Pressure Chamber Technique Research Articles

The Ecophysiological Response of Olive Trees under Different Fruit Loads.

Olive trees have a unique reproductive pattern marked by biennial fruiting. This study examined the repercussions of alternate fruit bearing on the water relations of olive trees and the associated ecophysiological mechanisms. The experiment spanned two consecutive years: the "ON" year, characterized by a high crop load, and the "OFF" year, marked by minimal fruit production. Key ecophysiological parameters, including sap flow, stomatal conductance, and photosynthetic rate, were monitored in both years. Pre-dawn water potential was measured using continuous stem psychrometers and the pressure chamber technique. Biochemical analyses focused on non-structural carbohydrate concentrations (starch, sucrose, and mannitol) and olive leaves' carbon-stable isotope ratio (δ13C). Results revealed a higher leaf gas exchange rate during the "ON" year, leading to an average 29.3% increase in water consumption and a 40.78% rise in the photosynthetic rate. Higher water usage during the "ON" year resulted in significantly lower (43.22% on average) leaf water potential. Sucrose and starch concentrations were also increased in the "ON" year, while there were no significant differences in mannitol concentration. Regarding the carbon-stable isotope ratio, leaves from the "OFF" year exhibited significantly higher δ13C values, suggesting a higher resistance to the CO2 pathway from the atmosphere to carboxylation sites compared to the "ON" year plants.

Field measurement of tree fine root hydraulic conductivity using a root pressure chamber technique

Field measurement of tree fine root hydraulic conductivity using a root pressure chamber technique

A root functional-structural model allows assessment of the effects of water deficit on water and solute transport parameters.

Root water uptake is driven by a combination of hydrostatic and osmotic forces. Water transport was characterized in primary roots of maize seedlings grown hydroponically under standard and water deficit (WD) conditions, as induced by addition of 150 g l-1 polyethylene glycol 8000 (water potential= -0.336 MPa). Flow measurements were performed using the pressure chamber technique in intact roots or on progressively cut root system architectures. To account for the concomitant transport of water and solutes in roots under WD, we developed within realistic root system architectures a hydraulic tree model integrating both solute pumping and leak. This model explains the high spontaneous sap exudation of roots grown in standard conditions, the non-linearity of pressure-flow relationships, and negative fluxes observed under WD conditions at low external hydrostatic pressure. The model also reveals the heterogeneity of driving forces and elementary radial flows throughout the root system architecture, and how this heterogeneity depends on both plant treatment and water transport mode. The full set of flow measurement data obtained from individual roots grown under standard or WD conditions was used in an inverse modeling approach to determine their respective radial and axial hydraulic conductivities. This approach allows resolution of the dramatic effects of WD on these two components.

Phenotyping and modeling of root hydraulic architecture reveal critical determinants of axial water transport.

Water uptake by roots is a key adaptation of plants to aerial life. Water uptake depends on root system architecture (RSA) and tissue hydraulic properties that, together, shape the root hydraulic architecture. This work investigates how the interplay between conductivities along radial (e.g. aquaporins) and axial (e.g. xylem vessels) pathways determines the water transport properties of highly branched RSAs as found in adult Arabidopsis (Arabidopsis thaliana) plants. A hydraulic model named HydroRoot was developed, based on multi-scale tree graph representations of RSAs. Root water flow was measured by the pressure chamber technique after successive cuts of a same root system from the tip toward the base. HydroRoot model inversion in corresponding RSAs allowed us to concomitantly determine radial and axial conductivities, providing evidence that the latter is often overestimated by classical evaluation based on the Hagen–Poiseuille law. Organizing principles of Arabidopsis primary and lateral root growth and branching were determined and used to apply the HydroRoot model to an extended set of simulated RSAs. Sensitivity analyses revealed that water transport can be co-limited by radial and axial conductances throughout the whole RSA. The number of roots that can be sectioned (intercepted) at a given distance from the base was defined as an accessible and informative indicator of RSA. The overall set of experimental and theoretical procedures was applied to plants mutated in ESKIMO1 and previously shown to have xylem collapse. This approach will be instrumental to dissect the root water transport phenotype of plants with intricate alterations in root growth or transport functions.

