*2.2. Leaf Pigment and Nitrogen Content*

Chlorophyll a (Chl a), Chlorophyll b (Chl b), carotenoids (Car), Chl a + b, Chl a/b and leaf nitrogen content (LN) were significantly affected by light treatment (Table S4). Increased light intensity from L100 to L500 decreased Chl a, Chl b, Chl a + b and Car contents, while Chl a/b increased (Table 3). Chl a, Chl b, Chl a + b and Car contents for plants in the L500 treatment were decreased by 27.8%, 49.5%, 32.9% and 25.9% (*p* < 0.01), respectively, compared to L100, but those for plants grown at L400 and L500 did not differ significantly. Chl a/b was 17.5% (*p* < 0.01) higher in L500 than in L100. In addition, increased light intensity decreased LN content, and at L500, LN content decreased by 50.6% (*p* < 0.001) compared to L100.

**Table 3.** Effect of light treatments on Chlorophyll a (Chl a, µg cm−<sup>2</sup> ), Chlorophyll b (Chl b, µg cm−<sup>2</sup> ), carotenoids (Car, µg cm−<sup>2</sup> ), Chl a + b (µg cm−<sup>2</sup> ), Chl a/b and leaf nitrogen content (LNC, mg/g) of alfalfa plants.


<sup>a</sup> L100, L200, L300, L400 and L500 refer 100, 200, 300, 400 and 500 µmol m−<sup>2</sup> s −1 , respectively. Within a column, values followed by different letters are significantly different (*p* < 0.05). Values within parentheses are the standard errors of the means (*n* = 4).

### *2.3. Photosynthetic and Chlorophyll Fluorescence Characteristics 2.3. Photosynthetic and Chlorophyll Fluorescence Characteristics* The photosynthetic characteristics of alfalfa plants varied among light treatments

rentheses are the standard errors of the means (*n* = 4).

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<sup>a</sup> L100, L200, L300, L400 and L500 refer 100, 200, 300, 400 and 500 µmol m−2 s

The photosynthetic characteristics of alfalfa plants varied among light treatments (Table S5, Figure 2). The maximum net photosynthetic rate (*P*n), transpiration rate (*T*r) and stomatal conductance (*g*s) values of alfalfa plants were at L400 and L500, whereas intercellular CO<sup>2</sup> concentration (*C*<sup>i</sup> ) was highest at L100 to L300. On average, the net photosynthetic rates, *T*<sup>r</sup> and *g*<sup>s</sup> of alfalfa plants, were significantly increased by 230, 62 and 52%, respectively, (*p* < 0.01) at L400 and L500 compared to L100. However, intercellular CO<sup>2</sup> concentration at L400 and L500 decreased by 8.9 and 10.1% (*p* < 0.001), respectively, compared to L100. The photosynthetic characteristics of alfalfa leaves did not differ significantly at L400 and L500. The increased *P*<sup>n</sup> at L400 and L500 suggests that high light intensity was positively related to increased *g*<sup>s</sup> and *T*r, but negatively related to decreased *C*i in alfalfa plants. (Table S5, Figure 2). The maximum net photosynthetic rate (*P*n), transpiration rate (*T*r) and stomatal conductance (*g*s) values of alfalfa plants were at L400 and L500, whereas intercellular CO<sup>2</sup> concentration (*C*i) was highest at L100 to L300. On average, the net photosynthetic rates, *T*<sup>r</sup> and *g*<sup>s</sup> of alfalfa plants, were significantly increased by 230, 62 and 52%, respectively, (*p* < 0.01) at L400 and L500 compared to L100. However, intercellular CO<sup>2</sup> concentration at L400 and L500 decreased by 8.9 and 10.1% (*p* < 0.001), respectively, compared to L100. The photosynthetic characteristics of alfalfa leaves did not differ significantly at L400 and L500. The increased *P*<sup>n</sup> at L400 and L500 suggests that high light intensity was positively related to increased *g*<sup>s</sup> and *T*r, but negatively related to decreased *C*<sup>i</sup> in alfalfa plants.

