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Grain size is a crucial agronomic trait that affects stable yield, appearance, milling quality, and domestication in rice. However, the molecular and genetic relationships among QTL genes (QTGs) underlying natural variation for grain size remain elusive. Here, we identified a novel QTG SGW5 (suppressor of gw5) by map-based cloning using an F2 segregation population by fixing same genotype of the master QTG GW5. SGW5 positively regulates grain width by influencing cell division and cell size in spikelet hulls. Two nearly isogenic lines exhibited a significant differential expression of SGW5 and a 12.2% increase in grain yield. Introducing the higher expression allele into the genetic background containing the lower expression allele resulted in increased grain width, while its knockout resulted in shorter grain hulls and dwarf plants. Moreover, a cis-element variation in the SGW5 promoter influenced its differential binding affinity for the WRKY53 transcription factor, causing the differential SGW5 expression, which ultimately leads to grain size variation. GW5 physically and genetically interacts with WRKY53 to suppress the expression of SGW5. These findings elucidated a new pathway for grain size regulation by the GW5-WRKY53-SGW5 module and provided a novel case for generally uncovering QTG interactions underlying the genetic diversity of an important trait in crops.

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The transcription factor WRKY53 of the model plant Arabidopsis thaliana is an important regulator of leaf senescence. Its expression, activity and degradation are tightly controlled by various mechanisms and feedback loops. Hydrogen peroxide is one of the inducing agents for WRKY53 expression, and a long-lasting intracellular increase in H2O2 content accompanies the upregulation of WRKY53 at the onset of leaf senescence. We have identified different antioxidative enzymes, including catalases (CATs), superoxide dismutases (SODs) and ascorbate peroxidases (APXs), as protein interaction partners of WRKY53 in a WRKY53-pulldown experiment at different developmental stages. The interaction of WRKY53 with these enzymes was confirmed in vivo by bimolecular fluorescence complementation assays (BiFC) in Arabidopsis protoplasts and transiently transformed tobacco leaves. The interaction with WRKY53 inhibited the activity of the enzyme isoforms CAT2, CAT3, APX1, Cu/ZuSOD1 and FeSOD1 (and vice versa), while the function of WRKY53 as a transcription factor was also inhibited by these complex formations. Other WRKY factors like WRKY18 or WRKY25 had no or only mild inhibitory effects on the enzyme activities, indicating that WRKY53 has a central position in this crosstalk. Taken together, we identified a new additional and unexpected feedback regulation between H2O2, the antioxidative enzymes and the transcription factor WRKY53.

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Large amounts of potash fertilizer are often applied to apple (Malus domestica) orchards to enhance fruit quality and yields, but this treatment aggravates KCl-based salinity stress. Melatonin (MT) is involved in a variety of abiotic stress responses in plants. However, its role in KCl stress tolerance is still unknown. In the present study, we determined that an appropriate concentration (100 µm) of MT significantly alleviated KCl stress in Malus hupehensis by enhancing K+ efflux out of cells and compartmentalizing K+ in vacuoles. Transcriptome deep-sequencing analysis identified the core transcription factor gene MdWRKY53, whose expression responded to both KCl and MT treatment. Overexpressing MdWRKY53 enhanced KCl tolerance in transgenic apple plants by increasing K+ efflux and K+ compartmentalization. Subsequently, we characterized the transporter genes MdGORK1 and MdNHX2 as downstream targets of MdWRKY53 by ChIP-seq. MdGORK1 localized to the plasma membrane and enhanced K+ efflux to increase KCl tolerance in transgenic apple plants. Moreover, overexpressing MdNHX2 enhanced the KCl tolerance of transgenic apple plants/callus by compartmentalizing K+ into the vacuole. RT-qPCR and LUC activity analyses indicated that MdWRKY53 binds to the promoters of MdGORK1 and MdNHX2 and induces their transcription. Taken together, our findings reveal that the MT-WRKY53-GORK1/NHX2-K+ module regulates K+ homeostasis to enhance KCl stress tolerance in apple. These findings shed light on the molecular mechanism of apple response to KCl-based salinity stress and lay the foundation for the practical application of MT in salt stress.

