Characterization of the BnA10.tﬂ1 Gene Controls Determinate Inﬂorescence Trait in Brassica napus L.

: Determinate inﬂorescences have a signiﬁcant e ﬀ ect on the genetic improvement of rapeseed, so understanding the molecular function underlying the inﬂorescence trait may be beneﬁcial to oilseed breeding. A previous study found candidate gene BnTFL1 ( Terminal Flower 1 ) for control of the inﬂorescence trait on Brassica napus chromosome A10 (16,627–16,847 kb). However, little is known about the function of the BnTFL1 gene in B. napus . In this study, we ﬁrstly studied the formation of the shoot apical meristem and gene expression in indeterminate and determinate inﬂorescences; the results showed that the inﬂorescence architecture and BnA10.TFL1 expression showed signiﬁcant di ﬀ erences in the shoot apex at the budding stage. Then, two alleles (named BnA10.TFL1 gene from indeterminate and BnA10.tﬂ1 gene from determinate) were cloned and sequence-analyzed; the results suggest that the open reading frame of the alleles comprises 537 bp, encodes 178 amino acids containing a conserved phosphatidylethanolamine-binding protein (PEBP) domain, and shares high similarity with Arabidopsis thaliana TFL1. To analyze the function of BnA10.TFL1 , the BnA10.TFL1 gene was introduced into the determinate A. thaliana tﬂ1 mutant and B. napus 571 line by complementation experiment. The determinate traits were restored to indeterminate, and expression of BnA10.TFL1 was increased in the indeterminate shoot apex. These results reveal that BnA10.tﬂ1 is a gene controlling the determinate inﬂorescence trait. Moreover, the BnA10.TFL1 protein was localized to the nucleus, cytoplasm, and plasma membrane. Collectively, the results of this study help us to understand the molecular mechanism of determinate inﬂorescences and will provide a reliable research basis for the application of determinate inﬂorescences in B. napus . function of TFL1 in B. napus . In the present study, we mainly identiﬁed the function of BnA10.TFL1 from indeterminate inﬂorescence plants by the complementation test. The full-length sequence containing the BnA10.TFL1 gene was introduced into the determinate A. thaliana tﬂ1 mutant and the determinate B. napus 571 line. The results indicated that the BnA10.tﬂ1 gene may control the determinate inﬂorescence trait in B. napus . electron microscopy, para ﬃ n sectioning techniques, and qRT-PCR. The results showed that BnA10.tﬂ1 may be speciﬁcally expressed in the shoot apex, and down-regulation of its expression was observed in determinate inﬂorescence plants. Phylogenetic analysis showed that the BnA10.TFL1 protein has high similarity with A. thaliana TFL1. Further, when the BnA10.TFL1 gene from an indeterminate inﬂorescence plant was transferred to determinate inﬂorescence A. thaliana and B. napus , the determinate traits were turned to indeterminate. These results indicate that BnA10.tﬂ1 may control the determinate inﬂorescence trait. All these results taken together, this study may provide new insights into the molecular mechanism of and oilseed breeding for determinate inﬂorescences.


Plant Materials
In this study, two B. napus lines, the indeterminate inflorescence line 2014 and the determinate inflorescence line 4769, were used as materials. Using 2014 as the donor parent and 4769 as the recurrent parent, near-isogenic lines (NIL) were constructed after three consecutive selective backcrosses and selfing for two generations. These offspring were then divided into homozygous groups for indeterminate and determinate NIL. These plants were grown in an experimental field at Qinghai University, Xining, China. All required routine field management was carried out.
An early maturing B. napus line with determinate inflorescences, named 571 (carrying the 4769 allele at the BnA10.tfl1 locus), was used as the transformation recipient. We also purchased the A. thaliana CS3091 mutant (tfl1-2, determinate inflorescences) from the Arabidopsis Biological Resource Center (ABRC). Transgenic plants were grown in growth chambers (10,000 Lux) under 16 h light at 24 • C and 8 h dark at 16 • C. The total DNA of the plants was isolated using the CTAB (cetyltrimethylammonium bromide) method [22,23].

