A PIP1 aquaporin contributes to hydrostatic pressure-induced water transport in both the root and rosette of Arabidopsis

Abstract

Aquaporins are channel proteins that facilitate the transport of water across plant cell membranes. In this work, we used a combination of pharmacological and reverse genetic approaches to investigate the overall significance of aquaporins for tissue water conductivity in Arabidopsis (Arabidopsis thaliana). We addressed the function in roots and leaves of AtPIP1;2, one of the most abundantly expressed isoforms of the plasma membrane intrinsic protein family. At variance with the water transport phenotype previously described in AtPIP2;2 knockout mutants, disruption of AtPIP1;2 reduced by 20% to 30% the root hydrostatic hydraulic conductivity but did not modify osmotic root water transport. These results document qualitatively distinct functions of different PIP isoforms in root water uptake. The hydraulic conductivity of excised rosettes (K(ros)) was measured by a novel pressure chamber technique. Exposure of Arabidopsis plants to darkness increased K(ros) by up to 90%. Mercury and azide, two aquaporin inhibitors with distinct modes of action, were able to induce similar inhibition of K(ros) by approximately 13% and approximately 25% in rosettes from plants grown in the light or under prolonged (11-18 h) darkness, respectively. Prolonged darkness enhanced the transcript abundance of several PIP genes, including AtPIP1;2. Mutant analysis showed that, under prolonged darkness conditions, AtPIP1;2 can contribute to up to approximately 20% of K(ros) and to the osmotic water permeability of isolated mesophyll protoplasts. Therefore, AtPIP1;2 can account for a significant portion of aquaporin-mediated leaf water transport. The overall work shows that AtPIP1;2 represents a key component of whole-plant hydraulics.

Figure 1.

Expression analysis of a PIP1;2-GUS construct. The figure shows the GUS staining of a whole 5-d-old plant grown in vitro (A). GUS staining of a cross section at > 5 mm from a root tip (B; bar =50 μm), of an entire leaf (C), or of a leaf cross section (D; bar = 50 μm) was made in a 21-d-old plant grown in hydroponic conditions.

Figure 2.

Molecular characterization of the pip1;2-1 and pip1;2-2 insertion mutants. A, Physical map of the AtPIP1;2 gene with schematic position of the T-DNA insertions identified in the Salk_145347 and Salk_19794 lines and corresponding to pip1;2-1 and pip1;2-2, respectively (see text). The initiating (ATG) and STOP codons are indicated, with exons shown in black. The numbering of nucleotides refers to the genomic sequence of AtPIP1;2 in BAC clone F4I18. Horizontal arrowheads indicate the positions and orientations of primer sequences used for PCR analysis of the gDNA and resulting cDNA in the genotypes indicated below. B, PCR analysis of gDNA of wild-type (WT), pip1;2-1, and pip1;2-2 plants using a pair of AtPIP1;1-specific primers (1;1f/1;1r) (1), a pair of AtPIP1;2-specific primers (1;2fa/1;2ra) (2), and two primers specific for AtPIP1;2 (1;2ra) and the T-DNA (LBb1), respectively (3). C, RT-PCR analysis of AtPIP1;2 mRNA expression in wild-type, pip1;2-1, pip1;2-2, and pip1;2-1Comp plants. Expression of AtPIP1;2 cDNA was probed with primers (1;2fb/1;2ra) (1) located downstream of the T-DNA insertion site. Amplification of an Elongation Factor1α cDNA fragment was performed for controlling cDNA integrity (2). [See online article for color version of this figure.]

Figure 3.

Mean hydrostatic hydraulic conductivity of roots (Lp_r-h) from wild-type (WT), pip1;2-1, and pip1;2-1Comp plants. Lp_r-h was measured during the daytime in plants grown under a normal photoperiodic regime. Data were pooled from four independent cultures and the indicated number of plants. Values are means ± se, and the asterisk indicates a statistically significant difference (probability < 0.002) from the wild-type value.

