RNA-Seq analysis reveals an essential role of tyrosine metabolism pathway in response to root-rot infection in Gerbera hybrida
Autoři:
Nigarish Munir aff001; Chunzhen Cheng aff001; Chaoshui Xia aff002; Xuming Xu aff002; Muhammad Azher Nawaz aff003; Junaid Iftikhar aff004; Yukun Chen aff001; Yuling Lin aff001; Zhongxiong Lai aff001
Působiště autorů:
Institute of Horticultural Biotechnology, Fujian Agriculture and Forestry University, Fuzhou, China
aff001; Sanming Academy of Agricultural Sciences, Sanming, Fujian, China
aff002; Department of Horticulture, College of Agriculture, University of Sargodha, Sargodha, Pakistan
aff003; Fujian Provincial Key Labortary of Plant Functional Biology, College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, China
aff004
Vyšlo v časopise:
PLoS ONE 14(10)
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pone.0223519
Souhrn
Gerbera hybrida is one of the top five cut flowers across the world, it is host for the root rot causing parasite called Phytophthora cryptogea. In this study, plantlets of healthy and root-rot pathogen-infected G. hybrida were used as plant materials for transcriptome analyis using high-throughput Illumina sequencing technique. A total 108,135 unigenes were generated with an average length of 727 nt and N50 equal to 1274 nt out of which 611 genes were identified as DEGs by DESeq analyses. Among DEGs, 228 genes were up-regulated and 383 were down-regulated. Through this annotated data and Kyoto encyclopedia of genes and genomes (KEGG), molecular interaction network, transcripts accompanying with tyrosine metabolism, phenylalanine, tyrosine, and tryptophan biosynthesis, phenylpropanoid and flavonoid biosynthesis, and plant hormone signal transduction pathways were thoroughly observed considering expression pattern. The involvement of DEGs in tyrosine metabolism pathway was validated by real-time qPCR. We found that genes related with tyrosine metabolism were activated and up-regulated against stress response. The expression of GhTAT, GhAAT, GhHPD, GhHGD and GhFAH genes was significantly increased in the leaves and petioles at four and six dpi (days post inoculation) as compared with control. The study predicts the gene sequences responsible for the tyrosine metabolism pathway and its responses against root-rot resistance in gerbera plant. In future, identification of such genes is necessary for the better understanding of rot resistance mechanism and to develop a root rot resistance strategy for ornamental plants.
Klíčová slova:
Gene expression – Biosynthesis – Leaves – Gene ontologies – Metabolic pathways – Amino acid metabolism – Tyrosine – Aminotransferases
Zdroje
1. Fu Y, Esselink GD, Visser RGF, van Tuyl JM, Arens P. Transcriptome analysis of Gerbera hybrida including in silico confirmation of defense genes found. Front Plant Sci. Frontiers; 2016;7: 247.
2. Kuang Q, Li L, Peng J, Sun S, Wang X. Transcriptome analysis of Gerbera hybrida ray florets: putative genes associated with gibberellin metabolism and signal transduction. PLoS One. Public Library of Science; 2013;8: e57715. doi: 10.1371/journal.pone.0057715 23472101
3. Laitinen RAE, Broholm S, Albert VA, Teeri TH, Elomaa P. Patterns of MADS-box gene expression mark flower-type development in Gerbera hybrida (Asteraceae). BMC Plant Biol. BioMed Central; 2006;6: 11.
4. Hansen H V. taxonomic revision of the genus Gerbera (Compositae, Mutisieae) sections Gerbera, Parva, Piloselloides (in Africa), and Lasiopus. Council for Nordic Publications in Botany; 1985.
