| Identification of auxin-deficient mutants by forward genetic screen has been hindered by the complexity, redundancy or vitality of indole-3-acetic acid (IAA) biosynthesis routes. Although alleles of a few low auxin mutants were recently identified by screening altered responses to shade, ethylene, N-1-naphthylphthalamicacid (NPA) or cytokinin (CK), systematic genetic screen for isolating auxin-deficient mutants is still lacking. CK can induce root curling in auxin biosynthesis mutant CK-induced root curling 1/tryptophan aminotransferase of Arabidopsis 1 (ckrcl/taal), which can be used as a phenotypic’marker’for isolating auxin-deficient mutants. Phenotypic observations, genetic analyses and biochemical complementation after large-scale genetic screens showed that this screen system was capable for isolating mutants related to auxin biosynthesis, transport and signaling. Unlike transport/signaling mutants, the curled/waved phenotypes of auxin deficient mutants were significantly induced by CKs and could be rescued by exogenous auxins (except ckrwl), and mutants in some known biosynthesis genes were re-isolated. In addition, new auxin deficient mutants were also successfully isolated, suggesting that ckrc screening system is effective for discovering additional genes involved in auxin biosynthesis or homeostasis.CK-induced root waving 1(ckrwl), as a ckrc-like mutant, was isolated from the T-DNA mutant pools. Compared to other typical ckrc/w mutants, it shows different phenotypes, such as CK-induced root curling in ckrwl was rescue only by 2,4-dichlorophenoxyacetic acid (2,4-D). ckrwl was detected to be allelic to wavy growth 3 (wav3) which shows a greater curvature of root bending in response to gravity in previous studies, ckrw1 shows decreased endogenous free IAA and its metabolites, which are measured by LC-MRM-MS method. These results suggest that this mutant is probably not simply auxin-deficient. Cytokinins (CKs) regulate plant development and growth via a two-component signaling pathway. By forward genetic screening, we isolated an Arabidopsis mutant named grow fast on cytokinins 1 (gfc1), whose seedlings grew larger aerial parts on MS medium with CK. gfcl is allelic to a previously reported cutin mutant defective in cuticular ridges (dcr). GFC1/DCR encodes a soluble BAHD acyltransferase (a name based on the first four enzymes characterized in this family:Benzylalcohol O-acetyltransferase, Anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase and Deacetylvindoline 4-O-acetyltransferase) with diacylglycerol acyltransferase (DGAT) activity in vitro and is necessary for normal cuticle formation on epidermis in vivo.Here we show that gfcl was a CK-insensitive mutant, as revealed by its low regeneration frequency in vitro and resistance to CK in adventitious root formation and dark-grown hypocotyl inhibition assays. In addition, gfc1 had de-etiolated phenotypes in darkness and was therefore defective in skotomorphogenesis. The background expression levels of most type-A Arabidopsis Response Regulator (ARK) genes were higher in the gfcl mutant. The gfc1-associated phenotypes were also observed in the cutin-deficient glycerol-3-phosphate acyltransferase 4/8 (gpat4/8) double mutant [defective in glycerol-3-phosphate (G3P) acyltransferase enzymes GPAT4 and GPAT8, which redundantly catalyze the acylation of G3P by hydroxyl fatty acid (OH-FA)], but not in the cutin-deficient mutant cytochrome p450, family 86, subfamily A, polypeptide 2/aberrant induction of type three 1 (cyp86A2/att1), which affects the biosynthesis of some OH-FAs. Our results indicate that some acyltransferases associated with cutin formation are involved in CK responses and skotomorphogenesis in Arabidopsis. |
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