Aquaporins and their function in root water transport under salt stress conditions in Eutrema salsugineum

Eutrema salsugineum is considered as extremophile model species. To gain insights into the root hydraulic conductivity and the role played by aquaporins in E. salsugineum, we investigated the aquaporin family profiles, plant water status and root hydraulic conductivity under standard (salt-free) and salt stress conditions. We found that there was no variation in the relative electric conductivity of the leaves when the salt concentration was less than 200 mM NaCl, and the transpiration rate dropped to 60.6% at 100 mM NaCl for 14 days compared to that at standard conditions. The pressure chamber techniques indicated that the root hydraulic conductivity of E. salsugineum was repressed by salt stress. However, propionic acid, usually used as an aquaporin inhibitor, unexpectedly enhanced the root hydraulic conductivity of E. salsugineum. The aquaporin family in E. salsugineum was profiled and the PIP aquaporin expression was investigated at the transcriptional and translational levels. Finally, two EsPIPs were identified to play a role in salt stress. The overall study provides evidence on how halophytes maintain their water status and aquaporin regulation pattern under salt stress conditions.

Measurements and simulation of leaf xylem water potential and root water uptake in heterogeneous soil water contents

The relationship between leaf water potential, transpiration rate and soil water potential is complex, particularly when the soil water potential in the root zone is not uniform, which is the rule rather than the exception in soils. Our objectives were: 1) to measure the effect of heterogeneous soil water potentials on the relation between leaf water potential and transpiration rate and 2) to test whether root water uptake models could predict this relation. To this end, we combined the root pressure chamber technique, which allows measuring the suction in the leaves of transpiring plants, with two models of root water uptake, a simple one where soil and roots are presented as resistances in series and a more detailed 3D root architecture model. The experiments were carried out with lupines grown in sandy soil, for which the root architecture and root hydraulic properties had been previously estimated. The soil was partitioned in two layers separated by a coarse sand layer that allowed the roots to grow through but limited the water redistribution between the layers. Three scenarios (wet-wet, dry-wet, dry-dry) were tested. The results showed that the relation between transpiration and leaf water potential was linear in all scenarios. As the upper soil layer severely dried, the conductance of the soil-plant system decreased by ca. 1.65 times compared to the conductance of the plant-soil system in a uniform wet soil. As both layers dried, the conductivity was 8.26 times lower compared to the uniform-wet case. The combination of the experiment and modelling showed that a simple model is capable to reproduce the relation between transpiration, leaf water potential and soil water potential (despite an offset in the leaf water potential). Both simplified and the 3D root architecture models were capable of reproducing the measured changes in hydraulic conductance of the plant-soil system due to the soil drying. However, both models overestimated the measured leaf water potential by 0.1 MPa, probably because of a gradient in osmotic potential between the xylem and the soil. The simulations predicted the occurrence of hydraulic lift, even at day time conditions, although the hydraulic lift was relatively more important at low transpiration rates. The simulation suggested that a root architecture model is needed to estimate the variations of water uptake along the individual roots and this might be crucial to properly model hydraulic lift.

The impact of xylem cavitation on water potential isotherms measured by the pressure chamber technique in Metasequoia glyptostroboides Hu & W.C. Cheng.

Pressure-volume (PV) curve analysis is the most common and accurate way of estimating all components of the water relationships in leaves (water potential isotherms) as summarized in the Höfler diagram. PV curve analysis yields values of osmotic pressure, turgor pressure, and elastic modulus of cell walls as a function of relative water content. It allows the computation of symplasmic/apoplastic water content partitioning. For about 20 years, cavitation in xylem has been postulated as a possible source of error when estimating the above parameters, but, to the best of the authors' knowledge, no one has ever previously quantified its influence. Results in this paper provide independent estimates of osmotic pressure by PV curve analysis and by thermocouple psychrometer measurement. An anatomical evaluation was also used for the first time to compare apoplastic water fraction estimates from PV analysis with anatomical values. Conclusions include: (i) PV curve values of osmotic pressure are underestimated prior to correcting osmotic pressure for water loss by cavitation in Metasequoia glyptostroboides; (ii) psychrometer estimates of osmotic pressure obtained in tissues killed by freezing or heating agreed with PV values before correction for apoplastic water dilution; (iii) after correction for dilution effects, a solute concentration enhancement (0.27MPa or 0.11 osmolal) was revealed. The possible sources of solute enhancement were starch hydrolysis and release of ions from the Donnan free space of needle cell walls.

