The role of galacturonosyltransferase12/irregular xylem8 in Arabidopsis secondary cell wall formation and anther dehiscence
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GAUT12 (GAlactUronosylTransferase12)/IRX8 (IRregular Xylem8) is a putative glycosyltransferase involved in Arabidopsis secondary cell wall biosynthesis. Prior work has shown that Arabidopsis mutant irregular xylem8 (irx8) has collapsed xylem due to the lack of xylan and a small fraction of homogalacturonan (HG). The initial goal of this thesis research was to identify the biochemical function of GAUT12 by establishing an in vitro enzymatic reaction. Two hypotheses were tested: 1) GAUT12 is a galacturonosyltransferase (GalAT) that adds the galacturonic acid (GalA) into the xylan reducing-end oligosaccharide sequence, and 2) GAUT12 is an HG:GalAT that synthesizes a subfraction of HG. An anti-GAUT12 polyclonal antibody that specifically recognizes GAUT12 was generated and used to immunoabsorb GAUT12. The immunoabsorbed-GAUT12 fraction contained little activity for incorporation of D-[14C]GalA from UDP-D-[14C]GalA onto HG oligosaccharides or heat-inactivated irx8 microsomes, although it remains possible that GAUT12 utilizes other pectic structures as acceptors. During the course of this study, further characterization of the irx8 mutant revealed a reduction in lignin biosynthesis and deposition in mutant stems, which was also reflected in irx8 suspension culture cells grown on xylogenic medium. A reduction of lignin and xylan was also observed in the anther endothecium causing indehiscent anthers in irx8. The pollen grains of irx8, despite their smaller size compared to wild type, were viable upon manual release. Therefore, the irx8 sterility was solely due to indehiscent anthers. Detailed analyses of lignin using NMR spectroscopy confirmed the loss of G lignin and altered lignin extractability in irx8 stem cell walls, revealing potential changes in xylan and G lignin in the irx8 mutant. In addition, known genes involved in xylan biosynthesis were investigated for their expression levels by qPCR in irx8 suspension culture cells grown on xylogenic medium. Genes tightly correlated with tracheary element formation and those that may associate with GAUT12 function in xylan biosynthesis were identified.