**1. Introduction**

Light is one of the most important environmental factors influencing plant growth and development. Changes in light intensity, light quality and the photoperiod have impacts on plant morphology and metabolism [1]. Subsequently, plants can exhibit numerous adaptative strategies in response to the light environment [2]. When grown in the shade, many shade-intolerant plants (e.g., *Arabidopsis thaliana*) exhibit a well-known shade avoidance syndrome (SAS) that increases their adaptive and competitive ability [3]. The SAS is triggered by a reduction in light intensity perceived by photoreceptor cryptochromes, which in turn control adaptive responses [4]. These SAS responses range from development changes, such as increased leaf hyponasty, specific leaf area and ratio of palisade/spongy tissues; hypocotyl, petiole and stem elongation; reduced tillering (monocots)/branching (dicots); and increased internode length [5]. Physiological changes, such as decreased leaf carbon assimilation and enzyme activity, also occur [6]. The morphological changes in response to shading allow the plant to elongate and thereby gain access to unfiltered sunlight [7].

**Citation:** Tang, W.; Guo, H.; Baskin, C.C.; Xiong, W.; Yang, C.; Li, Z.; Song, H.; Wang, T.; Yin, J.; Wu, X.; et al. Effect of Light Intensity on Morphology, Photosynthesis and Carbon Metabolism of Alfalfa (*Medicago sativa*) Seedlings. *Plants* **2022**, *11*, 1688. https://doi.org/ 10.3390/plants11131688

Academic Editors: Valeria Cavallaro and Rosario Muleo

Received: 31 May 2022 Accepted: 23 June 2022 Published: 25 June 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

However, plant elongation due to shading comes at a cost. Plant carbon resources must be redirected to stems or petioles to promote their elongation at the expense of production of new leaves. Additionally, excessive stem elongation leads to plant lodging or mechanical injury, which decreases plant fitness [8]. In crop production, shading occurs for the low-tier plants, which decreases light intensity and changes the light quality to a low ratio of red light, especially in intercropping system, [9]. Subsequently, these plants respond to shade by inducing a series of adaptive morphological and physiological changes at the cost of assimilated resources, which eventually negatively affects yield [10]. Thus, gaining a better understanding of how crops adapt and respond to shade stress could help guide the design of crop cultivation in agriculture systems.

A range of light levels is a common approach for exploring how shading stress affects pigment accumulation and the photosynthetic capacity of leaves [1]. Light intensity can directly affect light harvesting by plants and lead to changes in the abundance of chlorophyll pigments and differences in the health status of PSII. Rascher et al. (2010) [11] found that low light led to higher levels of Chl a, b, an improved maximal PSII quantum yield (Fv/Fm) and an early onset of nonphotochemical quenching (NPQ), which increased light-capturing capacity. Similar results were obtained for seedlings of Chinese cabbage (*Brassica campestris*) [12] and sweet pepper (*Capsicum annuum*) [13]. These significant differences in photochemical efficiency can be viewed as adaptations to low light; therefore, their regulatory mechanisms have long been important areas of research.

Photosynthesis allows plants to convert light energy into chemical energy. The Calvin cycle is a series of biochemical redox reactions that take place in the stroma of chloroplasts, and they play a vital role in photosynthetic carbon fixation [14]. In shade-intolerant species, low photosynthesis due to low light reduces expression of genes and activity of the Calvin cycle enzymes involved in CO<sup>2</sup> fixation and regeneration of rubisco-1, 5-bisphosphate (RuBP), thereby decreasing the potential for carbon assimilation in plants [15]. RuBP carboxylase or oxygenase (Rubisco) is the rate-limiting step of photosynthesis, and it catalyzes CO<sup>2</sup> fixation in C<sup>3</sup> plants [16]. Previously, it was reported that shade-associated with downregulation of the net photosynthetic rate was due to reduction in the amount or activity of Rubisco [17]. Photosynthesis is also catalyzed by other key enzymes, e.g., Rubisco activase (RCA) and fructose-1, 6-bisphosphatase (FBPase) [18]. Recent studies on soybean (*Glycine max*) and tomato (*Lycopersicon esculentum*) have shown that gene expression of the key enzymes involved in the Calvin cycle was downregulated in low but not high light [18,19]. However, the specific effects of light intensity on the photosynthesis processes in plants remain largely unknown. Therefore, levels of gene expression of the key enzymes of the Calvin cycle of plants grown at different light intensities need to be studied to elucidate the molecular mechanism of plant response to shading stress.

Alfalfa (*Medicago sativa* L.) is a high-quality forage for dairy cows and other livestock because of its high dry matter accumulation and high protein and soluble sugar content [20,21]. With increasing demand for food and the decreasing availability of arable land, grass/legume forage intercropping is gaining in popularity as a sustainable practice for low-input or resource-limited agricultural systems, such as maize–alfalfa and oat– alfalfa [22]. However, intercropped alfalfa plants often suffer from shade stress due to the reduced amount of intercepted sunlight. Subsequently, shading increases plants height and internodal distance, and reduces stems strength, which makes alfalfa plants susceptible to lodging, thereby reducing forage yield [23]. The SAS effects on alfalfa could be of high practical importance for intercropping systems, but the minimum amount of light required for alfalfa growth and development has received little research attention. To date, only one study has indicated that shade-intolerant alfalfa plants will delay flowering when grown in the shade (i.e., low ratio of red to far-red light) [24]. Research-based information is lacking on the effect of shading on the growth and physiological metabolism of *M. sativa* seedlings. Thus, it is important to investigate the adaptability of alfalfa responses to low light intensity, which would be useful information for determining proper plant spacing and strip configuration in intercropping systems.

