Drought stress globally poses a significant threat to maize (Zea mays L.) productivity and the underlying molecular mechanisms of drought tolerance remain elusive. In this study, we characterized ZmbHLH47, a basic helix-loop-helix (bHLH) transcription factor, as a positive regulator of drought tolerance in maize. ZmbHLH47 expression was notably induced by both drought stress and abscisic acid (ABA). Transgenic plants overexpressing ZmbHLH47 displayed elevated drought tolerance and ABA responsiveness, while the zmbhlh47 mutant exhibited increased drought sensitivity and reduced ABA sensitivity. Mechanistically, it was revealed that ZmbHLH47 could directly bind to the promoter of ZmSnRK2.9 gene, a member of the subgroup III SnRK2 kinases, activating its expression. Furthermore, ZmSnRK2.9-overexpressing plants exhibited enhanced ABA sensitivity and drought tolerance, whereas the zmsnrk2.9 mutant displayed a decreased sensitivity to both. Notably, overexpressing ZmbHLH47 in the zmsnrk2.9 mutant closely resembled the zmsnrk2.9 mutant, indicating the importance of the ZmbHLH47-ZmSnRK2.9 module in ABA response and drought tolerance. These findings provided valuable insights and a potential genetic resource for enhancing the environmental adaptability of maize.

BACKGROUND: Abscisic acid (ABA) receptor pyrabactin resistance 1/PYR1-like/regulatory components of ABA receptor proteins (PYR/PYL/RCARs) have been demonstrated to play pivotal roles in ABA signaling and in response to diverse environmental stimuli including drought, salinity and osmotic stress in Arabidopsis. However, whether and how GhPYL9-5D and GhPYR1-3A, the homologues of Arabidopsis PYL9 and PYR1 in cotton, function in responding to ABA and abiotic stresses are still unclear.
RESULTS: GhPYL9-5D and GhPYR1-3A were targeted to the cytoplasm and nucleus. Overexpression of GhPYL9-5D and GhPYR1-3A in Arabidopsis wild type and sextuple mutant pyr1pyl1pyl2pyl4pyl5pyl8 plants resulted in ABA hypersensitivity in terms of seed germination, root growth and stomatal closure, as well as seedling tolerance to water deficit, salt and osmotic stress. Moreover, the VIGS (Virus-induced gene silencing) cotton plants, in which GhPYL9-5D or GhPYR1-3A were knocked down, showed clearly reduced tolerance to polyethylene glycol 6000 (PEG)-induced drought, salinity and osmotic stresses compared with the controls. Additionally, transcriptomic data revealed that GhPYL9-5D was highly expressed in the root, and GhPYR1-3A was strongly expressed in the fiber and stem. GhPYL9-5D, GhPYR1-3A and their homologs in cotton were highly expressed after treatment with PEG or NaCl, and the two genes were co-expressed with redox signaling components, transcription factors and auxin signal components. These results suggest that GhPYL9-5D and GhPYR1-3A may serve important roles through interplaying with hormone and other signaling components in cotton adaptation to salt or osmotic stress.
CONCLUSIONS: GhPYL9-5D and GhPYR1-3A positively regulate ABA-mediated seed germination, primary root growth and stomatal closure, as well as tolerance to drought, salt and osmotic stresses likely through affecting the expression of multiple downstream stress-associated genes in Arabidopsis and cotton.

Pectin is a major component of the plant cell wall, forming a network that contributes to cell wall integrity and flexibility. Pectin methylesterase (PME) catalyzes the removal of methylester groups from the homogalacturonan backbone, the most abundant pectic polymer, and contributes to intercellular adhesion during plant development and different environmental stimuli stress. In this study, we identified and characterized an Arabidopsis type-II PME, PME53, which encodes a cell wall deposited protein and may be involved in the stomatal lineage pathway and stomatal functions. We demonstrated that PME53 is expressed explicitly in guard cells as an abscisic acid (ABA)-regulated gene required for stomatal movement and thermotolerance. The expression of PME53 is significantly affected by the stomatal differentiation factors SCRM and MUTE. The null mutation in PME53 results in a significant increase in stomatal number and susceptibility to ABA-induced stomatal closure. During heat stress, the pme53 mutant highly altered the activity of PME and significantly lowered the expression level of the calmodulin AtCaM3, indicating that PME53 may be involved in Ca2+-pectate reconstitution to render plant thermotolerance. Here, we present evidence that the PME53-mediated de-methylesterification status of pectin is directed toward stomatal development, movement, and regulation of the flexibility of the guard cell wall required for the heat response.

