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Title: Vacuolar Proton Pyrophosphatase Is Required for High Magnesium Tolerance in Arabidopsis
Magnesium (Mg2+) is an essential nutrient in all organisms. However, high levels of Mg2+ in the environment are toxic to plants. In this study, we identified the vacuolar-type H+-pyrophosphatase, AVP1, as a critical enzyme for optimal plant growth under high-Mg conditions. The Arabidopsis avp1 mutants displayed severe growth retardation, as compared to the wild-type plants upon excessive Mg2+. Unexpectedly, the avp1 mutant plants retained similar Mg content to wild-type plants under either normal or high Mg conditions, suggesting that AVP1 may not directly contribute to Mg2+ homeostasis in plant cells. Further analyses confirmed that the avp1 mutant plants contained a higher pyrophosphate (PPi) content than wild type, coupled with impaired vacuolar H+-pyrophosphatase activity. Interestingly, expression of the Saccharomyces cerevisiae cytosolic inorganic pyrophosphatase1 gene IPP1, which facilitates PPi hydrolysis but not proton translocation into vacuole, rescued the growth defects of avp1 mutants under high-Mg conditions. These results provide evidence that high-Mg sensitivity in avp1 mutants possibly resulted from elevated level of cytosolic PPi. Moreover, genetic analysis indicated that mutation of AVP1 was additive to the defects in mgt6 and cbl2 cbl3 mutants that are previously known to be impaired in Mg2+ homeostasis. Taken together, our results suggest AVP1 is required for more » cellular PPi homeostasis that in turn contributes to high-Mg tolerance in plant cells. « less
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International Journal of Molecular Sciences
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National Science Foundation
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  1. Abstract

    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 andmore »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.

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  2. Abstract

    Multiple transporters and channels mediate cation transport across the plasma membrane and tonoplast to regulate ionic homeostasis in plant cells. However, much less is known about the molecular function of transporters that facilitate cation transport in other organelles such as Golgi. We report here thatArabidopsisKEA4, KEA5, and KEA6, members of cation/proton antiporters‐2 (CPA2) superfamily were colocalized with the known Golgi marker, SYP32‐mCherry. Although singlekea4,5,6mutants showed similar phenotype as the wild type under various conditions,kea4/5/6triple mutants showed hypersensitivity to low pH, high K+, and high Na+and displayed growth defects in darkness, suggesting that these three KEA‐type transporters function redundantly in controlling etiolated seedling growth and ion homeostasis. Detailed analysis indicated that thekea4/5/6triple mutant exhibited cell wall biosynthesis defect during the rapid etiolated seedling growth and under high K+/Na+condition. The cell wall‐derived pectin homogalacturonan (GalA)3partially suppressed the growth defects and ionic toxicity in thekea4/5/6triple mutants when grown in the dark but not in the light conditions. Together, these data support the hypothesis that the Golgi‐localized KEAs play key roles in the maintenance of ionic and pH homeostasis, thereby facilitating Golgi function in cell wall biosynthesis during rapid etiolated seedling growth and in coping with high K+/Na+stress.

  3. Summary

    Two types of tonoplast proton pumps, H+‐pyrophosphatase (V‐PPase) and the H+‐ATPase (V‐ATPase), establish the proton gradient that powers molecular traffic across the tonoplast thereby facilitating turgor regulation and nutrient homeostasis. However, how proton pumps regulate development remains unclear.

    In this study, we investigated the function of two types of proton pumps in Arabidopsis embryo development and pattern formation. While disruption of either V‐PPase or V‐ATPase had no obvious effect on plant embryo development, knocking out both resulted in severe defects in embryo pattern formation from the early stage.

    While the first division in wild‐type zygote was asymmetrical, a nearly symmetrical division occurred in the mutant, followed by abnormal pattern formation at all stages of embryo development. The embryonic defects were accompanied by dramatic differences in vacuole morphology and distribution, as well as disturbed localisation of PIN1. The development of mutant cotyledons and root, and the auxin response of mutant seedlings supported the hypothesis that mutants lacking tonoplast proton pumps were defective in auxin transport and distribution.

    Taking together, we proposed that two tonoplast proton pumps are required for vacuole morphology and PIN1 localisation, thereby controlling vacuole and auxin‐related developmental processes in Arabidopsis embryos and seedlings.

  4. Abstract

    Cellular pyrophosphate (PPi) homeostasis is vital for normal plant growth and development. Plant proton‐pumping pyrophosphatases (H+‐PPases) are enzymes with different tissue‐specific functions related to the regulation of PPi homeostasis. Enhanced expression of plant H+‐PPases increases biomass and yield in different crop species. Here, we emphasise emerging studies utilising heterologous expression in yeast and plant vacuole electrophysiology approaches, as well as phylogenetic relationships and structural analysis, to showcase that the H+‐PPases possess a PPi synthesis function. We postulate this synthase activity contributes to modulating and promoting plant growth both in H+‐PPase‐engineered crops and in wild‐type plants. We propose a model where the PPi synthase activity of H+‐PPases maintains the PPi pool when cells adopt PPi‐dependent glycolysis during high energy demands and/or low oxygen environments. We conclude by proposing experiments to further investigate the H+‐PPase‐mediated PPi synthase role in plant growth.

  5. Abstract

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