Molecular characterization and expression analysis of the critical floral genes in hickory (Carya cathayensis Sarg.).

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PLAPHY4016_proof ■ 4 August 2014 ■ 1/9

Plant Physiology and Biochemistry xxx (2014) 1e9

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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy

Research article

Molecular characterization and expression analysis of the critical floral genes in hickory (Carya cathayensis Sarg.) Q2

Chen Shen, Yingwu Xu, Jianqin Huang, Zhengjia Wang, Jiani Qiu, Youjun Huang* The Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Lin'an, Zhejiang 311300, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 May 2014 Accepted 23 July 2014 Available online xxx

The full ORFs of three floral genes in hickory (Carya cathayensis Sarg.), CcAGL24 (the AGAMOUS-LIKE24 homolog), CcSOC1 (the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 homolog) and CcAP1 (the APETALA1 homolog) are derived using a 5' RACE PCR protocol. Through sequence alignment and phylogenetic analysis, it is demonstrated that the three genes belong to the MADS-Box family. According to the evolutionary trees of the three genes, the homologous genes from the same family cluster well together, while those from different orders doesn't match evolutionary regularity of individual organisms. The result of Quantitative RT-PCR analysis shows that the transcriptional levels of the three genes are upregulated in early stage and down-regulated in late stage in pistillate floral development. However, it takes different time to reach respective expression peak among the three genes. In staminate floral development, the transcription trend of the three genes is up-regulated, subsequently down-regulated, and then up-regulated again. Nevertheless, those trajectories, peaks, expression levels, inflection points are different in pistillate floral development. The result suggests that their functions are different in between pistillate and staminate floral development. The probable ordinal site of the three genes in the flowering network from top down is CcAGL24, CcSOC1, and CcAP1, which is identical to that in herbaceous plants. Moreover, several adverse environmental factors trigger several negative genes and then confine the development of staminate floral buds. Our results suggest the possible relationship among the three critical floral genes and their functions throughout the floral development in hickory. © 2014 Published by Elsevier Masson SAS.

Keywords: AGL24 AP1 Carya cathayensis Sarg. Expression analysis SOC1

1. Introduction Flowering is a switch from vegetative to reproductive growth in angiosperms, through which shoot apical meristems turn into floral meristems and then develop floral organs (Weigel and Nilsson, 1995; Fitter et al., 2002). Five major pathways in flowering process have been designated i.e., the photoperiod pathway, the autonomous pathway, the gibberellin pathway, the vernalization pathway and the thermosensory pathway in Arabidopsis thaliana n et al., 1999; Mouradov et al., 2002; Bla zquez et al., 2003; (Rolda Kumar et al., 2012). Each route responds to endogenous or environmental cues independently but intertwines a comprehensive network via several floral integrators during late stages. In this network, the inner-signals (age, gibberellin, etc.) and outer-signals (e.g. photoperiod, atmosphere temperature) are perceived by several receptors and transferred to their downstream genes. Subsequently, these genes trigger several floral integrators such as

* Corresponding author. Tel.: þ86 57163732761; fax: þ86 57163730809. E-mail addresses: [email protected], [email protected] (Y. Huang).

Flowering locus T (FT), AGAMOUS-LIKE24 (AGL24), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), and lead to floral organ development finally (Corbesier et al., 2007; Tamaki et al., 2007). As a transmissible signal, FT protein is induced via inner-signals and outer-signals and is translocated from leaf phloem to shoot apex where it binds to the transcription factor Flowering locus D (FD) to form a dimer, then stimulates transcription of the floral meristem identity gene APETALA1 (AP1) (Laurent et al., 2007; Tamaki et al., 2007; Turck et al., 2008). Another floral integrator AGL24 is also regulated by inner-signals and outer-signals via each pathway. As AGL24 is activated in one shoot apex, it promotes target gene SOC1. Subsequently, both genes combine together and form the AGL24-SOC1 dimer. The dimer activates directly the floral meristem identity gene LEAFY (LFY) finally (Lee et al., 2008; Liu et al., 2008). SOC1 is regulated not only by AGL24, but also by other direct target genes of key flowering genes such as CONSTANS (CO) and FLOWERING LOCUS C (FLC) (Hepworth et al., 2002; Seo et al., 2009). SOC1 plays a crucial role in regulating the floral organ development by interacting with several floral meristem genes APETALA1 (AP1), SEPALLATA1 (SEP1), SEPALLATA2 (SEP2), and SEPALLATA3 (SEP3) (Krizek and Meyerowitz, 1996; Pelaz et al., 2001;

