Dr Harvey Millar
ARC QEII Research Fellow
Plant Molecular Biology Group
School of Biomedical & Chemical Sciences
The University of Western Australia

Personal Website Address: http://www.mitoz.bcs.uwa.edu.au
Email: hmillar@cyllene.uwa.edu.au

Research Aims

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The Big Question

Determining the composition and function of mitochondria in plants and their interaction with the cell.

The Short-term goals

• In-depth mitochondrial proteome analysis by mass spectrometry.
• Differential proteome analysis during environmental stresses.
• Defining signals that influence mitochondrial gene expression.
• Finding novel components of multi-protein complexes in plant mitochondria.

Group (lab) Members and areas of research

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• Joshua Heazlewood (Post-doc) - mitochondrial mass spectrometry
• Pia Sappl (PhD student) - GST proteomics in plants
• Jasun Ito (PhD student) - phosphoproteomics
• Julian Tonti-Filippini (Webdesigner) - webdesign and database integration.

Funding

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Current

ARC Discovery Project "Functional Genomics of Plant Mitochondria"

Applications submitted or planned

ARC Discovery - Subcellular proteomics in Arabidopsis

 

Techniques in the Laboratory

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Currently active

• Arabidopsis and rice organelle isolation
• Arabidopsis cell culture maintenance and manipulation
• 1D, 2D and native gel electrophoresis
• Spectrophotometric enzyme analysis
• Western blot analysis
• In-gel protein digestion
• MALDI-TOF peptide analysis
• ESI-Q-TOF peptide analysis
• MALDI-Q-TOF peptide analysis
• Mascot and ProID peptide matching to genomic resources
• MySQL databasing of proteomic and genomic resources

In development

• mass spectrometry analysis of post-translational modifications
• multidimensional LC - MS/MS analysis
• higher order analysis of transcriptomic and proteomic data integration
• Clustering analysis of genomic data

Top Three;

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areas of knowledge

• Mitochondrial metabolism and function
• Proteome analysis
• Arabidopsis genomic resources

technical skills

• Subcellular fractionation
• Protein separation by electrophoresis and chromatography
• Peptide mass spectrometry

A statement on most significant contributions to this research field

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My major contribution has been the integration of a range of techniques from protein purification , enzyme kinetics and whole plant respiratory studies with molecular biology, to further our understanding of the role mitochondria play in the primary carbon and nitrogen metabolism of a range of plants. This approach has led to the discovery of allosteric activation of the alternative oxidase and the multigene family encoding the alternative oxidase of plant mitochondria, the primary publication on this work (Millar et al. 1993) has been cited in the literature 125 times to date. This research has changed the way other workers in the field measure AOX and interpret their results. I have undertaken the first work on the differential effect of nitric oxide on plant respiratory pathways (1995-1997). I have initiated a biochemical and compositional analysis of the mitochondrial lipoic acid containing complexes in plants linking expressed proteins with genes from genome sequencing, and uncovering their acute sensitivity to environmental stresses (1998-). I have used soybean, maize and now rice to consider the role of respiratory metabolism during the early growth stages of seedling embryo shoots and roots - this has integrated whole plant respiratory measurements quantitatively correlated to enzymology, bioenergetics, immunology and transcript abundance data (1997-). I have undertaken a plant proteome analysis programme to assess in an even more holistic way and at a molecular level the function of mitochondria in plants (2001-). I have also used these techniques to uncover changes induced by oxidative stress, functionally assess import machinery components, identify novel components of electron transport chain complexes in plants and compare mitochondria in plants to their counterparts in mammals and yeast (2001-). With an increasing interest in the expression and regulation of multigene families in Arabidopsis, I have been using bioinformatic analysis, microarray data and proteomics to analyse superfamilies involved in membrane transport and plant defense/detoxification in this model plant (2001-). Proteomic analysis has the potential to answer a range of unanswered questions in plant metabolism and for the first time to understand and assess the full range of these in plants.

