Cytokinin Metabolic Genes and Enzymes

细胞分裂素是一种调节细胞分裂和分化的植物激素。最重要的自然产生的细胞分裂素是玉米素，一种具有高生物活性的化合物。在植物组织中发现了多种玉米素代谢物，其中一些是活性的，而另一些则不是，因此，了解玉米素代谢是如何调控的是至关重要的。该项目的长期目标是确定和描述实施这种控制的因素。以前的研究已经分离出玉米素特异的酶，包括将糖基转移到玉米素的O-糖基转移酶，并从豆类、拟南芥和玉米中克隆了与这些酶相对应的基因家族。O-葡萄糖化的玉米素广泛存在于植物组织中，是细胞分裂素的一种不活跃的、可逆的(通过水解)储存形式。与玉米素不同，O-葡萄糖化的玉米素能抵抗酶的破坏。因此，葡萄糖转移酶基因的活性可能会对活性细胞分裂素的水平产生重大影响，因此也会对植物的发育产生重大影响。另一种重要的酶是玉米素还原酶，它将玉米素转化为二氢玉米素，这是一种比玉米素更稳定的活性细胞分裂素。虽然二氢玉米素存在于所有植物中，而且已经从植物组织中分离出了这种还原酶，但与这种类型的酶相对应的基因还没有被鉴定出来。拟议的研究集中在玉米素O-葡萄糖基转移酶和还原酶上，包括一系列的分子、酶、生理和发育研究。本研究的具体目标是：(1)研究玉米素O-糖基化在种子萌发、运输和衰老过程中的作用；(2)确定玉米素O-葡萄糖基转移酶在拟南芥中的表达和细胞定位；(3)鉴定与底物专一性有关的酶上的氨基酸序列；以及(4)分离编码玉米素还原酶的基因。至于更广泛的影响，该项目涉及与捷克科学院的R·万科娃博士进行国际合作。该项目将研究与教育相结合，重点是本科生的培训，包括让学生在布拉格的R.Vankova实验室进行国际体验。拟议的研究还包括与私营行业的合作，这进一步扩大了研究的影响，并加强了新发现的流动。

Cytokinins are plant hormones mediating cell division and differentiation. The most important naturally-occurring cytokinin is zeatin, a compound with high biological activity. Various zeatin metabolites are found in plant tissues, some of which are active while others are not, and therefore, it is crucial to understand how zeatin metabolism is regulated. The long-term goal of this project is to identify and characterize elements exerting such controls. Previous research has led to isolation of zeatin-specific enzymes, including O-glycosyltransferases, which transfer a sugar group to zeatin, and cloning of a family of genes corresponding to these enzymes from beans, Arabidopsis, and maize. O-glucosylated zeatin occurs widely in plant tissues where it functions as an inactive, reversible (via hydrolysis) storage form of cytokinin. In contrast to zeatin, O-glucosylated zeatin is resistant to enzymatic breakdown. Consequently, the activities of the glucosyltransferase genes are likely to have significant impact on the level of active cytokinins and, as a consequence, also on plant development. Another important enzyme is zeatin reductase, which converts zeatin to dihydrozeatin, an active cytokinin that is much more stable than zeatin. Although dihydrozeatin occurs in all plants and the reductase has been isolated from plant tissue, genes corresponding to this type of enzyme have not yet been identified. The proposed research focuses on zeatin O-glucosyltransferases and reductases and includes a range of molecular, enzymatic, physiological, and developmental investigations. The specific objectives are to: (1) examine the role of zeatin O-glucosylation in seed germination, transport, and senescence; 2) determine the expression and cytolocalization of zeatin O-glucosyltransferases in Arabidopsis; 3) identify amino acid sequences on the enzymes critical to substrate specificity; and 4) isolate genes encoding zeatin reductases. As to broader impacts, the project involves international collaboration, with Dr. R. Vankova of the Czech Academy of Sciences. The project integrates research with education, with an emphasis on undergraduate training that includes an international experience for students in the laboratory of R. Vankova in Prague. The proposed research also includes collaboration with private industry, which further broadens the impact of the research and enhances the flow of new findings.