Measurement of physiological traits and mesophyll conductance
The measurements of net photosynthetic rates (Anet), stomatal conductance to water vapor (gsw), intercellular CO2 concentrations (Ci), transpiration (E), intrinsic WUE (Anet/ gsw), instantaneous WUE (Anet/E) and mesophyll conductance to CO2 (gm) were previously described in Pathare et al ., (2020). Briefly, isotopologs of CO2 and H2O were measured using the LI-6400XT infrared gas analyzer (LiCor, Lincoln, NE, USA) coupled to a tunable diode laser absorption spectroscope (TDLAS, model TGA 200A, Campbell Scientific, Logan, UT, USA) and a cavity-ring down absorption spectroscope (Picarro, Sunnyvale, CA, USA) as described previously (Ubierna et al ., 2017). The entire LI6400XT, the 2 cm x 6 cm leaf chamber (6400-11, Li-Cor), and LI-6400-18-RGB light source were placed in a growth cabinet (model EF7, Conviron; Controlled Environments Inc., MN, USA) with fluorescent lamps (F48T12/CW/ VHO; Sylvania, Wilmington, MA, USA) set at a PPFD of ~250 µmol photons m-2 s-1 and air temperature was maintained at 25 oC. In current study, gm was estimated using the method described by Ogeeet al. , (2018) as discussed in Pathare et al ., (2020). This method utilizes a newly developed model of C4photosynthetic discrimination that provides an estimate of the isotopic equilibration between CO2 and H2O inside the leaf and gm.