Alternative Splicing Underpins the ALMT9 Transporter Function for Vacuolar Malic Acid Accumulation in Apple.

Vacuolar malic acid accumulation largely determines fruit acidity, a key trait for the taste and flavor of apple and other fleshy fruits. Aluminum-activated malate transporter 9 (ALMT9/Ma1) underlies a major genetic locus, Ma, for fruit acidity in apple, but how the protein transports malate across the tonoplast is unclear. Here, it is shown that overexpression of the coding sequence of Ma1 (Ma1α) drastically decreases fruit acidity in "Royal Gala" apple, leading to uncovering alternative splicing underpins Ma1's function. Alternative splicing generates two isoforms: Ma1ß is 68 amino acids shorter with much lower expression than the full-length protein Ma1α. Ma1ß does not transport malate itself but interacts with the functional Ma1α to form heterodimers, creating synergy with Ma1α for malate transport in a threshold manner (When Ma1ß/Ma1α ≥ 1/8). Overexpression of Ma1α triggers feedback inhibition on the native Ma1 expression via transcription factor MYB73, decreasing the Ma1ß level well below the threshold that leads to significant reductions in Ma1 function and malic acid accumulation in fruit. Overexpression of Ma1α and Ma1ß or genomic Ma1 increases both isoforms proportionally and enhances fruit malic acid accumulation. These findings reveal an essential role of alternative splicing in ALMT9-mediated malate transport underlying apple fruit acidity.

Exploring the Interplay between Polyphenols and Lysyl Oxidase Enzymes for Maintaining Extracellular Matrix Homeostasis.

Collagen, the most abundant structural protein found in mammals, plays a vital role as a constituent of the extracellular matrix (ECM) that surrounds cells. Collagen fibrils are strengthened through the formation of covalent cross-links, which involve complex enzymatic and non-enzymatic reactions. Lysyl oxidase (LOX) is responsible for catalyzing the oxidative deamination of lysine and hydroxylysine residues, resulting in the production of aldehydes, allysine, and hydroxyallysine. These intermediates undergo spontaneous condensation reactions, leading to the formation of immature cross-links, which are the initial step in the development of mature covalent cross-links. Additionally, non-enzymatic glycation contributes to the formation of abnormal cross-linking in collagen fibrils. During glycation, specific lysine and arginine residues in collagen are modified by reducing sugars, leading to the creation of Advanced Glycation End-products (AGEs). These AGEs have been associated with changes in the mechanical properties of collagen fibers. Interestingly, various studies have reported that plant polyphenols possess amine oxidase-like activity and can act as potent inhibitors of protein glycation. This review article focuses on compiling the literature describing polyphenols with amine oxidase-like activity and antiglycation properties. Specifically, we explore the molecular mechanisms by which specific flavonoids impact or protect the normal collagen cross-linking process. Furthermore, we discuss how these dual activities can be harnessed to generate properly cross-linked collagen molecules, thereby promoting the stabilization of highly organized collagen fibrils.

The Surprising Dynamics of Electrochemical Coupling at Membrane Sandwiches in Plants.

Transport processes across membranes play central roles in any biological system. They are essential for homeostasis, cell nutrition, and signaling. Fluxes across membranes are governed by fundamental thermodynamic rules and are influenced by electrical potentials and concentration gradients. Transmembrane transport processes have been largely studied on single membranes. However, several important cellular or subcellular structures consist of two closely spaced membranes that form a membrane sandwich. Such a dual membrane structure results in remarkable properties for the transport processes that are not present in isolated membranes. At the core of membrane sandwich properties, a small intermembrane volume is responsible for efficient coupling between the transport systems at the two otherwise independent membranes. Here, we present the physicochemical principles of transport coupling at two adjacent membranes and illustrate this concept with three examples. In the supplementary material, we provide animated PowerPoint presentations that visualize the relationships. They could be used for teaching purposes, as has already been completed successfully at the University of Talca.

Transporter networks can serve plant cells as nutrient sensors and mimic transceptor-like behavior.

