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Heterologous expression of an RNA-binding protein affects flowering time as well as other developmental processes in Solanaceae 5 Hyun Min Kim1,3, Jeong Hwan Lee2,3, Ah-Young Kim1, Se Hee Park1, Sang Hoon Ma1, Sanghyeob Lee2,**, Young Hee Joung1,* 1 School of Biological Sciences and Technology, Chonnam National University, Gwangju, 500-757, South Korea 10 2 Department of Bioresource Engineering and Plant Engineering Research Institute, Sejong University, 98 Gunja-diong, Gwangjin-gu, Seoul, 143-747, South Korea; 3 Hyun Min Kim and Jeong Hwan Lee contributed equally to this work. *Corresponding 15 authors: Young Hee Joung, Ph.D. E-mail: [email protected] Phone: +82-62-530-5202 Fax: +82-62-530-2199 20 **Corresponding authors: Sanghyeob Lee, Ph.D. E-mail: [email protected] Phone: +82-2-3408-4375 Fax: +82-2-3408-4318 25 1 Abbreviations: CDFs, Cycling Dof Factors; CO, CONSTANS; COL, CONSTANSLike; FBH, FLOWERING BHLH; FHA domain, forkhead associated domain; FKF1, FLAVIN-BINDING, KELCH REPEAT, F-BOX 1; FT, FLOWERING LOCUS T; LD, long day; GI, GIGANTEA; LFY, LEAFY; NIFK, nucleolar protein interacting 5 with the FHA domain of pKI-67; ORFs, open reading frames; PCR, polymerase chain reaction; RBP, RNA-binding protein; RRM, RNA recognition motif; RT–qPCR, real time quantitative polymerase chain reaction; SAM, shoot apical meristem; SOC1, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1; SVP, SHORT VEGETATIVE PAHSE; SFT, SINGLE FLOWER TRUSS; SP, SELF PRUNING; 10 TFL1, TERMINAL FLOWER1; TSF, TWIN SISTER OF FT 2 Abstract Flowering time in members of the Solanaceae plant family, such as pepper (Capsicum spp.) and tomato (Solanum lycopersicum), is an important agronomic trait for 5 controlling shoot architecture and improving yield. To investigate the feasibility of flowering time regulation in tomato, an RNA-binding protein (RBP) encoding gene homologous to human Nucleolar protein interacting with the forkhead associated (FHA) domain of pKI-67 (NIFK), CaRBP, was isolated from hot pepper. The function of CaRBP was determined in transgenic tomato. The deduced amino acid sequence 10 includes an RNA recognition motif (RRM) and showed most similarity to the RRM present in a putative RBP encoded by human NIFK. CaRBP was highly expressed in the vegetative and reproductive tissues, such as leaves and fruits, respectively. Subcellular localization analysis indicated that CaRBP is a nucleolar protein. Heterologous expression of CaRBP under 35S promoter in tomato plants induced 15 severe alteration of flowering with additional defects of vegetative organs. This floral retardation was associated with the alteration of SFT/SP3D and SlSOC1s as floral integrators. Furthermore, CaRBP reduces the expression levels of SlCOLs/TCOLs via changes in the expression of SlCDF3, SlFBHs, and SlFKF1s. This indicates a repressive effect of CaRBP on the regulation of flowering time in tomato. Overall, 20 these results suggest that alteration in CaRBP expression levels may provide an effective means of controlling flowering time in day-neutral Solanaceae. 25 Key words: CaRBP, Flowering time, Hot pepper, RNA recognition motif, RNAbinding protein, Tomato 30 3 Introduction The overall architecture of higher plants is determined by many physiological and genetic pathways that give rise to species-unique morphologies (Sussex and Kerk 5 2001). In Arabidopsis exhibiting monopodial growth, the shoot apical meristem (SAM) begins to produce leaves and later converts to a reproductive meristem to form flowers. However, Solanaceae plants such as tomato (Solanum lycopersicum) and pepper (Capsicum spp.) show a sympodial growth architecture (Lifschitz and Eshed 2006). In sympodial growth, the development of a SAM is terminated by a flower or 10 an inflorescence, and further growth continues from the uppermost axillary meristems. Finally, a short shoot segment known as the sympodial unit, consisting of three leaves and an inflorescence in tomato, and two leaves and a solitary flower in pepper, is reiterated in the life cycle of Solanaceae. Regulation of plant architecture has a profound impact on the agronomic performance of crops, as can be seen in the 15 development of semi-dwarf wheat and rice varieties bringing about a remarkable increase in yield known as the green revolution (Peng et al. 1999). Studies of plant architecture or flowering time mutants have revealed that these two developmental processes are significantly interconnected. In the model Arabidopsis, genetic and molecular studies on the regulation of flowering time from 20 the early 1990s revealed that different environmental signals influencing flowering time, such as day length (photoperiod), vernalization, and ambient temperature, are perceived through a complicated network of molecular pathways in the plant (Boss et al. 2004; Putterill et