Arabidopsis subtilase SASP is involved in the regulation of ABA signaling and drought tolerance by interacting with OPEN STOMATA 1

Arabidopsis subtilase SASP is involved in the regulation of ABA signaling and drought tolerance... Abstract Arabidopsis Senescence-Associated Subtilisin Protease (SASP) has previously been reported to participate in leaf senescence and in the development of inflorescences and siliques. Here, we describe a new role of SASP in the regulation of abscisic acid (ABA) signaling. SASP encodes a subtilase and its expression was considerably induced by darkness, ABA, and ethylene treatments. sasp knockout mutants displayed obvious developmental phenotypes such as early flowering and smaller leaves. In particular, the sasp mutants exhibited enhanced ABA sensitivity during seed germination and seedling growth, heightened ABA-mediated leaf senescence, and increased production of reactive oxygen species (ROS). Importantly, the sasp mutants also showed remarkably increased tolerance to drought, with expression of six ABA signaling-related genes being either up- or down-regulated following ABA treatment. Interaction assays demonstrated that SASP physically interacts with OPEN STOMATA 1 (OST1) at the cell periphery. Co-expression of SASP and OST1 led to degradation of OST1, whereas this degradation was impaired in sasp-1 protoplasts. ROS attenuation assays demonstrated that in sasp-1 mutant guard cells the attenuation rate markedly decreased. Taken together, the results demonstrate that SASP plays an important role in regulating ABA signaling and drought tolerance through interaction with OST1. Abscisic acid (ABA) signaling, Arabidopsis thaliana, drought tolerance, OPEN STOMATA 1 (OST1), reactive oxygen species (ROS), senescence-associated subtilisin protease (SASP) Introduction Subtilases (SBTs) consist of a large family of serine peptidases, which differ from other types of proteases by the specific arrangement of the Asp, His, and Ser residues in their catalytic triad (Lang-Mladek et al., 2010). Compared to animals and micro-organisms, plants possess more SBTs (Srivastava et al., 2008). For example, the Arabidopsis genome encodes 56 SBTs, which are categorized into six distinct subfamilies, namely SBT1–6 (Rautengarten et al., 2005; Srivastava et al., 2008). Plant SBTs have been reported to be involved in protein turnover, seed and fruit development, modifications of cell wall, post-translational processing of protein precursors, regulation of stomatal density and distribution, abiotic and biotic responses, and programmed cell death (PCD) (Tripathi and Sowdhamini, 2006). Plant SBTs are known to play important roles in several biological processes. First, plant SBTs can catalyse non-selective protein degradation. For instance, cucumisin was the first SBT isolated from plant species (Kaneda and Tominaga, 1975; Yamagata et al., 1994) and is an extracellular alkaline protease that exhibits broad substrate specificity toward peptides and protein substrates (Yamagata et al., 1994). AtSBT1.7 shows little selectivity for synthetic peptide substrates and is able to degrade a wide assortment of extracellular proteins (Hamilton et al., 2003). Second, plant SBTs can act as proprotein convertases. AtSBT6.1 was found to specifically process pro-RALF23 to release a C-terminal growth-inhibiting RALF peptide (Srivastava et al., 2009). Third, plant SBTs are implicated in plant-specific developmental processes (Acharya et al., 2013). AtSBT1.4, referred to as Senescence-Associated Subtilisin Protease (SASP), was isolated by a proteomic method and found to be involved in the process of leaf senescence (Martinez et al., 2015). sasp-1 mutant plants produce 25% more inflorescence branches and siliques than either wild-type Col-0 or complementation lines (Martinez et al., 2015). Thus, the importance of SBTs in numerous biological processes is clear. It is well known that proteolysis-dependent protein breakdown and recycling play important roles in plant responses to environmental stresses. A number of studies have shown that proteases, such as serine and aspartic proteases, play an essential part in plant responses to drought stress. For example, Hieng et al. (2004) examined three European Phaseolus vulgaris cultivars under conditions of severe water deficit and found that the most sensitive one exhibited a marked increase in activities of two different serine proteases, while the most resistant cultivar showed a significant decrease in the activities of these enzymes, indicating a negative correlation between protease activity and drought tolerance. Similarly, a study in peanut subjected to progressive drought stress conditions also showed that expression of the Arachis hypogaea serine protease (AHSP) gene decreased more rapidly in a drought-tolerant cultivar than in a drought-susceptible one (Dramé et al., 2007), which helped to reduce drought-induced hydrolytic processes in the tolerant cultivar, and hence to avoid severe damage to cellular structures such as the membrane systems. Taken together, it is evident that proteases, especially serine proteases, are implicated in drought stress responses, and that they appear to be negatively correlated with drought tolerance. Recent studies have demonstrated that abscisic acid (ABA) biosynthesis and signaling are subject to regulation by protein degradation process. The Arabidopsis U-box E3 ubiquitin ligase SENESCENCE-ASSOCIATED E3 UBIQUITIN LIGASE 1 (SAUL1) has been shown to target AAO3 (Arabidopsis aldehyde oxidase 3, a key enzyme for ABA biosynthesis) for degradation; in the saul1 mutant the AAO3 protein is over-accumulated and as a consequence the ABA content is substantially increased, which ultimately leads to rapid ABA-induced leaf senescence (Raab et al., 2009). ABA INSENSITIVE 1 (ABI1) is a phosphatase that binds to OPEN STOMATA 1 (OST1) to inhibit its activity in the absence of ABA. ABI1 has been found to be targeted and ubiquitinated by the U-box E3 ligases PLANT U-BOX 12 (PUB12) and PUB13 for degradation after ABA-bound PYRABACTIN RESISTANCE 1 (PYR1) interacts with ABI1, ultimately leading to degradation of ABI1 by the 26S proteasome (Kong et al., 2015). In maize, ZmOST1 was found to be phosphorylated by CASEIN KINASE 2 (CK2), a pleiotropic protein kinase, after which the kinase activity of ZmOST1 was turned off (Vilela et al., 2015). The phosphorylated ZmOST1 was highly unstable and it was ultimately degraded through the 26S proteasome. In summary, these studies demonstrate that the major constituents of ABA signaling are targeted by ubiquitination, which brings them under tight control of protein degradation through the 26S proteasome (Vilela et al., 2015). Using a natural-gradient polyacrylamide gel electrophoresis method, we previously isolated a subtilase named TaSSP1 (Senescence-associated Subtilisin Protease 1; Genbank accession number AGN03879) from senescent leaves of wheat (Triticum aestivum), induced by darkness or ABA, and found that the enzymatic activity of TaSSP1 was strongly induced in the senescent leaves, suggesting that it may play an essential part in the process of leaf senescence (Qi and Xu, 2003). Another study found that during leaf senescence, expression of SASP (the Arabidopsis ortholog of TaSSP1) was significantly induced and the proteolytic activity of SASP was obviously increased (Martinez et al., 2015), further suggesting the possible involvement of TaSSP1/SASP in leaf senescence. In order to better understand the roles of TaSSP1/SASP in leaf senescence and/or other biological processes, we have focused on Arabidopsis SASP and characterized its function in detail. During the course of the current study, however, we discovered that SASP (AtSBT1.4) is implicated in the regulation of ABA signaling and drought tolerance. We further found that knockout of SASP does not result in any visible phenotype associated with leaf senescence, but causes increased ABA signaling and higher drought tolerance. Interaction assays and degradation analyses revealed physical interaction between SASP and OST1 as well as degradation of OST1 by SASP. Taken together, our results provide a direct demonstration that SASP plays important roles in regulating ABA signaling and drought tolerance by targeting OST1 for degradation. Materials and methods Plant materials and growth conditions All Arabidopsis materials used in this study were in the Columbia-0 (Col-0) genetic background. Two T-DNA insertional mutants, sasp-1 and sasp-3, were obtained from the Arabidopsis Biological Resource Center (ABRC, www.abrc.osu.edu). Seeds were surface-sterilized with 0.8% sodium hypochlorite (NaClO) and sown on half-strength Murashige and Skoog (MS) medium containing 0.8% (w/v) agar and 2% (w/v) sucrose. On the 15th day after germination, seedlings were transferred to soil and grown in a growth room at 22 °C under a 16/8 h light/dark cycle. Expression analysis Briefly, approximately 1 μg of the total RNA from the different treated genotypes was used to synthesize the first-strand cDNA using One-Step gDNA Removal and cDNA Synthesis SuperMix kits (Transgen, Beijing, China). The cDNAs were then used for RT-PCR and qRT-PCR assays (ABI 7500, Applied Biosystems) with primers corresponding to each gene (Supplementary Table S1 at JXB online). Histochemical β-glucuronidase (GUS) staining and subcellular localization A 2046-bp SASP promoter fragment plus the entire 5′UTR (5′-untranslated region) sequence was amplified from wild-type DNA, and it was then cloned into the pCXGUS vector immediately upstream of the GUS coding sequence. The resulting fusion construct was introduced into wild-type Col-0 to obtain stable transgenic lines. Subsequently, the transgenic plants were subjected to GUS staining according to the method described by Merlot et al. (2002). For subcellular localization, the entire coding sequence of SASP was inserted into the pC1300-221-GFP (green fluorescent protein) vector to generate a fusion construct pC1300-221-SASP-GFP. It was then introduced into Nicotiana benthamiana leaf epidermal cells by agro-infiltration. After 3 d at 22 °C under 16/8 h light/dark conditions, the leaves were observed under a Zeiss LSM750 confocal laser-scanning microscope to determine the subcellular localization of SASP-GFP. ABA and drought treatments More than 100 seeds were sown on MS medium plates supplemented without (control) or with ABA (Sigma) at concentrations of 0.1 or 0.2 μM. After a 2-d period of cold stratification at 4 °C, the seeds were transferred to a growth room and germinated for an additional 2 d under long-day conditions (18/6 h light/dark) at 22 °C and the germination rates were determined. To test the responsiveness of roots to ABA treatment, the germinated seeds were transferred to vertical agar plates containing different concentrations of ABA [0 (control), 20, or 30 μM] and left to grow for another 7 d, after which root length was determined. For the drought stress treatment, 3-week-old plants were subjected to water withdrawal for 14 d as described previously (Osakabe et al., 2010). They were then rewatered, and after a further 7 d survival rates were determined. Detection of ROS accumulation and attenuation in guard cells Detached 5-week-old cauline leaves from wild-type Col-0, sasp mutants, and SASP complementation lines were soaked in distilled water without or with 50 μM ABA under light conditions for 3 d prior to observations of leaf senescence. The leaves were then subjected to diaminobenzidine (DAB) staining (Du et al., 2013). For measurements of ROS accumulation in guard cells, peeled epidermal strips of 4-week-old leaves from Col-0 and sasp mutants were soaked in an incubation buffer containing 30 mM KCl and 10 mM Mes-KOH (pH 6.15) for 2 h, and then the same buffer was supplemented with 100 μM ABA and the strips were incubated for 15 min. Following brief rinses, the strips were transferred to fresh incubation buffer containing 25 μM 2′, 7′-dichlorofluorescin diacetate (H2DCF-DA, Sigma) for 15 min. After being washed with distilled water, the strips were observed under a Zeiss LSM 750 microscope to inspect the accumulation of ROS in the guard cells (Lee et al., 1999). To determine ROS attenuation rates, peeled epidermal strips from Col-0 and sasp-1 were treated as described above, and then photographed under the microscope with repeated imaging seven times at 5-min intervals. Measurement of stomatal aperture For measurement of stomatal aperture following ABA treatment, peeled epidermal strips from cauline leaves (sampled from 4-week-old plants) were immersed in a stomata-opening buffer with 10 mM KCl, 0.2 mM CaCl2, and 10 mM Mes-KOH (pH 6.15) for 2 h under continuous white light at 22 °C (Du et al., 2013). ABA was then added into the same buffer to reach a final concentration of 10 μM and the epidermal strips were continuously incubated for another 2 h under the same conditions. Guard cells were photographed with a Zeiss Axio Scope A1 microscope, and stomatal lengths and widths were measured. For determination of stomatal aperture following drought treatment, peeled epidermal strips from cauline leaves sampled from 4-week-old plants that had undergone 7 d of water being withheld were examined. Determination of ABA content Briefly, 200 mg of leaf material from 4-week-old plants of Col-0 and sasp mutants that had been treated without or with either ABA or ethephon (by spraying each hormone solution onto the leaf surfaces) were harvested and ground to a fine powder in liquid nitrogen. Then, 2 ml of 80% methanol solution containing 1% acetic acid and 19% pure water was added to the powder. After centrifugation of the powder suspension, the supernatant was collected and used for determination of the ABA content by HPLC-MS (Agilent) as described previously (Pan et al., 2010). Luciferase complementation imaging (LCI) assays To screen for the candidate proteins (involved in ABA signaling) that were able to interact with SASP, the complete coding sequence of each of the tested genes was amplified and each PCR product was inserted into the pCAMBIA1300-nLUC vector; similarly, the cDNA of SASP was cloned into the pCAMBIA1300-cLUC vector (Chen et al., 2008). The genes tested were as follows: OST1, PYR1, PYL1 (PYRABACTIN RESISTANCE LIKE 1), PYL4, PYL8, PYL9, ABI1, RBOHD (RESPIRATORY BURST OXIDASE HOMOLOGUE D), RBOHF (RESPIRATORY BURST OXIDASE HOMOLOGUE F), SLAC1 (SLOW ANION CHANNEL-ASSOCIATED 1), and KAT1 (POTASSIUM CHANNEL IN ARABIDOPSIS THALIANA 1). They were separately transformed into Agrobacterium strain GV3101, and the positive clones were cultured and subsequently co-infiltrated together with Agrobacterium p19 strain into 4-week-old Nicotiana benthamiana leaves. After 3 d, the leaves were injected with D-Luciferin (Perkin Elmer) solution and placed in the dark for 30 min. Bioluminescence was detected with a CCD camera (Tanon 5200, Shanghai). Yeast two-hybrid (Y2H) assays Briefly, the nucleotide sequence (1–78 bp) corresponding to the predicted N-terminal signal peptide of the SASP coding sequence was deleted, and the resulting truncated sequence was subcloned into the pGBKT7 vector to generate the pGBKT7-SASP construct. The pGBKT7-SASP construct was then co-transformed with pGADT7-OST1 into yeast strain AH109. The co-transformed yeast cells were first spotted onto a synthetic dropout medium without Leu and Trp (SD/–Leu/–Trp) and then transferred to a stringent selection medium [synthetic dropout medium lacking Leu, Trp, and adenine (SD/–Leu/–Trp/–Ade), but supplemented with 5 mM 3-AT (3-amino-1, 2, 4-triazole)] and allowed to grow for 4 d at 28 °C prior to observation (He et al., 2009). Bimolecular fluorescence complementation (BiFC) assays The entire coding sequences of SASP and OST1 were cloned into YN and YC vectors, respectively. The resulting constructs were individually introduced into Agrobacterium strain GV3101, and the positive clones were cultured and co-infiltrated into N. benthamiana leaves (Acharya et al., 2013). After 3 d, fluorescence signals were visualized using a Zeiss LSM750 confocal laser-scanning microscope. In vivo degradation assay The entire coding sequences of SASP, OST1, and the plasma-membrane marker gene Plasma membrane Intrinsic Protein 2a (PIP2a) were cloned into the pCAMBIA1300-221-GFP, pCAMBIA1300-221-mCherry, and pCAMBIA1300-221-YFP vectors, respectively, to generate SASP-GFP, OST1-mCherry, and PIP2a-YFP constructs. An Agrobacterium culture carrying the OST1-mCherry construct was mixed separately with different concentrations of Agrobacterium cultures bearing the SASP-GFP construct or the PIP2a-YFP construct plus Agrobacterium p19 strain for co-infiltration. At 3 d after co-infiltration, fluorescence signals were detected by using a Zeiss LSM750 confocal laser-scanning microscope. Transient expression assays in Arabidopsis protoplasts Briefly, 3-week-old leaves from Col-0 and sasp-1 mutant plants were cut into small pieces and treated with lysis buffer to isolate intact protoplasts. The pOST1::OST1-mCherry construct was transfected into the protoplasts using the PEG-mediated transformation method (Miao et al., 2006). After incubation for 16–20 h at room temperature, the protoplasts were observed using a Zeiss LSM750 confocal laser-scanning microscope. Results SASP expression is induced by dark and plant hormone treatments To determine whether SASP expression is induced by hormones or environmental cues, we examined the expression patterns of SASP in wild-type Col-0 after treatment in the dark (darkness is capable of inducing rapid leaf senescence), or treatment