1) A comprehensive set of tools required for functional analysis of the gene set will be created. These include (a) a series of mutants each defective for one gene in the network, (b) isoform-specific antibodies for each protein in the set, and (c) E. coli strains overexpressing each protein..
These tools will enable any research groups addressing starch metabolism to produce Arabidopsis plants lacking one or more members of the gene set. Individual proteins among the numerous starch metabolizing factors could be identified in fractionated extracts. Detailed biochemical characterizations various biochemical reconstitutions could be approached using purified factors.
2) Starch
biosynthesis and degradation in each mutant will be characterized regarding
starch levels, rates of accumulation and mobilization, and the molecular
architecture of amylopectin.
A broad characterization of starch metabolism will describe the effects of eliminating each protein in the study set. Leaf starch content will be measured over the course of diurnal cycles. Starch in roots and siliques also will be examined. Detailed structural analysis of amylopectin will be performed when altered starch metabolism is indicated.
3) The
effects of each mutation on the complement of specific isoforms of each enzyme
in the network will be determined using high-resolution, two-dimensional
zymogram techniques.
Zymograms that separate as many 30 different starch metabolism enzymes will be used to correlate activities with gene products. Using the mutant collection, this technique will determine whether loss of one protein affects activity of other enzymes in the network. These investigations are based on known functional interactions between components of the granule assembly machinery in other plant species.
4) Specific
proteins in the study set will be tested using affinity chromatography methods
for physical interaction with other components of the network. Additional proteins that interact with
components of the network will be identified.
Multisubunit complexes involving members of the study set are a likely explanation for the many known functional interactions between starch metabolizing-enzymes. Affinity columns bearing selected proteins will be used to purify interacting polypeptides from crude extracts, or to identify binding of other recombinant proteins. Isoform-specific antibodies and mass spectrometry will identify bound proteins.
5) Expression
of each gene will be characterized generally regarding tissue specificity and
the diurnal cycle.
Immunblot and quantitative RT-PCR methods will be used to examine expression in leaf, silique, and root. Expression in leaf will be determined over the course of the diurnal cycle.
6) The
effects of eliminating each member of the network on expression at the level of
mRNA accumulation of other Arabidopsis genes will be examined by global mRNA
profiling.
Changes in starch metabolism are likely to engender global responses that result in altered transcriptional profiles. Genes whose mRNA level is altered in each of the mutants will be identified in an effort to identify such global transcriptional networks, and also as a means of finding other candidate genes potentially involved in starch granule metabolism.