Joint Research Laboratory in Genomics and Nutriomics

University of Western Australia - Zhejiang University

Current Projects:

1. Molecular Profiling of Rice Seed Germination

Seed germination represents a crucial stage in the life cycle of a plant, where upon appropriate signals, changes occur within the dormant embryo to result in the elongation of the radicle. The result of these processes is that in as little as 24 h there are large changes in gene expression, cellular morphology and metabolic activity. Seed germination is one of the most intensively studied aspects of plant biology, in part due to the fact that often it is the seed that is the commercial end product of many plants, and contributes a major part to the global calorie intake.

We are undertaking an extensive study of the process of seed germination using rice, with particular reference to the biogenesis of the three energy organelles in plants cells, mitochondria, peroxisomes and plastids. Upon imbibition a series of molecular events occurs that results in an activation of the activities of these organelles, that are necessary to power or fuel the process of germination. Additionally rice has the ability to germinate in the absence of oxygen, the latter usually required by almost all other plants. In the absence of oxygen, energy to power germination cannot be produced via oxidative metabolism, and thus it is interesting, but unknown, how rice overcomes this potential barrier and continues to germinate. Several projects relating to rice germination are currently being undertaken:

1) Examination of the morphology, number and changes in mitochondria, peroxisomes and plastids during rice germination in the presence and absence of oxygen. These investigations are being carried out using a combination of electron microscopy and imaging of cells where the organelles are tagged with fluorescent proteins to allow them to be visualised.
2) Molecular profiling of changes in transcript, protein and metabolite abundance during rice germination in the present and absence of oxygen. These investigations are being carried out using a variety of functional genomic approaches.
3) Analysis of gene promoters to define cis elements (i.e. sequence elements in promoters) and trans acting factors (i.e. transcription factors) that are responsible for regulating gene expression in response to changing oxygen conditions. These investigations are being carried out using plant transformation and yeast hybrid assays.

Howell KA, Millar AH, Whelan, J. (2007). Building the Powerhouse. What are the signals involved in plant mitochondrial biogenesis. Plant Signalling and Behaviour 2: 311-313.

Howell KA, Cheng K, Murcha MW, Jenkin LE, Millar AH, Whelan J. (2007) Oxygen initiation of respiration and mitochondrial biogenesis in rice. Journal of Biological Chemistry  282:15619-15631

2. Functional characterisation of the PRAT protein transporter family in Arabidopsis

Mitochondria and Chloroplasts are estimated to contain approximately 2000 and 4000 proteins respectively (1,2). Whilst both organelles contain their own genome the majority of these proteins and encoded in the nucleus, synthesised in the cytosol and imported into their respective organelle (3-5). One of the extensively characterised protein trafficking systems is that of mitochondrial import in yeast (Saccharomyces cerevisiae). Proteins that are destined to the mitochondria are recognised by a multimeric translocase subunits on the outer membrane (TOM), passed through a membrane pore formed by a 40 kDa protein (TOM40) to the translocase of the inner membrane (TIM). Proteins are subsequently recognised by the TIM17/23 or TIM22 complex that subsequently insert the protein into or across the inner membrane to the mitochondrial matrix (6,7). These three TIM protein are essential for viability in yeast. In yeast, TIM17, TIM22 and TIM23 are encoded by single genes. In contrast analysis of the rice and Arabidopsis genomes indicate 24 and 17 genes for this family of protein respectively. Even in higher animals such as mammals the number of genes is more limited, 4 in humans and 6 in mouse and they simply represent more recent duplications http://millar3.biochem.uwa.edu.au/MPRIC/. In contrast in Arabidopsis and rice the genes represent ancient divergences before monocots and dicots diverged over 140 million years ago and many genes cannot be classified as encoding either TIM17, 22 or 23 like proteins (8).

TIM17, 22 and 23 belong to the Preprotein and amino acid transporter (PRAT) family of proteins. This family of proteins is defined by yeast TIM17, 22 and 23 and an outer envelope protein of 16 kDa from pea (OEP16). They contain four predicted transmembrane regions and the sequence signature [G/A]X₂[F/Y}X₁₀RX₃Dx₆[G/A/S]GX₃G where X is any amino acid (9). It has been proposed that they have being derived from amino acid permease in bacteria, Liv H in E. coli.

After the endosymbiosis that led to the formation of mitochondria gene transfer resulted in relocation of most genes from the mitochondrion to the nucleus (10,11). This resulted in the need to target  protein from their site of synthesis in the cytosol across one of two mitochondrial membranes. The machinery to achieve this, referred to as the mitochondrial protein import apparatus, is in part derived from components that existed in the endosymbiont. In particular the pore forming subunits of the translocases, TOM40 and TOB55 in the outer membrane and TIM17, 22 and 23 are all proposed to be derived from pre-existing bacterial components (12,13). With time other components of the import apparatus, such as the receptor subunits TOM20 and TOM70 evolved, the selection pressure was increased specificity to produce mitochondria of eukaryotic cells (4).

