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Identification and functional characterization of SOC1-like genes in Pyrus bretschneideri
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Identification and functional characterization of SOC1-like genes in Pyrus bretschneideri Zhe Liu1, Xiaoping Wu1, Mengyu Cheng, Zhihua Xie, Changlong Xiong, Shaoling Zhang, ⁎ Juyou Wu2, Peng Wang ,2 Centre of Pear Engineering Technology Research, State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Pear SOC1 Flower bud Flowering time Photoperiod
Flowering is a prerequisite for pear fruit production. Therefore, the development of flower buds and the control of flowering time are important for pear trees. However, the molecular mechanism of pear flowering is unclear. SOC1, a member of MADS-box family, is known as a flowering signal integrator in Arabidopsis. We identified eight SOC1-like genes in Pyrus bretschneideri and analyzed their basic information and expression patterns. Some pear SOC1-like genes were regulated by photoperiod in leaves. Moreover, the expression patterns were diverse during the development of pear flower buds. Two members of the pear SOC1-like genes, PbSOC1d and PbSOC1g, could lead to early flowering phenotype when overexpressed in Arabidopsis. PbSOC1d and PbSOC1g were identified as activators of the floral meristem identity genes AtAP1 and AtLFY and promote flowering time. These results suggest that PbSOC1d and PbSOC1g are promoters of flowering time and may be involved in flower bud development in pear.
1. Introduction Flowering is coordinated by endogenous factors, such as gibberellin (GA) and aging, and environmental conditions, such as temperature and photoperiod [1]. Photoperiod-related genes, such as homologous genes of CRY, FKF1, CDF, CO, FT and TFL1, participate in flowering time regulation in apple and pear [2–5]. MADS-box transcription factors play critical roles in plant reproductive processes, including floral transition and floral organ formation, male and female gametophyte development, and seed and fruit development [6,7]. The study of the MADS-box proteins has considerably expanded the membership of this superfamily, which is divided into type I and II [8]. Type I genes mostly contain a single exon that encodes the MADS-box domain and are divided into four subfamilies, Mα, Mβ, Mγ and Mδ; Type II proteins are plant-specific MIKC proteins with conserved MADS (M), intervening (I), keratin-like (K),
and C-terminal (C) domains [9]. The MIKC group members consist of MIKC⁎ and MIKCc. MIKC* is involved in regulating pollen tube development in Arabidopsis [10]. While MIKCc can be separated into 13 major clades in the phylogeny reconstruction, they are involved in almost all processes in plant reproductive development [11], such as floral organ formation [12] and flowering time control [13]. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), belonging to MIKCc clades, is one of the flowering signal integrators and promotes the floral transition [13]. SOC1-like genes, such as AGAMOUS-LIKE 42 (AGL42), AGAMOUS-LIKE 71 (AGL71) and AGAMOUSLIKE 72 (AGL72), which are close to SOC1 in phylogenetic relationships and similarly encode MADS-box transcription factors, are also notably involved in regulating the floral transition in plants [14]. The soc1 mutation causes late flowering, while SOC1 overexpression causes early flowering in Arabidopsis [15]. It has been reported in recent years that multiple SOC1-like genes accelerate flowering in plants when
Abbreviations: AP1, APETALA1; AGL, AGAMOUS-LIKE; At, Arabidopsis thaliana; CaMV, Cauliflower mosaic virus; CO, CONSTANS; DAPI, 4′, 6-Diamidino-2-phenylindole; Do, Dendrobium orchid; Fa, Fragaria × ananassa; FT, FLOWERING LOCUS T; Fv, Fragaria vesca; GA, gibberellin; GFP, green fluorescent protein; Gh, Gossypium hirsutum L; LFY, LEAFY; MEME, Multiple Em for Motif Elicitation; MS, Murashige and Skoog; OE, Overexpression lines; Pb, Pyrus bretschneideri; Ps, Pisum sativum; RT-qPCR, Quantitative RT-PCR; SOC1, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1; UTR, untranslated region; Vc, Vaccinium corymbosum; ZT, Zeitgeber time ⁎ Corresponding author. E-mail addresses: [email protected] (J. Wu), [email protected] (P. Wang). 1 Zhe Liu and Xiaoping Wu contributed equally to this work. 2 Communicated by Juyou Wu and Peng Wang. https://doi.org/10.1016/j.ygeno.2019.09.011 Received 20 August 2019; Received in revised form 11 September 2019; Accepted 13 September 2019 0888-7543/ © 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Zhe Liu, et al., Genomics, https://doi.org/10.1016/j.ygeno.2019.09.011
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Fig. 1. Phylogenetic analysis and genomic structures of PbSOC1-like members. (A) Phylogenetic analysis of SOC1-like genes of pear and other plant species. The tree can be classified into three clades and covered by three colors (pink, yellow and blue shading). The blue and red branches correspond to the SOC1-like genes of Arabidopsis and pear, respectively. (B) The logos of protein sequence alignment of SOC1-like members show the conserved MADS, intervening, keratin-like and C-terminal domain (SOC1-motif). (C) Exon-intron structure of PbSOC1-like genes. Exons (CDSs) and introns are represented by yellow rectangles and black lines, respectively. Untranslated regions (UTRs) are represented by blue rectangles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
2. Materials and methods
overexpressed, for example, cotton GhSOC1 [16], orchid DoSOC1 [17] and tree peony PsSOC1 [18]. Similarly, in such fruits as blueberries and strawberries, the SOC1 homologous genes also play roles in flowering time regulation. For example, overexpression of the K-domain of a blueberry SOC1-like (VcSOC1-K) in tobacco can lead to early flowering [19]. The SOC1-like genes in different varieties of strawberry play diverse roles. FaSOC1 of cultivated strawberry is a flowering promoter, whereas FvSOC1 of the Fragaria vesca represses flowering and promotes vegetative growth [20,21]. However, ectopic expression of a SOC1 homolog in the Gerbera hybrid has been found to lead to partial loss of floral organ identity but does not affect flowering time [22]. Therefore, identifying pear SOC1-like genes and defining their function is necessary. SOC1 takes part in the photoperiod, temperature, age and gibberellin pathways to promote floral transition, specifically by interacting with key factors in different pathways [13]. In Arabidopsis, SOC1 is activated by CONSTANS (CO), which is one of the central regulators in the photoperiod pathway, to promote flowering [23]. However, FLC, a component of the vernalization pathway, directly represses SOC1 expression by binding to its promoter [24]. In addition, SOC1 integrates the photoperiod and GA signals to promote flowering via the SPL3, SPL4 and SPL5 genes at the shoot apex [25]. SOC1 interacts with multiple MADS-box members, such as AGL24, FUL and AP1, through direct binding to the regulatory sequences to coordinate the expression of several flowering genes, such as SVP, AGL15, and AGL18, thereby providing positive feedback from inductive floral cues [26–28]. Pear (Pyrus bretschneideri) is a widely cultivated perennial fruit tree. Flowering is the necessary basis for pear fruit formation and harvest; thus, the control and regulation of floral induction is an important issue for pear production. In this study, we identified eight SOC1-like genes in pear and analyzed their structures and expression patterns. Moreover, we characterized the function of two PbSOC1-like genes by ectopic transgenic analysis in Arabidopsis. Our results suggest that PbSOC1d and PbSOC1g act as activators of flowering time.
2.1. Identification and classification of SOC1-like genes in pear The protein sequences of SOC1-like genes were collected from 23 species that have been reported, including common monocot and eudicot (Table S2). Except for Actinidia chinensis and Medicago truncatula information, all the databases were surveyed with known ID number via the NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Actinidia chinensis and Medicago truncatula information were collected from UniProt (https://www.uniprot.org) and JCVI (http://blast.jcvi.org/MedicagoBlast/index.cgi) databases, respectively. The sequences of SOC1-like members from Arabidopsis were acquired from the Arabidopsis Information Resource (http://www.Arabidopsis.org/). The sequences of pear were searched by BLASTP against the pear proteins in NCBI database [29]. The protein sequences were detected by Conserved Domains software (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb. cgi) by the criterion that they must contain the SOC1 conserved domain. Detailed information on SOC1-like genes identified in this study is given in Tables S1 and S2. All protein sequences were aligned by MUSCLE. After alignment, the phylogenetic trees were all reconstructed by RaxML, and all the bootstrap values were examined by Bayes [30–32]. These trees were viewed and edited with the iTOL program (http://itol.embl.de/login. cgi). Gene structure analysis of SOC1-like genes used the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/) by aligning the coding sequences with the corresponding genomic sequences. Multiple sequence alignments of the full-length protein sequences, including the highly conserved amino acid residues, were determined by Clustal Omega (www.clustal.org) [33]. To identify the conserved motifs of SOC1-like proteins, their complete amino acid sequences were utilized to carry out the MEME analysis using an online tool (http://meme-suite.org) [34]. The parameters were set as follows: optimum motif width ranges from 6 to 50 and a maximum number of motifs of 10. All other parameters were set as 2
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Fig. 2. Tissue-specific expression assay of PbSOC1-like genes. Eight PbPbSOC1-like gene expression patterns were detected by RT-qPCR in root, young stem, leaf bud, leaf, phloem, flower bud, petal, style, anther and ovary, and they were normalized with PbUBQ as a reference gene. The black columns represent five vegetative growth organs (including root, young stem, leaf bud, leaf and phloem) and the gray columns represent five reproductive growth organs (including flower bud, petal, style, anther and ovary). Error bars represent the standard error. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Thirty-day-old seedlings that had completed the stratification at 4 °C in sand were used for diurnal rhythmic and photoperiodic response expression analyses. The seedlings were grown under three conditions, 12 h light/12 h dark, short-day conditions (8 h light/16 h dark) and long-day conditions (16 h light/8 h dark) in a greenhouse kept at 22 ± 1 °C with 65 ± 5% relative humidity. After 30 days of light-dark treatment, young leaves from the seedlings were sampled from four randomly selected plants every 4 h during the 24 h photoperiodic cycle. All samples were immediately frozen in liquid nitrogen and stored at −80 °C until use. The experiments were performed with three independent biological replicates. Wild-type Arabidopsis Columbia-0 (Col-0) was used for overexpression studies. Seed germination and plant growth management followed the Arabidopsis growth standard protocol published on the TAIR website (http://www.Arabidopsis.org). Nicotiana benthamiana was grown and used for transient expression experiments. Arabidopsis and tobacco were both grown under long-day conditions (16 h light/8 h dark) at 22 °C in the greenhouse.