Figure 4.

Effects of the irradiance regime on rosette hydraulic conductivity (K_ros). A, Representative pressure-to-flow relationships measured in rosettes from plants grown under a normal photoperiodic regime and collected around midday (photosynthetically active radiation = 250 μmol photons m⁻² s⁻¹; white circles) or plants exposed to a prolonged night (11–21 h darkness; extended darkness; black circles). In both cases, excised rosettes were submerged into a bathing solution, inserted into a pressure chamber, and the flow of sap exuding from the sectioned hypocotyl [J_v(P)] was measured at the indicated pressure as described in “Materials and Methods.” The slope of the regression line is indicative of rosette hydraulic conductance, and together with the cumulated leaf surface allows calculating rosette hydraulic conductivity values (white circles, K_ros = 141.2 μL s⁻¹ m⁻² MPa⁻¹; black circles, K_ros = 223.4 μL s⁻¹ m⁻² MPa⁻¹). B, K_ros from plants grown under a normal photoperiodic regime (16 h light/8 h dark) and measured around midday (light) or during the night (dark). K_ros was also measured in plants exposed to an extended darkness (see text and schematic representation above). K_ros was expressed as percentage of the mean control value measured in the light (K_ros = 149.5 μL s⁻¹ m⁻² MPa⁻¹). The number of individual plants measured in each condition is indicated, and the asterisk indicates a statistically significant difference from control values (probability = 0.009). [See online article for color version of this figure.]

Figure 5.

Time-dependent effects of aquaporin blocking treatments on pressure-induced water transport in rosettes from plants grown under extended darkness. A, Effects of exposure to mercury. An excised rosette was subjected to a constant pressure (P = 0.32 MPa), and J_v(P) was measured over time. The bathing solution was complemented with 50 μm HgCl₂ at time t = 0. Fit of the kinetic data by a first-order exponential function indicated a final inhibition of 26.7% with a half-time (t_1/2) of 11.4 min. B, Effects of exposure to azide. Same procedure as in A except that 2 mm NaN₃, instead of mercury, was added to the bathing solution at t = 0 and was removed after 60 min. The fitted data indicate a maximal inhibition of 19.9%, with a t_1/2 = 6.2 min. Reversion occurred with a fitted amplitude of 98.9% and t_1/2 of 14.4 min.

Figure 6.

Effects of aquaporin blockers on K_ros of plants grown under extended darkness (A) or in the light under a normal photoperiodic regime (B). K_ros was measured in excised rosettes before (none) and after treatment for 60 min with mercury (50 μm HgCl₂) or azide (2 mm NaN₃). Data were cumulated from at least four independent cultures, with the indicated number of plants.

Figure 7.

Effects of the light regime on the expression of AtPIP genes in the Arabidopsis rosette. The transcript abundance of each AtPIP gene in whole rosettes was measured by real-time RT-PCR as explained in “Materials and Methods.” The figure shows the mean expression ratio between plants grown under extended darkness and plants grown in the light under a normal photoperiodic regime. Cumulated data (±se) are from three independent biological experiments, each with duplicate PCR reactions. Asterisks indicate significant effects (probability < 0.05) of the light regime.

Figure 8.

K_ros of wild-type (WT), pip1;2-1, and pip1;2-1Comp plants grown under extended darkness conditions. K_ros was measured on the indicated number of plants, as exemplified in Figure 4. Values are means ± se, and the asterisk indicates a statistically significant difference from the wild-type value.

Figure 9.

P_f of mesophyll protoplasts isolated from wild-type (WT), pip1;2-1, and pip1;2-2 plants grown under extended darkness conditions. Cumulated data are from four protoplast preparations from two independent plant cultures (wild type, n = 45; pip1;2-1, n = 32 ; pip1;2-2, n = 35). A, Relative distribution of P_f values in wild-type (black bars), pip1;2-1 (empty bars), and pip1;2-2 (gray bars) protoplasts. B, Mean P_f values (±se).