al. 2004; Sung and Amasino 2005; Lee et al. 2008; Song et al. 2013; Verhage et al. 2014; Capovilla et al. 2015). These pathways ultimately 25 converge at FLOWERING LOCUS T (FT), TWIN SISTER OF FT (TSF), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), and LEAFY (LFY), which are known floral integrator genes (Weigel et al. 1992; Kardailsky et al. 1999; Kobayashi et al. 1999; Lee et al. 2000; Onouchi et al. 2000; Yamaguchi et al. 2005). In addition, CONSTANS (CO), FLOWERING LOCUS C (FLC), SHORT 30 VEGETATIVE PAHSE (SVP), and FLOWERING LOCUS M (FLM) have been revealed as genes involved in photoperiod, vernalization, and ambient temperature pathways (Putterill et al. 1995; Michaels and Amasino 1999; Lee et al. 2007; Lee et al. 2013; Pose et al. 2013). However, the conservation of the molecular mechanisms that underlie flowering time in important crops remains unknown. 4 Some components involved in the regulation of flowering time in solanaceous crops have been identified by genetic interaction analyses. In tomato, SINGLE FLOWER TRUSS (SFT)/SP3D, an Arabidopsis FT homologue, regulates flowering time and shoot architecture (Molinero-Rosales et al. 2004; Lifschitz and Eshed 2006). 5 Overexpression of SFT in tomato plants induces accelerated flowering as well as modification in the sympodial pattern, suggesting that the action of FT homologues is plant species-specific. In addition, tomato SELF PRUNING (SP), an Arabidopsis TERMINAL FLOWER1 (TFL1) homologue, antagonistically functions in determining plant shoot determinacy (Pnueli et al. 1998). Subsequently, FASCICULATE, a 10 TFL1/SP homologue in pepper, functions in flowering time, plant architecture, and growth (Elitzur et al. 2009). In contrast to FT and TFL1 homologues, overexpression of Arabidopsis CO or two tomato CONSTANS-Like (COL) homologues (TCOL1/SlCOL1 and TCOL3/SlCOL3) does not affect flowering time in day-neutral tomato or tobacco plants (Ben-Naim et al. 2006). However, both TCOL1/SlCOL1 and 15 TCOL3/SlCOL3 showed diurnal expression patterns. These suggest that tomato COL homologues may be linked to other photoperiodic responses, but not regulation of flowering time. In addition, overexpression of tomato Cycling Dof Factors (SlCDFs) in Arabidopsis, which repress CO transcription via direct binding to the CO promoter in Arabidopsis (Imaizumi et al. 2005), results in late flowering through regulation of 20 CO and FT (Corrales et al. 2014). These findings suggest evolutionary conservation and divergence of components acting within the photoperiod pathway in day-neutral plants. RNA-binding proteins (RBPs) in plants play crucial roles in all aspects of posttranscriptional gene regulation, including pre-mRNA splicing, polyadenylation, RNA 25 stability, RNA export, and chromatin modification (Lorkovic 2009; Ambrosone et al. 2012). They are an important class of emerging factors affecting diverse processes of plant growth and development as well as plant adaptation to various environmental conditions (Aneeta et al. 2002; Li et al. 2002; Lim et al. 2004; Mockler et al. 2004; Zhao et al. 2004; Kim et al. 2007). One well-known example is the regulation of 30 flowering time by inhibiting FLOWERING LOCUS C (FLC) expression via two plant-specific RNA recognition motifs (RRMs) containing FCA and FPA proteins (Macknight et al. 1997; Schomburg et al. 2001). Recent reports have revealed that RRMs function in chromatin modification at the FLC locus with FLOWERING LOCUS D (FLD), a homologue of human lysine-specific demethylase 1 (Liu et al. 5 2007; Baurle and Dean 2008). In addition, they affect chromatin modification at loci involved in siRNA-dependent chromatin silencing, suggesting a more general role of FCA and FPA in plant growth and development (Baurle et al. 2007). Although plants such as Arabidopsis and rice have more than 200 and 250 putative RBP genes, 5 respectively (Lorkovic 2009; Cook et al. 2011), little is known about the roles of many plant RBPs due to the lack of in vivo and in vitro biochemical systems in plants for studying their mechanisms. We report here the characterization of CaRBP, an RNA-binding protein encoding gene cloned from hot pepper (C. annuum cv. Bukwang) and subsequently 10 determined to be homologous to human Nucleolar protein interacting with the forkhead associated (FHA) domain of pKI-67 (NIFK). We found that CaRBP was abundantly expressed in the leaves and fruits, and CaRBP localized to the nucleolus. We examined the function of CaRBP by assessing the expression of CaRBP under 35S promoter in tomato plants and revealed that overexpression of CaRBP in tomato 15 plants delayed flowering and altered vegetative organ formation. The delayed flowering was associated with altered expression of SFT/SP3D and SlSOC1s. In addition, CaRBP negatively controls SlCOLs/TCOLs via modulations in the expression of SlCDF3, tomato FLOWERING BHLH (SlFBHs), and tomato FLAVINBINDING, KELCH REPEAT, F-BOX 1 (SlFKF1). Our results suggest that CaRBP 20 functions in the control of various developmental processes such as flowering time and vegetative organ formation in plants. 