defaults. In order to characterize the evolution pattern of PbSOC1-like genes, the non-synonymous (Ka) and synonymous (Ks) substitutions, as well as the ratio of Ka to Ks (Ka/Ks) in their homologs, were estimated. The coding sequences of eight PbSOC1-like genes were aligned using Clustalw. Ka and Ks were calculated by Dnasp v6 [35]. For analyzing the cis-elements of each PbSOC1-like gene, we acquired the sequence of 2000 bp ahead the initiation codon ATG from NCBI database (https://www.ncbi.nlm.nih.gov/) and submitted to the PlantRegMap online program [36], a database to provide a comprehensive, high-quality resource of plant transcription factors (TFs) and regulatory elements (http://plantregmap.cbi.pku.edu.cn). 2.2. Plant materials and growth conditions The ten-year-old trees of P. bretschneideri Rehd. cultivar ‘Dangshansuli’ used in this study were grown in a natural environment experimental field (Nanjing Agriculture University, Nanjing, China). Seeds of these trees were vernalized in sand for two months at 4 °C and transferred to flowerpots containing soil and vermiculite in a growth chamber with an air temperature of 22 °C. Roots, young stems and leaves of seedlings were collected for tissue-specific expression analysis after 40 days of transplanting to flowerpots. Leaf bud, phloem, flower buds, petals, styles, anthers and ovaries were harvested a few days before flower fully opening from the 10-year-old pear trees. For temporal gene expression analysis during June to November of flower bud development, the flower bud samples are collected from the 10-year-old pear trees on the 15th day of each month.
2.3. RNA isolation and quantitative RT-PCR (RT-qPCR) analyses A kit (FOREGENE, Chengdu, China) specifically designed to purify total RNA from plant samples with high polysaccharides and polyphenols was used, and total RNA was extracted from frozen tissues according to the manufacturer's instructions. The quality of the total RNA was assessed by gel electrophoresis, and optical density readings were obtained with a Nanodrop 2000 (Thermo Scientific, US). Firststrand cDNA was synthesized by a RevertAid First Strand cDNA 3
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Fig. 3. Dynamic expression pattern of PbSOC1-like genes during pear flower bud development. (A) Flower bud developmental progress during six months of pear. Samples were collected from ten-year-old trees on the 15th day of each month. Figures were observed with a stereomicroscope in a bright field. The black arrows indicate morphological development at different times. Scale bars, 2 mm. (B) Temporal expression assay of PbSOC1-like genes at six developmental stages of pear flower buds. The PbUBQ gene was used as an internal control. Error bars on each dot represent the standard error (S.E.) of three replicates.
Synthesis Kit (Thermo Scientific, US) using 1 μg of total RNA and oligo (dT) primers according to the manufacturer's instructions. The products of the first-strand cDNA were subjected to PCR amplification. SYBR Green I Master Mix (Roche, Germany) in a Light Cycler 480 II (Roche, Germany) was used for RT-qPCR of the collected samples to determine relative gene expression levels. Gene-specific primers (Table S2) were designed using the NCBI primer-designing tool and were checked by BLAST. To determine the specific amplification of each primer, melt curve analysis and electrophoresis of the test reaction were performed. For RT-qPCR, the reactions were prepared in a total volume of 20 μL containing 0.1 μL of cDNA sample as the template, 5 μL of 0.05 μM gene-specific primer premix, 10 μL of 2× SYBR Green Master Mix and 4.9 μL of water. The Light Cycler 480 II (Roche, Germany) was programmed as follows: 5 min of preincubation at 95 °C followed by 55 cycles of 3 s at 95 °C, 10 s at 60 °C, and 30 s at 72 °C of amplification. Data were analyzed using the 2−ΔCT method, and the expression levels
of target genes were normalized using the UBQ gene of pear or Arabidopsis Actin. RT-qPCR was performed using three independent biological replicates and three technical replicates for each sample. All RT-qPCR experiments were designed following MIQE guidelines [37]. The expression data were analyzed and graphed by Graph Pad Prism 5. 2.4. Subcellular localization analysis of proteins The full-length coding sequences without the stop codon of PbSOC1d and PbSOC1g genes were amplified by PCR from RNA with the appropriate specific primers (Table S3). The amplified products were cloned into the pEASY vector using a pEASY-T5 Zero Cloning Kit (TransGen Biotech, China) and sequenced. Then, the inserts were subcloned into the modified binary vector pCAMBIA1300-35S:CDS-GFP [38] with the XbaI and BamHI restriction sites. To observe the subcellular localization of PbSOC1d and PbSOC1g, Agrobacterium4
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Fig. 4. Diurnal rhythms of PbSOC1-like genes in pear leaves. The illumination regime is illustrated by white (light) and black (dark) bars. Pear leaves were exposed to 12 h light and 12 h dark conditions. PbUBQ was used as a reference gene in the RT-qPCR assay, and the expression was normalized. Error bars represent the standard error. ZT: zeitgeber time; the time of lights on usually defines zeitgeber time zero (ZT0).
3. Result
mediated transient expression in N. benthamiana leaves was performed as reported [39]. The expression vector was transformed into epidermal cells of 40-day-old tobacco plants. Two days after infiltration, the fluorescence in the transformed cells was observed with DAPI as a nuclei marker under a laser scanning confocal microscope LSM780 (Zeiss, Germany).
3.1. Identification and classification of SOC1-like genes in pear SOC1 and its homologous AGL14, AGL19, AGL42, AGL71, and AGL72 are in the same clade with SOC1 encoding MIKCC-type proteins in Arabidopsis [9]. Using the six MIKCC-type protein sequences in Arabidopsis as a reference, we searched against pear protein and nucleotide databases at NCBI (http://www.ncbi.nlm.nih.gov) by BLAST. We obtained 8 SOC1-like genes from pear and designated them as PbSOC1a to PbSOC1h (Table S1). The shortest amino acid sequence among the eight proteins is PbSOC1a, with a 642-bp open reading frame (ORF) that encodes a peptide of 213 amino acid residues with a predicted molecular mass of 24.64 kDa. The longest one is PbSOC1f with 253 amino acids. To describe the relationships and to classify the SOC1 gene family in pear and other species, we assembled fifty genes from 23 reported species and pear into a phylogenetic tree (Fig. 1A and Table S2). The phylogenetic tree inferred from the amino acid sequences of SOC1-like proteins clearly resolved three main subclades, AtSOC1, AtAGL14/ AGL19 and AtAGL42/71/72 (Fig. 1A). PbSOC1a was in the cluster branch of AtSOC1 and shared an amino acid sequence ~67% identity with AtSOC1. PbSOC1b and PbSOC1c were most closely related to AGL14 and AGL19, and PbSOC1d, PbSOC1f, PbSOC1e, PbSOC1g and
2.5. Arabidopsis transformation For Arabidopsis transformation, the pCAMBIA1300-35S: CDS-GFP vectors were also transformed into Agrobacterium tumefaciens strain GV3101. Arabidopsis (Col-0) plants were transformed using the floral dip method [40]. Seeds of transgenic plants were selected on Murashige and Skoog (MS) medium with 20 mg/L hygromycin and cultured in a growth chamber under the long-day condition (16 h light/8 h dark, 22 °C) for ten days, and resistant plants were transferred to soil. The presence of the transgene in progeny was confirmed by PCR using genespecific and vector primers (Table S2). Transgenic Arabidopsis plants in the T2 and T3 generations were selected to examine flowering time and other phenotypes.
