Cloning, Characterization, and Overexpression in Arabidopsis to Determine the Function of SPL Genes from Woodland Strawberry (Fragaria Vesca)

doi:10.21203/rs.3.rs-620026/v1

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Cloning, Characterization, and Overexpression in Arabidopsis to Determine the Function of SPL Genes from Woodland Strawberry (Fragaria Vesca)

https://doi.org/10.21203/rs.3.rs-620026/v1

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SQUAMOSA promoter binding protein-like (SPL) proteins is a class of plant specific transcription factors that play important roles during plant development. However, the majority of SPL genes in strawberry are functionally uncharacterized. In this study, three SPL genes, i.e. FvSPL1, FvSPL2, and FvSPL11 (FvSPL1/2/11), from woodland strawberry were cloned and characterized. Phylogenetic analysis with SPL genes from Arabidopsis, tomato and chrysanthemum indicated that FvSPL1/2/11 were clustered into the same group with those of miR156 target site located at the 3’-untranslated region (UTR). Further biochemical analysis indicated that FvSPL1 was exclusively localized in the nucleus. Electrophoretic mobility shift assay demonstrated FvSPL1 could specifically recognized the GTAC motif. Transcriptional activity analysis showed FvSPL1 was a transcriptional activator that could activate the expression of FvAP1 gene. Finally, all of the transgenic Arabidopsis that overexpression the three FvSPL genes were exhibited significantly early flowering phenotype. Taken together, our study indicated that FvSPL1/2/11 similar to their orthologs in Arabidopsis mainly functions in regulating plant flowering. These results enriched our understanding to the functions of SPL genes in strawberry and might be utilized for strawberry flowering time manipulation in the future.

Plant Molecular Biology and Genetics

Woodland strawberry

FvSPL genes

flowering

transcription factors

transcriptional activation

MiR156-targeted woodland strawberry SPL genes, FvSPL1, FvSPL2, FvSPL11, regulate plant flowering.

Floral transition is the prerequisite for plants to complete reproductive growth, which is regulated by both external environmental factors and internal genetic factors (He et al. 2010). At present, a number of floral induction pathways, such as autonomous pathway (Wu et al. 2020), photoperiod pathway (Song et al. 2015), gibberellin (GA) pathway (He et al. 2020), age pathway (Wang 2014) and ambient temperature pathway (Fornara et al. 2010), have been identified in various plants. Numerous studies have indicated that these floral induction pathways are not separated but act synergistically to regulate plant flowering. Many key integrators that link different pathways have been identified. For example, several studies have demonstrated GA could promote the expression of FLOWERING LOCUS T (FT), the key regulator of photoperiod pathway, under long day condition (Song et al. 2013; Yamaguchi 2008). In addition, SQUAMOSA promoter binding like proteins (SPLs), the key regulators of age pathway were also found integrated with GA pathway through interaction with DELLA proteins (Yu et al. 2012). Moreover, Jung et al identified several SPLs regulate flowering through integration with photoperiod pathway by directly interaction with FD protein (Jung et al. 2016).