Other evidence of impact and contributions to the field

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I was awarded the University Medal from the Australian National University in 1994 for my BSc (Hons) degree and supported, by an APA, was awarded my PhD in June 1997. To date I have been an author on 48 articles in refereed international journals, I have co-authored 10 book chapters or journal reviews, have been invited to contribute four in 2004 as senior author. In 1997 I was offered a Marie Curie Post-doctoral Training Award, an EMBO Postdoctoral Fellowship, and an HFSPO Long-Term Post-Doctoral Fellowship to undertake post-doctoral work in a number of laboratories in Europe . I accepted the HFSPO fellowship to work at the University of Oxford, UK. In 1998 as an HFSPO fellow, I was appointed a Junior Research Fellow of Linacre College, Oxford . In 1999 I was offered a 10-year University Research Fellowship from the Royal Society, and the Glasstone Research Fellowship in Science from the University of Oxford , to continue by research in the UK . I declined these offers to take up a University of Western Australia (UWA) Post-Doctoral Fellowship from Nov 1999. I was then awarded an ARC Australian Post-doctoral Fellowship commencing in June 2000 and awarded a QEII Research Fellowship, before the completion of by APDF, commencing in Jan 2002. During 2000 I was awarded one of 12 Young Researcher Awards from the ARC/DISR to attend the Australia 's Science Future Symposium in Canberra organised by the Australian Academy of Science (AAS), and was included as one of 12 young scientists interviewed by the AAS for their video archive of Australian Scientists in 2001. I was a co-organiser of the 8 th International Congress on Plant Mitochondria that was held in Perth in July 2002. I was an invited speaker in 2002 at a Plant Gordon Conference in the USA , and in 2003 at the Society of Experimental Biology Meeting, Southhampton , UK . In 2003, I was awarded the Peter Goldacre Medal from the Australian Society for Plant Scientists (ASPS), for excellence in plant science by a researcher under 35. I was also awarded the 2003 Premiers' Prize for Early Career Acheivement in Science in Western Australia . Whilst at UWA I have been awarded four small research grants from the ARC Small Grant (2000) and then the UWA Grant Scheme (2001-2003), I am now a member of the small grant committee for biological sciences at UWA. I was a joint CI with Assoc/Prof. J. Whelan on a successful ARC large grant application in 2000, this was then rolled-into a combined successful grant application in 2001 with others for a five year Discovery Project on plant mitochondria (2002-2006). I have also been a joint-CI on a series of successful ARC REIF bid to fund proteomic and transgenic facilities in WA in 2000-2002. I have supervised 9 honour students (2000-2003) and currently supervise 5 PhD students at UWA. I am also a member of the American Society of Plant Biologists, The Australian Society of Plant Scientists and the Australian Society for Biochemistry and Molecular Biology.

Publications - Last 5 years

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Book Chapters

• * Heazlewood, J.L ., Millar A.H. ( 2004) Proteomics of Plant Mitochondria In: Plant Functional Genomics, Haworth Press, Binghamton , New York . (invited chapter), Dario Leister (ed) in press (accepted 16 th July 2003).

• Millar, A.H. , Day. D.A., Mathieu C. (2002) Nitric oxide synthesis by plants and its potential impact on nitrogen and respiratory metabolism. In: Photosynthetic nitrogen assimilation and associated carbon metabolism (Eds C. Foyer and G. Noctor), Advances in Photosynthesis series, Vol. 12, Kluwer Academic Publishers, The Netherlands, pp 193-2004

• *Millar, A.H. , Liddell, A., Leaver, C.J. (2001) Isolation and Sub-Fractionation of Mitochondria from Plants. In 'Methods in Cell Biology on Mitochondria' (Ed. E. Schon), Academic Press. Vol 65, pp. 53-74.

