Orphan gene BR2 positively regulates bolting resistance through the vernalization pathway in Chinese cabbage

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Although OGs are associated with stress responses as well as their high specificity of expressions have been extensively studied, the functions of OGs in the B. rapa require further exploration.
In plants, flowering is a significant developmental event, which modulates the transition from vegetative to reproductive growth.Currently, five genetically defined pathways are known to regulate floral transition, mainly including the photoperiod, age, vernalization, autonomous, and gibberellin (GA) pathways [24,25].The two crucial vernalization pathway genes include FLOWERING LOCUS C (FLC) and FRIGIDA (FRI) [26,27].FLC is a transcription factor of MADS-box that can suppress flower transformation, whereas FRI acts upstream of FLC and positively inhibits flowering [26].
Overexpression of B. rapa FLC homologs in Arabidopsis delayed flowering for nearly 4-7 weeks [34].However, FLC's cold-sensing mechanism in B. rapa has not been comprehensively studied.
Chinese cabbage (Brassica rapa ssp.pekinensis) is one of the leafy vegetables with wide planting area and the highest total yield in China.However, premature bolting leading to non-heading is a major problem in Chinese cabbage production, which severely affects the yield, especially during autumn at high latitudes and altitudes regions, as well as in spring in northern areas of China [35,36].Therefore, breeding bolting-resistant Chinese cabbage varieties have always been the primary concern of breeders.Genome triplication event of B. rapa has produced several flowering-related genes, which regulate mechanisms much more complicated than that of A. thaliana [37].
Altogether, four FLC paralogues (BrFLC1, BrFLC2, BrFLC4, BrFLC5) have been cloned in B. rapa [38].Furthermore, BrFLC2 has been indicated as a primary regulator gene of flowering time and vernalization response QTL in B. rapa [39,40].Moreover, natural splicing mutation at BrFLC1 intron site has been markedly linked with altered flowering time in B. rapa [41].Previous study indicates that BrFLC5 (Bra022771) is expressed at lower levels than two other BrFLC genes (BrFLC1, Bra009055 and BrFLC2, Bra028599) indicating that BrFLC5 is more conducive to cultivating varieties in B. rapa which are resistant to bolting [42].In addition, a histone H4 protein encoded by BrHIS4.A04 (Bra035673) has previously been shown to inhibit drought-mediated bolting in Chinese cabbage by attenuating the flowering genes expression [43].Furthermore,

Overexpressing BR2 delayed flowering in Arabidopsis
The flowering time of BR2 overexpressing Arabidopsis plants was significantly delayed under long-day (LD) conditions, with an average delay of 33 days (Fig. 1a and Supplementary Table S1).Furthermore, the leaves of the 'BR2OE' plants were all curled inward (Fig. 1a).The 'BR2OE' rosette radius, silique length, and leaves length were all shorter than the wild-type (WT) plants.The number of seeds in each silique of 'BR2OE' plants was significantly lower than that in WT plants.The length of tetradynamous stamens and pistils of the 'BR2OE' plants were all much longer than those of WT plants (Fig. 1b and c).In the 'BR2OE' plants, the tetradynamous stamens were lower than the pistils, while the tetradynamous stamens in WT plants were flush with or below the pistils.
To investigate whether the flowering time delay is related to the levels of its modulatory factors, the expression of some flowering key genes in the leaves and shoot apex (SA) of 'BR2OE' and WT plants were compared by qRT-PCR (Fig. 1d).It was revealed that the expression levels of the flowering pathway genes were similar to that observed in the delayed flowering phenotype.In the leaves and SA of the 'BR2OE' plant, the expressions of AtFT and AtSOC1 were markedly down-regulated, and AtLFY in SA of the 'BR2OE' was similar to AtFT and AtSOC1 expression.Furthermore, the AtLFY expression in the leaves of the 'BR2OE' and WT was not detected.Interestingly, AtFLC in leaves and SA increased by nearly 58 and 13 times compared with WT.Moreover, AtFRI and AtVIN3 were notably down-regulated in SA, suggesting that BR2 may mainly function through the vernalization pathway.

