A Mitochondrial Localized Chaperone Regulator OsBAG6 Functions in Saline-Alkaline Stress Tolerance in Rice.

B-cell lymphoma 2 (Bcl-2)-associated athanogene (BAG) family genes play prominent roles in regulating plant growth, development, and stress response. Although the molecular mechanism underlying BAG's response to abiotic stress has been studied in Arabidopsis, the function of OsBAG underlying saline-alkaline stress tolerance in rice remains unclear. In this study, OsBAG6, a chaperone regulator localized to mitochondria, was identified as a novel negative regulator of saline-alkaline stress tolerance in rice. The expression level of OsBAG6 was induced by high concentration of salt, high pH, heat and abscisic acid treatments. Overexpression of OsBAG6 in rice resulted in significantly reduced plant heights, grain size, grain weight, as well as higher sensitivity to saline-alkaline stress. By contrast, the osbag6 loss-of-function mutants exhibited decreased sensitivity to saline-alkaline stress. The transcriptomic analysis uncovered differentially expressed genes related to the function of "response to oxidative stress", "defense response", and "secondary metabolite biosynthetic process" in the shoots and roots of OsBAG6-overexpressing transgenic lines. Furthermore, cytoplasmic levels of Ca2+ increase rapidly in plants exposed to saline-alkaline stress. OsBAG6 bound to calcium sensor OsCaM1-1 under normal conditions, which was identified by comparative interactomics, but not in the presence of elevated Ca2+. Released OsCaM1-1 saturated with Ca2+ is then able to regulate downstream stress-responsive genes as part of the response to saline-alkaline stress. OsBAG6 also interacted with energy biosynthesis and metabolic pathway proteins that are involved in plant growth and saline-alkaline stress response mechanisms. This study reveals a novel function for mitochondrial localized OsBAG6 proteins in the saline-alkaline stress response alongside OsCaM1-1.

OsWRKY76 positively regulates drought stress via OsbHLH148-mediated jasmonate signaling in rice.

Drought stress is a major environmental threat that limits plant growth and crop productivity. Therefore, it is necessary to uncover the molecular mechanisms behind drought tolerance in crops. Here, OsWRKY76 positively regulated drought stress in rice. OsWRKY76 expression was induced by PEG treatment, dehydration stress, and exogenous MeJA rather than by no treatment. Notably, OsWRKY76 knockout weakened drought tolerance at the seedling stage and decreased MeJA sensitivity. OsJAZ12 was significantly induced by drought stress, and its expression was significantly higher in OsWRKY76-knockout mutants than in wild-type ZH11 under drought stress. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that OsWRKY76 interacted with OsJAZ12. OsWRKY76 weakened the interaction between OsbHLH148 and OsJAZ12 in yeast cells. The OsJAZ12 protein repressed the transactivation activity of OsbHLH148, and this repression was partly restored by OsWRKY76 in rice protoplasts. Moreover, OsDREB1E expression was lower in OsWRKY76-knockout mutants than in wild-type ZH11 under drought stress, but it was upregulated under normal growth conditions. Yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase assays showed that OsWRKY76 and OsbHLH148 bound directly to the OsDREB1E promoter and activated OsDREB1E expression in response to drought stress. These results suggest that OsWRKY76 confers drought tolerance through OsbHLH148-mediated jasmonate signaling in rice, offering a new clue to uncover the mechanisms behind drought tolerance.

OsWRKY28 positively regulates salinity tolerance by directly activating OsDREB1B expression in rice.

