Impact of light and sucrose supplementation on cellular differentiation, metabolic shift and modulation of gene expression in hairy roots of Daucus carota

Abstract

Green hairy root cultures of Daucus carota were subjected to gradually lowered sucrose concentrations to understand the modulation of secondary metabolism upon carbon-starved conditions. An apparent negative correlation with the sucrose concentrations in the media was observed with chloroplast development, as confirmed by both histological studies and relevant biochemical assays. Expansion of relative vascular area and well-developed multi-layered endodermis were evidenced in green hairy roots with low sucrose concentrations as compared to normal hairy roots cultivated with 2% sucrose under dark condition. Thus, anatomical modulation was evident in photo-oxidatively stressed green hairy roots because of their maintenance under continuous illumination. This was demonstrated further by the expression analysis of glutathione peroxidase and NADPH reductase genes which showed upregulation in green hairy roots maintained in ½ MS media containing 0.5% sucrose as compared to normal hairy roots. The amount of phenolic marker compound p-hydroxybenzoic acid was reduced to half, while enhanced levels of emitted terpenoid volatiles were noticed in these carbon-starved green hairy roots. Analysis of primary metabolites in green hairy roots showed increased levels of amino acids and sugar alcohols accumulation. Further, analysis revealed the elevated accumulation of organic acids which have significant bearing towards redirection of primary metabolites leading to the formation of enhanced terpenoid volatiles in green hairy roots. Thus, our study revealed the impact of light and sucrose supplementation towards cellular differentiation and redirection of secondary metabolism in hairy roots of D. carota.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Abbreviations

VLCFA:

Variable long chain fatty acids

CCR:

Cinnamoyl Co-A reductase

RBCL:

Large subunit of RuBisCo

PAL:

Phenylalanine ammonia lyase

LHCB:

Light harvesting complex b

TPS:

Terpene synthase

GPX:

Glutathione peroxidase

DXR:

1-Deoxy-d-xylulose 5-phosphate reductoisomerase

NADPHR:

NADPH reductase

OPPP:

Oxidative pentose phosphate pathway

References

  1. Aloni R (1980) Role of auxin and sucrose in the differentiation of sieve and tracheary elements in plant tissue cultures. Planta 150:255–263

    Article  CAS  PubMed  Google Scholar 

  2. Asuming WA, Beauchamp PS, Descalzo JT, Dev BC, Dev V, Frost S, Ma CW (2005) Essential oil composition of four Lomatium Raf. species and their chemotaxonomy. Biochem Syst Ecol 33:17–26

    Article  CAS  Google Scholar 

  3. Behnke K, Kaiser A, Zimmer I, Brüggemann N, Janz D, Polle A, Hampp R, Häansch R, Popko J, Schmitt-Kopplin P, Ehlting B, Rennenberg H, Barta C, Loreto F, Schnitzler J (2010) RNAi mediated suppression of isoprene emission in poplar transiently impacts phenolic metabolism under high temperature and high light intensities: a transcriptomic and metabolomic analysis. Plant Mol Biol 74:61–75

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Bera P, Mukherjee C, Mitra A (2017) Enzymatic production and emission of floral scent volatiles in Jasminum sambac. Plant Sci 256:25–38

    Article  CAS  PubMed  Google Scholar 

  5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  6. Chiou TJ, Bush DR (1998) Sucrose is a signal molecule in assimilate partitioning. Proc Natl Acad Sci USA 95:4784–4788

    Article  CAS  PubMed  Google Scholar 

  7. da Silva MHL, Andrade EHA, Zoghbi MGB, Luz AIR, da Silva JD, Maia JGS (1999) The essential oils of Lantana camara L. occurring in North Brazil. Flavour Frag J 14:208–210

    Article  Google Scholar 

  8. Daferera DL, Ziogas BN, Polissiou MG (2003) The effectiveness of plant essential oils on the growth of Botrytis cinerea, Fusarium sp. and Clavibacter michiganensis subsp. michiganensis. Crop Prot 22:39–44

    Article  CAS  Google Scholar 

  9. El-Afry MM, El-Nady MF, Abdelmonteleb EB, Metwaly MMS (2012) Anatomical studies on drought-stressed wheat plants (Triticum aestivum L.) treated with some bacterial strains. Acta Biol Szeged 56:165–174

    Google Scholar 

  10. Fraser PD, Bramley PM (2004) The biosynthesis and nutritional uses of carotenoids. Prog Lipid Res 43:228–265

    Article  CAS  Google Scholar 

  11. Gelvin BS (1990) Crown gall disease and hairy root disease. Plant Physiol 92:281–285

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gueguen V, Macherel D, Jaquinod M, Douce R, Bourguignon J (2000) Fatty acid and lipoic acid biosynthesis in higher plant mitochondria. J Biol Chem 275:5016–5025

    Article  CAS  PubMed  Google Scholar 

  13. Guerra D, Anderson AJ, Salisbury FB (1985) Reduced phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities and lignin synthesis in wheat grown under low pressure sodium lamps. Plant Physiol 78:126–130

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Häkkinen ST, Raven N, Henquet M, Laukkanen M, Anderlei T, Pitkänen J, Twyman RM, Bosch D, Oksman-Caldentey K, Schillberg S, Ritala A (2014) Molecular farming in tobacco hairy roots by triggering the secretion of a pharmaceutical antibody. Biotechnol Bioeng 111:336–346

    Article  CAS  PubMed  Google Scholar 

  15. Hall DO, Rao KK (1994) Photosynthesis, 5th edn. Cambridge University Press, Cambridge, pp 39–51

    Google Scholar 

  16. Hemm MR, Rider SD, Ogas J, Murry DJ, Chapple C (2004) Light induces phenylpropanoid metabolism in Arabidopsis roots. Plant J 38:765–778

    Article  CAS  PubMed  Google Scholar 

  17. Hilal M, Parrado MF, Rosa M, Gallardo M, Orce L, Massa ED et al (2004) Epidermal lignin deposition in quinoa cotyledons in response to UV-B radiation. Photochem Photobiol 79:205–210

    Article  CAS  PubMed  Google Scholar 

  18. Hill R (1937) Oxygen evolution by isolated chloroplasts. Nature 139:881–882

    Article  CAS  Google Scholar 

  19. Hoecker U, Toledo-Ortiz G, Bender J, Quail PH (2004) The photo-morphogenesis-related mutant red1 is defective in CYP83B1, a red light-induced gene encoding a cytochrome P450 required for normal auxin homeostasis. Planta 219:195–200

    Article  CAS  PubMed  Google Scholar 

  20. Igamberdiev AU, Eprintsev AT (2016) Organic acids: the pools of fixed carbon involved in redox regulation and energy balance in higher plants. Front Plant Sci 7:1042

    Article  PubMed  PubMed Central  Google Scholar 

  21. Igamberdiev AU, Lea PJ (2002) The role of peroxisomes in the Integration of metabolism and evolutionary diversity of photosynthetic organisms. Phytochemistry 60:651–674

    Article  CAS  PubMed  Google Scholar 

  22. Jacob-Velázquez D, González-Agüero M, Cisneros-Zevallos L (2015) Cross-talk between signalling pathways: the link between plant secondary metabolite production and wounding stress response. Sci Rep 5:8608. https://doi.org/10.1038/srep08608

    Article  CAS  Google Scholar 

  23. Joubès J, Raffaele S, Bourdenx B, Garcia C, Laroche-Traineau J, Moreau P et al (2008) The VLCFA elongase gene family in Arabidopsis thaliana: Phylogenetic analysis, 3D modelling and expression profiling. Plant Mol Biol 67:547–566

    Article  CAS  PubMed  Google Scholar 

  24. Kang YH, Parker CC, Smith AC, Waldron KW (2008) Characterization and distribution of phenolics in carrot cell walls. J Agric Food Chem 56:8858–8864

    Google Scholar 

  25. Kjeldsen F, Christensen LP, Edelenbos M (2001) Quantitative analysis of aroma compounds in carrot ( Daucus carota L.) cultivars by capillary gas chromatography using large-volume injection technique. J Agric Food Chem 49:4342–4348

    Article  CAS  PubMed  Google Scholar 

  26. Koch K (2004) Sucrose metabolism: regulatory mechanisms and pivotal roles in sugar sensing and plant development. Curr Opin Plant Biol 7:235–246

    Article  CAS  PubMed  Google Scholar 

  27. Maiti S, Mitra A (2017) Morphological, physiological and ultrastructural changes in flowers explain the spatio-temporal emission of scent volatiles in Polianthes tuberosa L. Plant Cell Physiol 58:2095–2011

    Article  CAS  PubMed  Google Scholar 

  28. Matsui A, Ishida J, Morosawa T et al (2008) Arabidopsis transcriptome analysis under drought, cold, high-salinity and ABA treatment conditions using a tiling array. Plant Cell Physiol 49:1135–1149

    Article  CAS  PubMed  Google Scholar 

  29. Mukherjee C, Sircar D, Chatterjee M, Mitra A (2014) Combating photo-oxidative stress in green hairy roots of Daucus carota cultivated under light irradiation. J Plant Physiol 171:179–187

    Article  CAS  PubMed  Google Scholar 

  30. Mukherjee C, Samanta T, Mitra A (2016) Redirection of metabolite biosynthesis from hydroxybenzoates to volatile terpenoids in green hairy roots of. Daucus carota Planta 243:305–320

    CAS  PubMed  Google Scholar 

  31. Nakabayashi R, Yonekura-Sakaibara K, Urano K, Suzuki M, Yamada Y, Nishizawa T, Matsuda F, Kojima M, Sakakibara H, Shinozaki K et al (2014) Enhancement of oxidative and drought tolerance in Arabidopsis by over-accumulation of antioxidant flavonoids. Plant J 77:367–379

    Article  CAS  PubMed  Google Scholar 

  32. Parr AJ, Ng A, Waldron KW (1997) Ester-linked phenolic components of carrot cell walls. J Agric Food Chem 45:2468–2471

    Article  CAS  Google Scholar 

  33. Rosa M, Prado C, Podazza G, Interdonato R, González JA, Hilal M, Prado FE (2009) Sugars-Metabolism, sensing and abiotic stress. Plant Signal Behav 4::388–393

    Article  Google Scholar 

  34. Samanta T, Kotamreddy JNR, Ghosh BC, Mitra A (2017) Changes in targeted metabolites, enzyme activities and transcripts at different developmental stages of tea leaves: a study for understanding the biochemical basis of tea shoot plucking. Acta Physiol Plant 39:11. https://doi.org/10.1007/s11738-016-2298-0

    Article  CAS  Google Scholar 

  35. Sircar D, Mitra A (2008) Evidence for p-hydroxybenzoate formation involving phenyl-propanoid chain-cleavage in hairy roots of Daucus carota. J Plant Physiol 165:407–414

    Article  CAS  PubMed  Google Scholar 

  36. Sircar D, Mitra A (2009) Accumulation of p-hydroxybenzoic acid in hairy roots of Daucus carota 2: Confirming biosynthetic steps through feeding of inhibitors and precursors. J Plant Physiol 166:1370–1380

    Article  CAS  PubMed  Google Scholar 

  37. Sircar D, Roychowdhury A, Mitra A (2007a) Accumulation of p-hydroxybenzoic acid in hairy roots of Daucus carota. J Plant Physiol 164:1358–1366

    Article  CAS  PubMed  Google Scholar 

  38. Sircar D, Dey G, Mitra A (2007b) A validated HPLC method for simultaneous determination of 2-hydroxy-4-methoxybenzaldehyde and 2-hydroxy-4-methoxybenzoic acid in root organs of Hemidesmus indicus. Chromatographia 65:349–353

    Article  CAS  Google Scholar 

  39. Smeekens S, Rook F (1997) Sugar sensing and sugar-mediated signal transduction in plants. Plant Physiol 115:7–13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Souret FF, Weathers PJ, Wobbe KK (2002) The mevalonate independent pathway is expressed in transformed roots of Artemisia annua and regulated by light and culture age. In Vitro Cell Dev Biol Plant 38:581–588

    Article  CAS  Google Scholar 

  41. Tone S, Taya M, Kino-Oka M (1997) Alteration of metabolite formation and morphological properties of hairy roots by environmental stimuli. In: Doran PM (ed) Hairy roots: culture and applications. Harwood Academic Publishers, Amsterdam, pp 65–72

    Google Scholar 

  42. Wetmore RH, Rier JP (1963) Experimental induction of vascular tissues in callus of angiosperms. Amer J Bot 50:418–430

    Article  CAS  Google Scholar 

  43. Xia J, Wishart DS (2011) Web-based inference of biological patterns, functions and pathways from metabolomic data using MetaboAnalyst. Nat Protoc 6:743–760

    Article  CAS  Google Scholar 

  44. Yahyaa M, Tholl D, Cormier G, Jensen R, Simon PW, Ibdah M (2015) Identification and characterization of terpene synthases potentially involved in the formation of volatile terpenes in carrot (Daucus carota L.) roots. J Agric Food Chem 63:4870–4878

    Article  CAS  PubMed  Google Scholar 

  45. Zhong JJ, Seki T, Kinoshita S, Yoshida T (1991) Effect of light irradiation on anthocyanin production by suspended culture of Perilla frutescens. Biotechnol Bioeng 38:653–658

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported from a research grant [38(1201)/08/EMR-II to A. Mitra] obtained from the Council of Scientific and Industrial Research (CSIR), India. S Mukherjee was a recipient of Joint M. Tech -PhD fellowship from the institute. N N Kutty was a recipient of individual doctoral fellowship from CSIR, India.

Author information

Affiliations

Authors

Contributions

SM and AM conceived and designed research. SM conducted experiments. SM and NK did HPLC and GC–MS analysis. SM and PB did molecular biology experiments. SM, NK, PB and AM analyzed data. SM and AM wrote the final manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Adinpunya Mitra.

Ethics declarations

Conflict of interest

Authors declared no competing interest.

Additional information

Communicated by Nokwanda Pearl Makunga.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 332 KB)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Mukherjee, S., Kutty, N.N., Bera, P. et al. Impact of light and sucrose supplementation on cellular differentiation, metabolic shift and modulation of gene expression in hairy roots of Daucus carota. Plant Cell Tiss Organ Cult 136, 383–397 (2019). https://doi.org/10.1007/s11240-018-1523-5

Download citation

Keywords

  • Daucus carota
  • Green hairy roots
  • Volatile terpenoid
  • p-Hydroxybenzoic acid
  • Sucrose
  • Organic acids