The zinc finger protein DHHC09 S-acylates the kinase STRK1 to regulate H2O2 homeostasis and promote salt tolerance in rice.

Soil salinity results in oxidative stress and heavy losses to crop production. The S-acylated protein SALT TOLERANCE RECEPTOR-LIKE CYTOPLASMIC KINASE 1 (STRK1) phosphorylates and activates CATALASE C (CatC) to improve rice (Oryza sativa L.) salt tolerance, but the molecular mechanism underlying its S-acylation involved in salt signal transduction awaits elucidation. Here, we show that the DHHC-type zinc finger protein DHHC09 S-acylates STRK1 at Cys5, Cys10, and Cys14 and promotes salt and oxidative stress tolerance by enhancing rice H2O2-scavenging capacity. This modification determines STRK1 targeting to the plasma membrane or lipid nanodomains and is required for its function. DHHC09 promotes salt signaling from STRK1 to CatC via transphosphorylation, and its deficiency impairs salt signal transduction. Our findings demonstrate that DHHC09 S-acylates and anchors STRK1 to the plasma membrane to promote salt signaling from STRK1 to CatC, thereby regulating H2O2 homeostasis and improving salt stress tolerance in rice. Moreover, overexpression of DHHC09 in rice mitigates grain yield loss under salt stress. Together, these results shed light on the mechanism underlying the role of S-acylation in RLK/RLCK-mediated salt signal transduction and provide a strategy for breeding highly salt-tolerant rice.

GROWTH REGULATING FACTOR 7-mediated arbutin metabolism enhances rice salt tolerance.

Salt stress is an environmental factor that limits plant growth and crop production. With the rapid expansion of salinized arable land worldwide, investigating the molecular mechanisms underlying the salt stress response in plants is urgently needed. Here, we report that GROWTH REGULATING FACTOR 7 (OsGRF7) promotes salt tolerance by regulating arbutin (hydroquinone-ß-D-glucopyranoside) metabolism in rice (Oryza sativa). Overexpression of OsGRF7 increased arbutin content, and exogenous arbutin application rescued the salt-sensitive phenotype of OsGRF7 knockdown and knockout plants. OsGRF7 directly promoted the expression of the arbutin biosynthesis genes URIDINE DIPHOSPHATE GLYCOSYLTRANSFERASE 1 (OsUGT1) and OsUGT5, and knockout of OsUGT1 or OsUGT5 reduced rice arbutin content, salt tolerance, and grain size. Furthermore, OsGRF7 degradation through its interaction with F-BOX AND OTHER DOMAINS CONTAINING PROTEIN 13 reduced rice salinity tolerance and grain size. These findings highlight an underexplored role of OsGRF7 in modulating rice arbutin metabolism, salt stress response, and grain size, as well as its broad potential use in rice breeding.

Local cryptic diversity in salinity adaptation mechanisms in the wild outcrossing Brassica fruticulosa.

It is normally supposed that populations of the same species should evolve shared mechanisms of adaptation to common stressors due to evolutionary constraint. Here, we describe a system of within-species local adaptation to coastal habitats, Brassica fruticulosa, and detail surprising strategic variability in adaptive responses to high salinity. These different adaptive responses in neighboring populations are evidenced by transcriptomes, diverse physiological outputs, and distinct genomic selective landscapes. In response to high salinity Northern Catalonian populations restrict root-to-shoot Na+ transport, favoring K+ uptake. Contrastingly, Central Catalonian populations accumulate Na+ in leaves and compensate for the osmotic imbalance with compatible solutes such as proline. Despite contrasting responses, both metapopulations were salinity tolerant relative to all inland accessions. To characterize the genomic basis of these divergent adaptive strategies in an otherwise non-saline-tolerant species, we generate a long-read-based genome and population sequencing of 18 populations (nine inland, nine coastal) across the B. fruticulosa species range. Results of genomic and transcriptomic approaches support the physiological observations of distinct underlying mechanisms of adaptation to high salinity and reveal potential genetic targets of these two very recently evolved salinity adaptations. We therefore provide a model of within-species salinity adaptation and reveal cryptic variation in neighboring plant populations in the mechanisms of adaptation to an important natural stressor highly relevant to agriculture.