with ABA or ethephon. qRT-PCR analysis revealed that SASP transcript levels increased with prolonged dark treatment, peaking at 48 h after treatment, but then declining at 72 h (Fig. 1A). ABA induced progressive accumulation of the SASP transcripts after 0.5, 12, and 24 h of treatment (Fig. 1B). Ethephon up-regulated SASP expression immediately after treatment, with the mRNA levels peaking at 0.5 h but declining gradually with prolonged treatment (Fig. 1C). Similar to ABA treatment, drought also led to up-regulation of SASP, and the expression level increased gradually with prolonged treatment (Fig. 1D). Further analysis of the conserved domain suggested that SASP possesses three domains, namely Inhibitor I9, Peptidases_S8_3, and Protease Associated (PA), and a 26-bp signal peptide is located at the N-terminus of this protein (Supplementary Fig. S1; Marchler-Bauer et al., 2002). Phylogenetic analysis and sequence alignment revealed that orthologous proteins of SASP exist widely in many plant species, including monocots (such as rice, wheat) and dicots (such as cotton, cabbage) (Supplementary Fig. S2), suggesting that SASP and its orthologous proteins may perform a particular function in plants. Thus, the results suggest that SASP can be induced by darkness, ABA and ethephon treatments, and it may serve particular functions during leaf senescence. Fig. 1. View largeDownload slide Induction of SASP expression by darkness, exogenous ABA and ethylene treatments, and drought stress. Relative expression levels of SASP in the dark (A), with application of 100 μM ABA (B) or 100 μM ethephon (ETN) (C), and with drought stress (D). Treatments were applied to 4-week-old Arabidopsis wild-type (Col-0) seedlings. Total RNAs were isolated at the indicated time points, and then subjected to qRT-PCR analysis to detect SASP expression levels. Actin 2 was used as the internal control. Data are means (±SD) of three biological replicates. Significant differences from time-point 0 were determined by Student’s t-test: *P<0.05, **P<0.01. Fig. 1. View largeDownload slide Induction of SASP expression by darkness, exogenous ABA and ethylene treatments, and drought stress. Relative expression levels of SASP in the dark (A), with application of 100 μM ABA (B) or 100 μM ethephon (ETN) (C), and with drought stress (D). Treatments were applied to 4-week-old Arabidopsis wild-type (Col-0) seedlings. Total RNAs were isolated at the indicated time points, and then subjected to qRT-PCR analysis to detect SASP expression levels. Actin 2 was used as the internal control. Data are means (±SD) of three biological replicates. Significant differences from time-point 0 were determined by Student’s t-test: *P<0.05, **P<0.01. Knockout of SASP does not change the progression of leaf senescence but accelerates the transition of Arabidopsis plants from vegetative to reproductive growth To better understand the roles of SASP in leaf senescence and plant growth as well as in development, we obtained two T-DNA insertional lines, sasp-1 (SALK_147962) and sasp-3 (SALK_001802), from the Arabidopsis Biological Research Center (Fig. 2A). RT-PCR analysis demonstrated that neither of the two lines was capable of producing full-length transcripts, indicating that SASP was indeed knocked out and thus that they are both knockout alleles (Fig. 2B). Surprisingly, the sasp mutants did not exhibit visible phenotypes associated with leaf senescence under either regular or dark conditions during the progression of development. Nevertheless, both mutants showed early-flowering phenotypes as they bolted and flowered earlier than the wild-type Col-0 plants, and generated fewer rosette leaves (Fig. 2C, D). In addition, the leaves of both mutants were smaller in size than those of the wild type at the onset of the bolting stage (Fig. 2E). At the maturity stage, the sasp mutants showed a significant increase in plant height and produced smaller seeds when compared to the wild type (Supplementary Fig. S3). A genomic DNA fragment of wild-type SASP driven by its native promoter fully rescued the early-flowering phenotype of the sasp-1 mutant plants, and partially restored the total number of rosette leaves and leaf size to the level of the wild-type plants (Fig. 2D, E). In parallel, the genetic complementation also fully rescued the smaller seed-size phenotype and partially decreased the plant height that was seen in the sasp-1 mutant plants (Supplementary Fig. S3). Collectively, these results suggest that knockout of SASP shortens the vegetative growth and accelerates the transition of plants from vegetative to reproductive growth. In addition, qRT-PCR analysis indicated that expression of two senescence-associated genes (SAGs), SAG29A and SAG12, was up-regulated by more than 14 and 38 times, respectively, in the sasp mutants compared to the wild type plants, implying that senescence-associated pathways were activated in the sasp mutants (Supplementary Fig. S4). Thus, the fact that knockout of SASP accelerates the transition of Arabidopsis plants from vegetative to reproductive growth possibly results from activated senescence-associated pathways that might, however, be insufficient to produce visible phenotypes associated with leaf senescence. Taken together, the results indicate that knockout of SASP has no visible effect on leaf senescence but it does accelerate the transition of Arabidopsis plants from vegetative to reproductive growth. Fig. 2. View largeDownload slide Characterization of sasp T-DNA insertional mutants and developmental phenotypes. (A) Schematic representation of the SASP gene and the positions of the T-DNA insertions. The shaded box represents the exon, open boxes represent untranslated regions (UTRs), and the T-DNA insertions are represented by triangles. The arrows denote the PCR primers used for genotyping and RT-PCR. (B) Semi-quantitative RT-PCR analysis of SASP expression in the sasp-1 and sasp-3 mutants. Actin 2 served as the constitutively expressed control. The primers used for PCR are shown in (A). (C, D) Comparison of flowering behavior between the wild-type Col-0, sasp mutants, and SASP complementation lines. (C) Flowering phenotypes of Col-0 and sasp mutants, and total rosette leaf number of each genotype at flowering. (D) Flowering phenotypes of Col-0, sasp-1, and SASP complementation lines (COM1 and COM2), and total rosette leaf number of each genotype at flowering. All the genotypes were grown in pots under long-day conditions (16/8 h light/dark), and the images were taken when the first genotype began to flower. Data are means (±SD) of three biological replicates; for each genotype, nine plants were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05. (E) Comparison of all leaves from one representative plant of each genotype from Col-0, sasp mutants, and SASP complementation lines. Scale bars are 1 cm. Fig. 2. View largeDownload slide Characterization of sasp T-DNA insertional mutants and developmental phenotypes. (A) Schematic representation of the SASP gene and the positions of the T-DNA insertions. The shaded box represents the exon, open boxes represent untranslated regions (UTRs), and the T-DNA insertions are represented by triangles. The arrows denote the PCR primers used for genotyping and RT-PCR. (B) Semi-quantitative RT-PCR analysis of SASP expression in the sasp-1 and sasp-3 mutants. Actin 2 served as the constitutively expressed control. The primers used for PCR are shown in (A). (C, D) Comparison of flowering behavior between the wild-type Col-0, sasp mutants, and SASP complementation lines. (C) Flowering phenotypes of Col-0 and sasp mutants, and total rosette leaf number of each genotype at flowering. (D) Flowering phenotypes of Col-0, sasp-1, and SASP complementation lines (COM1 and COM2), and total rosette leaf number of each genotype at flowering. All the genotypes were grown in pots under long-day conditions (16/8 h light/dark), and the images were taken when the first genotype began to flower. Data are means (±SD) of three biological replicates; for each genotype, nine plants were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05. (E) Comparison of all leaves from one representative plant of each genotype from Col-0, sasp mutants, and SASP complementation lines. Scale bars are 1 cm. sasp mutants are more sensitive to ABA at both seed germination and seedling stages Given that SASP transcripts were induced by ABA, we wondered whether sasp knockout mutants would display a distinct responsiveness to ABA compared to wild-type plants. To test this, we germinated sasp mutant seeds together with wild-type seeds on filter paper containing three different concentrations of ABA: 0 (control), 0.1, or 0.2 μM. We found that the germination rates of the sasp mutants were significantly lower than those of the wild type when seeds were germinated on media containing ABA (Fig. 3A, B). In addition, when seedlings were grown vertically on MS media containing 0 (control), 20, or 30 μM ABA, the root growth of the sasp mutants was obviously inhibited when compared with that of wild type (Fig. 3C, D). Overall, the results indicate that sasp knockout mutants are more sensitive to ABA at the seed germination and seedling growth stages, suggesting that ABA signaling might be enhanced in the sasp mutants. Fig. 3. View largeDownload slide Increased ABA sensitivity of sasp mutants during seed germination and seedling growth. (A, B) Germination assays of Col-0 and sasp mutants with or without ABA treatment. Seeds were germinated on filter paper soaked with 0 (control), 0.1, or 0.2 μM ABA solution. After 4 d, images were taken (A) and germination rates were calculated (B). Data are means (±SD) for three biological replicates; for each genotype, 50 seeds were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05, **P<0.0. Scale bars are 1 cm. (C, D) Effects of ABA treatment on root growth of Col-0 and sasp mutants. Seeds were germinated on MS medium plates for 5 d, and then the seedlings were transferred to new plates containing 0 (control), 20, or 30 μM ABA, and left to vertically grow for a further 12 d. Images were then taken (C) and root length was calculated relative to the control for each genotype (D). Data are means (±SD) for three biological replicates; for each genotype, 10 seedlings were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05, t-test. Scale bars are 1 cm. Fig. 3. View largeDownload slide Increased ABA sensitivity of sasp mutants during seed germination and seedling growth. (A, B) Germination assays of Col-0 and sasp mutants with or without ABA treatment. Seeds were germinated on filter paper soaked with 0 (control), 0.1, or 0.2 μM ABA solution. After 4 d, images were taken (A) and germination rates were calculated (B). Data are means (±SD) for three biological replicates; for each genotype, 50 seeds were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05, **P<0.0. Scale bars are 1 cm. (C, D) Effects of ABA treatment on root growth of Col-0 and sasp mutants. Seeds were germinated on MS medium plates for 5 d, and then the seedlings were transferred to new plates containing 0 (control), 20, or 30 μM ABA, and left to vertically grow for a further 12 d. Images were then taken (C) and root length was calculated relative to the control for each genotype (D). Data are means (±SD) for three biological replicates; for each genotype, 10 seedlings were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05, t-test. Scale bars are 1 cm. sasp mutants display enhanced ABA-mediated leaf senescence and increased ROS production In light of the fact that ABA is capable of promoting senescence in detached organs (Du et al., 2013) and that the senescence-associated pathways might have been activated in the sasp mutants, we hypothesized that sasp leaves may become senescent faster than those of wild-type plants under ABA treatment. To test this hypothesis, we treated detached leaves with 50 μM ABA for 3 d and found that the leaves of the sasp mutants did indeed display greater senescence than those of the wild type (Fig. 4). The ABA treatment appeared to be quite effective as SAG12 expression was significantly induced relative to untreated controls (Supplementary Fig. S5). Detached leaves from the SASP complementation lines exhibited similar leaf senescence phenotypes to the wild type under the same treatment conditions (Fig. 4). By contrast, under normal or dark conditions, detached sasp mutant leaves did not display any obvious early or delayed senescence when compared with the wild type (Supplementary Fig. S6). DAB staining revealed that the detached leaves from the sasp mutants showed increased ROS production compared with the wild-type and the SASP complementation plants upon ABA treatment (Fig. 4). In contrast to leaves treated with ABA, untreated leaves from both the wild type and the sasp mutants showed no notable differences from each other in senescence and ROS production (Fig. 4). Thus, it is clear that knockout of SASP promotes ABA-mediated leaf senescence and results in more ROS production in comparison with wild-type plants, further reinforcing the notion that ABA signaling is increased in the sasp mutants. Fig. 4. View largeDownload slide Enhanced leaf senescence and production of reactive oxygen species (ROS) in sasp mutants. Detached leaves from 5-week-old Col-0, sasp mutants, and SASP complementation lines were incubated without (control) or with 50 μM ABA for 3 d. Images were then taken, and the leaves were subjected to DAB staining for 8 h. Representative leaves are shown. Scale bars are 1 cm. Fig. 4. View largeDownload slide Enhanced leaf senescence and production of reactive oxygen species (ROS) in sasp mutants. Detached leaves from 5-week-old Col-0, sasp mutants, and SASP complementation lines were incubated without (control) or with 50 μM ABA for 3 d. Images were then taken, and the leaves were subjected to DAB staining for 8 h. Representative leaves are shown. Scale bars are 1 cm. sasp mutants display remarkably higher tolerance to drought stress Given the increased ROS production in the sasp mutant leaves when exposed to ABA, we wondered whether they might have increased drought tolerance. To test this, sasp mutants, SASP complementation lines, and wild-type plants were simultaneously grown to the onset of flowering in pots, and then water was withheld for a period of 14 d. The plants were then rewatered for 7 d before being examined. It was obvious that the sasp mutants were more tolerant to drought stress and their survival rates were nearly 80%, whereas the wild-type plants were more sensitive and their survival rates were approximately 20% (Fig. 5A, B). Complementation of the sasp-1 mutants with wild-type SASP gDNA, however, decreased the survival rates to around 35–42% (Fig. 5A, C). The stomata in the sasp mutants closed more than in the wild type under the drought conditions (Fig. 5D) and the stomatal apertures of the mutants were clearly reduced compared with the wild type (Fig. 5D). We then examined the expression of six important genes involved in ABA signaling in response to the drought stress treatment, and found that all of them except for PYL4 displayed elevated expression (Supplementary Fig. S7). Thus, the results indicated that SASP was involved in responsiveness to drought stress, and knockout of SASP led to a significant increase in drought tolerance in Arabidopsis. In addition, we also examined the stomatal apertures of leaves treated with 10 μM ABA and found that the apertures of the sasp mutants were approximately 43% of the wild-type level (Fig. 5E), suggesting that knockout of SASP significantly promotes stomatal closure under this ABA treatment. Taken together, the results indicate that SASP is involved in the regulation of drought tolerance, and loss of SASP function makes Arabidopsis plants more tolerant to drought stress. Fig. 5. View largeDownload slide Increased drought tolerance and stomatal closure of sasp mutants. (A) Increased drought tolerance of sasp mutants under 14-d drought stress treatment. Wild-type Col-0, sasp mutants, and SASP complementation lines (Com1 and Com2) were grown normally in pots for 4–5 weeks, followed by a 14-d drought stress treatment and subsequent rewatering for 7 d. Scale bars are 5 cm. (B, C) Survival rates of Col-0, sasp mutants, and SASP complementation lines calculated after 7 d of rewatering. Data are means (±SD) for three biological replicates; for each genotype, 12 plants were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: **P<0.01. (D) Changes in stomatal aperture in Col-0 and sasp mutants following drought treatment. The images show peeled leaf epidermal strips from 4-week-old Col-0 and sasp mutants following 7 d of drought, together with strips from watered controls. Representative guard cells are shown, and measurements of stomatal aperture (length, width) following the drought treatment are given in the graph. Data are means (±SD) for three biological replicates; for each genotype, 60 guard cells were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05. Scale bars are 20 μm. (E) Changes in stomatal aperture in Col-0 and sasp mutants after ABA treatment. Peeled leaf epidermal strips of 4-week-old plants grown under normal conditions were treated without (control) or with 10 μM ABA. Images of representative guard cells are shown, and measurements of stomatal aperture (length, width) following ABA treatment are given in the graph. Data are means (±SD) for three biological replicates; for each genotype, 50 guard cells were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05. Scale