Photosynthesis and Fluorescence Measurements

The net photosynthetic rate, transpiration rate and stomatal conductance were determined in situ using an LCi Portable Photosynthesis System (ADC BioScientific, Hoddesdon, the United Kingdom) with the following conditions in the measurement chamber: air temperature at 25°C, ambient CO₂ concentration at 550±50 μL L^-1, air flow rate at 205±30 μmol s^-1, and irradiance at 300 μmol m^-2 s^-1 [25 (link)].
The polyphasic rise of the chlorophyll fluorescence transient (OJIP) was measured at the upper surface of the dark-adapted (20 min) leaves in situ with the portable fluorometer FluorPen FP100max (Photon System Instruments, Brno, Czech Republic) as described in [26 (link)]. The parameters of the JIP test (see S1 Table) were calculated according to the theory of energy flow in the photosynthetic electron-transport chain [29 , 30 ]. The relative variable fluorescences W_OI, W_OJ, W_OK and W_IP (i.e., normalizations of the whole fluorescence transients) and the difference kinetics ΔW_OJ and ΔW_OK (as the differences between the drought-stressed and control plants) were also calculated according to [31 ] and their graphical representation was utilised to obtain further information on the primary photosynthetic processes.
The chlorophyll a and b contents and total carotenoids were determined spectrophotometrically [32 ] in the N,N-dimethylformamide extracts prepared as described in [33 (link)].