SPL gene family encodes a class of plant-specific transcription factor that have been identified nearly in all plant species. This class of TFs were found play vital roles in many aspects during plant growth and development, such as phase transition i.e. changes from vegetative stage to reproductive stage (Jung et al. 2011), phytohormone signal transduction (Zhang et al. 2007), embryo formation (Unte et al. 2003), plant architecture (Miura et al. 2010) and fruit development (Manning et al. 2006). In Arabidopsis, 11 SPLs were found targeted by miR156. Functional analysis indicated that nearly all these miR156 targeted SPLs except AtSPL6 and AtSPL13 were acted as phase transition accelerators (Wang et al. 2015). In addition, studies have found these miR156 targeted AtSPLs can be classed into two groups which represented by AtSPL3 and AtSPL9, respectively (Yu et al. 2012). The AtSPL3 group contains three relative small genes, i.e. AtSPL3, AtSPL4 and AtSPL5 (AtSPL3/4/5), that encode proteins less than 200 amino acids residues in length. Beside the size of AtSPL3/4/5 are relative smaller, the miR156 target site in the three genes was found all located at the 3’-untranslated region (UTR). AtSPL3/4/5 have been found mainly involved in phase transition. AtSPL3/4/5 was reported could directly combine to the promoter and activate the expression of floral meristem genes such as APETALA1 (AP1), FRUITFULL (FUL) and LEAFY (LFY) to promote plant flowering (Yamaguchi et al. 2009). Latter, AtSPL3/4/5 were found link age pathway and photoperiod pathway to mediate plant flowering through physically interaction with FD (Jung et al. 2016). However, genes in AtSPL9 group have been found possess diverse functions. For example, AtSPL9 not only play roles in phase transition (Schwarz et al. 2008; Wu et al. 2006), but also could negatively regulate anthocyanin biosynthesis (Gou et al. 2011). In chrysanthemum, several miR156 targeted SPL genes were also been identified play roles in regulation flowering. For instance, Wei et al found silencing a nuclear factor gene, CmNF-YB8, leading to down-regulation of miR156, which resulted in up-regulation of several CmSPLs such as CmSPL3, CmSPL5 and CmSPL9, consequently resulted in early flowering phenotype (Wei et al. 2017).

In our previous study, we identified nine miR156 targeted FvSPL genes in woodland strawberry. Further analysis found among the nine FvSPLs the miR156 target site of three FvSPLs, FvSPL1, FvSPL2 and FvSPL11 (FvSPL1/2/11), were located in the 3’-UTR and they were highly expressed in flower organs (Xiong et al. 2018). These characteristics were very similar to AtSPL3/4/5. As studies in Arabidopsis have demonstrated AtSPL3/4/5 were mainly function in regulating plants flowering (Yamaguchi et al. 2009). Therefore, we functionally analyzed FvSPL1/2/11 in this study. We first cloned the three genes from woodland strawberry, phylogenetic analysis showed FvSPL1/2/11 were closely related to the SPL genes with miR156 target site at 3’-UTR from Arabidopsis, tomato and chrysanthemum. Due to the functional redundancy of AtSPL3/4/5, we further analyzed the biological functions of FvSPL1 in detail as representative. Subcellular localization assay found FvSPL1 was exclusively localized in the nucleus. Both in vivo and in vitro assays demonstrated that FvSPL1 could recognize and bind to the GTAC motif, further transcriptional activity assay indicated that FvSPL1 is a transcriptional activator. Finally, we ectopically expressed the three FvSPLs in Arabidopsis, respectively. All the transgenic plants exhibit dramatically early flowering phenotype, several flowering related genes such as AtAP1, AtFUL, AtAGL42, AtCO and AtLFY were significantly upregulated. These results demonstrated the specific roles of FvSPL1/2/11 in plant phase transition, which provides valuable information for investigating their role plant flowering and fruit development of strawberry.

1. Plant materials and growth conditions

The woodland strawberry ‘Hawaii 4’ seeds were obtained from the National Clonal Germplasm Repository (Corvallis, Oregon). Nicotiana benthamiana was used for Agroinfiltration assay. The ecotype Col-0 of Arabidopsis thaliana was used for genetic transformation of FvSPL1/2/11. All plants were grown in greenhouse at 22℃ in 16h light/8h dark cycles. All plant materials were collected and stored at -80℃ after frozen by liquid nitrogen.

2. RNA extraction and the amplification of FvSPL genes

Plant total RNA was extracted using Plant Total RNA Isolation Kit Plus (Foregene, Chengdu, China) following the user manual instruction. RNA quality was evaluated by gel electrophoresis and spectrophotometry. First-strand cDNAs was synthesized by the Prime Script RT reagent kit with gDNA eraser (TAKARA, Dalian, China). Full-length CDS of FvSPL1/2/11 were amplified and further verified by sequencing. The primers were listed in Supplemental Table 1.

