Scanning ion‐selective electrode technique and X‐ray microanalysis provide direct evidence of contrasting Na+ transport ability from root to shoot in salt‐sensitive cucumber and salt‐tolerant pumpkin under NaCl stress

Grafting onto salt‐tolerant pumpkin rootstock can increase cucumber salt tolerance. Previous studies have suggested that this can be attributed to pumpkin roots with higher capacity to limit the transport of Na⁺ to the shoot than cucumber roots. However, the mechanism remains unclear. This study investigated the transport of Na⁺ in salt‐tolerant pumpkin and salt‐sensitive cucumber plants under high (200 mM) or moderate (90 mM) NaCl stress. Scanning ion‐selective electrode technique showed that pumpkin roots exhibited a higher capacity to extrude Na⁺, and a correspondingly increased H⁺ influx under 200 or 90 mM NaCl stress. The 200 mM NaCl induced Na⁺/H⁺ exchange in the root was inhibited by amiloride (a Na⁺/H⁺ antiporter inhibitor) or vanadate [a plasma membrane (PM) H⁺‐ATPase inhibitor], indicating that Na⁺ exclusion in salt stressed pumpkin and cucumber roots was the result of an active Na⁺/H⁺ antiporter across the PM, and the Na⁺/H⁺ antiporter system in salt stressed pumpkin roots was sufficient to exclude Na⁺. X‐ray microanalysis showed higher Na⁺ in the cortex, but lower Na⁺ in the stele of pumpkin roots than that in cucumber roots under 90 mM NaCl stress, suggesting that the highly vacuolated root cortical cells of pumpkin roots could sequester more Na⁺, limit the radial transport of Na⁺ to the stele and thus restrict the transport of Na⁺ to the shoot. These results provide direct evidence for pumpkin roots with higher capacity to limit the transport of Na⁺ to the shoot than cucumber roots.     

Changes in root development of Arabidopsis promoted by organic matter from oxisols

The presence of ramified and abundant lateral roots is fundamental to plant growth on highly weathered soils. In this work, the effects of humic acid (HA), fulvic acid and hexanic–methanolic (HM) extract, after alkaline extraction from topsoil of seven different oxisols, on the development of roots of Arabidopsis thaliana was evaluated. Furthermore, we used another emergent plant model, that is, micro‐tom (MT) tomatoes with size similar to that of Arabidopsis to test the effects of HA. It was observed that both humic fractions and the HM extract were able to change the root development, improving the number of lateral roots and their development in comparison to control plants. The promotion of root growth by the three organic matter fractions was higher than that observed by 10^?6 mol L^?1 indole acetic acid. The treatment of MT tomato mutant, less sensitive to auxin, with HA did not promote the emergence of lateral roots, being an additional indication of auxin‐like activities of HA. However, some organic matter fractions exhibited, besides promotion of lateral roots number, increase in the length of principal root, which is not a typical auxin effect, indicating that these substances could contain other physiologically active substances.     

Humic acid differentially improves nitrate kinetics under low‐ and high‐affinity systems and alters the expression of plasma membrane H+‐ATPases and nitrate transporters in rice