The Temporal Variation of Leaf Water Potential in Pistachio under Irrigated and Non-Irrigated Conditions

The present study was carried out in the experimental field of Pistachio Research Institute on pistachio trees which has uzun variety that was 30 years old. The aim of this research was to determine the Leaf Water Potential (LWP) of Pistacia vera L. under irrigated and non-irrigated conditions. In the study, the leaf water potential of pistachio was investigated under fully irrigated and non irrigated conditions. The leaf water potential values were measured one day before and after irrigation by using pressure chamber technique at the beginning, mid and end of irrigation season. According to the results obtained from measurements, the LWP value at the beginning of the irrigation season was -3.7 MPa at noon time due to relatively high temperature for both treatments. At the time of pre-dawn and sunset, this value increased and reached to -1.6 MPa due to relatively low temperature. In general, the LWP values during the mid of irrigation season, in the irrigated treatments, reached to almost -2.5 MPa in the non-irrigated treatment and the value was measured as -3.68 MPa.

Xylem sap calcium concentrations do not explain liming-induced inhibition of legume gas exchange

Liming is considered normal agricultural practise for remediating soil acidity and improving crop productivity; however recommended lime applications can reduce yield. We tested the hypothesis that elevated xylem sap Ca2+ limited gas exchange of Phaseolus vulgaris L. and Pisum sativum L. plants that exhibited reduced shoot biomass and leaf area when limed. We used Scholander and whole-plant pressure chamber techniques to collect root and leaf xylem sap, a calcium-specific ion-selective electrode to measure xylem sap Ca2+, infra-red gas analysis to measure gas exchange of limed and unlimed (control) plants, and a detached leaf transpiration bioassay to determine stomatal sensitivity to Ca2+. Liming reduced shoot biomass, leaf area and leaf gas exchange in both species. Root xylem sap Ca2+ concentration was only increased in P. vulgaris and not in P. sativum. Detached leaves of both species required 5 mM Ca2+ supplied to via the transpiration stream to induce stomatal closure, however, maximum in vivo xylem sap Ca2+ concentrations of limed plants was only 1.7 mM and thus not high enough to influence stomata. We conclude that an alternative xylem-borne antitranspirant other than Ca2+ decreases gas exchange of limed plants.

The water relations of two tropical rainforest species (<em>Virola surinamensis</em> and <em>Eperua falcata</em>): Is <em>Virola</em> unusual as previously reported?

The objective of this study was to examine the water relations and hydraulic architecture and vulnerability to cavitation in Virola surinamensis and V. michelii and to compare to similar measurements in Eperua falcata. In several previous reports Virola was seen to have a rather narrow range of xylem pressure potentials (Ψx) near zero in the course of a wet-season day while having water fluxes quite close to Eperua. We tested the hypothesis that the narrow range of Ψx might be consistent with very high hydraulic conductivities of stems, roots and shoots and high vulnerability to cavitation in Virola compared to Eperua. When this hypothesis proved false we concluded that the previous determinations of Ψx might be wrong in Virola due to latex. We re-examined the determination of Ψx in Virola by the pressure chamber technique and compared results to determination of Ψleaf by the thermocouple psychrometer technique and found that the likely range of Ψx are more negative than previously reported. Problems concerning the determination of Ψx in species with latex are discussed.

Comparative monitoring of temporal and spatial changes in tree water status using the non-invasive leaf patch clamp pressure probe and the pressure bomb

