Promoter analysis of the SPATULA (FvSPT) and SPIRAL (FvSPR) genes in the woodland diploid strawberry (Fragaria vesca L.)

The aim of this study was to identify transcription factor (TF) binding sites and cis-regulatory elements (CREs) on the promoters of FvSPR1-like2 (SPIRAL) and FvSPT (SPATULA) genes in the woodland diploid strawberry (Fragaria vesca L.). We identified: (1) MYB59, WRKY25 and WRKY8 TFs which play a role in ethylene signaling; (2) ARF family of TFs which play a role in ARF-mediated auxin signaling on the promoter of FvSPR1-like2 gene; (3) ARR family of TFs which play a role in cytokinin signaling; (4) ERF family of TFs which play a role in ethylene signaling on the promoter of FvSPT. This bioinformatic analysis of TFs and CREs may provide a better understanding of the function of genes involved in, and the mechanism underlying, non-climateric ripening during strawberry fruit maturation.


Introduction
Members of the SPR gene family encode small proteins that contribute to cell elongation by regulating microtubule organization (Nakajima et al. 2004). SPR genes in A. thaliana are classified into two main groups, SPR1 and SPR2 (Bichet et al. 2001;Burk and Ye 2002), and five subgroups, SPR1-like1 to SPR1-like5, all of which have been functionally characterized (Nakajima et al. 2004).
The SPR gene influences the elongation and development of plants at both cellular and organ levels (Furutani et al. 2000;Nakajima et al. 2004). Furutani et al. (2000) induced a mutant SPR gene in A. thaliana whose roots curved to the right unlike control roots that grew straight. The mutation resulted from the arrangement of cortical microtubules on the opposite side of the optimal direction in epidermal root cells, also effecting helical handedness. Overexpression of the SPR gene did not stimulate root skewing since its main function is to maintain the straight elongation of root cells. In addition, the SPR gene enhanced the rapid elongation of cells, resulting in the lengthwise enlargement of tissues. Moreover, SPR genes interact with cellular molecules to control anisotropic growth (Nakajima et al. 2004).
The SPT transcription factor positively indicates and controls cytokinin output in the medial region of the ovary (Reyes-Olalde et al. 2017a). The SPT gene regulates auxin signaling in gynoecium and style-sigma development (Moubayidin and Ostergaard 2014;Schuster et al. 2015). The SPT gene is expressed in non-climacteric strawberry (Fragaria × ananassa Duch.) when treated with auxin and ethylene and is regulated by four ethylene responsive elements (EREs) in the SPT promoter region (Tisza et al. 2010).
Transcriptional regulation of gene expression is fundamental to biological processes, such as cell growth, development, differentiation, fruit ripening and responses to environmental signals (Meshi and Iwabuchi 1995). Given its importance as a transcriptional regulator of genes, the analysis of plant promoters may provide important information that would better guide the construction of biotechnological systems because regulated gene expression systems can increase the function of genetically modified organisms (Corrado and Karali 2009). As internal physiological control regulators, plant hormones also have important roles in the transcriptional regulation of genes, such as development and fruit ripening , for example, ethylene and auxin control different steps of the flower-to-fruit transition (Bapat et al. 2010;Kumar et al. 2014;Ziliotto et al. 2012). An antagonistic effect can be observed between ethylene and auxin during tomato fruit ripening (Li et al. 2017).
In this study, tomato (Solanum lycopersicum L.) cv. Micro-Tom and tobacco (Nicotiana benthamiana) were selected as model plants, as these plants are often chosen in genetic studies to examine and observe differences in gene expression using the green fluorescence protein (GFP) marker gene (Hoshikawa et al. 2019;Reed and Osbourn 2018). Agrobacterium tumefaciens-mediated transfer, together with an agroinfiltration (also known as agroinjection) method, was used in this study. Agroinfiltration is an Agrobacterium-mediated transient recombinant protein expression method which can be used to avoid labor-intensive and time-consuming methods to produce stable transgenic plants (Hoshikawa et al. 2019). Infiltration is achieved by delivering the Agrobacterium with the target genes into extracellular leaf space by physical infiltration (Norkunas et al. 2018). Physical infiltration in this study was performed with a needleless syringe.
The aim of this study was to characterize the SPT and SPR gene promoters which were isolated from Fragaria vesca L., the woodland diploid strawberry, by finding specific motifs. The putative promoter region was identified with the JASPAR 2020 plantae algorithm (Fornes et al. 2020) for TFs as well as a promoter motifs database (http:// jaspar. gener eg. net/) allowing us to predict the promoter regions of the tomato (Solanum lycopersicum L.) cv. Micro-Tom SPT, , as well as F. vesca SPT,  genes, which show selective complementation in A. thaliana (Hidvégi et al. 2020). We compared these promoter sequences with promoters of A. thaliana SPT and SPR1-like2 (AtSPT and AtSPR1-like2) reference genes. Moreover, we used PCR amplification of different lengths of upstream regions of the FvSPT and FvSPR1-like2 coding sequences and insertion of putative promoter fragments into a binary vector (pGWB604) carrying the sGFP reporter gene. Tomato and tobacco were agroinjected with the pGWB604 and pGWB405 (CaMV35S::sGFP, as positive control) binary vectors that included a fusion of the promoter deletion lines and the sGFP reporter gene. Promoter deletion lines can be used to identify the presence of genetic regulatory elements (TFs or CREs) such as enhancers and silencers in the region upstream of the start codon.