Humic acids (HAs) have a major effect on nutrient uptake, metabolism, growth and development in plants. Here, we evaluated the effect of HA pretreatment applied with a nutrient solution on the uptake kinetics of nitrate nitrogen (N‐NO₃^?) and the metabolism of nitrogen (N) in rice under conditions of high and low NO₃^? supply. In addition, the kinetic parameters of NO₃^? uptake, N metabolites, and nitrate transporters (NRTs) and the plasma membrane (PM) H⁺‐ATPase gene expression were examined. The plants were grown in a growth chamber with modified Hoagland and Arnon solution until 21 days after germination (DAG), and they were then transferred to a solution without N for 48 h and then to another solution without N and with and without the addition of HAs for another 48 h. After this period of N deprivation, the plants received new nutrient solutions containing 0.2 and 2.0 mM N‐NO₃^?. Treatment of rice plants with HA promoted the induction of the genes OsNRT2.1‐2.2/OsNAR2.1 and some isoforms PM H⁺‐ATPase in roots. The application of HAs differentially modified the parameters of the uptake kinetics of NO₃^? under both concentrations. When grown with 0.2 mM NO₃^?, the plants pretreated with HA had lower K_m and C_min values as well as a higher V_max/K_m ratio. When grown with 2 mM NO₃^?, the plants pretreated with HA had a higher V_max value, a greater root and shoot mass, and a lower root/shoot ratio. The N fractions were also altered by pretreatment with HA, and a greater accumulation of NO₃^? and N‐amino was observed in the roots and shoots, respectively, of plants pretreated with HA. The results suggest that pretreatment with HA modifies root morphology and gene expression of PM H⁺‐ATPases and NO₃^? transporters, resulting in a greater efficiency of NO₃^? acquisition by high‐ and low‐affinity systems.     

Molecular identification and functional characterization of GhAMT1.3 in ammonium transport with a high affinity from cotton (Gossypium hirsutum L.)

Ammonium (NH₄⁺) represents a primary nitrogen source for many plants, its effective transport into and between tissues and further assimilation in cells determine greatly plant nitrogen use efficiency. However, biological components involved in NH₄⁺ movement in woody plants are unclear. Here, we report kinetic evidence for cotton NH₄⁺ uptake and molecular identification of certain NH₄⁺ transporters (AMTs) from cotton (Gossypium hirustum). A substrate‐influx assay using ¹⁵N‐isotope revealed that cotton possessed a high‐affinity transport system with a K_m of 58 μM for NH₄⁺. Sequence analysis showed that GhAMT1.1–1.3 encoded respectively a membrane protein containing 485, 509 or 499 amino acids. Heterologous functionality test demonstrated that GhAMT1.1–1.3 expression mediated NH₄⁺ permeation across the plasma membrane (PM) of yeast and/or Arabidopsis qko‐mutant cells, allowing a growth restoration of both mutants on NH₄⁺. Quantitative PCR measurement showed that GhAMT1.3 was expressed in roots and leaves and markedly up‐regulated by N‐starvation, repressed by NH₄⁺ resupply and regulated diurnally and age‐dependently, suggesting that GhAMT1.3 should be a N‐responsive gene. Importantly, GhAMT1.3 expression in Arabidopsis improved plant growth on NH₄⁺ and enhanced total nitrogen accumulation (～50% more), conforming with the observation of 2‐fold more NH₄⁺ absorption by GhAMT1.3‐transformed qko plant roots during a 1‐h root influx period. Together with its targeting to the PM and saturated transport kinetics with a K_m of 72 μM for NH₄⁺, GhAMT1.3 is suggested to be a high‐affinity NH₄⁺ permease that may play a significant role in cotton NH₄⁺ acquisition and utilization, adding a new member in the plant AMT family.     

Conserved V‐ATPase c subunit plays a role in plant growth by influencing V‐ATPase‐dependent endosomal trafficking

In plant cells, the vacuolar‐type H⁺‐ATPases (V‐ATPase) are localized in the tonoplast, Golgi, trans‐Golgi network and endosome. However, little is known about how V‐ATPase influences plant growth, particularly with regard to the V‐ATPase c subunit (VHA‐c). Here, we characterized the function of a VHA‐c gene from Puccinellia tenuiflora (PutVHA‐c) in plant growth. Compared to the wild‐type, transgenic plants overexpressing PutVHA‐c in Arabidopsis thaliana exhibit better growth phenotypes in root length, fresh weight, plant height and silique number under the normal and salt stress conditions due to noticeably higher V‐ATPase activity. Consistently, the Arabidopsis atvha‐c5 mutant shows reduced V‐ATPase activity and retarded plant growth. Furthermore, confocal and immunogold electron microscopy assays demonstrate that PutVHA‐c is mainly localized to endosomal compartments. The treatment of concanamycin A (ConcA), a specific inhibitor of V‐ATPases, leads to obvious aggregation of the endosomal compartments labelled with PutVHA‐c‐GFP. Moreover, ConcA treatment results in the abnormal localization of two plasma membrane (PM) marker proteins Pinformed 1 (AtPIN1) and regulator of G protein signalling‐1 (AtRGS1). These findings suggest that the decrease in V‐ATPase activity blocks endosomal trafficking. Taken together, our results strongly suggest that the PutVHA‐c plays an important role in plant growth by influencing V‐ATPase‐dependent endosomal trafficking.     