KEY MESSAGE: OsWRKY28 confers salinity tolerance by directly binding to OsDREB1B promoter and increasing its transcriptional activity, and negatively regulates abscisic acid mediated seedling establishment in rice. WRKY transcription factors have been reported to play a vital role in plants growth, development, abiotic and biotic stress responses. In this study, we explored the functions of a transcription factor OsWRKY28 in rice. The transcript level of OsWRKY28 was strikingly increased under drought, chilling, salt and abscisic acid treatments. The OsWRKY28 overexpression lines showed enhanced salinity stress tolerance, whereas the oswrky28 mutants displayed salt sensitivity compared to wild-type plants. Under salt stress treatment, the expression levels of OsbZIP05, OsHKT1;1 and OsDREB1B were significantly lower yet the level of OsHKT2;1 was significantly higher in oswrky28 mutants than those in wide type plants. Our data of yeast one-hybrid assay and dual-luciferase assay supported that OsWRKY28 could directly bind to the promoter of OsDREB1B to enhance salinity tolerance in rice. In addition, OsWRKY28 overexpression lines displayed hyposensitivity and the oswrky28 mutants showed hypersensitivity compared to wild-type plants under exogenous abscisic acid treatment. Based on the results of yeast two-hybrid assay and GAL4-dependent chimeric transactivation assay, OsWRKY28 physically interacts with OsMPK11 and its transcriptional activity could be regulated by OsMPK11. Together, OsWRKY28 confers salinity tolerance through directly targeting OsDREB1B promoter and further activating its transcription in rice.

The OsWRKY63-OsWRKY76-OsDREB1B module regulates chilling tolerance in rice.

Rice (Oryza sativa) is sensitive to low temperatures, which affects the yield and quality of rice. Therefore, uncovering the molecular mechanisms behind chilling tolerance is a critical task for improving cold tolerance in rice cultivars. Here, we report that OsWRKY63, a WRKY transcription factor with an unknown function, negatively regulates chilling tolerance in rice. OsWRKY63-overexpressing rice lines are more sensitive to cold stress. Conversely, OsWRKY63-knockout mutants generated using a CRISPR/Cas9 genome editing approach exhibited increased chilling tolerance. OsWRKY63 was expressed in all rice tissues, and OsWRKY63 expression was induced under cold stress, dehydration stress, high salinity stress, and ABA treatment. OsWRKY63 localized in the nucleus plays a role as a transcription repressor and downregulates many cold stress-related genes and reactive oxygen species scavenging-related genes. Molecular, biochemical, and genetic assays showed that OsWRKY76 is a direct target gene of OsWRKY63 and that its expression is suppressed by OsWRKY63. OsWRKY76-knockout lines had dramatically decreased cold tolerance, and the cold-induced expression of five OsDREB1 genes was repressed. OsWRKY76 interacted with OsbHLH148, transactivating the expression of OsDREB1B to enhance chilling tolerance in rice. Thus, our study suggests that OsWRKY63 negatively regulates chilling tolerance through the OsWRKY63-OsWRKY76-OsDREB1B transcriptional regulatory cascade in rice.

Rice OsWRKY50 Mediates ABA-Dependent Seed Germination and Seedling Growth, and ABA-Independent Salt Stress Tolerance.

Plant WRKY transcription factors play crucial roles in plant growth and development, as well as plant responses to biotic and abiotic stresses. In this study, we identified and characterized a WRKY transcription factor in rice, OsWRKY50. OsWRKY50 functions as a transcriptional repressor in the nucleus. The transcription of OsWRKY50 was repressed under salt stress conditions, but activated after abscisic acid (ABA) treatment. OsWRKY50-overexpression (OsWRKY50-OX) plants displayed increased tolerance to salt stress compared to wild type and control plants. The expression of OsLEA3, OsRAB21, OsHKT1;5, and OsP5CS1 in OsWRKY50-OX were much higher than wild type and control plants under salt stress. Furthermore, OsWRKY50-OX displayed hyposensitivity to ABA-regulated seed germination and seedling establishment. The protoplast-based transient expression system and yeast hybrid assay demonstrated that OsWRKY50 directly binds to the promoter of OsNCED5, and thus further inhibits its transcription. Taken together, our results demonstrate that rice transcription repressor OsWRKY50 mediates ABA-dependent seed germination and seedling growth and enhances salt stress tolerance via an ABA-independent pathway.

Involvement of rice transcription factor OsERF19 in response to ABA and salt stress responses.

