The miR156-Targeted SQUAMOSA PROMOTER BINDING PROTEIN (PmSBP) Transcription Factor Regulates the Flowering Time by Binding to the Promoter of SUPPRESSOR OF OVEREXPRESSION OF CO1 (PmSOC1) in Prunus mume

Prunus mume, a famous perennial ornamental plant and fruit tree in Asia, blooms in winter or early spring in the Yangtze River area. The flowering time directly determines its ornamental and economic value, so it is of great significance to study the molecular mechanism of flowering time. SQUAMOSA PROMOTER BINDING PROTEIN (SBP), often regulated by miR156, is an important flowering regulator, although its function is unknown in P. mume. Here, 11 miR156 precursors were analyzed and located in five chromosomes of the P. mume genome. The expression pattern showed that PmSBP1/6 was negatively correlated with miR156. The promoters of PmSBP1/6 were specifically expressed in the apical meristem. Overexpression of PmSBP1/6 in tobacco promoted flowering and changed the length ratio of pistil and stamen. Moreover, PmSBP1 also affected the number and vitality of pollen and reduced the fertility of transgenic tobacco. Furthermore, ectopic expression of PmSBP1/6 caused up-regulated expression of endogenous SUPPRESSOR OF OVEREXPRESSION OF CO1 (NtSOC1). The yeast-one hybrid assay showed that PmSBP1 was bonded to the promoters of PmSOC1s. In conclusion, a miR156-PmSBP1-PmSOC1s pathway was formed to participate in the regulation of flowering time in P. mume, which provided references for the molecular mechanism of flowering time regulation and molecular breeding of P. mume.


Introduction
Flowering time is a very important agronomic trait, which directly determines the ornamental value and economic benefits of ornamental plants. Plant flowering marks the end of vegetative growth and the beginning of reproductive growth, which is a very complex and important link in the process of plant growth and development. It is not only affected by external environmental factors (light, temperature, etc.) but also regulated by internal physiological factors. So far, six pathways regulating the flowering time of plants have been identified. These pathways include environmental factor pathways: photoperiod pathway, vernalization pathway, and temperature pathway; and the physiological factor pathways: autonomous pathway, gibberellin pathway, and aging pathway. Flowering time is jointly regulated by these pathways, which can affect each other and integrated by a series of genes, such as SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), LEAFY (LFY), FLOWERING LOCUS T (FT), CONSTANS (CO), FLOWERING LOCUS C (FLC), etc. [1].

Chromosomal Location, Phylogenetic Analysis and Sequence Analysis of miR156 from P. mume
In P. mume, the precursors of pmu-miR156 were encoded by 11 genomic sequences (pmu-MIR156a-k), which were located in five chromosomes (Supplementary Figure S1A) and cut into four types of mature sequences (pmu-miR156a-d, e-f, g-j, k). An ML phylogenetic tree of 36 precursor sequences (11 from P. mume, 10 from Arabidopsis, and 15 from tobacco) was built and the miR156 family was divided into three subgroups (Supplementary Figure S1B). Among them, the pmu-MIR156i/j/g/h were clustered with ath-MIR156a-f and nta-MIR156c-i, and the pmu-MIR156a/b/c/d/f /k were clustered with ath-MIR157a-d and nta-MIR156a/b/j-o, but the pmu-MIR156e was a subgroup alone. Sequence alignment showed that only two differentiated bases were found between the miR156 mature sequences from P. mume, Arabidopsis, and tobacco (Supplementary Figure S1C). The pmu-miR156f can fully bind to the miR156 site on the mRNA of PmSBP1, and they are located on the same chromosome.

