Two transporters mobilize magnesium from vacuolar stores to enable plant acclimation to magnesium deficiency.

Magnesium (Mg) is an essential metal for chlorophyll biosynthesis and other metabolic processes in plant cells. Mg is largely stored in the vacuole of various cell types and remobilized to meet cytoplasmic demand. However, the transport proteins responsible for mobilizing vacuolar Mg2+ remain unknown. Here, we identified two Arabidopsis (Arabidopsis thaliana) Mg2+ transporters (MAGNESIUM TRANSPORTER 1 and 2; MGT1 and MGT2) that facilitate Mg2+ mobilization from the vacuole, especially when external Mg supply is limited. In addition to a high degree of sequence similarity, MGT1 and MGT2 exhibited overlapping expression patterns in Arabidopsis tissues, implying functional redundancy. Indeed, the mgt1 mgt2 double mutant, but not mgt1 and mgt2 single mutants, showed exaggerated growth defects as compared to the wild type under low-Mg conditions, in accord with higher expression levels of Mg-starvation gene markers in the double mutant. However, overall Mg level was also higher in mgt1 mgt2, suggesting a defect in Mg2+ remobilization in response to Mg deficiency. Consistently, MGT1 and MGT2 localized to the tonoplast and rescued the yeast (Saccharomyces cerevisiae) mnr2Δ (manganese resistance 2) mutant strain lacking the vacuolar Mg2+ efflux transporter. In addition, disruption of MGT1 and MGT2 suppressed high-Mg sensitivity of calcineurin B-like 2 and 3 (cbl2 cbl3), a mutant defective in vacuolar Mg2+ sequestration, suggesting that vacuolar Mg2+ influx and efflux processes are antagonistic in a physiological context. We further crossed mgt1 mgt2 with mgt6, which lacks a plasma membrane MGT member involved in Mg2+ uptake, and found that the triple mutant was more sensitive to low-Mg conditions than either mgt1 mgt2 or mgt6. Hence, Mg2+ uptake (via MGT6) and vacuolar remobilization (through MGT1 and MGT2) work synergistically to achieve Mg2+ homeostasis in plants, especially under low-Mg supply in the environment.

Two tonoplast MATE proteins function as turgor-regulating chloride channels in Arabidopsis.

The central vacuole in a plant cell occupies the majority of the cellular volume and plays a key role in turgor regulation. The vacuolar membrane (tonoplast) contains a large number of transporters that mediate fluxes of solutes and water, thereby adjusting cell turgor in response to developmental and environmental signals. We report that two tonoplast Detoxification efflux carrier (DTX)/Multidrug and Toxic Compound Extrusion (MATE) transporters, DTX33 and DTX35, function as chloride channels essential for turgor regulation in Arabidopsis Ectopic expression of each transporter in Nicotiana benthamiana mesophyll cells elicited a large voltage-dependent inward chloride current across the tonoplast, showing that DTX33 and DTX35 each constitute a functional channel. Both channels are highly expressed in Arabidopsis tissues, including root hairs and guard cells that experience rapid turgor changes during root-hair elongation and stomatal movements. Disruption of these two genes, either in single or double mutants, resulted in shorter root hairs and smaller stomatal aperture, with double mutants showing more severe defects, suggesting that these two channels function additively to facilitate anion influx into the vacuole during cell expansion. In addition, dtx35 single mutant showed lower fertility as a result of a defect in pollen-tube growth. Indeed, patch-clamp recording of isolated vacuoles indicated that the inward chloride channel activity across the tonoplast was impaired in the double mutant. Because MATE proteins are widely known transporters of organic compounds, finding MATE members as chloride channels expands the functional definition of this large family of transporters.

Tonoplast CBL-CIPK calcium signaling network regulates magnesium homeostasis in Arabidopsis.

Although Mg(2+) is essential for a myriad of cellular processes, high levels of Mg(2+) in the environment, such as those found in serpentine soils, become toxic to plants. In this study, we identified two calcineurin B-like (CBL) proteins, CBL2 and CBL3, as key regulators for plant growth under high-Mg conditions. The Arabidopsis mutant lacking both CBL2 and CBL3 displayed severe growth retardation in the presence of excess Mg(2+), implying elevated Mg(2+) toxicity in these plants. Unexpectedly, the cbl2 cbl3 mutant plants retained lower Mg content than wild-type plants under either normal or high-Mg conditions, suggesting that CBL2 and CBL3 may be required for vacuolar Mg(2+) sequestration. Indeed, patch-clamp analysis showed that the cbl2 cbl3 mutant exhibited reduced Mg(2+) influx into the vacuole. We further identified four CBL-interacting protein kinases (CIPKs), CIPK3, -9, -23, and -26, as functionally overlapping components downstream of CBL2/3 in the signaling pathway that facilitates Mg(2+) homeostasis. The cipk3 cipk9 cipk23 cipk26 quadruple mutant, like the cbl2 cbl3 double mutant, was hypersensitive to high-Mg conditions; furthermore, CIPK3/9/23/26 physically interacted with CBL2/3 at the vacuolar membrane. Our results thus provide evidence that CBL2/3 and CIPK3/9/23/26 constitute a multivalent interacting network that regulates the vacuolar sequestration of Mg(2+), thereby protecting plants from Mg(2+) toxicity.

