The function of the Medicago truncatula ZIP transporter MtZIP14 is linked to arbuscular mycorrhizal fungal colonization.

Soil micronutrient availability, including zinc (Zn), is a limiting factor for crop yield. Arbuscular mycorrhizal (AM) fungi can improve host plant growth and nutrition through the mycorrhizal pathway of nutrient uptake. Although the physiology of Zn uptake through the mycorrhizal pathway is well established, the identity of the related molecular components are unknown. Here, RNA-seq analysis was used to identify genes differentially-regulated by AM colonization and soil Zn concentration in roots of Medicago truncatula. The putative Zn transporter gene MtZIP14 was markedly up-regulated in M. truncatula roots when colonized by Rhizophagus irregularis. MtZIP14 restored yeast growth under low Zn availability. Loss-of-function mutant plants (mtzip14) had reduced shoot biomass compared to the wild-type when colonized by AM fungi and grown under low and sufficient soil Zn concentration; at high soil Zn concentration, there were no genotypic differences in shoot biomass. The vesicular and arbuscular colonization of roots was lower in the mtzip14 plants regardless of soil Zn concentration. We propose that MtZIP14 is linked to AM colonization in M. truncatula plants, with the possibility that MtZIP14 function with AM colonization is linked to plant Zn nutrition.

Aluminum-Activated Malate Transporters Can Facilitate GABA 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 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.

Novel Ion Channel Targets and Drug Delivery Tools for Controlling Glioblastoma Cell Invasiveness.

Comprising more than half of all brain tumors, glioblastoma multiforme (GBM) is a leading cause of brain cancer-related deaths worldwide. A major clinical challenge is presented by the capacity of glioma cells to rapidly infiltrate healthy brain parenchyma, allowing the cancer to escape control by localized surgical resections and radiotherapies, and promoting recurrence in other brain regions. We propose that therapies which target cellular motility pathways could be used to slow tumor dispersal, providing a longer time window for administration of frontline treatments needed to directly eradicate the primary tumors. An array of signal transduction pathways are known to be involved in controlling cellular motility. Aquaporins (AQPs) and voltage-gated ion channels are prime candidates as pharmacological targets to restrain cell migration in glioblastoma. Published work has demonstrated AQPs 1, 4 and 9, as well as voltage-gated potassium, sodium and calcium channels, chloride channels, and acid-sensing ion channels are expressed in GBM and can influence processes of cell volume change, extracellular matrix degradation, cytoskeletal reorganization, lamellipodial and filopodial extension, and turnover of cell-cell adhesions and focal assembly sites. The current gap in knowledge is the identification of optimal combinations of targets, inhibitory agents, and drug delivery systems that will allow effective intervention with minimal side effects in the complex environment of the brain, without disrupting finely tuned activities of neuro-glial networks. Based on published literature, we propose that co-treatments using AQP inhibitors in addition to other therapies could increase effectiveness, overcoming some limitations inherent in current strategies that are focused on single mechanisms. An emerging interest in nanobodies as drug delivery systems could be instrumental for achieving the selective delivery of combinations of agents aimed at multiple key targets, which could enhance success in vivo.

Malate Transport and Metabolism in Nitrogen-Fixing Legume Nodules.

Legumes form a symbiosis with rhizobia, a soil bacterium that allows them to access atmospheric nitrogen and deliver it to the plant for growth. Biological nitrogen fixation occurs in specialized organs, termed nodules, that develop on the legume root system and house nitrogen-fixing rhizobial bacteroids in organelle-like structures termed symbiosomes. The process is highly energetic and there is a large demand for carbon by the bacteroids. This carbon is supplied to the nodule as sucrose, which is broken down in nodule cells to organic acids, principally malate, that can then be assimilated by bacteroids. Sucrose may move through apoplastic and/or symplastic routes to the uninfected cells of the nodule or be directly metabolised at the site of import within the vascular parenchyma cells. Malate must be transported to the infected cells and then across the symbiosome membrane, where it is taken up by bacteroids through a well-characterized dct system. The dicarboxylate transporters on the infected cell and symbiosome membranes have been functionally characterized but remain unidentified. Proteomic and transcriptomic studies have revealed numerous candidates, but more work is required to characterize their function and localise the proteins in planta. GABA, which is present at high concentrations in nodules, may play a regulatory role, but this remains to be explored.

Role of TaALMT1 malate-GABA transporter in alkaline pH tolerance of wheat.

Malate exudation through wheat (Triticum aestivum L) aluminium-activated malate transporter 1 (TaALMT1) confers Al3+ tolerance at low pH, but is also activated by alkaline pH, and is regulated by and facilitates significant transport of gamma-aminobutyric acid (GABA, a zwitterionic buffer). Therefore, TaALMT1 may facilitate acidification of an alkaline rhizosphere by promoting exudation of both malate and GABA. Here, the performance of wheat near isogenic lines ET8 (Al+3 -tolerant, high TaALMT1 expression) and ES8 (Al+3 -sensitive, low TaALMT1 expression) are compared. Root growth (at 5 weeks) was higher for ET8 than ES8 at pH 9. ET8 roots exuded more malate and GABA at high pH and acidified the rhizosphere more rapidly. GABA and malate exudation was enhanced at high pH by the addition of aluminate in both ET8 and transgenic barley expressing TaALMT1. Xenopus laevis oocytes expressing TaALMT1 acidified an alkaline media more rapidly than controls corresponding to higher GABA efflux. TaALMT1 expression did not change under alkaline conditions but key genes involved in GABA turnover changed in accordance with a high rate of GABA synthesis. We propose that TaALMT1 plays a role in alkaline tolerance by exuding malate and GABA, possibly coupled to proton efflux.

γ-Aminobutyric acid (GABA) signalling in plants.

The role of γ-aminobutyric acid (GABA) as a signal in animals has been documented for over 60 years. In contrast, evidence that GABA is a signal in plants has only emerged in the last 15 years, and it was not until last year that a mechanism by which this could occur was identified-a plant 'GABA receptor' that inhibits anion passage through the aluminium-activated malate transporter family of proteins (ALMTs). ALMTs are multigenic, expressed in different organs and present on different membranes. We propose GABA regulation of ALMT activity could function as a signal that modulates plant growth, development, and stress response. In this review, we compare and contrast the plant 'GABA receptor' with mammalian GABAA receptors in terms of their molecular identity, predicted topology, mode of action, and signalling roles. We also explore the implications of the discovery that GABA modulates anion flux in plants, its role in signal transduction for the regulation of plant physiology, and predict the possibility that there are other GABA interaction sites in the N termini of ALMT proteins through in silico evolutionary coupling analysis; we also explore the potential interactions between GABA and other signalling molecules.

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.

Characterization of human aquaporin ion channels in a yeast expression system as a tool for novel ion channel discovery.

Aquaporin (AQP) channels found in all domains of life are transmembrane proteins which mediate passive transport of water, glycerol, signaling molecules, metabolites, and charged solutes. Discovery of new classes of ion-conducting AQP channels has been slow, likely reflecting time- and labor-intensive methods required for traditional electrophysiology. Work here defines a sensitive mass-throughput system for detecting AQP ion channels, identified by rescue of cell growth in the K+-transport-defective yeast strain CY162 following genetic complementation with heterologously expressed cation-permeable channels, using the well characterized human AQP1 channel for proof of concept. Results showed AQP1 conferred transmembrane permeability to cations which rescued survival in CY162 yeast. Comprehensive testing showed that growth response properties fully recapitulated AQP1 pharmacological agonist and antagonist profiles for activation, inhibition, dose-dependence, and structure-function relationships, demonstrating validity of the yeast screening tool for AQP channel identification and drug discovery efforts. This method also provided new information on divalent cation blockers of AQP1, pH sensitivity of antagonists, and ion permeability of human AQP6. Site-directed mutagenesis of AQP1 channel regulatory domains confirmed that yeast growth rescue was mediated by the introduced channels. Optical monitoring with a lithium-sensitive photoswitchable probe in living cells independently demonstrated monovalent cation permeability of AQP1 channels in yeast plasma membrane. Ion channel properties of AQP1 expressed in yeast were consistent with those of AQP1 expressed in Xenopus laevis oocyte and K+-transport defective Escherichia coli. Outcomes here establish a powerful new approach for efficient screening of phylogenetically diverse AQPs for yet untested functions as cation channels.

Ecological phage therapy: Can bacteriophages help rapidly restore the soil microbiome?

Soil microbiota underpin ecosystem functionality yet are rarely targeted during ecosystem restoration. Soil microbiota recovery following native plant revegetation can take years to decades, while the effectiveness of soil inoculation treatments on microbiomes remains poorly explored. Therefore, innovative restoration treatments that target soil microbiota represent an opportunity to accelerate restoration outcomes. Here, we introduce the concept of ecological phage therapy-the application of phage for the targeted reduction of the most abundant and dominant bacterial taxa present in degraded ecosystems. We propose that naturally occurring bacteriophages-viruses that infect bacteria-could help rapidly shift soil microbiota towards target communities. Bacteriophages sculpt the microbiome by lysis of specific bacteria, and if followed by the addition of reference soil microbiota, such treatments could facilitate rapid reshaping of soil microbiota. Here, we experimentally tested this concept in a pilot study. We collected five replicate pre-treatment degraded soil samples, then three replicate soil samples 48 hours after phage, bacteria, and control treatments. Bacterial 16S rDNA sequencing showed that phage-treated soils had reduced bacterial diversity; however, when we combined ecological phage therapy with reference soil inoculation, we did not see a shift in soil bacterial community composition from degraded soil towards a reference-like community. Our pilot study provides early evidence that ecological phage therapy could help accelerate the reshaping of soil microbiota with the ultimate aim of reducing timeframes for ecosystem recovery. We recommend the next steps for ecological phage therapy be (a) developing appropriate risk assessment and management frameworks, and (b) focussing research effort on its practical application to maximise its accessibility to restoration practitioners.

Bioenergetic mapping of 'healthy microbiomes' via compound processing potential imprinted in gut and soil metagenomes.

Despite mounting evidence of their importance in human health and ecosystem functioning, the definition and measurement of 'healthy microbiomes' remain unclear. More advanced knowledge exists on health associations for compounds used or produced by microbes. Environmental microbiome exposures (especially via soils) also help shape, and may supplement, the functional capacity of human microbiomes. Given the synchronous interaction between microbes, their feedstocks, and micro-environments, with functional genes facilitating chemical transformations, our objective was to examine microbiomes in terms of their capacity to process compounds relevant to human health. Here we integrate functional genomics and biochemistry frameworks to derive new quantitative measures of in silico potential for human gut and environmental soil metagenomes to process a panel of major compound classes (e.g., lipids, carbohydrates) and selected biomolecules (e.g., vitamins, short-chain fatty acids) linked to human health. Metagenome functional potential profile data were translated into a universal compound mapping 'landscape' based on bioenergetic van Krevelen mapping of function-level meta-compounds and corresponding functional relative abundances, reflecting imprinted genetic capacity of microbiomes to metabolize an array of different compounds. We show that measures of 'compound processing potential' associated with human health and disease (examining atherosclerotic cardiovascular disease, colorectal cancer, type 2 diabetes and anxious-depressive behavior case studies), and displayed seemingly predictable shifts along gradients of ecological disturbance in plant-soil ecosystems (three case studies). Ecosystem quality explained 60-92 % of variation in soil metagenome compound processing potential measures in a post-mining restoration case study dataset. With growing knowledge of the varying proficiency of environmental microbiota to process human health associated compounds, we might design environmental interventions or nature prescriptions to modulate our exposures, thereby advancing microbiota-oriented approaches to human health. Compound processing potential offers a simplified, integrative approach for applying metagenomics in ongoing efforts to understand and quantify the role of microbiota in environmental- and human-health.

Pharmacological Inhibition of Membrane Signaling Mechanisms Reduces the Invasiveness of U87-MG and U251-MG Glioblastoma Cells In Vitro.

