Citrus phenylpropanoids and defense against pathogens. Part I: Metabolic 1 profiling in elicited fruits 2 3 Ana-Rosa Ballester a,b,c , M. Teresa Lafuente a , Ric C. H. de Vos b,c , Arnaud G. 4 Bovy b,c , Luis González-Candelas a,* 5 6 7 a Instituto de Agroquímica y Tecnología de Alimentos. Consejo Superior de 8 Investigaciones Científicas (IATA-CSIC). Av. Agustín Escardino 7. Paterna, 9 46980-Valencia. Spain. 10 b Plant Research International. P.O. Box 16. 6700 AA Wageningen, The 11 Netherlands 12 c Centre for Biosystems Genomics, 6700 PB, Wageningen, The Netherlands 13 14 *Corresponding author: 15 Tel: +34 963900022; fax; +34 963636301 16 e-mail address: [email protected]17 18
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Citrus phenylpropanoids and defense against pathogens. Part I: Metabolic 1
profiling in elicited fruits 2
3
Ana-Rosa Ballestera,b,c, M. Teresa Lafuentea, Ric C. H. de Vosb,c, Arnaud G. 4
Bovyb,c, Luis González-Candelasa,* 5
6
7
aInstituto de Agroquímica y Tecnología de Alimentos. Consejo Superior de 8
Investigaciones Científicas (IATA-CSIC). Av. Agustín Escardino 7. Paterna, 9
46980-Valencia. Spain. 10
bPlant Research International. P.O. Box 16. 6700 AA Wageningen, The 11
Netherlands 12
cCentre for Biosystems Genomics, 6700 PB, Wageningen, The Netherlands 13
However, this is the first report linking citrusnin A with the resistance of citrus 469
fruit to infection caused by P. digitatum. Furthermore, as far as we know, this 470
compound has not been yet related to the resistance of citrus or other fruits to 471
pathogens causing postharvest losses. 472
Compound 20 showed a similar λmax at 267.6 nm, but an accurate mass of m/z 473
215.1076 [M-H]-, corresponding to a molecular formula of C14H16O2. Based on 474
comparison with different metabolite databases, this compound was putatively 475
identified as drupanin aldehyde (i.e. 3-[4-hydroxy,3-(3-methyl-2-butenyl)-476
phenyl]-2-(E)-propenal or 4-hydroxy-3-prenylcinnamaldehyde) (Fig. 2D). This 477
compound was previously isolated from the peel of wounded grapefruits (Citrus 478
paradise) and oranges (C. sinensis) (Stange, Midland, Eckert, & Sims, 1993). It 479
is also known that drupanin itself, isolated from Baccharis sp., has antifungal 480
and antibacterial activity (Bisogno, Mascoti, Sanchez, Garibotto, Giannini, 481
Kurina-Sanz et al., 2007; Feresin, Tapia, Gimenez, Ravelo, Zacchino, Sortino et 482
al., 2003). However, its involvement in the resistance of citrus fruits to 483
pathogenic fungi has not been reported until now. Moreover, it has to be noted 484
that although citrusnin A and drupanin aldehyde levels increased in response to 485
the elicitor treatment, none of these compounds were detected in response to 486
P. digitatum infection (Ballester, Lafuente, & González-Candelas, Submitted). In 487
light of their structures both citrusnin A and drupanin could be biochemically 488
derived from precursors in the first part of the phenylpropanoid pathway, but the 489
genes and enzymes involved in their synthesis are unknown yet. The study of 490
the possible antifungal activity of these compounds against P. digitatum has not 491
been undertaken because they are not commercially available and their 492
concentration in the peel of citrus fruits is very low. However, the results 493
presented in this work encourage further research in this direction. 494
Since the HPLC-FD analysis of phenolic metabolites revealed the induction of 495
fluorescent compounds in the peel of elicited fruits, we checked the presence of 496
fluorescence in elicited oranges using a stereoscopic zoom microscope 497
SMZ800 with Epi-fluorescence attachment (Nikon) (Fig. 3). The amount of 498
