Analyzing Metabolic Changes in ETH3 Seeds

Seeds of ETH3 and control were collected at the fruit mature stage of ‘Huashuo’. The soluble sugar content was determined using anthrone colorimetry (Liu et al., 2015 (link)). The contents of sucrose and reducing sugars were evaluated using the 3,5-dinitrosalicylic acid method (Yang et al., 2017 (link)). Endogenous ethylene content was evaluated by the ACC content (Hu et al., 2021 (link)). The grinded samples of 0.5 g were homogenized in phosphate-buffered saline, and then centrifuged at for 20 min (4°C, 12000 rpm). These supernatants were used to measure the ACC contents. The ACC contents of the seed and shell were measured according to the Plant 1-aminocyclopropane carboxylic acid ACC kit (Shanghai Jingkang Bioengineering, Co., Ltd., Shanghai, China) instructions (Hu et al., 2021 (link)). The OD450 value was determined using a microplate reader (BioTek, Winooski, Vermont, USA).
Ten leaves from one tree were randomly selected to measure the chlorophyll content for each biological replicate. Leaves of ethephon treatment and control were cut into filaments. The filaments of 0.2 g were immersed in an acetone–ethanol mixture (2:1, v/v) for 24 h (4°C, darkness). The samples were shaken several times during the experiment. The absorbance indexes at 663 and 645 nm of the solution were assessed by a spectrophotometer (UV-1100, Mapada, China). The chlorophyll a and chlorophyll b contents were calculated, referring to the method of Zhang et al. (Zhang et al., 2021 (link)).