Yuan Youxi, Teng Quincy, Zhong Ruiqin, Haghighat Marziyeh, Richardson Elizabeth A, Ye Zheng-Hua
Department of Plant Biology, University of Georgia, Athens, GA, 30602, United States of America.
Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA, 30602, United States of America.
PLoS One. 2016 Jan 8;11(1):e0146460. doi: 10.1371/journal.pone.0146460. eCollection 2016.
Xylan is a major acetylated polymer in plant lignocellulosic biomass and it can be mono- and di-acetylated at O-2 and O-3 as well as mono-acetylated at O-3 of xylosyl residues that is substituted with glucuronic acid (GlcA) at O-2. Based on the finding that ESK1, an Arabidopsis thaliana DUF231 protein, specifically mediates xylan 2-O- and 3-O-monoacetylation, we previously proposed that different acetyltransferase activities are required for regiospecific acetyl substitutions of xylan. Here, we demonstrate the functional roles of TBL32 and TBL33, two ESK1 close homologs, in acetyl substitutions of xylan. Simultaneous mutations of TBL32 and TBL33 resulted in a significant reduction in xylan acetyl content and endoxylanase digestion of the mutant xylan released GlcA-substituted xylooligomers without acetyl groups. Structural analysis of xylan revealed that the tbl32 tbl33 mutant had a nearly complete loss of 3-O-acetylated, 2-O-GlcA-substituted xylosyl residues. A reduction in 3-O-monoacetylated and 2,3-di-O-acetylated xylosyl residues was also observed. Simultaneous mutations of TBL32, TBL33 and ESK1 resulted in a severe reduction in xylan acetyl level down to 15% of that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth. In particular, the S2 layer of secondary walls in xylem vessels of tbl33 esk1 and tbl32 tbl33 esk1 exhibited an altered structure, indicating abnormal assembly of secondary wall polymers. These results demonstrate that TBL32 and TBL33 play an important role in xylan acetylation and normal deposition of secondary walls.
木聚糖是植物木质纤维素生物质中的一种主要乙酰化聚合物，它可以在木糖基残基的O - 2和O - 3位进行单乙酰化和二乙酰化，以及在O - 2位被葡萄糖醛酸（GlcA）取代的木糖基残基的O - 3位进行单乙酰化。基于拟南芥DUF231蛋白ESK1特异性介导木聚糖2 - O - 和3 - O - 单乙酰化这一发现，我们之前提出木聚糖区域特异性乙酰取代需要不同的乙酰转移酶活性。在这里，我们证明了ESK1的两个紧密同源物TBL32和TBL33在木聚糖乙酰取代中的功能作用。TBL32和TBL33的同时突变导致木聚糖乙酰含量显著降低，突变木聚糖的内切木聚糖酶消化释放出没有乙酰基团的GlcA取代的木寡糖。木聚糖的结构分析表明，tbl32 tbl33突变体几乎完全丧失了3 - O - 乙酰化、2 - O - GlcA取代的木糖基残基。还观察到3 - O - 单乙酰化和2,3 - 二 - O - 乙酰化木糖基残基减少。TBL32、TBL33和ESK1的同时突变导致木聚糖乙酰水平严重降低至野生型的15%，同时，导管严重塌陷，植物生长受阻。特别是，tbl33 esk1和tbl32 tbl33 esk1木质部导管次生壁的S2层结构发生改变，表明次生壁聚合物组装异常。这些结果表明，TBL32和TBL33在木聚糖乙酰化和次生壁的正常沉积中起重要作用。
