Nitrate-dependent shoot sodium accumulation and osmotic functions of sodium in Arabidopsis under saline conditions.

Improving crop plants to be productive in saline soils or under irrigation with saline water would be an important technological advance in overcoming the food and freshwater crises that threaten the world population. However, even if the transformation of a glycophyte into a plant that thrives under seawater irrigation was biologically feasible, current knowledge about Na+ effects would be insufficient to support this technical advance. Intriguingly, crucial details about Na+ uptake and its function in the plant have not yet been well established. We here propose that under saline conditions two nitrate-dependent transport systems in series that take up and load Na+ into the xylem constitute the major pathway for the accumulation of Na+ in Arabidopsis shoots; this pathway can also function with chloride at high concentrations. In nrt1.1 nitrate transport mutants, plant Na+ accumulation was partially defective, which suggests that NRT1.1 either partially mediates or modulates the nitrate-dependent Na+ transport. Arabidopsis plants exposed to an osmotic potential of -1.0 MPa (400 mOsm) for 24 h showed high water loss and wilting in sorbitol or Na/MES, where Na+ could not be accumulated. In contrast, in NaCl the plants that accumulated Na+ lost a low amount of water, and only suffered transitory wilting. We discuss that in Arabidopsis plants exposed to high NaCl concentrations, root Na+ uptake and tissue accumulation fulfil the primary function of osmotic adjustment, even if these processes lead to long-term toxicity.

Salt intolerance in Arabidopsis: shoot and root sodium toxicity, and inhibition by sodium-plus-potassium overaccumulation.

MAIN CONCLUSION: Arabidopsis plants in NaCl suffering half growth inhibition do not suffer osmotic stress and seldom shoot Na (+) toxicity; overaccumulation of Na (+) plus K (+) might trigger the inhibition. It is widely assumed that salinity inhibits plant growth by osmotic stress and shoot Na(+) toxicity. This study aims to examine the growth inhibition of Arabidopsis thaliana by NaCl concentrations that allow the completion of the life cycle. Unaffected Col-0 wild-type plants were used to define nontoxic Na(+) contents; Na(+) toxicities in shoots and roots were analyzed in hkt1 and sos1 mutants, respectively. The growth inhibition of Col-0 plants at 40 mM Na(+) was mild and equivalent to that produced by 8 and 4 mM Na(+) in hkt1 and sos1 plants, respectively. Therefore, these mutants allowed to study the toxicity of Na(+) in the absence of an osmotic challenge. Col-0 and Ts-1 accessions showed very different Na(+) contents but similar growth inhibitions; Ts-1 plants showed very high leaf Na(+) contents but no symptoms of Na(+) toxicity. Ak-1, C24, and Fei-0 plants were highly affected by NaCl showing evident symptoms of shoot Na(+) toxicity. Increasing K(+) in isotonic NaCl/KCl combinations dramatically decreased the Na(+) content in all Arabidopsis accessions and eliminated the signs of Na(+) toxicity in most of them but did not relieve growth inhibition. This suggested that the dominant inhibition in these conditions was either osmotic or of an ionic nature unspecific for Na(+) or K(+). Col-0 and Ts-1 plants growing in sorbitol showed a clear osmotic stress characterized by a notable decrease of their water content, but this response did not occur in NaCl. Overaccumulation of Na(+) plus K(+) might trigger growth reduction in NaCl-treated plants.

The potassium transporters HAK2 and HAK3 localize to endomembranes in Physcomitrella patens. HAK2 is required in some stress conditions.

