In Arabidopsis, a tonoplast-localized proton-pumping pyrophosphatase AVP1 was shown to be the key enzyme for cytosolic PPi metabolism in different cell types of various plants. This enzyme activity has been correlated with the important function that AVP1 plays in many physiological processes. Arabidopsis fugu5 mutants lacking functional AVP1 show elevated levels of cytosolic PPi and display heterotrophic growth defects resulting from the inhibition of gluconeogenesis. This important role in controlling PPi level in plant cells is reinforced by a recent study showing that higher-order mutants defective in both tonoplast and cytosolic pyrophosphatases display much severe phenotypes including plant dwarfism, ectopic starch accumulation, decreased cellulose and callose levels, and structural cell wall defects. Moreover, the tonoplast-localized H+ -PPase AVP1 appears to be a predominant contributor to the regulation of cellular PPi levels because the quadruple knockout mutant lacking cytosolic PPase isoforms ppa1 ppa2 ppa4 ppa5 showed no obvious phenotypes. 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,nft vertical farming 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, and homeostasis associated with their different sub-cellular 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. The originally reported T-DNA insertional mutant avp1-1 contains an additional T-DNA insertion causing phenotypes unrelated to AVP1 mutation. We thus characterized another T-DNA insertion line avp1-4for this study. The avp1-4 mutant carried a T-DNA insertion in the third exon of AVP1 as further confirmed by PCR analysis and DNA sequencing . The avp1-4 homozygous mutants lacked detectable AVP1 transcripts , and its tonoplast PPi hydrolysis activity was considerably diminished, to only 10% of wild type . Compared with wild-type plants , avp1-4 mutants exhibited no obvious phenotypic changes during the life cycle including vegetative and reproductive periods , which is quite different from avp1-1, because pleiotropic phenotypes observed in avp1-1 are caused by mutation in the GNOMgene. We examined the phenotype of avp1-4 plants under multiple ionic stress conditions and found that avp1-4 mutant and wild-type seedlings grew similarly on the MS medium and did not show hypersensitive response to most of the ionic stresses such as 60 mM Na+ , 60 mM K+ , 40 mM Ca2+, 100 µM Zn2+, 40 µM Cu2+, or 100 µM Fe3+ . However, the growth of avp1-4 seedlings were severely impaired when 20 mM MgCl2 was supplemented . To validate the hypersensitivity of avp1-4 to MgCl2, we grew the seedlings of the mutant together with the wild-type plants on the 1/6 MS medium containing various levels of Mg2+, the avp1-4 mutant plants were clearly stunted as compared with Col-0 , although the primary root length of avp1 was comparable to that of Col-0 . In addition, we also studied one more mutant allele of AVP1 gene in the Wassilewskija background, designated as avp1-3, and another three mutant alleles of AVP1, fugu5-1, fugu5-2, and fugu5-3 in the Col-0 background. Measurements of seedling fresh weight confirmed a severe growth inhibition by 8 mM MgCl2 in both avp1-4 and avp1-3 mutants, as compared with their respective wild-type counterparts . Consistently, we also found that high-Mg sensitivity phenotypes in the three fugu5 mutants were comparable to those in avp1-4 . Together, these results suggested that AVP1 is required for Mg2+ tolerance in Arabidopsis. To verify that the observed phenotypes in the avp1 mutants are caused by a defect in AVP1, we conducted a complementation test in avp1-4 background. A coding sequence fragment of AVP1 was introduced into the avp1-4 mutant, and several homozygous transgenic lines were obtained . Phenotypic analysis of two representative lines showed that oblong-shaped cotyledons of avp1-4 when germinated on MS media containing low sucrose or in soil were fully restored to normal shape .
In addition, seedling growth defects of avp1-4 under high-Mg conditions were also completely rescued . Root length and shoot fresh weight of the transgenic lines under high Mg conditions were similar to those of the wild type . These data further confirmed that loss-of-function in AVP1 was indeed the causal mutation for the high-Mg hypersensitive phenotype of avp1-4.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 .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,indoor vertical farming the mutant plants exhibited a pronounced growth defect than wild-type plants in the hydroponic solutions supplemented with 15 mM external Mg2+, as revealed bymuch 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. To address the contribution of PPi hydrolysis activity to high-Mg tolerance, we directly measured V-PPase activity and PPi content under normal and high-Mg conditions. Under normal conditions, PPi hydrolysis activity of two avp1 mutant alleles was reduced by ∼85%, whereas activity from two complementary lines was comparable to the wild-type control . Consistently, the amount of PPi from both mutants was increased by ∼50% . After grown for three days on 15 mM Mg2+, all the plants displayed reduced PPi hydrolysis activity and higher PPi content. However, thePPi elevation of mutant plants during high Mg2+ stress was significantly higher than that of wild type . Altogether, these results strongly indicate that the dampened hydrolysis of cytosolic PPi is the major reason for the increased Mg sensitivity in the avp1 mutants.To assess whether increased Mg2+ sensitivity in the avp1 mutant is associated with Mg2+ homeostasis, we measured the Mg content in wild-type and mutant plants using ICP-MS. When 8 mM Mg2+ was added to the growth medium, Mg content in either shoot or root in all the plants was strikingly elevated, but no significant difference between wild-type and mutant plants in Mg content was observed. . Considering Ca and Mg often affect each other in their uptake and transport, we also measured the Ca content in the same plants.
Consistent with Mg-Ca antagonism, the Ca content in both wild-type and avp1 mutant plants was evidently lower when plants were grown under high external Mg2+ conditions, but Ca content in the shoots and roots in avp1 mutants was similar to that in wild-type plants . These data suggest that both Mg and Ca homeostasis are not altered in the avp1 mutants, which are consistent with the earlier conclusion that PPi hydrolysis rather than vacuolar acidification is responsible for AVP1 function under high-Mg stress.In Arabidopsis, the magnesium transporter MGT6 is important for controlling plant Mg2+ homeostasis and adaptation to both low- and high-Mg conditions. To investigate the functional interaction between AVP1 and MGT6, we created a double mutant that lacks both AVP1 and MGT6transcripts . We next tested the sensitivity of avp1-4 mgt6 double mutant to high external Mg conditions. When grown on the 1/6 MS medium containing 0.25 mM Mg2+, the mgt6 and avp1-4 mgt6 plants showed obvious growth retardation compared with Col-0 and avp1-4 seedlings, resulting from mgt6 mutation that renders plants hypersensitive to low Mg2+ . When the medium Mg2+ levels reached 1 mM, the growth of mgt6 and avp1-4 mgt6 mutants appeared comparable to that of wild-type . Notably, in the presence of high Mg levels such as 4 mM and 6 mM Mg2+ , avp1-4 mgt6 double mutant exhibited more severe inhibition of shoot growth with significantly lower fresh weight and more reduced chlorophyll content as compared to either mgt6 or avp1 single mutant. The enhanced sensitivity of the avp1-4 mgt6 double mutant suggest that AVP1 and MGT6 may represent two independent functions that are required for plant tolerance to high Mg2+ stresses. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 15 resulting from mgt6 mutation that renders plants hypersensitive to low Mg2+ . When the medium Mg2+ levels reached 1 mM, the growth of mgt6 and avp1-4 mgt6 mutants appeared comparable to that of wild-type . Notably, in the presence of high Mg levels such as 4 mM and 6 mM Mg2+ , avp1-4 mgt6 double mutant exhibited more severe inhibition of shoot growth with significantly lower fresh weight and more reduced chlorophyll content as compared to either mgt6 or avp1 single mutant. 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.