bars are 20 μm. Fig. 5. View largeDownload slide Increased drought tolerance and stomatal closure of sasp mutants. (A) Increased drought tolerance of sasp mutants under 14-d drought stress treatment. Wild-type Col-0, sasp mutants, and SASP complementation lines (Com1 and Com2) were grown normally in pots for 4–5 weeks, followed by a 14-d drought stress treatment and subsequent rewatering for 7 d. Scale bars are 5 cm. (B, C) Survival rates of Col-0, sasp mutants, and SASP complementation lines calculated after 7 d of rewatering. Data are means (±SD) for three biological replicates; for each genotype, 12 plants were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: **P<0.01. (D) Changes in stomatal aperture in Col-0 and sasp mutants following drought treatment. The images show peeled leaf epidermal strips from 4-week-old Col-0 and sasp mutants following 7 d of drought, together with strips from watered controls. Representative guard cells are shown, and measurements of stomatal aperture (length, width) following the drought treatment are given in the graph. Data are means (±SD) for three biological replicates; for each genotype, 60 guard cells were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05. Scale bars are 20 μm. (E) Changes in stomatal aperture in Col-0 and sasp mutants after ABA treatment. Peeled leaf epidermal strips of 4-week-old plants grown under normal conditions were treated without (control) or with 10 μM ABA. Images of representative guard cells are shown, and measurements of stomatal aperture (length, width) following ABA treatment are given in the graph. Data are means (±SD) for three biological replicates; for each genotype, 50 guard cells were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05. Scale bars are 20 μm. SASP is involved in regulation of expression of ABA-signaling genes To examine whether expression of ABA-signaling genes was changed in sasp mutants in response to ABA, we used qRT-PCR to measure the transcript levels of 24 genes that are implicated in, or associated with, ABA biosynthesis or signaling (ABA1, NCED3, PYR1, PYL4, ABI1, HAB1, RBOHD, RBOHF, OST1, etc.). We found that two negative regulator genes of ABA-signaling, ABI1 and HAB1, displayed similar expression patterns: without ABA treatment (control), the expression levels in the sasp mutants were both lower than in the wild type (Fig. 6), and although the expression in all genotypes was up-regulated after treatment with 100 μM ABA, the expression levels of the sasp mutants were still lower than those of the wild type. RBOHD, RBOHF, PYL4, and OST1 have been identified as positive regulators for ABA signaling, and up-regulation of these genes contributes to activation of the ABA signaling (Raghavendra et al., 2010; Acharya et al., 2013; Raghavendra et al, 2010). Our results showed that the expression of RBOHD, RBOHF, and PYL4 were all up-regulated in the sasp mutants irrespective of ABA treatment (Fig. 6), suggesting that knockout of SASP enhances the expression of these genes. It appeared that OST1 expression was slightly increased in the sasp mutants, although its expression was not induced by ABA. The ABA treatment was effective, as the transcript levels of the marker gene RD29A were up-regulated in all genotypes following treatment (Supplementary Fig. S8). In summary, the results indicated that knockout of SASP in the presence or absence of 100 μM ABA increases the expression of positive regulator genes of ABA signaling (RBOHD, RBOHF, PYL4, and OST1) but decreases the expression of negative regulator genes (ABI1 and HAB1), supporting the idea that ABA signaling is activated as well as increased in the sasp mutants. Fig. 6. View largeDownload slide Expression analyses of six essential genes involved in ABA signaling under control and ABA treatments. Leaves of 4-week-old Col-0 and sasp mutants were sprayed with 0 (control) or 100 μM ABA solution prior to sampling. After 3 h, total RNAs were isolated and reverse-transcribed into cDNAs, which were then used for quantitative PCR with primers corresponding to specific regions of ABI1, HAB1, RBOHD, RBOHF, PYL4, and OST1. Data are means (±SD) of three biological replicates. Significant differences from Col-0 within each treatment were determined by Student’s t-test: *P<0.05. Fig. 6. View largeDownload slide Expression analyses of six essential genes involved in ABA signaling under control and ABA treatments. Leaves of 4-week-old Col-0 and sasp mutants were sprayed with 0 (control) or 100 μM ABA solution prior to sampling. After 3 h, total RNAs were isolated and reverse-transcribed into cDNAs, which were then used for quantitative PCR with primers corresponding to specific regions of ABI1, HAB1, RBOHD, RBOHF, PYL4, and OST1. Data are means (±SD) of three biological replicates. Significant differences from Col-0 within each treatment were determined by Student’s t-test: *P<0.05. Another possibility is that the increased ROS production and the higher tolerance to drought stress in the sasp mutants could result from elevated ABA content. However, using LC-MS, we did not find any significant differences in ABA content between the wild type (19.00 ng g–1 DW) and the sasp mutants (23.41–24.60 ng g–1 DW) (Supplementary Fig. S9). It is worth noting that the ABA content in all the genotypes increased to over 250 ng g–1 DW after they were treated with ABA or ethephon, and the ABA content in the sasp mutants was significantly higher than in the corresponding wild-type Col-0 (Supplementary Fig. S9). However, after examining the expression levels of five key genes involved in ABA biosynthesis (ABA1, ABA2, ABA3, NCED3, and AAO3), we found that there were no significant differences in transcript levels among the genotypes under either control or treatment conditions (although the expression of all genes was obviously induced by ABA and ethephon; Supplementary Fig. S10). These results suggest that the significantly elevated ABA content in the sasp mutants under ABA and ethephon treatments resulted from impaired degradation of ABA. Taken together, the results demonstrate that it is ABA signaling that is increased in the sasp mutants under normal growth conditions. Expression patterns and subcellular localization of SASP RT-PCR analysis demonstrated that SASP was ubiquitously expressed in all the tissues examined, namely the roots, stem, rosette leaf, cauline leaf, siliques, and flowers (Fig. 7A). The expression levels in the stem, rosette, and cauline leaf were relatively higher than those in roots, siliques, and flowers. Histochemical localization showed that in transgenic plants containing the PSASP::GUS construct, GUS staining could be detected throughout all the tissues examined, which was in agreement with the RT-PCR results; however, the overall GUS signals were weak, suggesting that the SASP expression levels were relatively low in Arabidopsis (Fig. 7B–H). It is worth noting that relatively high levels of GUS expression were observed at the base of the stem as well as at the tip of leaf and in the anther (Fig. 7C, F, H). Subcellular localization indicated that SASP-GFP proteins were predominantly associated with the plasma membrane and were mainly located at the cell periphery, because the merged images displayed bright yellow signals due to the overlap of the green (SASP-GFP) and red (plasma membrane marker PIP2a-mCherry) channels (Fig. 7I). When the transformed cells were treated with 30% sucrose to induce plasmolysis, the SASP-GFP proteins remained associated with the plasma membrane. These results lead us to the conclusion that SASP is extensively expressed in various tested tissues and that SASP is a plasma membrane-associated protein that is largely localized at the cell periphery. Fig. 7. View largeDownload slide Expression patterns and subcellular localization of SASP. (A) Expression patterns of SASP in various tissues as determined by RT-PCR. Total RNAs were isolated from 6-week-old plants, and then used for reverse transcription and PCR reactions. (B–H) Detection of SASP promoter activity in different tissues by histochemical GUS staining. A 6-week-old whole plant (B) harboring the PSASP::GUS transgene was subjected to GUS staining and the following tissues were examined: roots (C), stem (D), rosette leaf (E), cauline leaf (F), silique (G), and mature flower (H). (I) Subcellular localization of SASP-GFP fusion protein. Agrobacterium strain GV3101 cultures separately carrying the SASP-GFP or PIP2a-mCherry fusion constructs were mixed and then co-infiltrated into Nicotiana benthamiana leaves. For plasmolysis, the detached co-infiltrated leaves were treated with 30% of sucrose for 15 min prior to observation. Scale bars are 25 μm Fig. 7. View largeDownload slide Expression patterns and subcellular localization of SASP. (A) Expression patterns of SASP in various tissues as determined by RT-PCR. Total RNAs were isolated from 6-week-old plants, and then used for reverse transcription and PCR reactions. (B–H) Detection of SASP promoter activity in different tissues by histochemical GUS staining. A 6-week-old whole plant (B) harboring the PSASP::GUS transgene was subjected to GUS staining and the following tissues were examined: roots (C), stem (D), rosette leaf (E), cauline leaf (F), silique (G), and mature flower (H). (I) Subcellular localization of SASP-GFP fusion protein. Agrobacterium strain GV3101 cultures separately carrying the SASP-GFP or PIP2a-mCherry fusion constructs were mixed and then co-infiltrated into Nicotiana benthamiana leaves. For plasmolysis, the detached co-infiltrated leaves were treated with 30% of sucrose for 15 min prior to observation. Scale bars are 25 μm SASP physically interacts with OST1 and co-localizes with it at the cell periphery As SASP is a subtilase and the sasp mutants displayed increased ABA signaling, we wondered whether certain components of ABA signaling are able to interact with SASP. To explore this possibility, we first performed protein interaction assays between SASP and major ABA signaling components using the luciferase complementation imaging (LCI) method to screen for the proteins that are able to interact with SASP (a total of 11 proteins were tested). Ultimately, only OST1 was discovered to have strong physical interaction with SASP in N. benthamiana leaves, because co-expression of SASP with OST1 exhibited a high luciferase signal compared to the negative controls (Fig. 8A). Consistent with the LCI assay, Y2H assays also demonstrated an interaction between SASP and OST1 (Fig. 8B). In addition, BiFC assays again confirmed the interaction between SASP and OST1 (Fig. 8C), and it was obvious that the interaction occurred at the cell periphery as the green and red fluorescence signals overlapped at this region (Fig. 8C). Co-localization analysis of SASP-GFP and OST1-mCherry demonstrated that the green fluorescence signals were mainly distributed at the cell periphery, while the red fluorescence signals were observed ubiquitously across the entire cell, including the nucleus (Fig. 8D). It was clear that the yellow fluorescence signals occurred predominantly at the cell periphery where the green and red signals overlapped, as demonstrated by the merged image, again suggesting that SASP and OST1 are co-localized at the cell periphery. Taken together, these data support the conclusion that SASP interacts physically with OST1 at the cell periphery and it is possible that OST1 serves as a substrate for SASP. Fig. 8. View largeDownload slide Physical interactions between SASP and OST1. (A) Luciferase complementation imaging analysis of the interaction between SASP and OST1. SASP and OST1 coding sequences were fused to nLUC and nLUC vectors, respectively, and then the resulting fusion constructs were separately transformed into Agrobacterium strain GV3101. The Agrobacterium cultures separately bearing SASP-nLUC and OST1-cLUC constructs were then mixed and co-infiltrated into Nicotiana benthamiana leaves. A construct combination of SGT1a-nLUC and RAR1-cLUC was used as a positive control, while two construct combinations, OST1-nLUC plus cLUC and SASP-cLUC plus nLUC, were used as negative controls for the co-infiltration. Scale bars are 1 cm. (B) Yeast two-hybrid analysis of the interaction between SASP and OST1. SASP and OST1 coding sequences were fused to the pGBKT7 and pGADT7 vectors, respectively, and then the resulting fusion constructs were co-transformed into yeast strain AH109. The positive transformants were first spotted onto control medium (SD/–Trp–Leu) to grow for 4–6 d, and then transferred to selective medium (SD/–Trp–Leu–Ade + 3-AT) to grow for 4–6 d before observation. (C) Bimolecular fluorescence complementation analysis of the interaction between SASP and OST1. SASP and OST1 coding sequences were fused to YN and YC vectors, respectively, and then the resulting fusion constructs were separately transformed into Agrobacterium strain GV3101 for co-infiltration. Two construct combinations, YN plus OST1-YC and SASP-YN plus YC, were used as negative controls for the co-infiltration. Images were taken under a confocal microscope. Scale bars are 25 μm. (D) Co-localization of SASP-GFP and OST1-mCherry. SASP and OST1 coding sequences were fused to the pC1305-d35S-GFP and pC1305-d35S-mCherry vectors, respectively, and then the resulting fusion constructs were separately transformed into Agrobacterium strain GV3101 for co-infiltration. Images were taken under a confocal microscope. Scale bars are 25 μm. Fig. 8. View largeDownload slide Physical interactions between SASP and OST1. (A) Luciferase complementation imaging analysis of the interaction between SASP and OST1. SASP and OST1 coding sequences were fused to nLUC and nLUC vectors, respectively, and then the resulting fusion constructs were separately transformed into Agrobacterium strain GV3101. The Agrobacterium cultures separately bearing SASP-nLUC and OST1-cLUC constructs were then mixed and co-infiltrated into Nicotiana benthamiana leaves. A construct combination of SGT1a-nLUC and RAR1-cLUC was used as a positive control, while two construct combinations, OST1-nLUC plus cLUC and SASP-cLUC plus nLUC, were used as negative controls for the co-infiltration. Scale bars are 1 cm. (B) Yeast two-hybrid analysis of the interaction between SASP and OST1. SASP and OST1 coding sequences were fused to the pGBKT7 and pGADT7 vectors, respectively, and then the resulting fusion constructs were co-transformed into yeast strain AH109. The positive transformants were first spotted onto control medium (SD/–Trp–Leu) to grow for 4–6 d, and then transferred to selective medium (SD/–Trp–Leu–Ade + 3-AT) to grow for 4–6 d before observation. (C) Bimolecular fluorescence complementation analysis of the interaction between SASP and OST1. SASP and OST1 coding sequences were fused to YN and YC vectors, respectively, and then the resulting fusion constructs were separately transformed into Agrobacterium strain GV3101 for co-infiltration. Two construct combinations, YN plus OST1-YC and SASP-YN plus YC, were used as negative controls for the co-infiltration. Images were taken under a confocal microscope. Scale bars are 25 μm. (D) Co-localization of SASP-GFP and OST1-mCherry. SASP and OST1 coding sequences were fused to the pC1305-d35S-GFP and pC1305-d35S-mCherry vectors, respectively, and then the resulting fusion constructs were separately transformed into Agrobacterium strain GV3101 for co-infiltration. Images were taken under a confocal microscope. Scale bars are 25 μm. Increased ABA signaling in the sasp mutants may be caused by abolishment of SASP activity on OST1 A previous study showed that overexpression of OST1 in ost1 mutant led to an ABA-hypersensitivity phenotype as ABA signaling was increased as a result of over-accumulation of OST1 (Acharya et al., 2013). To test whether or not SASP had enzymatic activity on OST1, we co-expressed SASP-GFP and OST1-mCherry in N. benthamiana leaves to examine the protein stability of OST1. It was apparent that although the different combinations exhibited gradually decreasing fluorescence, the signals from the combination of OST1-mCherry and SASP-GFP were reduced considerably compared to those from the combination of OST1-mCherry and PIP2a-YFP with increasing concentrations of SASP-GFP or PIP2a-YFP (Fig. 9A), suggesting that SASP-GFP might have degraded the OST1-mCherry in planta upon co-infiltration. Closer inspection revealed that the cells with high green fluorescence signal (containing highly expressed SASP-GFP construct) displayed quite weak red fluorescence signal (‘1’ in Fig. 9B), whilst the cells with weak green fluorescence signal (containing low-expressed SASP-GFP construct) exhibited high red fluorescence signal (‘2’ in Fig. 9B). These observations further support the notion that SASP plays a role in degrading the OST1-mCherry protein. In addition, we transfected the POST1::OST1-mCherry construct separately into Col-0 and sasp-1 protoplasts in order to check fluorescence signals. As shown in Fig. 9C, the signals at the cell periphery of the transfected Col-0 protoplasts were indistinct compared to those of the transfected sasp-1 protoplasts, which had clear and sharp signals. This suggests the possibility that OST1-mCherry in the Col-0 protoplasts had been subject to more severe degradation. Taken together, these results support the conclusion that SASP promotes degradation of OST1-mCherry in vivo, whilst knockout of SASP clearly impairs its degradation. Fig. 9. View largeDownload slide In vivo assays of SASP enzymatic activity against OST1. (A) In vivo tests of SASP-GFP enzymatic activity against OST1-mCherry in Nicotiana benthamiana leaves. SASP and OST1 coding sequences were positioned upstream of the GFP and mCherry genes, respectively, and the resulting fusion constructs were separately introduced into Agrobacterium strain GV3101. The Agrobacterium GV3101 culture harboring OST1-mCherry construct (OD600=0.6) was then mixed at a ratio of 1:1 (v/v) with each of the GV3101 cultures harboring the SASP-GFP or PIP2a-YFP construct with different OD600 values (0.3, 0.6, and 1.2), and then the mixtures in combination with the Agrobacterium p19 strain were infiltrated into N. benthamiana leaves. Control: leaves infiltrated only with Agrobacterium culture containing the OST1-mCherry construct plus the p19 strain. Fluorescence was observed under a confocal microscope at 3 d following the co-infiltration. Scale bars are 20 μm. (B) Detailed examination of the transformed cells of N. benthamiana leaves containing both the OST-mCherry and SASP-GFP constructs. The co-infiltrated leaves in (A, upper panel) were examined under a confocal microscope to check fluorescence intensity. 