Expression Analysis and Interaction Protein Screening of CRY1 in Strawberry

: Cryptochrome 1 (CRY1), a main blue light receptor protein, plays a signiﬁcant role in several biological processes. However, the expression patterns and function of CRY1 in strawberry have not been identiﬁed. Here, the expression proﬁle of CRY1 in different tissues and developmental stages of strawberry fruit, and expression patterns response to abiotic stresses (low temperature, salt and drought) were analyzed. Its subcellular localization, interaction proteins and heterologous overexpression in tobacco were also investigated. The results showed that CRY1 was mainly expressed in leaves and fruits with an expression peak at the initial red stage in strawberry fruit. Abiotic stresses could signiﬁcantly induce the expression of CRY1 . The CRY1 protein was located in both nucleus and cytoplasm. Five proteins (CSN5a-like, JAZ5, eIF3G. NF-YC9, and NDUFB9) interacting with CRY1 were discovered. Genes related ﬂowering times, such as HY5 and CO , in three overexpressed FaCRY1 tobacco lines, were signiﬁcantly upregulated. Taken together, our results suggested CRY1 have a broad role in biological processes in strawberry.


Introduction
The strawberry (Fragaria × ananassa) is an important economic fruit crop world-wide, which has rich nutrition and health-care functions [1]. Breeders have long been working in increasing the yield and quality of strawberry fruit [2,3], which were constrained by numerous factors; For this reason, photomorphogenesis [4][5][6], flowering time regulation [6,7], and adaptability to various stresses [8,9] of strawberry were also the focus of researchers. CRY1 (Cryptochrome 1), as one of the main blue light receptors, senses blue light and transmits signals, which plays a key role in multiple biological processes in plants.
In Arabidopsis, CRY1 showed the function of inhibiting hypocotyl elongation; the latest research showed that the nuclear CRY1, but not cytoplasmic CRY1, mediates blue light inhibition of hypocotyl elongation [10]. AtCRYs directly interacted with E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and inhibited the degradation of downstream ELONGATED HYPOCOTYL 5 (HY5) and CONSTANS (CO) protein, mainly resulting in positive regulation of photomorphogenesis and flowering [11,12]. The latest researches showed that AtCRY1 promotes H2A.Z deposition to regulate HY5 target gene expression and photomor phogenesis in blue light through the enhancement of both SWR1 complex activity and HY5 recruitment of SWR1 complex to HY5 target loci, which was likely mediated by interactions of CRY1 with SWC6 and ARP6, and CRY1 stabilization of HY5, respectively [13]. AtCRY1 undergoes COP1 and LRBs-dependent degradation in response to high blue light [14]. In addition, CRY1 had been identified in responding to multiple abiotic stresses, such as low temperature [15], drought [16,17], and salt stress [17][18][19]. Impaired response of cry1 mutants reflected participation of these photoreceptors in acquiring freezing tolerance in Arabidopsis [15]. Meanwhile, Arabidopsis cry1cry2 double mutants, which lack CRY proteins, showed higher drought tolerance compared to controls, while the opposite was found in plants overexpressing CRY1 gene due to the excessive water loss through stomata [20]. In rice, suppression of CRY1b decreases both melatonin and expression of brassinosteroid biosynthetic genes resulting in salt tolerance [18]. For strawberry researchers and breeders, application of varieties with stronger tolerance to various abiotic stresses holds a great potential in production.
In recent years, several interacting proteins of CRY1 involving various biological processes had been identified, such as PIFs [21], BES1 [22], SWC6, ARP6 [13], and GID1 and DELLA proteins [23,24], which were involved in plant development and hormone signal regulation. Even in Arabidopsis, the protein interacting with CRY1 identified in previous studies could only reveal a small part of mechanism. CRY1 was becoming more and more important as the intersection of light, hormone and stress signal pathway. However, little was known about the role of CRY1 in photomorphogenesis, stress response and flowering time regulation in strawberry.
In this study, we found that CRY1 was mainly expressed in functional leaves and fruits of strawberry. With the highest expression level in fruits at the initial red stage; Low temperature, drought and salt stress could all significantly induce the expression of CRY1 in strawberry. The CRY1 protein was localized in cytoplasm and nucleus. Five interacting proteins (CSN5a-like, JAZ5, NDUFB9, eIF3G, NF-YC9) of CRY1 were identified through yeast two-hybrid library screening. Three independent tobacco mutants with heterologous overexpression of strawberry CRY1 were obtained. The qRT-PCR of mutants showed that CRY1 can promote the expression of downstream transcription factor HY5 and flowering gene CO. These results will help to further improve the mechanism of CRY1 in horticultural crops. All of the efforts were carried out to have a better understanding of the role of CRY1 in the various biological processes in strawberry.