http://dx.doi.org/10.1016/j.plaphy.2014.07.020 0981-9428/© 2014 Published by Elsevier Masson SAS.

Please cite this article in press as: Shen, C., et al., Molecular characterization and expression analysis of the critical floral genes in hickory (Carya cathayensis Sarg.), Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.07.020

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C. Shen et al. / Plant Physiology and Biochemistry xxx (2014) 1e9

Wigge et al., 2005). LEAFY (LFY) is a flower-meristem-identity gene by regulating inflorescence development in Arabidopsis. The expression of LFY promotes activating another flower-meristemidentity gene AP1 which protein in turn represses the expression of LFY and SOC1-AGL24 complex. AP1 is also required as a floral organ gene for sepal and petal development. AGL24, SOC1 and AP1 belong to MADS-box family for each amino acid sequence containing a MADS-box domain. These three MADS-box members play important roles in regulating flowering time, floral meristem identity, and development of floral organ during the floral transi et al., 2003). tion (Hayama et al., 2003; Parenicova It is a long period for woody plants from vegetative growth to reproductive growth (Rottmann et al., 2000; Jansson and Douglas, 2007). Once entering reproductive, woody plants can bloom every year (i.e., flowering seasonally) (Hsu et al., 2006, 2011). Hickory (Carya cathayensis Sarg.) is a well-known nut tree in China, as it contains rich unsaturated fatty acids which benefit human health. This species is a woody deciduous, monecious, achlamydeous, and catkin-bearing plant with a long juvenile stage. Upon entering adult, short reproductive shoots in the tree develop to pistillate flowers. At the apex of the short branches, CcLFY expression reaches the highest level in midMarch (Huang et al., 2013). Subsequently, the primordia of pistillate flowers emerge in late March. Finally, macroscopic pistillate floral buds are developed in the middle of April (Huang et al., 2007). According to the data of transcriptome sequencing and gene chip in our previous research, it is suggested that seasonal flowering in hickory is a comprehensive event which includes the photoperiod pathway, the autonomous pathway, the gibberellin pathway, the vernalization pathway, and the thermosensory pathway longitudinally, and undergoes three stages latitudinally, that is signal transduction, signal integration and floral organ development (Huang et al., 2013). Staminate flower buds in hickory were differentiated from axillary buds in a short reproductive shoot in late May. Then the staminate floral buds remain dormant from July to next spring. In following year, the staminate inflorescences resume development in early April and wither finally after scattering pollen in early May (Huang et al., 2006). Many miRNAs play a pivotal role in regulating flowering (Wang et al., 2012). Moreover, the data of the whole genome sequencing of hickory (data not published) provides a significant genetic platform for further elucidating molecular mechanism of flowering of woody plants. In short, hickory is a good experimental material for studying the transition of juvenile to adult, seasonal flowering and sex differentiation. And this study also provides a necessary theoretical support for promoting early blossoming, early bearing and high yielding in the forestry production in hickory. In this paper, the open-reading frames (ORFs) of CcAGL24, CcSOC1 and CcAP1 were obtained by RACE technology. The characteristics of the three proteins and the relationships among their homologous proteins from different species were analyzed using bioinformatic methods. The expression of the three genes in the process of staminate and pistillate floral development was studied by Quantitative RT-PCR (qRT-PCR) method. This paper aims to explore the biological functions of the three genes and to elucidate the flowering molecular mechanisms in hickory. 2. Materials and methods 2.1. Plant materials Terminal buds from reproductive shoots in an about 30-year-old hickory tree were sampled in Lin'an city (30 N, 119 W), Zhejiang province, China. The sequence of sample dates is February 21st,

