Photoperiod modulates growth, morphoanatomy, and linalool content in Lippia alba L. (Verbenaceae) cultured in vitro

  • Kamila Motta de Castro
  • Diego Silva Batista
  • Evandro Alexandre Fortini
  • Tatiane Dulcineia Silva
  • Sérgio Heitor Sousa Felipe
  • Amanda Mendes Fernandes
  • Raysa Mayara de Jesus Sousa
  • Laís Stehling de Queiroz Nascimento
  • Victória Rabelo Campos
  • Richard Michael Grazul
  • Lyderson Facio Viccini
  • Wagner Campos OtoniEmail author
Original Article


Interactions between circadian clock regulation and metabolic responses are believed to explain the importance of rhythmic behavior in plant growth and survival. Lippia alba is an important species because of the medicinal properties of its essential oil extract. The objective of this work was to evaluate the effect of photoperiod on anatomy, growth, essential oil profile, and the expression of genes related to the synthesis of monoterpenes, sesquiterpenes, and the circadian clock in L. alba grown in vitro. The plants were cultured in vitro under different photoperiods (4, 8, 16, and 24 h of light) and irradiance of 41 μmol m−2 s−1. After 40 days of culture, results showed that L. alba presented high physiological plasticity under different photoperiods, with improved performance when exposed to continuous light. The best growth; anatomical organization of the mesophyll, stem, roots, and bundles; amount of photosynthetic pigments; photosynthetic rate; and protein synthesis occurred under a photoperiod of 24 h. The biosynthesis of linalool, the major compound, was increased under the 24-h photoperiod, possibly due to reduced geraniol synthesis. These findings allow a better understanding of how photoperiod acts in the regulation of primary and secondary metabolism, and especially with regard to the composition of essential oils.

Key message

Photoperiod modulates primary metabolism, growth, morphoanatomy, photosynthesis, and essential oil content in the medicinal plant Lippia alba cultured in vitro under 4, 8, 16 or 24 h of light.


Circadian rhythm Geraniol synthase Internal clock Medicinal plant Photosynthesis 



The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, DF, Brazil; grants 432412/2016-6 and 313740/2017-8 to LFV), and Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior, CAPES, DF, Brazil; Finance Code 001), for financial support. Dr. Roberto Fontes Vieira (Embrapa Recursos Genéticos e Biotecnologia—Embrapa/Cenargen, Brasília, DF, Brazil) is also acknowledged for providing the Lippia alba accession. We would like to thank Editage ( for English language editing.

Author contributions

KMC, DSB and WCO conceived and designed the experiments; KMC, TDS, EAF and SHSF performed the experiments, collected and analyzed the data; KMC, TDS, EAF, SHSF and RMJS performed the anatomical, physiological and biochemical analyses; LSQN, VRC and RMG carried out the microextraction and qualitative analysis of essential oils; DSB performed the gene expression analysis by RT-qPCR; KMC and DSB performed the statistical analysis; KMC, DSB, AMF, RMG, LFV and WCO contributed to the interpretation of the research and to the writing of the paper. All authors read and approved the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.