Soil salinity is a major environmental stressor that restricts the growth and yield of crops. Plants have evolved more complicated and precise mechanisms to cope with salt stress, as they cannot escape from harmful environments. In the current study we identified and characterized an AP2/ERF transcription factor in rice, OsERF19. The expression of OsERF19 was slightly repressed by salt stress or abscisic acid (ABA) treatment. OsERF19-overexpression (OsERF19-OX) plants displayed enhanced tolerance to salt stress and ABA hypersensitivity compared to wild type and control plants. Furthermore, OsLEA3, OsNHX1, OsHKT6, and OsOTS1 were upregulated in OsERF19-OX plants when the plants were subjected to salt stress. OsRAB21, OsNCED5, and OsP5CS1 were also upregulated in OsERF19-OX plants treated with ABA. Yeast one-hybrid and dual luciferase reporter assays demonstrated that OsERF19 directly targets the promoters of OsOTS1 and OsNCED5 and further increases their transcription. These results suggest that the transcription factor OsERF19 plays a positive role in salt stress and ABA responses in rice.

JMJ17-WRKY40 and HY5-ABI5 modules regulate the expression of ABA-responsive genes in Arabidopsis.

Abscisic acid (ABA) plays a crucial role in the adaptation of young seedlings to environmental stresses. However, the role of epigenetic components and core transcriptional machineries in the effect of ABA on seed germination and seedling growth remain unclear. Here, we show that a histone 3 lysine 4 (H3K4) demethylase, JMJ17, regulates the expression of ABA-responsive genes during seed germination and seedling growth. Using comparative interactomics, WRKY40, a central transcriptional repressor in ABA signaling, was shown to interact with JMJ17. WRKY40 facilitates the recruitment of JMJ17 to the ABI5 chromatin, which removes gene activation marks (H3K4me3) from the ABI5 chromatin, thereby repressing its expression. Additionally, WRKY40 represses the transcriptional activation activity of HY5, which can activate ABI5 expression by directly binding to its promoter. An increase in ABA concentrations decreases the affinity of WRKY40 for the ABI5 promoter. Thus, WRKY40 and JMJ17 are released from the ABI5 chromatin, activating HY5. The accumulated ABI5 protein further shows heteromeric interaction with HY5, and thus synergistically activates its own expression. Our findings reveal a novel transcriptional switch, composed of JMJ17-WRKY40 and HY5-ABI5 modules, which regulates the ABA response during seed germination and seedling development in Arabidopsis.

Arabidopsis BRCA1 represses RRTF1-mediated ROS production and ROS-responsive gene expression under dehydration stress.

Reactive oxygen species (ROS) act as important secondary messengers in abscisic acid (ABA) signaling and induce stomatal closure under dehydration stress. The breast cancer susceptibility gene 1 (BRCA1), an important tumor suppressor in animals, functions primarily in the maintenance of genome integrity in animals and plants. However, whether and how the plant BRCA1 regulates intracellular ROS homeostasis in guard cells under dehydration stress remains unknown. Here, we found that Arabidopsis atbrca1 loss-of-function mutants showed dehydration stress tolerance. This stress tolerant phenotype of atbrca1 was a result of ABA- and ROS-induced stomatal closure, which was enhanced in atbrca1 mutants compared with the wild-type. AtBRCA1 downregulated the expression of ROS-responsive and marker genes. Notably, these genes were also the targets of the AP2/ERF transcriptional activator RRTF1/ERF109. Under normal conditions, AtBRCA1 physically interacted with RRTF1 and inhibited its binding to the GCC-box-like sequence in target gene promoters. Under dehydration stress, the expression of AtBRCA1 was dramatically reduced and that of RRTF1 was activated, thus inducing the expression of ROS-responsive genes. Overall, our study reveals a novel molecular function of AtBRCA1 in the transcriptional regulation of intracellular ROS homeostasis under dehydration stress.

Rice plastidial NAD-dependent malate dehydrogenase 1 negatively regulates salt stress response by reducing the vitamin B6 content.

