Bc0700

A commercial kit (BC1230, Solarbio) was applied to determine AsA content based on the oxidation rate of AsA catalyzed by AAO (ascorbic acid oxidase) (spectrophotometric measurement at OD_{265 nm}). A commercial kit (BC1240, Solarbio) was applied to determine DHA content based on the production rate of AsA from DHA by DTT (1,4-dithiothreitol) (spectrophotometric measurement at OD_{265 nm}). A commercial kit (BC1170, Solarbio) was applied to determine GSH content based on spectrophotometric measurement of the product from the reaction between GSH and DTNB (5,5’-dithiobis-2-nitrobenoic acid) (OD_{265 nm}). A commercial kit (BC1180, Solarbio) was applied to determine GSSG content based on the production rate of GSH (measurement of OD_{265 nm} for the reaction between GSH and DTNB) from GSSG catalyzed by GR (GSH reductase). A commercial kit (BC0290, Solarbio) was applied to determine proline content based on spectrophotometric measurement of the product from the reaction between proline and ninhydrin (OD_{520 nm}). A commercial kit (BC0035, Solarbio) was applied to determine plant soluble sugar content based on anthrone colorimetric method (OD_{620 nm}). A commercial kit (BC0700, Solarbio) was applied to determine starch content based on the separation of starch with 80% ethanol and acid hydrolyzation to glucose, followed by anthrone colorimetric method (OD_{620 nm}) (Chen et al., 2020b (link)).

The phenotype of excessive starch and premature senescence of lses1 leaves appeared from three-leaf stage to tillering stage. Therefore, the leaves of WT and lses1 at six-leaf stage which is the transition period between seedling stage and tillering stage were used for the following analyses. The second leaf from the top of WT and lses1 seedlings at the sixth leaf stage was harvested at the end of the night (6:00) and at the end of the day (18:00), frozen in liquid nitrogen and stored at − 80 °C. Approximately 0.2 g frozen leaf samples were ground using a mortar and pestle under cryogenic conditions. The leaf powder was measured by using a starch content assay kit (BC0700, Solarbio, China) according to the manufacturer’s assay procedure. At the same time, the first, second, and third leaves from the top of the WT and lses1 seedlings were sampled and stained with I₂-KI solution as described by Hagen et al. (2008) with minor modifications [20 ]. The leaves were incubated in an acetone/ethanol (1:1, v/v) mixture and placed at room temperature for 24-36 h until the chlorophyll was removed. After bleaching, the leaves were put in I₂-KI solution for approximately 20 min and then removed and placed on a table. Photographs were taken and saved with a camera. Three biological replicates of each sample were performed.

Mature leaves of ~3-week-old plants were infected with Pst DC3000 and were then ground to powder in liquid nitrogen. After that, chlorophyll was extracted in the dark with 1 ml of ethanol (95%) at 4 °C for 24 h. The precipitate was washed three to five times with 95% ethanol until it was completely white. The supernatant was transferred to a new tube, and the content of chlorophyll was calculated using spectrophotometric absorbance (A) at light wavelengths of 665 and 649 nm, with 95% ethanol as a control⁷⁴ . The chlorophyll content is shown as milligrams of chlorophyll per gram of freshly infected leaves
Chlorophyll a (mg g⁻¹) = 13.95 × A₆₆₅ − 6.88 × A₆₄₉Chlorophyll b (mg g⁻¹) = 24.96 × A₆₄₉ − 7.32 × A_665.The content of starch was measured from leaves inoculated with Pst DC3000 (~150 mg of fresh weight). The leaves were ground to powder in liquid nitrogen and incubated at 80 °C for 30 min in 80% ethanol to separate the soluble sugar from the starch. The total starch in each sample was then quantified using a kit from Solarbio (BC0700, China) according to the manual.