Depletion of UDP-D-apiose/UDP-D-xylose synthases results in rhamnogalacturonan-II deficiency, cell wall thickening, and cell death in higher plants

(D)- Apiose serves as the binding site for borate cross- linking of rhamnogalacturonan II ( RG- II) in the plant cell wall, and biosynthesis of (D)- apiose involves UDP- (D)- apiose/ UDP- (D)- xylose synthase catalyzing the conversion of UDP- (D)- glucuronate to a mixture of UDP- (D)- apiose and UDP- (D)- xylose. In this study we have analyzed the cellular effects of depletion of UDP- D- apiose/ UDP- (D)- xylose synthases in plants by using virus- induced gene silencing ( VIGS) of NbAXS1 in Nicotiana benthamiana. The recombinant NbAXS1 protein exhibited UDP- (D)-apiose/ UDP- (D)- xylose synthase activity in vitro. The NbAXS1 gene was expressed in all major plant organs, and an NbAXS1- green fluorescent protein fusion protein was mostly localized in the cytosol. VIGS of NbAXS1 resulted in growth arrest and leaf yellowing. Microscopic studies of the leaf cells of the NbAXS1 VIGS lines revealed cell death symptoms including cell lysis and disintegration of cellular organelles and compartments. The cell death was accompanied by excessive formation of reactive oxygen species and by induction of various protease genes. Furthermore, abnormal wall structure of the affected cells was evident including excessive cell wall thickening and wall gaps. The mutant cell walls contained significantly reduced levels of D- apiose as well as 2- O- methyl- (L)- fucose and 2- O- methyl- (D)- xylose, which serve as markers for the RG- II side chains B and A, respectively. These results suggest that VIGS of NbAXS1 caused a severe deficiency in the major side chains of RG- II and that the growth defect and cell death was likely caused by structural alterations in RG- II due to a (D)- apiose deficiency.