA) following the manufacturer’s instructions. Samples were sequenced on an Illumina HiSeq 2000 platform (Illumina). Each sample yielded more than 6 Gb of data. Raw data in Fastq format were firstly processed using in-house Perl scripts. Clean data were obtained by removing reads containing adapters, and poly-N and low-quality reads from raw data. Filtered RNA-seq clean reads were aligned to the reference tomato genome (release SL2.50) using TopHat v2.0.13 (Kim et al., 2013). HTSeq v0.5.3 (https://htseq.readthedocs.io/en/master/) was used to count the number of reads mapped to each gene, and reads per kilobase of transcript per million mapped reads (RPKM) values were calculated based on the length of the gene and the number of reads mapped. Differential expression analysis was performed using the DEGSeq R package (1.12.0) and Cufflinks (Trapnell et al., 2012). Corrected P-values of 0.001 and log2 (fold change) ≥1 were set as the thresholds for significant differential expression. Statistical analysis Data were statistically analysed by SPSS software using one-way analysis of variance (ANOVA) and Duncan’s test of significance (*t-test, P-value <0.05; **t-test, P-value <0.01). Results Phylogenetic analyses of tomato Group A PP2C family members To explore all members of the group A PP2C family in tomato, predicted Arabidopsis PP2C protein sequences (Schweighofer et al., 2004) were used as queries for BLAST searches against the tomato genome database (https://solgenomics.net/). A total of 93 putative protein phosphatase 2C family members were identified in the tomato genome. Phylogenetic analyses indicated that tomato PP2Cs were divided into 12 groups (A−K; Supplementary Fig. S4), consistent with previous studies in Arabidopsis and rice (Schweighofer et al., 2004; Xue et al., 2008; Fuchs et al., 2013). Fourteen tomato PP2Cs were identified as members of group A, which have been shown to participate in ABA signaling. We named these 14 tomato members as SlPP2C1−14 (Supplementary Table S1). Further amino acid alignments indicated the presence of the conserved catalytic domain in both PP2Cs of tomato and Arabidopsis with diverse N-termini (Supplementary Fig. S5). All tomato PP2Cs possess conserved active site residues and PYL-interaction residues, except for SlPP2C7, in which some motifs are missing from the catalytic domain. Interestingly, similar to Arabidopsis AtAHG1, the amino acid sequences of SlPP2C11−14 lack the conserved tryptophan residue which acts as a ‘lock’ in the interaction with the ABA receptor (Antoni et al., 2012). It is worth noting that SlPP2C3 is the single orthologue of Arabidopsis AtHAI 1/2/3 (HIGHLY ABA-INDUCED 1/2/3; Supplementary Fig. S6). In Arabidopsis, three AtHAIs perform redundant functions during drought resistance, leaf senescence and seed dormancy (Bhaskara et al., 2012; Kim et al., 2013), which implies the non-redundant function of SlPP2C3 in tomato physiological processes. SlPP2C3 selectively interacts with monomeric ABA receptors and SlSnRK2.8 In our previous study on the interactions between SlPP2C1-5 and SlPYLs or SlSnRK2s, SlPP2C3 was the only member that interacted with SlSnRK2 in the Y2H assay (Chen et al., 2016). In the present work, we further investigated the manner of the interaction between SlPP2C3 and ABA receptors. Full-length SlPP2C3 interacted with sub-family I and sub-family II monomeric receptors in yeast cells. Interactions between SlPP2C3 and SlPYL1, 4, 7, 9, and 11 were ABA-dependent, while interactions with SlPYL2, 3, 10, and 13 were ABA-independent. In addition, SlPP2C3 did not interact with sub-family III dimeric receptor members SlPYL5 and 8, even in the presence of 10 μM ABA (Fig. 1A). Regarding SlSnRK2s, SlPP2C3 could only interact with SlSnRK2.8 (Fig. 1B), consistent with our previous results (Chen et al., 2016). Fig. 1. Open in new tabDownload slide Interactions of SlPP2C3 with SlPYLs and SlSnRK2s in yeast two hybrid (Y2H) assays. (A) Interaction of SlPP2C3-AD and SlPYLs-BD receptors. Lane 1, full-length SlPP2C3; lane 2, SlPP2C3 with Gly-160 mutated to Asp (SlPP2C3G160D); lane 3, the catalytic core (residues 105−411) of SlPP2C3 (ΔNSlPP2C3); lane 4, pGADT7 empty vector control. (B) Interaction of SlPP2C3-AD and SlSnRK2s-BD. Interactions were determined using growth assays on media lacking Leu, Trp, His and Ade (-LWHA), with or without 10 μM ABA. Yeast growth on media lacking Leu and Trp (-LW) served as controls. Fig. 1. Open in new tabDownload slide Interactions of SlPP2C3 with SlPYLs and SlSnRK2s in yeast two hybrid (Y2H) assays. (A) Interaction of SlPP2C3-AD and SlPYLs-BD receptors. Lane 1, full-length SlPP2C3; lane 2, SlPP2C3 with Gly-160 mutated to Asp (SlPP2C3G160D); lane 3, the catalytic core (residues 105−411) of SlPP2C3 (ΔNSlPP2C3); lane 4, pGADT7 empty vector control. (B) Interaction of SlPP2C3-AD and SlSnRK2s-BD. Interactions were determined using growth assays on media lacking Leu, Trp, His and Ade (-LWHA), with or without 10 μM ABA. Yeast growth on media lacking Leu and Trp (-LW) served as controls. The catalytic domain of SlPP2C3 (residues 105−411) acted in a similar manner to the full-length protein. However, the Gly160Asp (corresponding to AtABI1G180D encoded by abi1-1) mutation abolished the interaction between SlPP2C3 and all SlPYLs (Fig. 1A). Furthermore, a BiFC assay was performed to confirm the SlPP2C3-SlPYLs/SlSnRK2.8 interaction in planta. A fluorescence signal was detected in tobacco leaves co-expressing SlPP2C3 with SlPYL1, 3, 9, 10, 13 or SlSnRK2.8, but not in leaves co-expressing SlPP2C3 with dimeric PYL5 and PYL8 receptors (Fig. 2A). The interaction between SlPP2C3 and SlPYL1/9 may be due to appreciable levels of endogenous ABA in leaves. Most interactions occur in