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 overexpression 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 electro chemical 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,hydroponic vertical farming 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. 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.Arabidopsis thaliana ecotype Columbia and Wassilewskija were used as wild type in this study. The mutants fugu5-1, fugu5-2, fugu5-3, and transgenic plants fugu5-1+IPP1 were offered and characterize by Ferjani. The cbl2 cbl3 double mutant was described in previous studies. The T-DNA insertion mutants avp1-4and mgt6were obtained from the European Arabidopsis Stock Centre and the Arabidopsis Biological Resource Center. The mutant avp1-3was a T-DNA insertion mutant in the Wassilewskija background and obtained from INRA Arabidopsis T-DNA mutant library. Mutants with multiple gene-knockout events were generated by genetic crosses, and homozygous mutant plants were screened from F2 generation and identifified by genomic PCR using primers listed in Supplementary Table S1.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. Two-week-old hydroponically grown plants were treated with 1/6 MS solutions containing 0 or 15 mM MgCl2. After two-day treatment, leaves of all the plants were collected to prepare crude membrane as described previously.Plant materials were ground at 4 ◦C with cold homogenization buffer containing 350 mM sucrose, 70 mM Tris-HCl , 3 mM Na2EDTA, 0.2% BSA, 1.5% PVP-40, 5 mM DTT, 10% glycerol, 1 mM PMSF and 1×protease inhibitor mixture . The homogenate was filtered through four layers of cheesecloth and centrifuged at 4000× g for 20 min at 4 ◦C.
The supernatant was then centrifuged at 100,000× g for 1 h. The obtained pellet was suspended in 350 mM sucrose, 10 mM Tris-Mes , 2 mM DTT and 1× protease inhibitor mixture. Pyrophosphate hydrolysis was measured as described in previous studies. The assay solution for PPi hydrolysis activity contained 25 mM Tris-Mes , 2mM MgSO4, 100 µM Na2MoO4, 0.1% Brij 58, and 200 µM Na4P2O7. PPase activity was expressed as the difference of phosphate release measured in the absence and the presence of 50 mM KCl. After incubation at 28 ◦C for 40 min, 40 mM citric acid was added to terminate reactions. For the measurement of inorganic Pi amount, freshly prepared AAM solution acetone, 2.5 mM ammoniummolybdate, 1.25 M H2SO4 was added to the reaction solution, vortexed and colorimetrically examined at 355 nm.Two-week-old hydroponically grown plants were transferred to 1/6 MS solutions containing 0 or 15 mM MgCl2. After two-day treatment, leaves of all the plants were collected and PPi was extracted from leaf tissue as described previously. Leaf samples were ground to powder in liquid nitrogen, suspended with three volumes of pure water, heated at 85 ◦C for 15 min,vertical hydroponic garden and then centrifuged at 15,000 rpm for 10 min. The supernatants were collected and then centrifuged at 40,000 rpm for 10 min. The obtained supernatants were diluted with pure water and subjected to PPi assay using a PPi Assay Kit according to the manufacturer’s instructions. Fluorescence was monitored with a Safire 2 plate reader set at 316 nm for excitation and 456 nm for emission .The Bio-remediation, Education, Science and Technology partnership provides a sustainable and contemporary approach to developing new bio-remedial technologies for U. S. Department of Defense priority contaminants while increasing the representation of underrepresented minorities and women in an exciting new bio-technical field. This comprehensive and innovative bio-remediation education program provides underrepresented groups with a cross-disciplinary bio-remediation curriculum and financial support, coupled with relevant training experiences at advanced research laboratories and field sites. These programs are designed to provide a stream of highly trained minority and women professionals to meet national environmental needs. The BEST partnership of institutions and participants benefit from a unique central strategy— shared resources across institutional boundaries. By integrating diffuse resources, BEST forms a specialized “learning institution without walls,” where participants can receive advanced training at any BEST site, and where research capabilities flow freely among the participating institutions. Ongoing faculty and student exchange programs, video taped lectures, the Rotating Scholars program, and the BEST web-site ensure that all participants are empowered with opportunities to excel. The BEST partnership consists of Lawrence Berkeley National Laboratory in Berkeley, Calif., Jackson State University in Jackson, Miss., Ana G. Méndez University System in Puerto Rico, University of Texas at El Paso , University of Southern Mississippi Gulf Coast Research Lab, and University of California at Berkeley . The BEST program contract to the partnership is managed by LBNL for the Army Corps of Engineers, Waterways Experiment Station in Vicksburg, Miss.
WES manages the contract for the Army Corps of Engineers and is the contracting entity for DoD. The partnership formed by these participating institutions leverages existing institutional resources by strengthening intramural bio-remediation education and research capabilities, and through outreach programs, to disseminate training and scientific enhancement to other Historically Black Colleges and Universities and Minority Institutions . The BEST institutions are focal points for the development and dissemination of cutting-edge research and technology for the bio-remediation of nitro-aromatic compounds, polycyclic aromatic hydrocarbons and toxic metals. The multidisciplinary BEST partnership strategy creates a flask-to-field solution that develops laboratory research into technology, and technology into field-scale environmental applications required for the cost-effective restoration of damaged environments. This year saw the addition of the University of Southern Mississippi’s Gulf Coast Research Lab and the University of Texas at El Paso as partners in the BEST program.The USM Gulf Coast Research Lab investigators’ focus on PAH and heavy metal phytoremediation along shorelines provides an exciting new focus with increased field opportunities for students. The UTEP investigators are focusing on exciting new metal phytoremediation techniques using desert plants and exciting new techniques to determine risk assessment with PAHs. This year also saw the passage of the program directorship at LBNL from Dr. Jenny Hunter-Cevera to Dr. Terry C. Hazen in October 1999. Dr. Hunter-Cevera, who has managed the BEST program at LBNL since its inception, will be sorely missed, but her new position as president of the Maryland Biotechnology Institutes may provide increased opportunities for collaboration for the entire BEST program. Dr. Hazen, who specializes in bio-remediation field applications, has demonstrated or deployed bio-remediation technologies at more than 50 sites around the United States and in Europe. He has five patents in bio-remediation technologies that are licensed by more than 40 companies in the U.S. and Europe. During the past year, the BEST program has provided minority research training for five high school students, 74 undergraduates, 32 graduate students, three post-doctoral fellows and 10 faculty. Students and faculty investigators have given 43 presentations on BEST research at scientific meetings and have published 17 scientific papers. The program produced a full color brochure and flyers in 1999 for use in recruiting more students, and also sponsored 32 lecture/seminars on bio-remediation. Fourteen videotapes of BEST seminars at LBNL/UCB were distributed to the partner institutions.