H2O2-dependent oxidation of the transcription factor GmNTL1 promotes salt tolerance in soybean.

Reactive oxygen species (ROS) play an essential role in plant growth and responses to environmental stresses. Plant cells sense and transduce ROS signaling directly via hydrogen peroxide (H2O2)-mediated posttranslational modifications (PTMs) on protein cysteine residues. Here, we show that the H2O2-mediated cysteine oxidation of NAC WITH TRANS-MEMBRANE MOTIF1-LIKE 1 (GmNTL1) in soybean (Glycine max) during salt stress promotes its release from the endoplasmic reticulum (ER) membrane and translocation to the nucleus. We further show that an oxidative posttranslational modification on GmNTL1 residue Cys-247 steers downstream amplification of ROS production by binding to and activating the promoters of RESPIRATORY BURST OXIDASE HOMOLOG B (GmRbohB) genes, thereby creating a feed-forward loop to fine-tune GmNTL1 activity. In addition, oxidation of GmNTL1 Cys-247 directly promotes the expression of CATION H+ EXCHANGER 1 (GmCHX1)/SALT TOLERANCE-ASSOCIATED GENE ON CHROMOSOME 3 (GmSALT3) and Na+/H+ Antiporter 1 (GmNHX1). Accordingly, transgenic overexpression of GmNTL1 in soybean increases the H2O2 levels and K+/Na+ ratio in the cell, promotes salt tolerance, and increases yield under salt stress, while an RNA interference-mediated knockdown of GmNTL1 elicits the opposite effects. Our results reveal that the salt-induced oxidation of GmNTL1 promotes its relocation and transcriptional activity through an H2O2-mediated posttranslational modification on cysteine that improves resilience of soybean against salt stress.

The protein phosphatase PC1 dephosphorylates and deactivates CatC to negatively regulate H2O2 homeostasis and salt tolerance in rice.

Catalase (CAT) is often phosphorylated and activated by protein kinases to maintain hydrogen peroxide (H2O2) homeostasis and protect cells against stresses, but whether and how CAT is switched off by protein phosphatases remains inconclusive. Here, we identified a manganese (Mn2+)-dependent protein phosphatase, which we named PHOSPHATASE OF CATALASE 1 (PC1), from rice (Oryza sativa L.) that negatively regulates salt and oxidative stress tolerance. PC1 specifically dephosphorylates CatC at Ser-9 to inhibit its tetramerization and thus activity in the peroxisome. PC1 overexpressing lines exhibited hypersensitivity to salt and oxidative stresses with a lower phospho-serine level of CATs. Phosphatase activity and seminal root growth assays indicated that PC1 promotes growth and plays a vital role during the transition from salt stress to normal growth conditions. Our findings demonstrate that PC1 acts as a molecular switch to dephosphorylate and deactivate CatC and negatively regulate H2O2 homeostasis and salt tolerance in rice. Moreover, knockout of PC1 not only improved H2O2-scavenging capacity and salt tolerance but also limited rice grain yield loss under salt stress conditions. Together, these results shed light on the mechanisms that switch off CAT and provide a strategy for breeding highly salt-tolerant rice.

Phytochromes enhance SOS2-mediated PIF1 and PIF3 phosphorylation and degradation to promote Arabidopsis salt tolerance.

