Arabidopsis 2010 Project
This Arabidopsis 2010 project will use biochemistry and genetics to unlock mysteries of Coenzyme A (CoA)-linked substrates central to intermediary metabolism, protection from stress, and production of plant hormones.
Unraveling the Mysteries of CoA
The goal of this project is to identify functions for 59 genes of CoA metabolism, 44 encoding carboxy-CoA ligases and 15 encoding carboxy-CoA thioesterases. Only 11 of these 59 genes have known functions. CoA-linked substrates are central to intermediary metabolism and to pathways that produce structural building blocks of the cell and its organelles, many chemicals that protect plants against biotic and abiotic stresses, and several hormones. Catabolic pathways that recycle carbon also depend upon CoA.
This project will provide biochemical and genetic access points in many pathways to which the genes collectively contribute. Assigning definitive functions for the genes will require parallel/integrated deployment of four strategies:
- Bioinformatics approaches including transcript profiling, promoter motif analysis, and modeling of protein structures.
- High-throughput screens and detailed biochemical assays of recombinant proteins will identify substrates and define enzyme kinetics.
- Tissue-specific expression and subcellular-targeting information will distinguish pathways utilizing different isozymes.
- Reverse genetics using knockouts, RNAi, TILLING, and overexpression will define the broader biological context.
Why CoA Metabolism?
Coenzyme A is an essential participant in a vast range of primary and secondary biochemical pathways that supply the core building blocks and energy for the cell, and allow the environmentally responsive flexibility that is essential to plants as sessile life forms. These pathways include some that are the first we ever learn as biochemists. Others are barely alluded to in textbooks because they have proved largely intractable to both biochemical and genetic analysis. The turnover of chloroplast isoprenoid components is a typical example in this respect. Also, there are pathways in which CoA clearly must be involved but this fact, and the requisite enzyme activities, are simply not referred to in most descriptions. For example, in the conversion of linolenic acid to jasmonate (JA), 3-oxo-2(2′[Z]-pentenyl)-cyclopentane-1-octanoic acid (OPC:8) needs to be activated to the CoA thioester before b-oxidation to jasmonoyl-CoA, which in turn must be acted on by an acyl-CoA thioesterase isozyme to release jasmonic acid.
We are using two sets of genes – those encoding carboxyl-CoA ligases and thioesterases – as entry points to identify and elucidate pathways involving CoA intermediates. High-throughput approaches will provide information that will define the biochemical roles of these pathways and act as attractive entry points for other researchers interested in investigating these pathways further. Just as importantly, reverse genetics and mutant-screening approaches will reveal the broader biological relevance of each pathway and provide the means for additional biochemical, genetic and genomic studies.
Our current investigations of nine long-chain acyl-CoA synthetases (LACSs) and four acyl-CoA thioesterases (ACHs) in Arabidopsis have produced both answers and new puzzles. We will use the knowledge and tools developed in these projects to guide and enable high-throughput analyses to define the encoded biochemical functions and broader biological roles of all 59 genes in this CoA metabolism set.