'BR2OE' was responsive to vernalization
The vernalized 'BR2OE' plants bolting at about 23 days and flowering at about 34 days, which was much earlier than the non-vernalized plants (About 45 and 52 days); interestingly, this was still later than the WT plants in both vernalization and non-vernalization treatments (Fig. 2a, c and d).Furthermore, the average rosette leaf number of 'BR2OE' plants was more than that of WT plants under non-vernalization treatment, while there was no significant difference between 'BR2OE' and WT plants under vernalization condition (Fig. 2b).These observations showed that 'BR2OE' plants positively respond to vernalization treatment.

'BR2OE' indicated late flowering under both LD and SD conditions
To assess if the late flowering phenotype of Arabidopsis 'BR2OE' plants was linked with the photoperiod pathway, the flowering time of WT and 'BR2OE' plants under LD and SD conditions was measured (Fig. 2c and d).The bolting and flowering times of 'BR2OE' plants were much later than WT plants under both LD and SD conditions.
Moreover, it has been indicated that late-flowering plants form more rosette leaves when 'BR2OE' plants flowering under different conditions.Thus, 'BR2OE' plants bolting and flowering later than WT and had more rosette leaves under LD and SD conditions.This suggests that BR2 may be not involved in the photoperiodic pathway of flowering control.

BR2 gene promoter expression analysis
The BR2 promoter sequence was cloned and constructed upstream of the GUS gene of the pCAMBIA1305.1 vector.For GUS staining, homozygous plants of A. thaliana were obtained by using the floral-dip method.Histochemical staining for the transgenic line's GUS activity indicated high expression in the SA, leaves, leaf veins, stamens, stigmas and pedicel-silique junction (Supplementary Data Fig.S1a).But the expression of the BR2 gene promoter was very low in siliques and seeds.These data indicated that the levels of BR2 promoter expression are spatiotemporally regulated.

Subcellular localization of BR2
The BR2 protein's subcellular localization was assessed by injecting the tobacco leaves with a mixture of Agrobacterium tumefaciens carrying 35S::BR2::GFP and YFP cell membrane marker plasmids, and fluorescence was observed by confocal microscope.
The distribution of fluorescence signal of the fusion protein transiently expressed in cells suggested that BR2 was present on the cell membrane (Supplementary Data Fig.S1b).

Chinese cabbage
Since the overexpression of BR2 showed late flowering in Arabidopsis, the expression of BR2 in Chinese cabbage bolting resistant type (BR type) and bolting non-resistant type (BN type) varieties was analyzed.Chinese cabbage indicating short stem with rounded apices belongs to the BR type, while BN type has pointed apices [46,47].The expression pattern of BR2 in the heading stage was analyzed by selecting 10 BR type and 10 BN type varieties (Fig. 3a).The data revealed that BR2 expression was notably increased in all BR type, but was markedly down-regulated in the BN type (Fig. 3a).These results further confirmed that BR2 was related to the bolting resistance of Chinese cabbage.Moreover, using Chinese cabbage inbred line 'GT-24', the expression pattern of BR2 was analyzed under chilling stress (4℃).After the treatment, BR2 expression of was substantially decreased compared with the control (Fig. 3c), which may promote vegetative growth transformation to reproductive growth.

BR2 overexpression in Chinese cabbage caused delayed flowering
To further assess the effect of the BR2 on the phenotype of Chinese cabbage, T2 generation BR2 overexpressing homozygous plants were obtained (Supplementary Data Fig.S2) and referred to as 'GTBR2OE'.The phenotypes of wild-type Chinese cabbage 'GT-24' and 'GTBR2OE' plants under vernalization and non-vernalization conditions were observed (Fig. 4a).The number of rosette leaves of 'GTBR2OE' plants were more than those of the 'GT-24' plants in both vernalization and non-vernalization conditions (Fig. 4b and e).Under vernalization conditions, the bolting and flowering times of 'GTBR2OE' plants (About 68 and 77 days, respectively) were later than those of the 'GT-24' plants (About 61 and 64 days, respectively) (Fig. 4c and d).Similarly, the bolting and flowering times of 'GTBR2OE' plants (About 97 and 107 days, respectively) were later than those of the 'GT-24' plants (About 81 and 90 days, respectively) under non-vernalization conditions (Fig. 4f and g).
The morphological traits including plant weight, head weight, plant height, plant width, outer leaf length, outer leaf width, head height, head width, and shortened stem width of 'GT-24' and 'GTBR2OE' plants during heading stage were significantly different (Supplementary Data Fig.S3).Overall, BR2 regulates the bolting resistance of Chinese cabbage; however, the possible pathways need further analysis.

