RNA-sequencing reveals early, dynamic transcriptome changes in the corollas of pollinated petunias

BMC Plant Biology

Volumn 14, Issue 1, 2014, Pages

  Broderick, Shaun R ^a   Wijeratne, Saranga ^a   Wijeratn, Asela J ^a   Chapin, Laura J ^a   Meulia, Tea ^a   Jones, Michelle L ^a  

^a OHIO STATE UNIVERSITY   (United States)

Author keywords

Autophagy; Calcium signaling; Ethylene; KEGG; Petal senescence; RNA seq; String; Trinity; WGCNA, de novo assembly

Indexed keywords

ETHYLENE; ETHYLENE DERIVATIVE; PHYTOHORMONE; TRANSCRIPTOME;

AUTOPHAGY; CALCIUM SIGNALING; CELL AGING; DOWN REGULATION; FLOWER; GENE EXPRESSION REGULATION; GENETICS; HIGH THROUGHPUT SEQUENCING; METABOLISM; NUCLEOTIDE SEQUENCE; PETUNIA; PHYSIOLOGY; POLLINATION; SEQUENCE ANALYSIS; UPREGULATION;

AUTOPHAGY; BASE SEQUENCE; CALCIUM SIGNALING; CELL AGING; DOWN-REGULATION; ETHYLENES; FLOWERS; GENE EXPRESSION REGULATION, PLANT; HIGH-THROUGHPUT NUCLEOTIDE SEQUENCING; PETUNIA; PLANT GROWTH REGULATORS; POLLINATION; SEQUENCE ANALYSIS, RNA; TRANSCRIPTOME; UP-REGULATION;

EID: 84988700496     PISSN: None     EISSN: 14712229     Source Type: Journal    
DOI: 10.1186/s12870-014-0307-2     Document Type: Article

References (102)

1. Ashman TL, Schoen DJ: How long should flowers live? Nature 1994, 371(6500):788-791.
2. Chapin L, Jones M: Nutrient remobilization during pollination-induced corolla senescence in petunia. Acta Hortic 2007, 755:181-190.
3. Stead A: Pollination-induced flower senescence: a review. Plant Growth Regul 1992, 11(1):13-20.
4. Jones ML: Ethylene signaling is required for pollination-accelerated corolla senescence in petunias. Plant Sci 2008, 175(1-2):190-196.
5. Jones ML: Mineral nutrient remobilization during corolla senescence in ethylene-sensitive and-insensitive flowers. AoB Plants 2013, 5:plt023.
6. Wilkinson JQ, Lanahan MB, Clark DG, Bleecker AB, Chang C, Meyerowitz EM, Klee HJ: A dominant mutant receptor from Arabidopsis confers ethylene insensitivity in heterologous plants. Nat Biotechnol 1997, 15(5):444-447.
7. Jones M, Stead A, Clark D: Petunia flower senescence. In Petunia. Edited by Gerats T, Strommer J. New York: Springer; 2009:301-324.
8. Hoekstra FA, Weges R: Lack of control by early pistillate ethylene of the accelerated wilting of Petunia hybrida flowers. Plant Physiol 1986, 80(2):403-408.
9. Shibuya K, Barry KG, Ciardi JA, Loucas HM, Underwood BA, Nourizadeh S, Ecker JR, Klee HJ, Clark DG: The central role of PhEIN2 in ethylene responses throughout plant development in petunia. Plant Physiol 2004, 136(2):2900-2912.
10. Langston BJ, Bai S, Jones ML: Increases in DNA fragmentation and induction of a senescence-specific nuclease are delayed during corolla senescence in ethylene-insensitive (etr1-1) transgenic petunias. J Exp Bot 2005, 56(409):15-23.
11. Gilissen LJW, Hoekstra FA: Pollination-induced corolla wilting in Petunia hybrida rapid transfer through the style of a wilting-inducing substance. Plant Physiol 1984, 75(2):496-498.
12. Singh A, Evensen KB, Kao TH: Ethylene synthesis and floral senescence following compatible and incompatible pollinations in Petunia inflata. Plant Physiol 1992, 99(1):38-45.
13. Whitehead CS, Halevy AH: Ethylene sensitivity: the role of short-chain saturated fatty-acids in pollination-induced senescence of Petunia hybrida flowers. Plant Growth Regul 1989, 8(1):41-54.
14. Liu D, Sui S, Ma J, Li Z, Guo Y, Luo D, Yang J, Li M: Transcriptomic analysis of flower development in wintersweet (Chimonanthus praecox). PLoS One 2014, 9(1):e86976.
15. Hoeberichts FA, van Doorn WG, Vorst O, Hall RD, van Wordragen MF: Sucrose prevents up-regulation of senescence-associated genes in carnation petals. J Exp Bot 2007, 58(11):2873-2885.
16. Wang Y, Huang H, Ma Y, Fu J, Wang L, Dai S: Construction and de novo characterization of a transcriptome of Chrysanthemum lavandulifolium: analysis of gene expression patterns in floral bud emergence. Plant Cell Tiss Org 2014, 116(3):297-309.
17. Wang H, Stier G, Lin J, Liu G, Zhang Z, Chang Y, Reid MS, Jiang C: Transcriptome changes associated with delayed flower senescence on transgenic petunia by inducing expression of etr1-1, a mutant ethylene receptor. PLoS One 2013, 8(7):e65800.
18. Jones ML: Changes in gene expression during senescence. In Plant Cell Death Processes. Edited by Nooden L. San Diego, California: Elsevier Science; 2004:51-72.
19. Monteiro F, Sebastiana M, Figueiredo A, Sousa L, Cotrim HC, Pais MS: Labellum transcriptome reveals alkene biosynthetic genes involved in orchid sexual deception and pollination-induced senescence. Funct Integr Genomics 2012, 12(4):693-703.
20. Breeze E, Wagstaff C, Harrison E, Bramke I, Rogers H, Stead A, Thomas B, Buchanan-Wollaston V: Gene expression patterns to define stages of post-harvest senescence in Alstroemeria petals. Plant Biotechnol J 2004, 2(2):155-168.