6 Materials and methods Plant material and growth conditions 5 Hot pepper (C. annuum cv. Bukang) and transgenic tomato plants (S. lycopersicon cv. Micro-Tom) were grown in soil or Murashige and Skoog (MS) medium at 22–24°C under long day (LD) conditions [16/8-h (light/dark)] with light supplied at an intensity of 120 µmol m-2 s-1. 10 Isolation of CaRBP from hot pepper Total RNA was extracted from the leaf tissues of hot pepper plants using the QiagenRNeasy kit (Qiagen, Hilden, Germany). A full-length open reading frame (ORF) of CaRBP was cloned via Reverse Transcription (RT)–Polymerase Chain 15 Reaction (PCR) in accordance with the manufacturer’s instructions (Intron, Seongnamm, South Korea). The following conditions were used in the RT–PCR: 1 cycle of 95°C for 2 min; 40 cycles of 95°C for 20 s, 57°C for 20 s, 72°C for 1 min; 1 cycle of 72°C for 7 min. The amplified PCR products were cloned into a pBI121 binary vector harboring the 35S promoter for overexpression. Oligonucleotide primer 20 sequences used for cloning are provided in Supplementary Table S1. Generation of transgenic tomato plants Tomato seeds were sterilized in 4% NaOCl for 10 min and rinsed three times with 25 sterile distilled water. Seeds were germinated on MS medium and kept in a plant growth chamber under a 16 h photoperiod at 24°C for 2 weeks. Cotyledons from the 2-week-old tomato seedlings were cut to approximately 0.5 cm long. The cotyledon pieces were soaked into Agrobacterium tumefaciens culture media for 10 min and then placed on co-culture media (MS medium with 30 g/L sucrose, 10 μM zeatin, 1 30 μM IAA, and 200 μM acetosyringone, pH 5.2). After co-cultivation for 2 days, the cotyledon pieces were transferred to shoot induction medium (MS medium with 30 g/L sucrose, 1 μM IAA, 10 μM zeatin, 50 mg/L kanamycin, and 250 mg/L cefotaxime, pH 6.0) and transferred to fresh medium every 3 weeks until shoots regenerated. Regenerated shoots were excised from the callus and transferred to rooting medium 7 (MS medium with 15 g/L sucrose, 0.2 mg/L IBA, 50 mg/L kanamycin, and 250 mg/L cefotaxime, pH 6.0). The regenerated plants were transferred to soil. Genomic Southern blot analysis 5 Genomic DNA for Southern blot analysis was isolated from leaf tissue of transgenic tomato plants using the DNeasy Plant Mini Kit (Qiagen) in accordance with the manufacturer’s instructions. Twenty micrograms of genomic DNA were digested with HindIII, electrophoresed on a 0.8% agarose gel, and transferred to a nitrocellulose 10 membrane. An 840-bp PCR fragment of CaRBP, which was amplified with RBP5′UTR and RBP-3′UTR primers, was used as a probe and labeled using the PCR DIG Probe Synthesis kit (Roche Applied Science, Madison, WI, USA). The procedures of hybridization and detection were performed in accordance with the manufacturer’s instructions. 15 Inverse PCR analysis Two micrograms of genomic DNA were isolated from transgenic tomato plants, digested by appropriate restriction enzymes (AluI, HhaI, PvuII, TaqI, BssHII), and 20 inverse PCR was performed as previously described (Offringa and van der Lee 1995). In the first round of PCR, T-DNA right (BV1 and BV2) and left (BV4 and BV5) border-specific primers were used. In the second round of PCR, a 50-fold dilution of the first PCR products was used as a template and nested PCR primers (BV1nest, 2nest, 4nest, and 5nest) were used. Subsequently, final PCR products were cloned and 25 sequenced. Oligonucleotide primer sequences used for inverse PCR analysis are provided in Supplementary Table S1. RNA expression analyses 30 For real time (RT)–quantitative polymerase chain reaction (qPCR) analysis, total RNA was extracted from several tissues of hot pepper plants or the leaf tissue of transgenic tomato plants using the QiagenRNeasy kit (Qiagen). RNA quality was determined with a Nanodrop ND-2000 spectrophotometer (Nanodrop Technologies, 8 USA), and only high-quality RNA samples (A260/A230 >2.0 and A260/A280 >1.8) were used for subsequent experiments. To remove possible DNA contamination, RNA samples were treated with DNaseI (New England Biolabs, Ipswich, MA, USA) for 60 min at 37C. One to two micrograms of RNA were used for complementary 5 DNA (cDNA) synthesis in accordance with the manufacturer’s instructions (Roche Applied Science). The RT–qPCR analysis was carried out using a Rotor-Gene 6000 real-time amplification operator (Corbett Research, UK) using Roche SYBR Green Master mixture (Qiagen). The actin gene was used as the reference gene. All RT– qPCR experiments were carried out in two biological replicates (independently 10 harvested samples) with three technical triplicates each. Oligonucleotide primer sequences used for expression analysis are provided in Supplementary Table S2. The procedure used for determining the relative abundance of transcripts has been described previously (Pfaffl et al. 2002). For northern blot analysis, 50 µg of total RNA extracted from the leaf tissues of 15 transgenic tomato plants was used. An 840-bp PCR fragment of CaRBP amplified with RBP-5′UTR and RBP-3′UTR primers was used as a hybridization probe. The procedures of hybridization and detection were performed in accordance with the manufacturer’s instructions (Roche Applied Science). 