a column, values followed by different letters are significantly different (*p* <0.05). Values within pa-

−1, respectively. Within

**Figure 2.** Photosynthetic characteristics of alfalfa leaves under different light treatments. L100, L200, L300, L400 and L500 refer 100, 200, 300, 400 and 500 µmol m<sup>−</sup><sup>2</sup> s −1 , respectively. Net photosynthetic rate (*P*n) (**A**), transpiration rate (*T*r) (**B**), intercellular CO<sup>2</sup> concentration (*C*i) (**C**), stomatal **Figure 2.** Photosynthetic characteristics of alfalfa leaves under different light treatments. L100, L200, L300, L400 and L500 refer 100, 200, 300, 400 and 500 µmol m−<sup>2</sup> s −1 , respectively. Net photosynthetic rate (*P*n) (**A**), transpiration rate (*T*r) (**B**), intercellular CO<sup>2</sup> concentration (*C*<sup>i</sup> ) (**C**), stomatal conductance and (*g*s) (**D**). Vertical bars indicate 1 s.e. of the mean (*n* = 4). Different lowercase letters on the different bar mean significant differences (*p* < 0.05).

Chlorophyll fluorescence characteristics, including maximal PSII quantum yield (Fv/Fm), effective PSII quantum yield (ΦPSII), non-photochemical quenching (NPQ) and the electron transport rate (ETR), were significantly affected by treatment (Table S6, Figure 3). Figure 3 shows the difference in absorbed radiation energy of alfalfa leaves in response to light treatments. The Fv/Fm, ΦPSII and NPQ of alfalfa plants grown in the low-light treatments were significantly higher than those in the high-light treatments. Furthermore, L100 increased the Fv/Fm, ΦPSII, and NPQ by 12.0, 24.9 and 60.8%, respectively, but it decreased the ETR by 71.2% (*p* < 0.001) compared to L500. These results indicate that the original activity of the PSII reaction center was increased, and the transformation efficiency of primary light energy was improved in the low-light-intensity adaption of alfalfa. (Fv/Fm), effective PSII quantum yield (ΦPSII), non-photochemical quenching (NPQ) and the electron transport rate (ETR), were significantly affected by treatment (Table S6, Figure 3). Figure 3 shows the difference in absorbed radiation energy of alfalfa leaves in response to light treatments. The Fv/Fm, ΦPSII and NPQ of alfalfa plants grown in the low-light treatments were significantly higher than those in the high-light treatments. Furthermore, L100 increased the Fv/Fm, ΦPSII, and NPQ by 12.0, 24.9 and 60.8%, respectively, but it decreased the ETR by 71.2% (*p* < 0.001) compared to L500. These results indicate that the original activity of the PSII reaction center was increased, and the transformation efficiency of primary light energy was improved in the low-light-intensity adaption of alfalfa.

conductance and (*g*s) (**D**). Vertical bars indicate 1 s.e. of the mean (*n* = 4). Different lowercase letters

Chlorophyll fluorescence characteristics, including maximal PSII quantum yield

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on the different bar mean significant differences (*p* < 0.05).

**Figure 3.** Chlorophyll fluorescence characteristics of alfalfa leaves under different light treatments. L100, L200, L300, L400 and L500 refer 100, 200, 300, 400 and 500 µmol m<sup>−</sup><sup>2</sup> s −1 , respectively. Maximal PSII quantum yield (Fv/Fm) (**A**), effective PSII quantum yield (ΦPSII) (**B**), non-photochemical quenching (NPQ) (**C**) and electron transport rate (ETR) (**D**). Vertical bars indicate 1 s.e. of the mean (*n* = 4). Different lowercase letters on the different bar mean significant differences (*p* < 0.05). **Figure 3.** Chlorophyll fluorescence characteristics of alfalfa leaves under different light treatments. L100, L200, L300, L400 and L500 refer 100, 200, 300, 400 and 500 µmol m−<sup>2</sup> s −1 , respectively. Maximal PSII quantum yield (Fv/Fm) (**A**), effective PSII quantum yield (ΦPSII) (**B**), non-photochemical quenching (NPQ) (**C**) and electron transport rate (ETR) (**D**). Vertical bars indicate 1 s.e. of the mean (*n* = 4). Different lowercase letters on the different bar mean significant differences (*p* < 0.05).

