Mına KAZEMIAN, Elham Mohajel KAZEMI, Maryam KOLAHI, Valıollah Ghasemı OMRAN

Identification and in silico analysis of PHANTASTICA gene in Saintpaulia ionantha H. Wendl Gesneriaceae

Saintpaulia ionantha H. Wendl synonym Streptocarpus ionanthus has been considered an ornamental plant. The PHANTASTICA PHAN gene has a significant role in the formation of plant organs adaxial?abaxial polarity. In the present study the PHAN gene was identified in Saintpaulia for the first time using PCR. To determine the expression pattern of the PHAN gene, gene expression was compared at 3 developmental stages using real time PCR. For in silico analyses, protein characteristics, secondary and 3D structures of protein, intracellular localization, and the phylogenetic tree were investigated using bioinformatics tools. The partial CDS of PHAN gene contained 756 nucleotides, a predicted protein encoded with 252 amino acids with a molecular weight of 28,474 Da. The presence of the HTH MYB-type motif in this sequence indicated that the PHAN protein belonged to the MYB family. The secondary structure of the PHAN protein was composed of 45% ?-helix. The predicted PHAN protein 3D structure revealed that this protein can bind to nucleic acids. The binding site sequence included amino acids 10 R , 12 W , 13 H , 66 W , and 67 K . The intracellular localization of PHAN protein is believed to be in the nucleus. Phylogenetic analysis revealed that species belonging to each family were grouped in same clade. Gene expression revealed that in the later stages of petal development, expression was reduced. The results suggest that the expression pattern of the PHAN gene is that of adaxial?abaxial symmetry initiation during petal development as well as in the leaves of Saintpaulia. It appears that genetic engineering using biotechnology would be beneficial for improving the ornamental value of Saintpaulia.

Keywords:

Adaxial-abaxial DNA binding site, HTH domain, MYB family, ornamental plant,

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Aida R, Ohmiya A, Onozaki T (2018). Current researches in ornamental plant breeding. Breeding Science 68 (1): 1-2. doi: 10.1270/jsbbs.68.1
Almagro Armenteros JJ, Sonderby CK, Sonderby SK, Nielsen H, Winther O (2017). DeepLoc: prediction of protein subcellular localization using deep learning. Bioinformatics 33 (21): 3387- 3395. doi: 10.1093/bioinformatics/btx431
Ambawat S, Sharma P, Yadav N, Yadav R (2013). MYB transcription factor genes as regulators for plant responses: an overview. Physiology and Molecular Biology of Plants 19 (3): 307-321. doi: 10.1007/s12298-013-0179-1
Azadi P, Bagheri H, Nalousi AM, Nazari F, Chandler SF (2016). Current status and biotechnological advances in genetic engineering of ornamental plants. Biotechnology Advances 34 (6): 1073-1090.
Burko Y, Shleizer-Burko S, Yanai O, Shwartz I, Zelnik ID et al.(2013). A role for APETALA1/fruitful transcription factors in tomato leaf development. Plant Cell 25 (1): 2070-2083.
Bustamante M, Matus J, Riechmann JL (2016). Genome-wide analyses for dissecting gene regulatory networks in the shoot apical meristem. Journal of Experimental Botany 67 (6): 1639- 1648. doi: 10.1093/jxb/erw058
Byrne M, Simorowski J, Martienssen R (2002). ASYMMETRIC LEAVES1 reveals Knox gene redundancy in Arabidopsis. Development 129: 1957-1965.
Du H, Zhang L, Liu L, Tang X, Yang W et al. (2009). Biochemical and molecular characterization of plant MYB transcription factor family. Biochemistry 74 (1): 1-11. doi: 10.1134/ s0006297909010015
Endress PK, Matthews ML (2006). First steps toward a floral structural characterization of the major Rosid subclades. Plant Systematics and Evolution 260 (1): 223-51.
Feng K, Xu ZS, Que F, Liu JX, Wang F et al. (2018). An R2R3.MYB transcription factor, OjMYB1, functions in anthocyanin biosynthesis in Oenanthe javanica. Planta 247 (2): 301-315.
Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY et al. (2013). Pfam: the protein families’ database. Nucleic Acids Research 42 (1): 222-230.
Fukushima K, Hasebe M (2014). Adaxial–abaxial polarity: the developmental basis of leaf shape diversity. Genesis 52 (1): 1-18.
Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD et al. (2003). ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Research 31 (13): 3784-3788.
Ge L, Peng J, Berbel A, Madueno F, Chen R (2014). Regulation of compound leaf development by PHANTASTICA in Medicago truncatula. Plant Physiology 164 (1): 216-228.
Goldberg T, Hecht M, Hamp T, Karl T, Yachdav G et al. (2014). LocTree3 prediction of localization. Nucleic Acids Research 42 (1): 350-355.
Gubert C, Christy M, Ward D, Groner W, Liljegren S (2014). ASYMMETRIC LEAVES1 regulates abscission zone placement in Arabidopsis flowers. BMC Plant Biology 14 (2): 195-202.
Haston E, De Craene LP (2007). Inflorescence and floral development in Streptocarpus and Saintpaulia (Gesneriaceae) with particular reference to the impact of bracteole suppression. Plant Systematics and Evolution 265 (2): 13-25.
Hepworth J, Lenhard M (2014). Regulation of plant lateral-organ growth by modulating cell number and size. Current Opinion in Plant Biology 17 (1): 36-42.
Huang T, Irish VF (2016). Gene networks controlling petal organogenesis. Journal of Experimental Botany 67 (1): 61-68.
Huang W, Sun W, Lv H, Xiao G, Zeng S et al. (2013). Isolation and molecular characterization of thirteen R2R3-MYB
transcription factors from Epimedium sagittatum. International Journal of Molecular Sciences 14 (2): 594-610.
Hugouvieux V, Silva CS, Jourdain A, Stigliani A, Charras Q et al. (2018). Tetramerization of MADS family transcription factors SEPALLATA3 and AGAMOUS is required for floral meristem determinacy in Arabidopsis. Nucleic Acids Research 46 (2): 4966-4977.
Husbands AY, Benkovics AH, Nogueira FT, Lodha M, Timmermans MC (2015). The ASYMMETRIC LEAVES complex employs multiple modes of regulation to affect adaxial–abaxial patterning and leaf complexity. Plant Cell 27 (2): 3321-3335.
Ikezaki M, Kojima M, Sakakibara H, Kojima S, Ueno Y et al. (2009) Genetic networks regulated by ASYMMETRIC LEAVES1 (AS1) and AS2 in leaf development in Arabidopsis thaliana: KNOX genes control five morphological events. Plant Journal 61 (1): 70-82.
Iwasaki M, Takahashi H, Iwakawa H, Nakagawa A, Ishikawa T et al. (2013). Dual regulation of ETTIN (ARF3) gene expression by AS1-AS2, which maintains the DNA methylation level, is involved in stabilization of leaf adaxial–abaxial partitioning in Arabidopsis. Development 140 (2): 1958-1969. doi: 10.1242/ dev.085365
Kalpana K, Manjuvani S, Shoba K (2018). In silico comparative modeling of maturase K protein in Cymbopogon martinii plant. Research & Reviews: A Journal of Bioinformatics 5 (3): 30-36. doi: 10.1016/S1672-0229(08)60048-0
Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ (2015). The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols 10 (6): 845-855.
Ko ER, Ko D, Chen C, Lipsick JS (2008). A conserved acidic patch in the MYB domain is required for activation of an endogenous target gene and for chromatin binding. Molecular Cancer 7 (1): 77-79.
Kolehmainen J, Korpelainen H, Mutikainen P (2010). Inbreeding and inbreeding depression in a threathened endemic plant, the African violet (Saintpaulia ionantha ssp. grotei), of the East Usambara Mountains, Tanzania. African Journal of Ecology 48 (1): 576-587.
Kumar S, Nei M, Dudley J, Tamura K (2008). MEGA: a biologistcentric software for evolutionary analysis of DNA and protein sequences. Briefings in Bioinformatics 9 (1): 299-306.