Plant materials and growth conditions
Diploid strawberry (Fragaria vesca 'Rügen') was used as the template to amplify the putative promoter regions of FvSPT and FvSPR1-like2. Seeds of tomato (cv. Micro-Tom), diploid strawberry and tobacco (Nicotiana benthamiana) were sown ex vitro in 50 mm Jiffy-7® pots (1 seed/ pot). Jiffy pots were placed in a climate room at 22 °C and kept under an 8-h photoperiod at a photosynthetic photon flux density (PPFD) of 37 µmol m −2 s −1 provided by Biolux tubes (Osram L58W, Markham, Canada). When seedlings formed two fully developed leaves, rooted plantlets were transferred to plastic pots (9 cm) into soil and grown under the same conditions as seedlings. No fertilizers or additional supplements (e.g., pest control agents) were added.
The promoters of FvSPR1-like1, FvSPR1-like2 and FvSPT genes were isolated and aligned with the A. thaliana and S. lycopersicum sequences by using NCBI BLAST (https:// blast. ncbi. nlm. nih. gov/ Blast. cgi) analysis to find similarities or homologies. The promoter regions of the genes were examined with JASPAR 2020 (Fornes et al. 2020) and PLACE 30.0 (database of plant cis-acting regulatory DNA elements; Higo et al. 1999) to determine the transcriptional factor binding sites (TFBS) and CREs to develop promoter deletion lines of promoters of FvSPR1-like2 and FvSPT genes.

Agrobacterium-mediated transformation
Agrobacterium tumefaciens GV3101 strain (Intact Genomics, Creve Coeur, MI, USA) was incubated in an LB plate with a working concentration of 10 µg/mL gentamycin (10 mg/mL stock; Duchefa) at 28 °C for 2 d. A single colony of A. tumefaciens from the LB plate was incubated in 5 mL of liquid LB with 5 µL of spectinomycin (50 mg/mL stock; Duchefa) and 5 µL of gentamycin (10 mg/mL stock) overnight in a MaxQ 4000 Benchtop Orbital Shaker (ThermoFischer Scientific, Waltham, MA, USA) at 140 rpm and 28 °C. Cultures were placed on ice for 30 min then centrifuged for 10 min at 4000 rpm and at 4 °C. The supernatant was discarded, and the pellet was resuspended in 5.0 mL of 20 mM CaCl 2 on ice then centrifuged again for 5 min at 4000 rpm and 4 °C. The supernatant was discarded and 1.0 mL of icecold 20 mM CaCl 2 was added to the pellet in ice water. A 200 µL aliquot as competent A. tumefaciens cells was prechilled in 1.5-mL microcentrifuge tubes (Eppendorf, Hamburg, Germany). Plasmid DNA (3 µL; 500 ng) was added from the pGWB604 vector containing the promoter region into each tube containing competent A. tumefaciens cells and kept on ice for 20 min, placed in liquid nitrogen for 5 min, heat shocked at 37 °C for 5 min, then added to ice for 5 min. Liquid LB media (1.0 mL) was added to each heatshocked colony and incubated in a shaker at 28 °C and at 140 rpm for 3-4 h. Sample (100-150 µL) was pipetted onto an LB plate supplemented with 10 µg/mL of gentamycin (10 mg/mL stock) and 50 µg/mL of spectinomycin (50 mg/ mL stock). Based on the Bergkessel and Guthrie (2013) protocol, colony PCR was performed to confirm the success of transformation using the same conditions used for TOPO ® and Gateway ® LR cloning.

