{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,7]],"date-time":"2026-05-07T12:10:16Z","timestamp":1778155816110,"version":"3.51.4"},"reference-count":43,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,5,6]],"date-time":"2026-05-06T00:00:00Z","timestamp":1778025600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Key Laboratory of Seed Creation and Breeding and Sustainable Utilization of Grassland Resources in Inner Mongolia Autonomous Region","award":["2025KYPT0033"],"award-info":[{"award-number":["2025KYPT0033"]}]},{"name":"Key Laboratory of Seed Creation and Breeding and Sustainable Utilization of Grassland Resources in Inner Mongolia Autonomous Region","award":["2025KYPT0040"],"award-info":[{"award-number":["2025KYPT0040"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>Background: Cytokinin oxidase\/dehydrogenase (CKX) can irreversibly degrade cytokinin, regulating the growth and development of plants and helping them cope with environmental stress. Methods: To understand the expression characteristics and the biological functions of CKX in alfalfa (Medicago sativa), a novel gene, designated as MsCKX5 (GenBank: PV934228), was cloned, characterized and overexpressed in Arabidopsis thaliana. Results: qRT-PCR results showed that MsCKX5 gene expression was clearly tissue-specific and had the highest expression level in the stems. In addition, the expression level of MsCKX5 was significantly induced by cold treatments. Heterologous expression of the MsCKX5 gene in A. thaliana could enhance cold tolerance by regulating the activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and adjust the relative conductivity and malondialdehyde (MDA) content. The combined analysis of transcriptome and metabolome results indicated that flavone and flavonol biosynthesis as well as the plant hormone signal transduction pathways were the main enriched pathways between wild-type and MsCKX5 overexpressed lines. Conclusions: These results provide an important molecular basis for further elucidating the molecular mechanism of plant cold resistance and the breeding of cold-resistant crop varieties.<\/jats:p>","DOI":"10.3390\/genes17050557","type":"journal-article","created":{"date-parts":[[2026,5,7]],"date-time":"2026-05-07T10:39:47Z","timestamp":1778150387000},"page":"557","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Overexpression of an Alfalfa Gene, MsCKX5, Confers Cold Tolerance in Transgenic Arabidopsis thaliana"],"prefix":"10.3390","volume":"17","author":[{"given":"Lin","family":"Bian","sequence":"first","affiliation":[{"name":"Key Laboratory of Grassland Resources of the Ministry of Education, Engineering Center of Drought and Cold-Resistant Grass Breeding in the North of the National Forestry and Grassland Administration, Technology College of Grassland Science, Inner Mongolia Agricultural University, Hohhot 010010, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaowei","family":"Huo","sequence":"additional","affiliation":[{"name":"Key Laboratory of Grassland Resources of the Ministry of Education, Engineering Center of Drought and Cold-Resistant Grass Breeding in the North of the National Forestry and Grassland Administration, Technology College of Grassland Science, Inner Mongolia Agricultural University, Hohhot 010010, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qi","family":"Chen","sequence":"additional","affiliation":[{"name":"Key Laboratory of Grassland Resources of the Ministry of Education, Engineering Center of Drought and Cold-Resistant Grass Breeding in the North of the National Forestry and Grassland Administration, Technology College of Grassland Science, Inner Mongolia Agricultural University, Hohhot 010010, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yan","family":"Zhang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Grassland Resources of the Ministry of Education, Engineering Center of Drought and Cold-Resistant Grass Breeding in the North of the National Forestry and Grassland Administration, Technology College of Grassland Science, Inner Mongolia Agricultural University, Hohhot 010010, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Na","family":"Guo","sequence":"additional","affiliation":[{"name":"Key Laboratory of Grassland Resources of the Ministry of Education, Engineering Center of Drought and Cold-Resistant Grass Breeding in the North of the National Forestry and Grassland Administration, Technology College of Grassland Science, Inner Mongolia Agricultural University, Hohhot 