OsMas1,a novel maspardin protein gene,confers tolerance to salt and drought stresses by regulating ABA signaling in rice

Drought and salt stresses,the major environmental abiotic stresses in agriculture worldwide,affect plant growth,crop productivity,and quality.Therefore,developing crops with higher drought and salt tolerance is highly desirable.This study reported the isolation,biological function,and molecular characterization of a novel maspardin gene,OsMas1,from rice.The OsMas1 protein was localized to the cytoplasm.The expression levels of OsMas1 were up-regulated under mannitol,PEG6000,NaCl,and abscisic acid(ABA) treatments in rice.The OsMas1 gene was introduced into the rice cultivar Zhonghua 11(wild type,WT).OsMas1-overexpression(OsMas1-OE) plants exhibited significantly enhanced salt and drought tolerance;in contrast,OsMas1-interference(OsMas1-RNAi) plants exhibited decreased tolerance to salt and drought stresses,compared with WT.OsMas1-OE plants exhibited enhanced hypersensitivity,while OsMas1-RNAi plants showed less sensitivity to exogenous ABA treatment at both germination and post-germination stages.ABA,proline and K+ contents and superoxide dismutase(SOD),catalase(CAT),peroxidase(POD),and photosynthesis activities were significantly increased.In contrast,malonaldehyde(MDA),hydrogen peroxide(H2O2),superoxide anion radical(O2-··),and Na+ contents were significantly decreased in OsMas1-OE plants compared with OsMas1-RNAi and WT plants.Overexpression of OsMas1 up-regulated the genes involved in ABA signaling,proline biosynthesis,reactive oxygen species(ROS)-scavenging system,photosynthesis,and ion transport under salt and drought stresses.Our results indicate that the OsMas1 gene improves salt and drought tolerance in rice,which may serve as a candidate gene for enhancing crop resistance to abiotic stresses.

Mepiquat chloride priming confers the ability of cotton seed to tolerate salt by promoting ABA-operated GABA signaling control of the ascorbate–glutathione cycle

Background Ensuring that seeds germinate and emerge normally is a prerequisite for cotton production,esp.in areas with salinized soil.Priming with mepiquat chloride(MC)can promote seed germination and root growth under salt stress,but its mechanism has not been fully elucidated.In this study,physiological and biochemical experiments revealed that MC-priming promotes the tolerance of cotton seeds to salt stress by increasing the ability of antioxidant enzymes related to the ascorbate-glutathione(AsA-GSH)cycle to scavenge reactive oxygen species(ROS).Results Results revealed that treatment with inhibitors of abscisic acid(ABA)and γ-aminobutyric acid(GABA)biosynthesis reduced the positive effects of MC-priming.Similarly,MC-priming increased the contents of ABA and GABA under salt stress by stimulating the expression levels of GhNCED2 and GhGAD4 and the activity of calmodulin-binding(CML)glutamate decarboxylase(GAD).Further analysis showed that an inhibitor of ABA synthesis reduced the positive impacts of MC-priming on the content of GABA under salt stress,but the content of ABA was not affected by the GABA synthesis inhibitor.Furthermore,a multi-omics analysis revealed that MC-priming increased the abundance and phosphorylation levels of the proteins related to ABA signaling,CML,and Ca^(2+)channels/transporters in the MC-primed treatments,which resulted in increased oscillations in Ca^(2+)in the MC-primed cotton seeds under salt stress.Conclusion In summary,these results demonstrate that MC-mediated ABA signaling operates upstream of the GABA synthesis generated by GAD by activating the oscillations of Ca^(2+)and then enhancing activity of the AsA-GSH cycle,which ensures that cotton seeds are tolerant to salt stress.