Expression Patterns of PmSBP1/6 and miR156f during the Flower Development Period
The flower development period of P. mume consists of two phases with overlapping parts: flower bud morphological differentiation and dormancy, which were divided into nine stages (S1-S9): flower primordium forming stage (S1), sepal forming stage (S2), petal forming stage (S3), stamen forming stage (S4), pistil forming stage (S5), anther and ovule forming stage (S6), morphological differentiation finished stage (S7), alabastrum intumescence stage (S8), and flower upcoming to bloom stage (S9) ( Figure 1A,B). Among them, S1-S7 belong to the flower bud morphological differentiation phase, and S6-S9 belong to the flower bud dormancy phase. To verify the function of PmSBP1/6 and the regulation relationship with miR156f, their expression patterns in flower development were analyzed. The expression level of PmSBP1 gradually increased (S1-S4) and remained stable (S4-S6), but decreased sharply when flower bud morphological differentiation was completed (S7), then recovered slightly at S8 and then maintained a very low level before blooming (S9) ( Figure 1C). The expression level of PmSBP6 increased first (S1-S2) and decreased gradually from S2 to S4, then suddenly raised at pistil forming stage (S5) and then dropped when flower bud morphological differentiation was completed (S7), and maintained at a low level (S8-S9) ( Figure 1D). In general, PmSBP1/6 shows relatively high expression in the flower bud morphological differentiation phase, but low expression after differentiation. On the other hand, the expression level of pmu-miR156f decreased from S1 to S4, but suddenly increased at S5, then fell back and remained at a comparatively stable level (S6-S9). In conclusion, the expression patterns of PmSBP1/6 and pmu-miR156f were negatively correlated. stages (S1-S9): flower primordium forming stage (S1), sepal forming stage (S2), petal forming stage (S3), stamen forming stage (S4), pistil forming stage (S5), anther and ovule forming stage (S6), morphological differentiation finished stage (S7), alabastrum intumescence stage (S8), and flower upcoming to bloom stage (S9) ( Figure 1A,B). Among them, S1-S7 belong to the flower bud morphological differentiation phase, and S6-S9 belong to the flower bud dormancy phase. To verify the function of PmSBP1/6 and the regulation relationship with miR156f, their expression patterns in flower development were analyzed. The expression level of PmSBP1 gradually increased (S1-S4) and remained stable (S4-S6), but decreased sharply when flower bud morphological differentiation was completed (S7), then recovered slightly at S8 and then maintained a very low level before blooming (S9) ( Figure 1C). The expression level of PmSBP6 increased first (S1-S2) and decreased gradually from S2 to S4, then suddenly raised at pistil forming stage (S5) and then dropped when flower bud morphological differentiation was completed (S7), and maintained at a low level (S8-S9) ( Figure 1D). In general, PmSBP1/6 shows relatively high expression in the flower bud morphological differentiation phase, but low expression after differentiation. On the other hand, the expression level of pmu-miR156f decreased from S1 to S4, but suddenly increased at S5, then fell back and remained at a comparatively stable level (S6-S9). In conclusion, the expression patterns of PmSBP1/6 and pmu-miR156f were negatively correlated.  (A,B) Flower buds at different development stages. S1: flower primordium forming stage, S2: sepal forming stage, S3: petal forming stage, S4: stamen forming stage, S5: pistil forming stage, S6: anther and ovule forming stage, S7: morphological differentiation finished stage, S8: alabastrum intumescence stage, S9: flower upcoming to bloom. S1-S7 belong to the flower bud morphological differentiation phase, and S6-S9 belong to the flower bud dormancy phase, the two phases have overlapping parts. The minimum scale in (A) = 1 mm, and the scale bar in (B) = 400 µm. (C,D) Expression patterns of PmSBP1, PmSBP6 and miR156f during the flower development period.

The Activity Analysis of PmSBP1/6 Promoters
To verify the activity of the PmSBP1/6 promoters, the 2000 bp upstream sequences of PmSBP1/6 were cloned and fused with the β-glucuronidase (GUS) gene and transiently transformed into the N. benthamiana leaves by A. tumefaciens. Finally, clear blue spots were observed in the tobacco leaves with PmSBP1/6 promoters (Figure 2A,B) compared with the control (Figure 2C,D). For further study of the tissue spatial expression profile of PmSBP1/6 promoters, the promoters with GUS were stably overexpressed in tobacco. In the PmSBP1-promoter transgenic tobacco, obvious blue tissues were detected in the apical meristem ( Figure 2E). While in the PmSBP6-promoter transgenic tobacco, the blue tissues were detected in both the apical meristem and lateral bud ( Figure 2F). cence stage, S9: flower upcoming to bloom. S1-S7 belong to the flower bud morphological differentiation phase, and S6-S9 belong to the flower bud dormancy phase, the two phases have overlapping parts. The minimum scale in (A) = 1 mm, and the scale bar in (B) = 400 μm. (C,D) Expression patterns of PmSBP1, PmSBP6 and miR156f during the flower development period.

The Activity Analysis of PmSBP1/6 Promoters
To verify the activity of the PmSBP1/6 promoters, the 2000 bp upstream sequences of PmSBP1/6 were cloned and fused with the β-glucuronidase (GUS) gene and transiently transformed into the N. benthamiana leaves by A. tumefaciens. Finally, clear blue spots were observed in the tobacco leaves with PmSBP1/6 promoters (Figure 2A,B) compared with the control (Figure 2C,D). For further study of the tissue spatial expression profile of PmSBP1/6 promoters, the promoters with GUS were stably overexpressed in tobacco. In the PmSBP1-promoter transgenic tobacco, obvious blue tissues were detected in the apical meristem ( Figure 2E). While in the PmSBP6-promoter transgenic tobacco, the blue tissues were detected in both the apical meristem and lateral bud ( Figure 2F).

The Promoter Cis-Elements Composition of PmSBP1/6 and Their Response to GA3
The promoter cis-elements can affect the gene expression. The cloned promoter sequences of PmSBP1/6 (about 2000 bp) were predicted on the PlantCARE website (Supplementary Table S2 and Supplementary Data S1). Statistical results of cis-elements showed that gibberellin response elements, anaerobic induction elements, and light response elements were contained in the promoters of both PmSBP1 and PmSBP6 ( Figure 2G,H). In addition, the promoter of PmSBP6 also contained meristem expression elements ( Figure 2H). To analyze whether the expression of PmSBP1/6 is regulated by gibberellin, the qRT-PCR was carried out with the flower buds treated with 100 mg/L GA3 solutions. The expression patterns of PmSBP1 and PmSBP6 are shown in Figure 2I

Subcellular Localization of PmSBP1/6 Proteins
The subcellular localization of PmSBP1/6 was detected by the transient transformation of the PmSBP1/6-GFP fusion proteins into tobacco leaves. In the control 35S::GFP, the GFP fluorescence signals were examined in the cytoplasm and nucleus. In the 35S::PmSBP1/6-GFP, although the GFP fluorescence signals were detected mainly in the nucleus and cytoplasm, the fluorescence intensity is weaker than the control ( Figure 3).