3. Sequence analysis

The protein sequences of FvSPL1/2/11 were identified from F.vesca v1.1. The protein sequences of Solanum lycopersicum were retrieved from phytozome v12 (https://phytozome.jgi.doe.gov/pz/portal.html). For Chrysanthemum morifolium, the protein of SPLs were obtained according to Song et al. (Song et al. 2016). The protein sequences of Arabidopsis SPLs were collected from TAIR database (https://www.arabidopsis.org). Then, the SBP domain (PF03110) of SPL proteins were retrieved using Pfam (http://pfam.xfam.org). The phylogenetic tree based on SBP domain was constructed by MEGA7.0 with 1000 bootstrap replicates (Kumar. et al. 2016).

4. Subcellular localization of FvSPL1

The FvSPL1 CDS was cloned in frame with green fluorescent protein (GFP) tag in the binary vector pJX002. Subcellular localization analysis of FvSPL1 was performed as described by Xiong et al (Xiong et al. 2019). GFP fluorescence signal was observed by laser scanning microscopy (LSM800, Zeiss, Germany) within 2-5 days after injection, transient expression assays were repeated at least three times.

5. Transcriptional activity analysis

The transcriptional activity of FvSPL1 was determined by dual-luciferase assay system in tobacco leaves. Vectors construction and transcriptional activity assay were performed as described by Cheng et al (Cheng et al. 2017).

To analyze the trans-activation of FvSPL1 to FvAP1 gene. A 1000 bp fragment of the FvAP1 promoter that immediately upstream from the start codon was inserted into the reporter vector pGreenII 0800-LUC, and FvSPL1 was cloned into the effector vector pEAQ (Fig. 5a). The trans-activation activity of FvSPL1 to FvAP1 was analyzed by dual-luciferase system as described by Cheng et al (Cheng et al. 2017).

After two days of co-cultivation, the ratio of LUC/REN was measured using the Dual-Luciferase^®Reporter Assay System kit (Promega, USA), the ratio of LUC/REN was detected by GloMax 20 ~ 20 photoluminescence detector (Promega, USA). Each set of data was repeated three times.

6. Protein extraction and Electrophoretic mobility shift assay

The CDS of FvSPL1 was inserted into prokaryotic expression vector pGEX-4T-1. The FvSPL1-GST fusion protein was induced by 0.3 mM isopropyl thio-β-D-galactoside (IPTG) at 37°C for 4 hours and further purified by glutathione-super flow resin (Fig. 3a).

A 47 bp fragment of FvAP1 promoter containing the second GTAC motif was chosen as probe to verify the specific binding ability of FvSPL1 to this motif. Probe was labeled by biotin at 3’ end. The EMSA experiment was performed using the Chemiluminescent EMSA Kit (GS009) according to the manufacturer’s instructions (Beyotime, China). Labeled probes were detected by chemiluminescence method.

7. Overexpression of FvSPLs in Arabidopsis

Full-length cDNA of FvSPL1/2/11 was respectively inserted into binary vector pJX001 under the control of 35S promoter to generate overexpression constructs. The constructs were transformed into Arabidopsis (Col-0) using floral-dip method (Topping et al. 1995). The seeds of transgenic Arabidopsis were harvested and screened on 1/2 MS medium containing 50 mg/L hygromycin. T₃ generation homozygous transgenic lines were selected for further analysis. Primers are listed in Supplemental Table 1.

8. Phenotypic and gene expression analysis of the transgenic Arabidopsis

The homozygous T3 generation FvSPL1/2/11 overexpression plants were used for phenotypic analysis. Flowering time and leaves numbers was counted at the day when the first bud emergence after germination.

Two week olds Arabidopsis plants were collected to detect the expression level of flowering-related genes. qRT-PCR was performed with Light Cycler480 II (Roche, Switzerland) using SYBR Premix Ex Taq II (TAKARA, Dalian, China). Atβ-tubulin2 was used as an internal reference gene to normalize the expression of flowering-related genes. Two-step PCR amplification standard procedure through the following steps: 95℃/30s to predenaturation, 95℃/5s, 60℃/30s, 40 cycles. Relative expression levels of the detected genes were calculated by 2^-^△△^Ctmethod. All quantitative real-time primers were listed in Supplemental Table 1.

