Vacuolar Proton Pyrophosphatase Is Required for High Magnesium Tolerance in Arabidopsis

Abstract

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 cellular PPi homeostasis that in turn contributes to high-Mg tolerance in plant cells.

Keywords: AtAVP1; cellular PPi homeostasis; high-Mg tolerance; vacuolar H+-pyrophosphatase.

Figure 1

The avp1 mutant plants are hypersensitive to high-Mg conditions. (a) Schematic diagram of the AVP1 gene. Coding regions are depicted as black boxes, and the 5’ or 3’ UTRs are shown as shaded boxes and lines represent introns to scale. The molecular lesion in each of the five loss-of-function avp1 alleles is indicated by open triangles or asterisks. (b) Growth phenotype of wild types Col-0 and Ws and corresponding mutant plants avp1-4 and avp1-3 under different ionic stress conditions. Five-day-old seedlings were transferred onto 1/6 MS medium or 1/6 MS medium supplemented with 8 mM MgCl₂. Photographs were taken on the 10th day after transfer. (c) Growth phenotype of Col-0 and three fugu5 mutant alleles on 1/6 MS medium or 1/6 MS medium supplemented with 5 mM MgCl₂. (d) Root length and (e) fresh weight of seedlings on the 10th day after transfer. Data are presented as the mean ± SD of four replicate experiments. Asterisks indicate statistically significant differences compared with the Col-0 or Ws (Student’s t-test, * p < 0.05).

Figure 2

Functional complementation of avp1-4 and fugu5-1 under high-Mg stress grown on agar plates or in hydroponic culture. (a) Growth phenotype of wild type Col-0, avp1-4 and corresponding complementary line avp1-4+AVP1 on 1/6 MS medium or 1/6 MS medium supplemented with 8 mM MgCl₂. Photographs were taken on the 10th day after transfer. (b) Root length and (c) fresh weight of seedlings described in (a) on the 10th day after transfer. (d) Growth phenotype of wild type (Col-0), fugu5-1 and corresponding complementary line fugu5-1+IPP1 on 1/6 MS medium or 1/6 MS medium supplemented with 5 mM MgCl₂. Photographs were taken on the 10th day after transfer. (e) Root length and (f) fresh weight of seedlings described in (d) on the 10th day after transfer. (g) Effect of Mg²⁺ concentration on growth of Col-0, avp1-4, fugu5-1, and corresponding complemented lines. Two-week-old plants grown in 1/6 MS hydroponic culture were transferred onto 1/6 MS or 1/6 MS hydroponic culture supplemented with 15 mM MgCl₂. Photographs were taken on the 5th day after transfer. (h) Fresh weight and (i) chlorophyll content of the plants of various genotypes described in (g). Data are presented as the mean ± SD of four replicate experiments. Asterisks indicate statistically significant differences compared with the Col-0 (Student’s t-test, * p < 0.05).

Figure 3

V-PPase activities and the contents of PPi under different Mg²⁺ conditions. (a) H⁺-PPase hydrolysis activity was determined from microsomal membranes of two-week old plants of wild type (Col-0), mutants (avp1-4 and fugu5-1) and complementary lines (avp1-4+AVP1 and fugu5-1+IPP1) grown in hydroponic culture. Results are shown as percentage of the Col-0 control activity. Values are mean ± SD of three replicate experiments. (b) PPi content of seedlings under different Mg²⁺ conditions. Seedlings grown under the same conditions and collected at the same stage as described in (a) were used for the quantification of PPi. Values are mean ± SD from triplicate experiments. Asterisk indicates significant difference compared with the wild type (Student’s t-test, * p < 0.05).

Figure 4

Mg and Ca content in the avp1 mutant under different Mg²⁺ conditions. (a,b) Mg content in the root (a) and shoot (b) under different Mg²⁺ regimes. (c,d) Ca content in the root (c) and shoot (d) under different Mg²⁺ regimes. Data are presented as the mean ± SD of triplicate experiments.

Figure 5

Phenotypic analysis of Mg²⁺ sensitivity in the avp1-4 mgt6 double mutant. (a) RT-PCR analysis of AVP1 and MGT6 gene expression in wild-type Col-0, homozygous avp1-4 or mgt6 single mutant and the avp1-4 mgt6 double mutant. (b–e) Phenotypic analysis of Mg²⁺ sensitivity in avp1-4, mgt6, and avp1-4 mgt6 mutants. (f) Fresh weight of seedlings on the 10th day after transfer. (g) Chlorophyll content of seedlings on the 10th day after transfer. Data are mean ± SD from triplicate experiments. Any pair of genotypes/treatments that do not share the same letter are significantly different (p < 0.05) based on a Duncan’s multiple range test.

Figure 6

Phenotypic analysis of Mg²⁺ sensitivity in the avp1-4 cbl2 cbl3 triple mutant. (a) RT-PCR analysis of AVP1, CBL2, and CBL3 gene expression in wild type Col-0, avp1-4 single mutant, cbl2 cbl3 double mutant and avp1-4 cbl2 cbl3 triple mutant. (b–e) Phenotypic analysis of Mg²⁺ sensitivity in avp1-4, cbl2 cbl3, and avp1-4 cbl2 cbl3 mutants. (f) Fresh weight of seedlings on the 10th day after transfer. (g) Chlorophyll content of seedlings on the 10th day after transfer. Data are mean ± SD from triplicate experiments. Any pair of genotypes/treatments that do not share the same letter are significantly different (p < 0.05) based on a Duncan’s multiple range test.