A revised and unified pressure-clamp/relaxation theory for studying plant cell water relations with pressure probes: In-situ determination of cell volume for calculation of volumetric elastic modulus and hydraulic conductivity T. Knipfer a,n , J. Fei a , G.A. Gambetta a,1 , K.A. Shackel b , M.A. Matthews a a Department of Viticulture and Enology, University of California, Davis, USA b Department of Plant Sciences/Pomology, University of California, Davis, USA HIGHLIGHTS  Accurate estimates of plant cell volume (ν o ) can be determined in-situ using cell-pressure-probes.  A revised ν o -theory was developed that is valid for the pressure-clamp and pressure-relaxation methods.  For the same cell, the pressure-clamp method gave a systematically lower (21%) ν o as compared to the pressure-relaxation method.  Effects of solute mixing could only explain a potential error in calculated ν o of o3%.  The results suggest that both methods are differentially affected by cell osmotic behavior (i.e. solute reflection coefficient, s) in response to turgor changes. article info Article history: Received 28 May 2013 Received in revised form 19 May 2014 Accepted 22 May 2014 Available online 5 June 2014 Keywords: Convection Micromanipulation Solute diffusion Solute reflection coefficient Tradescantia virginiana abstract The cell-pressure-probe is a unique tool to study plant water relations in-situ. Inaccuracy in the estimation of cell volume (ν o ) is the major source of error in the calculation of both cell volumetric elastic modulus (ε) and cell hydraulic conductivity (Lp). Estimates of ν o and Lp can be obtained with the pressure-clamp (PC) and pressure-relaxation (PR) methods. In theory, both methods should result in comparable ν o and Lp estimates, but this has not been the case. In this study, the existing ν o -theories for PC and PR methods were reviewed and clarified. A revised ν o -theory was developed that is equally valid for the PC and PR methods. The revised theory was used to determine ν o for two extreme scenarios of solute mixing between the experimental cell and sap in the pressure probe microcapillary. Using a fully automated cell-pressure-probe (ACPP) on leaf epidermal cells of Tradescantia virginiana, the validity of the revised theory was tested with experimental data. Calculated ν o values from both methods were in the range of optically determined ν o ( ¼1.1–5.0 nL) for T. virginiana. However, the PC method produced a systematically lower (21%) calculated ν o compared to the PR method. Effects of solute mixing could only explain a potential error in calculated ν o of o3%. For both methods, this discrepancy in ν o was almost identical to the discrepancy in the measured ratio of ΔV/ΔP (total change in microcapillary sap volume versus corresponding change in cell turgor) of 19%, which is a fundamental parameter in calculating ν o . It followed from the revised theory that the ratio of ΔV/ΔP was inversely related to the solute reflection coefficient. This highlighted that treating the experimental cell as an ideal osmometer in both methods is potentially not correct. Effects of non-ideal osmotic behavior by transmembrane solute movement may be minimized in the PR as compared to the PC method. & 2014 Elsevier Ltd. All rights reserved. 1. Introduction The cell-pressure-probe has been used for more than 30 years to study water relations in-situ at the individual cell level (Hüsken et al., 1978; Steudle, 1993; Tomos and Leigh, 1999), mostly in higher plants (Tomos et al., 1981; Tyerman and Steudle, 1982; Zhu and Steudle, 1991; Moore and Cosgrove, 1991) but also in algal Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/yjtbi Journal of Theoretical Biology http://dx.doi.org/10.1016/j.jtbi.2014.05.035 0022-5193/& 2014 Elsevier Ltd. All rights reserved. Abbreviations: ACPP, automated cell pressure probe; PC, pressure-clamp; PR, pressure-relaxation n Corresponding author. Tel.: þ1 530 752 7185. E-mail address: knipfer.thorsten@yahoo.de (T. Knipfer). 1 Present address: Institut des Sciences de la Vigne et du Vin, Bordeaux, France Journal of Theoretical Biology 359 (2014) 80–91