the nucleus and cytoplasm, except for SlPP2C3 and SlPYL3, which only occur in the nucleus. The localization of the interaction was further investigated by exploring the sub-cellular location of SlPP2C3 (Fig. 2B). Both in vitro and in planta assays indicated that SlPP2C3 could not interact with dimeric receptors belonging to sub-family III (Fig. 1A; Fig. 2A). Fig. 2. Open in new tabDownload slide Interaction of SlPP2C3 with SlPYLs and SlSnRK2s in tobacco leaves. (A) SlPP2C3-YFPC and SlPYLs-YFPN or SlSnRK2s-YFPN were co-expressed in N. benthamiana leaves through A. tumefaciens infiltration. The YFP signal was monitored 2 d after co-infiltration. (B) Sub-cellular location of SlPP2C3. SlPP2Cs-GFP was transiently expressed in N. benthamiana leaves through A. tumefaciens infiltration. The GFP signal was monitored 2 d after infiltration. The left column shows the fluorescence signal; the middle column shows the bright-field image; the right column shows the merged image. Fig. 2. Open in new tabDownload slide Interaction of SlPP2C3 with SlPYLs and SlSnRK2s in tobacco leaves. (A) SlPP2C3-YFPC and SlPYLs-YFPN or SlSnRK2s-YFPN were co-expressed in N. benthamiana leaves through A. tumefaciens infiltration. The YFP signal was monitored 2 d after co-infiltration. (B) Sub-cellular location of SlPP2C3. SlPP2Cs-GFP was transiently expressed in N. benthamiana leaves through A. tumefaciens infiltration. The GFP signal was monitored 2 d after infiltration. The left column shows the fluorescence signal; the middle column shows the bright-field image; the right column shows the merged image. Our previous study showed that sub-family III receptors SlPYL5 and SlPYL8 interact with SlPP2C5, an orthologue of Arabidopsis HAB1 (Chen et al., 2016). To better understand the molecular mechanism underlying the selective receptor interaction of SlPP2C3, the amino acid sequences of SlPP2C3 and SlPP2C5 were compared. Some differences were found in the PP2C-PYL interaction regions (Fig. 3A; Melcher et al., 2009; Miyazono et al., 2009). We therefore conducted site-directed mutagenesis of SlPP2C3 to switch these residues to their counterparts in SlPP2C5, reasoning that the mutants may mimic SlPP2C5 (Fig. 3A). Our results revealed that the S295F mutation in SlPP2C3 strengthened the SlPP2C3-SlPYL5/8 interaction, while the R285K mutation strengthened the SlPP2C3-SlPYL8 interaction in the presence of ABA (Fig. 3B). These results partially explain the interaction preferences of SlPP2Cs and SlPYLs. Fig. 3. Open in new tabDownload slide SlPP2C3 mutants interact with dimeric receptors SlPYL5 and SlPYL8 in Y2H assays. (A) Alignment of SlPP2C3 and SlPP2C5 amino acid sequences. The locations of the SlPP2C3 mutations are marked by black asterisks. Black triangles indicate residues involved in PYLs interaction (Miyazono et al., 2009). (B) Interaction of SlPP2C3, SlPP2C5 or SlPP2C3 mutants (fused to the activating domain) and either SlPYL5 or SlPYL8 (fused to the binding domain). Interaction was determined by growth assay on medium lacking Leu, Trp, His and Ade (-LWHA), with or without 10 μM ABA. Photographs were taken after 5 d of growth at 30 °C. Fig. 3. Open in new tabDownload slide SlPP2C3 mutants interact with dimeric receptors SlPYL5 and SlPYL8 in Y2H assays. (A) Alignment of SlPP2C3 and SlPP2C5 amino acid sequences. The locations of the SlPP2C3 mutations are marked by black asterisks. Black triangles indicate residues involved in PYLs interaction (Miyazono et al., 2009). (B) Interaction of SlPP2C3, SlPP2C5 or SlPP2C3 mutants (fused to the activating domain) and either SlPYL5 or SlPYL8 (fused to the binding domain). Interaction was determined by growth assay on medium lacking Leu, Trp, His and Ade (-LWHA), with or without 10 μM ABA. Photographs were taken after 5 d of growth at 30 °C. SlPP2C3 expression is induced by ABA and negatively correlated with fruit ripening SlPP2C3 was highly transcribed in seeds, but its expression in seedlings, roots and stems were relatively low. During flowering and fruit development, SlPP2C3 expression was high in flowers and immature fruits, but this decreased during ripening, indicating that SlPP2C3 might play a negative role in fruit ripening (Fig. 4A). Fig. 4. Open in new tabDownload slide Expression patterns of SlPP2Cs genes in tomato. (A) Relative expression of SlPP2C3 in various tomato tissues. S, seed; Se, seedling; R, root; St, stem; L, leaf; Fl, flower; fruit pericarp at IM [(immature, 15 days after anthesis (DAA)], MG (mature green, 35 DAA), B (breaker), B5 (5 d after breaker) and B10 (10 d after breaker). Error bars represent mean ± standard deviation (SD) of biological triplicates. (B) Relative expression of SlPP2C3 in tomato seeds following imbibition of water for specified times. (C) Relative expression of SlPP2C3 in detached leaves of tomato. (D) Relative expression of SlPP2C3 in tomato seeds treated with exogenous ABA or NaCl for 48 h. (E) Relative expression of SlPP2C3 in MG tomato pericarp disks treated with 100 μM exogenous ABA. For all expression analyses, SlSAND, SlEXP and SlCAC were used as references. Error bars indicate mean ± SD of biological triplicates (*P-value, t-test <0.05; **P-value, t-test <0.01). Fig. 4. Open in new tabDownload slide Expression patterns of SlPP2Cs genes in tomato. (A) Relative expression of SlPP2C3 in various tomato tissues. S, seed; Se, seedling; R, root; St, stem; L, leaf; Fl, flower; fruit pericarp at IM [(immature, 15 days after anthesis (DAA)], MG (mature green, 35 DAA), B (breaker), B5 (5 d after breaker) and B10 (10 d after breaker). Error bars represent mean ± standard deviation (SD) of biological triplicates. (B) Relative expression of SlPP2C3 in tomato seeds following imbibition of water for specified