Accepted Manuscript Title: Molecular Cloning and Expression Analysis of Cryptochrome Gene PsCRY2 in Tree Peony Author: Ren Xiuxia, Wang Shunli, Xue Jingqi, Zhu Fuyong, Liu Chuanjiao, Zhang Xiuxin PII: DOI: Reference: S2468-0141(17)30033-X http://dx.doi.org/doi: 10.1016/j.hpj.2016.10.003 HPJ 49 To appear in: Horticultural Plant Journal Received date: Revised date: Accepted date: 10-8-2016 14-9-2016 27-10-2016 Please cite this article as: Ren Xiuxia, Wang Shunli, Xue Jingqi, Zhu Fuyong, Liu Chuanjiao, Zhang Xiuxin, Molecular Cloning and Expression Analysis of Cryptochrome Gene PsCRY2 in Tree Peony, Horticultural Plant Journal (2017), http://dx.doi.org/doi: 10.1016/j.hpj.2016.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain Horticultural Plant Journal Available online at www.sciencedirect.com The journal’s homepage: http://www.journals.elsevier.com/horticultural-plant-journal Molecular Cloning and Expression Analysis of Cryptochrome Gene PsCRY2 in Tree Peony REN Xiuxia, WANG Shunli, XUE Jingqi, ZHU Fuyong, LIU Chuanjiao, and ZHANG Xiuxin * Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, China, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Science, Beijing 100081, China Received 10 August 2016; Received in revised form 14 September 2016; Accepted 27 October 2016 * Corresponding author Tel.: +86 10 82105944 E-mail address: zhangxiuxin@caas.cn The Chinese version of this paper is published in Acta Horticulturae Sinica DOI: 10.16420/j.issn.0513-353x.2015-0252 http://dx.doi.org/10.1016/j.hpj.2016 2468-0141 ©2016 Chinese Society for Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS) Abstract Cryptochromes are blue/ultraviolet-A (UV-A) light receptors involved in regulating various aspects of plant growth and development Investigations of the structure and functions of cryptochromes in plants have largely focused on herbaceous plants However, few data on the function of CRY2 are available in woody plants In this study, a cryptochrome (CRY2) gene was isolated from Paeonia suffruticosa by Reverse Transcription Polymerase Chain Reaction (RT-PCR) Sequence alignment and motif analysis showed that the deduced amino acids contained a PHR domain near the amino terminus and a CCT domain near the carboxy terminus PsCRY2 showed high identity with AtCRY2 of Arabidopsis Phylogenetic analysis indicated that it was closely related to Citrus sinensis Gene expression analysis revealed that the highest expression levels of PsCRY2 occurred in the bud and seed embryo of P suffruticosa, followed by the roots, stems, and leaves PsCRY2 was upregulated during the entire process of bud differentiation, whereas this was downregulated during the early stage of bud development and upregulated in the middle and late stages The highest level of PsCRY2 expression was observed in the big bell-like flower buds These results suggested that PsCRY2 plays an important role in both bud differentiation and bud development The expression patterns of PsCRY2 in the buds of ‘Luoyanghong’ and ‘Qiufa 1’ were similar, whereas that in the buds of ‘Qiufa 1’ was significantly higher than in the buds of ‘Luoyanghong’ The buds of plants subjected to different photoperiod treatments exhibited variations in PsCRY2 expression patterns The expression of PsCRY2 decreased during bud sprouting and in the small bell-like flower buds that were subjected to short-day photoperiod compared to that observed under long-day photoperiod Page of Keywords: tree peony; photoperiod pathway; cryptochromes; PsCRY2; qRT-PCR Introduction Flowering pertains to the transitory period from the vegetative stage to reproductive growth and is regulated by both environmental and endogenous cues There are four major floral-inductive pathways in Arabidopsis, namely, photoperiod, autonomous, vernalization, and gibberellin (GA)-induced pathways The photoperiod and vernalization pathways