fluorescence in the transversal cut of peel oranges was higher in elicited fruits 499
than in non-treated fruits. The fluorescence was concentric around the 500
inoculation point, which reinforces the idea that the elicitor treatment induced 501
only local disease resistance and that the effect is limited to only a small area 502
around the origin of infection (1-4 mm distance from the inoculation site). 503
Metabolic profiling results of this study strongly suggest an implication of 504
phenylpropanoids, flavonoids and their derivatives in the induction of resistance 505
in citrus fruit, being especially relevant the induction of scoparone and three 506
other fluorescent phenolic compounds that have not been previously related to 507
the resistance of citrus fruit against disease caused by P. digitatum. Two of 508
them, citrusnin A and drupanin aldehyde, were putatively identified and showed 509
very relevant increases in elicited fruits. Therefore, their implication in citrus fruit 510
responses deserves further investigation. Finally, our results indicate that the 511
highest inductions in phenylpropanoids were found in the albedo, whereas the 512
highest metabolite concentrations were detected in the external tissue. These 513
results reinforce the idea that the internal tissue is more susceptible to 514
P. digitatum infection and it is the one that should increase to a greater extent 515
the defensive barriers in order to avoid the progression of the fungus. 516
517
Acknowledgements 518
We thank Drs J. Sendra, E. Sentandreu (IATA-CSIC, Valencia-Spain) and Bert 519
Schipper (Plant Research International, Wageningen-The Netherlands) for their 520
assistance with HPLC and LC-PDA-QTOF-MS analyses, and María Dolores 521
Gómez for her help with the microscopy at the Instituto de Biología Molecular y 522
Celular de Plantas (IBMCP-CSIC-UPV, Valencia- Spain). The technical 523
assistance of Ana Izquierdo (IATA-CSIC, Valencia-Spain) is gratefully 524
acknowledged. ARB, RdV and AB acknowledge the Centre for Biosystems 525
Genomics, which is part of the Netherlands Genomics Initiative, for additional 526
funding. This work was supported by Research Grants AGL2008-04828-C03-527
02, AGL2009-11969 and CONSOLIDER FUNC-FOOD from the Spanish 528
Ministry of Science and Technology, and PROMETEO/2010/010 from the 529
Generalitat Valenciana. 530
531
References 532
Afek, U., Orenstein, J., Carmeli, S., Rodov, V., & Joseph, M. B. (1999). 533 Umbelliferone, a phytoalexin associated with resistance of immature 534 Marsh grapefruit to Penicillium digitatum. Phytochemistry, 50(7), 1129-535 1132. 536
Almada-Ruiz, E., Martínez-Téllez, M. A., Hernández-Alamos, M. M., Vallejo, S., 537 Primo-Yúfera, E., & Vargas-Arispuro, I. (2003). Fungicidal potential of 538 methoxylated flavones from citrus for in vitro control of Colletotrichum 539 gloeosporioides, causal agent of anthracnose disease in tropical fruits. 540 Pest Management Science, 59 1245-1249. 541
Arcas, M. C., Botía, J. M., Ortuño, A., & Del Río, J. A. (2000). UV irradiation 542 alters the levels of flavonoids involved in the defence mechanism of 543 Citrus aurantium fruits against Penicillium digitatum. European Journal of 544 Plant Pathology, 106(7), 617-622. 545
Arras, G. (1996). Mode of action of an isolate of Candida famata in biological 546 control of Penicillium digitatum in orange fruits. Postharvest Biology and 547 Technology, 8(3), 191-198. 548
Ballester, A. R., Izquierdo, A., Lafuente, M. T., & González-Candelas, L. (2010). 549 Biochemical and molecular characterization of induced resistance 550 against Penicillium digitatum in citrus fruit. Postharvest Biology and 551 Technology, 56, 31-38. 552
