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RAV1 (Related to ABI3/VP1) is a plant‐specific B3 and AP2 domain‐containing transcription factor that acts as a negative regulator of growth in many plant species. The expression of RAV1 is downregulated by brassinosteroids (BRs); large‐scale transcriptome analyses have shown that the expression of RAV1 was previously targeted by BRI1‐EMS‐SUPPRESOR1 (BES1) and BRASSINAZOLE‐RESISTANT1 (BZR1), which are critical transcription factors for the BR‐signaling process. Using RAV1‐overexpressing transgenic plants, we showed that RAV1 overexpression reduced the BR signaling capacity, resulting in the downregulation of BR biosynthetic genes and BES1 expression. Furthermore, we demonstrated that BES1, not BZR1, is directly bound to the RAV1 promoter and repressed RAV1 expression, and vice versa; RAV1 is also bound to the BES1 promoter and repressed BES1 expression. This mutual inhibition was specific to RAV1 and BES1 because RAV1 exhibited binding activity to the BZR1 promoter but did not repress BZR1 expression. We observed that constitutively activated BR signaling phenotypes in bes1‐D were attenuated by the repression of endogenous BES1 expression in transgenic bes1‐D plants overexpressing RAV1. RNA‐sequencing analysis of RAV1‐overexpressing transgenic plants and bes1‐D mutant plants revealed differentially expressed genes by RAV1 and BES1 and genes that were oppositely co‐regulated by RAV1 and BES1. RAV1 and BES1 regulated different transcriptomes but co‐regulated a specific set of genes responsible for the balance between growth and defense. These results suggested that the mutual inhibitory transcriptional activities of RAV1 and BES1 provide fine regulatory mechanisms for plant growth and development. [ABSTRACT FROM AUTHOR]

SUMMARY: Root growth dynamics is an outcome of complex hormonal crosstalk. The primary root meristem size, for example, is determined by antagonizing actions of cytokinin and auxin. Here we show that RAV1, a member of the AP2/ERF family of transcription factors, mediates cytokinin signaling in roots to regulate meristem size. The rav1 mutants have prominently longer primary roots, with a meristem that is significantly enlarged and contains higher cell numbers, compared with wild‐type. The mutant phenotype could be restored on exogenous cytokinin application or by inhibiting auxin transport. At the transcript level, primary cytokinin‐responsive genes like ARR1, ARR12 were significantly downregulated in the mutant root, indicating impaired cytokinin signaling. In concurrence, cytokinin induced regulation of SHY2, an Aux/IAA gene, and auxin efflux carrier PIN1 was hindered in rav1, leading to altered auxin transport and distribution. This effectively altered root meristem size in the mutant. Notably, CRF1, another member of the AP2/ERF family implicated in cytokinin signaling, is transcriptionally repressed by RAV1 to promote cytokinin response in roots. Further associating RAV1 with cytokinin signaling, our results demonstrate that cytokinin upregulates RAV1 expression through ARR1, during post‐embryonic root development. Regulation of RAV1 expression is a part of secondary cytokinin response that eventually represses CRF1 to augment cytokinin signaling. To conclude, RAV1 functions in a branch pathway downstream to ARR1 that regulates CRF1 expression to enhance cytokinin action during primary root development in Arabidopsis. Significance Statement: Interplay of auxin and cytokinin is known to regulate primary root meristem size in Arabidopsis, and here we show that RAV1 augments cytokinin signaling to control primary root meristem size. Cytokinin regulates RAV1 expression through ARR1, and RAV1 downregulates CRF1 expression to promote cytokinin actions and antagonize auxin response to determine proper root meristem size during post‐embryonic root development. [ABSTRACT FROM AUTHOR]

Abstract Background Early branching or syllepsis has been positively correlated with high biomass yields in short-rotation coppice (SRC) poplar plantations, which could represent an important lignocellulosic feedstock for the production of second-generation bioenergy. In prior work, we generated hybrid poplars overexpressing the chestnut gene RELATED TO ABI3/VP1 1 (CsRAV1), which featured c. 80% more sylleptic branches than non-modified trees in growth chambers. Given the high plasticity of syllepsis, we established a field trial to monitor the performance of these trees under outdoor conditions and a SRC management. Results We examined two CsRAV1-overexpression poplar events for their ability to maintain syllepsis and their potential to enhance biomass production. Two poplar events with reduced expression of the CsRAV1 homologous poplar genes PtaRAV1 and PtaRAV2 were also included in the trial. Under our culture conditions, CsRAV1-overexpression poplars continued developing syllepsis over two cultivation cycles. Biomass production increased on completion of the first cycle for one of the overexpression events, showing unaltered structural, chemical, or combustion wood properties. On completion of the second cycle, aerial growth and biomass yields of both overexpression events were reduced as compared to the control. Conclusions These findings support the potential application of CsRAV1-overexpression to increase syllepsis in commercial elite trees without changing their wood quality. However, the syllepsis triggered by the introduction of this genetic modification appeared not to be sufficient to sustain and enhance biomass production.

