Regulation of Arabidopsis leaf hydraulics involves light-dependent phosphorylation of aquaporins in veins

Abstract

The water status of plant leaves depends on the efficiency of the water supply, from the vasculature to inner tissues. This process is under hormonal and environmental regulation and involves aquaporin water channels. In Arabidopsis thaliana, the rosette hydraulic conductivity (Kros) is higher in darkness than it is during the day. Knockout plants showed that three plasma membrane intrinsic proteins (PIPs) sharing expression in veins (PIP1;2, PIP2;1, and PIP2;6) contribute to rosette water transport, and PIP2;1 can fully account for Kros responsiveness to darkness. Directed expression of PIP2;1 in veins of a pip2;1 mutant was sufficient to restore Kros. In addition, a positive correlation, in both wild-type and PIP2;1-overexpressing plants, was found between Kros and the osmotic water permeability of protoplasts from the veins but not from the mesophyll. Thus, living cells in veins form a major hydraulic resistance in leaves. Quantitative proteomic analyses showed that light-dependent regulation of Kros is linked to diphosphorylation of PIP2;1 at Ser-280 and Ser-283. Expression in pip2;1 of phosphomimetic and phosphorylation-deficient forms of PIP2;1 demonstrated that phosphorylation at these two sites is necessary for Kros enhancement under darkness. These findings establish how regulation of a single aquaporin isoform in leaf veins critically determines leaf hydraulics.

Figure 1.

Contribution to Rosette Water Transport and Expression Patterns of Individual PIP Genes. (A) K_ros of single pip knockout mutants (Da Ines et al., 2010; Postaire et al., 2010; Péret et al., 2012) (see Supplemental Figure 2 online) grown in L (yellow bars) or D (gray bars) conditions. For comparison of mutants in various accessions, values were normalized to the corresponding wild type (WT; Col-0, L, K_ros = 221 ± 17 μL s⁻¹ MPa⁻¹ m⁻²; D, K_ros = 275 ± 9 μL s⁻¹ MPa⁻¹ m⁻²; P < 0.05) (Ler, L, K_ros = 201.6 ± 13.9 μL s⁻¹ MPa⁻¹ m⁻²; D, K_ros = 292.0 ± 11.9 μL s⁻¹ MPa⁻¹ m⁻²; P < 0.05). Averaged data (±se) are from five independent cultures and the indicated number of plants. Asterisks indicate significant difference from the wild type (P < 0.004). (B) ProPIP:GUS reporter gene expression in transgenic plants. Cross sections show intense staining in the veins of plants expressing ProPIP2;1:GUS or ProPIP2;6-GUS constructs (black bars = 2.5 mm; red bars = 0.1 mm).

Figure 2.

Effects of Deregulated Expression of PIP2;1 on K_ros. (A) K_ros and PIP2;1 expression in two d35S:PIP2;1 lines (-1 and -3), one d35S:PIP2;1ko line, and their untransformed controls (Col-0 and pip2;1-2, respectively). The top panel shows averaged K_ros values (±se) from three independent cultures, with the indicated number of plants. Plant were grown in L (yellow bars) or D (gray bars) conditions. Letters indicate statistically different values at P < 0.05. The bottom panel shows immunodetection of PIP2 in leaf extracts of the indicated lines grown in L. (B) Imaging of an entire leaf (top) and secondary vein region (bottom) of Col-0 plants expressing a ProGLDPA:GFP construct, using epifluorescence and biphoton fluorescence microscopy, respectively (bars = 50 μm) (C) Characterization of Col-0, pip2;1-2, and two independent pip2;1-2 lines expressing a ProGLDPA:PIP2;1 construct. Same conventions as in (A).

Figure 3.

Effects of Light Regime on P_f Coefficients of Mesophyll or Vein-Specific Protoplasts. (A) Mesophyll protoplasts were purified from Col-0 or d35S:PIP2;1 (lines -1 and -3) plants grown under L (yellow bars) or D (gray bars) conditions. P_f was characterized using an osmotic swelling assay as described in the text. Averaged data (±se) from the indicated number of protoplasts are obtained from at least 12 independent preparations and three plant cultures. Asterisks indicate significant effect of light on a genotype (P < 0.05). (B) Fluorescent protoplasts were sorted out using a micropipette from a leaf digest of transgenic Col-0 or d35S:PIP2;1-1 plants expressing a ProGLDPA:GFP construct and grown under L (yellow bars) or D (gray bars) conditions. Experimental conditions for measuring P_f and conventions are as in (A). (C) Leaf fluorescent protoplasts purified from transgenic Col-0 expressing a ProGLDPA:GFP construct were characterized under transmission light microscopy according to their diameter (Ø) and chloroplast content (see Supplemental Figure 5A online). The figure shows protoplast images representative of three distinct classes: (I) small sized (mean Ø = 12.7 ± 0.4 µm), translucent (with approximately two chloroplasts) protoplasts, (II) large (mean Ø = 17.3 ± 1.0 µm) protoplasts containing at most three chloroplasts, (III) large protoplasts (mean Ø = 22.0 ± 0.5 µm) with more than three chloroplasts. The same three subclasses could also be observed in d35S:PIP2;1-1 plants expressing the same ProGLDPA:GFP construct (see Supplemental Figure 5B online). Bars = 8 µm. (D) Effects of light on the P_f of the three subclasses of leaf vein protoplasts. Protoplasts were isolated from Col-0 (left panel) and d35S:PIP2;1-1 plants (right panel) expressing ProGLDPA:GFP and grown under L (yellow bars) or D (black bars) conditions. Experimental conditions for measuring P_f and conventions are as in (A). [See online article for color version of this figure.]

Figure 4.

Effects of Light Regime on PIP Abundance. The figure shows a quantitative proteomic profiling of PIPs in rosettes of Col-0 plants grown under L (yellow bars) or D (gray bars) conditions. Absolute abundance of each isoform (±se) was determined with three technical replicates. [See online article for color version of this figure.]

Figure 5.

Effects of Light Regime on PIP2;1/PIP2;2 C-Terminal Phosphorylation. All studies were performed in rosettes from Col-0 or pip2;1-1 plants grown under L (yellow bars) or D (gray bars) conditions. Phosphoproteomics analysis reveals the existence of three unmodified or phosphorylated forms for a C-terminal tryptic peptide (Ser-277 to Val-287) of PIP2;1/PIP2;2 (top). Absolute abundance of each form (±se) was determined with three technical replicates from three (Col-0) or two (pip2;1-2) independent plant cultures. Letters indicate statistically different values at P < 0.05. [See online article for color version of this figure.]

Figure 6.

Effects of Light Regime on K_ros of pip2;1-2 Plants Expressing Phosphorylation Mutants of PIP2;1. Plants expressing the indicated PIP2;1 mutant forms were grown under L (yellow bars) or D (gray bars) conditions, and their K_ros was compared with values in the wild type (Col-0) and pip2;1-2. Averaged data (±se) are from the indicated number of plants and three independent cultures. Letters indicate statistically different values at P < 0.05. The figure shows K_ros data obtained in one individual clone representative of each genotype, with corresponding immunoblot analysis of PIP2 expression in the bottom panel. Complementary analyses are shown in Supplemental Figure 8 online. [See online article for color version of this figure.]