Sensing of external mineral nutrient concentrations is essential for plants to colonize environments with a large spectrum of nutrient availability. Here, we analyzed transporter networks in computational cell biology simulations to understand better the initial steps of this sensing process. The networks analyzed were capable of translating the information of changing external nutrient concentrations into cytosolic H+ and Ca2+ signals, two of the most ubiquitous cellular second messengers. The concept emerging from the computational simulations was confirmed in wet-lab experiments. We document in guard cells that alterations in the external KCl concentration were translated into cytosolic H+ and Ca2+ transients as predicted. We show that transporter networks do not only serve their primary task of transport, but can also take on the role of a receptor without requiring conformational changes of a transporter protein. Such transceptor-like phenomena may be quite common in plants.

Plant HKT Channels: An Updated View on Structure, Function and Gene Regulation.

HKT channels are a plant protein family involved in sodium (Na+) and potassium (K+) uptake and Na+-K+ homeostasis. Some HKTs underlie salt tolerance responses in plants, while others provide a mechanism to cope with short-term K+ shortage by allowing increased Na+ uptake under K+ starvation conditions. HKT channels present a functionally versatile family divided into two classes, mainly based on a sequence polymorphism found in the sequences underlying the selectivity filter of the first pore loop. Physiologically, most class I members function as sodium uniporters, and class II members as Na+/K+ symporters. Nevertheless, even within these two classes, there is a high functional diversity that, to date, cannot be explained at the molecular level. The high complexity is also reflected at the regulatory level. HKT expression is modulated at the level of transcription, translation, and functionality of the protein. Here, we summarize and discuss the structure and conservation of the HKT channel family from algae to angiosperms. We also outline the latest findings on gene expression and the regulation of HKT channels.

An extracellular cation coordination site influences ion conduction of OsHKT2;2.

BACKGROUND: HKT channels mediate sodium uniport or sodium and potassium symport in plants. Monocotyledons express a higher number of HKT proteins than dicotyledons, and it is only within this clade of HKT channels that cation symport mechanisms are found. The prevailing ion composition in the extracellular medium affects the transport abilities of various HKT channels by changing their selectivity or ion transport rates. How this mutual effect is achieved at the molecular level is still unknown. Here, we built a homology model of the monocotyledonous OsHKT2;2, which shows sodium and potassium symport activity. We performed molecular dynamics simulations in the presence of sodium and potassium ions to investigate the mutual effect of cation species. RESULTS: By analyzing ion-protein interactions, we identified a cation coordination site on the extracellular protein surface, which is formed by residues P71, D75, D501 and K504. Proline and the two aspartate residues coordinate cations, while K504 forms salt bridges with D75 and D501 and may be involved in the forwarding of cations towards the pore entrance. Functional validation via electrophysiological experiments confirmed the biological relevance of the predicted ion coordination site and identified K504 as a central key residue. Mutation of the cation coordinating residues affected the functionality of HKT only slightly. Additional in silico mutants and simulations of K504 supported experimental results. CONCLUSION: We identified an extracellular cation coordination site, which is involved in ion coordination and influences the conduction of OsHKT2;2. This finding proposes a new viewpoint in the discussion of how the mutual effect of variable ion species may be achieved in HKT channels.

Yeast strain Saccharomyces cerevisiae BYT45 lacking the cation extrusion systems ENA1-5 and NHA1 is suitable for the characterization of TASK-3 potassium channel antagonists.

Finding new potential antagonists of potassium channels is a continuing task. TASK potassium channels operate over a large physiological range of membrane voltages, why they are thought to contribute to the excitability and resting potential of mammalian membrane potentials. Additionally, they are regulated by extracellular stimuli like changes in pH and K+ concentrations. TASK malfunctions are associated with diseases, which makes them popular targets for the search of new antagonists. Identification of channel inhibitors can be a time-consuming and expensive project. Here, we present an easy-to-use and inexpensive yeast system for the expression of the two-pore domain K+ channel TASK-3, and for the characterization of TASK-3 antagonists. The Saccharomyces cerevisiae strain BYT45 was used to express guinea pig TASK-3. The system allowed the expression and characterization of TASK-3 at variable pH values and K+ concentrations. Three known TASK-3 antagonists have been tested in the BYT45 yeast system: PK-THPP, ZnCl2 and Bupivacaine. Their inhibitory effect on TASK-3 was tested in solid and liquid media assays, and half maximal inhibitory concentrations were estimated. Although the system is less sensitive than more refined systems, the antagonistic activity could be confirmed for all three inhibitors.

