Tropical Plant Biology

, Volume 12, Issue 1, pp 12–20 | Cite as

Network Analysis of Differentially Expressed Genes across Four Sweet Orange Varieties Reveals a Conserved Role of Gibberellin and Ethylene Responses and Transcriptional Regulation in Expanding Citrus Fruits

  • Minghao Cao
  • Jian Zheng
  • Yihong Zhao
  • Zhiqiang Zhang
  • Zhi-Liang ZhengEmail author


Citrus represents the most important non-climacteric fruits and thus understanding transcriptional control during fruit development is important for improving fruit yield and quality. Compared to relatively intensive transcriptomic studies of ripening citrus fruits, much less is known regarding expanding fruits. To provide a systems view of hormone response and transcriptional regulation in citrus fruit development from Stage I (slow fruit growth) to Stage II (rapid growth), we re-analyzed the fruit transcriptomes which we previously collected from the sweet orange varieties, Newhall, Xinhui, Bingtang, and Succari (Citrus sinensis L. Osbeck). A total of 3145 genes were differentially expressed across all four varieties, indicating that they likely have conserved functions in orange fruit development. Using a gene coexpression network-based systems approach, we constructed the subnetworks respectively for gibberellin response, ethylene response, transcription factors and chromatin modifications. Analysis of these subnetworks has led to the identification of more than a dozen major hub genes, such as EXPA1, GASA1/14, ERF13, HB22, ATK1, and TOPII, which represent the most promising candidates for future functional characterization.


Citrus Fruit development Gene coexpression network Hormone response Transcription and chromatin modification 



Days post anthesis


Ethylene response factor




False discovery rate




GA-Stimulated in Arabidopsis


Gene ontology


Weighted gene coexpression network analysis



This research was mainly supported by a grant from Chongqing Science and Technology Commission (Grant No. cstc2012gg-yyjsB80004).

Author Contributions

M.C., J. Z., Z.Z and Z.-L.Z. performed bioinformatic analyses, Y.Z. performed systems biology analysis, and all authors discussed the results. Y.Z. and Z.-L.Z. wrote the article.

Supplementary material

12042_2018_9213_MOESM1_ESM.pdf (43 kb)
Fig. S1 GA response subnetwork with all gene nodes shown. (PDF 42 kb)
12042_2018_9213_MOESM2_ESM.pdf (47 kb)
Fig. S2 Ethylene response subnetwork with all gene nodes shown. (PDF 47 kb)
12042_2018_9213_MOESM3_ESM.pdf (32 kb)
Fig. S3 Transcription factor gene subnetwork with all gene nodes shown. (PDF 32 kb)
12042_2018_9213_MOESM4_ESM.pdf (298 kb)
Fig. S4 Chromatin modification gene network with all gene nodes shown. (PDF 297 kb)
12042_2018_9213_MOESM5_ESM.xlsx (90 kb)
Table S1 List of 3145 commonly regulated genes in four sweet orange varieties and the result of GO enrichment analysis. (XLSX 89 kb)
12042_2018_9213_MOESM6_ESM.xlsx (61 kb)
Table S2 List of genes in the four subnetworks. (XLSX 61 kb)


