ELONGATED HYPOCOTYL 5a modulates FLOWERING LOCUS T2 and gibberellin levels to control dormancy and bud break in poplar.

Photoperiod is a crucial environmental cue for phenological responses, including growth cessation and winter dormancy in perennial woody plants. Two regulatory modules within the photoperiod pathway explain bud dormancy induction in poplar (Populus spp.): the circadian oscillator LATE ELONGATED HYPOCOTYL 2 (LHY2) and GIGANTEA-like genes (GIs) both regulate the key target for winter dormancy induction FLOWERING LOCUS T2 (FT2). However, modification of LHY2 and GIs cannot completely prevent growth cessation and bud set under short-day (SD) conditions, indicating that additional regulatory modules are likely involved. We identified PtoHY5a, an orthologs of the photomorphogenesis regulatory factor ELONGATED HYPOCOTYL 5 (HY5) in poplar (Populus tomentosa), that directly activates PtoFT2 expression and represses the circadian oscillation of LHY2, indirectly activating PtoFT2 expression. Thus, PtoHY5a suppresses SD-induced growth cessation and bud set. Accordingly, PtoHY5a knockout facilitates dormancy induction. PtoHY5a also inhibits bud-break in poplar by controlling gibberellic acid (GA) levels in apical buds. Additionally, PtoHY5a regulates the photoperiodic control of seasonal growth downstream of phytochrome PHYB2. Thus, PtoHY5a modulates seasonal growth in poplar by regulating the PtoPHYB2-PtoHY5a-PtoFT2 module to determine the onset of winter dormancy, and by fine-tuning GA levels to control bud-break.

Salicylic acid-activated BIN2 phosphorylation of TGA3 promotes Arabidopsis PR gene expression and disease resistance.

The plant defense hormone, salicylic acid (SA), plays essential roles in immunity and systemic acquired resistance. Salicylic acid induced by the pathogen is perceived by the receptor nonexpressor of pathogenesis-related genes 1 (NPR1), which is recruited by TGA transcription factors to induce the expression of pathogenesis-related (PR) genes. However, the mechanism by which post-translational modifications affect TGA's transcriptional activity by salicylic acid signaling/pathogen infection is not well-established. Here, we report that the loss-of-function mutant of brassinosteroid insensitive2 (BIN2) and its homologs, bin2-3 bil1 bil2, causes impaired pathogen resistance and insensitivity to SA-induced PR gene expression, whereas the gain-of-function mutant, bin2-1, exhibited enhanced SA signaling and immunity against the pathogen. Our results demonstrate that salicylic acid activates BIN2 kinase, which in turn phosphorylates TGA3 at Ser33 to enhance TGA3 DNA binding ability and NPR1-TGA3 complex formation, leading to the activation of PR gene expression. These findings implicate BIN2 as a new component of salicylic acid signaling, functioning as a key node in balancing brassinosteroid-mediated plant growth and SA-induced immunity.

The HAT1 transcription factor regulates photomorphogenesis and skotomorphogenesis via phytohormone levels.

