Identification of Signal Compound Involved in Root Development

University Of Arizona, Tucson AZ

Investigators

Abstract

Most of what is known about mitosis in plants is based on extrapolation from how cell division functions in other systems, such as yeast. Identification of new genes that control this multicellular process is hampered by the lack of a system in which cell division can be controlled precisely and synchronized among individuals. Recent studies in our laboratory have made it possible to synchronize cell division in root cap meristems by the experimental manipulation of root cap development. This system has been exploited to characterize a previously unknown enzyme from pea roots whose expression appears to be needed for the control of cell cycle in plants. The objective of this proposal is to identify the metabolic product(s) of this UDP-glucuronosyltransferase (UGT). The identity of the gene, PsUGT1, with sequences common to mammalian UGT-encoding genes, has been confirmed by in vitro enzyme activity. Expression of this microsomal-membrane bound UGT is tightly correlated with cell division, spatially and temporally. Moreover, a transient induction of PsUGT1 expression occurs in concert, within a time window measured in minutes, with the experimental activation of a transient increase in mitosis. Most important, meristem-localized inducible inhibition of PsUGT1 expression by antisense mRNA mutagenesis under the control of its own promoter is lethal in pea hairy roots as well as transgenic alfalfa plants. Fortuitously, however, constitutive expression of PsUGT1 antisense under the control of the CaMV35S promoter yields an intermediate, nonlethal phenotype in transgenic alfalfa plants. This provided a tool to demonstrate that the primary effect of PsUGT1 expression appears to be a reduced rate of cell division. Thus, a 2-3 fold reduction in plant growth is correlated with a 2-3 fold reduction in the rate of DNA synthesis. These observations are consistent with the following model: PsUGT1 expression regulates the activity of a factor controlling plant growth and development (Factor GD). Factor GD is constitutively present in meristematic cells, but its activity is modulated by reversible conjugation to glucuronide, catalyzed by PsUGT1. If this model is correct, completing the objectives will provide important insight into the ways mitosis is regulated in plants. In addition, transgenic plant systems may provide a new experimental approach to understanding mechanisms by which glycoconjugation regulates life processes by controlling biological levels of potent endogenous ligands as well as environmental poisons. The goal of this project is to determine the chemical nature of the metabolic product(s) of PsUGT1. Two approaches will be used: 1. Conjugation of known candidate molecules such as phytohormones with 14C-labelled-glycone by PsUGT1 will be tested directly by enzyme assay followed by separation of products on thin layer chromatography; and 2. The products of PsUGT1 activity obtained from recombinant strains of Neurospora crassa and plant tissue will be purified and analyzed by HPLC, mass spectroscopy and NMR. The identification and characterization of Factor GD will facilitate future studies to examine its role in cellular differentiation.