BcSOC1 Promotes Bolting and Stem Elongation in Flowering Chinese Cabbage

Flowering Chinese cabbage is one of the most economically important stalk vegetables. However, the molecular mechanisms underlying bolting, which is directly related to stalk quality and yield, in this species remain unknown. Previously, we examined five key stem development stages in flowering Chinese cabbage. Here, we identified a gene, BcSOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1), in flowering Chinese cabbage using transcriptome analysis, whose expression was positively correlated with bolting. Exogenous gibberellin (GA3) and low-temperature treatments significantly upregulated BcSOC1 and promoted early bolting and flowering. Additionally, BcSOC1 overexpression accelerated early flowering and stem elongation in both Arabidopsis and flowering Chinese cabbage, whereas its knockdown dramatically delayed bolting and flowering and inhibited stem elongation in the latter; the inhibition of stem elongation was more notable than delayed flowering. BcSOC1 overexpression also induced cell expansion by upregulating genes encoding cell wall structural proteins, such as BcEXPA11 (cell wall structural proteins and enzymes) and BcXTH3 (xyloglucan endotransglycosidase/hydrolase), upon exogenous GA3 and low-temperature treatments. Moreover, the length of pith cells was correlated with stem height, and BcSOC1 interacted with BcAGL6 (AGAMOUS-LIKE 6) and BcAGL24 (AGAMOUS-LIKE 24). Thus, BcSOC1 plays a vital role in bolting and stem elongation of flowering Chinese cabbage and may play a novel role in regulating stalk development, apart from the conserved function of Arabidopsis SOC1 in flowering alone.


Introduction
Brassica spp. (family Cruciferae) are cultivated worldwide because of their economically important traits, including flavor [1,2], leafy heads, and enlarged organs (roots, stems, and inflorescences) [3,4]. Although several studies have focused on the development of leafy heads, roots, and stem tubers, there have been only a few studies on stem development in Brassica spp. [5,6]. Flowering Chinese cabbage (Brassica campestris L. ssp. chinensis (L.) Makino var. utilis Tsenet Lee), a subspecies of Chinese cabbage from southern China, is a typical stalk vegetable [7]. It has 10 chromosomes, and its genome is of the AA type. The genome size of B. rapa, a representative species of the AA genome, is 353.14 Mb [8]. As a diploid, flowering Chinese cabbage is suitable for gene modification and genetic studies. The edible parts of flowering Chinese cabbage, which include flower stalks and leaves, are rich in anticarcinogenic compounds and antioxidants such as glucosinolates, phenols, vitamin C, and carotenoids [1,7,9]. Development of the stem (also referred to as stalk) includes bolting (stem elongation and thickening) and flowering, both of which are directly related to stalk quality and yield. However, the molecular mechanisms underlying stalk

Expression and Subcellular Localization of BcSOC1 in Flowering Chinese Cabbage
In the previous study, we examined five developmental stages (S1-S5) of the flowering Chinese cabbage cultivar 'Youlv 501 caixin' to gain insight into stalk development. With this aim, we performed RNA-seq using two biological replicates of shoot tip specimens from S1 (15 d after sowing, the seedling stage), S3 (31 d after sowing, the bolting stage), and S5 (37 d after sowing, the flowering or harvesting stage) [25]. We further analyzed differentially expressed flowering-related genes at these developmental stages ( Figure S1). Interestingly, BcSOC1 was significantly upregulated at stages S3 and S5, indicating that BcSOC1 was a potential regulator of bolting and flowering in this species. In addition, the flowering-related genes AP1, LFY, SPL3, SPL5, and SPL9 exhibited higher expression levels at stages S3 or S5 ( Figure S1). Given the specificity of BcSOC1 expression, we aimed to determine its role in bolting and flowering of flowering Chinese cabbage.
To further elucidate the role of BcSOC1, we examined the expression profile of BcSOC1 in the roots, shoot apex, leaves, and flowers at the blooming stage of flowering Chinese cabbage using qRT-PCR ( Figure 1C). BcSOC1-1 transcript levels were significantly higher in the shoot apex than in the other organs, whereas BcSOC1-2 was upregulated in the leaves and roots. Subcellular localization of BcSOC1 revealed that both BcSOC1-1 and BcSOC1-2 were localized in the nucleus (Figures 1D and S2).  To further elucidate the role of BcSOC1, we examined the expression profile of BcSOC1 in the roots, shoot apex, leaves, and flowers at the blooming stage of flowering Chinese cabbage using qRT-PCR ( Figure 1C). BcSOC1-1 transcript levels were significantly higher in the shoot apex than in the other organs, whereas BcSOC1-2 was upregulated in the leaves and roots. Subcellular localization of BcSOC1 revealed that both BcSOC1-1 and BcSOC1-2 were localized in the nucleus (Figures 1D and S2).