21. Price AM, Orellana DFA, Salleh FM, Stevens R, Acock R, Buchanan-Wollaston V, Stead AD, Rogers HJ: A comparison of leaf and petal senescence in wallflower reveals common and distinct patterns of gene expression and physiology. Plant Physiol 2008, 147(4):1898-1912.
22. van Doorn WG, Balk PA, van Houwelingen AM, Hoeberichts FA, Hall RD, Vorst O, van der Schoot C, van Wordragen MF: Gene expression during anthesis and senescence in Iris flowers. Plant Mol Biol 2003, 53(6):845-863.
23. van Doorn WG, Woltering EJ: Physiology and molecular biology of petal senescence. J Exp Bot 2008, 59(3):453-480.
24. Wang Z, Gerstein M, Snyder M: RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 2009, 10(1):57-63.
25. Wang Y, Ghaffari N, Johnson CD, Braga-Neto UM, Wang H, Chen R, Zhou H: Evaluation of the coverage and depth of transcriptome by RNA-seq in chickens. BMC Bioinformatics 2011, 12:S5.
26. Bai S, Willard B, Chapin LJ, Kinter MT, Francis DM, Stead AD, Jones ML: Proteomic analysis of pollination-induced corolla senescence in petunia. J Exp Bot 2010, 61(4):1089-1109.
27. Jones ML, Chaffin GS, Eason JR, Clark DG: Ethylene-sensitivity regulates proteolytic activity and cysteine protease gene expression in petunia corollas. J Exp Bot 2005, 56(420):2733-2744.
28. Kovaleva LV, Timofeeva GV, Zakharova EV, Voronkov AS, Rakitin VY: Ethylene synthesis in petunia stigma tissues governs the growth of pollen tubes in progamic phase of fertilization. Russ J Plant Physl 2011, 58(3):402-408.
29. Villarino GH, Bombarely A, Giovannoni JJ, Scanlon MJ, Mattson NS: Transcriptomic analysis of Petunia hybrida in response to salt stress using high throughput RNA sequencing. PLoS One 2014, 9(4):e94651.
30. Soetaert SSA, Van Neste CMF, Vandewoestyne ML, Head SR, Goossens A, Van Nieuwerburgh FCW, Deforce DLD: Differential transcriptome analysis of glandular and filamentous trichomes in Artemisia annua. BMC Plant Biol 2013, 13:UNSP 220.
31. 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: Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 2011, 29(7):644-652.
32. O'Neil ST, Dzurisin JDK, Carmichael RD, Lobo NF, Emrich SJ, Hellmann JJ: Population-level transcriptome sequencing of nonmodel organisms Erynnis propertius and Papilio zelicaon. BMC Genomics 2010, 11:310.
33. Anders S, Huber W: Differential expression analysis for sequence count data. Genome Biol 2010, 11:R106.
34. Langfelder P, Horvath S: Fast R functions for robust correlations and hierarchical clustering. J Stat Softw 2012, 46(11):1-17.
35. Langfelder P, Horvath S: WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 2008, 9:559.
36. Woltering EJ, Vrije T, Harren F, Hoekstra FA: Pollination and stigma wounding: same response, different signal? J Exp Bot 1997, 48(5):1027-1033.
37. Gilissen LJW: Style-controlled wilting of flower. Planta 1977, 133(3):275-280.
38. Yan H, Zhang H, Chen M, Jian H, Baudino S, Caissard J, Bendahmane M, Li S, Zhang T, Zhou N, Qiu X, Wang Q, Tang K: Transcriptome and gene expression analysis during flower blooming in Rosa chinensis 'Pallida'. Gene 2014, 540(1):96-103.
39. Breeze E, Harrison E, McHattie S, Hughes L, Hickman R, Hill C, Kiddle S, Kim Y, Penfold CA, Jenkins D, Zhang C, Morris K, Jenner C, Jackson S, Thomas B, Tabrett A, Legaie R, Moore JD, Wild DL, Ott S, Rand D, Beynon J, Denby K, Mead A, Buchanan-Wollaston V: High-resolution temporal profiling of transcripts during Arabidopsis leaf senescence reveals a distinct chronology of processes and regulation. Plant Cell 2011, 23(3):873-894.
40. Guo Y, Gan S: Convergence and divergence in gene expression profiles induced by leaf senescence and 27 senescence-promoting hormonal, pathological and environmental stress treatments. Plant Cell Environ 2012, 35(3):644-655.
41. Jansen R, Greenbaum D, Gerstein M: Relating whole-genome expression data with protein-protein interactions. Genome Res 2002, 12(1):37-46.