010010, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jing","family":"Xu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Grassland Resources of the Ministry of Education, Engineering Center of Drought and Cold-Resistant Grass Breeding in the North of the National Forestry and Grassland Administration, Technology College of Grassland Science, Inner Mongolia Agricultural University, Hohhot 010010, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jianwei","family":"Li","sequence":"additional","affiliation":[{"name":"Key Laboratory of Grassland Resources of the Ministry of Education, Engineering Center of Drought and Cold-Resistant Grass Breeding in the North of the National Forestry and Grassland Administration, Technology College of Grassland Science, Inner Mongolia Agricultural University, Hohhot 010010, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhiqiang","family":"Zhang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Grassland Resources of the Ministry of Education, Engineering Center of Drought and Cold-Resistant Grass Breeding in the North of the National Forestry and Grassland Administration, Technology College of Grassland Science, Inner Mongolia Agricultural University, Hohhot 010010, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1038\/s41438-021-00483-5","article-title":"The genome of Medicago polymorpha provides insights into its edibility and nutritional value as a vegetable and forage legume","volume":"8","author":"Cui","year":"2021","journal-title":"Hortic. Res.-Engl."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Pu, X., Fu, Y., Xu, C., Li, X., Wang, W., De, K., Wei, X., and Yao, X. (2024). Transcriptomic analyses provide molecular insight into the cold stress response of cold-tolerant alfalfa. BMC Plant Biol., 24.","DOI":"10.1186\/s12870-024-05136-y"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1104\/pp.112.1.281","article-title":"Nitrogen and Carbon Flows Estimated by 15N and 13C Pulse-Chase Labeling during Regrowth of Alfalfa","volume":"112","author":"Avice","year":"1996","journal-title":"Plant Physiol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1093\/aob\/mcl254","article-title":"Elevated atmospheric CO2 and strain of rhizobium alter freezing tolerance and cold-induced molecular changes in alfalfa (Medicago sativa)","volume":"99","author":"Bertrand","year":"2007","journal-title":"Ann. Bot."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Xu, H., Xu, L., and Hassan, M.A. (2025). Mitigating low-temperature stress in alfalfa by postponing phosphorus application and remodeling of antioxidant activities and carbon-nitrogen metabolism. Front. Plant Sci., 16.","DOI":"10.3389\/fpls.2025.1550026"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1146\/annurev-arplant-042817-040440","article-title":"The Oxylipin Pathways: Biochemistry and Function","volume":"69","author":"Wasternack","year":"2018","journal-title":"Annu. Rev. Plant Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/j.phytochem.2018.02.008","article-title":"Cytokinin activity of N(6)-benzyladenine derivatives assayed by interaction with the receptors in planta, in vitro, and in silico","volume":"149","author":"Savelieva","year":"2018","journal-title":"Phytochemistry"},{"key":"ref_8","unstructured":"Li, J., Li, C., and Smith, S.M. (2017). Hormone Metabolism and Signaling in Plants, Academic Press."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1104\/pp.16.00530","article-title":"Cytokinins Are Initial Targets of Light in the Control of Bud Outgrowth","volume":"172","author":"Roman","year":"2016","journal-title":"Plant Physiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1104\/pp.18.00519","article-title":"Initial Bud Outgrowth Occurs Independent of Auxin Flow from Out of Buds","volume":"179","author":"Chabikwa","year":"2019","journal-title":"Plant Physiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"dev149344","DOI":"10.1242\/dev.149344","article-title":"Cytokinin signaling in plant development","volume":"145","author":"Kieber","year":"2018","journal-title":"Development"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1016\/j.tplants.2018.10.012","article-title":"Cytokinin-A Developing Story","volume":"24","author":"Wybouw","year":"2019","journal-title":"Trends Plant Sci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"614","DOI":"10.1111\/pbi.13305","article-title":"Cytokinin dehydrogenase: A genetic target for yield improvement in wheat","volume":"18","author":"Chen","year":"2020","journal-title":"Plant Biotechnol. J."