Assimilate Partitioning

Nutrient Transport and Plant Productivity
(Patrick, Offler and McCurdy)

Fig. 1. Principal transport events for sucrose moving from sites of production in photosynthetic leaves (sources)to sites of utilisation in non-photosynthetic organs (sinks). The key membrane transport events are numbered.

Our research program focuses on transport of nutrients (carbon and mineral ions) in plants. Transport events play a central role in determining nutrient allocation between growth/storage organs and hence plant productivity and crop yield. Sucrose is the main form in which photosynthetically-reduced carbon is transported through the phloem from photosynthetic leaves (sources) to non-photosynthetic organs (sinks - see Fig. 1). Key steps in sucrose movement are the cell-to-cell transport events located in source and sink organs. Sucrose allocation patterns between competing sink organs, and hence crop yield, are strongly influenced by cell-to-cell transport events that take place in the recipient sink organs. These cell-to-cell transport events may involve sucrose movement across plasma membranes to and from the cell wall space (apoplasmic transport). Membrane movement is facilitated by transporters (Fig. 1; steps 3 and 4). Alternatively, cell-to-cell transport may occur through interconnecting plasmodesmata (symplasmic transport - see Fig. 1). Similar considerations apply to the transport of mineral ions.

In this context, our overall research goal is to discover the mechanisms controlling cell-to-cell transport of sucrose and ions in sinks using developing legume seeds and cereal grains as experimental models (Fig. 2). These organs provide an unique opportunity to readily access membrane transport events (Figs. 1 and 2). Our research focuses on these transport events and on the development of cells specialised for these transport functions. In addition, we address the relationship between membrane transport and subsequent storage of the transported nutrients. Fig. 2. Key morphological features of developing broad bean and wheat seeds in relation to the cellular pathway of nutrient transport illustrated diagrammatically.

Molecular Physiology of Nutrient Transporters

Using a molecular physiological approach, our group is using developing seeds of grain legumes (pea, broad and French bean) and cereals (wheat and rice) to investigate transport steps responsible for moving sucrose and mineral ions from the phloem to sites of storage (Fig. 2). Similar studies are being undertaken to discover the role of hexose transporters in determining sugar accumulation in developing tomato fruit. Current projects in this area are outlined below

Regulation of sucrose transporter expression and activity in developing legume seeds

If sucrose symporter activity is key to determining seed growth rates, it is imperative to understand regulation of its transport activity. Past studies have provided some evidence for transcriptional control of transporter activity with the possible involvement of sugar signals. However, there is a need for further investigation before firm conclusions can be drawn. Two experimental models are being used in these studies. These are in vitro induction of sucrose transporter expression in cotyledon cells that do not normally express the sucrose symporter gene and mutants of starch metabolism with altered sucrose demand and sucrose levels.

Chief Investigators: Prof John Patrick, A/Prof Tina (CE) Offler, T Wang (John Innes Research Institute, UK)
Contact: Prof Patrick

Characterisation of novel sucrose transporters in developing legume seeds

1. The missing link in our understanding of phloem unloading, and hence nutrient allocation, is how nutrient efflux across cell membranes occurs (Fig. 1, transport step 3;). Physiological approaches using legume seed coats, protoplasts and plasma membrane vesicles have demonstrated transport behaviours of sucrose efflux consistent facilitated diffusion and proton antiport. Further progress depends upon studying transport properties of membrane proteins responsible for sucrose efflux in isolation. Thus far four sucrose transporter homologs have been cloned and currently these are being functionally characterized. In addition, a novel functional cloning system for sucrose effluxers is being developed.

Chief Investigators: Prof John Patrick, A/Prof Tina (CE) Offler, Prof WB Frommer (Carnegie Institute, Stanford University, USA)
Contact: Prof Patrick

Ion and water transport in developing legume seeds

Fluxes of potassium and accompanying anions loaded into developing seeds match those of sucrose and collectively largely account for transported osmotic content. Electrophysiological studies have characterized ion channels located in the maternal and filial tissues. Their transport activities appear to be coordinated with that of the sucrose transporters. Water channels have been identified in native membrane prepared from maternal tissues. Three plasma membrane intrinsic proteins have been cloned and one of these is found to support water movement when expressed in Xenopus oocytes.

