Os1 fl

At the end of the trial, the chlorophyll content was determined according to Moran and Porath [43 (link)]. At the beginning and every 20 days [T1 (0 days), T2 (20 days), T3 (40 days), T4 (60 days)] of the experimental period, the net photosynthetic rate (AN) was measured on fully expanded leaves using a CO₂/H₂O IRGA (LCi, ADC Bioscientific Ltd., Hoddesdon, UK). The measurements were carried out in clear conditions from 10:00 to 14:00 (solar time). After measuring photosynthesis, in the same leaves, chlorophyll a fluorescence was measured using a modulated chlorophyll fluorimeter OS1-FL (Opti-Sciences Corporation, Tyngsboro, MA, USA). Each leaf was dark-adapted for 20 min using cuvette clips supplied by the company. Fluorescence was expressed as the Fv/Fm ratio, which indicates the maximum quantum yield of PSII, where F0 = the minimum fluorescence, Fm = the maximum fluorescence of the dark-adapted state, and Fv = the variable fluorescence [44 (link)].

Chlorophyll was extracted and quantified as described previously (Wang et al. 2022 (link)). Total fluorescence in leaves was measured using a portable modulated chlorophyll fluorometer (model OS1-FL) according to the manufacturer’s instructions (Opti-Sciences, Tyngsboro, MA). The variable and maximal fluorescence (F_v/F_m) of individual leaves was quantified directly using the fluorometer’s module 9 program (He and Gan 2002 (link)).

The chlorophyll fluorescence [variable fluorescence (Fv)/maximal fluorescence (Fm); μmol m^− 2 s^− 1] was measured using a modulated chlorophyll fluorometer OS1-FL (Opti-Sciences, ADC, BioScientific, Ltd., Hoddesdon, UK). The fresh leaf was obscured by clipping after 20 min to set up a dark-adapted state. Chlorophyll fluorescence was excited by a 660 nm solid-state light source, with filters blocking radiation longer than 690 nm. Saturation of the photosystem being measured was achieved by using a filtered 35 W halogen lamp (350–690 nm) with a pulse of 15,000 μmol m^− 2 s^− 1 during 0.8 s.

Chlorophyll fluorescence was measured using the modulated chlorophyll fluorometer OS1-FL (Opti-Sciences Inc, USA). Flag leaves of stressed and unstressed genotypes was placed in close contact with the photosynthetically active radiation clip, which provides basic data to the OS1-FL system on ambient conditions. The maximal quantum yield of PSII was calculated as Fv/Fm=(Fm−Fo)/Fm, where the minimum fluorescence (Fo) was recorded after dark adaptation for 10 min and the maximum fluorescence (Fm) was monitored by application of a 0.8-s saturating light pulse (6,000 μmol photons m⁻² s⁻¹) from white LED light. Gas-exchange measurements were done using a LI-6400XT (LI-COR Inc., NE, USA) in attached leaves of WT and HYR transgenic plants under well-watered and drought-stress conditions. CO₂ gas-exchange measurements were performed after 4 h of illumination with a daily photoperiodic cycle of 14 h light and 10 h dark at leaf temperature of 25 °C, CO₂ at 400 μmol s⁻¹ and RH of 55–60%. Instantaneous WUE (WUEi) was calculated as described4 (link) using the formula: WUEi=(Pn/E).

The maximum quantum efficiency of PSII photochemistry was measured on days 7, 14, 21, 28 of the experimental period, using a modulated chlorophyll fluorometer OS1-FL (Opti-Sciences Corporation, Tyngsboro, MA, USA). Leaves were dark-adapted for 30 min prior to measurements. Fv/Fm ratio was calculated using the formula (Fm-F0)/Fm, where Fm is maximal fluorescence yield of the dark-adapted state and F0 is minimum fluorescence yield (Maxwell and Johnson, 2000 (link)).

All six transgenic plant lines (S1–S6) were used to determine which has the highest sucrose content based on photosynthesis capability and chlorophyll content. Photosynthetic activity was measured at early morning in the tenth leaf from the top of the transgenic and WT plant was measured using the OS1-FL (OPTI—Sciences Co. Ltd., Hudson, NH, USA) as described previously (Hajirezaei et al., 2002 (link)). Chlorophyll was extracted from the frozen leaf powder using ethanol 90%, boiled for 5 min and measured absorbance at OD 620 nm to calculate chlorophyll concentration (Lichtenthaler, 1987 ).

At each harvesting time the cell density was measured by employing a Thoma counting chamber and the fresh weight was determined gravimetrically. The photosynthetic rate was measured with an imaging/pulse-amplitude modulation fluorimeter (OS1-FL, Opti-Sciences).
Total lipids were extracted from frozen pellets with 200 μL of a mixture of chloroform:isopropanol (1:1) and vigorous vortexing for 3 min. The samples were centrifuged (14.000 × g, 5 min, room temperature) and the supernatants were transferred to a new tube. The pellet was re-extracted with 500 μL of hexane and vigorous vortexing for 3 min. The samples were centrifuged, and the combined supernatants were dried in a speed vac. The amount of lipids was determined gravimetrically.