Maize seedlings produced from dry seeds exposed to liquid nitrogen display altered levels of shikimate pathway compounds

  • Rosmery Pereira
  • Melissa Arguedas
  • Julia Martínez
  • Lázaro Hernández
  • Byron Enrique Zevallos
  • Marcos Edel Martínez-Montero
  • Lourdes Yabor
  • Sershen
  • José Carlos LorenzoEmail author


In light of climate change and risks of food insecurity, it is becoming increasingly important to preserve plant germplasm in genebanks. Storage of seeds, particularly via cryopreservation, is one of the most proficient methods for ex situ plant germplasm conservation. Whilst seed cryo-banking can have little, to no, or even beneficial effects on subsequent seedling vigor in some species, it can lead to a number of plant abnormalities (morphological and physiological). This study investigated the effects of maize seed cryopreservation on seedling growth (until 14 d) and levels of selected amino acids produced in the shikimate pathway, a major link between primary and secondary metabolism. Seed cryopreservation reduced FW in recovered seedlings, reduced caffeic acid (2.5-fold decrease), and increased levels of all other shikimate pathway–related compounds assessed: phenylalanine (2.9-fold increase), tyrosine (2.6-fold increase), and shikimic (2.1-fold increase) and protocathecuic (3.1-fold increase) acids in cotyledons. Our results suggest that maize seed cryopreservation results in seedlings that exhibit signs of an ‘overly’ efficient and caffeic acid–deficient shikimate pathway, possibly related to their reduced growth during a highly vulnerable growth stage. However, these metabolic abnormalities manifested most severely in the maternal (cotyledonary), as opposed to vegetative (roots, stems, and leaves), tissues and hence are likely to disappear when the seedlings shed the cotyledons and become completely autotrophic.


Biochemical Cryopreservation Liquid nitrogen Shikimic acid pathway Phenolics Zea mays



This research was supported by the Institute of Botany (Leibniz University of Hannover, Germany), the Escuela Superior Politécnica Agropecuaria de Manabí Manuel Félix López (Ecuador), the University of KwaZulu-Natal (South Africa), and the Bioplant Centre (University of Ciego de Ávila, Cuba). It was also partially supported by the German Academic Exchange Service (DAAD) through a grant to Dr. José Carlos Lorenzo Feijoo. The authors are grateful to Prof. Dr. Jutta Papenbrock, Dr. Yvana Glasenapp, and Dr. Ariel Turcios for their excellent scientific suggestions; and to Mr. José Laguna for his skilled technical assistance.

Author contribution

RP, MA, JM, LH, BEZ, MEMM, LY, S, and JCL designed the research; RP, MA, JM, LH, and JCL conducted the experiments; BEZ, MEMM, LY, S, and JCL wrote the paper; S and JCL had primary responsibility for the final content. All authors have read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Human and animal rights

This research did not involve experiments with human or animal participants.

Informed consent

Informed consent was obtained from all individual participants included in the study. Additional informed consent was obtained from all individual participants for whom identifying information is included in this article.


