Background. All plant cells evolved from the ancient ocean, where they had to survive in a world rich in common salt, NaCl. When plants moved onto the land, they found themselves in a less saline world, either growing in the soil or living in rivers and ponds. All cells, animal, plant and bacterial, accumulate K⁺ and exclude Na⁺, which can have deleterious effects on the functioning of proteins, and can cause cells to lose water. It is thought that cells achieve this via protein channels and energy-consuming pumps in the cell membrane. Many modern land plants, including most crops, experience Na⁺ as a toxin and are unable to exclude it from their cells. Fortunately, some land and aquatic plants retained the ancient ability to tolerate NaCl. This involves many types of adaptations, such as salt-extruding glands. The challenge of high salt must be also met at cellular level. Plant cells are encased in cellulose walls. The cells accumulate K⁺, attracting water, which generates internal pressure (turgor) that is balanced by tension in the cell walls. Turgor enables non-woody plants to stand up. Salt-tolerant plants are able to exclude Na⁺ from the living cytoplasm, and change their internal concentration of salts to retain enough water and keep turgor at a comfortable level. All cells, from bacterial to animal to plant, employ K⁺ and usually Cl^- as the ions gained or lost during volume and/or turgor regulation. The ability to take up or expel these ions is central to surviving high salinity and/or salinity fluctuations. Turgor regulation essentially involves conversion of mechanical stimulus to electrochemical regulatory events, such as ion flows into or out of the cell. Survival in salty environments dates back to the earliest plants, yet we still do not understand how the structure of the cell and its electric circuitry work together to enable this to happen. Are the transporters different in salt-tolerant and salt-sensitive plants? Is more of some transporter expressed in salt-tolerant species at the time of stress? Or is it the way the transporters work together in salt tolerant species? One approach to studying this fundamental problem is to go back to the ancient group of giant celled algae, the charophytes. One branch of their family evolved into the land plants, whilst other branches became modern charophytes. Some charophytes (Lamprothamnium succinctum) survive in a fluctuating environment, from freshwater to twice the saltiness of seawater, whilst other charophytes (Chara australis) live in freshwater, and, like many crop plants, cannot cope with even moderately salty environments. The giant size of charophyte cells (of up to 1 mm in diameter and several cm in length) enables us to study salt tolerance at the level of single cells, with minimal disturbance to the extracellular matrix (ECM), a cell wall- membrane-cytoskeleton continuum.

Current projects
September 2009

Mr. Sabah Al Khazaaly (pictured) expects to submit his Ph. D. thesis early in 2010. His research involved both the salt tolerant Lamprothamnium and salt sensitive Chara australis. He resolved the two components of salt stress, reduction in turgor and sodium toxicity, by exposing the cells to sorbitol medium and saline medium of equivalent osmolarity. In both Chara and Lamprothamnium, the background membrane conductance did not change upon mild (non plasmolysing) turgor decrease, but it increased in a Ca²⁺ dependent manner in saline medium. The proton pump in salt tolerant charophyte cells was activated by a decrease in turgor (Al Khazaaly and Beilby, 2007) and must therefore sense the pressure change or receive information from a pressure sensor. This activation is transient, as Lamprothamnium cells regulate their turgor (Bisson and Kirst, 1980). The proton pump is also activated by an increase in Na⁺ concentration (Beilby and Shepherd, 2001), so cells must be able to monitor Na⁺ concentration. This activation persists as long as the cells stay in the high salt medium. The smaller Lamprothamnium plants in more saline environments are presumed to have less energy for growth (Shepherd, Beilby and Heslop, 1999). The proton pump in salt-sensitive charophyte cells does not respond to decrease in turgor (Beilby and Shepherd, 2006). It is transiently activated by an increase in Na⁺ concentration if Ca²⁺ concentration in the medium is sufficiently high. It is rapidly inactivated when Na⁺ concentration is high and Ca²⁺ concentration is low (Beilby and Shepherd 2006; Shepherd, Beilby, Al Khazaaly and Shimmen, 2008).  Thus, in charophytes higher Ca²⁺ content of saline media exerts its protective influence not only by blocking voltage independent channels (VICs), but also by keeping the pump running.

In salt-sensitive Chara the inactivation of the pump brings the membrane potential to the E_rev of the background current, which is near -100 mV and rather insensitive to changes in ionic composition or pH of the medium. (This is puzzling from thermodynamic considerations and needs more research.) Spontaneous repetitive action potentials (APs) are often observed with long duration in low calcium saline media, further depleting the cell of K⁺ and Cl^- (Shepherd et al. 2008). The involvement of APs in signalling saline stress from root to shoot may also be important in land plants (Felle and Zimmermann, 2007).

