Salinity tolerance in plants. Quantitative approach to ion transport starting from halophytes and stepping to genetic and protein engineering for manipulating ion fluxes

Abstract

Ion transport is the fundamental factor determining salinity tolerance in plants. The Review starts from differences in ion transport between salt tolerant halophytes and salt-sensitive plants with an emphasis on transport of potassium and sodium via plasma membranes. The comparison provides introductory information for increasing salinity tolerance. Effects of salt stress on ion transport properties of membranes show huge opportunities for manipulating ion fluxes. Further steps require knowledge about mechanisms of ion transport and individual genes of ion transport proteins. Initially, the Review describes methods to measure ion fluxes, the independent set of techniques ensures robust and reliable basement for quantitative approach. The Review briefly summarizes current data concerning Na(+) and K(+) concentrations in cells, refers to primary thermodynamics of ion transport and gives special attention to individual ion channels and transporters. Simplified scheme of a plant cell with known transport systems at the plasma membrane and tonoplast helps to imagine the complexity of ion transport and allows choosing specific transporters for modulating ion transport. The complexity is enhanced by the influence of cell size and cell wall on ion transport. Special attention is given to ion transporters and to potassium and sodium transport by HKT, HAK, NHX, and SOS1 proteins. Comparison between non-selective cation channels and ion transporters reveals potential importance of ion transporters and the balance between the two pathways of ion transport. Further on the Review describes in detail several successful attempts to overexpress or knockout ion transporters for changing salinity tolerance. Future perspectives are questioned with more attention given to promising candidate ion channels and transporters for altered expression. Potential direction of increasing salinity tolerance by modifying ion channels and transporters using single point mutations is discussed and questioned. An alternative approach from synthetic biology is to create new regulation networks using novel transport proteins with desired properties for transforming agricultural crops. The approach had not been widely used earlier; it leads also to theoretical and pure scientific aspects of protein chemistry, structure-function relations of membrane proteins, systems biology and physiology of stress and ion homeostasis. Summarizing, several potential ways are aimed at required increase in salinity tolerance of plants of interest.

Keywords: halophyte; ion channel; ion fluxes; ion transporter; protein engineering; salinity tolerance; synthetic biology; systems biology.

FIGURE 1

Halophytes Salicornia sp. (larger and bright green plants at the picture) and Suaeda maritima (smaller and grayish-green plants at the picture) are growing at the salt-affected soil near river Medway in the UK, where the area is flooded with mixed sea and river water under high tides (Beginning of June 2014). The size of the largest specimen is about 15 cm.

FIGURE 2

Proposed simplified model of several attractors for a plant cell with specific metabolic and regulatory networks determined by cytoplasmic ion concentrations and membrane potential. Changes in extracellular ion concentrations result in transition of cells from one state of concentrations-membrane potential-protein and DNA–RNA expression levels and activity pattern to another one. Stability of the physiological states and trajectories of transitions could be studied in more detail using complex approaches of ionomics–metabolomics–proteomics–nucleic acids expression arrays and biophysical methods.

FIGURE 3

Kinetics of initial unidirectional Na⁺ influx into roots of Arabidopsis thaliana (open circles) and Thellungiella halophila (closed circles) as determined from ²²Na⁺ accumulation of individual plants from ²²Na⁺ labeled nutrient solution with 100 mM NaCl and 0.1 mM CaCl₂. Error bars are SE (n = 4). Reproduced from Wang et al. (2006) with the permission from the publisher Oxford University Press.

FIGURE 4

Comparison of current–voltage curves for whole-cell instantaneous currents in root protoplasts of A. thaliana (open symbols) and T. halophila (closed symbols). Currents are normalized to protoplast surface. The pipette solution was always 100 mM KCl. The bath solution was 100 KCl (A) or 100 NaCl (B). Data are given as means ± SE (n = 6 for A. thaliana, n = 13 for T. halophila). Reproduced from Volkov and Amtmann (2006) with the permission from the publisher John Wiley and Sons. Note that IV curve for instantaneous current in T. halophila resembles (though not completely obeys) the expected one from Goldmann–Hodkin–Katz equation and the shift in reversal potential of ion current in 100 mM KCl/100 mM NaCl is reflected in slope of IV curve for voltages above and below the reversal potential. The electric current characterizes the ion transport properties of plasma membranes and their selectivity for K⁺ over Na⁺.

