Register      Login
Crop and Pasture Science Crop and Pasture Science Society
Plant sciences, sustainable farming systems and food quality
RESEARCH ARTICLE

Comparative transcriptome analysis of five Medicago varieties reveals the genetic signals underlying freezing tolerance

Lijun Xu A , Xuejuan Tang A , Bo Wang B , Xiaoping Xin https://orcid.org/0000-0002-2875-9970 A G , Qizhong Sun C G , Yalu Li D , Jinqiang Chen A , Gele Qing E and Mingying Guo F
+ Author Affiliations
- Author Affiliations

A Hulunber Grassland Ecosystem Observation and Research Station/Chinese Academy of Agricultural Sciences Institute of Agricultural Resources and Regional Planning, Beijing 100081, China.

B Conservation Program Center, Chinese Academy of Engineering, Beijing 100088, China.

C Chinese Academy of Agricultural Sciences Grassland Research Institute, Hohhot 010010, Inner Mongolia, China.

D College of Grassland, Resources and Environment/Inner Mongolia Agricultural University, Hohhot 010000, Inner Mongolia, China.

E Xilinhot Vocational School, Xinlinhot 026000, Inner Mongolia, China.

F Hulunber Grassland Station, Hulunber, Inner Mongolia 021000, China.

G Corresponding author. Email: xinxiaoping@caas.cn; sunqz@126.com

Crop and Pasture Science 70(3) 273-282 https://doi.org/10.1071/CP18165
Submitted: 20 April 2018  Accepted: 13 January 2019   Published: 12 March 2019

Abstract

Medicago plants such as M. sativa (lucerne, alfalfa) are important forage all over the world. Freezing-tolerance capacity is one of the key determinants of the survival and production of Medicago. In order to explore the molecular basis underlying freezing tolerance, we sequenced the root transcriptomes of five Medicago varieties belonging to two species, M. sativa and M. varia, and compared their gene expression and molecular evolution. A range of 19.5–23.8 Gb clean bases was obtained, and de novo transcriptome assembly generated 205 238–268 520 unigenes. The GO (Gene Ontology) terms of basic biological processes such as binding, cell and metabolism were most represented for the unigenes. In addition, a large number of unigenes related to GO terms and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways of membrane, signalling, transcription and response to stimulus were identified in functional annotation. In total, 12 455 orthologs were identified among the five Medicago varieties. Among the orthologs, many unigenes that directly related to freezing tolerance were highly expressed in all five varieties, including genes for WRKY transcription factors, calcium-binding factors, and antioxidant enzymes such as catalase and ascorbate peroxidase. Molecular evolution testing showed that the unigenes involved in membrane shared high Ka/Ks (non-synonymous/synonymous substitution rate) across all the five Medicago varieties. Positively selected genes were mainly involved in transcription regulation, metabolism and signal transduction. Our study provides a large transcriptome dataset in the Medicago genus and brings new insights into the freezing tolerance for Medicago species.

Additional keywords: APX, CAT, CBF.


References

Agarwal P, Reddy MP, Chikara J (2011) WRKY: its structure, evolutionary relationship, DNA-binding selectivity, role in stress tolerance and development of plants. Molecular Biology Reports 38, 3883–3896.
WRKY: its structure, evolutionary relationship, DNA-binding selectivity, role in stress tolerance and development of plants.Crossref | GoogleScholarGoogle Scholar | 21107718PubMed |

An D, Yang J, Zhang P (2012) Transcriptome profiling of low temperature-treated cassava apical shoots showed dynamic responses of tropical plant to cold stress. BMC Genomics 13, 64
Transcriptome profiling of low temperature-treated cassava apical shoots showed dynamic responses of tropical plant to cold stress.Crossref | GoogleScholarGoogle Scholar | 22321773PubMed |