Ballester, A. R., Lafuente, M. T., Forment, J., Gadea, J., De Vos, C. H. R., 553 Bovy, A. G., & González-Candelas, L. (2011). Transcriptomic profiling of 554 citrus fruit peel tissues reveals fundamental effects of phenylpropanoids 555 and ethylene on induced resistance. Molecular Plant Pathology, 12(9), 556 879-897. 557
Ballester, A. R., Lafuente, M. T., & González-Candelas, L. (2006). Spatial study 558 of antioxidant enzymes, peroxidase and phenylalanine ammonia-lyase in 559 the citrus fruit-Penicillium digitatum interaction. Postharvest Biology and 560 Technology, 39(2), 115-124. 561
Ballester, A. R., Lafuente, M. T., & González-Candelas, L. (Submitted). Citrus 562 phenylpropanoids and defense against pathogens. Part II: Gene 563 expression and metabolite accumulation in the response of fruits to 564 Penicillium digitatum infection. Accompanying manuscript submitted to 565 Food Chemistry. 566
Ben Yehoshua, S., Rodov, V., Kim, J. J., & Carmeli, S. (1992). Preformed and 567 induced antifungal materials of citrus fruit in relation to the enhancement 568 of decay resistance by heat and ultraviolet treatment. Journal of 569 Agricultural and Food Chemistry, 40, 1217-1221. 570
Bisogno, F., Mascoti, L., Sanchez, C., Garibotto, F., Giannini, F., Kurina-Sanz, 571 M., & Enriz, R. (2007). Structure−antifungal activity relationship of 572 cinnamic acid derivatives. Journal of Agricultural and Food Chemistry, 573 55(26), 10635-10640. 574
Bourgaud, F., Hehn, A., Larbat, R., Doerper, S., Gontier, E., Kellner, S., & 575 Matern, U. (2006). Biosynthesis of coumarins in plants: a major pathway 576 still to be unravelled for cytochrome P450 enzymes. Phytochemistry 577 Reviews, 5(2-3), 293-308. 578
D'Hallewin, G., Schirra, M., Manueddu, E., Piga, A., & Ben Yehoshua, S. 579 (1999). Scoparone and scopoletin accumulation and ultraviolet-C 580 induced resistance to postharvest decay in oranges as influenced by 581 harvest date. Journal of the American Society for Horticultural Science, 582 124(6), 702-707. 583
Del Río, J. A., Arcas, M. C., Benavente-García, O., & Ortuño, A. (1998). Citrus 584 polymethoxylated flavones can confer resistance against Phytophthora 585 citrophthora, Penicillium digitatum, and Geotrichum species. Journal of 586 Agricultural and Food Chemistry, 46(10), 4423-4428. 587
Del Río, J. A., Gómez, P., Báidez, A., Arcas, M. C., Botía, J. M., & Ortuño, A. 588 (2004). Changes in the levels of polymethoxyflavones and flavanones as 589 part of the defense mechanism of Citrus sinensis (cv. Valencia Late) 590
fruits against Phytophthora citrophthora. Journal of Agricultural and Food 591 Chemistry, 52(7), 1913-1917. 592
Dixon, R. A., & Paiva, N. L. (1995). Stress-induced phenylpropanoid 593 metabolism. The Plant Cell, 7(7), 1085-1097. 594
Droby, S., Chalutz, E., Horev, B., Cohen, L., Gaba, V., Wilson, C. L., & 595 Wisniewski, M. (1993). Factors affecting UV-induced resistance in 596 grapefruit against the green mold decay caused by Penicillium digitatum. 597 Plant Pathology, 42(3), 418-424. 598
Droby, S., Vinokur, V., Weiss, B., Cohen, L., Daus, A., Goldschmidt, E. E., & 599 Porat, R. (2002). Induction of resistance to Penicillium digitatum in 600 grapefruit by the yeast biocontrol agent Candida oleophila. 601 Phytopathology, 92(4), 393-399. 602
Fajardo, J. E., McCollum, T. G., McDonald, R. E., & Mayer, R. T. (1998). 603 Differential induction of proteins in orange flavedo by biologically based 604 elicitors and challengen by Penicillium digitatum Sacc. Biological Control, 605 13(3), 143-151. 606
Feresin, G. E., Tapia, A., Gimenez, A., Ravelo, A. G., Zacchino, S., Sortino, M., 607 & Schmeda-Hirschmann, G. (2003). Constituents of the Argentinian 608 medicinal plant Baccharis grisebachii and their antimicrobial activity. 609 Journal of Ethnopharmacology, 89(1), 73-80. 610