GA 3 and Low-Temperature Treatments Affect Bolting and BcSOC1 Expression in Flowering Chinese Cabbage
Previous studies have shown that both GA 3 and low-temperature treatments promote bolting and flowering in flowering Chinese cabbage [24]. Therefore, to determine whether BcSOC1 is involved in the regulation of bolting and flowering, the seeds of flowering Chinese cabbage were treated with GA 3 (200 mg/L) and low temperatures (4 • C or 15 • C), and then cultured in perlite. As shown in Figure S3, plant height slowly increased during the early stages and rapidly after bolting. Furthermore, GA 3 treatment promoted bolting, as indicated by increased height, during the entire growth period. The stem diameter of the control (CK) plants increased throughout the growth period, whereas GA 3 treatment significantly reduced stem diameter. In the CK plants, budding and flowering time were 25 d and 30 d after sowing, respectively, whereas the budding time and flowering time were advanced to 21 d and 26 d after sowing, respectively, upon GA 3 treatment. Additionally, budding and flowering rates were higher in the GA 3 -treated plants than in the CK plants.
BcSOC1-1 expression was slightly affected by the GA 3 treatment during the vegetative phase; however, GA 3 notably affected the expression of BcSOC1-1 during the reproductive phase (Figure 2A,B). BcSOC1-1 expression remained low 8-18 d after the GA 3 treatment and was not significantly different from that in the CK plants. However, 21-31 d after the GA 3 treatment, the expression level of BcSOC1-1 in the leaves rapidly increased 2.82-7.26 times and in the shoot tips 2.16-2.67 times compared with that in the CK plants. Furthermore, the expression level of BcSOC1-2 in the leaves was upregulated 7.00 and 47.95 times compared with that in the CK leaves after 21 d and 31 d of treatment, respectively ( Figure 2C). However, the expression level of BcSOC1-2 in the shoot tips was upregulated 18.0, 35.82, 2.82, and 2.09 times compared to the CK plants at 13,18,25, and 31 d after treatment, respectively ( Figure 2D). Therefore, the expression of BcSOC1-2 was more significantly affected by GA 3 treatment than that of BcSOC1-1.
Similarly, low temperatures promoted bolting in flowering Chinese cabbage, thereby increasing plant height and reducing stalk diameter ( Figure S3). The expression levels of BcSOC1-1 and BcSOC1-2 in the leaves were significantly higher at low temperatures than in the CK leaves at 20, 22, and 25 d after treatment ( Figure 2E,G). Similarly, BcSOC1-1 and BcSOC1-2 were significantly upregulated in the shoot tip at each sampling period, especially 20 d after treatment, which indicated the critical period of bolting and stem elongation in flowering Chinese cabbage ( Figure 2F,H). In addition, the upregulation of BcSOC1-2 expression in shoot tips was much higher than that of BcSOC1-1 at 20 d after the low-temperature treatment, indicating that BcSOC1-2 expression was more significantly affected by low temperature compared with that of BcSOC1-1. These results indicate that BcSOC1 may be involved in the regulation of bolting, flowering, and stem elongation in flowering Chinese cabbage, as its expression is affected by both GA 3 and low temperature. and BcSOC1-2 in the leaves and shoot apex exposed to GA3. (E-H) Expression levels of BcSOC1-1 and BcSOC1-2 in the leaves and shoot apex exposed to low temperature (4 °C and 15 °C). CK as the control. Error bars represent standard errors. Different letters (a, b and c) indicate significant differences (p < 0.05) determined using ANOVA. * represents the reproductive phase. and BcSOC1-2 in the leaves and shoot apex exposed to GA 3 . (E-H) Expression levels of BcSOC1-1 and BcSOC1-2 in the leaves and shoot apex exposed to low temperature (4 • C and 15 • C). CK as the control. Error bars represent standard errors. Different letters (a, b and c) indicate significant differences (p < 0.05) determined using ANOVA. * represents the reproductive phase.