Auxin response factor gene MdARF2 is involved in ABA signaling and salt stress response in apple

Auxin response factors(ARFs)play key roles throughout the whole process of plant growth and development,and mediate auxin response gene transcription by directly binding with auxin response elements(AuxREs).However,their functions in abiotic stresses are largely limited,especially in apples.Here,the auxin response factor gene MdARF2(HF41569)was cloned from apple cultivar‘Royal Gala’(Malus×domestica Borkh.).Phylogenetic analysis showed that ARF2 proteins are highly conserved among different species and MdARF2 is the closest relative to PpARF2 of Prunus persica,but they differ at the DNA level.MdARF2 contains three typical conserved domains including the B3 DNAbinding domain,Auxin_resp domain and AUX_IAA domain.The subcellular localization demonstrated that MdARF2 is localized in the nucleus.The three-dimensional structure prediction of the proteins showed that MdARF2 is highly similar with AtARF2,and they contain helices,folds,and random coils.The promoter of MdARF2 contains cis-acting elements which respond to various stresses,as well as environmental and hormonal signals.Expression analysis showed that MdARF2 is widely expressed in all tissues of apple,with the highest expression of MdARF2 in root.Functional analysis with a series of MdARF2 transgenic apple calli indicated that MdARF2 can reduce the sensitivity to ABA signaling and enhance salt tolerance in apple.In summary,the results of this research provide a new basis for studying the regulation of abiotic stresses by ARFs.

Tyrosylprotein sulfotransferase suppresses ABA signaling via sulfation of SnRK2.2/2.3/2.6

Phytohormone abscisic acid(ABA)plays vital roles in stress tolerance,while long-term overactivation of ABA signaling suppresses plant growth and development.However,the braking mechanism of ABA responses is not clear.Protein tyrosine sulfation catalyzed by tyrosylprotein sulfotransferase(TPST)is a critical post-translational modification.Through genetic screening,we identified a tpst mutant in Arabidopsis that was hypersensitive to ABA.In-depth analysis revealed that TPST could interact with and sulfate SnRK2.2/2.3/2.6,which accelerated their degradation and weakened the ABA signaling.Taken together,these findings uncovered a novel mechanism of desensitizing ABA responses via protein sulfation.

LJbiquitin--Proteasome System in ABA Signaling： From Perception to Action

Protein post-translational modification （PTM） by ubiquitination has been observed during many aspects of plant growth, development, and stress responses. The ubiquitin-proteasome system precisely regulates phytohormone signaling by affecting protein activity, localization, assembly, and interaction ability. Absci- sic acid （ABA） is a major phytohormone, and plays important roles in plants under normal or stressed growth conditions. The ABA signaling pathway is composed of phosphatases, kinases, transcription fac- tors, and membrane ion channels. It has been reported that multiple ABA signaling transducers are sub- jected to the regulations by ubiquitination. In particular, recent studies have identified different types of E3 ligases that mediate ubiquitination of ABA receptors in different cell compartments. This review focuses on modulation of these components by monoubiquitination or polyubiquitination that occurs in the plasma membrane, endomembranes, and from the cytosol to the nucleus; this implies the existence of retrograde and trafficking processes that are regulated by ubiquitination in ABA signaling. A number of single-unit E3 ligases, components of multi-subunit E3 ligases, E2s, and specific subunits of the 26S proteasome involved in ABA signal regulation are discussed. Dissecting the precise functions of ubiquitination in the ABA pathway may help us understand key factors in the signaling of other phytohormones regulated by ubiqui- tination and other types of PTMs.

ROP11 GTPase Negatively Regulates ABA Signaling by Protecting ABI1 Phosphatase Activity from Inhibition by the ABA Receptor RCAR1/PYL9 in Arabidopsis

The phytohormone abscisic acid （ABA） regulates many key processes in plants, such as seed germina- tion, seedling growth, and abiotic stress tolerance. In recent years, a minimal set of core components of a major ABA signaling pathway has been discovered. These components include a RCAR/PYR/PYL family of ABA receptors, a group of PP2C phosphatases, and three SnRK2 kinases. However, how the interactions between the receptors and their targets are regulated by other proteins remains largely unknown. In a companion paper published in this issue, we showed that ROP11, a member of the plant- specific Rho-like small GTPase family, negatively regulates multiple ABA responses in Arabidopsis. The current work demonstrated that the constitutively active ROP11 （CA-ROP11） can modulate the RCAR1/PYL9-mediated ABA signaling pathway based on reconstitution assays in Arabidopsis thaliana protoplasts. Furthermore, using luciferase complementation imaging, yeast two-hybrid assays, co- immunoprecipitation assays in Nicotiana benthamiana and bimolecular fluorescence complementation assays, we demonstrated that CA-ROP11 directly interacts with ABI1, a signaling component downstream of RCAR1/PYL9. Finally, we provided biochemical evidence that CA-ROP11 protects ABI1 phosphatase activity from inhibition by RCAR1/PYL9 and thus negatively regulates ABA signaling in plant cells. A model of how ROP11 acts to negatively regulate ABA signaling is presented.

