Cytokinin (6-benzylaminopurine) elevates lignification and the expression of genes involved in lignin biosynthesis of carrot

Carrot is a root crop consumed worldwide and has great nutritional qualities. It is considered as one of the ten most important vegetable crops. Cytokinins are an essential class of the plant hormones that regulate many processes of plant growth. Till now, the effects of cytokinin, BAP, on lignin biosynthesis and related gene expression profiles in carrot taproot is unclear. In order to investigate the effect of applied BAP on lignin-related gene expression profiles, lignin accumulation, anatomical structures, and morphological characters in carrot taproots. Carrot roots were treated with different concentrations of BAP (0, 10, 20, and 30 mg L−1). The results showed that the application of BAP significantly increased plant length, shoot fresh weight, root fresh weight, and taproot diameter. In addition, BAP at 20 mg L−1 or 30 mg L−1 enhanced the average number of petioles. BAP treatment led to increased number and width of xylem vessels. The parenchyma cell numbers of pith were significantly induced in taproots treated with the BAP at a concentration of 30 mg L−1. BAP significantly upregulated most of the expression levels of lignin biosynthesis genes, caused elevated lignin accumulation in carrot taproots. Our results indicate that BAP may play important roles in growth development and lignification in carrot taproots. Our results provide a valuable database for more studies, which may focus on the regulation of root lignification via controlling cytokinin levels in carrot taproots.

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4-Coumarate-CoA ligase




p-Coumaroyl shikimate/quinate 3-hydroxylase


Cinnamate 4-hydroxylase


Cinnamyl alcohol dehydrogenase


Caffeoyl-CoA O-methyltransferase


Cinnamoyl-CoA reductase


Caffeic acid O-methyltransferase


Dry weight


Ferulate 5-hydroxylase


Hydroxycinnamoyl-CoA shikimate/quinate




Phenylalanine ammonia lyase




Real-time quantitative PCR


  1. Aimaretti NR, Ybalo CV, Rojas ML, Plou FJ, Yori JC (2012) Production of bioethanol from carrot discards. Bioresour Technol 123:727–732

    CAS  PubMed  Google Scholar 

  2. Ali MB, McNear DH (2014) Induced transcriptional profiling of phenylpropanoid pathway genes increased flavonoid and lignin content in Arabidopsis leaves in response to microbial products. BMC Plant Biol 14(1):1–14

    Google Scholar 

  3. Aloni R (1982) Role of cytokinin in differentiation of secondary xylem fibers. Plant Physiol 70(6):1631–1633

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Aloni R (1995) The induction of vascular tissues by auxin and cytokinin. Plant hormones. Springer, In, pp 531–546

    Google Scholar 

  5. Aloni R, Aloni E, Langhans M, Ullrich CI (2006) Role of cytokinin and auxin in shaping root architecture: regulating vascular differentiation, lateral root initiation, root apical dominance and root gravitropism. Ann Bot 97(5):883–893

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Baum FS, Aloni R, Peterson CA (1991) Role of cytokinin in vessel regeneration in wounded Coleus internodes. Ann Bot 67(6):543–548

    CAS  Google Scholar 

  7. Bertell G, Bolander E, Eliasson L (1990) Factors increasing ethylene production enhance the sensitivity of root growth to auxins. Physiol Plant 79(2):255–258

    CAS  Google Scholar 

  8. Bertell G, Eliasson L (1992) Cytokinin effects on root growth and possible interactions with ethylene and indole-3-acetic acid. Physiol Plant 84(2):255–261

    CAS  Google Scholar 

  9. Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Annu Rev Plant Biol 54(1):519–546

    CAS  PubMed  Google Scholar 

  10. Cervilla L, Rosales M, Rubio-Wilhelmi M, Sánchez-Rodríguez E, Blasco B, Ríos J, Romero L, Ruiz J (2009) Involvement of lignification and membrane permeability in the tomato root response to boron toxicity. Plant Sci 176(4):545–552

    CAS  PubMed  Google Scholar 

  11. Davies PJ (2010) The plant hormones: their nature, occurrence, and functions. Plant hormones. Springer, In, pp 1–15

    Google Scholar 

  12. Duan AQ, Feng K, Wang GL, Liu JX, Xu ZS, Xiong AS (2019) Elevated gibberellin enhances lignin accumulation in celery (Apium graveolens L.) leaves. Protoplasma 256(3):777–788

    CAS  PubMed  Google Scholar 

  13. Franceschini A, Szklarczyk D, Frankild S, Kuhn M, Simonovic M, Roth A, Lin J, Minguez P, Bork P, von Mering C (2013) STRING v9. 1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res 41(D808)

  14. Food and Agriculture Organization of the United Nations (FAO) (2018) FAOSTAT Statistic Database.

  15. Girgžde E, Samsone I (2017) Effect of cytokinins on shoot proliferation of silver birch (Betula pendula) in tissue culture. Environ Exp Biol 15(1):1–5