The Promoter Cis-Elements Composition of PmSBP1/6 and Their Response to GA3
The promoter cis-elements can affect the gene expression. The cloned promoter sequences of PmSBP1/6 (about 2000 bp) were predicted on the PlantCARE website (Supplementary Table S2 and Supplementary Data S1). Statistical results of cis-elements showed that gibberellin response elements, anaerobic induction elements, and light response elements were contained in the promoters of both PmSBP1 and PmSBP6 ( Figure 2G,H). In addition, the promoter of PmSBP6 also contained meristem expression elements ( Figure 2H). To analyze whether the expression of PmSBP1/6 is regulated by gibberellin, the qRT-PCR was carried out with the flower buds treated with 100 mg/L GA3 solutions. The expression patterns of PmSBP1 and PmSBP6 are shown in Figure 2I,J. The expression level of PmSBP1 and PmSBP6 significantly decreased after gibberellin treatment for 48 h and 2 h, respectively ( Figure 2I,J).

Subcellular Localization of PmSBP1/6 Proteins
The subcellular localization of PmSBP1/6 was detected by the transient transformation of the PmSBP1/6-GFP fusion proteins into tobacco leaves. In the control 35S::GFP, the GFP fluorescence signals were examined in the cytoplasm and nucleus. In the 35S::PmSBP1/6-GFP, although the GFP fluorescence signals were detected mainly in the nucleus and cytoplasm, the fluorescence intensity is weaker than the control ( Figure 3).

Overexpression of PmSBP1/1tb and PmSBP6 in Tobacco Promotes Flowering and Affects Fertility
The PmSBP1, PmSBP1tb, and PmSBP6 were overexpressed in tobacco under CaMV35S. To confirm the function of PmSBP1 and miR156, the miR156 site of PmSBP1 was synonymously mutated and named PmSBP1tb. The schematic map of PmSBP1/1tb recombinant vectors is shown in Figure 4A. More than 10 transgenic lines of each gene were obtained and confirmed by a reverse transcription polymerase chain reaction (RT-PCR) assay, and three lines were selected for subsequent analyses. In the juvenile period of transgenic T3 generation plants, the vegetative growth of the 35S::PmSBP1tb transgenic seedlings was significantly less than that of the wild type (WT) ( Figure 4C), but the 35S::PmSBP1 transgenic lines were slightly larger than that of the WT ( Figure 4B). Besides, the vegetative growth of the three 35S::PmSBP6 transgenic lines was also a little larger than that of the WT ( Figure  5A). As plants grew older, the difference in vegetative growth between all the transgenic seedlings and wild type gradually disappeared (Figures 4D,E and 5B).

Overexpression of PmSBP1/1tb and PmSBP6 in Tobacco Promotes Flowering and Affects Fertility
The PmSBP1, PmSBP1tb, and PmSBP6 were overexpressed in tobacco under CaMV35S. To confirm the function of PmSBP1 and miR156, the miR156 site of PmSBP1 was synonymously mutated and named PmSBP1tb. The schematic map of PmSBP1/1tb recombinant vectors is shown in Figure 4A. More than 10 transgenic lines of each gene were obtained and confirmed by a reverse transcription polymerase chain reaction (RT-PCR) assay, and three lines were selected for subsequent analyses. In the juvenile period of transgenic T 3 generation plants, the vegetative growth of the 35S::PmSBP1tb transgenic seedlings was significantly less than that of the wild type (WT) ( Figure 4C), but the 35S::PmSBP1 transgenic lines were slightly larger than that of the WT ( Figure 4B). Besides, the vegetative growth of the three 35S::PmSBP6 transgenic lines was also a little larger than that of the WT ( Figure 5A). As plants grew older, the difference in vegetative growth between all the transgenic seedlings and wild type gradually disappeared ( Figures 4D,E Figure 4I). Interestingly, the flowering time of the PmSBP1tb transgenic lines was significantly earlier than that of WT (Figure 4J).
In the reproductive growth stage, the 35S::PmSBP1tb transgenic tobacco was normal, but the 35S::PmSBP1 and 35S::PmSBP6 transgenic tobacco were different from the wildtype tobacco. In the wild-type tobacco, the length of style and filaments were the same, allowing them to be self-bred and bear fruit ( Figure 5G). While in the 35S::PmSBP1 ( Figure  6A) and 35S::PmSBP6 ( Figure 5H) transgenic tobacco, the length of style and filaments  Figure 4H) and PmSBP6 ( Figure 5E, F) transgenic lines was similar to that of WT ( Figure 4I). Interestingly, the flowering time of the PmSBP1tb transgenic lines was significantly earlier than that of WT ( Figure 4J).
In the reproductive growth stage, the 35S::PmSBP1tb transgenic tobacco was normal, but the 35S::PmSBP1 and 35S::PmSBP6 transgenic tobacco were different from the wild-type tobacco. In the wild-type tobacco, the length of style and filaments were the same, allowing them to be self-bred and bear fruit ( Figure 5G). While in the 35S::PmSBP1 ( Figure 6A) and 35S::PmSBP6 ( Figure 5H) transgenic tobacco, the length of style and filaments were inconsistent. In the 35S::PmSBP1 transgenic tobacco, the length ratio of style to filament showed two phenotypes: one was the high-style (the style was higher than the filament) and the other was the short-style (the style was shorter than the filament). Among them, the high-style flower was the main phenotype (96%), with smaller size, withered anthers, less pollen, and fast-drying filaments ( Figure 6A,B), which could not be self-pollinated. The pods became smaller and the seed number in one pod was less after artificial self-pollination ( Figure 6C). The pollen viability of the high-style flower was tested by TTC staining and pollen germination in vitro. As shown in Figure 6D and Supplementary Figure S2, in the wild-type tobacco and three 35S::PmSBP1 transgenic lines S1-3, S1-5, and S1-6, the pollen staining rates were 85.8%, 41%, 32.5%, and 13.8%, respectively, and the pollen germination rates were 78.8%, 18.2%, 13.7%, and 12.5%, respectively. In short, the results of the two methods showed that the pollen viability of 35S::PmSBP1 transgenic plants was lower than that of WT. The flower phenotype in the 35S::PmSBP6 transgenic tobacco was partly different from the 35S::PmSBP1 transgenic tobacco. In the 35S::PmSBP6 transgenic tobacco, all the flowers showed a high-style phenotype with normal size, but the flowers could bear fruit normally after artificial self-pollination ( Figure 5H). were inconsistent. In the 35S::PmSBP1 transgenic tobacco, the length ratio of style to filament showed two phenotypes: one was the high-style (the style was higher than the filament) and the other was the short-style (the style was shorter than the filament). Among them, the high-style flower was the main phenotype (96%), with smaller size, withered anthers, less pollen, and fast-drying filaments ( Figure 6A,B), which could not be self-pollinated. The pods became smaller and the seed number in one pod was less after artificial self-pollination ( Figure 6C). The pollen viability of the high-style flower was tested by TTC staining and pollen germination in vitro. As shown in Figure 6D and Supplementary Figure  S2, in the wild-type tobacco and three 35S::PmSBP1 transgenic lines S1-3, S1-5, and S1-6, the pollen staining rates were 85.8%, 41%, 32.5%, and 13.8%, respectively, and the pollen germination rates were 78.8%, 18.2%, 13.7%, and 12.5%, respectively. In short, the results of the two methods showed that the pollen viability of 35S::PmSBP1 transgenic plants was lower than that of WT. The flower phenotype in the 35S::PmSBP6 transgenic tobacco was partly different from the 35S::PmSBP1 transgenic tobacco. In the 35S::PmSBP6 transgenic tobacco, all the flowers showed a high-style phenotype with normal size, but the flowers could bear fruit normally after artificial self-pollination ( Figure 5H).