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Receptor-like kinases (RLKs) are the most important class of cell surface receptors, and play crucial roles in plant development and stress responses. However, few studies have been reported about the biofunctions of RLKs in leaf senescence. Here, we characterized a novel Arabidopsis RLK-encoding gene, SENESCENCE-RELATED RECEPTOR KINASE 1 (SENRK1), which was significantly down-regulated during leaf senescence. Notably, the loss-of-function senrk1 mutants displayed an early leaf senescence phenotype, while overexpression of SENRK1 significantly delayed leaf senescence, indicating that SENRK1 negatively regulates age-dependent leaf senescence in Arabidopsis. Furthermore, the senescence-promoting transcription factor WRKY53 repressed the expression of SENRK1. While the wrky53 mutant showed a delayed senescence phenotype as previously reported, the wrky53 senrk1-1 double mutant exhibited precocious leaf senescence, suggesting that SENRK1 functions downstream of WRKY53 in regulating age-dependent leaf senescence in Arabidopsis.

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Flowering is a crucial stage for plant reproductive success; therefore, the regulation of plant flowering has been widely researched. Although multiple well-defined endogenous and exogenous flowering regulators have been reported, new ones are constantly being discovered. Here, we confirm that a novel plant growth regulator guvermectin (GV) induces early flowering in Arabidopsis. Interestingly, our genetic experiments newly demonstrated that WRKY41 and its homolog WRKY53 were involved in GV-accelerated flowering as positive flowering regulators. Overexpression of WRKY41 or WRKY53 resulted in an early flowering phenotype compared to the wild type (WT). In contrast, the w41/w53 double mutants showed a delay in GV-accelerated flowering. Gene expression analysis showed that flowering regulatory genes SOC1 and LFY were upregulated in GV-treated WT, 35S:WRKY41, and 35S:WRKY53 plants, but both declined in w41/w53 mutants with or without GV treatment. Meanwhile, biochemical assays confirmed that SOC1 and LFY were both direct targets of WRKY41 and WRKY53. Furthermore, the early flowering phenotype of 35S:WRKY41 lines was abolished in the soc1 or lfy background. Together, our results suggest that GV plays a function in promoting flowering, which was co-mediated by WRKY41 and WRKY53 acting as new flowering regulators by directly activating the transcription of SOC1 and LFY in Arabidopsis.

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INTRODUCTION: Rhizoctonia solani, the causative agent of the sheath blight disease (ShB), invades rice to obtain nutrients, especially sugars; however, the molecular mechanism via which R. solani hijacks sugars from rice remains unclear. OBJECTIVES: In this study, rice-R. solani interaction model was used to explore whether pathogen effector proteins affect plant sugar absorption during infection. METHODS: Yeast one-hybrid assay was used to identify Activator of SWEET2a (AOS2) from R. solani. Localization and invertase secretion assays showed that nuclear localization and secreted function of AOS2. Hexose transport assays verified the hexose transporter activity of SWEET2a and SWEET3a. Yeast two-hybrid assays, Bimolecular fluorescence complementation (BiFC) and transactivation assay were conducted to verify the AOS2-WRKY53-Grassy tiller 1 (GT1) transcriptional complex and its activation of SWEET2a and SWEET3a. Genetic analysis is used to detect the response of GT1, WRKY53, SWEET2a, and SWEET3a to ShB infestation. Also, the soluble sugar contents were measured in the mutants and overexpression plants before and after the inoculation of R. solani. RESULTS: The present study found that R. solani protein AOS2 activates rice SWEET2a and localized in the nucleus of tobacco cells and secreted in yeast. AOS2 interacts with rice transcription factor WRKY53 and GT1 to form a complex that activates the hexose transporter gene SWEET2a and SWEET3a and negatively regulate rice resistance to ShB. CONCLUSION: These data collectively suggest that AOS2 secreted by R. solani interacts with rice WRKY53 and GT1 to form a transcriptional complex that activates SWEETs to efflux sugars to apoplast; R. solani acquires more sugars and subsequently accelerates host invasion.