27th, March 1st, 5th, 11st, 14th, 18th, 22nd, 26th, 29th, 31st, April 6th, 13rd, denoted as S1eS13, respectively. These samples from terminal buds were used to study the development of pistillate floral buds. It is worth mentioning that the expression of CcLFY reaches the highest level at S7 (March 18th). It is known that most primordia of pistillate flowers emerge at S9 (March 26th), and macroscopic pistillate floral buds are developed at S13 (April 13rd). Furthermore, the staminate samples were collected from axillary buds of reproductive shoots, which develop from vegetative buds to staminate floral ones. They were sampled successively on May 9th, 17th, 22nd, 25th, 29th, June 2nd, July 25th, August 16th, September 22nd, October 23rd, November 22nd, December 25th in 2012 and January 22nd, February 25th, March 23rd, April 2nd in next year, denoted as S14eS29. Particularly, most primordia of pistillate flowers emerge at S18 (May 29th). As the staminate floral buds remain dormant from S20 (July 25th) to next spring, and the staminate inflorescences go on developing from S29 (April 2nd). All samples were immediately frozen in liquid nitrogen and stored at 70 C. Other experimental reagents such as reverse transcription kits, gel extraction kits and ampicillin (Amp), restriction enzymes, rTaq DNA polymerases and PMD18-T carriers were bought from ShengGong biological engineering (Shanghai) Co., LTD. 2.2. Methods 2.2.1. Total RNA extraction and cDNA synthesis The total RNA was extracted using CTAB method and then reversely transcribed using Prime ScriptRT Reagent Kit (TaKaRa, Shanghai, China) according to the manufacturer's instructions. 2.2.2. Cloning and sequence analysis of CcAGL24, CcSOC1 and CcAP1 Fragments of the three genes were assembled from the reads data of 454 transcriptome sequencing of hickory. According to conservative regions of the three genes, we designed their primers respectively for the following 5’ RACE protocol. The forward and reverse primers of CcAGL24 are 5'-ATGGCGAGGGAGAAGAT CAAGATCA-3', 5'-GAGAAGAGAGAGCCCTAATTTGAGA-3' respectively. The forward primer of CcSOC1 is 5'-ATGGTGAGAGGAAAGA CTCAGATGA-3', and the reverse primer of CcSOC1 is 5'-ATTT GGTGGGGGTAAACGCTTTGTT-3'. Similarly, the forward and reverse primers of CcAP1 are 5'-ATAGAGAACAAAATCAACCGTCAGG-3', 5'-C ATTTCACAGGGCAAAGCATCC-3', respectively. The ORFs were cloned to pMD18-T (TaKaRa) and sequenced for confirmation. Homology searches of CcAGL24, CcSOC1 and CcAP1 were performed using BLASTP (http://www.ncbi.nlm.nih.gov/blast/). The physicochemical property and the hydrophilicity of the three encoding proteins were performed via ProParam of ExPASy server. Multiple sequence alignments of the three proteins were performed by Clustal 1.83 software. Base and amino acid Sequences of three genes were further aligned and the overall similarity of homologous genes was calculated using DNAMAN 7.0 software. Phylogenetic trees of SOC1 and AP1 were constructed through maximum likelihood of MEGA 6.06 software (no. of bootstrap replication ¼ 500, JoneseTayloreThornton model). 2.2.3. Quantitative RT-PCR Specific primers of the three genes for quantitative RT-PCR (qRTPCR) were designed using Primer express software (Applied Biosystems). The primer sequences were as follows: CcSOC1 (forward: 5'-ACGAACTACACAAGATAGAACAAC-3', reverse: 5'-GGGTAAACGCTTTGTTCTCC-3'), CcAP1 (foward: 5'-CC AAGTTATGCACGAATCCA-3', reverse: 5'-ACGCAGTCCCAATAGTG TCC-3'), CcLFY (forward: 5'-CCAAATGCGACACTAGGTGC-3', reverse:


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5'-TCCACGCCCCAACATTTTC-3'). The forward and reverse primers of internal standard hickory actin are 5'-GCTGAACGGGAAATTGGTC-3' and 5'-AGAGATGGCTGGAAGAG-3', respectively. Real-time RT-PCR was performed with the SYBR Green PCR kit (TaKaRa) on the BioRAD quantitative PCR instrument. Five micrograms RNA was used for cDNA synthesis using oligo dT-primer and Superscript II RnaseReverse Transcriptase (Invitrogen) according to the manufacturer's instructions. Amplification of cDNA was performed in the presence of gene-specific primers and the SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA) in MicroAmp Optical 96well reaction plates with optical covers using an ABI Prism 7500 Sequence Detector (Applied Biosystems). Each sample was analyzed in biological triplicate, using individual plants and treatments to test for reproducibility. The reaction conditions were 50 C for 2 min, 94 C for 10 min, and then 40 cycles of 94 C for 15 s and 60 C for 1 min. All cDNA samples were included in triplicate in all assays. Relative quantification of gene transcript abundance data was carried out with the 2DDCT or comparative CT method (Livak and Schmittgen, 2001), where the threshold cycle (CT) indicates the cycle number at which the amount of amplified transcript reaches a fixed threshold. Transcript levels were normalized with the CT values obtained from hickory actin. And variance analysis

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and multiple comparisons of the qRT-PCR data from all periods were analyzed by SPSS17.0 (Duncan test, a ¼ 0.05). 3. Results 3.1. Clone, sequencing and structural analysis of CcAGL24, CcSOC1 and CcAP1 The ORFs of the three genes were obtained by 5' RACE PCR. CcAGL24 has an open reading frame of 681 bp, which encodes a polypeptide of 226 amino acids, with 28.8 kDa molecular mass and deduced isoelectric point of 5.70 (Fig. 1a) (GenBank Accession KF918308). CcSOC1 has an open reading frame of 651 bp encoding a polypeptide of 217 amino acids with a predicted molecular mass of 24.9 kDa and isoelectric point o of 9.37 (Fig. 1a) (GenBank Accession KF918307). The full-length of CcAP1 cDNA is 750 bp, which encodes a polypeptide of 248 amino acids with predicted molecular mass of 25.4 kDa and isoelectric point of 8.65 (Fig. 1b) (GenBank Accession KF918309). All the three genes contain a typical MADS-box domain, with proteins of the MIKC type. Besides, both CcSOC1 and CcAP1 have K-box conserved domain. It is predicted that the encoding three proteins are highly hydrophilic especially in amino terminal.

Fig. 1. Sequence analysis of CcAGL24, CcSOC1 and CcAP1 genes. (a) Cloning of full ORFs CcAGL24 and CcSOC1. Lane 1e3: CcSOC1; lane 4e6: CcAGL24; lane 8: 100 bp Standard molecular weight marker. (b) Cloning of full ORFs CcAP1. Lane1: CcAP1; lane 3: 100 bp Standard molecular weight marker. (cee) Encoding domain of nucleotide sequence and its amino acid sequence of CcAGL24, CcSOC1 and CcAP1 respectively, showing their MADS-boxs domains and (or) K-boxs.