Plant Materials
The octoploid cultivated strawberry 'Benihoppe' (Fragaria × ananassa Duch.), diploid strawberry 'Ruegen' (Fragaria vesca), and tobacco (Nicotiana benthamiana) were cultured in the plant tissue culture room and greenhouse under long day condition (16 h of light, 5000 lx, 8 h of darkness, 22 • C. Plants were grown in plastic basin with a mixture of peat soil, perlite and vermiculite (Volume ratio = 4/1/1), regular irragation, fertilization with compound fertilizer solution. In vitro plants were cultured in 1/2 MS solid medium. The plants were cultured in 1/2 MS solid medium for 40 d before transplant.

Gene Cloning, Sequence Analysis and Phylogenetic Tree Construction
The full length of coding sequences (CDS) of FaCRY1 were cloned from octoploid cultivated strawberry 'Benihoppe' with primers (Table S1)  The cis-acting elements in the promoter region were predicted with Plant-Care (http: //bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 23 December 2019).

The Expression Pattern of CRY1 Analyzed with qRT-PCR
Different tissues (roots, stems, young leaves, function leaves, flowers, mature full red fruit and runner) of 'Benihoppe' were collected from the plants in soil; They were used as materials for tissue-specific expression profile analysis. Meanwhile, different development stages of fruits: little green (LG, seven days after fruit set), big green (BG, 15 days after fruit set), white (W), turn red (TR,1/4 red), half red (HR, 1/2 red) and mature full red (FR) [7,25]; They were collected as materials for fruits development periods expression profile analysis. All tissues were set up with three biological replicates, all replicates were taken from plants that cultured under the same conditions. Each biological replicate contained at least six samples, and three to four samples were collected from each plant.
The in vitro plants of diploid strawberry 'Ruegen' were put into the artificial climate chamber at 4 • C for low temperature treatment [26,27], the relative humidity was 75%. Plants grown at 25 • C were used as the control. The leaf tissues were sampled at 0 h, 3 h, 6 h, 9 h and 12 h interval respectively. As for drought and salt stress treatment, the tissue culture seedlings of diploid strawberry 'Ruegen' were put into Hogland nutrient solution. After acclimatization for four days, plants were divided into three groups, and transferred into either 10% PEG-6000 + Hogland solution, 200 mm NaCl + Hogland solution or Hogland solution respectively to mimic drought stress [28,29], salt stress [29,30], or regular grown condition. The leaf and root tissues were sampled at 0 h, 3 h, 6 h, 12 h, and 24 h interval respectively.
The total RNA of all samples were extracted with a modified CTAB method [31]. The quality of RNA was evaluated by electrophoresis on a 1% agarose gel and scanned using a NanoDrop spectrophotometer [7]. A reverse transcription PCR reaction was conducted with Prime-Script TM RT Reagent Kit with gDNA Eraser (Takara, Dalian, China) (Confirmed: Takara is a Japanese company. The kit we used was produced by its subordinate company in Dalian, China) using 1 µg total RNA, following the manufacturer's protocol. The cDNA was used as the template in sequential 20 µL qRT-PCR reaction system as the template on the basis of a SYBR Premix ExTaqTM Kit (Takara, Dalian, China).
The relative gene expression was determined by qRT-PCR. The qRT-PCRs were performed on the CFX96 real-time PCR system (Bio-Rad, CFX connect). Each reaction was performed with three technical replicates. The calculation method for qRT-PCR was 2 −∆∆CT . The Actin was used as housekeeping gene for the calibration. IBM SPSS Statistics (Version 23) was used for one-way ANOVA test, Duncan test and multiple comparison (p < 0.05). Origin (Version 9) was used for drafting Figures. Primers were listed in Table S1.

