*5.7. Enzyme Activity*

The second fully expanded leaf of alfalfa seedlings was harvested and used for enzymatic assays. The activity of enzymes, including ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), ribulose-1,5-bisphosphate carboxylase/oxygenase activase (RCA, EC was not found), fructose-1, 6-bisphosphatase (FBPase, EC 3.1.3.11), thioredoxin reductase (TRXs, EC 1.8.1.9), sucrose synthase (SS, EC 2.4.1.13), sucrose phosphate synthase (SPS, EC 2.4.1.14), ADP-glucose pyrophosphorylase (AGPase, EC 2.7.7.27), soluble starch enzyme (SSE, EC 2.4.1.21), starch-branching enzyme (SBE, EC 2.4.1.18) and starch phosphorylase (SP, EC 2.4.1.1), was determined using plant-enzyme-linked im-

munosorbent assay (ELISA) kits. A frozen leaf sample (0.1 g) was homogenized in 1 mL of phosphate buffer (0.01 mol L−<sup>1</sup> , pH = 7.4) using a cold mortar and pestle and centrifuged at 5000× *g* and 4 ◦C for 10 min. The clear supernatant was then stored at 4 ◦C for 24 h pending analyses for the activity of enzymes. Firstly, 50 µL of standard or sample was added to the appropriate well of a microplate (except the blank wells). Secondly, 100 µL of HRP conjugate reagent was added, and the wells were covered with an adhesive plate membrane and incubated for 60 min at 37 ◦C. Thirdly, the liquid was discarded, and the wells were washed with 350 µL of wash buffer, and this procedure was repeated five times. Fourthly, a mixture of 50 µL of substrate A and 50 µL of substrate B was added to each well, mixed gently and incubated at 37 ◦C for 15 min in the dark. Finally, 50 µL of stop solution was added to each well, and the optical density was measured within 15 min at 450 nm using a microtiter plate reader (Infinite MPlex, Tecan, Austria). All the activity of enzymes was calculated using the methods reported by Pan et al. (2020) [18].

The protein concentration of each enzyme extraction solution was measured according to Li et al. (2020) [65]. The results are expressed as U/mL of protein.