20 Subcellular localization analysis The ORF of CaRBP was cloned into pBI121-EGFP vector harboring 35S promoter for the subcellular localization assay. Agrobacterium strain GV3101 was used for infiltration of tobacco (N. benthamiana). Subsequently, epidermal cells of infiltrated 25 tobacco leaves were examined for fluorescence using a confocal microscope (Leica TCS SP5, Leica Microscope Systems, Germany). The oligonucleotide primer sequences used for cloning are shown in Supplementary Table S1. 9 Results Isolation of CaRBP homologous to human NIFK from hot pepper 5 We found one putative gene showing a higher expression pattern in early vegetative development from microarray analysis of hot pepper (C. annuum cv. Bukwang) (Lee et al. 2010; Lee and Choi 2013), and amplified a full-length cDNA (949 nucleotides) using a hot pepper cDNA library as a template. DNA sequencing of this clone showed that it encodes a predicted protein of 231 amino acids. Comparison of the gene with 10 sequences in the GenBank database (http://www.ncbi.nlm.nih.gov) revealed significant homology to a class of proteins harboring RRMs, also referred to as RBDs. The deduced amino acid sequence of its ORF region contains an RRM (amino acid residues 54-117) that shows most similarity to the RRM present in a putative RBP encoded by NIFK (Fig. 1a). Studies suggest that human NIFK may be required for cell 15 cycle progression (Takagi et al. 2001), and similar genes have been found in fruit fly Drosophila melanogaster and budding yeast Saccharomyces cerevisiae (Winzeler et al. 1999). Nucleolar protein 15 (Nop15p), a homologue of human NIFK, is known to be involved in the cellular control of cytokinesis (Oeffinger and Tollervey 2003; Colau et al. 2004; Dez and Tollervey 2004). In addition to the RRM, this clone has a 20 bipartite nuclear localization signal (NLS) at the C-terminus (amino acid residues 177-210) (Fig. 1a), suggesting that CaRBP localizes to the nucleus (Dingwall et al. 1988). The overall amino acid identity of this clone to those of human, Drosophila, and yeast was 25%, 25%, and 31%, respectively. However, its RRM domain exhibited approximately 55% identity (Fig. 1b). We also found other plant genes 25 homologous to the clone isolated from hot pepper in the GenBank database (Fig. 1b). The deduced amino acid sequences and RRM domains among plant genes were about 66% and 83% identical, respectively. Interestingly, the amino acid sequence of this clone was 94% identical to that of tomato (S. lycopersicum), another member of the Solanaceae family. Phylogenetic analysis showed that this clone clustered in a clade 30 that was distinct from other RBPs with RRM domains (e.g. FCA and FPA) (Fig. 1c). We designated this clone isolated from hot pepper as CaRBP. The nucleotide sequence of CaRBP was deposited into the GenBank database as accession number KP699580. 10 The expression patterns of CaRBP and subcellular localization of CaRBP In order to investigate the temporal and spatial expression patterns of CaRBP in hot pepper, we performed a RT–qPCR analysis in a variety of tissues and fruit 5 developmental stages in young and mature hot pepper plants. CaRBP transcripts were abundantly expressed in leaves, and to a lesser extent, in flowers and ovaries (female organ) (Fig. 2). However, CaRBP transcripts were poorly expressed in stems, roots, and anthers (male organ). We then evaluated the expression level of CaRBP in the fruit developmental stages. CaRBP transcripts were observed in all stages of 10 development, but the breaking green stage showed the highest expression level. This expression pattern is consistent with those of other RBP genes such as FCA and FVE in Arabidopsis and rice (Macknight et al. 1997; Ausin et al. 2004; Kim et al. 2004; Lee et al. 2005; Baek et al. 2008). When we compared expression levels of CaRBP with other RRM-containing RBP genes found in the hot pepper genome (Kim et al. 15 2014); CaRBP expression was much higher than any of other RRM-containing RBP genes. These data indicate that CaRBP is broadly expressed in vegetative and reproductive tissues. Since many RBPs are known to localize predominantly in the nucleus (Ambrosone et al. 2012) and CaRBP has a bipartite NLS at the C-terminus (Fig. 1a), 20 we carried out an experiment to investigate whether CaRBP is a nuclear protein. The coding sequence of the CaRBP gene was fused with that of EGFP driven by a 35S promoter, and this construct was transiently expressed in tobacco (Nicotiana benthamiana) leaves. The CaRBP-EGFP protein was localized in the nucleus (I panel in Fig. 3). Because the deduced amino acid sequence of CaRBP has significant 25 homologies with human NIFK and yeast Nop15p as nucleolar proteins (Fig. 1), we examined the subcellular localization pattern of CaRBP in more detail. As expected, the CaRBP-EGFP protein was predominantly restricted to the nucleolus (II panel in Fig. 3). These data demonstrate the nucleolar localization of CaRBP. 