Soil salinity is one of the most detrimental abiotic stresses affecting plant survival, and light is a core environmental signal regulating plant growth and responses to abiotic stress. However, how light modulates the plant's response to salt stress remains largely obscure. Here, we show that Arabidopsis (Arabidopsis thaliana) seedlings are more tolerant to salt stress in the light than in the dark, and that the photoreceptors phytochrome A (phyA) and phyB are involved in this tolerance mechanism. We further show that phyA and phyB physically interact with the salt tolerance regulator SALT OVERLY SENSITIVE2 (SOS2) in the cytosol and nucleus, and enhance salt-activated SOS2 kinase activity in the light. Moreover, SOS2 directly interacts with and phosphorylates PHYTOCHROME-INTERACTING FACTORS PIF1 and PIF3 in the nucleus. Accordingly, PIFs act as negative regulators of plant salt tolerance, and SOS2 phosphorylation of PIF1 and PIF3 decreases their stability and relieves their repressive effect on plant salt tolerance in both light and dark conditions. Together, our study demonstrates that photoactivated phyA and phyB promote plant salt tolerance by increasing SOS2-mediated phosphorylation and degradation of PIF1 and PIF3, thus broadening our understanding of how plants adapt to salt stress according to their dynamic light environment.

CycC1;1-WRKY75 complex-mediated transcriptional regulation of SOS1 controls salt stress tolerance in Arabidopsis.

SALT OVERLY SENSITIVE1 (SOS1) is a key component of plant salt tolerance. However, how SOS1 transcription is dynamically regulated in plant response to different salinity conditions remains elusive. Here, we report that C-type Cyclin1;1 (CycC1;1) negatively regulates salt tolerance by interfering with WRKY75-mediated transcriptional activation of SOS1 in Arabidopsis (Arabidopsis thaliana). Disruption of CycC1;1 promotes SOS1 expression and salt tolerance in Arabidopsis because CycC1;1 interferes with RNA polymerase II recruitment by occupying the SOS1 promoter. Enhanced salt tolerance of the cycc1;1 mutant was completely compromised by an SOS1 mutation. Moreover, CycC1;1 physically interacts with the transcription factor WRKY75, which can bind to the SOS1 promoter and activate SOS1 expression. In contrast to the cycc1;1 mutant, the wrky75 mutant has attenuated SOS1 expression and salt tolerance, whereas overexpression of SOS1 rescues the salt sensitivity of wrky75. Intriguingly, CycC1;1 inhibits WRKY75-mediated transcriptional activation of SOS1 via their interaction. Thus, increased SOS1 expression and salt tolerance in cycc1;1 were abolished by WRKY75 mutation. Our findings demonstrate that CycC1;1 forms a complex with WRKY75 to inactivate SOS1 transcription under low salinity conditions. By contrast, under high salinity conditions, SOS1 transcription and plant salt tolerance are activated at least partially by increased WRKY75 expression but decreased CycC1;1 expression.

The transmembrane protein LbRSG from the recretohalophyte Limonium bicolor enhances salt gland development and salt tolerance.

Salt glands are the unique epidermal structures present in recretohalophytes, plants that actively excrete excess Na+ by salt secretory structures to avoid salt damage. Here, we describe a transmembrane protein that localizes to the plasma membrane of the recretohalophyte Limonium bicolor. As virus-induced gene silencing of the corresponding gene LbRSG in L. bicolor decreased the number of salt glands, we named the gene Reduced Salt Gland. We detected LbRSG transcripts in salt glands by in situ hybridization and transient transformation. Overexpression and silencing of LbRSG in L. bicolor pointed to a positive role in salt gland development and salt secretion by interacting with Lb3G16832. Heterologous LbRSG expression in Arabidopsis enhanced salt tolerance during germination and the seedling stage by alleviating NaCl-induced ion stress and osmotic stress after replacing or deleting the (highly) negatively charged region of extramembranous loop. After screened by immunoprecipitation-mass spectrometry and verified using yeast two-hybrid, PGK1 and BGLU18 were proposed to interact with LbRSG to strengthen salt tolerance. Therefore, we identified (highly) negatively charged regions in the extramembrane loop that may play an essential role in salt tolerance, offering hints about LbRSG function and its potential to confer salt resistance.