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Fig. 5. Photoperiodic response of PbSOC1-like genes in pear. The expression pattern of partial PbSOC1-like genes showed a diurnal rhythm in plants grown in short days (8 h light/16 h dark) and long days (16 h light/8 h dark) in 48 h. Expression as determined by RT-qPCR was normalized to PbUBQ in all cases. Blue squares represent short-day conditions, and red dots represent long-day conditions. Light conditions are indicated by the colored bar at the bottom of the graph. Black boxes indicate darkness, and white boxes indicate light. Error bars represent the standard error. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
amino acid changes at key sites (Fig. S1). Therefore, these proteins all belong to the MIKCC type MADS-box transcription factors. MEME program was also used to identify the conserved motifs of SOC1-like proteins in Arabidopsis and pear, and all the 14 protein sequences were found to contain 1, 2, 3, 4, 5 and 6 motifs (Figs. S2 and S3). Motif 1 for M (MADS-box), motif 3 and motif 6 for I (intervening-domain), motif 2 and motif 5 for K (keratin-like domain), and the motifs corresponding to C have diversity. Interestingly, the C-terminal diversity of PbSOC1-like proteins was associated with 3 subclades of the phylogenetic tree. For example, protein of PbSOC1a only contains motif 4, proteins of PbSOC1b and PbSOC1c contain motif 4 and motif 9, while proteins of PbSOC1d-1h contain motif 4 and motif 7. When analyzing the evolution rate and pattern of a protein, the comparison between the number of non-synonymous substitutions (Ka) and synonymous substitutions (Ks) were required [45]. The Ka/Ks ratios of the PbSOC1-like members were examined, and it was figured out that all the results were < 1 (Table S4), indicating that they were undergoing purifying selective pressure. Meanwhile, structural analysis between the full-length cDNA and genomic DNA sequence revealed that most PbSOC1-like genes contain seven conserved exons and six variable introns that are identical to AtSOC1 and its homology genes in Arabidopsis, except for PbSOC1e (Fig. 1C). However, the intron lengths of PbSOC1-like genes are longer
PbSOC1h were more closely related to AGL42, AGL71, and AGL72 (Fig. 1A). The amino acid sequence identity between PbSOC1b and PbSOC1c was as high as 82%, but compared with AtAGL14 and AtAGL19, it was only ~55%. In addition, five other SOC1 homologous proteins of pear (PbSOC1d-h) are highly similar to each other, sharing 70% to 80% identity. Notably, seven species of Rosaceae were selected, including pear, and their SOC1 homologs are well-clustered (Fig. 1A), suggesting a closer evolutionary relationship in the Rosaceae family. SOC1 and SOC1-like genes have been reported to have four conserved domains [18,41,42]. The logo diagrams of four conserved domains were obtained by protein alignment analysis of fifty SOC1-like members indicated in Fig. 1A. The genes all share the conserved MADSbox, intervening-domain, keratin-like domain, and SOC1-motif (Fig. 1B). The MADS-box domain, composed of 58 amino acids at the Nterminal, is mainly responsible for DNA binding but is also involved in dimerization reactions and the binding of accessory factors [9]. The intervening-domain is approximately 30 amino acids with weakly conserved residues. Both the keratin-like domain and SOC1-motif function in the formation of higher-order complexes, and the coiled coil structure in the K-domain participate in protein-protein interactions, and the C domain is critical for the specificity of interactions [43,44]. All eight PbSOC1-like proteins have four conserved domains and no 6
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DAPI
Bright Field
Merge
PbSOC1g-GFP
PbSOC1d-GFP
GFP(Vector)
GFP
Fig. 6. Subcellular localization of PbSOC1d-GFP and PbSOC1g-GFP in leaf epidermal cells of tobacco. The fluorescence of GFP was observed by laser scanning confocal microscopy, and the nucleus was labeled with DAPI and the empty vector of GFP was used as a control. GFP, GFP fluorescence; BF, bright field; Merged, merged image of GFP and BF. Bar = 20 μm.
Fig. 7. Overexpression of PbSOC1d causes early flowering in Arabidopsis. (A) Control plants and transgenic lines were grown in soil under LD conditions. (B) The number of rosette leaves and number of days required to reach flowering in control plants and PbPbSOC1d-OE transgenic lines. (C) RT-qPCR analyses of PbSOC1d, AtFT, AtAP1 and AtLFY in vector control plants and transgenic lines and normalized by AtACT. Error bars indicate standard error (S.E.) of the mean. Asterisks indicate significant differences between control and transgenic lines (n ≥ 10, * means p < .05, **means the p value ranges from 0.05 and 0.001, *** means p < .001, by t-test, compared with vector control).
higher plants including Arabidopsis. The identification of cis-elements in the promoters of PbSOC1-like genes could contribute to predict and understand the potential regulatory functions [27]. Many cis-elements were found to be associated with plants reproductive development, such as AP2, MIKC_MADS, SBP, TALE and ZF-HD (Table S5). For
than those of their corresponding genes in Arabidopsis. These results suggest that the functions of PbSOC1-like genes are potentially similar to those in Arabidopsis. Characterized as a transcription factor, SOC1 has been reported to play an important role in the reproductive development of various 7
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Fig. 8. Overexpression of PbSOC1g promotes flowering in Arabidopsis. (A) The phenotype of PbSOC1g transgenic lines under LD conditions (16/8 h light/dark, 22 °C). (B) The number of rosette leaves and number of days during flowering in transgenic plants and control plants. C) The expression levels of PbSOC1g, AtFT, AtAP1 and AtLFY in seedlings after 12 days were measured by RT-qPCR and normalized by AtACT. Error bars indicate standard error (S.E.) of the mean. Asterisks indicate significant differences between control and transgenic lines (n ≥ 10, * means p < .05, **means the p value ranges from 0.05 and 0.001, *** means p < .001, by t-test, compared with vector control).
largely unchanged in the first four months but decreased significantly in October and November. In the process from flower bud differentiation to gradually entering dormancy, PbSOC1a, PbSOC1d and PbSOC1g show a relatively regular pattern of initially increasing and then decreasing transcription. These results indicate that the expression pattern of PbSOC1-like genes is different and dynamic during flower bud differentiation and development in pear.
instance, the CArG-box belong to MIKC_MADS elements has been studied in depth as a crucial binding site of SOC1 in Arabidopsis [26,46]. Similarly, each of the eight PbSOC1-like genes contains more than one CArG-box binding site in promoter regions (Table S6). These results suggest that the transcription of PbSOC1-like genes may be affected by these factors and participate in the pear development pathway.
3.2. Temporal and spatial expression patterns of PbSOC1-like genes
3.3. Expression pattern of PbSOC1-like genes in leaves under different photoperiods
To characterize the biological function of PbSOC1-like genes, we first examined their spatial expression in various tissues in pear by quantitative real-time PCR. Overall, PbSOC1-like genes have similar expression patterns that can be detected at high levels in leaf buds and leaves but at relatively low levels in flower buds and flower tissues. (Fig. 2). PbSOC1a transcripts were almost undetectable in flower samples, except flower buds, but were predominantly expressed in leaves. The relative expression levels of different PbSOC1-like genes were varied in the same organ. PbSOC1a, PbSOC1b and PbSOC1e accumulated in pear roots (Fig. 2). All eight PbSOC1-like genes were expressed in flower buds, suggesting that these PbSOC1-like gene functions might be associated with flower bud differentiation and development in pear. Previous studies have suggested that the pear, as a perennial woody plant, undergoes flower bud differentiation and development from June to November every year in China and then enters the dormancy state [47]. To investigate genes involved in pear flower bud differentiation and development, flower bud samples of pear were collected on the 15th day of each month between June and November. Flower bud differentiation and development of pear can be divided into two stages, physiological and morphological differentiation (https://www. actahort.org/books/872/872_14.htm). Morphological differentiation was observed from August and became clear over time (Fig. 3A). PbSOC1-like genes showed four different expression patterns (Fig. 3B). First, the transcription levels of PbSOC1a, PbSOC1b and PbSOC1c were relatively consistent, and the expression gradually increased from June to July, reaching a peak in July, and then continuously decreased. However, PbSOC1c is slightly different because the expression rebounded slightly in September and November. Second, both PbSOC1d and PbSOC1g show a steady trend of rising first and then falling. The difference is that one peak is in August and one peak is in September. Then, PbSOC1e and PbSOC1f show opposite changes to the first type. The expression levels of these genes fell sharply from June to July and then continued to rise. The expression level of PbSOC1h remained