Plants dynamically modulate their growth and development to acclimate to the fluctuating light environment via a complex phytohormone network. However, the dynamic molecular regulatory mechanisms underlying how plants regulate phytohormones during skotomorphogenesis and photomorphogenesis are largely unknown. Here, we identified a HD-ZIP II transcription factor, HOMEODOMAIN ARABIDOPSIS THALIANA1 (HAT1), as a key node that modulates the dose effects of brassinosteroids (BR) and auxin on hypocotyl growth during skotomorphogenesis and photomorphogenesis. Compared with the wild-type (Col-0), both HAT1 loss of function and its overexpression led to disrupted photomorphogenic and skotomorphogenic hypocotyl growth. HAT1 overexpression (HAT1OX) plants displayed longer hypocotyls in the light but shorter hypocotyls in darkness, whereas the triple mutant hat1hat2hat3 showed the opposite phenotype. Furthermore, we found that CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) interacted with dephosphorylated HAT1 and facilitated the degradation of HAT1 by ubiquitination in darkness, while HAT1 was phosphorylated and stabilized by BRASSINOSTEROID INSENSITIVE2 (BIN2) in the light. Interestingly, we observed distinct dose-dependent effects of BR and auxin on hypocotyl elongation under varying light conditions and that HAT1 functioned as a key node in this process. The shorter hypocotyl of HAT1OX in darkness was due to the inhibition of BR biosynthetic gene BRASSINOSTEROID-6-OXIDASE2 (BR6OX2) expression to reduce BRs content, while brassinolide (BL) treatment alleviated this growth repression. In the light, HAT1 inhibited BR biosynthesis but enhanced auxin signaling by directly repressing IAA3/SHORT HYPOCOTYL 2 (SHY2) expression. Our findings uncover a dual function of HAT1 in regulating BR biosynthesis and auxin signaling that is crucial for ensuring proper skotomorphogenic and photomorphogenic growth.

Ethylene activates poplar defense against Dothiorella gregaria Sacc by regulating reactive oxygen species accumulation.

Populus canker is a widespread disease that seriously threatens the survival of trees. Phytohormones are considered as effective chemical molecules improving plant resistance to various diseases. Ethylene is an important phytohormone that is extensively involved in the regulation of plant growth, development, and stress responses, but how ethylene and ethylene signaling regulates defense responses in woody plants is still unclear. Here, we showed that ethylene positively regulates the responses of poplar to canker caused by the hemibiotrophic fungus Dothiorella gregaria. Treatment of Populus tomentosa with 1-aminocyclopropane-1-carboxylic acid (ACC, the biosynthetic precursor of ethylene) significantly enhanced disease resistance, accompanied by the induction of pathogen-related protein (PR) gene expression and H2 O2 accumulation. Blocking ethylene biosynthesis using aminoethoxyvinyl glycine (AVG, a specific inhibitor of ethylene biosynthesis) repressed the disease resistance. Overexpression of the ethylene biosynthesis gene PtoACO7 in Populus tomentosa promoted defense responses and disease resistance. Furthermore, we demonstrated that the ethylene-induced defense response is independent of the salicylic acid pathway, but needs ROS signaling. ACC or PtoACO7 overexpression induced expressions of PtoRbohD/RbohF, which encode NADPH oxidases, and elevated H2 O2 levels in poplar. Inhibition of the NADPH oxidase compromised ethylene-induced disease resistance and PR gene expressions, while H2 O2 application could completely rescue the AVG-caused disease hypersensitivity. Therefore, the involvement of ethylene in disease resistance is done by activation of PR gene expressions and ROS production. Our results also showed that modifying ethylene biosynthesis or its signaling pathway has a great potential for improving disease resistance in woody plants.

Regulation of anthocyanin accumulation via MYB75/HAT1/TPL-mediated transcriptional repression.

Anthocyanin is part of secondary metabolites, which is induced by environmental stimuli and developmental signals, such as high light and sucrose. Anthocyanin accumulation is activated by the MYB-bHLH-WD40 (MBW) protein complex in plants. But the evidence of how plants maintain anthocyanin in response to signals is lacking. Here we perform molecular and genetic evidence to display that HAT1 plays a new breaker of anthocyanin accumulation via post-translational regulations of MBW protein complex. Loss of function of HAT1 in the Arabidopsis seedlings exhibits increased anthocyanin accumulation, whereas overexpression of HAT1 significantly repressed anthocyanin accumulation. We found that HAT1 interacted with MYB75 and thereby interfered with MBW protein complex. Overexpression of HAT1 suppresses abundant anthocyanin phenotype of pap1-D plant. HAT1 is characterized as a transcriptional repressor possessing an N-terminal EAR motif, which determines to interact with TOPLESS corepressor. Repression activity of HAT1 in regulation of gene expression and anthocyanin accumulation can be abolished by deletion or mutation of the EAR motif 1. Chromatin immunoprecipitation assays revealed that MYB75 formed a transcriptional repressor complex with HAT1-TPL by histone H3 deacetylation in target genes. We proposed that HAT1 restrained anthocyanin accumulation by inhibiting the activities of MBW protein complex through blocking the formation of MBW protein complex and recruiting the TPL corepressor to epigenetically modulate the anthocyanin late biosynthetic genes (LBGs).