The function of HAK transporters in high-affinity K+ uptake in plants is well established; this study aims to demonstrate that some transporters of the same family play important roles in endomembranes. The PpHAK2-PpHAK4 genes of Physcomitrella patens encode three transporters of high sequence similarity. Quantitative PCR showed that PpHAK2 and PpHAK3 transcripts are expressed at approximately the same level as the PpACT5 gene, while the expression of PpHAK4 seems to be restricted to specific conditions that have not been determined. KHA1 is an endomembrane K+/H+ antiporter of Saccharomyces cerevisiae, and the expression of the PpHAK2 cDNA, but not that of PpHAK3, suppressed the defect of a kha1 mutant. Transient expression of the PpHAK2-green fluorescent protein (GFP) and PpHAK3-GFP fusion proteins in P. patens protoplasts localized to the endoplasmic reticulum and Golgi complex, respectively. To determine the function of PpHAK2 and PpHAK3 in planta, we constructed ΔPphak2 and ΔPphak2 ΔPphak3 plants. ΔPphak2 plants were normal under all of the conditions tested except under K+ starvation or at acidic pH in the presence of acetic acid, whereupon they die. The defect observed under K+ starvation was suppressed by the presence of Na+. We propose that PpHAK2 may encode either a K(+)-H(+) symporter or a K+/H+ antiporter that mediates the transfer of H+ from the endoplasmic reticulum lumen to the cytosol. PpHAK2 may be a model of the second function of HAK transporters in plant cells. The disruption of the PpHAK3 gene in ΔPphak2 plants showed no effect.

Knockouts of Physcomitrella patens CHX1 and CHX2 transporters reveal high complexity of potassium homeostasis.

This study aims to increase our understanding of the functions of CHX transporters in plant cells using the model plant Physcomitrella patens, in which four CHX genes have been identified, PpCHX1-PpCHX4. Two of these genes, PpCHX1 and PpCHX2, are expressed at approximately the same level as the PpACT5 gene, but the other two genes show an extremely low expression. PpCHX1 and PpCHX2 restored growth of Escherichia coli mutants on low K(+)-containing media, suggesting that they mediated K+ uptake that may be energized by symport with H+. In contrast, these genes suppressed the defect associated with the kha1 mutation in Saccharomyces cerevisiae, which suggests that they might mediate K+/H+ antiport. PpCHX1-green fluorescent protein (GFP) fusion protein transiently expressed in P. patens protoplasts co-localized with a Golgi marker. In similar experiments, the PpCHX2-GFP protein appeared to localize to tonoplast and plasma membrane. We constructed the ΔPpchx1 and ΔPpchx2 single mutant lines, and the ΔPpchx2 ΔPphak1 double mutant. Single mutant plants grew normally under all the conditions tested and exhibited normal K+ and Rb+ influxes; the ΔPpchx2 mutation did not increase the defect of ΔPphak1 plants. In long-term experiments, ΔPpchx2 plants showed slightly higher Rb+ retention than wild-type plants, which suggests that PpCHX2 mediates the transfer of Rb+ either from the vacuole to the cytosol or from the cytosol to the external medium in parallel with other transporters. The distinction between these two possibilities is technically difficult. We suggest that K+ transporters of several families are involved in the pH homeostasis of organelles by mediating either K+/H+ antiport or K(+)-H(+) symport.

Counting highly cited papers for university research assessment: conceptual and technical issues.

A Kuhnian approach to research assessment requires us to consider that the important scientific breakthroughs that drive scientific progress are infrequent and that the progress of science does not depend on normal research. Consequently, indicators of research performance based on the total number of papers do not accurately measure scientific progress. Similarly, those universities with the best reputations in terms of scientific progress differ widely from other universities in terms of the scale of investments made in research and in the higher concentrations of outstanding scientists present, but less so in terms of the total number of papers or citations. This study argues that indicators for the 1% high-citation tail of the citation distribution reveal the contribution of universities to the progress of science and provide quantifiable justification for the large investments in research made by elite research universities. In this tail, which follows a power low, the number of the less frequent and highly cited important breakthroughs can be predicted from the frequencies of papers in the upper part of the tail. This study quantifies the false impression of excellence produced by multinational papers, and by other types of papers that do not contribute to the progress of science. Many of these papers are concentrated in and dominate lists of highly cited papers, especially in lower-ranked universities. The h-index obscures the differences between higher- and lower-ranked universities because the proportion of h-core papers in the 1% high-citation tail is not proportional to the value of the h-index.

Phylogenetic analysis of k(+) transporters in bryophytes, lycophytes, and flowering plants indicates a specialization of vascular plants.

As heritage from early evolution, potassium (K(+)) is absolutely necessary for all living cells. It plays significant roles as stabilizer in metabolism and is important for enzyme activation, stabilization of protein synthesis, and neutralization of negative charges on cellular molecules as proteins and nucleic acids. Land plants even enlarged this spectrum of K(+) utilization after having gone ashore, despite the fact that K(+) is far less available in their new oligotrophic habitats than in sea water. Inevitably, plant cells had to improve and to develop unique transport systems for K(+) accumulation and distribution. In the past two decades a manifold of K(+) transporters from flowering plants has been identified at the molecular level. The recently published genome of the fern ally Selaginella moellendorffii now helps in providing a better understanding on the molecular changes involved in the colonization of land and the development of the vasculature and the seeds. In this article we present an inventory of K(+) transporters of this lycophyte and pigeonhole them together with their relatives from the moss Physcomitrella patens, the monocotyledon Oryza sativa, and two dicotyledonous species, the herbaceous plant Arabidopsis thaliana, and the tree Populus trichocarpa. Interestingly, the transition of green plants from an aqueous to a dry environment coincides with a dramatic reduction in the diversity of voltage-gated potassium channels followed by a diversification on the basis of one surviving K(+) channel class. The first appearance of K(+) release (K(out)) channels in S. moellendorffii that were shown in Arabidopsis to be involved in xylem loading and guard cell closure coincides with the specialization of vascular plants and may indicate an important adaptive step.

HAK transporters from Physcomitrella patens and Yarrowia lipolytica mediate sodium uptake.

The widespread presence of Na(+)-specific uptake systems across plants and fungi is a controversial topic. In this study, we identify two HAK genes, one in the moss Physcomitrella patens and the other in the yeast Yarrowia lipolytica, that encode Na(+)-specific transporters. Because HAK genes are numerous in plants and are duplicated in many fungi, our findings suggest that some HAK genes encode Na(+) transporters and that Na(+) might play physiological roles in plants and fungi more extensively than is currently thought.

HKT2;2/1, a K⁺-permeable transporter identified in a salt-tolerant rice cultivar through surveys of natural genetic polymorphism.

We have investigated OsHKT2;1 natural variation in a collection of 49 cultivars with different levels of salt tolerance and geographical origins. The effect of identified polymorphism on OsHKT2;1 activity was analysed through heterologous expression of variants in Xenopus oocytes. OsHKT2;1 appeared to be a highly conserved protein with only five possible amino acid substitutions that have no substantial effect on functional properties. Our study, however, also identified a new HKT isoform, No-OsHKT2;2/1 in Nona Bokra, a highly salt-tolerant cultivar. No-OsHKT2;2/1 probably originated from a deletion in chromosome 6, producing a chimeric gene. Its 5' region corresponds to that of OsHKT2;2, whose full-length sequence is not present in Nipponbare but has been identified in Pokkali, a salt-tolerant rice cultivar. Its 3' region corresponds to that of OsHKT2;1. No-OsHKT2;2/1 is essentially expressed in roots and displays a significant level of expression at high Na⁺ concentrations, in contrast to OsHKT2;1. Expressed in Xenopus oocytes or in Saccharomyces cerevisiae, No-OsHKT2;2/1 exhibited a strong permeability to Na⁺ and K⁺, even at high external Na⁺ concentrations, like OsHKT2;2, and in contrast to OsHKT2;1. Our results suggest that No-OsHKT2;2/1 can contribute to Nona Bokra salt tolerance by enabling root K⁺ uptake under saline conditions.

A simple index for the high-citation tail of citation distribution to quantify research performance in countries and institutions.