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Jasmonic acid (JA) is an important hormone that functions in plant defense. cam1 and wrky53 mutants were more resistant to Spodoptera littoralis than in the wild-type (WT) Arabidopsis group. In addition, JA concentration in cam1 and wrky53 mutants was higher compared with the WT group. To explore how these two proteins affect the resistance of Arabidopsis plants, we used a yeast two-hybrid assay, firefly luciferase complementation imaging assay and in vitro pull-down assay confirming that calmodulin 1 (CAM1) interacted with WRKY53. However, these two proteins separate when calcium concentration increases in Arabidopsis leaf cells. Then, electrophoretic mobility shift assay and luciferase activation assay were used to verify that WRKY53 could bind to lipoxygenases 3 (LOX3) and lipoxygenases 4 (LOX4) gene promoters and negatively regulate gene expression. This study reveals that CAM1 and WRKY53 negatively regulate plant resistance to herbivory by regulating the JA biosynthesis pathway via the dissociation of CAM1-WRKY53, then the released WRKY53 binds to the LOXs promoters to negatively regulate LOXs gene expression. This study reveals WRKY53's mechanism in insect resistance, a new light on the function of WRKY53.

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Saline stress is the most limiting condition impacting the plant growth, development, and productivity. In this present study, jasmonic acid (JA) was used as a foliar spray on the rice seedlings grown under saline stress. Increase in photosynthetic pigments, anthocyanin, and total protein content was observed with JA treatment while NaCl showed reduction in biochemical constituents and enhanced antioxidant enzyme activity. The leaf cells of NaCl-treated seedlings accumulated more ROS and had more fragmented nuclei, whereas JA decreased the accumulation and fragmentation during saline stress. In NaCl treatment, gene expression analysis showed many fold upregulation in comparison with other treatments. The results suggest that JA acts as a promoter for growth, physiological, biochemical, and cellular contents, as well as ameliorate the effects of saline stress. The expression of genes demonstrated that saline stress may promote autophagy, which leads to autophagic cell death, and improve tolerance to saline stress in rice seedlings via the jasmonic acid signaling pathway. However, the mechanism by which jasmonate signaling induces autophagy and cell death is unknown and requires further exploration.

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Sheath blight (ShB) is one of the most common diseases in rice that significantly affects yield production. However, the underlying mechanisms of rice defense remain largely unknown. Our previous transcriptome analysis identified that infection with Rhizoctonia solani significantly induced the expression level of SWEET2a, a member of the SWEET sugar transporter. The sweet2a genome-editing mutants were less susceptible to ShB. Further yeast-one hybrid, ChIP, and transient assays demonstrated that WRKY53 binds to the SWEET2a promoter to activate its expression. WRKY53 is a key brassinosteroid (BR) signaling transcription factor. Similar to the BR receptor gene BRI1 and biosynthetic gene D2 mutants, the WRKY53 mutant and overexpressor were less and more susceptible to ShB compared to wild-type, respectively. Inoculation with R. solani induced expression of BRI1, D2, and WRKY53, but inhibited MPK6 (a BR-signaling regulator) activity. Also, MPK6 is known to phosphorylate WRKY53 to enhance its transcription activation activity. Transient assay results indicated that co-expression of MPK6 and WRKY53 enhanced WRKY53 trans-activation activity to SWEET2a. Furthermore, expression of WRKY53 SD (the active phosphorylated forms of WRKY53) but not WRKY53 SA (the inactive phosphorylated forms of WRKY53), enhanced WRKY53-mediated activation of SWEET2a compared to expression of WRKY53 alone. Taken together, our analyses showed that R. solani infection may activate BR signaling to induce SWEET2a expression via WRKY53 through negative regulation of ShB resistance in rice.