Drought is the most serious abiotic stress, which significantly reduces crop productivity. The phytohormone ABA plays a pivotal role in regulating stomatal closing upon drought stress. Here, we characterized the physiological function of AtBBD1, which has bifunctional nuclease activity, on drought stress. We found that AtBBD1 localized to the nucleus and cytoplasm, and was expressed strongly in trichomes and stomatal guard cells of leaves, based on promoter:GUS constructs. Expression analyses revealed that AtBBD1 and AtBBD2 are induced early and strongly by ABA and drought, and that AtBBD1 is also strongly responsive to JA. We then compared phenotypes of two AtBBD1-overexpression lines (AtBBD1-OX), single knockout atbbd1, and double knockout atbbd1/atbbd2 plants under drought conditions. We did not observe any phenotypic difference among them under normal growth conditions, while OX lines had greatly enhanced drought tolerance, lower transpirational water loss, and higher proline content than the WT and KOs. Moreover, by measuring seed germination rate and the stomatal aperture after ABA treatment, we found that AtBBD1-OX and atbbd1 plants showed significantly higher and lower ABA-sensitivity, respectively, than the WT. RNA sequencing analysis of AtBBD1-OX and atbbd1 plants under PEG-induced drought stress showed that overexpression of AtBBD1 enhances the expression of key regulatory genes in the ABA-mediated drought signaling cascade, particularly by inducing genes related to ABA biosynthesis, downstream transcription factors, and other regulatory proteins, conferring AtBBD1-OXs with drought tolerance. Taken together, we suggest that AtBBD1 functions as a novel positive regulator of drought responses by enhancing the expression of ABA- and drought stress-responsive genes as well as by increasing proline content.

Stomatal densities, aperture openness and their responsiveness to environmental change determine plant water loss and regulate entry of pathogens. Stomatal responsiveness is usually assessed on restricted areas of leaves or isolated epidermal peels floated in solution. Analyzing these responses in the whole plant context could give valuable additional information, for example on the role of mesophyll in stomatal responses. We analyzed stomatal responses to the phytohormone abscisic acid (ABA) and pathogenic elicitors in intact plants by dynamic measurement of leaf temperature. We tested whether ABA-induced stomatal closure in wheat requires external nitrate and whether bacterial elicitor-induced stomatal closure can be detected by dynamic thermal imaging in intact Arabidopsis. We found that wheat was hypersensitive to all applied treatments, as even mock-treated leaves showed a strong increase in leaf temperature. Nevertheless, ABA activated stomatal closure in wheat independent of exogenous nitrate. Pathogenic elicitors triggered a fast and transient increase in leaf temperature in intact Arabidopsis, indicating short-term stomatal closure. The data suggest that the dynamics of pathogen-induced stomatal closure is different in whole plants compared to epidermal peels, where elicitor-induced stomatal closure persists longer. We propose that dynamic thermal imaging could be applied to address the effect of pathogenic elicitors on stomatal behavior in whole plants to complement detached sample assays and gain a better understanding of stomatal immunity.

Hydrogen sulfide (H2S) is a signaling molecule that regulates plant hormone and stress responses. The phytohormone abscisic acid (ABA) plays an important role in plant adaptation to unfavorable environmental conditions and induces the persulfidation of L-CYSTEINE DESULFHYDRASE1 (DES1) and the production of H2S in guard cells. However, it remains largely unclear how H2S and protein persulfidation participate in the relay of ABA signals. In this study, we discovered that ABSCISIC ACID INSENSITIVE 4 (ABI4) acts downstream of DES1 in the control of ABA responses in Arabidopsis. ABI4 undergoes persulfidation at Cys250 that is triggered in a time-dependent manner by ABA, and loss of DES1 function impairs this process. Cys250 and its persulfidation are essential for ABI4 function in the regulation of plant responses to ABA and the H2S donor NaHS during germination, seedling establishment, and stomatal closure, which are abolished in the ABI4Cys250Ala mutated variant. Introduction of the ABI4Cys250Ala variant into the abi4 des1 mutant did not rescue its hyposensitivity to ABA. Cys250 is critical for the binding of ABI4 to its cognate motif in the promoter of Mitogen-Activated Protein Kinase Kinase Kinase 18 (MAPKKK18), which propagates the MAPK signaling cascade induced by ABA. Furthermore, the DES1-mediated persulfidation of ABI4 enhances the transactivation activity of ABI4 toward MAPKKK18, and ABI4 can bind the DES1 promoter, forming a regulatory loop. Taken together, these findings advance our understanding of a post-translational regulatory mechanism and suggest that ABI4 functions as an integrator of ABA and MAPK signals through a process in which DES1-produced H2S persulfidates ABI4 at Cys250.

UNASSIGNED: The Arabidopsis transcription factor NAC103 is up-regulated and its encoding protein is stabilized by ABA treatment, which positively regulates several ABA-responsive downstream genes during seed germination and seedlings growth. The Arabidopsis transcription factor NAC103 was previously found to be involved in endoplasmic reticulum (ER) stress and DNA damage responses. In this study, we report the new biological function of NAC103 in abscisic acid (ABA) response during seed germination and seedling growth in Arabidopsis. The expression of NAC103 was up-regulated and the NAC103 protein was stabilized by ABA treatment. Both the loss-of-function mutants of NAC103, created by targeted gene-editing, and the over-expression plants of NAC103 have no obvious germination-related phenotype under normal growth conditions. However, under exogenous ABA treatment conditions, the NAC103 mutants were less sensitive to ABA during seed germination; in contrast, the NAC103 over-expression plants were more sensitive to ABA during seed germination and young seedling growth. Further, NAC103 regulated several ABA-responsive downstream genes including MYB78, MYB3, PLP3, AMY1, and RGL2. These results demonstrate that NAC103 positively regulates ABA response in Arabidopsis.