9. Statistical analyses

IBM SPSS Statistics v25 was used to analyzed the data by Student’s t-test. Data were presented as the mean ± SD. * and ** represent P < 0.05 and P < 0.01 respectively.

1. Sequence analysis of FvSPL1/2/11

Our previous study indicated that FvSPL1/2/11 were highly expressed in flower organs. Therefore, we cloned the three genes from flower of woodland strawberry ‘Hawaii 4’. Sequence analysis showed that FvSPL1/2/11 encodes relative small protein containing 187, 157 and 178 amino acid, respectively. To further identify the evolutionary relationship between FvSPL1/2/11 and SPL genes of other species, the phylogenetic analysis was performed based on SBP domain of the SPL genes from woodland strawberry, chrysanthemum, tomato and Arabidopsis. The results indicated that FvSPL1/2/11 were divided into the same group with CNR, SlySBP3, SlySBP4 from tomato, AtSPL3/4/5 from Arabidopsis and CmSPL4, CmSPL5 from chrysanthemum (Fig. 1).

2. Subcellular localization and transcriptional activity of FvSPL1

In Arabidopsis, AtSPL3/4/5 were found redundantly regulate vegetative to productive phase transition. Here, we toke FvSPL1, the homolog of AtSPL3, as representative to analyze its biochemical functions. To determine the express location of FvSPL1, we transiently expressed GFP fused FvSPL1 in tobacco leaves through Agroinfiltration. As shown in Fig. 2a, FvSPL1-GFP is exclusively localized in the nucleus, while the GFP control is distributed both at cytoplasm and nucleus. We further analyzed the transcriptional activity of FvSPL1 by dual-luciferase assay system. The results indicated that the value of LUC/REN ratio of FvSPL1 was 1.5 times higher compared to the empty vector control (Fig. 2c). These results demonstrated that FvSPL1 is a nuclear protein possesses transcriptional activation activity.

3. FvSPL1 recognizes and binds to the GTAC motif both in vitro

The first identified two SPL genes, SBP1 and SBP2, were isolated from Antirrhinum majus as they specifically bind to the core GTAC motif in the promoter of SQUAMOSA (Klein et al. 1996). Here, we also observed the existence of the same core motifs in the promoter region of several flowering genes. For example, there are four GTAC motifs in the promoter of FvAP1 (Fig. 3b). Therefore, we synthesized the second fragment of FvAP1 promoter that contains the GTAC motif to detect whether FvSPL1 could specifically bind to it by electrophoretic mobility shift assay (EMSA). The results showed that GST-FvSPL1 recombinant protein could specifically recognize and bind to the GTAC motif in the promoter of FvAP1 in vitro (Fig. 3b).

4. FvSPL1 activates the transcription of FvAP1 in vivo

As we have confirmed FvSPL1 is a transcriptional activator that could specifically binds to the GTAC motif in the promoter of FvAP1. Therefore, we further applied dual-luciferase assay system to determine whether FvSPL1 could regulate the expression of FvAP1 in vivo. As shown in Fig. 4b, the value of LUC/REN was significantly higher in sample that co-transformed FvSPL1 and the promoter of FvAP1 than those of in empty effector control. These results verified FvSPL1 could positively regulate the transcription of FvAP1 in vivo, and supports the premise that FvSPL1 has functional role in the regulation plants flowering.

5. Ectopic expression of FvSPLs in Arabidopsis accelerates plant flowering

To further analyze the biological functions of three FvSPLs in plants, we respectively generated overexpression constructs of the three genes that under the control of CaMV35 promoter then further genetically transformed them in Arabidopsis. All transgenic lines that overexpression of the three genes exhibited dramatically early floweirng phenotype under long day conditions compared to the wild type (Fig. 5; Supplementary Fig. 1 and Supplementary Fig. 2). For example, transgenic lines overexpression FvSPL1, FvSPL2 and FvSPL11 flowering approximate at the 16th, 17th and 17th days, while the wild type plants flowering at the 23th days.

As the transgenic plants significantly promoted flowering, we therefore detected the expression levels of several flowering-related genes in Arabidopsis by qRT-PCR. As shown in Fig. 5c, the expression levels of AtFUL, AtGL42, AtLFY and AtAP1 increased significantly in FvSPL1 overexpression lines, especially AtAP1, which was up to 14 times higher than that of the control. However, in FvSPL1 overexpression lines AtCO was only up-regulated about three folds higher. Similarly, all these five detected genes were up regulated in FvSPL2 and FvSPL11 overexpression lines, despite the expression level of individual gene was quite different in each lines. For example, the expression level of AtCO in FvSPL2 overexpression lines were much higher than those of in other two lines, but the expression level of AtAP1 in the three transgenic plant lines were quite similar.

As a class of plant specific transcription factors SPLs has been extensively studied in model plants. For example, in Arabidopsis, AtSPL7 was found regulating Cu homeostasis (Zhang et al. 2014). AtSPL8 play roles in pollen sac development (Unte et al. 2003). AtSPL1 and AtSPL12 functions in regulating plant thermotolerance at reproductive stage (Chao et al. 2017), and almost all the miR156 targeted AtSPLs play role in phase transition (Wang et al. 2015). In rice, OsSPL3 regulates crown root development (Shao et al. 2019), OsSPL13 and OsSPL16 function in regulating grain size (Si et al. 2016; Wang et al. 2012), and OsSPL14 was identified play important role in regulating plant architecture (Jiao et al. 2010; Miura et al. 2010). In tomato, Colorless nonripening (CNR), the ortholog of AtSPL3, was found essential for fruit ripening (Manning et al. 2006). However, the function of SPLs in strawberry are still largely elusive. In our previous study, we observed FvSPL1/2/11 were specifically expressed in flower organs. In addition, the target site of miR156 in FvSPL1/2/11 also located at their 3’-UTR region similar to AtSPL3/4/5 (Xiong et al. 2018). Therefore, we cloned these three gene from woodland strawberry for further analysis. Phylogenic analysis with SPL genes from Arabidopsis, tomato and chrysanthemum indicated that all SPLs with miR156 target site at 3’-UTR were classed into the same group. This information implying FvSPL1/2/11 might possess similar functions with AtSPL3/4/5.

Due to the functional redundancy between AtSPL3/4/5, here, we only take FvSPL1 as representative for detail analysis. We first detected the subcellular localization of FvSPL1 in tobacco epidermal cells by Agroinfiltration. The results shown that FvSPL1 was exclusively localized in the nucleus (Fig. 2a). Further transcriptional activity assay indicated that FvSPL1 has transcriptional activation capacity (Fig. 2c), which confirmed FvSPL1 is a transcriptional activator that could activate the expression of its downstream genes. SPL genes were originally cloned from A. majus because they were found binding to the GTAC core motif of the promoter of SQAUMSA gene (Klein et al. 1996). Following studies about SPL genes in plants also demonstrated that the SBP domain in required and sufficient binding to the GTAC motif. For example, in Arabidopsis, SPL3/4/5 were found could directly bind to GTAC motif in the promoters of several flowering genes such as LFY, FUL and AP1 to regulate plant flowering (Yamaguchi et al. 2009). In this study, we also observed the existence of several GTAC motifs in the promoter region of several flowering homolog genes in woodland strawberry. Therefore, we utilized EMSA to verify the specific binding ability of FvSPL1 to the GTAC motif in the promoter of FvAP1. In addition, in vivo assay by luciferase system also confirmed FvSPL1 could bind the FvAP1 promoter and activate its expression.