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KEY MESSAGE: MiWRKY53 is expressed in response to various stresses and hormones. Although it is localized in the nucleus, it shows no transcriptional activation. Role of SA-mediated plant defence response is demonstrated. WRKY transcription factors are one the largest gene families in plants involved in almost every process in plants including development, physiological processes, and stress response. Salicylic acid (SA) is key regulator of biotic stress against various pathogens in plants acting via its multiple mechanisms to induce defence response. Herein, we have identified and functionally validated WRKY53 from mulberry (Morus indica var. K2). MiWRKY53 expressed differentially in response to different stress and hormonal treatments. MiWRKY53 belongs to group III of WKRY gene family, localized in nucleus, and lacks transcriptional activation activity in yeast. Hormone responsive behaviour of MiWRKY53 Arabidopsis overexpression (OE) transgenics preferentially was noted in root growth assay in response to Salicylic acid (SA). Arabidopsis overexpression plants also displayed alteration in leaf phenotype having wider leaves than the wild-type plants. PR-1 transcripts were higher in MiWRKY53 Arabidopsis OE plants and they displayed resistance towards biotrophic pathogen Pseudomonas syringae PstDC3000. MiWRKY53 Mulberry OE transgenics also depicted SA-responsive behaviour. Several hormones and stress-related cis-acting elements were also identified in the 1.2-Kb upstream regulatory region (URR) of MiWRKY53. Functional characterization of full-length promoter region revealed that it is induced by SA and further analysis of deletion constructs helped in the identification of minimal promoter responsible for its inducibility by SA. Altogether, the findings from this study point towards the SA preferential behaviour of MiWRKY53 and its function as regulator of plant defence response through SA-mediated mechanisms.

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Plants tightly control gene transcription to adapt to environmental conditions and steer growth and development. Different types of epigenetic modifications are instrumental in these processes. In recent years, an important role for the chromatin-modifying RPD3/HDA1 class I HDAC HISTONE DEACETYLASE 9 (HDA9) emerged in the regulation of a multitude of plant traits and responses. HDACs are widely considered transcriptional repressors and are typically part of multiprotein complexes containing co-repressors, DNA, and histone-binding proteins. By catalyzing the removal of acetyl groups from lysine residues of histone protein tails, HDA9 negatively controls gene expression in many cases, in concert with interacting proteins such as POWERDRESS (PWR), HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 15 (HOS15), WRKY53, ELONGATED HYPOCOTYL 5 (HY5), ABA INSENSITIVE 4 (ABI4), and EARLY FLOWERING 3 (ELF3). However, HDA9 activity has also been directly linked to transcriptional activation. In addition, following the recent breakthrough discovery of mutual negative feedback regulation between HDA9 and its interacting WRKY-domain transcription factor WRKY53, swift progress in gaining understanding of the biology of HDA9 is expected. In this review, we summarize knowledge on this intriguing versatile-and long under-rated-protein and propose novel leads to further unravel HDA9-governed molecular networks underlying plant development and environmental biology.

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Epigenetic regulation of gene expression is important for plant adaptation to environmental changes. Previous results showed that Arabidopsis RPD3-like histone deacetylase HDA9 is known to function in repressing plant response to stress in Arabidopsis. However, how HDA9 targets to specific chromatin loci and controls gene expression networks involved in plant response to stress remains largely unclear. Here, we show that HDA9 represses stress tolerance response by interacting with and regulating the DNA binding and transcriptional activity of WRKY53, which functions as a high-hierarchy positive regulator of stress response. We found that WRKY53 is post-translationally modified by lysine acetylation at multiple sites, some of which are removed by HDA9, resulting in inhibition of WRKY53 transcription activity. Conversely, WRKY53 negatively regulates HDA9 histone deacetylase activity. Collectively, our results indicate that HDA9 and WRK53 are reciprocal negative regulators of each other's activities, illustrating how the functional interplay between a chromatin regulator and a transcription factor regulates stress tolerance in plants.