  1. Adams RP (1997) Identification of essential oil components by gas chromatography/mass spectroscopy. J Am Soc Mass Spectrom 6:671–672Google Scholar
  2. Alabadí D, Oyama T, Yanovsky MJ, Harmon FG, Más P, Kay SA (2001) Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock. Science 293:880–883. CrossRefGoogle Scholar
  3. Amin B, Hosseinzadeh H (2016) Black cumin (Nigella sativa) and its active constituent, thymoquinone: an overview on the analgesic and anti-inflammatory effects. Planta Med 82:8–16. Google Scholar
  4. Baerenfaller K, Massonnet C, Hennig L, Russenberger D, Sulpice R, Walsh S, Stitt M, Granier C, Gruissem W (2015) A long photoperiod relaxes energy management in Arabidopsis leaf six. Curr Plant Biol 2:34–45. CrossRefGoogle Scholar
  5. Barbosa QP, da Câmara CA, Ramos CS, Nascimento DC, Lima-Filho JV, Guimarães EF (2012) Chemical composition, circadian rhythm and antibacterial activity of essential oils of Piper divaricatum: a new source of safrole. Quím Nova 35:1806–1808. CrossRefGoogle Scholar
  6. Barros PM, Cherian S, Costa M, Sapeta H, Saibo NJM, Oliveira MM (2017) The identification of almond GIGANTEA gene and its expression under cold stress, variable photoperiod, and seasonal dormancy. Biol Plant 61:631–640. CrossRefGoogle Scholar
  7. Batista DS, Castro KM, Silva AR, Teixeira ML, Sales TA, Soares LI, Cardoso MG, Santos MO, Viccini LF, Otoni WC (2016) Light quality affects in vitro growth and essential oil profile in Lippia alba (Verbenaceae). In Vitro Cell Dev Biol-Plant 52:276–282. CrossRefGoogle Scholar
  8. Batista DS, Castro KM, Koehler AD, Porto BN, Silva AR, Souza VC, Teixeira ML, Cardoso MG, Santos MO, Viccini LF, Otoni WC (2017a) Elevated CO2 improves growth, modifies anatomy, and modulates essential oil qualitative production and gene expression in Lippia alba (Verbenaceae). Plant Cell Tiss Organ Cult 128:357–368. CrossRefGoogle Scholar
  9. Batista DS, Dias LLC, Rêgo MMD, Saldanha CW, Otoni WC (2017b) Flask sealing on in vitro seed germination and morphogenesis of two types of ornamental pepper explants. Ciên Rural 47:1–6. Google Scholar
  10. Benelli G, Pavela R, Giordani C, Casettari L, Curzi G, Cappellacci L, Petrelli R, Maggi F (2018) Acute and sub-lethal toxicity of eight essential oils of commercial interest against the filariasis mosquito Culex quinquefasciatus and the housefly Musca domestica. Ind Crops Prod 112:668–680. CrossRefGoogle Scholar
  11. Bilger W, Bjorkman O (1990) Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of Hedera canariensis. Photosynth Res 25:173–185. CrossRefGoogle Scholar
  12. Böhme K, Barros-Velázquez J, Calo-Mata P, Aubourg SP (2014) Antibacterial, antiviral and antifungal activity of essential oils: mechanisms and applications. Antimicrobial compounds. In: Villa T, Veiga-Crespo P (eds) Antimicrobial compounds. Springer, Berlin, pp 51–81CrossRefGoogle Scholar
  13. Bordage S, Sullivan S, Laird J, Millar AJ, Nimmo HG (2016) Organ specificity in the plant circadian system is explained by different light inputs to the shoot and root clocks. New Phytol 212:136–149. CrossRefGoogle Scholar
  14. Castro EM, Pinto JEBP, Melo HCD, Soares AM, Alvarenga AA, Lima Júnior EC (2005) Aspectos anatômicos e fisiológicos de plantas de guaco submetidas a diferentes fotoperíodos. Hortic Bras 23:846–850. CrossRefGoogle Scholar
