ABA Accumulation in Dehydrating Leaves Is Associated with Decline in Cell Volume, Not Turgor Pressure.

Identifying the mechanisms for cell responses as plants dehydrate is crucial for analyzing and predicting crop and ecosystem responses to climate change (Blum, 1996, 2017; Bartlett et al., 2016), for isolating the proteins and the genes underlying the responses (Christmann et al., 2013), and for the design ofmodel plants and crops with increased water use efficiency and/or drought tolerance (e.g. Nemhauser and Torii, 2016; Yang et al., 2016). The dehydration-sensingmechanisms involved in driving the accumulation of the hormone abscisic acid (ABA; see symbols in Table I) are of special importance as it is implicated in stomatal closure during drought (Rodriguez-Dominguez et al., 2016) or increasing vapor pressure deficit (McAdam et al., 2016), and may contribute to the decline of leaf hydraulic conductance (Shatil-Cohen et al., 2011; Pantin et al., 2013). Cellular ABA accumulation during dehydration may occur due to modulation of transport from cellular or apoplastic stores, de novo synthesis, and/or turnover (Finkelstein, 2013). However, disentangling the factors that leaf cells sense during dehydration is difficult as many changes typically occur in tandem: turgor is lost, solute concentrations increase, relative water content (RWC) decreases, and cell membranes shrink, altering interactions with the cytoskeleton and cell wall (Haswell and Verslues, 2015). Two recent articles (McAdam and Brodribb, 2016; Sussmilch et al., 2017) have argued based on applying external pressure to leaves that turgor loss provides the endogenous signal triggeringABAaccumulation and that species differ greatly in the turgor loss threshold that triggers ABA accumulation. We derived new equations from plant water relations theory enabling the calculation of turgor, solute potential, and RWC for the experimental leaves in those studies. These calculations establish that the accumulation of ABA in these artificially dehydrated leaves was not due to decline of turgor pressure but instead was associated with the decline of RWC. These analyses further show that the RWC loss associated with ABA accumulation varied by approximately 10% across the diverse angiosperm species, indicating functional convergence in cellular drought sensing and providing clues for identification of the components of the signaling pathway. The debate on the precise determinants of ABA accumulation began decades ago. In a dehydrating leaf, cell volume, turgor, osmotic potential, and leaf water potential decline together, and making a distinction among these may seem at first semantic. However, it is critical to distinguish exactly which of these or related physical properties is ultimately sensed and leads to ABA accumulation. For example, changes in cell volume independently of turgor may affect sensors of cytoskeletal properties, ion concentrations or ion transport rates, or cell membrane interactionswith the cell wall, whereas sensing of membrane tension might be affected by volume and/or turgor. The idea that turgor loss was the driver for ABA accumulation arose from early experiments showing the hormone levels increased in drying leaves as leaf water potential (Cleaf) declined (e.g. Zabadal, 1974; Beardsell andCohen, 1975;Wright, 1977), and was later further supported circumstantially by the finding that in many species, stomatal closure, known to be driven by ABA levels, apparently coincides roughly, on average, with turgor loss point (global data recently synthesized in Bartlett et al., 2016). Subsequent experiments took the necessary next step by dehydrating leaves of several species on the bench top andmeasuring ABA accumulation, and used pressure volume curves to estimate solute and pressure potentials from leaf water potentials (Pierce and Raschke, 1980). These calculations showed that increases in ABA accumulation correlated more closely with the decline of turgor pressure (CP) than with the declines of either osmotic potential (CS) or Cleaf. Yet, those studies did not consider the decline of RWC as a potential driver. Subsequent experiments confirmed that CS did not driveABAaccumulation: leaf sections of spinach (Spinacia oleracea) and maize (Zea mays) accumulated ABA if incubated in mannitol or polyethylene glycol, which 1 This work was supported by the U.S. National Science Foundation (award nos. 1457279 and 1557906), the Australian Research Council (DP150103863 and LP130101183), and International Wheat Yield Partnership/Grains Research and Development Corporation US00082. 2 Address correspondence to [email protected]. L.S., G.P.J., and T.N.B. designed the study, conducted the analyses, and wrote the article. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.17.01097