Patellin1 Negatively Modulates Salt Tolerance by Regulating PM Na+/H+ Antiport Activity and Cellular Redox Homeostasis in Arabidopsis

Patellin1 Negatively Modulates Salt Tolerance by Regulating PM Na+/H+ Antiport Activity and... Abstract Soil salinity significantly represses plant development and growth. Mechanisms involved sodium (Na+) extrusion and compartmentation, intracellular membrane trafficking as well as redox homeostasis regulation play important roles in plant salt tolerance. In this study, we report that Patellin1 (PATL1), a membrane trafficking-related protein, modulates salt tolerance in Arabidopsis. The T-DNA insertion mutant of PATL1 (patl1) with an elevated PATL1 transcription level displays a salt-sensitive phenotype. PATL1 partially associates with the plasma membrane (PM) and endosomal system, and might participate in regulating membrane trafficking. Interestingly, PATL1 interacts with SOS1, a PM Na+/H+ antiporter in the Salt-Overly-Sensitive (SOS) pathway, and the PM Na+/H+ antiport activity is lower in patl1 than in Col-0. Furthermore, the reactive oxygen species (ROS) content is higher in patl1 and the redox signaling of antioxidants is partially disrupted in patl1 under salt stress conditions. Artificial elimination of ROS could partially rescue the salt-sensitive phenotype of patl1. Taken together, our results indicate that PATL1 participates in plant salt tolerance by regulating Na+ transport at least in part via SOS1, and by modulating cellular redox homeostasis during salt stress. Introduction Soil salinity is a significant stress for crop growth and development worldwide (Tuteja 2007, Munns and Tester 2008). Accumulated sodium ions in soil cause both osmotic and ionic stresses to plants (Hasegawa et al. 2000, Munns and Tester 2008, Zhu 2016). As sessile organisms, plants have evolved efficient strategies to cope with salt stress. Plant cells perceive the salt stress signal which in turn induces release of cellular second messengers such as Ca2+, inositol phosphates as well as reactive oxygen species (ROS) (Hasegawa et al. 2000, Xiong et al. 2002, Mahajan and Tuteja 2005). The Salt-Overly-Sensitive (SOS) pathway is induced by such signals and participates in plant salt tolerance by modulating ion homeostasis. SOS1, a plasma membrane (PM)-localized Na+/H+ antiporter, transports Na+ out of the cell through its Na+/H+ antiport activity (Shi et al. 2000, Qiu et al. 2002, Shi et al. 2002, Quan et al. 2007, Lin et al. 2009, Quintero et al. 2011, Zhu 2016). Several regulators of the SOS pathway including ABI2, GIGANTEA (GI) and 14-3-3 proteins have been identified recently, which together fine-tune SOS activity for proper plant salt adaption (Ohta et al. 2003, Kim et al. 2013, Zhou et al. 2014, Tan et al. 2016). Furthermore, Na+ compartmentation in the vacuole is also important for regulation of salt tolerance. Vacuolar membrane-localized Na+/H+ antiporters transport Na+ into the vacuole driven by the H+ gradient established by vacuolar H+-ATPase and/or the H+-pyrophosphatase, and repress the toxic accumulation of Na+ in the cytoplasm (Apse et al. 1999, Gaxiola et al. 2001, Yokoi et al. 2002, An et al. 2007, Barragán et al. 2012). Salt stress also disrupts cellular redox homeostasis and causes oxidative stress to plants, leading to cell death and growth inhibition (Mittler 2002, Miller et al. 2010). Soil salinity induces excessive generation of ROS including superoxide radicals, hydrogen peroxide and hydroxyl radicals by destroying the electron transport chain in chloroplast and/or mitochondria as well as through membrane-bound NADPH oxidases (Miller et al. 2010, Dietz et al. 2016, Gilroy et al. 2016, Huang et al. 2016, Takagi et al. 2016). ROS are highly reactive and capable of causing lipid peroxidation, protein denaturation and DNA damage (Gill and Tuteja 2010, Farmer and Mueller 2013). In fact, lipid peroxidation will generate lipid peroxide, another destructive species, in plant cells (Farmer and Mueller 2013, Reiter et al. 2014). Mitogen-activated protein kinase (MAPK) cascades have been identified to be involved in regulating ROS generation under abiotic stress (Zhang and Klessig 2001, Xiong et al. 2002). Besides being toxic molecules; however, ROS have also been identified as signaling molecules in plant development and stress response (Gechev et al. 2006, Van Breusegem and Dat 2006, Zhou et al. 2016). Rapid elevation of ROS in plant cells under salt stress might serve as signals for stress acclimation (Gechev et al. 2006, Miller et al. 2010). A recent study revealed that specific ROS accumulation mediated by the NADPH oxidase AtrbohF in the vasculature contributes to Na+ translocation regulation and salt tolerance in Arabidopsis (Jiang et al. 2012). A study has also reported that ROS produced by both AtrbohD and AtrbohF function as signals to regulate K+/Na+ homeostasis and enhanced salt tolerance (Ma et al. 2012). Plants have also evolved effective strategies to remove cellular toxic ROS. Previous studies revealed that enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX) function in enzymatic ROS scavenging in plants (Mittler 2002, Blokhina et al. 2003, Apel and Hirt 2004). In addition, reduced glutathione (GSH) and ascorbate contribute to non-enzymatic ROS scavenging by directly interacting with and detoxifying oxygen free radicals (Apel and Hirt 2004). The balance between ROS generation and scavenging during salt stress is critical for cellular redox homeostasis and plant salt tolerance (Moon et al. 2003, Chung et al. 2008, Miller et al. 2010). Intracellular membrane trafficking participates in modulating salt tolerance. During salt stress, cycling of PM proteins between the PM and endosomes is enhanced, indicating that membrane trafficking and redistribution of PM proteins represent the initial responses of plant cells under abiotic conditions such as soil salinity (Jurgens 2004, Murphy et al. 2005, Luu et al. 2012, Garcia de la Garma et al. 2015, Munns and Gilliham 2015). In fact, ROS production and membrane trafficking are reported to be co-ordinated during plant salt tolerance regulation. PM endocytosis is involved in intracellular ROS production, while defects in endocytosis disrupt ROS generation in endosomes during salt stress (Leshem et al. 2006, Leshem et al. 2007, Liu et al. 2012). Repression of VAMP711, an Arabidopsis v-SNARE, leads to repression of fusion of H2O2-containing vesicles with the vacuole, leading to enhanced vacuolar H+-ATPase activity and increased salt tolerance (Leshem et al. 2006). The plant-unique Rab GTPase ARA6 regulates SNARE complex formation between VAMP727 and SYP121. The ARA6 loss-of-function mutant ara6 conferred salt hypersensitivity (Ebine et al. 2011). In addition, overexpression of OsRab7 enhanced accumulation of trafficking vesicles in root tips during salt stress, and transgenic rice exhibited enhanced salt tolerance (Peng et al. 2014). The Arabidopsis Patellin (PATL) protein family consists of six members, i.e. PATL1, 2, 3, 4, 5 and 6, and might play a role in membrane trafficking (Peterman et al. 2004). PATL1 contains a variable N-terminal domain followed by a Sec14 domain and a Golgi dynamics domain (GOLD), both of which are conserved in other membrane trafficking-related proteins (Peterman et al. 2004, Peterman et al. 2006, Mousley et al. 2007). PATL1 specifically binds to phosphoinositides which function in regulating membrane trafficking (Peterman et al. 2004, Mousley et al. 2007, Thole and Nielsen 2008). Interestingly, PATL1 might participate in vesicle trafficking mediated by the deubiquitinating enzyme AMSH3, possibly via binding and recruiting AMSH3 to the PM in Arabidopsis (Isono et al. 2010). PATL1 can be modified with ubiquitin, suggesting that ubiquitination might play a role in PATL1 function (Igawa et al. 2009, Saracco et al. 2009). PATL2 is a possible substrate of MPK4, and phosphorylation of PATL2 by MPK4 alters its binding affinity for phosphoinositides (Suzuki et al. 2016). Interestingly, salt stress promotes phosphorylation of PATL2, indicating a link between PATL2 and regulation of salt tolerance (Hsu et al. 2009). PATL3 and PATL6 are involved in regulation of plant immunity. They restrict the intracellular transport of Alfalfa mosaic virus viral movement protein-containing vesicles and enhance plant immunity (Peiro et al. 2014). Therefore, PATLs function in regulating plant abiotic as well as biotic stress tolerance; however, the underlying mechanisms still needs to be understood in detail. To fine-tune plant growth and its stress response in a challenging environment, efficient negative feedback regulation mechanisms are needed to avoid over-response to environmental stresses. For example, ABA promotes S-nitrosylation and subsequent inactivation of SnRK2.6, a positive regulator of ABA signaling, leading to attenuated ABA signaling (Wang et al. 2015). Actually, several other studies have also revealed this negative feedback regulation in plants during stress response (Zhao et al. 2017). We proposed that there might also exist such a mechanism for plant salt tolerance, through which plant growth and stress response can be well balanced upon soil salinity. Here we identify Arabidopsis PATL1 as a negative regulator of salt tolerance by regulating Na+ transport at least in part via SOS1, and by modulating cellular redox homeostasis. Membrane trafficking is partially defective in the PATL1 T-DNA insertion mutant patl1, which displays a salt-sensitive phenotype compared with wild-type plants. Seedlings overexpressing PATL1 exhibit higher salt sensitivity. PATL1 interacts with SOS1 directly in planta and negatively regulates its PM Na+/H+ antiport activity. Cellular redox homeostasis is disrupted in patl1 under saline condition, and artificial elimination of ROS could partially rescue the salt-sensitive phenotype of patl1. PATL1 transcription is salt induced, which might be a negative feedback mechanism in regulation of plant salt tolerance. These results provide new evidences supporting that membrane trafficking is critical for cellular ion and ROS homeostasis during salt stress and confers plant salt tolerance. Results The Arabidopsis patl1 mutant is hypersensitive to salt stress To investigate the function of membrane trafficking in regulation of plant salt tolerance, a pool of Arabidopsis T-DNA insertion mutants related to membrane trafficking obtained from the Arabidopsis Biological Resource Center (ABRC) were used for screening. In this way, a collection of about 120 mutant lines and their relative wild-type plant [Columbia-0 (Col-0)] was sown in Murashige and Skoog (MS) medium containing 125 mM NaCl for salt sensitivity analysis. We identified that the SALK_103668 plant displayed a salt-hypersensitive phenotype (Fig. 1A, D). The T-DNA was inserted in the 5'-untranslated region (5'-UTR) of the At1g72150 gene, which encodes PATL1 (Fig. 1A), a possible membrane trafficking-related protein that might participate in cell plate formation during cytokinesis (Peterman et al. 2004). We then named SALK_103668 as patl1 (Fig. 1A). The full-length transcript of PATL1 was enriched at least 10-fold in the patl1 mutant (Fig. 1B;Supplementary Fig. S1A). We have analyzed the genome structure of the PATL1 promoter region in patl1. The data showed that there exists a Cauliflower mosaic virus (CaMV) 35S promoter upstream of the translation start site (ATG) due to T-DNA insertion (Supplementary Fig. S2A, B), which might explain the elevated PATL1 transcription level in the patl1 mutant compared with that in Col-0. Furthermore, 5'-rapid amplification of cDNA ends (5'-RACE) assay has also been performed using seedlings of Col-0 and patl1, and the result revealed that the transcription initiation site of PATL1 in Col-0 was at –298 bp relative to the translation start site (ATG), while in the patl1 background the transcription initiation site was –922 bp (Supplementary Fig. S2C, D). Fig. 1 View largeDownload slide The patl1 mutant is hypersensitive to salt stress. (A) Schematic representation of the structure of PATL1. Filled gray boxes, UTRs; empty boxes, exons; lines between boxes, introns; triangle, T-DNA insertion site. (B, C) Determination of PATL1 transcription. RT–PCR was used to monitor the expression of PATL1 in Col-0 and the patl1 mutant as well as PATL1-overexpressing plants. ACTIN2 (ACT2) was used as a loading control. Similar results were obtained in three independent replicated experiments. (D) Analysis of salt sensitivity in Col-0, patl1 and two PATL1 transgenic plants. Seeds were germinated and grown on MS medium with or without 75 or 125 mM NaCl. Seedlings were photographed after 2 weeks of growth. (E, F) Analysis of the germination rate, cotyledon greening rate and fresh weight of seedlings in (D). Error bars represent the SD (n >10). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). Fig. 1 View largeDownload slide The patl1 mutant is hypersensitive to salt stress. (A) Schematic representation of the structure of PATL1. Filled gray boxes, UTRs; empty boxes, exons; lines between boxes, introns; triangle, T-DNA insertion site. (B, C) Determination of PATL1 transcription. RT–PCR was used to monitor the expression of PATL1 in Col-0 and the patl1 mutant as well as PATL1-overexpressing plants. ACTIN2 (ACT2) was used as a loading control. Similar results were obtained in three independent replicated experiments. (D) Analysis of salt sensitivity in Col-0, patl1 and two PATL1 transgenic plants. Seeds were germinated and grown on MS medium with or without 75 or 125 mM NaCl. Seedlings were photographed after 2 weeks of growth. (E, F) Analysis of the germination rate, cotyledon greening rate and fresh weight of seedlings in (D). Error bars represent the SD (n >10). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). When Col-0 and patl1 seeds were germinated on MS medium with 75 or 125 mM NaCl, respectively, significant differences between the two genotypes were observed. At 5 d post-germination on MS with 75 mM NaCl, 78% seeds of Col-0 germinated while for patl1 seedlings, only 50% were able to germinate (Fig. 1D, E). The inhibitory effect on germination was more pronounced for patl1 than for Col-0 when the concentration of NaCl in MS was elevated to 125 mM. Under this condition, 61% seeds of Col-0 germinated, while only 38% seeds of patl1 seeds were able to germinate (Fig. 1D, E). Furthermore, the subsequent cotyledon greening was also significantly repressed in patl1 compared with Col-0 (Fig. 1D, F). In addition, when 5-day-old patl1 mutant and Col-0 seedlings were transferred to MS medium containing 125 mM NaCl, shoot tissue and the primary root of patl1 showed a significant reduction in growth compared with Col-0 (Supplementary Fig. S3A). The primary root length of patl1 decreased at least 30% compared with that of Col-0 (Supplementary Fig. S3B). We have also determined the plant salt sensitivity in soil. In this way, 2-week-old seedlings of Col-0 and patl1 grown under long-day conditions were irrigated with water containing 200 mM NaCl. Salt stress significantly inhibited plant growth; however, the inhibitory effect on patl1 was more severe than that on Col-0 (Supplementary Fig. S3C). Consistently, the Chl content in patl1 was much lower than that in Col-0 during soil salinity (Supplementary Fig. S3D). Since PATL1 transcription was significantly elevated in patl1 and as we lacked a second allele of this mutant, the Pro35S::GFP-PATL1 construct was transformed into the Col-0 background to generate PATL1 transgenic plants (Pro35S::GFP-PATL1/Col-0) (Fig. 1C;Supplementary Fig. S1B). Independent homozygous T2 lines (#80 and #86) were used for the salt sensitivity assay as described. We found that Pro35S::GFP-PATL1/Col-0 seedlings displayed a salt-sensitive phenotype similar to that of the patl1 mutant (Fig. 1D–F;Supplementary Fig. S3). Furthermore, to determine the possible effect of salt stress on PATL1, about 2,000 bp of the PATL1 promoter was fused to the β-glucuronidase (GUS) reporter gene. The resulting construct was transformed into Col-0. Independent T2 transgenic lines were used for GUS staining under control or salt stress conditions. Data showed that GUS expression driven by the PATL1 promoter was induced by salt stress (Supplementary Fig. S4A). Consistently, a reverse transcription–PCR (RT–PCR) assay revealed that PATL1 was induced in response to salt stress, suggesting a negative feedback regulation mechanism in plants (Supplementary Fig. S4B). Taken together, these results indicate that the salt-sensitive phenotype of the patl1 mutant is due to elevated PATL1 transcription and that PATL1 could be a negative regulator of salt tolerance in Arabidopsis. PATL1 associates with the PM and endosomes in plants As already described, PATL1 contains a variable N-terminal domain followed by a Sec14 domain and a GOLD domain, which are also found in other membrane trafficking-related proteins (Peterman et al. 2004, Mousley et al. 2007), suggesting that PATL1 might function as a putative membrane trafficking-related protein and have a regulatory role in membrane trafficking. To determine the subcellular localization of PATL1, PATL1–green fluorescent protein (GFP) fusion protein was transiently expressed in Nicotiana benthamiana leaf cells via agroinfiltration. Three dafter the inoculation, infiltrated tobacco leaves were incubated with FM4-64, a lipophilic styryl dye staining the PM and endosomes (Vida and Emr 1995), and then observed using confocal microscopy. The results revealed that PATL1–GFP mainly localized at the PM (Supplementary Fig. S5A). Interestingly, co-localization between PATL1–GFP and vesicles stained by FM4-64, the putative endosomes, was also observed (Supplementary Fig. S5A). When 3-week-old soil-grown Pro35S::GFP-PATL1/Col-0 (line #80) seedlings were used for protoplast isolation and confocal microscopy, we also found that PATL1–GFP mainly localized at the PM and endosomes, indicated by FM4-64 staining (Supplementary Fig. S5B). Furthermore, data revealed that PATL1–GFP also co-localized with the red fluorescent protein (RFP)-tagged VSR2, a marker of pre-vacuolar compartments (PVCs) (Fig. 2). In summary, these data together suggest that PATL1 partially associates with the PM and endosomal system, and it might function in regulating membrane trafficking in plants. Fig. 2 View largeDownload slide PATL1 associates with the PM and endosomes in plants. (A–C) Co-localization of PATL1 and VSR2, a marker of pre-vacuolar compartments (PVCs) in Arabidopsis leaf protoplasts. The fluorescence signal was detected using a Leica SP5 confocal fluorescence microscope. (A) GFP; (B) RFP; and (C) merged image. Scale bar = 5 µm. Fig. 2 View largeDownload slide PATL1 associates with the PM and endosomes in plants. (A–C) Co-localization of PATL1 and VSR2, a marker of pre-vacuolar compartments (PVCs) in Arabidopsis leaf protoplasts. The fluorescence signal was detected using a Leica SP5 confocal fluorescence microscope. (A) GFP; (B) RFP; and (C) merged image. Scale bar = 5 µm. PATL1 is involved in regulation of membrane trafficking It has been reported that PATL1 might function as a membrane trafficking-related protein (Peterman et al. 2004; Fig. 2; Supplementary Fig. S5). To investigate whether PATL1 plays a regulatory role in membrane trafficking during salt stress, we first examined the effect of brefeldin A (BFA), an inhibitor of membrane trafficking, in root cells of the Col-0 and patl1 seedlings. The result showed that the number of BFA bodies was significantly lower in the patl1 mutant compared with Col-0, indicating that membrane trafficking was partially repressed in patl1 (Fig. 3A, B). Interestingly, we found that PATL1 might also be implicated in regulating the expression of genes related to membrane trafficking during salt stress. Candidate genes, Rab7, PI3K, SYP61 and ZATA-1, were selected since all these genes have been previously found to be involved in membrane trafficking and salt tolerance (Zhu et al. 2002, Mazel et al. 2004, Leshem et al. 2007, Oh et al. 2010, Tian et al. 2015). Ten-day-old seedlings of Col-0 and patl1 as well as two transgenic plants (OX-#80 and #86) were left untreated or treated with 100 mM NaCl for different periods of time, and expression of selected genes was determined quantitatively. The results revealed that transcription levels of Rab7, PI3K, SYP61 and ZATA-1 were significantly lower in patl1, OX-#80 and #86 compared with Col-0 during salt stress (Fig. 3C–F). However, the mechanism by which PATL1 participates in regulating expression of these genes still needs to be determined. Together, these results indicate that PATL1 might be involved in regulation of membrane trafficking during salt stress and play a regulatory role in plant salt tolerance. Fig. 3 View largeDownload slide PATL1 participates in regulation of membrane trafficking. (A) Confocal assay of the effect of BFA on protein trafficking in root cells from 5-day-old Col-0 and patl1 seedlings. Seedlings were briefly washed after incubation in liquid MS medium with 50 µM BFA and 5 mg ml–1 FM4-64 for 30 min. Scale bar = 10 µm. (B) Quantification of the BFA bodies in root cells for (A). Error bars represent the SD (n >6). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). (C–F) Relative expression of genes encoding membrane trafficking-related proteins in Col-0, patl1, OX-#80 and OX-#86 seedlings. Gene transcription was measured by quantitative real-time PCR in 10-day-old seedlings without or with 2 and 6 h treatment with 100 mM NaCl with ACT2 as the internal control. Data were normalized by the gene expression level in Col-0 under control conditions. Error bars represent the SD (n >6). Statistical significance was determined by a Student’s t test; significant differences are indicated by asterisks (*P ≤ 0.05; **P ≤ 0.01). Fig. 3 View largeDownload slide PATL1 participates in regulation of membrane trafficking. (A) Confocal assay of the effect of BFA on protein trafficking in root cells from 5-day-old Col-0 and patl1 seedlings. Seedlings were briefly washed after incubation in liquid MS medium with 50 µM BFA and 5 mg ml–1 FM4-64 for 30 min. Scale bar = 10 µm. (B) Quantification of the BFA bodies in root cells for (A). Error bars represent the SD (n >6). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). (C–F) Relative expression of genes encoding membrane trafficking-related proteins in Col-0, patl1, OX-#80 and OX-#86 seedlings. Gene transcription was measured by quantitative real-time PCR in 10-day-old seedlings without or with 2 and 6 h treatment with 100 mM NaCl with ACT2 as the internal control. Data were normalized by the gene expression level in Col-0 under control conditions. Error bars represent the SD (n >6). Statistical significance was determined by a Student’s t test; significant differences are indicated by asterisks (*P ≤ 0.05; **P ≤ 0.01). PATL1 interacts with SOS1 in planta To substantiate the underlying mechanism by which PATL1 modulates plant salt tolerance, we used Pro35S:Flag-HA-PATL1/Col-0 transgenic plants to identify possible PATL1-interacting proteins using mass spectrometry as described (Zhou et al. 2014). The data revealed that SOS1 might be a putative interacting protein of PATL1. In fact, SOS1 is a PM-localized Na+/H+ antiporter of the SOS pathway, which functions in ion homeostasis during salt stress in plants (Zhu 2016). We then verified the interaction between PATL1 and SOS1 using bimolecular fluorescence complementation (BiFC) analysis in N. benthamiana leaves. The result showed that the yellow fluorescence protein (YFP) fluorescence signal was only detected in leaves transiently expressing PATL1–YFPN and YFPC–SOS1 but not in leaves expressing PATL1–YFPN and YFPC, or YFPN and YFPC–SOS1, respectively (Fig. 4A;Supplementary Fig. S6A). Previous studies have demonstrated that SOS1 contains 12 transmembrane domains and a long autoinhibitory C-terminus in the cytosol (Shi et al. 2000, Quintero et al. 2011). Our BiFC assay revealed that it was the C-terminus but not the N-terminus of SOS1 which interacted with PATL1 in tobacco leaves (Supplementary Fig. S6B). Fig. 4 View largeDownload slide PATL1 interacts with SOS1. (A) BiFC analysis in N. benthamiana. PATL1–YFPN and YFPC–SOS1 were co-expressed in tobacco leaves and the yellow fluorescent protein (YFP) fluorescence signal was detected 3 d after infiltration via a Leica SP5 confocal microscope. Scale bar = 100 µm. (B) Schematic diagram of motifs of PATL1. Black line box, variable N-terminal domain; green line box, Sec14 domain; purple line box, Golgi dynamics domain (GOLD); filled gray box, coiled-coil region; vertical lines, PXXP motif. PATL1 was divided into four fragments according to its predicted structure: PATL1N1 and PATL1N2 for two parts of the variable N-terminal domain, PATL1Sec14 for the Sec14 domain and PATL1GOLD for the GOLD domain, respectively. (C) Co-IP assay of the interaction between PATL1 and SOS1. Myc-tagged full-length or truncated PATL1 was transiently co-expressed with the Flag-SOS1 C-terminus in Arabidopsis leaf protoplasts. Proteins were immunoprecipitated with anti-C-Myc antibody-conjugated agarose. Immunoblot assays with anti-C-Myc and anti-Flag antibodies were used to detect Myc-PATL1 and the PATL1-interacting SOS1 C-terminus, respectively. Experimental details are provided in the Materials and Methods. IP, immunoprecipitation; IB, immunoblot. (D) Y2H analysis of the interaction between PATL1 and SOS1. Yeast strains expressing the indicated constructs were grown on synthetic complete medium without tryptophan and leucine (SC-W/L; left panel) and on synthetic complete medium without histidine, tryptophan and leucine (SC-H/W/L; right panel). Photographs were taken after 4–5 d of growth on the indicated medium. Fig. 4 View largeDownload slide PATL1 interacts with SOS1. (A) BiFC analysis in N. benthamiana. PATL1–YFPN and YFPC–SOS1 were co-expressed in tobacco leaves and the yellow fluorescent protein (YFP) fluorescence signal was detected 3 d after infiltration via a Leica SP5 confocal microscope. Scale bar = 100 µm. (B) Schematic diagram of motifs of PATL1. Black line box, variable N-terminal domain; green line box, Sec14 domain; purple line box, Golgi dynamics domain (GOLD); filled gray box, coiled-coil region; vertical lines, PXXP motif. PATL1 was divided into four fragments according to its predicted structure: PATL1N1 and PATL1N2 for two parts of the variable N-terminal domain, PATL1Sec14 for the Sec14 domain and PATL1GOLD for the GOLD domain, respectively. (C) Co-IP assay of the interaction between PATL1 and SOS1. Myc-tagged full-length or truncated PATL1 was transiently co-expressed with the Flag-SOS1 C-terminus in Arabidopsis leaf protoplasts. Proteins were immunoprecipitated with anti-C-Myc antibody-conjugated agarose. Immunoblot assays with anti-C-Myc and anti-Flag antibodies were used to detect Myc-PATL1 and the PATL1-interacting SOS1 C-terminus, respectively. Experimental details are provided in the Materials and Methods. IP, immunoprecipitation; IB, immunoblot. (D) Y2H analysis of the interaction between PATL1 and SOS1. Yeast strains expressing the indicated constructs were grown on synthetic complete medium without tryptophan and leucine (SC-W/L; left panel) and on synthetic complete medium without histidine, tryptophan and leucine (SC-H/W/L; right panel). Photographs were taken after 4–5 d of growth on the indicated medium. To determine the SOS1-ineracting region(s) in PATL1, we further divided PATL1 into four fragments according to its predicted structure (Peterman et al. 2004; Fig. 4B). Full-length and truncated PATL1s were translationally fused with the Myc tag and transiently co-expressed with the Flag-tagged SOS1 C-terminus (Flag-SOS1C) in Arabidopsis leaf protoplasts for co-immunoprecipitation (Co-IP) assay. The results showed that Flag-SOS1C was co-immunoprecipitated with Myc-PATL1 in vivo (Fig. 4C). Furthermore, it was the N2 part in the variable N-terminal domain but not the Sec14 or GOLD domain in PATL1 that mediated the interaction between PATL1 and SOS1, indicating that the variable N-terminal domain is responsible for protein–protein interaction for PATL1, linking PATL1 to diverse biological functions (Fig. 4C). In addition, we have