5. Laitinen RAE, Immanen J, Auvinen P, Rudd S, Alatalo E, Paulin L, et al. Analysis of the floral transcriptome uncovers new regulators of organ determination and gene families related to flower organ differentiation in Gerbera hybrida (Asteraceae). Genome Res. Cold Spring Harbor Lab; 2005;15: 475–486. doi: 10.1101/gr.3043705 15781570
6. Zhang T, Zhao Y, Juntheikki I, Mouhu K, Broholm SK, Rijpkema AS, et al. Dissecting functions of SEPALLATA‐like MADS box genes in patterning of the pseudanthial inflorescence of Gerbera hybrida. New Phytol. Wiley Online Library; 2017;216: 939–954. doi: 10.1111/nph.14707 28742220
7. Deng X, Bashandy H, Ainasoja M, Kontturi J, Pietiäinen M, Laitinen RAE, et al. Functional diversification of duplicated chalcone synthase genes in anthocyanin biosynthesis of Gerbera hybrida. New Phytol. Wiley Online Library; 2014;201: 1469–1483. doi: 10.1111/nph.12610 24266452
8. Koskela S, Söderholm PP, Ainasoja M, Wennberg T, Klika KD, Ovcharenko V V, et al. Polyketide derivatives active against Botrytis cinerea in Gerbera hybrida. Planta. Springer; 2011;233: 37–48. doi: 10.1007/s00425-010-1277-8 20878179
9. Brisco-McCann EI, Hausbeck MK. Diseases of Gerbera. Handb Florists’ Crop Dis. Springer; 2018; 533–559.
10. Nawaz MA, Chen C, Shireen F, Zheng Z, Sohail H, Afzal M, et al. Genome-wide expression profiling of leaves and roots of watermelon in response to low nitrogen. BMC Genomics. BioMed Central; 2018;19: 456.
11. Mizuno H, Kawahara Y, Sakai H, Kanamori H, Wakimoto H, Yamagata H, et al. Massive parallel sequencing of mRNA in identification of unannotated salinity stress-inducible transcripts in rice (Oryza sativa L.). BMC Genomics. BioMed Central; 2010;11: 683.
12. Schilmiller AL, Miner DP, Larson M, McDowell E, Gang DR, Wilkerson C, et al. Studies of a biochemical factory: tomato trichome deep expressed sequence tag sequencing and proteomics. Plant Physiol. Am Soc Plant Biol; 2010;153: 1212–1223.
13. Timperio AM D’Alessandro A, Fagioni M, Magro P, Zolla L. Production of the phytoalexins trans-resveratrol and delta-viniferin in two economy-relevant grape cultivars upon infection with Botrytis cinerea in field conditions. Plant Physiol Biochem. Elsevier; 2012;50: 65–71. doi: 10.1016/j.plaphy.2011.07.008
14. Fagard M, Launay A, Clément G, Courtial J, Dellagi A, Farjad M, et al. Nitrogen metabolism meets phytopathology. J Exp Bot. Oxford University Press; 2014;65: 5643–5656. doi: 10.1093/jxb/eru323 25080088
15. Rojas CM, Senthil-Kumar M, Tzin V, Mysore K. Regulation of primary plant metabolism during plant-pathogen interactions and its contribution to plant defense. Front Plant Sci. Frontiers; 2014;5: 17.
16. Wang M, Gu Z, Wang R, Guo J, Ling N, Firbank LG, et al. Plant Primary Metabolism Regulated by Nitrogen Contributes to Plant–Pathogen Interactions. Plant Cell Physiol. Oxford University Press; 2018;60: 329–342.
17. Brauc S, De Vooght E, Claeys M, Höfte M, Angenon G. Influence of over-expression of cytosolic aspartate aminotransferase on amino acid metabolism and defence responses against Botrytis cinerea infection in Arabidopsis thaliana. J Plant Physiol. Elsevier; 2011;168: 1813–1819. doi: 10.1016/j.jplph.2011.05.012 21676488
18. Lecompte F, Abro MA, Nicot PC. Contrasted responses of Botrytis cinerea isolates developing on tomato plants grown under different nitrogen nutrition regimes. Plant Pathol. Wiley Online Library; 2010;59: 891–899.
