Transcriptome Analysis of the Cytokinin Response in Medicago truncatula

Abstract

As an important legume plant, Medicago truncatula is a preeminent model for the study of the processes of nitrogen fixation, symbiosis, and legume genomics. The regulatory mechanism of the cytokinin response has been studied in many plants, such as rice, Arabidopsis, tomato, and barley, but information about regulatory pathways and genes involved in the cytokinin response in Medicago truncatula is notably limited. In this study, to better understand the cytokinin response in Medicago truncatula, transcriptome analysis of seedlings grown with 6-benzylaminopurine or lovastatin was performed using RNA-Seq. In this study, 3627 and 3093 transcripts were differentially expressed in cytokinin-induced/control (Cyto/Ctrl) and cytokinin-inhibited/control (Inh/Ctrl) groups, respectively, and differentially expressed genes were tested by quantitative real-time PCR (qRT-PCR). Analysis of the cytokinin response in Medicago truncatula revealed a large number of transcripts involved in signal transduction, metabolic process, secondary metabolite biosynthesis, transport and catabolism, growth and development, defense mechanisms, and transcription. There were 43 transcription factor families, including 1845 transcription factor (TF) genes with 2147 TF transcripts, as detected by RNA-Seq. Additionally, 216 TF genes with 220 transcripts were differentially expressed in Cyto/Ctrl, and 185 TF genes with 189 transcripts were in Inh/Ctrl. A total of 289 and 260 DETs involved in biosynthesis, metabolism, and transduction of plant hormones were identified in the Cyto/Ctrl and Inh/Ctrl groups, respectively. Furthermore, 15 transcripts, including A-ARR, IPT, and CKX, were demonstrated to play roles in cytokinin regulation or signal transduction. These findings were associated with the cytokinin response in other plants. The resulting data provide the first cytokinin transcriptome analysis in Medicago truncatula. Further analysis and identification of cytokinin-regulated transcripts or signal transduction transcripts may help to elucidate the regulatory mechanisms governing the cytokinin response in Medicago truncatula.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Harris MA (2000) Gene ontology: tool for the unification of biology. Nat Genet 25:25–29

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. Bar M, Israeli A, Levy M, Gera H, Jiménez-Gómez J, Kouril S (2016) CLAUSA Is a MYB transcription factor that promotes leaf differentiation by attenuating cytokinin signaling. Plant Cell 28(7):1602–1615

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Bhargava A, Clabaugh I, To JP, Maxwell BB, Chiang YH, Schaller GE, Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in arabidopsis. Plant Physiol 162:272–294

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. Branca A, Paape TD, Zhou P, Briskine R, Farmer AD, Mudge J, Ben C (2011) Whole-genome nucleotide diversity, recombination, and linkage disequilibrium in the model legume Medicago truncatula. P Natl Acad Sci USA 108:E864–E870

    CAS  Article  Google Scholar 

  5. Brzobohaty B, Moore I, Kristoffersen P, Bako L, Campos N, Schell J, Palme K (1993) Release of active cytokinin by a beta-glucosidase localized to the maize root meristem. Science 262:1051–1054

    CAS  PubMed  Article  Google Scholar 

  6. Buchfink B, Xie C, Huson DH (2015) Fast and sensitive protein alignment using DIAMOND. Nat Methods 12:59–60

    CAS  PubMed  Article  Google Scholar 

  7. Chao Y, Xie L, Yuan J, Guo T, Li Y, Liu F, Han L (2018) Transcriptome analysis of leaf senescence in red clover (Trifolium pratense L.). Physiol Mol Biol Pla 24:753–765

    CAS  Article  Google Scholar 

  8. Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676

    CAS  PubMed  Article  Google Scholar 

  9. Crowell DN, Salaz MS (1992) Inhibition of growth of cultured tobacco cells at low concentrations of lovastatin is reversed by cytokinin. Plant Physiol 100:2090–2095

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. D’Agostino IB, Kieber JJ (1999) Molecular mechanisms of cytokinin action. Curr Opin Plant Bio 2(5):359–364