also investigated the interaction between PATL1 and SOS1 through yeast two-hybrid (Y2H) assay. Consistently, the PATL1 N2 fragment specifically interacted with the SOS1 C-terminus in yeast (Fig. 4D). Taken together, these results demonstrated that PATL1 interacts with SOS1, suggesting that PATL1 might regulate salt tolerance at least in part through SOS1. PATL1 negatively regulates SOS1 activity and ion homeostasis during salt stress Since PATL1 interacts with SOS1 directly in planta, we then determined whether PATL1 participates in regulating SOS1 biological function. It is well known that salt stress impairs ion homeostasis in plant cells and SOS1 contributes to regulation of K+/Na+ homeostasis during salt stress (Xiong et al. 2002, Zhu 2016). PM-localized Na+/H+ antiporters (including SOS1) mainly mediate Na+ efflux (to transport Na+ out of the cell) during salt stress (Aronson 1985, Shi et al. 2002). We first analyzed the PM Na+/H+ antiport activity by isolation of PM vesicles. Thus, 3-week-old soil-grown seedlings of Col-0 and patl1 were treated with 250 mM NaCl for 3 d for PM H+-ATPase activation as described (Yang et al. 2010, Zhou et al. 2012), and then PM vesicles were isolated to determine Na+/H+ antiport activity. The results showed that PM Na+/H+ antiport activity was significantly lower in patl1 than that in Col-0 at all concentrations of NaCl assayed (Fig. 5A). To examine whether PATL1 participates in regulating plant cellular K+/Na+ homeostasis during salt stress, 3-week-old soil-grown Col-0, patl1 and two independent PATL1 transgenic plants (OX-#80 and #86) were exposed to 150 mM NaCl for 10 d, and Na+ and K+ content was measured via atomic absorption spectroscopy (AAS). We found that the Na+ content increased in all the plants during salt stress, while patl1, OX-#80 and #86 accumulated a higher level of Na+ than Col-0 (Fig. 5B). In contrast, K+ content was similar in all the genotypes under both control and salt stress conditions (Fig. 5C). Therefore, the K+/Na+ ratio was much lower in patl1, OX-#80 and #86 plants compared with Col-0 upon salt treatment (Fig. 5D). These data demonstrated that PATL1 participates in regulation of PM Na+/H+ antiport activity and ion homeostasis in plants during salt stress. Fig. 5 View largeDownload slide PATL1 negatively regulates SOS1 activity during salt stress. (A) Comparison of PM Na+/H+ antiport activity in Col-0 and the patl1 mutant. Error bars represent the SD (n = 5) of at least three replicate experiments, each from an independent isolation of PMs. Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). (B, C) Na+ and K+ accumulation in Col-0, patl1, OX-#80 and OX-#86 plants was determined with an atomic absorption spectrophotometer (Hitachi Z-5000). Details are provided in the Materials and Methods. Error bars represent the SD (n >6). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). (D) Analysis of the K+/Na+ ratio in Col-0, patl1, OX-#80 and OX-#86 plants under control or salt stress conditions on the basis of the ion accumulation data in (B) and (C). Error bars represent the SD (n >6). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). Fig. 5 View largeDownload slide PATL1 negatively regulates SOS1 activity during salt stress. (A) Comparison of PM Na+/H+ antiport activity in Col-0 and the patl1 mutant. Error bars represent the SD (n = 5) of at least three replicate experiments, each from an independent isolation of PMs. Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). (B, C) Na+ and K+ accumulation in Col-0, patl1, OX-#80 and OX-#86 plants was determined with an atomic absorption spectrophotometer (Hitachi Z-5000). Details are provided in the Materials and Methods. Error bars represent the SD (n >6). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). (D) Analysis of the K+/Na+ ratio in Col-0, patl1, OX-#80 and OX-#86 plants under control or salt stress conditions on the basis of the ion accumulation data in (B) and (C). Error bars represent the SD (n >6). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). PATL1 regulates plant cellular redox homeostasis during salt stress Salt stress induces ROS generation and imposes oxidative damage on plants (Mittler 2002). Many components of membrane trafficking have been identified as being involved in redox homeostasis regulation under salt conditions (Mazel et al. 2004, Leshem et al. 2006, Leshem et al. 2007, Türkan and Demiral 2009). We then investigated whether PATL1 also plays a regulatory role in ROS generation and scavenging during salt stress. The H2O2 content in Col-0 and patl1 seedlings was determined using 3,3-diaminobenzidine (DAB) staining, and the data showed that patl1 accumulated a higher level of H2O2 than Col-0 after salt treatment (Supplementary Fig. S7A). In addition, we quantitatively measured the H2O2 content with an Amplex Red Kit (Invitrogen). Salt stress significantly increased H2O2 content in both plants, and patl1 accumulated a higher level during salt stress (Supplementary Fig. S7C). We further quantified superoxide radicals in Col-0 and patl1 seedlings under salt stress through histochemical staining with nitroblue tetrazolium (NBT). The result showed that the content of superoxide radicals was higher in patl1 than in Col-0 (Supplementary Fig. S7B). In all of these assays, no detectable difference was observed between seedlings of patl1 and Col-0 under control conditions (Supplementary Fig. S7A–C). These results suggested that PATL1 participates in ROS generation during salt stress. Interestingly, we found that expression of AtrbohD, which encodes a PM-bound NADPH oxidase mediating ROS production in Arabidopsis, was induced in patl1 in response to salt stress (Supplementary Fig. S7D), suggesting that PATL1 is involved in salt-induced ROS production possibly through AtrbohD. ROS generated by abiotic stress such as soil salinity specifically induce scavenging signaling in plants (Blokhina et al. 2003, Apel and Hirt 2004, Gill and Tuteja 2010). We thus analyzed the enzymatic activities of SOD, CAT, POX and APX in patl1 and Col-0 to investigate whether PATL1 plays a role in regulating enzymatic ROS scavenging signaling. The results showed that salt stress enhanced the activities of SOD, CAT, POX and APX to different extents in both plants, and higher enzymatic activity was observed in patl1 compared with that in Col-0 (Supplementary Fig. S8A–D). We proposed that the higher enzymatic activity in patl1 was due to a more severe oxidative stress imposed by salt stress (Supplementary Fig. S7). Interestingly, when a synthetic antioxidant, dimethylthiourea (DMTU), was used for ROS detoxification, the enzymatic activity decreased in both Col-0 and patl1 plants (Supplementary Fig. S8A–D). However, the decreased level of enzymes in patl1 was lower than that in Col-0, indicating that regulation of redox homeostasis in patl1 is partially disrupted (Supplementary Fig. S8E). Consistently, we found that at this concentration (0.5 mM) of DMTU, accumulation of H2O2 deceased in both plants, but patl1 still accumulated a higher level of H2O2 compared with Col-0 (Supplementary Fig. S8F). These data suggested that PATL1 is involved in regulation of plant cellular redox homeostasis during salt stress by modulating ROS generation and the enzymatic ROS scavenging machinery. The ratio of reduced GSH to oxidized GSH (GSSG) was also determined, and the result showed that the GSH:GSSG ratio in patl1 was significantly lower than that in Col-0 under salt conditions, indicating that patl1 seedlings were exposed to a more severe oxidative stress during salt stress (Fig. 6A). To further determine whether PATL1 participates in regulating plant salt tolerance partially through modulating cellular redox homeostasis, we artificially eliminated ROS induced by salt stress using the antioxidant DMTU and then analyzed the cellular redox state and the phenotype in response to salt stress. When 0.75 mM DMTU was added in the culture medium, the GSH:GSSG ratio in patl1 increased to a level similar to that in Col-0 (Fig. 6A), indicating rescue of the cellular redox homeostasis. Consistently, the salt-sensitive phenotype of patl1 was partially rescued when DMTU was added in the culture medium (Fig. 6B, C). Therefore, we concluded that PATL1 also functions in regulating plant cellular redox homeostasis during salt stress. Fig. 6 View largeDownload slide Antioxidant partially rescues the salt-sensitive phenotype of patl1. (A) Determination of the GSH:GSSG ratio. Five-day-old seedlings were left untreated or treated with 100 mM NaCl for 12 h and analyzed for reduced or oxidized glutathione levels using an A061-1 Kit (Nanjing Jiancheng). Error bars represent the SD (n = 3). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05; **P ≤ 0.01). (B) Salt sensitivity assays of patl1 and Col-0 when DMTU was added. Col-0 and patl1 mutant seeds were germinated and grown vertically on MS medium with or without 100 mM NaCl. When grown on NaCl-containing medium, 0, 0.5 or 0.75 mM DMTU was added. Seedlings were photographed after 10 d of growth. (C) Analysis of primary root length of seedlings in (B). Error bars represent the SD (n >10). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05; **P ≤ 0.01). Fig. 6 View largeDownload slide Antioxidant partially rescues the salt-sensitive phenotype of patl1. (A) Determination of the GSH:GSSG ratio. Five-day-old seedlings were left untreated or treated with 100 mM NaCl for 12 h and analyzed for reduced or oxidized glutathione levels using an A061-1 Kit (Nanjing Jiancheng). Error bars represent the SD (n = 3). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05; **P ≤ 0.01). (B) Salt sensitivity assays of patl1 and Col-0 when DMTU was added. Col-0 and patl1 mutant seeds were germinated and grown vertically on MS medium with or without 100 mM NaCl. When grown on NaCl-containing medium, 0, 0.5 or 0.75 mM DMTU was added. Seedlings were photographed after 10 d of growth. (C) Analysis of primary root length of seedlings in (B). Error bars represent the SD (n >10). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05; **P ≤ 0.01). Discussion Membrane trafficking is essential for plant growth, development and stress response (Geldner et al. 2001, Jurgens 2004). Studies have found that many components of membrane trafficking are involved in plant salt stress response (Mazel et al. 2004, Leshem et al. 2006, Leshem et al. 2007). In this study, we reported that the membrane trafficking-related protein PATL1 participates in regulation of salt tolerance in Arabidopsis. PATL1 negatively regulates membrane trafficking and PM Na+/H+ antiport activity during salt stress. PATL1 interacts with SOS1, indicating that its regulatory role in salt tolerance might be partially through SOS1. In addition, PATL1 modulates cellular redox homeostasis by regulating ROS generation and scavenging under salt conditions. PATL1 functions as a negative regulator of plant salt tolerance (Fig. 7, a working model). Fig. 7 View largeDownload slide A working model of PATL1 in regulation of salt tolerance. Plant PATL1 impairs membrane trafficking, which is important for regulation of cellular redox homeostasis and ion homeostasis via affecting ROS generation/detoxification and PM Na+/H+ antiporter localization. In this way, excessive ROS and Na+ were accumulated in plant cells during salt stress through disrupting ROS-generating/detoxifying signaling and impairing PM Na+/H+ antiport, respectively. PATL1 is induced during salt stress. It might be a negative feedback mechanism, which helps plants to optimize their growth and stress response to challenging environments. On the other hand, PATL1 might turn down normal vegetative growth for the salt stress response, contributing to maintenance of the balance between growth and stress response for plant survival. However, this was achieved at the cost of disruption of ion homeostasis, since PATL1 interacts with SOS1 and functions as a negative regulator of SOS1. Fig. 7 View largeDownload slide A working model of PATL1 in regulation of salt tolerance. Plant PATL1 impairs membrane trafficking, which is important for regulation of cellular redox homeostasis and ion homeostasis via affecting ROS generation/detoxification and PM Na+/H+ antiporter localization. In this way, excessive ROS and Na+ were accumulated in plant cells during salt stress through disrupting ROS-generating/detoxifying signaling and impairing PM Na+/H+ antiport, respectively. PATL1 is induced during salt stress. It might be a negative feedback mechanism, which helps plants to optimize their growth and stress response to challenging environments. On the other hand, PATL1 might turn down normal vegetative growth for the salt stress response, contributing to maintenance of the balance between growth and stress response for plant survival. However, this was achieved at the cost of disruption of ion homeostasis, since PATL1 interacts with SOS1 and functions as a negative regulator of SOS1. The transcription of PATL1 in the patl1 mutant background is significantly elevated compared with that in Col-0 (Fig. 1B), possibly due to the T-DNA insertion which results in a 35S promoter in the promoter region of PATL1. Furthermore, 5'-RACE data indicate that the transcription initiation site of PATL1 was different in Col-0 and patl1 (Supplementary Fig. S2C, D). Since there was only one translation start site (ATG) identical to that in Col-0 which could result in full-length PATL1 protein in patl1 and as overexpression of PATL1 leads to a salt-sensitive phenotype similar to that of the patl1 mutant (Fig. 1C–F;Supplementary Fig. S3), T-DNA insertion in the patl1 mutant might only alter the transcription level of PATL1, without any change in its protein structure. Therefore, patl1 might not be a gain-of-function mutant. Our data also showed that PATL1 transcription increased during salt stress (Supplementary Fig. S4). It might be a negative feedback mechanism, which helps plants optimize their growth and stress response in challenging environments. We propose that multiple pathways (including the SOS pathway) were activated for ion and redox homeostasis