### *2.4. Leaf Non-Structural Carbohydrate Contents 2.4. Leaf Non-Structural Carbohydrate Contents* Soluble sugar (SS), sucrose and starch (St) were significantly affected by light treat-

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Soluble sugar (SS), sucrose and starch (St) were significantly affected by light treatments (*p* < 0.001) (Table S7). As expected, the content of SS, sucrose and St in leaves increased significantly with increased light intensity (Figure 4). The highest SS, sucrose and St contents in leaves were measured in the high-light treatments (i.e., L400 and L500) compared to low-light treatments (i.e., L100 and L200). ments (*p* < 0.001) (Table S7). As expected, the content of SS, sucrose and St in leaves increased significantly with increased light intensity (Figure 4). The highest SS, sucrose and St contents in leaves were measured in the high-light treatments (i.e., L400 and L500) compared to low-light treatments (i.e., L100 and L200).

**Figure 4.** Changes in carbon balance of alfalfa plants under different light treatments. L100, L200, L300, L400 and L500 refer 100, 200, 300, 400 and 500 µmol m<sup>−</sup><sup>2</sup> s −1 , respectively. Soluble sugar content (**A**), sucrose content (**B**) and starch content (**C**). Vertical bars indicate 1 s.e. of the mean (*n* = 4). Different lowercase letters on the different bar mean significant differences (*p* < 0.05). **Figure 4.** Changes in carbon balance of alfalfa plants under different light treatments. L100, L200, L300, L400 and L500 refer 100, 200, 300, 400 and 500 µmol m−<sup>2</sup> s −1 , respectively. Soluble sugar content (**A**), sucrose content (**B**) and starch content (**C**). Vertical bars indicate 1 s.e. of the mean (*n* = 4). Different lowercase letters on the different bar mean significant differences (*p* < 0.05).

### *2.5. Gene Expression and Enzymatic Activity 2.5. Gene Expression and Enzymatic Activity*

than at L100 (*p* < 0.001).

The expression levels of genes encoding sucrose synthase (*SS*), sucrose phosphate synthase (*SPS*), starch synthase (*AGPase*, *SSS*, *SBE* and *SP*) and those involved in the Calvin cycle (such as *RCA*, *RbcL*, *RbcS*, *FBPase*, *TK* and *PGK*) were quantitatively analyzed, and they were significantly affected by the light treatments (Table S8). The relative expression levels of these genes were upregulated with increasing light intensity up to L500 compared to the L100 treatment (Figure 5A–L). In addition, the relative expression of *RbcS* The expression levels of genes encoding sucrose synthase (*SS*), sucrose phosphate synthase (*SPS*), starch synthase (*AGPase*, *SSS*, *SBE* and *SP*) and those involved in the Calvin cycle (such as *RCA*, *RbcL*, *RbcS*, *FBPase*, *TK* and *PGK*) were quantitatively analyzed, and they were significantly affected by the light treatments (Table S8). The relative expression levels of these genes were upregulated with increasing light intensity up to L500 compared to the L100 treatment (Figure 5A–L). In addition, the relative expression of *RbcS* in the L400 treatment was 2.6 (*p* < 0.001) times higher than that in the L100 treatment.