BACKGROUND: Conventional scientometric predictors of research performance such as the number of papers, citations, and papers in the top 1% of highly cited papers cannot be validated in terms of the number of Nobel Prize achievements across countries and institutions. The purpose of this paper is to find a bibliometric indicator that correlates with the number of Nobel Prize achievements. METHODOLOGY/PRINCIPAL FINDINGS: This study assumes that the high-citation tail of citation distribution holds most of the information about high scientific performance. Here I propose the x-index, which is calculated from the number of national articles in the top 1% and 0.1% of highly cited papers and has a subtractive term to discount highly cited papers that are not scientific breakthroughs. The x-index, the number of Nobel Prize achievements, and the number of national articles in Nature or Science are highly correlated. The high correlations among these independent parameters demonstrate that they are good measures of high scientific performance because scientific excellence is their only common characteristic. However, the x-index has superior features as compared to the other two parameters. Nobel Prize achievements are low frequency events and their number is an imprecise indicator, which in addition is zero in most institutions; the evaluation of research making use of the number of publications in prestigious journals is not advised. CONCLUSION: The x-index is a simple and precise indicator for high research performance.

Potassium and sodium uptake systems in fungi. The transporter diversity of Magnaporthe oryzae.

In this study, we report an inventory of the K(+) uptake systems in 62 fungal species for which the complete genome sequences are available. This inventory reveals that three types of K(+) uptake systems, TRK and HAK transporters and ACU ATPases, are widely present in several combinations across fungal species. PAT ATPases are less frequently present and are exceptional in Ascomycota. The genome of Magnaporthe oryzae contains four TRK, one HAK, and two ACU genes. The study of the expression of these genes at high K(+), K(+) starvation, and in infected rice leaves revealed that the expression of four genes, ACU1, ACU2, HAK1, and TRK1 is much lower than that of TRK2, TRK3, and TRK4, except under K(+) starvation. The two ACU ATPases were cloned and functionally identified as high-affinity K(+) or Na(+) uptake systems. These two ATPases endow Saccharomyces cerevisiae with the capacity to grow for several generations in low Na(+) concentrations when K(+) was absent, which produces a dramatic increase of cellular Na(+)/K(+) ratio.

The SOS1 transporter of Physcomitrella patens mediates sodium efflux in planta.

• SOS1 is an Na(+)/H(+) antiporter that plays a central role in Na(+) tolerance in land plants. SOS1 mediation of Na(+) efflux has been studied in plasma-membrane vesicles and deduced from the SOS1 suppression of the Na(+) sensitivity of yeast mutants defective in Na(+) -efflux. However, SOS1-mediated Na(+) efflux has not been characterized in either plant or yeast cells. Here, we use Physcomitrella patens to investigate the function of SOS1 in planta. • In P. patens, a nonvascular plant in which the study of ion cellular fluxes is technically simple, the existence of two SOS1 genes suggests that the Na(+) efflux remaining after the deletion of the ENA1 ATPase is mediated by a SOS1 system. Therefore, we cloned the P. patens SOS1 and SOS1B genes (PpSOS1 and PpSOS1B, respectively) and complementary DNAs, and constructed the PpΔsos1 and PpΔena1/PpΔsos1 deletion lines by gene targeting. • Comparison of wild-type, and PpΔsos1 and PpΔena1/PpΔsos1 mutant lines revealed that PpSOS1 is crucial for Na(+) efflux and that the PpΔsos1 line, and especially the PpΔena1/PpΔsos1 lines, showed excessive Na(+) accumulation and Na(+)-triggered cell death. The PpΔsos1 and PpΔena1/PpΔsos1 lines showed impaired high-affinity K(+) uptake. • Our data support the hypothesis that PpSOS1 mediates cellular Na(+) efflux and that PpSOS1 enhances K(+) uptake by an indirect effect.

Sodium or potassium efflux ATPase a fungal, bryophyte, and protozoal ATPase.