1, a cell containing strongly expressed SASP-GFP but weakly expressed OST1-mCherry; 2, a cell containing strongly expressed OST1-mCherry but weakly expressed SASP-GFP. Scale bars are 20 μm. (C) Effects of the sasp-1 mutation on degradation of OST1-mCherry in Arabidopsis protoplasts. Intact protoplasts were isolated from 4-week-old wild-type Col-0 and sasp-1 leaves, and then transfected with the POST1::OST1-mCherry construct. The transfected protoplasts were imaged under a confocal microscope. For each transfection, at least 20 cells were observed and more than 70% of cells displayed similar results. Two representative cells from each transfection are shown. Scale bars are 70 μm. Fig. 9. View largeDownload slide In vivo assays of SASP enzymatic activity against OST1. (A) In vivo tests of SASP-GFP enzymatic activity against OST1-mCherry in Nicotiana benthamiana leaves. SASP and OST1 coding sequences were positioned upstream of the GFP and mCherry genes, respectively, and the resulting fusion constructs were separately introduced into Agrobacterium strain GV3101. The Agrobacterium GV3101 culture harboring OST1-mCherry construct (OD600=0.6) was then mixed at a ratio of 1:1 (v/v) with each of the GV3101 cultures harboring the SASP-GFP or PIP2a-YFP construct with different OD600 values (0.3, 0.6, and 1.2), and then the mixtures in combination with the Agrobacterium p19 strain were infiltrated into N. benthamiana leaves. Control: leaves infiltrated only with Agrobacterium culture containing the OST1-mCherry construct plus the p19 strain. Fluorescence was observed under a confocal microscope at 3 d following the co-infiltration. Scale bars are 20 μm. (B) Detailed examination of the transformed cells of N. benthamiana leaves containing both the OST-mCherry and SASP-GFP constructs. The co-infiltrated leaves in (A, upper panel) were examined under a confocal microscope to check fluorescence intensity. 1, a cell containing strongly expressed SASP-GFP but weakly expressed OST1-mCherry; 2, a cell containing strongly expressed OST1-mCherry but weakly expressed SASP-GFP. Scale bars are 20 μm. (C) Effects of the sasp-1 mutation on degradation of OST1-mCherry in Arabidopsis protoplasts. Intact protoplasts were isolated from 4-week-old wild-type Col-0 and sasp-1 leaves, and then transfected with the POST1::OST1-mCherry construct. The transfected protoplasts were imaged under a confocal microscope. For each transfection, at least 20 cells were observed and more than 70% of cells displayed similar results. Two representative cells from each transfection are shown. Scale bars are 70 μm. Given that OST1 degradation was impaired in the sasp mutants, we examined whether ROS production was enhanced in the mutant guard cells due to higher accumulation of OST1 in the sasp mutants than in the wild type. Without ABA treatment, there were no significant differences in ROS production in the guard cells between the wild type and the sasp mutants after incubation with H2DCF-DA (Fig. 10A). However, in the presence of 100 μM ABA, the guard cells of the mutants accumulated remarkably more ROS than those of wild type, which resembled the phenotypes of OST1-overexpressing plants (Acharya et al., 2013). Thus, these results provided a direct demonstration that knockout of SASP facilitated increased ROS production upon treatment with ABA, which was presumably due to the over-accumulation of OST1. Furthermore, the ROS attenuation assays indicated that the DCF fluorescence intensity of sasp-1 remained relatively constant over the 35-min time frame of the experiment (Fig. 10B, C). By contrast, the DCF fluorescence intensity of the wild type decreased rapidly once the ABA treatment was removed, and it had become very weak by the 30-min time point. Therefore, it seems that in the sasp mutants ROS attenuation was markedly impaired in the guard cells, presumably due to the persistent activation of ROS production by the accumulating OST1. Taken together, it appears that the increased ABA signaling in the sasp mutants may result from over-accumulation of OST1, which presumably arises from the abolishment of SASP activity on OST1. Fig. 10. View largeDownload slide Comparison of attenuation rates of reactive oxygen species (ROS) in guard cells between Col-0 and sasp mutants. (A) Comparison of ROS production in guard cells with or without ABA treatment as shown by the fluorescent ROS indicator H2DCF-DA. Peeled epidermal strips from Col-0 and the sasp mutants were first incubated without (control) or with 100 μM ABA for 15 min, followed by three rinses with distilled water. They were then transferred to 25 μM H2DCF-DA solution for incubation for 30 min. After brief rinses, the strips were observed under a confocal microscope and representative images of guard cells are shown. Scale bars are 70 μm. (B) Comparison of ROS attenuation between Col-0 and sasp-1 in guard cells. Peeled epidermal strips from Col-0 and sasp-1 were treated with 100 μM ABA and 25 μM H2DCF-DA as described in (A) and then imaged under a confocal microscope (0 min). A further seven images were taken at 5-min intervals. Scale bars are 70 μm. (C) Kinetics of ROS attenuation rates between the Col-0 and sasp-1 in guard cells illustrated in (B). Fluorescence intensity was quantified using Adobe Photoshop 5.0, and the values are expressed as means (±SD) for three biological replicates. Fig. 10. View largeDownload slide Comparison of attenuation rates of reactive oxygen species (ROS) in guard cells between Col-0 and sasp mutants. (A) Comparison of ROS production in guard cells with or without ABA treatment as shown by the fluorescent ROS indicator H2DCF-DA. Peeled epidermal strips from Col-0 and the sasp mutants were first incubated without (control) or with 100 μM ABA for 15 min, followed by three rinses with distilled water. They were then transferred to 25 μM H2DCF-DA solution for incubation for 30 min. After brief rinses, the strips were observed under a confocal microscope and representative images of guard cells are shown. Scale bars are 70 μm. (B) Comparison of ROS attenuation between Col-0 and sasp-1 in guard cells. Peeled epidermal strips from Col-0 and sasp-1 were treated with 100 μM ABA and 25 μM H2DCF-DA as described in (A) and then imaged under a confocal microscope (0 min). A further seven images were taken at 5-min intervals. Scale bars are 70 μm. (C) Kinetics of ROS attenuation rates between the Col-0 and sasp-1 in guard cells illustrated in (B). Fluorescence intensity was quantified using Adobe Photoshop 5.0, and the values are expressed as means (±SD) for three biological replicates. Discussion SASP appears to be involved in specifically regulating ABA signaling rather than participating in general protein breakdown during leaf senescence SASP was previously isolated from senescent Arabidopsis leaves using a zymogram approach, and SASP expression and proteolytic activity were both found to be induced during leaf senescence (Martinez et al., 2015). Our results also showed that the SASP transcript level was up-regulated during darkness-induced rapid leaf senescence (Fig. 1A). In addition, a previous study had demonstrated that the endopeptidase activity of TaSSP1, an ortholog of Arabidopsis SASP, was strongly induced in naturally and ABA-induced senescent wheat leaves (Qi and Xu, 2003). All these data point to a possible role of SASP in leaf senescence. However, in the current study we found that knockout of SASP did not result in any visible senescence-related phenotype; in contrast, our results support a new role of SASP in regulating ABA signaling and drought tolerance in Arabidopsis. SASP expression was strongly induced by ABA, and the sasp mutants were more sensitive to ABA than wild-type plants at both seed germination and seedling stages (Fig. 3). Upon ABA treatment, detached leaves of the sasp mutants displayed greater senescence and over-production of ROS than the wild type (Fig. 4). Moreover, the sasp mutant plants demonstrated higher resistance to drought stress than the wild type (Fig. 5). More importantly, in the sasp mutants the ABA-signaling genes ABI1 and HAB1 were down-regulated while RBOHD, ROBHF, PYL4, and OST1 were up-regulated, suggesting increased ABA signaling in the sasp mutants (Fig. 6). Given that SASP not only co-localized with OST1 but also interacted with it at the cell periphery (Fig. 8), we speculate that OST1 is a substrate for SASP. Further in vivo enzymatic activity assays indicated that co-transfection of the OST1-mCherry construct with the SASP-GFP construct in N. benthamiana leaves appeared to result in the degradation of OST1 (Fig. 9A, B). In addition, when the OST1-mCherry construct was transformed into sasp-1 protoplasts, the degradation of OST1-mCherry was impaired to some extent, confirming a role of SASP in degrading OST1 and regulating ABA signaling (Fig. 9C). It has been reported that during leaf senescence endogenous ABA levels increase dramatically and that a subset of genes implicated in the key steps of ABA biosynthesis and signaling are significantly upregulated, implying that ABA plays essential roles in the progression of leaf senescence (Tan et al., 2003; Lim et al., 2007). Indeed, ABA is regarded as a hormonal trigger controlling the onset of leaf senescence (Gao et al., 2016; Zhao et al., 2016). Arabidopsis SAUL1 was discovered to be induced by ABA and it targets AAO3 for degradation; in the saul1 mutant the AAO3 protein is over-accumulated and hence the ABA content substantially increases, which results in rapid ABA-induced leaf senescence (Hoth et al., 2002; Raab et al., 2009). Thus, the role of SAUL1 is to counteract the over-production of AAO3 and hence maintain a particular level of endogenous ABA. Similarly, SASP presumably targets OST1 (and perhaps a few additional proteins) for degradation to weaken ABA signaling. It therefore seems that during leaf senescence ABA biosynthesis and signaling are increased and hence induce higher production of SASP, which in turn targets the activated OST1 for degradation, thus reducing the OST1 protein level and attenuating ABA signaling (Fig. 11). Hence, it appears that SASP acts as a buffer against increased ABA signaling rather than serving a function in general protein breakdown during leaf senescence. Although we are as yet unable to determine if the substrate specificity of SASP is indeed narrow, it is likely that SASP exhibits quite stringent substrate specificity because in our assays it only physically interacted with OST1 out of a total of 11 proteins involved in ABA signaling (Fig. 8A–C). In addition, we also identified another three proteins (ERS1, ACBP2, and ACBP3) that interacted with SASP through large-scale protein-protein interaction assays, and they are all involved in the regulation of ABA signaling or stomatal movement. Therefore, it seems that SASP is largely responsible for degradation of OST1 and three other proteins associated with ABA signaling, although we cannot completely rule out its action in general protein breakdown. Fig. 11. View largeDownload slide A model for SASP regulation of ABA signaling. Exogenous ABA, drought, darkness, and leaf senescence are all able to induce expression of some key ABA-biosynthesis and ABA-signaling genes, which leads to an increase in endogenous ABA levels and triggers protein activities of ABA-signaling components, such as PYR1, PYL, RCAR, and OST1. The activated OST1 subsequently targets downstream proteins such as RBOHD, RBOHF, and SLAC1 for phosphorylation. In parallel, SASP expression is also induced by either exogenous or endogenous ABA, which ultimately results in higher accumulation of SASP. The SASP targets activated-OST1 by protein–protein interaction and degrades it, and thus the activity of OST1 is attenuated. It therefore appears that SASP acts as a buffer against the increased ABA signaling by weakening OST1 activity to counteract the potency of ABA. Fig. 11. View largeDownload slide A model for SASP regulation of ABA signaling. Exogenous ABA, drought, darkness, and leaf senescence are all able to induce expression of some key ABA-biosynthesis and ABA-signaling genes, which leads to an increase in endogenous ABA levels and triggers protein activities of ABA-signaling components, such as PYR1, PYL, RCAR, and OST1. The activated OST1 subsequently targets downstream proteins such as RBOHD, RBOHF, and SLAC1 for phosphorylation. In parallel, SASP expression is also induced by either exogenous or endogenous ABA, which ultimately results in higher accumulation of SASP. The SASP targets activated-OST1 by protein–protein interaction and degrades it, and thus the activity of OST1 is attenuated. It therefore appears that SASP acts as a buffer against the increased ABA signaling by weakening OST1 activity to counteract the potency of ABA. Enhanced drought tolerance of sasp mutants is derived from increased ABA signaling The fact that the sasp mutants displayed enhanced tolerance to drought stress points to the involvement of SASP in stress responses (Fig. 5A). A few studies have shown that, for some plant species, drought stress usually leads to changes in gene expression or proteolytic activities of several categories of proteases (especially serine and aspartic proteases), which in turn determines the resistance or susceptibility of the species to the stress. Drought stress may stimulate/repress the gene expression of serine/aspartic proteases, and thus increase/decrease the enzymatic activities of these proteases (Hieng et al., 2004; Drame et al., 2007; Budič et al., 2013), or bring about combinations of both effects. In general, according to studies on several crop species with different drought tolerance, under severe stress conditions drought-tolerant cultivars usually display reduced gene expression or proteolytic activities of serine or aspartic proteases, whereas drought-susceptible cultivars commonly exhibit increased gene expression or proteolytic activities of these proteases (Hieng et al., 2004; Drame et al., 2007), suggesting that an increase in proteolytic activity diminishes drought tolerance and that a decrease in proteolytic activity improves drought tolerance. A possible explanation for this phenomenon is that the increased proteolytic activities speed up the breakdown of cellular proteins and thus disrupt normal metabolic processes, which ultimately leads to damaged membranes, cell death, and leaf senescence. In comparison, we found that the enhanced drought tolerance in sasp mutants comes from increased ABA signaling, which eventually reduces water loss in the mutants by triggering rapid stomatal closure, and thus improves their survival rates. It therefore seems that the mechanism of enhanced drought tolerance in the Arabidopsis sasp mutants is probably quite different from that of the drought-tolerant cultivars examined in previous studies. Supplementary data Supplementary data are available at JXB online. Table S1. Primers used in this study. Fig. S1. Schematic diagram of domain structures of SASP. Fig. S2. Phylogenetic relationships among SASP and orthologous proteins from other species. Fig. S3. Phenotypic analyses of sasp mutants. Fig. S4. Detection of transcriptional activation of senescence-associated genes in sasp mutants. Fig. S5. Expression analyses of SAG12 in sasp mutants with and without ABA treatment. Fig. S6. Images of 4-week-old leaves from wild type and sasp-1 subjected to dark-induced senescence treatment. Fig. S7. Expression analyses of six essential genes implicated in ABA signaling in wild-type and sasp mutants in response to drought. Fig S8. Expression analyses of RD29A in wild type and sasp mutants in response to ABA. Fig. S9. ABA content in wild type and sasp mutants treated with ABA or ETN. Fig. S10. Expression analyses of five key genes for ABA biosynthesis in wild type and sasp mutants treated with ABA or ETN. Acknowledgements This work was supported by grants from the Natural Science Foundation of Jiangsu Province (BK20151425), The Fundamental Research Funds for the Central Universities (KYTZ201402, KYRC201409), The Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Jiangsu Science Fund for Distinguished Young Scholars (BK20150027). References Acharya BR , Jeon BW , Zhang W , Assmann SM . 2013 . 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For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Botany Oxford University Press