    Article  Google Scholar 

  11. Day RC, Herridge RP, Ambrose BA, Macknight RC (2008) Transcriptome analysis of proliferating Arabidopsis endosperm reveals biological implications for the control of syncytial division, cytokinin signaling, and gene expression regulation. Plant Physiol 148:1964–1984

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Durán-Medina Y, Serwatowska J, Reyes-Olalde JI, De Folter S, Marsch-Martínez N (2017) The AP2/ERF transcription factor DRNL modulates gynoecium development and affects its response to cytokinin. Front Plant Sci 8:1841

    PubMed  PubMed Central  Article  Google Scholar 

  13. Eddy SR (1998) Profile hidden Markov models. Bioinformatics 14:755–763

    CAS  PubMed  Article  Google Scholar 

  14. Finn RD, Clements J, Eddy SR (2011) HMMER web server: interactive sequence similarity searching. Nucleic Acids Res 39:W29–W37

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. Franco-Zorrilla JM, Martín AC, Leyva A, Paz-Ares J (2005) Interaction between phosphate-starvation, sugar, and cytokinin signaling in Arabidopsis and the roles of cytokinin receptors CRE1/AHK4 and AHK3. Plant Physiol 138(2):847–857

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. Gomi K, Sasaki A, Itoh H, Ueguchi-Tanaka M, Ashikari M, Kitano H, Matsuoka M (2004) GID2, an F-box subunit of the SCF E3 complex, specifically interacts with phosphorylated SLR1 protein and regulates the gibberellin-dependent degradation of SLR1 in rice. Plant J 37(4):626–634

    CAS  PubMed  Article  Google Scholar 

  17. Hashizume T, Matsubara S, Endo A (1983) Compactin (ML-236B) as a new growth inhibitor of plant callus. Agric Biol Chem 47:1401–1403

    CAS  Google Scholar 

  18. Hata S, Takagishi H, Kouchi H (1987) Variation in the content and composition of sterols in alfalfa seedlings treated with compactin (ML-236B) and mevalonic acid. Plant Cell Physiol 28:709–714

    CAS  Article  Google Scholar 

  19. Hwang I, Sheen J (2001) Two-component circuitry in Arabidopsis cytokinin signal transduction. Nature 413:383–389

    CAS  PubMed  Article  Google Scholar 

  20. Jameson PE, Song J (2016) Cytokinin: a key driver of seed yield. J Exp Bot 67:593–606

    CAS  PubMed  Article  Google Scholar 

  21. Jin JP, Tian F, Yang DC, Meng YQ, Kong L, Luo JC, Gao G (2017) PlantTFDB 4.0: toward a central hub for transcription factors and regulatory interactions in plants. Nucleic Acids Res 45:D1040–D1045

    CAS  PubMed  Article  Google Scholar 

  22. Kanehisa M, Goto S, Kawashima S, Okuno Y, Hattori M (2004) The KEGG resource for deciphering the genome. Nucleic Acids Res 32:D277–D280

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Kant S, Burch D, Badenhorst P, Palanisamy R, Mason J, Spangenberg G (2015) Regulated expression of a cytokinin biosynthesis gene IPT delays leaf senescence and improves yield under rainfed and irrigated conditions in canola (Brassica napus L.). PLoS ONE 10:e0116349

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  24. Keshishian EA, Rashotte AM (2015) Plant cytokinin signalling. Essays Biochem 58:13–27

    PubMed  Article  Google Scholar 

  25. Kim D, Langmead B, Salzberg SL (2015) HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12:357–360

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. Kiran NS, Polanská L, Fohlerová R, Mazura P, Válková M, Šmeral M, Brzobohatý B (2006) Ectopic over-expression of the maize beta-glucosidase Zm-p60.1 perturbs cytokinin homeostasis in transgenic tobacco. J Exp Bot 57:985–996

    CAS  PubMed  Article  Google Scholar 

  27. Kollmer I, Novak O, Strnad M, Schmulling T, Werner T (2014) Overexpression of the cytosolic cytokinin oxidase/dehydrogenase (CKX7) from Arabidopsis causes specific changes in root growth and xylem differentiation. Plant J 78:359–371