42. Verlinden S: Flower senescence. In The Molecular Biology and Biotechnology of Flowering. Edited by Jordan B. Cambridge, MA: CABI Publishing; 2006:404.
43. Jones ML, Woodson WR: Pollination-induced ethylene in carnation: role of stylar ethylene in corolla senescence. Plant Physiol 1997, 115(1):205-212.
44. Lovell PJ, Lovell PH, Nichols R: The control of flower senescence in petunia (Petunia hybrida). Ann Bot 1987, 60(1):49-59.
45. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G, Gene Ontology Consortium: Gene Ontology: tool for the unification of biology. Nat Genet 2000, 25(1):25-29.
46. Avila-Ospina L, Moison M, Yoshimoto K, Masclaux-Daubresse C: Autophagy, plant senescence, and nutrient recycling. J Exp Bot 2014, 65(14):3799-3811.
47. Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25(17):3389-3402.
48. Fromm J, Hajirezaei M, Wilke I: The biochemical response of electrical signaling in the reproductive system of Hibiscus plants. Plant Physiol 1995, 109(2):375-384.
49. Wedzony M, Filek M: Changes of electric potential in pistils of Petunia hybrida Hort. and Brassica napus L. during pollination. Acta Physiol Plant 1998, 20(3):291-297.
50. Dumas C, Gaude T: Fertilization in plants: is calcium a key player? Semin Cell Dev Biol 2006, 17(2):244-253.
51. 2+ in the pistil of Petunia hybrida Hort. during growth of the pollen tube: cytochemical and radiographic studies. Acta Biol Cracov Bot 1997, 39:79-89.
52. Tintor N, Ross A, Kanehara K, Yamada K, Fan L, Kemmerling B, Nuernberger T, Tsuda K, Saijo Y: Layered pattern receptor signaling via ethylene and endogenous elicitor peptides during Arabidopsis immunity to bacterial infection. Proc Natl Acad Sci U S A 2013, 110(15):6211-6216.
53. Elleman CJ, Dickinson HG: Commonalties between pollen/stigma and host/pathogen interactions: calcium accumulation during stigmatic penetration by Brassica oleracea pollen tubes. Sex Plant Reprod 1999, 12(3):194-202.
54. Boutrot F, Segonzac C, Chang KN, Qiao H, Ecker JR, Zipfel C, Rathjen JP: Direct transcriptional control of the Arabidopsis immune receptor FLS2 by the ethylene-dependent transcription factors EIN3 and EIL1. Proc Natl Acad Sci U S A 2010, 107(32):14502-14507.
55. Waithaka K, Dodge LL, Reid MS: Carbohydrate traffic during opening of gladiolus florets. Acta Hortic 2001, 543:217-226.
56. Wiemken V, Wiemken A, Matile P: Physiology of flowers of Ipomoea tricolor (Cav): studies on excised flowers and recovery of a phloem exudate. Biochem Physiol Pfl 1976, 169(4):363-376.
57. Bieleski RL, Reid MS: Physiological changes accompanying senescence in the ephemeral daylily flower. Plant Physiol 1992, 98(3):1042-1049.
58. Nichols R, Ho LC: Effects of ethylene and sucrose on translocation of drymatter and 14C-sucrose in cut flower of glasshouse carnation (Dianthus caryophyllus) during senescence. Ann Bot 1975, 39(160):287-296.
59. Yanagisawa S, Yoo SD, Sheen J: Differential regulation of EIN3 stability by glucose and ethylene signalling in plants. Nature 2003, 425(6957):521-525.
60. van der Meulen-Muisers JJM, van Oeveren JC, van der Plas LHW, van Tuyl JM: Postharvest flower development in Asiatic hybrid lilies as related to tepal carbohydrate status. Postharvest Biol Tec 2001, 21(2):201-211.
61. Lenartowska M, Rodriguez-Garcia MI, Bednarska E: Immunocytochemical localization of esterified and unesterified pectins in unpollinated and pollinated styles of Petunia hybrida Hort. Planta 2001, 213(2):182-191.
62. Lenartowska M, Krzeslowska M, Bednarska E: Pectin dynamic and distribution of exchangeable Ca2+ in Haemanthus albiflos hollow style during pollen-pistil interactions. Protoplasma 2011, 248(4):695-705.
