Molecular and Functional Analysis of Pectins in Intercellular Attachment by T-DNA Tagging Using a Haploid Tobacco Tissue Culture System

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Intercellular attachment is an essential process in the morphogenesis of multicellular organisms. A novel mutant, nolac-H18 (non-organogenic callus with loosely attached cells), generated by T -DNA transformation using leaf-disk-cultures of haploid Nicotiana plumbaginifolia, lost the ability to form tight intercellular attachments and adventitious shoots. The gene tagged with T -DNA, named NpGUT1 (glucuronyltransferase 1), was similar to the gene for the catalytic domains of animal glucuronyltransferases and was predominantly expressed in shoot and root apical meristems. The transformation of NpGUT1 complemented the nolac-H18 mutation and the expression of antisense NpGUT1 RNA produced crumbled shoots. The mutation caused defects in the glucuronic acid of rhamnogalacturonan II (RG-II) of pectin, which drastically reduced the formation of borate cross-linking of RG-II. NpGUT1, which encodes a novel glucuronyltransferase, is the first glycosyltransferase gene identified in pectin biosynthesis and is essential for intercellular attachment in plant meristems and tissues. In another mutant line nolac-H14, no neutral-sugar side chains, composed mainly of linear arabinan, were present. The gene tagged with T-DNA was cloned and named LARA1 (long arabinan related protein 1). The gene encodes a membrane protein and was predominantly expressed in shoot apex and root primordia. When nolac-H14 callus was cultured with arabinose, the phenotype of the mutant was recovered. These results suggest that LARA1 might be involved in the supply of UDP-arabinose to be putative arabinosyltransferase.