BcSOC1 Overexpression Accelerates Stem Elongation and Flowering in Arabidopsis and Flowering Chinese Cabbage
To investigate whether BcSOC1 plays the same role as AtSOC1 in Arabidopsis, we ectopically expressed BcSOC1-1 and BcSOC1-2 under the constitutive CaMV 35S promoter in Arabidopsis accession Col. We obtained 25 pBI121:BcSOC1-1 and 30 pBI121:BcSOC1-2 T1 transgenic lines. The bolting and flowering time of all the transgenic lines with the WT background was decreased, with different severity of the phenotype ( Figure 3A, Table S7). The pBI121:BcSOC1-1 transgenic plants (OE-1, 2, and 3) flowered 3-4 d earlier than the WT plants, and the phenotype of pBI121:BcSOC1-2 transgenic plants (OE-4 and 5) was similar to that of pBI121:BcSOC1-1 plants. However, a considerably early flowering phenotype was also observed in pBI121:BcSOC1-2 transgenic plants, with the OE-6 line exhibiting the strongest phenotype (flowering 8 d earlier than the WT) ( Table 1 and Table S7). Transgenic lines were also significantly taller than the WT, with OE-6 line being the tallest ( Figure 3A, Table 1). Interestingly, qRT-PCR analyses revealed that the earliest flowering line (OE-6) exhibited the highest level of transgene expression, and the severity of the phenotype positively correlated with the expression of BcSOC1-1 and BcSOC1-2 ( Figure 3B,C). These results suggest that BcSOC1 plays a role similar to that of AtSOC1, which promotes bolting and flowering in Arabidopsis. In addition, pBI121:BcSOC1-2 lines had earlier bolting and flowering times than pBI121:BcSOC1-1 did (Table 1). Compared with WT, the expression levels of pBI121:BcSOC1-1 and pBI121:BcSOC1-2 were increased 12-20 and 30-44 times, respectively. This suggests that the overexpression level of BcSOC1-2 was higher than that of BcSOC1-1. To genetically characterize BcSOC1, we optimized Agrobacterium-mediated transformation in flowering Chinese cabbage ( Figure S4) and expressed BcSOC1 under the CaMV 35S promoter. All six pBI121:BcSOC1 T1 transgenic lines exhibited early bolting, accelerated stem elongation before flowering, and a significantly taller phenotype compared with the WT. Moreover, the first flower buds appeared markedly earlier in the transgenic lines compared with those in the WT plants, and pBI121:BcSOC1-2 transgenic lines exhibited a more significant phenotype compared to the pBI121:BcSOC1-1 lines ( Figure 3D, Table 1). Consistent with these results, BcSOC1 and its downstream gene BcLFY were significantly upregulated in the transgenic lines ( Figure 3E-G), indicating that BcSOC1 overexpression promoted early bolting, flowering, and stem elongation in flowering Chinese cabbage. (E-G) Expression of BcSOC1-1, BcSOC1-2, and BcLFY in the overexpression lines. WT as the control (CK). Significant differences were determined using Student's t-test (* represents p < 0.05 and ** represents p < 0.01).

BcSOC1 Silencing Delays Bolting and Stem Elongation in Flowering Chinese Cabbage
We generated BcSOC1 knockdown lines using VIGS ( Figure 4) and obtained nine single and six double knockdown lines. We observed and analyzed the phenotypes of control (WT and pTRV2) and knockdown plants when control plants began to flower. Compared with the WT and empty vector control, the knockdown lines exhibited delayed bolting and flowering, increased leaf area and leaf number, and prolonged vegetative growth ( Figures 4A-D and S5). Among them, the phenotype of pTRV2:BcSOC1-2 knockdown lines was more prominent than that of pTRV2:BcSOC1-1 lines, and the phenotype of the double knockdown lines was more notable than that of the single knockdown lines. The expression of BcSOC1 in the knockdown lines was significantly reduced compared with that in the CK plants, and the transgene expression was more significantly decreased in the double knockdown lines than in the single knockdown lines ( Figure 4E-G). These results demonstrate that reduced BcSOC1 expression in the knockdown lines resulted in delayed bolting in flowering Chinese cabbage, and the functions of the two homologous genes were overlapping and complementary. (E-G) Expression of BcSOC1-1, BcSOC1-2, and BcLFY in the overexpression lines. WT as the control (CK). Significant differences were determined using Student's t-test (* represents p < 0.05 and ** represents p < 0.01).

