Characterization of PIC7 gene functions in Arabidopsis hormone response
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As the major plant hormone, auxin regulates many aspects of plant growth and development. Recent studies indicate that the auxin response pathway interacts with other plant hormone signaling pathways such as cytokinin, ethylene, ABA and GA in multiple ways. Therefore, the final outcome of plant growth responses is due to the crosstalk among several plant hormones. To identify novel genes involved in the auxin response, our laboratory used a forward genetics approach to isolate ethyl methanesulfonate (EMS) mutagenized Arabidopsis mutants that were resistant to synthetic auxin picloram. The objective of the research described here was to characterize one of the auxin (picloram) resistant mutants, pic7-1. The mutant gene pic7-1 was identified as adenine phosphoribosyltransferase1 (APT1). APT1 involves in purine metabolism by converting adenine into adenosine monophosphate (AMP) through transferring a phosphoribosyl group from 5-phosphoribosyl 1-pyrophosphate (PRPP) to adenine. The mutation in pic7-1 lies on the substrate-PRPP (5-Phosphoribosyl 1-pyrophosphate) binding domain of APT1, suggesting that mutation may alter the catalytic activity of the enzyme. This idea is further supported by the resistance of pic7-1 to 2,6-diaminopurine (DAP) which is metabolized by APT1 to its toxic nucleotide, 2,6-DAP riboside. APT1 is also suggested to be involved in cytokinin metabolism by converting active cytokinin bases to cytokinin ribosides. Therefore, loss of APT1 activity might lead to locally high level of active cytokinin. In pic7-1, transcript levels of cytokinin induced auxin biosynthetic genes, CYP79B2, CYP79B3 and NIT3 are elevated. In addition, morphological phenotypes between pic7-1 and auxin over-producing mutants are similar, suggesting that auxin level is increased in pic7-1 because of elevated active cytokinin. Resistance of pic7-1 to exogeneous auxin as well as altered auxin signaling components indicates that auxin signaling may also be altered in pic7-1. Moreover, mis-regulated type-B Arabidopsis Response Regulators (ARRs) and cytokinin related responses in pic7-1 indicate hampered cytokinin signaling in the mutant. Physiological characterization reveals that APT1 is also involved in several other plant hormone signaling pathways such as abscisic acid, gibberellic acid and ethylene. Arabidopsis expressing the reporter transgenes APT1::APT1-GUS and APT1::APT1-GFP reveals highly localized expression of APT1 in vasculature, pericycle, root tip and guard cells. APT1 is localized to cytoplasm, nucleus and the endoplasmic reticulum at the subcellular level. Expression of APT1::APT1-GFP complements the mutant phenotypes of pic7-1, further confirming that observed mutant phenotypes are caused by the mutation in APT1. The APT1 gene generates at least two transcripts through alternative splicing although biological significance of these splice variants is to be characterized.