SOC1-like genes in Arabidopsis and homologies from other plant species display diurnal and circadian patterns of expression [42,48–50]. The leaf is the major site for perception of day length, and all analyzed PbSOC1-like genes were expressed in leaves. Therefore, further experiments analyzed the diurnal rhythms and photoperiodic response of PbSOC1-like genes in the leaves of pear. The diurnal rhythms of these genes exhibited different patterns and can be divided into three categories (Fig. 4). The expression patterns of PbSOC1a, PbSOC1b and PbSOC1c peaked at dusk (zeitgeber time 13; ZT 13). The expression of PbSOC1d was the highest at dawn (ZT 1). The other four PbSOC1-like genes were highly expressed at night (Fig. 4). The transition from vegetative to reproductive growth is mainly controlled by day length in many plant species. Moreover, to study the effects of photoperiod on the expression of PbSOC1-like genes, the transcription levels of these genes were measured under long days (16 h light/8 h dark) and short days (8 h light/16 h dark). The expression of PbSOC1a, PbSOC1d and PbSOC1g showed an obvious response to photoperiod. Under long-day conditions, the peak expression of PbSOC1a, PbSOC1d and PbSOC1g occurred during ZT21, while the peak occurrence of these gene expression was shown at ZT13 under short-day conditions. The expression of five other PbSOC1-like genes was not regularly responsive to photoperiod (Fig. 5). Taken together, these results indicate that PbSOC1a, PbSOC1d and PbSOC1g might be the key genes in the regulation of flowering in pear. Therefore, PbSOC1d and PbSOC1g were selected for further functional testing in flowering regulation. 3.4. Subcellular localization of PbSOC1d and PbSOC1g proteins To examine the subcellular localization of PbSOC1d and PbSOC1g proteins, we fused each gene's full coding sequence to green fluorescent protein (GFP). The resulting constructs driven by the 35S promoter 8
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species to form three distinct branches (Fig. 1A). PbSOC1-like family members are highly conserved in gene structures and protein domains in pear compared with Arabidopsis, indicating that they are typical MIKCC proteins with a highly conserved MADS-box at the N-terminus, a K-box in the middle, a specific SOC1 motif (Fig. 1 and Fig. S1) [52]. Therefore, PbSOC1-like genes may play a conserved role in the regulation of flowering. PbSOC1-like genes are expressed in a range of tissues, though at differing levels (Fig. 2). All eight PbSOC1-like gene transcripts reached predominant levels in the leaves, indicating that they may all play crucial roles in pear leaves. Meanwhile, PbSOC1-like genes are expressed in flower buds, consistent with a potential role in the control of flower bud differentiation and development. PbSOC1d-PbSOC1h are also clearly expressed in phloem, suggesting that they might also play a similar role to that of their homologs [14], such as promoting flowering in the shoot apical and axillary meristems. However, another commonality observed for pear SOC1-like genes is notably low expression levels in the floral organ, including petals, styles, anthers and ovaries. Likewise, many SOC1-like genes in diverse species were detected mainly in vegetative organs and almost not in floral organs [16,49], indicative of a common role during vegetative development as general regulators of plant organogenesis. Some fruit trees, such as mulberry, mei and pear, the formation and development of their inflorescence primordia occur in the buds in summer and then stop growing in the dormant period until the bud continues to flower in the spring [53,54]. During flower bud development in summer, mulberry FT expression first increases and then decreases over time, plummeted in November before endodormancy of the buds [53]. A similar situation has been reported in pear, except that the expression of PpFTs was not clearly induced, but the rapid decrease of PpTFL1s expression may be involved in floral induction [55]. In our study, differential PbSOC1-like gene expression was observed in different flower buds during the course of flower bud differentiation and development (Fig. 3A and B). The three genes PbSOC1a, PbSOC1d and PbSOC1g show a trend of rising first and then falling in the process of flower bud development. The difference is that the time of reaching the peak is diverse, with PbSOC1a peaking in July, PbSOC1d peaking in August and PbSOC1g peaking in September, indicating that these three genes may play potential roles in different stages of flower bud morphological differentiation (Fig. 3B). PbSOC1a, PbSOC1b and PbSOC1h were expressed relatively higher at earlier stages of flower bud, possibly reflecting that they are involved in the early stage during the vegetative to the reproductive transition. In apricot, ParSOC1 has been associated with cold response and chilling requirements during dormancy [42]. Among the eight PbSOC1-like genes, only PbSOC1f still had a high expression level in November and showed an upward trend, which may be involved in the regulation process of dormancy. The expression analysis showed that almost all PbSOC1-like genes had a diurnal rhythm at a cycle of 24 h under 12 h light and 12 h dark conditions (Fig. 4). A connection between the internal rhythm and response to environment cues by SOC1 was shown in Arabidopsis [56] and Japanese apricot [42]. Our results suggest that PbSOC1-like genes may also be involved in the cross-talk between clock rhythm and environmental signaling in pear. The function of SOC1, initially named AGL20, in controlling flowering is reported in Arabidopsis [15]. The regulation of SOC1 in flowering is associated with photoperiod in Arabidopsis [13]. Under longday and short-day conditions, the response of the PbSOC1a gene to photoperiod was similar to that of SOC1 in Arabidopsis [57] and ZmMADS1 in maize [48]. PbSOC1d and PbSOC1g also responded to photoperiod changes, and the expression pattern was more stable under long-day conditions (Fig. 5). PbSOC1d and PbSOC1g belong to the same subclade of AGL42, AGL71 and AGL72, which are Arabidopsis SOC1 subfamily genes, in the phylogenetic analysis (Fig. 1C). Meanwhile, AGL42, AGL71 and AGL72 are produced by gene duplication during evolution, and they can also promote flowering in Arabidopsis [14].
were then transiently expressed in leaf epidermal cells of tobacco. Confocal microscopy observations suggested that both PbSOC1d and PbSOC1g fusion proteins were localized exclusively in the nucleus (Fig. 6). Thus, these proteins appear to be nuclear proteins, suggesting that they can participate in transcriptional regulation. 3.5. Overexpression of PbSOC1d and PbSOC1g promotes flowering in Arabidopsis Because we lacked a dependable transformation protocol for pears and there was an extremely long period from seedling to flower, the functionality of PbSOC1d and PbSOC1g was analyzed in Arabidopsis. Arabidopsis lines overexpressing PbSOC1d and PbSOC1g were generated, and two independent lines were selected for flowering time analysis. To avoid the fluctuation of gene expression by other environmental factors, all plants grew under the same long-day conditions. First, the transgenic lines were assayed by RT-qPCR to validate the presence and transcript levels of the introduced PbSOC1d and PbSOC1g (Figs. 7C and 8C). The results showed that the expression levels of genes were significantly higher in corresponding transgenic lines than in the vector control plants. According to the results (Figs. 7A and 8A), PbSOC1d and PbSOC1g-overexpressing lines exhibited strongly early flowering traits, consistent with the phenotypes of AtAGL71 and AtAGL72 [14]. Under long-day conditions, the control plants transformed with empty vector required 25.1 ± 0.27 days until the time of flowering, compared with 22.9 ± 0.30 and 22.1 ± 0.31 days in the PbSOC1d overexpression lines OE1 and OE2, respectively (Fig. 7B). The shortened vegetative phase was also manifested as a decrease in the number of rosette leaves in transgenic lines, ranging from 8.89 ± 0.21 to 8.41 ± 0.21 leaves on average, compared to the 9.85 ± 0.18 leaves in control plants (Fig. 7B). Similar statistical results were found in PbSOC1g-overexpressing lines. The transgenic lines began to bolt at approximately 22 days after light and had 8 rosette leaves on average, whereas the vector control plants required approximately 25 days to reach bolting and had 9.85 rosette leaves on average (Fig. 8B). These results indicate that both PbSOC1d and PbSOC1g play conserved roles in promoting flowering in Arabidopsis. Since the transgenic plants overexpressing PbSOC1d and PbSOC1g have an early flowering phenotype, the results suggested that they may be involved in the regulation of flowering time-related genes. Thus, three flowering time-related genes, including AtFT, AtAP1 and AtLFY, were tested in the transgenic lines. The expression of all three flowering time-related genes was significantly upregulated in the PbSOC1d-OE and PbSOC1g-OE transgenic lines in Arabidopsis (Figs. 7C and 8C). These observations suggest that PbSOC1d and PbSOC1g play roles in the promotion of flowering, partly through engaging similar downstream regulators in Arabidopsis. 4. Discussion Flowering is an important economic characteristic of fruit production. The switch of floral induction is triggered by a variety of environmental cues and endogenous signals [1]. The SOC1 gene in Arabidopsis and its homology in multiple species, such as orchids, rice, maize, tobacco, soybeans, kiwifruit, grapes, and apples (Table S2), have been demonstrated to play an important role in promotion flowering by integrating multiple floral cues [51]. In this study, we analyzed this gene family in pear and confirmed that PbSOC1d and PbSOC1g promote flowering. Based on the pear genome sequencing database [29], we perform a comprehensive analysis of the SOC1-like family, including their phylogenetic, exon/intron structures, conserved domains and protein sequences; these findings make it possible for us to predict the potential functions and evolutionary relationships among uncharacterized members of this gene family. We identified eight SOC1-like genes in pear (Table S1), which were clustered with homologous genes in other 9
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Thus, PbSOC1d and PbSOC1g likely have similar functions to AGL42, AGL71 and AGL72 in promoting flowering in pear. The nuclear localization of PbSOC1d and PbSOC1g may explain their role in transcriptional regulation (Fig. 6). Moreover, both PbSOC1d and PbSOC1g are able to promote flowering in Arabidopsis (Fig. 7A and 8A). In Arabidopsis, SOC1 upregulates AP1 expression via a pathway mediated by LFY or FT [13,28]. The expression of AtFT, AtAP1 and AtLFY was upregulated in the PbSOC1d-OE and PbSOC1g-OE lines (Fig. 8C and 9C). These results suggest that a conserved pathway for SOC1-like genes regulates flowering through the promotion of flowering time genes and flower organ identity genes.