Impairing the motility of glioblastoma multiforme (GBM) cells is a compelling goal for new approaches to manage this highly invasive and rapidly lethal human brain cancer. Work here characterized an array of pharmacological inhibitors of membrane ion and water channels, alone and in combination, as tools for restraining glioblastoma spread in human GBM cell lines U87-MG and U251-MG. Aquaporins, AMPA glutamate receptors, and ion channel classes (shown to be upregulated in human GBM at the transcript level and linked to mechanisms of motility in other cell types) were selected as pharmacological targets for analyses. Effective compounds reduced the transwell invasiveness of U87-MG and U251-MG glioblastoma cells by 20-80% as compared with controls, without cytotoxicity. The compounds and doses used were: AqB013 (14 µM); nifedipine (25 µM); amiloride (10 µM); apamin (10 µM); 4-aminopyridine (250 µM); and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; 30 µM). Invasiveness was quantified in vitro across transwell filter chambers layered with extracellular matrix. Co-application of each of the ion channel agents with the water channel inhibitor AqB013 augmented the inhibition of invasion (20 to 50% greater than either agent alone). The motility impairment achieved by co-application of pharmacological agents differed between the GBM proneural-like subtype U87-MG and classical-like subtype U251-MG, showing patterns consistent with relative levels of target channel expression (Human Protein Atlas database). In addition, two compounds, xanthurenic acid and caelestine C (from the Davis Open Access Natural Product-based Library, Griffith University QLD), were discovered to block invasion at micromolar doses in both GBM lines (IC50 values from 0.03 to 1 µM), without cytotoxicity, as measured by full mitochondrial activity under conditions matching those in transwell assays and by normal growth in spheroid assays. Mechanisms of action of these agents based on published work are likely to involve modulation of glutamatergic receptor signaling. Treating glioblastoma by the concurrent inhibition of multiple channel targets could be a powerful approach for slowing invasive cell spread without cytotoxic side effects, potentially enhancing the effectiveness of clinical interventions focused on eradicating primary tumors.

Proteoliposomes reconstituted with human aquaporin-1 reveal novel single-ion-channel properties.

Human aquaporin 1 (hAQP1) forms homotetrameric channels that facilitate fluxes of water and small solutes across cell membranes. In addition to water channel activity, hAQP1 displays non-selective monovalent cation-channel activity gated by intracellular cyclic GMP. Dual water and ion-channel activity of hAQP1, thought to regulate cell shape and volume, could offer a target for novel therapeutics relevant to controlling cancer cell invasiveness. This study probed properties of hAQP1 ion channels using proteoliposomes, which, unlike conventional cell-based systems such as Xenopus laevis oocytes, are relatively free of background ion channels. Histidine-tagged recombinant hAQP1 protein was synthesized and purified from the methylotrophic yeast, Pichia pastoris, and reconstituted into proteoliposomes for biophysical analyses. Osmotic water channel activity confirmed correct folding and channel assembly. Ion-channel activity of hAQP1-Myc-His6 was recorded by patch-clamp electrophysiology with excised patches. In symmetrical potassium, the hAQP1-Myc-His6 channels displayed coordinated gating, a single-channel conductance of approximately 75 pS, and multiple subconductance states. Applicability of this method for structure-function analyses was tested using hAQP1-Myc-His6 D48A/D185A channels modified by site-directed mutations of charged Asp residues estimated to be adjacent to the central ion-conducting pore of the tetramer. No differences in conductance were detected between mutant and wild-type constructs, suggesting the open-state conformation could differ substantially from expectations based on crystal structures. Nonetheless, the method pioneered here for AQP1 demonstrates feasibility for future work defining structure-function relationships, screening pharmacological inhibitors, and testing other classes in the broad family of aquaporins for previously undiscovered ion-conducting capabilities.

Aquaporin ion conductance properties defined by membrane environment, protein structure, and cell physiology.

