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Functional analysis of KNOTTED-like homeobox and OVATE family proteins involved in secondary cell wall development in Arabidopsis Wang, Shumin

Abstract

The formation of plant secondary cell walls requires a complex network of transcriptional regulation, culminating in a coordinated suite of biosynthetic genes depositing walls, in a spatial and temporal fashion. The transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) is a Class II KNOTTED1-like homeobox (KNOX2) gene, that acts as a negative regulator of secondary cell wall biosynthesis in interfascicular fibers. Previously, members of Ovate Family Proteins (OFP1 and OFP4), were shown to interact with KNAT7 to negatively regulate wall formation. However, the function of other closely related KNOX2 and OFP genes in secondary wall formation remains unclear. Herein, I showed that knat3knat7 double mutants possess an enhanced irregular xylem (irx) phenotype relative to single mutants, and decreased interfascicular fiber cell wall thickness. Additionally, unlike the increased lignin content characteristic of knat7 mutants, knat3knat7 had no change in lignin content, while the monomeric lignin composition was substantially reduced relative to the wild-type plants. In contrast, KNAT3 overexpression resulted in thicker interfascicular fiber secondary walls, suggesting a positive regulation of KNAT3 in wall development. A thorough examination of OFP mutants showed that none of the single mutants revealed any wall defects, including ofp4, which was previously shown to interact with KNAT7. However, they do display leaf phenotypes. In contrast, plants overexpressing OFP isoforms consistently exhibited cell swelling, disordered microtubules, and dark-grown de-etiolated phenotypes, resembling phenotypes common to brassinosteroid deficient mutants. Using yeast two-hybrid and bimolecular fluorescence complementation assays, I identified two genes that interacted with OFP4, NAP1;1 and NAP1;2, members of the Nucleosome Assembly Protein 1 (NAP1) family. Higher-order, loss-of-function NAP1 and OFP mutants also exhibit altered cotyledon shape and a reduced cotyledon width:length ratio. The kidney-shaped cotyledon phenotype apparent in OFP4 overexpressing plants was suppressed in the nap1;1 nap1;2 nap1;3 triple mutant background. Together, my research suggests that in addition to KNAT7, KNAT3 also contributes to cell wall deposition, and that a complex network of positive and negative regulation governed by KNOX2 proteins regulates secondary wall formation. Moreover, the complex of OFP4 and NAP1 plays a significant role in the cotyledon development.

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