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

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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.