ARAD proteins associated with pectic Arabinan biosynthesis form complexes when transiently overexpressed in planta
ARAD proteins associated with pectic Arabinan biosynthesis form complexes when transiently overexpressed in planta
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DOI：
10.1007/s00425-012-1592-3
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发表时间：
2012-07-01
期刊：
影响因子：
4.3
通讯作者：
Scheller, Henrik Vibe
中科院分区：
文献类型：
--
作者：
Harholt, Jesper;Jensen, Jacob Krueger;Scheller, Henrik Vibe
已知糖基转移酶复合物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多大程度上也参与了果胶的生物合成。为了解决这个问题，研究人员开始研究糖基转移酶家族GT47中的ARAD1 （ARABINAN DEFICIENT 1）及其同源物ARAD2。利用双分子荧光互补、福斯特共振能量转移和非还原凝胶电泳，我们发现ARAD1和ARAD2定位在同一个高尔基室中，并在烟叶中短暂表达时形成同源和异二聚体的分子间二聚体。对arad2细胞壁或部分进行生化分析，单糖组成与野生型比较无差异。双突变体arad1 - arad2具有arad1细胞壁表型，arad2的过表达不与arad1表型互补，说明arad1和arad2不是冗余酶。为了详细研究突变体的细胞壁结构，利用阿拉伯特异性单克隆抗体LM13对arad1、arad2和arad1进行了免疫组化分析。在根中，arad2的标记模式与野生型、arad1和arad1都不同。同样，在花序茎的表皮细胞壁中，arad2与野生型、arad1或arad1与arad2的LM13结合也不同。总之，这些数据表明ARAD2与阿拉伯糖的生物合成有关，而不是ARAD1的冗余，并且这两种糖基转移酶可能在由二硫桥连接在一起的复合物中起作用。
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, Forster 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.