30Once sentence summary: The contrasting roles of BBX21 and BBX24 in photomorphogenesis arise 31 through their different C-terminal regions and how they alter HY5 function at the post-transcriptional level.
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Abstract
34The Arabidopsis thaliana BBX family comprises several positive and negative regulators of 35 photomorphogenesis. BBX24, a member of BBX structural group IV, acts as a negative regulator of 36 photomorphogenesis, while another member from the same group, BBX21, is a positive regulator. The 37 molecular basis for the functional diversity shown by these related BBX family members is unknown.
38Using domain-swap lines, we show that the C-terminal regions of BBX24 and BBX21 specify their
Cotyledon opening is a key morphological change that occurs in seedlings during de-etiolation. Brassinosteroids (BR) inhibit opening of the cotyledons in darkness while light promotes cotyledon opening. The molecular regulation of the interplay between light and BR to regulate cotyledon opening is not well understood. Here, we show the B-box protein BBX32 negatively regulates light signaling and promotes BR signaling to inhibit cotyledon opening in Arabidopsis (Arabidopsis thaliana). BBX32 is highly expressed in the cotyledons of seedlings during de-etiolation. bbx32 and 35S:BBX32 seedlings exhibit enhanced and reduced cotyledon opening, respectively, in response to both light and brassinazole (BRZ) treatment in the dark, suggesting that BBX32 mediates cotyledon opening through both light and BR signaling pathways. BBX32 expression is induced by exogenous BR and is upregulated in bzr1-1D (BRASSINAZOLE RESISTANT1-1D). Our in vitro and in vivo interaction studies suggest that BBX32 physically interacts with BZR1. Further, we found that PIF3 (PHYTOCHROME INTERACTING FACTOR 3) interacts with BBX32 and promotes BR-mediated cotyledon closure. BBX32, BZR1, and PIF3 regulate the expression of common target genes that modulate the opening and closing of cotyledons. Our work suggests BBX32 integrates light and BR signals to regulate cotyledon opening during de-etiolation.
Summary
Greening of cotyledons during de‐etiolation is critical for harvesting light energy and sustaining plant growth. PIF3 and HY5 antagonistically regulate protochlorophyllide synthesis in the dark. However, the mechanism by which the PIF3/HY5 module regulates genes involved in protochlorophyllide synthesis is not clear.
Using genetic, molecular and biochemical techniques we identified that the B‐BOX protein BBX11 acts directly downstream of PIF3 and HY5 to transcriptionally modulate genes involved in protochlorophyllide synthesis.
Dark‐grown bbx11 and 35S:BBX11 seedlings exhibit an enhanced and reduced ability to green, respectively, when exposed to light. Transcript levels of HEMA1 and CHLH are upregulated in 35S:BBX11 seedlings that accumulate high levels of protochlorophyllide in the dark and undergo photobleaching upon illumination. PIF3 inhibits BBX11 in the dark by directly binding to its promoter. bbx11 suppresses the cotyledon greening defect of pif3 after prolonged dark, indicating that the PIF3‐mediated regulation of greening is dependent on BBX11. The enhanced greening of hy5 is also suppressed in hy5 lines overexpressing BBX11. In light, HY5 directly binds to the promoter of BBX11 and activates its expression to regulate BBX11‐mediated hypocotyl inhibition.
We show that a PIF3/HY5 module regulates BBX11 expression in opposite ways to optimise protochlorophyllide accumulation in the dark and promote photomorphogenesis in light.
BBX proteins are a family of zinc finger transcription factors that are versatile regulators of plant development. The 32 BBX proteins in Arabidopsis are subdivided into five structural groups based on their domain structure. Members of group IV play important and diverse roles in light-regulated development. The N-terminal B-box domains mediate DNA binding and transcriptional regulation. The C-terminal region determines the functional diversity of the structurally similar group IV members as reported in our recent study investigating the basis of functional diversification between BBX21 and BBX24. We also found that multi-layered regulation of HY5 by the BBX proteins leads to a diverse repertoire of developmental effects. Here we provide a comprehensive structure-function analysis of the group IV BBX proteins.
UV-B radiation acts as a developmental cue and a stress factor for plants, depending on dose. Activation of the transcription factor ELONGATED HYPOCOTYL 5 (HY5) in a UV RESISTANCE LOCUS 8 (UVR8)-dependent manner leads to the induction of a broad set of genes under UV-B. However, the underlying molecular mechanisms regulating this process are less understood. Here, we use molecular, biochemical, genetic, and metabolomic tools to identify the B-BOX transcription factor B-BOX PROTEIN 11 (BBX11) as a component of the molecular response to UV-B in Arabidopsis (Arabidopsis thaliana). BBX11 expression is induced by UV-B in a dose-dependent manner. Under low UV-B, BBX11 regulates hypocotyl growth suppression, whereas it protects plants exposed to high UV-B radiation by promoting the accumulation of photo-protective phenolics and antioxidants, and inducing DNA repair genes. Our genetic studies indicate that BBX11 regulates hypocotyl elongation under UV-B partially dependent on HY5. Overexpression of BBX11 can partially rescue the high UV-B sensitivity of hy5, suggesting that HY5-mediated UV-B stress tolerance is partially dependent on BBX11. HY5 regulates the UV-B-mediated induction of BBX11 by directly binding to its promoter. BBX11 reciprocally regulates the mRNA and protein levels of HY5. We report here the role of a BBX11-HY5 feedback loop in regulating photomorphogenesis and stress tolerance under UV-B.
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