BcSOC1 Silencing Delays Bolting and Stem Elongation in Flowering Chinese Cabbage
We generated BcSOC1 knockdown lines using VIGS ( Figure 4) and obtained nine single and six double knockdown lines. We observed and analyzed the phenotypes of control (WT and pTRV2) and knockdown plants when control plants began to flower. Compared with the WT and empty vector control, the knockdown lines exhibited delayed bolting and flowering, increased leaf area and leaf number, and prolonged vegetative growth ( Figures 4A-D and S5). Among them, the phenotype of pTRV2:BcSOC1-2 knockdown lines was more prominent than that of pTRV2:BcSOC1-1 lines, and the phenotype of the double knockdown lines was more notable than that of the single knockdown lines. The expression of BcSOC1 in the knockdown lines was significantly reduced compared with that in the CK plants, and the transgene expression was more significantly decreased in the double knockdown lines than in the single knockdown lines ( Figure 4E We also examined the expression of BcLFY, which acts downstream of BcSOC1, in the knockdown lines [17] and observed that BcLFY was significantly downregulated in the knockdown lines compared with that in the CK plants. Furthermore, the downregulation was more remarkable in the double knockdown lines ( Figure 4H).
We also examined the expression of BcLFY, which acts downstream of BcSOC1, in the knockdown lines [17] and observed that BcLFY was significantly downregulated in the knockdown lines compared with that in the CK plants. Furthermore, the downregulation was more remarkable in the double knockdown lines ( Figure 4H).
We selected three knockdown lines (T2, T5, and T7) for further characterization. Notably, as the CK plants entered the end of flowering, the knockdown lines were just blooming and were significantly shorter than the CK plants. This indicates that stem elongation was inhibited before flowering in the knockdown lines ( Figure 4I,J) and stems reached optimal height only at the final flowering stage ( Figure 4J). These results demonstrate that BcSOC1 knockdown not only delayed bolting and flowering but also inhibited stem elongation to some extent. Additionally, stem elongation inhibition was more significant than delayed flowering, and the phenotype was most significant in T7, followed by T5, which suggested that BcSOC1-2 had the more effective silencing compared with BcSOC1-1. These findings are consistent with the results of overexpression of BcSOC1-2 in Arabidopsis and flowering Chinese cabbage.

BcSOC1 Overexpression Probably Induces Cell Expansion by Upregulating BcEXPA11 and BcXTH3
To determine whether cell size was affected in the transgenic plants, we compared the size of pith cells in longitudinal sections of the shoot apex at the blooming stage of both WT and overexpression lines ( Figure 5A). Compared to the WT, cell length and area in the pBI121:BcSOC1-1 and pBI121:BcSOC1-2 transgenic plants were significantly increased ( Figure 5B,C). In a previous study, RNA-seq of flowering Chinese cabbage exposed to 15 • C for 5 d [26] revealed a significant upregulation of cell wall structural proteins and enzymes, including BcEXPA11 and xyloglucan endotransglycosidase/hydrolase BcXTH3, at low temperature ( Figure S6). Therefore, we determined the expression of BcEXPA11 and BcXTH3 in the shoot apex of pBI121:SOC1 and pTRV2:BcSOC1 lines. Both genes were upregulated in BcSOC1 overexpression lines OE-3 and OE-6 and downregulated in the knockdown lines T2, T5, and T7 ( Figure 5D,E). These results suggest that the increase in stalk length was positively correlated with increased pith cell size, which was promoted by the overexpression of BcSOC1 through a significant upregulation of the BcEXPA11 and BcXTH3 expression.
We also examined the expression of BcEXPA11 and BcXTH3 after GA 3 and lowtemperature (15 • C) treatments ( Figure 5F,G). In the CK group, both genes were significantly upregulated in the shoot apex after 25 or 30 d of sowing. However, GA 3 and low-temperature treatments significantly upregulated BcEXPA11 and BcXTH3, with the expression levels rapidly increasing after 25 or 30 d of sowing, which is the critical period for stem elongation in flowering Chinese cabbage.
including BcEXPA11 and xyloglucan endotransglycosidase/hydrolase BcXTH3, at low tem-perature ( Figure S6). Therefore, we determined the expression of BcEXPA11 and BcXTH3 in the shoot apex of pBI121:SOC1 and pTRV2:BcSOC1 lines. Both genes were upregulated in BcSOC1 overexpression lines OE-3 and OE-6 and downregulated in the knockdown lines T2, T5, and T7 ( Figure 5D,E). These results suggest that the increase in stalk length was positively correlated with increased pith cell size, which was promoted by the overexpression of BcSOC1 through a significant upregulation of the BcEXPA11 and BcXTH3 expression.  Error bars represent standard errors. Significant differences were determined using Student's t-test (** represents p < 0.01). (F,G) Expression of BcEXPA11 and BcXTH3 under GA 3 and low-temperature (15 • C) treatments. CK as the control. Significant differences were determined using ANOVA.