    PubMed  Article  CAS  Google Scholar 

  28. Kopecny D, Pethe C, Sebela M, Houba-Herin N, Madzak C, Majira A, Laloue M (2005) High-level expression and characterization of Zea mays cytokinin oxidase/dehydrogenase in Yarrowia lipolytica. Biochimie 87:1011–1022

    CAS  PubMed  Article  Google Scholar 

  29. Kuderova A, Urbankova I, Valkova M, Malbeck J, Brzobohaty B, Nemethova D, Hejatko J (2008) Effects of conditional IPT-dependent cytokinin overproduction on root architecture of Arabidopsis seedlings. Plant Cell Physiol 49:570–582

    CAS  PubMed  Article  Google Scholar 

  30. Kuppu S, Mishra N, Hu R, Sun L, Zhu X, Shen G, Zhang H (2013) Water-deficit inducible expression of a cytokinin biosynthetic gene IPT improves drought tolerance in cotton. PLoS ONE 8:e64190

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. Kuroha T, Tokunaga H, Kojima M, Ueda N, Ishida T, Nagawa S, Sakakibara H (2009) Functional analyses of LONELY GUY cytokinin-activating enzymes reveal the importance of the direct activation pathway in Arabidopsis. Plant Cell 21:3152–3169

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. Latchman DS (1997) Transcription factors: an overview. NT J Biochem Cell B 29:1305–1312

    CAS  Article  Google Scholar 

  33. Li B, Dewey CN (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinform 12(1):323

    CAS  Article  Google Scholar 

  34. Li YJ, Wang B, Dong RR, Hou BK (2015) AtUGT76C2, an Arabidopsis cytokinin glycosyltransferase is involved in drought stress adaptation. Plant Sci 236:157–167

    CAS  PubMed  Article  Google Scholar 

  35. Liu P, Zhang C, Ma JQ, Zhang LY, Yang B, Tang XY, Li JN (2018) Genome-wide identification and expression profiling of cytokinin oxidase/dehydrogenase (CKX) genes reveal likely roles in pod development and stress responses in oilseed rape (Brassica napus L.). Genes 9(3):168

    PubMed Central  Article  CAS  Google Scholar 

  36. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative pcr and the 2−ΔΔCT method. Methods 25:402–408

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15(12):550

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  38. Mantiri FR, Kurdyukov S, Lohar DP, Sharopova N, Saeed NA, Wang XD, Rose RJ (2008) The transcription factor MtSERF1 of the ERF subfamily identified by transcriptional profiling is required for somatic embryogenesis induced by auxin plus cytokinin in Medicago truncatula. Plant Physiol 146:1622–1636

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. Mao XZ, Cai T, Olyarchuk JG, Wei LP (2005) Automated genome annotation and pathway identification using the KEGG orthology (KO) as a controlled vocabulary. Bioinformatics 21:3787–3793

    CAS  PubMed  Article  Google Scholar 

  40. Mason MG, Mathews DE, Argyros DA, Maxwell BB, Kieber JJ, Alonso JM, Schaller GE (2005) Multiple type-B response regulators mediate cytokinin signal transduction in Arabidopsis. Plant Cell 17(11):3007–3018

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. Miyawaki K, Tarkowski P, Matsumoto-Kitano M, Kato T, Sato S, Tarkowska D, Kakimoto T (2006) Roles of arabidopsis ATP/ADP isopentenyltransferases and tRNA isopentenyltransferases in cytokinin biosynthesis. P Natl Acad Sci USA 103:16598–16603

    CAS  Article  Google Scholar 

  42. Mok DW, Mok MC (2001) Cytokinin metabolism and action. Annu Rev Plant Physiol Plant Mol Biol 52:89–118

    CAS  PubMed  Article  Google Scholar 

  43. Moll KM, Zhou P, Ramaraj T, Fajardo D, Devitt NP, Sadowsky MJ, Silverstein KA (2017) Strategies for optimizing BioNano and dovetail explored through a second reference quality assembly for the legume model Medicago truncatula. BMC Genomics 18(1):578

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  44. Muller B, Sheen J (2008) Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis. Nature 453:1094–1097