The K(+) and Na(+) concentrations in living cells are strictly regulated at almost constant concentrations, high for K(+) and low for Na(+). Because these concentrations correspond to influx-efflux steady states, K(+) and Na(+) effluxes and the transporters involved play a central role in the physiology of cells, especially in environments with high Na(+) concentrations where a high Na(+) influx may be the rule. In eukaryotic cells two P-type ATPases are crucial in these homeostatic processes, the Na,K-ATPase of animal cells and the H(+)-ATPase of fungi and plants. In fungi, a third P-type ATPase, the ENA ATPase, was discovered nineteen years ago. Although for many years it was considered to be exclusively a fungal enzyme, it is now known to be present in bryophytes and protozoa. Structurally, the ENA (from exitus natru: exit of sodium) ATPase is very similar to the sarco/endoplasmic reticulum Ca(2+) (SERCA) ATPase, and it probably exchanges Na(+) (or K(+)) for H(+). The same exchange is mediated by Na(+) (or K(+))/H(+) antiporters. However, in eukaryotic cells these antiporters are electroneutral and their function depends on a DeltapH across the plasma membrane. Therefore, the current notion is that the ENA ATPase is necessary at high external pH values, where the antiporters cannot mediate uphill Na(+) efflux. This occurs in some fungal environments and at some points of protozoa parasitic cycles, which makes the ENA ATPase a possible target for controlling fungal and protozoan parasites. Another technological application of the ENA ATPase is the improvement of salt tolerance in flowering plants.

High-affinity sodium uptake in land plants.

High-affinity Na(+) uptake in plants and its mediation by HKT transporters have been studied in very few species. This study expands the knowledge of high-affinity Na(+) uptake in land plants for both uptake characteristics and involvement of HKT transporters. In non-flowering plants, we analyzed the Na(+) content of wild mosses, carried out experiments on K(+) and Na(+) uptake in the micromolar range of concentrations with the moss Physcomitrella patens and the liverwort Riccia fluitans, studied a Deltahkt1 mutant of P. patens and identified the HKT genes of the lycopodiophyta (clubmoss) Selaginella moellendorffii. In flowering plants we studied Na(+) uptake in the micromolar range of concentrations in 16 crop plant species, identified the HKT transporters that could mediate high-affinity Na(+) uptake in several species of the Triticeae tribe, and described some characteristics of high-affinity Na(+) uptake in other species. Our results suggest that high-affinity Na(+) uptake occurs in most land plants. In very few of them, rice and species in the Triticeae and Aveneae tribes of the Poaceae family, it is probably mediated by HKT transporters. In other plants, high-affinity Na(+) uptake is mediated by one or several transporters whose responses to the presence of K(+) or Ba(2+) are fundamentally different from those of HKT transporters.

Role of ENA ATPase in Na(+) efflux at high pH in bryophytes.

Potassium or Na(+) efflux ATPases, ENA ATPases, are present in all fungi and play a central role in Na(+) efflux and Na(+) tolerance. Flowering plants lack ENA ATPases but two ENA ATPases have been identified in the moss Physcomitrella patens, PpENA1 and PpENA2. PpENA1 mediates Na(+) efflux in Saccharomyces cerevisiae. To propose a general function of ENA ATPases in bryophytes it was necessary to demonstrate that these ATPases mediate Na(+) efflux in planta and that they exist in more bryophytes than P. patens. For these demonstrations (1) we cloned a third ATPase from P. patens, PpENA3, and studied the expression pattern of the three PpENA genes; (2) we constructed and studied the single and double Deltappena1 and Deltappena2 mutants; and (3) we cloned two ENA ATPases from the liverwort Marchantia polymorpha, MpENA1 and MpENA2, and expressed them in S. cerevisiae. The results from the first two approaches revealed that the expression of ENA ATPases was greatly enhanced at high pH and that Na(+) efflux at high pH depended on PpENA1. The ENA1 ATPase of M. polymorpha suppressed the defective growth of a S. cerevisiae mutant at high K(+) or Na(+) concentrations, especially at high K(+).

Diversity in expression patterns and functional properties in the rice HKT transporter family.