  1. Acosta Y, Hernández L, Mazorra C, Quintana N, Zevallos BE, Cejas I, Sershen LJC, Martínez-Montero ME, Fontes D (2019) Seed cryostorage enhances subsequent plant productivity in the forage species Teramnus labialis (L.F.) Spreng. CryoLetters 40:36–44Google Scholar
  2. Aharoni A, Galili G (2011) Metabolic engineering of the plant primary secondary metabolism interface. Curr Opin Biotechnol 22:239–244CrossRefGoogle Scholar
  3. Arguedas M, Perez A, Abdelnour A, Hernandez M, Engelmann F, Martínez ME, Yabor L, Lorenzo JC (2016) Short-term liquid nitrogen storage of maize, common bean and soybean seeds modifies their biochemical composition. Agric Sci 4:6–12Google Scholar
  4. Arguedas M, Gómez D, Hernández L, Engelmann F, Garramone R, Cejas I, Yabor L, Martínez-Montero ME, Lorenzo JC (2018a) Maize seed cryo-storage modifies chlorophyll, carotenoid, protein, aldehyde and phenolics levels during early stages of germination. Acta Physiol Plant 40:118CrossRefGoogle Scholar
  5. Arguedas M, Villalobos A, Gómez D, Hernández L, Zevallos B, Cejas I, Yabor L, Martínez-Montero ME, Lorenzo JC (2018b) Field performance of cryopreserved seed-derived maize plants. CryoLetters 39:366–370Google Scholar
  6. Berjak P, Pammenter NW (2014) Cryostorage of germplasm of tropical recalcitrant-seeded species: approaches and problems. Int J Plant Sci 175:29–39CrossRefGoogle Scholar
  7. Bourgaud F, Gravot A, Milesi S, Gontier E (2001) Production of plant secondary metabolites: a historical perspective. Plant Sci 161:839–851CrossRefGoogle Scholar
  8. Cejas I, Vives K, Laudat T, González-Olmedo J, Engelmann F, Martínez-Montero ME, Lorenzo JC (2012) Effects of cryopreservation of Phaseolus vulgaris L. seeds on early stages of germination. Plant Cell Rep 31:2065–2073CrossRefGoogle Scholar
  9. Cejas I, Méndez R, Villalobos A, Palau F, Aragón C, Engelmann F, Carputo D, Aversano R, Martínez ME, Lorenzo JC (2013) Phenotypic and molecular characterization of Phaseolus vulgaris plants from non-cryopreserved and cryopreserved seeds. Am J Plant Sci 4:844–849CrossRefGoogle Scholar
  10. Cejas I, Rivas M, Nápoles L, Marrero P, Yabor L, Aragón C, Pérez A, Engelmann F, Martínez-Montero ME, Lorenzo JC (2015) Impact of liquid nitrogen exposure on selected biochemical and structural parameters of hydrated Phaseolus vulgaris L. seeds. CryoLetters 36:149–157Google Scholar
  11. Cejas I, Rumlow A, Turcios A, Engelmann F, Martínez ME, Yabor L, Papenbrock J, Lorenzo JC (2016) Exposure of common bean seeds to liquid nitrogen modifies mineral composition of young plantlet leaves. Am J Plant Sci 7:1612–1617CrossRefGoogle Scholar
  12. CIMMYT (2016) Maize genetic resources. Available via: CIMMYT,. Accessed 20/10 2017
  13. Coelho SVB, Rosa SDVF, Fernandes JS (2017) Cryopreservation of coffee seeds: a simplified method. Seed Sci Technol 45:638–649Google Scholar
  14. de Oliveira MV, Jin X, Chen X, Griffith D, Batchu S, Maeda HA (2019) Imbalance of tyrosine by modulating TyrA arogenate dehydrogenases impacts growth and development of Arabidopsis thaliana. Plant J 97:901–922CrossRefGoogle Scholar
  15. Dixon RA, Strack D (2003) Phytochemistry meets genome analysis, and beyond. Phytochemistry 62:815–816CrossRefGoogle Scholar
  16. Gonzalez-Arnao MT, Martinez-Montero ME, Cruz-Cruz CA, Engelmann F (2014) Advances in cryogenic techniques for the long-term preservation of plant biodiversity 4. Springer, Cham
  17. Harding K, Marzalina M, Krishnapillay B, Nashatul Z, Normah M, Benson E (2000) Molecular stability assessments of trees regenerated from cryopreserved Mahogany (Swietenia macrophylla King.) seed germplasm using non-radioactive techniques to examine the chromatin structure and DNA methylation status of the ribosomal RNA genes. J Trop For Sci 12:149–163Google Scholar