BcSOC1 Interacts with BcAGL6, BcAGL24, and BcSOC1
AtSOC1 forms dimers with AtAGL6 and AtAGL24 to regulate bolting and flowering in Arabidopsis [27]. To determine the putative protein interactions of BcSOC1 in flowering Chinese cabbage, yeast two-hybrid experiments were performed using 53-BD+AD-T and LAM-BD+AD-T as positive and negative controls, respectively. We observed that BcSOC1-2 interacted with BcSOC1-1 and BcSOC1-2 to form homologous dimers and with BcAGL6 and BcAGL24 to form heterologous dimers ( Figure 6A). However, BcSOC1-1 did not interact with BcAGL6 and BcAGL24 ( Figure 6A).
To verify the results of the yeast two-hybrid experiments, bimolecular fluorescence complementation experiments were performed in vivo. Yellow fluorescent protein (YFP) fluorescence in the nuclei of plant cells confirmed the interactions of BcSOC1-2 with BcSOC1-1, BcSOC1-2, BcAGL6, and BcAGL24 ( Figures 6B and S7).
To verify the results of the yeast two-hybrid experiments, bimolecular fluorescence complementation experiments were performed in vivo. Yellow fluorescent protein (YFP) fluorescence in the nuclei of plant cells confirmed the interactions of BcSOC1-2 with BcSOC1-1, BcSOC1-2, BcAGL6, and BcAGL24 ( Figures 6B and S7).

SOC1 Expression Pattern Correlates with Bolting in Flowering Chinese Cabbage
Gene expression patterns are closely related to gene function. Previous studies have shown that SOC1 expression in leaves promotes flowering, and it is necessary for the initiation of flowering in the meristem [28][29][30]. In this study, BcSOC1-1 and BcSOC1-2 were expressed in the same tissues but at different levels. In flowering Chinese cabbage, BcSOC1-1 was upregulated in the shoot apex ( Figure 1D), similar to Chrysanthemum [31], whereas BcSOC1-2 was upregulated in the leaves ( Figure 1D), similar to Arabidopsis, rice, and wheat [29,32,33]. Furthermore, BcSOC1 was primarily expressed in vegetative organs, unlike FaSOC1 in strawberry, which is upregulated in the reproductive organs, such as the shoot apex, flower bud, stamen, and sepal [34]. Previous studies on Arabidopsis, strawberry, and soybean suggest that SOC1-like proteins are localized in the cytoplasm [17,34,35]. However, in flowering Chinese cabbage, BcSOC1 was localized in the nucleus ( Figure 2E,F), similar to ZmMAD1 and LsSOC1 [36], indicating that SOC1 plays different roles in different species. Figure 6. Yeast two-hybrid and bimolecular fluorescence complementation assays to determine protein interactions. (A) Yeast two-hybrid assay for protein-protein interactions between BcSOC1 and BcAGL6, BcAGL24, and BcSOC1. BD and AD represent empty pGBKT7 and pGADT7 vectors, respectively. SD/-Trp-Leu, the synthetic dextrose medium lacked tryptophan and leucine; the SD/-Trp-His-Leu-Ade medium lacked tryptophan, histidine, leucine, and adenine. Positive bacteria were stained using X-α-Gal. (B) Bimolecular fluorescence complementation assay was used to detect the interactions of BcSOC1 (fused with N-terminal fragment of YFP) with BcAGL6, BcAGL24, and BcSOC1-1 (fused with C-terminal fragment of YFP). The nuclei were stained with DsRed. Bar = 50 µm.

SOC1 Expression Pattern Correlates with Bolting in Flowering Chinese Cabbage
Gene expression patterns are closely related to gene function. Previous studies have shown that SOC1 expression in leaves promotes flowering, and it is necessary for the initiation of flowering in the meristem [28][29][30]. In this study, BcSOC1-1 and BcSOC1-2 were expressed in the same tissues but at different levels. In flowering Chinese cabbage, BcSOC1-1 was upregulated in the shoot apex ( Figure 1D), similar to Chrysanthemum [31], whereas BcSOC1-2 was upregulated in the leaves ( Figure 1D), similar to Arabidopsis, rice, and wheat [29,32,33]. Furthermore, BcSOC1 was primarily expressed in vegetative organs, unlike FaSOC1 in strawberry, which is upregulated in the reproductive organs, such as the shoot apex, flower bud, stamen, and sepal [34]. Previous studies on Arabidopsis, strawberry, and soybean suggest that SOC1-like proteins are localized in the cytoplasm [17,34,35]. However, in flowering Chinese cabbage, BcSOC1 was localized in the nucleus ( Figure 2E,F), similar to ZmMAD1 and LsSOC1 [36], indicating that SOC1 plays different roles in different species.
ClSOC1-1 and ClSOC1-2 are induced throughout the growth period in Chrysanthemum [31]. The transcript levels of SOC1 in rice and wheat are significantly increased during transition from vegetative to reproductive growth, and then maintained at high levels [32,33]. In the present study, the transcript levels of BcSOC1-1 and BcSOC1-2 were high throughout growth and development of flowering Chinese cabbage, and were significantly increased during floral transition ( Figure S1). This expression profile is consistent with previous reports, indicating that BcSOC1-1 and BcSOC1-2 may be involved in the regulation of bolting and flowering in flowering Chinese cabbage.

