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.
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References
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
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
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
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 57:289–300
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
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
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
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
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
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
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
Feder A et al (2015) A kelch domain-containing F-box coding gene negatively regulates flavonoid accumulation in muskmelon. Plant Physiol 169:1714
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
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
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
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
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
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
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
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
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
Holland D, Hatib K, Bar-Ya’akov I (2009) Pomegranate: botany, horticulture, breeding. Hort Rev 35:127–191
Holton TA, Cornish EC (1995) Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 7:1071–1083
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
Jurenka J (2008) Therapeutic applications of pomegranate (Punica granatum L.): a review. Altern Med Rev 13:128–144
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
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
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
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
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
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 38:83–88
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
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
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
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
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
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
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
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
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
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
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
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
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
Seeram NP, Schulman RN, Heber D (2006) Pomegranates: ancient roots to modern medicine. CRC Press Taylor & Francis Group, Boca Raton
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+
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
Winkel-Shirley B (2001) Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol 126:485–493
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
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
Acknowledgements
We thank Kamel Hatib for orchard management.
Data archiving statement
The raw sequencing data is archived in NCBI No. PRJNA521857.
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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.
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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.
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Communicated by A. M. Dandekar
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Harel-Beja, R., Tian, L., Freilich, S. et al. Gene expression and metabolite profiling analyses of developing pomegranate fruit peel reveal interactions between anthocyanin and punicalagin production. Tree Genetics & Genomes 15, 22 (2019). https://doi.org/10.1007/s11295-019-1329-6
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DOI: https://doi.org/10.1007/s11295-019-1329-6