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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

Growth of leaves in late stage. WT, fugu5-3 and overexpressors were grown on MS plates with 1% sucrose for 11 days and then on rockwool pots for another 14 days. Leaves were collected from 25-days-old plants and their leaf surface areas were measured. (A) Rosette leaves were taken from WT and mutant lines and were ordered from youngest to oldest ones from the left to the right. Bar = 5 mm. (B) Surface area of 1st to 9th rosette leaves. (C) Sum area of all rosette leaves of each genotype. (D) Cell size of 7th rosette leaves. (E) Cell number of 7th rosette leaves. Error bars show SD; n = 8. Asterisk indicates significant difference at P < 0.05 compared with WT (Student’s t-test).
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Figure 5: Growth of leaves in late stage. WT, fugu5-3 and overexpressors were grown on MS plates with 1% sucrose for 11 days and then on rockwool pots for another 14 days. Leaves were collected from 25-days-old plants and their leaf surface areas were measured. (A) Rosette leaves were taken from WT and mutant lines and were ordered from youngest to oldest ones from the left to the right. Bar = 5 mm. (B) Surface area of 1st to 9th rosette leaves. (C) Sum area of all rosette leaves of each genotype. (D) Cell size of 7th rosette leaves. (E) Cell number of 7th rosette leaves. Error bars show SD; n = 8. Asterisk indicates significant difference at P < 0.05 compared with WT (Student’s t-test).

Mentions: On the other hand, to determine the effects of loss-of-function and overexpression of H+-PPase on leaf development, we examined the leaves from the 1st to the 9th positions at 25 DAS (Figures 5A,B). While the total surface area of all nine leaves of OX8 and OX30 lines was ∼12% larger, the area of fugu5-3 was about 15% smaller than that of WT (Figure 5C). This is consistent with the differences we found in shoot fresh weight (Figure 4). Also, the leaf surface in the three overexpressors showed a particularly significant increase in the rosette leaves from 7th to 9th positions. Yet, this could also reflect the fact that these leaves are not yet fully expanded at 25 DAS.


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)

Growth of leaves in late stage. WT, fugu5-3 and overexpressors were grown on MS plates with 1% sucrose for 11 days and then on rockwool pots for another 14 days. Leaves were collected from 25-days-old plants and their leaf surface areas were measured. (A) Rosette leaves were taken from WT and mutant lines and were ordered from youngest to oldest ones from the left to the right. Bar = 5 mm. (B) Surface area of 1st to 9th rosette leaves. (C) Sum area of all rosette leaves of each genotype. (D) Cell size of 7th rosette leaves. (E) Cell number of 7th rosette leaves. Error bars show SD; n = 8. Asterisk indicates significant difference at P < 0.05 compared with WT (Student’s t-test).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Growth of leaves in late stage. WT, fugu5-3 and overexpressors were grown on MS plates with 1% sucrose for 11 days and then on rockwool pots for another 14 days. Leaves were collected from 25-days-old plants and their leaf surface areas were measured. (A) Rosette leaves were taken from WT and mutant lines and were ordered from youngest to oldest ones from the left to the right. Bar = 5 mm. (B) Surface area of 1st to 9th rosette leaves. (C) Sum area of all rosette leaves of each genotype. (D) Cell size of 7th rosette leaves. (E) Cell number of 7th rosette leaves. Error bars show SD; n = 8. Asterisk indicates significant difference at P < 0.05 compared with WT (Student’s t-test).
Mentions: On the other hand, to determine the effects of loss-of-function and overexpression of H+-PPase on leaf development, we examined the leaves from the 1st to the 9th positions at 25 DAS (Figures 5A,B). While the total surface area of all nine leaves of OX8 and OX30 lines was ∼12% larger, the area of fugu5-3 was about 15% smaller than that of WT (Figure 5C). This is consistent with the differences we found in shoot fresh weight (Figure 4). Also, the leaf surface in the three overexpressors showed a particularly significant increase in the rosette leaves from 7th to 9th positions. Yet, this could also reflect the fact that these leaves are not yet fully expanded at 25 DAS.

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