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Author contributions P. W., J. W, S. Z. initiated and supervised the project. Z. L., X. W., M. C., C. X., and Z. X. performed the experiments. Z. L. and P. W. wrote the manuscript. All of the authors were involved in data analysis and interpretation. Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgments This work is supported by the grant from the National Natural Science Foundation of China (31501715, 31772256, 31772276), Fundamental Research Funds for the Central Universities (KYZ201836, KYZ201888) and Jiangsu Province Science and Technology Support Program (BE2018389). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ygeno.2019.09.011. References [1] F. Fornara, A. de Montaigu, G. Coupland, SnapShot: control of flowering in Arabidopsis, Cell 141 (2010) 550 (550 e551-552). [2] L. Xing, D. Zhang, S. Qi, X. Chen, N. An, Y. Li, C. Zhao, M. Han, J. Zhao, Transcription profiles reveal the regulatory mechanisms of spur bud changes and flower induction in response to shoot bending in apple (Malus domestica Borkh.), Plant Mol. Biol. 99 (2019) 45–66. [3] X. Liu, Z. Liu, Z. Hao, G. Chen, K. Qi, H. Zhang, H. Jiao, X. Wu, S. Zhang, J. Wu, P. Wang, Characterization of Dof family in Pyrus bretschneideri and role of PbDof9.2 in flowering time regulation, Genomics (2019), https://doi.org/10.1016/j.ygeno. 2019.05.005. [4] P. Wang, Z. Liu, P. Cao, X. Liu, X. Wu, K. Qi, S. Zhang, J. Wu, PbCOL8 is a clockregulated flowering time repressor in pear, Tree Genet. Genomes 13 (2017) 107. [5] L. Xing, D. Zhang, X. Song, K. Weng, Y. Shen, Y. Li, C. Zhao, J. Ma, N. An, M. Han, Genome-wide sequence variation identification and floral-associated trait comparisons based on the re-sequencing of the ’Nagafu no. 2′ and ‘Qinguan’ varieties of apple (Malus domestica Borkh.), Front. Plant Sci. 7 (2016) 908. [6] L. Gramzow, G. Theissen, Phylogenomics of MADS-box genes in plants - two opposing life styles in one gene family, Biology (Basel) 2 (2013) 1150–1164. [7] S. Masiero, L. Colombo, P.E. Grini, A. Schnittger, M.M. Kater, The emerging importance of type I MADS box transcription factors for plant reproduction, Plant Cell 23 (2011) 865–872. [8] S. De Bodt, J. Raes, Y. Van de Peer, G. Theissen, And then there were many: MADS goes genomic, Trends Plant Sci. 8 (2003) 475–483. [9] L. Parenicova, Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis: new openings to the MADS world, Plant Cell 15 (2003) 1538–1551. [10] B.J. Adamczyk, D.E. Fernandez, MIKC* MADS domain heterodimers are required for pollen maturation and tube growth in Arabidopsis, Plant Physiol. 149 (2009) 1713–1723. [11] C. Smaczniak, R.G. Immink, G.C. Angenent, K. Kaufmann, Developmental and evolutionary diversity of plant MADS-domain factors: insights from recent studies, Development 139 (2012) 3081–3098. [12] C. Ferrandiz, Q. Gu, R. Martienssen, M.F. Yanofsky, Redundant regulation of meristem identity and plant architecture by FRUITFULL, APETALA1 and CAULIFLOWER, Development 127 (2000) 725–734. [13] J. Lee, I. Lee, Regulation and function of SOC1, a flowering pathway integrator, J.
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Genomics journal homepage: www.elsevier.com/locate/ygeno
Identification and functional characterization of SOC1-like genes in Pyrus bretschneideri Zhe Liu1, Xiaoping Wu1, Mengyu Cheng, Zhihua Xie, Changlong Xiong, Shaoling Zhang, ⁎ Juyou Wu2, Peng Wang ,2 Centre of Pear Engineering Technology Research, State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Pear SOC1 Flower bud Flowering time Photoperiod
Flowering is a prerequisite for pear fruit production. Therefore, the development of flower buds and the control of flowering time are important for pear trees. However, the molecular mechanism of pear flowering is unclear. SOC1, a member of MADS-box family, is known as a flowering signal integrator in Arabidopsis. We identified eight SOC1-like genes in Pyrus bretschneideri and analyzed their basic information and expression patterns. Some pear SOC1-like genes were regulated by photoperiod in leaves. Moreover, the expression patterns were diverse during the development of pear flower buds. Two members of the pear SOC1-like genes, PbSOC1d and PbSOC1g, could lead to early flowering phenotype when overexpressed in Arabidopsis. PbSOC1d and PbSOC1g were identified as activators of the floral meristem identity genes AtAP1 and AtLFY and promote flowering time. These results suggest that PbSOC1d and PbSOC1g are promoters of flowering time and may be involved in flower bud development in pear.
1. Introduction Flowering is coordinated by endogenous factors, such as gibberellin (GA) and aging, and environmental conditions, such as temperature and photoperiod [1]. Photoperiod-related genes, such as homologous genes of CRY, FKF1, CDF, CO, FT and TFL1, participate in flowering time regulation in apple and pear [2–5]. MADS-box transcription factors play critical roles in plant reproductive processes, including floral transition and floral organ formation, male and female gametophyte development, and seed and fruit development [6,7]. The study of the MADS-box proteins has considerably expanded the membership of this superfamily, which is divided into type I and II [8]. Type I genes mostly contain a single exon that encodes the MADS-box domain and are divided into four subfamilies, Mα, Mβ, Mγ and Mδ; Type II proteins are plant-specific MIKC proteins with conserved MADS (M), intervening (I), keratin-like (K),
and C-terminal (C) domains [9]. The MIKC group members consist of MIKC⁎ and MIKCc. MIKC* is involved in regulating pollen tube development in Arabidopsis [10]. While MIKCc can be separated into 13 major clades in the phylogeny reconstruction, they are involved in almost all processes in plant reproductive development [11], such as floral organ formation [12] and flowering time control [13]. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), belonging to MIKCc clades, is one of the flowering signal integrators and promotes the floral transition [13]. SOC1-like genes, such as AGAMOUS-LIKE 42 (AGL42), AGAMOUS-LIKE 71 (AGL71) and AGAMOUSLIKE 72 (AGL72), which are close to SOC1 in phylogenetic relationships and similarly encode MADS-box transcription factors, are also notably involved in regulating the floral transition in plants [14]. The soc1 mutation causes late flowering, while SOC1 overexpression causes early flowering in Arabidopsis [15]. It has been reported in recent years that multiple SOC1-like genes accelerate flowering in plants when
Abbreviations: AP1, APETALA1; AGL, AGAMOUS-LIKE; At, Arabidopsis thaliana; CaMV, Cauliflower mosaic virus; CO, CONSTANS; DAPI, 4′, 6-Diamidino-2-phenylindole; Do, Dendrobium orchid; Fa, Fragaria × ananassa; FT, FLOWERING LOCUS T; Fv, Fragaria vesca; GA, gibberellin; GFP, green fluorescent protein; Gh, Gossypium hirsutum L; LFY, LEAFY; MEME, Multiple Em for Motif Elicitation; MS, Murashige and Skoog; OE, Overexpression lines; Pb, Pyrus bretschneideri; Ps, Pisum sativum; RT-qPCR, Quantitative RT-PCR; SOC1, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1; UTR, untranslated region; Vc, Vaccinium corymbosum; ZT, Zeitgeber time ⁎ Corresponding author. E-mail addresses: [email protected] (J. Wu), [email protected] (P. Wang). 1 Zhe Liu and Xiaoping Wu contributed equally to this work. 2 Communicated by Juyou Wu and Peng Wang. https://doi.org/10.1016/j.ygeno.2019.09.011 Received 20 August 2019; Received in revised form 11 September 2019; Accepted 13 September 2019 0888-7543/ © 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Zhe Liu, et al., Genomics, https://doi.org/10.1016/j.ygeno.2019.09.011
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Fig. 1. Phylogenetic analysis and genomic structures of PbSOC1-like members. (A) Phylogenetic analysis of SOC1-like genes of pear and other plant species. The tree can be classified into three clades and covered by three colors (pink, yellow and blue shading). The blue and red branches correspond to the SOC1-like genes of Arabidopsis and pear, respectively. (B) The logos of protein sequence alignment of SOC1-like members show the conserved MADS, intervening, keratin-like and C-terminal domain (SOC1-motif). (C) Exon-intron structure of PbSOC1-like genes. Exons (CDSs) and introns are represented by yellow rectangles and black lines, respectively. Untranslated regions (UTRs) are represented by blue rectangles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
2. Materials and methods
overexpressed, for example, cotton GhSOC1 [16], orchid DoSOC1 [17] and tree peony PsSOC1 [18]. Similarly, in such fruits as blueberries and strawberries, the SOC1 homologous genes also play roles in flowering time regulation. For example, overexpression of the K-domain of a blueberry SOC1-like (VcSOC1-K) in tobacco can lead to early flowering [19]. The SOC1-like genes in different varieties of strawberry play diverse roles. FaSOC1 of cultivated strawberry is a flowering promoter, whereas FvSOC1 of the Fragaria vesca represses flowering and promotes vegetative growth [20,21]. However, ectopic expression of a SOC1 homolog in the Gerbera hybrid has been found to lead to partial loss of floral organ identity but does not affect flowering time [22]. Therefore, identifying pear SOC1-like genes and defining their function is necessary. SOC1 takes part in the photoperiod, temperature, age and gibberellin pathways to promote floral transition, specifically by interacting with key factors in different pathways [13]. In Arabidopsis, SOC1 is activated by CONSTANS (CO), which is one of the central regulators in the photoperiod pathway, to promote flowering [23]. However, FLC, a component of the vernalization pathway, directly represses SOC1 expression by binding to its promoter [24]. In addition, SOC1 integrates the photoperiod and GA signals to promote flowering via the SPL3, SPL4 and SPL5 genes at the shoot apex [25]. SOC1 interacts with multiple MADS-box members, such as AGL24, FUL and AP1, through direct binding to the regulatory sequences to coordinate the expression of several flowering genes, such as SVP, AGL15, and AGL18, thereby providing positive feedback from inductive floral cues [26–28]. Pear (Pyrus bretschneideri) is a widely cultivated perennial fruit tree. Flowering is the necessary basis for pear fruit formation and harvest; thus, the control and regulation of floral induction is an important issue for pear production. In this study, we identified eight SOC1-like genes in pear and analyzed their structures and expression patterns. Moreover, we characterized the function of two PbSOC1-like genes by ectopic transgenic analysis in Arabidopsis. Our results suggest that PbSOC1d and PbSOC1g act as activators of flowering time.