63. O'Donoghue EM, Somerfield SD, Watson LM, Brummell DA, Hunter DA: Galactose metabolism in cell walls of opening and senescing petunia petals. Planta 2009, 229(3):709-721.
64. O'Donoghue EM, Somerfield SD, Heyes JA: Vase solutions containing sucrose result in changes to cell walls of sandersonia (Sandersonia aurantiaca) flowers. Postharvest Biol Tec 2002, 26(3):285-294.
65. O'Donoghue EM, Somerfield SD, Heyes JA: Organization of cell walls in Sandersonia aurantiaca floral tissue. J Exp Bot 2002, 53(368):513-523.
66. De Vetten NC, Huber DJ: Cell wall changes during the expansion and senescence of carnation Dianthus caryophyllus petals. Physiol Plantarum 1990, 78(3):447-454.
67. Shibuya K, Niki T, Ichimura K: Pollination induces autophagy in petunia petals via ethylene. J Exp Bot 2013, 64(4):1111-1120.
68. Eisinger W: Role of cytokinins in carnation flower senescence. Plant Physiol 1977, 59(4):707-709.
69. Saks Y, Vanstaden J, Smith MT: Effect of gibberellic-acid on carnation flower senescence: evidence that the delay of carnation flower senescence by gibberellic acid depends on the stage of flower development. Plant Growth Regul 1992, 11(1):45-51.
70. Porat R, Halevy AH: Enhancement of petunia and dendrobium flower senescence by jasmonic acid methyl ester is via the promotion of ethylene production. Plant Growth Regul 1993, 13(3):297-301.
71. Jofuku KD, Denboer BGW, Vanmontagu M, Okamuro JK: Control of Arabidopsis flower and seed development by the homeotic gene APETALA2. Plant Cell 1994, 6(9):1211-1225.
72. Yan X, Zhang L, Chen B, Xiong Z, Chen C, Wang L, Yu J, Lu C, Wei W: Functional identification and characterization of the Brassica Napus transcription factor gene BnAP2, the ortholog of Arabidopsis thaliana APETALA2. PLoS One 2012, 7(3):e33890.
73. Karlova R, Rosin FM, Busscher-Lange J, Parapunova V, Do PT, Fernie AR, Fraser PD, Baxter C, Angenent GC, de Maagd RA: Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening. Plant Cell 2011, 23(3):923-941.
74. Izumi M, Wada S, Makino A, Ishida H: The autophagic degradation of chloroplasts via Rubisco-containing bodies is specifically linked to leaf carbon status but not nitrogen status in Arabidopsis. Plant Physiol 2010, 154(3):1196-1209.
75. Guiboileau A, Yoshimoto K, Soulay F, Bataille M, Avice J, Masclaux-Daubresse C: Autophagy machinery controls nitrogen remobilization at the wholeplant level under both limiting and ample nitrate conditions in Arabidopsis. New Phytol 2012, 194(3):732-740.
76. Guiboileau A, Avila-Ospina L, Yoshimoto K, Soulay F, Azzopardi M, Marmagne A, Lothier J, Masclaux-Daubresse C: Physiological and metabolic consequences of autophagy deficiency for the management of nitrogen and protein resources in Arabidopsis leaves depending on nitrate availability. New Phytol 2013, 199(3):683-694.
77. Ono Y, Wada S, Izumi M, Makino A, Ishida H: Evidence for contribution of autophagy to Rubisco degradation during leaf senescence in Arabidopsis thaliana. Plant Cell Environ 2013, 36(6):1147-1159.
78. Wang Y, Yu B, Zhao J, Guo J, Li Y, Han S, Huang L, Du Y, Hong Y, Tang D, Liu Y: Autophagy contributes to leaf starch degradation. Plant Cell 2013, 25(4):1383-1399.
79. van Doorn WG, Beers EP, Dangl JL, Franklin-Tong VE, Gallois P, Hara-Nishimura I, Jones AM, Kawai-Yamada M, Lam E, Mundy J, Mur LAJ, Petersen M, Smertenko A, Taliansky M, Van Breusegem F, Wolpert T, Woltering E, Zhivotovsky B, Bozhkov PV: Morphological classification of plant cell deaths. Cell Death Differ 2011, 18(8):1241-1246.
80. Chen WQ, Provart NJ, Glazebrook J, Katagiri F, Chang HS, Eulgem T, Mauch F, Luan S, Zou GZ, Whitham SA, Budworth PR, Tao Y, Xie ZY, Chen X, Lam S, Kreps JA, Harper JF, Si-Ammour A, Mauch-Mani B, Heinlein M, Kobayashi K, Hohn T, Dangl JL, Wang X, Zhu T: Expression profile matrix of Arabidopsis transcription factor genes suggests their putative functions in response to environmental stresses. Plant Cell 2002, 14(3):559-574.