Baxter A, Mittler R, Suzuki N (2014) ROS as key players in plant stress signalling. Journal of Experimental Botany 65, 1229–1240.
ROS as key players in plant stress signalling.Crossref | GoogleScholarGoogle Scholar | 24253197PubMed |

Beike AK, Lang D, Zimmer AD, Wust F, Trautmann D, Wiedemann G, Beyer P, Decker EL, Reski R (2015) Insights from the cold transcriptome of Physcomitrella patens: global specialization pattern of conserved transcriptional regulators and identification of orphan genes involved in cold acclimation. New Phytologist 205, 869–881.
Insights from the cold transcriptome of Physcomitrella patens: global specialization pattern of conserved transcriptional regulators and identification of orphan genes involved in cold acclimation.Crossref | GoogleScholarGoogle Scholar | 25209349PubMed |

Bela K, Horvath E, Galle A, Szabados L, Tari I, Csiszar J (2015) Plant glutathione peroxidases: emerging role of the antioxidant enzymes in plant development and stress responses. Journal of Plant Physiology 176, 192–201.
Plant glutathione peroxidases: emerging role of the antioxidant enzymes in plant development and stress responses.Crossref | GoogleScholarGoogle Scholar | 25638402PubMed |

Benjamini Y, Yekutieli D (2001) The control of the false discovery rate in multiple testing under dependency. Annals of Statistics 29, 1165–1188.

Biazzi E, Nazzicari N, Pecetti L, Brummer EC, Palmonari A, Tava A, Annicchiarico P (2017) Genome-wide association mapping and genomic selection for alfalfa (Medicago sativa) forage quality traits. PLoS One 12, e0169234
Genome-wide association mapping and genomic selection for alfalfa (Medicago sativa) forage quality traits.Crossref | GoogleScholarGoogle Scholar | 28068350PubMed |

Chen X, Zhang J, Liu Q, Guo W, Zhao T, Ma Q, Wang G (2014) Transcriptome sequencing and identification of cold tolerance genes in hardy Corylus species (C. heterophylla Fisch) floral buds. PLoS One 9, e108604
Transcriptome sequencing and identification of cold tolerance genes in hardy Corylus species (C. heterophylla Fisch) floral buds.Crossref | GoogleScholarGoogle Scholar | 25544987PubMed |

Chen J, Yang X, Huang X, Duan S, Long C, Chen J, Rong J (2017) Leaf transcriptome analysis of a subtropical evergreen broadleaf plant, wild oil-tea camellia (Camellia oleifera), revealing candidate genes for cold acclimation. BMC Genomics 18, 211
Leaf transcriptome analysis of a subtropical evergreen broadleaf plant, wild oil-tea camellia (Camellia oleifera), revealing candidate genes for cold acclimation.Crossref | GoogleScholarGoogle Scholar | 28241790PubMed |

Chinnusamy V, Zhu J, Zhu JK (2007) Cold stress regulation of gene expression in plants. Trends in Plant Science 12, 444–451.
Cold stress regulation of gene expression in plants.Crossref | GoogleScholarGoogle Scholar | 17855156PubMed |

Choudhary R, Saroha AE, Swarnkar PL (2012) Effect of abscisic acid and hydrogen peroxide on antioxidant enzymes in Syzygium cumini plant. Journal of Food Science and Technology 49, 649–652.
Effect of abscisic acid and hydrogen peroxide on antioxidant enzymes in Syzygium cumini plant.Crossref | GoogleScholarGoogle Scholar | 24082280PubMed |

Deng X, Zhou S, Hu W, Feng J, Zhang F, Chen L, Huang C, Luo Q, He Y, Yang G, He G (2013) Ectopic expression of wheat TaCIPK14, encoding a calcineurin B-like protein-interacting protein kinase, confers salinity and cold tolerance in tobacco. Physiologia Plantarum 149, 367–377.