Lai Y, Li H, Yamagishi M (2013). A review of target gene specificity of flavonoid R2R3-MYB transcription factors and a discussion of factors contributing to the target gene selectivity. Frontiers in Biology 8 (2): 577-598.
Lou YC, Wei SY, Rajasekaran M, Chou CC, Hsu HM et al. (2009). Solution NMR structure of the R2R3 DNA binding domain of Myb1 protein from protozoan parasite Trichomonas vaginalis. Nucleic Acids Research 37 (2): 2381-2394.
Lowenstein DM, Matteson KC, Minor ES (2019). Evaluating the dependence of urban pollinators on ornamental, non-native, and ‘weedy’ floral resources. Urban Ecosystems 22 (2): 293- 302.
Machida C, Nakagawa A, Kojima S, Takahashi H, Machida Y (2015). The complex of ASYMMETRIC LEAVES (AS) proteins plays a central role in antagonistic interactions of genes for leaf polarity specification in Arabidopsis. Wiley Interdisciplinary Reviews-Developmental Biology 4 (1): 655-671.
Nhut DT, Trinh DB, Cuong D.M, Tung HT, Huy NP et al. (2018). Study on silver nanoparticles as a novel explant disinfectant for micropropagation of African violet (Saintpaulia ionantha H. Wendl.). Vietnam Journal of Biotechnology 16 (1): 1-10.
Ogata K, Morikawa S, Nakamura H, Sekikawa A, Inoue T et al. (1994). Solution structure of a specific DNA complex of the MYB DNA-binding domain with cooperative recognition helices. Cell 79 (2): 639-648. doi: 10.1016/0092-8674(94)90549-5
Ori N, Eshed Y, Chuck G, Bowman J, Hake S (2000). Mechanisms that control knox gene expression in the Arabidopsis shoot. Development 127 (2): 5523-5532.
Peykari N, Zamani K (2019). Cloning and characterization of a constitutive promoter of polyubiquitin gene from Cicer ariethinum. Journal of Crop Science and Biotechnology 9 (25): 35-45.
Roberts WR, Roalson EH (2017). Comparative transcriptome analyses of flower development in four species of Achimenes (Gesneriaceae). BMC Genomics 18 (1): 1-26. doi: 10.1186/ s12864-017-3623-8
Sobral R, Costa MM (2017). Role of floral organ identity genes in the development of unisexual flowers of Quercus suber L. Scientific reports 7 (1): 10368. doi: 10.1038/s41598-017-10732-0
Stephan L, Tilmes V, Hulskamp M (2019). Selection and validation of reference genes for quantitative Real-Time PCR in Arabis alpina. PLoS ONE 14 (3): e0211172. doi: 10.1371/journal. pone.0211172
Sun Y, Zhou Q, Zhang W, Fu Y, Huang H (2002). ASYMMETRIC LEAVES1, an Arabidopsis gene that is involved in the control of cell differentiation in leaves. Planta 214 (1): 694-702. doi: 10.1007/s004250100673
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013). MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30 (2): 2725-2729. doi: 10.1093/molbev/mst197
Tattersall AD, Turner L, Knox MR, Ambrose MJ, Ellis TH et al. (2005). The mutant crispa reveals multiple roles for PHANTASTICA in pea compound leaf development. Plant Cell 17 (3): 1046-1060. doi: 10.1105/tpc.104.029447
Tian X, Zou P, Miao M, Ning Z, Liao J (2018). RNA-Seq analysis reveals the distinctive adaxial–abaxial polarity in the asymmetric one-theca stamen of Canna indica. Molecular Genetics and Genomics 293 (4): 391-400. doi: 10.1007/s00438-017-1392-3
Timmermans MC, Hudson A, Becraft PW, Nelson T (1999). ROUGH SHEATH2: A Myb protein that represses knox homeobox genes in maize lateral organ primordia. Science 284 (1): 151-153.
Truskina J, Vernoux T (2017). The growth of a stable stationary structure: coordinating cell behavior and patterning at the shoot apical meristem. Current Opinion in Plant Biology 41 (1): 83-88. doi: 10.1016/j.pbi.2017.09.011
Waites R, Hudson A (1995). Phantastica: a gene required for dorsoventrality of leaves in Antirrhinum majus. Development 121 (3): 2143-2154.
Weber A, Clark JL, Moller M (2013). A new formal classification of Gesneriaceae. Scientific Journal of the Marie Selby Botanical Gardens 31 (2): 68-94.
Wei S, Lou Y, Tsai J, Ho M, Chou C et al. (2012). Structure of the Trichomonas vaginalis MYB3 DNA-binding domain bound to a promoter sequence reveals a unique C-terminal beta-hairpin conformation. Nucleic Acids Research 40 (1): 449-459.
Wilkins O, Nahal H, Foong J, Provart NJ, Campbell MM (2009). Expansion and diversification of the Populus R2R3–MYB family of transcription factors. Plant Physiology 149 (1): 981- 993. doi: 10.1104/pp.108.132795
Wu G, Lin WC, Huang T, Poethig RS, Springer PS et al. (2008). KANADI1 regulates adaxial–abaxial polarity in Arabidopsis by directly repressing the transcription of ASYMMETRIC LEAVES2. Proceedings of the National Academy of Sciences USA 105 (1): 16392-16397.
Xu B, Li Z, Zhu Y, Wang H, Ma H et al. (2008). Arabidopsis genes AS1, AS2, and JAG negatively regulate boundary-specifying genes to promote sepal and petal development. Plant Physiology 146 (2): 566-575. doi: 10.1104/pp.107.113787
Xu W, Dubos C, Lepiniec L (2015). Transcriptional control of flavonoid biosynthesis by MYB–bHLH–WDR complexes. Trends in Plant Science 20 (3): 176-185. doi: 10.1016/j.tplants.2014.12.001
Xu ZS, Feng K, Que F, Wang F, Xiong AS (2017). A MYB Transcription Factor DcMYB6, is involved in regulating anthocyanin biosynthesis in purple carrot taproots. Scientific Reports 7 (1): 45-54.
Yang J, Yan R, Roy A, Xu D, Poisson J et al. (2015). The I-TASSER Suite: protein structure and function prediction. Nature Methods 12 (1): 7-15. doi: 10.1038/nmeth.3213
Yang T, Wang Y, Teotia S, Zhang Zh, Tang G (2018). The making of leaves: how small RNA networks modulate leaf development. Frontiers in Plant Science 9 (1): 1-7. doi: 10.3389/ fpls.2018.00824