    Google Scholar 

  16. Hu D, Liu X, Wang C, Yang H, Li H, Ruan R, Yuan X, Yi Z (2015) Expression analysis of key enzyme genes in lignin synthesis of culm among different lodging resistances of common buckwheat (Fagopyrum esculentum Moench). Sci Agric Sin 48:1864–1872

    CAS  Google Scholar 

  17. Kappler R, Kristen U (1986) Exogenous cytokinins cause cell separation and cell expansion in the root tip cortex of Zea mays. Bot Gaz 147(3):247–251

    CAS  Google Scholar 

  18. Khadr A, Wang Y, Que F, Li T, Xu Z, Xiong A (2020) Exogenous abscisic acid suppresses the lignification and changes the growth, root anatomical structure and related gene profiles of carrot. Acta Biochim Biophys Sin 52(1):97–100

    PubMed  Google Scholar 

  19. Li T, Huang Y, Khadr A, Wang YH, Xu ZS, Xiong AS (2020) DcDREB1A, a DREB-binding transcription factor from Daucus carota, enhances drought tolerance in transgenic Arabidopsis thaliana and modulates lignin levels by regulating lignin-biosynthesis-related genes. Environ Exp Bot 169:103896

    CAS  Google Scholar 

  20. Ogita S, Nomura T, Kato Y, Uehara-Yamaguchi Y, Inoue K, Yoshida T, Sakurai T, Shinozaki K, Mochida K (2018) Transcriptional alterations during proliferation and lignification in Phyllostachys nigra cells. Sci Rep 8(1):1–11

    CAS  Google Scholar 

  21. Ogita S, Nomura T, Kishimoto T, Kato Y (2012) A novel xylogenic suspension culture model for exploring lignification in Phyllostachys bamboo. Plant Methods 8:40

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Pathak M, Singh B, Sharma A, Agrawal P, Pasha SB, Das HR, Das RH (2006) Molecular cloning, expression, and cytokinin (6-benzylaminopurine) antagonist activity of peanut (Arachis hypogaea) lectin SL-I. Plant Mol Biol 62(4):529–545

    CAS  PubMed  Google Scholar 

  23. Que F, Hou XL, Wang GL, Xu ZS, Tan GF, Li T, Wang YH, Khadr A, Xiong AS (2019) Advances in research on the carrot, an important root vegetable in the Apiaceae family. Hortic Res 6(1):1–15

    Google Scholar 

  24. Roberts LW, Gahan PB, Aloni R (2012) Vascular differentiation and plant growth regulators. Springer Science & Business Media

    Google Scholar 

  25. Rogers LA, Campbell MM (2004) The genetic control of lignin deposition during plant growth and development. New Phytol 164(1):17–30

    CAS  Google Scholar 

  26. Sajjad Y, Jaskani MJ, Ashraf MY, Qasim M, Ahmad R (2014) Response of morphological and physiological growth attributes to foliar application of plant growth regulators in gladiolus ‘white prosperity’. Pak J Agric Sci 51(1):123–129

    Google Scholar 

  27. Shimizu-Sato S, Tanaka M, Mori H (2009) Auxin–cytokinin interactions in the control of shoot branching. Plant Mol Biol 69:429–435

    CAS  PubMed  Google Scholar 

  28. Simmons BA, Loqué D, Ralph J (2010) Advances in modifying lignin for enhanced biofuel production. Curr Opin Plant Biol 13:312–319

    Google Scholar 

  29. Somerville C, Bauer S, Brininstool G, Facette M, Hamann T, Milne J, Osborne E, Paredez A, Persson S, Raab T (2004) Toward a systems approach to understanding plant cell walls. Science 306:2206–2211

    CAS  PubMed  Google Scholar 

  30. Stiebeling B, Neumann KH (1987) Identification and concentration of endogenous cytokinins in carrots (Daucus carota L.) as influenced by development and a circadian rhythm. J Plant Physiol 127:111–121

    CAS  Google Scholar 

  31. Stiebeling B, Pauler B, Neumann KH (1987) The influence of 6-BA application on yield, phosphorus and nitrogen uptake and endogenous cytokinin concentrations in carrots (Daucus carota L.) grown in phosphorusor nitrogendepleted soil. J Plant Nutr Soil Sci 150:69–74

    CAS  Google Scholar 

  32. Svensson SB (1972) A comparative study of the changes in root growth, induced by coumarin, auxin, ethylene, kinetin and gibberellic acid. Physiol Plant 26:115–135

    CAS  Google Scholar 

  33. Tang Y, Liu F, Xing H, Mao K, Chen G, Guo Q, Chen J (2019) Correlation analysis of lignin accumulation and expression of key genes involved in lignin biosynthesis of ramie (Boehmeria nivea). Genes 10(5):389

    CAS  PubMed Central  Google Scholar 

  34. Tian C, Jiang Q, Wang F, Wang GL, Xu ZS, Xiong AS (2015) Selection of suitable reference genes for qPCR normalization under abiotic stresses and hormone stimuli in carrot leaves. PLoS One:10(2)

