Identification of somatic embryogenesis (SE) related proteins through label-free shotgun proteomic method and cellular role in Catharanthus roseus (L.) G. Don

  • Basit Gulzar
  • Abdul MujibEmail author
  • Manchikatla V. Rajam
  • Arajmand Frukh
  • Nadia Zafar
Original Article


Somatic embryogenesis (SE) is an intricate in vitro multi-step biotechnological tool used to develop embryos/plants from a single or a group of somatic cells. It is a model technique for understanding various plant developmental pathways. A lot of research is going on to elucidate the mechanism underlying the process of SE. This study was aimed at the identification of SE related proteins in a medicinally important plant, Catharanthus roseus via label free liquid chromatography–mass spectroscopy (LC–MS). LC–MS is a sensitive and reliable technique than the gel based techniques, using LC–MSMS in tandem for separation and identification of proteins. Here, we are reporting for the first time SE related proteins in C. roseus by using gel free shotgun proteomic approach. The non embryogenic and embryogenic calli of C. roseus were used for comparative proteome analysis. A total of 3573 proteins were identified in both embryogenic and non embryogenic calli of which 1511 proteins were found to be common in both the calli. In non embryogenic callus 982 proteins while in embryogenic callus 1079 proteins were exclusively identified, which were associated with varied cellular functions. The most of these proteins function in different metabolic processes and stress responses. More than 72 stress responsive proteins and isoforms were observed exclusively in embryogenic callus including glutathione S transferase, ascorbate peroxidase, catalase, superoxide dismutase, alkylhydro peroxidase, SOD Fe N domain containing protein, pyridine nucleotide disulphide oxidoreductase, thioredoxin reductase. The role of plant growth regulators (PGRs) in inducing stress cause switching on/off of several genes has been discussed, led biochemical and molecular alterations in acquiring somatic embryogenic competence.

Key message

Proteomic map of Catharanthus roseus was prepared. A total of 3573 proteins were identified, of which 1079 were embryogenic. These proteins have role in metabolic and stress responses.


Embryogenic-non embryogenic callus Gel-free proteomics Somatic embryogenesis Catharanthus roseus 



The first author is thankful to University Grant Commission (UGC) for providing financial assistance with (Grant No. 2061530497). The authors are also thankful to the Department of Botany, Jamia Hamdard, and University of Delhi for providing laboratory and other research facilities for this research.

Author contributions

BG, AF, NZ conducted most of the experiments. BG also made the draft of article. AM and MVR edited the article.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest in this research.

Supplementary material

11240_2019_1563_MOESM1_ESM.xlsx (103 kb)
Supplementary Table 1. Proteins/isoforms (accession numbers) exclusively found in F1 i.e. embryogenic tissue of Catharanthus roseus—Supplementary material 1 (XLSX 102 KB)
11240_2019_1563_MOESM2_ESM.xlsx (45 kb)
Supplementary Table 2. Accession numbers of the proteins found in embrogenic callus (F1) having high abundance (twice or more) than that of non embryogenic callus (F2). The ratio (F1/F2) protein abundances are listed in decreasing order—Supplementary material 2 (XLSX 45 KB)


