Honours Project Opportunities in Plant Molecular Biology

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Who are we?

Dr Penny Smith
Contact details: Room 248, Macleay Building
Phone: 9036 7169
Email:

Research Interests

Plant Molecular and Cell Biology. Symbiotic interactions between legumes and rhizobia, long distance signaling in plants, allergenic proteins in plant seeds.


Potential Honours Projects

1. Symbiotic interactions between legumes and rhizobia

Penny Smith, Patrick Loughlin, David Day

Symbiotic interactions between legumes and rhizobia

When legumes are growing in soils with low levels of nitrogen they are able to form a symbiosis with bacteria of the rhizobia spp. that fix atmospheric nitrogen and provide it to the plant in return for a supply of reduced carbon. Consequently crop legumes such as soybean and lucerne are able to grow with less applied nitrogen fertilizer which has obvious economical and environmental benefits. To develop the symbiosis a new organ, the nodule, develops to house the rhizobia. The development of the nodule requires a complicated signaling process between the rhizobia and the plant that establishes the interaction. During infection the rhizobia are contained within the infection thread and once inside the nodule they differentiate into obligate symbionts called bateroids. The bacteroids are surrounded by a symbiotic membrane of plant origin, called the symbiosome membrane, which effectively segregates them from the plant cytoplasm. Our group studies mechanisms for transport of nutrients across the symbiosome membrane and the assimilation of the nitrogen fixed by rhizobia as well as the mechanism by which proteins translated in the cytoplasm move to their subcellular location in nodules.

Transport proteins on the symbiosome membrane

Bacteroids (the symbiotic form of rhizobia) inside the infected nodule cells are surrounded by a membrane of plant origin that effectively segregates the bacteroids from the plant cytoplasm and controls the type and quantity of compounds that pass between the partners. The bacteroids enclosed by the plant envelope form the symbiosome, a facultative organelle (part prokaryote, part eukaryote), which is the fundamental N2-fixing unit within legume nodules. Rhizobia are totally dependent upon their plant hosts for nutrients when living within the nodule. The symbiosome membrane (SM) has selective permeability to metabolites by grace of a unique suite of proteins encoded in the host cell nucleus and targeted to the symbiosome.

The major exchange of nutrients across the SM is the provision of reduced carbon from the plant, to fuel the bacteroid, and the efflux of fixed nitrogen from the bacteroid to the plant. It is believed that nitrogen is transported out of the symbiosome as ammonium and carbon into the symbiosome as malate, although the transporters have not been identified. There is evidence for a range of other transport proteins on the symbiosome membrane including zinc and iron transporters. We are currently undertaking a proteomic analysis of the symbiosome membrane and have identified a number of undescribed intrinsic membrane proteins. An honours project would involve characterisation of one or more of these transport proteins, confirming their symbiosome membrane localisation using GFP fusions and determining whether they are essential for N-fixation using RNAi to knock-down expression.

Are rhizobia in soybean nodules auxotrophs for amino acids? (with Bruce Lyon)

When rhizobia interact with legumes their metabolism changes making them dependent on their plant host for their energy source. The bacteroid also stops assimilating nitrogen and supplies fixed nitrogen to the plant. A recent finding in the pea- Rhizobium leguminosarum symbiosis is that bacteroids are effectively auxotrophs for amino acid synthesis and the plant controls the symbiosis by essentially 're-selling' fixed nitrogen, as branched chain amino acids, back to the bacteroid. Structurally and metabolically, pea nodules are somewhat different from soybean, being indeterminant whereas soybean nodules are determinant. Furthermore the pea rhizobial symbiont is R. leguminosarum and soybean's is Bradyrhizobium japonicum. This project aims to determine whether the soybean symbiont B. japonicum is also an auxotroph for branched chain amino acids. Mutant strains of B. japonicum that cannot transport particular groups of amino acids will be created and their ability to form an effective symbiosis with soybean will be monitored.

Targeting of proteins to organelles in nodules

Eukaryotic cells are divided into a number of distinct compartments (organelles) which include plastids, mitochondria and peroxisomes. This compartmentalisation is essential to allow the thousands of biochemical reactions necessary for the cell to function to be regulated correctly. In infected cells of nodules of sub-tropical legumes a biosynthetic pathway commonly used to synthesis nucleotides (purine biosysnthesis) has been hijacked to allow the products of nitrogen fixation to be assimilated by the plant. Purine biosynthesis occurs in plastids in most tissues but in nodules it occurs in both plastids and mitochondria. This project is to investigate how the enzymes required for purine biosynthesis are targeted to both organelles. Most proteins are directed to only one organelle but it has recently been shown that some proteins have targeting sequences that direct them to both plastids and mitochondria. These proteins are said to be "dual targeted". We would like to know whether any of the purine biosynthesis enzymes are dual targeted in soybean nodules. To do this the region of the genes encoding the enzymes will be fused to GFP and expressed in a soybean hairy root transformation system. The localization of the proteins- plastid, mitochondrial or in both organelles-will be determined by confocal microscopy. Once the localization of the proteins has been determined the regions required for this targeting will be dissected using site directed mutagenesis followed by expression in soybean nodules.

Immunolocalisation of the purine biosynthesis enzyme, AIR synthetase, in nodule cells
Immunolocalisation of the purine biosynthesis enzyme, AIR synthetase, in nodule cells


2. Long distance signaling in plants: proteins, mRNAs and microRNAs

Aniline blue staining of lupin pedicel showing vascular tissue including phloem

Aniline blue staining of lupin pedicel showing vascular tissue including phloem

When something changes in their environment plants can't just get up and move to another place like animals. They need to be able to sense changes in their environment and respond so they can make the most of the light, nutrient and climate conditions. If their roots sense that the nutrients in the soil are becoming depleted they need to change the way they use these nutrients so that they channel them into processes that allow them to produce seeds. Plants are also able to modulate nutrient uptake from the soil solution through gene regulation and in some instances may form a symbiosis with another organism to help them access nutrients; if the day length is correct for flowering they need to be able to sense this and start producing flowers. These processes involve long distance signaling - the signals that help the plant respond move in the phloem. We are investigating different types of signals that move in phloem including proteins, mRNAs and microRNAs and also the mechanisms that are used to move the signals. Our work uses white lupin as a model for identifying the components of phloem which we then study the translocation of in the model plant Arabidopsis. The project below looks at miRNAs in phloem but I can also provide projects on proteomics of phloem or transcription factors that may move in phloem.


Which microRNAs are translocated in phloem?

The discovery of microRNAs revolutionised the way we thought about gene regulation. These small RNA molecules bind to target mRNAs, a process which either directs the mRNA for destruction or reduces its translation. miR399 is a microRNA that moves in phloem to initiate a response to phosphorus deficiency. It seems likely there are other microRNAs that help plants respond to other environmental stimuli. In this project we are focusing on identifying these microRNAs in both Arabidopsis, due to the huge genetic resources available, and lupin as phloem is relatively easy to isolate from this species. We have developed an Arabidopsis micrografting procedure that allows us to detect miRNAs moving in phloem and this will be used to test whether specific miRNAs move between shoots and roots and/or vice versa. miRNAs will be detected by real-time quantitative PCR. Changes in miRNA abundance in phloem in response to nutrient deficiency will also be studied in white lupin.

Phloem bleeding from lupin pods

Phloem bleeding from lupin pods.