  15. Carvalho IS, Cavaco T, Carvalho LM, Duque P (2010) Effect of photoperiod on flavonoid pathway activity in sweet potato (Ipomoea batatas (L.) Lam.) leaves. Food Chem 118:384–390. CrossRefGoogle Scholar
  16. Chen JW, Bai KD, Cao KF (2009) Inhibition of monoterpene biosynthesis accelerates oxidative stress and leads to enhancement of antioxidant defenses in leaves of rubber tree (Hevea brasiliensis). Acta Physiol Plant 31:95. CrossRefGoogle Scholar
  17. Costa AC, Rosa M, Megguer CA, Silva FG, Pereira FG, Otoni WC (2014) A reliable methodology for assessing the in vitro photosynthetic competence of two Brazilian savanna species: Hyptis marrubioides and Hancornia speciosa. Plant Cell Tiss Organ Cult 117(3):443–454. CrossRefGoogle Scholar
  18. Cross JM, Von Korff M, Altmann T, Bartzetko L, Sulpice R, Gibon Y, Palacios N, Stitt M (2006) Variation of enzyme activities and metabolite levels in 24 Arabidopsis accessions growing in carbon-limited conditions. Plant Physiol 142:1574–1588. CrossRefGoogle Scholar
  19. Cruz CD (2016) Genes software-extended and integrated with the R, Matlab and Selegen. Acta Sci Agron 38:547–552. CrossRefGoogle Scholar
  20. Dodd AN, Salathia N, Hall A, Kévei E, Tóth R, Nagy F, Hibberd JM, Millar AJ, Webb AAR (2005) Plant circadian clocks increase photosynthesis, growth, survival, and competitive advantage. Science 309:630–633. CrossRefGoogle Scholar
  21. Farré EM, Harmer SL, Harmon FG, Yanovsky MJ, Kay SA (2005) Overlapping and distinct roles of PRR7 and PRR9 in the Arabidopsis circadian clock. Curr Biol 15:47–54CrossRefGoogle Scholar
  22. Fernie AR, Roscher A, Ratcliffe RG, Kruger NJ (2001) Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells. Planta 212:250–263. CrossRefGoogle Scholar
  23. Fréchette E, Chang CYY, Ensminger I (2016) Photoperiod and temperature constraints on the relationship between the photochemical reflectance index and the light use efficiency of photosynthesis in Pinus strobus. Tree Physiol 36:311–324. CrossRefGoogle Scholar
  24. Gendron JM, Pruneda-Paz JL, Doherty CJ, Gross AM, Kang SE, Kay SA (2012) Arabidopsis circadian clock protein, TOC1, is a DNA-binding transcription factor. Proc Natl Acad Sci 109:3167–3172. CrossRefGoogle Scholar
  25. Hädrich N, Hendriks JH, Kötting O, Arrivault S, Feil R, Zeeman SC, Gibon Y, Schulze WX, Stitt M, Lunn JE (2012) Mutagenesis of cysteine 81 prevents dimerization of the APS1 subunit of ADP-glucose pyrophosphorylase and alters diurnal starch turnover in Arabidopsis thaliana leaves. Plant J 70:231–242. CrossRefGoogle Scholar
  26. Harmer SL, Hogenesch JB, Straume M, Chang HS, Han B, Zhu T, Wang X, Kreps JA, Kay SA (2000) Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. Science 290:2110–2113. CrossRefGoogle Scholar
  27. Haydon MJ, Hearn TJ, Bell LJ, Hannah MA, Webb AA (2013) Metabolic regulation of circadian clocks. Semin Cell Dev Biol 24:414–421. CrossRefGoogle Scholar
  28. Hennebelle T, Sahpaz S, Joseph H, Bailleul F (2008) Ethnopharmacology of Lippia alba. J Ethnopharmacol 116:211–222. CrossRefGoogle Scholar
  29. Hsie BS, Bueno AIS, Bertolucci SKV, Carvalho AA, Cunha SHB, Martins ER, Pinto JEBP (2019) Study of the influence of wavelengths and intensities of LEDs on the growth, photosynthetic pigment, and volatile compounds production of Lippia rotundifolia Cham in vitro. J Photochem Photobiol B 198:111577. CrossRefGoogle Scholar
