Professor Christopher Grof
(02) 4921 5858
General Background to Grof Lab Research
The use of Sorghum and Setaria as model C4 plant species
Increasing fuel costs, finite resources and the need to develop more carbon neutral and cleaner fuels have created a need for renewable resources. Sorghum bicolor (Sorghum) is a crop plant adapted to the hot water limited environments of northern Australia, both as a grain and forage crop. As a result of its rapid growth rate, Sorghum is an ideal crop for biofuel production from grain, sugar and biomass accumulation. However, in order to accelerate fundamental understanding of the complex biology and mechanisms underpinning carbon partitioning towards soluble carbohydrate and structural cell wall components, a simple model C4 system such as Setaria (foxtail and green millet) is required. The development of Setariainto a robust genetic and transformation platform will be a crucial piece of the puzzle.
Setaria, an ideal model for dissecting biomass quality traits
Lignocellulosic bioethanol derived from plant biomass will provide a cost effective contribution to environmental sustainability and energy security. Setaria italica (foxtail millet) is an ideal genetic model to dissect biomass quality traits. A large number of plant lines exhibiting broad genetic diversity will be screened using Fourier Transform Infra-Red (FTIR) spectral analysis to identify those ecotypes with differing cell wall composition. Quantitative expression analysis of key genes using RT-qPCR, will be undertaken on the most divergent ecotypes as a platform to unravel the pathways and mechanisms contributing to variation in cell wall composition. Outcomes of this project will contribute significantly to our understanding of the capacity to tailor cell wall attributes to maximise digestibility of plant biomass.
Development of key tools for effective transformation of Setaria.
The goal of this project is to establish a high throughput system for construct preparation, transformation and cell wall composition analysis of Setaria transgenic lines engineered for overexpression or down regulation of candidate genes. Initially the focus will be on establishing the system by preparing cloning 'friendly' binary vectors, testing of key promoter/terminator sequences and reporter genes as a first step in utilising the system for assessing cell wall modification strategies.
Sorghum, a biofuel feedstock for arid environments
As a result of its rapid growth rate, Sorghum is an ideal crop for biofuel production from grain, sugar and biomass accumulation. The overall strategy of this project will be to identify candidate genes associated with sugar metabolism, biomass accumulation and cell wall composition in a series of segregating Sorghum populations using a combination of DArT (Diversity Array Technology) and Next Generation Sequencing. Validation of the role these genes play in key metabolic processes will be undertaken by genetic manipulation in the model C4 grass Setaria.
Genetic regulation of C4 grass internode development with specific emphasis on secondary cell wall composition
C4monocot grass shoots are composed of a repeating phytomeric unit constituting a node to which a leaf is attached and a subtending internode. Within the internode, three developmental regions can be identified namely meristematic, cell expansion and maturation zones. Within the cell expansion zone only primary cell wall deposition takes place, whereas within the maturation zone lignified secondary cell wall deposition occurs. In order to dissect the molecular changes underpinning primary/secondary cell wall deposition and composition, hence digestibility of the C4 plant biomass, the following strategies will be pursued focusing on Setariaas the model C4 plant:
- The three developmental zones as well as thetransitional region between elongation/maturation zones of an elongating internode will be separated and both the high (mRNA) and low (small RNA) molecular weight RNA fractions isolated. High throughput RNA sequencing will be undertaken to establish a detailed database and bioinformatic analysis will provide a comprehensive molecular genetic platform for the project.
- Screen a mutant Setariapopulation generated by ionising radiation using the high throughput FTIR approach (Martin et al. under review for Biotechnology for Biofuels) to identify lines exhibiting changes in cell wall composition. High throughput RNA sequencing of this small subset of mutant lines to identify genetic changes leading to alterations in primary and secondary cell wall composition.
Optimisation of an Agrobacterium Mediated Transformation System for Setaria viridis
One significant impediment to understanding cell wall complexity and biomass quality is the inadequacy of current model genetic systems. Arabidopsis although a true model plant, is a dicot and too far removed on an evolutionary scale from candidate C4biofuel feedstock grasses, particularly evident when analysing cell wall composition. Maize and Sorghum, both possess C4photosynthesis and contain a wealth of genomic resources, however their larger size, large genome, long generation time and inefficient transformation systems limit their utility as a model system. Recently Setaria species italica and viridis have been proposed as potential new models to fill this gap. Members of the Panicoideae clade, Setaria species are closely related to several of the major C4 bioenergy grasses. Setaria italica (foxtail millet) and its wild ancestor Setaria viridis (green foxtail) both possess features inherent in ideal model genetic systems.