A HAT1-DELLA signaling module regulates trichome initiation and leaf growth by achieving gibberellin homeostasis.

Trichome initiation and leaf growth are two critical developmental processes in the plant life cycle, which need to be optimized in accordance with developmental stage and immediate surroundings. To a large extent, this optimization is achieved by fine-tuning of hormonal pathways, including the gibberellin (GA) pathway. However, the mechanism by which plants control GA homeostasis to optimize these two developmental processes is unknown. Here, we report that HAT1, a HD-ZIP II transcription factor, negatively regulates GA-mediated trichome initiation and cotyledon expansion. Both protein and transcript levels indicated that HAT1 was induced by GA, while an increased abundance of HAT1, in turn, was found to suppress GA biosynthesis and signaling, thus forming a regulatory negative feedback loop that controls GA homeostasis to fine-tune trichome development and cotyledon expansion. We also found that HAT1 interacts with DELLAs, including GAI and RGA. GAI inhibits both protein stability and the binding activity of HAT1 to its target genes. Overexpression of HAT1 in della5 can completely suppress the enhanced trichome initiation and enlarged cotyledon of della5. Our findings demonstrate that HAT1 functions as a critical repressor to regulate GA-mediated trichome initiation and cotyledon growth; in addition, we describe a novel mechanism by which the plant regulates trichome initiation and cotyledon expansion through a HAT1-DELLA regulatory module under various GA concentrations.

Transcription factor HAT1 is a substrate of SnRK2.3 kinase and negatively regulates ABA synthesis and signaling in Arabidopsis responding to drought.

Drought is a major threat to plant growth and crop productivity. The phytohormone abscisic acid (ABA) plays a critical role in plant response to drought stress. Although ABA signaling-mediated drought tolerance has been widely investigated in Arabidopsis thaliana, the feedback mechanism and components negatively regulating this pathway are less well understood. Here we identified a member of Arabidopsis HD-ZIP transcription factors HAT1 which can interacts with and be phosphorylated by SnRK2s. hat1hat3, loss-of-function mutant of HAT1 and its homolog HAT3, was hypersensitive to ABA in primary root inhibition, ABA-responsive genes expression, and displayed enhanced drought tolerance, whereas HAT1 overexpressing lines were hyposensitive to ABA and less tolerant to drought stress, suggesting that HAT1 functions as a negative regulator in ABA signaling-mediated drought response. Furthermore, expression levels of ABA biosynthesis genes ABA3 and NCED3 were repressed by HAT1 directly binding to their promoters, resulting in the ABA level was increased in hat1hat3 and reduced in HAT1OX lines. Further evidence showed that both protein stability and binding activity of HAT1 was repressed by SnRK2.3 phosphorylation. Overexpressing SnRK2.3 in HAT1OX transgenic plant made a reduced HAT1 protein level and suppressed the HAT1OX phenotypes in ABA and drought response. Our results thus establish a new negative regulation mechanism of HAT1 which helps plants fine-tune their drought responses.

Hydrogen Peroxide and Nitric Oxide Crosstalk Mediates Brassinosteroids Induced Cold Stress Tolerance in Medicago truncatula.

