Interestingly, in companion cells of the phloem, AVP1 was also shown to be localized to the plasma membrane and function as a PPi synthase that contribute to phloem loading, photosynthate partitioning, and energy metabolism. On the other hand, AVP1 is also believed to contribute to the establishment of electrochemical potential across the vacuole membrane, which is important for subsequent vacuolar secondary transport and ion sequestration. Constitutive over expression of AVP1 improves the growth and yield of diverse transgenic plants under various abiotic stress conditions—including drought, salinity, as well as phosphorus and nitrogen deficiency—although the mechanism remains to be fully understood. Taken together, AVP1 serves as a multi-functional protein involved a variety of physiological processes in plants, some of which await to be fully understood. Magnesium is an essential macro-nutrient for plant growth and development, functioning in numerous biological processes and cellular functions, including chlorophyll biosynthesis and carbon fixation. Either deficiency or excess of Mg in the soil could be detrimental to plant growth and therefore plants have evolved multiple adaptive mechanisms to maintain cellular Mg concentration within an optimal range. In higher plants, the most well-documented Mg2+ transporters belong to homologues of bacterial CorA super family and are also called “MRS2” based on their similarity to yeast Mitocondrial RNA splicing 2 protein. Several members of the MGT family mediate Mg2+ transport in bacteria or yeast as indicated by functional complementation as well as 63Ni tracer assay . In plants, they have been shown to play vital roles in Mg2+ uptake, translocation,square planter pots and homeostasis associated with their different subcellular localizations and diverse tissue-specific expression patterns. For instance, MGT2 and MGT3 are tonoplast localized and possibly involved in
Mg2+ partitioning into mesophyll vacuoles; MGT4, MGT5, and MGT9 are strongly expressed in mature anthers and play a crucial role in pollen development and male fertility. MGT6 and MGT7 are shown to be most directly involved in Mg homeostasis because knocking-down or knocking-out either of the genes leads to hypersensitivity to low Mg conditions. MGT6 encodes a plasma membrane-localized high-affinity Mg2+ transporter and mediates Mg2+ uptake in root hairs, particularly under Mg-limited conditions . MGT7 is also preferentially expressed in roots and loss-of-function of MGT7 caused poor seed germination and severe growth retardation under low-Mg conditions. Double mutant of mgt6 and mgt7 displayed a stronger phenotype than single mutants, suggesting that MGT6 and MGT7 may be synergistic in controlling Mg homeostasis in low-Mg environment conditions. In contrast to considerable research on Mg transport and homeostasis under Mg deficient conditions, the regulatory mechanisms required for adaptation to excessive external Mg remain poorly understood. Recent studies suggested that MGT6 and MGT7 are essential for plants to adapt to both normal and high Mg conditions. The mgt6 mutant displayed dramatic growth defects with a decrease in cellular Mg content in the shoot, when grown under high Mg2+. Grafting experiments further suggested a shoot-based mechanism for Mg2+ detoxification although the exact role of MGT6 in this process is still not clear. More importantly, a core regulatory pathway consisting of two calcineurin B-like Ca sensors partnering with four CBL-interacting protein kinases has been established that allows plant cells to sequester Mg2+ into plant vacuoles, thereby protecting plant cells from high Mg2+ toxicity. In this study, we identified the tonoplast pyrophosphatase, AVP1, as an important component in high Mg2+ tolerance in Arabidopsis. Furthermore, by analyzing the avp1-4 mgt6 double mutant and avp1-4 cbl2 cbl3 triple mutant, we showed that the role of AVP1 in high-Mg tolerance was independent of previously reported MGT6 or CBL/CIPK-mediated pathway. Instead, our results suggested a novel link between high Mg2+ stress and PPi homeostasis in plants. Reducing the PPi concentration in the cytoplasm and increasing the acidification of vacuoles represent the two main biochemical functions of AVP1.