The role of plasma membrane H+‐ATPase in jasmonate‐induced ion fluxes and stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) elicits stomatal closure in many plant species. Stomatal closure is accompanied by large ion fluxes across the plasma membrane (PM). Here, we recorded the transmembrane ion fluxes of H⁺, Ca²⁺ and K⁺ in guard cells of wild‐type (Col‐0) Arabidopsis, the CORONATINE INSENSITIVE1 (COI1) mutant coi1‐1 and the PM H⁺‐ATPase mutants aha1‐6 and aha1‐7, using a non‐invasive micro‐test technique. We showed that MeJA induced transmembrane H⁺ efflux, Ca²⁺ influx and K⁺ efflux across the PM of Col‐0 guard cells. However, this ion transport was abolished in coi1‐1 guard cells, suggesting that MeJA‐induced transmembrane ion flux requires COI1. Furthermore, the H⁺ efflux and Ca²⁺ influx in Col‐0 guard cells was impaired by vanadate pre‐treatment or PM H⁺‐ATPase mutation, suggesting that the rapid H⁺ efflux mediated by PM H⁺‐ATPases could function upstream of the Ca²⁺ flux. After the rapid H⁺ efflux, the Col‐0 guard cells had a longer oscillation period than before MeJA treatment, indicating that the activity of the PM H⁺‐ATPase was reduced. Finally, the elevation of cytosolic Ca²⁺ concentration and the depolarized PM drive the efflux of K⁺ from the cell, resulting in loss of turgor and closure of the stomata.     

Adaptation of plasma membrane H+ ATPase and H+ pump to P deficiency in rice roots

The plasma membrane (PM) H⁺ ATPase is involved in the plant response to nutrient deficiency. However, adaptation of this enzyme in monocotyledon plants to phosphorus (P) deficiency lacks direct evidence. In this study, we detected that P deficient roots of rice (Oryza Sativa L.) could acidify the rhizosphere. We further isolated the PM from rice roots and analyzed the activity of PM H⁺ ATPase. In vitro, P deficient rice roots showed about 30% higher activity of PM H⁺ ATPase than the P sufficient roots at assay of pH 6.0. The P deficiency resulted in a decrease of the substrate affinity value (K _m ) of PM H⁺ ATPase. The proton pumping activity of membrane vesicles from the P deficient roots was about 70% higher than that from P sufficient roots. Western blotting analysis indicated that higher activity of PM H⁺ ATPase in P deficient roots was related to a slightly increase of PM H⁺ ATPase protein abundance in comparison with that in P sufficient roots. Taken together, our results demonstrate that the P deficiency enhanced activities of both PM H⁺-ATPase and H⁺ pump, which contributed to the rhizosphere acidification in rice roots.     

