Award details

Nutrient ion homeostasis in plant cells: studies with ion- selective microelectrodes

Reference	BBS/E/C/00011427
Principal Investigator / Supervisor	Professor Tony Miller
Co-Investigators / Co-Supervisors
Institution	Rothamsted Research
Department	Rothamsted Research Department
Funding type	Research
Value (£)	401,190
Status	Completed
Type	Institute Project
Start date	01/04/1997
End date	31/03/1999
Duration	24 months

Abstract

The intracellular measurement of nutrient ions is essential for an understanding of transport and compartmentation. There are several techniques for measuring such concentrations in single cells but ion-selective microelectrode measurements are the best approach. The measurements are non-destructive, they can report from both the cytosol and the vacuole, and they give information on the electrical and chemical gradients across membranes. This information yields insights into transport mechanisms at the plasma membrane and tonoplast. Ion-selective sensors are available for several ions, including potassium, calcium, sodium, protons, chloride and nitrate. The manufacture of triple- barrelled ion- selective microelectrodes allows the simultaneous measurement of intracellular pH and the activities of other ions such as potassium or nitrate from the same cellular compartment. This approach has been used to measure how the compartmentation of nutrient ions between the vacuole and cytosol changes during conditions of surplus and deficiency. The measurements have established that there is homeostasis of both potassium and nitrate ion concentrations in the cytoplasm of barley root cells. Cytosolic potassium is maintained at an activity of 70 mM, only in severely K-starved root cells is cytosolic potassium decreased. This is accompanied by an acidification of the cytosol, and these changes occur in the epidermal and expanding root cells but not cortical cells. Cytosolic nitrate is kept at 4 mM but this may be related to the observation that the tonoplast ATPase proton pump is inhibited by nitrate concentrations greater than 4 mM. Only seedlings starved of nitrate for 3 days showed a decrease in the cytosolic nitrate. With these facts established, the aim is to investigate how cytosolic nutrient ion homeostasis is regulated using transgenic plants. In other work, sodium- and chloride-selective microelectrodes are being used to determine if there are differences in the compartmentation of these ions in cells of a salt-tolerant and a salt-sensitive species of barley. Triple-barrelled nitrate and pH-selective microelectrodes will be used to investigate how long seed reserves can maintain cytosolic nitrate concentrations and how this related to the point at which growth ceases. Treatments will be applied to barley seedlings which should acidify cytosolic pH in order to investigate the link between cytosolic pH and potassium concentration. Both potassium- and nitrate- selective microelectrodes will be used to characterise compartmentation of these nutrients in wild- type barley suspension cultures. These measurements will be necessary before similar measurements on transgenic cells with altered expression of membrane transporters can begin. Sodium- and chloride-selective microelectrodes suitable for measurements in barley root cells will be developed. Intracellular nitrate pools will be measured in transgenic Arabidopsis plants growing at high and low external nitrate concentrations. These plants will have increased expression of a low affinity nitrate transporter, or decreased expression of a high affinity nitrate transporter. An electrophysiological assay of the nitrate-elicited change in membrane potential in transgenic Arabidopsis root cells will also be used to establish that there is altered transporter activity in these plants. Sodium-selective microelectrodes will be used to measure the intracellular sodium concentration in root cells of two varieties of barley which have very different degrees of salt tolerance. Also, potassium- selective microelectrodes will also be used to measure the cytosolic potassium concentration of root cells of these two varieties of barley plants growing under salt stress. These electrodes will also be used to measure intracellular K pools in barley root cells of seedlings growing under severe K-deficiency to determine the relationship between protein synthesis and changes in cytosolic K and pH.

Summary

unavailable

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search