Brassinosteroids (BRs) play pivotal roles in modulating plant growth, development, and stress responses. In this study, a Medicago truncatula plant pretreated with brassinolide (BL, the most active BR), enhanced cold stress tolerance by regulating the expression of several cold-related genes and antioxidant enzymes activities. Previous studies reported that hydrogen peroxide (H2O2) and nitric oxide (NO) are involved during environmental stress conditions. However, how these two signaling molecules interact with each other in BRs-induced abiotic stress tolerance remain largely unclear. BL-pretreatment induced, while brassinazole (BRZ, a specific inhibitor of BRs biosynthesis) reduced H2O2 and NO production. Further, application of dimethylthiourea (DMTU, a H2O2 and OH- scavenger) blocked BRs-induced NO production, but BRs-induced H2O2 generation was not sensitive to 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO, a scavenger of NO). Moreover, pretreatment with DMTU and PTIO decreased BL-induced mitochondrial alternative oxidase (AOX) and the photosystem capacity. However, pretreatment with PTIO was found to be more effective than DMTU in reducing BRs-induced increases in Valt, Vt, and MtAOX1 gene expression. Similarly, BRs-induced photosystem II efficiency was found in NO dependent manner than H2O2. Finally, we conclude that H2O2 was involved in NO generation, whereas NO was found to be crucial in BRs-induced AOX capacity, which further contributed to the protection of the photosystem under cold stress conditions in Medicago truncatula.

Nitric oxide as a signaling molecule in brassinosteroid-mediated virus resistance to Cucumber mosaic virus in Arabidopsis thaliana.

Brassinosteroids (BRs) are growth-promoting plant hormones that play a crucial role in biotic stress responses. Here, we found that BR treatment increased nitric oxide (NO) accumulation, and a significant reduction of virus accumulation in Arabidopsis thaliana. However, the plants pre-treated with NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-1-oxyl-3-oxide (PTIO)] or nitrate reductase (NR) inhibitor (tungstate) hardly had any NO generation and appeared to have the highest viral replication and suffer more damages. Furthermore, the antioxidant system and photosystem parameters were up-regulated in brassinolide (BL)-treated plants but down regulated in PTIO- or tungstate-treated plants, suggesting NO may be involved in BRs-induced virus resistance in Arabidopsis. Further evidence showed that NIA1 pathway was responsible for BR-induced NO accumulation in Arabidopsis. These results indicated that NO participated in the BRs-induced systemic resistance in Arabidopsis. As BL treatment could not increase NO levels in nia1 plants in comparison to nia2 plants. And nia1 mutant exhibited decreased virus resistance relative to Col-0 or nia2 plants after BL treatment. Taken together, our study addressed that NIA1-mediated NO biosynthesis is involved in BRs-mediated virus resistance in A. thaliana.

Role of Transcription Factor HAT1 in Modulating Arabidopsis thaliana Response to Cucumber mosaic virus.

Arabidopsis thaliana homeodomain-leucine zipper protein 1 (HAT1) belongs to the homeodomain-leucine zipper (HD-Zip) family class II that plays important roles in plant growth and development as a transcription factor. To elucidate further the role of HD-Zip II transcription factors in plant defense, the A. thaliana hat1, hat1hat3 and hat1hat2hat3 mutants and HAT1 overexpression plants (HAT1OX) were challenged with Cucumber mosaic virus (CMV). HAT1OX displayed more susceptibility, while loss-of-function mutants of HAT1 exhibited less susceptibility to CMV infection. HAT1 and its close homologs HAT2 and HAT3 function redundantly, as the triple mutant hat1hat2hat3 displayed increased virus resistance compared with the hat1 and hat1hat3 mutants. Furthermore, the induction of the antioxidant system (the activities and expression of enzymatic antioxidants) and the expression of defense-associated genes were down-regulated in HAT1OX but up-regulated in hat1hat2hat3 when compared with Col-0 after CMV infection. Further evidence showed that the involvement of HAT1 in the anti-CMV defense response might be dependent on salicylic acid (SA) but not jasmonic acid (JA). The SA level or expression of SA synthesis-related genes was decreased in HAT1OX but increased in hat1hat2hat3 compared with Col-0 after CMV infection, but there were little difference in JA level or JA synthesis-related gene expression among HAT1OX or defective plants. In addition, HAT1 expression is dependent on SA accumulation. Taken together, our study indicated that HAT1 negatively regulates plant defense responses to CMV.

