Dissecting the causal polymorphism of the Lr67res multipathogen resistance gene.

Partial resistance to multiple biotrophic fungal pathogens in wheat (Triticum aestivum L.) is conferred by a variant of the Lr67 gene, which encodes a hexose-proton symporter. Two mutations (G144R, V387L) differentiate the resistant and susceptible protein variants (Lr67res and Lr67sus). Lr67res lacks sugar transport capability and was associated with anion transporter-like properties when expressed in Xenopus laevis oocytes. Here, we extended this functional characterization to include yeast and in planta studies. The Lr67res allele, but not Lr67sus, induced sensitivity to ions in yeast (including NaCl, LiCl, KI), which is consistent with our previous observations that Lr67res expression in oocytes induces novel ion fluxes. We demonstrate that another naturally occurring single amino acid variant in wheat, containing only the Lr67G144R mutation, confers rust resistance. Transgenic barley plants expressing the orthologous HvSTP13 gene carrying the G144R and V387L mutations were also more resistant to Puccinia hordei infection. NaCl treatment of pot-grown adult wheat plants with the Lr67res allele induced leaf tip necrosis and partial leaf rust resistance. An Lr67res-like function can be introduced into orthologous plant hexose transporters via single amino acid mutation, highlighting the strong possibility of generating disease resistance in other crops, especially with gene editing.

Barley Nodulin 26-like intrinsic protein permeates water, metalloids, saccharides, and ion pairs due to structural plasticity and diversification.

Aquaporins can facilitate the passive movement of water, small polar molecules, and some ions. Here, we examined solute selectivity for the barley Nodulin 26-like Intrinsic Protein (HvNIP2;1) embedded in liposomes and examined through stopped-flow light scattering spectrophotometry and Xenopus laevis oocyte swelling assays. We found that HvNIP2;1 permeates water, boric and germanic acids, sucrose, and lactose but not d-glucose or d-fructose. Other saccharides, such as neutral (d-mannose, d-galactose, d-xylose, d-mannoheptaose) and charged (N-acetyl d-glucosamine, d-glucosamine, d-glucuronic acid) aldoses, disaccharides (cellobiose, gentiobiose, trehalose), trisaccharide raffinose, and urea, glycerol, and acyclic polyols, were permeated to a much lower extent. We observed apparent permeation of hydrated KCl and MgSO4 ions, while CH3COONa and NaNO3 permeated at significantly lower rates. Our experiments with boric acid and sucrose revealed no apparent interaction between solutes when permeated together, and AgNO3 or H[AuCl4] blocked the permeation of all solutes. Docking of sucrose in HvNIP2;1 and spinach water-selective SoPIP2;1 aquaporins revealed the structural basis for sucrose permeation in HvNIP2;1 but not in SoPIP2;1, and defined key residues interacting with this permeant. In a biological context, sucrose transport could constitute a novel element of plant saccharide-transporting machinery. Phylogenomic analyses of 164 Viridiplantae and 2993 Archaean, bacterial, fungal, and Metazoan aquaporins rationalized solute poly-selectivity in NIP3 sub-clade entries and suggested that they diversified from other sub-clades to acquire a unique specificity of saccharide transporters. Solute specificity definition in NIP aquaporins could inspire developing plants for food production.

Arabidopsis plasma membrane intrinsic protein (AtPIP2;1) is implicated in a salinity conditional influence on seed germination.

