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Investigation of a KNAT7-BLH-OFP transcription factor complex involved in regulation of secondary cell wall biosynthesis in Arabidopsis thaliana Liu, Yuanyuan


The plant secondary cell wall is a composite network of complex polymers (cellulose, lignin, and hemicellulose) that provides protective and structural properties to the cell wall. Based on previous research, the Arabidopsis KNOX gene KNAT7 has been shown to act as a transcription factor that regulates secondary wall formation in Arabidopsis inflorescence stems in coordination with Ovate Family Proteins (OFPs). Co-expression and yeast two-hybrid analyses suggest that BEL1-LIKE HOMEODOMAIN (BLH) transcription factors could be part of a KNOX-BLH-OVATE transcription factor complex regulating aspects of secondary cell wall formation, together with KNAT7 and OFP1/4. I investigated the interactions of BLH partners with KNAT7 and OFP proteins through yeast two-hybrid and in planta bimolecular fluorescence complementation analyses, and have identified a BLH protein BLH6 (At4g34610), from among six candidate BLH proteins as a BLH interacting partner of KNAT7. In addition, I demonstrated that OFP4 interacts with homeodomain of KNAT7 and BLH6 interacts with the KNAT7 MEINOX domain by yeast two-hybrid analyses. Furthermore, I investigated the function of BLH6 and an additional BLH protein, BLH5 (At2g27220), by characterizing the phenotypic effects of blh loss of function and BLH overexpression on stem anatomy. Phenotype analysis showed that blh5 knockout mutant and BLH5 overexpression mutant are indistinguishable from wild type. blh6 knock out mutant displayed slightly thicker cell walls in interfascicular fibers. In addition, I employed protoplast transfection assay to demonstrate that BLH6 is a transcriptional repressor. This study provides new information regarding the existence of a BLH6-KNAT7-OFP complex and insights into the biological function of BLH6.

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