Plant growth under low K(+) availability or salt stress requires tight control of K(+) and Na(+) uptake, long-distance transport, and accumulation. The family of membrane transporters named HKT (for High-Affinity K(+) Transporters), permeable either to K(+) and Na(+) or to Na(+) only, is thought to play major roles in these functions. Whereas Arabidopsis (Arabidopsis thaliana) possesses a single HKT transporter, involved in Na(+) transport in vascular tissues, a larger number of HKT transporters are present in rice (Oryza sativa) as well as in other monocots. Here, we report on the expression patterns and functional properties of three rice HKT transporters, OsHKT1;1, OsHKT1;3, and OsHKT2;1. In situ hybridization experiments revealed overlapping but distinctive and complex expression patterns, wider than expected for such a transporter type, including vascular tissues and root periphery but also new locations, such as osmocontractile leaf bulliform cells (involved in leaf folding). Functional analyses in Xenopus laevis oocytes revealed striking diversity. OsHKT1;1 and OsHKT1;3, shown to be permeable to Na(+) only, are strongly different in terms of affinity for this cation and direction of transport (inward only or reversible). OsHKT2;1 displays diverse permeation modes, Na(+)-K(+) symport, Na(+) uniport, or inhibited states, depending on external Na(+) and K(+) concentrations within the physiological concentration range. The whole set of data indicates that HKT transporters fulfill distinctive roles at the whole plant level in rice, each system playing diverse roles in different cell types. Such a large diversity within the HKT transporter family might be central to the regulation of K(+) and Na(+) accumulation in monocots.

Growth at high pH and sodium and potassium tolerance in media above the cytoplasmic pH depend on ENA ATPases in Ustilago maydis.

Potassium and Na(+) effluxes across the plasma membrane are crucial processes for the ionic homeostasis of cells. In fungal cells, these effluxes are mediated by cation/H(+) antiporters and ENA ATPases. We have cloned and studied the functions of the two ENA ATPases of Ustilago maydis, U. maydis Ena1 (UmEna1) and UmEna2. UmEna1 is a typical K(+) or Na(+) efflux ATPase whose function is indispensable for growth at pH 9.0 and for even modest Na(+) or K(+) tolerances above pH 8.0. UmEna1 locates to the plasma membrane and has the characteristics of the low-Na(+)/K(+)-discrimination ENA ATPases. However, it still protects U. maydis cells in high-Na(+) media because Na(+) showed a low cytoplasmic toxicity. The UmEna2 ATPase is phylogenetically distant from UmEna1 and is located mainly at the endoplasmic reticulum. The function of UmEna2 is not clear, but we found that it shares several similarities with Neurospora crassa ENA2, which suggests that endomembrane ENA ATPases may exist in many fungi. The expression of ena1 and ena2 transcripts in U. maydis was enhanced at high pH and at high K(+) and Na(+) concentrations. We discuss that there are two modes of Na(+) tolerance in fungi: the high-Na(+)-content mode, involving ENA ATPases with low Na(+)/K(+) discrimination, as described here for U. maydis, and the low-Na(+)-content mode, involving Na(+)-specific ENA ATPases, as in Neurospora crassa.

Effects of polylinker uATGs on the function of grass HKT1 transporters expressed in yeast cells.

HvHKT1 mediates K(+) or Na(+) uniport in yeast cells if the expression promoter is joined directly to the HvHKT1 cDNA, and Na(+)-K(+) symport if a 59 nucleotide polylinker is inserted. Our results show that three ATG triplets in the polylinker decreased the synthesis of the transporter and that the lower amount of transporter caused the functional change. With the rice HKT1 cDNA, the 59 nt polylinker changed the mode of Na(+) uptake from K(+)-insensitive to K(+)-inhibitable. These two modes of Na(+) uptake also occurred in rice plants.

Na+ accumulation in root symplast of sunflower plants exposed to moderate salinity is transpiration-dependent.

Twenty-day-old sunflower plants (Helianthus annuus L. cv Sun-Gro 380) grown hydroponically under controlled conditions were used to study the effect of transpiration on Na(+) compartmentalization in roots. The plants were exposed to low Na(+) concentrations (25 mM NaCl) and different environmental humidity conditions over a short time period (8.5 h). Under these conditions, Na(+) was accumulated primarily in the root, but only the Na(+) accumulated in the root symplast was dependent on transpiration, while the Na(+) accumulated in both the shoot and the root apoplast exhibited a low transpiration dependence. Moreover, Na(+) content in the root apoplast was reached quickly (0.25 h) and increased little with time. These results suggest that, in sunflower plants under moderate salinity conditions, Na(+) uptake in the root symplast is mediated by a transport system whose activity is enhanced by transpiration.