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We functionally characterized the grape (Vitis vinifera) VvPIP2;4N (for Plasma membrane Intrinsic Protein) aquaporin gene. Expression of VvPIP2;4N in Xenopus laevis oocytes increased their swelling rate 54-fold. Northern blot and quantitative reverse transcription-polymerase chain reaction analyses showed that VvPIP2;4N is the most expressed PIP2 gene in root. In situ hybridization confirmed root localization in the cortical parenchyma and close to the endodermis. We then constitutively overexpressed VvPIP2;4N in grape 'Brachetto', and in the resulting transgenic plants we analyzed (1) the expression of endogenous and transgenic VvPIP2;4N and of four other aquaporins, (2) whole-plant, root, and leaf ecophysiological parameters, and (3) leaf abscisic acid content. Expression of transgenic VvPIP2;4N inhibited neither the expression of the endogenous gene nor that of other PIP aquaporins in both root and leaf. Under well-watered conditions, transgenic plants showed higher stomatal conductance, gas exchange, and shoot growth. The expression level of VvPIP2;4N (endogenous + transgene) was inversely correlated to root hydraulic resistance. The leaf component of total plant hydraulic resistance was low and unaffected by overexpression of VvPIP2;4N. Upon water stress, the overexpression of VvPIP2;4N induced a surge in leaf abscisic acid content and a decrease in stomatal conductance and leaf gas exchange. Our results show that aquaporin-mediated modifications of root hydraulics play a substantial role in the regulation of water flow in well-watered grapevine plants, while they have a minor role upon drought, probably because other signals, such as abscisic acid, take over the control of water flow.

Plants provide an excellent system to study CO(2) diffusion because, under light saturated conditions, photosynthesis is limited by CO(2) availability. Recent findings indicate that CO(2) diffusion in leaves can be variable in a short time range. Mesophyll CO(2) conductance could change independently from stomata movement or CO(2) fixing reactions and it was suggested that, beside others, the membranes are mesophyll CO(2) conductance limiting components. Specific aquaporins as membrane intrinsic pore proteins are considered to have a function in the modification of membrane CO(2) conductivity. Because of conflicting data, the mechanism of membrane CO(2) diffusion in plants and animals is a matter of a controversy vivid debate in the scientific community. On one hand, data from biophysics are in favor of CO(2) diffusion limiting mechanisms completely independent from membrane structure and membrane components. On the other, there is increasing evidence from physiology that a change in membrane composition has an effect on CO(2) diffusion., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

CO(2) faces a series of resistances while diffusing between the substomatal cavities and the sites of carboxylation within chloroplasts. The absence of techniques to measure the resistance of individual steps makes it difficult to define their relative importance. Resistance to diffusion through intercellular airspace differs between leaves, but is usually of minor importance. Leaves with high photosynthetic capacity per unit leaf area reduce mesophyll resistance by increasing the surface area of chloroplasts exposed to intercellular airspace per unit leaf area, S(c). Cell walls impose a significant resistance. Assuming an effective porosity of the cell wall of 0.1 or 0.05, then cell walls could account for 25% or 50% of the total mesophyll resistance, respectively. Since the fraction of apoplastic water that is unbound and available for unhindered CO(2) diffusion is unknown, it is possible that the effective porosity is <0.05. Effective porosity could also vary in response to changes in pH or cation concentration. Consequently, cell walls could account for >50% of the total resistance and a variable proportion. Most of the remaining resistance is imposed by one or more of the three membranes as mesophyll resistance can be altered by varying the expression of cooporins. The CO(2) permeability of vesicles prepared from chloroplast envelopes has been reduced by RNA interference (RNAi) expression of NtAQP1, but not those prepared from the plasma membrane. Carbonic anhydrase activity also influences mesophyll resistance. Mesophyll resistance is relatively insensitive to the manipulation of any step in the pathway because it represents only part of the total and may also be countered by pleiotropic compensatory changes. The parameters in greatest need of additional measurements are S(c), mesophyll cell wall thickness, and the permeabilities of the plasma membrane and chloroplast envelope.