GOLDEN2-LIKE transcription factors coordinate the tolerance to Cucumber mosaic virus in Arabidopsis.

Arabidopsis thaliana GOLDEN2-LIKE (GLKs) transcription factors play important roles in regulation of photosynthesis-associated nuclear genes, as well as participate in chloroplast development. However, the involvement of GLKs in plants resistance to virus remains largely unknown. Here, the relationship between GLKs and Cucumber mosaic virus (CMV) stress response was investigated. Our results showed that the Arabidopsis glk1glk2 double-mutant was more susceptible to CMV infection and suffered more serious damages (such as higher oxidative damages, more compromised in PSII photochemistry and more reactive oxygen species accumulation) when compared with the wild-type plants. Interestingly, there was little difference between single mutant (glk1 or glk2) and wild-type plants in response to CMV infection, suggesting GLK1 and GLK2 might function redundant in virus resistance in Arabidopsis. Furthermore, the induction of antioxidant system and defense-associated genes expression in the double mutant were inhibited when compared with single mutant or wild-type plants after CMV infection. Further evidences showed that salicylic acid (SA) and jasmonic acid (JA) might be involved in GLKs-mediated virus resistance, as SA or JA level and synthesis-related genes transcription were impaired in glk1glk2 mutant. Taken together, our results indicated that GLKs played a positively role in virus resistance in Arabidopsis.

Nitric oxide is involved in brassinosteroid-induced alternative respiratory pathway in Nicotiana benthamiana seedlings' response to salt stress.

Recent studies reported that brassinosteroids (BRs) can induce plant tolerance to different environmental stresses via the nitric oxide (NO) signaling pathway. Previous reports have indicated that alternative oxidase (AOX) plays an important role in plants under various stresses. The mechanisms governing how NO is involved as a signal molecule which connects BR with AOX in regulating stress tolerance are still unknown. Recently, we found that Nicotiana benthamiana seedlings which were pretreated with BR have more tolerance to salt stress, accompanied with an increase of CN-resistant respiration. Our results suggested that pretreatment with 0.1 µM brassinolide (BL, the most active brassinosteroid) alleviated salt-induced oxidative damage and increased the NbAOX1 transcript level. Application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-1-oxyl-3-oxide (cPTIO, an NO scavenger) or virus-induced gene silencing of nitrate reductase (NR) and nitric oxide synthase (NOS)-like enzyme compromised the BRs-induced alternative respiratory pathway. Furthermore, pretreatment with specific chemical inhibitors of NR and NOS or gene silencing experiments decreased plant resistance to salt stress which also compromised BRs-induced salt stress tolerance. In conclusion, NO is involved in BRs-induced AOX capability which plays essential roles in salt tolerance in N. benthamiana seedlings.

The heat response regulators HSFA1s promote Arabidopsis thermomorphogenesis via stabilizing PIF4 during the day.

During summer, plants often experience increased light inputs and high temperatures, two major environmental factors with contrasting effects on thermomorphological traits. The integration of light and temperature signaling to control thermomorphogenesis in plants is critical for their acclimation in such conditions, but the underlying mechanisms remain largely unclear. We found that heat shock transcription factor 1d (HSFA1d) and its homologs are necessary for plant thermomorphogenesis during the day. In response to warm daytime temperature, HSFA1s markedly accumulate and move into the nucleus where they interact with phytochrome-interacting factor 4 (PIF4) and stabilize PIF4 by interfering with phytochrome B-PIF4 interaction. Moreover, we found that the HSFA1d nuclear localization under warm daytime temperature is mediated by constitutive photomorphogenic 1-repressed GSK3-like kinase BIN2. These results support a regulatory mechanism for thermomorphogenesis in the daytime mediated by the HSFA1s-PIF4 module and uncover HSFA1s as critical regulators integrating light and temperature signaling for a better acclimation of plants to the summer high temperature.