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"371","DOI":"10.1055\/s-2006-923928","article-title":"New insights into the biology of cytokinin degradation","volume":"8","author":"Werner","year":"2006","journal-title":"Plant Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1139\/o75-006","article-title":"3-Methyl-2-butenal: An enzymatic degradation product of the cytokinin, N-6-(delta-2 isopentenyl)adenine","volume":"53","author":"Brownlee","year":"1975","journal-title":"Can. J. Biochem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2532","DOI":"10.1105\/tpc.014928","article-title":"Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity","volume":"15","author":"Werner","year":"2003","journal-title":"Plant Cell"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"741","DOI":"10.1126\/science.1113373","article-title":"Cytokinin oxidase regulates rice grain production","volume":"309","author":"Ashikari","year":"2005","journal-title":"Science"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3552","DOI":"10.1093\/jxb\/erac088","article-title":"Cytokinin oxidase\/dehydrogenase family genes exhibit functional divergence and overlap in rice growth and development, especially in control of tillering","volume":"73","author":"Rong","year":"2022","journal-title":"J. Exp. Bot."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Du, Y., Zhang, Z., Gu, Y., Li, W., Wang, W., Yuan, X., Zhang, Y., Yuan, M., Du, J., and Zhao, Q. (2023). Genome-wide identification of the soybean cytokinin oxidase\/dehydrogenase gene family and its diverse roles in response to multiple abiotic stress. Front. Plant Sci., 14.","DOI":"10.3389\/fpls.2023.1163219"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1105\/tpc.110.079079","article-title":"Cytokinin regulates the activity of reproductive meristems, flower organ size, ovule formation, and thus seed yield in Arabidopsis thaliana","volume":"23","author":"Bartrina","year":"2011","journal-title":"Plant Cell"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2169","DOI":"10.1105\/tpc.111.087395","article-title":"Analysis of cytokinin mutants and regulation of cytokinin metabolic genes reveals important regulatory roles of cytokinins in drought, salt and abscisic acid responses, and abscisic acid biosynthesis","volume":"23","author":"Nishiyama","year":"2011","journal-title":"Plant Cell"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"374","DOI":"10.1007\/s12374-019-0141-z","article-title":"Overexpression of the Cytokinin Oxidase\/dehydrogenase (CKX) from Medicago sativa Enhanced Salt Stress Tolerance of Arabidopsis","volume":"62","author":"Li","year":"2019","journal-title":"J. Plant Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2805","DOI":"10.1093\/jxb\/ert131","article-title":"Enhanced drought and heat stress tolerance of tobacco plants with ectopically enhanced cytokinin oxidase\/dehydrogenase gene expression","volume":"64","author":"Motyka","year":"2013","journal-title":"J. Exp. Bot."},{"key":"ref_24","first-page":"125","article-title":"Identification and abiotic stress response of CKX gene family in alfalfa","volume":"34","author":"Bian","year":"2025","journal-title":"Acta Prataculturae Sin."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2745","DOI":"10.1016\/S2095-3119(18)61926-4","article-title":"Assessment of suitable reference genes for qRT-PCR analysis in Adelphocoris suturalis","volume":"17","author":"Luo","year":"2018","journal-title":"J. Integr. Agric."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1767","DOI":"10.1093\/nar\/gkp1137","article-title":"The Sanger FASTQ file format for sequences with quality scores, and the Solexa\/Illumina FASTQ variants","volume":"38","author":"Cock","year":"2010","journal-title":"Nucleic Acids Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"290","DOI":"10.1038\/nbt.3122","article-title":"StringTie enables improved reconstruction of a transcriptome from RNA-seq reads","volume":"33","author":"Pertea","year":"2015","journal-title":"Nat. Biotechnol."