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Identification and Characterization of IAMH1 Gene in Biosynthesis of Plant Hormone Auxin

Abstract

The plant hormone auxin plays indispensable roles in every aspect of plant life. This dissertation centered on the major natural form of auxin, Indole-3-acetic acid (IAA). In Chapter 1, we reviewed the biosynthesis and degradation of auxin. In Chapter 2 we covered an unexpected finding when crossing an auxin biosynthesis mutant, yuc1, to a floral homeotic gene mutant, agamous (ag). The severe phenotypes of ag were suppressed in the F2 population, and the suppression could be stably transmitted to later generations even without the presence of yuc1 mutation. We demonstrated that this suppression was mediated by T-DNA insertions in the two genes, and it was related to epigenetic xiv changes in the ag mutant. We also demonstrated that this phenomenon is not rare, and advised caution when interpreting results obtained from T-DNA mutants. In Chapter 3, we introduced a novel technology for improved control of guide RNA (gRNA) production when using CRISPR systems for genome editing or gene activation /suppression. We employed self-cleaving ribozymes and attached them to both ends of a gRNA sequence to enable the expression of gRNAs using RNA polymerase II promoters. We showed that the ribozyme-flanked gRNA design is functional both in vitro and in yeast, and this opens new doors for controlled gRNA expression which was not possible using RNA polymerase III promoters. In Chapter 4, we reported a surprising finding that a null mutant of an auxinrelated gene abp1 generated using our ribozyme-based CRISPR technology described in Chapter 3 did not show any developmental defects, which is contrary to previous findings that abp1 null mutants were embryonic lethal. We continued to identify another null abp1 mutant which also had no obvious defects under normal growth conditions, and raised questions about the previous claims that ABP1 is an essential gene and it encodes an auxin-binding protein which may function as an auxin receptor. In Chapter 5, we described the identification and characterization of auxin -biosynthesis genes IAMH1 and IAMH2 involved in converting Indole-3-acetamide (IAM) into IAA. We showed that IAMH1 could hydrolyze IAM into IAA both in vitro and in vivo and this finding added another missing piece in the auxin biosynthesis pathways

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