Reduced apoplastic barriers in tissues of shoot-proximal rhizomes of Oryza coarctata are associated with Na+ sequestration.

Oryza coarctata is the only wild rice species with significant salinity tolerance. The present work examines the role of the substantial rhizomatous tissues of O. coarctata in conferring salinity tolerance. Transition to an erect phenotype (shoot emergence) from prostrate growth of rhizome tissues is characterized by marked lignification and suberization of supporting sclerenchymatous tissue, epidermis, and bundle sheath cells in aerial shoot-proximal nodes and internodes in O. coarctata. With salinity, however, aerial shoot-proximal internodal tissues show reductions in lignification and suberization, most probably related to re-direction of carbon flux towards synthesis of the osmporotectant proline. Concurrent with hypolignification and reduced suberization, the aerial rhizomatous biomass of O. coarctata appears to have evolved mechanisms to store Na+ in these specific tissues under salinity. This was confirmed by histochemical staining, quantitative real-time reverse transcription-PCR expression patterns of genes involved in lignification/suberization, Na+ and K+ contents of internodal tissues, as well as non-invasive microelectrode ion flux measurements of NaCl-induced net Na+, K+, and H+ flux profiles of aerial nodes were determined. In O. coarctata, aerial proximal internodes appear to act as 'traffic controllers', sending required amounts of Na+ and K+ into developing leaves for osmotic adjustment and turgor-driven growth, while more deeply positioned internodes assume a Na+ buffering/storage role.

Biochemical pH clamp: the forgotten resource in membrane bioenergetics.

Solute uptake and release by plant cells are frequently energized by coupling to H+ influx supported by the proton motive force (pmf). The pmf results from a stable pH difference between the apoplast and the cytosol, with bulk values ranging from 4.9 to 5.8 and from 7.1 to 7.5, respectively, in combination with a negative electrical membrane potential. The P-type H+ ATPases pumping H+ from the cytosol into the apoplast at the expense of ATP hydrolysis are generally viewed as the only pmf source, exclusively linking membrane transport to energy metabolism. However, recent evidence suggests that pump activity may be insufficient to energize transport, particularly under stress conditions. Indeed, cytosolic H+ scavenging and apoplastic H+ generation by metabolism (denoted as 'active' buffering in contrast to the readily exhausted 'passive' matrix buffering) also stabilize the pH gradient. In the cytosol, H+ scavenging is mainly associated with malate decarboxylation catalyzed by malic enzyme, and via the GABA shunt of the tricarboxylic acid (TCA) cycle involving glutamate decarboxylation. In the apoplast, formation of bicarbonate from CO2 , the end-product of respiration, generates H+ at pH ≥ 6. Membrane potential is stabilized by K+ release and/or by anion uptake via ion channels. Finally, thermodynamic aspects of active buffering are discussed.

Microsensors in plant biology: in vivo visualization of inorganic analytes with high spatial and/or temporal resolution.

This Expert View provides an update on the recent development of new microsensors, and briefly summarizes some novel applications of existing microsensors, in plant biology research. Two major topics are covered: (i) sensors for gaseous analytes (O2, CO2, and H2S); and (ii) those for measuring concentrations and fluxes of ions (macro- and micronutrients and environmental pollutants such as heavy metals). We show that application of such microsensors may significantly advance understanding of mechanisms of plant-environmental interaction and regulation of plant developmental and adaptive responses under adverse environmental conditions via non-destructive visualization of key analytes with high spatial and/or temporal resolution. Examples included cover a broad range of environmental situations including hypoxia, salinity, and heavy metal toxicity. We highlight the power of combining microsensor technology with other advanced biophysical (patch-clamp, voltage-clamp, and single-cell pressure probe), imaging (MRI and fluorescent dyes), and genetic techniques and approaches. We conclude that future progress in the field may be achieved by applying existing microsensors for important signalling molecules such as NO and H2O2, by improving selectivity of existing microsensors for some key analytes (e.g. Na, Mg, and Zn), and by developing new microsensors for P.