2.1. Identification and classification of SOC1-like genes in pear The protein sequences of SOC1-like genes were collected from 23 species that have been reported, including common monocot and eudicot (Table S2). Except for Actinidia chinensis and Medicago truncatula information, all the databases were surveyed with known ID number via the NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Actinidia chinensis and Medicago truncatula information were collected from UniProt (https://www.uniprot.org) and JCVI (http://blast.jcvi.org/MedicagoBlast/index.cgi) databases, respectively. The sequences of SOC1-like members from Arabidopsis were acquired from the Arabidopsis Information Resource (http://www.Arabidopsis.org/). The sequences of pear were searched by BLASTP against the pear proteins in NCBI database [29]. The protein sequences were detected by Conserved Domains software (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb. cgi) by the criterion that they must contain the SOC1 conserved domain. Detailed information on SOC1-like genes identified in this study is given in Tables S1 and S2. All protein sequences were aligned by MUSCLE. After alignment, the phylogenetic trees were all reconstructed by RaxML, and all the bootstrap values were examined by Bayes [30–32]. These trees were viewed and edited with the iTOL program (http://itol.embl.de/login. cgi). Gene structure analysis of SOC1-like genes used the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/) by aligning the coding sequences with the corresponding genomic sequences. Multiple sequence alignments of the full-length protein sequences, including the highly conserved amino acid residues, were determined by Clustal Omega (www.clustal.org) [33]. To identify the conserved motifs of SOC1-like proteins, their complete amino acid sequences were utilized to carry out the MEME analysis using an online tool (http://meme-suite.org) [34]. The parameters were set as follows: optimum motif width ranges from 6 to 50 and a maximum number of motifs of 10. All other parameters were set as 2
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Fig. 2. Tissue-specific expression assay of PbSOC1-like genes. Eight PbPbSOC1-like gene expression patterns were detected by RT-qPCR in root, young stem, leaf bud, leaf, phloem, flower bud, petal, style, anther and ovary, and they were normalized with PbUBQ as a reference gene. The black columns represent five vegetative growth organs (including root, young stem, leaf bud, leaf and phloem) and the gray columns represent five reproductive growth organs (including flower bud, petal, style, anther and ovary). Error bars represent the standard error. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Thirty-day-old seedlings that had completed the stratification at 4 °C in sand were used for diurnal rhythmic and photoperiodic response expression analyses. The seedlings were grown under three conditions, 12 h light/12 h dark, short-day conditions (8 h light/16 h dark) and long-day conditions (16 h light/8 h dark) in a greenhouse kept at 22 ± 1 °C with 65 ± 5% relative humidity. After 30 days of light-dark treatment, young leaves from the seedlings were sampled from four randomly selected plants every 4 h during the 24 h photoperiodic cycle. All samples were immediately frozen in liquid nitrogen and stored at −80 °C until use. The experiments were performed with three independent biological replicates. Wild-type Arabidopsis Columbia-0 (Col-0) was used for overexpression studies. Seed germination and plant growth management followed the Arabidopsis growth standard protocol published on the TAIR website (http://www.Arabidopsis.org). Nicotiana benthamiana was grown and used for transient expression experiments. Arabidopsis and tobacco were both grown under long-day conditions (16 h light/8 h dark) at 22 °C in the greenhouse.
defaults. In order to characterize the evolution pattern of PbSOC1-like genes, the non-synonymous (Ka) and synonymous (Ks) substitutions, as well as the ratio of Ka to Ks (Ka/Ks) in their homologs, were estimated. The coding sequences of eight PbSOC1-like genes were aligned using Clustalw. Ka and Ks were calculated by Dnasp v6 [35]. For analyzing the cis-elements of each PbSOC1-like gene, we acquired the sequence of 2000 bp ahead the initiation codon ATG from NCBI database (https://www.ncbi.nlm.nih.gov/) and submitted to the PlantRegMap online program [36], a database to provide a comprehensive, high-quality resource of plant transcription factors (TFs) and regulatory elements (http://plantregmap.cbi.pku.edu.cn). 2.2. Plant materials and growth conditions The ten-year-old trees of P. bretschneideri Rehd. cultivar ‘Dangshansuli’ used in this study were grown in a natural environment experimental field (Nanjing Agriculture University, Nanjing, China). Seeds of these trees were vernalized in sand for two months at 4 °C and transferred to flowerpots containing soil and vermiculite in a growth chamber with an air temperature of 22 °C. Roots, young stems and leaves of seedlings were collected for tissue-specific expression analysis after 40 days of transplanting to flowerpots. Leaf bud, phloem, flower buds, petals, styles, anthers and ovaries were harvested a few days before flower fully opening from the 10-year-old pear trees. For temporal gene expression analysis during June to November of flower bud development, the flower bud samples are collected from the 10-year-old pear trees on the 15th day of each month.
2.3. RNA isolation and quantitative RT-PCR (RT-qPCR) analyses A kit (FOREGENE, Chengdu, China) specifically designed to purify total RNA from plant samples with high polysaccharides and polyphenols was used, and total RNA was extracted from frozen tissues according to the manufacturer's instructions. The quality of the total RNA was assessed by gel electrophoresis, and optical density readings were obtained with a Nanodrop 2000 (Thermo Scientific, US). Firststrand cDNA was synthesized by a RevertAid First Strand cDNA 3
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Fig. 3. Dynamic expression pattern of PbSOC1-like genes during pear flower bud development. (A) Flower bud developmental progress during six months of pear. Samples were collected from ten-year-old trees on the 15th day of each month. Figures were observed with a stereomicroscope in a bright field. The black arrows indicate morphological development at different times. Scale bars, 2 mm. (B) Temporal expression assay of PbSOC1-like genes at six developmental stages of pear flower buds. The PbUBQ gene was used as an internal control. Error bars on each dot represent the standard error (S.E.) of three replicates.
Synthesis Kit (Thermo Scientific, US) using 1 μg of total RNA and oligo (dT) primers according to the manufacturer's instructions. The products of the first-strand cDNA were subjected to PCR amplification. SYBR Green I Master Mix (Roche, Germany) in a Light Cycler 480 II (Roche, Germany) was used for RT-qPCR of the collected samples to determine relative gene expression levels. Gene-specific primers (Table S2) were designed using the NCBI primer-designing tool and were checked by BLAST. To determine the specific amplification of each primer, melt curve analysis and electrophoresis of the test reaction were performed. For RT-qPCR, the reactions were prepared in a total volume of 20 μL containing 0.1 μL of cDNA sample as the template, 5 μL of 0.05 μM gene-specific primer premix, 10 μL of 2× SYBR Green Master Mix and 4.9 μL of water. The Light Cycler 480 II (Roche, Germany) was programmed as follows: 5 min of preincubation at 95 °C followed by 55 cycles of 3 s at 95 °C, 10 s at 60 °C, and 30 s at 72 °C of amplification. Data were analyzed using the 2−ΔCT method, and the expression levels
of target genes were normalized using the UBQ gene of pear or Arabidopsis Actin. RT-qPCR was performed using three independent biological replicates and three technical replicates for each sample. All RT-qPCR experiments were designed following MIQE guidelines [37]. The expression data were analyzed and graphed by Graph Pad Prism 5. 2.4. Subcellular localization analysis of proteins The full-length coding sequences without the stop codon of PbSOC1d and PbSOC1g genes were amplified by PCR from RNA with the appropriate specific primers (Table S3). The amplified products were cloned into the pEASY vector using a pEASY-T5 Zero Cloning Kit (TransGen Biotech, China) and sequenced. Then, the inserts were subcloned into the modified binary vector pCAMBIA1300-35S:CDS-GFP [38] with the XbaI and BamHI restriction sites. To observe the subcellular localization of PbSOC1d and PbSOC1g, Agrobacterium4
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Fig. 4. Diurnal rhythms of PbSOC1-like genes in pear leaves. The illumination regime is illustrated by white (light) and black (dark) bars. Pear leaves were exposed to 12 h light and 12 h dark conditions. PbUBQ was used as a reference gene in the RT-qPCR assay, and the expression was normalized. Error bars represent the standard error. ZT: zeitgeber time; the time of lights on usually defines zeitgeber time zero (ZT0).
3. Result
mediated transient expression in N. benthamiana leaves was performed as reported [39]. The expression vector was transformed into epidermal cells of 40-day-old tobacco plants. Two days after infiltration, the fluorescence in the transformed cells was observed with DAPI as a nuclei marker under a laser scanning confocal microscope LSM780 (Zeiss, Germany).
3.1. Identification and classification of SOC1-like genes in pear SOC1 and its homologous AGL14, AGL19, AGL42, AGL71, and AGL72 are in the same clade with SOC1 encoding MIKCC-type proteins in Arabidopsis [9]. Using the six MIKCC-type protein sequences in Arabidopsis as a reference, we searched against pear protein and nucleotide databases at NCBI (http://www.ncbi.nlm.nih.gov) by BLAST. We obtained 8 SOC1-like genes from pear and designated them as PbSOC1a to PbSOC1h (Table S1). The shortest amino acid sequence among the eight proteins is PbSOC1a, with a 642-bp open reading frame (ORF) that encodes a peptide of 213 amino acid residues with a predicted molecular mass of 24.64 kDa. The longest one is PbSOC1f with 253 amino acids. To describe the relationships and to classify the SOC1 gene family in pear and other species, we assembled fifty genes from 23 reported species and pear into a phylogenetic tree (Fig. 1A and Table S2). The phylogenetic tree inferred from the amino acid sequences of SOC1-like proteins clearly resolved three main subclades, AtSOC1, AtAGL14/ AGL19 and AtAGL42/71/72 (Fig. 1A). PbSOC1a was in the cluster branch of AtSOC1 and shared an amino acid sequence ~67% identity with AtSOC1. PbSOC1b and PbSOC1c were most closely related to AGL14 and AGL19, and PbSOC1d, PbSOC1f, PbSOC1e, PbSOC1g and
2.5. Arabidopsis transformation For Arabidopsis transformation, the pCAMBIA1300-35S: CDS-GFP vectors were also transformed into Agrobacterium tumefaciens strain GV3101. Arabidopsis (Col-0) plants were transformed using the floral dip method [40]. Seeds of transgenic plants were selected on Murashige and Skoog (MS) medium with 20 mg/L hygromycin and cultured in a growth chamber under the long-day condition (16 h light/8 h dark, 22 °C) for ten days, and resistant plants were transferred to soil. The presence of the transgene in progeny was confirmed by PCR using genespecific and vector primers (Table S2). Transgenic Arabidopsis plants in the T2 and T3 generations were selected to examine flowering time and other phenotypes.