Expression Pattern of Endogenous Flowering-Related Genes in PmSBP1/1tb and PmSBP6 Transgenic Tobacco
To investigate the early flowering transgenic tobacco, the expression level of eight endogenous flowering-related genes (NtSOC1, NtCO, NtAP1, NtMADS3, NtMADS4, Nt-MADS11, NtNFL1, and NtNFL2) in the shoot tip of tobacco (90 d in short-day conditions, 100 d in long-day conditions) was detected by qRT-PCR. The expression level of these eight endogenous flowering-related genes in the three PmSBP1 transgenic tobacco lines is shown in Figure 7A. In short-day conditions, their expression was higher than that of the wild-type tobacco; while in long-day conditions, the expression level was lower than that of wild-type tobacco, except for NtSOC1 showing similar expression to wild-type tobacco. As shown in Figure 7B, the expression level of the endogenous NtSOC1 in the PmSBP1tb transgenic tobacco was increased under both short-day and long-day conditions. In short-day conditions, the expression of the other seven flowering-related genes was similar to WT. In long-day conditions, the expression of NtCO only increased sharply in the PmSBP1tb transgenic tobacco line S1tb-7; however, it changed slightly in the other two lines. The expression level of NtAP1, NtMADS3, NtMADS4, and NtMADS11 in all three lines decreased; however, the expression of NtNFL1 and NtNFL2 fluctuated marginally. As shown in Figure 8, the expression level of these eight flowering-related genes in the three PmSBP6 transgenic lines under short-day conditions was higher than that of WT. Under long-day conditions, their expression levels in the three lines fluctuated up and down at the relative expression value of WT. The expression level of NtSOC1 was directly proportional to the phenotype of early flowering in these transgenic plants.

PmSBP1 Binding to the PmSOC1s Promoters
To verify the regulatory relationship between PmSBP1 and PmSOC1s, the promoter sequences of PmSOC1s (about 850 bp) were cloned and analyzed (Supplementary Data S1). Among them, except for PmSOC1-1, the promoters of PmSOC1-2 and PmSOC1-3 contained four SBP binding sites 'GTAC' respectively ( Figure 9A). The 100 bp fragments of PmSOC1s promoters containing SBP binding sites were used as baits. The B1 fragment of PmSOC1-2 (C2B1) and the B2 fragment of PmSOC1-3 (C3B2) contained two and three SBP binding sites, respectively. The interactions between PmSBP1 and the fragments of PmSOC1s promoters were detected by yeast one-hybrid ( Figure 9B). All transformed yeast grew normally on the SD/-Leu solid medium. As shown in Figure 9B, PmSBP1 could bind to the B2 fragments of PmSOC1-2 (C2B2) and PmSOC1-3 promoters (C3B2) but could not associate with the other fragments (C2B1, C2B3, and C3B1). This result suggested that PmSBP1 could activate PmSOC1-2 and PmSOC1-3 by binding to the B2 sites of their promoters. Furthermore, we compared the eight SBP-binding fragments containing the core 'GTAC' (11 bp) with the SBPbinding sequence of Arabidopsis AtSPL9 (ID: MA1322.1, homologous with PmSBP1), and found that the three bases adjacent to the core 'GTAC' in C2B2 and C3B2 were consistent with the high-frequency bases in the SBP-binding sequence of AtSPL9 ( Figure 9C), which indicated that the three bases adjacent to 'GTAC' were critical for the binding with PmSBP1. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 10 of 19 in PmSBP1 transgenic tobacco. S1-3, S1-6, and S1-7 were three transgenic lines. (B) The expression of flowering-related genes in PmSBP1tb transgenic tobacco. S1tb-2, S1tb-4, and S1tb-7 were three transgenic lines. in PmSBP1 transgenic tobacco. S1-3, S1-6, and S1-7 were three transgenic lines. (B) The expression of flowering-related genes in PmSBP1tb transgenic tobacco. S1tb-2, S1tb-4, and S1tb-7 were three transgenic lines.