• Atkin, O.K., Millar, A.H. , Gardeström, P., Day, D.A. (2000) Relationships between photosynthesis, carbohydrate metabolism and respiration. In Photosynthesis: Physiology and Metabolism (Advances in Photosynthesis) Leegood RC, Sharkey TD, von Caemmerer S (eds). Kluwer Academic Publishers, The Netherlands . pp153-175.

Journal Articles

• *Sappl PG, Onãte-Sanchez L, Singh K, Millar AH (2004) Proteomic analysis of glutathione S-transferases of Arabidopsis thaliana reveals differential salicylic acid induced expression of the plant specific Phi and Tau classes. Plant Molecular Biology, in press (acceptance 28 th January 2004).

• * Taylor NL, Heazlewood JL, Day DA, Millar AH (February 5, 2004). Lipoic acid-dependent oxidative catabolism of 2-oxo acids in mitochondria provides evidence of branched chain amino acid catabolism in Arabidopsis. Plant Physiology, in press (acceptance 1 st Dec 2003) doi/10.1104/pp.103.035675.

• *Orinda Chew O, Lister R, Qbadou S, Heazlewood JL, Soll J, Schleiff E, Millar AH , Whelan J (2004) A plant outer mitochondrial membrane protein with high amino acid sequence identity to a chloroplast protein import receptor. FEBS Letters, 557: 109-114.

• *Lister R, Chew O, Heazlewood JL, Lee M-N, Millar AH and Whelan J ( January 15, 2004 ) A Transcriptomic and Proteomic Characterization of the Arabidopsis Mitochondrial Protein Import Apparatus and Its Response to Mitochondrial Dysfunction. Plant Physiology; doi/10.1104/pp.103.033910

• Norman C, Howell KA, Cawthray G, Millar AH , Whelan J, Day DA (2004) Salicylic acid is an uncoupler ands inhibitor of mitochondrial electron transport. Plant Physiology 134: 492-501

• Taylor N.L., Day D.A., Millar A.H. (2004) The impact of lipid peroxidation on the function of mitochondria in integrated cellular carbon/nitrogen metabolism. J. Exp Bot 55: 1-10.

• *JL Heazlewood, JS Tonti-Filippini, A Gout, DA Day, JM Whelan and AH Millar (2004) Experimental Analysis of the Arabidopsis Mitochondrial Proteome Highlights Signalling and Regulatory Components, Provides Assessment of Targeting Prediction Programs and Points to Plant Specific Mitochondrial Proteins. The Plant Cell 16:241-256.

• Daley DO, Considine MJ, Howell KA, Millar AH , Day DA and Whelan J (2003). Analysis of respiratory gene expression in soybean cotyledons during postgerminative development. Plant Molecular Biology 51: 745-755.

• Goggin DE, Lipscombe R, Fedorova E, Millar AH , Atkins CA and Smith PMC (2003) Intracellular localisation and targeting of aminoimidazole ribonucleotide synthetase in cowpea. Plant Physiology 131:1033-1041.

• Taylor NL, Rudhe C, Hulett JM , Lithgow T, Glaser E, Day DA, Millar AH and Whelan J (2003) Environmental Stresses Inhibit and Stimulate Different Protein Import Pathways in Plant Mitochondria. FEBS Letters 547:125-130

• *Giegé P, Heazlewood JL, Roessner-Tunali U, Millar AH , Fernie AR , Leaver CJ, and Sweetlove LJ (2003) Enzymes of glycolysis are functionally associated with the mitochondrion in Arabidopsis cells. The Plant Cell 15: 2140-2151 .

• Millar AH , Mittova V, Kiddle G, Heazlewood JL, Bartoli CG, Guiamet JJ, Theodoulou FL , Foyer CH (2003) Control of ascorbate synthesis by respiration and its implications for stress responses. Plant Physiology 133: 443-447.

• Holtzapffel RC, Castelli J, Finnegan PM, Millar AH , Whelan J, Day DA (2003) A tomato alternative oxidase protein with altered regulatory properties. BBA - Bioenergetics 1606:153-162.

