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Contribution of PPi-Hydrolyzing Function of Vacuolar H(+)-Pyrophosphatase in Vegetative Growth of Arabidopsis: Evidenced by Expression of Uncoupling Mutated Enzymes.

Asaoka MM, Segami S, Ferjani A, Maeshima M - Front Plant Sci (2016)

Bottom Line: Overexpressors with high enzymatic activity grew more vigorously with fresh weight increased by more than 24 and 44%, compared to the wild type and fugu5-3, respectively.When uncoupling mutated variants of H(+)-PPase, that could hydrolyze PPi but could not translocate protons, were introduced into the fugu5-3 mutant background, shoot growth defects recovered to the same levels as when a normal H(+)-PPase was introduced.Taken together, our findings clearly demonstrate that additional expression of H(+)-PPase improves plant growth by increasing cell number, predominantly as a consequence of the PPi-hydrolyzing activity of the enzyme.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University Nagoya, Japan.

ABSTRACT
The vacuolar-type H(+)-pyrophosphatase (H(+)-PPase) catalyzes a coupled reaction of pyrophosphate (PPi) hydrolysis and active proton translocation across the tonoplast. Overexpression of H(+)-PPase improves growth in various plant species, and loss-of-function mutants (fugu5s) of H(+)-PPase in Arabidopsis thaliana have post-germinative developmental defects. Here, to further clarify the physiological significance of this important enzyme, we newly generated three varieties of H(+)-PPase overexpressing lines with different levels of activity that we analyzed together with the loss-of-function mutant fugu5-3. The H(+)-PPase overexpressors exhibited enhanced activity of H(+)-PPase during vegetative growth, but no change in the activity of vacuolar H(+)-ATPase. Overexpressors with high enzymatic activity grew more vigorously with fresh weight increased by more than 24 and 44%, compared to the wild type and fugu5-3, respectively. Consistently, the overexpressors had larger rosette leaves and nearly 30% more cells in leaves than the wild type. When uncoupling mutated variants of H(+)-PPase, that could hydrolyze PPi but could not translocate protons, were introduced into the fugu5-3 mutant background, shoot growth defects recovered to the same levels as when a normal H(+)-PPase was introduced. Taken together, our findings clearly demonstrate that additional expression of H(+)-PPase improves plant growth by increasing cell number, predominantly as a consequence of the PPi-hydrolyzing activity of the enzyme.

No MeSH data available.


Related in: MedlinePlus

Expression level and enzyme activities of H+-PPase and V-ATPase in WT and mutant lines. H+-PPase overexpressors were grown on MS plates for 15 days (A,B) or WT, fugu5-3 and three representative H+-PPase overexpressors (OX8, OX28, and OX30) were grown on MS plates with 1% sucrose for 11 days, transplanted to rockwool pots, and then grown for another 14 days (C). (A) Crude membrane fractions were prepared and aliquots (0.5 μg) were subjected to immunoblot analysis with specific antibodies against H+-PPase. Representative blots from two independent experiments are shown. (B) The intensities of immunostained bands shown in (A) are expressed as relative values to those of WT. (C) Crude membrane fractions were prepared from plants (0.4–1.0 g of fresh weight) at indicated growth stages and subjected to assays of PPi hydrolysis activity by H+-PPase and ATP hydrolysis activity by V-ATPase. Bars indicate SD from three independent experiments. The activities are shown as relative values on the basis of membrane protein to that of WT at 7 DAS. DAS, days after sowing.
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Figure 1: Expression level and enzyme activities of H+-PPase and V-ATPase in WT and mutant lines. H+-PPase overexpressors were grown on MS plates for 15 days (A,B) or WT, fugu5-3 and three representative H+-PPase overexpressors (OX8, OX28, and OX30) were grown on MS plates with 1% sucrose for 11 days, transplanted to rockwool pots, and then grown for another 14 days (C). (A) Crude membrane fractions were prepared and aliquots (0.5 μg) were subjected to immunoblot analysis with specific antibodies against H+-PPase. Representative blots from two independent experiments are shown. (B) The intensities of immunostained bands shown in (A) are expressed as relative values to those of WT. (C) Crude membrane fractions were prepared from plants (0.4–1.0 g of fresh weight) at indicated growth stages and subjected to assays of PPi hydrolysis activity by H+-PPase and ATP hydrolysis activity by V-ATPase. Bars indicate SD from three independent experiments. The activities are shown as relative values on the basis of membrane protein to that of WT at 7 DAS. DAS, days after sowing.

Mentions: We introduced a 2 × 35SΩpro::VHP1 construct into WT to evaluate the effect of H+-PPase overexpression on plant growth. Then, we selected 14 independent lines in the T3 generation that were both homozygous for a single T-DNA insertion and grew normally under standard growth conditions. The amount of H+-PPase protein in shoots was quantified by immunoblotting (Figures 1A,B). Among the 14 transgenic lines, OX8, OX28 and OX30, showed relatively high expression levels compared to the WT. OX8, OX30, and OX28 were selected as representative lines with high-, mid-, and low-expression levels, respectively.