Dynamic changes in aquaporin gene expression occur during seed germination. One example is the ~30-fold increase in Arabidopsis thaliana PIP2;1 transcripts within 24h of seed imbibition. To investigate whether AtPIP2;1 can influence seed germination wild-type Columbia-0, single (Atpip2;1 ) and double (Atpip2;1-Atpip2;2 ) loss-of-function mutants, along with transgenic 2x35S::AtPIP2;1 over-expressing (OE) lines and null-segregant controls, were examined. The various genotypes were germinated in control and saline (75mM NaCl treatment) conditions and tested for germination efficiency, imbibed seed maximum cross sectional (MCS) area, imbibed seed mass, and seed Na+ and K+ content. Seed lacking functional AtPIP2;1 and/or AtPIP2;2 proteins or constitutively over-expressing AtPIP2;1 , had delayed germination in saline conditions relative to wild-type and null-segregant seed, respectively. Exposure to saline germination conditions resulted in Atpip2;1 mutants having greater imbibed seed mass and less accumulated Na+ than wild-type, whereas lines over-expressing AtPIP2;1 had reduced imbibed seed mass and greater seed K+ content than null-segregant control seed. The results imply a role for AtPIP2;1 in seed germination processes, whether directly through its capacity for water and ion transport or H2 O2 signalling, or indirectly through potentially triggering dynamic differential regulation of other aquaporins expressed during germination. Future research will aid in dissecting the aquaporin functions influencing germination and may lead to novel solutions for optimising germination in sub-optimal conditions, such as saline soils.

A Metagenomic Investigation of the Viruses Associated with Shiraz Disease in Australia.

Shiraz disease (SD) is an economically important virus-associated disease that can significantly reduce yield in sensitive grapevine varieties and has so far only been reported in South Africa and Australia. In this study, RT-PCR and metagenomic high-throughput sequencing was used to study the virome of symptomatic and asymptomatic grapevines within vineyards affected by SD and located in South Australia. Results showed that grapevine virus A (GVA) phylogroup II variants were strongly associated with SD symptoms in Shiraz grapevines that also had mixed infections of viruses including combinations of grapevine leafroll-associated virus 3 (GLRaV-3) and grapevine leafroll-associated virus 4 strains 5, 6 and 9 (GLRaV-4/5, GLRaV-4/6, GLRaV-4/9). GVA phylogroup III variants, on the other hand, were present in both symptomatic and asymptomatic grapevines, suggesting no or decreased virulence of these strains. Similarly, only GVA phylogroup I variants were found in heritage Shiraz grapevines affected by mild leafroll disease, along with GLRaV-1, suggesting this phylogroup may not be associated with SD.

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.

Expression of the wheat multipathogen resistance hexose transporter Lr67res is associated with anion fluxes.

Many disease resistance genes in wheat (Triticum aestivum L.) confer strong resistance to specific pathogen races or strains, and only a small number of genes confer multipathogen resistance. The Leaf rust resistance 67 (Lr67) gene fits into the latter category as it confers partial resistance to multiple biotrophic fungal pathogens in wheat and encodes a Sugar Transport Protein 13 (STP13) family hexose-proton symporter variant. Two mutations (G144R, V387L) in the resistant variant, Lr67res, differentiate it from the susceptible Lr67sus variant. The molecular function of the Lr67res protein is not understood, and this study aimed to broaden our knowledge on this topic. Biophysical analysis of the wheat Lr67sus and Lr67res protein variants was performed using Xenopus laevis oocytes as a heterologous expression system. Oocytes injected with Lr67sus displayed properties typically associated with proton-coupled sugar transport proteins-glucose-dependent inward currents, a Km of 110 ± 10 µM glucose, and a substrate selectivity permitting the transport of pentoses and hexoses. By contrast, Lr67res induced much larger sugar-independent inward currents in oocytes, implicating an alternative function. Since Lr67res is a mutated hexose-proton symporter, the possibility of protons underlying these currents was investigated but rejected. Instead, currents in Lr67res oocytes appeared to be dominated by anions. This conclusion was supported by electrophysiology and 36Cl- uptake studies and the similarities with oocytes expressing the known chloride channel from Torpedo marmorata, TmClC-0. This study provides insights into the function of an important disease resistance gene in wheat, which can be used to determine how this gene variant underpins disease resistance in planta.

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.

Characterisation of internal oxygen concentration of strawberry (Fragaria × ananassa) and blueberry (Vaccinium corymbosum).

