A147 1 1

35S::BoLSU1, 35S::BoLSU2 and wild-type Arabidopsis seedlings were treated with sulfur deficiency for 7 d. 100 mg seedlings were frozen separately in liquid nitrogen and homogenized with liquid nitrogen. Chlorophyll content was determined using an assay kit (Nanjing Jiancheng Bioengineering Institute, A147-1-1, Nanjing, China), and chlorophyll was extracted with a mixed solution of ethanol and acetone (1:2). Measurements were made at 645 nm and 663 nm. Chlorophyll content was calculated in accordance with manufacturer’s instruction.

After the treatment period (63 days), the whole plant was uprooted, washed, and the fresh weight, root weight, root length, and plant height were measured. A root scanner (WinRHIZO STD4800 LA2400, Regent, Canada) was used to determine the root surface area. Forage leaves and soil samples were collected to determine enzyme activity. Leaf catalase, peroxidase, superoxide dismutase, malondialdehyde, H₂O₂, proline, and chlorophyll contents were determined using the corresponding kits (A007-1-1, A084-3-1, A001-1-2, A003-1-2, A064-1-1, A107-1-1, and A147-1-1, Nanjing Jiancheng Bio-Engineering Institute Co., Ltd., China). The activity of soil alkaline protease (ALPT), urease (UE), neutral phosphatase (NP), acid phosphatase (ACP), polyphenol oxidase (PPO), and sucrase (SC) were determined using soil enzyme activity kits (BC0885, BC0125, BC0465, BC0145, BC0115, and BC0245, Beijing Solarbio Science & Technology Co., Ltd., China).