PmSBP1 Binding to the PmSOC1s Promoters
To verify the regulatory relationship between PmSBP1 and PmSOC1s, the promoter sequences of PmSOC1s (about 850 bp) were cloned and analyzed (Supplementary Data S1). Among them, except for PmSOC1-1, the promoters of PmSOC1-2 and PmSOC1-3 contained four SBP binding sites 'GTAC' respectively ( Figure 9A). The 100 bp fragments of PmSOC1s promoters containing SBP binding sites were used as baits. The B1 fragment of PmSOC1-2 (C2B1) and the B2 fragment of PmSOC1-3 (C3B2) contained two and three SBP binding sites, respectively. The interactions between PmSBP1 and the fragments of PmSOC1s promoters were detected by yeast one-hybrid ( Figure 9B). All transformed yeast grew normally on the SD/-Leu solid medium. As shown in Figure 9B, PmSBP1 could bind to the B2 fragments of PmSOC1-2 (C2B2) and PmSOC1-3 promoters (C3B2) but could not associate with the other fragments (C2B1, C2B3, and C3B1). This result suggested that PmSBP1 could activate PmSOC1-2 and PmSOC1-3 by binding to the B2 sites of their promoters. Furthermore, we compared the eight SBP-binding fragments containing the core 'GTAC' (11 bp) with the SBP-binding sequence of Arabidopsis AtSPL9 (ID: MA1322.1, homologous with PmSBP1), and found that the three bases adjacent to the core 'GTAC' in C2B2 and C3B2 were consistent with the high-frequency bases in the SBP-binding sequence of AtSPL9 ( Figure 9C), which indicated that the three bases adjacent to 'GTAC' were critical for the binding with PmSBP1.

Discussion
Plant microRNAs (miRNAs), 19-25 nt long, are highly conserved small non-coding RNAs, and play important role in juvenile-to-adult phase transition and flowering time in Arabidopsis [22,23]. The miR156 is quite conservative among plant species [24]. In this