  18. Hatfield JL, Prueger JH (2015) Temperature extremes: effect on plant growth and development. Weath Clim Ext 10:4–10CrossRefGoogle Scholar
  19. Humphreys JM, Chapple C (2002) Rewriting the lignin roadmap. Curr Opin Plant Biol 5:224–229CrossRefGoogle Scholar
  20. ISTA (2005) International rules for seed testing international seed testing association, Bassersdorf, Switzerland, pp 35Google Scholar
  21. Kantardzic M (2003) Data mining: concepts, models, methods and algorithms. Wiley, New JerseyGoogle Scholar
  22. Kulus D, Abratowska A (2017) (CRYO) Conservation of Ajania pacifica (Nakai) Bremer et Humphries shoot tips via encapsulation-dehydration technique. Cryo Letters 38(5):387–398Google Scholar
  23. Lakhanpaul S, Babrekar P, Chandel K (1996) Monitoring studies in onion (Allium cepa L.) seeds retrieved from storage at −20C and −180C. Cryo Letters 17:219–232Google Scholar
  24. Lewis NG (2017) Plant phenolics. Antioxidants in higher plants. CRC Press, Boca Raton, pp 135–169CrossRefGoogle Scholar
  25. Lorenzo JC, Yabor L, Medina N, Quintana N, Wells V (2015) Coefficient of variation can identify the most important effects of experimental treatments. Not Bot Horti Agrobo Cluj-Nap 43:287–291. Google Scholar
  26. Macheroux P, Schmid J, Amrhein N, Schaller A (1999) A unique reaction in a common pathway: mechanism and function of chorismate synthase in the shikimate pathway. Planta 207:325–334CrossRefGoogle Scholar
  27. Mikuła A, Makowski D, Walters C, Rybczyński JJ (2010) Exploration of cryo-methods to preserve tree and herbaceous fern gametophytes. In: Kumar A, Fernández H, Revilla MA (eds) Working with ferns: issues and applications. Springer, New York, pp 173–192Google Scholar
  28. Mira S, Estrelles E, González-Benito ME, Bekker R (2015) Effect of water content and temperature on seed longevity of seven Brassicaceae species after 5 years of storage. Plant Biol 17:153–162CrossRefGoogle Scholar
  29. Naik S, Shaanker RU, Ravikanth G, Dayanandan S (2019) How and why do endophytes produce plant secondary metabolites? Symbiosis:1–9Google Scholar
  30. Nguyen HT, Leipner J, Stamp P, Guerra-Peraza O (2009) Low temperature stress in maize (Zea mays L.) induces genes involved in photosynthesis and signal transduction as studied by suppression subtractive hybridization. Plant Physiol Biochem 47:116–122CrossRefGoogle Scholar
  31. Palmer-Young EC, Farrell IW, Adler LS, Milano NJ, Egan PA, Irwin RE, Stevenson PC (2019) Secondary metabolites from nectar and pollen: a resource for ecological and evolutionary studies. Ecology 100:e02621CrossRefGoogle Scholar
  32. Pérez-Rodríguez JL, Escriba RCR, González GYL, Olmedo JLG, Martínez-Montero ME (2017) Effect of desiccation on physiological and biochemical indicators associated with the germination and vigor of cryopreserved seeds of Nicotiana tabacum L. cv. Sancti Spíritus 96. In Vitro Cell Dev Biol Plant:1–9Google Scholar
  33. Prasad NR, Karthikeyan A, Karthikeyan S, Reddy BV (2011) Inhibitory effect of caffeic acid on cancer cell proliferation by oxidative mechanism in human HT-1080 fibrosarcoma cell line. Mol Cell Biochem 349:11–19CrossRefGoogle Scholar
  34. Qian Y, Lynch JH, Guo L, Rhodes D, Morgan JA, Dudareva N (2019) Completion of the cytosolic post-chorismate phenylalanine biosynthetic pathway in plants. Nat Commun 10:15CrossRefGoogle Scholar
  35. Salisbury FB, Ross CW (1992) In: Spanish I (ed) Plant physiology. Wadsworth Publishing, California, p 759Google Scholar