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  45. Naito T, Yamashino T, Kiba T, Koizumi N, Kojima M, Sakakibara H, Mizuno T (2007) A link between cytokinin and ASL9 (ASYMMETRIC LEAVES 2 LIKE 9) that belongs to the AS2/LOB (LATERAL ORGAN BOUNDARIES) family genes in Arabidopsis thaliana. Biosci Biotech Bioch 71:1269–1278

    CAS  Article  Google Scholar 

  46. Nguyen KH, HaC V, Nishiyama R, Watanabe Y, Leyva-González MA, Fujita Y, Schaller GE (2016) Arabidopsis type B cytokinin response regulators ARR1, ARR10, and ARR12 negatively regulate plant responses to drought. P Natl Acad Sci USA 113(11):3090–3095

    CAS  Article  Google Scholar 

  47. Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SL (2015) StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol 33:290–295

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. Polanska L, Malbeck J, Travnickova A, Vankova R, Machackova I (2004) Cytokinin occurrence in chloroplasts of tobacco plants harboring zeatin O-glucosyl transferase gene (Zog1). Acta Physiol Plant 26:39–39

    Google Scholar 

  49. Raines T, Blakley IC, Tsai YC, Worthen JM, Franco-Zorrilla JM, Solano R, Kieber JJ (2016) Characterization of the cytokinin-responsive transcriptome in rice. BMC Plant Biol 16:260

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  50. Rashotte AM, Carson SD, To JP, Kieber JJ (2003) Expression profiling of cytokinin action in Arabidopsis. Plant Physiol 132:1998–2011

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. Reid DE, Heckmann AB, Novak O, Kelly S, Stougaard J (2016) CYTOKININ OXIDASE/DEHYDROGENASE3 maintains cytokinin homeostasis during root and nodule development in lotus japonicus. Plant Physiol 170:1060–1074

    CAS  PubMed  Article  Google Scholar 

  52. Reyes-Olalde JI, Zúñiga-Mayo VM, Serwatowska J, Montes RAC, Lozano-Sotomayor P, Herrera-Ubaldo H, Paolo D (2017) The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium. PLoS Genet 13:e1006726

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  53. Roche J, Love J, Guo Q, Song J, Cao M, Fraser K, Jameson PE (2016) Metabolic changes and associated cytokinin signals in response to nitrate assimilation in roots and shoots of Lolium perenne. Physiol Plant 156:497–511

    CAS  PubMed  Article  Google Scholar 

  54. Rogers EE, Ausubel FM (1997) Arabidopsis enhanced disease susceptibility mutants exhibit enhanced susceptibility to several bacterial pathogens and alterations in PR-1 gene expression. Plant Cell 9(3):305–316

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Ross EJ, Stone JM, Elowsky CG, Arredondo-Peter R, Klucas RV, Sarath G (2004) Activation of the Oryza sativa non-symbiotic haemoglobin-2 promoter by the cytokinin-regulated transcription factor, ARR1. J Exp Bot 55:1721–1731

    CAS  PubMed  Article  Google Scholar 

  56. Schaller GE, Street IH, Kieber JJ (2014) Cytokinin and the cell cycle. Curr Opin Plant Biol 21:7–15

    CAS  PubMed  Article  Google Scholar 

  57. Schmulling T, Werner T, Riefler M, Krupkova E, Bartrina y Manns I (2003) Structure and function of cytokinin oxidase/dehydrogenase genes of maize, rice, Arabidopsis and other species. J Plant Res 116:241–252

    PubMed  Article  CAS  Google Scholar 

  58. Shi X, Gupta S, Lindquist IE, Cameron CT, Mudge J, Rashotte AM (2013) Transcriptome analysis of cytokinin response in tomato leaves. PLoS ONE 8:e55090

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  59. Tadege M, Ratet P, Mysore KS (2005) Insertional mutagenesis: a Swiss army knife for functional genomics of Medicago truncatula. Trends Plant Sci 10:229–235

    CAS  PubMed  Article  Google Scholar 

  60. Tadege M, Wen J, He J, Tu H, Kwak Y, Eschstruth A, Ratet P (2008) Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula. Plant J 54:335–347