Plant biotechnology offers a means to re-engineer plant cell wall composition. By using a model system such as Setaria, putative gene candidates for manipulation of cell wall composition can be rapidly tested. The goal of this project is to establish such a high throughput system for construct preparation, transformation and cell wall composition analysis of Setaria transformants engineered for overexpression or down-regulation of candidate genes.
Initially this project will focus on establishing the system by preparing cloning 'friendly' binary vectors, testing of key promoter/terminator sequences and reporter genes. As a first step in utilising this system for assessing cell wall modification strategies we will endeavour to down-regulate lignin biosynthesis. It is well established that lignin concentration negatively correlated with digestibility of cell walls. This is true in natural populations and also experimentally validated using transgenic approaches. Recently, knockdown experiments were conducted in switchgrass for the lignin biosynthesis genes caffeic acid O-methyltransferase (COMT) (Fu et al. 2011) and Cinnamyl-Alcohol Dehydrogenase (CAD) (Saathoff et al. 2011). The successful down regulation of either COMT or CAD biosynthesis genes in Setaria will provide a proof of concept and lays a foundation for high throughput analysis of novel gene candidates identified through transcriptome comparisons.
Tailoring cell wall composition of the dominant C4 forage grass in northern Australia to improve digestibility - Translational validation of fundamental research
The key phenotypic trait of biomass digestibility is important in two different contexts. From a biofuels point of view, increased digestibility correlates directly with increased efficiency of biomass deconstruction if enzymatic hydrolysis is the strategy for release of fermentable sugars. Similarly, the rate of ruminant live weight gain is directly correlated with forage digestibility. Globally, C4 grasses are the principal biofuels feedstock targets due to their increased photosynthetic, water and nutrient use efficiency. In Australia, C4 grasses are the principal forage source of beef cattle in Northern Australia. Furthermore, with the predicted temperature increases attributed to climate change, the distribution and importance of C4 grasses in the Australian landscape will only increase. The project will be based on the C4 model system, Setaria, but fundamental understanding of the genetics/genomics directing cell wall composition, hence influencing digestibility, will be rapidly applied to the improvement of Cenchrus (buffel grass), the principal C4forage in northern Australia.
Associate Professor David McCurdy|
(02) 4921 5879
General Background to McCurdy Lab Research
Transfer cells are a unique plant cell type characterized by invaginated wall ingrowths that protrude into the cell volume and act to increase surface area of plasma membrane, hence achieving enhanced densities of plasma membrane nutrient transporters. These cells develop at apoplasmic/symplasmic bottlenecks in nutrient transport pathways in plants, and thus can strongly influence nutrient delivery to source organs such as fruits and seeds. The main focus of the McCurdy Lab is to identify transcriptional regulators of transfer cell development using the model species Arabidopsis thaliana. A range of molecular and cellular biology approaches including Next Generation Sequencing, gene cloning and mutant analysis and fluorescence confocal microscopy of GFP-tagged proteins are being employed in a number of different projects focused on this aim.
Honours and RHD projects
Genetic regulation of transfer cell development
This project will use the model species Arabidopsis thaliana to investigate genetic control of transfer cell development. Several novel transcription factors identified as putative regulators of transcriptional cascades required for transfer cell development have been identified, and this project will join a larger team using amiRNA interference and overexpression approaches to investigate the role of these genes in this process. Future goals will involve ChIP RNA-Seq to identify downstream targets of these transcription factors, and thus identify elements of the transcriptional cascade(s) leading to wall ingrowth deposition.
RNA-Seq analysis of transfer cell development
This project will contribute to an on-going study using Illumina-based sequencing (RNA-Seq) to analyse transcriptional changes occurring during induction and wall ingrowth building of transfer cells. The experimental approach uses both induction of epidermal transfer cells in Vicia faba cotyledons and mutants in Arabidopsis that display altered levels of phloem parenchyma transfer cell development. Candidate genes identified by either approach will be tested by analysing insertional knockout mutants and amiRNA knockdown lines in Arabidopsis.
