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Exploring three PIPs and three TIPs of grapevine for transport of water and atypical substrates through heterologous expression in aqy- yeast.

Sabir F, Leandro MJ, Martins AP, Loureiro-Dias MC, Moura TF, Soveral G, Prista C - PLoS ONE (2014)

Bottom Line: All functional aquaporins were found to be sensitive to HgCl2, since their water conductivity was reduced (24-38%) by the addition of 0.5 mM HgCl2.Our results also indicate that Vitis aquaporins are putative transporters of other small molecules of physiological importance.The phenotypic growth variations of yeast cells showed that heterologous expression of Vitis aquaporins increased susceptibility to externally applied boron and H2O2, suggesting the contribution of Vitis aquaporins in the transport of these species.

View Article: PubMed Central - PubMed

Affiliation: Centre for Botany Applied to Agriculture (CBAA), Instituto Superior de Agronomia, University of Lisbon, Lisbon, Portugal; Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal.

ABSTRACT
Aquaporins are membrane channels that facilitate the transport of water and other small molecules across the cellular membranes. We examined the role of six aquaporins of Vitis vinifera (cv. Touriga nacional) in the transport of water and atypical substrates (other than water) in an aqy- strain of Saccharomyces cerevisiae. Their functional characterization for water transport was performed by stopped-flow fluorescence spectroscopy. The evaluation of permeability coefficients (Pf) and activation energies (Ea) revealed that three aquaporins (VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2) are functional for water transport, while the other three (VvTnPIP1;4, VvTnPIP2;3 and VvTnTIP4;1) are non-functional. TIPs (VvTnTIP1;1 and VvTnTIP2;2) exhibited higher water permeability than VvTnPIP2;1. All functional aquaporins were found to be sensitive to HgCl2, since their water conductivity was reduced (24-38%) by the addition of 0.5 mM HgCl2. Expression of Vitis aquaporins caused different sensitive phenotypes to yeast strains when grown under hyperosmotic stress generated by KCl or sorbitol. Our results also indicate that Vitis aquaporins are putative transporters of other small molecules of physiological importance. Their sequence analyses revealed the presence of signature sequences for transport of ammonia, boron, CO2, H2O2 and urea. The phenotypic growth variations of yeast cells showed that heterologous expression of Vitis aquaporins increased susceptibility to externally applied boron and H2O2, suggesting the contribution of Vitis aquaporins in the transport of these species.

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Activation energies (Ea) of water transport in V. vinifera aquaporins expressed in yeast.(A) Arrhenius plot of Pf at temperature range (9–37°C), where T is temperature in Kelvin. Ea was evaluated from the slopes. Strains expressing VvTnPIP1;4, VvTnTIP4;1 and empty plasmid (pUG35) showed steeper slope, while strains expressing VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2 exhibited shallow slope. (B) Calculated Ea from the slope showed that VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2 expressing strains exhibited lower Ea, while VvTnPIP1;4, VvTnPIP2;3 and VvTnTIP4;1 expressing strains showed were almost equal to control strain (pUG35). Data are mean ± SD of three independent experiments with at least five traces (ten traces in case of Pf at 23°C) at each temperature.
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pone-0102087-g005: Activation energies (Ea) of water transport in V. vinifera aquaporins expressed in yeast.(A) Arrhenius plot of Pf at temperature range (9–37°C), where T is temperature in Kelvin. Ea was evaluated from the slopes. Strains expressing VvTnPIP1;4, VvTnTIP4;1 and empty plasmid (pUG35) showed steeper slope, while strains expressing VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2 exhibited shallow slope. (B) Calculated Ea from the slope showed that VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2 expressing strains exhibited lower Ea, while VvTnPIP1;4, VvTnPIP2;3 and VvTnTIP4;1 expressing strains showed were almost equal to control strain (pUG35). Data are mean ± SD of three independent experiments with at least five traces (ten traces in case of Pf at 23°C) at each temperature.

Mentions: To evaluate the activation energy for water transport, Pf values of all yeast strains were analyzed in a range of temperatures (9–37°C) and Arrhenius plots were drawn (Figure 5A). As expected, transport of water was strongly dependent on temperature in control yeast strain, as reflected by higher activation energy Ea (14.05±0.01 kcal mol−1) (Figure 5B and Table 2). Arrhenius plots for VvTnPIP1;4, VvTnPIP2;3 and VvTnTIP4;1 expressing yeast strains exhibited parallel steep slopes, overlapped with control strain (Figure 5A) and almost similar activation energies (14.74±0.62, 14.53±0.55, 14.86±0.22 kcal mol−1, respectively) (Figure 5B and Table 2). On the other hand, expression of VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2 led to drastic reduction in the activation energies to 10.84±0.83, 8.8±0.77 and 8.77±0.62 kcal mol−1, respectively (Figure 5B and Table 2), demonstrating that transport of water was majorly mediated by aquaporins in these yeast strains.


Exploring three PIPs and three TIPs of grapevine for transport of water and atypical substrates through heterologous expression in aqy- yeast.

Sabir F, Leandro MJ, Martins AP, Loureiro-Dias MC, Moura TF, Soveral G, Prista C - PLoS ONE (2014)

Activation energies (Ea) of water transport in V. vinifera aquaporins expressed in yeast.(A) Arrhenius plot of Pf at temperature range (9–37°C), where T is temperature in Kelvin. Ea was evaluated from the slopes. Strains expressing VvTnPIP1;4, VvTnTIP4;1 and empty plasmid (pUG35) showed steeper slope, while strains expressing VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2 exhibited shallow slope. (B) Calculated Ea from the slope showed that VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2 expressing strains exhibited lower Ea, while VvTnPIP1;4, VvTnPIP2;3 and VvTnTIP4;1 expressing strains showed were almost equal to control strain (pUG35). Data are mean ± SD of three independent experiments with at least five traces (ten traces in case of Pf at 23°C) at each temperature.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4128642&req=5

pone-0102087-g005: Activation energies (Ea) of water transport in V. vinifera aquaporins expressed in yeast.(A) Arrhenius plot of Pf at temperature range (9–37°C), where T is temperature in Kelvin. Ea was evaluated from the slopes. Strains expressing VvTnPIP1;4, VvTnTIP4;1 and empty plasmid (pUG35) showed steeper slope, while strains expressing VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2 exhibited shallow slope. (B) Calculated Ea from the slope showed that VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2 expressing strains exhibited lower Ea, while VvTnPIP1;4, VvTnPIP2;3 and VvTnTIP4;1 expressing strains showed were almost equal to control strain (pUG35). Data are mean ± SD of three independent experiments with at least five traces (ten traces in case of Pf at 23°C) at each temperature.
Mentions: To evaluate the activation energy for water transport, Pf values of all yeast strains were analyzed in a range of temperatures (9–37°C) and Arrhenius plots were drawn (Figure 5A). As expected, transport of water was strongly dependent on temperature in control yeast strain, as reflected by higher activation energy Ea (14.05±0.01 kcal mol−1) (Figure 5B and Table 2). Arrhenius plots for VvTnPIP1;4, VvTnPIP2;3 and VvTnTIP4;1 expressing yeast strains exhibited parallel steep slopes, overlapped with control strain (Figure 5A) and almost similar activation energies (14.74±0.62, 14.53±0.55, 14.86±0.22 kcal mol−1, respectively) (Figure 5B and Table 2). On the other hand, expression of VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2 led to drastic reduction in the activation energies to 10.84±0.83, 8.8±0.77 and 8.77±0.62 kcal mol−1, respectively (Figure 5B and Table 2), demonstrating that transport of water was majorly mediated by aquaporins in these yeast strains.

Bottom Line: All functional aquaporins were found to be sensitive to HgCl2, since their water conductivity was reduced (24-38%) by the addition of 0.5 mM HgCl2.Our results also indicate that Vitis aquaporins are putative transporters of other small molecules of physiological importance.The phenotypic growth variations of yeast cells showed that heterologous expression of Vitis aquaporins increased susceptibility to externally applied boron and H2O2, suggesting the contribution of Vitis aquaporins in the transport of these species.

View Article: PubMed Central - PubMed

Affiliation: Centre for Botany Applied to Agriculture (CBAA), Instituto Superior de Agronomia, University of Lisbon, Lisbon, Portugal; Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal.

ABSTRACT
Aquaporins are membrane channels that facilitate the transport of water and other small molecules across the cellular membranes. We examined the role of six aquaporins of Vitis vinifera (cv. Touriga nacional) in the transport of water and atypical substrates (other than water) in an aqy- strain of Saccharomyces cerevisiae. Their functional characterization for water transport was performed by stopped-flow fluorescence spectroscopy. The evaluation of permeability coefficients (Pf) and activation energies (Ea) revealed that three aquaporins (VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2) are functional for water transport, while the other three (VvTnPIP1;4, VvTnPIP2;3 and VvTnTIP4;1) are non-functional. TIPs (VvTnTIP1;1 and VvTnTIP2;2) exhibited higher water permeability than VvTnPIP2;1. All functional aquaporins were found to be sensitive to HgCl2, since their water conductivity was reduced (24-38%) by the addition of 0.5 mM HgCl2. Expression of Vitis aquaporins caused different sensitive phenotypes to yeast strains when grown under hyperosmotic stress generated by KCl or sorbitol. Our results also indicate that Vitis aquaporins are putative transporters of other small molecules of physiological importance. Their sequence analyses revealed the presence of signature sequences for transport of ammonia, boron, CO2, H2O2 and urea. The phenotypic growth variations of yeast cells showed that heterologous expression of Vitis aquaporins increased susceptibility to externally applied boron and H2O2, suggesting the contribution of Vitis aquaporins in the transport of these species.

Show MeSH
Related in: MedlinePlus