1. Introduction
Climate change has modified weather patterns with increasingly extreme events. Rainfall is one of the climatic components with an environmental impact causing extreme events in the world [
1]. In addition, the world’s rising population, now pegged at more than 8 billion, is putting intense pressure on using natural resources for agri-food production [
2]. Livestock is a supplier of agri-food products and an essential protein source in the family diet. Consumers’ high demand for meat, milk, and other derivatives has increased animal production and led to a greater need for forage [
3]. Forage production consumes 76% of the freshwater available for productive activities on the planet [
4], which is a significant problem for regions due to the scarcity of this natural resource.
Drought and intensive agri-food production activity cause environmental deterioration in arid areas [
5] where the productivity is with a high-water demand. This makes it necessary to perform strategies to produce forage through alternative crops with a lower water consumption and the ability to compete with other crops in quality and quantity [
6,
7]. Alfalfa (
Medicago sativa L.) is the main forage crop, with the best nutritional quality and quantity of fresh and dry biomass produced as food for livestock. Paradoxically, however, it is one of the crops with the highest water consumption [
8].
Several species are studied as alternative forage crops in the leading livestock areas of the world, with studies related to the evaluation of water efficiency, environmental adaptation, and productivity. Among these species are white clover (
Trifolium repens L.), red clover (
T. pretense L.), pink clover (
T. fragiferum), and birdsfoot treefoil (
Lotus corniculatus Lam.) [
9]. The
Lotus genus has more than two hundred annual and perennial species with different growth habits, such as prostrate, erect, and semi-erect forms [
10]. Most ecotypes and varieties of
L. corniculatus are grown in regions with a humid temperate climate [
11]. Few studies have been conducted under extreme conditions, such as those of arid lands [
10]. However,
Lotus has considerable genetic diversity, which is an opportunity to explore the response capacity of the genetic resources of this genus, which could adapt to conditions of low water availability and extreme temperatures.
The plants have several morphological, physiological, and chemical mechanisms to tolerate extreme environments [
12]. Some physiological indicators, such as phytochemicals produced through secondary metabolism, named secondary metabolites, are highly reactive in plants under environmental stress, such as a water deficit, soil salinity, extreme temperatures, and a nutrient deficiency [
13,
14]. They play a crucial role in the survival of plants in marginal conditions since they are activated in response to environmental stress and function as agents and physiological regulators [
15]. Secondary metabolites, such as phenolic compounds, alkaloids, and terpenoids, have been shown to have antioxidant and antimicrobial properties, which help mitigate oxidative damage caused by water scarcity [
16,
17].
Furthermore, reactive oxygen species (ROS) are increased under water stress as molecular signals that activate adaptive responses in plants [
18]. ROS and secondary metabolites’ activity interaction is a complex process that allows plants to survive in extreme environments [
19]. This study aimed to evaluate different ecotypes and one variety of
Lotus corniculatus L. in terms of its ability to produce secondary metabolites with antioxidant activity under a water deficit through other seasonal times.
2. Materials and Methods
2.1. Geographic Location
The experiment was conducted at the experimental field of the Unidad Regional Universitaria de Zonas Aridas of the Universidad Autonoma Chapingo at Bermejillo Mapimí, Durango, Mexico. The region is located at 25.8° NL and 103.6° WL and an elevation of 1130 m. The area has a desert climate with rain in summer and cool winters, with an average annual rainfall of 258 mm, average potential evaporation of 2000 mm, and an average temperature of 21 °C, with a maximum of 33.7 °C and a minimum of 7.5 °C [
20]. Temperatures recorded inside the shade mesh were extremely high in summer, low in winter, and moderate during spring and autumn. The experimental area within the shade mesh was covered in advance with a plastic cover to avoid alterations in the soil moisture content in the pots during rainy periods.
2.2. Experimental Setup
A randomized block experimental design with three replicates was used under shade mesh conditions. The treatments of soil water content were as follows: (1) no water deficit (NWD) with 100% of field capacity (FC), and (2) water deficit (WD) with 89% of FC in four ecotypes identified with ID numbers 255301, 255305, 202700, and 226792, and one variety named Estanzuela Ganador of
L. corniculatus, which was obtained from different areas, such as Europe, North America, and South America, with a temperate humid climate (
Table 1).