19. Olea F, Pérez-García A, Cantón FR, Rivera ME, Cañas R, Ávila C, et al. Up-regulation and localization of asparagine synthetase in tomato leaves infected by the bacterial pathogen Pseudomonas syringae. Plant cell Physiol. Oxford University Press; 2004;45: 770–780. doi: 10.1093/pcp/pch092 15215512
20. Pageau K, Reisdorf-Cren M, Morot-Gaudry J-F, Masclaux-Daubresse C. The two senescence-related markers, GS1 (cytosolic glutamine synthetase) and GDH (glutamate dehydrogenase), involved in nitrogen mobilization, are differentially regulated during pathogen attack and by stress hormones and reactive oxygen species in Nicoti. J Exp Bot. Oxford University Press; 2005;57: 547–557. doi: 10.1093/jxb/erj035 16377736
21. van Baarlen P, Legendre L, van Kan JAL. Plant defence compounds against Botrytis infection. Botrytis: Biology, pathology and control. Springer; 2007. pp. 143–161.
22. Moe LA. Amino acids in the rhizosphere: from plants to microbes. Am J Bot. Wiley Online Library; 2013;100: 1692–1705. doi: 10.3732/ajb.1300033 23956051
23. Watanabe M, Balazadeh S, Tohge T, Erban A, Giavalisco P, Kopka J, et al. Comprehensive dissection of spatiotemporal metabolic shifts in primary, secondary, and lipid metabolism during developmental senescence in Arabidopsis. Plant Physiol. Am Soc Plant Biol; 2013;162: 1290–1310.
24. Galili G, Amir R, Fernie AR. The regulation of essential amino acid synthesis and accumulation in plants. Annu Rev Plant Biol. Annual Reviews; 2016;67: 153–178.
25. Kochevenko A, Araújo WL, Maloney GS, Tieman DM, Do PT, Taylor MG, et al. Catabolism of branched chain amino acids supports respiration but not volatile synthesis in tomato fruits. Mol Plant. Elsevier; 2012;5: 366–375. doi: 10.1093/mp/ssr108 22199237
26. Kruk J, Szymańska R, Cela J, Munne-Bosch S. Plastochromanol-8: fifty years of research. Phytochemistry. Elsevier; 2014;108: 9–16. doi: 10.1016/j.phytochem.2014.09.011 25308762
27. Szabados L, Savoure A. Proline: a multifunctional amino acid. Trends Plant Sci. Elsevier; 2010;15: 89–97. doi: 10.1016/j.tplants.2009.11.009 20036181
28. Häusler RE, Ludewig F, Krueger S. Amino acids–a life between metabolism and signaling. Plant Sci. Elsevier; 2014;229: 225–237.
29. Singh SK. Explorations of plant’s chemodiversity: role of nitrogen-containing secondary metabolites in plant defense. Molecular Aspects of Plant-Pathogen Interaction. Springer; 2018. pp. 309–332.
30. Block A, Widhalm JR, Fatihi A, Cahoon RE, Wamboldt Y, Elowsky C, et al. The origin and biosynthesis of the benzenoid moiety of ubiquinone (coenzyme Q) in Arabidopsis. Plant Cell. Am Soc Plant Biol; 2014;26: 1938–1948.
31. Wang M, Maeda HA. Aromatic amino acid aminotransferases in plants. Phytochem Rev. Springer; 2018;17: 131–159.
32. Hildebrandt TM, Nesi AN, Araújo WL, Braun H-P. Amino acid catabolism in plants. Mol Plant. Elsevier; 2015;8: 1563–1579.
33. Schenck CA, Maeda HA. Tyrosine biosynthesis, metabolism, and catabolism in plants. Phytochemistry. Elsevier; 2018;149: 82–102. doi: 10.1016/j.phytochem.2018.02.003 29477627
34. Lee E-J, Facchini PJ. Tyrosine aminotransferase contributes to benzylisoquinoline alkaloid biosynthesis in opium poppy. Plant Physiol. Am Soc Plant Biol; 2011;157: 1067–1078.
35. Wang M, Toda K, Maeda HA. Biochemical properties and subcellular localization of tyrosine aminotransferases in Arabidopsis thaliana. Phytochemistry. Elsevier; 2016;132: 16–25.