Agroinfiltration in tomato and tobacco
A single A. tumefaciens colony was cultured in 5 mL of LB medium supplemented with 5 µL gentamycin (10 mg/ mL stock) and 5 µL spectinomycin (50 mg/mL) overnight at 28 °C on a shaker at 140 rpm. Cultures were transferred to 50 mL of induction medium (10.5 g K 2 HPO 4 , 4.5 g KH 2 PO 4 , 1 g (NH 4 ) 2 SO 4 , 0.5 g Na-citrate, 1 g glucose, 1 g fructose, 4 mL glycerol, 0.12 g MgSO 4 , 1.95 g MES (10 mM); pH 5.6; Singer et al. 2012) containing 100 µM acetosyringone (Duchefa), which was added after autoclaving (121 °C, 60 min). Cells were incubated in induction medium at 30 °C for 5-6 h at 140 rpm. After incubation, cells were centrifuged at 4000 rpm for 10 min, and then the pellet was resuspended in infiltration medium (10 mM MgSO 4 , 10 mM MES; pH 5.6; Singer et al. 2012) supplemented with 200 µM acetosyringone. Green and ripening tomato fruits (age: about 60 d after germination; sample number: 20 fruits/vector construct, 2 fruits/plant) and tobacco leaves (age: about 45 d after sowing; sample number: 20 leaves/ vector construct, 2 leaves/plant) were agroinjected by using a 1 mL syringe (Z683531; Sigma-Aldrich, St. Louis, MI, USA) with a 0.5 × 1.6 mm needle (Sigma-Aldrich). Infiltration solution was injected (5-6 mm deep) into tomato fruit through stylar apex, while leaves were injected by slightly injuring the epithelium tissue of the abaxial surface. Plants were tested 3 d later with the Phire Plant Direct PCR Kit (ThermoFischer Scientific). The PCR mixture consisted of 10 µL of Phire Plant Buffer (2 ×), 40 pmol of each primer pair (specific to the sGFP gene and the GlyA gene of A. tumefaciens GV3101), 0.4 µL of Phire Hot Start II DNA Pol and 0.5 µL of diluted plant tissue. PCR conditions were 98 °C for 5 min followed by 40 cycles at 98 °C for 5 s, 60 °C for 5 s and 72 °C for 20 s. Cycling was followed by a final incubation of 72 °C for 1 min. PCR products were separated by gel electrophoresis based on the same protocol that was used for promoter PCR. Only sGFP-positive plants were selected for GFP fluorescence and RT-qPCR analysis.