Investigating a role for POLAR in wall ingrowth deposition in transfer cells
Recent investigations have identified POLARas a possible regulator of wall ingrowth deposition in phloem parenchyma transfer cells. POLAR is a membrane-localized protein involved in defining cell polarity in meristemoid (guard cell progenitor) cells, and thus may also have a role in determining polarized wall ingrowth deposition in transfer cells. This project will use a combination of phloem parenchyma targeted over-expression and amiRNA knockdown to directly test a role for POLAR in wall ingrowth deposition, and confocal microscopy to follow the subcellular localization of GFP-POLAR fusion proteins expressed specifically in phloem parenchyma transfer cells. Additional studies will attempt to identify proteins that interact with POLAR and may therefore also possibly be involved in directing wall ingrowth deposition in transfer cell.
Investigating signals regulating transfer cell development in Arabidopsis thaliana
We have developed high-throughput procedures using fluorescence microscopy to assess the extent of transfer cell development in minor veins of Arabidopsis leaves. This development provides a platform to analyse signals which regulate not only the trans-differentiation of phloem parenchyma (PP) cells into PP transfer cells, but also provides a tool to assess possible plasticity of phloem loading mechanisms in this species. This project will test a range of mutants defective in signal transduction pathways and/or altered leaf sugar levels to examine the effects of these signals on transfer cell development. The project will use a combination of molecular biology, fluorescence confocal microscopy and electron microscopy to assess the effects of these mutants on wall ingrowth deposition in transfer cells.
Associate Professor Yong-Ling Ruan|
(02) 4921 7958
General Background to Ruan Lab Research
Sugars are the building blocks of plant growth and development
Plants use solar energy to produce sugars as building blocks and signals to fuel, feed and regulate growth and development. As such, sugar allocation and signaling play pivotal roles in plant performance. With this background, the Ruan Lab focuses on elucidating mechanisms by which sugar
metabolism, transport and signaling regulate plant development, yield formation and stress tolerance. We use molecular, cellular, biochemical and physiological approaches to address these fundamental questions. Our research has been published in the top-ranked plant-specific journals, such
as Plant Cell, and is currently funded by the Australian Research Council (ARC). For details, please visit the following website: http://www.newcastle.edu.au/profile/yong-ling-ruan
Honours and RHD projects
Identifying key genes controlling resource partitioning in plants for higher yield
This project will identify regulatory genes controlling carbon nutrient partitioning in plants, thereby developing innovative molecular solutions to improve crop yield and quality. Contemporary molecular, cellular and biochemical approaches will be utilised to address a set of hypotheses.
Improving food security: seed and fruit development using gene technology
Seed and fruit are organs of major agronomical importance. We recently identified a key regulatory gene, INVINH1, which controls seed size and fruit sugar level by repressing the activity of invertase (INV) that converts sucrose into glucose and fructose (Jin et al 2009 Plant Cell; Wang & Ruan 2012
Plant Physiology). Further work aims to elucidate the molecular mechanisms and signalling pathways that controls the coexpression and interaction of the INVINH1 and its target, INV. These questions will be addressed by using contemporary molecular, cellular and biochemical approaches.
Manipulating genes to sustain reproductive development under abiotic stress
Similar to animals, reproductive development in plants is extremely sensitive to stress, including heat and drought, that often causes floral, seed and fruit abortion, and ultimately irreversible yield loss. We have recently identified biochemical and cellular bottlenecks that could control seed and/or
fruit set, or their abortion under stress (Ruan et al 2012 Trends in Plant Science). This project will identify the genes underlying these processes for manipulation to sustain plant reproduction, hence yield, under stress.
Searching for SWEETS: Identifying novel sugar transporters and sensors
In many sink organs such as developing tomato fruit, photosynthesis-derived sucrose is unloaded from phloem into the extracellular space for hydrolysis into hexoses before being taken up by recipient cells. Recent exciting development in the field (e.g. Chen et al 2012 Nature) suggests this
process is likely mediated by set of sugar effluxers, influxers and sensors. This project aims to identify these novel SWEETS genes through collaboration with leading experts in USA.
Live longer for a better life: Understanding cell death in plant for high productivity
The lifespan of plant cells and tissues determines plant productivity. Plant cells can die from 'suicide' through programmed cell death or be 'murdered' by adverse factors such as heat, cold or lack of nutrients. This project aims to understand the cellular and molecular basis underlying
plant cell death and to identify the 'death' gene for genetic improvement of plant longevity, hence yield and quality.
