Expressions and functions of poplar peroxidase related to lignin biosynthesis and stress responses.

本研究对杨树体内过氧化物酶的表达和功能进行了较为详尽的研究。首先，从杨树基因组数据库中筛选出杨树过氧化物酶基因，从中筛选出83个完整的过氧化物酶基因序列。根据所选择的序列，分离出31个个体过氧化物酶基因，并用实时荧光定量聚合酶链式反应(Real-time PCR)技术分析其转录水平。8种过氧化物酶在多种器官中广泛表达，另外7种过氧化物酶基因在叶片中被过氧化氢强烈诱导。其次，对杨树各器官中的过氧化物酶蛋白进行了蛋白质组学分析。高表达的过氧化物酶蛋白为酸性异构体，属于小克拉斯特蛋白。两种过氧化物酶同工酶在发育中的木质部中特异表达。另外两种过氧化物酶在胁迫条件下高度诱导，表明这两种过氧化物酶可能参与大豆油菜素的合成或清除活性氧物种(ROS)。一种分离的过氧化物酶同工酶CWPO-C对芥子醇和合成木质素聚合物具有很高的氧化能力。这些动力学数据有力地表明CWPO-C是一种木质化特异的过氧化物酶。因此，用免疫组织化学技术研究了CWPO-C在杨树茎木质部发育过程中的定位。CWPO-C的标记强度从细胞壁加厚期到纤维细胞成熟期逐渐增强，这与发育中的木质部木质素含量的增加非常一致。这些结果有力地支持了CWPO-C对次生木质部木质化的作用。有趣的是，CWPO-C的免疫标记也在射线实质细胞内观察到，而在发育中的纤维细胞内没有检测到信号。这表明CWPO-C是在薄壁细胞中生物合成的，并提供给中间片层、细胞角和细胞壁，实现木质素聚合。

In this study, expressions and functions of peroxidases in poplar were exhaustively investigated. Firstly, poplar peroxidase genes were screened from Populus Genome Database, and 83 genes were selected as complete peroxidase sequences. Based on the selected sequences, 31 of individual peroxidase cDNA were isolated, and their transcriptional profiles were investigated by Real-time PCR technique. Eight peroxidases were extensively expressed in many organs and other 7 peroxidase genes were strongly induced in the leaf by hydrogen peroxide treatment.Secondary, proteomic analysis of peroxidase proteins in poplar organs was performed. The highly expressed peroxidase proteins were acidic isoforms and belong to a small claster. Two peroxidase isoforms were specifically expressed in the developing xylem. Other two peroxidases were highly induced by stress treatments, suggesting that these two peroxidases may be involved in the suberin synthesis or in the elimination of reactive oxygen species (ROS).An isolated peroxidase isoenzyme, CWPO-C, showed high oxidation ability toward sinapyl alcohol and synthetic lignin polymer. These kinetic data strongly suggest that CWPO-C is a lignification specific peroxidase. Therefore, localization of CWPO-C in the developing xylem of poplar stem was investigated by an immunohistochemical technique. The intensity of the CWPO-C labeling increased gradually from the cell wall thickening stage to mature stage of fiber cells, which is very consistent with the increase of lignin content in the developing xylem. These results strongly support that CWPO-C is responsible for the lignification of the secondary xylem. Interestingly, immuno-labeling of CWPO-C was also observed inside of the ray parenchyma cells instead no signals were detected within the developing fiber cells. This suggests that CWPO-C is biosynthesized in the parenchyma cells and provided to the middle lamellae, the cell corners, and the cell walls to achieve lignin polymerization.