Key message: The two predicted WD40 propellers on TOPLESS function as protein–protein interaction domains. The 1st WD40 propeller mediates interaction with RAV1, and the 2nd WD40 propeller mediates interaction with VRN5. The TOPLESS/TOPLESS-RELATED (TPL/TPR) co-repressor family proteins are known to interact with a wide variety of proteins including transcription factors, Mediator subunits, histone deacetylases, and histone tails. Through these interactions, TPL/TPR act to repress transcription in an increasingly diverse array of plant pathways. Proteins that bind TPL/TPR typically contain one or more Repression Domains (RDs) that mediate the interaction. For example, the well-characterized Ethylene response factor-associated Amphiphilic Repression (EAR) motif is known to facilitate interaction by binding the TOPLESS Domain (TPD) located in the N-terminus. Here we show that in yeast two-hybrid assays, the non-EAR protein, Related to ABI3/VP1-1 (RAV1), binds a novel region located within the first nine WD40-repeats of TPL. Protein modeling and in silico analysis suggest that these nine WD40 repeats may form the first of two WD40 propellers located on C-terminus of TPL. The interaction between RAV1 and the 1st WD40 propeller is conserved with another RAV family member, TEMPRANILLO1 (TEM1) and is mediated by the B3 Repression Domain (BRD) located on both RAV1 and TEM1. Also, the predicted 2nd WD40 propeller was shown in yeast cells to bind Vernalization 5 (VRN5), which contains several unconfirmed partial RDs. Furthermore, we demonstrate that the 1st WD40 propeller of TPL can form a complex with RAV1 both in yeast and in Arabidopsis protoplasts. [ABSTRACT FROM AUTHOR]

Background: Early branching or syllepsis has been positively correlated with high biomass yields in short-rotation coppice (SRC) poplar plantations, which could represent an important lignocellulosic feedstock for the production of second-generation bioenergy. In prior work, we generated hybrid poplars overexpressing the chestnut gene RELATED TO ABI3/VP1 1 (CsRAV1), which featured c. 80% more sylleptic branches than non-modified trees in growth chambers. Given the high plasticity of syllepsis, we established a field trial to monitor the performance of these trees under outdoor conditions and a SRC management. Results: We examined two CsRAV1-overexpression poplar events for their ability to maintain syllepsis and their potential to enhance biomass production. Two poplar events with reduced expression of the CsRAV1 homologous poplar genes PtaRAV1 and PtaRAV2 were also included in the trial. Under our culture conditions, CsRAV1-overexpression poplars continued developing syllepsis over two cultivation cycles. Biomass production increased on completion of the first cycle for one of the overexpression events, showing unaltered structural, chemical, or combustion wood properties. On completion of the second cycle, aerial growth and biomass yields of both overexpression events were reduced as compared to the control. Conclusions: These findings support the potential application of CsRAV1-overexpression to increase syllepsis in commercial elite trees without changing their wood quality. However, the syllepsis triggered by the introduction of this genetic modification appeared not to be sufficient to sustain and enhance biomass production. [ABSTRACT FROM AUTHOR]

RAV1 ( Related to ABI3/ VP1) has been widely studied in plants such as Arabidopsis, pepper and rice; however, the functions of RAVs in maize have not been described. In this study, a novel gene ZmRAV1 was amplified from a Zea mays inbred line H21. This gene was predicted to encode a transcription factor with 2 distinct DNA-binding domains, AP2 and B3, which are both present in members of the RAV family. Transient expression assays of a 35S:: YFP-ZmRAV1 fusion construct in onion epidermal cells revealed that the ZmRAV1 protein is localized in the nucleus. ZmRAV1 expression was upregulated in maize by dehydration, salt, and ABA stress. Overexpression of ZmRAV1 in transgenic Arabidopsis plants enhanced salt and osmotic stress tolerance compared to the wild type, further confirmed by increased survival rate, longer primary roots and physiological characteristics such as lower relative electrolyte leakages. Illumina sequencing revealed that a number of salt responsive genes, primarily involved in reactive oxygen species scavenging, were upregulated in the ZmRAV1 transgenic line compared to the wild-type plants. The detection of the activity of antioxidant enzyme in WT and 35S:: ZmRAV1 plants under salt stress shown that, higher maintenance of POD contributes to the salt tolerance of Arabidopsis transgenic lines. These data suggest that ZmRAV1 functions as a transcriptional activator that may be involved in the salt and osmotic resistance signaling pathways in plants. [ABSTRACT FROM AUTHOR]

Leaf senescence is a developmentally programmed cell death process that constitutes the final step of leaf development and involves the extensive reprogramming of gene expression. Despite the importance of senescence in plants, the underlying regulatory mechanisms are not well understood. This study reports the isolation and functional analysis of RAV1, which encodes a RAV family transcription factor. Expression of RAV1 and its homologues is closely associated with leaf maturation and senescence. RAV1 mRNA increased at a later stage of leaf maturation and reached a maximal level early in senescence, but decreased again during late senescence. This profile indicates that RAV1 could play an important regulatory role in the early events of leaf senescence. Furthermore, constitutive and inducible overexpression of RAV1 caused premature leaf senescence. These data strongly suggest that RAV1 is sufficient to cause leaf senescence and it functions as a positive regulator in this process. [ABSTRACT FROM PUBLISHER]