Verification of GFP fluorescence by UV light
GFP fluorescence was verified with a FastGene ® blue/green LED flashlight (FG-11; NIPPON Genetics, Tokyo, Japan), which was used to irradiate (excitation: 489 nm; emission: 520 nm) leaves and fruit at the mature red ripening stage (about three days after agroinjection) at a distance of ~ 10 cm from each organ in the dark. To photograph the irradiated leaves, a yellow UV filter (NIPPON Genetics) was mounted to the camera (Nikon Coolpix B500, Tokyo, Japan) lens to filter out blue light, and to allow GFP fluorescence to be visualized. Fluorescence was also verified in controls at the same time. The location of GFP fluorescence was visually assessed and confirmed. Three controls were used for both methods at the same time as the agroinjection into ripe fruits and leaves using infiltration solution: (a) without any A. tumefaciens; (b) A. tumefaciens without any plasmid; (c) A. tumefaciens with a constitutive promoter (CaMV-35S) + sGFP in the plasmid.

Quantification of sGFP expression by real-time PCR
After confirming the possible presence of the sGFP gene using UV fluorescence, tomato fruits and tobacco leaves (two per plant; 20 plants/line) showing fluorescence following UV light detection were picked after 3 d. To measure sGFP intensity, RT-qPCR was used (Wang et al. 2004). Total RNA was isolated using Direct-zol™ (Zymo Research, Irvine, CA, USA) with TRIzol reagent based on the manufacturer's protocol. After purifying total RNA, three quality control methods were applied: 1) microcapillary electrophoresis with an Implen n50 spectrophotometer (Implen, Munich, Germany) for preliminary quantification; 2) agarose gel electrophoresis to assess total RNA degradation and potential contamination; 3) Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA) to check the quality and quantity of total RNA. cDNA was amplified from 120 ng of total RNA with reverse transcription using the FIREScript RT cDNA Synthesis MIX (Solis BioDyne, Tartu, Estonia). qPCR was performed with the 5 × HOT FIREPol EvaGreen qPCR Supermix (Solis BioDyne) on the ABI 7300 real-time PCR system (ThermoFischer Scientific) to detect the intensity of sGFP expression. Specific primers (Suppl. Table 1) for RT-qPCR were used to detect sGFP and normalizing (reference) genes (MtGAPDH: At1g13440, FvGAPDH: ID07104 and NbGAPDH: At1g12900) which were selected based on the stability of housekeeping gene expression level (Expósito-Rodríguez et al. 2008;Liu et al. 2012Liu et al. , 2020. In the RT-qPCR analysis, we used the 2 −ΔΔCt method to quantify the relative changes in gene expression (Livak and Schmittgen 2001). To compare the intensity of sGFP gene expression between the positive control (CaMV35S::sGFP) and promoter deletion line::sGFP constructs, gene expression logarithmic fold change (log2LFC) was calculated. The 2 −ΔΔCt method and log2LFC were calculated by HTqPCR v3.11 (Dvinge and Bertone 2009) in R software (Gentleman et al. 2004;Huber et al. 2015). The Student's t test was performed using ΔΔCt values, and a p-value less than 0.05 was considered to be significant. Statistical analyses were conducted in GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). Results were exported into Microsoft Excel 365.
We compared the different promoter regions (TF and CREs) related to flowering, fruit development and ripening in tomato, A. thaliana and F. vesca (Suppl. Table 2, Suppl. Table 5). Table 1 shows the frequency of TFBS in the promoter sequences that play a role in flowering and fruit ripening. There were 16, 25, 7, 5, 34, 24 and 29 TFBS in the promoter sequences of MtSPR1-like2, FvSPR1-like2, FvSPR1-like1, AtSPR1-like2, MtSPT, FvSPT and AtSPT genes, respectively (Table 1). Table 2 shows the frequency of CREs in the promoter sequences that played a role in flowering and fruit ripening. There were 11, 25, 6, 1, 27, 26 and 16 CREs in the promoter sequences of MtSPT,FvSPT and AtSPT genes, respectively (Table 2). Based on the PLACE 30.0 database, CREs that were regulated by auxin, ethylene, GA 3 and cytokinin were classification. We identified 1, 8 and 1 CREs that were promoted by ethylene, auxin and GA 3 , respectively. Cytokinin did not promote CREs in these promoter regions.
The FvSPR500::pGWB604 and FvSPR1000:pGWB604 constructs did not work in tobacco leaves, but FvSPR2000:pGWB604 did. FvSPR2000::pGWB604 also expressed the sGFP gene in Micro-Tom fruit. We found ARF1, ARF2, ARF5 and ARF8 sites in the − 1067 to − 1059 bp region of the promoter deletion lines of the FvSPR1-like2 gene. These sites were not in the − 500 to − 1 bp and − 501 to − 1000 bp regions. The ARF family of TFs play a role in ARFmediated auxin signaling in the maturation of reproductive organs , perhaps, explaining why FvSPR2000::pGWB604 was the only construct that induced sGFP in tobacco leaves and tomato fruit. The FvSPT1000::pGWB604, FvSPT2000::pGWB604 and FvSPT3000::pGWB604 constructs worked in tobacco leaves, but FvSPT1000::pGWB604 did not work in Micro-Tom tomato fruit. The FvSPR500::pGWB604 construct did not work in Micro-Tom tomato fruit or in tobacco leaves. The FvSPR1000::pGWB604 construct had a lower sGFP gene expression intensity than FvSPR2000::pGWB604. This differential expression may have been caused by MYB59, WRKY25 and WRKY8 sites, which are regulated by ethylene (Li et al. 2006(Li et al. , 2011Chen et al. 2013). The ethylene-auxin interaction might have a role in regulating the promoter of the FvSPR gene, as occurs in tomato where there is an antagonistic effect between ethylene and auxin during tomato fruit ripening (Li et al. 2017). The FvSPR1000::pGWB604 construct does not have the MYB59, WRKY25 and WRKY8 sites because these are only found between the −1256 and − 1248 bp, − 1609 to − 1602 and − 1610 to − 1602 regions (Suppl . Table 2), respectively, which do not exist in the FvSPR2000::pGWB604 construct. The FvSPT1000::pGWB604, FvSPT2000::pGWB604 and FvSPT3000::pGWB604 constructs worked in tobacco leaves (Suppl. Figure 1), but the FvSPT1000::pGWB604 construct did not work in Micro-Tom tomato fruit (Suppl. Figure 2).