Proton pumping by tomato roots. Effect of Fe deficiency and hormones on the activity and distribution of plasma membrane H+-ATPase in rhizodermal cells

The immunocytochemical localization of the plasma membrane H⁺‐ATPase in epidermal cells of tomato roots was studied using a monoclonal antibody raised against purified maize P‐type H⁺‐ATPase. Plants subjected to iron starvation exhibited increased proton extrusion that was confined to the root elongation zones. Immunogold labelling of the H⁺‐ATPase on the plasma membrane was considerably higher in rhizodermal cells within zones with intense proton extrusion than in non‐acidifying areas of the roots. Transfer cells were formed in rhizodermal cells of Fe‐deficient plants. Quantitative determination of immunolabelling revealed that the density of PM H⁺‐ATPase in transfer cells was about twice that of ordinary epidermal cells. In transfer cells, H⁺‐ATPase was most abundant on the plasma membrane lining the labyrinthine invaginations of the peripheral cell wall. While the number of immunologically detectable ATPase molecules in transfer cells was not spatially correlated with proton extrusion activity, the frequency of transfer cells was considerably higher in acidifying root areas relative to non‐active segments. Split‐root experiments indicated that both the steady‐state level of plasma membrane H⁺‐ATPase and proton extrusion activity are systemically regulated, indicating inter‐organ regulation of rhizosphere acidification. Exogenous application of the auxin analog 2,4‐dichlorophenoxyacetic acid and the ethylene precursor 1‐aminocyclopropane‐1‐carboxlic acid caused the formation of transfer cells at a frequency similar to that observed in Fe‐deficient roots. However, the number of proton pumps was not affected by the hormone treatment, suggesting that both responses are regulated independently. It is concluded that transfer cells in the rhizodermis may be important but not crucial for rhizosphere acidification.     

Aluminum‐dependent dynamics of ion transport in Arabidopsis: specificity of low pH and aluminum responses

Low‐pH and Al³⁺ stresses are the major causes of poor plant growth in acidic soils. However, there is still a poor understanding of plant responses to low‐pH and Al³⁺ toxicity. Low‐pH or combined low‐pH and Al³⁺ stress was imposed in order to measure rhizosphere pH, ion fluxes, plasma membrane potential and intracellular H⁺ concentration in distal elongation and mature zones (MZs) along the longitudinal axis of Arabidopsis thaliana roots. Low‐pH stress facilitated H⁺ influx into root tissues and caused cytoplasmic acidification; by contrast, combined low‐pH/Al³⁺ treatment either decreased H⁺ influx in the distal elongation zone (DEZ) or induced H⁺ efflux in the MZ, leading to cytoplasmic alkalinization in both zones. Low‐pH stress induced an increase in rhizosphere pH in the DEZ, whereas combined low‐pH/Al³⁺ stress resulted in lower rhizosphere pH in both root zones compared with the low‐pH treatment alone. Low‐pH stress facilitated K⁺ efflux; the presence of Al³⁺ diminished K⁺ efflux or favored K⁺ influx into root tissues. In both zones, low‐pH treatment induced plasma membrane (PM) depolarization, which was significantly diminished (P≤ 0.05) when combined stresses (low‐pH/100 µM Al³⁺) were imposed. After 60 min of exposure, low pH caused PM depolarization, whereas low pH/100 µM Al³⁺ caused PM hyperpolarization. Thus, low pH and Al³⁺ toxicity differentially affect root tissues and, consequently, the rhizosphere, which might underpin the differential mechanisms of plant adaptation to these abiotic stresses.     

Auxin biosynthetic gene TAR2 is involved in low nitrogen‐mediated reprogramming of root architecture in Arabidopsis

In plants, the plasticity of root architecture in response to nitrogen availability largely determines nitrogen acquisition efficiency. One poorly understood root growth response to low nitrogen availability is an observed increase in the number and length of lateral roots (LRs). Here, we show that low nitrogen‐induced Arabidopsis LR growth depends on the function of the auxin biosynthesis gene TAR2 (tryptophan aminotransferase related 2). TAR2 was expressed in the pericycle and the vasculature of the mature root zone near the root tip, and was induced under low nitrogen conditions. In wild type plants, low nitrogen stimulated auxin accumulation in the non‐emerged LR primordia with more than three cell layers and LR emergence. Conversely, these low nitrogen‐mediated auxin accumulation and root growth responses were impaired in the tar2‐c null mutant. Overexpression of TAR2 increased LR numbers under both high and low nitrogen conditions. Our results suggested that TAR2 is required for reprogramming root architecture in response to low nitrogen conditions. This finding suggests a new strategy for improving nitrogen use efficiency through the engineering of TAR2 expression in roots.     