Counteraction of ABA-Mediated Inhibition of Seed Germination and Seedling Establishment by ABA Signaling Terminator in Arabidopsis

Seed germination and seedling establishment are important for the reproductive success of plants,but seeds and seedlings typically encounter constantly changing environmental conditions.By inhibiting seed germination and post-germinative growth through the PYR1/PYL/RCAR ABA receptors and PP2C co-receptors,the phytohormone abscisic acid(ABA)prevents premature germination and seedling growth under unfavorable conditions.However,little is known about how the ABA-mediated inhibition of seed germination and seedling establishment is thwarted.Here,we report that ABA Signaling Terminator(ABT),a WD40 protein,efficiently switches off ABA signaling and is critical for seed germination and seedling establishment.ABT is induced by ABA in a PYR1/PYL/RCAR-PP2C-dependent manner.Overexpression of ABT promotes seed germination and seedling greening in the presence of ABA,whereas knockout of ABT has the opposite effect.We found that ABT interacts with the PYR1/PYL/RCAR and PP2C proteins,interferes with the interaction between PYR1/PYL4 and ABI1/ABI2,and hampers the inhibition of ABI1/ABI2 by ABA-bound PYR1/PYL4,thereby terminating ABA signaling.Taken together,our results reveal a core mechanism of ABA signaling termination that is critical for seed germination and seedling establishment in Arabidopsis.

Rheostatic Control of ABA Signaling through HOS15-Mediated OST1 Degradation

Dehydrating stresses trigger the accumulation of abscisic acid(ABA),a key plant stress-signaling hormone that activates Snf1-Related Kinases(SnRK2s)to mount adaptive responses.However,the regulatory circuits that terminate the SnRK2s signal relay after acclimation or post-stress conditions remain to be defined.Here,we show that the desensitization of the ABA signal is achieved by the regulation of OST1(SnRK2.6)protein stability via the E3-ubiquitin ligase HOS15.Upon ABA signal,HOS15-induced degradation of OST1 is inhibited and stabilized OST1 promotes the stress response.When the ABA signal terminates,protein phosphatases ABI1/2 promote rapid degradation of OST1 via HOS15.Notably,we found that even in the presence of ABA,OST1 levels are also depleted within hours of ABA signal onset.The unexpected dynamics of OST1 abundance are then resolved by systematic mathematical modeling,demonstrating a desensitizing feedback loop by which OST1-induced upregulation of ABI1/2 leads to the degradation of OST1.This model illustrates the complex rheostat dynamics underlying the ABA-induced stress response and desensitization.

NUA and ESD4 negatively regulate ABA signaling during seed germination

The phytohormone abscisic acid(ABA)plays important roles in plant growth,development and adaptative responses to abiotic stresses.SNF1-related protein kinase 2s(SnRK2)are key components that activate the ABA core signaling pathway.NUCLEAR PORE ANCHOR(NUA)is a component of the nuclear pore complex(NPC)that involves in deSU-MOylation through physically interacting with the EARLY IN SHORT DAYS 4(ESD4)SUMO protease.However,it is not clear how NUA functions with SnRK2 and ESD4 to regulate ABA signaling.In our study,we found that nua loss-of-function mutants exhibited pleiotropic ABA-hypersensitive phenotype.We also found that ABA-responsive genes remarkably up-regulated in nua by exogenous ABA.The nua snrk2.2 snrk2.3 triple mutant and nua abi5 double mutant partially rescued the ABA-hypersensitive phenotype of nua,thereby suggesting that NUA is epistatic to SnRK2s.Additionally,we observed that esd4-3 mutant was also ABA-hypersensitive.NUA and ESD4 were further demonstrated to physically interact with SnRK2s and negatively regulate ABA signaling by reducing SnRK2s stability.Taken together,our findings uncover a new regulatory mechanism that can modulate ABA signaling.