  1. Aprile A, Federici C, Close TJ, De Bellis L, Cattivelli L, Roose ML (2011) Expression of the H+-ATPase AHA10 proton pump is associated with citric acid accumulation in lemon juice sac cells. Funct Integr Genomics 11:551–563. CrossRefGoogle Scholar
  2. Ben-Cheikh W, Perez-Botella J, Tadeo FR, Talon M, Primo-Millo E (1997) Pollination increases gibberellin levels in developing ovaries of seeded varieties of Citrus. Plant Physiol 114:557–564CrossRefGoogle Scholar
  3. Cercos M, Soler G, Iglesias DJ, Gadea J, Forment J, Talon M (2006) Global analysis of gene expression during development and ripening of citrus fruit flesh. A proposed mechanism for citric acid utilization. Plant Mol Biol 62:513–527. CrossRefGoogle Scholar
  4. Chen C et al (2002) The Arabidopsis ATK1 gene is required for spindle morphogenesis in male meiosis. Development 129:2401–2409CrossRefGoogle Scholar
  5. Du D, Rawat N, Deng Z, Gmitter FG Jr (2015) Construction of citrus gene coexpression networks from microarray data using random matrix theory. Hortic Res 2:15026. CrossRefGoogle Scholar
  6. Esmon CA, Tinsley AG, Ljung K, Sandberg G, Hearne LB, Liscum E (2006) A gradient of auxin and auxin-dependent transcription precedes tropic growth responses. Proc Natl Acad Sci U S A 103:236–241. CrossRefGoogle Scholar
  7. Fujii H, Shimada T, Sugiyama A, Nishikawa F, Endo T, Nakano M, Ikoma Y, Shimizu T, Omura M (2007) Profiling ethylene-responsive genes in mature mandarin fruit using a citrus 22K oligoarray. Plant Sci 173:340–348CrossRefGoogle Scholar
  8. Fujita K, Horiuchi H, Takato H, Kohno M, Suzuki S (2012) Auxin-responsive grape Aux/IAA9 regulates transgenic Arabidopsis plant growth. Mol Biol Rep 39:7823–7829. CrossRefGoogle Scholar
  9. Gapper NE, McQuinn RP, Giovannoni JJ (2013) Molecular and genetic regulation of fruit ripening. Plant Mol Biol 82:575–591. CrossRefGoogle Scholar
  10. Hu W, Wang Y, Bowers C, Ma H (2003) Isolation, sequence analysis, and expression studies of florally expressed cDNAs in Arabidopsis. Plant Mol Biol 53:545–563. CrossRefGoogle Scholar
  11. Huang D, Zhao Y, Cao M, Qiao L, Zheng Z-L (2016) Integrated systems biology analysis of transcriptomes reveals candidate genes for acidity control in developing fruits of sweet orange (Citrus sinensis L. Osbeck). Front Plant Sci 7:486. Google Scholar
  12. Katz E, Lagunes PM, Riov J, Weiss D, Goldschmidt EE (2004) Molecular and physiological evidence suggests the existence of a system II-like pathway of ethylene production in non-climacteric Citrus fruit. Planta 219:243–252. CrossRefGoogle Scholar
  13. Kieffer M, Master V, Waites R, Davies B (2011) TCP14 and TCP15 affect internode length and leaf shape in Arabidopsis. Plant J 68:147–158. CrossRefGoogle Scholar
  14. Kim HJ, Hong SH, Kim YW, Lee IH, Jun JH, Phee BK, Rupak T, Jeong H, Lee Y, Hong BS, Nam HG, Woo HR, Lim PO (2014) Gene regulatory cascade of senescence-associated NAC transcription factors activated by ETHYLENE-INSENSITIVE2-mediated leaf senescence signalling in Arabidopsis. J Exp Bot 65:4023–4036. CrossRefGoogle Scholar
  15. Kumar R, Khurana A, Sharma AK (2014) Role of plant hormones and their interplay in development and ripening of fleshy fruits. J Exp Bot 65:4561–4575. CrossRefGoogle Scholar
  16. Langfelder P, Horvath S (2008) WGCNA: an R package for weighted correlation network analysis. BMC Bioinf 9:559. CrossRefGoogle Scholar
  17. Lee S, Chung EJ, Joung YH, Choi D (2010a) Non-climacteric fruit ripening in pepper: increased transcription of EIL-like genes normally regulated by ethylene. Funct Integr Genomics 10:135–146. CrossRefGoogle Scholar
  18. Lee SJ, Park JH, Lee MH, Yu JH, Kim SY (2010b) Isolation and functional characterization of CE1 binding proteins. BMC Plant Biol 10:277. CrossRefGoogle Scholar
