Departamento de Biologia Vegetal, Instituto de Biologia, CP 6109, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil.
Plant Physiol Biochem. 2013 Jan;62:1-10. doi: 10.1016/j.plaphy.2012.10.015. Epub 2012 Nov 6.
Secreted class III peroxidases (EC 1.11.1.7) are implicated in a broad range of physiological processes throughout the plant life cycle. However, the unambiguous determination of the precise biological role of an individual class III peroxidase isoenzyme is still a difficult task due to genetic redundancy and broad substrate specificity in vitro. In addition, many difficulties are encountered during extraction and analysis of cell wall proteins. Since class III peroxidases are also secreted into the apoplast, the use of suspension cell cultures can facilitate isolation and functional characterization of individual isoforms. Here, we report on the characterization of class III peroxidases secreted in the spent medium of sugarcane suspension cell cultures. After treatment with specific inducers of cell wall lignification, peroxidases were isolated and activities assayed with guaiacol, syringaldazine and coniferyl alcohol. Enzymatic activity was not significantly different after treatments, regardless of the substrate, with the exception of methyl-jasmonate treatment, which led to a decreased guaiacol peroxidase activity. Remarkably, peroxidases isolated from the medium were capable of oxidizing syringaldazine, an analog to sinapyl alcohol, suggesting that sugarcane cultures can produce peroxidases putatively correlated to lignification. A proteomic approach using activity staining of 2-DE gels revealed a complex isoperoxidase profile, composed predominantly of cationic isoforms. Individual spots were excised and analyzed by LC-ESI-Q-TOF and homology-based search against the Sugarcane EST Database resulted in the identification of several proteins. Spatio-temporal expression pattern of selected genes was determined for validation of identified class III peroxidases that were preferentially expressed during sugarcane stem development.
分泌型 III 过氧化物酶（EC 1.11.1.7）参与植物整个生命周期中的广泛的生理过程。然而，由于遗传冗余和体外广泛的底物特异性，明确确定单个 III 类过氧化物酶同工酶的确切生物学作用仍然是一项艰巨的任务。此外，在细胞壁蛋白的提取和分析过程中会遇到许多困难。由于 III 类过氧化物酶也分泌到质外体中，悬浮细胞培养物的使用可以促进单个同工型的分离和功能表征。在这里，我们报告了从甘蔗悬浮细胞培养物的废培养基中分泌的 III 类过氧化物酶的特性。用细胞壁木质素化的特定诱导剂处理后，用愈创木酚、丁香醛和松柏醇醛分离过氧化物酶，并测定其活性。无论底物如何，处理后酶活性没有显着差异，除了茉莉酸甲酯处理导致愈创木酚过氧化物酶活性降低。值得注意的是，从中介质分离的过氧化物酶能够氧化丁香醛，丁香醛是松柏醇的类似物，这表明甘蔗培养物可以产生可能与木质化相关的过氧化物酶。使用 2-DE 凝胶的活性染色的蛋白质组学方法揭示了复杂的同工酶图谱，主要由阳离子同工酶组成。将单个斑点切除并通过 LC-ESI-Q-TOF 进行分析，并根据 Sugarcane EST Database 进行基于同源性的搜索，鉴定出几种蛋白质。选择基因的时空表达模式用于鉴定在甘蔗茎发育过程中优先表达的鉴定的 III 类过氧化物酶的验证。