30 Overexpression of CaRBP in tomato plants causes pleiotropic phenotypes including severe delayed flowering In order to investigate the function of the CaRBP using a transgenic approach, a fulllength CaRBP ORF under the control of 35S promoter was constructed. This 11 construct was then introduced into tomato plants (S. lycopersicon cv. Micro-Tom) (Supplementary Fig. S1a), as the genus Capsicum yields low frequency and reproducibility of stable transformation by Agrobacterium (Lee et al. 2004) and the heterologous overexpression usually gives higher expression level of introduced 5 genes. For 35S::CaRBP construct, five regenerated plantlets were obtained from 100 cotyledon discs. When 35S::GUS construct used as a negative control was also transformed, two or three regenerated plantlets were obtained from 20 cotyledon discs. This indicate that transformation efficiency in 35S::CaRBP construct (5%) was lower than that in 35S::GUS construct (approximately 12.5%). Of five regenerated plantlets, 10 four transgenic lines survived in soil and two transgenic lines failed to make flowers due to long vegetative growth. Finally, we obtained only two independent transgenic lines (TG-1 and TG-2) in the T0 generation. This severe phenomenon is consistent with the observation that high overexpression of RRMs-containing RBPs such as FCA of Arabidopsis and rice resulted in more severe defects in growth and development, 15 which eventually caused to premature senescence and death (Quesada et al. 2003; Lee et al. 2005). However, the transgenic plants harboring 35S::GUS vector did not show any morphological changes (data not shown). Subsequently, genomic Southern hybridization analysis revealed that two individual plants from two independent transgenic lines (TG-1 and TG-2) have a single transgene (Supplementary Fig. S1b). 20 Inverse PCR analysis also showed that the insertion sites of TG-1 and TG-2 lines in the tomato genome were SGN-U581138 and BAC C02SLm0065M14.1, located on chromosomes 1 and 2, respectively (Supplementary Fig. S2). In order to determine CaRBP function in more detail, we analyzed the phenotype of two transgenic lines (TG-1 and TG-2) in the T1 generation. Individual 25 plants from TG-1 or TG-2 lines were grouped into two classes (I and II), based on the degrees of their flowering times and morphological defects (Fig. 4; Fig. 5; Supplementary Fig. S3). Under LD conditions, Class I plants exhibited markedly delayed flowering, thereby leading to continuous vegetative growth over one year (Fig. 4b, e). Thus, they did not show any inflorescence, compared to wild-type and 30 35S::GUS plants (Fig. 5a). They also showed a dwarf phenotype (Fig. 4b, e; Fig. 5b) and severe morphological defects of the vegetative organs (i.e., curled and nonserrated leaves) (Fig. 4g, h). Furthermore, length of stems, roots and terminal leaflets of the leaves was shorter in Class I plants than in the wild-type plants (Fig. 5c, d, e). In contrast, Class II plants did not show any morphological defects or reduced length 12 of leaves, stems, and roots in vegetative growth, compared to wild-type tomato or 35S::GUS plants (Fig. 4c, f, g, h; Fig. 5b, c, d, e). However, more delayed flowering was also observed in Class II plants, compared to wild-type or 35S::GUS plants (Fig. 4c, f; Fig. 5a). Subsequent PCR-based DNA genotyping analysis revealed that Class I 5 and II plants from TG-2 line were homozygous and hemizygous genotypes, respectively (Supplementary Fig. S4a). Furthermore, northern blot analysis showed that the different phenotypes of Class I and II plants from TG-1 or TG-2 lines were directly correlated with the level of CaRBP expression (Supplementary Fig. S4b). However, we cannot dismiss the possibility that these phenotypic differences between 10 Class I and II plants may result from genetic instability during plant tissue cultures (Phillips et al. 1994). Although CaRBP RNA expression in Class I plants from TG1 line was strong, compared with that in Class I plants from TG-2 line (Supplementary Fig. S4b), the phenotype of TG-2 line was more severe than that of TG-1 line (Fig. 4; Fig. 5; Supplementary Fig. S3). This suggests that the levels of 15 CaRBP protein may be important for severe phenotypes. Taken together, these data suggest that increased CaRBP expression is likely responsible for this severely retarded flowering and severe morphological defect phenotypes of 35S::CaRBP plants and also that CaRBP may function in the regulation of flowering time as well as other vegetative organ development. 