times. (C) Relative expression of SlPP2C3 in detached leaves of tomato. (D) Relative expression of SlPP2C3 in tomato seeds treated with exogenous ABA or NaCl for 48 h. (E) Relative expression of SlPP2C3 in MG tomato pericarp disks treated with 100 μM exogenous ABA. For all expression analyses, SlSAND, SlEXP and SlCAC were used as references. Error bars indicate mean ± SD of biological triplicates (*P-value, t-test <0.05; **P-value, t-test <0.01). To examine whether SlPP2C3 transcription is regulated by ABA, we performed qRT–PCR to investigate the expression of SlPP2C3 during seed imbibition and leaf dehydration. These two physiological processes are accompanied by a decrease and increase in endogenous ABA concentrations, respectively. Expression of SlPP2C3 was decreased during seed imbibition; on the other hand, SlPP2C3 expression was induced in dehydrated leaves (Fig. 4B, C). Furthermore, SlPP2C3 expression was induced under exogenous ABA treatment in seeds and mature green fruits (Fig. 4D, E). These results indicate that expression of SlPP2C3 was positively regulated by ABA. Altering SlPP2C3 expression affects ABA sensitivity Given the expression and interaction pattern, we were interested in the physiological function of SlPP2C3 in planta. To gain insight into the role of SlPP2C3, SlPP2C3 was overexpressed or silenced by RNAi in ‘Micro-Tom’ tomato plants. Independent SlPP2C3-OE (OE4 and OE33) and SlPP2C3-RNAi (RNAi4 and RNAi13) transgenic lines with dramatically increased or decreased SlPP2C3 expression were confirmed and subjected to further investigation (Supplementary Fig. S7A, B). SlPP2C3-OE and SlPP2C3-RNAi plants were similar to WT plants in terms of overall architecture (Supplementary Fig. S8A−D); the leaves of transgenic lines exhibited similar shape, color and size to WT leaves (Supplementary Fig. S8F, G). These results suggest that SlPP2C3 likely plays a limited role in plant vegetative growth. SlPP2C3 is a putative core component in ABA signaling, hence we wondered whether ABA sensitivity could be altered by SlPP2C3 manipulation. ABA-mediated inhibition of seed germination was compared in WT and transgenic lines. Germination of SlPP2C3-RNAi seeds was delayed, and was more sensitive to exogenous ABA, while seeds of SlPP2C3-OE lines behaved in an opposite manner (Fig. 5A, B). Furthermore, the primary root growth of SlPP2C3-OE seedlings was less sensitive to exogenous ABA treatment compared with that of WT seedlings. However, SlPP2C3-RNAi seedlings were more sensitive to exogenous ABA treatment (Fig. 5C, D). These results indicate that SlPP2C3 might act as a negative regulator in ABA signaling. Fig. 5. Open in new tabDownload slide Altered sensitivity to ABA-mediated inhibition of seed germination, and primary root growth in WT and SlPP2C3 transgenic lines. (A, B) Approximately 50 seeds of WT, SlPP2C3-OE and SlPP2C3-RNAi lines (three independent experiments) were sown on half-strength MS medium with or without 3 μM ABA. Seed germination was scored every day. Values are mean ± standard error (n=3; *t-test, P-value <0.05; **t-test, P-value <0.01). (C, D) Primary root length and photographs of tomato seedlings 5 d after transferring three-day-old seedlings to half-strength MS medium with or without 10 μM ABA. OE, OE4 seeds mixed with OE33 seeds. RNAi, RNAi4 seeds mixed with RNAi13 seeds. OE-0/OE-10, OE seedlings 5 d after transferring three-day-old seedlings to half-strength MS medium with or without 10 μM ABA. Percentages represent the ratio of root length of seedlings treated with 10 μM ABA to those not treated with ABA. Values are mean ± SD (n=3; *t-test, P-value <0.05; **t-test, P-value <0.01). Fig. 5. Open in new tabDownload slide Altered sensitivity to ABA-mediated inhibition of seed germination, and primary root growth in WT and SlPP2C3 transgenic lines. (A, B) Approximately 50 seeds of WT, SlPP2C3-OE and SlPP2C3-RNAi lines (three independent experiments) were sown on half-strength MS medium with or without 3 μM ABA. Seed germination was scored every day. Values are mean ± standard error (n=3; *t-test, P-value <0.05; **t-test, P-value <0.01). (C, D) Primary root length and photographs of tomato seedlings 5 d after transferring three-day-old seedlings to half-strength MS medium with or without 10 μM ABA. OE, OE4 seeds mixed with OE33 seeds. RNAi, RNAi4 seeds mixed with RNAi13 seeds. OE-0/OE-10, OE seedlings 5 d after transferring three-day-old seedlings to half-strength MS medium with or without 10 μM ABA. Percentages represent the ratio of root length of seedlings treated with 10 μM ABA to those not treated with ABA. Values are mean ± SD (n=3; *t-test, P-value <0.05; **t-test, P-value <0.01). SlPP2C3 plays a negative role in drought tolerance To determine whether SlPP2C3 plays a role in ABA-mediated drought tolerance, 16-day-old WT and transgenic plants were subjected to a 22 day drought stress treatment. At 15 days after the treatment, SlPP2C3-OE plants had more wilted leaves than WT plants, while SlPP2C3-RNAi plants exhibited a significant improvement in drought tolerance compared with WT plants (Fig. 6A). At five days after rewatering, SlPP2C3-OE plants recorded a 6% survival rate compared with 30% for WT plants, while the survival rate of SlPP2C3-RNAi plants was nearly 60% (Fig. 6B). Consistent with these results, the relative water content in SlPP2C3-OE leaves was significantly lower than in WT leaves, and the relative water content in SlPP2C3-RNAi leaves was highest (Fig. 6C). Leaves of SlPP2C3-RNAi plants exhibited lower rates of water loss than WT leaves, which implies a lower transpiration rate (Fig. 6D). These results suggest that SlPP2C3 plays a negative role in plant drought tolerance. Fig. 6. Open in new tabDownload