In order to dissect if both activities are required in this specific high Mg2+-associated process, we resorted to the transgenic line expressing yeast IPP1 gene under the control of the AVP1 promoter in the fugu5-1 mutant background. IPP1 is a cytosolic soluble protein which is not capable of translocating H+ , thus decoupling the hydrolysis and proton pump activities. Interestingly, our results showed that the severely retarded growth of fugu5-1 mutant plants under high-Mg conditions was completely recovered by expression of the IPP1 gene . The quantitative analysis of seedling fresh weight confirmed the complementation . To extend the phenotypic analysis of the avp1 mutants in mature plants, we examined the phenotype of avp1 mutants using hydroponic culture system. Consistent with the patterns of plant growth on agar plates, the mutant plants exhibited a pronounced growth defect than wild-type plants in the hydroponic solutions supplemented with 15 mM external Mg2+, as revealed by much lower fresh weight and lower chlorophyll content . The IPP1 transgenic line also behaved like wild-type plants but not avp1 mutant under this condition, suggesting that PPi hydrolysis is the key function that AVP1 plays in high-Mg adaptation. Although Mg is an essential macro-nutrient required for plant growth, high concentrations of environmental Mg2+ could be detrimental, and the targets underlying toxic effect of high-Mg are not well understood. In the present study, we characterized multiple avp1 mutant alleles and found they were hypersensitive to high external Mg2+. This finding has not only improved our understanding of the mechanism underlying Mg2+ tolerance but also uncovered a novel physiological function of AVP1 in plants. When the plants were confronted with high Mg stress, sequestration of excessive Mg2+ into the vacuole plays a vital role in detoxification of Mg excess from the cytoplasm . The AVP1 protein predominantly localized in the vacuolar membrane and was a highly abundant component of the tonoplast proteome. Encoded by AVP1, vacuolar H+ -PPase, together with vacuolar H+ -ATPase, plays a critical part in establishing the electrochemical potential by pumping H+ across the vacuolar membrane. This proton gradient, in turn, facilitates secondary fluxes of ions and molecules across the tonoplast. Based on this well-established idea, we hypothesized that avp1 mutants may be impaired in cellular ionic homeostasis and should thus exhibit hypersensitivity to a broad range of ions.
However, unexpectedly, we found that avp1 was hypersensitive only to high external Mg2+ but not to other cations . It was shown that over expression of AVP1 improved plant salt tolerance in quite a few species, which was interpreted as the result of increased sequestration of Na+ into the vacuole. It is thus reasonable to speculate that the tonoplast electrochemical potential generated by AVP1 would likewise favor Mg2+ transport into vacuoles via secondary Mg2+/H+ antiporter. Surprisingly, our subsequent experiments did not support this hypothesis and several lines of evidence suggested that the hypersensitivity of avp1 to high Mg2+ was not due to the compromised Mg2+ homeostasis in the mutant. First, unlike other high Mg2+-sensitive mutants such as mgt6 and the vacuolar cbl/cipk mutants, the Mg and Ca content in the avp1 mutant was not altered as compared with wild type, suggesting that AVP1 may not be directly involved in Mg2+ transport in plant cells. Second, higher order mutants of the avp1-4 mgt6 double mutant and avp1-4 cbl2 cbl3 triple mutant displayed a dramatic enhancement in Mg2+ sensitivity as compared to single mutants. These genetic data strongly suggest that AVP1 does not function in the same pathway mediated by MGT6 and does not serve as a target for vacuolar CBL-CIPK. Moreover,blueberry grow it was previously shown that either vacuolar H+ -ATPase double mutant vha-a2 vha-a3 or the mhx1 mutant defective in the proposed Mg2+/H+ antiporter was not hypersensitive to high Mg2+. These results implicate the vacuolar Mg2+ compartmentalization should be fulfilled by an unknown Mg2+ transporter/channel, whose activity is largely not dependent on the tonoplast ∆pH. Identification of this novel Mg2+ transport system across the tonoplast, which is probably targeted by vacuolar CBL-CIPK complexes, would be the key to understand the mechanism. Third, expression of the cytosolic soluble pyrophosphatase isoform IPP1 could fully rescue the Mg-hypersensitivity caused by AVP1 mutation. These lines of evidence pinpoint PPi hydrolysis, rather than ∆pH-assisted secondary ion transport and sequestration, as the major function of AVP1 in high Mg2+ adaptation. Under high Mg stress