Nutrient exchange in arbuscular mycorrhizal symbiosis from a thermodynamic point of view.

To obtain insights into the dynamics of nutrient exchange in arbuscular mycorrhizal (AM) symbiosis, we modelled mathematically the two-membrane system at the plant-fungus interface and simulated its dynamics. In computational cell biology experiments, the full range of nutrient transport pathways was tested for their ability to exchange phosphorus (P)/carbon (C)/nitrogen (N) sources. As a result, we obtained a thermodynamically justified, independent and comprehensive model of the dynamics of the nutrient exchange at the plant-fungus contact zone. The predicted optimal transporter network coincides with the transporter set independently confirmed in wet-laboratory experiments previously, indicating that all essential transporter types have been discovered. The thermodynamic analyses suggest that phosphate is released from the fungus via proton-coupled phosphate transporters rather than anion channels. Optimal transport pathways, such as cation channels or proton-coupled symporters, shuttle nutrients together with a positive charge across the membranes. Only in exceptional cases does electroneutral transport via diffusion facilitators appear to be plausible. The thermodynamic models presented here can be generalized and adapted to other forms of mycorrhiza and open the door for future studies combining wet-laboratory experiments with computational simulations to obtain a deeper understanding of the investigated phenomena.

The potassium battery: a mobile energy source for transport processes in plant vascular tissues.

Contents 1049 I. 1049 II. 1050 III. 1050 IV. 1050 V. 1051 VI. 1051 VII. 1052 VIII. 1052 1053 References 1053 SUMMARY: Plant roots absorb potassium ions from the soil and transport them in the xylem via the transpiration stream to the shoots. There, in source tissues where sufficient chemical energy (ATP) is available, K+ is loaded into the phloem and then transported with the phloem stream to other parts of the plant; in part, transport is also back to the roots. This, at first sight, futile cycling of K+ has been uncovered to be part of a sophisticated mechanism that (1) enables the shoot to communicate its nutrient demand to the root, (2) contributes to the K+ nutrition of transport phloem tissues and (3) transports energy stored in the K+ gradient between phloem cytosol and the apoplast. This potassium battery can be tapped by opening AKT2-like potassium channels and then enables the ATP-independent energization of other transport processes, such as the reloading of sucrose. Insights into these mechanisms have only been possible by combining wet-lab and dry-lab experiments by means of computational cell biology modeling and simulations.

The receptor-like pseudokinase MRH1 interacts with the voltage-gated potassium channel AKT2.

The potassium channel AKT2 plays important roles in phloem loading and unloading. It can operate as inward-rectifying channel that allows H+-ATPase-energized K+ uptake. Moreover, through reversible post-translational modifications it can also function as an open, K+-selective channel, which taps a 'potassium battery', providing additional energy for transmembrane transport processes. Knowledge about proteins involved in the regulation of the operational mode of AKT2 is very limited. Here, we employed a large-scale yeast two-hybrid screen in combination with fluorescence tagging and null-allele mutant phenotype analysis and identified the plasma membrane localized receptor-like kinase MRH1/MDIS2 (AT4G18640) as interaction partner of AKT2. The phenotype of the mrh1-1 knockout plant mirrors that of akt2 knockout plants in energy limiting conditions. Electrophysiological analyses showed that MRH1/MDIS2 failed to exert any functional regulation on AKT2. Using structural protein modeling approaches, we instead gathered evidence that the putative kinase domain of MRH1/MDIS2 lacks essential sites that are indispensable for a functional kinase suggesting that MRH1/MDIS2 is a pseudokinase. We propose that MRH1/MDIS2 and AKT2 are likely parts of a bigger protein complex. MRH1 might help to recruit other, so far unknown partners, which post-translationally regulate AKT2. Additionally, MRH1 might be involved in the recognition of chemical signals.