    CAS  PubMed  Article  Google Scholar 

  61. Talla SK, Panigrahy M, Kappara S, Nirosha P, Neelamraju S, Ramanan R (2016) Cytokinin delays dark-induced senescence in rice by maintaining the chlorophyll cycle and photosynthetic complexes. J Exp Bot 67:1839–1851

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  62. Tang H, Krishnakumar V, Bidwell S, Rosen B, Chan A, Zhou S, Mayer KF (2014) An improved genome release (version Mt4.0) for the model legume Medicago truncatula. BMC Genomics 15(1):312

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  63. Tatusov RL, Galperin MY, Natale DA, Koonin EV (2000) The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res 28:33–36

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  64. Thatcher LF, Williams AH, Garg G, Buck SG, Singh KB (2016) Transcriptome analysis of the fungal pathogen Fusarium oxysporum f. sp. medicaginis during colonisation of resistant and susceptible Medicago truncatula hosts identifies differential pathogenicity profiles and novel candidate effectors. BMC Genomics 17:860

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  65. Tokunaga H, Kojima M, Kuroha T, Ishida T, Sugimoto K, Kiba T, Sakakibara H (2012) Arabidopsis lonely guy (LOG) multiple mutants reveal a central role of the LOG-dependent pathway in cytokinin activation. Plant J 69:355–365

    CAS  PubMed  Article  Google Scholar 

  66. Vidal RO, do Nascimento LC, Mondego JM, Pereira GA, Carazzolle MF (2012) Identification of SNPs in RNA-seq data of two cultivars of glycine max (soybean) differing in drought resistance. Genet Mol Biol 35:331–334

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  67. Xia Z, Zeng X, Ma G (2009) Regulation of CPPU on dynamic changes of endogenous phytohormone in Momordica charantia L. Southwest China J Agricu Sci 22:5

    Google Scholar 

  68. Zalewski W, Galuszka P, Gasparis S, Orczyk W, Nadolska-Orczyk A (2010) Silencing of the HvCKX1 gene decreases the cytokinin oxidase/dehydrogenase level in barley and leads to higher plant productivity. J Exp Bot 61:1839–1851

    CAS  PubMed  Article  Google Scholar 

  69. Zhang S, Shi Y, Cheng N, Du H, Fan W, Wang C (2015) De novo characterization of fall dormant and nondormant alfalfa (Medicago sativa L.) leaf transcriptome and identification of candidate genes related to fall dormancy. PLoS ONE 10:0122170

    Google Scholar 

  70. Zhao J, Ba W, Zeng Q, Song S, Zhang M, Li X, Luo X (2015) Moderately enhancing cytokinin level by down-regulation of expression in cotton concurrently increases fiber and seed yield. Mol Beeding 35:60

    Article  CAS  Google Scholar 

  71. Zurcher E, Muller B (2016) Cytokinin synthesis, signaling, and function-advances and new insights. Int Rev Cel Mol Bio 324:1–38

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The work is supported by the Fundamental Research Funds for the Central Universities (2019ZY11) and Tibet Science and Technology Major Projects of Pratacultural Industry. The data were analyzed on the free online platform of Majorbio I-Sanger Cloud Platform (www.i-sanger.com).

Author information

Affiliations

Authors

Contributions

YHC and LBH planned and designed the experiments, and wrote the main manuscript text; ZXZ and HCL performed majorly the experiments and data acquisition; CNM participated partially the experiments and data analysis; YHC and LBH participated the figures preparation and MS English editing.

Corresponding author

Correspondence to Yuehui Chao.

Ethics declarations

Conflict of Interest

All the authors declare that they do not have conflict of interest.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhou, Z., Liu, H., Ma, C. et al. Transcriptome Analysis of the Cytokinin Response in Medicago truncatula. J. Plant Biol. 63, 189–202 (2020). https://doi.org/10.1007/s12374-020-09244-8

Download citation

Keywords

  • Medicago truncatula
  • Cytokinin regulation
  • Differentially expressed transcript
  • Transcriptome
  • 6-Benzylaminopurine
  • Lovastatin