A vacuolar phosphate transporter essential for phosphate homeostasis in Arabidopsis.

Inorganic phosphate (Pi) is stored in the vacuole, allowing plants to adapt to variable Pi availability in the soil. The transporters that mediate Pi sequestration into vacuole remain unknown, however. Here we report the functional characterization of Vacuolar Phosphate Transporter 1 (VPT1), an SPX domain protein that transports Pi into the vacuole in Arabidopsis. The vpt1 mutant plants were stunted and consistently retained less Pi than wild type plants, especially when grown in medium containing high levels of Pi. In seedlings, VPT1 was expressed primarily in younger tissues under normal conditions, but was strongly induced by high-Pi conditions in older tissues, suggesting that VPT1 functions in Pi storage in young tissues and in detoxification of high Pi in older tissues. As a result, disruption of VPT1 rendered plants hypersensitive to both low-Pi and high-Pi conditions, reducing the adaptability of plants to changing Pi availability. Patch-clamp analysis of isolated vacuoles showed that the Pi influx current was severely reduced in vpt1 compared with wild type plants. When ectopically expressed in Nicotiana benthamiana mesophyll cells, VPT1 mediates vacuolar influx of anions, including Pi, SO4(2-), NO3(-), Cl(-), and malate with Pi as that preferred anion. The VPT1-mediated Pi current amplitude was dependent on cytosolic phosphate concentration. Single-channel analysis showed that the open probability of VPT1 was increased with the increase in transtonoplast potential. We conclude that VPT1 is a transporter responsible for vacuolar Pi storage and is essential for Pi adaptation in Arabidopsis.

Conserved mechanism for vacuolar magnesium sequestration in yeast and plant cells.

Magnesium (Mg2+) is an essential nutrient for all life forms. In fungal and plant cells, the majority of Mg2+ is stored in the vacuole but mechanisms for Mg2+ transport into the vacuolar store are not fully understood. Here we demonstrate that members of ancient conserved domain proteins (ACDPs) from Saccharomyces cerevisiae and Arabidopsis thaliana function in vacuolar Mg2+ sequestration that enables plant and yeast cells to cope with high levels of external Mg2+. We show that the yeast genome (as well as other fungal genomes) harbour a single ACDP homologue, referred to as MAM3, that functions specifically in vacuolar Mg2+ accumulation and is essential for tolerance to high Mg. In parallel, vacuolar ACDP homologues were identified from Arabidopsis and shown to complement the yeast mutant mam3Δ. An Arabidopsis mutant lacking one of the vacuolar ACDP homologues displayed hypersensitivity to high-Mg conditions and accumulated less Mg in the vacuole compared with the wild type. Taken together, our results suggest that conserved transporters mediate vacuolar Mg2+ sequestration in fungal and plant cells to maintain cellular Mg2+ homeostasis in response to fluctuating Mg2+ levels in the environment.

Four plasma membrane-localized MGR transporters mediate xylem Mg2+ loading for root-to-shoot Mg2+ translocation in Arabidopsis.

Magnesium (Mg2+), an essential structural component of chlorophyll, is absorbed from the soil by roots and transported to shoots to support photosynthesis in plants. However, the molecular mechanisms underlying root-to-shoot Mg2+ translocation remain largely unknown. We describe here the identification of four plasma membrane (PM)-localized transporters, named Mg2+ release transporters (MGRs), that are critical for root-to-shoot Mg transport in Arabidopsis. Functional complementation assays in a Mg2+-uptake-deficient bacterial strain confirmed that these MGRs conduct Mg2+ transport. PM-localized MGRs (MGR4, MGR5, MGR6, and MGR7) were expressed primarily in root stellar cells and participated in the xylem loading step of the long-distance Mg2+ transport process. In particular, MGR4 and MGR6 played a major role in shoot Mg homeostasis, as their loss-of-function mutants were hypersensitive to low Mg2+ but tolerant to high Mg2+ conditions. Reciprocal grafting analysis further demonstrated that MGR4 functions in the root to determine shoot Mg2+ accumulation and physiological phenotypes caused by both low- and high-Mg2+ stress. Taken together, our study has identified the long-sought transporters responsible for root-to-shoot Mg2+ translocation in plants.

Spatio-temporal dynamics of arbuscular mycorrhizal fungi and soil organic carbon in coastal saline soil of China.

