Bc0290

The 3 different treatments on the transgenic lines and wild-type, including water, salt and simulated drought stress, were performed and kept for 7 days. The fresh leaves of wild-type and transgenic P. hopeiensis lines under water, salt and simulated drought stress were collected and cut into filaments with a width of 2 mm. The filaments were extracted with a mixture of acetone and ethanol (volume ratio 1:1) for 24 h. The absorbance was measured at 663 nm, 645 nm, and 470 nm by spectrophotometer, and the total chlorophyll content was calculated (Zhang 2016 ). A superoxide dismutase (SOD) activity, peroxidase (POD) activity, catalase (CAT) activity, malondialdehyde (MDA) content, and proline (PRO) content detection kit (BC0170, BC0090, BC0200, BC0020, and BC0290 Solarbio, China).

The plasma samples from the verification cohort were used to detect the levels of proline (Solarbio, BC0290) following the manufacturer’s protocols. The plasma level of PYCR1 was measured by ELISA according to standard protocols (LMAI Bio, LM-11556H). To evaluate the difference in the levels of metabolites and related enzymes between groups, Student’s t test or the Mann-Whitney U test was performed depending on the distribution of data, which were subsequently applied for ROC curve analysis to distinguish patients with asthma and controls.

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)).