Insights into plant salt stress signaling and tolerance.

Soil salinization is an essential environmental stressor, threatening agricultural yield and ecological security worldwide. Saline soils accumulate excessive soluble salts which are detrimental to most plants by limiting plant growth and productivity. It is of great necessity for plants to efficiently deal with the adverse effects caused by salt stress for survival and successful reproduction. Multiple determinants of salt tolerance have been identified in plants, and the cellular and physiological mechanisms of plant salt response and adaption have been intensely characterized. Plants respond to salt stress signals and rapidly initiate signaling pathways to re-establish cellular homeostasis with adjusted growth and cellular metabolism. This review summarizes the advances in salt stress perception, signaling, and response in plants. A better understanding of plant salt resistance will contribute to improving crop performance under saline conditions using multiple engineering approaches. The rhizosphere microbiome-mediated plant salt tolerance as well as chemical priming for enhanced plant salt resistance are also discussed in this review.

S-nitrosylation of the transcription factor MYB30 facilitates nitric oxide-promoted seed germination in Arabidopsis.

The gaseous signaling molecule nitric oxide (NO) plays an important role in breaking seed dormancy. NO induces a decrease in abscisic acid (ABA) content by transcriptionally activating its catabolic enzyme, the ABA 8'-hydroxylase CYP707A2. However, the underlying mechanism of this process remains unclear. Here, we report that the transcription factor MYB30 plays a critical role in NO-induced seed germination in Arabidopsis (Arabidopsis thaliana). MYB30 loss-of-function attenuates NO-mediated seed dormancy breaking. MYB30 triggers a NO-induced decrease in ABA content during germination by directly promoting CYP707A2 expression. NO induces S-nitrosylation at Cys-49 of MYB30 and enhances its transcriptional activity. Conversely, the ABA receptors PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) interact with MYB30 and repress its transcriptional activity. ABA promotes the interaction between PYL4 and MYB30, whereas S-nitrosylation releases the PYL4-mediated inhibition of MYB30 by interfering with the PYL4-MYB30 interaction. Genetic analysis showed that MYB30 functions downstream of PYLs during seed dormancy and germination in response to NO. Furthermore, MYB30 mutation significantly represses the reduced dormancy phenotype and the enhanced CYP707A2 expression of the pyr1 pyl1 pyl2 pyl4 quadruple mutant. Our findings reveal that S-nitrosylation of MYB30 precisely regulates the balance of seed dormancy and germination, providing insights into the underlying mechanism of NO-promoted seed germination.

RHA2b-mediated MYB30 degradation facilitates MYB75-regulated, sucrose-induced anthocyanin biosynthesis in Arabidopsis seedlings.

Anthocyanins play diverse roles in plant physiology and stress adaptation. In Arabidopsis, the MYB-bHLH-WD40 (MBW) complex has a crucial role in the regulation of anthocyanin synthesis. Here, we report that the R2R3-MYB transcription factor MYB30 and the ubiquitin E3 ligase RHA2b participate in anthocyanin biosynthesis through regulation of the MBW complex. MYB30 was found to negatively regulate sucrose-induced anthocyanin biosynthesis in Arabidopsis seedlings. Expression of multiple genes involved in flavonoid or anthocyanin biosynthesis was affected in the myb30 mutant, and MYB30 directly repressed the expression of MYB75, which encodes a core component of the MBW complex, by binding to its promoter. Moreover, MYB30 physically interacted with MYB75 to inhibit its activity by repressing MBW complex assembly. In addition, sucrose treatment significantly promoted MYB30 degradation via the action of RHA2b. The ubiquitination and degradation of MYB30 were significantly attenuated in the rha2b mutant under high-sucrose treatment, and further analysis showed that MYB75 directly promoted RHA2b expression in response to high sucrose. Our work thus reveals an anthocyanin biosynthetic regulatory module, RHA2b-MYB30, that controls the function of the MBW complex via MYB75. The repression of MYB75 by MYB30 is released by MYB75-induced RHA2b expression, thus ensuring the self-activation of MYB75 when anthocyanin synthesis is needed.

Ubiquitin negatively regulates ABA responses by inhibiting SnRK2.2 and SnRK2.3 kinase activity in Arabidopsis.