Recently, another parameter of salt stress was found that distinguishes Chara from Lamprothamnium: Chara exhibits salinity-induced noise in the membrane potential upon exposure to saline medium (Al Khazaaly, Walker, Beilby and Shepherd, 2009). At frequencies between 1 and 500 Hz classical noise analysis shows (1/f²) rise of noise power as frequency falls, and a marked increase in noise power when the cell is exposed to high salinity (but not to equivalent osmotic stress). Inspection of the time domain shows that as well as initiating depolarisation, exposure to high Na⁺ concentrations usually initiates a continuous but random series of small rapid depolarisations with a slower recovery. We postulate that high Na⁺ concentration activates proton (or hydroxyl) channels. After longer exposure to high salinity, the membrane potential of Chara australis cells continues to depolarise toward zero, while the noise diminishes (suggesting that progressively larger numbers of proton/hydroxyl channels are activated). The current-voltage (I/V) data after several hours of saline stress can be simulated with the action of proton/hydroxyl channels (Beilby and Al Khazaaly, 2009). The activation of these channels at the time of exposure to salt would be disastrous for plant cells, as both the negative membrane potential and the pH gradients between the cytoplasm, vacuole and the medium are necessary for the cell to survive in high salt. Interestingly, proton/hydroxyl channels are also present in roots of land plants, where they mediate circulating currents similar to those observed in charophyte cells (Raven, 1991; Tyerman et al, 2001).

Projects available in 2010

The research combines several techniques applied to the cells under osmotic/salt stress:

1. Charophytes: Current/voltage (I/V) characteristics measure ion currents across the cell membranes. The I/V characteristics of the various transporters are resolved by modeling. The H⁺/OH^- channels will be further investigated in salt-stressed Chara and Lamprothamnium cells. The Cl^-/2H⁺ symporter, responsible for importing Cl^- into the cells will be investigated in Lamprothamnium at the time of turgor regulation under osmotic/salt stress.
   
   The salt-induced noise in the membrane potential difference (PD) will be further investigated: speed of the response upon introduction or withdrawal of NaCl, substitution of different ions, spatial distribution along the cell.
   
   The concentration of Na⁺ in the cytoplasm as function of external Na⁺ will be investigated using Sodium green and confocal microscopy.
   
   
2. Land plants: We plan to investigate circulating currents in roots of land plants (cereals and legumes) and the effect of salinity. Tyerman et al (1997) observed salinity induced noise in wheat root protoplasts. We plan to detect this noise by measuring PDs across parts of the whole roots.
   
   We also want to investigate whether salinity induces trains of APs conducting from the roots to the shoots (Felle and Zimmermann, 2007).

The student can learn the whole range of techniques or concentrate on one of the strands, depending on their background and interests.

References

Al Khazaaly S, Walker NA, Beilby MJ and Shepherd VA, 2009, Membrane potential fluctuations in Chara australis: a  characteristic signature of high external sodium. European Biophysics Journal, in press, available online

Al Khazaaly S and Beilby MJ, 2007, Modeling ion transporters at the time of hypertonic regulation in Lamprothamnium succinctum. Charophytes 1: 28 – 47

Beilby MJ and Al Khazaaly S, 2009, The role of H⁺/OH^- channels in salt stress response of Chara australis. Journal of Membrane Biology 230: 21 - 34

Beilby MJ and Shepherd VA, 2006, The electrophysiology of salt tolerance in charophytes. Cryptogamie Algologie 27: 403 - 417

Beilby MJ and Shepherd VA, 2001, Modeling the current-voltage characteristics of charophyte membranes. II. The effect of salinity on membranes of Lamprothamnium papulosum. Journal of Membrane Biology 181: 77 - 89

Bisson MA and Kirst GO, 1980, Lamprothamnium, a euryhaline charophyte. I. Osmotic relations and membrane potential steady state. Journal of Experimental Botany 31: 1223 -1235

Felle HH and Zimmermann MR, 2007, Systemic signalling in barley through action potentials. Planta 226: 203 - 214

Shepherd VA, Beilby MJ, Al Khazaaly S and Shimmen T, 2008, Mechano-perception in Chara cells: the influence of salinity and calcium on touch-activated receptor potentials, action potentials and ion transport. Plant Cell Environment 31: 1575 -1591

Shepherd VA, Beilby MJ and Heslop DJ, 1999, Ecophysiology of the hypotonic response in the salt-tolerant charophyte alga Lamprothamnium papulosum. Plant Cell Environment 22: 333 - 346

Raven JA, 1991, Terrestrial rhizophytes and H⁺ currents circulating over at least a millimeter - an obligate relationship. New Phytologist 117: 177-185

Tyerman SD, Beilby MJ, Whittington J, Juswono U, Newman I and Shabala S, 2001, Oscillations in proton transport revealed from simultaneous measurements of net current and net proton fluxes from isolated root protoplasts: MIFE meets patch-clamp. Australian Journal of Plant Physiology 28: 591 - 604

Tyerman SD, Skerrett M, Garrill A, Findlay GP, Leigh RA, 1997, Pathways for the permeation of Na⁺ and Cl^- into protoplasts derived from the cortex of wheat roots. Journal of Experimental Botany 48: 459 - 480