slide Drought resistance of SlPP2C3 transgenic lines. (A) WT and SlPP2C3 transgenic 16-day-old plants were subjected to drought stress by withholding water for 22 days, followed by rewatering for 5 days. Experiments were repeated three times. (B) Survival rates scored 5 d after rewatering. (C) Relative water content in WT and SlPP2C3 transgenic leaves after drought stress. (D) Water loss analysis of detached leaves. Values are mean ± SD of three independent experiments (*t-test, P-value <0.05; **t-test, P-value <0.01). Fig. 6. Open in new tabDownload slide Drought resistance of SlPP2C3 transgenic lines. (A) WT and SlPP2C3 transgenic 16-day-old plants were subjected to drought stress by withholding water for 22 days, followed by rewatering for 5 days. Experiments were repeated three times. (B) Survival rates scored 5 d after rewatering. (C) Relative water content in WT and SlPP2C3 transgenic leaves after drought stress. (D) Water loss analysis of detached leaves. Values are mean ± SD of three independent experiments (*t-test, P-value <0.05; **t-test, P-value <0.01). SlPP2C3 affects the fruit ripening schedule Given the role of ABA in fruit ripening regulation, and the negative correlation between the expression pattern of SlPP2C3 and fruit ripening, we investigated whether SlPP2C3 plays a role in fruit ripening. SlPP2C3-OE fruits required an additional 2 d from anthesis to the breaker stage compared with WT fruits, while SlPP2C3-RNAi fruits required two to three days less (Fig. 7A, B). The breaker time of WT fruits was 37 to 42 days, compared with 39−44 days, and 34−40 days, for SlPP2C3-OE and SlPP2C3-RNAi fruits, respectively. Fig. 7. Open in new tabDownload slide Phenotypes of WT and SlPP2C3 transgenic fruits. (A) Ripening time of fruits from WT and SlPP2C3 transgenic lines. DAA, days after anthesis. (B) Days from anthesis to break. (C−E) Changes in ethylene release and ABA content in WT and SlPP2C3 transgenic fruits during fruit ripening (n >15). Values are mean ± SD (n=3). Letters represent significant differences (t-test, P-value <0.05). Fig. 7. Open in new tabDownload slide Phenotypes of WT and SlPP2C3 transgenic fruits. (A) Ripening time of fruits from WT and SlPP2C3 transgenic lines. DAA, days after anthesis. (B) Days from anthesis to break. (C−E) Changes in ethylene release and ABA content in WT and SlPP2C3 transgenic fruits during fruit ripening (n >15). Values are mean ± SD (n=3). Letters represent significant differences (t-test, P-value <0.05). The ethylene and ABA production peaks of SlPP2C3-OE fruits were delayed when compared with WT, while for SlPP2C3-RNAi fruits they were advanced (Fig.7C, D). Moreover, the expression of some 1-aminocyclopropane-1-carboxylic acid oxidase genes involved in ethylene biosynthesis, and several ethylene receptor genes, was up-regulated in SlPP2C3-RNAi fruits (Supplementary Fig. S9; Fig. S13B). To determine whether fruit quality was altered in SlPP2C3-OE and SlPP2C3-RNAi lines, several typical fruit quality parameters were measured. We observed that fruit shape index (the ratio of fruit vertical diameter to horizontal diameter) of SlPP2C3-OE (especially OE33) was higher than that of WT (Fig. 8A-C). The fruit weight and seed number of SlPP2C3-OE were lower than WT, while SlPP2C3-RNAi showed no significant differences (Fig. 8D, F). Fruit firmness, soluble solids content, total sugar content, and the titratable acid in transgenic fruits were similar to WT parameters (Fig. 8E, H; Supplementary Fig. S10D, E). Fig. 8. Open in new tabDownload slide Physiological parameters related to fruit in WT and SlPP2C3 transgenic lines. (A) Horizontal diameters of B10 fruits. (B) Vertical diameters of B10 fruits. (C) Fruit shape index of B10 fruits. (D) Single fruit weight of B10 fruits. (E) Soluble solid content (Brix) of B10 fruits. (F) Seed number of each fruit. (G) Locule number rate of fruits. (H) Fruit firmness during fruit ripening. Values are mean ± SD (n=3). Letters represent significant differences (t-test, P-value <0.05). Fig. 8. Open in new tabDownload slide Physiological parameters related to fruit in WT and SlPP2C3 transgenic lines. (A) Horizontal diameters of B10 fruits. (B) Vertical diameters of B10 fruits. (C) Fruit shape index of B10 fruits. (D) Single fruit weight of B10 fruits. (E) Soluble solid content (Brix) of B10 fruits. (F) Seed number of each fruit. (G) Locule number rate of fruits. (H) Fruit firmness during fruit ripening. Values are mean ± SD (n=3). Letters represent significant differences (t-test, P-value <0.05). Fruit size enlargement due to the increased number of seed compartments (locules) is a crucial feature of domesticated tomato (Lippman and Tanksley, 2001). Therefore, we investigated the number of locules in transgenic tomato fruits. The number of OE33 fruits with two locules was significantly higher than that of WT, while the number of OE33 fruits with three locules was less (Fig. 8G). Total carotenoid and lycopene concentrations in SlPP2C3-OE fruits were higher than in WT fruits, but those in SlPP2C3-RNAi and WT fruits were similar (Supplementary Fig. S10A−C). These results suggest that SlPP2C3 functions in fruit development and metabolic processes. SlPP2C3 controls fruit glossiness by altering the outer epidermis Notably, SlPP2C3-RNAi fruits exhibited a dull fruit surface compared to WT (Fig. 9A). Scanning electron microscopy analysis revealed a verrucous outer epidermis surface for SlPP2C3-RNAi fruits, indicating changes in cuticle/wax metabolism (Fig. 9B). To understand the underlying mechanism, a global gene expression analysis of SlPP2C3-RNAi fruit skin was performed (Dataset 1 at Dryad). Interestingly, 374 genes were up-regulated and 519 were down-regulated in SlPP2C3-RNAi