Deciphering salt stress responses in Solanum pimpinellifolium through high-throughput phenotyping.

Soil salinity is a major environmental stressor affecting agricultural productivity worldwide. Understanding plant responses to salt stress is crucial for developing resilient crop varieties. Wild relatives of cultivated crops, such as wild tomato, Solanum pimpinellifolium, can serve as a useful resource to further expand the resilience potential of the cultivated germplasm, S. lycopersicum. In this study, we employed high-throughput phenotyping in the greenhouse and field conditions to explore salt stress responses of a S. pimpinellifolium diversity panel. Our study revealed extensive phenotypic variations in response to salt stress, with traits such as transpiration rate, shoot mass, and ion accumulation showing significant correlations with plant performance. We found that while transpiration was a key determinant of plant performance in the greenhouse, shoot mass strongly correlated with yield under field conditions. Conversely, ion accumulation was the least influential factor under greenhouse conditions. Through a Genome Wide Association Study, we identified candidate genes not previously associated with salt stress, highlighting the power of high-throughput phenotyping in uncovering novel aspects of plant stress responses. This study contributes to our understanding of salt stress tolerance in S. pimpinellifolium and lays the groundwork for further investigations into the genetic basis of these traits, ultimately informing breeding efforts for salinity tolerance in tomato and other crops.

Whole-genome sequencing of allotetraploid bermudagrass reveals the origin of Cynodon and candidate genes for salt tolerance.

Bermudagrass (Cynodon dactylon) is a globally distributed, extensively used warm-season turf and forage grass with high tolerance to salinity and drought stress in alkaline environments. However, the origin of the species and genetic mechanisms for salinity tolerance in the species are basically unknown. Accordingly, we set out to study evolution divergence events in the Cynodon genome and to identify genes for salinity tolerance. We developed a 604.0 Mb chromosome-level polyploid genome sequence for bermudagrass 'A12359' (n = 18). The C. dactylon genome comprises 2 complete sets of homoeologous chromosomes, each with approximately 30 000 genes, and most genes are conserved as syntenic pairs. Phylogenetic study showed that the initial Cynodon species diverged from Oropetium thomaeum approximately 19.7-25.4 million years ago (Mya), the A and B subgenomes of C. dactylon diverged approximately 6.3-9.1 Mya, and the bermudagrass polyploidization event occurred 1.5 Mya on the African continent. Moreover, we identified 82 candidate genes associated with seven agronomic traits using a genome-wide association study, and three single-nucleotide polymorphisms were strongly associated with three salt resistance genes: RAP2-2, CNG channels, and F14D7.1. These genes may be associated with enhanced bermudagrass salt tolerance. These bermudagrass genomic resources, when integrated, may provide fundamental insights into evolution of diploid and tetraploid genomes and enhance the efficacy of comparative genomics in studying salt tolerance in Cynodon.

B. subtilis CNBG-PGPR-1 induces methionine to regulate ethylene pathway and ROS scavenging for improving salt tolerance of tomato.

Soil salinity severely threatens plant growth and crop yields. The utilization of PGPR is an effective strategy for enhancing plant salt tolerance, but the mechanisms involved in this process have rarely been reported. In this study, we investigated the effects of Bacillus subtilis CNBG-PGPR-1 on improving plant salt tolerance and elucidated the molecular pathways involved. The results showed that CNBG-PGPR-1 significantly improved the cellular homeostasis and photosynthetic efficiency of leaves and reduced ion toxicity and osmotic stress caused by salt in tomato. Transcriptome analysis uncovered that CNBG-PGPR-1 enhanced plant salt tolerance through the activation of complex molecular pathways, with plant hormone signal transduction playing an important role. Comparative analysis and pharmacological experiments confirmed that the ethylene pathway was closely related to the beneficial effect of CNBG-PGPR-1 on improving plant salt tolerance. Furthermore, we found that methionine, a precursor of ethylene synthesis, significantly accumulated in response to CNBG-PGPR-1 in tomato. Exogenous L-methionine largely mimicked the beneficial effects of CNBG-PGPR-1 and activated the expression of ethylene pathway-related genes, indicating CNBG-PGPR-1 induces methionine accumulation to regulate the ethylene pathway in tomato. Finally, CNBG-PGPR-1 reduced salt-induced ROS by activating ROS scavenger-encoding genes, mainly involved in GSH metabolism and POD-related genes, which were also closely linked to methionine metabolism. Overall, our studies demonstrate that CNBG-PGPR-1-induced methionine is a key regulator in enhancing plant salt tolerance through the ethylene pathway and ROS scavenging, providing a novel understanding of the mechanism by which beneficial microbes improve plant salt tolerance.