Subcellular Localization Analysis
The CDS of FaCRY1 was amplified and homologous recombinated into a plasmid vector (pYTSL-16), which was modified from pMDC83-35S and pSITE-2NB [7]. The FaCRY1 gene was inserted frame with the GFP (Green fluorescent protein) gene on the vector. The fusion protein contained FaCRY1 and GFP (FaCRY1::GFP) was driven by the 35S promoter. The HY5-mCherry vector driven by the same 35S promoter was used as a nucleus maker [32]. The plasmid was further transformed into Agrobacterium tumefaciens strain GV3101. The plasmids were transiently expressed in the epidermal cells of tobacco (Nicotiana benthamiana) leaves as previously described [7,33]. These two vectors were transiently co-expressed in tobacco leaf epidermal cells. All of the fluorescence signals of the samples were detected by a confocal laser scanning microscopy system (FV3000 Olympus, Tokyo, Japan).

Yeast Two-Hybrid Screening Assay
The yeast two-hybrid cDNA library was constructed based on pGADT7 was entrusted to Shanghai Ouyi Biomedical Technology Co., Ltd., Shanghai, China. Yeast two-hybrid screening assay and the validation of potential interacting proteins analysis was conducted according to the manual of consulting instructions of Matchmaker™ Gold Yeast Two-Hybrid Screening Systems (Shanghai Yubo Biotechnology Co., Ltd., Shanghai, China). The SD/-Trp and SD/-Trp/-His/-Ade culture medium were used for auto-activation and toxicity test of bait plasmid.
About 260 monoclonal yeast strains screened by SD/-Leu/-Trp/-His/-Ade/X-α-Gal/AbA (QDO/X/A) culture medium were sequenced. The plasmid containing 68 gene fragments was extracted and checked by rotary experiments. Ten genes were selected, cloned and inserted into pGADT7 vector for the second rotary experiments. Primers were listed in Table S1.

Agrobacterium-Mediated Heterologous Expression of the FaCRY1 in Tobacco
A pCambia2301::d35S-FaCRY1-NOS vector, carrying the Kanamycin and GUS gene for later selection, was constructed for heterologous expression of the FaCRY1 in tobacco (Nicotiana benthamiana). Tobacco leaf disk was transformed with A. tumefaciens strain GV3101 according to the reported protocols [36], which carried out with minor modifications. The obtained plantlets were selected using 100 mg/L Kanamycin as antibiotic. After bacteriostatic culture, the explants were cultured in the medium containing 100 mg/L Kanamycin, 250 mg/L carboxybenzyl, 250 mg/L Timentin, 6-BA 2.0 mg/L + NAA 0.01 mg/L.
The transgenic lines were identified with the GUS histochemical staining. Genomic DNA were extracted from the plantlets. Two independently confirmed transformants were further used to detect the Kanamycin gene integration using PCR to validate insertion of the T-DNA region. The young leaves from tobacco were selected as samples. PCR amplicons were detected in a 1% agarose gel ( Figure S1). The qRT-PCR was used to detect the expression level of relative genes. Primers were listed in Table S1.

