PIP1 plasma membrane aquaporins in tobacco: from cellular effects to function in plants

Abstract

The molecular functions of several aquaporins are well characterized (e.g., by analysis of aquaporin-expressing Xenopus oocytes). However, their significance in the physiology of water transport in multicellular organisms remains uncertain. The tobacco plasma membrane aquaporin NtAQP1 was used to elucidate this issue. By comparing antisense plants that were inhibited in NtAQP1 expression with control plants, we found evidence for NtAQP1 function in cellular and whole-plant water relations. The consequences of a decrease in cellular water permeability were determined by measurement of transpiration rate and stem and leaf water potential as well as growth experiments under extreme soil water depletion. Plants impaired in NtAQP1 expression showed reduced root hydraulic conductivity and lower water stress resistance. In conclusion, our results emphasize the importance of symplastic aquaporin-mediated water transport in whole-plant water relations.

Figure 1.

RNA Gel Blot Hybridization Signals with RNA from Control or Antisense Plants. (A) Hybridization with a probe specific to NtAQP1 (top) or to 28S rRNA as a loading control (bottom). Root RNA was extracted from control plants (c) or from four independently transformed antisense NtAQP1–expressing lines (as1 to as4). (B) Hybridization with RNA from antisense line 4 (as4) or control (c). Radioactive probes comprising gene-specific regions were synthesized from tobacco aquaporins, which were named according to their Arabidopsis counterparts, NtPIP1a, NtPIP2a, and NtTIPa. As a control for equal lane loading, the complete actin cDNA was used as a probe.

Figure 2.

Protoplast Swelling Analysis. (A) Protoplasts were produced from roots of control plants (open circles) and antisense NtAQP1–expressing lines (closed circles) and subjected to hypoosmotic conditions. The mean volume increase of control protoplasts (n = 36) or antisense protoplasts (n = 36; n = 9 for each line) in the first 60 sec after osmolarity change is shown. In separate experiments with the same conditions, the osmolarity of aliquots taken from the chamber buffer solution was determined (diamonds; n = 10). V_i/V₀, volume increase/initial volume. (B) P_os value distribution of the investigated protoplasts obtained from control lines (white bars) and antisense lines (black bars).

Figure 3.

Determination of Root Hydraulic Conductivity by the High-Pressure Flowmeter Method. (A) Representative pressure/flow rate relationship of a control plant (open circles) and an antisense NtAQP1 line (closed circles). Both plants had similar root surface areas (control, 0.0605 m²; antisense, 0.0615 m²). The applied pressure was increased at 2-sec intervals. (B) Average specific root hydraulic conductivity of controls and antisense NtAQP1–expressing tobacco plants as measured by the high-pressure flowmeter method. Complete data are given in Table 1.

Figure 4.

Data for Control and Antisense NtAQP1 Tobacco. Transpiration rates (E; top), stem water potential (Ψ_stem; bottom left), and leaf water potential (Ψ_leaf; bottom right) of control (white bars) and antisense NtAQP1 (black bars) tobacco are shown. Plants were either watered or not watered for 1 week (drought). Complete data are given in Table 1.

Figure 5.

Water Stress Symptoms. (A) Plants were watered with a PEG solution, inducing an osmotic pressure of −0.35 MPa, and photographed 2 hr later. Left, control plant; right, antisense NtAQP1–expressing plant. (B) Average angles of leaves from plants treated with a PEG solution. Angles were measured in relation to the shoot axis for antisense NtAQP1 lines (closed circles; n = 8) and controls (open circles; n = 6).