An Anion Conductance, the Essential Component of the Hydroxyl-Radical-Induced Ion Current in Plant Roots.

Oxidative stress signaling is essential for plant adaptation to hostile environments. Previous studies revealed the essentiality of hydroxyl radicals (HO•)-induced activation of massive K⁺ efflux and a smaller Ca2+ influx as an important component of plant adaptation to a broad range of abiotic stresses. Such activation would modify membrane potential making it more negative. Contrary to these expectations, here, we provide experimental evidence that HO• induces a strong depolarization, from -130 to -70 mV, which could only be explained by a substantial HO•-induced efflux of intracellular anions. Application of Gd3+ and NPPB, non-specific blockers of cation and anion conductance, respectively, reduced HO•-induced ion fluxes instantaneously, implying a direct block of the dual conductance. The selectivity of an early instantaneous HO•-induced whole cell current fluctuated from more anionic to more cationic and vice versa, developing a higher cation selectivity at later times. The parallel electroneutral efflux of K⁺ and anions should underlie a substantial leak of the cellular electrolyte, which may affect the cell's turgor and metabolic status. The physiological implications of these findings are discussed in the context of cell fate determination, and ROS and cytosolic K⁺ signaling.

Tissue and cellular localization of condensed tannins in poplar roots and potential association with nitrogen uptake.

Condensed tannins are common in vegetative tissues of woody plants, including in roots. In hybrid poplar (Populus tremula x alba; also known as P. x canescens) CT assays indicated they were most concentrated in younger white roots and at the root tip. Furthermore, CT-specific staining of embedded tissue sections demonstrated accumulation in root cap cells and adjacent epidermal cells, as well as a more sporadic presence in cortex cells. In older, brown roots as well as roots with secondary growth (cork zone), CT concentration was significantly lower. The insoluble fraction of CTs was greatest in the cork zone. To determine if CT accumulation correlates with nutrient uptake in poplar roots, a microelectrode ion flux measurement (MIFE™) system was used to measure flux along the root axis. Greatest NH4 + uptake was measured near the root tip, but NO3- and Ca2+ did not vary along the root length. In agreement with earlier work, providing poplars with ample nitrogen led to higher accumulation of CTs across root zones. To test the functional importance of CTs in roots directly, CT-modified transgenic plants could be important tools.

AtCIPK16, a CBL-interacting protein kinase gene, confers salinity tolerance in transgenic wheat.

Globally, wheat is the major source of staple food, protein, and basic calories for most of the human population. Strategies must be adopted for sustainable wheat crop production to fill the ever-increasing food demand. Salinity is one of the major abiotic stresses involved in plant growth retardation and grain yield reduction. In plants, calcineurin-B-like proteins form a complicated network with the target kinase CBL-interacting protein kinases (CIPKs) in response to intracellular calcium signaling as a consequence of abiotic stresses. The AtCIPK16 gene has been identified in Arabidopsis thaliana and found to be significantly upregulated under salinity stress. In this study, the AtCIPK16 gene was cloned in two different plant expression vectors, i.e., pTOOL37 having a UBI1 promoter and pMDC32 having a 2XCaMV35S constitutive promoter transformed through the Agrobacterium-mediated transformation protocol, in the local wheat cultivar Faisalabad-2008. Based on their ability to tolerate different levels of salt stress (0, 50, 100, and 200 mM), the transgenic wheat lines OE1, OE2, and OE3 expressing AtCIPK16 under the UBI1 promoter and OE5, OE6, and OE7 expressing the same gene under the 2XCaMV35S promoter performed better at 100 mM of salinity stress as compared with the wild type. The AtCIPK16 overexpressing transgenic wheat lines were further investigated for their K+ retention ability in root tissues by utilizing the microelectrode ion flux estimation technique. It has been demonstrated that after 10 min of 100 mM NaCl application, more K+ ions were retained in the AtCIPK16 overexpressing transgenic wheat lines than in the wild type. Moreover, it could be concluded that AtCIPK16 functions as a positive elicitor in sequestering Na+ ions into the cell vacuole and retaining more cellular K+ under salt stress to maintain ionic homeostasis.