Fowler S, Thomashow MF (2002) Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. The Plant Cell 14, 1675–1690.
Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway.Crossref | GoogleScholarGoogle Scholar | 12172015PubMed |

Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnology 29, 644–652.
Full-length transcriptome assembly from RNA-Seq data without a reference genome.Crossref | GoogleScholarGoogle Scholar | 21572440PubMed |

Guo Z, Tan J, Zhuo C, Wang C, Xiang B, Wang Z (2014) Abscisic acid, H2O2 and nitric oxide interactions mediated cold-induced S-adenosylmethionine synthetase in Medicago sativa subsp. falcata that confers cold tolerance through up-regulating polyamine oxidation. Plant Biotechnology Journal 12, 601–612.
Abscisic acid, H2O2 and nitric oxide interactions mediated cold-induced S-adenosylmethionine synthetase in Medicago sativa subsp. falcata that confers cold tolerance through up-regulating polyamine oxidation.Crossref | GoogleScholarGoogle Scholar | 24517136PubMed |

He X, Sambe MA, Zhuo C, Tu Q, Guo Z (2015) A temperature induced lipocalin gene from Medicago falcata (MfTIL1) confers tolerance to cold and oxidative stress. Plant Molecular Biology 87, 645–654.
A temperature induced lipocalin gene from Medicago falcata (MfTIL1) confers tolerance to cold and oxidative stress.Crossref | GoogleScholarGoogle Scholar | 25744207PubMed |

Hu R, Zhu X, Xiang S, Zhan Y, Zhu M, Yin H, Zhou Q, Zhu L, Zhang X, Liu Z (2016) Comparative transcriptome analysis revealed the genotype specific cold response mechanism in tobacco. Biochemical and Biophysical Research Communications 469, 535–541.
Comparative transcriptome analysis revealed the genotype specific cold response mechanism in tobacco.Crossref | GoogleScholarGoogle Scholar | 26692485PubMed |

Kaye C, Guy CL (1995) Perspectives of plant cold tolerance: physiology and molecular responses. Science Progress 78, 271–299.

Kosová K, Vítámvás P, Prášil IT (2014) Wheat and barley dehydrins under cold, drought, and salinity—what can LEA-II proteins tell us about plant stress response? Frontiers of Plant Science 5, 343
Wheat and barley dehydrins under cold, drought, and salinity—what can LEA-II proteins tell us about plant stress response?Crossref | GoogleScholarGoogle Scholar |

Król A, Amarowicz R, Weidner S (2015) The effects of cold stress on the phenolic compounds and antioxidant capacity of grapevine (Vitis vinifera L.) leaves. Journal of Plant Physiology 189, 97–104.
The effects of cold stress on the phenolic compounds and antioxidant capacity of grapevine (Vitis vinifera L.) leaves.Crossref | GoogleScholarGoogle Scholar | 26555272PubMed |

Lee BH, Henderson DA, Zhu JK (2005) The Arabidopsis cold-responsive transcriptome and its regulation by ICE1. The Plant Cell 17, 3155–3175.
The Arabidopsis cold-responsive transcriptome and its regulation by ICE1.Crossref | GoogleScholarGoogle Scholar | 16214899PubMed |

Li B, Dewey CN (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12, 323
RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome.Crossref | GoogleScholarGoogle Scholar | 21816040PubMed |

Los DA, Murata N (2004) Membrane fluidity and its roles in the perception of environmental signals. Biochimica et Biophysica Acta 1666, 142–157.
Membrane fluidity and its roles in the perception of environmental signals.Crossref | GoogleScholarGoogle Scholar | 15519313PubMed |

NDong C, Danyluk J, Wilson KE, Pocock T, Huner NP, Sarhan F (2002) Cold-regulated cereal chloroplast late embryogenesis abundant-like proteins. Molecular characterization and functional analyses. Plant Physiology 129, 1368–1381.
Cold-regulated cereal chloroplast late embryogenesis abundant-like proteins. Molecular characterization and functional analyses.Crossref | GoogleScholarGoogle Scholar | 12114590PubMed |