  1. Campos NA, Panis B, Carpentier SC (2017) Somatic embryogenesis in coffee: the evolution of biotechnology and the integration of omics technologies offer great opportunities. Front Plant Sci 8:1460. CrossRefPubMedCentralGoogle Scholar
  2. Chen SL, Yu H, Luo HM, Wu Q, Li CF, Steinmetz A (2016) Conservation and sustainable use of medicinal plants: problems, progress, and prospects. Chin Med 11:37. CrossRefPubMedCentralGoogle Scholar
  3. Eng JK, McCormack AL, Yates JR (1994) An approach to correlate MS/MS data to amino acid sequences in a protein database. J Am Soc Mass Spectrum 5:976–989CrossRefGoogle Scholar
  4. Feher A (2015) Somatic embryogenesis—stress-induced remodeling of plant cell fate. Biochem Biophys Acta 1849:385–402Google Scholar
  5. Fehér A, Pasternak TP, Dudits D (2003) Transition of somatic plant cells to an embryogenic state. Plant Cell Tissue Org Cult 74:201–228CrossRefGoogle Scholar
  6. Fraga HP, Vieria LN, Heringer AS, Puttkammer CC, Silveira V, Guerra MP (2016) DNA methylation and proteome profiles of Araucaria angustfolia (Bertol) Kuntzeembryogenic cultures as affected by plant growth regulators supplementation. Plant Cell Tissue Org Cult 125(2):353–374CrossRefGoogle Scholar
  7. Ge F, Hu H, Huang X, Zhang Y, Wang Y, Li Z, Zou C, Peng H, Li L, Gao S, Pan G, Shen Y (2017) Metabolomic and proteomic analysis of maize embryonic callus induced from immature embryo. Sci Rep 7(1):1004. CrossRefPubMedCentralGoogle Scholar
  8. Guerra DD, Callis J (2012) Ubiquitin on the move: the ubiquitin modification system plays diverse roles in the regulation of endoplasmic reticulum- and plasma membrane-localized proteins. Plant Physiol 160(1):56–64. CrossRefPubMedCentralGoogle Scholar
  9. Helleboid S, Hendriks T, Bauw G, Inze D, Vasseur J, Hilbert JL (2000) Three major somatic embryogenesis related proteins in Cichorium identified as PR proteins. J Exp Bot 51:1189–1200CrossRefGoogle Scholar
  10. Heringer AS, Barroso T, Macedo AF, Santa-Catarina C, Souza GHMF, Floh EIS, Souza-Filho GA, Silveira V (2015) Label-free quantitative proteomics of embryogenic and non-embryogenic callus during sugarcane somatic embryogenesis. PLoS ONE. Google Scholar
  11. Heringer AS, Reis RS, Passaman LZ, de Souza-Filho GA, Santa-Catarina C, Silveira V (2017) Comparative proteomics analysis of the effect of combined red and blue lights on sugarcane somatic embryogenesis. Acta Physiol Plant 39:52. CrossRefGoogle Scholar
  12. Heringer AS, Santa-Catarina C, Silveira V (2018) Insights from proteomic studies into plant somatic embryogenesis. Proteomics. Google Scholar
  13. Hincha DK, Thalhammer A (2012) LEA proteins: IDPs with versatile functions in cellular dehydration tolerance. Biochem Soc T 40:1000–1003CrossRefGoogle Scholar
  14. Hoenemann C, Ambold J, Hohe A (2012) Gene expression of a putative glutathione S-transferase is responsive to abiotic stress in embryogenic cell cultures of Cyclamen persicum.. Electronic J Biotechnol 15:1–6Google Scholar
  15. Horstman A, Li M, Heidmann I, Weemen M, Chen B, Muino JM, Angenent GC, Boutilier K (2017) The Baby Boom transcription factor activates the LEC1-ABI3-FUS3-LEC2 network to induce somatic embryogenesis. Plant Physiol 175:848–857PubMedCentralGoogle Scholar
  16. Ikeda M, Umehara M, Kamada H (2006) Embryogenesis-related genes; its expression and roles during somatic and zygotic embryogenesis in carrot and Arabidopsis. Plant Biotechnol 23:153–161CrossRefGoogle Scholar
  17. Imin N, Nizamidin M, Daniher D, Nolan KE, Rose RJ, Rolfe BG (2005) Proteomic analysis of somatic embryogenesis in Medicago truncatula. Explant cultures grown under 6-benzylaminopurine and 1-naphthaleneacetic acid treatments. Plant Physiol 137:1250–1260. CrossRefPubMedCentralGoogle Scholar
  18. Isaacson T, Damasceno CM, Saravanan RS, He Y, Catala C, Saladie M, Rose JK (2006) Sample extraction techniques for enhanced proteomic analysis of plant tissues. Nat Protoc 1(2):769–774CrossRefGoogle Scholar
  19. Jiménez VM (2005) Involvement of plant hormones and plant growth regulators on in vitro somatic embryogenesis. Plant Growth Regul 47:91–110CrossRefGoogle Scholar