Arabidopsis subtilase SASP is involved in the regulation of ABA signaling and drought tolerance by interacting with OPEN STOMATA 1

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Abstract

Abstract Arabidopsis Senescence-Associated Subtilisin Protease (SASP) has previously been reported to participate in leaf senescence and in the development of inflorescences and siliques. Here, we describe a new role of SASP in the regulation of abscisic acid (ABA) signaling. SASP encodes a subtilase and its expression was considerably induced by darkness, ABA, and ethylene treatments. sasp knockout mutants displayed obvious developmental phenotypes such as early flowering and smaller leaves. In particular, the sasp mutants exhibited enhanced ABA sensitivity during seed germination and seedling growth, heightened ABA-mediated leaf senescence, and increased production of reactive oxygen species (ROS). Importantly, the sasp mutants also showed remarkably increased tolerance to drought, with expression of six ABA signaling-related genes being either up- or down-regulated following ABA treatment. Interaction assays demonstrated that SASP physically interacts with OPEN STOMATA 1 (OST1) at the cell periphery. Co-expression of SASP and OST1 led to degradation of OST1, whereas this degradation was impaired in sasp-1 protoplasts. ROS attenuation assays demonstrated that in sasp-1 mutant guard cells the attenuation rate markedly decreased. Taken together, the results demonstrate that SASP plays an important role in regulating ABA signaling and drought tolerance through interaction with OST1. Abscisic acid (ABA) signaling, Arabidopsis thaliana, drought tolerance, OPEN STOMATA 1 (OST1), reactive oxygen species (ROS), senescence-associated subtilisin protease (SASP) Introduction Subtilases (SBTs) consist of a large family of serine peptidases, which differ from other types of proteases by the specific arrangement of the Asp, His, and Ser residues in their catalytic triad (Lang-Mladek et al., 2010). Compared to animals and micro-organisms, plants possess more SBTs (Srivastava et al., 2008). For example, the Arabidopsis genome encodes 56 SBTs, which are categorized into six distinct subfamilies, namely SBT1–6 (Rautengarten et al., 2005; Srivastava et al., 2008). Plant SBTs have been reported to be involved in protein turnover, seed and fruit development, modifications of cell wall, post-translational processing of protein precursors, regulation of stomatal density and distribution, abiotic and biotic responses, and programmed cell death (PCD) (Tripathi and Sowdhamini, 2006). Plant SBTs are known to play important roles in several biological processes. First, plant SBTs can catalyse non-selective protein degradation. For instance, cucumisin was the first SBT isolated from plant species (Kaneda and Tominaga, 1975; Yamagata et al., 1994) and is an extracellular alkaline protease that exhibits broad substrate specificity toward peptides and protein substrates (Yamagata et al., 1994). AtSBT1.7 shows little selectivity for synthetic peptide substrates and is able to degrade a wide assortment of extracellular proteins (Hamilton et al., 2003). Second, plant SBTs can act as proprotein convertases. AtSBT6.1 was found to specifically process pro-RALF23 to release a C-terminal growth-inhibiting RALF peptide (Srivastava et al., 2009). Third, plant SBTs are implicated in plant-specific developmental processes (Acharya et al., 2013). AtSBT1.4, referred to as Senescence-Associated Subtilisin Protease (SASP), was isolated by a proteomic method and found to be involved in the process of leaf senescence (Martinez et al., 2015). sasp-1 mutant plants produce 25% more inflorescence branches and siliques than either wild-type Col-0 or complementation lines (Martinez et al., 2015). Thus, the importance of SBTs in numerous biological processes is clear. It is well known that proteolysis-dependent protein breakdown and recycling play important roles in plant responses to environmental stresses. A number of studies have shown that proteases, such as serine and aspartic proteases, play an essential part in plant responses to drought stress. For example, Hieng et al. (2004) examined three European Phaseolus vulgaris cultivars under conditions of severe water deficit and found that the most sensitive one exhibited a marked increase in activities of two different serine proteases, while the most resistant cultivar showed a significant decrease in the activities of these enzymes, indicating a negative correlation between protease activity and drought tolerance. Similarly, a study in peanut subjected to progressive drought stress conditions also showed that expression of the Arachis hypogaea serine protease (AHSP) gene decreased more rapidly in a drought-tolerant cultivar than in a drought-susceptible one (Dramé et al., 2007), which helped to reduce drought-induced hydrolytic processes in the tolerant cultivar, and hence to avoid severe damage to cellular structures such as the membrane systems. Taken together, it is evident that proteases, especially serine proteases, are implicated in drought stress responses, and that they appear to be negatively correlated with drought tolerance. Recent studies have demonstrated that abscisic acid (ABA) biosynthesis and signaling are subject to regulation by protein degradation process. The Arabidopsis U-box E3 ubiquitin ligase SENESCENCE-ASSOCIATED E3 UBIQUITIN LIGASE 1 (SAUL1) has been shown to target AAO3 (Arabidopsis aldehyde oxidase 3, a key enzyme for ABA biosynthesis) for degradation; in the saul1 mutant the AAO3 protein is over-accumulated and as a consequence the ABA content is substantially increased, which ultimately leads to rapid ABA-induced leaf senescence (Raab et al., 2009). ABA INSENSITIVE 1 (ABI1) is a phosphatase that binds to OPEN STOMATA 1 (OST1) to inhibit its activity in the absence of ABA. ABI1 has been found to be targeted and ubiquitinated by the U-box E3 ligases PLANT U-BOX 12 (PUB12) and PUB13 for degradation after ABA-bound PYRABACTIN RESISTANCE 1 (PYR1) interacts with ABI1, ultimately leading to degradation of ABI1 by the 26S proteasome (Kong et al., 2015). In maize, ZmOST1 was found to be phosphorylated by CASEIN KINASE 2 (CK2), a pleiotropic protein kinase, after which the kinase activity of ZmOST1 was turned off (Vilela et al., 2015). The phosphorylated ZmOST1 was highly unstable and it was ultimately degraded through the 26S proteasome. In summary, these studies demonstrate that the major constituents of ABA signaling are targeted by ubiquitination, which brings them under tight control of protein degradation through the 26S proteasome (Vilela et al., 2015). Using a natural-gradient polyacrylamide gel electrophoresis method, we previously isolated a subtilase named TaSSP1 (Senescence-associated Subtilisin Protease 1; Genbank accession number AGN03879) from senescent leaves of wheat (Triticum aestivum), induced by darkness or ABA, and found that the enzymatic activity of TaSSP1 was strongly induced in the senescent leaves, suggesting that it may play an essential part in the process of leaf senescence (Qi and Xu, 2003). Another study found that during leaf senescence, expression of SASP (the Arabidopsis ortholog of TaSSP1) was significantly induced and the proteolytic activity of SASP was obviously increased (Martinez et al., 2015), further suggesting the possible involvement of TaSSP1/SASP in leaf senescence. In order to better understand the roles of TaSSP1/SASP in leaf senescence and/or other biological processes, we have focused on Arabidopsis SASP and characterized its function in detail. During the course of the current study, however, we discovered that SASP (AtSBT1.4) is implicated in the regulation of ABA signaling and drought tolerance. We further found that knockout of SASP does not result in any visible phenotype associated with leaf senescence, but causes increased ABA signaling and higher drought tolerance. Interaction assays and degradation analyses revealed physical interaction between SASP and OST1 as well as degradation of OST1 by SASP. Taken together, our results provide a direct demonstration that SASP plays important roles in regulating ABA signaling and drought tolerance by targeting OST1 for degradation. Materials and methods Plant materials and growth conditions All Arabidopsis materials used in this study were in the Columbia-0 (Col-0) genetic background. Two T-DNA insertional mutants, sasp-1 and sasp-3, were obtained from the Arabidopsis Biological Resource Center (ABRC, www.abrc.osu.edu). Seeds were surface-sterilized with 0.8% sodium hypochlorite (NaClO) and sown on half-strength Murashige and Skoog (MS) medium containing 0.8% (w/v) agar and 2% (w/v) sucrose. On the 15th day after germination, seedlings were transferred to soil and grown in a growth room at 22 °C under a 16/8 h light/dark cycle. Expression analysis Briefly, approximately 1 μg of the total RNA from the different treated genotypes was used to synthesize the first-strand cDNA using One-Step gDNA Removal and cDNA Synthesis SuperMix kits (Transgen, Beijing, China). The cDNAs were then used for RT-PCR and qRT-PCR assays (ABI 7500, Applied Biosystems) with primers corresponding to each gene (Supplementary Table S1 at JXB online). Histochemical β-glucuronidase (GUS) staining and subcellular localization A 2046-bp SASP promoter fragment plus the entire 5′UTR (5′-untranslated region) sequence was amplified from wild-type DNA, and it was then cloned into the pCXGUS vector immediately upstream of the GUS coding sequence. The resulting fusion construct was introduced into wild-type Col-0 to obtain stable transgenic lines. Subsequently, the transgenic plants were subjected to GUS staining according to the method described by Merlot et al. (2002). For subcellular localization, the entire coding sequence of SASP was inserted into the pC1300-221-GFP (green fluorescent protein) vector to generate a fusion construct pC1300-221-SASP-GFP. It was then introduced into Nicotiana benthamiana leaf epidermal cells by agro-infiltration. After 3 d at 22 °C under 16/8 h light/dark conditions, the leaves were observed under a Zeiss LSM750 confocal laser-scanning microscope to determine the subcellular localization of SASP-GFP. ABA and drought treatments More than 100 seeds were sown on MS medium plates supplemented without (control) or with ABA (Sigma) at concentrations of 0.1 or 0.2 μM. After a 2-d period of cold stratification at 4 °C, the seeds were transferred to a growth room and germinated for an additional 2 d under long-day conditions (18/6 h light/dark) at 22 °C and the germination rates were determined. To test the responsiveness of roots to ABA treatment, the germinated seeds were transferred to vertical agar plates containing different concentrations of ABA [0 (control), 20, or 30 μM] and left to grow for another 7 d, after which root length was determined. For the drought stress treatment, 3-week-old plants were subjected to water withdrawal for 14 d as described previously (Osakabe et al., 2010). They were then rewatered, and after a further 7 d survival rates were determined. Detection of ROS accumulation and attenuation in guard cells Detached 5-week-old cauline leaves from wild-type Col-0, sasp mutants, and SASP complementation lines were soaked in distilled water without or with 50 μM ABA under light conditions for 3 d prior to observations of leaf senescence. The leaves were then subjected to diaminobenzidine (DAB) staining (Du et al., 2013). For measurements of ROS accumulation in guard cells, peeled epidermal strips of 4-week-old leaves from Col-0 and sasp mutants were soaked in an incubation buffer containing 30 mM KCl and 10 mM Mes-KOH (pH 6.15) for 2 h, and then the same buffer was supplemented with 100 μM ABA and the strips were incubated for 15 min. Following brief rinses, the strips were transferred to fresh incubation buffer containing 25 μM 2′, 7′-dichlorofluorescin diacetate (H2DCF-DA, Sigma) for 15 min. After being washed with distilled water, the strips were observed under a Zeiss LSM 750 microscope to inspect the accumulation of ROS in the guard cells (Lee et al., 1999). To determine ROS attenuation rates, peeled epidermal strips from Col-0 and sasp-1 were treated as described above, and then photographed under the microscope with repeated imaging seven times at 5-min intervals. Measurement of stomatal aperture For measurement of stomatal aperture following ABA treatment, peeled epidermal strips from cauline leaves (sampled from 4-week-old plants) were immersed in a stomata-opening buffer with 10 mM KCl, 0.2 mM CaCl2, and 10 mM Mes-KOH (pH 6.15) for 2 h under continuous white light at 22 °C (Du et al., 2013). ABA was then added into the same buffer to reach a final concentration of 10 μM and the epidermal strips were continuously incubated for another 2 h under the same conditions. Guard cells were photographed with a Zeiss Axio Scope A1 microscope, and stomatal lengths and widths were measured. For determination of stomatal aperture following drought treatment, peeled epidermal strips from cauline leaves sampled from 4-week-old plants that had undergone 7 d of water being withheld were examined. Determination of ABA content Briefly, 200 mg of leaf material from 4-week-old plants of Col-0 and sasp mutants that had been treated without or with either ABA or ethephon (by spraying each hormone solution onto the leaf surfaces) were harvested and ground to a fine powder in liquid nitrogen. Then, 2 ml of 80% methanol solution containing 1% acetic acid and 19% pure water was added to the powder. After centrifugation of the powder suspension, the supernatant was collected and used for determination of the ABA content by HPLC-MS (Agilent) as described previously (Pan et al., 2010). Luciferase complementation imaging (LCI) assays To screen for the candidate proteins (involved in ABA signaling) that were able to interact with SASP, the complete coding sequence of each of the tested genes was amplified and each PCR product was inserted into the pCAMBIA1300-nLUC vector; similarly, the cDNA of SASP was cloned into the pCAMBIA1300-cLUC vector (Chen et al., 2008). The genes tested were as follows: OST1, PYR1, PYL1 (PYRABACTIN RESISTANCE LIKE 1), PYL4, PYL8, PYL9, ABI1, RBOHD (RESPIRATORY BURST OXIDASE HOMOLOGUE D), RBOHF (RESPIRATORY BURST OXIDASE HOMOLOGUE F), SLAC1 (SLOW ANION CHANNEL-ASSOCIATED 1), and KAT1 (POTASSIUM CHANNEL IN ARABIDOPSIS THALIANA 1). They were separately transformed into Agrobacterium strain GV3101, and the positive clones were cultured and subsequently co-infiltrated together with Agrobacterium p19 strain into 4-week-old Nicotiana benthamiana leaves. After 3 d, the leaves were injected with D-Luciferin (Perkin Elmer) solution and placed in the dark for 30 min. Bioluminescence was detected with a CCD camera (Tanon 5200, Shanghai). Yeast two-hybrid (Y2H) assays Briefly, the nucleotide sequence (1–78 bp) corresponding to the predicted N-terminal signal peptide of the SASP coding sequence was deleted, and the resulting truncated sequence was subcloned into the pGBKT7 vector to generate the pGBKT7-SASP construct. The pGBKT7-SASP construct was then co-transformed with pGADT7-OST1 into yeast strain AH109. The co-transformed yeast cells were first spotted onto a synthetic dropout medium without Leu and Trp (SD/–Leu/–Trp) and then transferred to a stringent selection medium [synthetic dropout medium lacking Leu, Trp, and adenine (SD/–Leu/–Trp/–Ade), but supplemented with 5 mM 3-AT (3-amino-1, 2, 4-triazole)] and allowed to grow for 4 d at 28 °C prior to observation (He et al., 2009). Bimolecular fluorescence complementation (BiFC) assays The entire coding sequences of SASP and OST1 were cloned into YN and YC vectors, respectively. The resulting constructs were individually introduced into Agrobacterium strain GV3101, and the positive clones were cultured and co-infiltrated into N. benthamiana leaves (Acharya et al., 2013). After 3 d, fluorescence signals were visualized using a Zeiss LSM750 confocal laser-scanning microscope. In vivo degradation assay The entire coding sequences of SASP, OST1, and the plasma-membrane marker gene Plasma membrane Intrinsic Protein 2a (PIP2a) were cloned into the pCAMBIA1300-221-GFP, pCAMBIA1300-221-mCherry, and pCAMBIA1300-221-YFP vectors, respectively, to generate SASP-GFP, OST1-mCherry, and PIP2a-YFP constructs. An Agrobacterium culture carrying the OST1-mCherry construct was mixed separately with different concentrations of Agrobacterium cultures bearing the SASP-GFP construct or the PIP2a-YFP construct plus Agrobacterium p19 strain for co-infiltration. At 3 d after co-infiltration, fluorescence signals were detected by using a Zeiss LSM750 confocal laser-scanning microscope. Transient expression assays in Arabidopsis protoplasts Briefly, 3-week-old leaves from Col-0 and sasp-1 mutant plants were cut into small pieces and treated with lysis buffer to isolate intact protoplasts. The pOST1::OST1-mCherry construct was transfected into the protoplasts using the PEG-mediated transformation method (Miao et al., 2006). After incubation for 16–20 h at room temperature, the protoplasts were observed using a Zeiss LSM750 confocal laser-scanning microscope. Results SASP expression is induced by dark and plant hormone treatments To determine whether SASP expression is induced by hormones or environmental cues, we examined the expression patterns of SASP in wild-type Col-0 after treatment in the dark (darkness is capable of inducing rapid leaf senescence), or treatment with ABA or ethephon. qRT-PCR analysis revealed that SASP transcript