Paraffin Sectioning and Scanning Electron Microscopy Analysis
Previous studies have found that the determinate phenotype was caused by the development and differentiation of the shoot apical meristem (SAM) [3][4][5]14], To further understand the phenotypic differences of apical SAM, we observed the inflorescence SAM in homozygous indeterminate and determinate plants at different stage. Paraffin sectioning of the plant SAMs was performed as previously described [24]. The samples were treated with 50% FAA (Formalin-acetic acid-alcohol), then safranine-dyed, rinsed, dehydrated, cleared, infiltrated, embedded, sliced, sealed, and examined with a Nikon microscope (Nikon, Tokyo, Japan) [25]. Scanning electron microscopy (SEM) was performed following the previously described methods [26]: the SAMs were transferred to 2.5% glutaraldehyde and then rinsed, fixed again, and dehydrated. After drying and spraying with gold powder, they were imaged using a JSM-6610LV scanning electron microscope (JEOL Ltd., Tokyo, Japan).

Gene Cloning and Sequencing
Sequence of the gene was obtained in the BRAD (http://brassicadb.org/brad/) based on the homologous to TFL1 from A. thaliana (AT5G03840). Specific primers (TFL1-orf-F/TFL1-orf-R) were designed based on this sequence (Table S1). The gDNA and cDNA of BnA10.TFL1/BnA10.tfl1 were isolated from indeterminate and determinate plants, and the sequences were cloned using PMD18-T vector and E. coli DH5a methods. The six positive clones which were verified with M13-specific primers were sequenced by Sangon Biotech (Shanghai, China). To obtain proteins homologous to BnA10.TFL1 from other species, the BnA10.TFL1 amino acid sequence was deduced and downloaded using GeneDoc (http://www.nrbsc.org/gfx/genedoc/) and NCBI (https://blast.ncbi.nlm.nih.gov/) BlastP. The phylogenetic tree was analyzed using the neighbor-joining method and 1000 bootstrap replicates in MEGA 7.0 (https://www.megasoftware.net/).

Plasmid Construction
We explored the function of BnA10.TFL1 via complementation tests. The primers were designed with NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) using the BnaA10g26300D (TFL1 homologous) sequences from the BRAD. The enzyme restriction sites EcoRI and PstI and 15 bp sequences from both sides of the pCAMBIA2300 vector were added to the ends of the primers (Table S1). The fragment of genomic DNA that included the upstream, full-length gene, and downstream sequences was amplified using Phusion Hot Start High Fidelity DNA polymerase (NEB, Ipswich, MA, USA) with recombinant primers (CP525-F/CP525-R, Table S1) from the indeterminate inflorescence line 2014. The complementation plasmid pBnA10.TFL1:BnA10.TFL1 was produced using a sequenced genomic fragment and the pCAMBIA2300 vector that had been digested with EcoRI and PstI using a One Step Cloning Kit (Vazyme, Nanjing, China), as previously described [24]. The confirmed recombinant plasmid was transformed into GV3101 (Agrobacterium tumefaciens) and stored at −80 • C after adding sterilized glycerin.

Plant Transformation
For transformation of A. thaliana, we used the floral dip method described previously [27]. The fused construct was introduced into the A. thaliana tfl1-2 mutant. For transformation of B. napus, the complementation plasmid, pBnA10.TFL1:BnA10.TFL1, was transformed into the determinate inflorescence 571 through the Agrobacterium-tumefaciens-mediated hypocotyl method [28]. The positive transgenic plants were confirmed and identified by antibiotic selection and PCR (Table S1).

Subcellular Localization of the BnA10.TFL1 Protein
To analyze the subcellular localization of the BnA10.TFL1 protein, primers (without the stop codon "TAA") relating to subcellular localization were designed with Primer5 using the cDNA sequence. Then, the enzyme restriction sites XbaI and BamHI and the homologous sequences from the pCAMBIA3301-GFP vector were added to the ends of the primer. The cDNA of BnA10.TFL1 was amplified using Phusion Hot Start High Fidelity DNA polymerase (NEB, Ipswich, MA, USA) with recombinant primers (XWB-3301-XbaIF/XWB-3301-BamHIR, Table S1) from the indeterminate inflorescence line 2014. The confirmed sequence was inserted into the modified pCAMBIA3301-GFP vector (containing the GFP gene) that had been digested by XbaI and BamHI using a One Step Cloning Kit (Vazyme, Nanjing, China). The fusion plasmid BnA10.TFL1:GFP, transformed into GV3101 (Agrobacterium tumefaciens), was introduced into the leaf epidermal cells of Nicotiana benthamiana by injection methods, as previously described [31]. The GFP signals were obtained using a confocal laser scanning microscope (Leica, Wetzlar, Germany).

