Karrikins control seedling photomorphogenesis and anthocyanin biosynthesis through a HY5-BBX transcriptional module.

The butenolide molecule, karrikin (KAR), emerging in smoke of burned plant material, enhances light responses such as germination, inhibition of hypocotyl elongation, and anthocyanin accumulation in Arabidopsis. The KAR signaling pathway consists of KARRIKIN INSENSITIVE 2 (KAI2) and MORE AXILLARY GROWTH 2 (MAX2), which, upon activation, act in an SCF E3 ubiquitin ligase complex to target the downstream signaling components SUPPRESSOR OF MAX2 1 (SMAX1) and SMAX1-LIKE 2 (SMXL2) for degradation. How degradation of SMAX1 and SMXL2 is translated into growth responses remains unknown. Although light clearly influences the activity of KAR, the molecular connection between the two pathways is still poorly understood. Here, we demonstrate that the KAR signaling pathway promotes the activity of a transcriptional module consisting of ELONGATED HYPOCOTYL 5 (HY5), B-BOX DOMAIN PROTEIN 20 (BBX20), and BBX21. The bbx20 bbx21 mutant is largely insensitive to treatment with KAR2 , similar to a hy5 mutant, with regards to inhibition of hypocotyl elongation and anthocyanin accumulation. Detailed analysis of higher order mutants in combination with RNA-sequencing analysis revealed that anthocyanin accumulation downstream of SMAX1 and SMXL2 is fully dependent on the HY5-BBX module. However, the promotion of hypocotyl elongation by SMAX1 and SMXL2 is, in contrast to KAR2 treatment, only partially dependent on BBX20, BBX21, and HY5. Taken together, these results suggest that light- and KAR-dependent signaling intersect at the HY5-BBX transcriptional module.

Identification of BBX proteins as rate-limiting cofactors of HY5.

As a source of both energy and environmental information, monitoring of incoming light is crucial for plants to optimize growth throughout development1. Concordantly, the light signalling pathways in plants are highly integrated with numerous other regulatory pathways2,3. One of these signal integrators is the basic leucine zipper domain (bZIP) transcription factor LONG HYPOCOTYL 5 (HY5), which has a key role as a positive regulator of light signalling in plants4,5. Although HY5 is thought to act as a DNA-binding transcriptional regulator6,7, the lack of any apparent transactivation domain8 makes it unclear how HY5 is able to accomplish its many functions. Here we describe the identification of three B-box containing proteins (BBX20, BBX21 and BBX22) as essential partners for HY5-dependent modulation of hypocotyl elongation, anthocyanin accumulation and transcriptional regulation. The bbx20 bbx21 bbx22 (bbx202122) triple mutant mimics the phenotypes of hy5 in the light and its ability to suppress the cop1 mutant phenotype in darkness. Furthermore, 84% of genes that exhibit differential expression in bbx202122 are also regulated by HY5, and we provide evidence that HY5 requires the B-box proteins for transcriptional regulation. Finally, expression of a truncated dark-stable version of HY5 (HY5(ΔN77)) together with BBX21 mutated in its VP motif strongly promoted de-etiolation in dark-grown seedlings, demonstrating the functional interdependence of these factors. In sum, this work clarifies long-standing questions regarding HY5 action and provides an example of how a master regulator might gain both specificity and dynamicity through the obligate dependence of cofactors.

CONSTITUTIVELY PHOTOMORPHOGENIC1 promotes ABA-mediated inhibition of post-germination seedling establishment.

Under acute stress conditions, precocious seedling development may result in the premature death of young seedlings, before they switch to autotrophic growth. The phytohormone abscisic acid (ABA) inhibits seed germination and post-germination seedling establishment under unfavorable conditions. Various environmental signals interact with the ABA pathway to optimize these early developmental events under stress. Here, we show that light availability critically influences ABA sensitivity during early seedling development. In dark conditions, the ABA-mediated inhibition of post-germination seedling establishment is strongly enhanced. COP1, a central regulator of seedling development in the dark, is necessary for this enhanced post-germination ABA sensitivity in darkness. Despite their slower germination, cop1 seedlings establish faster than wild type in the presence of ABA in both light and dark. PHY and CRY photoreceptors that inhibit COP1 activity in light modulate ABA-mediated inhibition of seedling establishment in light. Genetically, COP1 acts downstream to ABI5, a key transcriptional regulator of ABA signaling, and does not influence the transcriptional and protein levels of ABI5 during the early post-germination stages. COP1 promotes post-germination growth arrest independent of the antagonistic interaction between ABA and cytokinin signaling pathways. COP1 facilitates the binding of ABI5 on its target promoters and the ABA-mediated upregulation of these target genes is reduced in cop1-4. Together, our results suggest that COP1 positively regulates ABA signaling to inhibit post-germination seedling establishment under stress.

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.