Contribution of PPi-Hydrolyzing Function of Vacuolar H(+)-Pyrophosphatase in Vegetative Growth of Arabidopsis: Evidenced by Expression of Uncoupling Mutated Enzymes.

Asaoka MM, Segami S, Ferjani A, Maeshima M - Front Plant Sci (2016)

Expression level and enzyme activities of H+-PPase and V-ATPase in WT and mutant lines. H+-PPase overexpressors were grown on MS plates for 15 days (A,B) or WT, fugu5-3 and three representative H+-PPase overexpressors (OX8, OX28, and OX30) were grown on MS plates with 1% sucrose for 11 days, transplanted to rockwool pots, and then grown for another 14 days (C). (A) Crude membrane fractions were prepared and aliquots (0.5 μg) were subjected to immunoblot analysis with specific antibodies against H+-PPase. Representative blots from two independent experiments are shown. (B) The intensities of immunostained bands shown in (A) are expressed as relative values to those of WT. (C) Crude membrane fractions were prepared from plants (0.4–1.0 g of fresh weight) at indicated growth stages and subjected to assays of PPi hydrolysis activity by H+-PPase and ATP hydrolysis activity by V-ATPase. Bars indicate SD from three independent experiments. The activities are shown as relative values on the basis of membrane protein to that of WT at 7 DAS. DAS, days after sowing.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814480&req=5

Figure 1: Expression level and enzyme activities of H+-PPase and V-ATPase in WT and mutant lines. H+-PPase overexpressors were grown on MS plates for 15 days (A,B) or WT, fugu5-3 and three representative H+-PPase overexpressors (OX8, OX28, and OX30) were grown on MS plates with 1% sucrose for 11 days, transplanted to rockwool pots, and then grown for another 14 days (C). (A) Crude membrane fractions were prepared and aliquots (0.5 μg) were subjected to immunoblot analysis with specific antibodies against H+-PPase. Representative blots from two independent experiments are shown. (B) The intensities of immunostained bands shown in (A) are expressed as relative values to those of WT. (C) Crude membrane fractions were prepared from plants (0.4–1.0 g of fresh weight) at indicated growth stages and subjected to assays of PPi hydrolysis activity by H+-PPase and ATP hydrolysis activity by V-ATPase. Bars indicate SD from three independent experiments. The activities are shown as relative values on the basis of membrane protein to that of WT at 7 DAS. DAS, days after sowing.
Mentions: We introduced a 2 × 35SΩpro::VHP1 construct into WT to evaluate the effect of H+-PPase overexpression on plant growth. Then, we selected 14 independent lines in the T3 generation that were both homozygous for a single T-DNA insertion and grew normally under standard growth conditions. The amount of H+-PPase protein in shoots was quantified by immunoblotting (Figures 1A,B). Among the 14 transgenic lines, OX8, OX28 and OX30, showed relatively high expression levels compared to the WT. OX8, OX30, and OX28 were selected as representative lines with high-, mid-, and low-expression levels, respectively.

Bottom Line: Overexpressors with high enzymatic activity grew more vigorously with fresh weight increased by more than 24 and 44%, compared to the wild type and fugu5-3, respectively.When uncoupling mutated variants of H(+)-PPase, that could hydrolyze PPi but could not translocate protons, were introduced into the fugu5-3 mutant background, shoot growth defects recovered to the same levels as when a normal H(+)-PPase was introduced.Taken together, our findings clearly demonstrate that additional expression of H(+)-PPase improves plant growth by increasing cell number, predominantly as a consequence of the PPi-hydrolyzing activity of the enzyme.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University Nagoya, Japan.

ABSTRACT
The vacuolar-type H(+)-pyrophosphatase (H(+)-PPase) catalyzes a coupled reaction of pyrophosphate (PPi) hydrolysis and active proton translocation across the tonoplast. Overexpression of H(+)-PPase improves growth in various plant species, and loss-of-function mutants (fugu5s) of H(+)-PPase in Arabidopsis thaliana have post-germinative developmental defects. Here, to further clarify the physiological significance of this important enzyme, we newly generated three varieties of H(+)-PPase overexpressing lines with different levels of activity that we analyzed together with the loss-of-function mutant fugu5-3. The H(+)-PPase overexpressors exhibited enhanced activity of H(+)-PPase during vegetative growth, but no change in the activity of vacuolar H(+)-ATPase. Overexpressors with high enzymatic activity grew more vigorously with fresh weight increased by more than 24 and 44%, compared to the wild type and fugu5-3, respectively. Consistently, the overexpressors had larger rosette leaves and nearly 30% more cells in leaves than the wild type. When uncoupling mutated variants of H(+)-PPase, that could hydrolyze PPi but could not translocate protons, were introduced into the fugu5-3 mutant background, shoot growth defects recovered to the same levels as when a normal H(+)-PPase was introduced. Taken together, our findings clearly demonstrate that additional expression of H(+)-PPase improves plant growth by increasing cell number, predominantly as a consequence of the PPi-hydrolyzing activity of the enzyme.

No MeSH data available.


Related in: MedlinePlus