mediate the response to environmental signals, whereas the autonomous and GA pathways apparently act independent of these signals (Simpson and Dean, 2002) Wang et al (2014, 2015) hypothesized that the autonomous and GA pathways regulate tree peony flowering, whereas Li (1999) proposed that light determines the flower quality of tree peony The photoperiod pathway is the most conservative flowering response pathway in both dicots and monocots (Yanovsky and Kay, 2003; Yang et al., 2013) In this pathway, day length is perceived by photoreceptors and along with entrainment factors, synchronizes its endogenous circadian clock with the environment Cryptochromes are soluble flavoproteins that are encoded by two genes in Arabidopsis, CRYPTOCHROME (CRY1) and CRY2 (Yanovsky and Kay, 2003) Cryptochrome (CRY2), which belongs to the flavoprotein subfamily, is a blue/UV receptor of the photoperiod pathway (Cashmore et al., 1999; Lin, 2002; Yasushi and Weigel, 2007) It is mainly involved in photomorphogenesis and photoperiodic regulation of flowering Homologous cryptochrome genes have been isolated from various species, including mosses, ferns, tomatoes, peas, rice, fruit flies, and mice (Liu et al., 2011) However, no homologous CRY2 gene has been reported in tree peony to date, and its function remains elusive Thus, cloning the homologous CRY2 gene of tree peony could reveal the genetic mechanisms underlying its flowering, which in turn may facilitate in methods of regulating tree peony flowering Molecular characterization and phylogenetic reconstruction of the PsCRY2 gene, which is homologous to the tree peony CRY2 gene, was performed in the present study Furthermore, the expression pattern of PsCRY2 in different tissue-organs, as well as in various developmental buds or buds subjected to different treatments, was analyzed The findings of the present study could provide theoretical basis for PsCRY2 gene function in floral induction Materials and methods 2.1 Materials A new tree peony variety ‘Qiufa 1’ (selected by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences) that re-blooms in autumn (Zhang et al., 2005) and a traditional cultivar ‘Luoyanghong’ were used as materials in this study Different developmental buds of 5-year-old ‘Luoyanghong’ and ‘Qiufa 1’ were collected from March 2014 to October 2014 Uniform seeds of ‘Qiufa 1’ were harvested in August 2014 Some of the harvested seeds were immediately frozen in liquid N2 and then stored at –80 ℃ for gene expression analysis, and the remaining seeds were sown in a pot to produce seedlings, which were then grown Page of within a temperature range of 15–28 ℃ in a greenhouse The seedlings were collected in March 2015 for gene expression analysis and stored using the same conditions as those used for seeds 2.2 Methods 2.2.1 mRNA extraction and cDNA synthesis Total mRNA from the buds, roots, stems, and leaves of ‘Qiufa 1’ and buds of ‘Luoyanghong’ was extracted according to Liu et al (2014) and Wang et al (2014) Total mRNA from the embryo of ‘Qiufa 1’ was extracted by using Trizol (Invitrogen, USA), following the procedure of Li et al (2010) High-quality RNA was selected, and cDNA was synthesized by using a QuantScript RT Kit (TIANGEN, Beijing, China), according to the manufacturer’s instructions 2.2.2 Primer design and PCR reaction system Based on the 5' and 3' terminal sequence of the PsCRY2 gene that was obtained by our laboratory, a pair of specific primers was designed using Primer 5.0 to amplify the complete open reading frame (ORF) of the PsCRY2 gene The primers were as follows: PsCRY2-1F: 5'-TATTTCCTATCCATTTCG-3'; and PsCRY2-1R: 5'-TTTCATTCTAATCAAGCAG-3' The PCR reaction system was performed in a 30 μL volume containing 20.8 μL of ddH2O, μL 10 × LA Taq buffer (Mg2+ Plus), μL dNTP Mixture (2.5 mmol·L-1 each), 0.2 μL LA Taq (5 U·μL-1), μL of each primer, and 36 ng of the template