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

细胞间粘附是多细胞生物形态发生的重要过程。利用单倍体烟草（Nicotianaplumbaginifolia）的叶盘培养物通过T-DNA转化产生的新突变体nolac-H18（具有松散附着细胞的非器官发生愈伤组织）丧失了形成紧密细胞间附着和不定芽的能力。用T-DNA标记的基因，命名为NpGUT 1（葡萄糖醛酸转移酶1），与动物葡萄糖醛酸转移酶催化结构域的基因相似，并且主要在茎和根的顶端分生组织中表达。NpGUT 1的转化补充了nolac-H18突变，反义NpGUT 1 RNA的表达产生了破碎的芽。该突变引起的缺陷，在葡糖醛酸的鼠李糖半乳糖醛酸聚糖II（RG-II）的果胶，这大大减少了形成的硼酸盐交联的RG-II。NpGUT 1编码一种新的葡萄糖醛酸转移酶，是果胶生物合成中鉴定的第一个糖基转移酶基因，对植物分生组织和组织中的细胞间附着至关重要。在另一个突变株系nolac-H14中，不存在主要由线性阿拉伯聚糖组成的中性糖侧链。用T-DNA标记的基因被克隆并命名为LARA 1（long arabinan related protein 1）。该基因编码一种膜蛋白，主要在茎尖和根原基中表达。当nolac-H14愈伤组织与阿拉伯糖一起培养时，突变体的表型得到恢复。这些结果表明，LARA 1可能参与供应UDP-阿拉伯糖是假定的阿拉伯糖基转移酶。

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.