GA 3 and Low-Temperature Treatments Accelerate Bolting and Flowering in Flowering Chinese
Cabbage by Mediating BcSOC1 Expression SOC1 responds to flowering signals from both endogenous and environmental stimuli and acts on the downstream floral meristem-specific genes to participate in the formation of plant floral meristems and the regulation of flowering time [12,15,37]. Moon et al. [20] reported that exogenous GA 3 promoted SOC1 expression in WT A. thaliana (Ler) and shortened flowering time. GA 3 treatment increased SOC1 expression and rescued the non-flowering phenotype of ga1-3, whereas SOC1 null mutant exhibited reduced sensitivity to GA 3 for flowering. In this study, BcSOC1-1 and BcSOC1-2 were upregulated in flowering Chinese cabbage upon exposure to exogenous GA 3 after budding (Figure 2A-D), which is consistent with the findings of Dorca-Fornell et al. [38] and suggests that BcSOC1-1 and BcSOC1-2 may mediate premature flowering caused by GA 3 . Moreover, rapid stem elongation and significant increase in plant height ( Figure S1) indicate that BcSOC1 regulates stem elongation in flowering Chinese cabbage.
Lee et al. [39] reported that SOC1 did not express in FRI FLC lines of Arabidopsis without vernalization, whereas SOC1 expression significantly increased after vernalization. In addition, SOC1 expression increased upon vernalization treatment in FLC-3 mutants [20]. In the present study, the expression profiles of BcSOC1-1 and BcSOC1-2 at 4 • C or 15 • C were the same as that under the GA 3 treatment and were rapidly upregulated after budding ( Figures 2E-H and S1). This suggests that BcSOC1-1 and BcSOC1-2 regulate bolting in flowering Chinese cabbage when induced at low temperatures.

BcSOC1 Plays a Vital Role in Regulating Bolting and Stem Elongation in Flowering Chinese Cabbage
AtSOC1 integrates different signaling pathways to promote bolting and flowering in Arabidopsis [28,39]. In the present study, BcSOC1 overexpression in Arabidopsis (Col) shortened its flowering time ( Figure 3, Table 1), suggesting that BcSOC1 exhibits a high degree of functional conservation in promoting flowering. Additionally, increased plant height in BcSOC1 overexpression lines indicated its role in stalk development. To test this hypothesis, we generated and phenotyped BcSOC1 overexpression lines in the background of flowering Chinese cabbage, and we observed that BcSOC1 overexpression significantly advanced bolting in flowering Chinese cabbage ( Figure 3, Table 1). Furthermore, pTRV2:BcSOC1 knockdown lines exhibited delayed bolting and inhibition of stem elongation before flowering (Figure 4), suggesting that BcSOC1 regulates both bolting and stem elongation in flowering Chinese cabbage. Similar findings were reported by Mauren et al. [40], who found that MtSOC1a promotes flowering and primary stem elongation in Medicago, further verifying the role of SOC1 in the regulation of stalk development. In addition, we determined that the two homologous genes were functionally redundant, and BcSOC1-2 was the key regulatory factor.
Several MADS-box transcription factors regulate the expression of downstream target genes in the form of dimers [12,15,37]. In Arabidopsis, the MADS-box transcription factors AGL6, AGL24, FUL, and SEP3 bind with the regulatory sequences of SOC1 [27,41]. In the present study, BcAGL6, BcAGL24, BcSOC1-1, and BcSOC1-2 formed dimers with BcSOC1-2 ( Figure 6). These data suggest that BcSOC1-2 may upregulate the expression of downstream meristem genes through interactions with other MDS-box proteins and thereby regulate bolting of flowering Chinese cabbage. However, BcSOC1-1 did not interact with BcAGL6, BcAGL24, and BcSOC1-1 ( Figure 6A). Nayar et al. [42] conducted deletion analysis and revealed that the KC region (K-box and C-terminal domain) plays a pivotal role in dimerization. However, K-box and C-terminal domains are not completely conserved relative to the highly conserved MADS-box. Alternative splicing events were found only at the borders of or within the K-box domain; a small change in the sequence of K-box domain transcript can have strong consequences for the interaction capacity and functioning of the encoded protein [43]. In the present study, amino acid mutations were detected between the K-box of BcSOC1-1 and BcSOC1-2, which may be the main reason for the distinct interaction patterns between the two BcSOC1 proteins with interacting partners.
Cell cycle regulation of the shoot apical meristem affects stem elongation and thickening, and BcEXPA11 and BcXTH3 are the key factors responsible for cell expansion [44,45]. EXPA11 promotes the sliding of polymers such as cell wall cellulose and hemicellulose by reducing the viscosity of polysaccharides between cell walls and enabling cell wall expansion [46]. XTH3 participates in the regulation of cell wall relaxation by cutting xyloglucan chains [47]. Investigation of the pith cell structure in BcSOC1 overexpression lines revealed that stem elongation was correlated with the increase in pith cell length and area ( Figure 5). Therefore, the rapid elongation of stalk in flowering Chinese cabbage may be attributed to increased cell expansion. The present study revealed that GA 3 and low temperatures accelerate bolting and flowering in flowering Chinese cabbage, probably by upregulating BcSOC1, BcEXPA11, and BcXTH3 (Figures 2 and 5), and the overexpression of hemicellulose by reducing the viscosity of polysaccharides between cell walls and enabling cell wall expansion [46]. XTH3 participates in the regulation of cell wall relaxation by cutting xyloglucan chains [47]. Investigation of the pith cell structure in BcSOC1 overexpression lines revealed that stem elongation was correlated with the increase in pith cell length and area ( Figure 5). Therefore, the rapid elongation of stalk in flowering Chinese cabbage may be attributed to increased cell expansion. The present study revealed that GA3 and low temperatures accelerate bolting and flowering in flowering Chinese cabbage, probably by upregulating BcSOC1, BcEXPA11, and BcXTH3 (Figures 2 and 5), and the overexpression of BcSOC1 induces cell expansion by upregulating BcEXPA11 and BcXTH3 ( Figure 5). Figure 7 demonstrates the role of BcSOC1 in the bolting and flowering of flowering Chinese cabbage. While numerous SOC1-like genes regulate flowering, others may exhibit both functional conservation and divergence with AtSOC1 [22,23]. Taken together, the results of the present study suggest that BcSOC1 may function as a floral integrator gene and promote stem elongation, providing new insights into stalk development in flowering Chinese cab- While numerous SOC1-like genes regulate flowering, others may exhibit both functional conservation and divergence with AtSOC1 [22,23]. Taken together, the results of the present study suggest that BcSOC1 may function as a floral integrator gene and promote stem elongation, providing new insights into stalk development in flowering Chinese cabbage. In addition, stalk development in flowering Chinese cabbage is accompanied by both vegetative and reproductive growth, which is different from other cruciferous leaf vegetables such as B. rapa, B. oleracea, and B. campestris. Therefore, further investigation is necessary to understand the synergistic mechanisms underlying vegetative and reproductive growth in regulating stalk development.

