Li 6400xt portable photosynthesis system

At the heading stage, LI-6400XT Portable Photosynthesis System (LI-COR, Inc., Lincoln, NE USA) was used to measure Pn of the flag leaves in the early, middle, and late seasons in 2017. The photosynthetically active radiation was controlled at 1200 μmol m⁻² s⁻¹ provided by a 6400-2B LED light source. Six representative flag leaves from each plot were measured and recorded, and the mean values were calculated for Pn.

Hydrated and dehydrated tissues were collected at certain intervals and their fresh weights (FWs) were measured immediately. The DW was measured after drying for at least 48 h in a 65 °C oven. Water content (WC) was calculated using the formula WC = (FW − DW)/DW. At least five biological replicates were included for each time point.
Electrolyte leakage and MDA contents were examined to assess membrane stability, soluble sugars and proline contents were determined to evaluate cellular structure protection, and GSH, POD and SOD activities were determined to quantify antioxidant capacity (ROS elimination). Electrolyte leakage was measured using a DDBJ-350 electrical conductivity meter (INESA Scientific Instruments Co., Shanghai, China). The contents of MDA, soluble sugars, and proline, and the activities of GSH, POD, and SOD were measured using antioxidant detection kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Photosynthesis and respiration rates were detected using a LI-6400XT portable photosynthesis system (LI-COR, Lincoln, NE, USA). Chlorophyll fluorescence was measured using a IMAGING-PAM M-series chlorophyll fluorometer (Heinz Walz, Effeltrich, Germany).

Leaf chlorophyll content was measured from the recently expanded leaves of the CK and GT-KD seedlings grew at 12 or 15 DAG, with a SPAD meter (SPAD-502 Plus, Konica Minolta, Inc., Tokyo, Japan) under a saturating actinic light (660 nm) with an intensity of 1,100 μmol m⁻²s⁻¹. The middle widest part of the recently expanded leaf of every seedling, that is the second leaf of the 12 DAG seedlings and the third leaf of the 15 DAG seedlings, was used for the SPAD value measurement. The net photosynthetic rate (Pn) and transpiration rate (TR) were measured from the latest expanded leaf (the third leaf) of the 15 DAG seedlings (with three leaves and a heart leaf) with a portable LI-6400XT Portable Photosynthesis System (LI-COR, Lincoln, NE, USA), recorded at a saturating actinic light (660 nm) with an intensity of 1,100 μmol m⁻²s⁻¹, at the time from 09:00 to 12:00 in the morning. All the measurements were conducted on the middle part of the latest expanded leaves following the instructions of the manufacturer. Five replicates were randomly taken for each genotype.

Leaf succulence on a dry weight basis was measured using the equation: $\text{Succulence}(\text{g}{\text{H}}_2\text{O}{\text{g}}^{-1}\text{DW})=\frac{(\text{FW}-\text{DW})}{\text{DW}}$
Relative water content (RWC) was found out with the help of the formula: $\text{RWC}(\mathrm{\%})=\frac{(\text{FW}-\text{DW})}{(\text{TW}-\text{DW})}\times 100$
where FW is the fresh weight; DW the dry weight and TW the turgid weight of leaves after rehydration in distilled water for 24 h at room temperature (∼25 °C).
Leaf osmotic potential was calculated using van't Hoff equation (Kramer and Boyer 1995 ) on osmolality of expressed leaf sap measured by a vapour pressure osmometer (VAPRO-5520; Wescor Inc., Logan, UT, USA) (Gucci et al. 1991 ). Xylem pressure potential (XPP) was measured on excised stems with a plant water status console Model 1000 (PMS Instrument Co., Albany, NY, USA). Instantaneous WUE was calculated from the rate of CO₂ fixation (A) which was measured using a Li-6400XT portable photosynthesis system (LICOR Biosciences) per amount of water transpired (E) from the leaf surface as $\text{WUE}(\mu \text{mol}{\text{CO}}_2{\text{mmol}}^{-1}{\text{H}}_2\text{O})=\frac{A}{E}$

A 0.1 g of leaves were digested in 5 mL alcohol (95%, v/v) overnight at 4 °C, and then centrifuged at 12,000 rpm (Centrifuge 5425/5425 R; Eppendorf, Germany) for 5 min at 4 °C. The supernatant was stored at 4 °C for Chl a, Chl b, Chl a+b, and Chl a/b ratio measurements [46 (link)]. Leaf photosynthetic parameters were measured between 10:30 and 11:30 AM using an LI-6400 XT portable photosynthesis system (LI-COR Inc., Lincoln, NE, USA). The Pn, Ci, Gs, and Tr were measured in a chamber at 1,500 μM m⁻² s⁻¹ photosynthetically active radiation (PAR) and 380 ± 5 μM CO₂ M⁻¹ [15 ]. The WUE and Rubisco activity [47 (link)] were estimated as follows:

For each line, all phenotypic traits were evaluated using five representative leading tillers. Plant height and its component traits, including spike length, internode number, and the length of the first internode to the sixth internode from the top, were measured during harvest.
Five photosynthesis traits, chlorophyll content, the net photosynthetic rate, intercellular CO₂ concentration, the transpiration rate, and stomatal conductance, were investigated at both the seedling and heading stages. The chlorophyll content was measured using a SPAD-502 Plus chlorophyll meter (Minolta, Osaka, Japan), and the other four photosynthesis traits were estimated using an LI-6400XT Portable Photosynthesis system (LI-COR, Lincoln, USA). Data from two consecutive years (E1 of 2015–2016 and E2 of 2016–2017) were combined and then used as phenotypic traits in E3.

Leaves from two weeks wild-type plants and two sapk3 mutant lines were fixed with 2.5% glutaraldehyde and observed by scanning electron microscopy (S-3400 N, Hitachi, Japan). The second leaves of two weeks rice plants were selected for the stomatal conductance analysis, which was performed using the LI-6400XT Portable Photosynthesis System (LI-COR, USA) before and after 7 days exposure to drought stress.