fruit skin compared with WT (Dataset 2 at Dryad). These included two transcription factors and 24 structural genes related to cuticle formation (Table 1). Expression of selected genes was verified by qRT–PCR (Fig. 9C). R2R3-type MYB transcription factor SlMYB96 (Solyc03g116100) was down-regulated in SlPP2C3-RNAi fruits, while SlMYB106 (Solyc02g088190) was up-regulated. Arabidopsis orthologues AtMYP96 and AtMYB106 were shown to regulate gene expression in cutin and wax biosynthesis (Seo et al., 2011; Oshima et al., 2013). MYB96 expression is significantly induced by ABA in Arabidopsis (Seo et al., 2009). The 18 up-regulated genes included cuticle transporter gene SlABCG32, cutin synthase gene SlCD1 (required for cutin polymerization; Bessire et al., 2011; Yeats et al., 2014; Fabre et al., 2016), 11 cutin biosynthesis genes including SlLACS2, which may be necessary for acyl activation of fatty acids (Schnurr et al., 2004), four CYP450 genes (SlCYP77A1, SlCYP77A2, SlCYP86A68 and SlCYP86A69) which may be involved in fatty acid mid-chain and terminal hydroxylation (Li-Beisson et al., 2009; Shi et al., 2013), four SlHTH genes for which orthologues are known to be important in the formation of dicarboxylic fatty acids in Arabidopsis (Kurdyukov et al., 2006), and two glycerol-3-phosphate acyltransferase genes (SlGAPT4 and SlGAPT6) involved in the final step of cutin monomer biosynthesis (Li et al., 2007). Meanwhile, seven out of nine down-regulated genes are involved in wax biosynthesis. These results indicate that low SlPP2C3 expression may alter the cuticle composition of the fruit outer epidermis. Fig. 9. Open in new tabDownload slide Reduced fruit brightness and altered expression of genes related to cuticle metabolism and ABA signaling of SlPP2C3-RNAi fruit skin. (A) Photographs of WT and SlPP2C3-RNAi fruits at MG and B+10 stages showing surface brightness. (B) Scanning electron microscopy images of the outer epidermis surface of WT and SlPP2C3-RNAi fruits at the B stage. Bar indicate 1 cm.(C) Expression of cuticle metabolism genes in WT and transgenic fruit skin at the MG stage. (D−F) Expression of SlPP2Cs (D), SlPYLs (E) and SlSnRK2s (F) in WT and transgenic fruit skin at the MG stage. SlSAND, SlEXP and SlCAC genes were used as references. Data are mean ± SD of three biological replicates (*t-test, P-value <0.05; **t-test P-value <0.01). Fig. 9. Open in new tabDownload slide Reduced fruit brightness and altered expression of genes related to cuticle metabolism and ABA signaling of SlPP2C3-RNAi fruit skin. (A) Photographs of WT and SlPP2C3-RNAi fruits at MG and B+10 stages showing surface brightness. (B) Scanning electron microscopy images of the outer epidermis surface of WT and SlPP2C3-RNAi fruits at the B stage. Bar indicate 1 cm.(C) Expression of cuticle metabolism genes in WT and transgenic fruit skin at the MG stage. (D−F) Expression of SlPP2Cs (D), SlPYLs (E) and SlSnRK2s (F) in WT and transgenic fruit skin at the MG stage. SlSAND, SlEXP and SlCAC genes were used as references. Data are mean ± SD of three biological replicates (*t-test, P-value <0.05; **t-test P-value <0.01). Table 1. Differentially expressed genes related to cuticle metabolism in transgenic fruit peel Gene ID . Gene name . Annotation . Pathway . Log2 fold change . Adjusted P-value . Up-regulated Solyc02g088190 MIXTA MYB transcription factor Regulation 1.70 7.30E-08 Solyc12g009490 SHN2 Ethylene-responsive transcription factor Regulation 1.88 2.32E-05 Solyc01g109180 LACS2 Long-chain fatty acid CoA ligase Cutin synthesis 2.51 7.18E-101 Solyc01g094750 CYP86A68 Cytochrome P450 Cutin synthesis 2.99 9.47E-05 Solyc08g081220 CYP86A69 Cytochrome P450 Cutin synthesis 2.46 8.12E-138 Solyc05g055400 CYP77A2 Cytochrome P450 Cutin synthesis 1.77 1.01E-152 Solyc11g007540 CYP77A1 Cytochrome P450 Cutin synthesis 2.77 1.49E-38 Solyc06g062600 HTH-like Choline dehydrogenase Cutin synthesis 2.63 9.18E-21 Solyc03g121600 HTH-like Choline dehydrogenase Cutin synthesis 2.01 5.85E-16 Solyc06g035580 HTH-like Choline dehydrogenase Cutin synthesis 2.08 4.99E-123 Solyc01g094700 GPAT4 Glycerol-3-phosphate acyltransferase Cutin synthesis 1.63 1.39E-36 Solyc09g014350 GPAT6 Glycerol-3-phosphate acyltransferase Cutin synthesis 3.06 4.99E-123 Solyc06g065670 ABCG32 ATP-binding cassette transporter Cutin transport 1.34 1.39E-08 Solyc11g006250 CD1/CUS1 GDSL esterase/lipase Cutin assembly 1.95 6.57E-191 Solyc01g088400 CER1-like CER1 fatty acid hydroxylase Wax synthesis 1.05 5.52E-06 Solyc03g005320 KCS-like Fatty acid elongase 3-ketoacyl-CoA synthase Wax synthesis 3.27 7.59E-05 Solyc08g067260 KCS10 Fatty acid elongase 3-ketoacyl-CoA synthase Wax synthesis 1.08 9.75E-35 Solyc11g067180 CER4-like Fatty acyl coA reductase Wax synthesis 5.99 2.44E-04 Down-regulated Solyc07g056320 GPAT1 ER glycerol-phosphate acyltransferase Cutin synthesis -2.40 1.17E-24 Solyc03g065250 CER1 CER1 fatty acid hydroxylase Wax synthesis -1.72 1.12E-22 Solyc03g117800 CER1-like CER1 fatty acid hydroxylase Wax synthesis -1.64 8.16E-28 Solyc10g080840 MAH1 Cytochrome P450 Wax synthesis -2.48 8.41E-27 Solyc09g083050 KCS2 Fatty acid elongase 3-ketoacyl-CoA synthase Wax synthesis -4.33 1.14E-65 Solyc10g009240 KCS1 Fatty acid elongase 3-ketoacyl-CoA synthase Wax synthesis -1.35 2.43E-04 Solyc06g074390 CER4/FAR3 Fatty acyl coA reductase Wax synthesis -3.19 3.74E-14 Solyc09g092270 CER2-like Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase Wax synthesis -1.59 8.39E-13 Gene ID . Gene name . Annotation . Pathway . Log2 fold change . Adjusted P-value . Up-regulated Solyc02g088190 MIXTA MYB transcription factor Regulation 1.70 7.30E-08 Solyc12g009490 SHN2 Ethylene-responsive transcription factor Regulation 1.88 2.32E-05 Solyc01g109180 LACS2 