What do organelles do?

The process of photosynthesis traps the energy of sunlight as chemical energy, which the plant uses for growth, defence and reproduction. The aim of this research program is to analyse the energy-handling metabolic pathways associated with plant organelles with the goal of discovering novel genes, proteins and mechanisms by which metabolite flux and energy flow is managed at the cellular level. By understanding these processes we can identify ways to optimize organelle function for improved plant growth, product quality and yield. We aim to:

• Identify the genes, proteins and metabolites involved in the metabolic pathways of the three “energy organelles”
• Investigate the function of newly discovered or poorly understood enzymes or transporters
• Discover how organelle energy metabolism contributes to plant growth, product quality and yield

Identifying Enzymes and Metabolites

One powerful approach to the discovery of new enzymes is proteomic analysis of energy organelles purified from plants. As part of this general strategy, research in the laboratory of CI Millar is identifying new proteins and protein complexes through ligand-binding. Such proteins are expected to bind to nucleotides, metals and other cofactors.

Equally important is the discovery, identification and quantification of the hundreds of metabolites produced by plant energy metabolism. Our metabolomics platform is being developed to be able to identify and measure sugars, organic acids, amino acids etc to give us powerful insights into the compounds that play key roles in the processes we are studying.

Research Highlight

ATP-binding proteins

PhD student Jun Ito has used ATP affinity chromatography to identify over 40 ATP-binding proteins with diverse roles in metabolism, metabolic regulation and organelle biogenesis. Selected proteins were separated by 1D and 2D gel electrophoresis and identified by mass spectrometry. A range of highly enriched proteins were identified from the mitochondrial proteome, including 14-3-3 proteins and RNA-binding proteins, as well as proteins known to contain nucleotide-binding domains and/or to be inhibited or stimulated by ATP.

Ito J, Heazlewood JL, Millar AH (2006) Analysis of the soluble ATP-binding proteome of plant mitochondria identifies new proteins and nucleotide triphosphate interactions within the matrix. Journal of Proteome Research 5:3459-69

Investigating the function of enzymes and transporters

Once new proteins have been discovered, we attempt to discover their function. For example, we have found at least 12 novel peroxisomal proteins, including some with potential functions in redox reactions and metabolism of unusual fatty acids. Their functions are under investigation through the isolation and phenotypic analysis of knock-out mutants.

The Centre focuses on photorespiration (see ‘Photorespiration’) and nitrogen re-assimilation as a fundamentally important component in energy metabolism. Photorespiration involves all three energy organelles and plays a role in carbon conservation and responses of the plant to oxidative stresses. A suite of antisense/RNAi plants is being isolated for each of 8 target genes, providing our research teams with plants having incremental changes in 8 key steps in the photorespiratory pathway.

To study the control that each enzyme exerts, plants will be grown under defined conditions and photosynthetic performance and gas exchange analysed. At the same time, knock-out mutants of key steps in photorespiration have been isolated to evaluate preconceived ideas about how photorespiration works. Already we have discovered that enzymes previously assumed to be essential for photorespiration can be dispensed with.

Another focus of the Centre is on Complex I, the first reaction complex in the electron transport chain of the mitochondrion. This, the largest and most enigmatic of the mitochondrial respiratory complexes, is responsible for a variety of reactions that link respiration with other aspects of cellular function including vitamin C production. We have isolated homozygous T-DNA knockout plant lines for several Complex I proteins. These often have severe seed germination frequency phenotypes and we are working to improve germination to allow proper study of these mutants.

Research Highlight

Peroxisomal malate dehydrogenase is dispensable

Itsara Pracharoenwattana at UWA has discovered that plants lacking peroxisomal malate dehydrogenase grow well in air whereas most photorespiratory mutants require elevated CO₂ for growth. Discovery of the explanation for this finding will reveal new insights into the carbon economy and stress responses of plants. Dr Asaph Cousins at ANU is now examining photosynthesis and CO₂ exchange properties in these plants.

Pracharoenwattana I, Cornah JE and Smith SM Arabidopsis peroxisomal malate dehydrogenase functions in beta-oxidation but not the glyoxylate cycle. Plant J, in press.

How does organelle energy metabolism contribute to plant growth, product quality and yield?

Our fundamental studies are accumulating information on the effects of various alterations in energy metabolism on plant growth and performance. It is anticipated that breakthroughs in our understanding of how to beneficially change plant energy will come from this research. As one example of how this may be achieved, we have discovered changes in resource allocation between organic acids and starch in leaves in plants with a modification to central carbon metabolism. We hope to build on this early success to rationally engineer plants with tissue-specific and developmentally regulated changes to resource allocation. Redirecting more resources into starch would be of great benefit in the biofuels sector.