Drought-triggered repression of miR166 promotes drought tolerance in soybean

Drought stress limits agricultural productivity worldwide.Identifying and characterizing genetic components of drought stress-tolerance networks may improve crop resistance to drought stress.We show that the regulatory module formed by miR166 and its target gene,ATHB14-LIKE,functions in the regulation of drought tolerance in soybean(Glycine max).Drought stress represses the accumulation of miR166,leading to upregulation of its target genes.Optimal knockdown of miR166 in the stable transgenic line GmSTTM166 conferred drought tolerance without affecting yield.Expression of ABA signaling pathway genes was regulated by the miR166-mediated regulatory pathway,and ATHB14-LIKE directly activates some of these genes.There is a feedback regulation between ATHB14-LIKE and MIR166 genes,and ATHB14-LIKE inhibits MIR166 expression.These findings reveal that drought-triggered regulation of the miR166-mediated regulatory pathway increases plants drought resistance,providing new insights into drought stress regulatory network in soybean.

Roles of ABA Signal Transduction during Higher Plant Seed Development and Germination

ABA is one of the 5 phytohormones in higher plants, which is also the most important hormone that regulates higher plants in response to environmental stress, by ABA signal transduction. Understanding ABA signal transduction at the molecular level is crucial to biology and ecology, and rational breeding complied with corresponding eco-environmental changes. Great advancements have taken place over the past 10 years by application of the Arabidopsis experimental system. Many components involved in ABA signal transduction have been isolated and identified and a clear overall picture of gene expression and control for this transduction has become an accepted fact. On the basis of the work in our laboratory, in conjunction with the data available at the moment, the authors have attempted to integrate ABA signal transduction pathways into a common one and give some insights into the relationship between ABA signal transduction and other hormone signal transduction pathways, with an emphasis upon the ABA signal transduction during higher plant seed development. A future challenge in this field is that different experimental systems are applied and various receptors and genes need to be characterized through the utilization of microarray chips.

TaSRO1 interacts with TaVP1 to modulate seed dormancy and pre-harvest sprouting resistance in wheat

Dormancy is an adaptive trait which prevents seeds from germinating under unfavorable environmental conditions.Seeds with weak dormancy undergo pre-harvest sprouting(PHS)which decreases grain yield and quality.Understanding the genetic mechanisms that regulate seed dormancy and resistance to PHS is crucial for ensuring global food security.In this study,we illustrated the function and molecular mechanism of TaSRO1 in the regulation of seed dormancy and PHS resistance by suppressing TaVP1.The tasro1 mutants exhibited strong seed dormancy and enhanced resistance to PHS,whereas the mutants of tavp1 displayed weak dormancy.Genetic evidence has shown that TaVP1 is epistatic to TaSRO1.Biochemical evidence has shown that TaSRO1 interacts with TaVP1 and represses the transcriptional activation of the PHS resistance genes TaPHS1 and TaSdr.Furthermore,TaSRO1 undermines the synergistic activation of TaVP1 and TaABI5 in PHS resistance genes.Finally,we highlight the great potential of tasro1 alleles for breeding elite wheat cultivars that are resistant to PHS.

