Molecular mechanism of flowering time regulation in Brassica rapa: similarities and differences with Arabidopsis

Abstract

Properly regulated flowering time is pivotal for successful plant reproduction. The floral transition from vegetative growth to reproductive growth is regulated by a complex gene regulatory network that integrates environmental signals and internal conditions to ensure that flowering takes place under favorable conditions. Brassica rapa is a diploid Cruciferae species that includes several varieties that are cultivated as vegetable or oil crops. Flowering time is one of the most important agricultural traits of B. rapa crops because of its influence on yield and quality. The transition to flowering in B. rapa is regulated by several environmental and developmental cues, which are perceived by several signaling pathways, including the vernalization pathway, the autonomous pathway, the circadian clock, the thermosensory pathway, and gibberellin (GA) signaling. These signals are integrated to control the expression of floral integrators BrFTs and BrSOCls to regulate flowering. In this review, we summarized current research advances on the molecular mechanisms that govern flowering time regulation in B. rapa and compare this to what is known in Arabidopsis.

Headnote

Received 1 May 2023; Received in revised form 13 June 2023; Accepted 18 September 2023

Available online 10 January 2024

ABSTRACT

Properly regulated flowering time is pivotal for successful plant reproduction. The floral transition from vegetative growth to reproductive growth is regulated by a complex gene regulatory network that integrates environmental signals and internal conditions to ensure that flowering takes place under favorable conditions. Brassica rapa is a diploid Cruciferae species that includes several varieties that are cultivated as vegetable or oil crops. Flowering time is one of the most important agricultural traits of B. rapa crops because of its influence on yield and quality. The transition to flowering in B. rapa is regulated by several environmental and developmental cues, which are perceived by several signaling pathways, including the vernalization pathway, the autonomous pathway, the circadian clock, the thermosensory pathway, and gibberellin (GA) signaling. These signals are integrated to control the expression of floral integrators BrFTs and BrSOCls to regulate flowering. In this review, we summarized current research advances on the molecular mechanisms that govern flowering time regulation in B. rapa and compare this to what is known in Arabidopsis.

Keywords: Flowering time; Brassica rapa; Vernalization; Photoperiod

1. Introduction

Flowering is one of the most important developmental events in the plant life cycle. During the transition from vegetative growth to flowering, the shoot apical meristem (SAM) is reprogrammed to produce an inflorescence meristem, which marks the transition from vegetative growth to reproductive growth. The precise timing of this transition is pivotal for successful plant reproduction (Amasino, 2010; Song et al., 2013). Before the transition to flowering can take place, plants must ensure that environmental conditions are suitable for seed formation and they must accumulate enough energy to support seed development (Amasino, 2010). Throughout the course of evolution, flowering plants have developed a complex gene regulatory network that enables them to perceive the environmental and internal conditions that are favorable for flowering.

Brassica rapa is a diploid species in the Cruciferae family that includes a number of subspecies cultivated as vegetable or oil crops. This includes the vegetable crops B. rapa ssp. pekinensis (Chinese cabbage), B. rapa ssp. chinensis (pak choi), B. rapa ssp. chinensis var. narinosa (wutacai), B. rapa ssp. chinensis var. nipposinica (mizuna), B. rapa ssp. rapa (turnip), B. rapa ssp. chinensis var. parachinensis (caixin), and B. rapa ssp. chinensis var. broccoletto (broccoletto) and oil seed crops B. rapa ssp. chinensis var. oleífera (turnip rape) and B. rapa ssp. chinensis var. tricolaris (yellow sarson) (Zhao et al., 2005). These subspecies have different growth habits and vary greatly in flowering time. The spring types (caixin and yellow sarson) and semi-winter types flower without vernalization. In contrast, the winter type requires a long period of lowtemperature exposure prior to the flowering transition.

Proper flowering time in B. rapa crops is pivotal for the optimal formation of harvestable leaves and seeds. Premature bolting reduces vegetable growth and results in poor yield and leaf and seed quality, and delayed flowering may also affect seed yield. For these reasons, a primary focus of B. rapa breeding efforts is on the development of varieties that bolt when seasonably appropriate. Understanding the genetic and molecular mechanisms of flowering time control in B. rapa is pivotal for crop improvement. In this review, we summarize current research advances in the molecular mechanisms of flowering time regulation in B. rapa and compare this to knowledge of Arabidopsis.

2. Overview of flowering time regulation

Studies in Arabidopsis have revealed that the transition to flowering is regulated by vernalization, the autonomous pathway, the photoperiod, the thermosensory pathway, gibberellin (GA), and plant age (Amasino, 2010; Song et al., 2013). These environmental and intrinsic signals are integrated to regulate the expression of a group of genes called floral integrators, including FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1)/AGAMOUS-LIKE 20 (AGL20), to control flowering (Amasino, 2010; Song et al., 2013; Whittaker and Dean, 2017) (Fig. 1).

Vernalization is the requirement for a prolonged period of cold temperatures before flowering can take place. For plant species in temperate climates, vernalization is especially pivotal to prevent flowering in the fall and to promote flowering in the spring (Xu et al., 2023). Vernalization pathways regulate the floral transition by modulating FLOWERING LOCUS C (FLC) expression (Kim and Sung, 2014a; Whittaker and Dean, 2017). FLC acts with its partner SHORT VEGETATIVE PHASE (SVP) to repress transcription of the flowering-promoting genes FT, FD, and SOCI (Amasino, 2010). Vernalization represses FLC expression to establish flowering competence. In addition, FLC expression is suppressed by the autonomous pathway, which refers to signaling that constitutively promotes flowering independent of environmental cues.