Evidence that exogenous urea acts as a potent cue to alleviate ammonium‐inhibition of root system growth of cotton plant (Gossypium hirsutum)

Many plants grown with low‐millimolar concentration of NH₄⁺ as a sole nitrogen source develop NH₄⁺‐toxicity symptoms. To date, crucial molecular identities and a practical approach involved in the improvement of plant NH₄⁺‐tolerance remain largely unknown. By phenotyping of upland cotton grown on varied nitrogen forms, we came across a phenomenon that caused sub‐millimolar concentrations of urea (e.g., up 50 μM) to repress the growth inhibition of roots and whole plant cultivated in a NH₄⁺‐containing nutrient solution. A growth‐recovery assay revealed that the relief in NH₄⁺‐inhibited growth required only a short‐term exposure (≧12 h) of the roots to urea, implying that urea could elicit an internal signaling and be involved in antagonizing NH₄⁺‐sensitivity. Intriguingly, split‐root experiments demonstrated that low urea occurrence in one root‐half could efficaciously stimulate not only supplied root but also the root‐half grown in NH₄⁺‐solution without urea, indicating the existence of urea‐triggered local and systemic long‐distance signaling. In the split‐root experiment we also observed high arginase activity, strong arginine reduction and remarkable upregulation of polyamine biosynthesis‐related genes (ADC1/2, SPDS and SPMS). Therefore, we suggest that external urea might serve as an effective cue (signal molecule) in an arginine‐/polyamine‐related process for ameliorating NH₄⁺‐suppressed root growth, providing a novel aspect for deeper exploring and understanding plant NH₄⁺‐tolerance.     

TFT6 and TFT7, two different members of tomato 14-3-3 gene family, play distinct roles in plant adaption to low phosphorus stress

14‐3‐3 proteins are a large family of proteins but exact roles of their members in plant response to abiotic stresses are not clear, especially under nutrient deficiency. We investigated the expressions of all the tomato 14‐3‐3 gene family members (TFT1–TFT12) under low phosphorus stress (LP) and found that TFT6 belongs to the later responsive gene while TFT7 belongs to the early responsive gene. When the two genes were separately introduced into Arabidopsis and overexpressed, their plant growth under LP was much enhanced compared with wild‐type plant. TFT6 overexpressing plants showed reduced starch synthase activity, reduced starch content but enhanced sucrose loading into phloem in the shoot under LP. TFT7 overexpressing plants had much enhanced H⁺ flux along their root tip and activity of plasma membrane H⁺‐ATPase in the roots under LP. Our results suggest that TFT6 and TFT7 play different roles in plant adaption to LP. TFT6 acts mainly in leaves and is involved in the systemic response to LP by regulating leaf carbon allocation and increasing phloem sucrose transport to promote root growth, while TFT7 directly functions in root by activating root plasma membrane H⁺‐ATPase to release more protons under LP.     

Plasma membrane ATPase and H+ transport activities in microsomal membranes from mycorrhizal tomato roots

ATPase activity, ATP-dependent H⁺ transport and the amount of antigenic tomato plasma membrane H⁺-APTase have been analysed in membrane vesicles isolated from Glomus mosseae- or Glomus intraradices-colonized roots and from non-mycorrhizal tomato roots. Microsomal protein content was higher in mycorrhizal than in control roots. The specific activity of the plasma membrane H⁺-ATPase was not affected by mycorrhizal colonization, although this activity increased in membranes isolated from mycorrhizal roots when expressed on a fresh weight basis. Western blot analysis of microsomal proteins using antibodies raised against the Arabidopsis thaliana plasma membrane H⁺ - ATPase showed that mycorrhizal colonization did not change the relative amount of tomato plasma membrane ATPase in the microsomes. However, on a fresh weight basis, there was a greater amount of this protein in roots of mycorrhizal plants. In addition, mycorrhizal membranes showed a higher specific activity of the vanadate-sensitive ATP-dependant H⁺ transport than membranes isolated from control roots. These results suggest that mycorrhiza might regulate the plasma membrane ATPase by increasing the coupling efficiency between H⁺ transport and ATP hydrolysis. The observed effects of mycorrhizal colonization on plasma membrane H⁺-ATPase were independent of the AM fungal species colonizing the root system.     

