Arabidopsis 2010: Comprehensive Functional Analysis of the Arabidopsis RCD Gene Family

脱辅基类胡萝卜素是一类由类胡萝卜素降解而产生的次生代谢产物。它们作为发展和环境反应信号具有重要作用。它们还作为风味和营养质量的贡献者发挥重要作用。当第一个编码类胡萝卜素双加氧酶的基因玉米Vp 14被克隆时，在阐明脱辅基类胡萝卜素合成方面取得了关键性的进展。Vp 14编码脱落酸合成中的限制酶9-顺式环氧类胡萝卜素双加氧酶（NCED）。拟南芥含有编码与NCED相关的酶的基因家族的九个成员，所述酶在下文中称为RCD（与类胡萝卜素二氧合酶相关）。该家族中只有一部分直接参与脱落酸的合成，而该家族的其他成员可能编码将一系列类胡萝卜素代谢为多种挥发性和非挥发性脱辅基类胡萝卜素的酶。家族成员之一，RCD 1，确实在与NCED不同的位置切割多种类胡萝卜素底物，支持这一假设。因此，RCD家族在类胡萝卜素底物和切割位置上都表现出变化。我们将建立完整的拟南芥RCD基因家族的功能。我们已经确定了每个RCD基因的敲除突变体，并将酌情构建双重和三重敲除组合。使用细菌表达和体外试验，将确定RCD酶家族每个成员的底物特异性和产物。将定量每个突变体以及适当的双突变体和三突变体的脱辅基类胡萝卜素代谢谱。还将检查突变体与改变的脱辅基类胡萝卜素谱相关的表型改变。在完成这个计画后，我们将定义一套完整的进化相关酵素的功能，这些酵素可以合成广泛的类胡萝卜素。我们将进一步分离出这些生物活性分子的特定亚群合成缺陷的突变株系，并确定脱辅基类胡萝卜素亚群缺失对植物生长和发育的影响。关于RCD基因家族和单个基因产物的表征的当前信息可在http://www.hos.ufl.edu/kleeweb/2010RCDprogram.htmAll基因敲除将在2002年底前保藏于俄亥俄州州立大学拟南芥属储备中心（http：//www.biosci.ohiostate.edu/plantbio/Facilities/abrc/ABRCHOME.HTM）。

Apocarotenoids are a diverse set of plant secondary metabolites derived from carotenoid breakdown. They have significant roles as developmental and environmental response signals. They also have important roles as contributors to flavor and nutritional quality. A critical advance in elucidating apocarotenoid synthesis occurred when the first gene encoding a carotenoid dioxygenase, maize Vp14, was cloned. Vp14 encodes the limiting enzyme in abscisic acid synthesis, 9-cis-epoxycarotenoid dioxygenase (NCED). Arabidopsis contains nine members of a gene family encoding enzymes related to NCED, referred to hereafter as RCDs (Related to Carotenoid Dioxgenase). Only a subset of the family is directly involved in abscisic acid synthesis and the other members of the family likely encode enzymes that metabolize a range of carotenoids to multiple volatile and non-volatile apocarotenoids. One of the family members, RCD1, does indeed cleave multiple carotenoid substrates at a different position than NCED, supporting this hypothesis. Thus, the RCD family exhibits variation both in carotenoid substrates and position of cleavage. We will establish the functions of the complete Arabidopsis RCD gene family. We have identified knock-out mutants in every RCD gene and will construct double and triple knockout combinations, as appropriate. Using bacterial expression and in vitro assays, the substrate specificities and products of each member of the RCD enzyme family will be determined. The apocarotenoid metabolic profiles of each mutant, as well as appropriate double and triple mutants, will be quantified. Mutants will also be examined for phenotypic alterations associated with altered apocarotenoid profiles. Upon completion of the project, we will have defined the functions of a complete set of evolutionarily related enzymes that synthesize a broad range of apocarotenoids. We will have further isolated mutant lines deficient in synthesis of specific subsets of these biologically active moleculaes and determined the consequences of loss of subsets of apocarotenoids on plant growth and development. Current information on the RCD gene family and characterization of the individual gene products may be obtained at http://www.hos.ufl.edu/kleeweb/2010RCDprogram.htmAll gene knockouts will be deposited with the Ohio State University Arabidopsis stock center (http://www.biosci.ohiostate.edu/~plantbio/Facilities/abrc/ABRCHOME.HTM) by the end of 2002.