Multilayered regulation of chloroplast development by single B-BOX CONSTANS-LIKE and GOLDEN2-LIKE proteins in Arabidopsis

Chloroplast development is primarily regulated by transcription factors, including those belonging to the GOLDEN2-LIKE (GLK) family, that alter the expression of genes encoding chloroplast proteins. However, the mechanisms by which multiple transcription factors coordinate to optimize chloroplast development remain unclear. In this study, we identified the single B-BOX (BBX) CONSTANS-LIKE (COL) proteins COL6 (also known as BBX14), COL7 (BBX16), COL8 (BBX17), and COL16 (BBX15) as novel regulators of chloroplast development in Arabidopsis thaliana. Notably, the col6 col16 (col-d) double mutant exhibited a chlorophyll-excess phenotype, which was further enhanced in the quadruple col6 col7 col8 col16 (col-q) mutant. Introduction of col-d and col-q mutations in the glk1 glk2 (glk-d) mutant partially restored the pale-green phenotype of the original glk-d mutant. COL proteins negatively and redundantly regulated a class of genes encoding chloroplast proteins, acting directly on their promoters. Moreover, COL6 and COL7 expression exhibited a diurnal cycle, with their expression patterns contrasting with those of GLK1 and GLK2, which are positive regulators of chloroplast development. Furthermore, GLK expression was markedly higher in the col-q mutants than in wild-type plants. Co-immunoprecipitation assays revealed that COL proteins interacted with the GARP DNA-binding domain in the N-terminus of GLK1, likely affecting its function. Moreover, COL proteins associate with each other to form homomeric and heteromeric complexes, suggesting functional redundancies at the protein level. In conclusion, these results suggest that the multilayered regulatory mechanisms of single BBX COL and GLK proteins optimize chloroplast development in Arabidopsis thaliana in fluctuating environments.