Cold tolerance is regulated by a variety of transcription factors (TFs) and their target genes. Except for the well-characterized C-repeat binding factors (CBFs)-dependent transcriptional cascade, the mechanisms of cold tolerance mediated by other transcriptional regulatory networks are still largely unknown. Here, we used the assay for transposase-accessible chromatin with sequencing (ATAC-seq) and RNA-seq to identify cold responsive TFs in Vitis amurensis, a grape species with high cold hardiness. Nine TFs, including CBF4, RAV1 and ERF104, were identified after cold treatment. Weighted gene co-expression network analysis (WGCNA) and gene ontology (GO) analysis revealed that these TFs may regulate cold response through different pathways. As a prime candidate TF, overexpression of VaRAV1 in grape cells improved its cold tolerance. The transgenic cells exhibited low electrolyte leakage and malondialdehyde content and high peroxidase activity. Moreover, the TF gene TCP8 and a gene involving in homogalacturonan biosynthesis were found to be regulated by VaRAV1, suggesting that the contribution of VaRAV1 to cold tolerance may be achieved by enhancing the stability of cell membrane and regulating the expression of target genes involved in plant cell wall composition. Our work provides novel insights into plant response to cold stress and demonstrates the utility of ATAC-seq and RNA-seq for the rapid identification of TFs in response to cold stress in grapevine. VaRAV1 may play an important role in adaption to cold stress., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Dehydration stress response is a complex mechanism in plants involving several factors and hormone signalling pathways. RAV1 is a member of the AP2/ERF family of transcription factors that works in various developmental pathways. Here we show that downregulation of RAV1 gene expression is important for efficient dehydration stress response. Interestingly, the B3-domain transcription factor ABI3 negatively regulates RAV1 expression. In absence of ABI3, RAV1 expression increases during dehydration stress compared to control. As a part of stress response, ABI3 occupancy increases in the RAV1 promoter region. Such regulation of RAV1 gene expression seems vital as absence of RAV1 leads to reduced water loss during dehydration stress and consequently faster recovery compared to wild type. rav1 mutant seedlings show more abundant root growth under control condition and higher primary root elongation compared to wild type when subjected to dehydration stress. Mutants also exhibit enhanced ABA sensitivity compared to wild type. At the transcript level, rooting genes like NAC1 , ARF16 , SLR and SLR -downstream genes like ARF7 , PLT3 , SHR show differential expression in rav1 mutant, compared to wild type. Additionally, ethylene-responsive genes ETR1 , EIN2 and ERF1 also get differentially expressed in presence and absence of RAV1 under control and stress conditions. This indicates an altered ethylene response in the rav1 mutant. All these features render rav1 seedlings better equipped for responding to dehydration stress. It thus becomes evident that ABI3 mediated regulation of RAV1 gene expression is a significant part of dehydration stress signalling for efficient stress management at the molecular and morphological level. • Regulation of RAV1 expression is critical for efficient dehydration stress response. • ABI3, the B3 domain transcription factor regulates RAV1 gene expression. • RAV1 repression leads to efficient rooting and altered hormone response. • RAV1 repression affects regulation of specific gene expression. [ABSTRACT FROM AUTHOR]

Dehydration stress response is a complex mechanism in plants involving several factors and hormone signalling pathways. RAV1 is a member of the AP2/ERF family of transcription factors that works in various developmental pathways. Here we show that downregulation of RAV1 gene expression is important for efficient dehydration stress response. Interestingly, the B3-domain transcription factor ABI3 negatively regulates RAV1 expression. In absence of ABI3, RAV1 expression increases during dehydration stress compared to control. As a part of stress response, ABI3 occupancy increases in the RAV1 promoter region. Such regulation of RAV1 gene expression seems vital as absence of RAV1 leads to reduced water loss during dehydration stress and consequently faster recovery compared to wild type. rav1 mutant seedlings show more abundant root growth under control condition and higher primary root elongation compared to wild type when subjected to dehydration stress. Mutants also exhibit enhanced ABA sensitivity compared to wild type. At the transcript level, rooting genes like NAC1, ARF16, SLR and SLR-downstream genes like ARF7, PLT3, SHR show differential expression in rav1 mutant, compared to wild type. Additionally, ethylene-responsive genes ETR1, EIN2 and ERF1 also get differentially expressed in presence and absence of RAV1 under control and stress conditions. This indicates an altered ethylene response in the rav1 mutant. All these features render rav1seedlings better equipped for responding to dehydration stress. It thus becomes evident that ABI3 mediated regulation of RAV1 gene expression is a significant part of dehydration stress signalling for efficient stress management at the molecular and morphological level., Competing Interests: Declaration of competing interest Contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the funding agency., (Copyright © 2020 Elsevier B.V. All rights reserved.)