Abscisic acid (ABA) is an essential phytohormone for plant responses to complex and variable environmental conditions. The molecular basis of the ABA signaling pathway has been well elucidated. SnRK2.2 and SnRK2.3 are key protein kinases participating in ABA responses, and the regulation of their activity plays an important role in signaling. Previous mass spectroscopy analysis of SnRK2.3 suggested that ubiquitin and homologous proteins may bind directly to the kinase. Ubiquitin typically recruits E3 ubiquitin ligase complexes to target proteins, marking them for degradation by the 26S proteasome. Here, we show that SnRK2.2 and SnRK2.3 interact with ubiquitin but are not covalently attached to the protein, resulting in the suppression of their kinase activity. The binding between SnRK2.2, SnRK2.3, and ubiquitin is weakened under prolonged ABA treatment. Overexpression of ubiquitin positively regulated the growth of seedlings exposed to ABA. Our results thus demonstrate a novel function for ubiquitin, which negatively regulates ABA responses by directly inhibiting SnRK2.2 and SnRK2.3 kinase activity.

MYB30 Regulates Submergence Tolerance by Repressing Ethylene Biosynthesis via ACS7 in Arabidopsis.

Floods impose detrimental effects on natural and agro-ecosystems, leading to a significant loss of worldwide crop production. Global climate change has even worsened this situation. Flooding is a continuous process including two stages of submergence and re-oxygenation, and both are harmful to plant growth and development, resulting in a serious decline in crop yield. Therefore, the understanding of plant flooding tolerance and developing flooding-resistant crops are of great significance. Here, we report that the Arabidopsis thaliana (Arabidopsis) R2R3-MYB transcription factor MYB30 participates in plant submergence response through 1-aminocyclopropane-1-carboxylic acid synthase 7 (ACS7) by repressing ethylene (ET) biosynthesis. The MYB30 loss-of-function mutant exhibits reduced submergence tolerance with a higher level of ET production, whereas the MYB30-overexpressing plant displays enhanced submergence tolerance and repressed ET production. The coding gene of ACS7 might be a direct target of MYB30 during the submergence response. MYB30 binds to the promoter of ACS7 and represses its transcription. The ACS7 loss-of-function mutant with defect in ET biosynthesis displays enhanced submergence tolerance, whereas plants overexpressing ACS7 exhibit a submergence-sensitive phenotype. Genetic analysis shows that ACS7 functions downstream of MYB30 in both ET biosynthesis and submergence response. Taken together, our work revealed a novel transcriptional regulation that modulates submergence response in plants.

The MIEL1-ABI5/MYB30 regulatory module fine tunes abscisic acid signaling during seed germination.

The transcription factor ABSCISIC ACID INSENSITIVE5 (ABI5) plays a crucial role in abscisic acid (ABA) signaling during seed germination. However, how ABI5 is regulated during this process is poorly understood. Here, we report that the ubiquitin E3 ligase MIEL1 and its target transcription factor MYB30 modulate ABA responses in Arabidopsis thaliana during seed germination and seedling establishment via the precise regulation of ABI5. MIEL1 interacts with and ubiquitinates ABI5 to facilitate its degradation during germination. The transcription factor MYB30, whose turnover is mediated by MIEL1 during seed germination, also interacts with ABI5 to interfere with its transcriptional activity. MYB30 functions downstream of MIEL1 in the ABA response, and both are epistatic to ABI5 in ABA-mediated inhibition of seed germination and postgerminative growth. ABA treatment induces the degradation of MIEL1 and represses the interaction between MIEL1 and ABI5/MYB30, thus releasing both ABI5 and MYB30. Our results demonstrate that MIEL1 directly mediates the proteasomal degradation of ABI5 and inhibits its activity via the release of its target protein MYB30, thus ensuring precise ABA signaling during seed germination and seedling establishment.

PAMP-INDUCED SECRETED PEPTIDE 3 modulates salt tolerance through RECEPTOR-LIKE KINASE 7 in plants.