Conclusion for future biology
In our experiment, we reported CREs specific to various TFs in regions of putative FvSPT and FvSPR1-like2 genes by bioinformatic analysis. The promoter of the FvSPR1-like2 gene has the following: (1) MYB59, WRKY25 and WRKY8 TFs, which play a role in ethylene signaling; (2) ARF family of TFs, which play a role in ARF-mediated auxin signaling. The promoter of the FvSPT gene has the following: (1) ARR family of TFs, which play a role in cytokinin signaling; (2) ERF family of TFs, which play a role in ethylene signaling. The function and names of these sites and elements, as defined in JAPAR2020 and PLACE 30.0 databases, were also identified. The function of TFs and CREs were confirmed with promoter deletion lines and sGFP reporter gene constructs in tobacco leaves or tomato fruit by agroinjection.
In recent years, the use of transgenic techniques has led to improvements in many plant species due to identification of a large number of genes. Molecular researchers have made efforts to isolate tissue-specific promoters to increase the added value of transgenes. Transcriptional regulation is a most important goal in the post-genomic era by understanding the transcriptional factors in the promoter regions. There are several databases about analyzing, identifying and characterizing promoters, which currently available from different plant species. With the analyzing progress of promoter and TFs that has been achieved in the agricultural sector through current biotechnological and bioinformatic techniques, might be open a new door to new tissue-and stage-specific promoters in new genetically modified (GM) cultivars.
Acknowledgements The research was financed by the Higher Education Institutional Excellence Program (NKFIH-1150-6/2019) of the Ministry of Innovation and Technology in Hungary, within the framework of the Biotechnology thematic program of the University of Debrecen, and that of Szent István University (NKFIH-1159-6/2019) and a grant from the Hungarian Research Fund K (101195) entitled "Functional analysis of genes and their promoters identified during the fruit ripening of strawberry".

Conflicts of interest
The authors declare no conflicts of interest regarding this paper.
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