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Fig. 5. Photoperiodic response of PbSOC1-like genes in pear. The expression pattern of partial PbSOC1-like genes showed a diurnal rhythm in plants grown in short days (8 h light/16 h dark) and long days (16 h light/8 h dark) in 48 h. Expression as determined by RT-qPCR was normalized to PbUBQ in all cases. Blue squares represent short-day conditions, and red dots represent long-day conditions. Light conditions are indicated by the colored bar at the bottom of the graph. Black boxes indicate darkness, and white boxes indicate light. Error bars represent the standard error. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
amino acid changes at key sites (Fig. S1). Therefore, these proteins all belong to the MIKCC type MADS-box transcription factors. MEME program was also used to identify the conserved motifs of SOC1-like proteins in Arabidopsis and pear, and all the 14 protein sequences were found to contain 1, 2, 3, 4, 5 and 6 motifs (Figs. S2 and S3). Motif 1 for M (MADS-box), motif 3 and motif 6 for I (intervening-domain), motif 2 and motif 5 for K (keratin-like domain), and the motifs corresponding to C have diversity. Interestingly, the C-terminal diversity of PbSOC1-like proteins was associated with 3 subclades of the phylogenetic tree. For example, protein of PbSOC1a only contains motif 4, proteins of PbSOC1b and PbSOC1c contain motif 4 and motif 9, while proteins of PbSOC1d-1h contain motif 4 and motif 7. When analyzing the evolution rate and pattern of a protein, the comparison between the number of non-synonymous substitutions (Ka) and synonymous substitutions (Ks) were required [45]. The Ka/Ks ratios of the PbSOC1-like members were examined, and it was figured out that all the results were < 1 (Table S4), indicating that they were undergoing purifying selective pressure. Meanwhile, structural analysis between the full-length cDNA and genomic DNA sequence revealed that most PbSOC1-like genes contain seven conserved exons and six variable introns that are identical to AtSOC1 and its homology genes in Arabidopsis, except for PbSOC1e (Fig. 1C). However, the intron lengths of PbSOC1-like genes are longer
PbSOC1h were more closely related to AGL42, AGL71, and AGL72 (Fig. 1A). The amino acid sequence identity between PbSOC1b and PbSOC1c was as high as 82%, but compared with AtAGL14 and AtAGL19, it was only ~55%. In addition, five other SOC1 homologous proteins of pear (PbSOC1d-h) are highly similar to each other, sharing 70% to 80% identity. Notably, seven species of Rosaceae were selected, including pear, and their SOC1 homologs are well-clustered (Fig. 1A), suggesting a closer evolutionary relationship in the Rosaceae family. SOC1 and SOC1-like genes have been reported to have four conserved domains [18,41,42]. The logo diagrams of four conserved domains were obtained by protein alignment analysis of fifty SOC1-like members indicated in Fig. 1A. The genes all share the conserved MADSbox, intervening-domain, keratin-like domain, and SOC1-motif (Fig. 1B). The MADS-box domain, composed of 58 amino acids at the Nterminal, is mainly responsible for DNA binding but is also involved in dimerization reactions and the binding of accessory factors [9]. The intervening-domain is approximately 30 amino acids with weakly conserved residues. Both the keratin-like domain and SOC1-motif function in the formation of higher-order complexes, and the coiled coil structure in the K-domain participate in protein-protein interactions, and the C domain is critical for the specificity of interactions [43,44]. All eight PbSOC1-like proteins have four conserved domains and no 6
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DAPI
Bright Field
Merge
PbSOC1g-GFP
PbSOC1d-GFP
GFP(Vector)
GFP
Fig. 6. Subcellular localization of PbSOC1d-GFP and PbSOC1g-GFP in leaf epidermal cells of tobacco. The fluorescence of GFP was observed by laser scanning confocal microscopy, and the nucleus was labeled with DAPI and the empty vector of GFP was used as a control. GFP, GFP fluorescence; BF, bright field; Merged, merged image of GFP and BF. Bar = 20 μm.
Fig. 7. Overexpression of PbSOC1d causes early flowering in Arabidopsis. (A) Control plants and transgenic lines were grown in soil under LD conditions. (B) The number of rosette leaves and number of days required to reach flowering in control plants and PbPbSOC1d-OE transgenic lines. (C) RT-qPCR analyses of PbSOC1d, AtFT, AtAP1 and AtLFY in vector control plants and transgenic lines and normalized by AtACT. Error bars indicate standard error (S.E.) of the mean. Asterisks indicate significant differences between control and transgenic lines (n ≥ 10, * means p < .05, **means the p value ranges from 0.05 and 0.001, *** means p < .001, by t-test, compared with vector control).
higher plants including Arabidopsis. The identification of cis-elements in the promoters of PbSOC1-like genes could contribute to predict and understand the potential regulatory functions [27]. Many cis-elements were found to be associated with plants reproductive development, such as AP2, MIKC_MADS, SBP, TALE and ZF-HD (Table S5). For
than those of their corresponding genes in Arabidopsis. These results suggest that the functions of PbSOC1-like genes are potentially similar to those in Arabidopsis. Characterized as a transcription factor, SOC1 has been reported to play an important role in the reproductive development of various 7
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Fig. 8. Overexpression of PbSOC1g promotes flowering in Arabidopsis. (A) The phenotype of PbSOC1g transgenic lines under LD conditions (16/8 h light/dark, 22 °C). (B) The number of rosette leaves and number of days during flowering in transgenic plants and control plants. C) The expression levels of PbSOC1g, AtFT, AtAP1 and AtLFY in seedlings after 12 days were measured by RT-qPCR and normalized by AtACT. Error bars indicate standard error (S.E.) of the mean. Asterisks indicate significant differences between control and transgenic lines (n ≥ 10, * means p < .05, **means the p value ranges from 0.05 and 0.001, *** means p < .001, by t-test, compared with vector control).
largely unchanged in the first four months but decreased significantly in October and November. In the process from flower bud differentiation to gradually entering dormancy, PbSOC1a, PbSOC1d and PbSOC1g show a relatively regular pattern of initially increasing and then decreasing transcription. These results indicate that the expression pattern of PbSOC1-like genes is different and dynamic during flower bud differentiation and development in pear.
instance, the CArG-box belong to MIKC_MADS elements has been studied in depth as a crucial binding site of SOC1 in Arabidopsis [26,46]. Similarly, each of the eight PbSOC1-like genes contains more than one CArG-box binding site in promoter regions (Table S6). These results suggest that the transcription of PbSOC1-like genes may be affected by these factors and participate in the pear development pathway.