  35. Vanholme R, Demedts B, Morreel K, Ralph J, Boerjan W (2010) Lignin biosynthesis and structure. Plant Physiol 153:895–905

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Vazquez-Cooz I, Meyer R (2002) A differential staining method to identify lignified and unlignified tissues. Biotech Histochem 77:277–282

    CAS  PubMed  Google Scholar 

  37. Wang GL, Que F, Xu ZS, Wang F, Xiong AS (2017) Exogenous gibberellin enhances secondary xylem development and lignification in carrot taproot. Protoplasma 254:839–848

    CAS  PubMed  Google Scholar 

  38. Wang GL, Sun S, Xing GM, Wu XJ, Wang F, Xiong AS (2015) Morphological characteristics, anatomical structure, and gene expression: novel insights into cytokinin accumulation during carrot growth and development. PLoS One 10(7)

  39. Wang J, Chen X, Gao Y, Zhang Y, Long S, Deng X, He D, Wang Y (2009) Expression of critical lignin metabolism genes in flax (Linum usitatissimum). Acta Agron Sin 35:1468–1473

    CAS  Google Scholar 

  40. Wang X, Ding J, Lin S, Liu D, Gu T, Wu H, Trigiano RN, McAvoy R, Huang J, Li Y (2020b) Evolution and roles of cytokinin genes in angiosperms 2: do ancient CKXs play housekeeping roles while non-ancient CKXs play regulatory roles? Hortic Res 7(1):29

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Wang X, Lin S, Liu D, Gan L, McAvoy R, Ding J, Li Y (2020a) Evolution and roles of cytokinin genes in angiosperms 1: do ancient IPTs play housekeeping while non-ancient IPTs play regulatory roles? Hortic Res 7(1):28

    PubMed  PubMed Central  Google Scholar 

  42. Wang YH, Li T, Zhang RR, Khadr A, Tian YS, Xu ZS, Xiong AS (2020c) Transcript profiling of genes involved in carotenoid biosynthesis among three carrot cultivars with various taproot colors. Protoplasma 257(3):1–15

    Google Scholar 

  43. Wang YH, Wu XJ, Sun S, Xing GM, Wang GL, Que F, Khadr A, Feng K, Li T, Xu ZS, Xiong AS (2018) DcC4H and DcPER are important in dynamic changes of lignin content in carrot roots under elevated carbon dioxide stress. J Agric Food Chem 66(30):8209–8220

    CAS  PubMed  Google Scholar 

  44. Wani SH, Kumar V, Shriram V, Sah SK (2016) Phytohormones and their metabolic engineering for abiotic stress tolerance in crop plants. Crop J 4(3):162–176

    Google Scholar 

  45. Webster BD, Radin JW (1972) Growth and development of cultured radish roots. Am J Bot 59(7):744–751

    CAS  Google Scholar 

  46. Werner T, Schmülling T (2009) Cytokinin action in plant development. Curr Opin Plant Biol 12(5):527–538

    CAS  PubMed  Google Scholar 

  47. Xu ZS, Tan HW, Wang F, Hou XL, Xiong AS (2014) CarrotDB: a genomic and transcriptomic database for carrot. Database 2014

  48. Yasmeen A, Nouman W, Basra SMA, Wahid A, Hussain N, Afzal I (2014) Morphological and physiological response of tomato (Solanum lycopersicum L.) to natural and synthetic cytokinin sources: a comparative study. Acta Physiol 36:3147–3155

    CAS  Google Scholar 

  49. Zhao Q, Dixon RA (2011) Transcriptional networks for lignin biosynthesis: more complex than we thought? Trends Plant Sci 16:227–233

    CAS  PubMed  Google Scholar 

  50. Zhou L, Li S, Huang P, Lin S, Addo-Danso SD, Ma Z, Ding G (2018) Effects of leaf age and exogenous hormones on callus initiation, rooting formation, bud germination, and plantlet formation in Chinese fir leaf cuttings. Forests 9:478

    Google Scholar 

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The research was supported by National Natural Science Foundation of China (31872098), the Open Fund of the State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University (ZW201905) and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Author information




Conceived and designed the experiments: ASX, AK. Performed the experiments: AK, YHW, RRZ, and XRW. Analyzed the data: AK, YHW, RRZ, and ZSX. Contributed reagents/materials/analysis tools: ASX. Wrote the paper: AK. Revised the paper: ASX. All authors read and approved the final manuscript.

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Correspondence to Ai-Sheng Xiong.

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Khadr, A., Wang, Y., Zhang, R. et al. Cytokinin (6-benzylaminopurine) elevates lignification and the expression of genes involved in lignin biosynthesis of carrot. Protoplasma (2020).

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  • Cytokinin
  • 6-benzylaminopurine
  • Lignification
  • Biosynthesis
  • Growth
  • Daucus carota L