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The stress hormone abscisic acid (ABA) induces expression of defence genes in many organs, modulates ion homeostasis and metabolism in guard cells, and inhibits germination and seedling growth. Concerning the latter effect, several mutants of Arabidopsis thaliana with improved capability for H⁺ efflux (wat1-1D, overexpression of AKT1 and ost2-1D) are less sensitive to inhibition by ABA than the wild type. This suggested that ABA could inhibit H⁺ efflux (H⁺-ATPase) and induce cytosolic acidification as a mechanism of growth inhibition. Measurements to test this hypothesis could not be done in germinating seeds and we used roots as the most convenient system. ABA inhibited the root plasma-membrane H⁺-ATPase measured in vitro (ATP hydrolysis by isolated vesicles) and in vivo (H⁺ efflux from seedling roots). This inhibition involved the core ABA signalling elements: PYR/PYL/RCAR ABA receptors, ABA-inhibited protein phosphatases (HAB1), and ABA-activated protein kinases (SnRK2.2 and SnRK2.3). Electrophysiological measurements in root epidermal cells indicated that ABA, acting through the PYR/PYL/RCAR receptors, induced membrane hyperpolarization (due to K⁺ efflux through the GORK channel) and cytosolic acidification. This acidification was not observed in the wat1-1D mutant. The mechanism of inhibition of the H⁺-ATPase by ABA and its effects on cytosolic pH and membrane potential in roots were different from those in guard cells. ABA did not affect the in vivo phosphorylation level of the known activating site (penultimate threonine) of H⁺-ATPase in roots, and SnRK2.2 phosphorylated in vitro the C-terminal regulatory domain of H⁺-ATPase while the guard-cell kinase SnRK2.6/OST1 did not.

Colorectal cancer (CRC) is the second leading cause of death worldwide, with 0.9 million deaths per year. The metastatic stage of the disease is identified in about 20% of cases at the first diagnosis and is associated with low patient-survival rates. Voltage-gated sodium channels (NaV) are abnormally overexpressed in several carcinomas including CRC and are strongly associated with the metastatic behavior of cancer cells. Acidification of the extracellular space by Na+/H+ exchangers (NHE) contributes to extracellular matrix degradation and cell invasiveness. In this study, we assessed the expression levels of pore-forming α-subunits of NaV channels and NHE exchangers in tumor and adjacent non-malignant tissues from colorectal cancer patients, CRC cell lines and primary tumor cells. In all cases, SCN5A (gene encoding for NaV1.5) was overexpressed and positively correlated with cancer stage and poor survival prognosis for patients. In addition, we identified an anatomical differential expression of SCN5A and SLC9A1 (gene encoding for NHE-1) being particularly relevant for tumors that originated on the sigmoid colon epithelium. The functional activity of NaV1.5 channels was characterized in CRC cell lines and the primary cells of colon tumors obtained using tumor explant methodologies. Furthermore, we assessed the performance of two new small-molecule NaV1.5 inhibitors on the reduction of sodium currents, as well as showed that silencing SCN5A and SLC9A1 substantially reduced the 2D invasive capabilities of cancer cells. Thus, our findings show that both NaV1.5 and NHE-1 represent two promising targetable membrane proteins against the metastatic progression of CRC.

This paper reviews strategies for manipulating plants and their root-associated microorganisms to improve plant health and productivity. Some strategies directly target plant processes that impact on growth, while others are based on our knowledge of interactions among the components of the rhizosphere (roots, microorganisms and soil). For instance, plants can be engineered to modify the rhizosphere pH or to release compounds that improve nutrient availability, protect against biotic and abiotic stresses, or encourage the proliferation of beneficial microorganisms. Rhizobacteria that promote plant growth have been engineered to interfere with the synthesis of stress-induced hormones such as ethylene, which retards root growth, and to produce antibiotics and lytic enzymes active against soilborne root pathogens. Rhizosphere engineering also can involve the selection by plants of beneficial microbial populations. For example, some crop species or cultivars select for and support populations of antibiotic-producing strains that play a major role in soils naturally suppressive to soil-borne fungal pathogens. The fitness of root-associated bacterial communities also can be enhanced by soil amendment, a process that has allowed the selection of bacterial consortia that can interfere with bacterial pathogens. Plants also can be engineered specifically to influence their associated bacteria, as exemplified by quorum quenching strategies that suppress the virulence of pathogens of the genus Pectobacterium. New molecular tools and powerful biotechnological advances will continue to provide a more complete knowledge of the complex chemical and biological interactions that occur in the rhizosphere, ensuring that strategies to engineer the rhizosphere are safe, beneficial to productivity, and substantially improve the sustainability of agricultural systems.

