Steve Tyerman

Characterization of the TaALMT1 protein as an Al3+ -activated anion channel in transformed tobacco (Nicotiana Tabacum L.) cells

TaALMT1 encodes a putative transport protein associated with Al(3+)-activated efflux of malate from wheat root apices. We expressed TaALMT1 in Nicotiana tabacum L. suspension cells and conducted a detailed functional analysis. Protoplasts were isolated for patch-clamping from cells expressing TaALMT1 and from control cells (empty vector transformed). With malate(2-) as the permeant anion in the protoplast, an inward current (anion efflux) that reversed at positive potentials was observed in protoplasts expressing TaALMT1 in the absence of Al(3+). This current was sensitive to the anion channel antagonist niflumate, but insensitive to Gd(3+). External AlCl(3) (50 microM), but not La(3+) and Gd(3+), increased the inward current in TaALMT1-transformed protoplasts. The inward current was highly selective to malate over nitrate and chloride (P(mal) >> P(NO3) >or= P(Cl), P(mal)/P(Cl) >or=18, +/-Al(3+)), under conditions with higher anion concentration internally than externally. The anion currents displayed a voltage and time dependent deactivation at negative voltages. Voltage ramps revealed that inward rectification was caused by the imposed anion gradients. Single channels with conductances between 10 and 17 pS were associated with the deactivation of the current at negative voltages, agreeing with estimates from voltage ramps. This study of the electrophysiological function of the TaALMT1 protein in a plant heterologous expression system provides the first direct evidence that TaALMT1 functions as an Al(3+)-activated malate(2-) channel. We show that the Al(3+)-activated currents measured in TaALMT1-transformed tobacco cells are identical to the Al(3+)-activated currents observed in the root cells of wheat, indicating that TaALMT1 alone is likely to be responsible for those endogenous currents.

Impact of flooding on the water use of semi-arid riparian eucalypts

The water use strategy of Eucalyptus largiflorens (F. Muell.) was investigated in response to flooding on the Chowilla Anabranch, a semi-arid floodplain of the Murray River, South Australia. Water use was measured using the heat pulse technique at six sites that varied in flood duration from 0 to 78 days. Soil chloride, plant water potential and surface root mass were also measured. Suppression of tree water use did not occur during flooding regardless of flood length and site health, suggesting that sufficient oxygen had been available to the trees. Increases in tree water use occurred at some sites after the flood because of increases in water availability due to leaching of salt from the soil profile. The soils with a higher clay content incurred little leaching of salts and therefore little change in tree water availability. In contrast, the sites with more sandy soils encountered greater leaching and greater increases in tree water availability. Despite differing soil type responses, all tree communities investigated showed a reduction in tree water stress in the period after flooding. These findings suggest that flooding in this environment improves the health of Eucalyptus largiflorens in the short-term. The implications of these findings are discussed with regard to the management of the Chowilla Anabranch.

Floodwater infiltration through root channels on a sodic clay floodplain and the influence on a local tree species Eucalyptus largiflorens

Dieback of riparian species on floodplains has been attributed to increased soil salinisation due to raised groundwater levels, resulting from irrigation and river regulation. This is exacerbated by a reduction in flooding frequency and duration of inundation. For the Chowilla floodplain on the River Murray raised water tables have increased the amount of salts mobilised in the soil profile, causing the trees to experience salt induced water stress. For the trees to survive in the long term, salts need to be leached from the root zone.This study investigated whether floodwater infiltrates through channels created by E. largiflorens (black box) roots, flushing salts away from roots, thereby allowing the trees to increase their water uptake. Trees at different sites on the floodplain were artificially flooded, by pumping 1.5 kL of creek water into impoundments constructed around the trees. Gas exchange parameters, and pre-dawn and midday water potential were measured the day before, the day after and one week after the artificial flood and compared against trees that were not flooded. Pre-dawn and midday water potentials were also measured one month after the flood. After flooding, the trees experienced less water stress, indicated by an increase in water potential of less than 0.2 MPa, in comparison to non-flooded control trees. However, this response was not evident one month after flooding. The response to flooding did not result in increased rates of transpiration, stomatal conductance or photosynthesis, even though flooding effectively doubled the trees yearly water supply.The infiltration of floodwater in the impoundments around E. largiflorens was also compared to that of impoundments on bare ground. Floodwater infiltrated 2 – 17 times faster around trees than on adjacent bare ground, for parts of the floodplain not grazed by livestock. Tracer dye experiments indicated that bulk flow of water through pores down the profile was the reason for the enhanced infiltration. Flooding leached salts in direct vicinity of tree roots, but only leached small amounts of salts from the bulk soil.