Gene Cloning
Total RNA was extracted from the leaves and shoot apex of flowering Chinese cabbage using a HiPure Plant RNA Mini Kit (Magen, Guangzhou, China) according to the manufacturer's instructions. First-strand cDNA was synthesized and subjected to inverse transcription using a HiScript QRT SuperMix for qPCR Reagent Kit (with gDNA Wiper; Vazyme, Nanjing, China). Coding sequences (CDS) of BcSOC1-1, BcSOC1-2, and BcLFY isolated from the shoot apex were cloned using gene-specific primers (Table S1).

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was isolated from the leaves and stems of plants exposed to exogenous GA 3 and low-temperature treatments. RNA of the WT and BcSOC1 transgenic plants was isolated from the fourth leaf, and qRT-PCR was performed using a ChamQ SYBR Color qPCR Master Mix (Vazyme) on a LightCycler 480 real-time PCR instrument (Roche Diagnostics, Rotkreutz, Switzerland). Three biological and three technical replicates were used for each sample, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference to normalize gene expression. The primers used in qRT-PCR analyses are listed in Table S2.

Subcellular Localization
Full-length BcSOC1-1 and BcSOC1-2 CDS were cloned into pEAQ-EGFP vectors and fused with green fluorescent protein (GFP) under the control of the CaMV 35S promoter. The primers used for cloning are listed in Table S3.
Nicotiana benthamiana leaves were first infiltrated with Agrobacterium tumefaciens strain GV3101 transformed with corresponding constructs and nuclear localization signal (NLS-DsRed). After three days, GFP fluorescence and DsRed protein were detected at 448 nm and 550 nm, respectively, using a laser-scanning confocal microscope. To further verify the results of subcellular localization, the plasmids were transformed into onion epidermal cells using a previously described method [49].