Doctor Andy Eamens|
(02) 4921 17784
General Background to Eamens Lab Research
Small RNAs and their regulatory roles in plant stress and development.
Small RNA (sRNAs), namely microRNAs (miRNAs) and trans-acting small-interfering RNAs (tasiRNAs) are processed in the plant cell nucleus from much larger double-stranded RNA (dsRNA) precursor transcripts. In the plant cell nucleus, miRNA and tasiRNA precursor transcripts are bound by dsRNA binding (DRB) proteins that deliver the bound dsRNA to a Dicer-like (DCL) endonuclease for accurate and efficient sRNA production. Following their nuclear maturation, miRNA and tasiRNA sRNAs are exported to the plant cell cytoplasm and loaded by an Argonaute (AGO) protein that uses the loaded sRNA as a sequence specificity guide to regulate the expression of complementary sRNA target genes. A large number of the experimentally-validated target genes of plant sRNAs encode; i) transcription factors that regulate the expression of developmentally-important genes, or; ii) proteins that are crucial to the plant to adapt to environmental stress.
Molecular characterisation of the drb2/ago2 double mutant
MicroRNAs (miRNAs) are a class of small RNA (sRNA), ~21 nucleotides in length that regulate the expression of developmentally important genes in plants and animals. In the model plant species Arabidopsis thaliana (Arabidopsis), I have generated a double knockout mutant line, the drb2/ago2 plant that expresses a severe developmental phenotype. The drb2/ago2 phenotype is a result of two disruptions to an alternate ArabidopsismiRNA pathway. The first to DRB2, a protein required for miRNA production in the plant cell nucleus, and the second to AGO2, a protein required for miRNA target gene expression regulation in the plant cell cytoplasm. Of the ~330 Arabidopsis miRNAs identified to date, the drb2/ago2 phenotype is a result of a loss of function of less than ten of these sRNAs. The aim of this project is to determine which of these ten putative DRB2/AGO2-dependent miRNAs cause drb2/ago2 plants to display such a severe and unique developmental phenotype.
Determining miR164 contribution to the drb235 developmental phenotype
In Arabidopsis, miR164 is a very well characterised miRNA, and in most tissues of the Arabidopsis plant, DRB1 is required for miR164 production. I have shown however, that in developmentally important tissues, namely the shoot and floral meristems, DRB2 in addition to DRB1, is also responsible for miR164 production. The drb2 single knockout mutant plant expresses a mild developmental phenotype due to tissue-specific increases in miR164 production. DRB3 and DRB5 are expressed in the same tissues as DRB2 in wild-type Arabidopsisplants, the shoot and floral meristem, but unlike drb2 plants, drb3, drb5 and drb35 mutant lines (plants defective in DRB3 and DRB5 activity) do not display phenotypes. However, the drb235 triple knockout mutant plant expresses a severe developmental phenotype to suggest that DRB3 and DRB5 are also required for miR164 production and/or miR164 target gene expression regulation. The aim of this project is to determine; i) miR164 contribution to the developmental phenotype expressed by drb235 plants, and; ii) if DRB3 and DRB5 are involved in miR164 production (nucleus) or regulating the expression of miR164 target genes (cytoplasm).
Cellular localisation of DRB3 and DRB5: static or dynamic?
DRB2, DRB3 and DRB5 expression overlaps in the developmentally important tissues of the shoot and floral meristems of wild-type Arabidopsisplants. Furthermore, DRB3 and DRB5, two predominantly cytoplasmic proteins, are required to regulate the expression of DRB2-dependent miRNA target genes. However, my recent molecular analyses of drb3, drb5 and drb35 knockout mutant plant lines (plants defective for DRB3 and/or DRB5 activity) suggest that for a small miRNA subset, unique to the DRB2-dependent miRNA subset, DRB3 and DRB5 are also involved in the plant cell nucleus-localised production stages of the ArabidopsismiRNA pathway. The aim of this project, via the molecular characterisation of drb knockout mutant plant lines and the use of fluorescently–labelled versions of DRB2 (nuclear localised control protein), DRB3 and DRB5 is to determine if DRB3 and DRB5 can be imported into the plant cell nucleus to assist in the production of this miRNA subset.