cDNA The reaction system was performed according to Wang et al (2015) The PCR products were separated by 1% agarose gel electrophoresis, and the expected products were purified from the gel and cloned with a pGEM-T vector (Promega) The identified positive clone was then sequenced by TaKaRa Biotechnology (Dalian) Co., LTD, China 2.2.3 Sequence analysis and phylogenetic study of the PsCRY2 gene in tree peony The sequence translation of the PsCRY2 gene, the molecular weight of the protein, the prediction of physical and chemical properties, subcellular localization, amino acid sequence alignment, two-dimensional (2D) and three-dimensional (3D) protein structure prediction and phylogenetic tree construction were performed as described by Zhu et al (2014) and Zhang et al (2014) Phosphorylation sites were predicted by using the online tool NetPhos 2.0 Server (http://www.cbs.dtu.dk/services/NetPhos/) 2.2.4 qRT-PCR A new pair of primers was designed for quantitative real-time RT-PCR (qRT-PCR) analysis, and the actin gene was used as reference The primers specific to the PsCRY2 gene that were used in qRT-PCR analysis were as follows: qPsCRY2-1F: 5'-CGTGCGAATAAAGCAGATA-3'; and qPsCRY2-1R: 5'-GAAACAAAGGTATCGGGAG-3' The length of the expected amplicon was 241 bp The detailed procedures for the qRT-PCR reactions were according to Wang et al (2014) Results 3.1 Full-length cDNA cloning and sequence analysis of the PsCRY2 gene in tree peony In the present study, one new PsCRY2 gene was cloned from the buds of P suffruticosa ‘Qiufa 1’ The full ORF length of the PsCRY2 gene was 094 bp, which encoded putative protein consisting of 633 amino acids (Fig 1) A BLASTN search on NCBI showed that the nucleotide sequence was very similar to the CRY2 gene Thus, the gene cloned in our work was designated as PsCRY2, with GenBank Accession Number KP982893 ExPaSy ProtParam online tools prediction showed that the PsCRY2 is a hydrophilic protein, with the molecular weight of approximately 72.46 kD, and a 5.81 isoelectric point 3.2 Homology comparison analysis of PsCRY2-encoded amino acid residues Page of Sequence alignment of the deduced amino acid sequence of PsCRY2 with two homologous CRY2 genes from Citrus sinensis and Arabidopsis thaliana indicated that PsCRY2 also contained the characteristic PHR and CCT domains (Fig 2), thereby suggesting that PsCRY2 belongs to the cryptochrome gene family Further studies showed that the PHR domain located at the amino terminal of PsCRY2 was a highly conserved domain, which included a DNA photolyase and FLAVINADENINEDINUCLEOTIDE (FAD)-binding domain On the other hand, three important DQXVP-acidic-TAES (DAS) domains in the carboxyl terminal were highly conserved, as these was a hallmark feature of cryptochromes, whereas the rest of the carboxyl terminal was not conserved (Lin and Shalitin, 2003) Based on these findings, we deduced that the biological function of the PsCRY2 gene may be similar to that of the other cryptochromes Besides, the phosphorylation sites and a nuclear localization signal (NLS) sequence were also detected in the carboxy terminus of PsCRY2 by using NetPhos 2.0 Server prediction (Fig 3) Based on these observations, we deduced that PsCRY2 was located in the nucleus, which was in agreement with its predicted subcellular location Secondary structure prediction of PsCRY2 showed that it consisted of 40.6% α-helices, 9.79% β-sheets, 15.48% the extended chains, and 34.12 % random coils Tertiary structure prediction of PsCRY2 and AtCRY2 showed that the PHR tertiary structures of PsCRY2 and AtCRY2 were similar, which consisted of several helical and β structures, which was in agreement with the above secondary structure prediction The CRY2 gene plays an important role in photoperiodic flowering regulation in Arabidopsis (Guo et al., 1998) The observed similarity in tertiary structure suggested that PsCRY2 regulates flowering in tree peony 3.3 Phylogenetic analysis of PsCRY2 To investigate the phylogenetic evolutionary relationships of PsCRY2, a phylogenetic reconstruction was performed by using 10 homologous CRY2 proteins from various species The results showed that PsCRY2 and CsCRY2 were clustered in one branch and was closely related to Citrus sinensis, whereas it was distantly related to the CRY2 proteins of monocot plants (Fig 4) 3.4 Expression analysis of the PsCRY2 gene in different tissues-organs and developmental buds The transcriptional expression pattern of PsCRY2 in specific tissues-organs were first investigated, which indicated an upregulation in seed embryos and buds, followed by seedling roots, stems, and leaves (Fig 5) Based on these findings, we deduced that the PsCRY2 gene plays a role in embryo development and flowering The expression profile of PsCRY2 in different differentiated and developmental buds showed that it was upregulated in the differentiated buds from the bract primordium phase to the pistil primordium phase, and followed a down-, up-, and down regulated trend in different developmental buds (Fig 6) In sum, the expression of the PsCRY2 gene was relatively high during the entire process of bud differentiation, and the highest expression was observed at the big bell-like flower bud and bell-like flower bud extending stages (Fig 6) Based on these observations, we deduced that PsCRY2 not only plays an important role in inducing flower bud differentiation, but also in regulating flowering in tree peony 3.5 The expression analysis of the PsCRY2 gene in the spring buds of ‘Luoyanghong’ and ‘Qiufa 1’ During bud development, the expression of the PsCRY2 gene followed an increasing trend in the spring buds of both ‘Luoyanghong’ and ‘Qiufa 1’, with the buds of ‘Qiufa 1’ showing a higher expression than that in ‘Luoyanghong’ (Fig 7) 3.6 The expression of the PsCRY2 gene in buds subjected to different photoperiod treatments Page of In the bud sprouting and small bell-like flower bud stages, the expression level of the PsCRY2 gene in ‘Qiufa 1’ was lower in the buds during autumn (short-day condition) than that in spring (long-day condition), whereas the expression during bud swelling was similar to that in spring (Fig 8) We also observed that the flowering quality of ‘Qiufa 1’ in spring was significantly better than the re-blooming quality in autumn Therefore, we deduced that tree peony flowering was controlled by the photoperiod pathway, and a higher level of PsCRY2 gene expression results in better tree peony flowering quality Discussion In the present study, the full-length of coding sequence of the PsCRY2 gene was cloned from the bud of ‘Qiufa 1’ Amino acid sequence analysis showed that the PsCRY2 consisted of two conserved domains, namely, the PHR and CCT domains, which were similar to that of AtCRY2 We also confirmed that PsCRY2 is the homologous gene of CRY2 Phylogenetic evolution analysis showed that PsCRY2 is highly conserved in plants, and may also be functionally conserved We also observed differences in the expression of PsCRY2 in various tissues of the ‘Qiufa 1’ plant, which was similar to that in other species, including A thaliana, Camellia sinensis, Brassica campestris, Malus sieversii, and Oryza sativa (Tóth et al., 2001; Platten et al., 2005; Chatterjee et al., 2006; Hirose et al., 2006; Mao, 2012) The upregulation of PsCRY2 in the buds and embryos suggest that PsCRY2 may affect flower bud differentiation, flowering, and seed development The observed similarity in the expression pattern of PsCRY2 in the spring buds of ‘Luoyanghong’ and ‘Qiufa 1’ was indicative of an identical gene function in tree peony, whereas its upregulation in the buds of ‘Qiufa 1’ suggests it regulates re-blooming in tree peony The expression levels of PsCRY2 under different photoperiod conditions and relatively poor re-blooming quality in autumn showed that this gene determines the quality of flowering These results suggest that the duration of exposure to light should be prolonged to improve the re-blooming quality of ‘Qiufa 1’ in autumn Successful CRY2 gene cloning provides additional information on the genetic mechanism underlying flower induction in tree peony as well as facilitates in the improvement of the forcing culture of tree peony flowering in autumn Acknowledgments The work is supported by the grants from Special Fund for Agro-scientific Research in the Public Interest (201203071), China Association for Science and Technology Foundation for Young Scholars (2016QRNC001), and the Agricultural Science and Technology Innovation Program (ASTIP) of the Chinese Academy of Agricultural Sciences References Cashmore, A.R., Jarillo, J.A., Wu, Y.J., Liu, D., 1999 Cryptochromes: Blue light receptors for plants and animals Science, 284: 760–765 Chatterjee, M., Sharma, P., Khurana, J.P., 2006 Cryptochrome from Brassica napus is up-regulated by blue light and controls hypocotyl/stem growth and anthocyanin accumulation Plant Physiology, 141: 61–74 Guo, H., Yang, H., Mockler, T.C., Lin, C., 1998 Regulation of flowering time by Arabidopsis photoreceptors Science, 279: 1360–1363 Hirose, F., Shinomura, T., Tanabata, T., Shimada, H., Takano, M., 2006 Involvement of rice cryptochromes in de-etiolation responses and flowering Plant and Cell Physiology, 47: 915–925 Li, J.J., 1999 Chinese tree peony and Paeonia lactiflora Beijing: China Forestry Publishing House (in Chinese) Li, X.H., Wang, K., Wang, S.L., Gao, L.Y., Xie, Z.Z., Hsam, S.L.K., Zeller, F.J., Yan, Y.M., 2010 Molecular characterization and comparative transcription alanalysis of LMW-m-type genes from wheat（Triticum aestivum L.）and Aegilops species Theoretical and Applied Genetics, 121: 845–856 Lin, C., 2002 Blue light receptors and signal transduction Plant Cell, 14: S207–S225 Page of Lin, C., Shalitin, D., 2003 Cryptochrome structure and signal transduction Annual Review of Plant Biology, 54: 469–496 Liu, C.J., Wang, S.L., Xue, J.Q., Zhu, F.Y., Ren, X.X., Li, M.Y., Zhang, X.X., 2014 Molecular cloning and expression analysis of the flowering-regulating transcription factor PrSOC1 gene in tree peony Acta Horticulturae Sinica, 44: 2259–2267 Liu, H., Liu, B., Zhao, C., Pepper, M., Lin, C., 2011 The action mechanisms of plant cryptochromes Trends in Plant Science, 16: 684–691 Mao, K., 2012 Molecular cloning and functional characterization of apple cryptochrome genes MCRY1 and MdCRY2 [Ph D Dissertation] Tai’an: Shandong Agricultural University (in Chinese) Platten, J.D., Foo, E., Foucher, F., Hecht, V., Reid, J.B., Weller, J.L., 2005 The cryptochrome gene family in pea includes two differentially expressed CRY2 genes Plant Molecular Biology, 59: 683–696 Simpson, G.G., Dean, C., 2002 Arabidopsis, the Rosetta stone of flowering time? Science, 296: 285–289 Tóth, R., Kevei, E., Hall, A., Millar, A.J., Nagy, F., Kozma-Bognár, L., 2001 Circadian clock-regulated expression of phytochrome and cryptochrome genesin Arabidopsis Plant Physiology, 127: 1607–1616 Wang, S.L., Xue, J.Q., Ahmadi, N., Patricia, H., Zhu, F.Y., Ren, X.X., Zhang, X.X., 2014 Molecular characterization and expression patterns of PsSVP genes reveal distinct roles in flower bud abortion and flowering in tree peony (Paeonia suffruticosa) Canadian Journal of Plant Science, 94: 1181–1193 Wang, S.L., Xue, J.Q., Zhu, F.Y., Zhang P., Ren, X.X., Liu, C.J., Zhang, X.X., 2014 Molecular cloning, expression and evolutionary analysis of the flowering-regulating transcription factor CONSTANS-like-4 gene in tree peony Acta Horticulturae Sinica, 41: 1409–1417 Wang, S.L., Beruto, M., Xue, J.Q., Zhu, F.Y., Liu, C.J., Yan, Y.M., Zhang, X.X., 2015 Molecular cloning and potential function prediction of homologous SOC1 genes in tree peony Plant Cell Reports, 34: 1459–1471 Yang, X.Q., Tang, Q.L., Song, M., Wang, Z.M., 2013 Flowering control mechanism of CO as a key factor in photoperiod pathway Plant Physiology Journal, (9): 861–868 (in Chinese) Yanovsky, M.J., Kay, S.A., 2003 Living by the calendar: how plants know when to flower Nature Reviews Molecular Cell Biology, 4: 265–275 Yasushi, K., Weigel, D., 2007 Move on up, it’s time for change mobile signals controlling photoperiod-dependent flowering Genes and Development, 21: 2371–2384 Zhang, P, Wang, S.L., Zhu, F.Y., Xue, J.Q., Liu, C.J., Ren, X.X., Zhang, X.X., 2014 Expression analysis of the PobZIP1 transcription factor gene in developmental seeds from tree peony and its relationship with ABA content Acta Horticulturae Sinica, 41: 1642–1650 Zhang, X.X, Wang, L.Y., Liu, A.Q., 2005 The study on the investigation of autumn-flowering tree peony varieties in Heze City, Shandong Province Northern Horticulture, (3): 42–44 (in Chinese) Zhu, F.Y., Liu, C.J., Xue, J.Q., Wang, S.L., Zhang, P., Ren, X.X., Zhang, X.X., 2014 Isolation of florigen gene PdFT and its effects on flowering of tree peony (Paeonia delavayi Franch) Scientia Agricultura Sinica, 47: 2613–2624 (in Chinese) Page of Fig PCR amplification result on an agarose gel of PsCRY2 Fig Alignment of the deduced amino acid sequence of PsCRY2 with other CRY2 proteins from plants The black and pink arrows indicate the regions of PHR and CCT domains, respectively; Conserved domains (DNA photolyase, FAD binding, DQXVP, acidic, and STAES domains) are indicated by boxes colored in red, blue, orange, green, and purple, respectively Fig Composition of the bipartite nuclear localization signal (NLS) of PsCRY2 Page of Fig Phylogenetic relationship analysis among CRY2 proteins and Arabidopsis cryptochrome proteins Boot-strap values from 000-replicates were indicated at each node Fig Expression analysis of the PsCRY2 gene in seedling roots, seedling stems, seedling leaves, adult plant buds, and seed embryos in tree peony by qRT-PCR a, b, c indicate significant difference at the 0.05 level Fig Expression pattern analysis of PsCRY2 gene in different developmental buds A Undifferentiated phase; B Bract primordium phase; C Sepal primordium phase; D Petal primordium phase; E Stamen primordium phase; F Pistil primordium phase; G Bud swelling; H Bud sprouting; I Leaflet emerging; J Flower bud emerging phase; K Small bell-like flower bud; L Big bell-like flower-bud; M Bell-like flower-bud extending; N Color exposing a, b, c indicate significant difference at the 0.05 level Page of Fig Expression analysis of the PsCRY2 gene in the spring buds of ‘Luoyanghong’ and ‘Qiufa 1’ a’, b’ indicate significant difference in the expression levels in the buds of ‘Luoyanghong’ and ‘Qiufa 1’ at the 0.05 level; a, b indicate significant difference in the expression levels in the buds at different developmental stages at the 0.05 level Fig Expression analysis of the PsCRY2 gene in the buds of tree peony under spring and autumn conditions a’, b’ indicate significant difference in the expression level of buds of ‘Qiufa 1’ under spring and autumn conditions at the 0.05 level; a, b indicate significant difference in the expression level of buds at different developmental stages at the 0.05 level Page of ... role in inducing flower bud differentiation, but also in regulating flowering in tree peony 3.5 The expression analysis of the PsCRY2 gene in the spring buds of ‘Luoyanghong’ and ‘Qiufa 1’ During... LTD, China 2.2.3 Sequence analysis and phylogenetic study of the PsCRY2 gene in tree peony The sequence translation of the PsCRY2 gene, the molecular weight of the protein, the prediction of physical... Page of Fig Expression analysis of the PsCRY2 gene in the spring buds of ‘Luoyanghong’ and ‘Qiufa 1’ a’, b’ indicate significant difference in the expression levels in the buds of ‘Luoyanghong’ and