Observations of the SAM Apex in B. napus
To find the developmental stage at which SAM architecture variation originates in the determinate trait, we observed the formation of the SAM in indeterminate and determinate plants by scanning electron microscopy and paraffin sectioning techniques ( Figure 1). The SAM arquitecture showed no differences between the indeterminate (Figure 1a,b,g,h) and determinate (Figure 1d,e,j,k) plants during both the vegetative meristem (two leaves) and early inflorescence meristem stages (four leaves). At the budding stage (eight leaves), we observed that SAM of indeterminate inflorescence (Figure 1c,i) was similar to that of two and four leaves stages. However SAM of the determinate inflorescence ( Figure 1f,l) showed morphology similar to the floral organ. These findings can help us to provide a reference for the observation and sampling of determinate inflorescences in B. napus.

Expression of BnA10.TFL1 in Different Tissues of B. napus
The expression levels of BnA10.TFL1 were examined using qRT-PCR in different tissues between the indeterminate and determinate NIL, including the shoot apex at the budding stage, roots, stems, leaves, and flowers ( Figure 2). The expression levels of BnA10.TFL1 in the shoot apex were higher than those in the other tissues. However, BnA10.TFL1 was significantly up-regulated in the shoot apex of the indeterminate NIL compared with the determinate NIL at the budding stage ( Figure 2, ** p < 0.01). The results indicated that BnA10.TFL1 may be specifically expressed in the shoot apex. This is similar to the expression of the TFL1 gene in A. thaliana. Thus, the expression levels of BnA10.TFL1 may cause changes in the determinate inflorescence trait. A previous study reported that BnTFL1, being homologous to the TFL1 gene of A. thaliana, is a candidate gene for the inflorescence trait in B. napus chromosome A10 [3]. This candidate gene was isolated from indeterminate and determinate plants, and the two alleles were named BnA10.TFL1 and BnA10.tfl1. The sequences of the BnA10.TFL1/BnA10.tfl1 gene were cloned from the indeterminate line 2014 and the determinate lines 4769/571 using the specific primer (TFL1-orf-F/TFL1-orf-R) by PCR amplification (Table S1). In 2014 (indeterminate) (Figure 3a), 1066 bp gDNA and 537 bp cDNA fragment sequences of BnA10.TFL1 were found, and these included four exons and three introns by Blastn analysis. In 4769/571 (determinate) (Figure 3a), the sequences of BnA10.tfl1 also had four exons and three introns; the gDNA and full-length cDNA of the gene comprised 1072 bp and 537 bp, respectively. Sequence analysis of the gDNA and cDNA of both BnA10.TFL1 and BnA10.tfl1 revealed that there are differences between 2014 (indeterminate) and 4769/571 (determinate) (Figure 3a): there are four single-nucleotide polymorphism (SNP) mutations in the exons and 18 SNP/Indel in the introns (15 SNP mutations, two insertions of 6 bp and 2 bp, and one instance of 2 bp deletion).
The amino acids were analyzed using GeneDoc and NCBI BlastP. The results indicated that the BnA10.TFL1 and BnA10.tfl1 alleles encode 178 amino acids; only two different amino acids were observed i.e., the phenylalanine (F) and leucine (L) in indeterminate 2014 becomes the leucine (L) and phenylalanine (F) in determinate 4769 (571) (Figure 3b). In addition, this protein contains a conserved phosphatidylethanolamine-binding protein (PEBP) domain and belongs to the PEBP family, and the amino acid sequence of BnA10.TFL1 showed 85% identity to A. thaliana TFL1 (AT5G03840) (Figure 3b). The phylogenetic analysis showed that the BnA10.TFL1 and TFL1 proteins from other plant species can be divided into two separate clades; BnA10.TFL1 was grouped closely with AtTFL1 (AAM27947.1) from A. thaliana (Figure 3c).  (Figure 4c,d). These results showed that BnA10.TFL1 has similar function to TFL1.