Q negatively regulates wheat salt tolerance through directly repressing the expression of TaSOS1 and reactive oxygen species scavenging genes.

The Q transcription factor plays important roles in improving multiple wheat domestication traits such as spike architecture, threshability and rachis fragility. However, whether and how it regulates abiotic stress adaptation remain unclear. We found that the transcriptional expression of Q can be induced by NaCl and abscisic acid treatments. Using the q mutants generated by CRISPR/Cas9 and Q overexpression transgenic lines, we showed that the domesticated Q gene causes a penalty in wheat salt tolerance. Then, we demonstrated that Q directly represses the transcription of TaSOS1-3B and reactive oxygen species (ROS) scavenging genes to regulate Na+ and ROS homeostasis in wheat. Furthermore, we showed that wheat salt tolerance protein TaWD40 interacts with Q to competitively interfere with the interaction between Q and the transcriptional co-repressor TaTPL. Taken together, our findings reveal that Q directly represses the expression of TaSOS1 and some ROS scavenging genes, thus causing a harmful effect on wheat salt tolerance.

The OsDIR55 gene increases salt tolerance by altering the root diffusion barrier.

The increased soil salinity is becoming a major challenge to produce more crops and feed the growing population of the world. In this study, we demonstrated that overexpression of OsDIR55 gene enhances rice salt tolerance by altering the root diffusion barrier. OsDIR55 is broadly expressed in all examined tissues and organs with the maximum expression levels at lignified regions in rice roots. Salt stress upregulates the expression of OsDIR55 gene in an abscisic acid (ABA)-dependent manner. Loss-function and overexpression of OsDIR55 compromised and improved the development of CS and root diffusion barrier, manifested with the decreased and increased width of CS, respectively, and ultimately affected the permeability of the apoplastic diffusion barrier in roots. OsDIR55 deficiency resulted in Na+ accumulation, ionic imbalance, and growth arrest, whereas overexpression of OsDIR55 enhances salinity tolerance and provides an overall benefit to plant growth and yield potential. Collectively, we propose that OsDIR55 is crucial for ions balance control and salt stress tolerance through regulating lignification-mediated root barrier modifications in rice.

Trehalose along with ABA promotes the salt tolerance of Avicennia marina by regulating Na+ transport.

Mangroves grow in tropical/subtropical intertidal habitats with extremely high salt tolerance. Trehalose and trehalose-6-phosphate (T6P) have an alleviating function against abiotic stress. However, the roles of trehalose in the salt tolerance of salt-secreting mangrove Avicennia marina is not documented. Here, we found that trehalose was significantly accumulated in A. marina under salt treatment. Furthermore, exogenous trehalose can enhance salt tolerance by promoting the Na+ efflux from leaf salt gland and root to reduce the Na+ content in root and leaf. Subsequently, eighteen trehalose-6-phosphate synthase (AmTPS) and 11 trehalose-6-phosphate phosphatase (AmTPP) genes were identified from A. marina genome. Abscisic acid (ABA) responsive elements were predicted in AmTPS and AmTPP promoters by cis-acting elements analysis. We further identified AmTPS9A, as an important positive regulator, that increased the salt tolerance of AmTPS9A-overexpressing Arabidopsis thaliana by altering the expressions of ion transport genes and mediating Na+ efflux from the roots of transgenic A. thaliana under NaCl treatments. In addition, we also found that ABA could promote the accumulation of trehalose, and the application of exogenous trehalose significantly promoted the biosynthesis of ABA in both roots and leaves of A. marina. Ultimately, we confirmed that AmABF2 directly binds to the AmTPS9A promoter in vitro and in vivo. Taken together, we speculated that there was a positive feedback loop between trehalose and ABA in regulating the salt tolerance of A. marina. These findings provide new understanding to the salt tolerance of A. marina in adapting to high saline environment at trehalose and ABA aspects.