20 Expression analysis of flowering time genes in 35S::CaRBP plants Change in ambient temperature is known to affect the transition to flowering in cultivated tomato and pepper plants (Samach and Lotan 2007). Although 25 overexpression of CO or tomato CO homologues has no significant effect on the flowering time in day-neutral tomato plants (Ben-Naim et al. 2006), CO regulatory genes such as CDFs acting within the photoperiod pathway are also present in tomato (Corrales et al. 2014). To investigate expression of flowering time genes, we selected a homozygous plant from one transgenic line (TG-2) that showed severely delayed 30 flowering phenotype under LD conditions in the T2 generation. The expression of CaRBP was abundantly detected in this plant (data not shown). We determined the expression levels of known genes within the photoperiod or ambient temperature pathways, and floral integrator genes in 35S::CaRBP plants. 13 Firstly, we searched a tomato genome database (http://solgenomics.net/) using Arabidopsis flowering time genes as queries and found several tomato genes homologous to CO, CDF, FBH, FKF1, FT/TFL1, GIGANTEA (GI), J3, LFY, SOC1, and SVP (Supplementary Table S3). Subsequent RT–qPCR analysis revealed that the 5 expression levels of TCOL1/SlCOL1, TCOL2/SlCOL2, TCOL3/SlCOL3, SlCOL4, SlCOL5, SlSOC1-1, and SlSOC1-2 (CO and SOC1 homologues) were reduced (Fig. 6a). Furthermore, SFT/SP3D (FT homologue) expression was significantly decreased (Fig. 6a), whereas SP or SP5G (TFL1 homologues) expression was not changed (Supplementary Fig. S5a). However, the expression levels of SlGIs, SlSVPs, SlJ3, and 10 FALSIFLORA (FA) (GI, SVP, J3, and LFY homologues) remained unaltered in these plants (Supplementary Fig. S5a). These data indicate that CaRBP regulates flowering via the repression of SlCOLs/TCOLs acting as an important factor within the photoperiod pathway with altered expression of SFT/SP3D and SlSOC1s. Because CO acts upstream of FT/TFL1 and SOC1 (Lee et al. 2000; Onouchi et 15 al. 2000; Yoo et al. 2005), and because CDF1 (Imaizumi et al. 2005), FKF1 (Nelson et al. 2000), and FBH (Ito et al. 2012) are known regulators of CO in photoperiodic flowering in Arabidopsis, we examined their expression in 35S::CaRBP plants. RT– qPCR analysis showed that SlCDF3 (a CDF1 homologue as a negative regulator of CO) expression was increased by 3-fold (Fig. 6b), whereas SlCDF2 and SlCDF5 20 expression was unchanged (Supplementary Fig. S5b). Furthermore, we found that the expression levels of SlFBH1, SlFBH3-1, SlFBH6, SlFBH7-2, SlFKF1-1, and SlFKF12 (FBH and FKF1 homologues) as positive regulators of CO were significantly reduced (Fig. 6b). However, some SlFBHs (SlFBH3-2, SlFBH4, SlFBH5, and SlFBH7-1) expression remained unaltered (Supplementary Fig. S5b). 25 Taken together, these data suggest that CaRBP may regulate transcription levels of SlCOLs/TCOLs via control of SlCDF3, SlFBHs, and SlFKF1s expression for modulation of flowering in tomato. 14 Discussion Post-transcriptional regulation by RBPs plays an important role in various developmental processes in plants. Although the roles of a few plant RBPs have been 5 identified, little is known about specific functions of many RBPs. The results of our study provide evidence that CaRBP homologous to human NIFK, encoding an RBP, modulate flowering time in tomato plants by controlling the expression levels of SFT/SP3D and SlSOC1s, and CaRBP negatively regulates SlCOLs/TCOLs via changes in the expression of SlCDF3, SlFBHs, and SlFKF1s. 10 CaRBP affects a variety of developmental processes RBPs are very important regulators in a variety of cellular processes including splicing, nuclear export, polyadenylation, RNA stability, decay, and translation 15 (Ambrosone et al. 2012). The CaRBP gene product corresponds to a predicted RBP with the RRM present in human NIFK protein and yeast Nop15p protein. Human NIFK protein as a nucleolar protein interacts with the FHA domain of Ki-67 protein in a mitosis-specific and phosphorylation-dependent manner, suggesting that this protein functions in cell cycle progression (Takagi et al. 2001). Yeast Nop15p 20 nucleolar protein involved in pre-rRNA processing is essential for cellular control of cytokinesis (Oeffinger and Tollervey 2003; Colau et al. 2004; Dez and Tollervey 2004). Given the homology between the RRMs of NIFK, Nop15p, and CaRBP, it is tempting to speculate that CaRBP also regulates developmental processes of vegetative organs via cell cycle or cell division control. BLAST results of a search 25 with the deduced amino acid sequence of CaRBP show that Arabidopsis contains an RBP encoded by the At5g04600 gene, which could also have a role in cell division (Fig. 1). Since T-DNA insertional alleles of this gene are currently available, phenotype analyses of these T-DNA mutant alleles in Arabidopsis will help determine the function of plant RBPs with RRMs shared by human NIFK and yeast Nop15p. 30 Possible role of CaRBP in the regulation of flowering time in day-neutral tomato CO/FT regulatory module controls flowering time in response to variations in day length in annual plants such as in Arabidopsis and rice (Song et al. 2015). In addition, 15 this module regulates seasonal growth cessation as well as timing of flowering in aspen trees (Bohlenius et al. 2006). However, Pharbitis (Pharbitis nil) as a short-day (SD) plant and tomato as a day-neutral plant in the same order (Solanales) show no relationship between the COL gene family and flowering (Ben-Naim et al. 2006; 5 Hayama et al. 2007), although FT-like genes such as PnFT and SFT/SP3D play a similar role in promoting flowering (Pnueli et al. 1998; Carmel-Goren et al. 2003). These data suggest that other COL genes are responsible for the regulation of FT-like genes in these plants. Give the previous result that the overexpression of TCOL1/SlCOL1 and TCOL3/SlCOL3 had no obvious effect on flowering time (Ben- 10 Naim et al. 2006), we propose that CaRBP may regulate SFT/SP3D and SlSOC1s via the control of some SlCOLs/TCOLs such as SlCOL4 and SlCOL5 under LD conditions in tomato plants. Several lines of evidence from this study support our hypothesis. Firstly, CaRBP was highly expressed in the leaves compared with other pepper genes encoding RBP (Fig. 2) (Kim et al. 2014). Secondly, overexpression of 15 CaRBP results in severely altered flowering (Fig. 4; Fig. 5). Thirdly, the expression levels of SFT/SP3D and SlSOC1s as FT and SOC1 homologues, downstream targets of CO in Arabidopsis (Yoo et al. 2005), are significantly reduced in 35S::CaRBP plants (Fig. 6). Lastly, the expression of SlCOL4 and SlCOL5 is significantly decreased in 35S::CaRBP plants (Fig. 6). However, we cannot dismiss the possibility 20 that SlCOLs/TCOLs mediate other photoperiodic or day-specific processes because TCOL1/SlCOL1 and TCOL3/SlCOL3 are under circadian control (Ben-Naim et al. 2006). Further elucidation of whether other SlCOLs/TCOLs regulate flowering time in tomato plants will provide a better understanding of the modulation of reproductive development in day-neutral plants. 25 Considering that CaRBP encodes RRM-containing RBPs such as FCA and FPA known as epigenetic regulators of FLC (Fig. 1) (Liu et al. 2007; Baurle and Dean 2008), it is likely that CaRBP negatively regulates the chromatin status of SlFBHs and that low expression of SlFBHs eventually results in reduced expression of several SlCOLs/TCOLs via a decrease of direct SlFBHs-binding to the promoter regions of 30 SlCOLs/TCOLs. Because CDFs negatively regulate flowering time by directly repressing floral activator genes like FT (Song et al. 2012), it seems that the level of unknown protein as upstream regulator(s) of SlCDF3 may be reduced by epigenetic regulation of its chromatin status by CaRBP, which would increase SlCDF3 RNA expression, eventually inducing an increase of direct SlCDF3’s binding to genomic 16 regions of SlCOLs/TCOLs, SFT/SP3D, or SlSOC1s, and thereby leading to reductions of their RNA expression. These mechanisms could explain why 35S::CaRBP plants show a delayed flowering phenotype. However, we cannot dismiss the possibility that CaRBP recruits other components to regulate the chromatin status of SlFBHs because 5 RRMs are involved in protein-protein interactions (Maris et al. 2005). Further investigation of whether CaRBP directly or indirectly binds to the genomic regions of SlFBHs to regulate their expression would provide a better understanding of photoperiodic flowering of day-neutral tomato plants. However, given that GI-FKF1 complexes regulate CDF and CO protein levels (Imaizumi et al. 2005; Sawa et al. 10 2007) and we did not investigate the expression levels of target proteins in this study, we cannot explain why the reduced RNA levels of SlFKF1 affect flowering time of 35S::CaRBP plants. Also, because tomato genome has a gene (SlRBP) homologous to CaRBP (Fig. 1), the study on knock-down or overexpression of SlRBP in tomato plants would be informative for the function of plant RBP homologous to human 15 NIFK. In summary, we have shown that an RBP encoded by CaRBP homologous to human NIFK controls SFT/SP3D and SlSOC1s expression via the reduced expression of some SlCOLs/TCOLs to regulate flowering time in day-neutral tomato plants. However, it is known that the dwarf and determinate phenotypes of Micro-tom 20 cultivar as a variety to carry out molecular work in tomato, are related with several mutations in brassinosteroid (BR) and gibberellic acid (GA) (Marti et al. 2006), and a mutation in SP gene (Pnueli et al. 1998), respectively. Thus, further investigation is required to observe whether the late flowering phenotype shown in this study is affected by genetic background. Furthermore, given that SlCDF genes regulate abiotic 25 stress responses as well as flowering time in Arabidopsis (Corrales et al. 2014), it will be challenging to determine whether the genetic manipulation of CaRBP results in various abiotic tolerances in day-neutral solanaceous crops. 