in the L400 treatment was 2.6 (*p* < 0.001) times higher than that in the L100 treatment. The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase activase (RCA), Rubisco, fructose-1, 6-bisphosphatase (FBPase), thioredoxin reductase (TRXs), sucrose synthase (SS), sucrose phosphate synthase (SPS), adenosine diphosphate glucose pyro-phosphorylase (AGPase), soluble starch synthase (SSS), starch-branching enzyme (SBE) and starch phosphorylase (SP) of alfalfa plants varied with the light treatment (Table S9). Rubisco, RCA, FBPase, TRXs, SS, SPS, AGPase, SSS, SBE and SP activities of alfalfa plants increased gradually with increasing light intensity from L100 to L500, and the highest values were at L400 and L500 (Figure 6A–J). On average, the activities of RCA, Rubisco, FBPase, TRXs, SS, AGPase, SSS, SBE and SP were higher (*p* < 0.001) at L500 than at L100. The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase activase (RCA), Rubisco, fructose-1, 6-bisphosphatase (FBPase), thioredoxin reductase (TRXs), sucrose synthase (SS), sucrose phosphate synthase (SPS), adenosine diphosphate glucose pyrophosphorylase (AGPase), soluble starch synthase (SSS), starch-branching enzyme (SBE) and starch phosphorylase (SP) of alfalfa plants varied with the light treatment (Table S9). Rubisco, RCA, FBPase, TRXs, SS, SPS, AGPase, SSS, SBE and SP activities of alfalfa plants increased gradually with increasing light intensity from L100 to L500, and the highest values were at L400 and L500 (Figure 6A–J). On average, the activities of RCA, Rubisco, FBPase, TRXs, SS, AGPase, SSS, SBE and SP were higher (*p* < 0.001) at L500 than at L100. In addition, SPS activity of alfalfa plants was the highest at L400, which was 40.8% higher than at L100 (*p* < 0.001).

In addition, SPS activity of alfalfa plants was the highest at L400, which was 40.8% higher

**Figure 5.** Changes in level of gene expression of alfalfa plants growing in different light treatments. L100, L200, L300, L400 and L500 refer 100, 200, 300, 400 and 500 µmol m<sup>−</sup><sup>2</sup> s −1 , respectively. Rubisco activase (*RCA*, (**A**)), Rubisco large subunit (*RbcL*, (**B**)), Rubisco small subunit (*RbcS*, (**C**)), Fructose-1,6-bisphosphatase (*FBPase*, (**D**)), Transketolase (*TK*, (**E**)), Phosphoglycerate kinase (*PGK*, (**F**)), sucrose synthase (*SS*, (**G**)), sucrose phosphate synthase (*SPS*, (**H**)), ADP-glucose pyrophosphorylase (*AGPase*, (**I**)), soluble starch synthase (*SSS*, (**J**)), starch-branching enzyme (*SBE*, (**K**)) and starch phosphorylase (*SP*, (**L**)). Vertical bars indicate 1 s.e. of the mean (*n* = 3). Different lowercase letters on the different bar mean significant differences (*p* < 0.05). **Figure 5.** Changes in level of gene expression of alfalfa plants growing in different light treatments. L100, L200, L300, L400 and L500 refer 100, 200, 300, 400 and 500 µmol m−<sup>2</sup> s −1 , respectively. Rubisco activase (*RCA*, (**A**)), Rubisco large subunit (*RbcL*, (**B**)), Rubisco small subunit (*RbcS*, (**C**)), Fructose-1,6 bisphosphatase (*FBPase*, (**D**)), Transketolase (*TK*, (**E**)), Phosphoglycerate kinase (*PGK*, (**F**)), sucrose synthase (*SS*, (**G**)), sucrose phosphate synthase (*SPS*, (**H**)), ADP-glucose pyrophosphorylase (*AGPase*, (**I**)), soluble starch synthase (*SSS*, (**J**)), starch-branching enzyme (*SBE*, (**K**)) and starch phosphorylase (*SP*, (**L**)). Vertical bars indicate 1 s.e. of the mean (*n* = 3). Different lowercase letters on the different bar mean significant differences (*p* < 0.05).

