Abstract Brassinosteroids (BRs) are a group of steroid hormones regulating plant growth and development. Since BRs do not undergo transport among plant tissues, their metabolism is tightly regulated by transcription factors (TFs) and feedback loops. BAS1 (CYP734A1, formerly CYP72B1) and SOB7 (CYP72C1) are two BR-inactivating cytochrome P450s identified in Arabidopsis thaliana. We previously found that a TF ATAF2 (ANAC081) suppresses BAS1 and SOB7 expression by binding to the Evening Element (EE) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)-binding site (CBS) on their promoters. Both the EE and CBS are known binding targets of the circadian regulatory protein CCA1. Here, we confirm that CCA1 binds the EE and CBS motifs on BAS1 and SOB7 promoters, respectively. Elevated accumulations of BAS1 and SOB7 transcripts in the CCA1 null mutant cca1-1 indicate that CCA1 is a repressor of their expression. When compared with either cca1-1 or the ATAF2 null mutant ataf2-2, the cca1-1 ataf2-2 double mutant shows higher SOB7 transcript accumulations and a stronger BR-insensitive phenotype of hypocotyl elongation in white light. CCA1 interacts with ATAF2 at both DNA–protein and protein–protein levels. ATAF2, BAS1, and SOB7 are all circadian regulated with distinct expression patterns. These results demonstrate that CCA1 and ATAF2 differentially suppress BAS1- and SOB7-mediated BR inactivation.
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Circadian regulation of the GLYCINE-RICH RNA-BINDING PROTEIN gene by the master clock protein CIRCADIAN CLOCK-ASSOCIATED 1 is important for plant innate immunity
Recent studies have demonstrated the importance of temporal regulation of pathogen defense by the circadian clock. However, our understanding of the molecular basis underlying this role of the circadian clock is still in its infancy. We report here the mechanism by which the Arabidopsis master clock protein CCA1 regulates an output target gene GRP7 for its circadian expression and function in pathogen defense. Our data firmly establish that CCA1 physically associates with the GRP7 promoter via the predicted CCA1-binding motif, evening element (EE). A site-directed mutagenesis study showed that while individual EE motifs differentially contribute to robust circadian expression of GRP7, abolishing all four EE motifs in the proximal GRP7 promoter disrupts rhythmicity of GRP7 expression and results in misalignment of defense signaling mediated by GRP7 and altered pathogen responses. This study provides a mechanistic link of the circadian regulation of an output gene to its biological function in pathogen defense, underscoring the importance of temporal control of plant innate immunity.
more » « less- NSF-PAR ID:
- 10395383
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Journal of Experimental Botany
- Volume:
- 74
- Issue:
- 3
- ISSN:
- 0022-0957
- Format(s):
- Medium: X Size: p. 991-1003
- Size(s):
- ["p. 991-1003"]
- Sponsoring Org:
- National Science Foundation
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CCA1 andTOC1 , two key clock genes, is altered in CHS‐deficient seedlings across the day/night cycle. Similar findings for a mutant line lacking flavonoid 3′‐hydroxylase (F3′H) activity, and thus able to synthesize mono‐ but not dihydroxylated B‐ring flavonoids, suggests that the latter are at least partially responsible; this was further supported by the ability of quercetin to enhanceCCA1 promoter activity in wild‐type and CHS‐deficient seedlings. The effects of flavonoids on circadian function were also reflected in photosynthetic activity, with chlorophyll cycling abolished in CHS‐ and F3′H‐deficient plants. Remarkably, the same phenotype was exhibited by plants with artificially high flavonoid levels, indicating that neither the antioxidant potential nor the light‐screening properties of flavonoids contribute to optimal clock function, as has recently also been demonstrated in animal systems. Collectively, the current experiments point to a previously unknown connection between flavonoids and circadian cycling in plants and open the way to better understanding of the molecular basis of flavonoid action. -
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Abstract PHYB ACTIVATION TAGGED SUPPRESSOR 1 (BAS1) and SUPPRESSOR OF PHYB‐4 7 (SOB7) are two cytochrome P450 enzymes that inactivate brassinosteroids (BRs) in
Arabidopsis . The NAC transcription factor (TF) ATAF2 (ANAC081) and the core circadian clock regulator CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) both suppress the expression ofBAS1 andSOB7 via direct promoter binding. Additionally, BRs cause feedback suppression onATAF2 expression. Here, we report that two ATAF‐subgroup TFs, ANAC102 and ATAF1 (ANAC002), also contribute to the transcriptional suppression ofBAS1 andSOB7 .ANAC102 andATAF1 gene‐knockout mutants exhibit elevated expression of bothBAS1 andSOB7 , expanded tissue‐level accumulation of their protein products and reduced hypocotyl growth in response to exogenous BR treatments. Similar toATAF2 , bothANAC102 andATAF1 are transcriptionally suppressed by BRs and white light. NeitherBAS1 norSOB7 expression is further elevated inATAF double or triple mutants, suggesting that the suppression effect of these three ATAFs is not additive. In addition,ATAF single, double, and triple mutants have similar levels of BR responsiveness with regard to hypocotyl elongation. ATAF2, ANAC102, ATAF1, and CCA1 physically interact with itself and each other, suggesting that they may coordinately suppressBAS1 andSOB7 expression via protein–protein interactions. Despite the absence of CCA1‐binding elements in their promoters,ANAC102 andATAF1 have similar transcript circadian oscillation patterns as that ofCCA1 , suggesting that these twoATAF genes may be indirectly regulated by the circadian clock.