  19. Liu Y, Wang L, Chen D, Wu X, Huang D, Chen L, Li L, Deng X, Xu Q (2014) Genome-wide comparison of microRNAs and their targeted transcripts among leaf, flower and fruit of sweet orange. BMC Genomics 15:695. CrossRefGoogle Scholar
  20. Lohse M et al (2014) Mercator: a fast and simple web server for genome scale functional annotation of plant sequence data. Plant Cell Environ 37:1250–1258. CrossRefGoogle Scholar
  21. Lu X, Cao X, Li F, Li J, Xiong J, Long G, Cao S, Xie S (2016) Comparative transcriptome analysis reveals a global insight into molecular processes regulating citrate accumulation in sweet orange (Citrus sinensis). Physiol Plant 158:463–482. CrossRefGoogle Scholar
  22. Martin RC, Asahina M, Liu PP, Kristof JR, Coppersmith JL, Pluskota WE, Bassel GW, Goloviznina NA, Nguyen TT, Martínez-Andújar C, Arun Kumar MB, Pupel P, Nonogaki H (2010) The regulation of post-germinative transition from the cotyledon- to vegetative-leaf stages by microRNA-targeted SQUAMOSA PROMOTER-BINDING PROTEIN LIKE13 in Arabidopsis. Seed Sci Res 20:89–96CrossRefGoogle Scholar
  23. Miyagishima SY, Kuwayama H, Urushihara H, Nakanishi H (2008) Evolutionary linkage between eukaryotic cytokinesis and chloroplast division by dynamin proteins. Proc Natl Acad Sci U S A 105:15202–15207. CrossRefGoogle Scholar
  24. Muller S, Fuchs E, Ovecka M, Wysocka-Diller J, Benfey PN, Hauser MT (2002) Two new loci, PLEIADE and HYADE, implicate organ-specific regulation of cytokinesis in Arabidopsis. Plant Physiol 130:312–324. CrossRefGoogle Scholar
  25. Oh SA, Johnson A, Smertenko A, Rahman D, Park SK, Hussey PJ, Twell D (2005) A divergent cellular role for the FUSED kinase family in the plant-specific cytokinetic phragmoplast. Curr Biol 15:2107–2111. CrossRefGoogle Scholar
  26. Osorio S, Alba R, Nikoloski Z, Kochevenko A, Fernie AR, Giovannoni JJ (2012) Integrative comparative analyses of transcript and metabolite profiles from pepper and tomato ripening and development stages uncovers species-specific patterns of network regulatory behavior. Plant Physiol 159:1713–1729. CrossRefGoogle Scholar
  27. Osorio S, Scossa F, Fernie AR (2013) Molecular regulation of fruit ripening. Front Plant Sci 4:198. Google Scholar
  28. Pagnussat GC, Yu HJ, Sundaresan V (2007) Cell-fate switch of synergid to egg cell in Arabidopsis eostre mutant embryo sacs arises from misexpression of the BEL1-like homeodomain gene BLH1. Plant Cell 19:3578–3592. CrossRefGoogle Scholar
  29. Petrovska B et al (2012) Plant Aurora kinases play a role in maintenance of primary meristems and control of endoreduplication. New Phytol 193:590–604. CrossRefGoogle Scholar
  30. Pignocchi C, Minns GE, Nesi N, Koumproglou R, Kitsios G, Benning C, Lloyd CW, Doonan JH, Hills MJ (2009) ENDOSPERM DEFECTIVE1 is a novel microtubule-associated protein essential for seed development in Arabidopsis. Plant Cell 21:90–105. CrossRefGoogle Scholar
  31. Pinheiro TT, Figueira A, Latado RR (2014) Early-flowering sweet orange mutant 'x11' as a model for functional genomic studies of Citrus. BMC Res Notes 7:511. CrossRefGoogle Scholar
  32. Qiao L, Cao M, Zheng J, Zhao Y, Zheng ZL (2017) Gene coexpression network analysis of fruit transcriptomes uncovers a possible mechanistically distinct class of sugar/acid ratio-associated genes in sweet orange. BMC Plant Biol 17:186. CrossRefGoogle Scholar
  33. Rauf M, Arif M, Fisahn J, Xue GP, Balazadeh S, Mueller-Roeber B (2013) NAC transcription factor speedy hyponastic growth regulates flooding-induced leaf movement in Arabidopsis. Plant Cell 25:4941–4955. CrossRefGoogle Scholar
  34. Rawat N, Kiran SP, Du D, Gmitter FG Jr, Deng Z (2015) Comprehensive meta-analysis, co-expression, and miRNA nested network analysis identifies gene candidates in citrus against Huanglongbing disease. BMC Plant Biol 15:184. CrossRefGoogle Scholar