**Figure 6.** Changes in enzymatic activity of alfalfa plants growing in different light treatments. L100, L200, L300, L400 and L500 refer 100, 200, 300, 400 and 500 µmol m<sup>−</sup><sup>2</sup> s −1 , respectively. Ribulose-1,5 bisphosphate carboxylase/oxygenase activase (RCA, (**A**)), ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, (**B**)), fructose-1, 6-bisphosphatase (FBPase, (**C**)), thioredoxin reductase (TRXs, (**D**)), sucrose synthase (SS, (**E**)), sucrose phosphate synthase (SPS, (**F**)), ADP-glucose pyrophosphorylase (AGPase, (**G**)), soluble starch synthase (SSS, (**H**)), starch-branching enzyme (SBE, (**I**)) and starch phosphorylase (SP, (**J**)), Vertical bars indicate 1 s.e. of the mean (*n* = 4). Different lowercase letters on the different bar mean significant differences (*p* < 0.05). **Figure 6.** Changes in enzymatic activity of alfalfa plants growing in different light treatments. L100, L200, L300, L400 and L500 refer 100, 200, 300, 400 and 500 µmol m−<sup>2</sup> s −1 , respectively. Ribulose-1,5-bisphosphate carboxylase/oxygenase activase (RCA, (**A**)), ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, (**B**)), fructose-1, 6-bisphosphatase (FBPase, (**C**)), thioredoxin reductase (TRXs, (**D**)), sucrose synthase (SS, (**E**)), sucrose phosphate synthase (SPS, (**F**)), ADP-glucose pyrophosphorylase (AGPase, (**G**)), soluble starch synthase (SSS, (**H**)), starch-branching enzyme (SBE, (**I**)) and starch phosphorylase (SP, (**J**)), Vertical bars indicate 1 s.e. of the mean (*n* = 4). Different lowercase letters on the different bar mean significant differences (*p* < 0.05).

#### **3. Discussion 3. Discussion**

The shade avoidance syndrome is an adaptive response that increases fitness in a shaded environment by reshaping the plant morphology and modifying physiological processes [25]. Our study examined the morphology, photosynthesis, carbohydrate metabolism and the expression of genes related to photosynthesis and carbon metabolism in leaves of alfalfa seedlings grown under low-to-high light intensities. Alfalfa seedlings displayed a degree of morphological adaptation, photosynthetic tolerance and carbon balance to the light intensity attenuation. Nevertheless, excessively low light significantly accelerated stem elongation and inhibited the photosynthetic process (e.g., net photosynthetic rate and Rubisco activity) of the seedlings. Low light intensity also negatively impacted production of photoassimilates (e.g., soluble sugar and starch), which in turn restricted growth and dry matter accumulation. Therefore, our results reveal the effects of simulated shade on phenotypic, physiological and expressional regulation in alfalfa, and thus provide insight into shade regulation in intercropping systems. The implications of The shade avoidance syndrome is an adaptive response that increases fitness in a shaded environment by reshaping the plant morphology and modifying physiological processes [25]. Our study examined the morphology, photosynthesis, carbohydrate metabolism and the expression of genes related to photosynthesis and carbon metabolism in leaves of alfalfa seedlings grown under low-to-high light intensities. Alfalfa seedlings displayed a degree of morphological adaptation, photosynthetic tolerance and carbon balance to the light intensity attenuation. Nevertheless, excessively low light significantly accelerated stem elongation and inhibited the photosynthetic process (e.g., net photosynthetic rate and Rubisco activity) of the seedlings. Low light intensity also negatively impacted production of photoassimilates (e.g., soluble sugar and starch), which in turn restricted growth and dry matter accumulation. Therefore, our results reveal the effects of simulated shade on phenotypic, physiological and expressional regulation in alfalfa, and thus provide insight into shade regulation in intercropping systems. The implications of these results are considered below.

these results are considered below.