OsNAC5 orchestrates OsABI5 to fine-tune cold tolerance in rice

Due to its tropical origins,rice(Oryza sativa)is susceptible to cold stress,which poses severe threats to production.OsNAC5,a NAC-type transcription factor,participates in the cold stress response of rice,but the detailed mechanisms remain poorly understood.Here,we demonstrate that OsNAC5 positively regulates cold tolerance at germination and in seedlings by directly activating the expression of ABSCISIC ACID INSENSITIVE 5(OsABI5).Haplotype analysis indicated that single nucleotide polymorphisms in a NAC-binding site in the OsABI5 promoter are strongly associated with cold tolerance.OsNAC5 also enhanced OsABI5 stability,thus regulating the expression of cold-responsive(COR)genes,enabling fine-tuned control of OsABI5 action for rapid,precise plant responses to cold stress.DNA affinity purification sequencing coupled with transcriptome deep sequencing identified several OsABI5 target genes involved in COR expression,including DEHYDRATION-RESPONSIVE ELEMENT BINDING FACTOR 1A(OsDREB1A),OsMYB20,and PEROXIDASE 70(OsPRX70).In vivo and in vitro analyses suggested that OsABI5 positively regulates COR gene transcription,with marked COR upregulation in OsNAC5-overexpressing lines and downregulation in osnac5 and/or osabi5 knockout mutants.This study extends our understanding of cold tolerance regulation via OsNAC5 through the OsABI5-CORs transcription module,which may be used to ameliorate cold tolerance in rice via advanced breeding.

The Arabidopsis spliceosomal protein SmEb modulates ABA responses by maintaining proper alternative splicing of HAB1

Abscisic acid(ABA)signaling is critical for seed germination and abiotic stress responses in terrestrial plants.PremRNA splicing is known to regulate ABA signaling.However,the involvement of canonical spliceosomal components in regulating ABA signaling is poorly understood.Here,we show that the spliceosome component Sm core protein SmEb plays an important role in ABA signaling.SmEb expression is up-regulated by ABA treatment,and analysis of Arabidopsis smeb mutant plants suggest that SmEb modulates the alternative splicing of the ABA signaling component HAB1 by enhancing the HAB1.1 splicing variant while repressing HAB1.2.Overexpression of HAB1.1 but not HAB1.2 rescues the ABA-hypersensitive phenotype of smeb mutants.Mutations in the transcription factor ABI3,4,or 5 also reduce the ABA hypersensitivity of smeb mutants during seed germination.Our results show that the spliceosomal component SmEb plays an important role in ABA regulation of seed germination and early seedling development.

Arabidopsis co-chaperonin CPN20 antagonizes Mg-chelatase H subunit to derepress ABA-responsive WRKY40 transcription repressor

Our previous study demonstrated that a chloroplast co-chaperonin 20(CPN20),one of the interaction partners of the magnesium-protoporphyrin IX chelatase H subunit(CHLH/ABAR),negatively regulates ABA signaling at the same node with ABAR but upstream of WRKY40 transcription repressor in Arabidopsis thaliana.In the present experiment,we showed that ABA directly inhibits the ABAR-CPN20 interaction,and also represses expression of CPN20,which depends on ABAR.CPN20 inhibits ABAR-WRKY40 interaction by competitively binding to ABAR.ABAR downregulates,but CPN20 upregulates,WRKY40 expression.The cpn20-1 mutation induces downregulation of WRKY40,and suppresses the upregulated level of WRKY40 due to the cch mutation in the ABAR gene.ABA-induced repressive effect of the WRKY40 gene is strengthened by downregulation of CPN20 but reduced by upregulation of CPN20.Together with our previously reported genetic data,we provide evidence that CPN20 functions through antagonizing the ABAR-WRKY40 coupled pathway,and ABA relieves this pathway of repression by inhibiting the ABAR-CPN20 interaction to activate ABAR-WRKY40 interaction.

HOS3, an ELO-Like Gene, Inhibits Effects of ABA and Implicates a S-1-P/Ceramide Control System for Abiotic Stress Responses in Arabidopsis thaliana

