Psychrometric Pressure-Volume Analysis of Osmoregulation

Osmotic and turgor adjustment in roots, shoots, and whole sporophytes of a NaCI-tolerant mutant and a NaCI-sensitive wildtype strain of the fern Ceratopteris richardii Brongn. were charactenzed following exposure to 60 millimolar NaCI, using a psychrometric approach to pressure-volume analysis. Water potential components of whole plants and shoots at full or ambient hydration were similar between strains. Roots of the mutant, however, had osmotic potentials at full turgor and water potentials at zero turgor which were lower (0.32 and 0.46 megapascal, respectively) than those in roots of the wild type after salinization. Although compromised in the absence of NaCI, sporophytes of the mutant strain were larger and much less necrotic in 60 millimolar NaCI after 17 days, compared to the wild type. Root growth in the mutant strain was unaffected by salinization and far exceeded root growth in the wild type. Correlation coefficients of the linear regression and the general consistency and precision ofthe pressure-volume data demonstrated the feasibility of using PV cuw for estimating water potential components of roots. The Iwtique should also provide a means of studying osmoregulaion in a variety of other rapidly equilibrating plant tissues. Generating V/2 isotherms by PV analysis is regarded as the most accurate method of estimating the symplastic 4',. of live plant tissue (33). A psychrometric or hygrometric determination of the 4', of plant extracts or frozen/thawed material is used frequently because of its speed and simplicity, but this assay presents several problems (5, 8, 13, 33, 35). PV curves also provide much more information about the dynamics of tissue water status than the one-time measurement of extracts or killed tissue, giving estimates of elasticity, water partition'Partially supported by NSF Grant DMB 88-03620 (L. G. H.). 2 Abbreviations: 4A, water potential; 4,, osmotic potential; RWC, relative water content; PV, pressure-volume; 4'p, turgor or pressure potential; 1 0a23, NaCl-tolerant mutant strain; Hn-n, wild-type strain; 4t, ambient water potential; 4',.a ambient osmotic potential; /,,,a, ambient pressure potential; 0,,'°", osmotic potential at full turgor; ,t0, water potential at the turgor loss point; ROWC, relative osmotic water content; R/S, root:shoot ratio; T/D, turgid weight:dry weight ratio; ROWC°, relative osmotic water content at the turgor loss point; RWC°, relative water content at the turgor loss point; ROWCa, ambient relative osmotic water content; RWCa, ambient relative water content; FW, fresh weight; DW, dry weight. ing, and component 4' changes across a broad range ofRWC (31, 33). The importance of roots in studies of plant growth and metabolism is obvious, yet the water relations of roots are routinely neglected. As a consequence, very little is known about osmotic and turgor regulation in roots. Use of the pressure chamber for PV analysis of roots has been attempted (16, 27, 32) but there have been some problems adapting this technique to roots. Root pressure apparently obscures the reading of balance points at high RWC (32), and 41 estimates at lower RWC are often erroneous because of tissue damage related to high pressurization. Theoretically, a psychrometric determination of root 4' offers important advantages. No pressurization is required, which allows reliable 4A estimates at much lower RWC than possible with the pressure chamber. Additionally, the problems related to root pressure with 4A estimates at high RWC are avoided. Moreover, a psychrometric determination of total ,6 may be more accurate. The pressure chamber gives only a measure of the 4,, of the apoplastic fluid; this is usually equated with total tissue 4A, based on the assumption that apoplastic solute content is negligible (6, 32). Recent evidence, however, indicates that apoplastic ',. of various tissues may be as low as -0.3 MPa under certain conditions (7, 19). The psychrometer, on the other hand, does furnish an estimate of total ,6 and is particularly useful for assaying 4' in rapidly equilibrating tissues. One objective of this study, therefore, was to determine the feasibility of using thermocouple psychrometry to generate PV curves of root tissue. Roots provide ideal candidates for psychrometry for three reasons: a lack of cuticle, a very high surface area-to-volume ratio and, typically, a very low ratio of cut surface-to-total surface area. The first two characteristics contribute to rapid sample equilibration within the psychrometer chamber, and the last obviates much of the 'cutedge' effect which sometimes confounds psychrometric estimates of leaf 41 (21, 39). Part of the objective was to study osmotic and turgor regulation in roots subjected to salinization. The ability of cells to adjust internal 4', and thus regulate water gain or loss in response to the osmotic and/or ionic stress of salt exposure has long been observed in aerial plant organs (1 1), yet reports of this protective phenomenon occurring in salinized roots are lacking. The homosporous fern Ceratopteris richardii forms a unique model system for an in vitro whole plant approach to