Aquaporins (AQPs) are multifunctional transmembrane channel proteins permeable to water and an expanding array of solutes. AQP-mediated ion channel activity was first observed when purified AQP0 from bovine lens was incorporated into lipid bilayers. Electrophysiological properties of ion-conducting AQPs since discovered in plants, invertebrates, and mammals have been assessed using native, reconstituted, and heterologously expressed channels. Accumulating evidence is defining amino acid residues that govern differential solute permeability through intrasubunit and central pores of AQP tetramers. Rings of charged and hydrophobic residues around pores influence AQP selectivity, and are candidates for further work to define motifs that distinguish ion conduction capability, versus strict water and glycerol permeability. Similarities between AQP ion channels thus far include large single channel conductances and long open times, but differences in ionic selectivity, permeability to divalent cations, and mechanisms of gating (e.g., by voltage, pH, and cyclic nucleotides) are unique to subtypes. Effects of lipid environments in modulating parameters such as single channel amplitude could explain in part the variations in AQP ion channel properties observed across preparations. Physiological roles of the ion-conducting AQP classes span diverse processes including regulation of cell motility, organellar pH, neural development, signaling, and nutrient acquisition. Advances in computational methods can generate testable predictions of AQP structure-function relationships, which combined with innovative high-throughput assays could revolutionize the field in defining essential properties of ion-conducting AQPs, discovering new AQP ion channels, and understanding the effects of AQP interactions with proteins, signaling cascades, and membrane lipids.

Picrotoxin Delineates Different Transport Configurations for Malate and γ Aminobutyric Acid through TaALMT1.

Plant-derived pharmacological agents have been used extensively to dissect the structure-function relationships of mammalian GABA receptors and ion channels. Picrotoxin is a non-competitive antagonist of mammalian GABAA receptors. Here, we report that picrotoxin inhibits the anion (malate) efflux mediated by wheat (Triticum aestivum) ALMT1 but has no effect on GABA transport. The EC50 for inhibition was 0.14 nM and 0.18 nM when the ALMTs were expressed in tobacco BY2 cells and in Xenopus oocytes, respectively. Patch clamping of the oocyte plasma membrane expressing wheat ALMT1 showed that picrotoxin inhibited malate currents from both sides of the membrane. These results demonstrate that picrotoxin inhibits anion efflux effectively and can be used as a new inhibitor to study the ion fluxes mediated by ALMT proteins that allow either GABA or anion transport.

Boron toxicity tolerance in barley through reduced expression of the multifunctional aquaporin HvNIP2;1.

Boron (B) toxicity is a significant limitation to cereal crop production in a number of regions worldwide. Here we describe the cloning of a gene from barley (Hordeum vulgare), underlying the chromosome 6H B toxicity tolerance quantitative trait locus. It is the second B toxicity tolerance gene identified in barley. Previously, we identified the gene Bot1 that functions as an efflux transporter in B toxicity-tolerant barley to move B out of the plant. The gene identified in this work encodes HvNIP2;1, an aquaporin from the nodulin-26-like intrinsic protein (NIP) subfamily that was recently described as a silicon influx transporter in barley and rice (Oryza sativa). Here we show that a rice mutant for this gene also shows reduced B accumulation in leaf blades compared to wild type and that the mutant protein alters growth of yeast (Saccharomyces cerevisiae) under high B. HvNIP2;1 facilitates significant transport of B when expressed in Xenopus oocytes compared to controls and to another NIP (NOD26), and also in yeast plasma membranes that appear to have relatively high B permeability. We propose that tolerance to high soil B is mediated by reduced expression of HvNIP2;1 to limit B uptake, as well as by increased expression of Bot1 to remove B from roots and sensitive tissues. Together with Bot1, the multifunctional aquaporin HvNIP2;1 is an important determinant of B toxicity tolerance in barley.

Emerging Roles of γ Aminobutyric Acid (GABA) Gated Channels in Plant Stress Tolerance.

The signaling role for γ-Aminobutyric acid (GABA) has been documented in animals for over seven decades. However, a signaling role for GABA in plants is just beginning to emerge with the discovery of putative GABA binding site/s and GABA regulation of anion channels. In this review, we explore the role of GABA in plant growth and development under abiotic stress, its interactions with other signaling molecules and the probability that there are other anion channels with important roles in stress tolerance that are gated by GABA.

Comparing Hydraulics Between Two Grapevine Cultivars Reveals Differences in Stomatal Regulation Under Water Stress and Exogenous ABA Applications.