Abscisic acid (ABA) plays an important role in plant growth, development, and stress responses. ABA regulates many aspects of plant growth and development, including seed maturation, dormancy, germination, the transition from vegetative to reproductive growth, leaf senescence and responses to environmental stresses, such as drought, high salinity and cold. It is also known that mitogen-activated protein kinase (MAPK) cascades function in ABA signaling. Recently, we and another group have identified the ABA-inducible MAP3Ks MAP3K17 and MAP3K18 as the upstream MAP3Ks of MKK3, implicating the MAP3K17/18-MKK3-MPK1/2/7/14 cascade in ABA signaling. It has also been reported that overexpression of MAP3K18 in Arabidopsis causes an early leaf senescence phenotype, ABA hypersensitive stomata closing, and drought tolerance. In this study, we generated transgenic plants overexpressing MAP3K17 (35S:MAP3K17) and its kinase-inactive form (35S:MAP3K17KN). The bolting of 35S:MAP3K17 was earlier than WT, and the fresh weights of the seedlings were smaller, whereas 35S:MAP3K17KN showed the opposite phenotype. These results indicate that the transition from vegetative to reproductive growth can be regulated by overexpression of MAP3K17 and its kinase-inactive form. Moreover, 35S:MAP3K17 showed lower sensitivity to ABA during post-germinated growth, whereas 35S:MAP3K17 KN showed the opposite phenotype, suggesting the negative roles of MAP3K17 in the response to ABA. Our work provides the possibility to regulate plant growth and development by the genetic manipulation of ABA-induced MAPK cascades, leading to improved crop growth and productivity.

The zinc finger protein (ZFP) transcription factor family plays an important role in regulating plant growth, development, and response to abiotic stress. In this study, we aimed to determine the role of GmZAT4, a C2H2-type transcription factor, in abiotic stress tolerance. The complete coding sequence of the GmZAT4 gene was isolated from soybean root RNA, which shows highest expression level compared with leaf, flower and other tissues. Using multiple sequence alignment and conserved domain analysis, we showed that GmZAT4 is a typical C2H2-type transcription factor which is comprised of two C2H2 domains, including a highly conserved QALGGH motif, and implied the regulation of abiotic stress tolerance in plant. A phylogenetic tree revealed that the soybean GmZAT4 gene clustered with ZAT4 from Glycine soja and AZF1, AZF2, and AZF3 from Arabidopsis thaliana. The mRNA expression levels of GmZAT4 were determined in two soybean cultivars by quantitative reverse transcription (qRT)-PCR and compared. The results showed higher expression (up to 60, 25 and 4 times, respectively) in the drought-tolerant type (Jinda 74) compared to the drought-sensitive soybean cultivar (Jinda 53) following treatment with 18% PEG, 150 mM NaCl, or 100 µM abscisic acid (ABA). GmZAT4 was ectopically over-expressed in A. thaliana to determine its role in abiotic stress tolerance. GmZAT4 overexpression enhanced the tolerance of A. thaliana to treatment with 20% PEG and 150 mM NaCl, and improved the germination rate following treatment with 1 µM or 2 µM ABA. The expression profiles of marker genes in the ABA signaling pathway, such as RD29A, RD29B, ABI, and RAD, indicated that GmZAT4 enhanced the abiotic stress tolerance of Arabidopsis. These results suggest that the C2H2-type ZFP encoded by GmZAT4 plays an important role in PEG and NaCl stress tolerance and ABA responses in soybean and A. thaliana.

Genomic imprinting is a form of epigenetic regulation resulting in differential gene expression that reflects the parent of origin. In plants, imprinted gene expression predominantly occurs in the seed endosperm. Maternal-specific DNA demethylation by the DNA demethylase DME frequently underlies genomic imprinting in endosperm. Whether other more ubiquitously expressed DNA demethylases regulate imprinting is unknown. Here, we found that the DNA demethylase ROS1 regulates the imprinting of DOGL4 DOGL4 is expressed from the maternal allele in endosperm and displays preferential methylation and suppression of the paternal allele. We found that ROS1 negatively regulates imprinting by demethylating the paternal allele, preventing its hypermethylation and complete silencing. Furthermore, we found that DOGL4 negatively affects seed dormancy and response to the phytohormone abscisic acid and that ROS1 controls these processes by regulating DOGL4 Our results reveal roles for ROS1 in mitigating imprinted gene expression and regulating seed dormancy.