Discussion
Plant microRNAs (miRNAs), 19-25 nt long, are highly conserved small non-coding RNAs, and play important role in juvenile-to-adult phase transition and flowering time in Arabidopsis [22,23]. The miR156 is quite conservative among plant species [24]. In this study, the sequence alignment results showed that the miR156 mature sequences in P. mume, Arabidopsis and tobacco were indeed highly conserved, with only two SNPs (Supplementary Figure S1A). The miR156-SPL is the famous age pathway; SPL is the target gene of miR156, and is regulated by miR156 mainly through complementation base-pairing at the posttranscriptional level [23]. In our previous study, the miR156-mediated PmSBPs mRNA cleavage was detected by 5 -RACE [25].
The spatiotemporal expression pattern of genes is closely related to gene function. So far, the expression pattern of SBP in the flower development period has been studied in some perennial plants. The expression profile of AtSPL9/15 homologous genes in some species is as follows. In loquat, the expression of EjSPL9 decreased with the development of flower bud [27]. In chestnut, the expression level of CmSPL9 was increased with the development of flower bud [28], and so does the JrSBP23 in walnut [29]. In Betula luminifera, BlSPL8 was highly expressed in the early and middle stages of male inflorescence [30]. Unlike them, the expression of PmSBP1 was relatively stable and high in the flower bud morphological differentiation phase of P. mume, which indicated that PmSBP1 may play a vital role in flower bud differentiation. The expression pattern of AtSPL3/4/5 homologous genes in other species is as follows. In loquat, the expression of EjSPL3 and EjSPL 4 reached the peak at flower bud initiation, the expression level of EjSPL4 and EjSPL5 was suddenly raised in the middle stage of flower bud development [27]. The BlSPL15 of Betula luminifera was significant highly expressed in the early stage of female inflorescence [30]. But partly like EjSPL4, EjSPL5 and BlSPL15, in this study, the expression level of PmSBP6 was significantly high at the stamen differentiation stage (S5), which implied that it may participate in the stamen differentiation or regulate fertility. Different expression patterns in different species indicated that they may have different functions during flower bud development.
The promoter is the switch of the gene, which can regulate the gene expression, or bind to the transcription factors to start or close the gene expression. In this study, the 2000 bp promoter sequences of PmSBP1/6 were cloned, both had typical promoter core structure regions, and the GUS staining results showed they had driven activity. Some fragment deletions were found in the cloned promoter sequences of PmSBP1/6 when compared with the genome sequence of wild P. mume. This may be the evolutionary result of the changes in environmental factors during the long-term cultivation process. In addition, the two promoter sequences contained multiple cis-acting elements, such as the light response elements, the anaerobic inducing elements, hormone response elements, and the endosperm expression element or meristem expression element. This suggested that PmSBP1 and PmSBP6 can be regulated by external environmental stimuli (light, water, and hormones), and may be expressed in some specific plant tissues. Besides, the exogenous GA3 downregulated the expression of PmSBP1/6, which was similar to the result of CmSPL9 in chestnut [28]. In addition, the tissue spatial expression profile showed that the PmSBP1 promoter was initiated in the apical meristem, and the PmSBP6 promoter was initiated in both the apical meristem and lateral bud. This displayed that they may play an important role in the initiation of flower buds.
Recently, the function of AtSPLs in clade II of Arabidopsis was extensively studied. AtSPL8 functions in both the male and female fertility of Arabidopsis by affecting the anther development [17,[31][32][33] and gynoecium patterning [33]. Based on the function research results, the other ten AtSPLs with miR156/157 sites are divided into three groups. Group 1 contains AtSPL2/9/10/11/13/15, which play role in both the transition from childhood to adulthood and from nutrition to reproduction, and AtSPL9/13/15 [34] play more important roles than AtSPL2/10/11 [10]. Group 2 contains AtSPL3/4/5, which can promote the transformation of flower meristem, but play a minor role in the change of vegetative stage or flower induction [16,35]. Group 3 contains AtSPL6, which does not play a major role in shoot morphogenesis, but may play an important role in some physiological processes [11]. As is known, the miR156-SPL is an important regulatory model in plant growth. Overexpression of miR156 in Arabidopsis prolongs childhood, while silencing miR156 makes plants mature early [3]. In Arabidopsis [2], tobacco [36], and several other plants [6,30,37], the content of miR156 was high and the content of SPL was low in childhood. With the increase of plant age, the content of miR156 decreased and the content of SPL increased. In addition, their expression can be regulated by each other [3,9].
In our study, PmSBP1 and its synonymous mutation PmSBP1tb and PmSBP6 were constitutively overexpressed in tobacco and showed their role in the regulation of plant growth, flowering time, and reproductive organ development. As our results showed, in the childhood stage, the 35S::PmSBP1tb transgenic tobacco was significantly smaller than that of the wild-type tobacco ( Figure 4C), while the 35S::PmSBP1 transgenic seedlings were only slightly larger than the wild-type tobacco ( Figure 4B). This may be related to the regulatory balance between the content of excessive SBP transcript and miR156. Unexpectedly, the 35S::PmSBP6 transgenic seedlings were also slightly larger than WT in the childhood stage ( Figure 5A). Like PmSBP1tb, The CDS region of PmSBP6 does not contain a miR156 binding site, which cannot be regulated by nta-miR156 in tobacco, but they do have opposite phenotypes (Figures 4B and 5A). We speculated that there was still a regulatory relationship between PmSBP6 and miR156 after removing the 3 UTR sequence (containing miR156 binding site); however, further research should be conducted to confirm the new findings.
The development of floral organs is very important for the sexual reproduction of plants, especially the stamen and pistil. In Arabidopsis, the AtSPL8 (without miR156 binding site) and other AtSPLs (with miR156 binding site) are necessary for the production of fully fertile flowers [23,26]. The knocking out of AtSPL8 resulted in abnormal anther development [17], but the overexpressing of AtSPL8 caused anther non-dehiscence [32]. When overexpressed miR156 in the spl8 mutant, the plant showed complete male sterility, but the overexpression of other AtSPL with miR156 binding sites in the spl8 mutant can alleviate the semi-sterile phenotype to a certain extent [26]. Another miR156 targeted gene AtSPL2 can affect plant fertility by affecting pollen production and fertilization rate. The fertility of both the gain-of-function mutant 35S::SPL2SRDX and the loss-of-function mutant spl2 decreased, and the fertility of the loss-of-function mutant was lower [38]. In this research, the overexpression of PmSBP1 (homologous to AtSPL9/15) and PmSBP6 (homologous to AtSPL3/4/5) in tobacco caused changes in the length of style and filament, and led to changes in the pollination mode, especially PmSBP1, which caused pollen abortion by reducing the number and vitality of pollen. However, overexpression of the PmSBP1tb gene neither caused changes in flower organs nor affected the pollen number and activity, and the fertility of PmSBP1tb transgenic plants was not affected. This indicated that both PmSBP1 and PmSBP6 can change the length ratio of style to the filament and affect the pollination mode, and the miR156 locus of PmSBP1 is indispensable in affecting plant fertility. Similar to Arabidopsis AtSPL8 [23,26] and AtSPL2 [38], all these genes affect plant fertility, but in different ways. Among these SBPs from different species, those not clustered together by phylogenetic analysis also have similar functions, this may be due to the species evolution.
Ectopic expression of PmSBP1 and PmSBP6 in tobacco caused early flowering, which was similar to their homologous genes in Arabidopsis [15,34,35]. However, in our study, the flowering time of each transgenic plant was not consistent under different photoperiod conditions. The flowering time of PmSBP1 and PmSBP6 transgenic tobacco was earlier than that of WT only in short-day conditions, but the flowering time of PmSBP1tb transgenic tobacco was earlier in both long-day and short-day conditions. This indicated that PmSBP1 and PmSBP6 were affected by miR156 in promoting flowering in tobacco through mediating the photoperiod pathway. Furthermore, we examined the expression pattern of eight endogenous flowering-related genes in transgenic plants under different photoperiod conditions. Combined with the phenotype and expression level, we found that the expression level of endogenous NtSOC1 in early flowering transgenic plants was significantly higher than that of wild-typeunder corresponding photoperiod conditions. This suggested that PmSBP1 and PmSBP6 can regulate flowering time by regulating NtSOC1. Furthermore, the results of the yeast-one hybrid showed that PmSBP1 can regulate PmSOC1-2 and PmSOC1-3 by directly binding to their promoters. However, the relationship between PmSBP6 and the promoter of PmSOC1s cannot be verified by yeast-one hybrid because the introduction of PmSBP6 makes yeast grow abnormally. In previous reports, AtSPL3/4/5 mediated flowering time regulation by cooperating with FT-FD complexes in the photoperiod pathway [15]. Moreover, the AtSPL3/4/5 can be regulated by AtSOC1 by directly binding to their promoters [13]. However, the study on how SBP protein regulates flowering time by directly binding to the promoter of SOC1 has not been reported. Our findings opened a new way for SBP to regulate flowering time and laid a foundation for molecular breeding of P. mume and its research in other species.