Pennycooke JC, Cheng H, Stockinger EJ (2008) Comparative genomic sequence and expression analyses of Medicago truncatula and alfalfa subspecies falcataCOLD-ACCLIMATION-SPECIFIC genes. Plant Physiology 146, 1242–1254.
Comparative genomic sequence and expression analyses of Medicago truncatula and alfalfa subspecies falcataCOLD-ACCLIMATION-SPECIFIC genes.Crossref | GoogleScholarGoogle Scholar | 18218976PubMed |

Saijo Y, Hata S, Kyozuka J, Shimamoto K, Izui K (2000) Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants. The Plant Journal 23, 319–327.
Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants.Crossref | GoogleScholarGoogle Scholar | 10929125PubMed |

Schulz E, Tohge T, Zuther E, Fernie AR, Hincha DK (2016) Flavonoids are determinants of freezing tolerance and cold acclimation in Arabidopsis thaliana. Scientific Reports 6, 34027
Flavonoids are determinants of freezing tolerance and cold acclimation in Arabidopsis thaliana.Crossref | GoogleScholarGoogle Scholar | 27658445PubMed |

Song L, Jiang L, Chen Y, Shu Y, Bai Y, Guo C (2016) Deep-sequencing transcriptome analysis of field-grown Medicago sativa L. crown buds acclimated to freezing stress. Functional & Integrative Genomics 16, 495–511.
Deep-sequencing transcriptome analysis of field-grown Medicago sativa L. crown buds acclimated to freezing stress.Crossref | GoogleScholarGoogle Scholar |

Streb P, Aubert S, Gout E, Bligny R (2003) Cold- and light-induced changes of metabolite and antioxidant levels in two high mountain plant species Soldanella alpina and Ranunculus glacialis and a lowland species Pisum sativum. Physiologia Plantarum 118, 96–104.
Cold- and light-induced changes of metabolite and antioxidant levels in two high mountain plant species Soldanella alpina and Ranunculus glacialis and a lowland species Pisum sativum.Crossref | GoogleScholarGoogle Scholar | 12702018PubMed |

Suzuki N, Koussevitzky S, Mittler R, Miller G (2012) ROS and redox signalling in the response of plants to abiotic stress. Plant, Cell & Environment 35, 259–270.
ROS and redox signalling in the response of plants to abiotic stress.Crossref | GoogleScholarGoogle Scholar |

Svensson JT, Crosatti C, Campoli C, Bassi R, Stanca AM, Close TJ, Cattivelli L (2006) Transcriptome analysis of cold acclimation in barley albina and xantha mutants. Plant Physiology 141, 257–270.
Transcriptome analysis of cold acclimation in barley albina and xantha mutants.Crossref | GoogleScholarGoogle Scholar | 16603669PubMed |

Thiel T, Michalek W, Varshney RK, Graner A (2003) Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theoretical and Applied Genetics 106, 411–422.
Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.).Crossref | GoogleScholarGoogle Scholar | 12589540PubMed |

Thomashow MF (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annual Review of Plant Physiology and Plant Molecular Biology 50, 571–599.
Plant cold acclimation: freezing tolerance genes and regulatory mechanisms.Crossref | GoogleScholarGoogle Scholar | 15012220PubMed |

Wang CT, Shao JM (2013) Characterization of the ZmCK1 gene encoding a calcium-dependent protein kinase responsive to multiple abiotic stresses in maize. Plant Molecular Biology Reporter 31, 222–230.
Characterization of the ZmCK1 gene encoding a calcium-dependent protein kinase responsive to multiple abiotic stresses in maize.Crossref | GoogleScholarGoogle Scholar |