NADPH oxidase-dependent H2O2 production mediates salicylic acid-induced salt tolerance in mangrove plant Kandelia obovata by regulating Na+/K+ and redox homeostasis.

Salicylic acid (SA) is known to enhance salt tolerance in plants. However, the mechanism of SA-mediated response to high salinity in halophyte remains unclear. Using electrophysiological and molecular biological methods, we investigated the role of SA in response to high salinity in mangrove species, Kandelia obovata, a typical halophyte. Exposure of K. obovata roots to high salinity resulted in a rapid increase in endogenous SA produced by phenylalanine ammonia lyase pathway. The application of exogenous SA improved the salt tolerance of K. obovata, which depended on the NADPH oxidase-mediated H2O2. Exogenous SA and H2O2 increased Na+ efflux and reduced K+ loss by regulating the transcription levels of Na+ and K+ transport-related genes, thus reducing the Na+/K+ ratio in the salt-treated K. obovata roots. In addition, exogenous SA-enhanced antioxidant enzyme activity and its transcripts, and the expressions of four genes related to AsA-GSH cycle as well, then alleviated oxidative damages in the salt-treated K. obovata roots. However, the above effects of SA could be reversed by diphenyleneiodonium chloride (the NADPH oxidase inhibitor) and paclobutrazol (a SA biosynthesis inhibitor). Collectively, our results demonstrated that SA-induced salt tolerance of K. obovata depends on NADPH oxidase-generated H2O2 that affects Na+/K+ and redox homeostasis in response to high salinity.

Metabolic adaptations leading to an enhanced lignification in wheat roots under salinity stress.

Analysis of salinity tolerance processes in wheat has focused on salt exclusion from shoots while root phenotypes have received limited attention. Here, we consider the varying phenotypic response of four bread wheat varieties that differ in their type and degree of salt tolerance and assess their molecular responses to salinity and changes in root cell wall lignification. These varieties were Westonia introgressed with Nax1 and Nax2 root sodium transporters (HKT1;4-A and HKT1;5-A) that reduce Na+ accumulation in leaves, as well as the 'tissue tolerant' Portuguese landrace Mocho de Espiga Branca that has a mutation in the homologous gene HKT1;5-D and has high Na+ concentration in leaves. These three varieties were compared with the relatively more salt-sensitive cultivar Gladius. Through the use of root histochemical analysis, ion concentrations, as well as differential proteomics and targeted metabolomics, we provide an integrated view of the wheat root response to salinity. We show different metabolic re-arrangements in energy conversion, primary metabolic machinery and phenylpropanoid pathway leading to monolignol production in a genotype and genotype by treatment-dependent manner that alters the extent and localisation of root lignification which correlated with an improved capacity of wheat roots to cope better under salinity stress.

Clock component OsPRR73 positively regulates rice salt tolerance by modulating OsHKT2;1-mediated sodium homeostasis.