Gene Cloning, Protein Property and Phylogenetic Analysis of FaCRY1
The full length of coding sequences of FaCRY1 were cloned with primers (Table S1). The analysis of conserved domain showed that CRY1 protein contained two conserved domains: the N-terminal PHR photolyase domain (contain MTHF region and FAD region) and the C-terminal CCE domain ( Figure 1a).
The phylogenetic tree showed that FaCRY1 had a close relationship with FvCRY1, the similarity in the amino acid sequence of FaCRY1 and FvCRY1 was 99.85%. FaCRY1 clustered with homologous proteins from other Rosaceae species, and the CRY1 from the octoploid cultivated strawberry (FaCRY1) was distantly related to the Arabidopsis (AtCRY1) and tomato (Solanum lycopersicum, SlCRY1).

Cis-Reglatory Elements in the Promoters of FaCRY1 Genes
The promoter sequences of CRY1, which were 1981 bp upstream of transcription start sit, were cloned from strawberry genomic DNA with primers listed in Table S1. In addition to the core elements of the basic transcription and regulation mechanism of higher plants, such as CAAT-box, TATA-box and A-box, as well as some elements with unknown functions. Twenty-six types of cis-regulatory elements were predicted in the CRY1 promoter sequences from octoploid cultivated strawberry ( Figure 1). We further classified them into four groups according to their functions annotated by Plant-Care and previous studies [7,[37][38][39][40]. The light response element was the largest category, followed by hormone response and stress-inducible element (Table 1), which indicates that the potential gene function of CRY1 in strawberry.

Cis-Reglatory Elements in the Promoters of FaCRY1 Genes
The promoter sequences of CRY1, which were 1981 bp upstream of transcription start sit, were cloned from strawberry genomic DNA with primers listed in Table S1. In addition to the core elements of the basic transcription and regulation mechanism of higher plants, such as CAAT-box, TATA-box and A-box, as well as some elements with unknown functions. Twenty-six types of cis-regulatory elements were predicted in the CRY1 promoter sequences from octoploid cultivated strawberry ( Figure 1). We further classified them into four groups according to their functions annotated by Plant-Care and previous studies [7,[37][38][39][40]. The light response element was the largest category, followed by hormone response and stress-inducible element (Table 1), which indicates that the potential gene function of CRY1 in strawberry.

The Expression Pattern of CRY1 in Strawberry
The qRT-PCR results showed the highest expression of CRY1 was detected in leaf and full red fruits tissues, with a relatively high expression level in young leaves, flowers and runner, but the lowest expression in the root and stem (Figure 2). During strawberry fruit development, the expression of CRY1 peaked at TR stage, and then decreased. The expression of CRY1 did not change significantly in the early or late stages in strawberry fruit development.
CCGTCC-box 1 related to meristem-specific activation MYC 1 N/A Myc 1 N/A ARE 1 cis-acting regulatory element essential for the anaerobic induction

The Expression Pattern of CRY1 in Strawberry
The qRT-PCR results showed the highest expression of CRY1 was detected in lea and full red fruits tissues, with a relatively high expression level in young leaves, flower and runner, but the lowest expression in the root and stem (Figure 2). During strawberry fruit development, the expression of CRY1 peaked at TR stage, and then decreased. The expression of CRY1 did not change significantly in the early or late stages in strawberry fruit development.

Effects of Abiotic Stress on CRY1 Expression
The expression patterns of CRY1 response to abiotic stresses (low temperature, sal and drought) were analyzed. The qRT-PCR showed that the transcript abundance o CRY1 in strawberry leaf exposed to 4 °C was significantly induced after 3 h, and then decreased significantly at 6 h. There was no significant difference of expression level be tween 4 °C and control from 6 to 12 h (Figure 3).

