75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

321 On the diversity of the Arabidopsis Fructokinase Gene Family
Borjana Arsova, Wolfgang Lein
Max-Planck-Institute of Molecular Plant Physiology, Wissenschaftspark Golm, Am Muhlenberg 1, 14476
Potsdam, Germany
In the post genomic era it is obvious that we know little or nothing about the majority of genes that are contained in the recently
sequenced genomes. Thus, the challenge exists to attach functions to the numerous genes of poorly described or totally unknown
function.
Also, the existence of multiple enzyme isoforms, whose precise properties and subcellular localizations are still unresolved,
disables the proper understanding of the structure and regulation even of basic metabolic pathways, such as glycolysis, with the
possibility that multiple isoforms provide the regulatory framework that is needed to adapt metabolism during development and in
response to the environment.
Free fructose, resulting e.g. from sucrose cleavage, is phosphorylated by Fructokinase (FK) providing fructose-6-phosphate
which can then be used for starch synthesis or glycolysis. The inhibition of this enzyme by free Fructose plays an important role in
maintaining the flux of carbon towards starch formation, thus having a regulatory role.
Surprisingly, the Arabidopsis-genome encodes 10 putative FK-isoforms (AtFK), which phylogenetically can be divided into
three subgroups. All related literature for FK-isoforms from maize, rice and tomato where biochemical analysis is linked to individual
genes belongs to two, closely related subgroups. In contrast, the third group represented by a distantly associated cluster, containing
two AtFK isoforms, remains uncharacterised in this or any other species. Sequence analysis showed, that all but one AtFK gene
are carrying all described conserved protein motives (1), that are characteristic for a fructokinase. Although the in silico analysis of
expression patterns in tissues and organs (2) didn't show a specific spatial separation, it did point to one isoform as the predominant
form. Similarly we could identify diurnally and substantially expressed forms, while a co-response analysis (3) allowed us to predict
three of these forms as plastid localized (including the two AtFKs from the third cluster, one of which was proved to be plastid
localised by an independent group(4)). This in itself was an interesting finding because a plastidial fructokinase has not been described
so far. Only two of the AtFKs showed co-expressing glycolysis related genes.
Recent results that will be presented include: biochemical analysis of 5 recombinant proteins (including the putative palstidial
forms) - all showing FK but not HK activity, assays querying the intracellular localisation of the proteins etc.
(1) Pego & Smeekens, 2000, Tips; (2) Steinhauser et al, 2004, Bioinformatics; (3) Thimm et al, 2004, Plant J; (4) Kleffman et al. (2004), Curr.
Biol
322 Transcriptomic and Metabolomic Analysis of Antisense ATP-Citrate Lyase Arabidopsis
thaliana Supplemented with Malonic Acid
Heather Babka, Beth Fatland, Basil Nikolau, Eve Syrkin Wurtele
Iowa State University
ATP-citrate lyase (ACL) catalyzes the production of acetyl-CoA in the cytosol of Arabidopsis thaliana. The cytosolic
pool of acetyl-CoA is required for the production of the stress-related phytochemicals, stilbenoids and flavonoids, and for
elongation of fatty acids. In addition, cytosolic acetyl-CoA is essential for the synthesis of membrane sterols. Antisense
ACL plants have reduced ACL activity, and a very distinct phenotype including miniature organs, smaller cells, reduced
cuticular wax, and an increased accumulation of starch. This phenotype is reversed by exogenous malonic acid, which
feeds into the carboxylation pathway of acetyl-CoA metabolism (Fatland et al., 2004). Antisense ACL and wildtype
plants with and without treatment with malonic acid were analyzed by transcriptomics and metabolomics. These analyses
are providing clues as to the mechanisms that Arabidopsis employs to cope with a decreased level of ACL activity in
planta.