regulation upon salt stress. After the initial response, salt-induced PATL1 might function in attenuating the SOS pathway to avoid over-response during the stress condition for sustained plant growth. Indeed, previous studies have reported similar negative feedback regulation mechanisms which are essential for plant stress adaption (Zhao et al. 2017). On the other hand, since PATL1 could associate with the cell plate, there also exists the possibility that salt-induced PATL1 represses plant growth, contributing to maintain the balance between growth and stress resistance. However, this is achieved at the cost of disruption of ion homeostasis, since PATL1 interacts with SOS1 and functions as a negative regulator of SOS1 (Figs. 4, 5). These findings together suggest the complexity of plant development and stress response. Soil salinity causes osmotic and ionic stresses to plants, and plants should decrease their cytoplasmic Na+ concentration to a non-toxic level via Na+ extrusion and/or compartmentation (Tester and Davenport 2003, Munns and Tester 2008, Zhu 2016). A PM Na+/H+ antiporter (such as SOS1) functions in Na+ extrusion by transporting Na+ out of plant cells (Shi et al. 2000, Yokoi et al. 2002, Zhu 2016). We found that there exists a direct interaction between PATL1 and SOS1 (Fig. 4), and PATL1 is involved in affecting regulation of SOS1 PM Na+/H+ antiport activity (Fig. 5). SOS1 is a membrane protein with 12 transmembrane domains and a long autoinhibitory cyotsolic C-terminus, and the proper localization of SOS1 at the PM is critical for its biological function. Membrane trafficking plays important regulatory roles in PM protein localization and cycling during plant development and stress response (Jurgens 2004). We hypothesize that PATL1-mediated membrane trafficking regulates SOS1 localization and cycling during salt stress. PATL1 might alter the cellular distribution of SOS1, resulting in dysfunction of SOS1 in response to salt stress (Fig. 5). Since PATL1 abundance in the patl1 mutant is elevated and patl1 displays a salt-sensitive phenotype (Fig. 1; Supplementary Figs. S1, S3), PATL1 might repress endocytosis of SOS1 from the PM, resulting in limited SOS1 turnover. In addition, salt stress could significantly enhance membrane trafficking such as endocytosis with SOS1 translocating in trafficking vesicle membranes. In this way, inside-out SOS1 might transport cytoplasmic Na+ into those vesicles and help maintain cellular ion homeostasis during salt stress. However, this Na+ compartmentation is repressed in patl1 due to limited endocytosis of SOS1 from the PM. On the other hand, since PATL1 interacts with the SOS1 C-terminus in vivo, we cannot exclude the possibility that PATL1 regulates SOS1 activity by directly affecting the configuration of SOS1 during salt stress. Dissecting the structure of the PATL1–SOS1 complex or analyzing the regulatory role of PATL1 in SOS1 activity in reconstituted liposomes in vitro will contribute to validatation of this hypothesis. Accumulated ROS during salt stress imposes oxidative stress on plants (Blokhina et al. 2003, Gill and Tuteja 2010). Studies have found that ROS might be generated in endocytic vesicles which will finally fuse with the central vacuole under salt stress conditions (Bassham et al. 2006, Leshem et al. 2006). Defects in vesicle generation or fusion during membrane trafficking disrupt cellular redox homeostasis in response to salt stress, leading to cell death. In fact, salt stress also stimulates endocytosis of NADPH oxidase (such as AtrbohD) in Arabidopsis (Hao et al. 2014). We hypothesize that PATL1 might be involved in regulation of redox homeostasis through modulating membrane trafficking in a similar way. Future work needs to be performed to elucidate the mechanism by which PATL1 modulates ROS production and scavenging under salt conditions. In addition, previous studies have demonstrated that ROS also function in PM Na+/H+ antiport activity and regulation of K+/Na+ homeostasis (Katiyar-Agarwal et al. 2006, Zhang et al. 2006, Chung et al. 2008, Ma et al. 2012). In this way, we hypothesize that PATL1 might also regulate K+/Na+ homeostasis by affecting PM Na+/H+ antiport activity through ROS directly for salt tolerance modulation. Materials and Methods Mutants and transgenic plants Arabidopsis thaliana Col-0 was used as the wild type in all experiments. patl1 was identified from a SALK line (103668) with a T-DNA insertion in the 5'-UTR of At1g72150. Transgenic plants overexpressing GFP-tagged PATL1 in Col-0 (lines #80 and #86) were used in this study. Plant growth and salt tolerance analysis Seeds were sterilized in a solution containing 20% sodium hypochlorite (NaClO) and 0.1% Triton X-100 for 15 min, washed five imes with sterilized water, and sown on MS medium with 0.3% (for horizontal growth) or 0.5% (for vertical growth) phytagel agar (Sigma-Aldrich). For salt sensitivity assay, Col-0, the patl1 mutant and the PATL1-overexpressing plants were germinated and grown vertically on MS medium without or with 75 or 125 mM NaCl. Seedlings were photographed after 2 weeks of growth. The fresh weight and primary root length were measured and statistically analyzed. For the germination assay, Col-0 and patl1 seeds were collected at the same time, stratified and grown as described above. The germination rate and cotyledon greening rate were scored at the indicated time point. The salt sensitivity analysis in soil was performed as described (Zhou et al. 2014). Confocal microscopy assay The coding sequence of PATL1 was amplified with the P1-Bf/P1-Sr primers and cloned into the pCAMBIA1205-GFP vector between the BamHI and SalI sites. The primer sequences are given in Supplementary Table S1. PATL1–GFP and a marker of specific endosomal compartments were co-expressed in Arabidopsis leaf protoplasts as described (Tian et al. 2015). The fluorescent signals of GFP or RFP were detected using a Leica SP5 confocal microscope according to the user manual. Co-immunoprecipitation assays in Arabidopsis leaf protoplasts The coding sequence of PATL1 was amplified with the P1-Bf/P1-Sr primers and cloned into the pCAMBIA1307-6×Myc vector between the BamHI and SalI sites. The coding sequences of PATL1N1, PATL1N2, PATL1Sec14 and PATL1GOLD were amplified with the P1n1-Bf/P1n1-Sr, P1n2-Bf/P1n2-Sr, P1s1-Bf/P1s1-Sr and P1g1-Bf/P1g1-Sr, respectively, and cloned into the pCAMBIA1307-6×Myc vector between the BamHI and SalI sites. The coding sequence of the SOS1 C-terminus (amino acids 557–1,146) was amplified with the SOS1C-Sf and SOS1C-Kr primers, and cloned into the pCAMBIA1307-Flag-HA vector between the SalI and KpnI sites. The primer sequences are given in Supplementary Table S1. The resulting plasmids were used for the Co-IP assay as described (Zhou et al. 2014). Bimolecular fluorescence complementation assay The coding sequence of PATL1 was amplified with the P1-Bf/P1-Sr primers while full-length SOS1, the SOS1 N-terminus and the C-terminus were amplified with the SOS1-Sf/SOS1-K(NS)r, SOS1-Sf/SOS1N-K(NS)r and SOS1C-Sf/SOS1-K(NS)r primers, respectively. The primer sequences are given in Supplementary Table S1. The resulting fragments were cloned into pSPYNE(R)173 and pSPYCE(M) at the BamHI/SalI and SalI/KpnI restriction sites, respectively (Walter et al. 2004, Waadt et al. 2008). Primer sequences are listed in Supplementary Table S1. Constructs containing PATL1-YFPN and YFPC-SOS1, YFPC-SOS1N or YFPC-SOS1C were introduced into Agrobacterium tumefaciens GV3101 through electroporation and infiltrated into N. benthamiana leaves. The YFP fluorescence signal was detected via a Leica SP5 confocal microscope 3 d after infiltration. Yeast two-hybrid assay The coding sequences of PATL1N1, PATL1N2, PATL1Sec14 and PATL1GOLD were amplified with P1n1-Ef/P1n1-Br, P1n2-Ef/P1n2-Br, P1s1-Ef/P1s1-Br and P1g1-Ef/P1g1-Br, respectively, and cloned into the pGBKT7 vector between the EcoRI and BamHI sites. The coding sequence of the SOS1 C-terminal amino acids (557–1,146) was amplified with the SOS1C-Ef and SOS1C-Br primers and cloned into the pGADT7 vector between the EcoRI and BamHI sites. The primer sequences are listed in Supplementary Table S1. Then, constructs pGBKT7-PATL1N1, pGBKT7-PATL1N2, pGBKT7-PATL1Sec14 and pGBKT7-PATL1GOLD as well as the pGBKT7 vector were co-transformed with pGADT7-SOS1C into yeast strain AH109. Yeast transformation and growth assays were performed as described in the Yeast Protocols Handbook (Clontech). 5'-RACE assay Ten-day-old seedlings of Col-0 and patl1 were harvested for total mRNA isolation. The resulting mRNA was subjected to 5'-RACE assay using the SMART™ RACE cDNA amplification kit (Clontech, Cat. No. 634914) according to the user manual. The primer sequences are given in Supplementary Table S3. PM isolation and Na+/H+ antiport activity assay and determination of ion content Three-week-old seedlings of Col-0 and patl1 were used for PM isolation by aqueous two-phase partitioning as described previously (Qiu et al. 2002). PM Na+/H+ antiport activity was determined as an Na+-induced dissipation of the pH gradient as described (Qiu et al. 2002). Col-0 and patl1 seedlings were grown in soil under long-day conditions for 3 weeks at 23°C, and the seedlings were treated with 150 mM NaCl for 10 d. Rosette leaves were harvested and dried at 80°C for 2 d. All the samples were accurately weighed and treated at 300°C for 1 h and at 575°C for 4 h in a muffle furnace. The resulting ground dry matter was dissolved in 0.1 M HCl. The Na+ and K+ contents of the samples were determined with an atomic absorption spectrophotometer (Hitachi Z-5000). BFA treatment and FM4-64 staining BFA (Invitrogen) treatment and FM4-64 (Invitrogen) staining were conducted as described (Tian et al. 2015) with modifications. Briefly, tobacco leaves transiently expressing PATL1–GFP were cut into small fragments and incubated in a medium containing 25 μM FM4-64 for 40 min, rinsed with water several times and observed under a confocal microscope (Leica SP5). For BFA treatment, roots of 5-day-old seedlings were briefly washed and incubated in 50 μM BFA (Sigma-Aldrich) and 1 μM FM4-64 for 25 min, then the BFA bodies of single cells were quantified via confocal microscopy. ROS accumulation and assays of ROS-scavenging enzyme activity ROS accumulation was measured using various approaches. For DAB staining assay, 10-day-old seedlings were left untreated or treated with 200 mM NaCl for 4 h and then incubated in staining buffer (0.1 mg ml–1 DAB dissolved in 0.1 M HCl, pH 5.0). Seedlings were destained in destaining buffer (acetic acid:glycerin:ethanol = 1:1:3) after staining for 8 h. For the Amplex Red H2O2/peroxidase assay, 10-day-old seedlings were left untreated or treated with 200 mM NaCl for 4 h and harvested. Detection of H2O2 accumulation was performed following the manufacturer’s instructions. NBT staining was used to determine the production of superoxide radicals as described (Wohlgemuth et al. 2002). To determine the activity of enzymes for ROS scavenging, 10-day-old seedlings of Col-0 and patl1 were left untreated or treated with 200 mM NaCl (with or without 0.5 mM DMTU) for 4 h. Then 0.5 g aliquots of fresh tissues were homogenized with 5 ml of ice-cold 25 mM HEPES buffer (pH 7.8) containing 0.2 mM EDTA, 2 mM ascorbate and 2% polyvinylpyrrolidone (PVP). The resulting homogenates were centrifuged at 12,000×g for 20 min at 4°C and the supernatants were used for determination of the enzymatic activities as described (Patra and Mishra 1979, Stewart and Bewley 1980, Nakano and Asada 1981, Hammerschmidt et al. 1982). Promoter activity analysis For GUS staining, a 2,000 bp genomic DNA fragment from 1 to 2,000 bp upstream of the translational start site (ATG) of PATL1 was amplified with the P1pro-Sf and P1pro-Br primers and then cloned into the SalI and BamHI sites of the pCM1391 vector. The plasmid was transformed into Col-0, and transgenic T2 lines were used for GFP observation or GUS staining as described (Zhou et al. 2014). The primer sequences are given in Supplementary Table S1. Quantitative real-time and RT–PCR assays Seedlings of Col-0, patl1 as well as PATL1-overexpressing lines were prepared as described. Total RNA was extracted with Trizol reagent (Invitrogen) and treated with RNase-free DNase I (TaKaRa) to remove genomic DNA. A 10 mg aliquot of RNA was used for reverse transcription with M-MLV reverse transcriptase (Promega) according to the manufacturer’s instructions. The resulting cDNAs were used for quantitative real-time PCR amplification or RT–PCR assay with the primers listed in Supplementary Table S2. ACTIN2 (ACT2) was used as an internal control. Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by National Natural Science Foundation of China (NSFC) [grant No. 31600201 to H.Z.] and the Start-Up Foundation of Sichuan University [to H.Z.]. Acknowledgments We thank the Arabidopsis Biological Resource Center (ABRC) for providing the T-DNA insertion lines. 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Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations APX ascorbate peroxidase BFA brefeldin A BiFC bimolecular fluorescence complementation CAT catalase Co-IP co-immunoprecipitation DAB 3,3-diaminobenzidine DMTU dimethylthiourea 5'-RACE 5'-rapid amplification of cDNA ends 5'-UTR 5'-untranslated region GFP green fluorescent protein GSH glutathione GSSG oxidized glutathione GUS β-glucuronidase MS Murashige and Skoog NBT nitroblue tetrazolium PATL1 Patellin 1 PM plasma membrane POD peroxidase PVC pre-vacuolar compartment RFP red fluorescent protein ROS reactive oxygen species RT–PCR reverse transcription–PCR SOD superoxide dismutase SOS Salt-Overly-Sensitive YFP yellow fluorescent protein Y2H yeast two-hybrid © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. 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/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

Patellin1 Negatively Modulates Salt Tolerance by Regulating PM Na+/H+ Antiport Activity and Cellular Redox Homeostasis in Arabidopsis

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com