Long-chain fatty acid CoA ligase Cutin synthesis 2.51 7.18E-101 Solyc01g094750 CYP86A68 Cytochrome P450 Cutin synthesis 2.99 9.47E-05 Solyc08g081220 CYP86A69 Cytochrome P450 Cutin synthesis 2.46 8.12E-138 Solyc05g055400 CYP77A2 Cytochrome P450 Cutin synthesis 1.77 1.01E-152 Solyc11g007540 CYP77A1 Cytochrome P450 Cutin synthesis 2.77 1.49E-38 Solyc06g062600 HTH-like Choline dehydrogenase Cutin synthesis 2.63 9.18E-21 Solyc03g121600 HTH-like Choline dehydrogenase Cutin synthesis 2.01 5.85E-16 Solyc06g035580 HTH-like Choline dehydrogenase Cutin synthesis 2.08 4.99E-123 Solyc01g094700 GPAT4 Glycerol-3-phosphate acyltransferase Cutin synthesis 1.63 1.39E-36 Solyc09g014350 GPAT6 Glycerol-3-phosphate acyltransferase Cutin synthesis 3.06 4.99E-123 Solyc06g065670 ABCG32 ATP-binding cassette transporter Cutin transport 1.34 1.39E-08 Solyc11g006250 CD1/CUS1 GDSL esterase/lipase Cutin assembly 1.95 6.57E-191 Solyc01g088400 CER1-like CER1 fatty acid hydroxylase Wax synthesis 1.05 5.52E-06 Solyc03g005320 KCS-like Fatty acid elongase 3-ketoacyl-CoA synthase Wax synthesis 3.27 7.59E-05 Solyc08g067260 KCS10 Fatty acid elongase 3-ketoacyl-CoA synthase Wax synthesis 1.08 9.75E-35 Solyc11g067180 CER4-like Fatty acyl coA reductase Wax synthesis 5.99 2.44E-04 Down-regulated Solyc07g056320 GPAT1 ER glycerol-phosphate acyltransferase Cutin synthesis -2.40 1.17E-24 Solyc03g065250 CER1 CER1 fatty acid hydroxylase Wax synthesis -1.72 1.12E-22 Solyc03g117800 CER1-like CER1 fatty acid hydroxylase Wax synthesis -1.64 8.16E-28 Solyc10g080840 MAH1 Cytochrome P450 Wax synthesis -2.48 8.41E-27 Solyc09g083050 KCS2 Fatty acid elongase 3-ketoacyl-CoA synthase Wax synthesis -4.33 1.14E-65 Solyc10g009240 KCS1 Fatty acid elongase 3-ketoacyl-CoA synthase Wax synthesis -1.35 2.43E-04 Solyc06g074390 CER4/FAR3 Fatty acyl coA reductase Wax synthesis -3.19 3.74E-14 Solyc09g092270 CER2-like Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase Wax synthesis -1.59 8.39E-13 Open in new tab Table 1. Differentially expressed genes related to cuticle metabolism in transgenic fruit peel Gene ID . Gene name . Annotation . Pathway . Log2 fold change . Adjusted P-value . Up-regulated Solyc02g088190 MIXTA MYB transcription factor Regulation 1.70 7.30E-08 Solyc12g009490 SHN2 Ethylene-responsive transcription factor Regulation 1.88 2.32E-05 Solyc01g109180 LACS2 Long-chain fatty acid CoA ligase Cutin synthesis 2.51 7.18E-101 Solyc01g094750 CYP86A68 Cytochrome P450 Cutin synthesis 2.99 9.47E-05 Solyc08g081220 CYP86A69 Cytochrome P450 Cutin synthesis 2.46 8.12E-138 Solyc05g055400 CYP77A2 Cytochrome P450 Cutin synthesis 1.77 1.01E-152 Solyc11g007540 CYP77A1 Cytochrome P450 Cutin synthesis 2.77 1.49E-38 Solyc06g062600 HTH-like Choline dehydrogenase Cutin synthesis 2.63 9.18E-21 Solyc03g121600 HTH-like Choline dehydrogenase Cutin synthesis 2.01 5.85E-16 Solyc06g035580 HTH-like Choline dehydrogenase Cutin synthesis 2.08 4.99E-123 Solyc01g094700 GPAT4 Glycerol-3-phosphate acyltransferase Cutin synthesis 1.63 1.39E-36 Solyc09g014350 GPAT6 Glycerol-3-phosphate acyltransferase Cutin synthesis 3.06 4.99E-123 Solyc06g065670 ABCG32 ATP-binding cassette transporter Cutin transport 1.34 1.39E-08 Solyc11g006250 CD1/CUS1 GDSL esterase/lipase Cutin assembly 1.95 6.57E-191 Solyc01g088400 CER1-like CER1 fatty acid hydroxylase Wax synthesis 1.05 5.52E-06 Solyc03g005320 KCS-like Fatty acid elongase 3-ketoacyl-CoA synthase Wax synthesis 3.27 7.59E-05 Solyc08g067260 KCS10 Fatty acid elongase 3-ketoacyl-CoA synthase Wax synthesis 1.08 9.75E-35 Solyc11g067180 CER4-like Fatty acyl coA reductase Wax synthesis 5.99 2.44E-04 Down-regulated Solyc07g056320 GPAT1 ER glycerol-phosphate acyltransferase Cutin synthesis -2.40 1.17E-24 Solyc03g065250 CER1 CER1 fatty acid hydroxylase Wax synthesis -1.72 1.12E-22 Solyc03g117800 CER1-like CER1 fatty acid hydroxylase Wax synthesis -1.64 8.16E-28 Solyc10g080840 MAH1 Cytochrome P450 Wax synthesis -2.48 8.41E-27 Solyc09g083050 KCS2 Fatty acid elongase 3-ketoacyl-CoA synthase Wax synthesis -4.33 1.14E-65 Solyc10g009240 KCS1 Fatty acid elongase 3-ketoacyl-CoA synthase Wax synthesis -1.35 2.43E-04 Solyc06g074390 CER4/FAR3 Fatty acyl coA reductase Wax synthesis -3.19 3.74E-14 Solyc09g092270 CER2-like Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase Wax synthesis -1.59 8.39E-13 Gene ID . Gene name . Annotation . Pathway . Log2 fold change . Adjusted P-value . Up-regulated Solyc02g088190 MIXTA MYB transcription factor Regulation 1.70 7.30E-08 Solyc12g009490 SHN2 Ethylene-responsive transcription factor Regulation 1.88 2.32E-05 Solyc01g109180 LACS2 Long-chain fatty acid CoA ligase Cutin synthesis 2.51 7.18E-101 Solyc01g094750 CYP86A68 Cytochrome P450 Cutin synthesis 2.99 9.47E-05 Solyc08g081220 CYP86A69 Cytochrome P450 Cutin synthesis 2.46 8.12E-138 Solyc05g055400 CYP77A2 Cytochrome P450 Cutin synthesis 1.77 1.01E-152 Solyc11g007540 CYP77A1 Cytochrome P450 Cutin synthesis 2.77 1.49E-38 Solyc06g062600 HTH-like Choline dehydrogenase Cutin synthesis 2.63 9.18E-21 Solyc03g121600 HTH-like Choline dehydrogenase Cutin synthesis 2.01 5.85E-16 Solyc06g035580 HTH-like Choline dehydrogenase Cutin synthesis 2.08 4.99E-123 Solyc01g094700 GPAT4 Glycerol-3-phosphate acyltransferase Cutin synthesis 1.63 1.39E-36 Solyc09g014350 GPAT6 Glycerol-3-phosphate acyltransferase Cutin synthesis 3.06 4.99E-123 Solyc06g065670 ABCG32 ATP-binding cassette transporter Cutin transport 1.34 1.39E-08 Solyc11g006250 CD1/CUS1 GDSL esterase/lipase Cutin assembly 1.95 6.57E-191 Solyc01g088400 CER1-like CER1 fatty acid hydroxylase Wax synthesis 1.05 5.52E-06 Solyc03g005320 KCS-like Fatty acid elongase 3-ketoacyl-CoA synthase Wax synthesis 3.27 7.59E-05 Solyc08g067260 KCS10 Fatty acid elongase 3-ketoacyl-CoA synthase Wax synthesis 1.08 