Background: Plant leaf movements can be mediated by specialized motor organs, the pulvini, or can be epinastic (i.e. based on different growth velocities of the adaxial and abaxial halves of the leaf). Both processes are associated with diurnally regulated increases in rates of membrane water transport, which in many cases has been shown to be facilitated by aquaporins. Rhythmic leaf movements are known from many plant species, but few papers deal with the involvement of aquaporins in such movements., Scope: Many details of the architecture and function of pulvini were worked out by Ruth Satter and co-workers using Samanea saman as a model organism. More recently a contribution of aquaporins to pulvinar movement in Samanea was demonstrated. Another model plant to study pulvinus-mediated leaf movements is Mimosa pudica. The contribution of both plasma membrane- and tonoplast-localized aquaporins to the seismonastic leaf movements in Mimosa was analysed. In tobacco, as an example of epinastic leaf movement, it was shown that a PIP1 aquaporin family member is an important component of the leaf movement mechanism.

The effects of short-term (minutes) variations of CO2 concentration on mesophyll conductance to CO2 (gm) were evaluated in six different C3 species by simultaneous measurements of gas exchange, chlorophyll fluorescence, online carbon isotope discrimination and a novel curve-fitting method. Depending on the species, gm varied from five- to ninefold, along the range of sub-stomatal CO2 concentrations typically used in photosynthesis CO2-response curves (AN)-Ci curves; where AN is the net photosynthetic flux and Ci is the CO2 concentrations in the sub-stomatal cavity), that is, 50 to 1500 micromol CO2 mol(-1) air. Although the pattern was species-dependent, gm strongly declined at high Ci, where photosynthesis was not limited by CO2, but by regeneration of ribulose-1,5-bisphosphate or triose phosphate utilization. Moreover, these changes on gm were found to be totally independent of the velocity and direction of the Ci changes. The response of gm to Ci resembled that of stomatal conductance (gs), but kinetic experiments suggested that the response of gm was actually faster than that of gs. Transgenic tobacco plants differing in the amounts of aquaporin NtAQP1 showed different slopes of the gm-Ci response, suggesting a possible role for aquaporins in mediating CO2 responsiveness of gm. The importance of these findings is discussed in terms of their effects on parameterization of AN-Ci curves.

The measurement of the response of net photosynthesis to leaf internal CO2 (i.e. A-Ci curves) is widely used for ecophysiological studies. Most studies did not consider CO2 exchange between the chamber and the surrounding air, especially at the two extremes of A-Ci curves, where large CO2 gradients are created, leading to erroneous estimations of A and Ci. A quantitative analysis of CO2 leakage in the chamber of a portable open gas exchange system (Li-6400, LI-COR Inc., NE, USA) was performed. In an empty chamber, the measured CO2 leakage was similar to that calculated using the manufacturer's equations. However, in the presence of a photosynthetically inactive leaf, the magnitude of leakage was substantially decreased, although still significant. These results, together with the analysis of the effects of chamber size, tightness, flow rate, and gasket material, suggest that the leakage is larger at the interface between the gaskets than through the gaskets. This differential leakage rate affects the parameterization by photosynthesis models. The magnitude of these errors was assessed in tobacco plants. The results showed that leakage results in a 10% overestimation of the leaf maximum capacity for carboxylation (Vc,max) and a 40% overestimation of day respiration (Rl). Using the manufacturer's equations resulted in larger, non-realistic corrections of the true values. The photosynthetic response to CO2 concentrations at the chloroplast (i.e. A-Cc curves) was significantly less affected by leakage than A-Ci curves. Therefore, photosynthetic parameterization can be improved by: (i) correcting A and Ci values for chamber leakage estimated using a photosynthetically inactive leaf; and (ii) using A-Cc instead of A-Ci curves.

Epinastic leaf movement of tobacco is based on differential growth of the upper and lower leaf surface and is distinct from the motor organ-driven mechanism of nyctinastic leaf movement of, for example, mimosa species. The epinastic leaf movement of tobacco is observed not only under diurnal light regimes but also in continuous light, indicating a control by light and the circadian clock. As the transport of water across membranes by aquaporins is an important component of rapid plant cell elongation, the role of the tobacco aquaporin Nt aquaporin (AQP)1 in the epinastic response was studied in detail. In planta NtAQP1-luciferase (LUC) activity studies, Northern and Western blot analyses demonstrated a diurnal and circadian oscillation in the expression of this plasma membrane intrinsic protein (PIP)1-type aquaporin in leaf petioles, exhibiting peaks of expression coinciding with leaf unfolding. Cellular water permeability of protoplasts isolated from leaf petioles was found to be high in the morning, i.e. during the unfolding reaction, and low in the evening. Moreover, diurnal epinastic leaf movement was shown to be reduced in transgenic tobacco lines with an impaired expression of NtAQP1. It is concluded that the cyclic expression of PIP1-aquaporin represents an important component of the leaf movement mechanism.