levels increased with prolonged dark treatment, peaking at 48 h after treatment, but then declining at 72 h (Fig. 1A). ABA induced progressive accumulation of the SASP transcripts after 0.5, 12, and 24 h of treatment (Fig. 1B). Ethephon up-regulated SASP expression immediately after treatment, with the mRNA levels peaking at 0.5 h but declining gradually with prolonged treatment (Fig. 1C). Similar to ABA treatment, drought also led to up-regulation of SASP, and the expression level increased gradually with prolonged treatment (Fig. 1D). Further analysis of the conserved domain suggested that SASP possesses three domains, namely Inhibitor I9, Peptidases_S8_3, and Protease Associated (PA), and a 26-bp signal peptide is located at the N-terminus of this protein (Supplementary Fig. S1; Marchler-Bauer et al., 2002). Phylogenetic analysis and sequence alignment revealed that orthologous proteins of SASP exist widely in many plant species, including monocots (such as rice, wheat) and dicots (such as cotton, cabbage) (Supplementary Fig. S2), suggesting that SASP and its orthologous proteins may perform a particular function in plants. Thus, the results suggest that SASP can be induced by darkness, ABA and ethephon treatments, and it may serve particular functions during leaf senescence. Fig. 1. View largeDownload slide Induction of SASP expression by darkness, exogenous ABA and ethylene treatments, and drought stress. Relative expression levels of SASP in the dark (A), with application of 100 μM ABA (B) or 100 μM ethephon (ETN) (C), and with drought stress (D). Treatments were applied to 4-week-old Arabidopsis wild-type (Col-0) seedlings. Total RNAs were isolated at the indicated time points, and then subjected to qRT-PCR analysis to detect SASP expression levels. Actin 2 was used as the internal control. Data are means (±SD) of three biological replicates. Significant differences from time-point 0 were determined by Student’s t-test: *P<0.05, **P<0.01. Fig. 1. View largeDownload slide Induction of SASP expression by darkness, exogenous ABA and ethylene treatments, and drought stress. Relative expression levels of SASP in the dark (A), with application of 100 μM ABA (B) or 100 μM ethephon (ETN) (C), and with drought stress (D). Treatments were applied to 4-week-old Arabidopsis wild-type (Col-0) seedlings. Total RNAs were isolated at the indicated time points, and then subjected to qRT-PCR analysis to detect SASP expression levels. Actin 2 was used as the internal control. Data are means (±SD) of three biological replicates. Significant differences from time-point 0 were determined by Student’s t-test: *P<0.05, **P<0.01. Knockout of SASP does not change the progression of leaf senescence but accelerates the transition of Arabidopsis plants from vegetative to reproductive growth To better understand the roles of SASP in leaf senescence and plant growth as well as in development, we obtained two T-DNA insertional lines, sasp-1 (SALK_147962) and sasp-3 (SALK_001802), from the Arabidopsis Biological Research Center (Fig. 2A). RT-PCR analysis demonstrated that neither of the two lines was capable of producing full-length transcripts, indicating that SASP was indeed knocked out and thus that they are both knockout alleles (Fig. 2B). Surprisingly, the sasp mutants did not exhibit visible phenotypes associated with leaf senescence under either regular or dark conditions during the progression of development. Nevertheless, both mutants showed early-flowering phenotypes as they bolted and flowered earlier than the wild-type Col-0 plants, and generated fewer rosette leaves (Fig. 2C, D). In addition, the leaves of both mutants were smaller in size than those of the wild type at the onset of the bolting stage (Fig. 2E). At the maturity stage, the sasp mutants showed a significant increase in plant height and produced smaller seeds when compared to the wild type (Supplementary Fig. S3). A genomic DNA fragment of wild-type SASP driven by its native promoter fully rescued the early-flowering phenotype of the sasp-1 mutant plants, and partially restored the total number of rosette leaves and leaf size to the level of the wild-type plants (Fig. 2D, E). In parallel, the genetic complementation also fully rescued the smaller seed-size phenotype and partially decreased the plant height that was seen in the sasp-1 mutant plants (Supplementary Fig. S3). Collectively, these results suggest that knockout of SASP shortens the vegetative growth and accelerates the transition of plants from vegetative to reproductive growth. In addition, qRT-PCR analysis indicated that expression of two senescence-associated genes (SAGs), SAG29A and SAG12, was up-regulated by more than 14 and 38 times, respectively, in the sasp mutants compared to the wild type plants, implying that senescence-associated pathways were activated in the sasp mutants (Supplementary Fig. S4). Thus, the fact that knockout of SASP accelerates the transition of Arabidopsis plants from vegetative to reproductive growth possibly results from activated senescence-associated pathways that might, however, be insufficient to produce visible phenotypes associated with leaf senescence. Taken together, the results indicate that knockout of SASP has no visible effect on leaf senescence but it does accelerate the transition of Arabidopsis plants from vegetative to reproductive growth. Fig. 2. View largeDownload slide Characterization of sasp T-DNA insertional mutants and developmental phenotypes. (A) Schematic representation of the SASP gene and the positions of the T-DNA insertions. The shaded box represents the exon, open boxes represent untranslated regions (UTRs), and the T-DNA insertions are represented by triangles. The arrows denote the PCR primers used for genotyping and RT-PCR. (B) Semi-quantitative RT-PCR analysis of SASP expression in the sasp-1 and sasp-3 mutants. Actin 2 served as the constitutively expressed control. The primers used for PCR are shown in (A). (C, D) Comparison of flowering behavior between the wild-type Col-0, sasp mutants, and SASP complementation lines. (C) Flowering phenotypes of Col-0 and sasp mutants, and total rosette leaf number of each genotype at flowering. (D) Flowering phenotypes of Col-0, sasp-1, and SASP complementation lines (COM1 and COM2), and total rosette leaf number of each genotype at flowering. All the genotypes were grown in pots under long-day conditions (16/8 h light/dark), and the images were taken when the first genotype began to flower. Data are means (±SD) of three biological replicates; for each genotype, nine plants were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05. (E) Comparison of all leaves from one representative plant of each genotype from Col-0, sasp mutants, and SASP complementation lines. Scale bars are 1 cm. Fig. 2. View largeDownload slide Characterization of sasp T-DNA insertional mutants and developmental phenotypes. (A) Schematic representation of the SASP gene and the positions of the T-DNA insertions. The shaded box represents the exon, open boxes represent untranslated regions (UTRs), and the T-DNA insertions are represented by triangles. The arrows denote the PCR primers used for genotyping and RT-PCR. (B) Semi-quantitative RT-PCR analysis of SASP expression in the sasp-1 and sasp-3 mutants. Actin 2 served as the constitutively expressed control. The primers used for PCR are shown in (A). (C, D) Comparison of flowering behavior between the wild-type Col-0, sasp mutants, and SASP complementation lines. (C) Flowering phenotypes of Col-0 and sasp mutants, and total rosette leaf number of each genotype at flowering. (D) Flowering phenotypes of Col-0, sasp-1, and SASP complementation lines (COM1 and COM2), and total rosette leaf number of each genotype at flowering. All the genotypes were grown in pots under long-day conditions (16/8 h light/dark), and the images were taken when the first genotype began to flower. Data are means (±SD) of three biological replicates; for each genotype, nine plants were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05. (E) Comparison of all leaves from one representative plant of each genotype from Col-0, sasp mutants, and SASP complementation lines. Scale bars are 1 cm. sasp mutants are more sensitive to ABA at both seed germination and seedling stages Given that SASP transcripts were induced by ABA, we wondered whether sasp knockout mutants would display a distinct responsiveness to ABA compared to wild-type plants. To test this, we germinated sasp mutant seeds together with wild-type seeds on filter paper containing three different concentrations of ABA: 0 (control), 0.1, or 0.2 μM. We found that the germination rates of the sasp mutants were significantly lower than those of the wild type when seeds were germinated on media containing ABA (Fig. 3A, B). In addition, when seedlings were grown vertically on MS media containing 0 (control), 20, or 30 μM ABA, the root growth of the sasp mutants was obviously inhibited when compared with that of wild type (Fig. 3C, D). Overall, the results indicate that sasp knockout mutants are more sensitive to ABA at the seed germination and seedling growth stages, suggesting that ABA signaling might be enhanced in the sasp mutants. Fig. 3. View largeDownload slide Increased ABA sensitivity of sasp mutants during seed germination and seedling growth. (A, B) Germination assays of Col-0 and sasp mutants with or without ABA treatment. Seeds were germinated on filter paper soaked with 0 (control), 0.1, or 0.2 μM ABA solution. After 4 d, images were taken (A) and germination rates were calculated (B). Data are means (±SD) for three biological replicates; for each genotype, 50 seeds were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05, **P<0.0. Scale bars are 1 cm. (C, D) Effects of ABA treatment on root growth of Col-0 and sasp mutants. Seeds were germinated on MS medium plates for 5 d, and then the seedlings were transferred to new plates containing 0 (control), 20, or 30 μM ABA, and left to vertically grow for a further 12 d. Images were then taken (C) and root length was calculated relative to the control for each genotype (D). Data are means (±SD) for three biological replicates; for each genotype, 10 seedlings were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05, t-test. Scale bars are 1 cm. Fig. 3. View largeDownload slide Increased ABA sensitivity of sasp mutants during seed germination and seedling growth. (A, B) Germination assays of Col-0 and sasp mutants with or without ABA treatment. Seeds were germinated on filter paper soaked with 0 (control), 0.1, or 0.2 μM ABA solution. After 4 d, images were taken (A) and germination rates were calculated (B). Data are means (±SD) for three biological replicates; for each genotype, 50 seeds were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05, **P<0.0. Scale bars are 1 cm. (C, D) Effects of ABA treatment on root growth of Col-0 and sasp mutants. Seeds were germinated on MS medium plates for 5 d, and then the seedlings were transferred to new plates containing 0 (control), 20, or 30 μM ABA, and left to vertically grow for a further 12 d. Images were then taken (C) and root length was calculated relative to the control for each genotype (D). Data are means (±SD) for three biological replicates; for each genotype, 10 seedlings were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05, t-test. Scale bars are 1 cm. sasp mutants display enhanced ABA-mediated leaf senescence and increased ROS production In light of the fact that ABA is capable of promoting senescence in detached organs (Du et al., 2013) and that the senescence-associated pathways might have been activated in the sasp mutants, we hypothesized that sasp leaves may become senescent faster than those of wild-type plants under ABA treatment. To test this hypothesis, we treated detached leaves with 50 μM ABA for 3 d and found that the leaves of the sasp mutants did indeed display greater senescence than those of the wild type (Fig. 4). The ABA treatment appeared to be quite effective as SAG12 expression was significantly induced relative to untreated controls (Supplementary Fig. S5). Detached leaves from the SASP complementation lines exhibited similar leaf senescence phenotypes to the wild type under the same treatment conditions (Fig. 4). By contrast, under normal or dark conditions, detached sasp mutant leaves did not display any obvious early or delayed senescence when compared with the wild type (Supplementary Fig. S6). DAB staining revealed that the detached leaves from the sasp mutants showed increased ROS production compared with the wild-type and the SASP complementation plants upon ABA treatment (Fig. 4). In contrast to leaves treated with ABA, untreated leaves from both the wild type and the sasp mutants showed no notable differences from each other in senescence and ROS production (Fig. 4). Thus, it is clear that knockout of SASP promotes ABA-mediated leaf senescence and results in more ROS production in comparison with wild-type plants, further reinforcing the notion that ABA signaling is increased in the sasp mutants. Fig. 4. View largeDownload slide Enhanced leaf senescence and production of reactive oxygen species (ROS) in sasp mutants. Detached leaves from 5-week-old Col-0, sasp mutants, and SASP complementation lines were incubated without (control) or with 50 μM ABA for 3 d. Images were then taken, and the leaves were subjected to DAB staining for 8 h. Representative leaves are shown. Scale bars are 1 cm. Fig. 4. View largeDownload slide Enhanced leaf senescence and production of reactive oxygen species (ROS) in sasp mutants. Detached leaves from 5-week-old Col-0, sasp mutants, and SASP complementation lines were incubated without (control) or with 50 μM ABA for 3 d. Images were then taken, and the leaves were subjected to DAB staining for 8 h. Representative leaves are shown. Scale bars are 1 cm. sasp mutants display remarkably higher tolerance to drought stress Given the increased ROS production in the sasp mutant leaves when exposed to ABA, we wondered whether they might have increased drought tolerance. To test this, sasp mutants, SASP complementation lines, and wild-type plants were simultaneously grown to the onset of flowering in pots, and then water was withheld for a period of 14 d. The plants were then rewatered for 7 d before being examined. It was obvious that the sasp mutants were more tolerant to drought stress and their survival rates were nearly 80%, whereas the wild-type plants were more sensitive and their survival rates were approximately 20% (Fig. 5A, B). Complementation of the sasp-1 mutants with wild-type SASP gDNA, however, decreased the survival rates to around 35–42% (Fig. 5A, C). The stomata in the sasp mutants closed more than in the wild type under the drought conditions (Fig. 5D) and the stomatal apertures of the mutants were clearly reduced compared with the wild type (Fig. 5D). We then examined the expression of six important genes involved in ABA signaling in response to the drought stress treatment, and found that all of them except for PYL4 displayed elevated expression (Supplementary Fig. S7). Thus, the results indicated that SASP was involved in responsiveness to drought stress, and knockout of SASP led to a significant increase in drought tolerance in Arabidopsis. In addition, we also examined the stomatal apertures of leaves treated with 10 μM ABA and found that the apertures of the sasp mutants were approximately 43% of the wild-type level (Fig. 5E), suggesting that knockout of SASP significantly promotes stomatal closure under this ABA treatment. Taken together, the results indicate that SASP is involved in the regulation of drought tolerance, and loss of SASP function makes Arabidopsis plants more tolerant to drought stress. Fig. 5. View largeDownload slide Increased drought tolerance and stomatal closure of sasp mutants. (A) Increased drought tolerance of sasp mutants under 14-d drought stress treatment. Wild-type Col-0, sasp mutants, and SASP complementation lines (Com1 and Com2) were grown normally in pots for 4–5 weeks, followed by a 14-d drought stress treatment and subsequent rewatering for 7 d. Scale bars are 5 cm. (B, C) Survival rates of Col-0, sasp mutants, and SASP complementation lines calculated after 7 d of rewatering. Data are means (±SD) for three biological replicates; for each genotype, 12 plants were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: **P<0.01. (D) Changes in stomatal aperture in Col-0 and sasp mutants following drought treatment. The images show peeled leaf epidermal strips from 4-week-old Col-0 and sasp mutants following 7 d of drought, together with strips from watered controls. Representative guard cells are shown, and measurements of stomatal aperture (length, width) following the drought treatment are given in the graph. Data are means (±SD) for three biological replicates; for each genotype, 60 guard cells were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05. Scale bars are 20 μm. (E) Changes in stomatal aperture in Col-0 and sasp mutants after ABA treatment. Peeled leaf epidermal strips of 4-week-old plants grown under normal conditions were treated without (control) or with 10 μM ABA. Images of representative guard cells are shown, and measurements of stomatal aperture (length, width) following ABA treatment are given in the graph. Data are means (±SD) for three biological replicates; for each genotype, 50 guard cells were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05. Scale bars are 20 μm. Fig. 5. View largeDownload slide