15th International Conference on Arabidopsis Research - TAIR

T07-083
Functional Analysis of Plant Nucleotide Metabolism:
The Nucleoside Mono- and Diphosphate Kinase
Gene Families in Arabidopsis thaliana
Claudia Kopka(1), Peter R. Lange(1), Ralf Boldt(2), Rita Zrenner(1)
1-Max-Planck-Institute of Molecular Plant Physiology, Golm, Germany
2-University of Rostock, Department of Bioscience - Plant Physiology, Rostock, Germany
Nucleotides have essential functions in a multitude of biochemical and
developmental processes during the life cycle of a plant. As components of
nucleic acids, phytohormones, and energy rich precursors for carbohydrate
metabolism (e.g. UDP-glucose) nucleotides are involved in whole plant
physiology. Predominantly in form of the purine ATP but especially important
for plant sucrose and cell wall metabolism in form of the pyrimidines UTP and
UDP nucleotides are co-substrates for most energy consuming or conserving
reactions.
Whereas the enzymes in the de novo synthesis of nucleotides are often
encoded by single genes the steps involved in salvaging and interconversion
processes are always encoded by multigene families. The aim of this project
is to elucidate the physiological function of genes necessary in supplying nucleoside
mono- and diphosphates for metabolism and growth in Arabidopsis.
We present a functional analysis of all nucleoside mono- and diphosphate
kinase genes with mRNA expression analysis using semiquantitative RT-PCR,
subcellular distribution studies using expression of GFP reportergene fusion
proteins, and by enzyme activity characterisation after heterologous expression
and purification of the respective proteins. In order to finally characterise
the in vivo function of all nucleotide kinases we are currently investigating
several mutants with defects in their nucleotide kinase gene expression.
T07 Metabolism (Primary, Secondary, Cross-Talk and Short Distance Metabolite Transport)
T07-084
Biosynthesis and distribution of glutathione in
developing Arabidopsis embryos
Andreas J Meyer(1), Narelle G Cairns(2), Christopher S Cobbett(2)
1-Heidelberg Institute of Plant Sciences, University of Heidelberg, Im Neuenheimer Feld 360,
69120 Heidelberg, Germany
2-Department of Genetics, The University of Melbourne, Australia 3010
Embryo development is a crucial part of the life cycle of plants, during which
the body plan of the daughter plant is established, storage products required
for germination are accumulated and desiccation tolerance develops, which
enables both seed and embryo to overcome prolonged periods with unfavourable
conditions. The tripeptide glutathione (GSH, gamma-glutamylcysteinyl
glycine) is an ubiquitous low molecular weight thiol in plant cells with cytosolic
concentrations between 0.1 and 3 mmol/L. Representing the major thiol
redox buffer in the cell GSH plays various important roles in plant metabolism
and is believed to be required during responses to environmental stress and
also for normal plant development. A single nucleotide substitution in the
gene AtGSH1 encoding of the first enzyme of GSH biosynthesis renders the
Arabidopsis thaliana mutant root meristemless1 (rml1) almost GSH-deficient
(Vernoux et al, 2000). The mutant lacks post-embryonic cell division, but despite
lack of GSH-biosynthesis capacity the embryo still develops to maturity
without any obvious phenotype. To investigate biosynthesis and distribution
of GSH in wild-type and mutant embryos, non-destructive GSH-imaging
techniques based on the GSH-specific fluorescent probe monochlorobimane
together with confocal laser scanning microscopy were used. Quantitative
image analysis showed that homozygous rml1 embryos from torpedo stage
onwards contain only very small amounts of GSH and thus are not supplied
with GSH from maternal tissues at this developmental stage. Additional
investigation of a T-DNA insertional mutation in AtGSH1 showed that this
knockout results in an embryonic lethal phenotype when homozygous. Thus,
the developing embryo requires at least a minimal amount of GSH in order
to complete embryogenesis to maturity and this GSH has to be synthesised
autonomously by the embryo itself.
Vernoux T et al. (2000) Plant Cell 12: 97-110
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin