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Article

Characterization of the FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 Homolog SlFKF1 in Tomato as a Model for Plants with Fleshy Fruit

by
Tomoki Shibuya
1,
Manabu Nishiyama
2,
Kazuhisa Kato
2,* and
Yoshinori Kanayama
2,*
1
Faculty of Life and Environmental Science, Shimane University, Matsue 690-8504, Japan
2
Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 980-8572, Japan
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(4), 1735; https://doi.org/10.3390/ijms22041735
Submission received: 11 December 2020 / Revised: 25 January 2021 / Accepted: 4 February 2021 / Published: 9 February 2021
(This article belongs to the Special Issue Cell Signaling in Model Plants 2.0)
Browse Figures
Versions Notes

Abstract

:
FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) is a blue-light receptor whose function is related to flowering promotion under long-day conditions in Arabidopsis thaliana. However, information about the physiological role of FKF1 in day-neutral plants and even the physiological role other than photoperiodic flowering is lacking. Thus, the FKF1 homolog SlFKF1 was investigated in tomato, a day-neutral plant and a useful model for plants with fleshy fruit. It was confirmed that SlFKF1 belongs to the FKF1 group by phylogenetic tree analysis. The high sequence identity with A. thaliana FKF1, the conserved amino acids essential for function, and the similarity in the diurnal change in expression suggested that SlFKF1 may have similar functions to A. thaliana FKF1. CONSTANS (CO) is a transcription factor regulated by FKF1 and is responsible for the transcription of genes downstream of CO. cis-Regulatory elements targeted by CO were found in the promoter region of SINGLE FLOWER TRUSS (SFT) and RIN, which are involved in the regulation of flowering and fruit ripening, respectively. The blue-light effects on SlFKF1 expression, flowering, and fruit lycopene concentration have been observed in this study and previous studies. It was confirmed in RNA interference lines that the low expression of SlFKF1 is associated with late flowering with increased leaflets and low lycopene concentrations. This study sheds light on the various physiological roles of FKF1 in plants.

1. Introduction

Cryptochrome (CRY) and phototropin are blue-light receptors in plants [1]. Studies using Arabidopsis thaliana have shown that these photoreceptors control plant responses, including de-etiolation, hypocotyl elongation, and photoperiodic flowering by CRY [2,3,4] and phototropism, stomatal opening, and chloroplast localization by phototropin [5,6,7,8], indicating that their functions are diverse. Even in long-day plants other than A. thaliana, flowering promotion by blue light, which is supposed to involve CRY, has been reported in Petunia and Eustoma [9,10].
There are other blue-light receptors, such as FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1), and ZEITLUPE (ZTL), whose function is reportedly related to flowering promotion under long-day conditions in A. thaliana. FKF1 and ZTL proteins control the function of CONSTANS (CO) antagonistically [11], and here, we focus on FKF1. FKF1 interacts with GIGANTEA (GI) in a blue-light-dependent manner in A. thaliana and induces the degradation of CYCLING DOF FACTOR 1 (CDF1), which suppresses CO transcription, and its family proteins [12,13,14,15]. FKF1 also stabilizes the CO protein by suppressing its degradation by CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) and SUPPRESSOR OF PHYA-105 (SPA) [16,17,18,19]. Furthermore, FKF1 promotes FLOWERING LOCUS T (FT) transcription by inducing the degradation of CDF1, which is the transcriptional repressor of FT [16]. In this way, FKF1 plays an essential role in flowering promotion by blue light. As FKF1 in long-day plants, it is suggested that the Gypsophila FKF1 homolog is involved in flowering promotion, besides A. thaliana [20,21]. Reportedly, OsFKF1 promotes flowering in rice, a short-day plant, regardless of the photoperiod condition [22]. The FKF1 homolog is also considered to play an important role in the developmental phase transition of liverwort [23]. In addition, FKF1 and ZTL regulate the clock period by ubiquitination [24].
Although FKF1 has been reported as a negative regulator of cellulose synthesis [25], there are still few reports on its physiological roles other than flowering promotion. FKF1 in A. thaliana is expressed in the vascular bundle sheath of leaves in relation to flowering control. However, its expression is also found in other tissues, including cotyledons, leaves, guard cells, and root tips, and their physiological importance remains unknown [26,27]. Additionally, FKF1-like sequences are conserved on the genomes of many plant species regardless of photoperiod responsiveness; short-day plants rice and soybean have phylogenetically FKF1-orthologous genes [22,28]. A few reports have suggested the possible functions of FKF1, such as stem and root growth and potassium response [29,30,31]. From these findings, it is expected that FKF1 plays various roles in plants.
Tomato is generally considered as one of the day-neutral plants, whose FKF1 has not been investigated well, and can also be used as a useful model for plants with fleshy fruit, which has accumulated data for bioinformatics. Hence, in this study, the sequence and physiological roles of the tomato FKF1 homolog SlFKF1 were analyzed.

2. Results

2.1. SlFKF cDNA Sequence

Using a BLAST search for a sequence orthologous to the amino acid sequence of A. thaliana FKF1, only one FKF1-like gene (XM_004228691.3) was found to be present in the tomato genome. This gene was labeled SlFKF1. The cDNA of SlFKF1 was prepared from cv. Micro-Tom by reverse transcription–polymerase chain reaction (RT-PCR) and sequenced. Consequently, its sequence was the same as that on the database. On the alignment based on the amino acid sequence, SlFKF1 was 75.1% identical to A. thaliana FKF1, and the amino acids essential for the function of FKF1 were also conserved in SlFKF1 [15,16,32] (Figure 1A). As a result of a phylogenetic tree analysis based on alignment, including ZTL groups having the same domain structure as FKF1 but different functions, it was deduced that SlFKF1 belongs to the FKF1 group (Figure 1B).

2.2. Expression Analysis of SlFKF1

SlFKF1 expression in the wild-type cv. Micro-Tom tomato showed clear diurnal change under a 16 h day length (Figure 2A). The expression was very low from the dark period to the first 6 h of the light period and then increased. The expression peak of SlFKF1 was shown at Zeitgeber time (ZT) 9. This expression pattern was similar to that of A. thaliana FKF1 [13]. SlFKF1 expression was observed in all organs tested in Figure 2B and was higher in mature leaves than in other organs. Furthermore, the effect of light quality on SlFKF1 expression was investigated in leaves and fruit (Figure 2C). High expression levels were found in the fruit, similar to those in the leaves. The expression levels were lower under blue light in both organs.

2.3. Effect of Light Quality on Lycopene Concentration

The effect of blue light on lycopene concentration, a major pigment in tomato fruit and a functional component [33], was investigated. Consequently, the lycopene concentration was lower under blue light than under white light, although it was not significantly different between white light and red light (Figure 3).

2.4. Analysis of SlFKF1 Promoter Sequences

Because FKF1 regulates downstream genes via the transcription factor CO [1,16], the possibility of regulating important factors related to flowering, ripening, and pigment synthesis was investigated. Putative CO-binding motifs were searched in the promoter regions of FT, RIN, and PSY tomato homologs, which are key factors for flowering promotion, fruit ripening control, and lycopene synthesis, respectively (Table 1). These genes were analyzed because SINGLE FLOWER TRUSS (SFT) is the tomato homolog of FT [34], and RIN and PSY are well-known important traits of tomato fruit development as factors governing the ripening process and carotenoid accumulation, respectively [35]. The motifs were found in SFT, PSY1, PSY2, and RIN promoters, whereas they were not found in the promoter of PSY3 in the tomato genome.

2.5. Transformation Experiments

SlFKF1 RNA interference (RNAi)-suppressed tomato plans were prepared to confirm the promoter analysis results above. The RNAi lines were differentiated from independent transformation events and produced normal seeds for phenotypic observations. The expression of each transgenic line at the expression peak of SlFKF1 (ZT9) was investigated, and it was confirmed that expression-suppressed lines with RNAi have lower expression than the wild type (Figure 4A). The flowering in RNAi lines was investigated, and the number of days and leaves until flowering increased in RNAi lines compared to the wild type, suggesting that flowering was delayed by the suppression of SlFKF1 expression (Figure 4D,F). Another interesting phenotype of RNAi lines was the increased number of leaflets (Figure 4E). The main stem length increased (Figure 4B), and this increase was not accompanied by an increase in internode length but the number of leaves (Figure 4C,F). In fact, because of the long main shoot length and a large number of leaves and leaflets, the RNAi line appeared to have a large plant volume (Figure S1). Regarding fruit coloration, the number of days from flowering to the breaker stage increased, and the degree of coloring was lower in RNAi lines than in the wild type 10 days after the beaker stage (Figure 5A,B).

3. Discussion

FKF1 has three domains: LOV, F-box, and KELCH repeat. In A. thaliana, it forms a gene family with ZTL [20]. ZTL has been reported to have a different function from FKF1 and is involved in the decomposition of TIMING OF CAB EXPRESSION 1 (TOC1), one of the components of the circadian clock [36,37,38]. The phylogenetic tree analysis results, including FKF1 and ZTL homologs, confirmed that SlFKF1 belongs to the FKF1 group. The high sequence identity with A. thaliana FKF1, the conserved amino acids essential for function, and the similarity in the diurnal change in expression suggested that SlFKF1 may have similar functions to A. thaliana FKF1. SlFKF1 was expressed not only in leaves, which are the photoreceptive organs for photoperiodic flowering control, but also in various organs, and its expression level was also high in fruit. Because the flowering of tomato is not affected by the photoperiod and the role of FKF1 in fruit has not been reported so far, in tomato, the physiological role of FKF1 other than in photoperiodic flowering should be examined.
CO is a transcription factor regulated by FKF1 and is responsible for the transcription of genes downstream of CO [1,16]. A CO-responsive element targeted by CO was reported by Tiwari et al. [39], and its CCACA core motif was identified by Gnesutta et al. [40]. SFT is the tomato homolog of FT involved in flowering promotion as a florigen [34], and RIN and PSY play important roles in governing the ripening process and carotenoid accumulation [35]. Therefore, it is possible that the FKF1-CO-mediated pathway regulates the expression of these genes and affects these developmental processes in tomato.
The heterozygotes of wild-type and mutant alleles of SFT in a determinant cultivar showed an approximately twofold increase in yield [41]. Thus, the physiological and agricultural importance of the FT-related flowering pathway in day-neutral plants is something of interest. A CO cis-element was found in the promoter region of SFT. Late flowering and increased leaflets were commonly observed in the sft mutant [42,43] and SlFKF1 RNAi lines, suggesting the relationship between SlFKF1 and SFT. In A. thaliana, FKF1 is considered a blue-light receptor responsible for promoting flowering under long-day conditions, whereas in rice, a short-day plant, the FKF1 homolog promotes flowering regardless of day length [22]. The results suggested that an FKF1 homolog may also function in the flowering pathway of day-neutral plants. A. thaliana FKF1 promotes flowering by suppressing the function of the COP1/SPA system that degrades the CO protein [17,18,19], and tomato likely has a similar mechanism. In contrast, it has been proposed in A. thaliana that FKF1 positively regulates the gibberellin (GA) signal through the degradation of the DELLA protein and activates the GA-dependent flowering promotion pathway [44]. However, in tomato, because GA rather suppresses flowering [45], it would not be possible to apply this A. thaliana model to tomato.
Because SlFKF1 expression was suppressed by blue light, the effect of blue light on lycopene concentration was investigated for comparison. In this study, the effect of blue-light irradiation on lycopene concentration in fruit was measured during cultivation. Although the effects of blue-light irradiation on lycopene concentration have been investigated in previous reports [46,47], their experimental conditions were different from this study. The effects of supplemental blue light over a long period after flowering in a greenhouse [46] and blue-light irradiation after harvest [47] have been investigated in previous reports. In addition, the effects of blue light on lycopene concentration are different between these reports. In this study, to limit the effect on vegetative growth, after cultivation under white light, the effect was investigated by irradiating blue light from the breaker only for 10 days. As a result, this study and a previous report [47] showed low fruit lycopene concentrations under blue light. Collectively, it is likely that blue light alone for a relatively short period negatively affects lycopene accumulation in tomato fruit.
The fruit lycopene concentration was low under blue light, as described above, and there are cis-regulatory elements targeted by CO in the promoter regions of RIN and PSY. Additionally, fruit SlFKF1 expression was low under blue light. Because there are reports that light quality regulates growth and environmental response through the regulation of photoreceptor gene expression [48,49], low SlFKF1 expression might be related to low lycopene concentration under blue light as one possible mechanism. It was confirmed in RNAi lines that low expression of SlFKF1 is associated with low concentrations of lycopene. So far, the effect of blue light on tomato fruit color has assumed the involvement of CRY [50,51,52], and there have been few reports on FKF1. A similar control mechanism is possible for flowering because blue light delays flowering in tomato [53,54], and leaf SlFKF1 expression is also low under blue light. It is interesting to note that blue light promotes flowering in A. thaliana and some other long-day plants in contrast to tomato [10,21,55].
A previous report suggested a relationship between auxin and FKF1 in the adventitious rooting of longan [30]. Auxin plays an important role in the growth and ripening of tomato fruit [56]. Another report suggested that potassium levels, which are important in fruit growth and quality, affect FKF1 expression in banana roots [31]. Therefore, the function of SlFKF1 may be related to auxin signaling and potassium nutrition in fruit. Because other photoreceptors such as CRY and PHY are considered important agronomic traits [57], the assumed role of FKF1 in fruit crops is promising based on this study.
FKF1 is present in the genomes of a wide variety of species, whereas so far, knowledge about its role has been limited. Its role in flowering in day-neutral plants and even the physiological role other than flowering is discussed in this study using tomato as a model plant, while preliminary transcriptome analysis has shown that blue light induces the expression of several transcriptional regulation- and signal transduction-related genes during tomato fruit ripening [58]. Starting with this study, the varying physiological roles of FKF1, which have been comparatively unknown compared to phytochrome and CRY, will be elucidated.

4. Materials and Methods

4.1. Plant Materials

Tomato cv. Micro-Tom wild type was cultivated for cloning and expression analysis in a growth chamber (LH240SP; Nihon Ika Co., Ltd., Osaka, Japan) at 25 °C with a white fluorescent lamp in a 16-h photoperiod. Photosynthetic photon flux density (PPFD) was 100 μmol m−2 s−1, and Sumisoil N150 (Sumika Agro-tech Co., Ltd., Osaka, Japan) was used as cultivation soil. The plants were supplemented with nutrient solution (Hyponex Japan) every week. The cultivation was conducted with reference to Tsunoda et al. [59].

4.2. Sequence Analysis of Tomato FKF1 Homolog SlFKF1 cDNA

A BLAST search was performed to search for a candidate protein of SlFKF1 and an open reading frame (ORF), encoding it based on the amino acid sequence of FKF1 (AT1G68050) of A. thaliana FKF1 in Tomato Genome CDS (ITAG release 2.40) of the Sol Genomics Network (SGN; https://solgenomics.net accessed on 8 February 2021) and NW_004194292.1 and XM_004228691.1 of the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov accessed on 8 February 2021). Using a primer set designed based on the candidate sequence obtained from the database, its ORF sequence was cloned by PCR with the cDNA obtained by reverse transcription of RNA extracted from cv. Micro-Tom tomato leaves and confirmed by sequencing.

4.3. Expression Analysis

For a diurnal change in expression, mature leaves that were fully developed and not senesced were randomly collected every 3 h from the start of the light period (ZT0) to ZT21 at approximately 30 days after germination. For the expression analysis in stems, flowers, immature leaves, and mature leaves, samples were collected on ZT8 approximately 50 days after germination. For the expression analysis in leaves under blue and red light, the plants at 2 months after sowing were cultivated for 1 day under a 16 h photoperiod with red or blue light emitting diodes (LEDs) (CCS Inc., Kyoto, Japan), and leaves were sampled for RNA extraction. For the expression analysis in fruit under blue and red light, the plants at the fruit breaker stage were cultivated for 10 days under a 16 h photoperiod with red or blue LEDs, and the pericarp was sampled for RNA extraction. PPFD was 100 μmol m−2 s−1, and the samples were collected on ZT10. Red and blue LEDs had peaks at 655 and 470 nm, respectively. RNA was prepared from these samples using the RNeasy Plant Mini Kit (Qiagen) for real-time PCR. The removal of genomic DNA and reverse transcription were performed using the Quantiscript Reverse Transcription Kit (Qiagen) and the ReverTra Ace qPCR RT Master Mix (Toyobo). Real-time PCR was performed using the QuantiTect SYBR Green PCR Kit (Qiagen) and the THUNDERBIRD SYBR qPCR Mix (Toyobo) according to Ikeda et al. [60]. SlACT and SlUBQ were used as reference genes [61,62], and Table S1 shows the nucleotide sequences for the primers.

4.4. Alignment, Phylogenetic Analysis, and Promoter Analysis

Using the amino acid sequences of AtFKF1 and AtZTL as queries, a BLAST search was performed for non-redundant protein sequences (nr) of each plant species in the NCBI to search for homologs. For tomato FKF1, a BLAST search was also performed on tomato genome protein sequences (ITAG release 2.40) on the SGN to obtain locus information about matching proteins, and the gene model was confirmed by referring to genomic detail. A phylogenetic tree was created using the neighbor-joining method for the alignment obtained by executing Clustal W2 on Genetyx version 10 for FKF1 and ZTL amino acid sequences. Bootstrap probabilities were calculated by 1000 trials.
For promoter analysis, each gene was searched by a keyword and BLAST on the SGN, and locus names were confirmed. Next, the locus was searched, and an upstream 3000-base sequence was obtained from the genomic sequence of genomic detail using the function of Get flanking sequences on SL2.50ch07. Additionally, the position of the start codon was confirmed from the cDNA sequence and protein sequence of the genomic detail, and the start codon upstream 1500-base sequence was determined. The CO-binding sequence CCACA was searched from the obtained 1500-base sequence by the Text Search function on Genetyx version 14.

4.5. Determination of Lycopene Concentration

The plants at the fruit breaker stage were cultivated for 10 days under a 16 h photoperiod with red LEDs, blue LEDs, or a white fluorescent lamp, and the pericarp was sampled for the determination of lycopene concentration. PPFD was 100 μmol m−2 s−1, and the samples were collected on ZT10. Red and blue LEDs (CCS Inc.) had peaks at 655 and 470 nm, respectively. According to Ito and Horie [63], lycopene was extracted using dimethyl ether/methanol (7:3), filtrated with DSMIC JP 13 (Advantec), and quantified by measuring the absorbance at 505 nm.

4.6. Transformation Experiment

RNAi was used for expression suppression. A partial fragment (341 bp) of SlFKF1 was amplified by RT-PCR using the cDNA of cv. Micro-Tom as a template and introduced into pBI-RNAi-GW (Inplanta Innovations, Inc., Yokohama, Japan), a vector for preparing an RNAi construct with the CaMV 35S promoter. Table S1 shows the nucleotide sequences for the primers used to prepare these vectors. The transformation was outsourced to Inplanta Innovations. The cultivation of transformed tomato plants and expression analysis were performed as described above.

Supplementary Materials

Can be found at https://www.mdpi.com/1422-0067/22/4/1735/s1. Table S1: List of primer sequences, Table S2: Accession numbers of proteins used for the phylogenetic tree analysis, Figure S1: Wild-type (WT) and SlFKF1 RNAi line (RI6) plants with ripe fruit.

Author Contributions

Conceptualization, K.K. and Y.K.; methodology, Y.K. and T.S.; investigation, T.S.; formal analysis, M.N. and T.S.; resources, T.S.; data curation, K.K.; writing—original draft preparation, T.S.; writing—review and editing, M.N. and Y.K.; visualization, T.S.; supervision, M.N. and Y.K.; project administration, Y.K.; funding acquisition, K.K. and Y.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by Grants-in-Aid for Scientific Research (16H02534, 19K06012).

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Shibuya, T.; Kanayama, Y. Flowering response to blue light and its molecular mechanisms in Arabidopsis and horticultural plants. Adv. Hort. Sci. 2014, 28, 179–183. [Google Scholar]
  2. Ahmad, M.; Cashmore, A.R. HY4 gene of A. thaliana encodes a protein with characteristics of a blue-light photoreceptor. Nature 1993, 366, 162–166. [Google Scholar] [CrossRef] [PubMed]
  3. Guo, H.; Yang, H.; Mockler, T.C.; Lin, C. Regulation of flowering time by Arabidopsis photoreceptors. Science 1998, 279, 1360–1363. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. El-Assal, S.; Alonso-Blanco, C.; Peeters, A.; Raz, V.; Koornneef, M. A QTL for flowering time in Arabidopsis reveals a novel allele of CRY2. Nat. Genet. 2001, 29, 435–440. [Google Scholar] [CrossRef]
  5. Kagawa, T.; Wada, M. Blue light-induced chloroplast relocation in Arabidopsis thaliana as analyzed by microbeam irradiation. Plant Cell Physiol. 2000, 41, 84–93. [Google Scholar] [CrossRef] [Green Version]
  6. Sakai, T.; Kagawa, T.; Kasahara, M.; Swartz, T.E.; Christie, J.M.; Briggs, W.R.; Wada, M. Arabidopsis nph1 and npl1: Blue light receptors that mediate both phototropism and chloroplast relocation. Proc. Natl. Acad. Sci. USA 2001, 98, 6969–6974. [Google Scholar] [CrossRef] [Green Version]
  7. Kinoshita, T.; Doi, M.; Suetsugu, N.; Kagawa, T.; Wada, M.; Shimazaki, K. Phot1 and phot2 mediate blue light regulation of stomatal opening. Nature 2001, 414, 656–660. [Google Scholar] [CrossRef] [PubMed]
  8. Wada, M.; Kagawa, T.; Sato, Y. Chloroplast movement. Annu. Rev. Plant Biol. 2003, 54, 455–468. [Google Scholar] [CrossRef] [PubMed]
  9. Fukuda, N.; Ishii, Y.; Ezura, H.; Olsen, J.E. Effects of light quality under red and blue light emitting diodes on growth and expression of FBP28 in petunia. Acta Hort. 2011, 907, 361–366. [Google Scholar] [CrossRef]
  10. Shibuya, T.; Takahashi, T.; Hashimoto, S.; Nishiyama, M.; Kanayama, Y. Effects of overnight radiation with monochromatic far-red and blue light on flower budding and expression of flowering-related and light quality-responsive genes in Eustoma grandiflorum. J. Agric. Meteorol. 2019, 75, 160–165. [Google Scholar] [CrossRef] [Green Version]
  11. Hwang, D.Y.; Park, S.; Lee, S.; Lee, S.S.; Imaizumi, T.; Song, Y.H. GIGANTEA regulates the timing stabilization of CONSTANS by altering the interaction between FKF1 and ZEITLUPE. Mol. Cells 2019, 42, 693–701. [Google Scholar]
  12. Fornara, F.; Panigrahi, K.C.S.; Gissot, L.; Sauerbrunn, N.; Rühl, M.; Jarillo, J.A.; Coupland, G. Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are essential for a photoperiodic flowering response. Dev. Cell 2009, 17, 75–86. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Imaizumi, T.; Tran, H.G.; Swartz, T.E.; Briggs, W.R.; Kay, S.A. FKF1 is essential for photoperiodic-specific light signaling in Arabidopsis. Nature 2003, 426, 302–306. [Google Scholar] [CrossRef] [PubMed]
  14. Imaizumi, T.; Schultz, T.F.; Harmon, F.G.; Ho, L.A.; Kay, S.A. FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in Arabidopsis. Science 2005, 309, 293–297. [Google Scholar] [CrossRef]
  15. Sawa, M.; Nusinow, D.A.; Kay, S.A.; Imaizumi, T. FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis. Science 2007, 318, 261–265. [Google Scholar] [CrossRef] [Green Version]
  16. Song, Y.H.; Smith, R.W.; To, B.J.; Millar, A.J.; Imaizumi, T. FKF1 conveys timing information for CONSTANS stabilization in photoperiodic flowering. Science 2012, 336, 1045–1049. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  17. Ponnu, J. Molecular mechanisms suppressing COP1/SPA E3 ubiquitin ligase activity in blue light. Physiol. Plant. 2020, 169, 418–429. [Google Scholar] [CrossRef] [PubMed]
  18. Lee, B.-D.; Cha, J.-Y.; Kim, M.R.; Shin, G.-I.; Paek, N.-C.; Kim, W.-Y. Light-dependent suppression of COP1 multimeric complex formation is determined by the blue-light receptor FKF1 in Arabidopsis. Biochem. Biophys. Res. Commun. 2019, 508, 191–197. [Google Scholar] [CrossRef] [PubMed]
  19. Lee, B.-D.; Kim, M.R.; Kang, M.-Y.; Cha, J.-Y.; Han, S.-H.; Nawkar, G.M.; Sakuraba, Y.; Lee, S.Y.; Imaizumi, T.; McClung, C.R.; et al. The F-box protein FKF1 inhibits dimerization of COP1 in the control of photoperiodic flowering. Nat. Commun. 2017, 8, 2259. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  20. Shibuya, T.; Murakawa, Y.; Nishidate, K.; Nishiyama, M.; Kanayama, Y. Characterization of flowering-related genes and flowering response in relation to blue light in Gypsophila paniculata. Hort. J. 2017, 86, 94–104. [Google Scholar] [CrossRef] [Green Version]
  21. Hori, Y.; Nishidate, K.; Nishiyama, M.; Kanahama, K.; Kanayama, Y. Flowering and expression of flowering-related genes under long-day conditions with light-emitting diodes. Planta 2011, 234, 321–330. [Google Scholar] [CrossRef]
  22. Han, S.-H.; Yoo, S.-C.; Lee, B.-D.; An, G.; Paek, N.-C. Rice FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (OsFKF1) promotes flowering independent of photoperiod. Plant Cell Environ. 2015, 38, 2527–2540. [Google Scholar] [CrossRef] [PubMed]
  23. Kubota, A.; Kita, S.; Ishizaki, K.; Nishihama, R.; Yamato, K.T.; Kohchi, T. Co-option of a photoperiodic growth-phase transition system during land plant evolution. Nat. Commun. 2014, 5, 3668. [Google Scholar] [CrossRef] [PubMed]
  24. Lee, C.-M.; Feke, A.; Li, M.-W.; Adamchek, C.; Webb, K.; Pruneda-Paz, J.; Bennett, E.J.; Kay, S.A.; Gendron, J.M. Decoys untangle complicated redundancy and reveal targets of circadian clock F-BOX proteins. Plant Physiol. 2018, 177, 1170–1186. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Yuan, N.; Balasubramanian, V.K.; Chopra, R.; Mendu, V. The photoperiodic flowering time regulator fkf1 negatively regulates cellulose biosynthesis. Plant Physiol. 2019, 180, 2240–2253. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Nelson, D.C.; Lasswell, J.; Rogg, L.; Cohen, M.A.; Bartel, B. FKF1, a Clock-controlled gene that regulates the transition to flowering in Arabidopsis. Cell 2000, 101, 331–340. [Google Scholar] [CrossRef] [Green Version]
  27. Obulareddy, N.; Panchal, S.; Melotto, M. Guard cell purification and RNA isolation suitable for high-throughput transcriptional analysis of cell-type responses to biotic stresses. Mol. Plant Microbe Interact. 2013, 26, 844–849. [Google Scholar] [CrossRef] [Green Version]
  28. Li, F.; Zhang, X.; Hu, R.; Wu, F.; Ma, J.; Meng, Y.; Fu, Y. Identification and molecular characterization of FKF1 and GI homologous genes in soybean. PLoS ONE 2013, 8, e79036. [Google Scholar] [CrossRef] [Green Version]
  29. Zeng, J.; Mo, Y.; Chen, J.; Li, C.; Zhao, L.; Liu, Y. Expression and interaction proteins analysis of BjuFKF1 in stem mustard. Sci. Hortic. 2020, 269, 109430. [Google Scholar] [CrossRef]
  30. Huang, F.; Fu, Z.; Zeng, L.; Morley-Bunker, M. Isolation and characterization of GI and FKF1 homologous genes in the subtropical fruit tree. Dimocarpus longan. Mol. Breed. 2017, 37, 90. [Google Scholar] [CrossRef]
  31. He, Y.; Li, R.; Lin, F.; Xiong, Y.; Wang, L.; Wang, B.; Guo, J.; Hu, C. Transcriptome changes induced by different potassium levels in banana roots. Plants 2020, 9, 11. [Google Scholar] [CrossRef] [Green Version]
  32. Han, L.; Mason, M.; Risseeuw, E.P.; Crosby, W.L.; Somers, D.E. Formation of an SCFZTL complex is required for proper regulation of circadian timing. Plant J. 2004, 40, 291–301. [Google Scholar] [CrossRef] [PubMed]
  33. Pesaresi, P.; Mizzotti, C.; Colombo, M.; Masiero, S. Genetic regulation and structural changes during tomato fruit development and ripening. Front. Plant Sci. 2014, 5, 124. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Lifschitz, E.; Eviatar, T.; Rozman, A.; Shalit, A.; Goldshmidt, A.; Amsellem, Z.; Alvarez, J.; Eshed, Y. The tomato FT ortholog triggers systemic signals that regulate growth and flowering and substitute for diverse environmental stimuli. Proc. Natl. Acad. Sci. USA 2006, 103, 6398–6403. [Google Scholar] [CrossRef] [Green Version]
  35. Liu, R.; How-Kit, A.; Stammittia, L.; Teyssier, E.; Rolin, D.; Mortain-Bertrand, A.; Halle, S.; Liu, M.; Kong, J.; Wu, C.; et al. A DEMETER-like DNA demethylase governs tomato fruit ripening. Proc. Natl. Acad. Sci. USA 2015, 112, 10804–10809. [Google Scholar] [CrossRef] [Green Version]
  36. Más, P.; Kim, W.-Y.; Somers, D.E.; Kay, S.A. Targeted degradation of TOC1 by ZTL modulates circadian function in Arabidopsis thaliana. Nature 2003, 426, 02163. [Google Scholar] [CrossRef]
  37. Kiba, T.; Henriques, R.; Sakakibara, H.; Chua, N.-H. Targeted degradation of PSEUDO-RESPONSE REGULATOR5 by an SCFZTL complex regulates clock function and photomorphogenesis in Arabidopsis thaliana. Plant Cell 2007, 19, 2516–2530. [Google Scholar] [CrossRef] [Green Version]
  38. Fujiwara, S.; Wang, L.; Han, L.; Suh, S.-S.; Salomé, P.A.; McClung, R.C.; Somers, D.E. Post-translational regulation of the Arabidopsis circadian clock through selective proteolysis and phosphorylation of pseudo-response regulator proteins. J. Biol. Chem. 2008, 283, 23073–23083. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  39. Tiwari, S.B.; Shen, Y.; Chang, H.-C.; Hou, Y.; Harris, A.; Ma, S.F.; McPartland, M.; Hymus, G.J.; Adam, L.; Marion, C.; et al. The flowering time regulator CONSTANS is recruited to the FLOWERING LOCUS T promoter via a unique cis-element. New Phytol. 2010, 187, 57–66. [Google Scholar] [CrossRef]
  40. Gnesutta, N.; Kumimoto, R.W.; Swain, S.; Chiara, M.; Siriwardana, C.S.; Horner, D.; Ben, F.; Holt, B.F.; Mantovania, R. CONSTANS imparts DNA sequence specificity to the histone fold NF-YB/NF-YC dimer. Plant Cell 2017, 29, 1516–1532. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  41. Krieger, U.; Lippman, Z.B.; Zamir, D. The flowering gene SINGLE FLOWER TRUSS drives heterosis for yield in tomato. Nat. Genet. 2010, 42, 459–463. [Google Scholar] [CrossRef]
  42. Shalit, A.; Rozman, A.; Goldshmidt, A.; Alvarez, J.P.; Bowman, J.; Eshed, Y.; Lifschitz, E. The flowering hormone florigen functions as a general systemic regulator of growth and termination. Proc. Natl. Acad. Sci. USA 2009, 106, 8392–8397. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Molinero-Rosales, N.; Latorre, A.; Jamilena, M.; Lozano, R. SINGLE FLOWER TRUSS regulates the transition and maintenance of flowering in tomato. Planta 2004, 218, 427–434. [Google Scholar] [CrossRef] [PubMed]
  44. Yan, J.; Li, X.; Zeng, B.; Zhong, M.; Yang, J.; Yang, P.; Li, X.; He, C.; Lin, J.; Liu, X.; et al. FKF1 F-box protein promotes flowering in part by negatively regulating DELLA protein stability under long-day photoperiod in Arabidopsis. J. Integr. Plant Biol. 2020, 62, 1717–1740. [Google Scholar] [CrossRef]
  45. Silva, G.F.F.; Silva, E.M.; Correa, J.P.O.; Vicente, M.H.; Jiang, N.; Notini, M.M.; Junior, A.C.; De Jesus, F.A.; Castilho, P.; Carrera, E.; et al. Tomato floral induction and flower development are orchestrated by the interplay between gibberellin and two unrelated microRNA-controlled modules. New Phytol. 2019, 221, 1328–1344. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Xie, B.-X.; Wei, J.-J.; Zhang, Y.-T.; Song, S.-W.; Su, W.; Sun, G.-W.; Hao, Y.-W.; Liu, H.-C. Supplemental blue and red light promote lycopene synthesis in tomato fruits. J. Integr. Agric. 2019, 18, 590–598. [Google Scholar] [CrossRef] [Green Version]
  47. Dhakal, R.; Baek, K.-H. Short period irradiation of single blue wavelength light extends the storage period of mature green tomatoes. Postharvest Biol. Technol. 2014, 90, 73–77. [Google Scholar] [CrossRef]
  48. Lin, C.; Yang, H.; Guo, H.; Mockler, T.; Chen, J.; Cashmore, A.R. Enhancement of blue-light sensitivity of Arabidopsis seedlings by a blue light receptor cryptochrome 2. Proc. Natl. Acad. Sci. USA 1998, 95, 2686–2690. [Google Scholar] [CrossRef] [Green Version]
  49. Novák, A.; Boldizsár, Á.; Ádám, É.; Kozma-Bognár, L.; Majláth, I.; Båga, M.; Tóth, B.; Chibbar, R.; Galiba, G. Light-quality and temperature-dependent CBF14 gene expression modulates freezing tolerance in cereals. J. Exp. Bot. 2016, 67, 1285–1295. [Google Scholar] [CrossRef] [Green Version]
  50. Kim, M.-J.; Kim, P.; Chen, Y.; Chen, B.; Yang, J.; Liu, X.; Kawabata, S.; Wang, Y.; Li, Y. Blue and UV-B light synergistically induce anthocyanin accumulation by co-activating nitrate reductase gene expression in Anthocyanin fruit (Aft) tomato. Plant Biol. 2020. [Google Scholar] [CrossRef]
  51. Giliberto, L.; Perrotta, G.; Pallara, P.; Weller, J.; Fraser, P.D.; Bramley, P.M.; Fiore, A.; Tavazza, M.; Giuliano, G. Manipulation of the blue light photoreceptor cryptochrome 2 in tomato affects vegetative development, flowering time, and fruit antioxidant content. Plant Physiol. 2005, 137, 199–208. [Google Scholar] [CrossRef] [Green Version]
  52. Liu, C.-C.; Ahammed, G.J.; Wang, G.-T.; Xu, C.-J.; Chen, K.-S.; Zhou, Y.-H.; Yu, J.-Q. Tomato CRY1a plays a critical role in the regulation of phytohormone homeostasis, plant development, and carotenoid metabolism in fruits. Plant Cell Environ. 2018, 41, 354–366. [Google Scholar] [CrossRef] [PubMed]
  53. Nanya, K.; Ishigami, Y.; Hikosaka, S.; Goto, E. Effects of blue and red light on stem elongation and flowering of tomato seedlings. Acta Hort. 2012, 956, 261–266. [Google Scholar] [CrossRef]
  54. Kim, H.M.; Hwang, S.J. The growth and development of ‘Mini Chal’ tomato plug seedlings grown under monochromatic or combined red and blue light-emitting diodes. Hort. Sci. Technol. 2019, 37, 190–205. [Google Scholar]
  55. Yoshida, H.; Mizuta, D.; Fukuda, N.; Hikosaka, S.; Goto, E. Effects of varying light quality from single-peak blue and red light-emitting diodes during nursery period on flowering, photosynthesis, growth, and fruit yield of everbearing strawberry. Plant Biotechnol. 2016, 33, 267–276. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  56. Kanayama, Y. Sugar metabolism and fruit development in tomato. Hort. J. 2017, 86, 417–425. [Google Scholar] [CrossRef] [Green Version]
  57. Mawphlang, O.I.L.; Kharshiing, E.V. Photoreceptor mediated plant growth responses: Implications for photoreceptor engineering toward improved performance in crops. Front. Plant Sci. 2017, 8, 1181. [Google Scholar] [CrossRef] [PubMed]
  58. Kawabata, S.; Miyamoto, K.; Li, Y. cDNA microarray analysis of differential gene expression in tomato fruits exposed to blue, UV-A, and UV-A+UV-B. Acta Hort. 2011, 907, 371–374. [Google Scholar] [CrossRef]
  59. Tsunoda, Y.; Hano, S.; Imoto, N.; Shibuya, T.; Ikeda, H.; Amagaya, K.; Kato, K.; Shirakawa, H.; Aso, H.; Kanayama, Y. Physiological roles of tryptophan decarboxylase revealed by overexpression of SlTDC1 in tomato. Sci. Hortic. 2021, 275, 109672. [Google Scholar] [CrossRef]
  60. Ikeda, H.; Shibuya, T.; Imanishi, S.; Aso, H.; Nishiyama, M.; Kanayama, Y. Dynamic metabolic regulation by a chromosome segment from a wild relative during fruit development in a tomato introgression line, IL8-3. Plant Cell Physiol. 2016, 57, 1257–1270. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  61. Mohammed, S.A.; Nishio, S.; Takahashi, H.; Shiratake, K.; Ikeda, H.; Kanahama, K.; Kanayama, Y. Role of vacuolar H+-inorganic pyrophosphatase in tomato fruit development. J. Exp. Bot. 2012, 63, 5613–5621. [Google Scholar] [CrossRef] [Green Version]
  62. Miura, K.; Shiba, H.; Ohta, M.; Kang, S.; Sato, A.; Yuasa, T.; Iwava-Inoue, M.; Kamada, H.; Ezura, H. SlICE1 encoding a MYC-type transcription factor controls cold tolerance in tomato, Solanum lycopersicum. Plant Biotechnol. 2012, 29, 253–260. [Google Scholar] [CrossRef] [Green Version]
  63. Ito, H.; Horie, H. Proper solvent selection for lycopene extraction in tomatoes and application to a rapid determination. Bull. Natl. Inst. Veg. Tea Sci. 2009, 8, 165–173. [Google Scholar]
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Figure 1. (A) Amino acid sequence alignment of SlFKF1 and A. thaliana FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1). Identical amino acids are shown in black boxes. Asterisks indicate the amino acids essential for the function of FKF1 [15,16,32]. (B) A phylogenetic tree based on the amino acid sequences of FKF1 and ZEITLUPE (ZTL) homologs from various species. LOV/PAS, F-box, and KELCH-repeat domain are shown on continuous, dotted, and broken lines, respectively, according to InterPro. The tree was constructed by the neighbor-joining method after sequence alignment using the Clustal W program. Branch numbers refer to the percentage of replicates that support the branch using the bootstrap method (1000 replicates). The scale bar corresponds to 0.1 amino acid substitutions per residue. Table S2 shows the accession numbers of the proteins used to construct the phylogenetic tree.
Figure 1. (A) Amino acid sequence alignment of SlFKF1 and A. thaliana FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1). Identical amino acids are shown in black boxes. Asterisks indicate the amino acids essential for the function of FKF1 [15,16,32]. (B) A phylogenetic tree based on the amino acid sequences of FKF1 and ZEITLUPE (ZTL) homologs from various species. LOV/PAS, F-box, and KELCH-repeat domain are shown on continuous, dotted, and broken lines, respectively, according to InterPro. The tree was constructed by the neighbor-joining method after sequence alignment using the Clustal W program. Branch numbers refer to the percentage of replicates that support the branch using the bootstrap method (1000 replicates). The scale bar corresponds to 0.1 amino acid substitutions per residue. Table S2 shows the accession numbers of the proteins used to construct the phylogenetic tree.
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Figure 2. Diurnal change in the expression level of SlFKF1 in leaves (A); expression level of SlFKF1 in stems (ST), flowers (FL), young leaves (YL), and mature leaves (ML) (B); and expression level of SlFKF1 in leaves and fruit under blue and red light (C). Total RNA was prepared from the wild-type plants of cv. Micro-Tom tomato. The relative expression levels were normalized against SlUBQ or SlACT with standard errors (n = 3), and the maximum level of the transcripts was set at 1.0. Values with ** are significantly different between blue and red light in each organ, according to Welch’s t-test (C).
Figure 2. Diurnal change in the expression level of SlFKF1 in leaves (A); expression level of SlFKF1 in stems (ST), flowers (FL), young leaves (YL), and mature leaves (ML) (B); and expression level of SlFKF1 in leaves and fruit under blue and red light (C). Total RNA was prepared from the wild-type plants of cv. Micro-Tom tomato. The relative expression levels were normalized against SlUBQ or SlACT with standard errors (n = 3), and the maximum level of the transcripts was set at 1.0. Values with ** are significantly different between blue and red light in each organ, according to Welch’s t-test (C).
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Figure 3. Lycopene concentration in fruit under white (W), blue (B), and red (R) light. Lycopene was extracted from the wild-type plants of cv. Micro-Tom tomato. Values indicate means with standard errors (n = 3). p < 0.01, values with different letters between treatments, according to Tukey’s test.
Figure 3. Lycopene concentration in fruit under white (W), blue (B), and red (R) light. Lycopene was extracted from the wild-type plants of cv. Micro-Tom tomato. Values indicate means with standard errors (n = 3). p < 0.01, values with different letters between treatments, according to Tukey’s test.
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Figure 4. SlFKF1 mRNA levels and growth of SlFKF1 RNA interference (RNAi) lines (RI) and wild type (WT). SlFKF1 mRNA levels (A) were measured with the main shoot length (B) and internode length (C). Days to flowering indicate the number of days to first inflorescences from sowing (D). The number of leaflets per leaf was also measured (E). The number of leaves indicates the number of leaves to first flowers (F). Values indicate means with standard errors (n = 3 in A and n = 9 in B–F). *** p < 0.001 and ** p < 0.01, significantly different from WT, according to Dunnett’s test.
Figure 4. SlFKF1 mRNA levels and growth of SlFKF1 RNA interference (RNAi) lines (RI) and wild type (WT). SlFKF1 mRNA levels (A) were measured with the main shoot length (B) and internode length (C). Days to flowering indicate the number of days to first inflorescences from sowing (D). The number of leaflets per leaf was also measured (E). The number of leaves indicates the number of leaves to first flowers (F). Values indicate means with standard errors (n = 3 in A and n = 9 in B–F). *** p < 0.001 and ** p < 0.01, significantly different from WT, according to Dunnett’s test.
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Figure 5. Number of days from flowering to the breaker stage (A) and fruit coloration 10 days after the breaker stage (B) in SlFKF1 RNAi lines (RI) and wild type (WT). Values indicate means with standard errors (n = 12–26). *** p < 0.01, significantly different from WT, according to Dunnett’s test.
Figure 5. Number of days from flowering to the breaker stage (A) and fruit coloration 10 days after the breaker stage (B) in SlFKF1 RNAi lines (RI) and wild type (WT). Values indicate means with standard errors (n = 12–26). *** p < 0.01, significantly different from WT, according to Dunnett’s test.
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Table
Table 1. CONSTANS (CO)-responsive elements on the promoters of SFT, RIN, and PSY homologs.
Table 1. CONSTANS (CO)-responsive elements on the promoters of SFT, RIN, and PSY homologs.
GeneLocus 1Putative CONSTANS Responsive Element 2Strand Position of 1st C from ATG
SFTSolyc03g063100TTTCCACAAAATop−379
PSY1Solyc03g031860TTCCCACACTGBottom−554
Solyc03g031860AAATGTGGTGTBottom−269
Solyc03g031860GTCTGTGGTCTBottom−186
PSY2Solyc02g081330TTGTGTGGTCABottom−274
PSY3Solyc01g005940not found
RINSolyc05g012020CTACCACAAGGTop−1049
Solyc05g012020ATGTGTGGCTABottom−701
1 Locus number in the Sol Genomics Network (SGN). 2 Bold letters indicate the core motif.
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Shibuya, T.; Nishiyama, M.; Kato, K.; Kanayama, Y. Characterization of the FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 Homolog SlFKF1 in Tomato as a Model for Plants with Fleshy Fruit. Int. J. Mol. Sci. 2021, 22, 1735. https://doi.org/10.3390/ijms22041735

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Shibuya T, Nishiyama M, Kato K, Kanayama Y. Characterization of the FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 Homolog SlFKF1 in Tomato as a Model for Plants with Fleshy Fruit. International Journal of Molecular Sciences. 2021; 22(4):1735. https://doi.org/10.3390/ijms22041735

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Shibuya, Tomoki, Manabu Nishiyama, Kazuhisa Kato, and Yoshinori Kanayama. 2021. "Characterization of the FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 Homolog SlFKF1 in Tomato as a Model for Plants with Fleshy Fruit" International Journal of Molecular Sciences 22, no. 4: 1735. https://doi.org/10.3390/ijms22041735

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