Cell-specific vacuolar calcium storage mediated by CAX1 regulates apoplastic calcium concentration, gas exchange, and plant productivity in Arabidopsis.

The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from Arabidopsis thaliana leaf cells differing in calcium concentration ([Ca], epidermis <10 mM versus mesophyll >60 mM) were compared using a microarray screen and single-cell quantitative PCR. Three tonoplast-localized Ca(2+) transporters, CAX1 (Ca(2+)/H(+)-antiporter), ACA4, and ACA11 (Ca(2+)-ATPases), were identified as preferentially expressed in Ca-rich mesophyll. Analysis of respective loss-of-function mutants demonstrated that only a mutant that lacked expression of both CAX1 and CAX3, a gene ectopically expressed in leaves upon knockout of CAX1, had reduced mesophyll [Ca]. Reduced capacity for mesophyll Ca accumulation resulted in reduced cell wall extensibility, stomatal aperture, transpiration, CO(2) assimilation, and leaf growth rate; increased transcript abundance of other Ca(2+) transporter genes; altered expression of cell wall-modifying proteins, including members of the pectinmethylesterase, expansin, cellulose synthase, and polygalacturonase families; and higher pectin concentrations and thicker cell walls. We demonstrate that these phenotypes result from altered apoplastic free [Ca(2+)], which is threefold greater in cax1/cax3 than in wild-type plants. We establish CAX1 as a key regulator of apoplastic [Ca(2+)] through compartmentation into mesophyll vacuoles, a mechanism essential for optimal plant function and productivity.

Magnesium transporters, MGT2/MRS2-1 and MGT3/MRS2-5, are important for magnesium partitioning within Arabidopsis thaliana mesophyll vacuoles.

• Magnesium accumulates at high concentrations in dicotyledonous leaves but it is not known in which leaf cell types it accumulates, by what mechanism this occurs and the role it plays when stored in the vacuoles of these cell types. • Cell-specific vacuolar elemental profiles from Arabidopsis thaliana (Arabidopsis) leaves were analysed by X-ray microanalysis under standard and serpentine hydroponic growth conditions and correlated with the cell-specific complement of magnesium transporters identified through microarray analysis and quantitative polymerase chain reaction (qPCR). • Mesophyll cells accumulate the highest vacuolar concentration of magnesium in Arabidopsis leaves and are enriched for members of the MGT/MRS2 family of magnesium transporters. Specifically, AtMGT2/AtMRS2-1 and AtMGT3/AtMRS2-5 were shown to be targeted to the tonoplast and corresponding T-DNA insertion lines had perturbed mesophyll-specific vacuolar magnesium accumulation under serpentine conditions. Furthermore, transcript abundance of these genes was correlated with the accumulation of magnesium under serpentine conditions, in a low calcium-accumulating mutant and across 23 Arabidopsis ecotypes varying in their leaf magnesium concentrations. • We implicate magnesium as a key osmoticum required to maintain growth in low calcium concentrations in Arabidopsis. Furthermore, two tonoplast-targeted members of the MGT/MRS2 family are shown to contribute to this mechanism under serpentine conditions.

Calcium delivery and storage in plant leaves: exploring the link with water flow.

Calcium (Ca) is a unique macronutrient with diverse but fundamental physiological roles in plant structure and signalling. In the majority of crops the largest proportion of long-distance calcium ion (Ca(2+)) transport through plant tissues has been demonstrated to follow apoplastic pathways, although this paradigm is being increasingly challenged. Similarly, under certain conditions, apoplastic pathways can dominate the proportion of water flow through plants. Therefore, tissue Ca supply is often found to be tightly linked to transpiration. Once Ca is deposited in vacuoles it is rarely redistributed, which results in highly transpiring organs amassing large concentrations of Ca ([Ca]). Meanwhile, the nutritional flow of Ca(2+) must be regulated so it does not interfere with signalling events. However, water flow through plants is itself regulated by Ca(2+), both in the apoplast via effects on cell wall structure and stomatal aperture, and within the symplast via Ca(2+)-mediated gating of aquaporins which regulates flow across membranes. In this review, an integrated model of water and Ca(2+) movement through plants is developed and how this affects [Ca] distribution and water flow within tissues is discussed, with particular emphasis on the role of aquaporins.

Genetic mapping of natural variation in potassium concentrations in shoots of Arabidopsis thaliana.