9.75E-35 Solyc11g067180 CER4-like Fatty acyl coA reductase Wax synthesis 5.99 2.44E-04 Down-regulated Solyc07g056320 GPAT1 ER glycerol-phosphate acyltransferase Cutin synthesis -2.40 1.17E-24 Solyc03g065250 CER1 CER1 fatty acid hydroxylase Wax synthesis -1.72 1.12E-22 Solyc03g117800 CER1-like CER1 fatty acid hydroxylase Wax synthesis -1.64 8.16E-28 Solyc10g080840 MAH1 Cytochrome P450 Wax synthesis -2.48 8.41E-27 Solyc09g083050 KCS2 Fatty acid elongase 3-ketoacyl-CoA synthase Wax synthesis -4.33 1.14E-65 Solyc10g009240 KCS1 Fatty acid elongase 3-ketoacyl-CoA synthase Wax synthesis -1.35 2.43E-04 Solyc06g074390 CER4/FAR3 Fatty acyl coA reductase Wax synthesis -3.19 3.74E-14 Solyc09g092270 CER2-like Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase Wax synthesis -1.59 8.39E-13 Open in new tab In order to assess the relationship between ABA signaling and cuticle formation, we examined the expression of ABA signaling core component genes in fruit skin of WT and SlPP2C3-RNAi lines (Fig. 9D-F; Fig. S13C). Expression of ABA receptor genes SlPYL3 and SlPYL8 was up-regulated in transgenic lines (Fig. 9E). SlPP2C1 was slightly down-regulated but expression of SlPP2C3 was not (Fig. 9D). Among eight SnRK2 members, expression of SlSnRK2.4, SlSnRK2.6 and SlSnRK2.8 was significantly up-regulated in transgenic lines (Fig. 9F). In addition, SlPYL3 and SlPP2C3 expression was elevated in the epidermis, and these genes were co-expressed with SlMYB96 and SlMYB106 (Supplementary Fig. S11). Compared with WT, expression of SlMYB96 was significantly up-regulated in the fruit skin of SlPP2C3-OE fruits at the MG stage, and expression of SlMYB106 was down-regulated (Supplementary Fig. S12A, B). However, in SlPP2C3-RNAi fruits, the aforementioned genes showed the opposite expression trend (Supplementary Fig. S12A, B). The promoter region of SlMYB96 and SlMYB106 includes an ACGT-containing ABA response element (Supplementary Fig. S12E). Expression of SlMYB96 in MG tomato pericarp was up-regulated under ABA treatment, while SlMYB106 expression was not responsive to ABA (Supplementary Fig. S12C, D). These results imply that altered ABA signal transduction in SlPP2C3-RNAi lines may be responsible for the changes in expression of genes involved in cuticle formation. Discussion Preferential interaction of SlPP2C3 with monomeric receptors ABA initiates signal transduction by forming an ABA-PYL-PP2C ternary complex. Both PYLs and PP2Cs are multigene families with redundant functions. According to oligomeric state, PYL receptors can be divided into dimeric and monomeric receptors (Hao et al., 2011). Group A PP2Cs are clustered into two sub-families (Supplementary Fig. S6) which determine the complexity of PYL-PP2C interactions. In the present work, SlPP2C3 did not interact with dimeric receptors SlPYL5 and SlPYL8 in either yeast or tobacco cells. Similar results were observed in our previous work (Chen et al., 2016). Phylogenetic analysis indicated that SlPP2C3 is the orthologue of Arabidopsis AtHAI1/2/3. In Arabidopsis, HAI PP2Cs exhibit a marked preference for interactions with monomeric PYLs, and they do not interact with dimeric receptors AtPYR1, AtPYL1, and AtPYL2 (Bhaskara et al., 2012). However, AtHAI1 activity can be inhibited by dimeric receptors in vitro (Antoni et al., 2012). The molecular mechanism of the selective interaction between HAI-like PP2Cs and dimeric receptors is unknown. In the present work, by generating mutants at key PP2C-PYL interaction residues of SlPP2C3, Arg-285 and Ser-295 were found to play an important role in preventing SlPP2C3 from interacting with dimeric receptors (Fig. 3). These results partially explain the mechanism of PP2C preference. However, more evidence from other members of sub-family II PP2Cs is required. SlPP2C3 negatively regulates ABA signaling and affects fruit ripening We have shown that SlPP2C3 acts as a negative regulator in tomato ABA signaling based on two pieces of evidence: (i) SlPP2C3 interacted with multiple SlPYLs and SlSnRK2.8 (Figs 1, 2A); and (ii) SlPP2C3-RNAi plants were hypersensitive to ABA, while SlPP2C3-OE plants were less sensitive in several ABA-mediated processes (Figs 5, 6). SlPP2C3 is similar to other group A PP2Cs reported to act negatively in ABA signaling in several plant species (Gosti et al., 1999; Rubio et al., 2009; Zhang et al., 2018). For example, Arabidopsis abi1-2 and abi1-3 showed enhanced responses to ABA in seed and vegetative tissues (Saez et al., 2006). Transpirational water loss under drought conditions was noticeably reduced in both hab1-1 abi1-2 and hab1-1 abi1-3 double mutants, which revealed cooperative negative regulation of ABA signaling by ABI1 and HAB1 (Saez et al., 2006). Notably, the fruit ripening schedule was affected by SlPP2C3 expression (Fig. 7A, B). Previous studies demonstrated the role of endogenous ABA concentrations and exogenous ABA treatment on tomato fruit ripening (Zhang et al., 2009; Sun et al., 2012b, 2017; Liang et al., 2020). More recent studies indicate that ABA signaling also plays a crucial role in regulating the fruit ripening process. For example, overexpression of tomato ABA receptor SlPYL9 or suppression of SlPP2C1 causes early fruit ripening (Zhang et al., 2018; Kai et al., 2019). In addition, ABA signaling inhibitor ABA ANTAGONIST1 inhibits tomato fruit ripening (Ye et al., 2017). Our current data indicate that tomato fruit ethylene production is affected by SlPP2C3 (Fig. 7C), and several previous studies demonstrated crosstalk between PP2C and ethylene production. For instance, in Arabidopsis, AtABI1 interacts with AtACS2 or AtACS6 and inhibits their activity by dephosphorylation (Ludwików et al., 2014). ABA inhibits ethylene production through