GA 3 and Low-Temperature Treatments
Seeds of flowering Chinese cabbage were sterilized using 70% ethanol for 3 min, washed 3-4 times with deionized water, and then placed in a petri dish lined with filter paper. Thereafter, 200 mg/L GA 3 solution and the same amount of deionized water were added to GA 3 and control (CK) treatments, respectively. Another set of sterilized seeds was exposed to low temperatures in a growth chamber, one group was placed at 4 • C and a second group at 15 • C. After five days of low-temperature treatment, the seeds were sown in perlite. Seedlings with three leaves and one core were transplanted into hydroponic containers and supplied with half-strength Hoagland nutrient solution.
The plants were harvested at 37-40 days after sowing. Plant height (cm) and stem thickness (mm) were measured using the cross method with ruler and vernier caliper, respectively. For GA 3

Ectopic Expression of BcSOC1 in Arabidopsis
The pBI121:BcSOC1 construct, generated by cloning BcSOC1 CDS into pBI121-GFP under the control of CaMV 35S promoter, was first transformed into A. tumefaciens strain GV3101, which was then transformed into Arabidopsis plants using the floral-dip method [50]. Transformed lines were selected on Murashige and Skoog (MS) medium (pH 5.8) containing 1% sucrose, 0.7% agar, and 50 mg/L kanamycin (Kan). The primer sequences are listed in Table S4.

Virus-Induced Gene Silencing (VIGS) Assay
BcSOC1-1 and BcSOC1-2 CDS were cloned into pTRV2 plasmids to generate silencing vectors pTRV2:BcSOC1-1 and pTRV2:BcSOC1-2, respectively, and VIGS experiments were performed as previously described [51]. The pTRV2:BcSOC1 and pTVR1 vectors were co-infiltrated into the cotyledons of young seedlings of the flowering Chinese cabbage. Two weeks after infection, the second true leaf was collected from each plant, and RNA was extracted for qRT-PCR analysis to evaluate the efficiency of gene silencing. Plants co-infiltrated with pTRV2 and pTRV1 were used as negative controls. Subsequently, plant morphological characteristics were determined. The primers used for VIGS assay are listed in Table S4.

Agrobacterium-Mediated Transformation in Flowering Chinese Cabbage
Cotyledon transformation of the 'Youlv 501 caixin' cultivar was performed using a previously described protocol with modifications [52]. After sowing on half-strength MS medium (pH 5.8) containing 1% sucrose and 0.6% agar, the sterilized seeds were placed in a tissue culture at 25/20 • C (day/night) under 16/8 h light/dark cycles and light intensity of 100 µmol m −2 s −1 . The cotyledons and hypocotyls of sterile seedlings were excised 3 d after emergence. The cotyledon and hypocotyl explants were then pre-cultured for callus initiation on MS medium (pH 5.8) containing 0.3 mg/L 1-naphthaleneacetic acid (NAA), 5.0 mg/L 6-benzylaminopurine (6-BA), 2.0 mg/L AgNO 3 , 3% sucrose, and 1% agar, in the dark for two days at 25 • C before inoculation and co-culturing with A. tumefaciens

Yeast Two-Hybrid Assay
BcSOC1-1, BcSOC1-2, BcAGL6, and BcAGL24 CDS were cloned into pGADT7 or pG-BKT7 plasmids to generate yeast two-hybrid vectors. The recombinant vector was then delivered into yeast strain Y2HGold to generate fusion proteins according to a previously described protocol [53]. Primer information is given in Table S5.

Bimolecular Fluorescence Complementation Assay
The vectors were produced by amplifying the CDS of BcSOC1-1, BcSOC1-2, BcAGL6, and BcAGL24 without the stop codons from their corresponding cDNAs. The fragments were cloned into the N-or C-terminus of pSPYNE-35S and pSPYCE-35S vectors to generate fusion proteins [54]. The Agrobacterium strain harboring the recombination plasmid and DsRed was co-infiltrated into young tobacco leaves as previously described [53]. After two days of incubation, yellow fluorescent protein (YFP) fluorescence was detected at 448 nm using a laser-scanning confocal microscope. To indicate the nuclei, DsRed protein was detected at 550 nm. Primer sequences are listed in Table S6.

Conclusions
In this study, we showed that GA 3 and low-temperature treatments significantly upregulated BcSOC1, BcEXPA11, and BcXTH3, thereby promoting early bolting and flowering in flowering Chinese cabbage. Furthermore, BcSOC1 knockdown dramatically delayed bolting, flowering, and inhibited stem elongation in this species. We also elucidated the molecular mechanism underlying stem elongation in flowering Chinese cabbage, wherein the overexpression of BcSOC1 induced cell expansion by upregulating BcEXPA11 and BcXTH3. We further demonstrated that BcAGL6 and BcAGL24 interacted with BcSOC1 to regulate bolting in flowering Chinese cabbage. These findings provide the foundation for further investigations on the mechanisms underlying stalk development in cruciferous stalk vegetables.