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Li, B., and Dewey, C.N. (2011). RSEM: Accurate transcript quantification from RNA-Seq data with or without a reference genome. Bmc Bioinform., 12.","DOI":"10.1186\/1471-2105-12-323"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1186\/s13059-014-0550-8","article-title":"Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2","volume":"15","author":"Love","year":"2014","journal-title":"Genome Biol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Xu, Y., Ran, S., Li, S., Lu, J., Huang, W., Zheng, J., Hou, M., and Zhong, F. (2024). Genome-Wide Identification and Abiotic Stress Expression Analysis of CKX and IPT Family Genes in Cucumber (Cucumis sativus L.). Plants, 13.","DOI":"10.3390\/plants13030422"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"100951","DOI":"10.1016\/j.crfs.2024.100951","article-title":"Melatonin alleviated chilling injury of cold-stored passion fruit by modulating cell membrane structure via acting on antioxidant ability and membrane lipid metabolism","volume":"10","author":"Lin","year":"2024","journal-title":"Curr. Res. Food Sci."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1505","DOI":"10.1016\/j.sjbs.2015.09.017","article-title":"Effects of water-deficit stress and paclobutrazol on growth, relative water content, electrolyte leakage, proline content and some antioxidant changes in Curcuma alismatifolia Gagnep. cv. Chiang Mai Pink","volume":"24","author":"Jungklang","year":"2017","journal-title":"Saudi J. Biol. Sci."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Song, Y., John Martin, J.J., Liu, X., Li, X., Hou, M., Zhang, R., Xu, W., Li, W., and Cao, H. (2025). Unraveling the response of secondary metabolites to cold tolerance in oil palm by integration of physiology and metabolomic analyses. BMC Plant Biol., 25.","DOI":"10.1186\/s12870-025-06292-5"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.tplants.2020.07.010","article-title":"Challenges and Opportunities in Machine-Augmented Plant Stress Phenotyping","volume":"26","author":"Singh","year":"2021","journal-title":"Trends Plant Sci."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1111\/tpj.12388","article-title":"Enhancement of oxidative and drought tolerance in Arabidopsis by overaccumulation of antioxidant flavonoids","volume":"77","author":"Nakabayashi","year":"2014","journal-title":"Plant J. For. Cell Mol. Biol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1016\/j.plaphy.2013.02.001","article-title":"The flavonoid biosynthetic pathway in Arabidopsis: Structural and genetic diversity","volume":"72","author":"Saito","year":"2013","journal-title":"Plant Physiol. Biochem. Ppb"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1005","DOI":"10.1016\/j.cell.2009.03.001","article-title":"Auxin: A trigger for change in plant development","volume":"136","author":"Vanneste","year":"2009","journal-title":"Cell"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"618","DOI":"10.1016\/j.devcel.2017.09.024","article-title":"MAP Kinase Cascades Regulate the Cold Response by Modulating ICE1 Protein Stability","volume":"43","author":"Zhao","year":"2017","journal-title":"Dev. Cell"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"651","DOI":"10.1146\/annurev-arplant-042809-112122","article-title":"Abscisic acid: Emergence of a core signaling network","volume":"61","author":"Cutler","year":"2010","journal-title":"Annu. Rev. Plant Biol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"23371","DOI":"10.1074\/jbc.M109.096644","article-title":"A subset of cytokinin two-component signaling system plays a role in cold temperature stress response in Arabidopsis","volume":"285","author":"Jeon","year":"2010","journal-title":"J. Biol. Chem."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/j.tplants.2007.11.005","article-title":"Cytokinin signaling: Two-components and more","volume":"13","author":"To","year":"2008","journal-title":"Trends Plant Sci."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"464","DOI":"10.1007\/s00344-020-10152-x","article-title":"Multifaceted Role of Salicylic Acid in Combating Cold Stress in Plants: A Review","volume":"40","author":"Saleem","year":"2021","journal-title":"J. Plant Growth Regul."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1146\/annurev.phyto.050908.135202","article-title":"Salicylic Acid, a multifaceted hormone to combat disease","volume":"47","author":"Vlot","year":"2009","journal-title":"Annu. Rev. Phytopathol."}],"container-title":["Genes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4425\/17\/5\/557\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,7]],"date-time":"2026-05-07T11:13:18Z","timestamp":1778152398000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4425\/17\/5\/557"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,6]]},"references-count":43,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["genes17050557"],"URL":"https:\/\/doi.org\/10.3390\/genes17050557","relation":{},"ISSN":["2073-4425"],"issn-type":[{"value":"2073-4425","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,6]]}}}