• *Chew O, Whelan J, Millar AH (2003) Molecular definition of the ascorbate-glutathione cycle in Arabidopsis mitochondria reveals dual-targeting of antioxidant defenses in plants. The Journal of Biological Chemistry 278: 46869-46877.

• *Millar AH , Heazlewood JL (2003). Genomic and Proteomic Analysis of Mitochondrial Carrier Proteins in Arabidopsis thaliana. Plant Physiology 131: 443-453.

• *Heazlewood, J.L., Millar A.H. (2003) Integrated plant proteomics: putting the green genomes to work. Functional Plant Biology 30: 471-482.

• *Heazlewood JL, Millar AH, Day DA, Whelan J (2003) What is a mitochondrion? Genome Biology 4:218.

• *Herald V, Heazlewood JL, Day DA, Millar AH (2003) Proteomic identification of divalent metal cation binding proteins in plant mitochondria. FEBS Letters 537, 96-100.

• *Heazlewood JL, Howell, KA, Whelan, J, Millar AH (2003) Towards the analysis of the rice mitochondrial proteome. Plant Physiology 132: 230-242.

• *Heazlewood JL, Whelan J, Millar AH (2003) The products of the mitochondrial ORF25 and ORFB genes are F O components of the plant F 1 F O ATP synthase. FEBS Letters 540:201-205.

• *Heazlewood JA, Howell KA, Millar AH (2003) Mitochondrial complex I from Arabidopsis and rice: orthologs of mammalian and yeast components coupled to plant-specific subunits. BBA - Bioenergetics 1604:159-169.

• Daley DO, Adams KL, Clifton R, Qualmann S, Millar AH , Palmer JD, Pratje E, Whelan J (2002) Gene transfer from the mitochondrion to the nucleus: Novel mechanisms for gene activation from Cox2. The Plant Journal 30:1-21.

• *Sweetlove LJ, Heazlewood JL, Herald V, Holtzapffel R, Day DA, Leaver CJ, Millar AH (2002) The impact of oxidative stress on Arabidopsis mitochondria. The Plant Journal 32: 891-904.

• *Lister R , Mowday B, Whelan J, Millar AH (2002) Zinc dependent intermembrane space proteins stimulate carrier import into plant mitochondria. The Plant Journal 30: 555-566.

• Considine M , Holtzapffel RC, Day DA, Whelan J, Millar AH (2002) Molecular distinction between alternative oxidase from monocots and dicots. Plant Physiology 129: 949-953.

• Holtzapffel RC, Finnegan PM, Millar AH , Badger MR, Day DA (2002) Mitochondrial protein expression in tomato fruit during on-vine ripening and cold storage. Functional Plant Biology 29:827-834.

• Taylor NL, Day DA and Millar AH . (2002) Environmental stress causes oxidative damage to plant mitochondria leading to inhibition of glycine decarboxylase. . The Journal of Biological Chemistry 277: 42663-42668.

• Logan, D.C., Millar, A.H. , Sweetlove L. J., Hill, S.A., Leaver, C.J. (2001) Mitochondrial assembly during germination of maize embryos. Plant Physiology 125:662-672.

• *Jenner HL, Winning BM, Millar AH , Tomlinson KL, Leaver CJ, and Hill SA (2001) NAD malic enzyme and the control of carbohydrate metabolism in potato tubers . Plant Physiology 126:1139-1149.

• *Sweetlove LJ, Mowday B, Heberstreit HF, Leaver CJ, Millar AH (2001) Nucleoside disphosphate kinase III is localized to the inter-membrane space in plant mitochondria. FEBS Letters 508: 272-276.

• *Millar AH , Sweetlove LJ, Giegé P, Leaver CJ. (2001) Analysis of the Arabidopsis Mitochondrial Proteome. Plant Physiology, 127:1711-1727.