3.2. Temporal and spatial expression patterns of PbSOC1-like genes
3.3. Expression pattern of PbSOC1-like genes in leaves under different photoperiods
To characterize the biological function of PbSOC1-like genes, we first examined their spatial expression in various tissues in pear by quantitative real-time PCR. Overall, PbSOC1-like genes have similar expression patterns that can be detected at high levels in leaf buds and leaves but at relatively low levels in flower buds and flower tissues. (Fig. 2). PbSOC1a transcripts were almost undetectable in flower samples, except flower buds, but were predominantly expressed in leaves. The relative expression levels of different PbSOC1-like genes were varied in the same organ. PbSOC1a, PbSOC1b and PbSOC1e accumulated in pear roots (Fig. 2). All eight PbSOC1-like genes were expressed in flower buds, suggesting that these PbSOC1-like gene functions might be associated with flower bud differentiation and development in pear. Previous studies have suggested that the pear, as a perennial woody plant, undergoes flower bud differentiation and development from June to November every year in China and then enters the dormancy state [47]. To investigate genes involved in pear flower bud differentiation and development, flower bud samples of pear were collected on the 15th day of each month between June and November. Flower bud differentiation and development of pear can be divided into two stages, physiological and morphological differentiation (https://www. actahort.org/books/872/872_14.htm). Morphological differentiation was observed from August and became clear over time (Fig. 3A). PbSOC1-like genes showed four different expression patterns (Fig. 3B). First, the transcription levels of PbSOC1a, PbSOC1b and PbSOC1c were relatively consistent, and the expression gradually increased from June to July, reaching a peak in July, and then continuously decreased. However, PbSOC1c is slightly different because the expression rebounded slightly in September and November. Second, both PbSOC1d and PbSOC1g show a steady trend of rising first and then falling. The difference is that one peak is in August and one peak is in September. Then, PbSOC1e and PbSOC1f show opposite changes to the first type. The expression levels of these genes fell sharply from June to July and then continued to rise. The expression level of PbSOC1h remained
SOC1-like genes in Arabidopsis and homologies from other plant species display diurnal and circadian patterns of expression [42,48–50]. The leaf is the major site for perception of day length, and all analyzed PbSOC1-like genes were expressed in leaves. Therefore, further experiments analyzed the diurnal rhythms and photoperiodic response of PbSOC1-like genes in the leaves of pear. The diurnal rhythms of these genes exhibited different patterns and can be divided into three categories (Fig. 4). The expression patterns of PbSOC1a, PbSOC1b and PbSOC1c peaked at dusk (zeitgeber time 13; ZT 13). The expression of PbSOC1d was the highest at dawn (ZT 1). The other four PbSOC1-like genes were highly expressed at night (Fig. 4). The transition from vegetative to reproductive growth is mainly controlled by day length in many plant species. Moreover, to study the effects of photoperiod on the expression of PbSOC1-like genes, the transcription levels of these genes were measured under long days (16 h light/8 h dark) and short days (8 h light/16 h dark). The expression of PbSOC1a, PbSOC1d and PbSOC1g showed an obvious response to photoperiod. Under long-day conditions, the peak expression of PbSOC1a, PbSOC1d and PbSOC1g occurred during ZT21, while the peak occurrence of these gene expression was shown at ZT13 under short-day conditions. The expression of five other PbSOC1-like genes was not regularly responsive to photoperiod (Fig. 5). Taken together, these results indicate that PbSOC1a, PbSOC1d and PbSOC1g might be the key genes in the regulation of flowering in pear. Therefore, PbSOC1d and PbSOC1g were selected for further functional testing in flowering regulation. 3.4. Subcellular localization of PbSOC1d and PbSOC1g proteins To examine the subcellular localization of PbSOC1d and PbSOC1g proteins, we fused each gene's full coding sequence to green fluorescent protein (GFP). The resulting constructs driven by the 35S promoter 8
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species to form three distinct branches (Fig. 1A). PbSOC1-like family members are highly conserved in gene structures and protein domains in pear compared with Arabidopsis, indicating that they are typical MIKCC proteins with a highly conserved MADS-box at the N-terminus, a K-box in the middle, a specific SOC1 motif (Fig. 1 and Fig. S1) [52]. Therefore, PbSOC1-like genes may play a conserved role in the regulation of flowering. PbSOC1-like genes are expressed in a range of tissues, though at differing levels (Fig. 2). All eight PbSOC1-like gene transcripts reached predominant levels in the leaves, indicating that they may all play crucial roles in pear leaves. Meanwhile, PbSOC1-like genes are expressed in flower buds, consistent with a potential role in the control of flower bud differentiation and development. PbSOC1d-PbSOC1h are also clearly expressed in phloem, suggesting that they might also play a similar role to that of their homologs [14], such as promoting flowering in the shoot apical and axillary meristems. However, another commonality observed for pear SOC1-like genes is notably low expression levels in the floral organ, including petals, styles, anthers and ovaries. Likewise, many SOC1-like genes in diverse species were detected mainly in vegetative organs and almost not in floral organs [16,49], indicative of a common role during vegetative development as general regulators of plant organogenesis. Some fruit trees, such as mulberry, mei and pear, the formation and development of their inflorescence primordia occur in the buds in summer and then stop growing in the dormant period until the bud continues to flower in the spring [53,54]. During flower bud development in summer, mulberry FT expression first increases and then decreases over time, plummeted in November before endodormancy of the buds [53]. A similar situation has been reported in pear, except that the expression of PpFTs was not clearly induced, but the rapid decrease of PpTFL1s expression may be involved in floral induction [55]. In our study, differential PbSOC1-like gene expression was observed in different flower buds during the course of flower bud differentiation and development (Fig. 3A and B). The three genes PbSOC1a, PbSOC1d and PbSOC1g show a trend of rising first and then falling in the process of flower bud development. The difference is that the time of reaching the peak is diverse, with PbSOC1a peaking in July, PbSOC1d peaking in August and PbSOC1g peaking in September, indicating that these three genes may play potential roles in different stages of flower bud morphological differentiation (Fig. 3B). PbSOC1a, PbSOC1b and PbSOC1h were expressed relatively higher at earlier stages of flower bud, possibly reflecting that they are involved in the early stage during the vegetative to the reproductive transition. In apricot, ParSOC1 has been associated with cold response and chilling requirements during dormancy [42]. Among the eight PbSOC1-like genes, only PbSOC1f still had a high expression level in November and showed an upward trend, which may be involved in the regulation process of dormancy. The expression analysis showed that almost all PbSOC1-like genes had a diurnal rhythm at a cycle of 24 h under 12 h light and 12 h dark conditions (Fig. 4). A connection between the internal rhythm and response to environment cues by SOC1 was shown in Arabidopsis [56] and Japanese apricot [42]. Our results suggest that PbSOC1-like genes may also be involved in the cross-talk between clock rhythm and environmental signaling in pear. The function of SOC1, initially named AGL20, in controlling flowering is reported in Arabidopsis [15]. The regulation of SOC1 in flowering is associated with photoperiod in Arabidopsis [13]. Under longday and short-day conditions, the response of the PbSOC1a gene to photoperiod was similar to that of SOC1 in Arabidopsis [57] and ZmMADS1 in maize [48]. PbSOC1d and PbSOC1g also responded to photoperiod changes, and the expression pattern was more stable under long-day conditions (Fig. 5). PbSOC1d and PbSOC1g belong to the same subclade of AGL42, AGL71 and AGL72, which are Arabidopsis SOC1 subfamily genes, in the phylogenetic analysis (Fig. 1C). Meanwhile, AGL42, AGL71 and AGL72 are produced by gene duplication during evolution, and they can also promote flowering in Arabidopsis [14].
were then transiently expressed in leaf epidermal cells of tobacco. Confocal microscopy observations suggested that both PbSOC1d and PbSOC1g fusion proteins were localized exclusively in the nucleus (Fig. 6). Thus, these proteins appear to be nuclear proteins, suggesting that they can participate in transcriptional regulation. 3.5. Overexpression of PbSOC1d and PbSOC1g promotes flowering in Arabidopsis Because we lacked a dependable transformation protocol for pears and there was an extremely long period from seedling to flower, the functionality of PbSOC1d and PbSOC1g was analyzed in Arabidopsis. Arabidopsis lines overexpressing PbSOC1d and PbSOC1g were generated, and two independent lines were selected for flowering time analysis. To avoid the fluctuation of gene expression by other environmental factors, all plants grew under the same long-day conditions. First, the transgenic lines were assayed by RT-qPCR to validate the presence and transcript levels of the introduced PbSOC1d and PbSOC1g (Figs. 7C and 8C). The results showed that the expression levels of genes were significantly higher in corresponding transgenic lines than in the vector control plants. According to the results (Figs. 7A and 8A), PbSOC1d and PbSOC1g-overexpressing lines exhibited strongly early flowering traits, consistent with the phenotypes of AtAGL71 and AtAGL72 [14]. Under long-day conditions, the control plants transformed with empty vector required 25.1 ± 0.27 days until the time of flowering, compared with 22.9 ± 0.30 and 22.1 ± 0.31 days in the PbSOC1d overexpression lines OE1 and OE2, respectively (Fig. 7B). The shortened vegetative phase was also manifested as a decrease in the number of rosette leaves in transgenic lines, ranging from 8.89 ± 0.21 to 8.41 ± 0.21 leaves on average, compared to the 9.85 ± 0.18 leaves in control plants (Fig. 7B). Similar statistical results were found in PbSOC1g-overexpressing lines. The transgenic lines began to bolt at approximately 22 days after light and had 8 rosette leaves on average, whereas the vector control plants required approximately 25 days to reach bolting and had 9.85 rosette leaves on average (Fig. 8B). These results indicate that both PbSOC1d and PbSOC1g play conserved roles in promoting flowering in Arabidopsis. Since the transgenic plants overexpressing PbSOC1d and PbSOC1g have an early flowering phenotype, the results suggested that they may be involved in the regulation of flowering time-related genes. Thus, three flowering time-related genes, including AtFT, AtAP1 and AtLFY, were tested in the transgenic lines. The expression of all three flowering time-related genes was significantly upregulated in the PbSOC1d-OE and PbSOC1g-OE transgenic lines in Arabidopsis (Figs. 7C and 8C). These observations suggest that PbSOC1d and PbSOC1g play roles in the promotion of flowering, partly through engaging similar downstream regulators in Arabidopsis. 4. Discussion Flowering is an important economic characteristic of fruit production. The switch of floral induction is triggered by a variety of environmental cues and endogenous signals [1]. The SOC1 gene in Arabidopsis and its homology in multiple species, such as orchids, rice, maize, tobacco, soybeans, kiwifruit, grapes, and apples (Table S2), have been demonstrated to play an important role in promotion flowering by integrating multiple floral cues [51]. In this study, we analyzed this gene family in pear and confirmed that PbSOC1d and PbSOC1g promote flowering. Based on the pear genome sequencing database [29], we perform a comprehensive analysis of the SOC1-like family, including their phylogenetic, exon/intron structures, conserved domains and protein sequences; these findings make it possible for us to predict the potential functions and evolutionary relationships among uncharacterized members of this gene family. We identified eight SOC1-like genes in pear (Table S1), which were clustered with homologous genes in other 9
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Thus, PbSOC1d and PbSOC1g likely have similar functions to AGL42, AGL71 and AGL72 in promoting flowering in pear. The nuclear localization of PbSOC1d and PbSOC1g may explain their role in transcriptional regulation (Fig. 6). Moreover, both PbSOC1d and PbSOC1g are able to promote flowering in Arabidopsis (Fig. 7A and 8A). In Arabidopsis, SOC1 upregulates AP1 expression via a pathway mediated by LFY or FT [13,28]. The expression of AtFT, AtAP1 and AtLFY was upregulated in the PbSOC1d-OE and PbSOC1g-OE lines (Fig. 8C and 9C). These results suggest that a conserved pathway for SOC1-like genes regulates flowering through the promotion of flowering time genes and flower organ identity genes.
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