Alkaliphiles grow under alkaline conditions that might be disadvantageous for the transmembrane pH gradient (ΔpH, outside acidic). In this study, the behaviors of extruded protons by the respiration of obligate alkaliphilic Bacillus clarkii K24-1U were investigated by comparison with those of neutralophilic Bacillus subtilis IAM 1026. Although whole-cell suspensions of both Bacillus species consumed oxygen immediately after the addition of air, there were lag times before the suspensions were acidified. Under alkaline conditions, the lag time for B. clarkii significantly increased, whereas that for B. subtilis decreased. In the presence of valinomycin or ETH-157, which disrupts the membrane electrical potential (Δψ), the cell suspensions of both Bacillus species acidified immediately after the addition of air. Artificial electroneutral antiporters (nigericin and monensin) that eliminate the ΔpH exhibited no significant effect on the lag times of the two Bacillus species except that monensin increased the lag times of B. clarkii. The inhibition of ATPase and the Na⁺ channel also exhibited little effects on the lag times. The increased lag time for B. clarkii may represent the Δψ-dependent proton retention on the outer surface of the cytoplasmic membrane to generate a sufficient ΔpH under alkaline conditions.

The impact of phosphorus (P) availability on root proliferation, proton efflux, and acid phosphatase activities in roots and leaves was investigated in two lines of common bean (Phaseolus vulgaris): BAT 477 and CocoT. Phosphorus was supplied as KH₂PO₄ at 0 and 60 μmol per plant (0P and 60P, respectively). Under P shortage, the plant growth was more restricted in CocoT than in BAT 477, shoots being more affected than roots. The root area increased significantly at 0P in both lines. Up to 1 week following P shortage, the proton efflux increased in both lines despite a higher extent in BAT 477 as compared to CocoT. Root acid phosphatase activity was significantly higher under P limitation in the both lines, this trend being more pronounced in BAT 477 than in CocoT. This was also true for the leaf acid phosphatase. Regardless of the bean line, higher values were recorded for the old leaves as compared to the young ones for this parameter. Interestingly, a significant correlation between Pi content in old leaves and their acid phosphatase activity was found in P-lacking (0P) plants of the both bean lines, suggesting that acid phosphatase may contribute to increase the phosphorus use efficiency in bean through the P remobilization from the old leaves. As a whole, our results highlight the significance of the root H+ extrusion and the acid phosphatase activity rather than the root proliferation in the relative tolerance of BAT 477 to severe P deficiency.

The effect of two Azospirillum strains (SP-7, Dol) was compared on root proton efflux and root enlargement of three wheat cultivars (Ghods, Omid and Roshan). Root colonization varied greatly among strain-plant combinations. Inoculation enhanced proton efflux and root elongation of wheat roots but this effect was directly dependent on the strain-plant combination. Strain SP-7 stimulated the greatest proton efflux and root elongation in cv. Roshan, whereas strain Dol induced the best effect on both these phenomena in cv. Ghods. Based on positive correlation between these two phenomena, it was suggests that proton efflux is related to increasing of root length by Azospirillum inoculation. The number of bacteria of both Azospirillum strains in root of cv. Omid was less than the other cultivars. Proton extrusion and root elongation of cv. Omid failed to respond significantly with these two strains. This may be due to incompatible host-strain combination. Thus compatible strains are necessary for increasing of proton efflux and root extension in wheat cultivars.[PUBLICATION ABSTRACT]

