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Salinity-mediated transcriptional and post-translational regulation of the Arabidopsis aquaporin PIP2;7

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Salt stress triggers a simultaneous transcriptional repression and aquaporin internalization to modify root cell water conductivity.

Abstract

Plasma membrane intrinsic proteins (PIPs) are involved in the adjustment of plant water balance in response to changing environmental conditions. In this study, Arabidopsis wild-type (Col-0) and transgenic lines overexpressing PIP2;7 were used to investigate and compare their response to salt stress. Hydraulic conductivity measurements using a high-pressure flowmeter (HPFM) revealed that overexpression of PIP2;7 induced a sixfold increase in root hydraulic conductivity of four week-old Arabidopsis thaliana plants compared to WT. Exposure to a high salt stress (150 mM NaCl) triggered a rapid repression of overall aquaporin activity in both genotypes. Response to salt stress was also investigated in 8 day-old seedlings. Exposure to salt led to a repression of PIP2;7 promoter activity and a significant decrease in PIP2;7 mRNA abundance within 2 h. Concomitantly, a rapid internalization of fluorescently-tagged PIP2;7 proteins was observed but removal from the cell membrane was not accompanied by further degradation of the protein within 4 h of exposure to salinity stress. These data suggest that PIP transcriptional repression and channel internalization act in concert during salt stress conditions to modulate aquaporin activity, thereby significantly altering the plant hydraulic parameters in the short term.

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Acknowledgments

We thank Ewelina Grzeskiewicz for her help in this work and Frank Van Breusegem (PSB, VIB, Ghent) for lending the Licor infrared gas-exchange system. Confocal microscopy was carried out at the UCL imaging platform IMABIOL. This work was supported by grants from the Belgian National Fund for Scientific Research (FNRS), the Interuniversity Attraction Poles Programme-Belgian Science Policy (IAP7/29), the Belgian French community ARC11/16–036 project and the Bauchau Award. A. P. was supported by an UCL Incoming post-doctoral Fellowship co-funded by the Marie Curie Actions of the European Commission. C.H. was a FNRS postdoctoral researcher. T.M. was a research fellow at the Fonds de Formation à la Recherche dans l’Industrie et l’Agriculture.

Author contributions

Alicia Pou: Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting and revising the article. Linda Jeanguenin: Acquisition of data, Analysis and interpretation of data, Drafting and revising the article. Thomas Milhiet: Acquisition of data, Analysis and interpretation of data. Henri Batoko: Drafting and revising the article. François Chaumont: Conception and design, Analysis and interpretation of data, Drafting and revising the article. Charles Hachez: Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting and revising the article.

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  1. Institut des Sciences de la Vie, Université catholique de Louvain, Croix du Sud 4-L7.07.14, 1348, Louvain-la-Neuve, Belgium

    Alicia Pou, Linda Jeanguenin, Thomas Milhiet, Henri Batoko, François Chaumont & Charles Hachez

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  1. Alicia Pou
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Correspondence to François Chaumont or Charles Hachez.

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11103_2016_542_MOESM1_ESM.eps

Lack of diurnal variation in transcript abundance of GUS gene under control conditions. RNA was extracted from an homozygous T3 line expressing the PIP2;7:nlsGFP-GUS construct (Hachez et al., 2014a) at different time points. The GUS transcript abundance showed no statistically significant (P>0.05) diurnal variation. The three genes used for normalization were the following ones: Elongation Factor (EF1α), SAND family protein (SFP) and protein phosphatase 2A3 (PP2A3). Data points represent means and standard deviation of four replicates from two independent biological replicates (EPS 755 KB)

11103_2016_542_MOESM2_ESM.eps

Transcript abundance of control genes under salt stress. Ct values of gene-specific primers used for data normalization in RT-qPCR. The transcript abundance remained unaltered during salt stress. Genes: Elongation Factor (EF1α), SAND family protein (SFP) and protein phosphatase 2A3 (PP2A3) (EPS 955 KB)

11103_2016_542_MOESM3_ESM.eps

Stomatal conductance, photosynthetic rate and transpiration in 30 day-old plants grown in soil. (A) Response of stomatal conductance for CO2 (g s ) after 4 h of salt stress in WT (Col-0) and 35S:Venus-PIP2;7 plants. Data points represent means and standard errors of at least six replicates. (B) Response of photosynthetic CO2 assimilation (A N ) after 4 h of salt stress in Col-0 (WT) and 35S:Venus-PIP2;7 plants. (C) Response of leaf transpiration (E) after 4 h of salt stress in Col-0 (WT) and Venus-PIP2;7 plants. Data points represent means and standard errors of at least six replicates. Different letters represent significant differences within treatments for both genotypes by two-way Anova with Tukey’s test (P < 0.05). (EPS 1063 KB)

Supplementary material 4 (DOCX 12 KB)

11103_2016_542_MOESM5_ESM.avi

Time-lapse analysis of the Venus-PIP2;7 fluorescence in root PM. Arabidopsis root cells expressing the PIP2;7:Venus-PIP2;7 construct were monitored by confocal microscopy and images were taken during an interval of 76 s. Note the presence of PM invaginations in cortical root cells (AVI 447 KB)

Forty seven min time-lapse analysis of the Venus-PIP2;7 fluorescence in root PM. Arabidopsis root cells expressing the PIP2;7:Venus-PIP2;7 construct were monitored by confocal microscopy and images were taken every 6 min during an interval of 48 min. Recording started at the onset of the stress. Note 1/the decrease in fluorescence of Venus-PIP2;7 in the PM 2/ the concomitant cell plasmolysis and 3/ the presence of PM invaginations in cortical root cells in response to this salt treatment (AVI 1064 KB)

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Pou, A., Jeanguenin, L., Milhiet, T. et al. Salinity-mediated transcriptional and post-translational regulation of the Arabidopsis aquaporin PIP2;7. Plant Mol Biol 92, 731–744 (2016). https://doi.org/10.1007/s11103-016-0542-z

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