Non‐selective cation channel activity of aquaporin AtPIP2;1 regulated by Ca2+ and pH

The aquaporin AtPIP2;1 is an abundant plasma membrane intrinsic protein in Arabidopsis thaliana that is implicated in stomatal closure, and is highly expressed in plasma membranes of root epidermal cells. When expressed in Xenopus laevis oocytes, AtPIP2;1 increased water permeability and induced a non-selective cation conductance mainly associated with Na+ . A mutation in the water pore, G103W, prevented both the ionic conductance and water permeability of PIP2;1. Co-expression of AtPIP2;1 with AtPIP1;2 increased water permeability but abolished the ionic conductance. AtPIP2;2 (93% identical to AtPIP2;1) similarly increased water permeability but not ionic conductance. The ionic conductance was inhibited by the application of extracellular Ca2+ and Cd2+ , with Ca2+ giving a biphasic dose-response with a prominent IC50 of 0.32 mм comparable with a previous report of Ca2+ sensitivity of a non-selective cation channel (NSCC) in Arabidopsis root protoplasts. Low external pH also inhibited ionic conductance (IC50 pH 6.8). Xenopus oocytes and Saccharomyces cerevisiae expressing AtPIP2;1 accumulated more Na+ than controls. Establishing whether AtPIP2;1 has dual ion and water permeability in planta will be important in understanding the roles of this aquaporin and if AtPIP2;1 is a candidate for a previously reported NSCC responsible for Ca2+ and pH sensitive Na+ entry into roots.

Root water transport under waterlogged conditions and the roles of aquaporins

Water flow through plants roots can be affected when the soil is waterlogged and oxygen deficient. For species not adapted to these conditions, water flow usually decreases within minutes to days, depending on the oxygen concentration in the root and rhizosphere. During this time, the decrease in water flow is attributed to decreased root hydraulic conductance, through an inhibition of plasma-membrane aquaporins. There is increasing evidence that aquaporins may also be involved in the transport of gases, end products of anaerobic respiration, and signalling molecules; all of which are relevant to oxygen-deficient conditions. Eventually, primary roots die if continually starved of oxygen, but may be replaced with adventitious roots that can maintain the supply of water to the shoot. Here, we review the effects of waterlogging and oxygen deficiency on root hydraulic conductance and aquaporin activity.

Effect of water stress and elevated temperature on hypoxia and cell death in the mesocarp of Shiraz berries: Berry hypoxia and death under water/heat stress

Background and Aim Berry shrivel during ripening is cultivar dependent and is correlated with berry cell death (CD). We hypothesised that under heat stress and water stress, regions of the pericarp in Shiraz berries would become hypoxic depending on berry porosity, and that this would induce CD. Methods and Results We measured CD and [O2] across the pericarp in berries developed under the factorial combination of two thermal regimes (ambient and heated) and two irrigation regimes (irrigated and non‐irrigated) in the Barossa Valley, South Australia. Heating increased ambient temperature by 0.6°C for irrigated and 1°C for non‐irrigated vines but had no effect on water relations, while non‐irrigation decreased stomatal conductance and stem water potential. Non‐irrigation decreased berry [O2] and increased both CD and ethanol concentration relative to irrigation. An association was established between mesocarp [O2] and CD. Berry respiration and total berry porosity decreased during berry ripening, but relative locule air‐space measured by X‐ray micro‐computed tomography increased late in ripening. Heating had little or no effect on CD or [O2] but decreased berry porosity, which was not affected by irrigation. Conclusion Water stress increased berry CD, which was associated with increased hypoxia. Significance of the Study The association between berry [O2] and CD provides insights into berry ripening with implications for yield and berry flavour.