The roles of clock components in salt stress tolerance remain incompletely characterized in rice. Here, we show that, among OsPRR (Oryza sativa Pseudo-Response Regulator) family members, OsPRR73 specifically confers salt tolerance in rice. Notably, the grain size and yield of osprr73 null mutants were significantly decreased in the presence of salt stress, with accumulated higher level of reactive oxygen species and sodium ions. RNA sequencing and biochemical assays identified OsHKT2;1, encoding a plasma membrane-localized Na+ transporter, as a transcriptional target of OsPRR73 in mediating salt tolerance. Correspondingly, null mutants of OsHKT2;1 displayed an increased tolerance to salt stress. Immunoprecipitation-mass spectrometry (IP-MS) assays further identified HDAC10 as nuclear interactor of OsPRR73 and co-repressor of OsHKT2;1. Consistently, H3K9ac histone marks at OsHKT2;1 promoter regions were significantly reduced in osprr73 mutant. Together, our findings reveal that salt-induced OsPRR73 expression confers salt tolerance by recruiting HDAC10 to transcriptionally repress OsHKT2;1, thus reducing cellular Na+ accumulation. This exemplifies a new molecular link between clock components and salt stress tolerance in rice.

The 14-3-3 protein GRF8 modulates salt stress tolerance in apple via the WRKY18-SOS pathway.

Salinity is a severe abiotic stress that limits plant survival, growth, and development. 14-3-3 proteins are phosphopeptide-binding proteins that are involved in numerous signaling pathways, such as metabolism, development, and stress responses. However, their roles in salt tolerance are unclear in woody plants. Here, we characterized an apple (Malus domestica) 14-3-3 gene, GENERAL REGULATORY FACTOR 8 (MdGRF8), the product of which promotes salinity tolerance. MdGRF8 overexpression improved salt tolerance in apple plants, whereas MdGRF8-RNA interference (RNAi) weakened it. Yeast 2-hybrid, bimolecular fluorescence complementation, pull-down, and coimmunoprecipitation assays revealed that MdGRF8 interacts with the transcription factor MdWRKY18. As with MdGRF8, overexpressing MdWRKY18 enhanced salt tolerance in apple plants, whereas silencing MdWRKY18 had the opposite effect. We also determined that MdWRKY18 binds to the promoters of the salt-related genes SALT OVERLY SENSITIVE 2 (MdSOS2) and MdSOS3. Moreover, we showed that the 14-3-3 protein MdGRF8 binds to the phosphorylated form of MdWRKY18, enhancing its stability and transcriptional activation activity. Our findings reveal a regulatory mechanism by the MdGRF8-MdWRKY18 module for promoting the salinity stress response in apple.

Acetylation of transcription factor BpTCP20 by acetyltransferase BpPDCE23 modulates salt tolerance in birch.

Teosinte branched 1/Cycloidea/Proliferating cell factor (TCP) transcription factors function in abiotic stress responses. However, how TCPs confer salt tolerance is unclear. Here, we characterized a TCP transcription factor, BpTCP20, that responds to salt stress in birch (Betula platyphylla Suk). Plants overexpressing BpTCP20 displayed increased salt tolerance, and Bptcp20 knockout mutants displayed reduced salt tolerance relative to the wild-type (WT) birch. BpTCP20 conferred salt tolerance by mediating stomatal closure and reducing reactive oxygen species (ROS) accumulation. Chromatin immunoprecipitation sequencing showed that BpTCP20 binds to NeuroD1, T-box, and two unknown elements (termed TBS1 and TBS2) to regulate target genes. In birch, salt stress led to acetylation of BpTCP20 acetylation at lysine 259. A mutated BpTCP20 variant (abolished for acetylation, termed BpTCP20259) was overexpressed in birch, which led to decreased salt tolerance compared with plants overexpressing BpTCP20. However, BpTCP20259-overexpressing plants still displayed increased salt tolerance relative to untransformed WT plants. BpTCP20259 showed reduced binding to the promoters of target genes and decreased target gene activation, leading to decreased salt tolerance. In addition, we identified dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex (BpPDCE23), an acetyltransferase that interacts with and acetylates BpTCP20 to enhance its binding to DNA motifs. Together, these results suggest that BpTCP20 is a transcriptional regulator of salt tolerance, whose activity is modulated by BpPDCE23-mediated acetylation.