Hydraulics of plants that have different strategies of stomatal regulation under water stress are relatively poorly understood. We explore how root and shoot hydraulics, stomatal conductance (g s), leaf and root aquaporin (AQP) expression, and abscisic acid (ABA) concentration in leaf xylem sap ([ABA]xylemsap) may be coordinated under mild water stress and exogenous ABA applications in two Vitis vinifera L. cultivars traditionally classified as near-isohydric (Grenache) and near-anisohydric (Syrah). Under water stress, Grenache exhibited stronger adjustments of plant and root hydraulic conductances and greater stomatal sensitivity to [ABA]xylemsap than Syrah resulting in greater conservation of soil moisture but not necessarily more isohydric behavior. Correlations between leaf (Ψleaf) and predawn (ΨPD) water potentials between cultivars suggested a "hydrodynamic" behavior rather than a particular iso-anisohydric classification. A significant decrease of Ψleaf in well-watered ABA-fed vines supported a role of ABA in the soil-leaf hydraulic pathway to regulate g s. Correlations between leaf and root AQPs expression levels under water deficit could explain the response of leaf (K leaf) and root (Lp r) hydraulic conductances in both cultivars. Additional studies under a wider range of soil water deficits are required to explore the possible differential regulation of g s and plant hydraulics in different cultivars and experimental conditions.

Common garden experiment reveals altered nutritional values and DNA methylation profiles in micropropagated three elite Ghanaian sweet potato genotypes.

Micronutrient deficiency is the cause of multiple diseases in developing countries. Staple crop biofortification is an efficient means to combat such deficiencies in the diets of local consumers. Biofortified lines of sweet potato (Ipomoea batata L. Lam) with enhanced beta-carotene content have been developed in Ghana to alleviate Vitamin A Deficiency. These genotypes are propagated using meristem micropropagation to ensure the generation of virus-free propagules. In vitro culture exposes micropropagated plants to conditions that can lead to the accumulation of somaclonal variation with the potential to generate unwanted aberrant phenotypes. However, the effect of micropropagation induced somaclonal variation on the production of key nutrients by field-grown plants has not been previously studied. Here we assessed the extent of in vitro culture induced somaclonal variation, at a phenotypic, compositional and genetic/epigenetic level, by comparing field-maintained and micropropagated lines of three elite Ghanaian sweet potato genotypes grown in a common garden. Although micropropagated plants presented no observable morphological abnormalities compared to field maintained lines, they presented significantly lower levels of iron, total protein, zinc, and glucose. Methylation Sensitive Amplification Polymorphism analysis showed a high level of in vitro culture induced molecular variation in micropropagated plants. Epigenetic, rather than genetic variation, accounts for most of the observed molecular variability. Taken collectively, our results highlight the importance of ensuring the clonal fidelity of the micropropagated biofortified lines in order to reduce potential losses in the nutritional value prior to their commercial release.

GABA signalling modulates plant growth by directly regulating the activity of plant-specific anion transporters.

The non-protein amino acid, gamma-aminobutyric acid (GABA) rapidly accumulates in plant tissues in response to biotic and abiotic stress, and regulates plant growth. Until now it was not known whether GABA exerts its effects in plants through the regulation of carbon metabolism or via an unidentified signalling pathway. Here, we demonstrate that anion flux through plant aluminium-activated malate transporter (ALMT) proteins is activated by anions and negatively regulated by GABA. Site-directed mutagenesis of selected amino acids within ALMT proteins abolishes GABA efficacy but does not alter other transport properties. GABA modulation of ALMT activity results in altered root growth and altered root tolerance to alkaline pH, acid pH and aluminium ions. We propose that GABA exerts its multiple physiological effects in plants via ALMT, including the regulation of pollen tube and root growth, and that GABA can finally be considered a legitimate signalling molecule in both the plant and animal kingdoms.

Comparison of ELISA and RT-PCR for the detection of Prunus necrotic ring spot virus and prune dwarf virus in almond (Prunus dulcis).

A technique based on the reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to detect the presence of Prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) simultaneously in almond. This paper presents the results of a 3-year study comparing both enzyme-linked immunosorbent assay (ELISA) and RT-PCR for the detection of PNRSV and PDV using 175 almond leaf samples. Multiplex RT-PCR was found to be more sensitive than ELISA, especially when followed by nested PCR for the detection of PDV. The RT-PCR technique has the added advantage that plant material can be tested at any time throughout the growing season.