  20. Jing D, Zhang J, Xia Y, Kong L, OuYang F, Zhang S, Zhang H, Wang J (2016) Proteomic analysis of stress-related proteins and metabolic pathways in Picea asperata somatic embryos during partial desiccation. Plant Biotechnol J. PubMedCentralGoogle Scholar
  21. Junaid A, Mujib A, Fatima S, Sharma MP (2008) Cultural conditions affect somatic embryogenesis in Catharanthus roseus L. (G.) Don. Plant Biotechnol Rep 2:179–189CrossRefGoogle Scholar
  22. Karpievitch YV, Ashoka DP, Gordon AA, Richard DS, Alan RD (2010) Liquid chromatography mass spectrometry-based proteomics: biological and technological aspects. Ann Appl Stat 4(4):1797–1823. CrossRefPubMedCentralGoogle Scholar
  23. Lu D, Wei W, Zhou W, Linda D, Xiao J, Yu L (2017) Establishment of a somatic embryo regeneration system and expression analysis of somatic embryogenesis-related genes in Chinese chestnut (Castanea mollissima Blume). Plant Cell Tissue Org Cult 130(3):601–616CrossRefGoogle Scholar
  24. Mahdavi-Darvari F, Noor NM, Ismanizan I (2014) Epigenetic regulation and gene markers as signals of early somatic embryogenesis. Plant Cell Tiss Org Cult 120(2):407–422CrossRefGoogle Scholar
  25. Mordhorst AP, Toonen MAJ, deVries SC (1997) Plant embryogenesis. Crit Rev Plant Sci 16(6):535–576CrossRefGoogle Scholar
  26. Mujib A, Ali M, Isah T, Dipti T (2014) Somatic embryo mediated mass production of Catharanthus roseus in culture vessel (bioreactor)—a comparative study. Saudi J Biol Sci 21(5):442–449CrossRefPubMedCentralGoogle Scholar
  27. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497CrossRefGoogle Scholar
  28. Nolan KE, Irwanto RR, Rose RJ (2003) Auxin up-regulates MtSERK1 expression in both Medicago truncatula root-forming and embryogenic cultures. Plant Physiol 133:218–230. CrossRefPubMedCentralGoogle Scholar
  29. Oh CS (2010) Characteristics of 14-3-3 proteins and their role in plant immunity. Plant Pathol J 26(1):1–7. CrossRefGoogle Scholar
  30. Orłowska A, Igielski R, Łagowska K, Pczyska EK (2017) Identification of LEC1, L1L and polycomb repressive Complex2 genes and their expression during the induction phase of Medicago truncatula Gaertn. somatic embryogenesis. Plant Cell Tissue Organ Cult 129:119–132. CrossRefGoogle Scholar
  31. Park CJ, Seo YS (2015) A review of the molecular chaperones for plant immunity. Plant Pathol J 31(4):323–333. CrossRefPubMedCentralGoogle Scholar
  32. Pulianmackal AJ, Kareem AV, Durgaprasad K, Trivedi ZB, Prasad K (2014) Competence and regulatory interactions during regeneration in plants. Front Plant Sci 5:142CrossRefPubMedCentralGoogle Scholar
  33. Rupps A, Raschke J, Rümmler M, Linke B, Zoglauer K (2016) Identification of putative homologs of Larix decidua to Babyboom (BBM), leafy cotyledon1 (LEC1), Wuschel-related Homeobox2 (WOX2) and somatic embryogenesis receptor-like kinase (SERK) during somatic embryogenesis. Planta 243:473–488CrossRefGoogle Scholar
  34. Schmidt ED, Guzzo F, Toonen MA, de Vries SC (1997) A leucine-rich repeat containing receptor-like kinase marks somatic plant cells competent to form embryos. Development 124:2049–2062Google Scholar
  35. Tolleter D, Hincha DK, Macherel D (2010) A mitochondrial late embryogenesis abundant protein stabilizes model membranes in the dry state. BBA Gen Sub 1798:1926–1933Google Scholar
  36. Verdeil JL, Alemanno L, Niemenak N, Tranbarger TJ (2007) Pluripotent versus totipotent plant stem cells: dependence versus autonomy? Trends Plant Sci 12:245–252CrossRefGoogle Scholar
  37. Washburn MP, Wolters D, Yates JR (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19(3):242–247. CrossRefGoogle Scholar
  38. Zhao J, Li H, Fu S, Chen B, Sun W, Zhang J (2015) An iTRAQ-based proteomics approach to clarify the molecular physiology of somatic embryo development in Prince Rupprecht’s larch (Larix principis-rupprechtii Mayr). PLoS ONE. Google Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Basit Gulzar
    • 1
  • Abdul Mujib
    • 1
    Email author
  • Manchikatla V. Rajam
    • 2
  • Arajmand Frukh
    • 1
  • Nadia Zafar
    • 1
  1. 1.Cellular Differentiation and Molecular Genetics Section, Department of BotanyJamia HamdardNew DelhiIndia
  2. 2.University of DelhiNew DelhiIndia

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