High soil salinity negatively affects plant growth and development, leading to a severe decrease in crop production worldwide. Here, we report that a secreted peptide, PAMP-INDUCED SECRETED PEPTIDE 3 (PIP3), plays an essential role in plant salt tolerance through RECEPTOR-LIKE KINASE 7 (RLK7) in Arabidopsis (Arabidopsis thaliana). The gene encoding the PIP3 precursor, prePIP3, was significantly induced by salt stress. Plants overexpressing prePIP3 exhibited enhanced salt tolerance, whereas a prePIP3 knockout mutant had a salt-sensitive phenotype. PIP3 physically interacted with RLK7, a leucine-rich repeat RLK, and salt stress enhanced PIP3-RLK7 complex formation. Functional analyses revealed that PIP3-mediated salt tolerance is dependent on RLK7. Exogenous application of synthetic PIP3 peptide activated RLK7, and salt treatment significantly induced RLK7 phosphorylation in a PIP3-dependent manner. Notably, MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3) and MPK6 were downstream of the PIP3-RLK7 module in salt response signaling. Activation of MPK3/6 was attenuated in pip3 or rlk7 mutants under saline conditions. Therefore, MPK3/6 might amplify salt stress response signaling in plants for salt tolerance. Collectively, our work characterized a novel ligand-receptor signaling cascade that modulates plant salt tolerance in Arabidopsis. This study contributes to our understanding of how plants respond to salt stress.

Plant salt response: Perception, signaling, and tolerance.

Salt stress is one of the significant environmental stressors that severely affects plant growth and development. Plant responses to salt stress involve a series of biological mechanisms, including osmoregulation, redox and ionic homeostasis regulation, as well as hormone or light signaling-mediated growth adjustment, which are regulated by different functional components. Unraveling these adaptive mechanisms and identifying the critical genes involved in salt response and adaption are crucial for developing salt-tolerant cultivars. This review summarizes the current research progress in the regulatory networks for plant salt tolerance, highlighting the mechanisms of salt stress perception, signaling, and tolerance response. Finally, we also discuss the possible contribution of microbiota and nanobiotechnology to plant salt tolerance.

Overexpression of PvWOX3a in switchgrass promotes stem development and increases plant height.

Switchgrass (Panicum virgatum L.) is an important perennial, noninvasive, tall ornamental grass that adds color and texture to gardens and landscapes. Moreover, switchgrass has been considered a forage and bioenergy crop because of its vigorous growth, low-input requirements, and broad geography. Here, we identified PvWOX3a from switchgrass, which encodes a WUSCHEL-related homeobox transcription factor. Transgenic overexpression of PvWOX3a in switchgrass increased stem length, internode diameter, and leaf blade length and width, all of which contributed to a 95% average increase in dry weight biomass compared with control plants. Yeast one-hybrid and transient dual-luciferase assays showed that PvWOX3a can repress the expression of gibberellin 2-oxidase and cytokinin oxidase/dehydrogenase through apparently direct interaction with their promoter sequences. These results suggested that overexpression of PvWOX3a could increase gibberellin and cytokinin levels in transgenic switchgrass plants, which promotes cell division, elongation, and vascular bundle development. We also overexpressed PvWOX3a in a transgenic miR156-overexpressing switchgrass line that characteristically exhibited more tillers, thinner internodes, and narrower leaf blades. Double transgenic switchgrass plants displayed significant increases in internode length and diameter, leaf blade width, and plant height but retained a tiller number comparable to that of plants expressing miR156 alone. Ultimately, the double transgenic switchgrass plants produced 174% more dry-weight biomass and 162% more solubilized sugars on average than control plants. These findings indicated that PvWOX3a is a viable potential genetic target for engineering improved shoot architecture and biomass yield of horticulture, fodder, and biofuel crops.

SIMP1 modulates salt tolerance by elevating ERAD efficiency through UMP1A-mediated proteasome maturation in plants.