36. Riewe D, Koohi M, Lisec J, Pfeiffer M, Lippmann R, Schmeichel J, et al. A tyrosine aminotransferase involved in tocopherol synthesis in Arabidopsis. Plant J. Wiley Online Library; 2012;71: 850–859. doi: 10.1111/j.1365-313X.2012.05035.x 22540282
37. Wang B, Sun W, Li Q, Li Y, Luo H, Song J, et al. Genome-wide identification of phenolic acid biosynthetic genes in Salvia miltiorrhiza. Planta. Springer; 2015;241: 711–725. doi: 10.1007/s00425-014-2212-1 25471478
38. Xiao Y, Zhang L, Gao S, Saechao S, Di P, Chen J, et al. The c4h, tat, hppr and hppd genes prompted engineering of rosmarinic acid biosynthetic pathway in Salvia miltiorrhiza hairy root cultures. PLoS One. Public Library of Science; 2011;6: e29713. doi: 10.1371/journal.pone.0029713 22242141
39. Seki M, Narusaka M, Ishida J, Nanjo T, Fujita M, Oono Y, et al. Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high‐salinity stresses using a full‐length cDNA microarray. Plant J. Wiley Online Library; 2002;31: 279–292. doi: 10.1046/j.1365-313x.2002.01359.x 12164808
40. Hwang IS, An SH, Hwang BK. Pepper asparagine synthetase 1 (CaAS1) is required for plant nitrogen assimilation and defense responses to microbial pathogens. Plant J. Wiley Online Library; 2011;67: 749–762. doi: 10.1111/j.1365-313X.2011.04622.x 21535260
41. Dornfeld C, Weisberg AJ, Ritesh KC, Dudareva N, Jelesko JG, Maeda HA. Phylobiochemical characterization of class-Ib aspartate/prephenate aminotransferases reveals evolution of the plant arogenate phenylalanine pathway. Plant Cell. Am Soc Plant Biol; 2014;26: 3101–3114.
42. Bauwe H, Hagemann M, Fernie AR. Photorespiration: players, partners and origin. Trends Plant Sci. Elsevier; 2010;15: 330–336. doi: 10.1016/j.tplants.2010.03.006 20403720
43. Hruz T, Laule O, Szabo G, Wessendorp F, Bleuler S, Oertle L, et al. Genevestigator v3: a reference expression database for the meta-analysis of transcriptomes. Adv Bioinformatics. Hindawi; 2008;2008.
44. Seifi HS, De Vleesschauwer D, Aziz A, Höfte M. Modulating plant primary amino acid metabolism as a necrotrophic virulence strategy: the immune-regulatory role of asparagine synthetase in Botrytis cinerea-tomato interaction. Plant Signal Behav. Taylor & Francis; 2014;9: e27995. doi: 10.4161/psb.27995 24521937
45. Asselbergh B, Curvers K, França SC, Audenaert K, Vuylsteke M, Van Breusegem F, et al. Resistance to Botrytis cinerea in sitiens, an abscisic acid-deficient tomato mutant, involves timely production of hydrogen peroxide and cell wall modifications in the epidermis. Plant Physiol. Am Soc Plant Biol; 2007;144: 1863–1877.
46. Wang R, Zhang M, Liu H, Xu J, Yu J, He F, et al. PsAAT3, an oomycete-specific aspartate aminotransferase, is required for full pathogenicity of the oomycete pathogen Phytophthora sojae. Fungal Biol. Elsevier; 2016;120: 620–630.