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Leaf senescence is an integral part of plant development aiming at the remobilization of nutrients and minerals out of the senescing tissue into developing parts of the plant. Sequential as well as monocarpic senescence maximize the usage of nitrogen, mineral, and carbon resources for plant growth and the sake of the next generation. However, stress-induced premature senescence functions as an exit strategy to guarantee offspring under long-lasting unfavorable conditions. In order to coordinate this complex developmental program with all kinds of environmental input signals, complex regulatory cues have to be in place. Major changes in the transcriptome imply important roles for transcription factors. Among all transcription factor families in plants, the NAC and WRKY factors appear to play central roles in senescence regulation. In this review, we summarize the current knowledge on the role of WRKY factors with a special focus on WRKY53. In contrast to a holistic multi-omics view we want to exemplify the complexity of the network structure by summarizing the multilayer regulation of WRKY53 of Arabidopsis.

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MAIN CONCLUSION: This paper highlighted a salicylic acid-inducible Caulimoviral promoter fragment from Cestrum yellow leaf curling virus (CmYLCV). Interaction of Arabidopsis transcription factors TGA3 and WRKY53 on CmYLCV promoter resulted in the enhancement of the promoter activity via NPR1-dependent salicylic acid signaling. Several transcriptional promoters isolated from plant-infecting Caulimoviruses are being presently used worldwide as efficient tools for plant gene expression. The CmYLCV promoter has been isolated from the Cestrum yellow leaf curling virus (Caulimoviruses) and characterized more than 12 years ago; also we have earlier reported a near-constitutive, pathogen-inducible CmYLCV promoter fragment (-329 to +137 from transcription start site; TSS) that enhances stronger (3×) expression than the previously reported fragments; all these fragments are highly efficient in monocot and dicot plants (Sahoo et al. Planta 240: 855-875, 2014). Here, we have shown that the full-length CmYLCV promoter fragment (-729 to +137 from TSS) is salicylic acid (SA) inducible. In this context, we have performed an in-depth study to elucidate the factors responsible for SA-inducibility of the CmYLCV promoter. We found that the as-1 1 and W-box1 elements (located at -649 and -640 from the TSS) of the CmYLCV promoter are required for SA-induced activation by recruiting Arabidopsis TGA3 and WRKY53 transcription factors. Consequently, as a nascent observation, we established the physical interaction between TGA3 and WYKY53; also demonstrated that the N-terminal domain of TGA3 is sufficient for the interaction with the full-length WRKY53. Such interaction synergistically activates the CmYLCV promoter activity in planta. Further, we found that activation of the CmYLCV promoter by SA through TGA3 and WRKY53 interaction depends on NPR1. Finally, the findings presented here provide strong support for the direct regulatory roles of TGA3 and WRKY53 in the SA and NPR1-dependent activation of a Caulimoviral promoter (CmYLCV).

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As sessile organisms, plants have to continuously adjust growth and development to ever-changing environmental conditions. At the end of the growing season, annual plants induce leaf senescence to reallocate nutrients and energy-rich substances from the leaves to the maturing seeds. Thus, leaf senescence is a means with which to increase reproductive success and is therefore tightly coupled to the developmental age of the plant. However, senescence can also be induced in response to sub-optimal growth conditions as an exit strategy, which is accompanied by severely reduced yield. Here, we show that class III homeodomain leucine zipper (HD-ZIPIII) transcription factors, which are known to be involved in basic pattern formation, have an additional role in controlling the onset of leaf senescence in Arabidopsis. Several potential direct downstream genes of the HD-ZIPIII protein REVOLUTA (REV) have known roles in environment-controlled physiological processes. We report that REV acts as a redox-sensitive transcription factor, and directly and positively regulates the expression of WRKY53, a master regulator of age-induced leaf senescence. HD-ZIPIII proteins are required for the full induction of WRKY53 in response to oxidative stress, and mutations in HD-ZIPIII genes strongly delay the onset of senescence. Thus, a crosstalk between early and late stages of leaf development appears to contribute to reproductive success.

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