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Transcriptomic changes triggered by carotenoid biosynthesis inhibitors and role of Citrus sinensis phosphate transporter 4;2 (CsPHT4;2) in enhancing carotenoid accumulation

  • Pengjun Lu
  • Shasha Wang
  • Don Grierson
  • Changjie Xu
Original Article
  • 220 Downloads
Part of the following topical collections:
  1. Terpenes and Isoprenoids

Abstract

Main conclusion

Carotenoid accumulation and chromoplast development in orange were perturbed by carotenoid inhibitors, and candidate genes were identified via transcriptomic analysis. The role of CsPHT4;2 in enhancing carotenoid accumulation was revealed.

Carotenoids are important plant pigments and their accumulation can be affected by biosynthesis inhibitors, but the genes involved were largely unknown. Here, application of norflurazon (NFZ), 2-(4-chlorophenylthio)-triethylamine hydrochloride (CPTA) and clomazone for 30 days to in vitro cultured sweet orange juice vesicles caused over-accumulation of phytoene (over 1000-fold), lycopene (2.92 μg g−1 FW, none in control), and deficiency in total carotenoids (reduced to 22%), respectively. Increased carotenoids were associated with bigger chromoplasts with enlarged plastoglobules or a differently crystalline structure in NFZ, and CPTA-treated juice vesicles, respectively. Global transcriptomic changes following inhibitor treatments were profiled. Induced expression of 1-deoxy-d-xylulose 5-phosphate synthase 1 by CPTA, hydroxymethylbutenyl 4-diphosphate reductase by both NFZ and CPTA, and reduced expression of chromoplast-specific lycopene β-cyclase by CPTA, as well as several downstream genes by at least one of the three inhibitors were observed. Expression of fibrillin 11 (CsFBN11) was induced following both NFZ and CPTA treatments. Using weighted correlation network analysis, a plastid-type phosphate transporter 4;2 (CsPHT4;2) was identified as closely correlated with high-lycopene accumulation induced by CPTA. Transient over-expression of CsPHT4;2 significantly enhanced carotenoid accumulation over tenfold in ‘Cara Cara’ sweet orange juice vesicle-derived callus. The study provides a valuable overview of the underlying mechanisms for altered carotenoid accumulation and chromoplast development following carotenoid inhibitor treatments and sheds light on the relationship between carotenoid accumulation and chromoplast development.

Keywords

Chromoplast Fibrillin (FBN) Lycopene Orange Weighted correlation network analysis (WGCNA) 

Abbreviations

CHRC

Chromoplast-specific carotenoid-associated protein

CLO

Clomazone

CPTA

2-(4-Chlorophenylthio)-triethylamine hydrochloride

CYCB

Chromoplast-specific lycopene β-cyclase

DXS

1-Deoxy-d-xylulose 5-phosphate synthase

FBN

Fibrillin

FPKM

Fragments per kilobase of transcript sequence per millions base pair

GO

Gene ontology

KEGG

Kyoto encyclopedia of genes and genomes

kME

Eigengene-based connectivity

HDR

Hydroxymethylbutenyl 4-diphosphate reductase

hp

High pigment

NFZ

Norflurazon

Or

Orange

PAP

Plastid lipid-associated protein

PHT

Phosphate transporter

PSY

Phytoene synthase

TEM

Transmission electron microscopy

TO

Topological overlap

WGCNA

Weighted correlation network analysis

Notes

Acknowledgements

This work was supported by the National Key Research and Development Program of China (2016YFD0400100) and the 111 project (B17039).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

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References

  1. Al-Babili S, Hartung W, Kleinig H, Beyer P (1999) CPTA modulates levels of carotenogenic proteins and their mRNAs and affects carotenoid and ABA content as well as chromoplast structure in Narcissus pseudonarcissus flowers. Plant Biol 1:607–612.  https://doi.org/10.1111/j.1438-8677.1999.tb00270.x CrossRefGoogle Scholar
  2. Botella-Pavía P, Besumbes O, Phillips M, Carretero-Paulet L, Boronat A, Rodríguez-Concepción M (2004) Regulation of carotenoid biosynthesis in plants: evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling the supply of plastidial isoprenoid precursors. Plant J 40:188–199.  https://doi.org/10.1111/j.1365-313X.2004.02198.x CrossRefPubMedPubMedCentralGoogle Scholar
  3. Breitenbach J, Zhu C, Sandmann G (2001) Bleaching herbicide norflurazon inhibits phytoene desaturase by competition with the cofactors. J Agric Food Chem 49:5270–5272CrossRefPubMedCentralGoogle Scholar
  4. Chen Y, Li FQ, Wurtzel ET (2010) Isolation and characterization of the Z-ISO gene encoding a missing component of carotenoid biosynthesis in plants. Plant Physiol 153:66–79.  https://doi.org/10.1104/pp.110.153916 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Chiu CJ, Taylor A (2007) Nutritional antioxidants and age-related cataract and maculopathy. Exp Eye Res 84:229–245.  https://doi.org/10.1016/j.exer.2006.05.015 CrossRefGoogle Scholar
  6. Coggins C, Henning G, Yokoyama H (1970) Lycopene accumulation induced by 2-(4-chlorophenylthio)-triethylamine hydrochloride. Science 168:1589–1590CrossRefPubMedCentralGoogle Scholar
  7. Cookson PJ, Kiano JW, Shipton CA, Fraser PD, Romer S, Schuch W et al (2003) Increases in cell elongation, plastid compartment size and phytoene synthase activity underlie the phenotype of the high pigment-1 mutant of tomato. Planta 217:896–903.  https://doi.org/10.1007/s00425-003-1065-9 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Enfissi E, Nogueira M, Bramley PM, Fraser PD (2017) The regulation of carotenoid formation in tomato fruit. Plant J 89:774–788.  https://doi.org/10.1111/tpj.13428 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Fantini E, Falcone G, Frusciante S, Giliberto L, Giuliano G (2013) Dissection of tomato lycopene biosynthesis through virus-induced gene silencing. Plant Physiol 163:986–998.  https://doi.org/10.1104/pp.113.224733 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Ford NA, Erdman JW Jr (2012) Are lycopene metabolites metabolically active? Acta Biochim Pol 59:1–4PubMedPubMedCentralGoogle Scholar
  11. Fraser PD, Bramley PM (2004) The biosynthesis and nutritional uses of carotenoids. Prog Lipid Res 43:228–265.  https://doi.org/10.1016/j.plipres.2003.10.002 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Fraser PD, Enfissi EM, Halket JM, Truesdale MR, Yu D, Gerrish C et al (2007) Manipulation of phytoene levels in tomato fruit: effects on isoprenoids, plastids, and intermediary metabolism. Plant Cell 19:3194–3211.  https://doi.org/10.1105/tpc.106.049817 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Fu XM, Kong WB, Peng G, Zhou JY, Azam M, Xu CJ et al (2012) Plastid structure and carotenogenic gene expression in red- and white-fleshed loquat (Eriobotrya japonica) fruits. J Exp Bot 63:341–354.  https://doi.org/10.1093/jxb/err284 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Galpaz N, Wang Q, Menda N, Zamir D, Hirschberg J (2008) Abscisic acid deficiency in the tomato mutant high-pigment 3 leading to increased plastid number and higher fruit lycopene content. Plant J 53:717–730.  https://doi.org/10.1111/j.1365-313X.2007.03362.x CrossRefPubMedPubMedCentralGoogle Scholar
  15. Guo B, Irigoyen S, Fowler TB, Versaw WK (2008a) Differential expression and phylogenetic analysis suggest specialization of plastid-localized members of the PHT4 phosphate transporter family for photosynthetic and heterotrophic tissues. Plant Signal Behav 3:784–790.  https://doi.org/10.4161/psb.3.10.6666 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Guo B, Jin Y, Wussler C, Blancaflor EB, Motes CM, Versaw WK (2008b) Functional analysis of the Arabidopsis PHT4 family of intracellular phosphate transporters. New Phytol 177:889–898.  https://doi.org/10.1111/j.1469-8137.2007.02331.x CrossRefPubMedPubMedCentralGoogle Scholar
  17. Irigoyen S, Karlsson PM, Kuruvilla J, Spetea C, Versaw WK (2011) The sink-specific plastidic phosphate transporter PHT4; 2 influences starch accumulation and leaf size in Arabidopsis. Plant Physiol 157:1765–1777.  https://doi.org/10.1104/pp.111.181925 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Kachanovsky DE, Filler S, Isaacson T, Hirschberg J (2012) Epistasis in tomato color mutations involves regulation of phytoene synthase 1 expression by cis-carotenoids. Proc Natl Acad Sci USA 109:19021–19026.  https://doi.org/10.1073/pnas.1214808109 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Kilambi HV, Kumar R, Sharma R, Sreelakshmi Y (2013) Chromoplast-specific carotenoid-associated protein appears to be important for enhanced accumulation of carotenoids in hp1 tomato fruits. Plant Physiol 161:2085–2101.  https://doi.org/10.1104/pp.112.212191 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Kilambi H, Manda K, Rai A, Charakana C, Bagri J, Sharma R et al (2017) Green-fruited Solanum habrochaites lacks fruit-specific carotenogenesis due to metabolic and structural blocks. J Exp Bot 68:4803–4819.  https://doi.org/10.1093/jxb/erx288 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Kolotilin I, Koltai H, Tadmor Y, Bar-Or C, Reuveni M, Meir A et al (2007) Transcriptional profiling of high pigment-2 dg tomato mutant links early fruit plastid biogenesis with its overproduction of phytonutrients. Plant Physiol 145:389–401.  https://doi.org/10.1104/pp.107.102962 CrossRefPubMedPubMedCentralGoogle Scholar
  22. La Rocca N, Rascio N, Oster U, Rudiger W (2007) Inhibition of lycopene cyclase results in accumulation of chlorophyll precursors. Planta 225:1019–1029.  https://doi.org/10.1007/s00425-006-0409-7 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Lange BM, Ketchum RE, Croteau RB (2001) Isoprenoid biosynthesis. Metabolite profiling of peppermint oil gland secretory cells and application to herbicide target analysis. Plant Physiol 127:305–314CrossRefPubMedCentralGoogle Scholar
  24. Langfelder P, Horvath S (2008) WGCNA: an R package for weighted correlation network analysis. BMC Bioinform 9:559.  https://doi.org/10.1186/1471-2105-9-559 CrossRefGoogle Scholar
  25. Leitner-Dagan Y, Ovadis M, Shklarman E, Elad Y, Rav David D, Vainstein A (2006) Expression and functional analyses of the plastid lipid-associated protein CHRC suggest its role in chromoplastogenesis and stress. Plant Physiol 142:233–244.  https://doi.org/10.1104/pp.106.082404 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Lieberman M, Segev O, Gilboa N, Lalazar A, Levin I (2004) The tomato homolog of the gene encoding UV-damaged DNA binding protein 1 (DDB1) underlined as the gene that causes the high pigment-1 mutant phenotype. Theor Appl Genet 108:1574–1581.  https://doi.org/10.1007/s00122-004-1584-1 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Liu LH, Shao ZY, Zhang M, Wang QM (2015) Regulation of carotenoid metabolism in tomato. Mol Plant 8:28–39.  https://doi.org/10.1016/j.molp.2014.11.006 CrossRefGoogle Scholar
  28. Lois L, Rodríguez-Concepción M, Gallego F, Campos N, Boronat A (2000) Carotenoid biosynthesis during tomato fruit development: regulatory role of 1-deoxy-d-xylulose 5-phosphate synthase. Plant J 22:503–513CrossRefPubMedCentralGoogle Scholar
  29. Lu S, Van Eck J, Zhou X, Lopez AB, O’Halloran DM, Cosman KM et al (2006) The cauliflower Or gene encodes a DnaJ cysteine-rich domain-containing protein that mediates high levels of beta-carotene accumulation. Plant Cell 18:3594–3605.  https://doi.org/10.1105/tpc.106.046417 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Lu YP, Liu SY, Sun H, Wu XM, Li JJ, Zhu L (2010) Neuroprotective effect of astaxanthin on H2O2-induced neurotoxicity in vitro and on focal cerebral ischemia in vivo. Brain Res 1360:40–48.  https://doi.org/10.1016/j.brainres.2010.09.016 CrossRefPubMedPubMedCentralGoogle Scholar
  31. Lu PJ, Wang CY, Yin TT, Zhong SL, Grierson D, Chen KS et al (2017) Cytological and molecular characterization of carotenoid accumulation in normal and high-lycopene mutant oranges. Sci Rep 7:761.  https://doi.org/10.1038/s41598-017-00898-y CrossRefPubMedPubMedCentralGoogle Scholar
  32. Maass D, Arango J, Wüst F, Beyer P, Welsch R (2009) Carotenoid crystal formation in Arabidopsis and carrot roots caused by increased phytoene synthase protein levels. PLoS One 4:e6373.  https://doi.org/10.1371/journal.pone.0006373 CrossRefPubMedPubMedCentralGoogle Scholar
  33. McQuinn RP, Wong B, Giovannoni JJ (2018) AtPDS overexpression in tomato: exposing unique patterns of carotenoid self-regulation and an alternative strategy for the enhancement of fruit carotenoid content. Plant Biotechnol J 16:482–494.  https://doi.org/10.1111/pbi.12789 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Mortimer CL, Misawa N, Perez-Fons L, Robertson FP, Harada H, Bramley PM, Fraser PD (2017) The formation and sequestration of nonendogenous ketocarotenoids in transgenic Nicotiana glauca. Plant Physiol 173:1617–1635.  https://doi.org/10.1104/pp.16.01297 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Mueller C, Schwender J, Zeidler J, Lichtenthaler H (2000) Properties and inhibition of the first two enzymes of the non-mevalonate pathway of isoprenoid biosynthesis. Biochem Soc Trans 28:792–793CrossRefPubMedCentralGoogle Scholar
  36. Murashige T, Tucker DPH (1969) Growth factor requirements of Citrus tissue culture. Proc First Int Citrus Symp 3:1155–1161Google Scholar
  37. Mustilli AC, Fenzi F, Ciliento R, Alfano F, Bowler C (1999) Phenotype of the tomato high pigment-2 mutant is caused by a mutation in the tomato homolog of DEETIOLATED1. Plant Cell 11:145–157.  https://doi.org/10.1105/tpc.11.2.145 CrossRefPubMedPubMedCentralGoogle Scholar
  38. Neuman H, Galpaz N, Cunningham FX, Zamir D, Hirschberg J (2014) The tomato mutation nxd1 reveals a gene necessary for neoxanthin biosynthesis and demonstrates that violaxanthin is a sufficient precursor for abscisic acid biosynthesis. Plant J 78:80–93.  https://doi.org/10.1111/tpj.12451 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Nisar N, Li L, Lu S, Khin NC, Pogson BJ (2015) Carotenoid metabolism in plants. Mol Plant 8:8–82.  https://doi.org/10.1016/j.molp.2014.12.007 CrossRefGoogle Scholar
  40. Nogueira M, Mora L, Enfissi EM, Bramley PM, Fraser PD (2013) Subchromoplast sequestration of carotenoids affects regulatory mechanisms in tomato lines expressing different carotenoid gene combinations. Plant Cell 25:4560–4579.  https://doi.org/10.1105/tpc.113.116210 CrossRefPubMedPubMedCentralGoogle Scholar
  41. Paolillo DJ Jr, Garvin DF, Parthasarathy MV (2004) The chromoplasts of Or mutants of cauliflower (Brassica oleracea L. var. botrytis). Protoplasma 224:245–253.  https://doi.org/10.1007/s00709-004-0059-1 CrossRefPubMedPubMedCentralGoogle Scholar
  42. Qin G, Gu H, Ma L, Peng Y, Deng XW, Chen Z et al (2007) Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis. Cell Res 17:471–482.  https://doi.org/10.1038/cr.2007.40 CrossRefPubMedPubMedCentralGoogle Scholar
  43. Ralley L, Schuch W, Fraser PD, Bramley PM (2016) Genetic modification of tomato with the tobacco lycopene β-cyclase gene produces high β-carotene and lycopene fruit. Z Naturforschung 71:295–301.  https://doi.org/10.1515/znc-2016-0102 CrossRefGoogle Scholar
  44. Rey P, Gillet B, Römer S, Eymery F, Massimino J, Peltier G et al (2000) Over-expression of a pepper plastid lipid-associated protein in tobacco leads to changes in plastid ultrastructure and plant development upon stress. Plant J 21:483–494.  https://doi.org/10.1046/j.1365-313x.2000.00699.x CrossRefPubMedPubMedCentralGoogle Scholar
  45. Rodríguez-Villalón A, Gas E, Rodríguez-Concepción M (2009) Phytoene synthase activity controls the biosynthesis of carotenoids and the supply of their metabolic precursors in dark-grown Arabidopsis seedlings. Plant J 60:424–435.  https://doi.org/10.1111/j.1365-313X.2009.03966.x CrossRefPubMedPubMedCentralGoogle Scholar
  46. Simkin AJ, Gaffé J, Alcaraz JP, Carde JP, Bramley PM, Fraser PD et al (2007) Fibrillin influence on plastid ultrastructure and pigment content in tomato fruit. Phytochemistry 68:1545–1556.  https://doi.org/10.1016/j.phytochem.2007.03.014 CrossRefPubMedPubMedCentralGoogle Scholar
  47. Singh DK, Maximova SN, Jensen PJ, Lehman BL, Ngugi HK, McNellis TW (2010) FIBRILLIN4 is required for plastoglobule development and stress resistance in apple and Arabidopsis. Plant Physiol 154:1281–1293.  https://doi.org/10.1104/pp.110.164095 CrossRefPubMedPubMedCentralGoogle Scholar
  48. Srinivasan R, Babu S, Gothandam KM (2017) Accumulation of phytoene, a colorless carotenoid by inhibition of phytoene desaturase (PDS) gene in Dunaliella salina V-101. Bioresour Technol 242:311–318.  https://doi.org/10.1016/j.biortech.2017.03.042 CrossRefPubMedPubMedCentralGoogle Scholar
  49. van Wijk KJ, Kessler F (2017) Plastoglobuli: plastid microcompartments with integrated functions in metabolism, plastid developmental transitions, and environmental adaptation. Annu Rev Plant Biol 68:253–289.  https://doi.org/10.1146/annurev-arplant-043015-111737 CrossRefPubMedPubMedCentralGoogle Scholar
  50. Walter MH, Strack D (2011) Carotenoids and their cleavage products: biosynthesis and functions. Nat Prod Rep 28:663–692.  https://doi.org/10.1039/c0np00036a CrossRefPubMedPubMedCentralGoogle Scholar
  51. Welsch R, Zhou X, Yuan H, Alvarez D, Sun T, Schlossarek D et al (2018) Clp protease and OR directly control the proteostasis of phytoene synthase, the crucial enzyme for carotenoid biosynthesis in Arabidopsis. Mol Plant 11:149–162.  https://doi.org/10.1016/j.molp.2017.11.003 CrossRefPubMedPubMedCentralGoogle Scholar
  52. Xu CJ, Fraser PD, Wang WJ, Bramley PM (2006) Differences in the carotenoid content of ordinary citrus and lycopene-accumulating mutants. J Agric Food Chem 54:5474–5481.  https://doi.org/10.1021/jf060702t CrossRefPubMedPubMedCentralGoogle Scholar
  53. Yuan H, Zhang J, Nageswaran D, Li L (2015) Carotenoid metabolism and regulation in horticultural crops. Hortic Res 2:15036.  https://doi.org/10.1038/hortres.2015.36 CrossRefPubMedPubMedCentralGoogle Scholar
  54. Zhang LC, Ma G, Kato M, Yamawaki K, Takagi T, Kiriiwa Y et al (2012) Regulation of carotenoid accumulation and the expression of carotenoid metabolic genes in citrus juice sacs in vitro. J Exp Bot 63:871–886.  https://doi.org/10.1093/jxb/err318 CrossRefPubMedPubMedCentralGoogle Scholar
  55. Zhang J, Guo SG, Ren Y, Zhang HY, Gong GY, Zhou M et al (2017) High-level expression of a novel chromoplast phosphate transporter ClPHT4;2 is required for flesh color development in watermelon. New Phytol 213:1208–1221.  https://doi.org/10.1111/nph.14257 CrossRefPubMedPubMedCentralGoogle Scholar
  56. Zhou XJ, Welsch R, Yang Y, Álvarez D, Riediger M, Yuan H et al (2015) Arabidopsis OR proteins are the major posttranscriptional regulators of phytoene synthase in controlling carotenoid biosynthesis. Proc Natl Acad Sci USA 112:3558–3563.  https://doi.org/10.1073/pnas.1420831112 CrossRefPubMedPubMedCentralGoogle Scholar
  57. Zhu CF, Bai C, Sanahuja G, Yuan D, Farre G, Naqvi S et al (2010) The regulation of carotenoid pigmentation in flowers. Arch Biochem Biophys 504:132–141.  https://doi.org/10.1016/j.abb.2010.07.028 CrossRefPubMedPubMedCentralGoogle Scholar

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© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative BiologyZhejiang UniversityHangzhouChina
  2. 2.Division of Plant and Crop Sciences, School of BiosciencesUniversity of NottinghamSutton BoningtonUK

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