  36. Song W, Liu L, Wang J, Wu Z, Zhang H, Tang J, Lin G, Wang Y, Wen X, Li W (2016) Signature motif-guided identification of receptors for peptide hormones essential for root meristem growth. Cell Res 26:674–685CrossRefGoogle Scholar
  37. Stanwood P, Bass L (1981) Seed germplasm preservation using liquid nitrogen. Seed Sci Technol 9:423Google Scholar
  38. Tzin V, Galili G (2010) Amino acids biosynthesis pathways in plants. Mol Plant 3:956–972CrossRefGoogle Scholar
  39. Uragami A, Lucas M, Ralambosoa J, Renard M, Dereuddre J (1993) Cryopreservation of microspore embryos of soilseed rape (Brassica napus) by dehydration in air with or without alginate encapsulation. Cryo Letters 14:83–90Google Scholar
  40. Villalobos-Olivera A, Martínez J, Quintana N, Zevallos BE, Cejas I, Lorenzo JC, González-Olmedo J, Montero MEM (2019) Field performance of micropropagated and cryopreserved shoot tips-derived pineapple plants grown in the field for 14 months. Acta Physiol Plant 41:34CrossRefGoogle Scholar
  41. Wang JP, Liu B, Sun Y, Chiang VL, Sederoff RR (2018) Enzyme-enzyme interactions in monolignol biosynthesis. Front Plant Sci 9:1942CrossRefGoogle Scholar
  42. Yang D, Huang Z, Jin W, Xia P, Jia Q, Yang Z (2018) DNA methylation: a new regulator of phenolic acids biosynthesis in Salvia miltiorrhiza. Ind Crop Prod 124:402–411CrossRefGoogle Scholar
  43. Young VR, Pellett PL (1994) Plant proteins in relation to human protein and amino acid nutrition. Am J Clin Nutr 59:1203S–1212SCrossRefGoogle Scholar
  44. Zevallos B, Cejas I, Rodríguez RC, Yabor L, Aragón C, González J, Engelmann F, Martínez ME, Lorenzo JC (2013a) Biochemical characterization of Ecuadorian wild Solanum lycopersicum Mill. plants produced from non-cryopreserved and cryopreserved seeds. Cryo Letters 34:413–421Google Scholar
  45. Zevallos B, Cejas I, Valle B, Yabor L, Aragón C, Engelmann F, Martínez ME, Lorenzo JC (2013b) Short-term liquid nitrogen storage of wild tomato (Solanum lycopersicum Mill.) seeds modifies the levels of phenolics in 7 day-old seedlings. Sci Hortic 160:264–267CrossRefGoogle Scholar
  46. Zevallos B, Cejas I, Engelmann F, Carputo D, Aversano R, Scarano M, Yanes-Paz E, Martínez-Montero M, Lorenzo JC (2014) Phenotypic and molecular characterization of plants regenerated from non-cryopreserved and cryopreserved wild Solanum lycopersicum Mill. seeds. Cryo Letters 35:216–225Google Scholar
  47. Zhang L, Li Y, Liang Y, Liang K, Zhang F, Xu T, Wang M, Song H, Liu X, Lu B (2019) Determination of phenolic acid profiles by HPLC-MS in vegetables commonly consumed in China. Food Chem 276:538–546CrossRefGoogle Scholar
  48. Zhou P, Li Q, Liu G, Xu N, Yang Y, Zeng W, Chen A, Wang S (2019) Integrated analysis of transcriptomic and metabolomic data reveals critical metabolic pathways involved in polyphenol biosynthesis in Nicotiana tabacum under chilling stress. Funct Plant Biol 46:30–43CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2019

Authors and Affiliations

  • Rosmery Pereira
    • 1
  • Melissa Arguedas
    • 1
  • Julia Martínez
    • 1
  • Lázaro Hernández
    • 1
  • Byron Enrique Zevallos
    • 2
  • Marcos Edel Martínez-Montero
    • 1
  • Lourdes Yabor
    • 1
  • Sershen
    • 3
  • José Carlos Lorenzo
    • 1
    Email author
  1. 1.Laboratory for Plant Breeding and Conservation of Genetic Resources, Bioplant CenterUniversity of Ciego de AvilaCiego de ÁvilaCuba
  2. 2.Escuela Superior Politécnica Agropecuaria de Manabí Manuel Félix López (ESPAMMFL)Campus Politécnico El LimónCalcetaEcuador
  3. 3.School of Life SciencesUniversity of KwaZulu-NatalDurbanSouth Africa

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