17 Author contribution statement YHJ and SL conceived and designed research. HMK, A-YL, SHP and SHM conducted experiments. HMK, JHL, SL and YHJ analyzed data. JHL, SL and YHJ 5 wrote the manuscript. All authors read and approved the manuscript. 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Asterisks and 5 double dots indicate identical and similar amino acid residues, respectively. c Phylogenetic relationships of RNA-binding proteins based on analyses of the deduced full-length amino acid sequences using the neighbor-joining (NJ) method. The scale bar indicates a divergence of 0.1 amino acid substitutions per site. The GenBank accession numbers of RNA binding proteins used in this study are as follows: SlRBP 10 (XM_004241249), NsRBP (XM_009760551), GmRBP (XM_003533869), MtRBP (XM_003592494), CusRBP (XM_004140846), CsRBP (XM_006492122), AtRBP (NM_120542), CasRBP (XM_010454060), Nop150 (O74978), DmRBP (NM_142809), hNIFK (Q9BYG3), FCA (AY854371), MtFCA (XM_003609755), OsFCA (AY274928), StFCA (XM_006354249), ZmFCA (NM_001175827), FPA 15 (NM_001084579), MtFPA (XM_003606774), OsFPA (NM_001070206), SlFPA (XM_004240936), and ZmFPA (XM_008654723). Fig. 2 Expression patterns of CaRBP in hot pepper plants. The lanes indicate leaves (L), stems (S), roots (R), flowers (FL), ovaries (OV), anthers (AN), immature fruit 20 stage [IF, 15 days after pollination (DAP)], mature green stage (MG, 28DAP), breaking green stage (BG, 30DAP), red-turning brown stage (RB, 42DAP), and red ripe stage (RR, 45DAP). Fruit developmental stages of hot pepper plants were previously described (Lee et al. 2010; Lee et al. 2014). The expression levels of CaRBP in leaves were set to one. The actin gene was employed as an internal control. 25 Error bars denote standard deviation of three technical replicates. Fig. 3 Subcellular localization of CaRBP in tobacco leaves. Tobacco (Nicotiana benthamiana) leaves were infiltrated with 35S::CaRBP:EGFP (I and II) and 35S::EGFP control (III and IV), and EGFP fluorescence was examined. II and IV 30 panels are the magnified images of I and III panels, respectively. 4', 6-Diamidino-2phenylindole (DAPI) was used for nuclear staining. Scale bars 10 µm. Fig. 4 Morphological phenotypes of 35S::CaRBP tomato plants. a to c Phenotypes of wild-type (a) and 35S::CaRBP tomato plants from TG-2 line (b and c) grown for 50 26 days under long-day (LD) conditions. The plants shown in b and c are Classes I and II, respectively. Scale bars 2 cm. d to f Phenotypes of wild-type (d) and 35S::CaRBP tomato plants from TG-2 line (e and f) grown for 90 days under LD conditions. The plants shown in e and f are Classes I and II, respectively. Class I plants were not 5 flowered. Scale bars 2 cm. g Stem and root phenotypes of 35S::CaRBP tomato plants from TG-2 line grown for 30 days under LD conditions. Scale bars 2 cm. h Leaflet phenotypes of the three leaves of 35S::CaRBP tomato plants from TG-2 line grown for 30 days under LD conditions. Scale bars 2 cm. 10 Fig. 5 Comparison of flowering time and size of vegetative organs between wild-type and 35S::CaRBP tomato plants. a Inflorescence number of wild-type and transgenic plants (TG-1 and TG-2 lines, n=5). Error bars denote standard deviation of individual plants. b Length of height from 90-d-old wild-type and transgenic plants (TG-1 and TG-2 lines, n=5). c Length of stem from 30-d-old wild-type and transgenic plants 15 (TG-2 line, n=10). d Length of root from 30-d-old wild-type and transgenic plants (TG-2 line, n=10). e Length of the terminal leaflet of three leaves from 30-d-old wildtype and transgenic plants (TG-2 line, n=10). Fig. 6 Expression levels of flowering time genes in 35S::CaRBP tomato plants. a 20 Expression levels of known floral integrator genes [CO, SOC1, and FT/TFL1 homologues (SlCOLs/TCOLs, SlSOC1s, and SFT/SP3D)] in the transgenic homozygous plants from TG-2 line grown for 90 days under LD conditions. The expression level of each gene in wild-type plants was set to one. The actin gene was employed as an internal control. Error bars denote standard deviation of three 25 technical replicates. b Expression levels of known CO regulator genes [CDF, FBH, and FKF1 homologues (SlCDF3, SlFBHs, and SlFKF1s)] in the transgenic homozygous plants from TG-2 line grown for 90 days under LD conditions. 30 27