Gas exchange mechanisms play crucial roles in maintaining fruit post-harvest quality in perishable fruit such as strawberry (Fragaria×ananassa Duch.) and blueberry (Vaccinium corymbosum L.). The internal oxygen concentration ([O2 ]) of strawberry and blueberry were measured using Clark-type oxygen sensing electrodes. The volume of intercellular voids in strawberry was obtained by micro-computed tomography (micro-CT). In both berries, internal [O2 ] was consistent and relatively high across measured tissues. The overall [O2 ] was well above the Michaelis constant (K m ) for cytochrome c oxidase in both fruit and different from previously examined grape (Vitis vinifera L.) berry mesocarp with near zero minimum [O2 ]. In strawberry and blueberry, cell vitality was also maintained at full maturity in the mesocarp. Higher storage temperature (i.e. 20 vs 4°C) reduced internal [O2 ] of strawberry. Pedicel detachment in blueberry was associated with greater fruit dehydration and lower internal [O2 ] after short-term storage of 12h. The results suggest that the intercellular voids of the fruit's mesocarp provide an efficient gas exchange route for maintaining high fruit internal [O2 ] post-harvest.

Enhancing crop yields through improvements in the efficiency of photosynthesis and respiration.

The rate with which crop yields per hectare increase each year is plateauing at the same time that human population growth and other factors increase food demand. Increasing yield potential ( Y p ) of crops is vital to address these challenges. In this review, we explore a component of Y p that has yet to be optimised - that being improvements in the efficiency with which light energy is converted into biomass ( ε c ) via modifications to CO2 fixed per unit quantum of light (α), efficiency of respiratory ATP production ( ε prod ) and efficiency of ATP use ( ε use ). For α, targets include changes in photoprotective machinery, ribulose bisphosphate carboxylase/oxygenase kinetics and photorespiratory pathways. There is also potential for ε prod to be increased via targeted changes to the expression of the alternative oxidase and mitochondrial uncoupling pathways. Similarly, there are possibilities to improve ε use via changes to the ATP costs of phloem loading, nutrient uptake, futile cycles and/or protein/membrane turnover. Recently developed high-throughput measurements of respiration can serve as a proxy for the cumulative energy cost of these processes. There are thus exciting opportunities to use our growing knowledge of factors influencing the efficiency of photosynthesis and respiration to create a step-change in yield potential of globally important crops.

Burning questions for a warming and changing world: 15 unknowns in plant abiotic stress.

We present unresolved questions in plant abiotic stress biology as posed by 15 research groups with expertise spanning eco-physiology to cell and molecular biology. Common themes of these questions include the need to better understand how plants detect water availability, temperature, salinity, and rising carbon dioxide (CO2) levels; how environmental signals interface with endogenous signaling and development (e.g. circadian clock and flowering time); and how this integrated signaling controls downstream responses (e.g. stomatal regulation, proline metabolism, and growth versus defense balance). The plasma membrane comes up frequently as a site of key signaling and transport events (e.g. mechanosensing and lipid-derived signaling, aquaporins). Adaptation to water extremes and rising CO2 affects hydraulic architecture and transpiration, as well as root and shoot growth and morphology, in ways not fully understood. Environmental adaptation involves tradeoffs that limit ecological distribution and crop resilience in the face of changing and increasingly unpredictable environments. Exploration of plant diversity within and among species can help us know which of these tradeoffs represent fundamental limits and which ones can be circumvented by bringing new trait combinations together. Better defining what constitutes beneficial stress resistance in different contexts and making connections between genes and phenotypes, and between laboratory and field observations, are overarching challenges.

Genetic Diversity of Grapevine Virus A in Three Australian Vineyards Using Amplicon High Throughput Sequencing (Amplicon-HTS).

Shiraz disease (SD) is one of the most destructive viral diseases of grapevines in Australia and is known to cause significant economic loss to local growers. Grapevine virus A (GVA) was reported to be the key pathogen associated with this disease. This study aimed to better understand the diversity of GVA variants both within and between individual SD and grapevine leafroll disease (LRD) affected grapevines located at vineyards in South Australia. Amplicon high throughput sequencing (Amplicon-HTS) combined with median-joining networks (MJNs) was used to analyze the variability in specific gene regions of GVA variants. Several GVAII variant groups contain samples from both vineyards studied, suggesting that these GVAII variants were from a common origin. Variant groups analyzed by MJNs using the overall data set denote that there may be a possible relationship between variant groups of GVA and the geographical location of the grapevines.