  30. Huang W, Pérez-García P, Pokhilko A, Millar AJ, Antoshechkin I, Riechmann JL, Mas P (2012) Mapping the core of the Arabidopsis circadian clock defines the network structure of the oscillator. Science 336:75–79. CrossRefGoogle Scholar
  31. Ito S, Kawamura H, Niwa Y, Nakamichi N, Yamashino T, Mizuno T (2009) A genetic study of the Arabidopsis circadian clock with reference to the timing of cab expression 1 (TOC1) gene. Plant Cell Physiol 50:290–303. CrossRefGoogle Scholar
  32. Karnovsky MJ (1965) A formaldehyde glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 27:137Google Scholar
  33. Kooten O, Snel JFH (1990) The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynth Res 25(3):147–150CrossRefGoogle Scholar
  34. Lazzarini LES, Bertolucci SKV, Pacheco FV, Santos J, Silva ST, Carvalho AA, Pinto JEBP (2018) Quality and intensity of light affect Lippia gracilis Schauer plant growth and volatile compounds in vitro. Plant Cell Tiss Organ Cult 135:367–379. CrossRefGoogle Scholar
  35. 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. CrossRefGoogle Scholar
  36. Loivamäki M, Louis S, Cinege G, Zimmer I, Fischbach RJ, Schnitzler JP (2007) Circadian rhythms of isoprene biosynthesis in grey poplar leaves. Plant Physiol 143:540–551. CrossRefGoogle Scholar
  37. Lorenzi H, Matos FJA (2008) Plantas medicinais no Brasil—nativas e exóticas. Instituto Plantarum de Estudos da Flora, Nova Odessa, p 512pGoogle Scholar
  38. Lu SX, Knowles SM, Andronis C, Ong MS, Tobin EM (2009) Circadian clock associated1 and late elongated hypocotyl function synergistically in the circadian clock of arabidopsis. Plant Physiol 150:834–843. CrossRefGoogle Scholar
  39. Mahmud KP, Holzapfel BP, Guisard Y, Smith JP, Nielsen S, Rogiers SY (2018) Circadian regulation of grapevine root and shoot growth and their modulation by photoperiod and temperature. J Plant Physiol 222:86–93. CrossRefGoogle Scholar
  40. Maurya JP, Bhalerao RP (2017) Photoperiod and temperature-mediated control of growth cessation and dormancy in trees: a molecular perspective. Ann Bot 120:351–360. CrossRefGoogle Scholar
  41. Maxwell K, Johnson GN (2000) Chlorophyll fluorescence—a practical guide. J Exp Bot 51:659–668. CrossRefGoogle Scholar
  42. Mengin V, Pyl ET, Moraes TA, Sulpice R, Krohn N, Encke B, Stitt M (2017) Photosynthate partitioning to starch in Arabidopsis thaliana is insensitive to light intensity but sensitive to photoperiod due to a restriction on growth in the light in short photoperiods. Plant Cell Environ 40:2608–2627. CrossRefGoogle Scholar
  43. Morais LAS (2009) Influência dos fatores abióticos na composição química dos óleos essenciais. Hortic Bras 27:50–63.  Accessed 10 Jan 2019
  44. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. CrossRefGoogle Scholar
  45. Nakamichi N, Kiba T, Henriques R, Mizuno T, Chua NH, Sakakibara H (2010) Pseudo-response regulators 9, 7, and 5 are transcriptional repressors in the Arabidopsis circadian clock. Plant Cell 22:594–605. CrossRefGoogle Scholar
  46. Nusinow DA, Helfer A, Hamilton EE, King JJ, Imaizumi T, SchultzTF Kay SA (2011) The ELF4-ELF3-LUX complex links the circadian clock to diurnal control of hypocotyl growth. Nature 475:398. CrossRefGoogle Scholar
