Metabolomic analyses reveal substances that contribute to the increased freezing tolerance of alfalfa (Medicago sativa L.) after continuous water deficit

Abstract

Background: Alfalfa is a high-quality forage cultivated widely in northern China. Recently, the failure of alfalfa plants to survive the winter has caused substantial economic losses. Water management has attracted considerable attention as a method for the potential improvement of winter survival. The aim of this study was to determine whether and how changes in the water regime affect the freezing tolerance of alfalfa.

Results: The alfalfa variety WL353LH was cultivated under water regimes of 80 and 25% of water-holding capacity, and all the plants were subjected to low temperatures at 4/0 °C (light/dark) and then - 2/- 6 °C (light/dark). The semi-lethal temperatures were lower for water-stressed than well-watered alfalfa. The pool sizes of total soluble sugars, total amino acids, and proline changed substantially under water-deficit and low-temperature conditions. Metabolomics analyses revealed 72 subclasses of differential metabolites, among which lipid and lipid-like molecules (e.g., fatty acids, unsaturated fatty acids, and glycerophospholipids) and amino acids, peptides, and analogues (e.g., proline betaine) were upregulated under water-deficit conditions. Some carbohydrates (e.g., D-maltose and raffinose) and flavonoids were also upregulated at low temperatures. Finally, Kyoto Encyclopedia of Genes and Genomes analyses revealed 18 significantly enriched pathways involved in the biosynthesis and metabolism of carbohydrates, unsaturated fatty acids, amino acids, and glycerophospholipids.

Conclusions: Water deficit significantly enhanced the alfalfa' freezing tolerance, and this was correlated with increased soluble sugar, amino acid, and lipid and lipid-like molecule contents. These substances are involved in osmotic regulation, cryoprotection, and the synthesis, fluidity, and stability of the cellular membrane. Our study provides a reference for improving alfalfa' winter survival through water management.

Keywords: Alfalfa; Forage grass; Freezing tolerance; LC-MS; Metabolomics; Water deficit.

Fig. 1

The four experimental phases in this research. The treatments are water-controlled treatments with substrate moisture at 80% (WW) and 25% of water-holding capacity (WD). Incubator temperatures were set for each of the four phases. Black dots S1 to S4 represent sampling time points at the ends of phases

Fig. 2

Dry weights of aboveground and belowground alfalfa parts in the four experimental phases. WW and WD: substrate moisture at 80 and 25% of water-holding capacity, respectively. The significances of differences between and within phases were evaluated using a multiple-range test. Different letters indicate significant differences (P < 0.05)

Fig. 3

Phenotypes of alfalfa after freezing in phase 4. WW and WD: substrate moisture at 80 and 25% of water-holding capacity, respectively. Phenotypes of alfalfa in WW (a and b) and in WD (c and d)

Fig. 4

Semi-lethal temperature, LT₅₀, of alfalfa crowns collected in WD and WW. WW and WD: substrate moisture at 80 and 25% of water-holding capacity, respectively. The significances of differences between and within phases were evaluated using a multiple-range test. Different letters indicate significant differences (P < 0.05)

Fig. 5

Changes in the contents of malondialdehyde, carbohydrates (starch and total soluble sugar), and nitrogenous compounds (total soluble protein, total amino acids, and proline) in the four phases. WW and WD: substrate moisture at 80 and 25% of water-holding capacity, respectively. The significances of differences between and within phases were evaluated using a multiple-range test. Different letters indicate significant differences (P < 0.05)

Fig. 6

Scatter plots of scores of the orthogonal partial least squares discriminant analysis for identified differential metabolites in the seven comparisons. WW and WD: substrate moisture at 80 and 25% of water-holding capacity, respectively. WD_1, 2, 3 and WW_1, 2, 3 represent samples collected at the ends of phases 2, 3, and 4, respectively

Fig. 7

Numbers of differential metabolites at the class and subclass levels. Names on the abscissa represent subclasses, gray parts above the plot represent classes. Within-phase and between-phase comparisons revealed metabolites affected by water deficit and low temperature, respectively. WW and WD: substrate moisture at 80 and 25% of water-holding capacity, respectively. WD_1, 2, 3 and WW_1, 2, 3 represent samples collected at the ends of phases 2, 3, and 4, respectively

Fig. 8

Enrichment ratios of 18 significantly enriched pathways. Asterisks represent significance at P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***). Within-phase and between-phase comparisons revealed pathways affected by water deficit and low temperatures, respectively. WW and WD: substrate moisture at 80 and 25% of water-holding capacity, respectively. WD_1, 2, 3 and WW_1, 2, 3 represent samples collected at the ends of phases 2, 3, and 4, respectively