Gonzalez-Candelas, L., Alamar, S., Sanchez-Torres, P., Zacarias, L., & Marcos, 611 J. (2010). A transcriptomic approach highlights induction of secondary 612 metabolism in citrus fruit in response to Penicillium digitatum infection. 613 BMC Plant Biology, 10(1), 194-211. 614
Goulas, V., & Manganaris, G. A. (2012). Exploring the phytochemical content 615 and the antioxidant potential of Citrus fruits grown in Cyprus. Food 616 Chemistry, 131(1), 39-47. 617
Hammerschmidt, R. (1999). Induced disease resistance: how do induced plants 618 stop pathogens? Physiological and Molecular Plant Pathology, 55(2), 77-619 84. 620
Hammerschmidt, R. (2009). Chapter 5 Systemic Acquired Resistance. In L. C. 621 V. Loon (Ed.), Advances in Botanical Research, vol. Volume 51 (pp. 173-622 222): Academic Press. 623
Harborne, J. B., & Williams, C. A. (2000). Advances in flavonoid research since 624 1992. Phytochemistry, 55(6), 481-504. 625
Hershkovitz, V., Ben-Dayan, C., Raphael, G., Pasmanik-Chor, M., Liu, J. I. A., 626 Belausov, E., Aly, R., Wisniewski, M., & Droby, S. (2011). Global 627 changes in gene expression of grapefruit peel tissue in response to the 628 yeast biocontrol agent Metschnikowia fructicola. Molecular Plant 629 Pathology, 13(4), 338–349. 630
Ibrahim, R. K., Bruneau, A., & Bantignies, B. (1998). Plant O-631 methyltransferases: Molecular analysis, common signature and 632 classification. Plant Molecular Biology, 36(1), 1-10. 633
Kavanagh, J. A., & Wood, R. K. S. (1967). The role of wounds in the infection of 634 oranges by Penicillium digitatum Sacc. Annals of Applied Biology, 60, 635 375-383. 636
Kim, H. G., Kim, G. S., Lee, J. H., Park, S., Jeong, W. Y., Kim, Y. H., Kim, J. H., 637 Kim, S. T., Cho, Y. A., Lee, W. S., Lee, S. J., Jin, J. S., & Shin, S. C. 638 (2011). Determination of the change of flavonoid components as the 639 defence materials of Citrus unshiu Marc. fruit peel against Penicillium 640
digitatum by liquid chromatography coupled with tandem mass 641 spectrometry. Food Chemistry, 128(1), 49-54. 642
Kim, J. J., Ben Yehoshua, S., Shapiro, B., Henis, Y., & Carmeli, S. (1991). 643 Accumulation of scoparone in heat-treated lemon fruit inoculated with 644 Penicillium digitatum Sacc. Plant Physiology, 97, 880-885. 645
Kuniga, T., Tsumura, T., Matsuo, Y., & Matsumoto, R. (2006). Changes in 646 scoparone concentrations in citrus cultivars after ultraviolet radiation. 647 Journal of the Japanese Society for Horticultural Science, 75(4), 328-648 330. 649
Lafuente, M. T., Ballester, A. R., Calejero, J., Zacarías, L., & González-650 Candelas, L. (2011). Effect of heat-conditioning treatments on quality and 651 phenolic composition of cold stored ‘Fortune’ mandarins. Food 652 Chemistry, 128(4), 1080-1086. 653
López-García, B., González-Candelas, L., Pérez-Payá, E., & Marcos, J. F. 654 (2000). Identification and characterization of a hexapeptide with activity 655 against phytopathogenic fungi that cause postharvest decay in fruits. 656 Molecular Plant-Microbe Interactions, 13(8), 837-846. 657
Moco, S., Bino, R. J., Vorst, O., Verhoeven, H. A., de Groot, J., van Beek, T. A., 658 Vervoort, J., & de Vos, C. H. R. (2006). A liquid chromatography-mass 659 spectrometry-based metabolome database for tomato. Plant Physiology, 660 141(4), 1205-1218. 661
Nafussi, B., Ben Yehoshua, S., Rodov, V., Peretz, J., Ozer, B. K., & D'Hallewin, 662 G. (2001). Mode of action of hot-water dip in reducing decay of lemon 663 fruit. Journal of Agricultural and Food Chemistry, 49(1), 107-113. 664
Nogata, Y., Sakamoto, K., Shiratsuchi, H., Ishii, T., Yano, M., & Ohta, H. (2006). 665 Flavonoid composition of fruit tissues of Citrus species. Bioscience, 666 Biotechnology, and Biochemistry, 70(1), 178-192. 667