The role of the PRAT family of protein in plants is largely unknown and has clearly expanded compared to the roles played in protein import in yeast and mammalian systems. The expansion is based on a number of observations, firstly OEP16 is a chloroplast protein. Initially it was defined as playing a role in metabolite transport of amino acids (14), but further studies in barley suggest a role in protein import into chloroplast of protochlorophyllide oxidoreductase (15). These conflicting reports have yet to be resolved. Secondly examination of the predicted protein sequences for TIM17 (3 proteins)  and 23 (3 proteins) from Arabidopsis indicates that all except 1 lacks the PRAT domain that defined this family (16). As outlined in more detail below the identity of TIM22 in plants cannot be clearly defined with no protein branching with the yeast protein with high confidence with phylogenetic analysis (Figure 1) (8). Finally and most strikingly one of the TIM17 proteins in plants links the inner and outer membrane, the first such protein in plants shown to have this topology (17). Examination of all the predicted PART sequences from Arabidopsis indicates that six predicted PRAT protein have C-terminal extensions of 50 to 100 amino acids relative to yeast Tim17, 22 or 23, and the role of these extensions are unclear (17). In complementation studies of yeast TIM17 or 23 mutants the orthologous plant genes could not complement unless these C-terminal extensions were removed (16). Examination of the rice sequences reveals a similar picture but additionally some predicted PART proteins have extensive N-terminal extensions and duplication of genes to produce proteins with 8 transmembrane regions has occurred. However given that the annotation of the rice genome is at an earlier stage than Arabidopsis these structures need to be confirmed. However at least it appears that the situation is similar to Arabidopsis indicating that this is likely a feature of most Angiosperms.

Figure 1. Phylogenetic analysis of predicted PRAT protein from Arabidopsis with the defining members of this family as comparison, TIM17, 22 and 23 from yeast and OEP16 from yeast.

References

Lister R, Carrie C, Duncan O, Ho LHM, Howell KA, Murcha MW, Whelan J. (2007). Functional definition of outer membrane proteins involved in preprotein import into mitochondria. Plant Cell  19: 3739-3759.

Murcha MW, Elhafez D, Lister R, Tonti-Filippini J, Baumgartner M, Philippar K, Carrie C, Mokranjac D, Soll J, Whelan J (2007) Characterisation of the preprotein and amino acid transporter family in Arabidopsis. Plant Physiology 143:199-212

Murcha, M.W., Elhafez, D., Millar, A.H. and Whelan, J. (2005). The C-terminal region of TIM 17 links the outer and inner mitochondrial membranes in Arabidopsis and is essential for protein import. Journal of Biological Chemistry 280: 16476-16483.

3. Interaction of AUX/Nutrient Signaling on Rice Development

Aim: To define AUX signaling regulation on rice root development

Background:

AUX polar distribution determines root components formation and cell identity. Previous studies in our laboratory have defined that Auxin transport OsPIN affects a variety of important growth and developmental processes in rice, including the root emergence and branching. To obtain a comprehensive knowledge of PIN genes in AUX pathway, we isolated a rice mutant with loss-function of an IAA gene and resistance to exogenous AUX (NAA/IAA), which showed defective in lateral root, root cap and disorder of epidermal cells, QC and stele structure. Under nitrogen starvation condition, the defect of mutant’s root cap can be rescued. QRT-PCR analysis has been carried out to detect expression of OsPIN gene family in rice mutant and wild type. It was found that OsPIN10a an AUX-inducible efflux-transporter, is repressed in the roots of heterozygous and homozygous mutants. The next stage of this project is to define the regulate changes in OsPIN10a expression in response to WT and mutant under the different nutrient condition.

Research Plan:

1) Predict cis-acting regulatory elements (CAREs) in the promoter regions of OsPIN gene family.
2) Detect if the promoter regions of these genes contain auxin response regions and nitrogen responsive regions.
3) Construct OsPIN10a overexpression and RNA interference transgenic plants.
4) Identify transcription factors that bind functional CAREs.

Background Reading:

Li J, Zhu S, Song X, Shen Y, Chen H, Yu J, Yi K, Karplus VJ, Wu P and Deng XW (2006) A rice glutamate receptor–like gene is critical for the division and survival of individual cells in the root apical meristem. Plant Cell. 18: 304-309
Xu M, Zhu L, Shou H and Wu P (2005) A PIN1 family gene, OsPIN1, involved in Auxin-dependent adventitious root emergence and tillering in Rice. Plant Cell Physiol. 46(10): 1674–1681
Qi X, Zhou J, Jia Q, Shou H, Chen H and P Wu (2005) A characterization of the response to Auxin Of the small gtpase, Rha1. Plant Science, 169: 1136-1145
Huang G, Wu Y, Zhu L, Qi X, Wang X and Wu P (2004) QTLs for nitrate induced elongation and initiation of lateral roots in rice (Oryza sativa L.). Plant and Soil. 263: 229-237