Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Molecular Biology, Šlechtitelů 11, Olomouc 783 71, Czech Republic.
Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Chemical Biology and Genetics, Šlechtitelů 11, Olomouc 783 71, Czech Republic.
Plant Physiol Biochem. 2014 Jan;74:283-93. doi: 10.1016/j.plaphy.2013.11.020. Epub 2013 Nov 28.
The cytokinin dehydrogenases (CKX; EC 1.5.99.12) are a protein family that maintains the endogenous levels of cytokinins in plants by catalyzing their oxidative degradation. The CKX family in maize (Zea mays L.) has thirteen members, only two of which--ZmCKX1 and ZmCKX10--have previously been characterized in detail. In this study, nine further maize CKX isoforms were heterologously expressed in Escherichia coli, purified by affinity and ion-exchange chromatography and biochemically characterized. ZmCKX6 and ZmCKX9 could only be expressed successfully after the removal of putative sequence-specific vacuolar sorting signals (LLPT and LPTS, respectively), suggesting that these proteins are localized to the vacuole. Substrate specificity analyses revealed that the CKX isoforms can be grouped into two subfamilies: members of the first strongly prefer cytokinin free bases while members of the second degrade a broad range of substrates. The most active isoform was found to be ZmCKX1. One of the studied isoforms, ZmCKX6, seemed to encode a nonfunctional enzyme due to a mutation in a conserved HFG protein domain at the C-terminus. Site-directed mutagenesis experiments revealed that this domain is essential for CKX activity. The roles of the maize CKX enzymes in the development of maize seedlings during the two weeks immediately after radicle emergence were also investigated. It appears that ZmCKX1 is a key regulator of active cytokinin levels in developing maize roots. However, the expression of individual CKX isoforms in the shoots varied and none of them seemed to have strong effects on the cytokinin pool.
细胞分裂素氧化酶（CKX；EC 1.5.99.12）是一类蛋白质家族，通过催化细胞分裂素的氧化降解来维持植物内源性细胞分裂素水平。玉米（Zea mays L.）中的 CKX 家族有 13 个成员，其中只有 2 个——ZmCKX1 和 ZmCKX10——之前已经被详细表征。在这项研究中，9 个进一步的玉米 CKX 同工型在大肠杆菌中异源表达，通过亲和和离子交换层析纯化，并进行了生化特性分析。只有去除假定的序列特异性液泡分拣信号（分别为 LLPT 和 LPTS）后，ZmCKX6 和 ZmCKX9 才能成功表达，这表明这些蛋白质定位于液泡。底物特异性分析表明，CKX 同工型可以分为两个亚家族：第一个亚家族的成员强烈偏好细胞分裂素游离碱基，而第二个亚家族的成员可以降解广泛的底物。最活跃的同工型是 ZmCKX1。研究的同工型之一 ZmCKX6 由于 C 末端保守的 HFG 蛋白结构域中的一个突变，似乎编码了一种无功能的酶。定点突变实验表明，该结构域对 CKX 活性至关重要。还研究了玉米 CKX 酶在胚根出现后两周内玉米幼苗发育过程中的作用。ZmCKX1 似乎是发育中的玉米根中活性细胞分裂素水平的关键调节因子。然而，各个 CKX 同工型在芽中的表达不同，它们似乎都没有对细胞分裂素库产生强烈影响。