Plant Materials
Prunus mume 'Sanlun Yudie', grown in the campus of Beijing Forestry University was used in this study. The flower buds and young leaves were collected for gene and promoter cloning, respectively. The developmental periods of flower buds were identified by hand sectioning and flower buds were collected from July (initiation of flower bud differentiation) to March (flowering) in the next year for expression pattern analysis. To detect the response of PmSBP1 on gibberellin, the 100 mg/L GA3 was sprayed on the flower buds of cut-off one-year-old branches after pistil formation and during bud dormancy, the buds were sampled after 0 h, 2 h, 6 h, 12 h, 24 h, 48 h, and 72 h. The Nicotiana tabacum and Nicotiana benthamiana were grown at 25 • C, 16 h light/8 h dark (long-day conditions) or 8 h light/16 h dark (short-day conditions) in the greenhouse. The stem tips of transgenic and wild-type tobacco were sampled for endogenous gene detection at 100 d in short-day conditions and 110 d in long-day conditions. All samples were collected in liquid nitrogen and stored at −80 • C for RNA isolation.

Bioinformatics Analysis of pmu-miR156
The mature sequences and precursor sequences of pmu-miR156 in P. mume, ath-miR156 in Arabidopsis, and nta-miR156 in Nicotiana tabacum were obtained from our previous study [25], the miRbase (www.mirbase.org/ftp.shtmlmL, accessed on 2 June 2021), and published article [39], respectively. The precursor sequences of pmu-miR156 were aligned to the genome data of P. mume by blastn, and the chromosome position was drawn through the MG2Cv2 (http://mg2c.iask.in/mg2c_v2.0, accessed on 2 June 2021). The DNAMAN software was used to analyze the differences of miR156 mature sequence in P. mume, Arabidopsis and tobacco. The phylogenetic tree of the precursor sequences of three species was obtained through the maximum likelihood method in MEGA software after Clustalx alignment.

Cloning and Analysis of PmSBP Gene and Promoter
The total RNA of flower buds was isolated by EASYspin Plus Plant RNA Kit (Aidlab, Beijing, China), and the first strand of cDNA was synthesized by TransScript RT Kit (TIAN-GEN, Beijing, China). The genome DNA of young leaves was extracted by DNAsecure Plant Kit (TIANGEN). The cDNA was used as a template to amplify the coding sequences (CDS) of PmSBP1 and PmSBP6. The promoter sequences of PmSBP1/6 (about 2000 bp) and PmSOC1s (about 850 bp) were cloned from the genome DNA by specific primers. The primers were designed by Oligo7 and are listed in Supplementary Table S1. The total volume of the 50 µL polymerase chain reaction (PCR) system includes 25 µL PrimeSTAR HS (Premix) (TaKaRa, Beijing, China), 1 µL forward primer, and 1 µL reverse primer, 2 µL cDNA or 1 µL genome DNA. The PCR procedure was 94 • C pre-denaturation for 2 min; 35 cycles of 98 • C for 10 s, 55 • C for 5 s, 72 • C for 90 s; and 72 • C extensions for 10 min. The PCR products were separated from 0.8% agarose gel using TIANgel Midi Purification Kit (TIANGEN) and cloned into the pCloneEZ-NRS-Omni-Amp/HC vector (Clone Smarter, Houston, TX, USA) and transformed into Escherichia coli DH5α for sequencing (TIANGEN). The cis-acting element composition of the PmSBP1/6 (about 2000 bp) promoter sequences was predicted on the PlantCARE website [40].

Expression Pattern Analysis
The total RNA of flower buds in different flower development stages and treated with GA3, and the stem tip of tobacco was isolated by the EASYspin Plus Plant RNA Kit (Aidlab). The FastQuant RT Kit (with gDNase) (TIANGEN) was used to synthesize the first strand of cDNA, and the SYBR Premix ExTaq II (TaKaRa) was used for qRT-PCR following the instructions. The miRNA RT/qPCR Detection kit (Aidlab) was used to synthesize cDNA and complete Poly(A) tailed qRT-PCR of miR156f. The qRT-PCR was performed on the PikoReal real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). The primers of the housekeeping gene (protein phosphatase 2A, PmPP2A) [41], miR156f [25], and PmSBP1/6 [21] of P. mume were referred to in the previous study. The reaction with 10 µL volume (1 µL of cDNA, 5 µL of SYBR Premix ExTaq II (Takara), and 0.2 µL of each primer) were conducted as follow: 30 s in 95 • C, 40 cycles of 5 s in 95 • C and 30 s in 60 • C, and finally end in 20 • C, and each reaction was repeated in triplicate. The qRT-PCR primers of the housekeeping gene (NtActin) and eight endogenous flowering-related genes in tobacco used were also from the previous study [42][43][44]. The relative expression level was calculated by the 2 −∆∆Ct method [45]. The error line was drawn according to the standard deviation (calculated by EXCEL) of the three technical repetitions. The significant difference analysis of the gene expression level under GA3 treatment was performed by F-test in EXCEL.