47. Stacey MG, Cahoon RE, Nguyen HT, Cui Y, Sato S, Nguyen CT, et al. Identification of Homogentisate Dioxygenase as a Target for Vitamin E Biofortification in Oilseeds. Plant Physiol. 2016;172: 1506–1518. doi: 10.1104/pp.16.00941 27660165
48. Miller GAD, Suzuki N, Ciftci‐Yilmaz S, Mittler RON. Reactive oxygen species homeostasis and signalling during drought and salinity stresses. Plant Cell Environ. Wiley Online Library; 2010;33: 453–467. doi: 10.1111/j.1365-3040.2009.02041.x 19712065
49. Falk J, Munné-Bosch S. Tocochromanol functions in plants: antioxidation and beyond. J Exp Bot. Oxford University Press; 2010;61: 1549–1566. doi: 10.1093/jxb/erq030 20385544
50. Maeda H, Dudareva N. The shikimate pathway and aromatic amino acid biosynthesis in plants. Annu Rev Plant Biol. Annual Reviews; 2012;63: 73–105.
51. Tzin V, Galili G. New insights into the shikimate and aromatic amino acids biosynthesis pathways in plants. Mol Plant. Elsevier; 2010;3: 956–972. doi: 10.1093/mp/ssq048 20817774
52. Cela J, Chang C, Munné-Bosch S. Accumulation of γ-rather than α-tocopherol alters ethylene signaling gene expression in the vte4 mutant of Arabidopsis thaliana. Plant cell Physiol. Oxford University Press; 2011;52: 1389–1400. doi: 10.1093/pcp/pcr085 21719428
53. Ji CY, Kim Y-H, Kim HS, Ke Q, Kim G-W, Park S-C, et al. Molecular characterization of tocopherol biosynthetic genes in sweetpotato that respond to stress and activate the tocopherol production in tobacco. Plant Physiol Biochem. Elsevier; 2016;106: 118–128. doi: 10.1016/j.plaphy.2016.04.037 27156136
54. Zhang C, Cahoon RE, Hunter SC, Chen M, Han J, Cahoon EB. Genetic and biochemical basis for alternative routes of tocotrienol biosynthesis for enhanced vitamin E antioxidant production. Plant J. Wiley Online Library; 2013;73: 628–639. doi: 10.1111/tpj.12067 23137278
55. Falk J, Andersen G, Kernebeck B, Krupinska K. Constitutive overexpression of barley 4‐hydroxyphenylpyruvate dioxygenase in tobacco results in elevation of the vitamin E content in seeds but not in leaves 1. FEBS Lett. Wiley Online Library; 2003;540: 35–40. doi: 10.1016/s0014-5793(03)00166-2 12681479
56. Dufourmantel N, Dubald M, Matringe M, Canard H, Garcon F, Job C, et al. Generation and characterization of soybean and marker‐free tobacco plastid transformants over‐expressing a bacterial 4‐hydroxyphenylpyruvate dioxygenase which provides strong herbicide tolerance. Plant Biotechnol J. Wiley Online Library; 2007;5: 118–133. doi: 10.1111/j.1467-7652.2006.00226.x 17207262
57. Ren W, Zhao L, Zhang L, Wang Y, Cui L, Tang Y, et al. Molecular cloning and characterization of 4-hydroxyphenylpyruvate dioxygenase gene from Lactuca sativa. J Plant Physiol. Elsevier; 2011;168: 1076–1083. doi: 10.1016/j.jplph.2010.12.017 21349599
58. Xiao Y, Gao S, Di P, Chen J, Chen W, Zhang L. Methyl jasmonate dramatically enhances the accumulation of phenolic acids in Salvia miltiorrhiza hairy root cultures. Physiol Plant. Wiley Online Library; 2009;137: 1–9. doi: 10.1111/j.1399-3054.2009.01257.x 19570133
59. Dixon DP, Edwards R. Enzymes of tyrosine catabolism in Arabidopsis thaliana. Plant Sci. Elsevier; 2006;171: 360–366. doi: 10.1016/j.plantsci.2006.04.008 22980205
60. Zhi T, Zhou Z, Huang Y, Han C, Liu Y, Zhu Q, et al. Sugar suppresses cell death caused by disruption of fumarylacetoacetate hydrolase in Arabidopsis. Planta. Springer; 2016;244: 557–571.
61. Locato V, De Gara L. Programmed cell death in plants: An overview. Plant Programmed Cell Death. Springer; 2018. pp. 1–8.