Abstract Real-time monitoring of plant water status under field conditions remains difficult to quantify. Here we give evidence that the magnetic-based leaf patch clamp pressure (LPCP) probe is a non-invasive and online-measuring method that can elucidate short- and long-term temporal and spatial dynamics of leaf water status of trees with high precision in real time. Measurements were controlled remotely by telemetry and data transfer to the Internet. Concomitant measurements using the pressure chamber technique (frequently applied for leaf water status monitoring) showed that both techniques yield in principle the same results despite of the high sampling variability of the pressure chamber data. There was a very good correlation between the output pressure signals of the LPCP probe and the balancing pressure values (on average r 2 = 0.90 ± 0.05; n = 8), i.e. the external pressure at which water appears at the cut end of a leaf under pressure chamber conditions. Simultaneously performed direct measurements of leaf cell turgor pressure using the well-established cell turgor pressure probe technique evidenced that both techniques measure relative changes in leaf turgor pressure. The output pressure signals of the LPCP probe and the balancing pressure values were inversely correlated to turgor pressure. Consistent with this, the balancing pressure values and the cell turgor pressure values could be fitted quite well by the same firm theoretical backing derived recently for the LPCP probe ( Zimmermann et al., 2008 ). This finding suggests that use of the LPCP probe technique in agricultural water management can be built up on the knowledge accumulated on spot leaf or stem water potential measurements.

Contrasting responses facing peak drought in seedlings of two co-occurring oak species

Summary To evaluate leaf physiological mechanisms involved in plant tolerance to water deficit between sites of varying overstorey density, we investigated leaf water relations of two broadleaved oaks (Quercus petraea (Matt.) Liebl. and Quercus pyrenaica Willd.) planted within a dense Scots pine stand, in a thinned adjacent area and in a nearby gap at a Mediterranean, mid-mountain field site. Leaf water parameters were estimated in established seedlings at the end of June and August over two consecutive years by measuring pressure–volume relationships with the pressure chamber technique. Plant water status was always similar in both species, and it was lower in August than June due to lower soil moisture at the end of summer. Higher light and diurnal water deficits in the gap were associated to more negative osmotic potentials. Impacts of overstorey density on some leaf water parameters depended on the species. While Q. pyrenaica showed active osmotic adjustment across sites, seedlings of Q. petraea were unable to cope with increased water deficit by osmotic adjustment in the unthinned dense area. While Q. pyrenaica showed consistent increases in the maximum bulk modulus of elasticity (emax) from June to August across sites, emax decreased in the unthinned dense area for seedlings of Q. petraea. These results could reflect distinct species strategies to cope with water deficit under the low-light conditions created by a dense overstorey. Higher leaf osmotic adjustment and bulk modulus of elasticity in Q. pyrenaica seedlings at peak summer drought might confer on them a competitive advantage during establishment in dry sub-Mediterranean understories. Opening of moderate canopy gaps in dense Scots pine stands improves some leaf mechanisms involved in drought tolerance in oak seedlings.

Response of a clingstone peach cultivar to regulated deficit irrigation

Regulated deficit irrigation (RDI) involves inducing water stress during specific fruit growth phases by irrigating at less than full evapotranspiration. The objectives of this research were to study the effects of RDI perfomed at stage II of fruit growth and postharvest, on productivity of clingstone peaches, fruit quality as well as photosynthetic rate and midday leaf water potential. The research was conducted in a commercial clingstone peach (Prunus persica L. Batch cv. A-37) orchard in Greece. Trees were irrigated by means of microsprinklers and their frequency was determined using local meteorological station data and the FAO 56 Pennman-Monteith method. Photosynthetic rate was measured by a portable infrared gas analyzer. Midday leaf water potential was measured by the pressure chamber technique. During the years 2005 and 2006, the treatment RDII with irrigation applied at growth stage II of the peach tree did not affect productivity, fresh and dry mass of fruits. RDII reduced preharvest fruit drop in comparison to the control. RDII as well as the combined treatment RDII plus RDIP with irrigation applied at postharvest, at both years reduced shoot length of the vigorous shoots inside the canopy. RDII in comparison to the control increased the soluble solids content of the fruits and the ratio soluble solids/acidity. However it did not affect fruit acidity and fruit firmness. RDII as well as RDII plus RDIP in 2006 increased 'double' fruits and fruits with open cavity in comparison to the control and RDIP. Water savings were considerable and associated with the climatic conditions of each year.

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

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.