ABI4-mediated transcriptional repression of AtACS4 and AtACS8 (Dong et al., 2015). In our expression data, several genes involved in ethylene biosynthesis (SlACOs) and signaling (SlETRs) were up-regulated in SlPP2C3-RNAi fruits (Supplementary Fig. S9). The timing of ethylene production varied in SlPP2C3-RNAi and SlPP2C3-OE fruits. However, SlPP2C3 silencing by RNAi or overexpression had no marked effect on amount of ethylene production (Fig. 7C). Based on these results, we propose that SlPP2C3 negatively affects fruit ripening by controlling the ethylene production time. SlPP2C3 affects fruit outer epidermis cuticle formation The outer epidermis of fruit is the first barrier protecting against desiccation and pathogen invasion, and it also plays a crucial role in fruit shelf life. ABA influences cuticle formation and cuticle-associated gene expression under water deficit conditions in Arabidopsis and tomato (Kosma et al., 2009; Curvers et al., 2010; Wang et al., 2011). A recent study indicated that ABA core signal components (PYL, PP2C and SnRK2) affect cuticle development in Arabidopsis. However, transcription factors ABF2/3/4, which are the direct targets of SnRK2, do not regulate the cuticle phenotype. This implies the existence of a divergent pathway operating downstream of SnRK2 kinase (Cui et al., 2016). In one study, tomato ABA-deficient mutants exhibited reduced cutin and wax composition, and down-regulated cuticle biosynthesis genes in leaves, but ABA deficiency affected the fruit cuticle much more than leaves (Martin et al., 2017). In our current study, SlPP2C3-RNAi fruits exhibited altered epidermis surface (Fig. 9A). Transcriptome data identified two MYB (SlMYB96 and SlMYB106) transcription factors and 25 cuticle metabolism genes, which were differentially expressed in SlPP2C3-RNAi fruit skin (Table 1). These MYB transcription factors may be downstream targets in ABA signaling pathways. Given the similar expression patterns of SlMYB96, SlMYB106, SlPYL3, and SlPP2C3, as well as the unique localization of the SlPYL3-SlPP2C3 complex (Fig. 2A; Supplementary Fig. S11), we speculate that SlPYL3-SlPP2C3 mediates upstream ABA signaling, and may be important in regulation of cuticle formation. However, the precise mechanism remains unknown. Overall, our results suggest that SlPP2C3-mediated ABA signaling plays a substantial role in fruit cuticle formation. Supplementary data The following supplementary data are available at JXB online. Fig. S1. Schematic diagram of SlPP2C3-OE and SlPP2C3-RNAi constructs. Fig. S2. Agarose gel electrophoresis of RNA used in this study. Fig. S3. Agilent 2100 analysis of fruit skin RNA. Fig. S4. Phylogenetic analysis of tomato and Arabidopsis type 2C protein phosphatase (PP2C) family members. Fig. S5. Alignment of the amino acid sequences of tomato and Arabidopsis group A PP2C members. Fig. S6. Phylogenetic analysis of tomato and Arabidopsis group A PP2C family members. Fig. S7. Expression of SlPP2C3 in various tomato tissues of SlPP2C3 transgenic lines. Fig. S8. Vegetative phenotypes of SlPP2C3 transgenic lines. Fig. S9. Expression of genes related to ethylene synthesis and signaling in WT and SlPP2C3-RNAi fruits. Fig. S10. Carotenoids, total sugars, and titratable acid in WT and SlPP2C3 transgenic fruits. Fig. S11. Spatiotemporal expression of SlPP2C3, SlPYL3, SlMYB96 and SlMYB106 during M82 tomato fruit development and ripening. Fig. S12. Information related to SlMYB96 and SlMYB106. Fig. S13. SlPP2C3 affects the expression of genes involved in ethylene and ABA pathways. Table S1. Basic information for tomato group A PP2Cs genes. Table S2. Primers used for SlPP2C3-OE and SlPP2C3-RNAi plasmid construction. Table S3. Primers used for bimolecular fluorescence complementation and sub-cellular localization constructs. Table S4. Primers used for yeast two-hybrid assay. Table S5. Primers used for site-directed mutagenesis. Table S6. Primers used for qRT–PCR analysis. Table S7. Parameters for qRT–PCR according to the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines derived from Bustin et al. (2010). Acknowledgements This work was financially supported by the Israel Science Foundation (ISF)–National Natural Science Foundation of China (NSFC) Joint Scientific Research Program (grant no. 31661143046) and the NSFC (grant nos. 31772270 and 31572095). Author contributions PL, QL, BL and YS designed the experimental plan and wrote the manuscript; BL and YS carried out experiments and analyzed the data; JW and YZ were responsible for tissue culture; WZ and YX assisted in experimental operations. All authors read and approved the final manuscript. Data availability The following data are available at Dryad Data Repository (https://doi.org/10.5061/dryad.4qrfj6q7m; Liang et al., 2021). Dataset 1. Total genes in WT and SlPP2C3-RNAi fruit skin at the mature green stage identified by RNA-seq. Dataset 2. Genes expressed differentially in WT and SlPP2C3-RNAi fruit skin according to RNA-seq. Primers and accession numbers of genes analysed in this study are listed in Supplementary Tables S1−S6. Seeds used in transgenic lines and all other data used in this study are available from the corresponding authors upon request. References Antoni R , Gonzalez-Guzman M, Rodriguez L, Rodrigues A, Pizzio GA, Rodriguez PL. 2012 . 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TI - Tomato protein phosphatase 2C influences the onset of fruit ripening and fruit glossiness
JF - Journal of Experimental Botany
DO - 10.1093/jxb/eraa593
DA - 2020-12-21
UR - https://www.deepdyve.com/lp/oxford-university-press/tomato-protein-phosphatase-2c-influences-the-onset-of-fruit-ripening-ksJ6ubzdeD
SP - 1
EP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -