Advertisement

Tree Genetics & Genomes

, 15:22 | Cite as

Gene expression and metabolite profiling analyses of developing pomegranate fruit peel reveal interactions between anthocyanin and punicalagin production

  • Rotem Harel-Beja
  • Li Tian
  • Shiri Freilich
  • Rida Habashi
  • Hamutal Borochov-Neori
  • Tamar Lahav
  • Taly Trainin
  • Adi Doron-Faigenboim
  • Ron Ophir
  • Irit Bar-Ya’akov
  • Rachel Amir
  • Doron HollandEmail author
Original Article
Part of the following topical collections:
  1. Genome Biology

Abstract

The fruit peel of pomegranate (Punica granatum L.) contains high concentrations of polyphenols, which play a critical role in determining the color and nutritional value of fruits. This study evaluated and compared the production of two major classes of polyphenols in the pomegranate fruit skin, i.e., anthocyanins, the main pigments in pomegranate, and punicalagin, a highly bioactive hydrolyzable tannin that is synthesized from an intermediate of the shikimate pathway. Gene expression and metabolite (anthocyanins and punicalagin) accumulation were determined, at three stages of fruit development, in the peel of red and pink cultivars, containing high and low levels of anthocyanins, respectively. Red and pink pomegranate cultivars showed the highest difference in gene expression during the transition from early to late fruit developmental stages, while differences between the cultivars were relatively small at each developmental stage. Positive correlations were found between anthocyanin and total punicalagin content. Of the differentially expressed contigs, 3093 and 312 contigs were correlated (Pearson’s r, |0.75|; P < = 0.02) with anthocyanins and punicalagin content, respectively. Interestingly, 143 contigs positively correlated with both anthocyanin and punicalagin. The differentially expressed contigs could be further divided into five groups representing a distinct characteristic correlation with each of the analyzed metabolites. Overall, the presented information provides a comprehensive view of the interplay between the hydrolyzable tannin and the anthocyanin pathways and points to genetic factors potentially involved in this interaction.

Keywords

Punica granatum Anthocyanin Punicalagin Fruit development Polyphenols Differential expression 

Notes

Acknowledgements

We thank Kamel Hatib for orchard management.

Data archiving statement

The raw sequencing data is archived in NCBI No. PRJNA521857.

Authors’ contribution

RHB and DH conducted the study and wrote the manuscript, DH LT and RA initiated the study, RHB and IBY collected and prepared the plant material, RH and RA did the punicalagin analysis, HBN did the AT analysis, TT purified the RNA, and SF, TL, ADF, and RO did the bioinformatics analysis.

Funding

This research was supported by research grant award No. IS-4822-15 R from BARD, The United States—Israel Binational Agricultural Research and Development Fund.

Compliance with ethical standards

Competing interests

The authors declare that they have no competing interests.

Supplementary material

11295_2019_1329_MOESM1_ESM.docx (64 kb)
ESM 1 (DOCX 63 kb)
11295_2019_1329_MOESM2_ESM.xlsx (412 kb)
ESM 2 (XLSX 411 kb)

References

  1. Adams LS, Seeram NP, Aggarwal BB, Takada Y, Sand D, Heber D (2006) Pomegranate juice, total pomegranate ellagitannins, and punicalagin suppress inflammatory cell signaling in colon cancer cells. J Agric Food Chem 54:980–985.  https://doi.org/10.1021/jf052005r CrossRefPubMedGoogle Scholar
  2. Ben-Simhon Z, Judeinstein S, Nadler-Hassar T, Trainin T, Bar-Ya’akov I, Borochov-Neori H, Holland D (2011) A pomegranate (Punica granatum L.) WD40-repeat gene is a functional homologue of Arabidopsis TTG1 and is involved in the regulation of anthocyanin biosynthesis during pomegranate fruit development. Planta 234:865–881.  https://doi.org/10.1007/s00425-011-1438-4 CrossRefPubMedGoogle Scholar
  3. Ben-Simhon Z, Judeinstein S, Trainin T, Harel-Beja R, Bar-Ya'akov I, Borochov-Neori H, Holland D (2015) A "white" anthocyanin-less pomegranate (Punica granatum L.) caused by an insertion in the coding region of the leucoanthocyanidin dioxygenase (LDOX; ANS) gene. PLoS One 10:e0142777.  https://doi.org/10.1371/journal.pone.0142777 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Series B Stat Methodol 57:289–300Google Scholar
  5. Bontpart T, Marlin T, Vialet S, Guiraud JL, Pinasseau L, Meudec E, Sommerer N, Cheynier V, Terrier N (2016) Two shikimate dehydrogenases, Vvsdh3 and Vvsdh4, are involved in gallic acid biosynthesis in grapevine. J Exp Bot 67:3537–3550.  https://doi.org/10.1093/jxb/erw184 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Borochov-Neori H, Judeinstein S, Harari M, Bar-Ya’akov I, Patil BS, Lurie S, Holland D (2011) Climate effects on anthocyanin accumulation and composition in the pomegranate (Punica granatum L.) fruit arils. J Agric Food Chem 59:5325–5334.  https://doi.org/10.1021/jf2003688 CrossRefPubMedGoogle Scholar
  7. Buchfink B, Xie C, Huson DH (2014) Fast and sensitive protein alignment using diamond. Nat Methods 12:59–60.  https://doi.org/10.1038/nmeth.3176 https://www.nature.com/articles/nmeth.3176#supplementary-information CrossRefPubMedGoogle Scholar
  8. Cao S, Hu Z, Zheng Y, Lu B (2010) Effect of bth on anthocyanin content and activities of related enzymes in strawberry after harvest. J Agric Food Chem 58:5801–5805.  https://doi.org/10.1021/jf100742v CrossRefPubMedGoogle Scholar
  9. Cheng H-Q, Han LB, Yang CL, Wu XM, Zhong NQ, Wu JH, Wang FX, Wang HY, Xia GX (2016) The cotton MYB108 forms a positive feedback regulation loop with CML11 and participates in the defense response against Verticillium dahliae infection. J Exp Bot 67:1935–1950.  https://doi.org/10.1093/jxb/erw016 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M (2005) Blast2go: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676.  https://doi.org/10.1093/bioinformatics/bti610 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Das PK, Geul B, Choi S-B, Yoo S-D, Park Y-I (2011) Photosynthesis-dependent anthocyanin pigmentation in Arabidopsis. Plant Signal Behav 6:23–25.  https://doi.org/10.4161/psb.6.1.14082 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Feder A et al (2015) A kelch domain-containing F-box coding gene negatively regulates flavonoid accumulation in muskmelon. Plant Physiol 169:1714PubMedPubMedCentralGoogle Scholar
  13. Freilich S, Lev S, Gonda I, Reuveni E, Portnoy V, Oren E, Lohse M, Galpaz N, Bar E, Tzuri G, Wissotsky G, Meir A, Burger J, Tadmor Y, Schaffer A, Fei Z, Giovannoni J, Lewinsohn E, Katzir N (2015) Systems approach for exploring the intricate associations between sweetness, color and aroma in melon fruits. BMC Plant Biol 15:71.  https://doi.org/10.1186/s12870-015-0449-x CrossRefPubMedPubMedCentralGoogle Scholar
  14. Gil MI, García-Viguera C, Artés F, Tomás-Barberán FA (1995) Changes in pomegranate juice pigmentation during ripening. J Sci Food Agric 68:77–81.  https://doi.org/10.1002/jsfa.2740680113 CrossRefGoogle Scholar
  15. Gil MI, Tomás-Barberán FA, Hess-Pierce B, Holcroft DM, Kader AA (2000) Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. J Agric Food Chem 48:4581–4589.  https://doi.org/10.1021/jf000404a CrossRefPubMedGoogle Scholar
  16. Götz S et al (2008) High-throughput functional annotation and data mining with the Blast2go suite. Nucleic Acids Res 36:3420–3435.  https://doi.org/10.1093/nar/gkn176 CrossRefPubMedPubMedCentralGoogle Scholar
  17. Gould KS (2004) Nature's Swiss army knife: the diverse protective roles of anthocyanins in leaves. J Biomed Biotechnol 2004:314–320.  https://doi.org/10.1155/S1110724304406147 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A (2011) Trinity: reconstructing a full-length transcriptome without a genome from RNA-seq data. Nat Biotechnol 29:644–652.  https://doi.org/10.1038/nbt.1883 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, Couger MB, Eccles D, Li B, Lieber M, MacManes MD, Ott M, Orvis J, Pochet N, Strozzi F, Weeks N, Westerman R, William T, Dewey CN, Henschel R, LeDuc RD, Friedman N, Regev A (2013) De novo transcript sequence reconstruction from RNA-seq using the trinity platform for reference generation and analysis. Nat Protoc 8:1494–1512.  https://doi.org/10.1038/nprot.2013.084 https://www.nature.com/articles/nprot.2013.084#supplementary-information CrossRefPubMedGoogle Scholar
  20. Han L, Yuan Z, Feng L, Yin Y (2015) Changes in the composition and contents of pomegranate polyphenols during fruit development. In: Yuan Z, Wilkins E, Wang D (eds) Proceedings of the third international symposium on pomegranate and minor mediterranean fruits. 1089 edn. International Society for Horticultural Science (ISHS), Leuven, Belgium, pp 53–61. doi: https://doi.org/10.17660/ActaHortic.2015.1089.5
  21. Holland D, Bar-Ya’akov I (2014) Pomegranate: aspects concerning dynamics of health beneficial phytochemicals and therapeutic properties with respect to the tree cultivar and the environment. In: Yaniv Z, Dudai N (eds) Medicinal and aromatic plants of the middle-east. Springer Netherlands, Dordrecht, pp 225–239.  https://doi.org/10.1007/978-94-017-9276-9_12 CrossRefGoogle Scholar
  22. Holland D, Hatib K, Bar-Ya’akov I (2009) Pomegranate: botany, horticulture, breeding. Hort Rev 35:127–191CrossRefGoogle Scholar
  23. Holton TA, Cornish EC (1995) Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 7:1071–1083CrossRefGoogle Scholar
  24. Jaakola L (2013) New insights into the regulation of anthocyanin biosynthesis in fruits. Trends Plant Sci 18:477–483.  https://doi.org/10.1016/j.tplants.2013.06.003 CrossRefPubMedGoogle Scholar
  25. Jurenka J (2008) Therapeutic applications of pomegranate (Punica granatum L.): a review. Altern Med Rev 13:128–144PubMedGoogle Scholar
  26. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25.  https://doi.org/10.1186/gb-2009-10-3-r25 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Lee JM, Joung JG, McQuinn R, Chung MY, Fei Z, Tieman D, Klee H, Giovannoni J (2012) Combined transcriptome, genetic diversity and metabolite profiling in tomato fruit reveals that the ethylene response factor SLERF6 plays an important role in ripening and carotenoid accumulation. The Plant J 70:191–204 doi:doi: https://doi.org/10.1111/j.1365-313X.2011.04863.x, 70, 191, 204
  28. Li B, Dewey CN (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12:323.  https://doi.org/10.1186/1471-2105-12-323 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550.  https://doi.org/10.1186/s13059-014-0550-8 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Maeda H, Dudareva N (2012) The shikimate pathway and aromatic amino acid biosynthesis in plants. Annu Rev Plant Biol 63:73–105.  https://doi.org/10.1146/annurev-arplant-042811-105439 CrossRefPubMedGoogle Scholar
  31. Meisel LEE, Fonseca B, González S, Baeza-Yates R, Cambiazo V, Campos R, Gonźalez M, Orellana A, Retamales J, Silva H (2005) A rapid and efficient method for purifying high quality total RNA from peaches (Prunus persica) for functional genomics analyses. Biol Res 38:83–88CrossRefGoogle Scholar
  32. Mengiste T, Chen X, Salmeron J, Dietrich R (2003) The BOTRYTIS SUSCEPTIBLE1 gene encodes an R2R3MYB transcription factor protein that is required for biotic and abiotic stress responses in Arabidopsis. Plant Cell 15:2551–2565.  https://doi.org/10.1105/tpc.014167 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Muir RM, Ibáñez AM, Uratsu SL, Ingham ES, Leslie CA, McGranahan GH, Batra N, Goyal S, Joseph J, Jemmis ED, Dandekar AM (2011) Mechanism of gallic acid biosynthesis in bacteria (Escherichia coli) and walnut (Juglans regia). Plant Mol Biol 75:555–565.  https://doi.org/10.1007/s11103-011-9739-3 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Niemetz R, Gross GG (2005) Enzymology of gallotannin and ellagitannin biosynthesis. Phytochemistry 66:2001–2011.  https://doi.org/10.1016/j.phytochem.2005.01.009 CrossRefGoogle Scholar
  35. Ono NN, Britton MT, Fass JN, Nicolet CM, Lin D, Tian L (2011) Exploring the transcriptome landscape of pomegranate fruit peel for natural product biosynthetic gene and ssr marker discoveryf. J Integ Plant Biol 53:800–813.  https://doi.org/10.1111/j.1744-7909.2011.01073.x CrossRefGoogle Scholar
  36. Ono NN, Qin X, Wilson AE, Li G, Tian L (2016) Two UGT84 family glycosyltransferases catalyze a critical reaction of hydrolyzable tannin biosynthesis in pomegranate (Punica granatum). PLoS One 11:e0156319.  https://doi.org/10.1371/journal.pone.0156319 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Ophir R, Sherman A, Rubinstein M, Eshed R, Sharabi Schwager M, Harel-Beja R, Bar-Ya'akov I, Holland D (2014) Single-nucleotide polymorphism markers from de-novo assembly of the pomegranate transcriptome reveal germplasm genetic diversity. PLoS One 9:e88998.  https://doi.org/10.1371/journal.pone.0088998 CrossRefPubMedPubMedCentralGoogle Scholar
  38. Orgil O, Schwartz E, Baruch L, Matityahu I, Mahajna J, Amir R (2014) The antioxidative and anti-proliferative potential of non-edible organs of the pomegranate fruit and tree. LWT Food Sci Technol 58:571–577.  https://doi.org/10.1016/j.lwt.2014.03.030 CrossRefGoogle Scholar
  39. Ossipov V, Salminen J-P, Ossipova S, Haukioja E, Pihlaja K (2003) Gallic acid and hydrolysable tannins are formed in birch leaves from an intermediate compound of the shikimate pathway. Biochem Syst Ecol 31:3–16.  https://doi.org/10.1016/S0305-1978(02)00081-9 CrossRefGoogle Scholar
  40. Poirier BC, Feldman MJ, Lange BM (2018) bHLH093/NFL and bHLH061 are required for apical meristem function in Arabidopsis thaliana. Plant Signal Behav 13:e1486146.  https://doi.org/10.1080/15592324.2018.1486146 CrossRefPubMedGoogle Scholar
  41. Qin G, Xu C, Ming R, Tang H, Guyot R, Kramer EM, Hu Y, Yi X, Qi Y, Xu X, Gao Z, Pan H, Jian J, Tian Y, Yue Z, Xu Y (2017) The pomegranate (Punica granatum L.) genome and the genomics of punicalagin biosynthesis. The Plant J 91:1108–1128.  https://doi.org/10.1111/tpj.13625 CrossRefPubMedGoogle Scholar
  42. Robinson MD, Oshlack A (2010) A scaling normalization method for differential expression analysis of RNA -seq data. Genome Biol 11:R25.  https://doi.org/10.1186/gb-2010-11-3-r25 CrossRefPubMedPubMedCentralGoogle Scholar
  43. Savoi S, Wong DCJ, Degu A, Herrera JC, Bucchetti B, Peterlunger E, Fait A, Mattivi F, Castellarin SD (2017) Multi-omics and integrated network analyses reveal new insights into the systems relationships between metabolites, structural genes, and transcriptional regulators in developing grape berries (Vitis vinifera L.) exposed to water deficit. Front Plant Sci 8.  https://doi.org/10.3389/fpls.2017.01124
  44. Schwartz E, Tzulker R, Glazer I, Bar-Ya’akov I, Wiesman Z, Tripler E, Bar-Ilan I, Fromm H, Borochov-Neori H, Holland D, Amir R (2009) Environmental conditions affect the color, taste, and antioxidant capacity of 11 pomegranate accessions’ fruits. J Agric Food Chem 57:9197–9209.  https://doi.org/10.1021/jf901466c CrossRefPubMedGoogle Scholar
  45. Seeram NP, Schulman RN, Heber D (2006) Pomegranates: ancient roots to modern medicine. CRC Press Taylor & Francis Group, Boca RatonGoogle Scholar
  46. Singh SA, Christendat D (2006) Structure of Arabidopsis dehydroquinate dehydratase-shikimate dehydrogenase and implications for metabolic channeling in the shikimate pathway. Biochemistry 45:7787–7796.  https://doi.org/10.1021/bi060366+ CrossRefPubMedGoogle Scholar
  47. Tzulker R, Glazer I, Bar-Ilan I, Holland D, Aviram M, Amir R (2007) Antioxidant activity, polyphenol content, and related compounds in different fruit juices and homogenates prepared from 29 different pomegranate accessions. J Agric Food Chem 55:9559–9570.  https://doi.org/10.1021/jf071413n CrossRefPubMedGoogle Scholar
  48. Winkel-Shirley B (2001) Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol 126:485–493CrossRefGoogle Scholar
  49. Wong DCJ, Matus JT (2017) Constructing integrated networks for identifying new secondary metabolic pathway regulators in grapevine: recent applications and future opportunities. Front Plant Sci 8.  https://doi.org/10.3389/fpls.2017.00505
  50. Yuan Z et al. (2017) The pomegranate (Punica granatum L.) genome provides insights into fruit quality and ovule developmental biology. Plant Biotechnol J:n/a-n/a doi: https://doi.org/10.1111/pbi.12875 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Rotem Harel-Beja
    • 1
  • Li Tian
    • 2
  • Shiri Freilich
    • 1
  • Rida Habashi
    • 3
  • Hamutal Borochov-Neori
    • 4
  • Tamar Lahav
    • 1
  • Taly Trainin
    • 1
  • Adi Doron-Faigenboim
    • 5
  • Ron Ophir
    • 5
  • Irit Bar-Ya’akov
    • 1
  • Rachel Amir
    • 3
  • Doron Holland
    • 1
    Email author
  1. 1.Newe Ya’ar Research Center, Agricultural Research OrganizationRamat YishayIsrael
  2. 2.Department of Plant SciencesUniversity of California DavisDavisUSA
  3. 3.MIGAL - Galilee Research InstituteKiryat ShmonaIsrael
  4. 4.Southern Arava R&DHevel EilotIsrael
  5. 5.Plant Sciences, Agricultural Research OrganizationBet DaganIsrael

Personalised recommendations