Subcellular Localization of the BnA10.TFL1 Protein
To confirm the subcellular localization of the BnA10.TFL1 protein in B. napus, the fusion plasmid BnA10.TFL1-GFP was transiently expressed in the leaves of tobacco (N. benthamiana). The BnA10.TFL1-GFP protein signals were localized to the nucleus, cytoplasm, and plasma membrane ( Figure 6). This provides a further basis for research into the molecular function of BnA10.TFL1.

Subcellular Localization of the BnA10.TFL1 Protein
To confirm the subcellular localization of the BnA10.TFL1 protein in B. napus, the fusion plasmid BnA10.TFL1-GFP was transiently expressed in the leaves of tobacco (N. benthamiana). The BnA10.TFL1-GFP protein signals were localized to the nucleus, cytoplasm, and plasma membrane ( Figure 6). This provides a further basis for research into the molecular function of BnA10.TFL1. The ER-mCherry was used as a marker for ER-rb CD-960, FIB2-mCherry was used as a nuclear marker, 35S-GFP + ER-mCherry and 35S-GFP + FIB2-mCherry were used as control; the hollow white arrow refers to the nuclear, the solid white arrow refers to the filamentous substance in the cytoplasm, the solid blue arrow refers to plasma membrane; scale bar = 20 µm.