Increased drought tolerance and stomatal closure of sasp mutants. (A) Increased drought tolerance of sasp mutants under 14-d drought stress treatment. Wild-type Col-0, sasp mutants, and SASP complementation lines (Com1 and Com2) were grown normally in pots for 4–5 weeks, followed by a 14-d drought stress treatment and subsequent rewatering for 7 d. Scale bars are 5 cm. (B, C) Survival rates of Col-0, sasp mutants, and SASP complementation lines calculated after 7 d of rewatering. Data are means (±SD) for three biological replicates; for each genotype, 12 plants were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: **P<0.01. (D) Changes in stomatal aperture in Col-0 and sasp mutants following drought treatment. The images show peeled leaf epidermal strips from 4-week-old Col-0 and sasp mutants following 7 d of drought, together with strips from watered controls. Representative guard cells are shown, and measurements of stomatal aperture (length, width) following the drought treatment are given in the graph. Data are means (±SD) for three biological replicates; for each genotype, 60 guard cells were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05. Scale bars are 20 μm. (E) Changes in stomatal aperture in Col-0 and sasp mutants after ABA treatment. Peeled leaf epidermal strips of 4-week-old plants grown under normal conditions were treated without (control) or with 10 μM ABA. Images of representative guard cells are shown, and measurements of stomatal aperture (length, width) following ABA treatment are given in the graph. Data are means (±SD) for three biological replicates; for each genotype, 50 guard cells were examined in each of the three replicates. Significant differences from Col-0 were determined by Student’s t-test: *P<0.05. Scale bars are 20 μm. SASP is involved in regulation of expression of ABA-signaling genes To examine whether expression of ABA-signaling genes was changed in sasp mutants in response to ABA, we used qRT-PCR to measure the transcript levels of 24 genes that are implicated in, or associated with, ABA biosynthesis or signaling (ABA1, NCED3, PYR1, PYL4, ABI1, HAB1, RBOHD, RBOHF, OST1, etc.). We found that two negative regulator genes of ABA-signaling, ABI1 and HAB1, displayed similar expression patterns: without ABA treatment (control), the expression levels in the sasp mutants were both lower than in the wild type (Fig. 6), and although the expression in all genotypes was up-regulated after treatment with 100 μM ABA, the expression levels of the sasp mutants were still lower than those of the wild type. RBOHD, RBOHF, PYL4, and OST1 have been identified as positive regulators for ABA signaling, and up-regulation of these genes contributes to activation of the ABA signaling (Raghavendra et al., 2010; Acharya et al., 2013; Raghavendra et al, 2010). Our results showed that the expression of RBOHD, RBOHF, and PYL4 were all up-regulated in the sasp mutants irrespective of ABA treatment (Fig. 6), suggesting that knockout of SASP enhances the expression of these genes. It appeared that OST1 expression was slightly increased in the sasp mutants, although its expression was not induced by ABA. The ABA treatment was effective, as the transcript levels of the marker gene RD29A were up-regulated in all genotypes following treatment (Supplementary Fig. S8). In summary, the results indicated that knockout of SASP in the presence or absence of 100 μM ABA increases the expression of positive regulator genes of ABA signaling (RBOHD, RBOHF, PYL4, and OST1) but decreases the expression of negative regulator genes (ABI1 and HAB1), supporting the idea that ABA signaling is activated as well as increased in the sasp mutants. Fig. 6. View largeDownload slide Expression analyses of six essential genes involved in ABA signaling under control and ABA treatments. Leaves of 4-week-old Col-0 and sasp mutants were sprayed with 0 (control) or 100 μM ABA solution prior to sampling. After 3 h, total RNAs were isolated and reverse-transcribed into cDNAs, which were then used for quantitative PCR with primers corresponding to specific regions of ABI1, HAB1, RBOHD, RBOHF, PYL4, and OST1. Data are means (±SD) of three biological replicates. Significant differences from Col-0 within each treatment were determined by Student’s t-test: *P<0.05. Fig. 6. View largeDownload slide Expression analyses of six essential genes involved in ABA signaling under control and ABA treatments. Leaves of 4-week-old Col-0 and sasp mutants were sprayed with 0 (control) or 100 μM ABA solution prior to sampling. After 3 h, total RNAs were isolated and reverse-transcribed into cDNAs, which were then used for quantitative PCR with primers corresponding to specific regions of ABI1, HAB1, RBOHD, RBOHF, PYL4, and OST1. Data are means (±SD) of three biological replicates. Significant differences from Col-0 within each treatment were determined by Student’s t-test: *P<0.05. Another possibility is that the increased ROS production and the higher tolerance to drought stress in the sasp mutants could result from elevated ABA content. However, using LC-MS, we did not find any significant differences in ABA content between the wild type (19.00 ng g–1 DW) and the sasp mutants (23.41–24.60 ng g–1 DW) (Supplementary Fig. S9). It is worth noting that the ABA content in all the genotypes increased to over 250 ng g–1 DW after they were treated with ABA or ethephon, and the ABA content in the sasp mutants was significantly higher than in the corresponding wild-type Col-0 (Supplementary Fig. S9). However, after examining the expression levels of five key genes involved in ABA biosynthesis (ABA1, ABA2, ABA3, NCED3, and AAO3), we found that there were no significant differences in transcript levels among the genotypes under either control or treatment conditions (although the expression of all genes was obviously induced by ABA and ethephon; Supplementary Fig. S10). These results suggest that the significantly elevated ABA content in the sasp mutants under ABA and ethephon treatments resulted from impaired degradation of ABA. Taken together, the results demonstrate that it is ABA signaling that is increased in the sasp mutants under normal growth conditions. Expression patterns and subcellular localization of SASP RT-PCR analysis demonstrated that SASP was ubiquitously expressed in all the tissues examined, namely the roots, stem, rosette leaf, cauline leaf, siliques, and flowers (Fig. 7A). The expression levels in the stem, rosette, and cauline leaf were relatively higher than those in roots, siliques, and flowers. Histochemical localization showed that in transgenic plants containing the PSASP::GUS construct, GUS staining could be detected throughout all the tissues examined, which was in agreement with the RT-PCR results; however, the overall GUS signals were weak, suggesting that the SASP expression levels were relatively low in Arabidopsis (Fig. 7B–H). It is worth noting that relatively high levels of GUS expression were observed at the base of the stem as well as at the tip of leaf and in the anther (Fig. 7C, F, H). Subcellular localization indicated that SASP-GFP proteins were predominantly associated with the plasma membrane and were mainly located at the cell periphery, because the merged images displayed bright yellow signals due to the overlap of the green (SASP-GFP) and red (plasma membrane marker PIP2a-mCherry) channels (Fig. 7I). When the transformed cells were treated with 30% sucrose to induce plasmolysis, the SASP-GFP proteins remained associated with the plasma membrane. These results lead us to the conclusion that SASP is extensively expressed in various tested tissues and that SASP is a plasma membrane-associated protein that is largely localized at the cell periphery. Fig. 7. View largeDownload slide Expression patterns and subcellular localization of SASP. (A) Expression patterns of SASP in various tissues as determined by RT-PCR. Total RNAs were isolated from 6-week-old plants, and then used for reverse transcription and PCR reactions. (B–H) Detection of SASP promoter activity in different tissues by histochemical GUS staining. A 6-week-old whole plant (B) harboring the PSASP::GUS transgene was subjected to GUS staining and the following tissues were examined: roots (C), stem (D), rosette leaf (E), cauline leaf (F), silique (G), and mature flower (H). (I) Subcellular localization of SASP-GFP fusion protein. Agrobacterium strain GV3101 cultures separately carrying the SASP-GFP or PIP2a-mCherry fusion constructs were mixed and then co-infiltrated into Nicotiana benthamiana leaves. For plasmolysis, the detached co-infiltrated leaves were treated with 30% of sucrose for 15 min prior to observation. Scale bars are 25 μm Fig. 7. View largeDownload slide Expression patterns and subcellular localization of SASP. (A) Expression patterns of SASP in various tissues as determined by RT-PCR. Total RNAs were isolated from 6-week-old plants, and then used for reverse transcription and PCR reactions. (B–H) Detection of SASP promoter activity in different tissues by histochemical GUS staining. A 6-week-old whole plant (B) harboring the PSASP::GUS transgene was subjected to GUS staining and the following tissues were examined: roots (C), stem (D), rosette leaf (E), cauline leaf (F), silique (G), and mature flower (H). (I) Subcellular localization of SASP-GFP fusion protein. Agrobacterium strain GV3101 cultures separately carrying the SASP-GFP or PIP2a-mCherry fusion constructs were mixed and then co-infiltrated into Nicotiana benthamiana leaves. For plasmolysis, the detached co-infiltrated leaves were treated with 30% of sucrose for 15 min prior to observation. Scale bars are 25 μm SASP physically interacts with OST1 and co-localizes with it at the cell periphery As SASP is a subtilase and the sasp mutants displayed increased ABA signaling, we wondered whether certain components of ABA signaling are able to interact with SASP. To explore this possibility, we first performed protein interaction assays between SASP and major ABA signaling components using the luciferase complementation imaging (LCI) method to screen for the proteins that are able to interact with SASP (a total of 11 proteins were tested). Ultimately, only OST1 was discovered to have strong physical interaction with SASP in N. benthamiana leaves, because co-expression of SASP with OST1 exhibited a high luciferase signal compared to the negative controls (Fig. 8A). Consistent with the LCI assay, Y2H assays also demonstrated an interaction between SASP and OST1 (Fig. 8B). In addition, BiFC assays again confirmed the interaction between SASP and OST1 (Fig. 8C), and it was obvious that the interaction occurred at the cell periphery as the green and red fluorescence signals overlapped at this region (Fig. 8C). Co-localization analysis of SASP-GFP and OST1-mCherry demonstrated that the green fluorescence signals were mainly distributed at the cell periphery, while the red fluorescence signals were observed ubiquitously across the entire cell, including the nucleus (Fig. 8D). It was clear that the yellow fluorescence signals occurred predominantly at the cell periphery where the green and red signals overlapped, as demonstrated by the merged image, again suggesting that SASP and OST1 are co-localized at the cell periphery. Taken together, these data support the conclusion that SASP interacts physically with OST1 at the cell periphery and it is possible that OST1 serves as a substrate for SASP. Fig. 8. View largeDownload slide Physical interactions between SASP and OST1. (A) Luciferase complementation imaging analysis of the interaction between SASP and OST1. SASP and OST1 coding sequences were fused to nLUC and nLUC vectors, respectively, and then the resulting fusion constructs were separately transformed into Agrobacterium strain GV3101. The Agrobacterium cultures separately bearing SASP-nLUC and OST1-cLUC constructs were then mixed and co-infiltrated into Nicotiana benthamiana leaves. A construct combination of SGT1a-nLUC and RAR1-cLUC was used as a positive control, while two construct combinations, OST1-nLUC plus cLUC and SASP-cLUC plus nLUC, were used as negative controls for the co-infiltration. Scale bars are 1 cm. (B) Yeast two-hybrid analysis of the interaction between SASP and OST1. SASP and OST1 coding sequences were fused to the pGBKT7 and pGADT7 vectors, respectively, and then the resulting fusion constructs were co-transformed into yeast strain AH109. The positive transformants were first spotted onto control medium (SD/–Trp–Leu) to grow for 4–6 d, and then transferred to selective medium (SD/–Trp–Leu–Ade + 3-AT) to grow for 4–6 d before observation. (C) Bimolecular fluorescence complementation analysis of the interaction between SASP and OST1. SASP and OST1 coding sequences were fused to YN and YC vectors, respectively, and then the resulting fusion constructs were separately transformed into Agrobacterium strain GV3101 for co-infiltration. Two construct combinations, YN plus OST1-YC and SASP-YN plus YC, were used as negative controls for the co-infiltration. Images were taken under a confocal microscope. Scale bars are 25 μm. (D) Co-localization of SASP-GFP and OST1-mCherry. SASP and OST1 coding sequences were fused to the pC1305-d35S-GFP and pC1305-d35S-mCherry vectors, respectively, and then the resulting fusion constructs were separately transformed into Agrobacterium strain GV3101 for co-infiltration. Images were taken under a confocal microscope. Scale bars are 25 μm. Fig. 8. View largeDownload slide Physical interactions between SASP and OST1. (A) Luciferase complementation imaging analysis of the interaction between SASP and OST1. SASP and OST1 coding sequences were fused to nLUC and nLUC vectors, respectively, and then the resulting fusion constructs were separately transformed into Agrobacterium strain GV3101. The Agrobacterium cultures separately bearing SASP-nLUC and OST1-cLUC constructs were then mixed and co-infiltrated into Nicotiana benthamiana leaves. A construct combination of SGT1a-nLUC and RAR1-cLUC was used as a positive control, while two construct combinations, OST1-nLUC plus cLUC and SASP-cLUC plus nLUC, were used as negative controls for the co-infiltration. Scale bars are 1 cm. (B) Yeast two-hybrid analysis of the interaction between SASP and OST1. SASP and OST1 coding sequences were fused to the pGBKT7 and pGADT7 vectors, respectively, and then the resulting fusion constructs were co-transformed into yeast strain AH109. The positive transformants were first spotted onto control medium (SD/–Trp–Leu) to grow for 4–6 d, and then transferred to selective medium (SD/–Trp–Leu–Ade + 3-AT) to grow for 4–6 d before observation. (C) Bimolecular fluorescence complementation analysis of the interaction between SASP and OST1. SASP and OST1 coding sequences were fused to YN and YC vectors, respectively, and then the resulting fusion constructs were separately transformed into Agrobacterium strain GV3101 for co-infiltration. Two construct combinations, YN plus OST1-YC and SASP-YN plus YC, were used as negative controls for the co-infiltration. Images were taken under a confocal microscope. Scale bars are 25 μm. (D) Co-localization of SASP-GFP and OST1-mCherry. SASP and OST1 coding sequences were fused to the pC1305-d35S-GFP and pC1305-d35S-mCherry vectors, respectively, and then the resulting fusion constructs were separately transformed into Agrobacterium strain GV3101 for co-infiltration. Images were taken under a confocal microscope. Scale bars are 25 μm. Increased ABA signaling in the sasp mutants may be caused by abolishment of SASP activity on OST1 A previous study showed that overexpression of OST1 in ost1 mutant led to an ABA-hypersensitivity phenotype as ABA signaling was increased as a result of over-accumulation of OST1 (Acharya et al., 2013). To test whether or not SASP had enzymatic activity on OST1, we co-expressed SASP-GFP and OST1-mCherry in N. benthamiana leaves to examine the protein stability of OST1. It was apparent that although the different combinations exhibited gradually decreasing fluorescence, the signals from the combination of OST1-mCherry and SASP-GFP were reduced considerably compared to those from the combination of OST1-mCherry and PIP2a-YFP with increasing concentrations of SASP-GFP or PIP2a-YFP (Fig. 9A), suggesting that SASP-GFP might have degraded the OST1-mCherry in planta upon co-infiltration. Closer inspection revealed that the cells with high green fluorescence signal (containing highly expressed SASP-GFP construct) displayed quite weak red fluorescence signal (‘1’ in Fig. 9B), whilst the cells with weak green fluorescence signal (containing low-expressed SASP-GFP construct) exhibited high red fluorescence signal (‘2’ in Fig. 9B). These observations further support the notion that SASP plays a role in degrading the OST1-mCherry protein. In addition, we transfected the POST1::OST1-mCherry construct separately into Col-0 and sasp-1 protoplasts in order to check fluorescence signals. As shown in Fig. 9C, the signals at the cell periphery of the transfected Col-0 protoplasts were indistinct compared to those of the transfected sasp-1 protoplasts, which had clear and sharp signals. This suggests the possibility that OST1-mCherry in the Col-0 protoplasts had been subject to more severe degradation. Taken together, these results support the conclusion that SASP promotes degradation of OST1-mCherry in vivo, whilst knockout of SASP clearly impairs its degradation. Fig. 9. View largeDownload slide In vivo assays of SASP enzymatic activity against OST1. (A) In vivo tests of SASP-GFP enzymatic activity against OST1-mCherry in Nicotiana benthamiana leaves. SASP and OST1 coding sequences were positioned upstream of the GFP and mCherry genes, respectively, and the resulting fusion constructs were separately introduced into Agrobacterium strain GV3101. The Agrobacterium GV3101 culture harboring OST1-mCherry construct (OD600=0.6) was then mixed at a ratio of 1:1 (v/v) with each of the GV3101 cultures harboring the SASP-GFP or PIP2a-YFP construct with different OD600 values (0.3, 0.6, and 1.2), and then the mixtures in combination with the Agrobacterium p19 strain were infiltrated into N. benthamiana leaves. Control: leaves infiltrated only with Agrobacterium culture containing the OST1-mCherry construct plus the p19 strain. Fluorescence was observed under a confocal microscope at 3 d following the co-infiltration. Scale bars are 20 μm. (B) Detailed examination of the transformed cells of N. benthamiana leaves containing both the OST-mCherry and SASP-GFP constructs. The co-infiltrated leaves in (A, upper panel) were examined under a confocal microscope to check fluorescence intensity. 