Methods for determining stomatal closure point appear linked to fundamentally different drivers

L. Turin Dickman, Kelsey R. Carter, Jeffrey Heikoop, John Heneghan, Dea Musa, Brent D. Newman, George Perkins and Sanna Sevanto, Earth & Environmental Sciences Division, Los Alamos National Laboratory, Los Alamos, NM, Christine Anderson-Cook, Anastasiia Kim, Nicholas Lubbers and Abby Nachtsheim, Computer, Computational and Statistical Sciences Division, Los Alamos National Laboratory, Los Alamos, NM, Louise H. Comas, Water Management Research Unit, USDA - Agricultural Research Service, Fort Collins, CO, Eric R. Moore, Sangeeta Negi, Michaeline Nelson Albright, Anthony Sabella, Christina Steadman, Scott Twary and John Dunbar, Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM

Background/Question/Methods
Stomatal closure point (SCP), the water potential (Ψleaf) at which stomata close to minimize water loss, is typically determined by one of two standard methods: 1) generating stomatal dehydration curves, where stomatal conductance (gs) and Ψleaf of excised tissue are measured successively as samples dehydrate to gs=0 (SCP_curve); or 2) imposing drought, monitoring daily gs under high-light, low-VPD conditions, and measuring Ψleaf when gs=0 (SCP_drought). Traditionally, results from these methods are considered interchangeable, but this has not been validated. As part of a larger study of soil microbiome effects on plant drought tolerance, we tested both methods on Zea mays grown in fritted clay treated with microbial inocula from two natural soils. To generate broad variation in SCP, we imposed a drought treatment on half of the pots (n=32) and kept the remainder watered to field capacity. Eight weeks after germination, we measured SCP_curve (n=24-32) and sampled soil pore water and leaf tissue for chemical and isotopic analysis (n=64). We then terminated watering and monitored gs between 8-10am daily until gs=0, at which point we measured Ψleaf (SCP_drought, n=64). Subsequently, we grew a second set of plants treated with soil inocula from the previous generation and repeated the experiment.
Results/Conclusions
SCPs measured with stomatal dehydration curves (SCP_curve) and under drought (SCP_ drought) were not significantly correlated in either generation (r=-0.10, p=0.65, n=24, and r=-0.09, p=0.65, n=32, respectively). In addition, preliminary analyses from the first generation indicate that relationships between SCP and other metrics differ between the two methods. While SCP_drought showed no correlation with leaf or soil water chemistry, SCP_curve was positively correlated with leaf δ15N (r=0.46, p=0.02, n=24) and negatively correlated with pore water NO3 (r=-0.54, p=0.01, n=21), suggesting a linkage between nitrogen cycling (plant uptake and accumulation) and SCP_curve, but not SCP_drought. Additionally, SCP_drought was negatively correlated with days to irreversible stomatal closure (r=-0.24, p=0.05, n=64) while SCP_curve was positively correlated with the same (r=0.65, p=0.001, n=24). These results suggest the SCPs derived from each method have fundamentally different drivers, and the methods used to evaluate SCP need to be carefully considered and distinguished in light of a study’s goals.