Transcriptome analysis of 'GTBR2OE' plants
To analyze the molecular mechanisms associated with the impact of BR2 on delayed flowering time, transcriptomic sequencing was performed by preparing the cDNA libraries from the SA of 'GT-24' and 'GTBR2OE' plants before bolting after vernalization treatment.All samples indicated Q20 and Q30 values > 97% and > 92%, respectively (Supplementary Table S2).The raw and clean read numbers for each sample ranged from 41,500,918 to 48,549,908 and 37,384,288 to 43,933,202, respectively, consistent with highly reliable transcriptome detection results.

Discussion
The OGs are crucial for species formation and evolution as they regulate mechanisms for generating new genes and functions [48].Furthermore, they exist in almost every genome and are associated with various biological pathways, such as immune regulation, species-specific adaptation processes, metabolism, stress biology, and traits regulation [49].However, most OGs are not well annotated because they lack identifiable functional domains, making their functional characterization difficult [1].The functional characterization of OGs is essential, which helps us understand the functional diversity of the genome and the evolution of complex traits [49].Here, an OG, BR2 was identified, which positively regulates bolting resistance and delayed flowering.Since the differentiation of Chinese cabbage from Arabidopsis, specific traits have been observed such as leafy head, and Chinese cabbage requires vernalization to enter reproductive growth.BR2 overexpression delays flowering, which further confirm the relationship between OGs and the formation of species-specific traits.
BR2 also regulates bolting resistance like previously reported [46].In Arabidopsis, BR1 overexpression markedly delays the flowering time.Furthermore, it has been verified that the nuclear BR1 protein acts via the photoperiod and vernalization pathways [46]. A.
thaliana species-specific OG Qua-Quine Starch (QQS) specifically modulates nitrogen and carbon allocation, decreases starch, and increases protein [5].Moreover, QQS has shown similar roles across species, including Arabidopsis, soybean, tobacco, and others [3,4,12].The protein-protein interactions analysis of NF-YC4, a QQS interaction protein, indicated that many proteins are a part of the transcription factor NF-Y complex, 3 of which are linked with flowering time [12].Previous study showed that an OG Prod1 was necessary for preaxial digit formation during salamander limb development, which provided new insights into species-specific traits regulation of OGs [50].The analysis of OGs in the Cucurbitaceae genomes indicated that many OGs are involved in reproductive growth and development processes [21].Brassica-specific OG BrLFM is proved to be cabbage.This investigation provided novel ideas and gene targets for the cultivation of new bolting-resistant varieties of Chinese cabbage, which will offer valuable insights into the functions of OGs in the regulation of species-specific traits.

Plant materials and cultivation
Plant materials include Chinese cabbage inbred line 'GT-24' and 'Chiffu', wild-type Agricultural University).N. benthamiana was cultivated as in prior studies [46].The plants were grown in conditions described in previous studies [46].Plants were grown under long-day (LD, 16 h light/8 h darkness), and short-day (SD, 8 h light/16 h darkness) conditions.

BR2 sequence analyses, vector construction and plant transformation
The analyses of the BR2 sequence were performed as detailed previously [46].For BR2 overexpression in Arabidopsis and Chinese cabbage, vector construction, plant transformation and selection, DNA isolation, and PCR amplification for positive transgenic plants detection were carried out as detailed in prior reports [23,46,62].
Primer pairs utilized are listed in Supplementary Table S7.

Vernalization and LD/SD treatments
For vernalization treatment, the seed sprouted WT and 'BR2OE' plants were grown at 4℃ for 6 weeks, and then transferred to LD conditions under normal growth temperature.For LD/SD treatments, WT and 'BR2OE' plants were grown in LD or SD conditions.The investigation of rosette leaf number, days to bolting and flowering of WT as well as 'BR2OE' plants were measured and compared at the same developmental stage by following previous methods [46].At least 30 plants were used for each experiment.