A comprehensive understanding of the relationship between arbuscular mycorrhizal (AM) fungi and coastal saline soil organic carbon (SOC) is crucial for analysis of the function of coastal wetlands in soil carbon sequestration. In a field experiment, the temporal and spatial dynamics of AM fungi, glomalin-related soil protein (GRSP) - which is described as a N-linked glycoprotein and the putative gene product of AM fungi, SOC, and soil aggregates were investigated in halophyte Kosteletzkya virginica rhizosphere soil of coastal saline areas of North Jiangsu, China. Soil samples were collected from a depth of up to 30 cm in two plantation regions from August 2012 to May 2013. Results showed K. virginica formed a strong symbiotic relationship to AM fungi. AM colonization and spore density were the highest in the 10-20 cm soil layer of Jinhai farm in August 2012, because of the presence of numerous fibrous roots in this soil layer. The total GRSP and SOC were the highest in the 0-10 cm soil layer in May 2013 and November 2012, respectively. Correlation coefficient analysis revealed that AM colonization and spore density were positively correlated with total GRSP. Meanwhile, total GRSP was significantly positively correlated with large macroaggregates (>3 mm), SOC, total P, Olsen P, and soil microbial biomass carbon (SMBC), but negatively correlated with microaggregates (<0.25 mm), soil EC, total N, and pH. SOC was positively correlated with spore density, large macroaggregates, small macroaggregates (2-0.25 mm), alkaline N, and SMBC and negatively correlated with microaggregates, EC, pH, and total K. Although it may be a statistical artifact, we found an interesting phenomenon that there was no significant correlation between soil aggregates and AM colonization or spore density. Hence, total GRSP is a vital source of saline soil C pool and an important biological indicator for evaluating coastal saline SOC pool and soil fertility, while AM colonization or spore density may not be.

A calcium signalling network activates vacuolar K+ remobilization to enable plant adaptation to low-K environments.

Potassium (K) is an essential nutrient, but levels of the free K ions (K+) in soil are often limiting, imposing a constant stress on plants. We have discovered a calcium (Ca2+)-dependent signalling network, consisting of two calcineurin B-like (CBL) Ca2+ sensors and a quartet of CBL-interacting protein kinases (CIPKs), which plays a key role in plant response to K+ starvation. The mutant plants lacking two CBLs (CBL2 and CBL3) were severely stunted under low-K conditions. Interestingly, the cbl2 cbl3 mutant was normal in K+ uptake but impaired in K+ remobilization from vacuoles. Four CIPKs-CIPK3, 9, 23 and 26-were identified as partners of CBL2 and CBL3 that together regulate K+ homeostasis through activating vacuolar K+ efflux to the cytoplasm. The vacuolar two-pore K+ (TPK) channels were directly activated by the vacuolar CBL-CIPK modules in a Ca2+-dependent manner, presenting a mechanism for the activation of vacuolar K+ remobilization that plays an important role in plant adaptation to K+ deficiency.

Author Correction: A calcium signalling network activates vacuolar K+ remobilization to enable plant adaptation to low-K environments.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Plant Membrane Transport Research in the Post-genomic Era.

Membrane transport processes are indispensable for many aspects of plant physiology including mineral nutrition, solute storage, cell metabolism, cell signaling, osmoregulation, cell growth, and stress responses. Completion of genome sequencing in diverse plant species and the development of multiple genomic tools have marked a new era in understanding plant membrane transport at the mechanistic level. Genes coding for a galaxy of pumps, channels, and carriers that facilitate various membrane transport processes have been identified while multiple approaches are developed to dissect the physiological roles as well as to define the transport capacities of these transport systems. Furthermore, signaling networks dictating the membrane transport processes are established to fully understand the regulatory mechanisms. Here, we review recent research progress in the discovery and characterization of the components in plant membrane transport that take advantage of plant genomic resources and other experimental tools. We also provide our perspectives for future studies in the field.

[Allelopathy of Phragmites australis on Spartina alterniflora under different salinity].

Using Phragmites australis litter as test material, two kinds of its mother liquid, i. e., water extract and decomposed products, were obtained by the methods of solution extraction and anaerobic decomposition. The two liquids with concentration 100% and 25% were used to treat the seeds and seedlings of Spartina alterniflora and the endemic beneficial fungi (Mortierella sp.) of S. alterniflora under 5 per thousand and 10 per thousand salinity, aimed to study the effects of different salinity on the germination and growth of S. alterniflora and the growth of the fungi. Salt stress promoted the seed germination and young root growth of Spartina alterniflora as well as the growth of Mortierella sp. significantly (P<0.05). The water extracts promoted the germination and growth of Spartina alterniflora and the growth of Mortierella sp. when the salinity was 10%o, but the promotion effect disappeared when the salinity was reduced to 5 per thousand. The decomposed products of Phragmites australis restrained the germination of Spartina alterniflora significantly (P<0.05) when the salinity was 5 per thousand, but promoted the growth of Mortierella sp. significantly (P<0.05) when the salinity was 10 per thousand. Under the two salinities, the decomposed products restrained the growth of Spartina alterniflora seedlings and young roots significantly (P<0.05).