Naturally-occurring variation in K(+) concentrations between plant genotypes is potentially exploitable in a number of ways, including altering the relationship between K(+) accumulation and growth, enhancing salinity resistance, or improving forage quality. However, achieving these requires greater insight into the genetic basis of the variation in tissue K(+) concentrations. To this end, K(+) concentrations were measured in the shoots of 70 Arabidopsis thaliana accessions and a Cape Verdi Island/Landsberg erecta recombinant inbred line (RIL) population. The shoot K(+) concentrations expressed on the basis of fresh matter (KFM) or dry matter (KDM) were both broadly and normally distributed as was the shoot dry matter content per unit fresh weight (DMC). Using the data from the RILs, four quantitative trait loci (QTL) were identified for KFM and three for KDM. These were located on chromosomes 2, 3, 4, and 5. Two of the QTLs for KFM overlapped with those for KDM. None of these QTLs overlapped with those for fresh weight or dry weight, but the QTL for KDM located on chromosome 3 overlapped with one for DMC. In silico analysis was used to identify known or putative K(+) and cation transporter genes whose loci overlapped with the QTLs. In most cases, multiple genes were identified and the possible role of their gene products in determining shoot K(+) concentrations is discussed.

Processes modulating calcium distribution in citrus leaves. An investigation using x-ray microanalysis with strontium as a tracer.

Citrus leaves accumulate large amounts of calcium that must be compartmented effectively to prevent stomatal closure by extracellular Ca2+ and interference with Ca(2+)-based cell signaling pathways. Using x-ray microanalysis, the distribution of calcium between vacuoles in different cell types of leaves of rough lemon (Citrus jambhiri Lush.) was investigated. Calcium was accumulated principally in palisade, spongy mesophyll, and crystal-containing idioblast cells. It was low in epidermal and bundle sheath cells. Potassium showed the reverse distribution. Rubidium and strontium were used as tracers to examine the pathways by which potassium and calcium reached these cells. Comparisons of strontium and calcium distribution indicated that strontium is a good tracer for calcium, but rubidium did not mirror the potassium distribution pattern. The amount of strontium accumulated was highest in palisade cells, lowest in bundle sheath and epidermal cells, and intermediate in the spongy mesophyll. Accumulation of strontium in palisade and spongy mesophyll was accompanied by loss of potassium from these cells and its accumulation in the bundle sheath. Strontium moved apoplastically from the xylem to all cell types, and manipulation of water loss from the adaxial leaf surface suggested that diffusion is responsible for strontium movement to this side of the leaf. The results highlight the importance of palisade and spongy mesophyll as repositories for calcium and suggest that calcium distribution between different cell types is the result of differential rates of uptake. This tracer technique can provide important information about the ion uptake and accumulation properties of cells in intact leaves.

THE PRESSURE PROBE: A Versatile Tool in Plant Cell Physiology.

This review discusses how the pressure probe has evolved from an instrument for measuring cell turgor and other water relations parameters into a device for sampling the contents of individual higher plant cells in situ in the living plant. Together with a suite of microanalytical techniques it has permitted the mapping of water and solute relations at the resolution of single cells and has the potential to link quantitatively the traditionally separate areas of water relations and metabolism. The development of the probe is outlined and its modification to measure root pressure and xylem tension described. The deployment of the pressure probe to determine and map turgor, hydraulic conductivity, reflection coefficient, cell rheological properties, solute concentrations and enzyme activities at the resolution of single cells is discussed. The controversy surrounding the interpretation of results obtained with the xylem-pressure probe is included. Possible further developments of the probe and applications of single cell sampling are suggested.

Nanolitre-scale assays to determine the activities of enzymes in individual plant cells.

There are a variety of methods for characterising gene expression at the level of individual cells and for demonstrating that the cells also contain the encoded proteins. However, measuring the activity of enzymes at the resolution of single cells in complex tissues, such as leaves, is problematic. We have addressed this by using single-cell sampling to extract 10-100 pl droplets of sap from individual plant cells and then measuring enzyme activities in these droplets with nanolitre-scale fluorescence-based assays. We have optimised these assays and used them to measure and characterise the activities of acid phosphatase, cysteine protease and nitrate reductase in sap samples from epidermal and mesophyll cells of barley (Hordeum vulgare L.) and Arabidopsis thaliana leaves exposed to different developmental and environmental conditions. During leaf senescence in barley, we found that the dynamics with which acid phosphatase and protease activities changed were different in each cell type and did not mirror the changes occurring at the whole-leaf level. Increases in nitrate reductase activities after exposure of barley plants to nitrate were large in mesophyll cells but small in epidermal cells. The technique was applied successfully to Arabidopsis and, as in barley, revealed cell-specific differences in the activities of both acid phosphatase and nitrate reductase. The assays add to the spectrum of techniques available for characterising cells within complex plant tissues, thus extending the opportunity to relate gene expression to biochemical activities at the single-cell level.

