Summary: Mitochondrial alternative oxidase (AOX) is involved in a large number of plant physiological processes, such as growth, development and stress responses; however, the exact role of AOX in response to drought remains unclear. In our study, we provide solid evidences that the activated AOX capacity positively involved in ethylene‐induced drought tolerance, in tomato (Solanum lycopersicum), accompanied by the changing level of hydrogen peroxide (H₂O₂) and autophagy. In AOX1a‐RNAi plants, the ethylene‐induced drought tolerance was aggravated and associated with decreasing level of autophagy. The H₂O₂ level was relatively higher in AOX1a‐RNAi plants, whereas it was lower in AOX1a‐overexpressing (35S‐AOX1a‐OE) plants after 1‐(aminocarbonyl)‐1‐cyclopropanecarboxylic acid (ACC) pretreatment in the 14th day under drought stress. Interestingly, the accumulation of autophagosome was accompanied by the changing level of reactive oxygen species (ROS) in AOX transgenic tomato under drought stress whether or not pretreated with ACC. Pharmacological scavenging of H₂O₂ accumulation in AOX1a‐RNAi (aox19) stimulated autophagy acceleration under drought stress, and it seems that AOX‐dependent ROS signalling is critical in triggering autophagy. Lower levels of ROS signalling positively induce autophagy activity, whereas higher ROS level would lead to rapid programmed cell death (PCD), especially in ethylene‐mediated drought tolerance. Moreover, ethylene‐induced autophagy during drought stress also can be through ERF5 binding to the promoters of ATG8d and ATG18h. These results demonstrated that AOX plays an essential role in ethylene‐induced drought tolerance and also played important roles in mediating autophagy generation via balancing ROS level.

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). Anthocyanins, a class of flavonoids distributed ubiquitously in the plant kingdom, are induced by environmental stimuli and developmental signals, such as high light and sucrose. It is well established that anthocyanin accumulation is regulated by the MYB-bHLH-WD40 (MBW) protein complex in plants. But little is known about the regulation of MBW protein complex by other factors. Here, we show that an HD-ZIP II transcription factor HAT1 negatively regulates anthocyanin accumulation via post-translational regulation 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 reveal that HAT1 interacted with MYB75 and thereby sequestered MBW protein complex. Overexpression of HAT1 in pap1-D mutant suppresses abundant anthocyanin phenotype of the pap1-D mutant. HAT1 identified was as a transcriptional repressor possessing an N-terminal EAR motif, which determines the interaction with TOPLESS corepressor. The deletion or mutation of the EAR motif 1 of HAT1 partially eliminates the repression activity of HAT1 in regulation of gene expression and anthocyanin accumulation. Our results illustrate a new repressor HAT1 which helps plants fine-tune anthocyanin accumulation.

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. Drought stress is a key environmental factor that severely reduces crop yield all over the world. The phytohormone abscisic acid (ABA) is known to mediate drought responses through regulating drought-responsive genes expression and stomatal closure, but the mechanisms that negatively regulate this process and prevent the adverse effects of excess drought responses on plant growth is less well studied. Here, we show that a HD-ZIP II transcription factor HAT1 negatively regulates ABA-mediated drought responses through suppressing ABA biosynthesis and signaling. The hat1hat3 mutant showed ABA-hypersensitive and drought-tolerant phenotypes, whereas the HAT1-overexpressing lines were insensitive to ABA and less tolerant to drought. Furthermore, we found SnRK2.3 kinase, a positive component of ABA signaling, interacts with and phosphorylates HAT1 to destabilize and suppress its binding activity. Overexpression of SnRK2.3 reduces HAT1 protein level and inhibits HAT1OX phenotypes in ABA and drought responses. Our results revealed a HAT1-mediated negative regulatory mechanism in attenuating the ABA signaling and drought response.