Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, CP 6109, 13083-970 Campinas, SP, Brazil.
Plant Physiol Biochem. 2012 Jun;55:66-76. doi: 10.1016/j.plaphy.2012.03.014. Epub 2012 Apr 3.
Class III peroxidases are present as large multigene families in all land plants. This large number of genes together with the diversity of processes catalyzed by peroxidases suggests possible functional specialization of each isoform. However, assigning a precise role for each individual peroxidase gene has continued to be a major bottleneck. Here we investigated the enzyme activity and translational profile of class III peroxidases during stem development of sugarcane as a first step in the estimation of physiological functions of individual isoenzymes. Internodes at three different developmental stages (young, developing and mature) were divided into pith (inner tissue) and rind (outer tissue) fractions. The rind of mature internodes presented the highest enzymatic activity and thus could be considered the ideal tissue for the discovery of peroxidase gene function. In addition, activity staining of 2DE gels revealed different isoperoxidase profiles and protein expression regulation among different tissue fractions. In-gel tryptic digestion of excised spots followed by peptide sequencing by LC-MS/MS positively matched uncharacterized peroxidases in the sugarcane database SUCEST. Multiple spots matching the same peroxidase gene were found, which reflects the generation of more than one isoform from a particular gene by post-translational modifications. The identified sugarcane peroxidases appear to be monocot-specific sequences with no clear ortholog in dicot model plant Arabidopsis thaliana.
III 类过氧化物酶作为一个大型的多基因家族存在于所有陆地植物中。大量的基因与过氧化物酶催化的多样性表明每个同工酶可能具有功能特异性。然而，为每个过氧化物酶基因赋予确切的功能一直是一个主要的瓶颈。在这里，我们研究了甘蔗茎发育过程中 III 类过氧化物酶的酶活性和翻译谱，作为估计单个同工酶生理功能的第一步。在三个不同发育阶段（幼嫩、发育中和成熟）的节间将内髓（内组织）和韧皮部（外组织）分开。成熟节间的韧皮部表现出最高的酶活性，因此可以认为是发现过氧化物酶基因功能的理想组织。此外，2DE 凝胶的活性染色显示不同的同工过氧化物酶图谱和不同组织部分之间的蛋白表达调控。经胶内胰蛋白酶消化后，通过 LC-MS/MS 对切下的斑点进行肽测序，与甘蔗数据库 SUCEST 中的未鉴定过氧化物酶阳性匹配。发现了多个与同一过氧化物酶基因匹配的斑点，这反映了特定基因通过翻译后修饰产生了不止一种同工酶。鉴定出的甘蔗过氧化物酶似乎是单子叶植物特有的序列，在双子叶模式植物拟南芥中没有明显的直系同源物。