Ortuño, A., Báidez, A., Gómez, P., Arcas, M. C., Porras, I., García-Lidón, A., & 668 Del Río, J. A. (2006). Citrus paradisi and Citrus sinensis flavonoids: Their 669 influence in the defence mechanism against Penicillium digitatum. Food 670 Chemistry, 98, 351-358. 671
Ortuño, A., Díaz, L., Alvarez, N., Porras, I., García-Lidón, A., & Del Río, J. A. 672 (2011). Comparative study of flavonoid and scoparone accumulation in 673 different Citrus species and their susceptibility to Penicillium digitatum. 674 Food Chemistry, 125(1), 232-239. 675
Porat, R., McCollum, T. G., Vinokur, V., & Droby, S. (2002). Effects of various 676 elicitors on the transcription of a β-1,3-endoglucanase gene in citrus fruit. 677 Journal of Phytopathology, 150, 70-75. 678
Porat, R., Vinokur, V., Holland, D., McCollum, T. G., & Droby, S. (2001). 679 Isolation of a citrus chitinase cDNA and characterization of its expression 680 in response to elicitation of fruit pathogen resistance. Journal of Plant 681 Physiology, 158(12), 1585-1590. 682
Rodov, V., Ben Yehoshua, S., Kim, J. J., Shapiro, B., & Ittah, Y. (1992). 683 Ultraviolet illumination induces scoparone production in kumquat and 684 orange fruit and improves decay resistance. Journal of the American 685 Society for Horticultural Science, 117(5), 788-792. 686
Ruelas, C., Tiznado-Hernández, M. E., Sánchez-Estrada, A., Robles-Burgueño, 687 M. R., & Troncoso-Rojas, R. (2006). Changes in phenolic acid content 688 during Alternaria alternata infection in tomato fruit. Journal of 689 Phytopathology, 154(4), 236-244. 690
Sánchez-Ballesta, M. T., Lluch, Y., Gosalbes, M. J., Zacarías, L., Granell, A., & 691 Lafuente, M. T. (2003). A survey of genes differentially expressed during 692 long-term heat-induced chilling tolerance in citrus fruit. Planta, 218(1), 693 65-70. 694
Sinbo, Y., Nakamura, Y., Altaf-Ul-Amin, M., Asahi, H., Kurokawa, K., Arita, M., 695 Saito, K., Ohta, D., Shibata, D., & Kanaya, S. (2006). KNApSAcK: A 696 comprehensive species-metabolite relationship database. In K. Saito, R. 697 A. Dixon & L. Willmitzer (Eds.), Biotechnology in Agriculture and 698 Forestry, vol. 57 (pp. 165-181). Springer-Verlag, Berlin Heilderberg. 699
Stange, Jr., Midland, S. L., Eckert, J. W., & Sims, J. J. (1993). An antifungal 700 compound produced by grapefruit and Valencia orange after wounding of 701 the peel. Journal of Natural Products, 56(9), 1627-1629. 702
van Loon, L. C., Rep, M., & Pieterse, C. M. J. (2006). Significance of inducible 703 defense-related proteins in infected plants. Annual Review of 704 Phytopathology, 44(1), 135-162. 705
Venditti, T., Molinu, M. G., Dore, A., Agabbio, M., & D'Hallewin, G. (2005). 706 Sodium carbonate treatment induces scoparone accumulation, structural 707 changes, and alkalinization in the albedo of wounded Citrus fruits. 708 Journal of Agricultural and Food Chemistry, 53(9), 3510-3518. 709
Watanabe, K., Myiyakado, M., Ohno, N., Ota, T., & Nonaka, F. (1985). 710 Citrusnin-A: A new antibacterial substance from leaves of Citrus 711 natsudaidai. Journal of Pesticide Science, 10, 137-140. 712
713 714
715
Figure Captions 716
Fig. 1. Flow chart of the experimental design. Solid vertical arrows indicate the 717
temperature and duration of the incubation period. The induction of resistance 718
treatment consisted of fruit inoculated with P. digitatum (indicated in the chart as 719
Pdig) and then incubated for 1 day at 20 ºC before being transferred at 37 ºC for 720
3 day to stop pathogen progress. At the end of this heat treatment, fruit were 721
maintained at 20 ºC. Tissue samples were taken from 15 fruits at 4, 5 and 7 d 722