• Millar AH , Considine MJ, Day, DA and Whelan J (2001) Unravelling the role of mitochondria during oxidative stress in plants. IUBMB Life 51:201-205.

• Millar, A.H. , Leaver, C.J. (2000) The cytotoxic lipid peroxidation product, 4-hydroxy-2-nonenal, specifically inhibits decarboxylating dehydrogenases in the matrix of plant mitochondria. FEBS Letters 481:117-121.

• Pádua, M., Aubert, S., Casimiro A, Bligny, R., Millar, A.H. and Day, D.A. (1999) Induction of alternative oxidase synthesis in copper-treated sycamore cells. Plant Physiology and Biochemistry 37: 131-137.

• Henry, B.K., Atkin, O.K., Day, D.A., Millar, A.H. , Menz , R.I. and Farquhar, G.D. (1999) The oxygen isotope discrimination technique for studying plant respiration: assessing sources of variability in its application. Aust. J. Plant Physiol. 26: 773-780.

• Millar, A.H. , Hill , S.A. , Leaver C.J. (1999) The plant mitochondrial 2-oxoglutarate dehydrogenase complex: purification and characterisation in potato. Biochemical Journal 334:571-576.

• Millar, A.H ., Leaver C.J., Hill S.A. (1999) Characterisation of the dihydrolipoamide acetyltransferase component of mitochondrial pyruvate dehydrogenase complex from potato and comparisons with similar components in diverse plant species. The European Journal of Biochemistry 264:973-981.

* directly relevant to this grant application.

Ten career-best publications

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1. *Heazlewood JL, Tonti-Filippini JS, Gout A, Day DA, Whelan JM, Millar AH (2004) Experimental Analysis of the Arabidopsis Mitochondrial Proteome Highlights Signalling and Regulatory Components, Provides Assessment of Targeting Prediction Programs and Points to Plant Specific Mitochondrial Proteins. The Plant Cell 16:241-256.

2. *Chew O, Whelan J, Millar AH (2003) Molecular definition of the ascorbate-glutathione cycle in Arabidopsis mitochondria reveals dual-targeting of antioxidant defenses in plants. The Journal of Biological Chemistry 278: 46869-46877.

3. *Heazlewood JL, Howell, KA, Whelan, J, Millar AH (2003) Towards the analysis of the rice mitochondrial proteome. Plant Physiology 132: 230-242.

4. * Millar AH , Heazlewood JL (2003). Genomic and Proteomic Analysis of Mitochondrial Carrier Proteins in Arabidopsis thaliana. Plant Physiology 131: 443-453.

5. Taylor NL, Day DA and Millar AH . (2002) Environmental stress causes oxidative damage to plant mitochondria leading to inhibition of glycine decarboxylase. The Journal of Biological Chemistry 277: 42663-42668.

6. *Sweetlove LJ, Heazlewood JL, Herald V, Holtzapffel R, Day DA, Leaver CJ, Millar AH (2002) The impact of oxidative stress on Arabidopsis mitochondria. The Plant Journal 32: 891-904

7. *Lister R, Mowday B, Whelan J, Millar AH (2002) Zinc dependent intermembrane space proteins stimulate carrier import into plant mitochondria. The Plant Journal 30: 555-566.

8. *Millar AH , Sweetlove LJ, Giegé P, Leaver CJ. (2001) Analysis of the Arabidopsis Mitochondrial Proteome. Plant Physiology, 127:1711-1727

9. Millar, A.H. , Hill , S.A. , Leaver C.J. (1999) The plant mitochondrial 2-oxoglutarate dehydrogenase complex: purification and characterisation in potato. Biochemical Journal 334:571-576.

10. Millar, A.H. , Knorpp, C., Leaver, C.J., Hill , S.A. (1998) Plant mitochondrial pyruvate dehydrogenase complex: purification and identification of catalytic components in potato. Biochemical Journal 334:571-576.