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3.2. Homology analysis of CcAGL24, CcSOC1 and CcAP1 Through sequence alignment, it is demonstrated that CcAGL24, CcSOC1 and CcAP1 share high homology with AGL24, SOC1 and AP1 homologs in other plant species. Specifically, CcAGL24 shares 76% identity with AGL24 in Arabidopsis (E value ¼ 7e-80). The CcAGL24 shares 32.48% similarity with AGL24 homologs in other species including Brassica napus (Identity ¼ 61.57%), A. thaliana (Identity ¼ 61.14%), Morus notabilis (Identity ¼ 42.79%). Their proteins all belong to MADSfamily because they include MADS-box domains (Fig. 1c; Fig. 2a). The CcSOC1 share 68.10% similarity with SOC1 homologs of other plant species including Theobroma cacao (Identity ¼ 80%), Glycine max (Identity ¼ 78%), Vigna unguiculata (Identity ¼ 78%), Prunus salicina (Identity ¼ 76%), Prunus armeniaca (Identity ¼ 76%), Prunus mume (Identity ¼ 76%), Vitis vinifera (Identity ¼ 74%), Spiraea cantoniensis (Identity ¼ 73%), Litchi chinensis (Identity ¼ 72%) and Citrus sinensis (Identity ¼ 72%). Their homologous proteins of SOC1 all belong to MADS-family and include MADS-box and K-box domains (Fig. 1d; Fig. 2b). CcAP1 averages 65.95% identity with its homologs in other species. In detail, CcAP1 shows 83% identity with JrAP1 of Juglans regia (E value ¼ 3e-136) and 73% identity with LcAP1 of L. chinensis. The homologous proteins of AP1 belong to MADS-family as well for containing MADS-box domain, besides K-box domain (Fig. 1e; Fig. 2c). Taken together, the 5-teminal regions of CcAGL24, CcSOC1, CcAP1 and their homologous proteins are conservative for containing MADS-box domains and other motifs, while their 3-terminal regions are less conservative without any domain or motif (Fig. 1cee). In the phylogenetic tree of SOC1, homologous proteins of SOC1 were well clustered in the same family plants. It is suggested that the phyletic evolution of SOC1 keeps consistency with the evolution of species at the family level. Fagales, Malvales, Sapindales, Brassicales, Fabales, Rosales, Caryophyllales and Magnoliales are clustered together, and belong to rosids except for Caryophyllales and Magnoliales. Furthermore, Gentianales, Solanales and Asterales are clustered to another clade, which all belong to asterids. Therefore, the clustering results are mostly anastomotic with the latest angiosperm classification system (APG III) at the order level (Fig. 3d). Interestingly, the Caryophyllales belongs to core eudicots, which is independent of rosids and asterids. However according to the molecular evolution tree of SOC1 homologs, it was clustered with rosids but not with asterids. The result suggests that Caryophyllales is closer to rosids than to asterids. CcSOC1 is closest to its homologs in T. cacao, Gossypium hirsutum, C. sinensis, Manglifera indica and L. chinensis (Fig. 3b). Similar to CcSOC1, the phylogenetic tree of CcAP1 showed that homologous proteins of AP1 are well clustered in the same family plants. However, the homologous proteins of AP1 cannot be clustered well at the order level (Fig. 3c). And the clustering result of AP1 is inferior to that of SOC1 homologs. That is, SOC1 is more highly conserved than AP1. CcAP1 has a closest relationship orderly with J. regia, Litchi chinenis, Hydrangea macrophylla and Neumbo nucifera (Fig. 3c). The rule also applies to CcAGL24 (Fig. 3a). It provides evidence to verify the result of sequence homology analysis as mentioned above. Through the phylogenetic trees of SOC1 and AP1 homologs, it is suggested that the research of genes' evolution is helpful to explore the evolution of species (Fig. 3d). Furthermore, since the law of gene evolution is not certainly same as that of the evolution of species, species taxonomy based on molecular technology cannot be merely reliant on one or several genes, but better on the information of its genome.

3.3. Expression of CcAGL24, CcSOC1 and CcAP1 in the floral development in hickory During pistillate flowering, the expression of CcAGL24, CcSOC1 and CcAP1 generally increased during the early stage and decreased in the late stage. The time points were different to reach their expression peaks of the three genes (Fig. 4aec). In detail, the maximum expression of CcAGL24 occurred at S6. And its high expression lasted till S9. Hereafter, the expression of CcAGL24 reduced continuously from S10 to S13 (Fig. 4a). CcSOC1 reached the highest expression at S7. And the high expression of CcSOC1 lasted till S10. Subsequently, the expression of CcSOC1 went continuously down (Fig. 4b). The expression of CcAP1 gradually increased in February and reached the maximum expression at S9 and then decreased in the late stage (Fig. 4c). Furthermore, we also detected the transcriptional expression of CcAGL24, CcSOC1, CcAP1 genes in the process of staminate flowering. The data showed that the expression tendency of the three genes increased in early stage then decreased at middle stage and finally increased again (Fig. 4eeg). Concretely speaking, the expression of CcAGL24 kept a high level from S15 to S20 and reached a peak at S15. Its expression remained low from S21 (Aug 16) to winter, and rose slightly after February (S27) of the next year (Fig. 4e). CcSOC1 maintained high expression in May with a maximum at S15 (May 17), but declined from June to the end of the year. It returned to high expression at the end of February (Fig. 4f). CcAP1 expressed high from late May to early June, with maximum expression at S18 (May 29), but expressed at a low level from July 25 (S20). Expression increased slowly at the end of February (S27) of the next year (Fig. 4g). In the process of staminate flower development, the general trend was consistent among the three genes. However, expression trajectory, maximum, and inflection points were different for each gene. It is suggested that the three genes play different roles in between pistillate and staminate floral development. 4. Discussion In the model plant Arabidopsis, AGL24 and SOC1 are the critical floral integrators in flowering. AGL24 protein activate its target gene SOC1 and fuse a dimer AGL24-SOC1, further directly activate downstream genes LFY and AP1, two critical flowering meristem genes, finally lead to the development of organogenesis (Lee et al., 2008). Studying functions of the three genes in the process of pistillate and staminate floral development is helpful to understand floral transition and flowering seasonally in woody plants. Floral meristem identity gene CcLFY is a switch of floral transition from vegetative to reproductive in hickory. Its high expression signifies that flowering enters floral organ development (Wang et al., 2011). In the process of pistillate flowering (from late February to middle of April), the expression of CcLFY increased gradually in early stage with a maximum expression at S7, and then gradually decreased (Fig. 4d). The maximum expression of CcAGL24, CcSOC1 and CcAP1 appeared at S6, S7 and S9, respectively. The chronological order of expression peaks of the three genes is CcAGL24, CcSOC1 and CcAP1. Hence, it is deduced that the order of the three genes is CcAGL24, CcSOC1 and CcAP1 via the upstream and downstream relationship in the pistillate flowering network in hickory. The relationship is similar to that in herbaceous model plants (Lee et al., 2008). The regulating relationship of the three genes is coincident between woody plant hickory and herbaceous model plants, such as A. thaliana, Antirrhinum majus, etc. It took nearly one year for the staminate floral development in hickory (Huang et al., 2006). According to qRT-PCR data, the


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Fig. 2. Sequence alignment of CcAGL24, CcSOC1 and CcAP1 homologs in angiosperm plants. (a) CcAGL24 homologs. (b) CcSOC1 homologs. (c) CcAP1 homologs.


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Fig. 3. Phylogenetic trees of AGL24, SOC1 and AP1 homologs. (a) Phylogenetic tree of AGL24 homologs. (b) Phylogenetic tree of SOC1 homologs. (c) Phylogenetic tree of AP1 homologs. (d) Phylogenetic tree of APG III system. Rectangle in blue indicates AGL24 homologs. Triangle in green indicates SOC1 homologs. And circle in red indicates AP1 homologs. Dashed circle indicates that homologs with a close relationship gather together. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


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Fig. 4. Timing Expression of floral genes in the process of flowering in hickory. (aed) Relative expression of CcAGL24, CcSOC1, CcAP1 and CcLFY genes in pistillate flowering in hickory, respectively. (eeg) Relative expression of CcAGL24, CcSOC1 and CcAP1 genes in staminate flowering in hickory, respectively.

maximum expression of CcAGL24 and CcSOC1 both appeared at S15 (May 17). While the maximum expression of CcAP1 emerged at S18 (May 29), it was 10 days later than that of CcAGL24 and CcSOC1. This result demonstrates that the three genes play one or several roles in the staminate flowering, and CcAGL24 and CcSOC1 probably locate in the upstream while CcAP1 locates in the downstream of the flowering network. Furthermore, the relationship among the three genes in staminate flowering is similar to that in pistillate flowering in hickory. Hereafter, the expression of the three genes kept a low level from August to January next year due to dormancy of staminate floral buds. From February, their expression rose gradually and promoted staminate floral buds differentiating further. Strikingly, the three genes expressed particularly in July. In detail, CcAGL24 expressed at a high level at S20 while CcSOC1 and CcAP1 expressed at low levels. It is inferred that the high expression of CcAGL24 is not enough to promote the high expression of CcSOC1 and CcAP1. And it

shows further that CcSOC1 and CcAP1 are not only positively regulated by CcAGL24, but also negatively regulated synchronously by other upstream genes such as homologous genes of SVP, TFL1 in hickory. According to the data of gene chip of hickory pistillate flowering in 2009 (The raw data of microarray analysis has been submitted to the website: http://www.cls.zju.edu.cn/binfo/hickory/), it was indicated that CcAGL24, CcSOC1 and CcAP1 expressed higher than SVP-like and TFL-like did, which expressed at a low level during pistillate flowering (Tao et al., 2012; Huang et al., 2013) (Fig. 5a). The result suggested that the positive floral genes like CcAGL24, CcSOC1 and CcAP1 were well triggered by several beneficial external environments (e.g. favorable light condition and rising temperature) and suitable internal environments (e.g. sufficient nutrition and dormancy releasing) while negative floral genes like SVP-like and TFL1-like expressed at a lower level (Huang et al., 2013). In contrast to pistillate floral development,


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Fig. 5. Expression and regulating relationship of critical floral genes in the process of flowering in hickory. (a) Expression of critical floral genes in the process of pistillate flowering in hickory. (b) Model of regulating relationship of floral genes in hickory. Full line shows the common regulating relationship in the process of pistillate and staminate flowering. Imaginary line shows the possible negative regulation in the process of staminate flowering. Solid arrow shows positive regulating relationship. The circle arrow shows negative Q1 regulating relationship.

the external environments and internal environments are benefit for staminate flowering in May and June. However, some adverse factors happened in late June and July, such as high atmosphere temperature, scorching sunshine, drought and nutrition competition from fruit development, staminate floral development, vegetative growth, and so on. These adverse environments inhibited positive floral genes expressing but promoted negative floral ones doing. Therefore, despite the upstream floral integrator CcAGL24 expressing at a high level, it couldn't trigger its downstream floral integrator CcSOC1 and floral organ gene CcAP1 expressing high enough due to some negative genes such as CcFLC (homolog of FLC in hickory), SVP-like and TFL1-like, etc. As a result, the staminate floral development had to be restricted finally. Here, a model was proposed to illustrate the possible regulative relationship among these critical floral genes in the process of staminate and pistillate flowering (Fig. 5b).

5. Conclusion Through sequence alignment and phylogenetic analyses, it is demonstrated that the three genes CcAGL24, CcSOC1, and CcAP1 belong to the MADS-Box family. According to the evolutionary trees of the three genes, the homologous genes from the same family cluster well together, while those from different orders do not match evolutionary regularity of individual organisms. The result of Quantitative RT-PCR analysis shows that the transcriptional levels of the three genes are up-regulated in early stage and downregulated in late stage in pistillate floral development. However, time varies to reach respective expression peak among the three genes. In staminate floral development, the transcription trend of the three genes is up-regulated, subsequently down-regulated, and then up-regulated again. Nevertheless, those trajectories, peaks,