Discussion
Plant growth habits (in particular, indeterminate or determinate inflorescences) have an important effect on the architecture and yield of plants such as oilseed [3,6,32], soybean [33,34], and tomato [35]. The development of the SAM determines the plant growth habit; the SAM is located at the top of the stem of the plant and is mainly composed of cells with unlimited growth capacity [36][37][38]. These SAM cells produce leaf buds, axillary buds, and flower buds that are closely related to the establishment of plant morphology and the economically important harvestable organs [39][40][41]. The determinate inflorescence trait was caused by development and differentiation of apex SAM [4,5,14]. In B. napus, the candidate gene for the inflorescence trait was identified [3]. In this study, the BnA10.TFL1 was differentially more expressed in the inflorescence apex than in other tissues ( Figure 2). Therefore, further research on the function of the BnA10.TFL1 gene is necessary for oilseed breeding.
In this study, we observed the morphological characteristics of determinate and indeterminate inflorescences at different growth stages of the SAM in B. napus. The results showed that the SAMs of determinate inflorescences (Figure 1d,e,j,k) were structurally similar to those of indeterminate inflorescences (Figure 1a,b,g,h) at the vegetative meristem (two leaves) and early inflorescence meristem stages (four leaves). However, the SAM of determinate inflorescences (Figure 1f,l) showed differences compared to indeterminate (Figure 1c,i) during the budding period (eight leaves). The apex of the determinate SAM (Figure 1f,l) showed an organ (similar to the shape of a floral organ) which may cause determinate growth, in contrast to the SAM with unlimited differentiation capacity seen in the indeterminate plants (Figure 1c,i). In other reports, the determinate SAMs were observed to form a terminal flower in the A. thaliana tfl1 mutant, halting SAM differentiation [4,8]. Meanwhile, the centroradialis mutant apex also developed a flower, terminating the normally indeterminate inflorescences in Antirrhinum [38]. In Sesamum indicum L., the shoot apex of determinate inflorescences showed a flower cluster [42]. Although the phenotypes of determinate inflorescences in these plants were different, they shared a common feature: the apex SAM continued to differentiate into new floral tissue, resulting in indeterminate plants becoming determinate.
Li et al. [3] reported that a candidate gene controlling plant growth habits, found on B. napus chromosome A10, was homologous to A. thaliana TFL1. In our research, the alleles BnA10.TFL1 and BnA10.tfl were isolated from indeterminate and determinate plants, respectively, by PCR. The BnA10.TFL1/BnA10.tfl had a 537 bp ORF (open reading frame) which included four exons and three introns. The amino acid sequences of the two alleles encoded a protein of 178 amino acids and contained a conserved PEBP domain (Figure 3a,b). The phylogenetic analysis showed that the BnA10.TFL1 and AtTFL1 proteins are highly homologous with 85% identities (Figure 3b,c). Cloned TFL1 in other plants, such as in rice [43], soybean [44], apple [45], dogwood [46], and Jatropha curcas [47], showed similar results. These studies indicate that BnA10.TFL1 may share a similar function with TFL1.
TFL1 has been reported to not only control inflorescence meristem identity but to also impact inflorescence architecture and the formation of the reproductive phase at the SAM [48,49]. The functions of TFL1 homologous were studied in some plants. In Vigna radiata, Li and Ding et al. found that the VrDet1 gene was transformed into the A. thaliana tfl1 mutant, the progeny transgenic plants showed indeterminate traits similar to wild-type A. thaliana, and the expression level of VrDet1 at the SAMs of indeterminate plants was higher than that in determinate plants [10]. In Punica granatum L., PgTFL1 was transformed into the tfl1-14 mutant, and the determinate trait restored growth to an indeterminate plant [50]. In this study, a full-length fragment of genomic DNA containing BnA10.TFL1 was cloned from indeterminate plants and transformed into the determinate tfl1-2 mutant (Figure 4b). The positive transgenic plants (Figure 4c,d) were restored to indeterminate inflorescences similar to wild-type A. thaliana (Figure 4a). The complementation plasmid pBnA10.TFL1:BnA10.TFL1 was also introduced into the determinate B. napus line 571 (Figure 5b), and the results showed that the abnormal organ at the apex of the determinate SAM was absent, confirming the restoration of the indeterminate phenotype (Figure 5c,d). Additionally, the expression of BnA10.TFL1 was also up-regulated in the indeterminate line 2014 and in the positive transgenic plants compared with in the determinate line 571 at the budding stage ( Figure 5e). Thus, BnA10.TFL1 may have similar functions to A. thaliana TFL1, which controls inflorescence architecture, as it restored determinate inflorescences in an A. thaliana tfl1 mutant that has a terminal flower. Importantly, it also restored determinate inflorescences in the B. napus line 571 that showed a floran organ at the SAM. The low expression of TFL1 gene (AT5G03840) in A. thaliana was caused by the single nucleotide substitution G to A at 3 -end of the second exon. In cucumber, what causes (mutation and deletion) to cause down-regulation of the CsTFL1 is unclear [20]. In this study, whether the amino acid, promoter and others changed between indeterminate and determinate may cause binding to downstream genes and expression differences that remains unknown. This will be subject to further study. Previous studies showed that TFL1 is expressed mainly in the SAM, and mobile TFL1 may play a role in vesicle trafficking [48,51]. Hanano and Goto [52] found that the TFL1 protein is localized to the nucleus and cytoplasm. However, CmTFL1c from Chrysanthemum morifolium also belongs to the nucleus and plasma membrane [16]. In this study, BnA10.TFL1 protein was found to localize to the nucleus, cytoplasm, and plasma membrane ( Figure 6). Therefore, our findings indicate that BnA10.tfl1 acts an important gene controlling determinate inflorescences in B. napus.

Conclusions
In summary, the present study revealed mainly that the function of BnA10.tfl1 controls determinate inflorescences in B. napus. First of all, we analyzed the morphological development of SAMs and gene expression by scanning electron microscopy, paraffin sectioning techniques, and qRT-PCR. The results showed that BnA10.tfl1 may be specifically expressed in the shoot apex, and down-regulation of its expression was observed in determinate inflorescence plants. Phylogenetic analysis showed that the BnA10.TFL1 protein has high similarity with A. thaliana TFL1. Further, when the BnA10.TFL1 gene from an indeterminate inflorescence plant was transferred to determinate inflorescence A. thaliana and B. napus, the determinate traits were turned to indeterminate. These results indicate that BnA10.tfl1 may control the determinate inflorescence trait. All these results taken together, this study may provide new insights into the molecular mechanism of and oilseed breeding for determinate inflorescences.