Chief Investigators: Prof John Patrick, Prof Stephen Tyerman (Waite Institute, University of Adelaide), Prof Wen-Hao Zhang (Beijing Agricultural University, China)
Contact: Prof Patrick

Molecular biology of hexose transporters in developing tomato fruit

Hexoses are accumulated to high concentrations in storage parenchyma cells of expanding tomato fruit. Phloem-imported sucrose enters the fruit apoplasm where it is cleaved into hexoses by an extracellular invertase. Subsequent uptake into storage parenchyma cells is mediated by hexose/H+ symport, the activity of which is a key determinant of fruit hexose levels. Three hexose transporter genes are temporally expressed in storage parenchyma cells during fruit development. Forward and reverse genetics has confirmed the role of the hexose transporters in fruit sugar accumulation. Current activities are focused on developing non-GMO approaches to manipulate hexose transporter activity and hence fruit sugar levels.

Chief Investigators: Dr David McCurdy, Prof John Patrick
Contact: Dr McCurdy

Molecular analysis & genetic manipulation of sucrose transport in cereals

Similar to grain legumes, phloem-imported sucrose moves symplasmically through developing grains except at their maternal/filial interfaces where it is exchanged to and from the grain apoplasm en route for endosperm storage sites. These membrane transport events are facilitated and, for uptake into filial tissues, occur by sucrose/H+ symport. Gene expression and membrane insertion of the protein product for a sucrose/H+ symporter and H+-ATPase are localised to the nucellar and aleurone layers. The physiological significance of these spatial expression patterns is being investigated.

Chief Investigators: A/Prof Tina (CE) Offler, Prof John Patrick, Dr RT Furbank (CSIRO)
Contact: A/Prof Offler

Cell and Molecular Biology of Transfer Cell Development

	In developing seeds of the grain legumes broad bean and pea, differentiation of cells specialized for membrane transport, "transfer cells", is a key regulatory process in facilitating nutrient import for seed growth. These transfer cells are characterized by an invaginated wall and amplified plasma membrane surface area (see Fig. 3). Deposition of the ingrowth wall commences at specific loci as small papillate ingrowths. These subsequently become branched and coalesce to form a fenestrated sheet of wall material. In a fully differentiated transfer cell, the ingrowth wall is comprised of a number of interconnected, fenestrated sheets. We can induce transfer cell differentiation in cotyledons both in culture and in vivo and have developed a procedure for viewing the cytoplasmic face of the ingrowth wall by scanning electron microscopy. Together these experimental procedures offer exciting possibilities for exploring the cell and molecular biology of transfer cells. Current projects are outlined below.
Fig. 3. Micrographs of transfer cells illustrating their wall morphology. A. Light micrograph of a broad bean cotyledon in transverse section showing the epidermal transfer cell complex. The characteristic wall ingrowths of the transfer cells are stained purple. B, C. Scanning electron micrographs of a transversely freeze-fractured transfer cell (B) illustrating the intricate wall labyrinth and a view of the cytoplasmic face (C) of wall ingrowths showing the multi-layered fenestrated sheets of wall material.

Discovering signaling cascades responsible for the induction of transfer cells

We have developed a novel bean seed culture system in which epidermal transfer cells can be induced to form in large numbers allowing a wide diversity of experimental questions to be readily explored. Within three hours of being exposed to the inductive signal, wall ingrowths can be visualized at the scanning electron microscope level and their formation depends upon gene expression. A research program is examining these early developmental events to identify the signal cascades responsible for transfer cell induction. A targeted approach is perturbing potential signaling cascades with various inhibitors and stimulators to test their effects on cell wall ingrowth formation. A non-targeted approach relies on applying molecular technologies to detect genes expressed during the early (less than one hour) inductive phases leading to cell wall ingrowth formation.

Chief Investigators: Prof John Patrick, Dr David McCurdy, A/Prof Tina (CE) Offler
Contact: Prof Patrick

Regulation of wall ingrowth deposition in transfer cells of Vicia faba cotyledons

There is evidence that the cytoskeleton has a role in regulating wall ingrowth deposition. Cortical microtubules (MTs) are redistributed towards the ingrowth wall and ingrowth deposition is abolished or modified in the presence of MT and actin depolymerizing drugs. A meshwork of fine fibres covering the cytoplasmic face of the ingrowth wall has been visualized by SEM. These observations are consistent with the hypothesis that localized disruption of a dense cytoskeletal meshwork (possibly actin) provides access to the plasma membrane for secretory vesicles to exocytose wall components at discrete loci thus forming the small papillate ingrowths which characterize the commencement of wall ingrowth deposition. An Honours project will test this hypothesis by verifying the nature of the fibrous meshwork and establishing the relationship between the meshwork and ingrowths in the presence and absence of cytoskeleton disrupting drugs.

Supervisors: A/Prof Tina (CE) Offler, Dr David W McCurdy
Contact: A/Prof Offler