Outward Rectification of Voltage-Gated K+ Channels Evolved at Least Twice in Life History.

Voltage-gated potassium (K+) channels are present in all living systems. Despite high structural similarities in the transmembrane domains (TMD), this K+ channel type segregates into at least two main functional categories-hyperpolarization-activated, inward-rectifying (Kin) and depolarization-activated, outward-rectifying (Kout) channels. Voltage-gated K+ channels sense the membrane voltage via a voltage-sensing domain that is connected to the conduction pathway of the channel. It has been shown that the voltage-sensing mechanism is the same in Kin and Kout channels, but its performance results in opposite pore conformations. It is not known how the different coupling of voltage-sensor and pore is implemented. Here, we studied sequence and structural data of voltage-gated K+ channels from animals and plants with emphasis on the property of opposite rectification. We identified structural hotspots that alone allow already the distinction between Kin and Kout channels. Among them is a loop between TMD S5 and the pore that is very short in animal Kout, longer in plant and animal Kin and the longest in plant Kout channels. In combination with further structural and phylogenetic analyses this finding suggests that outward-rectification evolved twice and independently in the animal and plant kingdom.

K2p channels in plants and animals.

Two-pore domain potassium (K2P) channels are membrane proteins widely identified in mammals, plants, and other organisms. A functional channel is a dimer with each subunit comprising two pore-forming loops and four transmembrane domains. The genome of the model plant Arabidopsis thaliana harbors five genes coding for K2P channels. Homologs of Arabidopsis K2P channels have been found in all higher plants sequenced so far. As with the K2P channels in mammals, plant K2P channels are targets of external and internal stimuli, which fine-tune the electrical properties of the membrane for specialized transport and/or signaling tasks. Plant K2P channels are modulated by signaling molecules such as intracellular H(+) and calcium and physical factors like temperature and pressure. In this review, we ask the following: What are the similarities and differences between K2P channels in plants and animals in terms of their physiology? What is the nature of the last common ancestor (LCA) of these two groups of proteins? To answer these questions, we present physiological, structural, and phylogenetic evidence that discards the hypothesis proposing that the duplication and fusion that gave rise to the K2P channels occurred in a prokaryote LCA. Conversely, we argue that the K2P LCA was most likely a eukaryote organism. Consideration of plant and animal K2P channels in the same study is novel and likely to stimulate further exchange of ideas between students of these fields.

Voltage-sensor transitions of the inward-rectifying K+ channel KAT1 indicate a latching mechanism biased by hydration within the voltage sensor.

The Kv-like (potassium voltage-dependent) K(+) channels at the plasma membrane, including the inward-rectifying KAT1 K(+) channel of Arabidopsis (Arabidopsis thaliana), are important targets for manipulating K(+) homeostasis in plants. Gating modification, especially, has been identified as a promising means by which to engineer plants with improved characteristics in mineral and water use. Understanding plant K(+) channel gating poses several challenges, despite many similarities to that of mammalian Kv and Shaker channel models. We have used site-directed mutagenesis to explore residues that are thought to form two electrostatic countercharge centers on either side of a conserved phenylalanine (Phe) residue within the S2 and S3 α-helices of the voltage sensor domain (VSD) of Kv channels. Consistent with molecular dynamic simulations of KAT1, we show that the voltage dependence of the channel gate is highly sensitive to manipulations affecting these residues. Mutations of the central Phe residue favored the closed KAT1 channel, whereas mutations affecting the countercharge centers favored the open channel. Modeling of the macroscopic current kinetics also highlighted a substantial difference between the two sets of mutations. We interpret these findings in the context of the effects on hydration of amino acid residues within the VSD and with an inherent bias of the VSD, when hydrated around a central Phe residue, to the closed state of the channel.

The role of K(+) channels in uptake and redistribution of potassium in the model plant Arabidopsis thaliana.

Potassium (K(+)) is inevitable for plant growth and development. It plays a crucial role in the regulation of enzyme activities, in adjusting the electrical membrane potential and the cellular turgor, in regulating cellular homeostasis and in the stabilization of protein synthesis. Uptake of K(+) from the soil and its transport to growing organs is essential for a healthy plant development. Uptake and allocation of K(+) are performed by K(+) channels and transporters belonging to different protein families. In this review we summarize the knowledge on the versatile physiological roles of plant K(+) channels and their behavior under stress conditions in the model plant Arabidopsis thaliana.