The Lotus genetic materials evaluated in this study were selected based on their ability to survive the environmental conditions in the study area, from a total of 12 original genetic materials: 4 varieties and 8 different accessions.
Ten treatments were established of the factorial 2 × 5. The experimental unit was a plant in a pot with three replicates. Seedlings were previously grown in 1 kg black plastic bags in soil mixed with compost at a 3:1 ratio for two months. After this time, plants of approximately 20 cm in height were transplanted into rigid 18 kg plastic pots containing a substrate prepared to have a ratio of 50:30:20 of soil, compost, and sand, respectively. According to a physical and chemical analysis of the substrate, it was 26% silt, 22% clay, and 52% sand, with a pH of 7.73, electric conductivity (EC) of 7.47 dS/m, and bulk density of 1.46 g/cm
3 (
Figure 1) The experiment was carried out from March 2021 to May 2022.
According to the determination using the membrane pot method [
21], the substrate used in the pots, the soil moisture content at the field capacity (FC), and the permanent wilting point (PWP) were 27.5% and 17.5%, respectively (
Figure 2).
2.3. Irrigation
Irrigation was performed manually, and during the first 15 days of the experiment, the soil moisture was maintained at FC (27.5%) for all treatments. Subsequently, the water content in the soil was restricted until maintaining the differentiated ranges of 26.5% ± 1 and 23.5% ± 1. The upper limit of soil water content in the first range was 27.5%, corresponding to 100% of FC, and the upper limit of the second range was 24.5%, corresponding to 89% of FC. This water decrease in the soil was decided since
L. corniculatus is a plant with a C3 photosynthetic pathway, which is sensitive to water stress [
22].
The different genetic resources of
L. corniculatus were homogenized in the plant canopy by cutting the biomass at 62 days after transplanting (DAT). Since the plant grows by forming a rhizome with several new shoots, the cuts of fresh matter on the different sampling dates were made with pruning shears 6 cm above soil level, using a plastic ring that allowed the cutting height to be uniform [
23]. From this date, seasonal cutting intervals were defined: two cuts at intervals of 42 days in each seasonal period (summer, autumn, and spring) and 92 days in winter (a single cut), the latter interval defined by the slow growth of the plant due to low temperatures.
The soil water content was measured regularly in real-time using a digital tensiometer (Model: MO750, Extech Instruments Co., Laredo, TX, USA). When the soil moisture content reached the lowest limit of a treatment, irrigation was resumed until the upper limit of each irrigation treatment. During the experimental time, the environmental temperature and humidity inside the shade were recorded daily using a digital thermometer-hygrometer (Model OUS-WA62, ORIA, Shanghai, China).
2.4. Measured Variables
Fresh tissue of
L. corniculatus samples was used to quantify secondary metabolites according to the method reported by Melgarejo et al. [
24]. All samples were stored at −40 °C until they were used. The total phenols concentration (TPC) and total tannins concentration (TTC) were measured using Folin–Cioucalteu reagent [
25]; the extract was prepared with 1 mg of plant tissue in 1000 µL of methanol, which was shaken for 1 h and subsequently centrifuged at 4000 rpm for 15 min. These variables were measured in mg Gallic Acid Equivalents per gram of fresh weight (GAE/gFW) [
26]. The total flavonoid concentration (TFC) was quantified using the method described by Maksimovíc et al. (2005) [
27] with modifications. Quercetin was the standard, so the result was expressed in mg Quercetin Equivalents (QE/gFW). At the same time, total saponins concentration (TSC) was calculated using colorimetry with Lieberman–Burchard reagent [
28] in mg of saponins/GFW. Radical Scavenging Activity (RSA) was measured using absorbance of the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical [
29] reported in Trolox Equivalent Antioxidant Capacity (TEAC)/gFW.
2.5. Data Analysis
Data were analyzed with normality tests, one-way ANOVA, Tukey’s range test, and simple linear regression using the PASW Statistics statistical program for Windows 18.0.0 Chicago, IL, USA, SPSS Inc.