Salinity is an important environmental factor that adversely impacts crop growth and productivity. Malate dehydrogenases (MDHs) catalyse the reversible interconversion of malate and oxaloacetate using NAD(H)/NADP(H) as a cofactor and regulate plant development and abiotic stress tolerance. Vitamin B6 functions as an essential cofactor in enzymatic reactions involved in numerous cellular processes. However, the role of plastidial MDH in rice (Oryza sativa) in salt stress response by altering vitamin B6 content remains unknown. In this study, we identified a new loss-of-function osmdh1 mutant displaying salt stress-tolerant phenotype. The OsMDH1 was expressed in different tissues of rice plants including leaf, leaf sheath, panicle, glume, bud, root and stem and was induced in the presence of NaCl. Transient expression of OsMDH1-GFP in rice protoplasts showed that OsMDH1 localizes to chloroplast. Transgenic rice plants overexpressing OsMDH1 (OsMDH1OX) displayed a salt stress-sensitive phenotype. Liquid chromatography-mass spectrometry (LC-MS) metabolic profiling revealed that the amount of pyridoxine was significantly reduced in OsMDH1OX lines compared with the NIP plants. Moreover, the pyridoxine content was higher in the osmdh1 mutant and lower in OsMDH1OX plants than in the NIP plants under the salt stress, indicating that OsMDH1 negatively regulates salt stress-induced pyridoxine accumulation. Furthermore, genome-wide RNA-sequencing (RNA-seq) analysis indicated that ectopic expression of OsMDH1 altered the expression level of genes encoding key enzymes of the vitamin B6 biosynthesis pathway, possibly reducing the level of pyridoxine. Together, our results establish a novel, negative regulatory role of OsMDH1 in salt stress tolerance by affecting vitamin B6 content of rice tissues.

Arabidopsis histone H3K4 demethylase JMJ17 functions in dehydration stress response.

Under dehydration in plants, antagonistic activities of histone 3 lysine 4 (H3K4) methyltransferase and histone demethylase maintain a dynamic and homeostatic state of gene expression by orientating transcriptional reprogramming toward growth or stress tolerance. However, the histone demethylase that specifically controls histone methylation homeostasis under dehydration stress remains unknown. Here, we document that a histone demethylase, JMJ17, belonging to the KDM5/JARID1 family, plays crucial roles in response to dehydration stress and abscisic acid (ABA) in Arabidopsis thaliana. jmj17 loss-of-function mutants displayed dehydration stress tolerance and ABA hypersensitivity in terms of stomatal closure. JMJ17 specifically demethylated H3K4me1/2/3 via conserved iron-binding amino acids in vitro and in vivo. Moreover, H3K4 demethylase activity of JMJ17 was required for dehydration stress response. Systematic combination of genome-wide chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) and RNA-sequencing (RNA-seq) analyses revealed that a loss-of-function mutation in JMJ17 caused an ectopic increase in genome-wide H3K4me3 levels and activated a plethora of dehydration stress-responsive genes. Importantly, JMJ17 bound directly to the chromatin of OPEN STOMATA 1 (OST1) and demethylated H3K4me3 for the regulation of OST1 mRNA abundance, thereby modulating the dehydration stress response. Our results demonstrate a new function of a histone demethylase under dehydration stress in plants.

Extensive allele-level remodeling of histone methylation modification in reciprocal F1 hybrids of rice subspecies.

Epigenetic mechanisms play a major role in heterosis, partly as a result of the remodeling of epigenetic modifications in F1 hybrids. Based on chromatin immunoprecipitation-sequencing (ChIP-Seq) analyses, we show that at the allele level extensive histone methylation remodeling occurred for a subset of genomic loci in reciprocal F1 hybrids of Oryza sativa (rice) cultivars Nipponbare and 93-11, representing the two subspecies japonica and indica. Globally, the allele modification-altered loci in leaf or root of the reciprocal F1 hybrids involved ˜12-43% or more of the genomic regions carrying either of two typical histone methylation markers, H3K4me3 (>21 000 genomic regions) and H3K27me3 (>11 000 genomic regions). Nevertheless, at the total modification level, the majority (from ˜43 to >90%) of the modification-altered alleles lay within the range of parental additivity in the hybrids because of concerted alteration in opposite directions, consistent with an overall attenuation of allelic differences in the modifications. Importantly, of the genomic regions that did show non-additivity in total modification level by either marker in the two tissues of hybrids, >80% manifested transgressivity, which involved genes enriched in specific functional categories. Extensive allele-level alteration of H3K4me3 alone was positively correlated with genome-wide changes in allele-level gene expression, whereas at the total level, both H3K4me3 and H3K27me3 remodeling, although affecting just a small number of genes, contributes to the overall non-additive gene expression to variable extents, depending on tissue/marker combinations. Our results emphasize the importance of allele-level analysis in hybrids to assess the remodeling of epigenetic modifications and their relation to changes in gene expression.