Comparative Analysis of Root Na+ Relation under Salinity between Oryza sativa and Oryza coarctata

Na+ toxicity is one of the major physiological constraints imposed by salinity on plant performance. At the same time, Na+ uptake may be beneficial under some circumstances as an easily accessible inorganic ion that can be used for increasing solute concentrations and maintaining cell turgor. Two rice species, Oryza sativa (cultivated rice, salt-sensitive) and Oryza coarctata (wild rice, salt-tolerant), demonstrated different strategies in controlling Na+ uptake. Glasshouse experiments and gene expression analysis suggested that salt-treated wild rice quickly increased xylem Na+ loading for osmotic adjustment but maintained a non-toxic level of stable shoot Na+ concentration by increased activity of a high affinity K+ transporter HKT1;5 (essential for xylem Na+ unloading) and a Na+/H+ exchanger NHX (for sequestering Na+ and K+ into root vacuoles). Cultivated rice prevented Na+ uptake and transport to the shoot at the beginning of salt treatment but failed to maintain it in the long term. While electrophysiological assays revealed greater net Na+ uptake upon salt application in cultivated rice, O. sativa plants showed much stronger activation of the root plasma membrane Na+/H+ Salt Overly Sensitive 1 (SOS1) exchanger. Thus, it appears that wild rice limits passive Na+ entry into root cells while cultivated rice relies heavily on SOS1-mediating Na+ exclusion, with major penalties imposed by the existence of the "futile cycle" at the plasma membrane.

An Improved Transwell Design for Microelectrode Ion-Flux Measurements.

The microelectrode ion flux estimation (MIFE) is a powerful, non-invasive electrophysiological method for cellular membrane transport studies. Usually, the MIFE measurements are performed in a tissue culture dish or directly with tissues (roots, parts of the plants, and cell tissues). Here, we present a transwell system that allows for MIFE measurements on a cell monolayer. We introduce a measurement window in the transwell insert membrane, which provides direct access for the cells to the media in the upper and lower compartment of the transwell system and allows direct cell-to-cell contact coculture. Three-dimensional multiphoton lithography (MPL) was used to construct a 3D grid structure for cell support in the measurement window. The optimal polymer grid constant was found for implementation in transwell MIFE measurements. We showed that human umbilical vein endothelial cells (HUVECs) efficiently grow and maintain their physiological response on top of the polymer structures.

MPM motifs of the yeast SKT protein Trk1 can assemble to form a functional K+-translocation system.

The yeast Trk1 polypeptide, like other members of the Superfamily of K Transporters (SKT proteins) consists of four Membrane-Pore-Membrane motifs (MPMs A-D) each of which is homologous to a single K-channel subunit. SKT proteins are thought to have evolved from ancestral K-channels via two gene duplications and thus single MPMs might be able to assemble when located on different polypeptides. To test this hypothesis experimentally we generated a set of partial gene deletions to create alleles encoding one, two, or three MPMs, and analysed the cellular localisation and interactions of these Trk1 fragments using GFP tags and Bimolecular Fluorescence Complementation (BiFC). The function of these partial Trk1 proteins either alone or in combinations was assessed by expressing the encoding genes in a K+-uptake deficient strain lacking also the K-channel Tok1 (trk1,trk2,tok1Δ) and (i) analysing their ability to promote growth in low [K+] media and (ii) by ion flux measurements using "microelectrode based ion flux estimation" (MIFE). We found that proteins containing only one or two MPM motifs can interact with each other and assemble with a polypeptide consisting of the rest of the Trk system to form a functional K+-translocation system.

Extracellular Spermine Triggers a Rapid Intracellular Phosphatidic Acid Response in Arabidopsis, Involving PLDδ Activation and Stimulating Ion Flux.

