Wendy Sullivan

Rapid shoot-to-root signalling regulates root hydraulic conductance via aquaporins

We investigated how root hydraulic conductance (normalized to root dry weight, Lo ) is regulated by the shoot. Shoot topping (about 30% reduction in leaf area) reduced Lo of grapevine (Vitis vinifera L.), soybean (Glycine max L.) and maize (Zea mays L.) by 50 to 60%. More detailed investigations with soybean and grapevine showed that the reduction in Lo was not correlated with the reduction in leaf area, and shading or cutting single leaves had a similar effect. Percentage reduction in Lo was largest when initial Lo was high in soybean. Inhibition of Lo by weak acid (low pH) was smaller after shoot damage or leaf shading. The half time of reduction in Lo was approximately 5 min after total shoot decapitation. These characteristics indicate involvement of aquaporins. We excluded phloem-borne signals and auxin-mediated signals. Xylem-mediated hydraulic signals are possible since turgor rapidly decreased within root cortex cells after shoot topping. There was a significant reduction in the expression of several aquaporins in the plasma membrane intrinsic protein (PIP) family of both grapevine and soybean. In soybean, there was a five- to 10-fold reduction in GmPIP1;6 expression over 0.5-1 h which was sustained over the period of reduced Lo .

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.

Aluminum-Activated Malate Transporters Can Facilitate GABA Transport

GABA and malate interact with ALMTs to regulate each other’s transport, suggesting a role for ALMTs, which transport both GABA and malate in planta, in communicating metabolic status. Plant aluminum-activated malate transporters (ALMTs) are currently classified as anion channels; they are also known to be regulated by diverse signals, leading to a range of physiological responses. Gamma-aminobutyric acid (GABA) regulation of anion flux through ALMT proteins requires a specific amino acid motif in ALMTs that shares similarity with a GABA binding site in mammalian GABAA receptors. Here, we explore why TaALMT1 activation leads to a negative correlation between malate efflux and endogenous GABA concentrations ([GABA]i) in both wheat (Triticum aestivum) root tips and in heterologous expression systems. We show that TaALMT1 activation reduces [GABA]i because TaALMT1 facilitates GABA efflux but GABA does not complex Al3+. TaALMT1 also leads to GABA transport into cells, demonstrated by a yeast complementation assay and via 14C-GABA uptake into TaALMT1-expressing Xenopus laevis oocytes; this was found to be a general feature of all ALMTs we examined. Mutation of the GABA motif (TaALMT1F213C) prevented both GABA influx and efflux, and resulted in no correlation between malate efflux and [GABA]i. We conclude that ALMTs are likely to act as both GABA and anion transporters in planta. GABA and malate appear to interact with ALMTs in a complex manner to regulate each other’s transport, suggestive of a role for ALMTs in communicating metabolic status.

Split personality of Aluminum Activated Malate Transporter family proteins: facilitation of both GABA and malate transport

Plant aluminum activated malate transporters (ALMTs) are currently classified as anion channels; they are also known to be regulated by diverse signals leading to a range of physiological responses. Gamma-aminobutyric acid (GABA) regulation of anion flux through ALMT proteins requires the presence of a specific amino acid motif in ALMTs that shares similarity with a GABA-binding site in mammalian GABAA receptors. Here, we explore why TaALMT1-activation leads to a negative correlation between malate efflux and endogenous GABA concentrations ([GABA]i) in both wheat root tips and in heterologous expression systems. We show that TaALMT1 activation reduces [GABA]i because TaALMT1 facilitates GABA efflux. TaALMT1-expression also leads to GABA transport into cells, demonstrated by a yeast complementation assay and via 14CGABA uptake into TaALMT1-expressing Xenopus laevis oocytes; this was found to be a general feature of all ALMTs we examined. Mutation of the GABA motif (TaALMT1F213C) prevented both GABA influx and efflux, and uncoupled the relationship between malate efflux and [GABA]i. We conclude that ALMTs are likely to act as both GABA and anion transporters in planta. GABA and malate appear to interact with ALMTs in a complex manner regulating each other’s transport, suggestive of a role for ALMTs in communicating metabolic status.

Rapid measurement of total non-structural carbohydrate concentration in grapevine trunk and leaf tissues using near infrared spectroscopy

Carbohydrate analysis of plants allow to understand the physiology and phenology of the crop.Near infrared (NIR) spectroscopy was collected from grapevine trunk and leaf tissues.NIR could predict starch and TNC concentration in grapevine trunk and leaf tissues. Carbohydrate assays are commonly used in crops and plant research to understand the way in which carbohydrates are allocated within the vine and to assess its influence on the physiology and phenology of the plant. Total non-structural carbohydrate (TNC) concentration is normally assessed by wet chemistry methods which are time consuming and costly, especially when studying carbohydrate dynamics over seasons. Near infrared (NIR) spectroscopy is a fast and easy technique that has lately gained wide acceptance for the analysis of the chemical composition of grain, food, wine, pharmaceutical products, among others. Near infrared is the region of the electromagnetic spectrum between 750nm and 2500nm and it is used to gather information on the relative proportions of CH, NH and OH bonds of the organic molecules. This study collected NIR spectra from grapevine trunk and leaf tissues, measured TNC concentration of the same samples using a wet chemical method and compared the results using multivariate data analysis to develop a rapid procedure for the estimation of TNC concentration in grapevine tissues. Results showed that NIR spectroscopy could be used to predict starch and TNC concentration in freeze-dried and ground grapevine trunk and leaf tissues. Moreover, it has been demonstrated that a robust universal model could be applied to the prediction of TNC in both leaves and trunks. Therefore, this method could be used as a practical tool for a rapid screening of TNC concentration for high temporal and spatial assessment of grapevine tissues at given phenological stages. The main advantages of this technique over traditional methods are the rapidity and the ease-of-use protocol in routine analysis, which allows a considerable reduction of costs and time.

A comparison of root and shoot hydraulics, aquaporin expression and leaf gas exchange between two grapevine cultivars reveals differences in hydraulic control mediated by aquaporins

Hydraulics of plants that take different strategies of stomatal control under water stress are still relatively poorly understood. Here we explore how root and shoot hydraulics, gas exchange, aquaporin expression and abscisic acid (ABA) concentration in leaf xylem sap ([ABA]xylem) may be involved and coordinated. A comparison in responses to mild water stress and ABA application was made between two cultivars of Vitis vinifera L. previously classified as isohydric (Grenache) and anisohydric (Syrah). Grenache showed stronger adjustments of leaf, plant, and root hydraulic conductances to decreased soil moisture and a steeper correlation of stomatal conductance (gs) to [ABA]xylem than Syrah resulting in greater conservation of soil moisture, but not necessarily more isohydric behaviour. Under well-watered conditions, changes in vapour pressure deficit (VPD) had a strong influence on gs in both cultivars with adjustments of leaf hydraulic conductance. Grenache was more sensitive to decreases in soil water availability compared to Syrah that rather responded to VPD. There were stronger correlations between plant hydraulic parameters and changes in aquaporin gene expression in leaves and roots of Grenache. Overall, the results reinforce the hypothesis that both hydraulic and chemical signals significantly contribute to the differences in water conservation behaviours of the two cultivars.