Subcellular Localization
To analyze the subcellular localization of PmSBP1/6, the CDSs of PmSBP1/6 were cloned into the vector pSuper1300-GFP between Sal I and Spe I restriction sites to generate the PmSBP1/6-GFP fusion gene driven by CaMV35S. The recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101, cultured in 15 mL liquid LB medium (with 50 mg/L kanamycin and 50 mg/L rifampicin) until the OD 600 = 0.8, then diluted to OD 600 = 0.5, and injected into the leaves of 4-6 weeks old Nicotiana benthamiana. After injection for 24-72 h, the leaves were sectioned and stained in 4 ,6-diamino-2-phenylindole (DAPI) solution for 10 min, and observed under TCS SP8 (Leica, Wetzlar, Germany) confocal laser scanning microscope. The fluorescence of GFP and DAPI were detected at 488 nm and 405 nm excitation wavelengths, respectively.

Promoter Activity Analysis
To analyze the promoter activity, the 2000 bp promoters of PmSBP1/6 were inserted into the vector pCAMBIA1305.4-GUS through Sal I/Bam HI restriction sites. After instantaneous transformation in N. benthamiana leaves, the leaves were cut and stained in histochemical GUS staining reagent for 24 h, and then rinsed in 75% alcohol to remove chlorophyll.

Overexpression of PmSBP1/6 and Their Promoters in Tobacco
The miR156-sensitive gene PmSBP1, miR156-insensitive genes PmSBP1tb, and PmSBP6 were cloned into the plant expression vector pSuper1300 using Spe I/Kpn I restriction sites under the CaMV35S. PmSBP1tb is a synonymous mutation in the miR156 binding site of PmSBP1, and this mutation was completed by Sangon Biotech Company (Shanghai, China). The recombinants 35S::PmSBP1/6 and 35S::PmSBP1tb were transformed into A. tumefaciens GV3101, respectively. The A. tumefaciens contain 35S::PmSBP1/6, 35S::PmSBP1tb, and PmSBP1/6-promoter::GUS vectors were separately cultured in 30 mL liquid LB medium (with 50 mg/L kanamycin and 50 mg/L rifampicin) until the OD 600 = 0.6-0.8, then diluted to OD 600 = 0.2-0.5 and infected tobacco by leaf disc method [46]. The phenotypes of T 3 generation of PmSBP1/6 and PmSBP1tb transgenic seedlings were observed and photographed. To determine the tissue expression specificity of the promoter of PmSBP1/6, different tissues of T 1 generation of PmSBP1/6-promoter transgenic seedlings were stained in GUS solution for GUS activity detection.

Phenotypic Statistical Analysis
The phenotype changes of these transgenic and wild-type tobacco were observed, recorded, and photographed. The flower morphology was observed throughout the flowering period, and the anthers were collected when the flower was just opened in each transgenic line. The 2,3,5-triphenyl tetrazolium chloride (TTC) staining and pollen germination in vitro were used to test the pollen viability. Three plants were used in each transgenic line. The TTC staining and pollen germination were observed under the microscope. The number of pollen grains in each field was more than 100, and six fields of each plant were counted. The average value and standard deviation of the three technical repetitions were calculated by EXCEL, and the error line was drawn according to the standard deviation. For transgenic plants of PmSBP1 and PmSBP6, artificial self-pollination was performed at the beginning of flower blooming. The pods were harvested when their color turned brown and cracked. The seeds and pods were photographed, and the seeds were stored in bags with desiccant.

Yeast One-Hybrid Assay
In the cloned promoter sequences of PmSOC1s, there are several 'GTAC' SBP binding sites. Fragments of about 100 bp including the SBP binding sites during the 850 bp promoters were inserted into the Sac I/Sal I-cleaved pAbAi vector as baits. The prey vector pGADT7-PmSBP1/6 was constructed by Bam H I/Eco R I sites. The primers were designed by Oligo7 and shown in Supplementary Table S1. The bait recombinants plasmid was transformed into the yeast Y1HGold strains after being digested by Bst BI, and their tolerance to Aureobasidin A (AbA) was detected in the SD/-Ura/AbA medium. Subsequently, the prey vector was transferred into the yeast-containing bait vectors by the Quick & Easy Yeast Transformation Mix (TaKaRa) following its procedure, which was selected on the SD/-Leu/AbA medium.

Conclusions
Overall, PmSBP1/6 were important regulatory genes in flowering time and fertility, negatively regulated by miR156 and exogenous gibberellin. Meanwhile, PmSBP1 can directly bind to the promoters of PmSOC1-2 and PmSOC1-3 to regulate flowering. In conclusion, a miR156-PmSBP1-PmSOC1s pathway was formed to participate in the regulation of flowering time in P. mume. This study lays a foundation for revealing the molecular mechanism of flowering time regulation in P. mume.