Salt stress significantly induces accumulation of misfolded or unfolded proteins in plants. Endoplasmic reticulum (ER)-associated protein degradation (ERAD) and other degradative machineries function in the degradation of these abnormal proteins, leading to enhanced salt tolerance in plants. Here we characterise that a novel receptor-like kinase, Salt-Induced Malectin-like domain-containing Protein1 (SIMP1), elevates ERAD efficiency during salt stress through UMP1A, a putative proteasome maturation factor in Arabidopsis. SIMP1 loss-of-function caused a salt-hypersensitive phenotype. SIMP1 interacts and phosphorylates UMP1A, and the protein stability of UMP1A is positively regulated by SIMP1. SIMP1 modulates the 26S proteasome maturation possibly through enhancing the recruitment of specific ß subunits of the core catalytic particle to UMP1A. Functionally, the SIMP1-UMP1A module plays a positive role in ERAD efficiency in Arabidopsis. The degradation of misfolded/unfolded proteins was impaired in both simp1 and ump1a mutants during salt stress. Consistently, both simp1 and ump1a plants exhibited reduced ER stress tolerance. Phenotypic analysis revealed that SIMP1 regulates salt tolerance through UMP1A at least in part. Taken together, our work demonstrated that SIMP1 modulates plant salt tolerance by promoting proteasome maturation via UMP1A, therefore mitigating ER stress through enhanced ERAD efficiency under saline conditions.

MYB30 and ETHYLENE INSENEITIVE3 antagonistically regulate root hair growth and phosphorus uptake under phosphate deficiency in Arabidopsis.

Phosphate (Pi) deficiency is one of the major adverse factors limiting plant growth and production. Enhanced RH development is thought to be the typical root morphological response under Pi deficiency, which will enhance the utilization of Pi resources from soil. Here, we report that MYB30-EIN3 module is functionally implicated in Pi deficiency-induced RH development in Arabidopsis. MYB30 and EIN3 antagonistically regulate RH growth via transcriptional regulation of RSL4 as well as other PSR genes, resulting in fine-tuned Pi uptake under Pi deficiency.

Regulation of Plant Responses to Salt Stress.

Salt stress is a major environmental stress that affects plant growth and development. Plants are sessile and thus have to develop suitable mechanisms to adapt to high-salt environments. Salt stress increases the intracellular osmotic pressure and can cause the accumulation of sodium to toxic levels. Thus, in response to salt stress signals, plants adapt via various mechanisms, including regulating ion homeostasis, activating the osmotic stress pathway, mediating plant hormone signaling, and regulating cytoskeleton dynamics and the cell wall composition. Unraveling the mechanisms underlying these physiological and biochemical responses to salt stress could provide valuable strategies to improve agricultural crop yields. In this review, we summarize recent developments in our understanding of the regulation of plant salt stress.

Protein kinase PpCIPK1 modulates plant salt tolerance in Physcomitrella patens.

KEY MESSAGE: This work demonstrates that PpCIPK1, a putative protein kinase, participates in regulating plant salt tolerance in moss Physcomitrella patens. Calcineurin B-Like protein (CBL)-interacting protein kinases (CIPKs) have been reported to be involved in multiple signaling networks and function in plant growth and stress responses, however, their biological functions in non-seed plants have not been well characterized. In this study, we report that PpCIPK1, a putative protein kinase, participates in regulating plant salt tolerance in moss Physcomitrella patens (P. patens). Phylogenetic analysis revealed that PpCIPK1 shared high similarity with its homologs in higher plants. PpCIPK1 transcription level was induced upon salt stress in P. patens. Using homologous recombination, we constructed PpCIPK1 knockout mutant lines (PpCIPK1 KO). Salt sensitivity analysis showed that independent PpCIPK1 KO plants exhibited severe growth inhibition and developmental deficiency of gametophytes under salt stress condition compared to that of wild-type P. patens (WT). Consistently, ionic homeostasis was disrupted in plants due to PpCIPK1 deletion, and high level of H2O2 was accumulated in PpCIPK1 KO than that in WT. Furthermore, PpCIPK1 functions in regulating photosynthetic activity in response to salt stress. Interestingly, we observed that PpCIPK1 could completely rescue the salt-sensitive phenotype of sos2-1 to WT level in Arabidopsis, indicating that AtSOS2 and PpCIPK1 are functionally conserved. In conclusion, our work provides evidence that PpCIPK1 participates in salt tolerance regulation in P. patens.

MYB30 and ETHYLENE INSENSITIVE3 antagonistically modulate root hair growth in Arabidopsis.