Reduced Plasma Membrane H+-ATPase Isoform OsA7 Expression and Proton Pump Activity Decrease Growth Without Affecting Nitrogen Accumulation in Rice

Plasma membrane H⁺-ATPase (PM H⁺-ATPase, EC 3.6.1.3.) is a proton pump that is necessary to promote cell growth and ion fluxes across the plasma membrane. The main goal of this study was to evaluate the role of PM H⁺-ATPase isoform OsA7 expression in rice growth and nitrogen (N) accumulation using three genetically engineered lineages with artificial micro RNA (amiRNA) targeting OsA7 (osa7.1, osa7.2, and osa7.3). PM H⁺-ATPase isoform expression in rice shoots and roots (wild-type) revealed that OsA7 is highly expressed in roots and is the most highly expressed PM H⁺-ATPase isoform. The three osa7 lineages had lower fresh weight, grain yield, height, and 1000-grain weight compared to control IRS plants. The hydroponic experiment comprised three NO₃⁻ levels over 30 days: 0.2 mM NO₃⁻–N, 2.0 mM NO₃⁻–N, and NO₃⁻ starvation for 3 days. The three osa7 lineages had lower PM H⁺-ATPase and V-H⁺-PPase activity as compared to the IRS plants. The root and shoot fresh weights were lower in osa7 lineages. The root/shoot ratio was lower in the osa7 lineages cultivated without nitrogen for 3 days and with 0.2 mM of NO₃⁻–N as compared to IRS, and did not change in plants cultivated with 2.0 mM NO₃⁻–N. The total N concentration did not change in the three osa7 lineages as compared to IRS. Overall, the results indicate that OsA7 is important for rice growth, grain production, and root growth, but does not affect N accumulation, highlighting the importance of other PM H⁺-ATPase isoforms in N uptake.

     

A novel fluorescence imaging approach to monitor salt stress‐induced modulation of ouabain‐sensitive ATPase activity in sunflower seedling roots

Seedlings exposed to salt stress are expected to show modulation of intracellular accumulation of sodium ions through a variety of mechanisms. Using a new methodology, this work demonstrates ouabain (OU)‐sensitive ATPase activity in the roots of sunflower seedlings subjected to salt stress (120 mM NaCl). 9‐Anthroylouabain (a derivative of ouabain known to inhibit Na⁺,K⁺‐ATPase activity in animal systems, EC 3.6.3.9) has been used as a probe to analyze OU‐sensitive ATPase activity in sunflower (Helianthus annuus) seedling roots by spectrofluorometric estimation and localization of its spatial distribution using confocal laser scanning microscopy. Salt stress for 48 h leads to a significant induction of OU‐sensitive ATPase activity in the meristematic region of the seedling roots. Calcium ions (10 mM) significantly inhibit enzyme activity and a parallel accumulation of sodium ions in the cytosol of the columella cells, epidermis and in the cells of the meristematic region of the roots is evident. As a rapid response to NaCl stress, the activity of OU‐sensitive ATPase gets localized in the nuclear membrane of root protoplasts and it gets inhibited after treatment with calcium ions. Nuclear membrane localization of the OU‐sensitive ATPase activity highlights a possible mechanism to efflux sodium ions from the nucleus. Thus, a correlation between OU‐sensitive ATPase activity, its modulation by calcium ions and accumulation of sodium ions in various regions of the seedling roots, has been demonstrated using a novel approach in a plant system.