Stress-Induced Volatile Emissions and Signalling in Inter-Plant Communication.

The sessile plant has developed mechanisms to survive the "rough and tumble" of its natural surroundings, aided by its evolved innate immune system. Precise perception and rapid response to stress stimuli confer a fitness edge to the plant against its competitors, guaranteeing greater chances of survival and productivity. Plants can "eavesdrop" on volatile chemical cues from their stressed neighbours and have adapted to use these airborne signals to prepare for impending danger without having to experience the actual stress themselves. The role of volatile organic compounds (VOCs) in plant-plant communication has gained significant attention over the past decade, particularly with regard to the potential of VOCs to prime non-stressed plants for more robust defence responses to future stress challenges. The ecological relevance of such interactions under various environmental stresses has been much debated, and there is a nascent understanding of the mechanisms involved. This review discusses the significance of VOC-mediated inter-plant interactions under both biotic and abiotic stresses and highlights the potential to manipulate outcomes in agricultural systems for sustainable crop protection via enhanced defence. The need to integrate physiological, biochemical, and molecular approaches in understanding the underlying mechanisms and signalling pathways involved in volatile signalling is emphasised.

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.

Oxygen uptake rates have contrasting responses to temperature in the root meristem and elongation zone.

Growing at either 15 or 25°C, roots of Arabidopsis thaliana, Columbia accession, produce cells at the same rate and have growth zones of the same length. To determine whether this constancy is related to energetics, we measured oxygen uptake by means of a vibrating oxygen-selective electrode. Concomitantly, the spatial distribution of elongation was measured kinematically, delineating meristem and elongation zone. All seedlings were germinated, grown, and measured at a given temperature (15 or 25°C). Columbia was compared to lines where cell production rate roughly doubles between 15 and 25°C: Landsberg and two Columbia mutants, er-105 and ahk3-3. For all genotypes and temperatures, oxygen uptake rate at any position was highest at the root cap, where mitochondrial density was maximal, based on the fluorescence of a reporter. Uptake rate declined through the meristem to plateau within the elongation zone. For oxygen uptake rate integrated over a zone, the meristem had steady-state Q10 values ranging from 0.7 to 2.1; by contrast, the elongation zone had values ranging from 2.6 to 3.3, implying that this zone exerts a greater respiratory demand. These results highlight a substantial energy consumption by the root cap, perhaps helpful for maintaining hypoxia in stem cells, and suggest that rapid elongation is metabolically more costly than is cell division.

Vascular Connections Into the Grape Berry: The Link of Structural Investment to Seededness.

Vascular bundles in the grape pedicel and berry contain the conduits, phloem and xylem, for transport of water, sugar, nutrients and signals into and through the grape berry and play a critical role in berry growth and composition. Here, we assess the vascular anatomy within the proximal region of the berry. Guided using a 3D berry model generated by micro-CT, differential staining of transverse sections of berries and receptacles was followed by fluorescent microscopy. Morphometric and vascular characteristics were analyzed within the central proximal region (brush zone, a fibrous extension from the pedicel vascular system into the berry) of the seeded cultivars Shiraz and Sauvignon Blanc, as well as the stenospermocarpic cultivars Ruby Seedless and Flame Seedless. Observations revealed a change in vascular arrangement from the receptacle into the berry brush zone and differences in xylem element size as well as xylem and phloem area relationships. Xylem anatomical and derived hydraulic parameters, as well as total tissue area of xylem and phloem varied between cultivars and in receptacle and berry components. Variation in vascular growth between grape pedicels and berries was independent of seededness. Differences in receptacle xylem vessel size and distribution could contribute to cultivar-dependent xylem backflow constraint.

An algal PIP-like aquaporin facilitates water transport and ionic conductance.

