Physiological and Metabolic Responses of Alfalfa to Cold Stress Under Saline–Alkaline Conditions

Sep 30, 2026

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Alfalfa (Medicago sativa L.) cultivars LJ and 218TR were subjected to saline–alkaline, cold, or combined saline–alkaline–cold conditions to compare visible traits, physiological responses, key metabolite profiles, and stress-responsive gene expression. Under single stresses, malondialdehyde, soluble sugars, proline, PAL activity, and antioxidant enzyme activities initially increased, but decreased under combined stress. Photosystem II efficiency and chlorophyll declined, while combined treatment raised cell mortality and superoxide anion levels. LJ showed greater tolerance, with salicylic acid as the most responsive metabolite and upregulated ICS and PAL supporting SA accumulation and improved stress resilience.

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CategoryResearch & Report

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1. Introduction

Article Physiological and Metabolic Responses of Alfalfa to Cold Stress Under Saline–Alkaline Conditions Xu Zhuang 1,†, Dongmei Zhang 1,†, Ying Yang 2, Weibo Han 1, Linlin Mu 1, Zhongbao Shen 1, Guili Di 3, Yaling Liu 4, Jia You 1,* and Jianli Wang 1, * Academic Editor: De-Guo Han Received: 12 November 2025 Revised: 16 December 2025 Accepted: 22 December 2025 Published: 26 December 2025 Copyright: © 2025 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. 1 Institute of Forage and Grassland Sciences, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China; [yangyangcaocao@haas.cn](yangyangcaocao@haas.cn) (X.Z.); [zhangdongmei@haas.cn](zhangdongmei@haas.cn) (D.Z.); [hanweibo@haas.cn](hanweibo@haas.cn) (W.H.); [budaoweng@haas.cn](budaoweng@haas.cn) (L.M.); [shenzhongbao@haas.cn](shenzhongbao@haas.cn) (Z.S.) 2 College of Life Sciences and Technology, Harbin Normal University, Harbin 150025, China; [yangying1212@126.com](yangying1212@126.com) 3 Institute of Economic Crops, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China; [diguili@haas.cn](diguili@haas.cn) 4 National Center of Pratacultural Technology Innovation (Under Preparation), Hohhot 010030, China; [liuyaling0719@163.com](liuyaling0719@163.com) * [Correspondence: uxiaoxia@haas.cn](Correspondence: uxiaoxia@haas.cn) (J.Y.); [jianli@haas.cn](jianli@haas.cn) (J.W.)† These authors contributed equally to this work. Abstract Alfalfa (Medicago sativa L.), a perennial leguminous herb, can tolerate cold and saline– alkaline conditions. In this study, alfalfa cultivars LJ and 218TR were exposed to saline– alkaline, cold, and saline–alkaline–cold conditions and compared in terms of phenotypes, physiological indices, key metabolite contents, and stress-responsive gene expression. Malondialdehyde, soluble sugar, proline contents and phenylalanine ammonia-lyase (PAL), superoxide dismutase, catalase, and peroxidase activities initially increased under individual stress conditions, but decreased when stresses were combined. Photosystem II maximum photochemical efficiency and chlorophyll contents decreased under individual and combined stress conditions. Nitroblue tetrazolium-stained leaves revealed that the combined stress treatment significantly increased cell mortality rates and superoxide anion levels. LJ was more tolerant to saline–alkaline, cold, and combined stress treatments than 218TR. Metabolite analyses indicated that for LJ and 218TR, salicylic acid (SA) was the most responsive metabolite to combined stress conditions. Additionally, the expression of isochorismate synthase (ICS) and PAL genes critical for SA biosynthesis was upregulated under single or combined stress conditions, leading to SA accumulation and improved tolerance to saline–alkaline–cold conditions. This study revealed the physiological indices and molecular changes underlying alfalfa responses to saline–alkaline stress combined with cold stress, providing a theoretical basis for breeding stress-tolerant cultivars. Keywords: alfalfa; saline-alkaline-cold stress; physiological characteristics; chlorophyll fluorescence; nitroblue tetrazolium staining; metabolites 1. Introduction Soil salinization–alkalinization, which is characterized by the accumulation of soluble salts from subsoil and groundwater layers in the surface layer, is a serious global ecological and agricultural issue that significantly inhibits plant growth and decreases crop yield and quality [1] . Saline–alkaline soils may adversely affect plant organ development, dry matter accumulation, and critical physiological processes [2], which can lead to damaged antioxidant defense systems; suppressed photosynthesis; imbalanced mineral/ion absorption, use, distribution, and translocation in roots [3]; and relatively inefficient nutrient uptake [4] . In saline–

FAQs

How were alfalfa cultivars evaluated under different stress treatments?

Cultivars LJ and 218TR were exposed to saline–alkaline, cold, and combined saline–alkaline–cold conditions, and then assessed for phenotypes, physiological indices, metabolite contents, and stress-responsive gene expression.

What physiological changes occurred under single versus combined stress?

Single stresses initially increased malondialdehyde, soluble sugars, proline, PAL activity, and antioxidant enzyme activities, whereas these markers decreased when stresses were combined. Photosystem II efficiency and chlorophyll contents declined under both single and combined stresses.

Which metabolite and genes were linked to improved tolerance?

Salicylic acid (SA) was the most responsive metabolite under combined stress for both cultivars. Expression of isochorismate synthase (ICS) and PAL genes increased under single or combined stress, promoting SA accumulation and improved tolerance to saline–alkaline–cold conditions.

Sep 30, 2026

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