The objective of our research was to determine how light intensity affects alfalfa seedling morphology and photosynthetic characteristics, as well as the key enzymes involved in the Calvin cycle and carbon metabolism coupled with expression of these genes. Here, alfalfa seedlings were exposed to five levels of light intensity for 14 days in a climate room, and their morphological and physiological responses were investigated. We hypothesized that a brief exposure to low light would increase leaf hyponasty and stem elongation but downregulate expression of genes for the key enzymes involved in the Calvin cycle and carbon metabolism, resulting in a synergistic decrease in photosynthetic rates and accumulation of dry matter. ling morphology and photosynthetic characteristics, as well as the key enzymes involvedin the Calvin cycle and carbon metabolism coupled with expression of these genes. Here, alfalfa seedlings were exposed to five levels of light intensity for 14 days in a climate room, and their morphological and physiological responses were investigated. We hypothesizedthat a brief exposure to low light would increase leaf hyponasty and stem elongation but downregulate expression of genes for the key enzymes involved in the Calvin cycle and carbon metabolism, resulting in a synergistic decrease in photosynthetic rates and accumulation of dry matter.

to low light intensity, which would be useful information for determining proper plant

The objective of our research was to determine how light intensity affects alfalfa seed-

*Plants* **2022**, *11*, x FOR PEER REVIEW 3 of 19

spacing and strip configuration in intercropping systems.

### **2. Results 2. Results**

#### *2.1. Morphological Characteristics 2.1. Morphological Characteristics*

Light treatment had a significant effect on alfalfa morphological characteristics (i.e., plant height, specific leaf area, abaxial leaf petiole angle and stem diameter) (*p* < 0.001) (Figure 1, Tables 1 and S2). Maximum plant height, specific leaf area and abaxial leaf petiole angle were measured in L100; these are the traits that decreased with increased light intensity. However, the highest and lowest stem diameters were measured for plants at L500 and L100, respectively. In addition, shoot dry matter (SDM), root dry matter (RDM) and the root-to-shoot ratio (RSR) were significantly affected by light treatments (*p* < 0.001) (Table S3). The SDM, RDM and RSR of alfalfa plants in L500 were significantly higher than those in L100. For the most part, the RSR did not differ significantly between L300, L400 and L500 (Table 1). Light treatment had a significant effect on alfalfa morphological characteristics (i.e., plant height, specific leaf area, abaxial leaf petiole angle and stem diameter) (*p* < 0.001) (Figure 1, Tables 1 and S2). Maximum plant height, specific leaf area and abaxial leaf petiole angle were measured in L100; these are the traits that decreased with increased light intensity. However, the highest and lowest stem diameters were measured for plants at L500 and L100, respectively. In addition, shoot dry matter (SDM), root dry matter (RDM) and the root-to-shoot ratio (RSR) were significantly affected by light treatments (*p* < 0.001) (Table S3). The SDM, RDM and RSR of alfalfa plants in L500 were significantly higher than those in L100. For the most part, the RSR did not differ significantly between L300, L400 and L500 (Table 1).

**Figure 1.** Changes in phenotype and plant traits of alfalfa as affected by light treatments. The plant height (**A**), stem diameter (**B**), abaxial leaf petiole angle (**C**) and plant phenotype (**D**) of alfalfa plants under different light intensity treatments. L100, L200, L300, L400 and L500 refer 100, 200, 300, 400 and 500 µmol m<sup>−</sup><sup>2</sup> s −1 , respectively. Vertical bars indicate 1 s.e. of the mean (*n* = 4). **Figure 1.** Changes in phenotype and plant traits of alfalfa as affected by light treatments. The plant height (**A**), stem diameter (**B**), abaxial leaf petiole angle (**C**) and plant phenotype (**D**) of alfalfa plants under different light intensity treatments. L100, L200, L300, L400 and L500 refer 100, 200, 300, 400 and 500 µmol m−<sup>2</sup> s −1 , respectively. Vertical bars indicate 1 s.e. of the mean (*n* = 4). Different lowercase letters on the different bar mean significant differences (*p* < 0.05).


**Table 1.** Effect of different light intensity treatments on specific leaf area (SLA, cm<sup>2</sup> mg−<sup>1</sup> ), shoot dry matter (SDM, mg plant−<sup>1</sup> ), root dry matter (RDM, mg plant−<sup>1</sup> ) and root-to-shoot ratio (RSR) 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).

Root morphology parameters, including root length (RL), surface area (RSA), volume (RV) and diameter (RD), varied among light treatments (Table S3). These parameters increased with increasing light up to L500 compared to the L100 treatment (Table 2). Increased light significantly increased RL by 22.8 to 182.5%, RSA by 26.0 to 353.2%, RV by 42.2 to 925.9% and RD by 4.3 to 84.5%. RD did not differ significantly from L300 to L500.

**Table 2.** Effect of light intensity treatments on root length (RL, cm), root surface area (RSA, cm<sup>2</sup> ), root volume (RV, cm<sup>3</sup> ) and root diameter (RD, mm) 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).