after the beginning of the experiment (IC4, IC5 and IC7, respectively), and other 723
15 oranges, with 4 wounds per fruit, were inoculated with P. digitatum to assess 724
the effectiveness of the treatment. Infection was allowed to progress for 6 d, 725
when disease severity was determined. Control non-treated fruits (NT) were 726
sampled at the beginning of the experiment. 727
728
Fig. 2. Metabolic profiling of elicited citrus-fruits. (A) Chromatogram of flavedo 729
(F) from non-treated (NT) an infected-cured oranges at 4 (IC4), 5 (IC5) and 7 730
(IC7) days after the beginning of the experiment obtained by HPLC-FD. (B) UV 731
spectra of induced compounds. (C) Mass spectra of compounds 18, 8 and 20. 732
(D) Chemical structure of compounds (18) citrusnin A, (8) scoparone, and (20) 733
drupanin aldehyde. 734
735
Fig. 3. Transversal cuts of the peel of citrus fruits using stereoscopy microscope 736
equipped with a fluorescence system. Photographs of non-treated (A, C) and 737
P. digitatum infected and cured (B, D) fruits using white light (A, B) and 738
fluorescence (C, D). Transversal cuts were made 7 days after the beginning of 739
the experiment. 740
1
Table 1. Phenylpropanoid and flavonoid concentration (µg g-1 fresh weight) in the flavedo of non-treated (FNT) and elicited Navelate oranges 4, 5 and 7 days after the beginning of the experiment (FIC4, FIC5 and FIC7, respectively). Results represent the mean of at least two biological replicates ± standard deviation (SD). Different letters among treatments indicate statistically significant differences according to the LSD test (p<0.05). Compound order based on families and retention time (Ballester, Lafuente, & González-Candelas, accompanying papper submitted to Food Chemistry).
3 Eriocitrin Flavanone 34.8 ± 0.3 a 17.1 ± 0.3 c 18.2 ± 1.5 c 24.6 ± 0.1 b 4 Narirutin Flavanone 33.1 ± 2.6 a nd nd 14.9 ± 17.5 a 7 Hesperidin Flavanone 1840.9 ± 74.8 b 2103.9 ± 118.9 a 2179.1 ± 41.4 a 1979.0 ± 83.2 ab 9 Didymin Flavanone 56.8 ± 9.3 b 67.1 ± 4.0 ab 67.3 ± 8.8 ab 78.6 ± 11.8 a 1 Chlorogenic acid Cinnamic acid 161.0 ± 26.4 a 134.0 ± 3.3 a 149.8 ± 3.5 a 152.9 ± 15.9 a 2 Caffeic acid Cinnamic acid 68.8 ± 16.7 a 64.3 ± 14.0 a 57.5 ± 2.3 a 60.5 ± 7.2 a 5 Isorhoifolin Flavone 65.8 ± 9.0 b 130.7 ± 29.6 a 59.5 ± 7.1 b 64.8 ± 1.1 b 6 Diosmin Flavone 26.5 ± 0.9 a 22.2 ± 3.2 a 26.4 ± 1.0 a 24.1 ± 9.6 a
10 Isosinensetin PMF 3.5 ± 1.1 a 2.7 ± 0.9 a 3.6 ± 0.4 a 4.0 ± 0.4 a 11 Hexamethyl-O-gossypetin PMF 1.0 ± 0.4 ab 0.7 ± 0.2 b 1.7 ± 0.2 a 1.3 ± 0.6 ab 12 Sinensetin PMF 100.5 ± 6.4 b 119.7 ± 10.0 a 102.4 ± 0.3 b 80.8 ± 3.6 c 13 Hexamethyl-O-quercetagetin* PMF 420.7 ± 11.1 b 428.7 ± 58.8 b 579.5 ± 3.9 a 532.3 ± 56.3 a 14 Nobiletin PMF 29.7 ± 1.4 b 29.0 ± 1.6 b 35.9 ± 1.2 a 30.9 ± 2.3 b 15 Tetramethyl-O-scutellarein PMF 140.8 ± 21.6 a 153.2 ± 9.2 a 141.8 ± 3.5 a 128.6 ± 10.9 a 16 Heptamethoxyflavone PMF 125.4 ± 11.0 b 132.2 ± 2.6 ab 142.8 ± 3.3 a 126.2 ± 2.9 ab 17 Tangeretin PMF 88.7 ± 14.1 c 110.1 ± 4.6 bc 138.2 ± 4.4 a 123.5 ± 9.1 ab
8 Scoparone (FD) Coumarin nd 29.7 ± 14.9 b 90.5 ± 7.7 a 54.0 ± 2.7 b 18 Citrusnin A (FD)* 28.4 ± 2.3 c 755.4 ± 51.6 bc 1121.2 ± 282.7 b 3249.7 ± 707.8 a 19 Compound 19 (FD)* 14.2 ± 5.5 c 189.2 ± 68.2 b 408.4 ± 25.0 a 325.4 ± 32.6 a 20 Drupanin aldehyde (FD)* 178.4 ± 17.4 c 930.0 ± 78.6 b 1538.5 ± 33.3 b 3310.5 ± 653.0 a
* values represent the area (mAU s) of the peak in the chromatogram (FD) indicates that those values were obtained with the fluorescent detector. nd. non-detected compound
2
Table 2. Phenylpropanoid and flavonoid concentration (µg g-1 fresh weight) in the albedo of non-treated (ANT) and elicited Navelate
oranges 4, 5 and 7 days after the beginning of the experiment (AIC4, AIC5 and AIC7, respectively) detected by HPLC-PDA-FD.
