Apoplastic water fraction and rehydration techniques introduce significant errors in measurements of relative water content and osmotic potential in plant leaves

Relative water content (RWC) and the osmotic potential (pi) of plant leaves are important plant traits that can be used to assess drought tolerance or adaptation of plants. We estimated the magnitude of errors that are introduced by dilution of pi from apoplastic water in osmometry methods and the errors that occur during rehydration of leaves for RWC and pi in 14 different plant species from trees, grasses and herbs. Our data indicate that rehydration technique and length of rehydration can introduce significant errors in both RWC and pi. Leaves from all species were fully turgid after 1-3 h of rehydration and increasing the rehydration time resulted in a significant underprediction of RWC. Standing rehydration via the petiole introduced the least errors while rehydration via floating disks and submerging leaves for rehydration led to a greater underprediction of RWC. The same effect was also observed for pi. The pi values following standing rehydration could be corrected by applying a dilution factor from apoplastic water dilution using an osmometric method but not by using apoplasticwater fraction (AWF) from pressure volume (PV) curves. The apoplastic water dilution error was between 5 and 18%, while the two other rehydration methods introduced much greater errors. We recommend the use of the standing rehydration method because (1) the correct rehydration time can be evaluated by measuring water potential, (2) overhydration effects were smallest, and (3) pi can be accurately corrected by using osmometric methods to estimate apoplastic water dilution.