Chemical Hydrophobicity and Uptake by Plant Roots

The transpiration stream concentration factor (TSCF), the ratio between a compound's concentration in the xylem to that in the solution adjacent to the roots, is commonly used to describe the relative ability of an organic compound to be passively transported from root to shoot. Widely cited bell-shaped curves relating TSCFto the octanol/water partition coefficient (log Kow) imply that significant root uptake and transfer into shoot tissues occurs only for compounds falling within an intermediate hydrophobicity range. However, recent laboratory and field data for relatively water soluble compounds such as sulfolane, methyl tert-butyl ether (MTBE), and 1,4-dioxane suggest that these relationships are not universally applicable, especiallyfor nonionizable, highly polar, water soluble organics. To re-evaluate the relationship between root uptake and chemical hydrophobicity, TSCFs were measured for 25 organic chemicals ranging in log Kow from -0.8 to 5 using a pressure chamber technique. Using the TSCF values measured in this study, a new empirical relationship between TSCF (0 and 1) and log Kow (-0.8 to 5) is presented that indicates that nonionizable, polar, highly water soluble organic compounds are most likely to be taken up by plant roots and translocated to shoot tissue.

Genotypic variation in drought response of silver birch (Betula pendula Roth): leaf and root morphology and carbon partitioning

Young trees of four clones of Betula pendula from origins with different amounts of precipitation were subjected to prolonged drought periods in two subsequent summers. The aims of this study were: (1) to investigate the drought response of silver birch in a multi-level approach and to elucidate the basis of the particularly wide physiological amplitude of this species and (2) to analyse variation in drought responses of different genotypes in order to identify potential genotypic differences with regard to their adaptability to drought. Responses of different physiological, biochemical, and morphological parameters to water shortage were regularly analysed: CO2 and H2O gas exchange were investigated under ambient and elevated CO2 concentration; leaf water status and osmotic adjustment were analysed with the pressure-chamber technique and pressure volume curves; leaves were analysed for chlorophyll and nitrogen content, and carbon isotopic composition; at the end of each drought period, trees were completely harvested and above- and belowground masses and areas determined.The following results were obtained: (1) Reduction of stomatal conductance was the first and most plastic response of silver birch to water shortage, thus effectively maintaining predawn leaf water potentials; non-stomatal limitations of photosynthesis were not detected. (2) Silver birch appeared to be capable of osmotic adjustment, which was mainly used during the phase of establishment. (3) Marked responses of total leaf area and the especially plastic phenology of this species have to be classified as adaptations to drought. (4) Both within-organ and between-organ allocation of biomass responded to drought. (5) Genotypes from drier locations displayed higher degrees of osmotic adjustment than those from origins with better water supply. (6) Genotypes from drier locations reached higher stomatal conductances under both well watered and drought stressed conditions. (7) Paticularly one genotype from a stand prone to summer drought displayed a more prodigal water use strategy than the genotypes from origins with higher precipitation. This strategy in combination with effective mechanisms of both drought avoidance and tolerance is classified as highly competitive and adapted to habitats with edaphic summer droughts.In conclusion, all genotypes responded to water shortage by marked mechanisms of drought avoidance on all levels of plant performance; The extent of response, however, appeared to be partly affected by adaptations to places of origin, with the genotypes from drier locations displaying a tendency for more prodigal water use strategies.

Some guidelines for monitoring desertification of Mediterranean drylands on the basis of plant water status

Drought stress can be considered as a dominant factor contributing to degradation of Mediterranean drylands. Therefore, there is a strong need to monitoring the level of water stress suffered by vegetation in the view of relating it to desertification risk of selected areas. In the present paper, some techniques for measuring and quantifying plant water stress are briefly reviewed and the advantages of using the pressure chamber technique to measure leaf water potential (ΨL) are highlighted. A new index (water stress impact on vegetation (WSIV)), based on the integral of the diurnal changes of ΨL, is proposed and its effectiveness for quantifying the amount of water stress suffered by vegetation is discussed on the basis of measurements performed in degraded areas of Turkey and Lebanon. The usefulness of WSIV as an ecophysiological tool for assessing the desertification risk of Mediterranean drylands is also discussed.

Measurement of xylem translocation of weak electrolytes with the pressure chamber technique.