  35. Schweizer F, Bodenhausen N, Lassueur S, Masclaux FG, Reymond P (2013) Differential contribution of transcription factors to Arabidopsis thaliana defense against Spodoptera littoralis. Front Plant Sci 4:13. CrossRefGoogle Scholar
  36. Sun S, Wang H, Yu H, Zhong C, Zhang X, Peng J, Wang X (2013) GASA14 regulates leaf expansion and abiotic stress resistance by modulating reactive oxygen species accumulation. J Exp Bot 64:1637–1647. CrossRefGoogle Scholar
  37. Talón M, Hedden P, Primo-Millo E (1990) Gibberellins inCitrus sinensis: a comparison between seeded and seedless varieties. J Plant Growth Regul 9:201–206CrossRefGoogle Scholar
  38. Tan QK, Irish VF (2006) The Arabidopsis zinc finger-homeodomain genes encode proteins with unique biochemical properties that are coordinately expressed during floral development. Plant Physiol 140:1095–1108. CrossRefGoogle Scholar
  39. Wang H et al (2005) The tomato Aux/IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis. Plant Cell 17:2676–2692. CrossRefGoogle Scholar
  40. Wang JH, Liu JJ, Chen KL, Li HW, He J, Guan B, He L (2017) Comparative transcriptome and proteome profiling of two Citrus sinensis cultivars during fruit development and ripening. BMC Genomics 18:984. CrossRefGoogle Scholar
  41. Wong DC, Sweetman C, Ford CM (2014) Annotation of gene function in citrus using gene expression information and co-expression networks. BMC Plant Biol 14:186. CrossRefGoogle Scholar
  42. Wu GA et al (2014a) Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication. Nat Biotechnol 32:656–662. CrossRefGoogle Scholar
  43. Wu J, Xu Z, Zhang Y, Chai L, Yi H, Deng X (2014b) An integrative analysis of the transcriptome and proteome of the pulp of a spontaneous late-ripening sweet orange mutant and its wild type improves our understanding of fruit ripening in citrus. J Exp Bot 65:1651–1671. CrossRefGoogle Scholar
  44. Xu Q et al (2013) The draft genome of sweet orange (Citrus sinensis). Nat Genet 45:59–66. CrossRefGoogle Scholar
  45. Xu J, Xu H, Liu Y, Wang X, Xu Q, Deng X (2015) Genome-wide identification of sweet orange (Citrus sinensis) histone modification gene families and their expression analysis during the fruit development and fruit-blue mold infection process. Front Plant Sci 6:607. Google Scholar
  46. Yin XR, Xie XL, Xia XJ, Yu JQ, Ferguson IB, Giovannoni JJ, Chen KS (2016) Involvement of an ethylene response factor in chlorophyll degradation during citrus fruit degreening. Plant J 86:403–412. CrossRefGoogle Scholar
  47. Yu K, Xu Q, Da X, Guo F, Ding Y, Deng X (2012) Transcriptome changes during fruit development and ripening of sweet orange (Citrus sinensis). BMC Genomics 13:10. CrossRefGoogle Scholar
  48. Zhang S, Shi Q, Albrecht U, Shatters RG, Stange R, McCollum G, Zhang S, Fan C, Stover E (2017) Comparative transcriptome analysis during early fruit development between three seedy citrus genotypes and their seedless mutants. Hortic Res 4:17041. CrossRefGoogle Scholar
  49. Zheng Q, Wang XJ (2008) GOEAST: a web-based software toolkit for gene ontology enrichment analysis. Nucleic Acids Res 36:W358–W363. CrossRefGoogle Scholar
  50. Zheng ZL, Zhao Y (2013) Transcriptome comparison and gene coexpression network analysis provide a systems view of citrus response to 'Candidatus Liberibacter asiaticus' infection. BMC Genomics 14:27. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Plant Nutrient Signaling and Fruit Quality Improvement Laboratory, National Citrus Engineering Research Center, Citrus Research InstituteSouthwest UniversityChongqingChina
  2. 2.Department of Health Policy & Health Services ResearchBoston UniversityBostonUSA
  3. 3.Department of Biological Sciences, Lehman CollegeCity University of New YorkBronxUSA

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