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0032-0781
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1471-9053
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10.1093/pcp/pcy081
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Abstract

Abstract Soil salinity significantly represses plant development and growth. Mechanisms involved sodium (Na+) extrusion and compartmentation, intracellular membrane trafficking as well as redox homeostasis regulation play important roles in plant salt tolerance. In this study, we report that Patellin1 (PATL1), a membrane trafficking-related protein, modulates salt tolerance in Arabidopsis. The T-DNA insertion mutant of PATL1 (patl1) with an elevated PATL1 transcription level displays a salt-sensitive phenotype. PATL1 partially associates with the plasma membrane (PM) and endosomal system, and might participate in regulating membrane trafficking. Interestingly, PATL1 interacts with SOS1, a PM Na+/H+ antiporter in the Salt-Overly-Sensitive (SOS) pathway, and the PM Na+/H+ antiport activity is lower in patl1 than in Col-0. Furthermore, the reactive oxygen species (ROS) content is higher in patl1 and the redox signaling of antioxidants is partially disrupted in patl1 under salt stress conditions. Artificial elimination of ROS could partially rescue the salt-sensitive phenotype of patl1. Taken together, our results indicate that PATL1 participates in plant salt tolerance by regulating Na+ transport at least in part via SOS1, and by modulating cellular redox homeostasis during salt stress. Introduction Soil salinity is a significant stress for crop growth and development worldwide (Tuteja 2007, Munns and Tester 2008). Accumulated sodium ions in soil cause both osmotic and ionic stresses to plants (Hasegawa et al. 2000, Munns and Tester 2008, Zhu 2016). As sessile organisms, plants have evolved efficient strategies to cope with salt stress. Plant cells perceive the salt stress signal which in turn induces release of cellular second messengers such as Ca2+, inositol phosphates as well as reactive oxygen species (ROS) (Hasegawa et al. 2000, Xiong et al. 2002, Mahajan and Tuteja 2005). The Salt-Overly-Sensitive (SOS) pathway is induced by such signals and participates in plant salt tolerance by modulating ion homeostasis. SOS1, a plasma membrane (PM)-localized Na+/H+ antiporter, transports Na+ out of the cell through its Na+/H+ antiport activity (Shi et al. 2000, Qiu et al. 2002, Shi et al. 2002, Quan et al. 2007, Lin et al. 2009, Quintero et al. 2011, Zhu 2016). Several regulators of the SOS pathway including ABI2, GIGANTEA (GI) and 14-3-3 proteins have been identified recently, which together fine-tune SOS activity for proper plant salt adaption (Ohta et al. 2003, Kim et al. 2013, Zhou et al. 2014, Tan et al. 2016). Furthermore, Na+ compartmentation in the vacuole is also important for regulation of salt tolerance. Vacuolar membrane-localized Na+/H+ antiporters transport Na+ into the vacuole driven by the H+ gradient established by vacuolar H+-ATPase and/or the H+-pyrophosphatase, and repress the toxic accumulation of Na+ in the cytoplasm (Apse et al. 1999, Gaxiola et al. 2001, Yokoi et al. 2002, An et al. 2007, Barragán et al. 2012). Salt stress also disrupts cellular redox homeostasis and causes oxidative stress to plants, leading to cell death and growth inhibition (Mittler 2002, Miller et al. 2010). Soil salinity induces excessive generation of ROS including superoxide radicals, hydrogen peroxide and hydroxyl radicals by destroying the electron transport chain in chloroplast and/or mitochondria as well as through membrane-bound NADPH oxidases (Miller et al. 2010, Dietz et al. 2016, Gilroy et al. 2016, Huang et al. 2016, Takagi et al. 2016). ROS are highly reactive and capable of causing lipid peroxidation, protein denaturation and DNA damage (Gill and Tuteja 2010, Farmer and Mueller 2013). In fact, lipid peroxidation will generate lipid peroxide, another destructive species, in plant cells (Farmer and Mueller 2013, Reiter et al. 2014). Mitogen-activated protein kinase (MAPK) cascades have been identified to be involved in regulating ROS generation under abiotic stress (Zhang and Klessig 2001, Xiong et al. 2002). Besides being toxic molecules; however, ROS have also been identified as signaling molecules in plant development and stress response (Gechev et al. 2006, Van Breusegem and Dat 2006, Zhou et al. 2016). Rapid elevation of ROS in plant cells under salt stress might serve as signals for stress acclimation (Gechev et al. 2006, Miller et al. 2010). A recent study revealed that specific ROS accumulation mediated by the NADPH oxidase AtrbohF in the vasculature contributes to Na+ translocation regulation and salt tolerance in Arabidopsis (Jiang et al. 2012). A study has also reported that ROS produced by both AtrbohD and AtrbohF function as signals to regulate K+/Na+ homeostasis and enhanced salt tolerance (Ma et al. 2012). Plants have also evolved effective strategies to remove cellular toxic ROS. Previous studies revealed that enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX) function in enzymatic ROS scavenging in plants (Mittler 2002, Blokhina et al. 2003, Apel and Hirt 2004). In addition, reduced glutathione (GSH) and ascorbate contribute to non-enzymatic ROS scavenging by directly interacting with and detoxifying oxygen free radicals (Apel and Hirt 2004). The balance between ROS generation and scavenging during salt stress is critical for cellular redox homeostasis and plant salt tolerance (Moon et al. 2003, Chung et al. 2008, Miller et al. 2010). Intracellular membrane trafficking participates in modulating salt tolerance. During salt stress, cycling of PM proteins between the PM and endosomes is enhanced, indicating that membrane trafficking and redistribution of PM proteins represent the initial responses of plant cells under abiotic conditions such as soil salinity (Jurgens 2004, Murphy et al. 2005, Luu et al. 2012, Garcia de la Garma et al. 2015, Munns and Gilliham 2015). In fact, ROS production and membrane trafficking are reported to be co-ordinated during plant salt tolerance regulation. PM endocytosis is involved in intracellular ROS production, while defects in endocytosis disrupt ROS generation in endosomes during salt stress (Leshem et al. 2006, Leshem et al. 2007, Liu et al. 2012). Repression of VAMP711, an Arabidopsis v-SNARE, leads to repression of fusion of H2O2-containing vesicles with the vacuole, leading to enhanced vacuolar H+-ATPase activity and increased salt tolerance (Leshem et al. 2006). The plant-unique Rab GTPase ARA6 regulates SNARE complex formation between VAMP727 and SYP121. The ARA6 loss-of-function mutant ara6 conferred salt hypersensitivity (Ebine et al. 2011). In addition, overexpression of OsRab7 enhanced accumulation of trafficking vesicles in root tips during salt stress, and transgenic rice exhibited enhanced salt tolerance (Peng et al. 2014). The Arabidopsis Patellin (PATL) protein family consists of six members, i.e. PATL1, 2, 3, 4, 5 and 6, and might play a role in membrane trafficking (Peterman et al. 2004). PATL1 contains a variable N-terminal domain followed by a Sec14 domain and a Golgi dynamics domain (GOLD), both of which are conserved in other membrane trafficking-related proteins (Peterman et al. 2004, Peterman et al. 2006, Mousley et al. 2007). PATL1 specifically binds to phosphoinositides which function in regulating membrane trafficking (Peterman et al. 2004, Mousley et al. 2007, Thole and Nielsen 2008). Interestingly, PATL1 might participate in vesicle trafficking mediated by the deubiquitinating enzyme AMSH3, possibly via binding and recruiting AMSH3 to the PM in Arabidopsis (Isono et al. 2010). PATL1 can be modified with ubiquitin, suggesting that ubiquitination might play a role in PATL1 function (Igawa et al. 2009, Saracco et al. 2009). PATL2 is a possible substrate of MPK4, and phosphorylation of PATL2 by MPK4 alters its binding affinity for phosphoinositides (Suzuki et al. 2016). Interestingly, salt stress promotes phosphorylation of PATL2, indicating a link between PATL2 and regulation of salt tolerance (Hsu et al. 2009). PATL3 and PATL6 are involved in regulation of plant immunity. They restrict the intracellular transport of Alfalfa mosaic virus viral movement protein-containing vesicles and enhance plant immunity (Peiro et al. 2014). Therefore, PATLs function in regulating plant abiotic as well as biotic stress tolerance; however, the underlying mechanisms still needs to be understood in detail. To fine-tune plant growth and its stress response in a challenging environment, efficient negative feedback regulation mechanisms are needed to avoid over-response to environmental stresses. For example, ABA promotes S-nitrosylation and subsequent inactivation of SnRK2.6, a positive regulator of ABA signaling, leading to attenuated ABA signaling (Wang et al. 2015). Actually, several other studies have also revealed this negative feedback regulation in plants during stress response (Zhao et al. 2017). We proposed that there might also exist such a mechanism for plant salt tolerance, through which plant growth and stress response can be well balanced upon soil salinity. Here we identify Arabidopsis PATL1 as a negative regulator of salt tolerance by regulating Na+ transport at least in part via SOS1, and by modulating cellular redox homeostasis. Membrane trafficking is partially defective in the PATL1 T-DNA insertion mutant patl1, which displays a salt-sensitive phenotype compared with wild-type plants. Seedlings overexpressing PATL1 exhibit higher salt sensitivity. PATL1 interacts with SOS1 directly in planta and negatively regulates its PM Na+/H+ antiport activity. Cellular redox homeostasis is disrupted in patl1 under saline condition, and artificial elimination of ROS could partially rescue the salt-sensitive phenotype of patl1. PATL1 transcription is salt induced, which might be a negative feedback mechanism in regulation of plant salt tolerance. These results provide new evidences supporting that membrane trafficking is critical for cellular ion and ROS homeostasis during salt stress and confers plant salt tolerance. Results The Arabidopsis patl1 mutant is hypersensitive to salt stress To investigate the function of membrane trafficking in regulation of plant salt tolerance, a pool of Arabidopsis T-DNA insertion mutants related to membrane trafficking obtained from the Arabidopsis Biological Resource Center (ABRC) were used for screening. In this way, a collection of about 120 mutant lines and their relative wild-type plant [Columbia-0 (Col-0)] was sown in Murashige and Skoog (MS) medium containing 125 mM NaCl for salt sensitivity analysis. We identified that the SALK_103668 plant displayed a salt-hypersensitive phenotype (Fig. 1A, D). The T-DNA was inserted in the 5'-untranslated region (5'-UTR) of the At1g72150 gene, which encodes PATL1 (Fig. 1A), a possible membrane trafficking-related protein that might participate in cell plate formation during cytokinesis (Peterman et al. 2004). We then named SALK_103668 as patl1 (Fig. 1A). The full-length transcript of PATL1 was enriched at least 10-fold in the patl1 mutant (Fig. 1B;Supplementary Fig. S1A). We have analyzed the genome structure of the PATL1 promoter region in patl1. The data showed that there exists a Cauliflower mosaic virus (CaMV) 35S promoter upstream of the translation start site (ATG) due to T-DNA insertion (Supplementary Fig. S2A, B), which might explain the elevated PATL1 transcription level in the patl1 mutant compared with that in Col-0. Furthermore, 5'-rapid amplification of cDNA ends (5'-RACE) assay has also been performed using seedlings of Col-0 and patl1, and the result revealed that the transcription initiation site of PATL1 in Col-0 was at –298 bp relative to the translation start site (ATG), while in the patl1 background the transcription initiation site was –922 bp (Supplementary Fig. S2C, D). Fig. 1 View largeDownload slide The patl1 mutant is hypersensitive to salt stress. (A) Schematic representation of the structure of PATL1. Filled gray boxes, UTRs; empty boxes, exons; lines between boxes, introns; triangle, T-DNA insertion site. (B, C) Determination of PATL1 transcription. RT–PCR was used to monitor the expression of PATL1 in Col-0 and the patl1 mutant as well as PATL1-overexpressing plants. ACTIN2 (ACT2) was used as a loading control. Similar results were obtained in three independent replicated experiments. (D) Analysis of salt sensitivity in Col-0, patl1 and two PATL1 transgenic plants. Seeds were germinated and grown on MS medium with or without 75 or 125 mM NaCl. Seedlings were photographed after 2 weeks of growth. (E, F) Analysis of the germination rate, cotyledon greening rate and fresh weight of seedlings in (D). Error bars represent the SD (n >10). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). Fig. 1 View largeDownload slide The patl1 mutant is hypersensitive to salt stress. (A) Schematic representation of the structure of PATL1. Filled gray boxes, UTRs; empty boxes, exons; lines between boxes, introns; triangle, T-DNA insertion site. (B, C) Determination of PATL1 transcription. RT–PCR was used to monitor the expression of PATL1 in Col-0 and the patl1 mutant as well as PATL1-overexpressing plants. ACTIN2 (ACT2) was used as a loading control. Similar results were obtained in three independent replicated experiments. (D) Analysis of salt sensitivity in Col-0, patl1 and two PATL1 transgenic plants. Seeds were germinated and grown on MS medium with or without 75 or 125 mM NaCl. Seedlings were photographed after 2 weeks of growth. (E, F) Analysis of the germination rate, cotyledon greening rate and fresh weight of seedlings in (D). Error bars represent the SD (n >10). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). When Col-0 and patl1 seeds were germinated on MS medium with 75 or 125 mM NaCl, respectively, significant differences between the two genotypes were observed. At 5 d post-germination on MS with 75 mM NaCl, 78% seeds of Col-0 germinated while for patl1 seedlings, only 50% were able to germinate (Fig. 1D, E). The inhibitory effect on germination was more pronounced for patl1 than for Col-0 when the concentration of NaCl in MS was elevated to 125 mM. Under this condition, 61% seeds of Col-0 germinated, while only 38% seeds of patl1 seeds were able to germinate (Fig. 1D, E). Furthermore, the subsequent cotyledon greening was also significantly repressed in patl1 compared with Col-0 (Fig. 1D, F). In addition, when 5-day-old patl1 mutant and Col-0 seedlings were transferred to MS medium containing 125 mM NaCl, shoot tissue and the primary root of patl1 showed a significant reduction in growth compared with Col-0 (Supplementary Fig. S3A). The primary root length of patl1 decreased at least 30% compared with that of Col-0 (Supplementary Fig. S3B). We have also determined the plant salt sensitivity in soil. In this way, 2-week-old seedlings of Col-0 and patl1 grown under long-day conditions were irrigated with water containing 200 mM NaCl. Salt stress significantly inhibited plant growth; however, the inhibitory effect on patl1 was more severe than that on Col-0 (Supplementary Fig. S3C). Consistently, the Chl content in patl1 was much lower than that in Col-0 during soil salinity (Supplementary Fig. S3D). Since PATL1 transcription was significantly elevated in patl1 and as we lacked a second allele of this mutant, the Pro35S::GFP-PATL1 construct was transformed into the Col-0 background to generate PATL1 transgenic plants (Pro35S::GFP-PATL1/Col-0) (Fig. 1C;Supplementary Fig. S1B). Independent homozygous T2 lines (#80 and #86) were used for the salt sensitivity assay as described. We found that Pro35S::GFP-PATL1/Col-0 seedlings displayed a salt-sensitive phenotype similar to that of the patl1 mutant (Fig. 1D–F;Supplementary Fig. S3). Furthermore, to determine the possible effect of salt stress on PATL1, about 2,000 bp of the PATL1 promoter was fused to the β-glucuronidase (GUS) reporter gene. The resulting construct was transformed into Col-0. Independent T2 transgenic lines were used for GUS staining under control or salt stress conditions. Data showed that GUS expression driven by the PATL1 promoter was induced by salt stress (Supplementary Fig. S4A). Consistently, a reverse transcription–PCR (RT–PCR) assay revealed that PATL1 was induced in response to salt stress, suggesting a negative feedback regulation mechanism in plants (Supplementary Fig. S4B). Taken together, these results indicate that the salt-sensitive phenotype of the patl1 mutant is due to elevated PATL1 transcription and that PATL1 could be a negative regulator of salt tolerance in Arabidopsis. PATL1 associates with the PM and endosomes in plants As already described, PATL1 contains a variable N-terminal domain followed by a Sec14 domain and a GOLD domain, which are also found in other membrane trafficking-related proteins (Peterman et al. 2004, Mousley et al. 2007), suggesting that PATL1 might function as a putative membrane trafficking-related protein and have a regulatory role in membrane trafficking. To determine the subcellular localization of PATL1, PATL1–green fluorescent protein (GFP) fusion protein was transiently expressed in Nicotiana benthamiana leaf cells via agroinfiltration. Three dafter the inoculation, infiltrated tobacco leaves were incubated with FM4-64, a lipophilic styryl dye staining the PM and endosomes (Vida and Emr 1995), and then observed using confocal microscopy. The results revealed that PATL1–GFP mainly localized at the PM (Supplementary Fig. S5A). Interestingly, co-localization between PATL1–GFP and vesicles stained by FM4-64, the putative endosomes, was also observed (Supplementary Fig. S5A). When 3-week-old soil-grown Pro35S::GFP-PATL1/Col-0 (line #80) seedlings were used for protoplast isolation and confocal microscopy, we also found that PATL1–GFP mainly localized at the PM and endosomes, indicated by FM4-64 staining (Supplementary Fig. S5B). Furthermore, data revealed that PATL1–GFP also co-localized with the red fluorescent protein (RFP)-tagged VSR2, a marker of pre-vacuolar compartments (PVCs) (Fig. 2). In summary, these data together suggest that PATL1 partially associates with the PM and endosomal system, and it might function in regulating membrane trafficking in plants. Fig. 2 View largeDownload slide PATL1 associates with the PM and endosomes in plants. (A–C) Co-localization of PATL1 and VSR2, a marker of pre-vacuolar compartments (PVCs) in Arabidopsis leaf protoplasts. The fluorescence signal was detected using a Leica SP5 confocal fluorescence microscope. (A) GFP; (B) RFP; and (C) merged image. Scale bar = 5 µm. Fig. 2 View largeDownload slide PATL1 associates with the PM and endosomes in plants. (A–C) Co-localization of PATL1 and VSR2, a marker of pre-vacuolar compartments (PVCs) in Arabidopsis leaf protoplasts. The fluorescence signal was detected using a Leica SP5 confocal fluorescence microscope. (A) GFP; (B) RFP; and (C) merged image. Scale bar = 5 µm. PATL1 is involved in regulation of membrane trafficking It has been reported that PATL1 might function as a membrane trafficking-related protein (Peterman et al. 2004; Fig. 2; Supplementary Fig. S5). To investigate whether PATL1 plays a regulatory role in membrane trafficking during salt stress, we first examined the effect of brefeldin A (BFA), an inhibitor of membrane trafficking, in root cells of the Col-0 and patl1 seedlings. The result showed that the number of BFA bodies was significantly lower in the patl1 mutant compared with Col-0, indicating that membrane trafficking was partially repressed in patl1 (Fig. 3A, B). Interestingly, we found that PATL1 might also be implicated in regulating the expression of genes related to membrane trafficking during salt stress. Candidate genes, Rab7, PI3K, SYP61 and ZATA-1, were selected since all these genes have been previously found to be involved in membrane trafficking and salt tolerance (Zhu et al. 2002, Mazel et al. 2004, Leshem et al. 2007, Oh et al. 2010, Tian et al. 2015). Ten-day-old seedlings of Col-0 and patl1 as well as two transgenic plants (OX-#80 and #86) were left untreated or treated with 100 mM NaCl for different periods of time, and expression of selected genes was determined quantitatively. The results revealed that transcription levels of Rab7, PI3K, SYP61 and ZATA-1 were significantly lower in patl1, OX-#80 and #86 compared with Col-0 during salt stress (Fig. 3C–F). However, the mechanism by which PATL1 participates in regulating expression of these genes still needs to be determined. Together, these results indicate that PATL1 might be involved in regulation of membrane trafficking during salt stress and play a regulatory role in plant salt tolerance. Fig. 3 View largeDownload slide PATL1 participates in regulation of membrane trafficking. (A) Confocal assay of the effect of BFA on protein trafficking in root cells from 5-day-old Col-0 and patl1 seedlings. Seedlings were briefly washed after incubation in liquid MS medium with 50 µM BFA and 5 mg ml–1 FM4-64 for 30 min. Scale bar = 10 µm. (B) Quantification of the BFA bodies in root cells for (A). Error bars represent the SD (n >6). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). (C–F) Relative expression of genes encoding membrane trafficking-related proteins in Col-0, patl1, OX-#80 and OX-#86 seedlings. Gene transcription was measured by quantitative real-time PCR in 10-day-old seedlings without or with 2 and 6 h treatment with 100 mM NaCl with ACT2 as the internal control. Data were normalized by the gene expression level in Col-0 under control conditions. Error bars represent the SD (n >6). Statistical significance was determined by a Student’s t test; significant differences are indicated by asterisks (*P ≤ 0.05; **P ≤ 0.01). Fig. 3 View largeDownload slide PATL1 participates in regulation of membrane trafficking. (A) Confocal assay of the effect of BFA on protein trafficking in root cells from 5-day-old Col-0 and patl1 seedlings. Seedlings were briefly washed after incubation in liquid MS medium with 50 µM BFA and 5 mg ml–1 FM4-64 for 30 min. Scale bar = 10 µm. (B) Quantification of the BFA bodies in root cells for (A). Error bars represent the SD (n >6). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). (C–F) Relative expression of genes encoding membrane trafficking-related proteins in Col-0, patl1, OX-#80 and OX-#86 seedlings. Gene transcription was measured by quantitative real-time PCR in 10-day-old seedlings without or with 2 and 6 h treatment with 100 mM NaCl with ACT2 as the internal control. Data were normalized by the gene expression level in Col-0 under control conditions. Error bars represent the SD (n >6). Statistical significance was determined by a Student’s t test; significant differences are indicated by asterisks (*P ≤ 0.05; **P ≤ 0.01). PATL1 interacts with SOS1 in planta To substantiate the underlying mechanism by which PATL1 modulates plant salt tolerance, we used Pro35S:Flag-HA-PATL1/Col-0 transgenic plants to identify possible PATL1-interacting proteins using mass spectrometry as described (Zhou et al. 2014). The data revealed that SOS1 might be a putative interacting protein of PATL1. In fact, SOS1 is a PM-localized Na+/H+ antiporter of the SOS pathway, which functions in ion homeostasis during salt stress in plants (Zhu 2016). We then verified the interaction between PATL1 and SOS1 using bimolecular fluorescence complementation (BiFC) analysis in N. benthamiana leaves. The result showed that the yellow fluorescence protein (YFP) fluorescence signal was only detected in leaves transiently expressing PATL1–YFPN and YFPC–SOS1 but not in leaves expressing PATL1–YFPN and YFPC, or YFPN and YFPC–SOS1, respectively (Fig. 4A;Supplementary Fig. S6A). Previous studies have demonstrated that SOS1 contains 12 transmembrane domains and a long autoinhibitory C-terminus in the cytosol (Shi et al. 2000, Quintero et al. 2011). Our BiFC assay revealed that it was the C-terminus but not the N-terminus of SOS1 which interacted with PATL1 in tobacco leaves (Supplementary Fig. S6B). Fig. 4 View largeDownload slide PATL1 interacts with SOS1. (A) BiFC analysis in N. benthamiana. PATL1–YFPN and YFPC–SOS1 were co-expressed in tobacco leaves and the yellow fluorescent protein (YFP) fluorescence signal was detected 3 d after infiltration via a Leica SP5 confocal microscope. Scale bar = 100 µm. (B) Schematic diagram of motifs of PATL1. Black line box, variable N-terminal domain; green line box, Sec14 domain; purple line box, Golgi dynamics domain (GOLD); filled gray box, coiled-coil region; vertical lines, PXXP motif. PATL1 was divided into four fragments according to its predicted structure: PATL1N1 and PATL1N2 for two parts of the variable N-terminal domain, PATL1Sec14 for the Sec14 domain and PATL1GOLD for the GOLD domain, respectively. (C) Co-IP assay of the interaction between PATL1 and SOS1. Myc-tagged full-length or truncated PATL1 was transiently co-expressed with the Flag-SOS1 C-terminus in Arabidopsis leaf protoplasts. Proteins were immunoprecipitated with anti-C-Myc antibody-conjugated agarose. Immunoblot assays with anti-C-Myc and anti-Flag antibodies were used to detect Myc-PATL1 and the PATL1-interacting SOS1 C-terminus, respectively. Experimental details are provided in the Materials and Methods. IP, immunoprecipitation; IB, immunoblot. (D) Y2H analysis of the interaction between PATL1 and SOS1. Yeast strains expressing the indicated constructs were grown on synthetic complete medium without tryptophan and leucine (SC-W/L; left panel) and on synthetic complete medium without histidine, tryptophan and leucine (SC-H/W/L; right panel). Photographs were taken after 4–5 d of growth on the indicated medium. Fig. 4 View largeDownload slide PATL1 interacts with SOS1. (A) BiFC analysis in N. benthamiana. PATL1–YFPN and YFPC–SOS1 were co-expressed in tobacco leaves and the yellow fluorescent protein (YFP) fluorescence signal was detected 3 d after infiltration via a Leica SP5 confocal microscope. Scale bar = 100 µm. (B) Schematic diagram of motifs of PATL1. Black line box, variable N-terminal domain; green line box, Sec14 domain; purple line box, Golgi dynamics domain (GOLD); filled gray box, coiled-coil region; vertical lines, PXXP motif. PATL1 was divided into four fragments according to its predicted structure: PATL1N1 and PATL1N2 for two parts of the variable N-terminal domain, PATL1Sec14 for the Sec14 domain and PATL1GOLD for the GOLD domain, respectively. (C) Co-IP assay of the interaction between PATL1 and SOS1. Myc-tagged full-length or truncated PATL1 was transiently co-expressed with the Flag-SOS1 C-terminus in Arabidopsis leaf protoplasts. Proteins were immunoprecipitated with anti-C-Myc antibody-conjugated agarose. Immunoblot assays with anti-C-Myc and anti-Flag antibodies were used to detect Myc-PATL1 and the PATL1-interacting SOS1 C-terminus, respectively. Experimental details are provided in the Materials and Methods. IP, immunoprecipitation; IB, immunoblot. (D) Y2H analysis of the interaction between PATL1 and SOS1. Yeast strains expressing the indicated constructs were grown on synthetic complete medium without tryptophan and leucine (SC-W/L; left panel) and on synthetic complete medium without histidine, tryptophan and leucine (SC-H/W/L; right panel). Photographs were taken after 4–5 d of growth on the indicated medium. To determine the SOS1-ineracting region(s) in PATL1, we further divided PATL1 into four fragments according to its predicted structure (Peterman et al. 2004; Fig. 4B). Full-length and truncated PATL1s were translationally fused with the Myc tag and transiently co-expressed with the Flag-tagged SOS1 C-terminus (Flag-SOS1C) in Arabidopsis leaf protoplasts for co-immunoprecipitation (Co-IP) assay. The results showed that Flag-SOS1C was co-immunoprecipitated with Myc-PATL1 in vivo (Fig. 4C). Furthermore, it was the N2 part in the variable N-terminal domain but not the Sec14 or GOLD domain in PATL1 that mediated the interaction between PATL1 and SOS1, indicating that the variable N-terminal domain is responsible for protein–protein interaction for PATL1, linking PATL1 to diverse biological functions (Fig. 4C). In addition, we have