Transcription factors BBX11 and HY5 interdependently regulate the molecular and metabolic responses to UV-B.

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.

B-box protein BBX32 integrates light and brassinosteroid signals to inhibit cotyledon opening.

Cotyledon opening is a key morphological change that occurs in seedlings during de-etiolation. Brassinosteroids (BRs) inhibit the opening of 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 treatment in 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 PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) 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.

PIF3/HY5 module regulates BBX11 to suppress protochlorophyllide levels in dark and promote photomorphogenesis in light.

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.

Two B-Box Proteins Regulate Photomorphogenesis by Oppositely Modulating HY5 through their Diverse C-Terminal Domains.

The Arabidopsis (Arabidopsis thaliana) BBX family comprises several positive and negative regulators of photomorphogenesis. BBX24, a member of BBX structural group IV, acts as a negative regulator of photomorphogenesis, whereas another member from the same group, BBX21, is a positive regulator. The molecular basis for the functional diversity shown by these related BBX family members is unknown. Using domain-swap lines, we show that the C-terminal regions of BBX24 and BBX21 specify their function. Because both BBX21 and BBX24 work in close association with HY5, we hypothesized that these proteins differentially regulate the levels or activity of HY5 to fulfill their opposite roles. We show that BBX21 can regulate HY5 post-transcriptionally and the two proteins can coordinate to promote photomorphogenesis. By contrast, BBX24 interferes with the binding of HY5 to the promoter of an anthocyanin biosynthetic gene, possibly by heterodimerizing with HY5 and preventing it from binding DNA. Our finding that both BBX21 and BBX24 regulate HY5 activity post-transcriptionally, in opposite ways, suggests that closely related B-box proteins execute contrasting functions through differential regulation of HY5.

The B-box bridge between light and hormones in plants.

Plant development is meticulously modulated by interactions between the surrounding environment and the endogenous phytohormones. Light, as an external signal coordinates with the extensive networks of hormones inside the plant to execute its effects on growth and development. Several proteins in plants have been identified for their crucial roles in mediating light regulated development. Among these are the B-box (BBX) family of transcription factors characterized by the presence of zinc-finger B-box domain in their N-terminal region. In Arabidopsis there are 32 BBX proteins that are divided into five structural groups on the basis of the domains present. Several BBX proteins play important roles in seedling photomorphogenesis, neighbourhood detection and photoperiodic regulation of flowering. There is increasing evidence that besides light signaling BBX proteins also play integral roles in several hormone signaling pathways in plants. Here we attempt to comprehensively integrate the roles of multiple BBX proteins in various light and hormone signaling pathways. We further discuss the role of the BBX proteins in mediating crosstalk between the two signaling pathways to harmonize plant growth and development. Finally, we try to analyse the conservation of BBX genes across species and discuss the role of BBX proteins in regulating economically important traits in crop plants.

Opposite roles of group IV BBX proteins: Exploring missing links between structural and functional diversity.

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.