4. Discussion
All the evaluated plant genetic resources maintained adequate phenolic compound production in the different seasons. However, the low temperatures in winter increased the output of this secondary metabolite, with the 255301 and 226792 ecotypes being the most susceptible as they produced more phenols to mitigate the cold stress in a WD. Kowalczewski et al. [
30] quantified the TPC from 5.00 to 8.16 mg GAE/gFW in
Hordeum vulgare L. var. KWS Olof, and although the water stress decreased the TPC, the photosynthetically active radiation (PAR) increased. Wong-Paz et al. [
16] pointed out that plants in semi-arid areas have high antioxidant capacity due to a high content of total phenols as a defense mechanism against plant stress. Baali et al. [
18] reported that in
L. corniculatus plants collected in March, the CTP was 87.1 ± 14.5 mg GAE/gFW, and the radical scavenging activity (RSA) was 26.9–79.7 mg TEAC/gFW.
The Estanzuela Ganador variety was susceptible to low winter temperatures, increasing the concentration of flavonoids as a defense mechanism against low temperatures and a water deficit. The 226792 ecotype was the most tolerant to winter conditions, with the most deficient production of this metabolite under NWD and without requiring the production of this metabolite to mitigate the WD. The TFC increased in response to low temperatures and a water deficit, with values higher than those -reported by Baali et al. (2019) [
18], who documented values of 36.5 ± 2.1 mg QE/gFW in TFC in
L. corniculatus extracts in response to environmental stress intensity. Additionally, the TFC in
L. corniculatus varies depending on the environmental temperature; for example, it increases at 10 °C and decreases at 30 °C [
31]. The decrease in TFC in summer, autumn, and spring could be due to the natural tolerance of most plant genetic resources evaluated in this study to the extreme temperatures in these seasons, as they do not require the antioxidant activity that promotes flavonoids. Cheng et al. [
32] reported that only low temperatures, and not high temperatures, encourage the expression of genes fitted to produce flavonoids and, therefore, their accumulation.
The response of the 255301 and 255792 ecotypes and the Estanzuela Ganador variety had high tannin concentrations in winter and spring, except for the 255792 ecotype, which only reacted this way in winter. All the mentioned responses were under a water deficit. This secondary metabolite signifies the importance of mitigating the abiotic stress associated with low temperatures and a water deficit. Previous studies in three
Lotus species (
L. glaber,
L. uliginosus, and
L. corniculatus) found a high tannin content in the leaves, stems, and flowers, which varied in the different seasons of the year, except for
L. glaber, for which there were no differences in spring, summer, and autumn [
33,
34]. The increase in tannins observed in winter could be due to the maturity of the plants and the increase in lignin, thereby decreasing the forage digestibility [
35].
Saponins were the most immediate secondary metabolite to the elevated temperatures reported in summer. At the same time, the TPC, TFC, and TTC were more sensitive to low temperatures in winter and high temperatures in summer under a water deficit, with different responses among the plant genetic resources of
Lotus. Szakiel et al. [
36] described that saponins could be involved in adapting plants to survive in adverse climatic conditions, with a positive correlation between the saponin content and the physiological phase throughout the year. Baali et al. [
18] detected the presence of saponins in
L. corniculatus using the qualitative foam test. Santacoloma [
37] reported the absence of saponins in the leaves, petioles, and stems of
L. corniculatus, which may be associated with the favorable environmental conditions of the study area.
The antioxidant activity results were correlated to the production of some secondary metabolites in the content of phenols, flavonoids, and tannins in winter under a water deficit, particularly in the 255301 and 255305 ecotypes, as well as the Estanzuela Ganador variety, but only in terms of flavonoid production. On the other hand, saponins did not contribute to the antioxidant activity in the winter phase. Still, they did contribute in the summer, which is the time of highest temperatures, in the 255301 and 266792 ecotypes, which were under a water deficit since they were the phylogenetic materials with the highest RSA in that season. This suggests that saponin production is activated as a defense mechanism against heat and a water deficit. Indeed, antioxidant compounds are produced as a defense mechanism to prevent growth inhibition and damage to the photosynthetic apparatus, cell membranes, and proteins, with a specific response to a water deficit dependent on the genotypes used [
38].
Finally, it is essential to point out that this study was under semi-controlled shade mesh conditions, so the results presented here require validation in the field.