Organelle biogenesis

How are organelles made and maintained?

The process of photosynthesis, trapping the energy of sunlight into chemical energy, occurs within chloroplasts. The interconversion of this chemical energy into the plant’s principal energy currencies (high-energy nucleotides) occurs predominantly in mitochondria. Still further chemical interconversions take place in peroxisomes. Yet in a dry seed, prior to its germination, none of these energy organelles are present in a functional form.

The biogenesis and relative abundance of these energy organelles has an enormous impact on plant growth and productivity. The aim of this research program is to understand the way in which such organelles are assembled inside plant cells, and the processes that control and coordinate these key events. We will generate Arabidopsis plants (see ‘Why Arabidopsis?’) with alterations in biogenesis of one organelle type and then use a combination of molecular profiling techniques to analyse the impact of these defined alterations on the biogenesis and activity of the other organelles, on nuclear gene expression, and on metabolite profiles.

To summarise, in Organelle Biogenesis we aim to:

• Identify which proteins accumulate as organelle components
• Understand how organelle proteins synthesized outside the organelles enter them
• Discover regulatory mechanisms controlling protein synthesis inside organelles
• Discover how organelle biogenesis varies with tissue and developmental stage

Identifying which proteins accumulate as organelle components

The Centre's methods for cell fractionation are constantly being refined to enable us to examine in greater detail the progression of organelle biogenesis, and the appearance of novel proteins that contribute to nascent organelle function. We are systematically identifying the proteins present in the three energy organelles using proteomics, protein-tagging and bioinformatics predictions. Non-dissociating electrophoresis techniques enable us to examine particular complexes that these proteins are associated with and to postulate how they are formed. Using such analyses we can identify which proteins are critical to the organelle for its maintenance and proper functioning.

Transport of proteins into organelles

Proteins encoded by the nuclear genome and destined for organelles must be imported into organelles using specific, selective machinery. We are studying proteins critical for protein transport into mitochondria, and more specifically the receptor proteins on the outer of the two mitochondrial membranes. Analysis of knockout plants for particular receptors is providing insights into pathways for protein import and has identified new import receptors in plants.

Research Highlight

Star researcher: Ryan Lister

Ryan Lister completed his PhD studies on "Characterisation of the Plant Mitochondrial Protein Import Machinery". Ryan has used the post-genomic resources that became available after the sequencing of the Arabidopsis genome to make substantial progress in our understanding of how plant mitochondria import proteins. Ryan's studies used a variety of 'omic' approaches and biochemical reconstitution experiments to gain the first global overview of the plant mitochondrial protein import apparatus and its regulation. Using gene inactivation approaches, Ryan carried out the first functional characterisation of all the known protein receptors on the mitochondrial surface. The results of these investigations have been published in several high-profile publications, with more to come. On the basis of his stellar research, Ryan has been awarded a prestigious Human Frontiers Science Program (HFSP) Fellowship to carry out research at the Salk Institute in California.

Regulatory mechanisms controlling protein synthesis inside energy organelles

The genomes of chloroplasts and mitochondria encode key components in energy metabolism. Research has indicated that both mitochondrial and chloroplast gene expression are largely regulated post-transcriptionally, although how this is achieved has remained an enigma. A key aspect of our analysis of the regulation of organelle gene expression focuses on the role of pentatricopeptide repeat (PPR) proteins, an extensive family of RNA-binding proteins. The putative RNA sequence-specificity of the PPR proteins suggests them to be excellent candidates for controllers of organelle gene expression. We are studying about 50 Arabidopsis PPR mutants, many of which exhibit novel phenotypes. Analysis of these plant lines is ongoing both in Perth (where the PPR research focus is on their mechanisms of action) and Canberra (focused on phenotype analysis of Arabidopsis PPR mutants). An approach being adopted in our investigations into the control of translation within organelles is the analysis of mRNAs bound to organellar ribosomes.

Research Hightlight

Rice and Arabidopsis share conserved PPR genes

Nick O'Toole's systematic comparison of the 927 PPR genes from rice and Arabidopsis has led to fascinating insights into the evolution of this gene family. Whereas animals and algae only have 10 or so PPR genes, plants have hundreds, suggesting a massive increase in the complexity of organelle gene expression concomitant with adaptation to life on dry land. These genes are highly conserved between rice and Arabidopsis, confirming they play similar, crucial roles in even distantly related plants. This study will provide a firm basis for all future research on this important group of genes. Preliminary results were presented by Ian Small in a Keynote Presentation at the International Plant Molecular Biology Conference in Adelaide in 2006. The full study will be published in 2007.

Variations in organelle biogenesis with tissue and developmental stage

Development and modification of organelle functions are inextricably linked to plant development, productivity and environmental tolerance. Intuitively one would expect differences in organelle composition and relative abundance, depending on the role of a particular plant tissue or its developmental stage. Extending our published work of 2006 we are undertaking a detailed study of organelle development during the germination process, including a comparison of organelles in different tissue types, to unravel some of the complexity involved in organelle biogenesis and plasticity.

Our preliminary results identify a shoot-specific mitochondrial proteome, and ongoing analysis of organelles purified from different tissue types will contribute significantly to our understanding of the relationships between organelle biogenesis and plant productivity.

Research Highlight

Organelle biogenesis during germination

Kate Howell's study of rice embryo germination from dry seed to 48 hrs post-imbibition revealed that the mitochondrial protein import machinery is vitally important at this crucial stage. Mitochondria at early stages of imbibition can be viewed as empty sacks coated in protein import channels. As imbibition proceeds, the proteins required for mitochondrial metabolic function are made and rapidly imported. Towards the end of the first 48 hours, the abundance of the components of the import apparatus declines dramatically as mitochondria attain their typical composition. We have initiated a similar study in Arabidopsis, with methods for analysis of transcripts, proteins and metabolites from Arabidopsis seeds being developed.

Howell, K.A., A.H. Millar, and J. Whelan. 2006. Ordered assembly of mitochondria during rice germination begins with pro-mitochondrial structures rich in components of the protein import apparatus. Plant Mol Biol 60: 201-223.