A hyper-osmotically sensitive mutant of Arabidopsis thaliana, designated hos3-1 （high expression of osmotically responsive genes）, was identified based on its hyper-luminescence of RD29A：LUC promoter fusion plants upon treatment with NaCI and ABA. These responses implicate the disrupted gene as a direct or indirect negative regulator of the RD29A stress-responsive pathway. By sequencing the flanking regions of the T-DNA borders, it was determined that the disrupted gene is at locus At4g36830, annotated as encoding a putative protein with high homology to CIG30 （ELO2/FEN1）. CIG30 has been implicated in synthesis of very long chain fatty acids （VLCFA）, which are essential precursors for sphingolipids and ceramides. Altered stress responses characteristic of ABA-hypersensitivity, including reduced root growth inhibition and reduced germination with ABA treatment and reduced water loss from leaves, were exhibited by allelic hos3-1 and hos3-2 mutants. The hos3-2 mutant is partially suppressed in its transcript abundance and is inherited as a recessive trait. Further, the HOS30RF under the control of the 35SCaMV promoter restored wild-type NaCI- and ABA-root growth sensitivity as well as RD29A：LUC luminescence in mutant plants. We also show here that the HOS3 wild-type gene functionally complements the sensitivity of elo2 and elo3 yeast mutants to monensin. Furthermore, both hos3-1 and hos3-2 alleles shared increased sensitivity to the herbicide Metolachlor, which inhibits acyl chain elongation in synthesis of VLCFA, and HOS3 functionally complemented both elo2 and elo3 and restored levels of VLCFA. Together, these data establish that HOS3 inhibits ABA-mediated stress responses and implicate the VLCFA pathway and products as control points for several aspects of abiotic stress signaling and responses. The results also provide support for a role of ceramide in the control of stomatal behavior.

Shoot-Specific Down-Regulation of Protein Farnesyltransferase （α-Subunit） for Yield Protection against Drought in Canola

Canola （Brassica napus L.） is one of the most important oilseed crops in the world and its seed yield and quality are significantly affected by drought stress. As an innate and adaptive response to water deficit, land plants avoid potential damage by rapid biosynthesis of the phytohormone abscisic acid （ABA）, which triggers stomatal closure to reduce transpirational water loss. The ABA-mediated stomatal response is a dosage-dependent process; thus, one genetic engineering approach for achieving drought avoidance could be to sensitize the guard cell＇s responsiveness to this hormone. Recent genetic studies have pinpointed protein farnesyltransferase as a key negative regulator controlling ABA sensitivity in the guard cells. We have previously shown that down-regulation of the gene encoding Arabidopsis β-subunit of farnesyltransferase （ERA1） enhances the plant＇s sensitivity to ABA and drought tolerance. Although the β-subunit of famesyltransferase （AtFTA） is also implicated in ABA sensing, the effectiveness of using such a gene target for improving drought tolerance in a crop plant has not been validated. Here, we report the identification and characterization of the promoter of Arabidopsis hydroxypyruvate reductase （AtHPR1）, which expresses specifically in the shoot and not in non-photosynthetic tissues such as root. The promoter region of AtHPR1 contains the core motif of the well characterized dehydration-responsive cis-acting element and we have confirmed thatAtHPR1 expression is inducible by drought stress. Conditional and specific down-regulation of FTA in canola using the AtHPR1 promoter driving an RNAi construct resulted in yield protection against drought stress in the field. Using this molecular strategy, we have made significant progress in engineering drought tolerance in this important crop species.

RAF22,ABI1 and OST1 form a dynamic interactive network that optimizes plant growth and responses to drought stress in Arabidopsis

Plants adapt to their ever-changing environment via positive and negative signals induced by environmental stimuli.Drought stress,for instance,induces accumulation of the plant hormone abscisic acid(ABA),triggering ABA signal transduction.However,the molecular mechanisms for switching between plant growth promotion and stress response remain poorly understood.Here we report that RAF(rapidly accelerated fibrosarcoma)-LIKE MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE 22(RAF22)in Arabidopsis tha/iana physically interacts with ABA INSENSITIVE 1(ABl1)and phosphorylates ABl1 at Ser416 residue to enhance its phosphatase activity.Interestingly,ABl1 can also enhance the activity of RAF22 through dephosphorylation,reciprocally inhibiting ABA signaling and promoting the maintenance of plant growth under normal conditions.Under drought stress,however,the ASA-activated OPEN STOMATA1(OST1)phosphorylates the Ser81 residue of RAF22 and inhibits its kinase activity,restraining its enhancement of ABl1 activity.Taken together,our study reveals that RAF22,ABl1,and OST1 form a dynamic regulatory network that plays crucial roles in optimizing plant growth and environmental adaptation under drought stress.