conditions, a number of adaptive responses are supposed to take place in plants, including the remodeling of plant morphogenesis as well as reprogramming of the gene expression and metabolite profile. However, very little is known so far and therefore, the molecular components targeted by excessive Mg2+ in plant cells remain obscure. Here, we suggest that the concentration of cellular PPi could be responsive to external Mg supply. Our results showed that extremely high levels of Mg2+ led to inhibition of the PPase activity in Arabidopsis, which in turn, resulted in the elevation of PPi content in the cytosol. Because high level of PPi is very toxic, the efficient removal of PPi by AVP1 under high Mg2+ conditions might become one of the limiting factors for optimal plant growth. This idea is supported by the observation that avp1 mutants accumulated significantly higher PPi content under high Mg2+ conditions compared with normal conditions . Most importantly, heterologous expression of the soluble PPase IPP1 gene rescued high Mg-sensitive phenotype of fugu5-1 , which strongly suggested that high Mg2+ hypersensitivity phenotype in avp1 mutants could primarily be attributed to impaired PPi homeostasis.It would be interesting to investigate how PPi concentrations vary in different Mg2+ conditions and during different plant growth stages.
Recently, cytosolic soluble pyrophosphatases were identified in Arabidopsis, and were shown to physiologically cooperate with the vacuolar H+ -PPase in regulating cytosolic PPi levels. Future studies should clarify if this type of soluble isoenzymes is also involved in the same high-Mg adaptation process. Collectively, our findings provide genetic and physiological evidence that AVP1 is a new component required for plant growth under high external Mg2+ concentrations and functions in regulating Mg2+ tolerance via PPi hydrolysis. For on-plate growth assays, seeds of different genotypes were sterilized with 75% ethanol for 10 min, washed in sterilized water for three times, and sown on Murashige and Skoog medium containing 2% sucrose and solidified with 0.8% phytoblend . The plates were incubated at 4 ◦C in darkness for two days and then were positioned vertically at 22 ◦C in growth chamber with a 14 h light/10 h dark photoperiod. After germination, five-day-old seedlings were transferred onto agarose-solidified media containing various ions as indicated in the figure legends and were grown under 14 h light/10 h dark photoperiod. For phenotypic assay in the hydroponics, 10-day-old seedlings geminated on MS plate were transferred to 1/6 strength MS solution and were grown under the 14 h light/10 h dark condition in the plant growth chamber. Fresh liquid solutions were replaced once a week. After two-week culture, the plants were treated with 1/6 MS solutions supplemented with 15 mM MgCl2. The majority of the current production, use, and disposal of engineered nanomaterials occur in terrestrial environments, and consequently terrestrial ecosystems are and will increasingly be some of the largest receptors of ENMs at all stages of their life cycles. In particular, soil is predicted to be one of the major receptors of ENMs due to ENM-contaminated bio-solid fertilizer and nanopesticide application to agricultural fields, runoff from landfills or ENM-bearing paints, or atmospheric deposition. Both agricultural and natural systems are at risk to ENM contamination via these release scenarios, which makes it necessary to understand the interactions between ENMs, soils, and soil organisms such as plants in order to predict their impacts in real-world scenarios. Gravity-driven vertical transport of TiO2, CeO2, and Cu2 engineered nanomaterials and their effects on soil pH and nutrient release were measured in three unsaturated soils. ENM transport was found to be highly limited in natural soils collected from farmland and grasslands, with the majority of particles being retained in the upper 0-3 cm of the soil profile, while greater transport depth was seen in a commercial potting soil. Physical straining appeared to be the primary mechanism of retention in natural soils as ENMs immediately formed micron-scale aggregates, which was exacerbated by coating particles with Suwannee River natural organic matter . Changes in soil pH were observed in natural soils contaminated with ENMs that were largely independent of ENM type and concentration. These changes may have been due to enhanced release of naturally present pH-altering ions in the soil, likely via substitution processes. This suggests ENMs will likely be highly retained near source zones in soil and may impact local communities sensitive to changes in pH or nutrient availability.