  47. O’Brien TP, McCully ME (1981) The study of plant structure: principles and selected methods. Melbourne, Termarcarphi Pty. Ltd, Scholar
  48. Otoni CG, Espitia PJ, Avena-Bustillos RJ, McHugh TH (2016) Trends in antimicrobial food packaging systems: emitting sachets and absorbent pads. Food Res Int 83:60–73. CrossRefGoogle Scholar
  49. Park DH, Somers DE, Kim YS, Choy YH, Lim HK, Soh MS, Kim HJ, Kay SA, Nam HG (1999) Control of circadian rhythms and photoperiodic flowering by the Arabidopsis GIGANTEA gene. Science 285:1579–1582. CrossRefGoogle Scholar
  50. Pascual ME, Slowing K, Carretero E, Sánches Mata D, Villar A (2001) Lippia: traditional uses, chemistry and pharmacology: a review. J Ethnopharmacol 76:201–214. CrossRefGoogle Scholar
  51. Pavela R, Govindarajan M (2017) The essential oil from Zanthoxylum monophyllum a potential mosquito larvicide with low toxicity to the non-target fish Gambusia affinis. J Pest Sci 90:369–378. CrossRefGoogle Scholar
  52. Peng Y, Li Y (2014) Combined effects of two kinds of essential oils on physical, mechanical and structural properties of chitosan films. Food Hydrocoll 36:287–293. CrossRefGoogle Scholar
  53. Pola CC, Medeiros EA, Pereira OL, Souza VG, Otoni CG, Camilloto GP, Soares NF (2016) Cellulose acetate active films incorporated with oregano (Origanum vulgare) essential oil and organophilic montmorillonite clay control the growth of phytopathogenic fungi. Food Pack Shelf Life 9:69–78. CrossRefGoogle Scholar
  54. Proestos C, Lytoudi K, Mavromelanidou OK, Zoumpoulakis P, Sinanoglou VJ (2014) Antioxidant capacity of selected plant extracts and their essential oils. Antioxidants 2:11–22. CrossRefGoogle Scholar
  55. Ragagnin RCG, Albuquerque CC, Oliveira FFM, Santos RG, Gurgel EP, Diniz JC, Viana FA (2014) Effect of salt stress on the growth of Lippia gracilis Schauer and on the quality of its essential oil. Acta Bot Brasilica 28:346–351. CrossRefGoogle Scholar
  56. Raut JS, Karuppayil SM (2014) A status review on the medicinal properties of essential oils. Ind Crops Prod 62:250–264. CrossRefGoogle Scholar
  57. Reis AC, Sousa SM, Vale AA, Pierre PMO, Franco AL, Campos JMS, Vieira RF, Viccini LF (2014) Lippia alba (Mill.) NE Br. (Verbenaceae): a new tropical autopolyploid complex? Am J Bot 101:1002–1012. CrossRefGoogle Scholar
  58. Saljoughian S, Shahin R, Alaa El-Din AB, Ralf G, Alireza O, Nooshin N, Amin MK (2018) The effects of food essential oils on cardiovascular diseases: a review. Crit Rev Food Sci Nutr 58:1688–1705. CrossRefGoogle Scholar
  59. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675. CrossRefGoogle Scholar
  60. Serrano-Bueno G, Romero-Campero FJ, Lucas-Reina E, Romero JM, Valverde F (2017) Evolution of photoperiod sensing in plants and algae. Curr Opin Plant Biol 37:10–17. CrossRefGoogle Scholar
  61. Shibaeva TG, Markovskaya EF (2013) Growth and development of cucumber Cucumis sativus L. in the prereproductive period under long photoperiods. Russ J Dev Biol 44:278–285. CrossRefGoogle Scholar
  62. Shin J, Sánchez-Villarreal A, Davis AM, Du SX, Berendzen KW, Koncz C, Ding Z, Li C, Davis SJ (2017) The metabolic sensor AKIN10 modulates the Arabidopsis circadian clock in a light-dependent manner. Plant Cell Environ 40:997–1008. CrossRefGoogle Scholar