Polyamines, such as putrescine (Put), spermidine (Spd), and spermine (Spm), are low-molecular-weight polycationic molecules found in all living organisms. Despite the fact that they have been implicated in various important developmental and adaptative processes, their mode of action is still largely unclear. Here, we report that Put, Spd, and Spm trigger a rapid increase in the signaling lipid, phosphatidic acid (PA) in Arabidopsis seedlings but also mature leaves. Using time-course and dose-response experiments, Spm was found to be the most effective; promoting PA responses at physiological (low µM) concentrations. In seedlings, the increase of PA occurred mainly in the root and partly involved the plasma membrane polyamine-uptake transporter (PUT), RMV1. Using a differential 32Pi-labeling strategy combined with transphosphatidylation assays and T-DNA insertion mutants, we found that phospholipase D (PLD), and in particular PLDδ was the main contributor of the increase in PA. Measuring non-invasive ion fluxes (MIFE) across the root plasma membrane of wild type and pldδ-mutant seedlings, revealed that the formation of PA is linked to a gradual- and transient efflux of K+. Potential mechanisms of how PLDδ and the increase of PA are involved in polyamine function is discussed.

Methods Related to Polyamine Control of Cation Transport Across Plant Membranes.

Polyamines (PAs) are unique polycationic metabolites, which modulate plants' growth, development, and stress responses. As polycations, PAs interfere with cationic transport systems as ion channels and ionotropic pumps. Here, we describe the application of two techniques, MIFE to study the effects of PAs on cation fluxes in vivo and conventional patch-clamp to evaluate the PA blockage of ion currents in isolated plant vacuoles. Preparation of vacuoles for patch-clamp assays is described and solutions and voltage protocols are given, which allow separate recordings of major vacuolar channel currents and quantify their blockage by PAs.

AtNPF2.5 Modulates Chloride (Cl-) Efflux from Roots of Arabidopsis thaliana.

The accumulation of high concentrations of chloride (Cl-) in leaves can adversely affect plant growth. When comparing different varieties of the same Cl- sensitive plant species those that exclude relatively more Cl- from their shoots tend to perform better under saline conditions; however, the molecular mechanisms involved in maintaining low shoot Cl- remain largely undefined. Recently, it was shown that the NRT1/PTR Family 2.4 protein (NPF2.4) loads Cl- into the root xylem, which affects the accumulation of Cl- in Arabidopsis shoots. Here we characterize NPF2.5, which is the closest homolog to NPF2.4 sharing 83.2% identity at the amino acid level. NPF2.5 is predominantly expressed in root cortical cells and its transcription is induced by salt. Functional characterisation of NPF2.5 via its heterologous expression in yeast (Saccharomyces cerevisiae) and Xenopus laevis oocytes indicated that NPF2.5 is likely to encode a Cl- permeable transporter. Arabidopsis npf2.5 T-DNA knockout mutant plants exhibited a significantly lower Cl- efflux from roots, and a greater Cl- accumulation in shoots compared to salt-treated Col-0 wild-type plants. At the same time, [Formula: see text] content in the shoot remained unaffected. Accumulation of Cl- in the shoot increased following (1) amiRNA-induced knockdown of NPF2.5 transcript abundance in the root, and (2) constitutive over-expression of NPF2.5. We suggest that both these findings are consistent with a role for NPF2.5 in modulating Cl- transport. Based on these results, we propose that NPF2.5 functions as a pathway for Cl- efflux from the root, contributing to exclusion of Cl- from the shoot of Arabidopsis.

Ion flux profiles and plant ion homeostasis control under salt stress.

The ability of a plant to maintain an ionic homeostasis is crucial in plant salt tolerance. Direct evidence based on data from the non-invasive measurement of ion fluxes would not only offer new insight about the function of the transporter but also provide a whole plant approach for dissecting salt adaptation mechanisms. Here, we review some reports using the ion-selective microelectrodes to characterize the net ion fluxes of tissues or cells.