62. Zhi T, Zhou Z, Qiu B, Zhu Q, Xiong X, Ren C. Loss of fumarylacetoacetate hydrolase causes light‐dependent increases in protochlorophyllide and cell death in Arabidopsis. Plant J. Wiley Online Library; 2019;
63. Li L, Steffens JC. Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance. Planta. Springer; 2002;215: 239–247. doi: 10.1007/s00425-002-0750-4 12029473
64. Aziz E, Akhtar W, Ilyas M, Rehman S, Koiwa H, Shinwari ZK, et al. Response of tobacco polyphenol oxidase gene to wounding, abscisic acid (ABA) and methyl jasmonate (MEJ). Pak J Bot. 2017;49: 499–502.
65. Jia H, Zhao P, Wang B, Tariq P, Zhao F, Zhao M, et al. Overexpression of polyphenol oxidase gene in strawberry fruit delays the fungus infection process. Plant Mol Biol Report. Springer; 2016;34: 592–606.
66. Chen X, Yang B, Huang W, Wang T, Li Y, Zhong Z, et al. Comparative Proteomic Analysis Reveals Elevated Capacity for Photosynthesis in Polyphenol Oxidase Expression-Silenced Clematis terniflora DC. Leaves. International Journal of Molecular Sciences. 2018. doi: 10.3390/ijms19123897 30563128
67. Wang J, Liu B, Xiao Q, Li H, Sun J. Cloning and expression analysis of litchi (Litchi Chinensis Sonn.) polyphenol oxidase gene and relationship with postharvest pericarp browning. PLoS One. Public Library of Science; 2014;9: e93982. doi: 10.1371/journal.pone.0093982 24763257
68. Chi M, Bhagwat B, Lane WD, Tang G, Su Y, Sun R, et al. Reduced polyphenol oxidase gene expression and enzymatic browning in potato (Solanum tuberosum L.) with artificial microRNAs. BMC Plant Biol. BioMed Central; 2014;14: 62.
69. Akram W, Anjum T. Quantitative changes in defense system of tomato induced by two strains of bacillus against Fusarium wilt. Indian J Fund Appl Life Sci. 2011;1: 7–13.
70. Abed-Ashtiani F, Arzanlou M, Nasehi A, Kadir J, Vadamalai G, Azadmard-Damirchi S. Plant tonic, a plant-derived bioactive natural product, exhibits antifungal activity against rice blast disease. Ind Crops Prod. Elsevier; 2018;112: 105–112.
71. Liu M-H, Yang B-R, Cheung W-F, Yang KY, Zhou H-F, Kwok JS-L, et al. Transcriptome analysis of leaves, roots and flowers of Panax notoginseng identifies genes involved in ginsenoside and alkaloid biosynthesis. BMC Genomics. BioMed Central; 2015;16: 265.
72. Thipyapong P, Hunt MD, Steffens JC. Antisense downregulation of polyphenol oxidase results in enhanced disease susceptibility. Planta. Springer; 2004;220: 105–117.
73. Aziz E, Batool R, Akhtar W, Rehman S, Gregersen PL, Mahmood T. Expression analysis of the polyphenol oxidase gene in response to signaling molecules, herbivory and wounding in antisense transgenic tobacco plants. 3 Biotech. Springer; 2019;9: 55. doi: 10.1007/s13205-019-1587-x 30729079
74. Beccari G, Covarelli L, Nicholson P. Infection processes and soft wheat response to root rot and crown rot caused by Fusarium culmorum. Plant Pathol. Wiley Online Library; 2011;60: 671–684.
75. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. Nature Publishing Group; 2011;29: 644. doi: 10.1038/nbt.1883 21572440
76. Götz S, García-Gómez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, et al. High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res. Oxford University Press; 2008;36: 3420–3435. doi: 10.1093/nar/gkn176 18445632
77. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. methods. Elsevier; 2001;25: 402–408. doi: 10.1006/meth.2001.1262 11846609
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