Root hair (RH) is essential for plant nutrient acquisition and the plant-environment communication. Here we report that transcription factors MYB30 and ETHYLENE INSENSITIVE3 (EIN3) modulate RH growth/elongation in Arabidopsis in an antagonistic way. The MYB30 loss-of-function mutant displays enhanced RH length, whereas the RH elongation in MYB30-overexpressing plants is highly repressed. MYB30 physically interacts with EIN3, a master transcription factor in ethylene signaling. MYB30 directly binds the promoter region of ROOT HAIR DEFECTIVE SIX-LIKE4 (RSL4) and represses its transcription. RSL4 loss-of-function suppresses the enhanced RH growth in myb30 mutant plants. Ethylene enhances MYB30-EIN3 complex formation, and reduces the association between MYB30 and RSL4 promotor via the action of EIN3. MYB30 and EIN3 antagonistically regulate the expression of RSL4 and a subset of core RH genes in a genome-wide way. Taken together, our work revealed a novel transcriptional network that modulates RH growth in plants.

The GSK3-like Kinase BIN2 Is a Molecular Switch between the Salt Stress Response and Growth Recovery in Arabidopsis thaliana.

Plant stress responses involve dynamic growth regulation. Growth is restricted in harsh environmental conditions and is rapidly restored when conditions improve. Here, we identified BIN2, a glycogen synthase kinase 3 (GSK3)-like kinase, as a molecular switch in the transition to robust growth after salt stress in Arabidopsis thaliana. In the rapid recovery phase after salt stress, the calcium sensors SOS3 and SCaBP8 perceive a calcium signal and promote BIN2 localization to the plasma membrane to repress the salt stress response, and BIN2 inhibits SOS2 activity and enhances growth by releasing BZR1/BES1 transcriptional activity. The expression of stress- and brassinosteroid-responsive genes is coordinately regulated during this process. bin2-3bil1 and bin2-3bil2 mutants defective in BIN2 and its homologs BIL1 and BIL2, respectively, are hyposensitive to salt stress. Our study suggests that salt signaling modulates the subcellular localization and interactions of BIN2. By phosphorylating different substrates, BIN2 regulates the salt stress response and growth recovery.

PpAOX regulates ER stress tolerance in Physcomitrella patens.

Severe environments disturb the folding or assembly of newly synthesized proteins, resulting in accumulation of misfolded or unfolded proteins in the endoplasmic reticulum (ER) as well as cytotoxic aggregation of abnormal proteins. Therefore, ER stress is evoked due to disturbed ER homeostasis. Alternative oxidase (AOX) plays an important role in coping with various abiotic stresses and plant growth. Our previous study has reported that PpAOX is involved in the regulation of salt tolerance in moss Physcomitrella patens (P. patens), but its biological functions in modulating ER stress remain unknown. Here we report that the gametophyte of P. patens displays severe growth inhibition and developmental deficiency under tunicamycin (Tm, an elicitor of ER stress)-induced ER stress conditions. PpAOX and selected ER stress response-like genes in P. patens were induced under Tm treatment. PpAOX knockout (PpAOX KO) plants exhibited decreased resistance to Tm-induced ER stress, whereas PpAOX-overexpressing lines (PpAOX OX) plants were more tolerant to Tm-induced ER stress. Data showed that PpAOX contributes to redox homeostasis under Tm treatment. In addition, we observed that PpAOX completely restores the Tm-sensitive phenotype of Arabidopsis AOX1a mutant (Ataox1a). Taken together, our work reveals a functional link between PpAOX and ER stress tolerance regulation in P. patens.

ABI5 modulates seed germination via feedback regulation of the expression of the PYR/PYL/RCAR ABA receptor genes.

As abscisic acid (ABA) receptors, PYR1/PYL/RCAR (PYLs) play important roles in ABA-mediated seed germination, but the regulation of PYLs in this process, especially at the transcriptional level, remains unclear. In this study, we found that expression of 11 of 14 PYLs changes significantly during seed germination and is affected by exogenous ABA. Two PYLs, PYL11 and PYL12, both of which are expressed specifically in mature seeds, positively modulate ABA-mediated seed germination. However, ABI5 was found to modulate the PYL11- and PYL12-mediated ABA response. In the abi5-7 mutant, ABA hypersensitivity caused by PYL11 and PYL12 overexpression was totally or partially blocked. By contrast, ABI5 regulates the expression of PYL11 and PYL12 by directly binding to their promoters. Moreover, the expression of eight other PYLs is also affected during the germination of abi5 mutants. Promoter analysis revealed that an ABI5-binding region is present next to the TATA box or initiator box. Together, our data demonstrate the role of PYL11 and PYL12 in seed germination. In addition, the identification of PYLs as targets of ABI5 reveals a role of ABI5 in the feedback regulation of ABA-mediated seed germination.