The pH sensor of the plant K+-uptake channel KAT1 is built from a sensory cloud rather than from single key amino acids.

The uptake of potassium ions (K+) accompanied by an acidification of the apoplasm is a prerequisite for stomatal opening. The acidification (approximately 2-2.5 pH units) is perceived by voltage-gated inward potassium channels (K(in)) that then can open their pores with lower energy cost. The sensory units for extracellular pH in stomatal K(in) channels are proposed to be histidines exposed to the apoplasm. However, in the Arabidopsis thaliana stomatal K(in) channel KAT1, mutations in the unique histidine exposed to the solvent (His267) do not affect the pH dependency. We demonstrate in the present study that His267 of the KAT1 channel cannot sense pH changes since the neighbouring residue Phe266 shifts its pKa to undetectable values through a cation-π interaction. Instead, we show that Glu240 placed in the extracellular loop between transmembrane segments S5 and S6 is involved in the extracellular acid activation mechanism. Based on structural models we propose that this region may serve as a molecular link between the pH- and the voltage-sensor. Like Glu240, several other titratable residues could contribute to the pH-sensor of KAT1, interact with each other and even connect such residues far away from the voltage-sensor with the gating machinery of the channel.

A minimal cysteine motif required to activate the SKOR K+ channel of Arabidopsis by the reactive oxygen species H2O2.

Reactive oxygen species (ROS) are essential for development and stress signaling in plants. They contribute to plant defense against pathogens, regulate stomatal transpiration, and influence nutrient uptake and partitioning. Although both Ca(2+) and K(+) channels of plants are known to be affected, virtually nothing is known of the targets for ROS at a molecular level. Here we report that a single cysteine (Cys) residue within the Kv-like SKOR K(+) channel of Arabidopsis thaliana is essential for channel sensitivity to the ROS H(2)O(2). We show that H(2)O(2) rapidly enhanced current amplitude and activation kinetics of heterologously expressed SKOR, and the effects were reversed by the reducing agent dithiothreitol (DTT). Both H(2)O(2) and DTT were active at the outer face of the membrane and current enhancement was strongly dependent on membrane depolarization, consistent with a H(2)O(2)-sensitive site on the SKOR protein that is exposed to the outside when the channel is in the open conformation. Cys substitutions identified a single residue, Cys(168) located within the S3 α-helix of the voltage sensor complex, to be essential for sensitivity to H(2)O(2). The same Cys residue was a primary determinant for current block by covalent Cys S-methioylation with aqueous methanethiosulfonates. These, and additional data identify Cys(168) as a critical target for H(2)O(2), and implicate ROS-mediated control of the K(+) channel in regulating mineral nutrient partitioning within the plant.

Distributed structures underlie gating differences between the kin channel KAT1 and the Kout channel SKOR.

The family of voltage-gated (Shaker-like) potassium channels in plants includes both inward-rectifying (K(in)) channels that allow plant cells to accumulate K(+) and outward-rectifying (K(out)) channels that mediate K(+) efflux. Despite their close structural similarities, K(in) and K(out) channels differ in their gating sensitivity towards voltage and the extracellular K(+) concentration. We have carried out a systematic program of domain swapping between the K(out) channel SKOR and the K(in) channel KAT1 to examine the impacts on gating of the pore regions, the S4, S5, and the S6 helices. We found that, in particular, the N-terminal part of the S5 played a critical role in KAT1 and SKOR gating. Our findings were supported by molecular dynamics of KAT1 and SKOR homology models. In silico analysis revealed that during channel opening and closing, displacement of certain residues, especially in the S5 and S6 segments, is more pronounced in KAT1 than in SKOR. From our analysis of the S4-S6 region, we conclude that gating (and K(+)-sensing in SKOR) depend on a number of structural elements that are dispersed over this approximately 145-residue sequence and that these place additional constraints on configurational rearrangement of the channels during gating.