Effects of Abiotic Stress on CRY1 Expression
The expression patterns of CRY1 response to abiotic stresses (low temperature, salt and drought) were analyzed. The qRT-PCR showed that the transcript abundance of CRY1 in strawberry leaf exposed to 4 • C was significantly induced after 3 h, and then decreased significantly at 6 h. There was no significant difference of expression level between 4 • C and control from 6 to 12 h (Figure 3).
The qRT-PCR showed that both salt stress and drought stress could significantly induce the expression of CRY1. In this study, the expression of CRY1 in strawberry roots was significantly up-regulated from 12 to 24 h (Figure 4a) and reached the peak at 6 h in leaf, and then decreased gradually, but there was no difference with control at 24 h under salt stress (Figure 4b). The expression of CRY1 in strawberry roots was significantly up-regulated only after 12 h (Figure 4c), and then had no difference compared to the control group thereafter. The expression of CRY1 in leaves was significantly induced in 3 h, and then tended to be stable, with no significant difference comparing to the control group.
These results showed that cold stress, salt stress, and drought stress can significantly induce the transcription of CRY1 in a short time, but they will gradually decline and tend to be stable in the end.  The qRT-PCR showed that both salt stress and drought stress could signific duce the expression of CRY1. In this study, the expression of CRY1 in strawbe was significantly up-regulated from 12 to 24 h (Figure 4a) and reached the peak leaf, and then decreased gradually, but there was no difference with control at 24 salt stress (Figure 4b). The expression of CRY1 in strawberry roots was significa regulated only after 12 h (Figure 4c), and then had no difference compared to th group thereafter. The expression of CRY1 in leaves was significantly induced in then tended to be stable, with no significant difference comparing to the control These results showed that cold stress, salt stress, and drought stress can sign induce the transcription of CRY1 in a short time, but they will gradually decline to be stable in the end.  The qRT-PCR showed that both salt stress and drought stress could significa duce the expression of CRY1. In this study, the expression of CRY1 in strawber was significantly up-regulated from 12 to 24 h (Figure 4a) and reached the peak leaf, and then decreased gradually, but there was no difference with control at 24 salt stress (Figure 4b). The expression of CRY1 in strawberry roots was significa regulated only after 12 h (Figure 4c), and then had no difference compared to the group thereafter. The expression of CRY1 in leaves was significantly induced in then tended to be stable, with no significant difference comparing to the control g These results showed that cold stress, salt stress, and drought stress can sign induce the transcription of CRY1 in a short time, but they will gradually decline a to be stable in the end.

Subcellular Localization of FaCRY1
The GFP fluorescence signals of FaCRY1::GFP fusion proteins were predominantly located in both nucleus and cytoplasm ( Figure 5), which indicated that the FaCRY1 was nucleus and cytoplasm localized protein.
under salt stress. (c) Relative expression level of CRY1 in strawberry root under drought stress. (d) Relative expression level of CRY1 in strawberry leaf under drought stress. The significance was annotated by letters. Different letters represent significant differences. p < 0.05.

Subcellular Localization of FaCRY1
The GFP fluorescence signals of FaCRY1::GFP fusion proteins were predominantly located in both nucleus and cytoplasm ( Figure 5), which indicated that the FaCRY1 was nucleus and cytoplasm localized protein.

Yeast Two-Hybrid Library Screening Assay
Auto-activation assay showed that full-length sequence of FaCRY1 (amino acids 1 to 646) could not be used as a bait as a result of strong auto-activation. Only pGBKT7-CRY1-BD-4 had no transcriptional activation and could be used for yeast two-hybrid screening assay.
A total of 260 monoclonal yeast and 68 gene fragment sequences were obtained. Ten genes were cloned and inserted into pGADT7 vector for the second rotary experiments. The final yeast two-hybrid validation results showed that only C1-4 (CSN5a-like, COP9

Yeast Two-Hybrid Library Screening Assay
Auto-activation assay showed that full-length sequence of FaCRY1 (amino acids 1 to 646) could not be used as a bait as a result of strong auto-activation. Only pGBKT7-CRY1-BD-4 had no transcriptional activation and could be used for yeast two-hybrid screening assay.

Heterologous Overexpression of Strawberry CRY1 in Tobacco
The validation of transgenic tobacco using GUS staining analysis showed that three independent lines were obtained. All three transgenic plants in the tissue culture bottle already gave flower (Figure 7). In contrast, the control plants were still at the vegetative growing stage. We selected OE-8 and OE-35 lines that had higher expression level of GUS gene for further analysis.
To confirm the potential function of FaCRY1 involving flowering time regulation, we performed qRT-PCR to examine some genes in the downstream photoperiodic flowering time pathway, such as COP1, HY5, and CO [7,11,12,[41][42][43]. The qRT-PCR results showed that the expression level of COP1 did not change significantly, but the important downstream transcription factor HY5 was significantly up-regulated. The CO gene, a key factor involved flowering, was also up-regulated, indicating that FaCRY1 may affect strawberry flowering time regulation.

Heterologous Overexpression of Strawberry CRY1 in Tobacco
The validation of transgenic tobacco using GUS staining analysis showed that three independent lines were obtained. All three transgenic plants in the tissue culture bottle already gave flower (Figure 7). In contrast, the control plants were still at the vegetative growing stage. We selected OE-8 and OE-35 lines that had higher expression level of GUS gene for further analysis.
To confirm the potential function of FaCRY1 involving flowering time regulation, we performed qRT-PCR to examine some genes in the downstream photoperiodic flowering time pathway, such as COP1, HY5, and CO [7,11,12,[41][42][43]. The qRT-PCR results showed that the expression level of COP1 did not change significantly, but the important downstream transcription factor HY5 was significantly up-regulated. The CO gene, a key factor involved flowering, was also up-regulated, indicating that FaCRY1 may affect strawberry flowering time regulation.

Discussion
The CRY1 genes were involved in various biological processes, which have been widely identified in various plants during the past 20 years. In our study, FaCRY1 had a very close relationship with FvCRY1 and other Rosaceae plants, which suggested a common ancient origin of the CRY1 proteins from these species (Figure 1); But also showed great variation compared with Arabidopsis or tomato (Figure 1). The promoter region was critical for gene expression regulation [7]. Cis-regulatory element analyses showed that FaCRY1 promoter distributed many elements related to the light, hormone, and stress response (Figure 2), which indicated that FaCRY1 participate in these signaling pathways as several previous studies have been reported [13,16,17,19,21,22,[44][45][46][47][48][49]. In this study, the expression peaks of FaCRY1 were observed in the leaf (Figure 2a) and fruit (Figure 2b). It has been demonstrated that CRY1 plays key role in the regulation of light signaling and

Discussion
The CRY1 genes were involved in various biological processes, which have been widely identified in various plants during the past 20 years. In our study, FaCRY1 had a very close relationship with FvCRY1 and other Rosaceae plants, which suggested a common ancient origin of the CRY1 proteins from these species (Figure 1); But also showed great variation compared with Arabidopsis or tomato (Figure 1). The promoter region was critical for gene expression regulation [7]. Cis-regulatory element analyses showed that FaCRY1 promoter distributed many elements related to the light, hormone, and stress response (Figure 2), which indicated that FaCRY1 participate in these signaling pathways as several previous studies have been reported [13,16,17,19,21,22,[44][45][46][47][48][49]. In this study, the expression peaks of FaCRY1 were observed in the leaf (Figure 2a) and fruit (Figure 2b). It has been demonstrated that CRY1 plays key role in the regulation of light signaling and fruit quality in Arabidopsis and tomato [21,[50][51][52]. However, CRY1 was also involved in the regulation of flowering time, which has functional redundancy with CRY2 [42,53,54]. These results indicated the potential function of CRY1 gene in strawberry growth and development.
Previous studies had demonstrated CRY1 also plays an important role in plant response to low temperature stress. Impaired response of cry1 mutants reflected participation of photoreceptor in acquiring freezing tolerance [15]. In this study, the qRT-PCR showed that the transcription of CRY1 in strawberry leaf was significantly induced from 3 h (Figure 3), which suggested that low temperature could up-regulate the expression of CRY1 in a short time. In addition to the fact that drought and salt stress can share a common signaling pathway mainly due to their osmotic effects [55]. The OE-CRY1 mutants had larger stomatal opening, which showed a more sensitive phenotype to drought, while cry1 mutants were more resistant to drought stress in Arabidopsis [17,20]. Transgenic lines of Arabidopsis overexpressing TaCRY1a and TaCRY2 (from T. aestivum) were more sensitive to high salt stress (120 mM NaCl) when compared with the watered control [19]. Strawberries were very sensitive to osmotic stress [56]. Interestingly, the expression of CRY1 in strawberry did not decrease to adapt to stress when strawberry was treated with osmotic stress (Figure 4). In this study, the expression of CRY1 in strawberry roots ( Figure 4c) and leaves (Figure 4d) was significantly up-regulated only at 12 h and 3 h under drought stress. Salt stress forced the expression of CRY1 in strawberry leaves to be significantly up-regulated for a long time from 6 h to 12 h (Figure 4b). While in roots, CRY1 was significantly induced from 12 h and still in a rising trend at 24 h (Figure 4a). Xu et al. [19]. found a remarkable increase in TaCRY2 transcripts in Arabidopsis roots treated with salt stress (250 mM NaCl) after 12 h, and transcription of TaCRY1a was induced by salt stress after 24 h of exposure to the stressor agent, which was similar to our results ( Figure 4). These results showed that CRY1 in strawberry leaves responded more rapid to stress than in roots ( Figure 4). Meanwhile, the CRY1 expression level was more amenable to salt stress than to drought stress ( Figure 4). In addition, the relative expression of CRY1 up-regulated to transmit stress signals in a short term, and then may regulated by a negative feedback mechanism similar to CRY2-COP1-HY5-BICs in Arabidopsis [57].
In this study, FaCRY1 protein was located in both cytoplasm and nucleus ( Figure 5). It displays a light-responsive nucleocytoplasmic shuttling pattern such as that of TaCRY1a (Triticum aestivum), LfCRY1 (Lilium formolongi) and AtCRY1 (Arabidopsis thaliana) [19,53,58]. which suggest FaCRY1 may participate in signal transmission and can complete its posttranslational life cycle inside the nucleus [59]. In addition, nucleocytoplasmic shuttling was used by the cell to control gene expression programs in response to the environment [19,60], such as osmotic stress.
Yeast two hybrid library screening assay was a technology to obtain new interacting proteins, which had been widely used to find new target proteins interacting with bait proteins nowadays. In this study, we identified five interacting proteins of CRY1: CSN5alike, JAZ5, NDUFB9, eIF3G, and NF-YC9 (Figure 6b). The yeast auto-activation assay showed that the area near the MTHF region of the PHR domain led to the activity in the FaCRY1 protein (Figure 6a). In Arabidopsis, the COOH-terminus of AtCRY (CCT1, amino acids 490 to 681) [35] and CRY1N493 (amino acids 1-493) [34] also has self-activation property in yeast. The pGBKT7-CRY1BD-4 retains part of the N-terminal PHR domains (responsible for sensing light) and a complete C-terminal structure (CCE), which plays a key role in the function of CRY. As a region of protein interaction, CCE domain transmits various signals of CRY to downstream factors [61]. In Arabidopsis, the loss of function of any member of CSN will cause constitutive photomorphogenetic, which was characterized by hypocotyl elongation, closed and undeveloped cotyledons, unable to yellowing, and deep purple seed coat [62]. Two conserved genes named CSN5A and CSN5B encode two isoforms of CSN Subunit 5 [63], of which CSN5A was considered to play the largest role in the role of CSN5 [64]. These results suggests that CRY1 may participate in the light morphogenesis through the interaction with CSN5a-like in strawberry. In this study, the two CRY1 interacting proteins played important roles in the stress response mechanism: JAZ5 and eIF3G (Figure 6b). Jaz family was a transcription inhibitor that plays a role in jasmonic acid signaling pathway [65], in which JAZ5, as an inhibitory protein of jasmonic acid signaling [66,67], can interact with MYB21/24 to participate in jasmonic acid mediated stamen development [68]. It can also bind to WRKY57 and WRKY33 to participate in jasmonic acid mediated resistance to Botrytis cinereal [69]. EIF3G was very important for the translation initiation pathway [70] and plays an important role in plant resistance to stress. TaeIF3G transgenic expression enhanced the tolerance of the TaeIF3G overexpressing parental yeast cells and Arabidopsis plants under different abiotic stress conditions [71]. In addition, eIF3G can also help plants resist virus infection in biological stress [72][73][74]. In this study, we found that CRY1 can be significantly induced by low temperature, drought and salt stress in strawberry (Figures 3 and 4), which suggested that CRY1 may participate in stress response through interaction with these proteins. NF-YC9 was mainly involved in flowering time regulation [75,76], ABA response transcription [77], actively regulates the speed of petal expansion, and mediates crosstalk between ethylene and GA [78]. These results suggested that CRY1 may be involved in flowering time regulation and hormone signal response in strawberry. We noticed that yeast two hybrid analysis was in vivo validation, and these potential interacting proteins of CRY1 need to be further verified by in vitro protein analysis before further analysis.
The qRT-PCR results showed that HY5 and CO gene was significantly up-regulated in the young leaves of tobacco tissue culture seedlings after heterologous overexpressing FaCRY1 (Figure 7), which suggested that CRY1 may regulate the expression of CO through HY5 [12]. In tomato, both OE-CRY1a and OE-CRY2 showed a phenotype of late flowering [51,79], however, cry1a, cry2 and cry1acry2 double mutants showed no significant difference in flowering time [52]. This evidence suggested that the role of CRYs in regulating flowering was divergent from different species. In tomato, the expression of COP1 in OE-CRY1a showed no changed; But significantly up-regulation in cry1a mutant; The expression of HY5 was significantly up-regulated in OE-CRY1a and down-regulated in cry1a mutant [80]. However, HY5 was significantly up-regulated and COP1 did not change significantly in strawberry (Figure 7). These results suggested that CRY1 mainly inhibited the degradation of HY5 by COP1 protein and did not affect the transcription level of COP1 to transmit signal in strawberry, which was similar to Arabidopsis [11]. Blue light may regulate CO, SOC1 gene expression and mediate flowering through BBX28c1 in our previous study [7]. We noticed that NF-YC9 plays an important role in the regulation of flowering time, NF-YC9 could form a complex with CO and drive the transcription of downstream genes, such FT and SOC1 [81,82]. These results implied a new flowering mechanism may exist in strawberry: CRY1-NF-YC9-CO, which needs to be further studied.

Conclusions
In this study, we found that CRY1 was mainly expressed in functional leaves and fruits of strawberry; With the highest expression level in fruits at the initial red stage; Low temperature, drought and salt stress could all significantly induce the expression of CRY1 in strawberry. The CRY1 protein was localized in cytoplasm and nucleus. Five interacting proteins (CSN5a-like, JAZ5, NDUFB9, eIF3G, NF-YC9) of CRY1 were identified through yeast two-hybrid library screening. Three independent tobacco mutants with heterologous overexpression of strawberry CRY1 were obtained. The qRT-PCR of mutants showed that CRY1 can promote the expression of downstream transcription factor HY5 and flowering gene CO. These results will help to further improve the mechanism of CRY1 in horticultural crops.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/horticulturae8050460/s1. Table S1: Primers used in this study; Table S2: Coding region sequences of FaCRY1. Figure S1: Detection the Kanamycin gene using PCR to validate insertion of the T-DNA region.