Wang L, Zhu W, Fang L, Sun X, Su L, Liang Z, Wang N, Londo JP, Li S, Xin H (2014) Genome-wide identification of WRKY family genes and their response to cold stress in Vitis vinifera. BMC Plant Biology 14, 103
Genome-wide identification of WRKY family genes and their response to cold stress in Vitis vinifera.Crossref | GoogleScholarGoogle Scholar | 24755338PubMed |

Winfield MO, Lu C, Wilson ID, Coghill JA, Edwards KJ (2010) Plant responses to cold: Transcriptome analysis of wheat. Plant Biotechnology Journal 8, 749–771.
Plant responses to cold: Transcriptome analysis of wheat.Crossref | GoogleScholarGoogle Scholar | 20561247PubMed |

Xu W, Zhang N, Jiao Y, Li R, Xiao D, Wang Z (2014) The grapevine basic helix-loop-helix (bHLH) transcription factor positively modulates CBF-pathway and confers tolerance to cold-stress in Arabidopsis. Molecular Biology Reports 41, 5329–5342.
The grapevine basic helix-loop-helix (bHLH) transcription factor positively modulates CBF-pathway and confers tolerance to cold-stress in Arabidopsis.Crossref | GoogleScholarGoogle Scholar | 24859977PubMed |

Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z, Wang J, Li S, Li R, Bolund L, Wang J (2006) WEGO: a web tool for plotting GO annotations. Nucleic Acids Research 34, W293
WEGO: a web tool for plotting GO annotations.Crossref | GoogleScholarGoogle Scholar | 16845012PubMed |

Zhang Z, Li J, Zhao X, Wang J, Wong GK, Yu J (2006) KaKs_Calculator: Calculating Ka and Ks through model selection and model averaging. Genomics, Proteomics & Bioinformatics 4, 259–263.
KaKs_Calculator: Calculating Ka and Ks through model selection and model averaging.Crossref | GoogleScholarGoogle Scholar |

Zhang Y, Yu H, Yang X, Li Q, Ling J, Wang H, Gu X, Huang S, Jiang W (2016) CsWRKY46, a WRKY transcription factor from cucumber, confers cold resistance in transgenic-plant by regulating a set of cold-stress responsive genes in an ABA-dependent manner. Plant Physiology and Biochemistry 108, 478–487.
CsWRKY46, a WRKY transcription factor from cucumber, confers cold resistance in transgenic-plant by regulating a set of cold-stress responsive genes in an ABA-dependent manner.Crossref | GoogleScholarGoogle Scholar | 27592172PubMed |

Zhou J, Liang Y, Niu Q, Chen L, Zhang X, Ye D (2013) The Arabidopsis general transcription factor TFIIB1 (AtTFIIB1) is required for pollen tube growth and endosperm development. Journal of Experimental Botany 64, 2205–2218.
The Arabidopsis general transcription factor TFIIB1 (AtTFIIB1) is required for pollen tube growth and endosperm development.Crossref | GoogleScholarGoogle Scholar | 23547107PubMed |

Zhou Y, Cheng Y, Yang Y, Li X, Supriyo B, Sun X, Yang Y (2016) Overexpression of SpCBL6, a calcineurin B-like protein of Stipa purpurea, enhanced cold tolerance and reduced drought tolerance in transgenic Arabidopsis. Molecular Biology Reports 43, 957–966.
Overexpression of SpCBL6, a calcineurin B-like protein of Stipa purpurea, enhanced cold tolerance and reduced drought tolerance in transgenic Arabidopsis.Crossref | GoogleScholarGoogle Scholar | 27393148PubMed |

Zhuo C, Wang T, Guo Z, Lu S (2016) Overexpression of MfPIP2-7 from Medicago falcata promotes cold tolerance and growth under NO3 – deficiency in transgenic tobacco plants. BMC Plant Biology 16, 138
Overexpression of MfPIP2-7 from Medicago falcata promotes cold tolerance and growth under NO3 deficiency in transgenic tobacco plants.Crossref | GoogleScholarGoogle Scholar | 27301445PubMed |