Phloem composition, translocation and carbon partitioning

We are examining in alterations in the composition of the phloem as plants respond to changes in the environment, particularly in understanding how extension growth is integrated with solute translocation and the effect of drought. We are examining the water and solute relations of cells along the whole translocation pathway, including root sieve elements, in order to determine the correlation with pressure driven flow throught the phloem from source to sink. These mechanisms must underlie the controlof carbon partitioning during development and following environmental change. We use aphids to extact sieve element sap and single cell sampling aproaches to extract cell sap for subsequent analysis.

This work is important in understand of carbon partitioning in agricultural situations plants and also has implications for control of aphids pests.

Regulation of phloem potassium concentration

This project aims to determine the relative contributions of the axial (phloem) and radial pathways to K⁺ uptake and distribution in roots.

Plants grow because cells are turgid, this turgor is generated by the accumulation of solutes. The major inorganic solutes in root tips are K⁺ salts whose deposition in root cells varies with growth. The regulation of K⁺ uptake is not well understood but is crucial to plant growth and development. In particular, it is unclear what proportion of K⁺ is obtained by uptake from the apoplastic space across the plasma membrane (the radial pathway) or by symplastic transport from the phloem (the axial pathway).

In collaboration with Dr. Phil White at HRI Wellesbourne we are using barley Arabidopsis seedlings for our studies since:

• Their roots have a simple anatomy and appropriate elongation zones have already been identified,
• A mutant (akt1-1) is available in which K⁺ influx to root cells from the apoplast is impaired,
• Their roots are amenable to single-cell sampling
• Sieve element sap can be extracted from leaves by aphid stylectomy
• Protoplasts from root cells are suitable for patch-clamp electrophysiology

Plants are grown either hydroponically or on agar to facilitate experimental manipulation. Growth rates will be determined by time-lapse photography. The pathways of K⁺ supply will be manipulated by altering K status internally in the phloem by foliar feeding or externally by altering the composition of the growth medium. The redistribution of K⁺ within the plant will be estimated from whole tissue analyses, sampling of xylem sap (root exudate), phloem sap (aphid stylectomy) and vacuolar sap (microsampling). The rates ofK⁺ deposition will be estimated from cell growth and K⁺content. Unidirectional fluxes of K⁺ across cell membranes (a component of the radial pathway) in specific regions will be measured using locally applied radiotracers (⁴²K, ⁸⁶Rb). K⁺ fluxes will be compared with deposition in cells differing in growth rate and development (root cap, zone of cell division, zone of cell elongation, mature root). Additionally, we will use the Arabidopsis akt1-1 mutant which does not grow at low external K⁺ since it lacks aK⁺ channel (AKT1) mediating K⁺ influx along the radial pathway from the apoplast. We will also determine the absolute capacity of cortical cells from contrasting regions of wild type and mutant to transport K⁺ across the plasma membrane (the radial route) using both radiometric and electrophysiological (membrane-patch voltage-clamp) techniques on isolated protoplasts.

This projects also providing us with information about the relative contribution of symplastic and apoplasticpathways form  water and solute flux into growing root cells.

Click the title for a powerpoint poster presented at the recent phloem meeting in Newcastle, Australia  entitled 'How Tight is Potassium Regulation in the Phloem in Comparison to Other Plant Cells?'

Control of Carbon partitioning in a Simplified Source-Sink System.

New approaches to understanding phloem regulation: Direct measurement of specific mRNA in sieve elements

We aremeasuring the levels of specific mRNAs in phloem sap. This is being achieved by obtaining sieve tube sap by aphid stylectomy, reverse transcription to produce cDNA and amplification by PCR using sequence-specific primers. mRNA species will be selected on the basis of their proposed role in regulating sieve element content. One outcome will be the prodution of a phlom specific cDMNA library. Successful completion of the project will link cellular physiology with molecular approaches with considerable opportunity for exploitation in both pure and applied research.

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