also investigated the interaction between PATL1 and SOS1 through yeast two-hybrid (Y2H) assay. Consistently, the PATL1 N2 fragment specifically interacted with the SOS1 C-terminus in yeast (Fig. 4D). Taken together, these results demonstrated that PATL1 interacts with SOS1, suggesting that PATL1 might regulate salt tolerance at least in part through SOS1. PATL1 negatively regulates SOS1 activity and ion homeostasis during salt stress Since PATL1 interacts with SOS1 directly in planta, we then determined whether PATL1 participates in regulating SOS1 biological function. It is well known that salt stress impairs ion homeostasis in plant cells and SOS1 contributes to regulation of K+/Na+ homeostasis during salt stress (Xiong et al. 2002, Zhu 2016). PM-localized Na+/H+ antiporters (including SOS1) mainly mediate Na+ efflux (to transport Na+ out of the cell) during salt stress (Aronson 1985, Shi et al. 2002). We first analyzed the PM Na+/H+ antiport activity by isolation of PM vesicles. Thus, 3-week-old soil-grown seedlings of Col-0 and patl1 were treated with 250 mM NaCl for 3 d for PM H+-ATPase activation as described (Yang et al. 2010, Zhou et al. 2012), and then PM vesicles were isolated to determine Na+/H+ antiport activity. The results showed that PM Na+/H+ antiport activity was significantly lower in patl1 than that in Col-0 at all concentrations of NaCl assayed (Fig. 5A). To examine whether PATL1 participates in regulating plant cellular K+/Na+ homeostasis during salt stress, 3-week-old soil-grown Col-0, patl1 and two independent PATL1 transgenic plants (OX-#80 and #86) were exposed to 150 mM NaCl for 10 d, and Na+ and K+ content was measured via atomic absorption spectroscopy (AAS). We found that the Na+ content increased in all the plants during salt stress, while patl1, OX-#80 and #86 accumulated a higher level of Na+ than Col-0 (Fig. 5B). In contrast, K+ content was similar in all the genotypes under both control and salt stress conditions (Fig. 5C). Therefore, the K+/Na+ ratio was much lower in patl1, OX-#80 and #86 plants compared with Col-0 upon salt treatment (Fig. 5D). These data demonstrated that PATL1 participates in regulation of PM Na+/H+ antiport activity and ion homeostasis in plants during salt stress. Fig. 5 View largeDownload slide PATL1 negatively regulates SOS1 activity during salt stress. (A) Comparison of PM Na+/H+ antiport activity in Col-0 and the patl1 mutant. Error bars represent the SD (n = 5) of at least three replicate experiments, each from an independent isolation of PMs. Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). (B, C) Na+ and K+ accumulation in Col-0, patl1, OX-#80 and OX-#86 plants was determined with an atomic absorption spectrophotometer (Hitachi Z-5000). Details are provided in the Materials and Methods. Error bars represent the SD (n >6). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). (D) Analysis of the K+/Na+ ratio in Col-0, patl1, OX-#80 and OX-#86 plants under control or salt stress conditions on the basis of the ion accumulation data in (B) and (C). Error bars represent the SD (n >6). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). Fig. 5 View largeDownload slide PATL1 negatively regulates SOS1 activity during salt stress. (A) Comparison of PM Na+/H+ antiport activity in Col-0 and the patl1 mutant. Error bars represent the SD (n = 5) of at least three replicate experiments, each from an independent isolation of PMs. Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). (B, C) Na+ and K+ accumulation in Col-0, patl1, OX-#80 and OX-#86 plants was determined with an atomic absorption spectrophotometer (Hitachi Z-5000). Details are provided in the Materials and Methods. Error bars represent the SD (n >6). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). (D) Analysis of the K+/Na+ ratio in Col-0, patl1, OX-#80 and OX-#86 plants under control or salt stress conditions on the basis of the ion accumulation data in (B) and (C). Error bars represent the SD (n >6). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05). PATL1 regulates plant cellular redox homeostasis during salt stress Salt stress induces ROS generation and imposes oxidative damage on plants (Mittler 2002). Many components of membrane trafficking have been identified as being involved in redox homeostasis regulation under salt conditions (Mazel et al. 2004, Leshem et al. 2006, Leshem et al. 2007, Türkan and Demiral 2009). We then investigated whether PATL1 also plays a regulatory role in ROS generation and scavenging during salt stress. The H2O2 content in Col-0 and patl1 seedlings was determined using 3,3-diaminobenzidine (DAB) staining, and the data showed that patl1 accumulated a higher level of H2O2 than Col-0 after salt treatment (Supplementary Fig. S7A). In addition, we quantitatively measured the H2O2 content with an Amplex Red Kit (Invitrogen). Salt stress significantly increased H2O2 content in both plants, and patl1 accumulated a higher level during salt stress (Supplementary Fig. S7C). We further quantified superoxide radicals in Col-0 and patl1 seedlings under salt stress through histochemical staining with nitroblue tetrazolium (NBT). The result showed that the content of superoxide radicals was higher in patl1 than in Col-0 (Supplementary Fig. S7B). In all of these assays, no detectable difference was observed between seedlings of patl1 and Col-0 under control conditions (Supplementary Fig. S7A–C). These results suggested that PATL1 participates in ROS generation during salt stress. Interestingly, we found that expression of AtrbohD, which encodes a PM-bound NADPH oxidase mediating ROS production in Arabidopsis, was induced in patl1 in response to salt stress (Supplementary Fig. S7D), suggesting that PATL1 is involved in salt-induced ROS production possibly through AtrbohD. ROS generated by abiotic stress such as soil salinity specifically induce scavenging signaling in plants (Blokhina et al. 2003, Apel and Hirt 2004, Gill and Tuteja 2010). We thus analyzed the enzymatic activities of SOD, CAT, POX and APX in patl1 and Col-0 to investigate whether PATL1 plays a role in regulating enzymatic ROS scavenging signaling. The results showed that salt stress enhanced the activities of SOD, CAT, POX and APX to different extents in both plants, and higher enzymatic activity was observed in patl1 compared with that in Col-0 (Supplementary Fig. S8A–D). We proposed that the higher enzymatic activity in patl1 was due to a more severe oxidative stress imposed by salt stress (Supplementary Fig. S7). Interestingly, when a synthetic antioxidant, dimethylthiourea (DMTU), was used for ROS detoxification, the enzymatic activity decreased in both Col-0 and patl1 plants (Supplementary Fig. S8A–D). However, the decreased level of enzymes in patl1 was lower than that in Col-0, indicating that regulation of redox homeostasis in patl1 is partially disrupted (Supplementary Fig. S8E). Consistently, we found that at this concentration (0.5 mM) of DMTU, accumulation of H2O2 deceased in both plants, but patl1 still accumulated a higher level of H2O2 compared with Col-0 (Supplementary Fig. S8F). These data suggested that PATL1 is involved in regulation of plant cellular redox homeostasis during salt stress by modulating ROS generation and the enzymatic ROS scavenging machinery. The ratio of reduced GSH to oxidized GSH (GSSG) was also determined, and the result showed that the GSH:GSSG ratio in patl1 was significantly lower than that in Col-0 under salt conditions, indicating that patl1 seedlings were exposed to a more severe oxidative stress during salt stress (Fig. 6A). To further determine whether PATL1 participates in regulating plant salt tolerance partially through modulating cellular redox homeostasis, we artificially eliminated ROS induced by salt stress using the antioxidant DMTU and then analyzed the cellular redox state and the phenotype in response to salt stress. When 0.75 mM DMTU was added in the culture medium, the GSH:GSSG ratio in patl1 increased to a level similar to that in Col-0 (Fig. 6A), indicating rescue of the cellular redox homeostasis. Consistently, the salt-sensitive phenotype of patl1 was partially rescued when DMTU was added in the culture medium (Fig. 6B, C). Therefore, we concluded that PATL1 also functions in regulating plant cellular redox homeostasis during salt stress. Fig. 6 View largeDownload slide Antioxidant partially rescues the salt-sensitive phenotype of patl1. (A) Determination of the GSH:GSSG ratio. Five-day-old seedlings were left untreated or treated with 100 mM NaCl for 12 h and analyzed for reduced or oxidized glutathione levels using an A061-1 Kit (Nanjing Jiancheng). Error bars represent the SD (n = 3). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05; **P ≤ 0.01). (B) Salt sensitivity assays of patl1 and Col-0 when DMTU was added. Col-0 and patl1 mutant seeds were germinated and grown vertically on MS medium with or without 100 mM NaCl. When grown on NaCl-containing medium, 0, 0.5 or 0.75 mM DMTU was added. Seedlings were photographed after 10 d of growth. (C) Analysis of primary root length of seedlings in (B). Error bars represent the SD (n >10). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05; **P ≤ 0.01). Fig. 6 View largeDownload slide Antioxidant partially rescues the salt-sensitive phenotype of patl1. (A) Determination of the GSH:GSSG ratio. Five-day-old seedlings were left untreated or treated with 100 mM NaCl for 12 h and analyzed for reduced or oxidized glutathione levels using an A061-1 Kit (Nanjing Jiancheng). Error bars represent the SD (n = 3). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05; **P ≤ 0.01). (B) Salt sensitivity assays of patl1 and Col-0 when DMTU was added. Col-0 and patl1 mutant seeds were germinated and grown vertically on MS medium with or without 100 mM NaCl. When grown on NaCl-containing medium, 0, 0.5 or 0.75 mM DMTU was added. Seedlings were photographed after 10 d of growth. (C) Analysis of primary root length of seedlings in (B). Error bars represent the SD (n >10). Statistical significance was determined by a Student’s t-test; significant differences are indicated by asterisks (*P ≤ 0.05; **P ≤ 0.01). Discussion Membrane trafficking is essential for plant growth, development and stress response (Geldner et al. 2001, Jurgens 2004). Studies have found that many components of membrane trafficking are involved in plant salt stress response (Mazel et al. 2004, Leshem et al. 2006, Leshem et al. 2007). In this study, we reported that the membrane trafficking-related protein PATL1 participates in regulation of salt tolerance in Arabidopsis. PATL1 negatively regulates membrane trafficking and PM Na+/H+ antiport activity during salt stress. PATL1 interacts with SOS1, indicating that its regulatory role in salt tolerance might be partially through SOS1. In addition, PATL1 modulates cellular redox homeostasis by regulating ROS generation and scavenging under salt conditions. PATL1 functions as a negative regulator of plant salt tolerance (Fig. 7, a working model). Fig. 7 View largeDownload slide A working model of PATL1 in regulation of salt tolerance. Plant PATL1 impairs membrane trafficking, which is important for regulation of cellular redox homeostasis and ion homeostasis via affecting ROS generation/detoxification and PM Na+/H+ antiporter localization. In this way, excessive ROS and Na+ were accumulated in plant cells during salt stress through disrupting ROS-generating/detoxifying signaling and impairing PM Na+/H+ antiport, respectively. PATL1 is induced during salt stress. It might be a negative feedback mechanism, which helps plants to optimize their growth and stress response to challenging environments. On the other hand, PATL1 might turn down normal vegetative growth for the salt stress response, contributing to maintenance of the balance between growth and stress response for plant survival. However, this was achieved at the cost of disruption of ion homeostasis, since PATL1 interacts with SOS1 and functions as a negative regulator of SOS1. Fig. 7 View largeDownload slide A working model of PATL1 in regulation of salt tolerance. Plant PATL1 impairs membrane trafficking, which is important for regulation of cellular redox homeostasis and ion homeostasis via affecting ROS generation/detoxification and PM Na+/H+ antiporter localization. In this way, excessive ROS and Na+ were accumulated in plant cells during salt stress through disrupting ROS-generating/detoxifying signaling and impairing PM Na+/H+ antiport, respectively. PATL1 is induced during salt stress. It might be a negative feedback mechanism, which helps plants to optimize their growth and stress response to challenging environments. On the other hand, PATL1 might turn down normal vegetative growth for the salt stress response, contributing to maintenance of the balance between growth and stress response for plant survival. However, this was achieved at the cost of disruption of ion homeostasis, since PATL1 interacts with SOS1 and functions as a negative regulator of SOS1. The transcription of PATL1 in the patl1 mutant background is significantly elevated compared with that in Col-0 (Fig. 1B), possibly due to the T-DNA insertion which results in a 35S promoter in the promoter region of PATL1. Furthermore, 5'-RACE data indicate that the transcription initiation site of PATL1 was different in Col-0 and patl1 (Supplementary Fig. S2C, D). Since there was only one translation start site (ATG) identical to that in Col-0 which could result in full-length PATL1 protein in patl1 and as overexpression of PATL1 leads to a salt-sensitive phenotype similar to that of the patl1 mutant (Fig. 1C–F;Supplementary Fig. S3), T-DNA insertion in the patl1 mutant might only alter the transcription level of PATL1, without any change in its protein structure. Therefore, patl1 might not be a gain-of-function mutant. Our data also showed that PATL1 transcription increased during salt stress (Supplementary Fig. S4). It might be a negative feedback mechanism, which helps plants optimize their growth and stress response in challenging environments. We propose that multiple