Aquaporins are water and solute channel proteins found throughout the kingdoms of life. Ion-conducting aquaporins (icAQPs) have been identified in both plants and animals indicating that this function may be conserved through evolution. In higher plants icAQP function has been demonstrated for isoforms from two of five aquaporin subfamilies indicating that this function could have existed before the divergence of higher plants from green algae. Here a PIP-like aquaporin from the charophytic alga Klebsormidium nitens was functionally characterised in Xenopus laevis oocytes and its expression was found to induce water and ion conductance.

Adaptable and Multifunctional Ion-Conducting Aquaporins.

Aquaporins function as water and neutral solute channels, signaling hubs, disease virulence factors, and metabolon components. We consider plant aquaporins that transport ions compared to some animal counterparts. These are candidates for important, as yet unidentified, cation and anion channels in plasma, tonoplast, and symbiotic membranes. For those individual isoforms that transport ions, water, and gases, the permeability spans 12 orders of magnitude. This requires tight regulation of selectivity via protein interactions and posttranslational modifications. A phosphorylation-dependent switch between ion and water permeation in AtPIP2;1 might be explained by coupling between the gates of the four monomer water channels and the central pore of the tetramer. We consider the potential for coupling between ion and water fluxes that could form the basis of an electroosmotic transducer. A grand challenge in understanding the roles of ion transporting aquaporins is their multifunctional modes that are dependent on location, stress, time, and development.

A Survey of Barley PIP Aquaporin Ionic Conductance Reveals Ca2+-Sensitive HvPIP2;8 Na+ and K+ Conductance.

Some plasma membrane intrinsic protein (PIP) aquaporins can facilitate ion transport. Here we report that one of the 12 barley PIPs (PIP1 and PIP2) tested, HvPIP2;8, facilitated cation transport when expressed in Xenopus laevis oocytes. HvPIP2;8-associated ion currents were detected with Na+ and K+, but not Cs+, Rb+, or Li+, and was inhibited by Ba2+, Ca2+, and Cd2+ and to a lesser extent Mg2+, which also interacted with Ca2+. Currents were reduced in the presence of K+, Cs+, Rb+, or Li+ relative to Na+ alone. Five HvPIP1 isoforms co-expressed with HvPIP2;8 inhibited the ion conductance relative to HvPIP2;8 alone but HvPIP1;3 and HvPIP1;4 with HvPIP2;8 maintained the ion conductance at a lower level. HvPIP2;8 water permeability was similar to that of a C-terminal phosphorylation mimic mutant HvPIP2;8 S285D, but HvPIP2;8 S285D showed a negative linear correlation between water permeability and ion conductance that was modified by a kinase inhibitor treatment. HvPIP2;8 transcript abundance increased in barley shoot tissues following salt treatments in a salt-tolerant cultivar Haruna-Nijo, but not in salt-sensitive I743. There is potential for HvPIP2;8 to be involved in barley salt-stress responses, and HvPIP2;8 could facilitate both water and Na+/K+ transport activity, depending on the phosphorylation status.

Plant transporters involved in combating boron toxicity: beyond 3D structures.

Membrane transporters control the movement and distribution of solutes, including the disposal or compartmentation of toxic substances that accumulate in plants under adverse environmental conditions. In this minireview, in the light of the approaching 100th anniversary of unveiling the significance of boron to plants (K. Warington, 1923; Ann. Bot.37, 629) we discuss the current state of the knowledge on boron transport systems that plants utilise to combat boron toxicity. These transport proteins include: (i) nodulin-26-like intrinsic protein-types of aquaporins, and (ii) anionic efflux (borate) solute carriers. We describe the recent progress made on the structure-function relationships of these transport proteins and point out that this progress is integral to quantitative considerations of the transporter's roles in tissue boron homeostasis. Newly acquired knowledge at the molecular level has informed on the transport mechanics and conformational states of boron transport systems that can explain their impact on cell biology and whole plant physiology. We expect that this information will form the basis for engineering transporters with optimised features to alleviate boron toxicity tolerance in plants exposed to suboptimal soil conditions for sustained food production.