Ciras 3

The chlorophyll content was determined 24 h before plant harvesting by means of a leaf-clip sensor (Dualex Force, Orsay, France). In each plant, three leaves of the third branch from the top were selected, and the average chlorophyll content was obtained from three measurements taken at the central position of each leaf.
The gas exchange measurements at 400 ppm CO₂, including stomatal conductance, CO₂ assimilation rate, and water use efficiency (WUE) were obtained from leaves of the third branch from the top of four randomly replicate plants per treatment, making use of a CIRAS-3 portable gas exchange system (PP-Systems, Amesbury, MA, USA) 24 h before plant harvesting. The leaves were pressed between the upper and lower gaskets of the leaf cuvette head of CIRAS-3 and pre-acclimated for 15−20 min.

The net photosynthesis rates of cotyledons of (4 DAG) seedlings were measured with CIRAS-3 (PP Systems, Amesbury, MA, United States). The analyzers act as absorption meters to measure infrared absorption. The optical bench is temperature-controlled and pressure-compensated to provide accurate CO₂ and H₂O measurement. Net photosynthesis (A) was determined from the difference between the CO₂ concentration entering (Cin) and exiting (Cout) the cuvette. IRGA CO₂ reading was corrected for water vapor, temperature, and atmospheric pressure. Since humidity dilutes the air leaving the cuvette (Cout), it was compensated using the following equation: net photosynthesis (A) = (Cin × W) – [Cout × (W+E)], where (A) is net photosynthesis, (W) is mass flow of air per unit leaf area into the cuvette, and (E) is transpiration rate.

The gas exchange analysis was performed on intact attached leaves of 21-day old plants using a Ciras 3 portable photosynthesis instrument equipped with a narrow (1.7 cm²) leaf cuvette (PP systems, Haverhill MA, United States). For ACCx3 pretreated plants treatments were done on days 18, 19, 20. The net assimilation rate (Pn), stomatal conductance (gs), and transpiration rate (E) were determined under two temperature conditions (22 and 37°C) and at steady state of photosynthesis using a CO₂ level of 400 µl l⁻¹ and light intensity of 700 µmol m⁻² s⁻¹.

The leaf gas exchange characteristics viz. photosynthetic rate (A), stomatal conductance (g_s), sub-stomatal conductance (C_i), and transpiration rate (E) were recorded from the second fully expanded young mature leaf from the top using portable photosynthesis system CIRAS-3 (PP Systems, Amesbury, U.S.A.). These observations were made in the morning between 8:00 and 10:00 with the adjustments as reported in Shehzad et al. (2020 ).

Six seedling leaves from three trays were measured for the photosynthetic parameters by using a narrow leaf chamber connected to a CIRAS-3 (PP Systems, Amesbury, MA, US) [26 (link)]. In this study, 400 µmol mol⁻¹ of carbon dioxide was constantly retained, and 800 µmol m⁻²s⁻¹ of light intensity was maintained with the light-emitting diode light sources placed in the leaf chamber.

Photosynthetic parameters, such as: leaf net photosynthesis (A), sub-stomatal CO₂ concentration (C_i), transpiration (E), stomatal conductance (gs), photosynthetic water use efficiency (WUE), and photosynthetic CO₂ response curve (A/C_i) were determined with a portable gas exchange analyzer (CIRAS-3; PP Systems, Amesbury, MA, USA). Measurements were performed 12 days after the administration of nanoparticles on fully expanded leaves at the fourth node. PARi levels at 1000 µmol/m²s were obtained from an LED Light Unit (RGBW) connected to the gas analyzer. The temperature within the chamber was kept at 25 °C, relative humidity at 80%, and reference CO₂ concentration at 390 µmol/mol. Photosynthetic CO₂ response curves were collected at a CO₂ concentration gradient ranging from 0 to 1500 µmol/mol. All measurements were performed on fully expanded leaves at the fourth node. The acclimatization time between measurements was 120 s. The results from each CO₂ level were recorded three times. Two biochemical parameters: maximum carboxylation rate (V_cmax) and maximum electron transport rate (J_max) were calculated in Rstudio (v.3.4.2; R Foundation for Statistical Computing, Vienna, Austria) [66 ] using the “plantecophys” package developed by Duursma [67 (link)].

Assimilation of CO₂, transpiration and stomatal conductance were measured using an infrared gas analyzer (Ciras-3, PP Systems, Hitchin, UK), with a Parkinson leaf chamber (PLC6) automatically controlling the measurement conditions (irradiance = 500 μmol (quanta) m⁻² s⁻¹, halogen lamps, RH = 30%, CO₂ concentration (400 μmol (CO₂) mol⁻¹ (air)). The airflow rate through the assimilation chamber was 350–400 cm³ min⁻¹. The measurements were made in the middle part of leaves and were performed for four replicates of each introgression form on one day before cold acclimation initiation and on the 7th, 14th, and 21st day of CA, in the temperature designed for these time-points according to Augustyniak et al. (2018) [38 (link)]. Based on the measured gas exchange parameters, water use efficiency was calculated as the ratio of the rate of CO₂ assimilation to the rate of transpiration.