Results represent the mean of at least two biological replicates ± standard deviation (SD). Different letters among treatments
indicate statistically significant differences according to the LSD test (p<0.05). Compound order based on families and retention
time (Ballester, Lafuente, & González-Candelas, accompanying papper submitted to Food Chemistry). ANT AIC4 AIC5 AIC7
No. Compound Family Conc. SD Conc. SD Conc. SD Conc. SD 3 Eriocitrin Flavanone 15.3 ± 6.1 a 14.3 ± 2.2 a 19.0 ± 0.1 a 11.8 ± 0.3 a 4 Narirutin Flavanone 434.3 ± 35.3 a 308.2 ± 65.9 b 404.7 ± 9.6 ab 373.2 ± 15.1 ab 7 Hesperidin Flavanone 2,027.1 ± 117.3 a 1,518.3 ± 107.8 b 1,818.1 ± 107.3 a 2,061.2 ± 67.0 a 9 Didymin Flavanone 348.6 ± 30.8 a 254.3 ± 47.3 b 327.7 ± 0.9 ab 307.1 ± 17.9 ab 1 Chlorogenic acid Cinnamic acid 14.2 ± 3.0 b 25.4 ± 5.0 a 24.7 ± 0.5 a 10.6 ± 1.4 b
12 Sinensetin PMF 3.9 ± 0.6 bc 6.7 ± 0.9 ab 8.9 ± 1.1 a 3.7 ± 1.6 c 13 Hexamethyl-O-quercetagetin* PMF 39.3 ± 3.9 b 58.8 ± 3.4 a 64.1 ± 1.6 a 63.0 ± 4.6 a 14 Nobiletin PMF 1.4 ± 0.0 b 2.8 ± 0.9 a 3.4 ± 0.2 a 2.6 ± 0.2 ab 15 Tetramethyl-O-scutellarein PMF 12.5 ± 2.5 a 13.0 ± 1.2 a 11.1 ± 1.6 a 13.7 ± 0.3 a 16 Heptamethoxyflavone PMF 20.4 ± 3.1 b 32.3 ± 3.2 a 37.1 ± 1.6 a 33.8 ± 2.8 a 17 Tangeretin PMF 5.9 ± 0.5 c 11.2 ± 2.1 b 16.2 ± 0.0 a 14.2 ± 2.3 ab
8 Scoparone (FD) Coumarin nd 6.2 ± 1.4 b 12.2 ± 0.7 ab 24.7 ± 7.6 a 18 Citrusnin A (FD)* nd 81.9 ± 39.8 b 177.8 ± 58.3 b 520.9 ± 34.3 a 19 Compound 19 (FD)* nd 120.6 ± 49.9 a 141.6 ± 35.4 a 147.4 ± 74.5 a 20 Drupanin aldehyde (FD)* nd 396.6 ± 74.9 b 518.1 ± 97.3 b 970.6 ± 127.7 a
* values represent the area (mAU s) of the peak in the chromatogram
(FD) indicates that those values are obtained from the fluorescent detector.