To explore the biological functions of FvSPL1/2/11, we ectopically expressed the three genes in Arabidopsis, respectively. As expected, transgenic plants overexpression the three genes were significantly accelerated flowering compared to the wild type plants under long day condition (Fig. 5; Supplementary Fig. 1 and Supplementary Fig. 2). Further qRT-PCR analysis indicated that several flowering genes such as AtFUL, AtCO, AtAGL42, AtLFY and AtAP1 were significantly up regulated in these transgenic lines (Fig. 5c; Supplementary Fig. 1c and Supplementary Fig. 2c), despite the expression level of these genes were a little bit different. These results confirmed that FvSPL1/2/11 have similar function to AtSPL3/4/5, and CmSPL5, because up regulation these genes lead to early flowering phenotype in the corresponding plant. Interestingly, no other phenotypic changes were observed in these transgenic Arabidopsis lines except early flowering. This phenomenon confirmed that the miR156 targeted small size group SPL genes mainly function in regulating plant flowering. As we have not obtained transgenic strawberry plants that overexpression FvSPL1/2/11, we could not make conclusions that the three genes have similar function to tomato CNR that could regulate strawberry fruit development. It would be worthy to investigating the function of FvSPL1/2/11 in strawberry plants in the future.

In this study, we cloned and characterized three miR156 targeted SPL genes, FvSPL1/2/11, from woodland strawberry. Functional analysis indicated that FvSPL1 is a transcription activator that upregulate the expression of flowering genes through specifically binding to the GTAC motif in their promoter. Further genetics analysis confirmed transgenic Arabidopsis overexpression the three genes all dramatically promoted flowering by up regulation flowering related genes. These results indicated that FvSPL1/2/11 specifically function in regulating plant flowering.

Acknowledgements We thank Dr. Jian-Ye Chen (South China Agricultural University) for presenting the Luciferase Assay System’s vectors. This study was supported in part by the open funds of the State Key Laboratory of Crop Genetics and Germplasm Enhancement (ZW201813) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Author contribution statement JSX and ZMC conceived and designed research. YBB performed the experiments and data analysis. MRL and CJM helped in experimental works and data analysis. YBB drafted the manuscript. JSX and ZMC revised and finalized the manuscript. All authors reviewed and approved the final manuscript.

Conflict of interest The authors declare that they have no conflict of interest.

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SupplementaryTable1.xls
Supplementary Table 1. Primers used in this study.
supplementaryFig.1.tif
Supplementary Figure 1. Overexpression of FvSPL2 in Arabidopsis accelerates flowering. (a) Phenotypes of WT and FvSPL2-OE transgenic Arabidopsis plants. Two FvSPL2 overexpression lines, L12 and L14, were selected as representatives. Total leaves numbers were calculated in WT and FvSPL2-OE lines. (b) Comparison of FvSPL2-OE lines and WT in flowering time. Ten FvSPL2-OE lines were calculated. (c) Relative expression levels of flowering-related genes in WT and FvSPL2-OE lines. Two FvSPL2 overexpression lines, L12 and L14, were selected as representatives. * Represents significant difference by Student’s t-test (n= 10, P < 0.05). ** Represents extremely significant difference by Student’s t-test (n=10, P < 0.01).
supplementaryFig.2.tif
Supplementary Figure 2. Overexpression of FvSPL11 in Arabidopsis accelerates flowering. (a) Phenotypes of WT and FvSPL11-OE transgenic Arabidopsis plants. Two FvSPL11 overexpression lines, L7 and L8, were selected as representatives. Total leaves numbers were calculated in WT and FvSPL11-OE lines. (b) Comparison of FvSPL11-OE lines and WT in flowering time. Eleven FvSPL11-OE lines were calculated. (c) Relative expression levels of flowering-related genes in WT and FvSPL11-OE lines. Two FvSPL11 overexpression lines, L7 and L8, were selected as representatives. * Represents significant difference by Student’s t-test (n= 10, P < 0.05). ** Represents extremely significant difference by Student’s t-test (n=10, P < 0.01).

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Cloning, Characterization, and Overexpression in Arabidopsis to Determine the Function of SPL Genes from Woodland Strawberry (Fragaria Vesca)

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