Czech J. Genet. Plant Breed., X:X | DOI: 10.17221/27/2025-CJGPB

Mechanisms of overwintering frost tolerance in alfalfa roots based on 4D-Label-free quantitative proteomicsOriginal Paper

Yajun Ma1, Yu Zhang3, Qiaoxian Zhang2, Guo Sun4, Yajun Yan5, Chuan Wang1, Wenbin Mian1, Tianhui Yang1, Ting Gao1*
1 Institute of Animal Science (Grass Livestock Engineering and Technology Research Center), Ningxia Academy of Agriculture and Forestry, Yinchuan,Ningxia, P.R. China
2 Ningxia Autonomous Region Animal Husbandry Workstation, Yinchuan, Ningxia, P.R.China
3 Ningxia Grassland Workstationr, Ningxia Autonomous Region Forestry and Grassland Bureau, Yinchuan,Ningxia, P.R. China
4 Yan Chi County Forestry and Grassland Bureau, Yan Chi, Ningxia, P.R. China
5 College of Life Sciences, Sichuan University, Chengdu, Sichuan, P.R. China

The present study investigates the differential protein expression levels between the frost-tolerant variety Qingda No.1 and the frost-sensitive variety Gannong No.9. The analysis was conducted using 4D-Label-free quantitative proteomics technology, with the samples collected prior to and after overwintering. The results showed that the protein expression of Qingda No.1 changed more significantly during the overwintering process, with 451 differentially expressed proteins (DEPs) being identified, of which 224 were up-regulated and 227 down-regulated. In contrast, the protein expression of Gannong No.9 differed from that of alfalfa roots of the frost-sensitive variety, with 204 DEPs being identified, of which 93 were up-regulated and 111 down-regulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the phenylpropanoid biosynthesis pathway was significantly enriched in both groups. Furthermore, enzymes such as phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (C4H), and chalcone isomerase (CHI) were significantly up-regulated in the freezing-tolerant varieties. Protein interaction network analysis revealed the synergistic regulation mode of DEPs. The present study elucidated the metabolic adaptive mechanism of overwintering in alfalfa roots at the protein level, thus providing a theoretical basis for the selection and breeding of frost-tolerant varieties.

Keywords: differentially expressed protein; enri­chment analysis; low-temperature stress; Medicago sativa; phenylpropanoid biosynthesis

Received: April 11, 2025; Revised: August 2, 2025; Accepted: August 4, 2025; Prepublished online: September 17, 2025 

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