Figure 1 .
Figure 1.Phenotypic characterization of 'BR2OE' lines.(a) The phenotype of 'BR2OE' plants.The images of 28-day-old leaves, scale bars = 2 cm (left part).The images of 50-day-old plants, scale bars = 10 cm (right part).(b) The phenotype of WT (upper part) and 'BR2OE' (lower part) flowers.The stamens and pistils were observed by removing one petal.Scale bars = 1 mm.(c) Tetradynamous stamens and pistils length of WT and 'BR2OE'.Data are means ± SE (n = 10).(d) Expression analysis of key flowering pathway genes in Arabidopsis 'BR2OE' and WT plants.WTL and BR2OEL represent the leaves of WT and 'BR2OE' plants, respectively.WTSA and BR2OESA represent shoot apex (SA) of WT and overexpression lines, respectively.The asterisk at the top of the column chart represents the significant differences detected by the Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3 .
Figure 3. Expression of BR2 gene in Chinese cabbage.(a) Phenotypes of bolting resistant type (BR type) and bolting non-resistant type (BN type) Chinese cabbage, and qRT-PCR assessment of BR2

Figure 5 .
Figure 5. Identification of DEGs between 'GTBR2OE' and 'GT-24' plants.(a) Heatmap of DEGs.(b) The top 20 enriched KEGG pathways are associated with the identified DEGs.Pathways are provided with the corresponding rich factors.(c) DEG gene ontology (GO) classification was carried out, with individual bars denoting the numbers of DEGs mapped to particular GO categories.Red: biological process (BP), blue: cellular component (CC), green: molecular function (MF).

Figure 6 .
Figure 6.Heatmap of flowering genes.Heatmap composed of log2 fold-change values of several flowering genes under different pathways.GA pathway represented gibberellin pathway.
R I P T Downloaded from https://academic.oup.com/hr/advance-article/doi/10.1093/hr/uhae216/7723781 by guest on 02 August 2024 involved in leafy head formation in Chinese cabbage, which will be vital for understanding the relationship between OGs and species-specific morphological features, such as the leafy head in Chinese cabbage [51].These data validate the crucial role of Downloaded from https://academic.oup.com/hr/advance-article/doi/10.1093/hr/uhae216/7723781 by guest on 02 August 2024 45, 57], consistent with the results of this research.The qRT-PCR and transcriptomic sequencing confirmed that BR2 overexpression in B. rapa markedly altered BrFLC5 expression and down-regulated key floral genes.After vernalization, 'GTBR2OE' still has stronger bolting resistance than 'GT-24' plants.These results indicate that the role of BR2 in the regulation of bolting resistance may involve the vernalization pathway.In B. rapa vegetables, flowering time is essential trait and is modulated by FLC [58].A recent study indicated that when overexpressed BrFLC5, which acts as a floral repressor in A. thaliana, and its increased expression promotes vernalization and might help develop an enhanced bolting-resistant breed of B. rapa vegetables [59].Another study showed that among the 3 analyzed BrFLC5 had the lowest expression, suggesting that it Downloaded from https://academic.oup.com/hr/advance-article/doi/10.1093/hr/uhae216/7723781 by guest on 02 August 2024 [28]t be a weak flowering time modulator in B. rapa; however, it might be more efficient for breeding premature bolting resistance in B. rapa[43].Furthermore, BrFLC5 has also been observed as a crucial candidate gene for a flowering time control in B. rapa[38, 60].The downregulation of BrVIN3.bexpression, a direct inhibitor of FLC, explained the increased expressions of BrFLC5.Studies showed that the mutations of AtVIN3 results in a vernalization-insensitive phenotype[28].These results strongly support the findings of this study.
. thaliana 'Col-0' (WT) and Arabidopsis BR2 overexpression plant 'BR2OE', Chinese cabbage BR2 overexpression plants 'GTBR2OE' and Nicotiana benthamiana.The two type varieties of Chinese cabbage (BR and BN types) were cultivated in our laboratory (Molecular Biology of Vegetable Laboratory, College of Horticulture, Shenyang