Potassium activities in cell compartments of salt-grown barley leaves.

Triple-barrelled microelectrodes measuring K(+) activity (a(K)), pH and membrane potential were used to make quantitative measurements of vacuolar and cytosolic a(K) in epidermal and mesophyll cells of barley plants grown in nutrient solution with 0 or 200 mM added NaCl. Measurements of a(K) were assigned to the cytosol or vacuole based on the pH measured. In epidermal cells, the salt treatment decreased a(K) in the vacuole from 224 to 47 mM and in the cytosol from 68 to 15 mM. In contrast, the equivalent changes in the mesophyll were from 235 to 150 mM (vacuole) and 79 to 64 mM (cytosol). Thus mechanisms exist to ameliorate the effects of salt on a(K) in compartments of mesophyll cells, presumably to minimize any deleterious consequences for photosynthesis. Thermodynamic calculations showed that K(+) is actively transported into the vacuole of both epidermal and mesophyll cells of salinized and non- salinized plants. Comparison of the values of a(K) in K(+)-replete, non-salinized leaf cells with those previously measured in root cells of plants grown under comparable conditions indicates that cytosolic a(K) is similar in cells of both organs, but vacuolar a(K) in leaf cells is approximately twice that in roots. This suggests differences in the regulation of vacuolar a(K), but not cytosolic a(K), in leaf and root cells.

Quantitative trait loci analysis of nitrate storage in Arabidopsis leading to an investigation of the contribution of the anion channel gene, AtCLC-c, to variation in nitrate levels.

Storage of excess nitrate in the vacuole and its subsequent remobilization is an important aspect of a plant's nitrogen economy, but the genes controlling the underlying processes have not all been identified and characterized. Cape Verdi Island (Cvi)/Landsberg erecta (Ler) and Columbia (Col)/Landsberg erecta recombinant inbred line (RIL) populations of Arabidopsis thaliana were used to identify quantitative trait loci (QTL) controlling natural variation in nitrate concentrations. One major and two minor QTLs were found for the Cvi/Ler population and one minor QTL for the Col/Ler RIL. These were designated NA1 to NA4. The major Cvi/Ler QTL (NA3) was located at the bottom of chromosome 5. No interaction among the QTLs was found by two-way ANOVA. By comparing in silico the locations of the QTLs with a physical map of the Arabidopsis genome, candidate genes for each QTL were identified. Several of these were anion channels of the AtCLC family. One of these, AtCLC-c, coincided with NA3 and its role was investigated using a mutant with a transposon insertion in AtCLC-c. Mutant plants homozygous for the insertion (designated clcc-1) had less than 5% of AtCLC-c mRNA compared with wild-type (WT) shoots. They also had significantly lower nitrate concentrations when grown at a range of external nitrate concentrations. The concentrations of chloride, malate, and citrate were also affected in the mutant. In wild-type plants, expression of AtCLC-c was down-regulated in the presence of nitrate, but ammonium had a much smaller effect while chloride and sulphate did not affect expression. These and published results suggest that multiple genes affect nitrate concentrations in plants and that AtCLC-c and other members of the AtCLC gene family play some role in this.

A role for HKT1 in sodium uptake by wheat roots.

The high affinity potassium transporter, HKT1 from wheat was introduced into Florida wheat in sense and antisense orientation under control of a ubiquitin promoter. Ten transgenic lines expressing the transgene were identified and two of these showed strong down-regulation of the native HKT1 transcript. One line (271) was expressing the antisense construct and the other (223) was expressing a truncated sense construct. The two lines were examined further for phenotype relating to cation transport. Membrane depolarisations were measured in low (0.1 mm) K+ and high (100 mm) NaCl. Under these conditions there was no difference between line 271 and the control at low K+, but at high Na+ there was a rapid depolarisation that was significantly larger in control plants. 22Na uptake was measured in this line and there was a significant decrease in uptake at 100 mm NaCl in the transgenic line when compared with the control. The two transgenic lines were grown at high NaCl (200 mm) and analysed for growth and root sodium content. Lines 271 and 223 showed enhanced growth under salinity when compared with the control and had lower sodium in the root. Secondary ion mass spectrometry (SIMS) analysis of transverse sections of the root showed that Na+ and K+ were strongly localised to stelar regions when compared with other ions, and that the Na+ : K+ ratios were reduced in salt-stressed transgenic tissue when compared with the control.