Interaction of BES1 and LBD37 transcription factors modulates brassinosteroid-regulated root forging response under low nitrogen in arabidopsis.

Brassinosteriod (BR) plays important roles in regulation of plant growth, development and environmental responses. BR signaling regulates multiple biological processes through controlling the activity of BES1/BZR1 regulators. Apart from the roles in the promotion of plant growth, BR is also involved in regulation of the root foraging response under low nitrogen, however how BR signaling regulate this process remains unclear. Here we show that BES1 and LBD37 antagonistically regulate root foraging response under low nitrogen conditions. Both the transcriptional level and dephosphorylated level of BES1, is significant induced by low nitrogen, predominantly in root. Phenotypic analysis showed that BES1 gain-of-function mutant or BES1 overexpression transgenic plants exhibits progressive outgrowth of lateral root in response to low nitrogen and BES1 negatively regulates repressors of nitrate signaling pathway and positively regulates several key genes required for NO3 - uptake and signaling. In contrast, BES1 knock-down mutant BES1-RNAi exhibited a dramatical reduction of lateral root elongation in response to low N. Furthermore, we identified a BES1 interacting protein, LBD37, which is a negative repressor of N availability signals. Our results showed that BES1 can inhibit LBD37 transcriptional repression on N-responsive genes. Our results thus demonstrated that BES1-LBD37 module acts critical nodes to integrate BR signaling and nitrogen signaling to modulate the root forging response at LN condition.

A BIN2-GLK1 Signaling Module Integrates Brassinosteroid and Light Signaling to Repress Chloroplast Development in the Dark.

Arabidopsis GLYCOGEN SYNTHASE KINASE 3 (GSK3)-like kinases play various roles in plant development, including chloroplast development, but the underlying molecular mechanism is not well defined. Here, we demonstrate that transcription factors GLK1 and GLK2 interact with and are phosphorylated by the BRASSINOSTEROID insensitive2 (BIN2). The loss-of-function mutant of BIN2 and its homologs, bin2-3 bil1 bil2, displays abnormal chloroplast development, whereas the gain-of-function mutant, bin2-1, exhibits insensitivity to BR-induced de-greening and reduced numbers of thylakoids per granum, suggesting that BIN2 positively regulates chloroplast development. Furthermore, BIN2 phosphorylates GLK1 at T175, T238, T248, and T256, and mutations of these phosphorylation sites alter GLK1 protein stability and DNA binding and impair plant responses to BRs/darkness. On the other hand, BRs and darkness repress the BIN2-GLK module to enhance BR/dark-mediated de-greening and impair the formation of the photosynthetic apparatus. Our results thus provide a mechanism by which BRs modulate photomorphogenesis and chloroplast development.

Altered Brain Activation in Early Drug-Naive Parkinson's Disease during Heat Pain Stimuli: An fMRI Study.

Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by motor and nonmotor signs and symptoms. To date, many studies of PD have focused on its cardinal motor symptoms. To study the nonmotor signs of early PD, we investigated the reactions solicited by heat pain stimuli in early untreated PD patients without pain using fMRI. The activation patterns of contact heat stimuli (51°C) were assessed in 14 patients and 17 age- and sex-matched healthy controls. Patients with PD showed significant decreases in activation of the superior temporal gyrus (STG) and insula compared with controls. In addition, a significant relationship between activation of the insula and STG and the pain scores was observed in healthy controls but not in PD. This study provided further support that the insula and STG are important parts of the somatosensory circuitry recruited during the period of pain. The hypoactivity of the STG and insula in PD implied that functions including affective, cognitive, and sensory-discriminative processes, which are associated with the insula and STG, were disturbed. This finding supports the view that leaving early PD untreated could be tied directly to central nervous system dysfunction.