1, a cell containing strongly expressed SASP-GFP but weakly expressed OST1-mCherry; 2, a cell containing strongly expressed OST1-mCherry but weakly expressed SASP-GFP. Scale bars are 20 μm. (C) Effects of the sasp-1 mutation on degradation of OST1-mCherry in Arabidopsis protoplasts. Intact protoplasts were isolated from 4-week-old wild-type Col-0 and sasp-1 leaves, and then transfected with the POST1::OST1-mCherry construct. The transfected protoplasts were imaged under a confocal microscope. For each transfection, at least 20 cells were observed and more than 70% of cells displayed similar results. Two representative cells from each transfection are shown. Scale bars are 70 μm. Fig. 9. View largeDownload slide In vivo assays of SASP enzymatic activity against OST1. (A) In vivo tests of SASP-GFP enzymatic activity against OST1-mCherry in Nicotiana benthamiana leaves. SASP and OST1 coding sequences were positioned upstream of the GFP and mCherry genes, respectively, and the resulting fusion constructs were separately introduced into Agrobacterium strain GV3101. The Agrobacterium GV3101 culture harboring OST1-mCherry construct (OD600=0.6) was then mixed at a ratio of 1:1 (v/v) with each of the GV3101 cultures harboring the SASP-GFP or PIP2a-YFP construct with different OD600 values (0.3, 0.6, and 1.2), and then the mixtures in combination with the Agrobacterium p19 strain were infiltrated into N. benthamiana leaves. Control: leaves infiltrated only with Agrobacterium culture containing the OST1-mCherry construct plus the p19 strain. Fluorescence was observed under a confocal microscope at 3 d following the co-infiltration. Scale bars are 20 μm. (B) Detailed examination of the transformed cells of N. benthamiana leaves containing both the OST-mCherry and SASP-GFP constructs. The co-infiltrated leaves in (A, upper panel) were examined under a confocal microscope to check fluorescence intensity. 1, a cell containing strongly expressed SASP-GFP but weakly expressed OST1-mCherry; 2, a cell containing strongly expressed OST1-mCherry but weakly expressed SASP-GFP. Scale bars are 20 μm. (C) Effects of the sasp-1 mutation on degradation of OST1-mCherry in Arabidopsis protoplasts. Intact protoplasts were isolated from 4-week-old wild-type Col-0 and sasp-1 leaves, and then transfected with the POST1::OST1-mCherry construct. The transfected protoplasts were imaged under a confocal microscope. For each transfection, at least 20 cells were observed and more than 70% of cells displayed similar results. Two representative cells from each transfection are shown. Scale bars are 70 μm. Given that OST1 degradation was impaired in the sasp mutants, we examined whether ROS production was enhanced in the mutant guard cells due to higher accumulation of OST1 in the sasp mutants than in the wild type. Without ABA treatment, there were no significant differences in ROS production in the guard cells between the wild type and the sasp mutants after incubation with H2DCF-DA (Fig. 10A). However, in the presence of 100 μM ABA, the guard cells of the mutants accumulated remarkably more ROS than those of wild type, which resembled the phenotypes of OST1-overexpressing plants (Acharya et al., 2013). Thus, these results provided a direct demonstration that knockout of SASP facilitated increased ROS production upon treatment with ABA, which was presumably due to the over-accumulation of OST1. Furthermore, the ROS attenuation assays indicated that the DCF fluorescence intensity of sasp-1 remained relatively constant over the 35-min time frame of the experiment (Fig. 10B, C). By contrast, the DCF fluorescence intensity of the wild type decreased rapidly once the ABA treatment was removed, and it had become very weak by the 30-min time point. Therefore, it seems that in the sasp mutants ROS attenuation was markedly impaired in the guard cells, presumably due to the persistent activation of ROS production by the accumulating OST1. Taken together, it appears that the increased ABA signaling in the sasp mutants may result from over-accumulation of OST1, which presumably arises from the abolishment of SASP activity on OST1. Fig. 10. View largeDownload slide Comparison of attenuation rates of reactive oxygen species (ROS) in guard cells between Col-0 and sasp mutants. (A) Comparison of ROS production in guard cells with or without ABA treatment as shown by the fluorescent ROS indicator H2DCF-DA. Peeled epidermal strips from Col-0 and the sasp mutants were first incubated without (control) or with 100 μM ABA for 15 min, followed by three rinses with distilled water. They were then transferred to 25 μM H2DCF-DA solution for incubation for 30 min. After brief rinses, the strips were observed under a confocal microscope and representative images of guard cells are shown. Scale bars are 70 μm. (B) Comparison of ROS attenuation between Col-0 and sasp-1 in guard cells. Peeled epidermal strips from Col-0 and sasp-1 were treated with 100 μM ABA and 25 μM H2DCF-DA as described in (A) and then imaged under a confocal microscope (0 min). A further seven images were taken at 5-min intervals. Scale bars are 70 μm. (C) Kinetics of ROS attenuation rates between the Col-0 and sasp-1 in guard cells illustrated in (B). Fluorescence intensity was quantified using Adobe Photoshop 5.0, and the values are expressed as means (±SD) for three biological replicates. Fig. 10. View largeDownload slide Comparison of attenuation rates of reactive oxygen species (ROS) in guard cells between Col-0 and sasp mutants. (A) Comparison of ROS production in guard cells with or without ABA treatment as shown by the fluorescent ROS indicator H2DCF-DA. Peeled epidermal strips from Col-0 and the sasp mutants were first incubated without (control) or with 100 μM ABA for 15 min, followed by three rinses with distilled water. They were then transferred to 25 μM H2DCF-DA solution for incubation for 30 min. After brief rinses, the strips were observed under a confocal microscope and representative images of guard cells are shown. Scale bars are 70 μm. (B) Comparison of ROS attenuation between Col-0 and sasp-1 in guard cells. Peeled epidermal strips from Col-0 and sasp-1 were treated with 100 μM ABA and 25 μM H2DCF-DA as described in (A) and then imaged under a confocal microscope (0 min). A further seven images were taken at 5-min intervals. Scale bars are 70 μm. (C) Kinetics of ROS attenuation rates between the Col-0 and sasp-1 in guard cells illustrated in (B). Fluorescence intensity was quantified using Adobe Photoshop 5.0, and the values are expressed as means (±SD) for three biological replicates. Discussion SASP appears to be involved in specifically regulating ABA signaling rather than participating in general protein breakdown during leaf senescence SASP was previously isolated from senescent Arabidopsis leaves using a zymogram approach, and SASP expression and proteolytic activity were both found to be induced during leaf senescence (Martinez et al., 2015). Our results also showed that the SASP transcript level was up-regulated during darkness-induced rapid leaf senescence (Fig. 1A). In addition, a previous study had demonstrated that the endopeptidase activity of TaSSP1, an ortholog of Arabidopsis SASP, was strongly induced in naturally and ABA-induced senescent wheat leaves (Qi and Xu, 2003). All these data point to a possible role of SASP in leaf senescence. However, in the current study we found that knockout of SASP did not result in any visible senescence-related phenotype; in contrast, our results support a new role of SASP in regulating ABA signaling and drought tolerance in Arabidopsis. SASP expression was strongly induced by ABA, and the sasp mutants were more sensitive to ABA than wild-type plants at both seed germination and seedling stages (Fig. 3). Upon ABA treatment, detached leaves of the sasp mutants displayed greater senescence and over-production of ROS than the wild type (Fig. 4). Moreover, the sasp mutant plants demonstrated higher resistance to drought stress than the wild type (Fig. 5). More importantly, in the sasp mutants the ABA-signaling genes ABI1 and HAB1 were down-regulated while RBOHD, ROBHF, PYL4, and OST1 were up-regulated, suggesting increased ABA signaling in the sasp mutants (Fig. 6). Given that SASP not only co-localized with OST1 but also interacted with it at the cell periphery (Fig. 8), we speculate that OST1 is a substrate for SASP. Further in vivo enzymatic activity assays indicated that co-transfection of the OST1-mCherry construct with the SASP-GFP construct in N. benthamiana leaves appeared to result in the degradation of OST1 (Fig. 9A, B). In addition, when the OST1-mCherry construct was transformed into sasp-1 protoplasts, the degradation of OST1-mCherry was impaired to some extent, confirming a role of SASP in degrading OST1 and regulating ABA signaling (Fig. 9C). It has been reported that during leaf senescence endogenous ABA levels increase dramatically and that a subset of genes implicated in the key steps of ABA biosynthesis and signaling are significantly upregulated, implying that ABA plays essential roles in the progression of leaf senescence (Tan et al., 2003; Lim et al., 2007). Indeed, ABA is regarded as a hormonal trigger controlling the onset of leaf senescence (Gao et al., 2016; Zhao et al., 2016). Arabidopsis SAUL1 was discovered to be induced by ABA and it targets AAO3 for degradation; in the saul1 mutant the AAO3 protein is over-accumulated and hence the ABA content substantially increases, which results in rapid ABA-induced leaf senescence (Hoth et al., 2002; Raab et al., 2009). Thus, the role of SAUL1 is to counteract the over-production of AAO3 and hence maintain a particular level of endogenous ABA. Similarly, SASP presumably targets OST1 (and perhaps a few additional proteins) for degradation to weaken ABA signaling. It therefore seems that during leaf senescence ABA biosynthesis and signaling are increased and hence induce higher production of SASP, which in turn targets the activated OST1 for degradation, thus reducing the OST1 protein level and attenuating ABA signaling (Fig. 11). Hence, it appears that SASP acts as a buffer against increased ABA signaling rather than serving a function in general protein breakdown during leaf senescence. Although we are as yet unable to determine if the substrate specificity of SASP is indeed narrow, it is likely that SASP exhibits quite stringent substrate specificity because in our assays it only physically interacted with OST1 out of a total of 11 proteins involved in ABA signaling (Fig. 8A–C). In addition, we also identified another three proteins (ERS1, ACBP2, and ACBP3) that interacted with SASP through large-scale protein-protein interaction assays, and they are all involved in the regulation of ABA signaling or stomatal movement. Therefore, it seems that SASP is largely responsible for degradation of OST1 and three other proteins associated with ABA signaling, although we cannot completely rule out its action in general protein breakdown. Fig. 11. View largeDownload slide A model for SASP regulation of ABA signaling. Exogenous ABA, drought, darkness, and leaf senescence are all able to induce expression of some key ABA-biosynthesis and ABA-signaling genes, which leads to an increase in endogenous ABA levels and triggers protein activities of ABA-signaling components, such as PYR1, PYL, RCAR, and OST1. The activated OST1 subsequently targets downstream proteins such as RBOHD, RBOHF, and SLAC1 for phosphorylation. In parallel, SASP expression is also induced by either exogenous or endogenous ABA, which ultimately results in higher accumulation of SASP. The SASP targets activated-OST1 by protein–protein interaction and degrades it, and thus the activity of OST1 is attenuated. It therefore appears that SASP acts as a buffer against the increased ABA signaling by weakening OST1 activity to counteract the potency of ABA. Fig. 11. View largeDownload slide A model for SASP regulation of ABA signaling. Exogenous ABA, drought, darkness, and leaf senescence are all able to induce expression of some key ABA-biosynthesis and ABA-signaling genes, which leads to an increase in endogenous ABA levels and triggers protein activities of ABA-signaling components, such as PYR1, PYL, RCAR, and OST1. The activated OST1 subsequently targets downstream proteins such as RBOHD, RBOHF, and SLAC1 for phosphorylation. In parallel, SASP expression is also induced by either exogenous or endogenous ABA, which ultimately results in higher accumulation of SASP. The SASP targets activated-OST1 by protein–protein interaction and degrades it, and thus the activity of OST1 is attenuated. It therefore appears that SASP acts as a buffer against the increased ABA signaling by weakening OST1 activity to counteract the potency of ABA. Enhanced drought tolerance of sasp mutants is derived from increased ABA signaling The fact that the sasp mutants displayed enhanced tolerance to drought stress points to the involvement of SASP in stress responses (Fig. 5A). A few studies have shown that, for some plant species, drought stress usually leads to changes in gene expression or proteolytic activities of several categories of proteases (especially serine and aspartic proteases), which in turn determines the resistance or susceptibility of the species to the stress. Drought stress may stimulate/repress the gene expression of serine/aspartic proteases, and thus increase/decrease the enzymatic activities of these proteases (Hieng et al., 2004; Drame et al., 2007; Budič et al., 2013), or bring about combinations of both effects. In general, according to studies on several crop species with different drought tolerance, under severe stress conditions drought-tolerant cultivars usually display reduced gene expression or proteolytic activities of serine or aspartic proteases, whereas drought-susceptible cultivars commonly exhibit increased gene expression or proteolytic activities of these proteases (Hieng et al., 2004; Drame et al., 2007), suggesting that an increase in proteolytic activity diminishes drought tolerance and that a decrease in proteolytic activity improves drought tolerance. A possible explanation for this phenomenon is that the increased proteolytic activities speed up the breakdown of cellular proteins and thus disrupt normal metabolic processes, which ultimately leads to damaged membranes, cell death, and leaf senescence. In comparison, we found that the enhanced drought tolerance in sasp mutants comes from increased ABA signaling, which eventually reduces water loss in the mutants by triggering rapid stomatal closure, and thus improves their survival rates. It therefore seems that the mechanism of enhanced drought tolerance in the Arabidopsis sasp mutants is probably quite different from that of the drought-tolerant cultivars examined in previous studies. Supplementary data Supplementary data are available at JXB online. Table S1. Primers used in this study. Fig. S1. Schematic diagram of domain structures of SASP. Fig. S2. Phylogenetic relationships among SASP and orthologous proteins from other species. Fig. S3. Phenotypic analyses of sasp mutants. Fig. S4. Detection of transcriptional activation of senescence-associated genes in sasp mutants. Fig. S5. Expression analyses of SAG12 in sasp mutants with and without ABA treatment. Fig. S6. Images of 4-week-old leaves from wild type and sasp-1 subjected to dark-induced senescence treatment. Fig. S7. Expression analyses of six essential genes implicated in ABA signaling in wild-type and sasp mutants in response to drought. Fig S8. Expression analyses of RD29A in wild type and sasp mutants in response to ABA. Fig. S9. ABA content in wild type and sasp mutants treated with ABA or ETN. Fig. S10. Expression analyses of five key genes for ABA biosynthesis in wild type and sasp mutants treated with ABA or ETN. Acknowledgements This work was supported by grants from the Natural Science Foundation of Jiangsu Province (BK20151425), The Fundamental Research Funds for the Central Universities (KYTZ201402, KYRC201409), The Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Jiangsu Science Fund for Distinguished Young Scholars (BK20150027). References Acharya BR , Jeon BW , Zhang W , Assmann SM . 2013 . 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Journal of Experimental BotanyOxford University Press

Published: Aug 17, 2018

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