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expression levels, inflection points are different in pistillate floral development. The result suggests that their functions are different in between pistillate and staminate floral development. The probable ordinal site of the three genes in the flowering network from top down is CcAGL24, CcSOC1, and CcAP1, which is identical to that in herbaceous plants. Moreover, several adverse environmental factors trigger several negative genes and then confine the development of staminate floral buds. Our results suggest the possible relationship among the three critical floral genes and their functions throughout the floral development in hickory. Competing interests The authors declare no competing interests. Acknowledgments We thank Yegen Tang for help in partial experiment. We also wish to thank the reviewers for their helpful comments and proposals on the manuscript. This work was supported by the National High Technology Research and Development program of China (863 Program, 2013AA102605), the National Natural Science Foundation of China (31170637). Author's contributions YWX, JQH, YJH and ZJW conceived and designed the experiments, and contributed reagents/materials. YJH and CS wrote the manuscript. CS and JNQ carried out the experiments. YJH and CS contributed to analysis of the data. References Bl azquez, M.A., Ahn, J.H., Weigel, D., 2003. A thermosensory pathway controlling flowering time in Arabidopsis thaliana. Nat. Genet. 33, 168e171. Corbesier, L., Vincent, C., Jang, S., et al., 2007. FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis. Science 316, 1030e1033. Fitter, A.H., Fitter, R.S., 2002. Rapid changes in flowering time in British plants. Science 296, 1689e1691. Hayama, R., Coupland, G., 2003. Shedding light on the circadian clock and the photoperiodic control of flowering. Curr. Opin. Plant Biol. 6, 13e19. Hepworth, S.R., Valverde, F., Ravenscroft, D., Mouradov, A., Coupland, G., 2002. Antagonistic regulation of flowering-time gene SOC1 by CONSTANS and FLC via separate promoter motifs. EMBO J. 21, 4327e4337. Hsu, C.Y., Adams, J.P., Kim, H., No, K., Ma, C., Strauss, S.H., Drnevich, J., Vandervelde, L., Ellis, J.D., Rice, B.M., et al., 2011. FLOWERING LOCUS T duplication coordinates reproductive and vegetative growth in perennial poplar. Proc. Natl. Acad. Sci. USA 108 (26), 10756e10761. Hsu, C.Y., Liu, Y., Luthe, D.S., Yuceer, C., 2006. Poplar FT2 shortens the juvenile phase and promotes seasonal flowering. Plant Cell. 18, 1846e1861. Huang, Y.J., Liu, L.L., Huang, J.Q., Wang, Z.J., Chen, F.F., Zhang, Q.X., Zheng, B.S., Chen, Ming, 2013. Use of transcriptome sequencing to understand the pistillate flowering in hickory (Carya cathayensis Sarg.). BMC Genomics 14, 691.

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Development of SSR Markers in Hickory (Carya cathayensis Sarg.) and Their Transferability to Other Species of Carya.

Transcriptional profiling by DDRT-PCR analysis reveals gene expression during seed development in Carya cathayensis Sarg.

Comparative Proteomic Analysis of the Graft Unions in Hickory (Carya cathayensis) Provides Insights into Response Mechanisms to Grafting Process.

The mechanism of high contents of oil and oleic acid revealed by transcriptomic and lipidomic analysis during embryogenesis in Carya cathayensis Sarg.

Effect of 26 years of intensively managed Carya cathayensis stands on soil organic carbon and fertility.

Inhibition of β-catenin signaling involved in the biological activities of a lignan E2S isolated from Carya cathayensis fruits.

Molecular characterization of the Arabidopsis floral homeotic gene APETALA1.

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Molecular characterization, expression analysis and heterologous expression of two translationally controlled tumor protein genes from Cucumis sativus.

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Molecular Characterization and Expression Profiles of Polygalacturonase Genes in Apolygus lucorum (Hemiptera: Miridae).

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Screening of critical genes in lung adenocarcinoma via network analysis of gene expression profile.

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Molecular Characterization and Expression Analysis of Ribosomal Protein S6 Gene in the Cashmere Goat (Capra hircus).