Effects of Combined Salt and Heat Stress on Agronomic Traits, Photosynthetic Parameters, and Physiological Biochemistry in Six Alfalfa (Medicago sativa L.) Cultivars

Abstract

Climate change due to global warming increases the susceptibility of plants to multiple combined stresses. Soil salinization and high temperature stresses that co-occur in arid/semiarid regions severely restrict the growth and development of plants. Although alfalfa (Medicago sativa L.) is an important forage grass, the physiological mechanisms driving its responses to combined salt and heat stress are not yet clear. This study aimed to reveal the physiological and biochemical response mechanisms of six alfalfa cultivars to different stresses by comparing plant morphology, agronomic traits, photosynthetic characteristics, and physiological and biochemical responses under control conditions, salt stress (200 mM NaCl), heat stress (38 °C), and combined salt and heat stress. Compared with single stresses, combined stress significantly inhibited the growth and biomass accumulation of alfalfa. Under combined stress, the cultivars presented decreases in plant height and total fresh biomass of 11.87-26.49% and 28.22-39.97%, respectively, compared with those of the control plants. Heat stress promoted alfalfa photosynthesis by increasing stomatal conductance, net photosynthetic rate, and transpiration rate, while salt stress and combined stress significantly suppressed these effects. Combined stress significantly increased the concentration of Na⁺ but decreased that of K⁺ and the relative water content in alfalfa leaves. Compared with the control and single stress treatments, combined stress significantly increased the level of membrane lipid peroxidation and accumulation of reactive oxygen species. The proline contents in the leaves of the different alfalfa cultivars were 2.79-11.26 times greater under combined stress than in the control. Combined stress causes alfalfa to redistribute energy from growth and development to stress defense pathways, ultimately leading to a reduction in biomass. Our study provides theoretical guidance for analyzing the mechanisms of grass resistance to combined salt and heat stress.

Keywords: Medicago sativa; combined salt and heat stress; photosynthesis; plant growth.

Figure 1

Changes in the phenotypes and agronomic traits of different alfalfa cultivars under single and combined stresses. (a), CT, SS, HS, and CSH represent the control, salt stress, heat stress, and combined stress, respectively; the same applies in the figures below. (b) Plant height, (c) branch number, (d) shoot fresh weight, (e) root fresh weight, (f) shoot dry weight, and (g) total fresh weight. Data are presented as the mean ± standard error (n = 10). Significant differences at the p < 0.05 level are indicated by different lowercase letters for the same cultivar.

Figure 2

Changes in the chlorophyll contents of the six alfalfa cultivars under single and combined stresses. (a) Chlorophyll a, (b) chlorophyll b, and (c) total chlorophyll. The data are presented as the mean ± standard error (n = 3). Significant differences at the p < 0.05 level are indicated by different lowercase letters for the same cultivar.

Figure 3

Changes in the photosynthetic parameters of different alfalfa cultivars under single and combined stresses. (a) Net photosynthetic rate, (b) transpiration rate, (c) stomatal conductance, and (d) intercellular CO₂ concentration. The data are presented as the mean ± standard error (n = 5). Significant differences at the p < 0.05 level are indicated by different lowercase letters for the same cultivar.

Figure 4

Changes in the leaf (a) Na⁺ concentration, (b) K⁺ concentration, and (c) Na⁺/K⁺ ratio of different alfalfa cultivars under single and combined stresses. The data are presented as the mean ± standard error (n = 3). Significant differences at the p < 0.05 level are indicated by different lowercase letters for the same cultivar.

Figure 5

Changes in (a) electrolyte leakage, (b) relative water content, (c) soluble sugar content, and (d) proline content of different alfalfa cultivars under single and combined stresses. The data are presented as the mean ± standard error (n = 3). Significant differences at the p < 0.05 level are indicated by different lowercase letters for the same cultivar.

Figure 6

Changes in malondialdehyde (MDA) content (a), ROS content (c,e), and the activities of SOD (b), CAT (d), and APX (f) in different alfalfa cultivars under single and combined stresses. The data are presented as the mean ± standard error (n = 3). Significant differences at the p < 0.05 level are indicated by different lowercase letters for the same cultivar.

Figure 7

Multivariate analyses of alfalfa responses to single and combined stresses. (a) Principal component analysis (PCA) of morpho−physiological and biochemical indicators under salt, heat, and combined stresses. (b) PCA of six alfalfa cultivars under different stress conditions. (c) Pearson’s correlation analysis of different response variables. (a) CT = control; SS = salt stress; HS = heat stress; and CSH = combined salt and heat stresses. (b) CT−XM, CT−520, CT−ZM, CT−5030, CT−Bar, and CT−358 indicate alfalfa cultivars of ‘Xinmu No. 4’, ‘Bara520YQ’, ‘Zhongmu No. 4’, ‘Power 5030’, ‘Barricade’, and ‘WL358HQ’ under the control condition, respectively. The same prefix coding applies to stress treatments (e.g., SS−XM for ‘Xinmu No. 4’ under salt stress). (c) ** and * represent extremely significant correlation (p < 0.01) and significant correlation (p < 0.05), respectively.

Figure 8

Cluster analysis of agronomic traits with physiological and biochemical indicators of six alfalfa cultivars under single stress and combined stress conditions. (a) Changes in agronomic and physiological traits under four treatments. (b) Changes in agronomic and physiological traits of six alfalfa cultivars under combined stress treatment.