pathways (including the SOS pathway) were activated for ion and redox homeostasis regulation upon salt stress. After the initial response, salt-induced PATL1 might function in attenuating the SOS pathway to avoid over-response during the stress condition for sustained plant growth. Indeed, previous studies have reported similar negative feedback regulation mechanisms which are essential for plant stress adaption (Zhao et al. 2017). On the other hand, since PATL1 could associate with the cell plate, there also exists the possibility that salt-induced PATL1 represses plant growth, contributing to maintain the balance between growth and stress resistance. However, this is achieved at the cost of disruption of ion homeostasis, since PATL1 interacts with SOS1 and functions as a negative regulator of SOS1 (Figs. 4, 5). These findings together suggest the complexity of plant development and stress response. Soil salinity causes osmotic and ionic stresses to plants, and plants should decrease their cytoplasmic Na+ concentration to a non-toxic level via Na+ extrusion and/or compartmentation (Tester and Davenport 2003, Munns and Tester 2008, Zhu 2016). A PM Na+/H+ antiporter (such as SOS1) functions in Na+ extrusion by transporting Na+ out of plant cells (Shi et al. 2000, Yokoi et al. 2002, Zhu 2016). We found that there exists a direct interaction between PATL1 and SOS1 (Fig. 4), and PATL1 is involved in affecting regulation of SOS1 PM Na+/H+ antiport activity (Fig. 5). SOS1 is a membrane protein with 12 transmembrane domains and a long autoinhibitory cyotsolic C-terminus, and the proper localization of SOS1 at the PM is critical for its biological function. Membrane trafficking plays important regulatory roles in PM protein localization and cycling during plant development and stress response (Jurgens 2004). We hypothesize that PATL1-mediated membrane trafficking regulates SOS1 localization and cycling during salt stress. PATL1 might alter the cellular distribution of SOS1, resulting in dysfunction of SOS1 in response to salt stress (Fig. 5). Since PATL1 abundance in the patl1 mutant is elevated and patl1 displays a salt-sensitive phenotype (Fig. 1; Supplementary Figs. S1, S3), PATL1 might repress endocytosis of SOS1 from the PM, resulting in limited SOS1 turnover. In addition, salt stress could significantly enhance membrane trafficking such as endocytosis with SOS1 translocating in trafficking vesicle membranes. In this way, inside-out SOS1 might transport cytoplasmic Na+ into those vesicles and help maintain cellular ion homeostasis during salt stress. However, this Na+ compartmentation is repressed in patl1 due to limited endocytosis of SOS1 from the PM. On the other hand, since PATL1 interacts with the SOS1 C-terminus in vivo, we cannot exclude the possibility that PATL1 regulates SOS1 activity by directly affecting the configuration of SOS1 during salt stress. Dissecting the structure of the PATL1–SOS1 complex or analyzing the regulatory role of PATL1 in SOS1 activity in reconstituted liposomes in vitro will contribute to validatation of this hypothesis. Accumulated ROS during salt stress imposes oxidative stress on plants (Blokhina et al. 2003, Gill and Tuteja 2010). Studies have found that ROS might be generated in endocytic vesicles which will finally fuse with the central vacuole under salt stress conditions (Bassham et al. 2006, Leshem et al. 2006). Defects in vesicle generation or fusion during membrane trafficking disrupt cellular redox homeostasis in response to salt stress, leading to cell death. In fact, salt stress also stimulates endocytosis of NADPH oxidase (such as AtrbohD) in Arabidopsis (Hao et al. 2014). We hypothesize that PATL1 might be involved in regulation of redox homeostasis through modulating membrane trafficking in a similar way. Future work needs to be performed to elucidate the mechanism by which PATL1 modulates ROS production and scavenging under salt conditions. In addition, previous studies have demonstrated that ROS also function in PM Na+/H+ antiport activity and regulation of K+/Na+ homeostasis (Katiyar-Agarwal et al. 2006, Zhang et al. 2006, Chung et al. 2008, Ma et al. 2012). In this way, we hypothesize that PATL1 might also regulate K+/Na+ homeostasis by affecting PM Na+/H+ antiport activity through ROS directly for salt tolerance modulation. Materials and Methods Mutants and transgenic plants Arabidopsis thaliana Col-0 was used as the wild type in all experiments. patl1 was identified from a SALK line (103668) with a T-DNA insertion in the 5'-UTR of At1g72150. Transgenic plants overexpressing GFP-tagged PATL1 in Col-0 (lines #80 and #86) were used in this study. Plant growth and salt tolerance analysis Seeds were sterilized in a solution containing 20% sodium hypochlorite (NaClO) and 0.1% Triton X-100 for 15 min, washed five imes with sterilized water, and sown on MS medium with 0.3% (for horizontal growth) or 0.5% (for vertical growth) phytagel agar (Sigma-Aldrich). For salt sensitivity assay, Col-0, the patl1 mutant and the PATL1-overexpressing plants were germinated and grown vertically on MS medium without or with 75 or 125 mM NaCl. Seedlings were photographed after 2 weeks of growth. The fresh weight and primary root length were measured and statistically analyzed. For the germination assay, Col-0 and patl1 seeds were collected at the same time, stratified and grown as described above. The germination rate and cotyledon greening rate were scored at the indicated time point. The salt sensitivity analysis in soil was performed as described (Zhou et al. 2014). Confocal microscopy assay The coding sequence of PATL1 was amplified with the P1-Bf/P1-Sr primers and cloned into the pCAMBIA1205-GFP vector between the BamHI and SalI sites. The primer sequences are given in Supplementary Table S1. PATL1–GFP and a marker of specific endosomal compartments were co-expressed in Arabidopsis leaf protoplasts as described (Tian et al. 2015). The fluorescent signals of GFP or RFP were detected using a Leica SP5 confocal microscope according to the user manual. Co-immunoprecipitation assays in Arabidopsis leaf protoplasts The coding sequence of PATL1 was amplified with the P1-Bf/P1-Sr primers and cloned into the pCAMBIA1307-6×Myc vector between the BamHI and SalI sites. The coding sequences of PATL1N1, PATL1N2, PATL1Sec14 and PATL1GOLD were amplified with the P1n1-Bf/P1n1-Sr, P1n2-Bf/P1n2-Sr, P1s1-Bf/P1s1-Sr and P1g1-Bf/P1g1-Sr, respectively, and cloned into the pCAMBIA1307-6×Myc vector between the BamHI and SalI sites. The coding sequence of the SOS1 C-terminus (amino acids 557–1,146) was amplified with the SOS1C-Sf and SOS1C-Kr primers, and cloned into the pCAMBIA1307-Flag-HA vector between the SalI and KpnI sites. The primer sequences are given in Supplementary Table S1. The resulting plasmids were used for the Co-IP assay as described (Zhou et al. 2014). Bimolecular fluorescence complementation assay The coding sequence of PATL1 was amplified with the P1-Bf/P1-Sr primers while full-length SOS1, the SOS1 N-terminus and the C-terminus were amplified with the SOS1-Sf/SOS1-K(NS)r, SOS1-Sf/SOS1N-K(NS)r and SOS1C-Sf/SOS1-K(NS)r primers, respectively. The primer sequences are given in Supplementary Table S1. The resulting fragments were cloned into pSPYNE(R)173 and pSPYCE(M) at the BamHI/SalI and SalI/KpnI restriction sites, respectively (Walter et al. 2004, Waadt et al. 2008). Primer sequences are listed in Supplementary Table S1. Constructs containing PATL1-YFPN and YFPC-SOS1, YFPC-SOS1N or YFPC-SOS1C were introduced into Agrobacterium tumefaciens GV3101 through electroporation and infiltrated into N. benthamiana leaves. The YFP fluorescence signal was detected via a Leica SP5 confocal microscope 3 d after infiltration. Yeast two-hybrid assay The coding sequences of PATL1N1, PATL1N2, PATL1Sec14 and PATL1GOLD were amplified with P1n1-Ef/P1n1-Br, P1n2-Ef/P1n2-Br, P1s1-Ef/P1s1-Br and P1g1-Ef/P1g1-Br, respectively, and cloned into the pGBKT7 vector between the EcoRI and BamHI sites. The coding sequence of the SOS1 C-terminal amino acids (557–1,146) was amplified with the SOS1C-Ef and SOS1C-Br primers and cloned into the pGADT7 vector between the EcoRI and BamHI sites. The primer sequences are listed in Supplementary Table S1. Then, constructs pGBKT7-PATL1N1, pGBKT7-PATL1N2, pGBKT7-PATL1Sec14 and pGBKT7-PATL1GOLD as well as the pGBKT7 vector were co-transformed with pGADT7-SOS1C into yeast strain AH109. Yeast transformation and growth assays were performed as described in the Yeast Protocols Handbook (Clontech). 5'-RACE assay Ten-day-old seedlings of Col-0 and patl1 were harvested for total mRNA isolation. The resulting mRNA was subjected to 5'-RACE assay using the SMART™ RACE cDNA amplification kit (Clontech, Cat. No. 634914) according to the user manual. The primer sequences are given in Supplementary Table S3. PM isolation and Na+/H+ antiport activity assay and determination of ion content Three-week-old seedlings of Col-0 and patl1 were used for PM isolation by aqueous two-phase partitioning as described previously (Qiu et al. 2002). PM Na+/H+ antiport activity was determined as an Na+-induced dissipation of the pH gradient as described (Qiu et al. 2002). Col-0 and patl1 seedlings were grown in soil under long-day conditions for 3 weeks at 23°C, and the seedlings were treated with 150 mM NaCl for 10 d. Rosette leaves were harvested and dried at 80°C for 2 d. All the samples were accurately weighed and treated at 300°C for 1 h and at 575°C for 4 h in a muffle furnace. The resulting ground dry matter was dissolved in 0.1 M HCl. The Na+ and K+ contents of the samples were determined with an atomic absorption spectrophotometer (Hitachi Z-5000). BFA treatment and FM4-64 staining BFA (Invitrogen) treatment and FM4-64 (Invitrogen) staining were conducted as described (Tian et al. 2015) with modifications. Briefly, tobacco leaves transiently expressing PATL1–GFP were cut into small fragments and incubated in a medium containing 25 μM FM4-64 for 40 min, rinsed with water several times and observed under a confocal microscope (Leica SP5). For BFA treatment, roots of 5-day-old seedlings were briefly washed and incubated in 50 μM BFA (Sigma-Aldrich) and 1 μM FM4-64 for 25 min, then the BFA bodies of single cells were quantified via confocal microscopy. ROS accumulation and assays of ROS-scavenging enzyme activity ROS accumulation was measured using various approaches. For DAB staining assay, 10-day-old seedlings were left untreated or treated with 200 mM NaCl for 4 h and then incubated in staining buffer (0.1 mg ml–1 DAB dissolved in 0.1 M HCl, pH 5.0). Seedlings were destained in destaining buffer (acetic acid:glycerin:ethanol = 1:1:3) after staining for 8 h. For the Amplex Red H2O2/peroxidase assay, 10-day-old seedlings were left untreated or treated with 200 mM NaCl for 4 h and harvested. Detection of H2O2 accumulation was performed following the manufacturer’s instructions. NBT staining was used to determine the production of superoxide radicals as described (Wohlgemuth et al. 2002). To determine the activity of enzymes for ROS scavenging, 10-day-old seedlings of Col-0 and patl1 were left untreated or treated with 200 mM NaCl (with or without 0.5 mM DMTU) for 4 h. Then 0.5 g aliquots of fresh tissues were homogenized with 5 ml of ice-cold 25 mM HEPES buffer (pH 7.8) containing 0.2 mM EDTA, 2 mM ascorbate and 2% polyvinylpyrrolidone (PVP). The resulting homogenates were centrifuged at 12,000×g for 20 min at 4°C and the supernatants were used for determination of the enzymatic activities as described (Patra and Mishra 1979, Stewart and Bewley 1980, Nakano and Asada 1981, Hammerschmidt et al. 1982). Promoter activity analysis For GUS staining, a 2,000 bp genomic DNA fragment from 1 to 2,000 bp upstream of the translational start site (ATG) of PATL1 was amplified with the P1pro-Sf and P1pro-Br primers and then cloned into the SalI and BamHI sites of the pCM1391 vector. The plasmid was transformed into Col-0, and transgenic T2 lines were used for GFP observation or GUS staining as described (Zhou et al. 2014). The primer sequences are given in Supplementary Table S1. Quantitative real-time and RT–PCR assays Seedlings of Col-0, patl1 as well as PATL1-overexpressing lines were prepared as described. Total RNA was extracted with Trizol reagent (Invitrogen) and treated with RNase-free DNase I (TaKaRa) to remove genomic DNA. A 10 mg aliquot of RNA was used for reverse transcription with M-MLV reverse transcriptase (Promega) according to the manufacturer’s instructions. The resulting cDNAs were used for quantitative real-time PCR amplification or RT–PCR assay with the primers listed in Supplementary Table S2. ACTIN2 (ACT2) was used as an internal control. Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by National Natural Science Foundation of China (NSFC) [grant No. 31600201 to H.Z.] and the Start-Up Foundation of Sichuan University [to H.Z.]. Acknowledgments We thank the Arabidopsis Biological Resource Center (ABRC) for providing the T-DNA insertion lines. 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Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations APX ascorbate peroxidase BFA brefeldin A BiFC bimolecular fluorescence complementation CAT catalase Co-IP co-immunoprecipitation DAB 3,3-diaminobenzidine DMTU dimethylthiourea 5'-RACE 5'-rapid amplification of cDNA ends 5'-UTR 5'-untranslated region GFP green fluorescent protein GSH glutathione GSSG oxidized glutathione GUS β-glucuronidase MS Murashige and Skoog NBT nitroblue tetrazolium PATL1 Patellin 1 PM plasma membrane POD peroxidase PVC pre-vacuolar compartment RFP red fluorescent protein ROS reactive oxygen species RT–PCR reverse transcription–PCR SOD superoxide dismutase SOS Salt-Overly-Sensitive YFP yellow fluorescent protein Y2H yeast two-hybrid © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. 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/about_us/legal/notices)

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Plant and Cell PhysiologyOxford University Press

Published: Aug 1, 2018

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