Characterisation of a tissue-specific stress-induced miRNA pathway in Arabidopsis.
The majority of the ~330 ArabidopsismiRNAs identified to date have been shown to require the DRB1/DCL1 protein partnership for their accurate and efficient production. Mature DRB1-dependent miRNAs are exported into the cytoplasm and loaded by AGO1. AGO1 uses the loaded miRNA as a guide to identify, bind and subsequently cleave highly complementary target gene mRNAs. However, in developmentally important tissues, namely the shoot and floral meristems, DRB2 is required for the nuclear-localised production stages of a subset of miRNAs. DRB3 and DRB5, two cytoplasmic DRB proteins, are required to regulate the expression of DRB2-dependent miRNA target genes, independently of mRNA cleavage. Messenger RNA cleavage-independent regulation of DRB2-depdendent miRNA target genes indicates the involvement of an additional AGO protein to AGO1. This project aims to fully characterise, at the molecular and phenotypic level, this tissue-specific non-canonical miRNA pathway.
Determining nuclear DRB contribution to miRNA and tasiRNA production.
Of the five Arabidopsis DRBs, DRB1, DRB2 and DRB4 are expressed in the plant cell nucleus. Analogous to the DRB1/DCL1 partnership in the miRNA pathway, DRB4 forms a functional partnership with DCL4 for efficient and accurate tasiRNA production. Furthermore, and as reported for the miRNA pathway, DRB2 is involved in the production of a subset of tasiRNAs in developmentally important tissues. DRB2 involvement in both the miRNA and tasiRNA pathway is limited by its tissue-restricted expression in wild-type Arabidopsis plants (shoot and floral meristems). However, when expressed constitutively and ubiquitously in the drb14 double knockout mutant plant (plant line defective in miRNA and tasiRNA production), DRB2 can fully compensate for the loss of DRB1 and DRB4 activity, restoring miRNA and tasiRNA accumulation to wild-type levels. The aim of this project is to develop a new series of drb knockout mutant plant lines defective in DRB1, DRB2 and DRB4 activity to establish the hierarchical and redundant roles of these three nuclear DRBs in the Arabidopsis miRNA and tasiRNA pathways.
Characterisation of reversible RNA silencing in Arabidopsis under stress conditions.
In Arabidopsis, miRNAs produced by the canonical DRB1/DCL1 functional partnership are exported to the plant cell cytoplasm and loaded by AGO1. AGO1 uses the loaded miRNA as a sequence specificity guide to identify, bind and subsequently cleave target gene mRNAs. Cleavage of target gene mRNAs is an endpoint reaction. The majority of miRNAs that required the non-canonical DRB2/DCL1 functional partnership are nutrient stress responsive miRNAs, that is: their production is altered under stress conditions to regulate the expression of genes required by the plant to mount an effective stress response. Furthermore, following their export to the cytoplasm, some DRB2-dependent miRNAs are loaded to alternate AGO proteins. These alternate AGOs encode defective mRNA cleavage domains to suggest that they regulate target gene expression via translational repression, and not mRNA cleavage, the predominant mode of sRNA-directed RNA silencing in plants. This projects aims to assess if stress responsive miRNAs regulate the expression of their target genes via translational repression, a reversible mode of RNA silencing.
Conjoint Professor Christina (Tina) Offler |
(02) 4921 5704
Emeritus Professor John Patrick
General Background to Offler-Patrick Lab Research
Transfer cell development.
Transfer cells have intricately-invaginated wall ingrowths that greatly amplify their plasma membranes which in turn contain high densities of nutrient transporters. These features confer transfer cells with a significant role in regulating biomass partitioning and accumulation as well as placing these specialised cells at the core of generating novel approaches to improve crop yield. The latter drives our research program on transfer cell biology using a unique Vicia faba cotyledon transfer cell induction system, developed in our laboratory. Our broad gaols are to discover mechanisms orchestrating construction of the exquisite wall ingrowths of transfer cells and processes targeting high densities of transporters to the plasma membranes lining these wall ingrowths.
Honours and RHD projects
Building transfer cell wall ingrowths and nutrient transport capacity
A range of projects, using contemporary cellular and molecular techniques, are available within this research program. These include discovering mechanisms controlling wall ingrowth construction, formation of polarized membrane domains and targeting proteins to plasma membranes as well as identifying transcriptomes underpinning these developmental processes.