FIGURE 5

Effect of salt stress on instantaneous current in protoplasts from the elongation zone and emerged blade portion of the developing leaf 3 of barley (Hordeum vulgare L.) (A–F). Averaged current–voltage (I–V) relationship in protoplasts from mesophyll (A,D) and epidermis (B,E) of the emerged blade, and from protoplasts of the elongation zone (C,F). Control (A–C) and salt treatment (D–F), n = 5–8 for control protoplasts and n = 3–6 for protoplasts for salt treatment; error bars are standard errors. Concentrations of KCl and NaCl in the bath are indicated. The pipette solution was always 100 mM KCl. Plants had been exposed to 100 mM NaCl for 3 days prior to protoplast isolation. Reproduced from Volkov et al. (2009), composed from Figures 1, 2, 4 and 5 with the permission from the publisher John Wiley and Sons.

FIGURE 6

Basic scheme of membrane potentials, potassium K⁺ and sodium Na⁺ concentrations and pH values in a generalized plant cell together with main ion transport systems ensuring potassium and sodium transport according to electrochemical forces. Different cell types usually have less transporters, though specialized for more determined ion transport functions. The concentrations and membrane potentials are rather indicative and change depending on conditions of mineral nutrition and are not the same for different cell types (see text and references for more details). KIRC are inward rectifying potassium channels (e.g., Hirsch et al., 1998); KORC are outward rectifying potassium channels; GluR are glutamate receptors; cngc are cyclic nucleotide gated ion channels; HAK is high affinity potassium transporter; CHXT are cation H⁺ exchange transporters (e.g., Evans et al., 2012); HKT are high K⁺ affinity transporters; C-Cl^--CT are cation chloride contrasnporters; SOS1 is well studied sodium-proton antiporter; H⁺-ATPase is proton pump of plasma membrane; V-H⁺-ATPase is vacuolar proton pump; V-PP-H⁺-ATPase is vacuolar pyrophosphatase, another vacuolar proton pump; NHX1 is vacuolar sodium (cation)/proton antiporter. For more details and description see text.

FIGURE 7

Simplified scheme demonstrating principles of ion transport via membrane. Voltage difference below -180 mV allows potassium transport against 1000-fold concentration difference via potassium-selective pore of ion channel 1. Similar thermodynamically favorable potassium transport with lower rates and specific mechanism is facilitated by potassium transporter 2. Transporters 2 and 3 are H⁺/K⁺-cotransporters, they co-transport one or two H⁺ per K⁺; H⁺ is transported according to voltage difference, hence adding energy for K⁺ transport. Transporters 2 and 3 can potentially lead to inward transport of K⁺ against over 1,000,000 concentration difference; their functioning depends also on pH difference across membrane. More details are in the text.

FIGURE 8

Simplified scheme of different plant cells and the ion fluxes for the cells. The same ion fluxes result in different changes of ion concentrations for cells of different sizes and surface/volume ratios. Ion fluxes are directly recalculated from number of ions/second to concentrations.

FIGURE 9

The scheme indicating expected and studied effects of cell walls on ion fluxes and cell plasma membrane potential. Plasma membrane of approximately 10 nm thickness is surrounded by the cell wall of about 250 nm thickness, the interior of the cell is under hydrostatic turgor pressure of several bars (1 bar = 0.1 MPa) due to concentration differences and, hence, differences in osmotic pressures inside the cell and outside of it. Cell wall has negative surface charge at pH around 6–7, hence influencing the voltage difference between the sides of plasma membrane. One of micro- or nanochannels in the porous cell wall is depicted with charged inner walls and potential ion-rectifying properties. More details are given in the text.

FIGURE 10

Single-channel recordings in outside-out patches of T. halophila root protoplasts. The pipette solution was 100 mM KCl. The bath solution contained 100 mM KCl (A), 10 mM KCl (B) or 100 mM NaCl (C). c, current level with no open channels; o, current levels of single-channel openings. Spiky openings of outward-rectifying channels allowing inward current are indicated with asterisks. Openings of a second type of channel are indicated with crosses. Reproduced from Volkov and Amtmann (2006) with the permission from the publisher John Wiley and Sons.

FIGURE 11

Novel artificially designed ion channels and ion transporters potentially provide an opportunity to avoid the present evolved protein regulation networks and may be useful for altering ion concentrations, membrane potential and signaling without essentially interacting with the fine-tuning of the existing regulatory networks. A schematic generalized plant potassium channel in a plasma membrane with several potentially known regulation factors and a model of artificially designed ion channel lacking the evolved known regulation feedbacks.