SUMOylation of MYB30 enhances salt tolerance by elevating alternative respiration via transcriptionally upregulating AOX1a in Arabidopsis.

Salt stress reduces crop growth and productivity globally. Here we report that a R2R3-MYB transcription factor MYB30 participates in salt tolerance in Arabidopsis. MYB30 can be SUMOylated by SIZ1 in response to salt stress and the lysine (K)283 of MYB30 is essential for its SUMOylation. In contrast to wild-type MYB30, the MYB30K283R mutant failed to rescue the salt-sensitive phenotype of the myb30-2 mutant, indicating that SUMOylation of MYB30 is required for the salt-stress response. Through transcriptomic analysis, we identified a MYB30 target, alternative oxidase 1a (AOX1a). MYB30 binds the promoter of AOX1a and upregulates its expression in response to salt stress; however, MYB30K283R cannot bind the promoter of AOX1a. The cyanide (CN)-resistant alternative respiration (Alt) mediated by AOX is significantly reduced in the myb30-2 mutant through the loss of function of MYB30. As a result, the redox homeostasis is disrupted in the myb30-2 mutant compared with that in wild-type seedlings (WT) under salt conditions. The artificial elimination of excess reactive oxygen species partially rescues the salt-sensitive phenotype of the myb30-2 mutant, whereas after the exogenous application of SHAM, an inhibitor of AOXs and Alt respiration, the salt tolerance of Col-0 and the complemented plants decreased to a level similar to that observed in myb30-2. Finally, overexpression of AOX1a in myb30-2 confers WT-like salt tolerance compared with that of the myb30-2 mutant. Taken together, our results revealed a functional link between MYB30 and AOX1a, and indicated that SIZ1-mediated SUMOylation of MYB30 enhances salt tolerance by regulating Alt respiration and cellular redox homeostasis via AOX1a in Arabidopsis.

Patellin1 negatively regulates plant salt tolerance by attenuating nitric oxide accumulation in Arabidopsis.

Salt stress adversely affects plant growth and development. Multiple adaptive mechanisms have been used for plant salt tolerance. We previously reported that membrane trafficking-related protein patellin1 (PATL1) negatively regulates plant salt tolerance. Here, we characterized that Arabidopsis PATL1 negatively modulates nitric oxide (NO) accumulation upon salt exposure. Our work revealed a functional link between salt response and NO signaling.

A Functional Alternative Oxidase Modulates Plant Salt Tolerance in Physcomitrella patens.

Alternative oxidase (AOX) has been reported to be involved in mitochondrial function and redox homeostasis, thus playing an essential role in plant growth as well as stress responses. However, its biological functions in nonseed plants have not been well characterized. Here, we report that AOX participates in plant salt tolerance regulation in moss Physcomitrella patens (P. patens). AOX is highly conserved and localizes to mitochondria in P. patens. We observed that PpAOX rescued the impaired cyanide (CN)-resistant alternative (Alt) respiratory pathway in Arabidopsis thaliana (Arabidopsis) aox1a mutant. PpAOX transcription and Alt respiration were induced upon salt stress in P. patens. Using homologous recombination, we generated PpAOX-overexpressing lines (PpAOX OX). PpAOX OX plants exhibited higher Alt respiration and lower total reactive oxygen species accumulation under salt stress condition. Strikingly, we observed that PpAOX OX plants displayed decreased salt tolerance. Overexpression of PpAOX disturbed redox homeostasis in chloroplasts. Meanwhile, chloroplast structure was adversely affected in PpAOX OX plants in contrast to wild-type (WT) P. patens. We found that photosynthetic activity in PpAOX OX plants was also lower compared with that in WT. Together, our work revealed that AOX participates in plant salt tolerance in P. patens and there is a functional link between mitochondria and chloroplast under challenging conditions.