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,square pot 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, 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. 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, the PPi 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,blueberries in containers 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. 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, 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.