*3.4. Effect of Light Intensity on Enzymatic Activity*

In C3 plants, photosynthesis is mostly regulated by the activity of Calvin cycle enzymes, including RCA, Rubisco, FBPase and TRXs, which are recognized as very early and fast responses of plants to shading stress [51,52]. Our results show that Rubisco activity in high-light-treated alfalfa plants was significantly higher than that in plants grown in low-light conditions. Similar results were reported by Feng et al. [19], suggesting that the activity of Rubisco increases with increasing light intensity, which could increase carbon assimilation and RuBP regeneration in the Calvin cycle [17]. We also found that decreased *P*<sup>n</sup> in alfalfa grown under low light intensity was accompanied by reductions in RAC and Rubisco activity and the transcriptional levels of most genes (*RCA*, *RbcL*, *RbcS*, *FBPase*, *TK* and *PGK*) involved in the Calvin cycle. Our results are in accordance with those of Zhang et al. (2020) [53], suggesting that restriction of CO<sup>2</sup> carboxylation in the Calvin cycle is a result of impaired activity of RCA. The RCA could remove inhibitors bound to Rubisco, and thus a decline in the activity of RCA indirectly causes the decreased CO<sup>2</sup> assimilation rate in low-light-grown seedlings [54]. Further, the activation state of RCA, which is controlled by the redox state of the cell, is sensitive to light intensity, and the proper regulation of RCA activity is also vital for acclimation to light fluctuation in *Arabidopsis* [55,56]. Therefore, depression of photosynthetic capacity induced by low light could be attributed to deceleration of the Calvin cycle [53].

Light intensity also plays a vital role in regulating the enzymes related to sucrose and starch biosynthesis [57,58]. The relative expression levels of *SPS*, *SS*, *AGPase*, *SSS*, *SBE* and *SP* were enhanced, and their encoding enzymes showed higher activities in the high-light treatments than low-light treatments, resulting in improved production of sucrose and starch [19]. Similar results also are reported for *Arabidopsis* [59] and soybean [19], suggesting that changes in light intensity equally promote the activities of SS, SPS and SSS and increase the sucrose and starch content, which improve plant growth and development. Therefore, the enzymatic activities of sucrose synthesis and starch synthesis enzymes play a vital role in regulating carbohydrate production, which is important in controlling storage of carbon reserves and growth of cells and tissues in plants under low light [60,61]. Therefore, the enzymatic activities for increasing sucrose and starch contents in alfalfa plants were the most effective in the L400 and L500 treatments.
