Leaf physiology
Pre-dawn leaf water potential (ΨPD), photosynthesis under saturated light intensity (A area, µmol m-2 s-1), stomatal conductance (g s, mol m-2s-1), and the maximum and operating efficiencies of photosystem II were measured between July 21 and August 26, 2018. All traits were measured during 12 sessions. Each session consisted of two steps that were executed on the same day. First, ΨPD was measured from each of three trees growing in one mesocosm of each treatment combination. The selected trees were evenly distributed within a mesocosm and ΨPD from at least one diploid and one triploid tree was measured per mesocosm. ΨPD was measured between 0400-0530 h using a pressure chamber (PMS Instrument Company, Albany OR, USA). In one additional session, we quantified ΨPD and leaf physiological traits from only two mesocosms, of which one was exposed to drought stress and defoliation and the other was exposed to drought stress only. During the experiment, every mesocosm was selected two or three times for measurements. Mesocosms subjected to drought stress were measured at different stages of soil dry down.
In a second step, leaf gas-exchange and variable chlorophyll fluorescence were measured on fully expanded, young leaves from all trees growing in the previously selected mesocosms using an LI-6400XT portable photosynthesis system with a broadleaf cuvette and a pulse amplitude modulation (PAM) fluorometer (Li-Cor Biosciences, Lincoln, NE, USA). During measurements, leaves in the cuvette were exposed to a photosynthetic photon flux of 1800 µmol m-2s-1. The reference CO2 partial pressure was 40 Pa, and the cuvette temperature was set at 25 °C. We did not control vapor pressure deficit between leaf and cuvette air, which ranged from 1.4–1.9 kPa. Measurements were taken on sunlit foliage between 0900-1200h. From August 18 through August 25, gas exchange was also measured at a cuvette CO2 partial pressure (p CO2) ranging from 7.5 to 25 Pa on sunlit foliage from all trees of three mesocosms that were neither drought stressed nor defoliated. The observed relationship betweenA area and intercellularp CO2 (C i) was used to estimate V cmax (µmol CO2m-2 s-1), the maximum velocity of RuBP carboxylation, employing a trend-fitting method that minimized the total sums of squares for differences between observed versus predictedA (Long & Bernacchi, 2003). Estimates relied on Michaelis–Menten constants for CO2(K c) and oxygen (K o), as well as photosynthetic compensation p CO2 (Γ*), derived using formulae from Long and Bernacchi (2003). Because we did not account for the influence of mesophyll conductance on CO2 diffusion into the chloroplast (Dillaway & Kruger, 2010), our derived V cmax values are “apparent” (Bernacchi et al., 2013) and based on C i rather than chloroplastic p CO2(C c).