Xylem translocation and root uptake of weak electrolytes were investigated with the pressure chamber technique (PCT) using de-topped soybean plants. Two compounds were organic bases (fenpropimorph and imazalil) and four were organic acids (bentazone, primisulfuron-methyl, rimsulfuron and triasulfuron). The compounds covered a wide range of log KOW and pKa values. Concentrations in external solution and in xylem sap were measured by HPLC at pH values in external solution of 4.5, 6.5 and 8.5. For weak bases, translocation was higher at low pH and the transpiration stream concentration factors (TSCF) were in the range 0.31-0.95. At pH 8.5, the concentrations in leaking xylem sap were very low for fenpropimorph, and steady-state was probably not reached. For weak acids, TSCF values derived with external pH from 4.5 to 8.5 were in the range 0.55-1.50 for primisulfuron-methyl, 0.64-1.35 for rimsulfuron, 0.81-0.93 for triasulfuron and 0.69-0.92 for bentazone. The variation of TSCF of the weak electrolytes was much smaller in these PCT experiments than in recent experiments with intact plants. The likely reason is that de-topped soybean plants in the pressure chamber seemed to be unable to regulate their xylem sap pH, which was almost identical to the pH in external solution. Without pH differences, the ion-trap process, which is responsible for accumulation or exclusion of weak acids and bases in the xylem of living plants, does not take place. Model simulations carried out for intact and de-topped plants supported this hypothesis. By variation of the pH of the xylem sap, good agreement between measurements and simulations could be achieved.

What are the driving forces for water lifting in the xylem conduit?

After Renner had shown convincingly in 1925 that the transpirational water loss generates tensions larger than 0.1 MPa (i.e. negative pressures) in the xylem of cut leafy twigs the Cohesion Theory proposed by Böhm, Askenasy, Dixon and Joly at the end of the 19th century was immediately accepted by plant physiologists. Introduction of the pressure chamber technique by Scholander et al. in 1965 enforced the general belief that tension is the only driving force for water lifting although substantial criticism regarding the technique and/or the Cohesion Theory was published by several authors. As typical for scientific disciplines, the advent of minimal- and non-invasive techniques in the last decade as well as the development of a new, reliable method for xylem sap sampling have challenged this view. Today, xylem pressure gradients, potentials, ion concentrations and volume flows as well as cell turgor pressure gradients can be monitored online in intact transpiring higher plants, and within a given physiological context by using the pressure probe technique and high-resolution NMR imaging techniques, respectively. Application of the pressure probe technique to transpiring plants has shown that negative absolute pressures (down to - 0.6 MPa) and pressure gradients can exist temporarily in the xylem conduit, but that the magnitude and (occasionally) direction of gradients contrasts frequently the belief that tension is the only driving force. This seems to be particularly the case for plants faced with problems of height, drought, freezing and salinity as well as with cavitation of the tensile water. Reviewing the current data base shows that other forces come into operation when exclusively tension fails to lift water against gravity due to environmental conditions. Possible candidates are longitudinal cellular and xylem osmotic pressure gradients, axial potential gradients in the vessels as well as gel- and gas bubble-supported interfacial gradients. The multiforce theory overcomes the problem of the Cohesion Theory that life on earth depends on water being in a highly metastable state.

A new validation of the Scholander pressure chamber technique based on stem diameter variations

The Scholander pressure chamber is one of the most widely used techniques for measuring plant water status. However, the technique has been the subject of recent controversies, and its validity awaits new experimental evidence. This paper presents a new test based on the analysis of the dependence on water potential difference (DeltaPsi) of stem diameter variation (DeltaD) in walnut (Juglans regia L.). The correlation between DeltaPsi and DeltaD was established (1) on transpiring potted trees, (2) on dehydrating cut branches, (3) by perfusing the xylem of branch segments with mannitol and sucrose solutions, and (4) by pressurizing segments in a pressure sleeve. The DeltaPsi was respectively assessed with a pressure chamber (1, 2), a freezing point osmometer (3) and an air pressure transducer (4). A single relationship was established between DeltaPsi (ranging from 0 to -2 MPa) and DeltaD for all the experiments. This shows that the measured changes of water potential were correlated to similar modifications of water content in the stems, irrespective of the technique used to induce these changes, and therefore validates the pressure chamber technique and confirms the occurrence of large negative pressures in the xylem of walnut branches.