Laboratory for Molecular Plant Biology, VKR Research Centre Pro-Active Plants, Department of Plant Biology and Biotechnology, University of Copenhagen, 1871, Frederiksberg C, Denmark.
Planta. 2012 Jul;236(1):115-28. doi: 10.1007/s00425-012-1592-3. Epub 2012 Jan 21.
Glycosyltransferase complexes are known to be involved in plant cell wall biosynthesis, as for example in cellulose. It is not known to what extent such complexes are involved in biosynthesis of pectin as well. To address this question, work was initiated on ARAD1 (ARABINAN DEFICIENT 1) and its close homolog ARAD2 of glycosyltransferase family GT47. Using bimolecular fluorescence complementation, Förster resonance energy transfer and non-reducing gel electrophoresis, we show that ARAD1 and ARAD2 are localized in the same Golgi compartment and form homo-and heterodimeric intermolecular dimers when expressed transiently in Nicotiana benthamiana. Biochemical analysis of arad2 cell wall or fractions hereof showed no difference in the monosaccharide composition, when compared with wild type. The double mutant arad1 arad2 had an arad1 cell wall phenotype and overexpression of ARAD2 did not complement the arad1 phenotype, indicating that ARAD1 and ARAD2 are not redundant enzymes. To investigate the cell wall structure of the mutants in detail, immunohistochemical analyses were carried out on arad1, arad2 and arad1 arad2 using the arabinan-specific monoclonal antibody LM13. In roots, the labeling pattern of arad2 was distinct from both that of wild type, arad1 and arad1 arad2. Likewise, in epidermal cell walls of inflorescence stems, LM13 binding differed between arad2 and WILD TYPE, arad1 or arad1 arad2. Altogether, these data show that ARAD2 is associated with arabinan biosynthesis, not redundant with ARAD1, and that the two glycosyltransferases may function in complexes held together by disulfide bridges.
糖基转移酶复合物已知参与植物细胞壁的生物合成，例如纤维素。目前尚不清楚这些复合物在果胶生物合成中参与的程度。为了解决这个问题，我们开始研究糖基转移酶家族 GT47 的 ARAD1（阿拉伯聚糖缺陷 1）及其密切同源物 ARAD2。使用双分子荧光互补、Förster 共振能量转移和非还原凝胶电泳，我们表明 ARAD1 和 ARAD2 定位于同一高尔基体隔室中，并且当在 Nicotiana benthamiana 中瞬时表达时，它们形成同源和异源二聚体的分子间二聚体。与野生型相比，对 arad2 细胞壁或其部分的生化分析显示单糖组成没有差异。arad1 arad2 双突变体具有 arad1 细胞壁表型，并且 ARAD2 的过表达不能补充 arad1 表型，表明 ARAD1 和 ARAD2 不是冗余酶。为了详细研究突变体的细胞壁结构，使用阿拉伯聚糖特异性单克隆抗体 LM13 对 arad1、arad2 和 arad1 arad2 进行了免疫组织化学分析。在根部，arad2 的标记模式与野生型、arad1 和 arad1 arad2 都不同。同样，在花序茎的表皮细胞壁中，LM13 结合在 arad2 和 WILD TYPE、arad1 或 arad1 arad2 之间存在差异。总之，这些数据表明 ARAD2 与阿拉伯聚糖生物合成有关，与 ARAD1 不冗余，并且这两种糖基转移酶可能在由二硫键结合在一起的复合物中发挥作用。