Light causes growth of dicotyledonous leaves by stimulating proton efflux, cell wall acidification and loosening, and solute accumulation for turgor maintenance. For cells still undergoing cell division at the base of expanding tobacco (Nicotiana tabacum L. cv. Xanthi) leaves, light-stimulated growth depends on K+ uptake, and is inhibited by the potassium channel blocker tetraethylammonium (TEA). The generality of this mechanism has been tested by comparing the effect of light on the growth-associated physiology of dividing and expanding cells in the base with cells at the tip growing by cell expansion only. The magnitude of the light-induced growth response of excised leaf discs is greatest at the leaf base and declines as cells mature. Basal tissue is more sensitive to exogenous potassium, which enhances light-stimulated growth at < 1 mM, whereas tip tissue requires higher levels (> 10 mM). Growth is inhibited by TEA similarly in tip and base. However, light-stimulated K+ uptake and proton efflux respond differently to TEA in tip and basal tissue. In basal tissue, TEA reduces light-stimulated K+ uptake by 60% and inhibits light-stimulated proton efflux. These results agree with those presented by M. Claussen et al. (1997, Planta 201:227—234) showing that auxin-stimulated H+ pump activity and growth in coleoptiles require K+ uptake as an electrical counterbalance to H+ efflux. In contrast, in tip tissue, TEA inhibits light-stimulated K+ uptake by only 17% and does not inhibit proton efflux. Our results suggest that the basipetal gradient in the effect of TEA on light-regulated growth physiology can be explained by TEA effects on K+ uptake: TEA inhibits light-stimulated H+ pump activity, wall acidification and membrane hyperpolarization only in cells dependent on TEA-sensitive channels for light-stimulated K+ uptake. Further, our data suggest that younger, basal tissue is dependent on TEA-sensitive, sucrose-stimulatable channels for light-stimulated K+ uptake whereas older, tip tissue is able to use an additional, TEA-insensitive K+ transporter to mediate light-stimulated K+ uptake.

This paper examines the effects of ionic gold on Saccharomyces cerevisiae, as determined by long-term (growth in gold-containing media) and short-term interactions (H+ efflux activity). An increasing gold concentration inhibited growth and at < 0.2 mM Au, growth was not observed. Transmission electron microscopy revealed no differences in ultrastructure but fine electron dense particles were observed in unstained preparations from gold-containing medium. After glucose addition (to 10 mM) to starved suspensions of S. cerevisiae, glucose-dependent reduction of external pH occurred as the cells extruded protons. In the presence of increasing gold concentrations, the lag time before proton extrusion did not change but the rate and duration decreased significantly with a marked influence on proton efflux rate being observed at < or = 10 microM. Extension of preincubation time of yeast cells in gold-containing medium resulted in a decreasing proton efflux rate and colloidal phase formation in the cell suspensions, the time between gold addition and the beginning of colloidal phase formation depending on the gold concentration used. Both Ca and Mg enhanced the inhibitory effect of gold on the yeast cells with Ca showing a stronger inhibitory effect than Mg.

The light-induced proton efflux and active carbon uptake are inhibited by mercury and cadmium ions in Anabaena flos-aquae. The inhibitory effects of these heavy metal ions are reversed by 40 mM concentration of sodium. Here we report that light-induced proton efflux is sodium-dependent which leads to a characteristic enhancement in the rate of photosynthetic oxygen generation and carbon fixation. A low concentration (10 mu M) of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) significantly inhibited the rate of oxygen generation while 10 mu M carbonyl cyanide-m-chlorophenylhydrazone (CCCP) completely blocked the oxygen generation activity in the organism. The chlorophyll-a fluorescence yield indicates that little fluorescence quenching occurred in the absence of sodium ion. Increasing the extracellular sodium ion accelerated both the initial rate and the extent of fluorescence quenching. These results support the assumption that metal-induced inhibition of the photosynthetic machinery may be mediated by the movement of protons.

Forest die-back and impaired tree vitality have frequently been ascribed to Al-toxicity and Al-induced nutritional disorders due to increased acidification of forest soils. Therefore, in this experiment effects of Al were studied on growth and nutrient uptake with seedlings of five different forest tree species. During growth in culture solutions with and without AI all five species proved to be very Al-tolerant, despite high accumulation of Al in roots. In the coniferous evergreens Douglas-fir and Scots pine shoot as well as root Al concentrations were significantly higher than in the deciduous broad-leaved species oak and birch. Larch showed intermediate Al levels. In none of the five species did Al reduce nutrient concentrations or the Ca/Al ratio to values below the critical level. Besides differences in Al accumulation, coniferous and broad-leaved species also differed with respect to uptake and assimilation of nitrogen. Due to extra $NH_4^ + $ uptake, oak and birch showed a much higher N uptake and higher $NH_4^ + $ preference than the coniferous species. Especially with oak this high $NH_4^ + $ preference in combination with a low specific root surface area resulted in a high root proton efflux density. In comparison to both broad-leaved trees and Scots pine the $NO_3^ - $ reduction capacity of larch and Douglas-fir was extremely low. This may have important consequences for both species if grown in $NO_3^ - $-rich soils.