Trithorax-group protein ATX5 mediates the glucose response via impacting the HY1-ABI4 signaling module.

KEY MESSAGE: Trithorax-group Protein ARABIDOPSIS TRITHORAX5 modulates the glucose response. Glucose is an evolutionarily conserved modulator from unicellular microorganisms to multicellular animals and plants. Extensive studies have shown that the Trithorax-group proteins (TrxGs) play essential roles in different biological processes by affecting histone modifications and chromatin structures. However, whether TrxGs function in the glucose response and how they achieve the control of target genes in response to glucose signaling in plants remain unknown. Here, we show that the Trithorax-group Protein ARABIDOPSIS TRITHORAX5 (ATX5) affects the glucose response and signaling. atx5 loss-of-function mutants display glucose-oversensitive phenotypes compared to the wild-type (WT). Genome-wide RNA-sequencing analyses have revealed that ATX5 impacts the expression of a subset of glucose signaling responsive genes. Intriguingly, we have established that ATX5 directly controls the expression of HY1 by trimethylating H3 lysine 4 of the Arabidopsis Heme Oxygenase1 (HY1) locus. Glucose signaling causes the suppression of ATX5 activity and subsequently reduces the H3K4me3 levels at the HY1 locus, thereby leading to the increased expression of ABSCISIC ACID-INSENSITIVE4 (ABI4). This result suggests that an important ATX5-HY1-ABI4 regulatory module governs the glucose response. This idea is further supported by genetic evidence showing that an atx5 hy1-100 abi4 triple mutant showed a similar glucose-insensitive phenotype as compared to that of the abi4 single mutant. Our findings show that a novel ATX5-HY1-ABI4 module controls the glucose response in Arabidopsis thaliana.

Trithorax-group proteins ARABIDOPSIS TRITHORAX4 (ATX4) and ATX5 function in abscisic acid and dehydration stress responses.

Trithorax-group proteins (TrxGs) play essential regulatory roles in chromatin modification to activate transcription. Although TrxGs have been shown to be extensively involved in the activation of developmental genes, how the specific TrxGs function in the dehydration and abscisic acid (ABA)-mediated modulation of downstream gene expression remains unknown. Here, we report that two evolutionarily conserved Arabidopsis thaliana TrxGs, ARABIDOPSIS TRITHORAX4 (ATX4) and ATX5, play essential roles in the drought stress response. atx4 and atx5 single loss-of-function mutants showed drought stress-tolerant and ABA-hypersensitive phenotypes during seed germination and seedling development, while the atx4 atx5 double mutant displayed further exacerbation of the phenotypes. Genome-wide RNA-sequencing analyses showed that ATX4 and ATX5 regulate the expression of genes functioning in dehydration stress. Intriguingly, ABA-HYPERSENSITIVE GERMINATION 3 (AHG3), an essential negative regulator of ABA signaling, acts genetically downstream of ATX4 and ATX5 in response to ABA. ATX4 and ATX5 directly bind to the AHG3 locus and trimethylate histone H3 of Lys 4 (H3K4). Moreover, ATX4 and ATX5 occupancies at AHG3 are dramatically increased under ABA treatment, and are also essential for RNA polymerase II (RNAPII) occupancies. Our findings reveal novel molecular functions of A. thaliana TrxGs in dehydration stress and ABA responses.