Abscisic Acid Mediates a Divergence in the Drought Response of Two Conifers

During water stress, stomatal closure occurs as water tension and levels of abscisic acid (ABA) increase in the leaf, but the interaction between these two drivers of stomatal aperture is poorly understood. We investigate the dynamics of water potential, ABA, and stomatal conductance during the imposition of water stress on two drought-tolerant conifer species with contrasting stomatal behavior. Rapid rehydration of excised shoots was used as a means of differentiating the direct influences of ABA and water potential on stomatal closure. Pinus radiata (Pinaceae) was found to exhibit ABA-driven stomatal closure during water stress, resulting in strongly isohydric regulation of water loss. By contrast, stomatal closure in Callitris rhomboidea (Cupressaceae) was initiated by elevated foliar ABA, but sustained water stress saw a marked decline in ABA levels and a shift to water potential-driven stomatal closure. The transition from ABA to water potential as the primary driver of stomatal aperture allowed C. rhomboidea to rapidly recover gas exchange after water-stressed plants were rewatered, and was associated with a strongly anisohydric regulation of water loss. These two contrasting mechanisms of stomatal regulation function in combination with xylem vulnerability to produce highly divergent strategies of water management. Species-specific ABA dynamics are proposed as a central component of drought survival and ecology.By guarding the interface between plant and atmosphere, the stomata of land plants occupy a uniquely important role that connects diverse aspects of plant biology with atmospheric processes. Capitalizing upon the potential for stomata to be used to modify plant growth and survival, or as a tool for interpreting environmental change, requires a mechanistic understanding of how these tiny valves operate. Yet, an integrated understanding of stomatal control remains elusive. Foremost in this uncertainty is an explanation for how complex signals from the environment are translated into guard cell movement. A particularly challenging feature of stomatal behavior is the fact that environmental perturbation induces both physical and chemical responses within the plant and that turgor-regulated stomata are responsive to both signals. Disentangling these distinct contributions to stomatal conductance (g_s) has been made more complicated by the limited communication between molecular-scaled disciplines of mutant characterization and membrane transport biology and researchers at the larger scale of plant water relations and xylem transport. As a result, two contrasting views of stomatal control exist. Molecular biologists view stomata as osmotically regulated valves uniquely responsive to plant hormone levels and the resultant movement of ions across the guard cell membranes (Schroeder et al., 2001; Roelfsema and Hedrich, 2005). By contrast, most process-based models assume a direct influence of soil water content on stomatal aperture (Buckley, 2005; Damour et al., 2010).The phytohormone abscisic acid (ABA) is seen as a cornerstone of stomatal function because it has been shown to trigger responses in guard cell membrane channels and transporters that cause a reduction in guard cell turgor, thereby closing stomata. ABA-mediated stomatal closure in seed plants (but not in ferns and lycophytes; Brodribb and McAdam, 2011) is broadly accepted as the explanation for stomatal closure during water stress (Zhang and Davies, 1989; Bauer et al., 2013); yet, there are very few studies that show a good correlation between the level of ABA and g_s during water stress in the field. The traditional explanation for this lack of a strong relationship suggests that ABA is a root-derived hormone that is delivered to the leaf in the transpiration stream (Zhang et al., 1987; Davies and Zhang, 1991) and hence that the xylem ABA flux, rather than the leaf level of ABA, should dictate the intensity of the stomatal response to soil drying (Tardieu et al., 1992; Tardieu and Davies, 1993). The flux-based model for ABA action in the leaf remains the most widely used interpretation of how stomata sense and respond to drying soil, despite the fact that there is mounting evidence for significant ABA synthesis in the leaf and guard cells, and short term responses to ABA that cannot be explained by xylem transport (Christmann et al., 2005; Lee et al., 2006; Georgopoulou and Milborrow, 2012). Furthermore, the ABA flux approach has never been successfully applied to explain variation in transpiration in trees (Sperry, 2000; Cochard et al., 2002), suggesting that there may be some benefit in reexamining some of the principles and assumptions used to link water stress, ABA, and transpiration.Here, we examine the dynamics of stomatal closure, leaf ABA levels, and xylem tension during the gradual imposition of water stress upon two conifer species, Pinus radiata and Callitris rhomboidea, known for having contrasting stomatal responses to desiccation. Our primary aim is to separate the interacting effects of ABA and water tension on guard cell turgor pressure and stomatal diffusive conductance and hence to reveal the relative importance of water tension and ABA levels during drought as effectors of stomatal closure. Conifers are particularly suitable for identifying different closing signals because they do not appear to produce hydropassive stomatal movements (McAdam and Brodribb, 2012). This makes them ideal for examining the direct effects of ABA and water tension without the mechanical interactions between subsidiary cells and guard cells (Franks and Farquhar, 2007) that greatly complicate the mechanics of angiosperm stomatal movements. Both conifer species examined grow naturally in low rainfall habitats, but P. radiata is strongly isohydric (meaning that stomata close in a very narrow range of leaf hydration), while C. rhomboidea is anisohydric (meaning that stomata have a relatively low sensitivity to leaf hydration).