81. Besseau S, Li J, Palva ET: WRKY54 and WRKY70 co-operate as negative regulators of leaf senescence in Arabidopsis thaliana. J Exp Bot 2012, 63(7):2667-2679.
82. Miao Y, Zentgraf U: A HECT E3 ubiquitin ligase negatively regulates Arabidopsis leaf senescence through degradation of the transcription factor WRKY53. Plant J 2010, 63(2):179-188.
83. Chang X, Donnelly L, Sun D, Rao J, Reid MS, Jiang C: A petunia homeodomain-leucine zipper protein, PhHD-Zip, plays an important role in flower senescence. PLoS One 2014, 9(2):e88320.
84. Liu J, Li J, Wang H, Fu Z, Liu J, Yu Y: Identification and expression analysis of ERF transcription factor genes in petunia during flower senescence and in response to hormone treatments. J Exp Bot 2011, 62(2):825-840.
85. Dietrich K, Weltmeier F, Ehlert A, Weiste C, Stahl M, Harter K, Droege-Laser W: Heterodimers of the Arabidopsis transcription factors bZIP1 and bZIP53 reprogram amino acid metabolism during low energy stress. Plant Cell 2011, 23(1):381-395.
86. Hummel M, Rahmani F, Smeekens S, Hanson J: Sucrose-mediated translational control. Ann Bot 2009, 104(1):1-7.
87. Yuasa T, Nagasawa Y, Osanai K, Kaneko A, Tajima D, Htwe NMPS, Nang MPS, Ishibashi Y, Iwaya-Inoue M: Induction of a bZIP type transcription factor and amino acid catabolism-related genes in soybean seedling in response to starvation stress. J Botany 2013, 2013(Article ID 935479):1-8.
88. Gerats T, Vandenbussche M: A model system comparative for research: Petunia. Trends Plant Sci 2005, 10(5):251-256.
89. Schneider CA, Rasband WS, Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nat Methods 2012, 9(7):671-675.
90. Zhong S, Joung J, Zheng Y, Chen Y, Liu B, Shao Y, Xiang JZ, Fei Z, Giovannoni JJ: High-throughput illumina strand-specific RNA sequencing library preparation. Cold Spring Harb Protoc 2011, 2011(8):940-949.
91. Kofler R, Orozco-terWengel P, De Maio N, Pandey RV, Nolte V, Futschik A, Kosiol C, Schloetterer C: PoPoolation: a toolbox for population genetic analysis of next generation sequencing data from pooled individuals. PLoS One 2011, 6(1):e15925.
92. Martin M: Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 2011, 17(1):10-12.
93. Fu L, Niu B, Zhu Z, Wu S, Li W: CD-HIT: accelerated for clustering the nextgeneration sequencing data. Bioinformatics 2012, 28(23):3150-3152.
94. Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M: Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 2005, 21(18):3674-3676.
95. The UniProt Consortium: Activities at the Universal Protein Resource (UniProt). Nucleic Acids Res 2014, 42(D1):D191-D198.
96. Zdobnov EM, Apweiler R: InterProScan-an integration platform for the signature-recognition methods in InterPro. Bioinformatics 2001, 17(9):847-848.
97. Li H, Durbin R: Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009, 25(14):1754-1760.
98. VENNY: An interactive tool for comparing lists with Venn Diagrams. In [http://bioinfogp.cnb.csic.es/tools/venny/index.html]
99. Chapin LJ, Jones ML: Ethylene regulates phosphorus remobilization and expression of a phosphate transporter (PhPT1) during petunia corolla senescence. J Exp Bot 2009, 60(7):2179-2190.
100. Benjamini Y, Hochberg Y: Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Stat Soc B 1995, 57(1):289-300.
101. Jensen LJ, Kuhn M, Stark M, Chaffron S, Creevey C, Muller J, Doerks T, Julien P, Roth A, Simonovic M, Bork P, von Mering C: STRING 8-a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res 2009, 37:D412-D416.
102. Guo AY, He K, Liu D, Bai SN, Gu XC, Wei LP, Luo JC: DATF: a database of Arabidopsis transcription factors. Bioinformatics 2005, 21(10):2568-2569.