  63. Song YH, Kubota A, Kwon MS, Covington MF, Lee N, Taagen ER, Laboy Cintrón D, Hwang DY, Akiyama R, Hodge SK, Huang H, Nguyen NH, Nusinow DA, Millar AJ, Shimizu KK, Imaizumi T (2018) Molecular basis of flowering under natural long-day conditions in Arabidopsis. Nat Plants 4:824–835. CrossRefGoogle Scholar
  64. Szczepanski S, Lipski A (2014) Essential oils show specific inhibiting effects on bacterial biofilm formation. Food Control 36:224–229. CrossRefGoogle Scholar
  65. Triozzi PM, Ramos-Sánchez JM, Hernández-Verdeja T, Moreno-Cortés A, Allona I, Perales M (2018) Photoperiodic regulation of shoot apical growth in poplar. Front Plant Sci 9:1030. CrossRefGoogle Scholar
  66. Viccini LF, Pierre PMO, Praça MM, Souza-Costa DC, Costa Romanel E, Sousa SM, Peixoto PHP, Salimena FRG (2006) Chromosome numbers in the genus Lippia (Verbenaceae). Plant System Evol 256:171–178. CrossRefGoogle Scholar
  67. Viccini LF, Silveira RS, Vale AA, Campos JMS, Reis AC, Santos MO, Campos VR, Carpanez AG, Grazul RM (2014) Citral and linalool content has been correlated to DNA content in Lippia alba (Mill.) NE Brown (Verbenaceae). Ind Crops Prod 59:14–19. CrossRefGoogle Scholar
  68. Vogg G, Heim R, Hansen J, Schäfer C, Beck E (1998) Frost hardening and photosynthetic performance of Scots pine (Pinus sylvestris L.) needles. I. Seasonal changes in the photosynthetic apparatus and its function. Planta 204:193–200. CrossRefGoogle Scholar
  69. Wagas KK, ErumD Tanveer A, HammadI Bushra M (2016) Evaluation of Ajuga bracteosa for antioxidant, anti-inflammatory, analgesic, antidepressant and anticoagulant activities. BMC Complem Altern Med 16:375. CrossRefGoogle Scholar
  70. Wang L, Han F, Zheng HQ (2018) Photoperiod-controlling guttation and growth of rice seedlings under microgravity on board Chinese spacelab TG-2. Microgravity Sci Technol 30:839–847. CrossRefGoogle Scholar
  71. Wei Y, Qian-liang M, Bing L, Khalid R, Cheng-Jian Z, Ting H, Lu-ping Q (2016) Medicinal plant cell suspension cultures: pharmaceutical applications and high-yielding strategies for the desired secondary metabolites. Crit Rev Biotechnol 36:215–232. CrossRefGoogle Scholar
  72. Welburn AR (1994) The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol 144:307–313. CrossRefGoogle Scholar
  73. Zuo Z, Wang B, Ying B, Zhou L, Zhang R (2017) Monoterpene emissions contribute to thermotolerance in Cinnamomum camphora. Trees 31:1759–1771. CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Kamila Motta de Castro
    • 1
  • Diego Silva Batista
    • 2
  • Evandro Alexandre Fortini
    • 1
  • Tatiane Dulcineia Silva
    • 1
  • Sérgio Heitor Sousa Felipe
    • 1
  • Amanda Mendes Fernandes
    • 1
  • Raysa Mayara de Jesus Sousa
    • 1
  • Laís Stehling de Queiroz Nascimento
    • 3
  • Victória Rabelo Campos
    • 3
  • Richard Michael Grazul
    • 4
  • Lyderson Facio Viccini
    • 3
  • Wagner Campos Otoni
    • 1
    Email author
  1. 1.Departamento de Biologia Vegetal/BIOAGROUniversidade Federal de ViçosaViçosaBrazil
  2. 2.Programa de Pós-Graduação em Agricultura e AmbienteUniversidade Estadual do Maranhão, Cidade Universitária Paulo VISão LuísBrazil
  3. 3.Laboratório de Genética e Biotecnologia, Departamento de BiologiaUniversidade Federal de Juiz de ForaJuiz de ForaBrazil
  4. 4.Departamento de QuímicaNúcleo Multifuncional de Pesquisas Químicas (NUPEQ), Universidade Federal de Juiz de ForaJuiz de ForaBrazil

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