Perception of day length, temperature and intrinsic developmental signals occurs in the leaves, and a long-distance signal, named florigen, is transmitted to the SAM via the phloem vascular system in order to promote floral transition. Florigen is encoded by FT. The FT protein is produced in leaves and travels to the SAM; there it forms a complex with the transcription factor FD to activate the downstream floral meristem identity genes LFY and API (Turek et al., 2008). This in turn leads to the reprogramming of the SAM into an inflorescence meristem. Thus, FT functions as a pivotal link between leaves that perceive environmental signals conducive to flowering and the SAM, where the floral meristem forms. FT expression in leaves is regulated by the photoperiodic, thermosensory, GA, and age pathways (Song et al., 2013; Golembeski and Imaizumi, 2015; Bao et al., 2020; Freytes et al., 2021). The photoperiodic pathway perceives seasonal changes by monitoring day length and transducing the information to the CONSTANS (CO) transcription factor. Under optimal long day conditions, increased CO protein activity enhances FT expression to accelerate flowering (Song et al., 2013; Golembeski and Imaizumi, 2015; Freytes et al., 2021) The thermosensory pathway activates FT in response to optimal temperatures conducive to flowering (Song et al., 2013). GA promotes flowering primarily through activating FT expression in leaves under long day conditions (Bao et al., 2020). Under short day conditions, GA can also accelerate flowering, primarily through inducing SOCI and LFY expression (Bao et al., 2020; Sheng et al., 2022). Additionally, flower competence increases with plant age, which is mediated by several microRNAs (miRNAs) and SQUAMOSA PROMOTER BINDING-LIKE (SPL) transcription factors to suppress FT and LFY and prevent premature flowering (Amasino, 2010).

Studies in Arabidopsis offer a good framework for understanding the genetic and molecular mechanisms of flowering time regulation in В. rapa. Relative studies have identified candidate flowering-regulating loci that are linked to the gene orthologs of key Arabidopsis flowering regulators using quantitative trait loci (QTL) and genome-wide association studies (GWAS) from different B. rapa accessions (Lou et al., 2007, 2011; Schiessl et al., 2017; Su et al., 2018; Kaur et al., 2021; Qu et al., 2022). Nevertheless, in contrast to Arabidopsis, a whole genome triplication event during the evolution of the Brassica species led to duplicate genes that were free to functionally diverge (Wang et al., 2011). Sequence variation and different expression patterns of these paralogs resulted in a complicated gene regulatory network of flowering time in B. rapa.

3. Vernalization and the autonomous pathway regulate flowering time by controlling FLC expression

Environmental requirements for vernalization vary among plant species and ecotypes within species. These differences determine the reproductive strategies of different plants. Winterannual species of Arabidopsis germinate in autumn, undergo vernalization during winter, and blossom in spring; meanwhile, single-season annuals can germinate and bloom in one season without vernalization. Studies of natural variation in Arabidopsis have identified FLC and FRIGIDA (FRI) as key regulators of vernalization (Amasino, 2010; Whittaker and Dean, 2017). FLC encodes a MADS-box transcription factor that prevents flowering by repressing the floral integrator genes FT and SOCI. Before cold exposure, the expression level of FLC is controlled by two antagonistic pathways, namely, FRI and associated regulators, which function as activators, and the autonomous pathway, which represses FLC. This initial level of FLC expression before cold exposure is the primary factor that determines the cold duration necessary for flowering (Whittaker and Dean, 2017). Throughout vernalization, FLC transcription is halted, and a series of chromatin modifications at the FLC locus lead to epigenetic silencing of FLC. This establishes flowering competence and enables flowering to take place when temperatures warm (Kim and Sung, 2014a; Berry and Dean, 2015; Whittaker and Dean, 2017; Li et al., 2023).

3.1. The role of BrFLCs in flowering time regulation

Brassica rapa is a typical vernalization-sensitive plant; however, the vernalization requirements vary among and within varieties. The B. rapa genome contains four copies of FLC (Table 1) (Schranz et al., 2002; Kim et al., 2006, 2007; Li et al., 2016; Ma et al., 2021). BrFLCl, BrFLC2, and BrFLCS are syn tenie to Arabidopsis FLC (Wang et al., 2011). Constitutive expression of BrFLCl, BrFLC2, and BrFLCS in Arabidopsis delayed flowering, thus indicating that they have a similar role in flowering time as Arabidopsis FLC (Kim et al., 2007; Huang et al., 2018). Knockdown of BrFLC2 in pak-choi accelerated flowering, while BrFLCS overexpression in Chinese cabbage resulted in delayed flowering (Kim et al., 2007; Gu et al., 2015; Huang et al., 2018).

The expression of all four of the BrFLCs is repressed by vernalization in a manner similar to that of Arabidopsis FLC (Kim et al., 2007; Huang et al., 2018; Xi et al., 2018; Takada et al., 2019; Xiao et al., 2019). Interestingly, FLC expression negatively correlated with the duration of cold required for vernalization in B. rapa and B. napus (Takada et al., 2019; Calderwood et al., 2021). BrFLC2 is a major QTL associated with flowering time in B. rapa, whereas BrFLCl and BrFLCS colocalize with other QTLs associated with flowering time (Schranz et al., 2002; Lou et al., 2007; Li et al., 2009; Yuan et al., 2009; Zhao et al., 2010; Xiao et al., 2013, 2014; Su et al., 2018; Kim et al., 2022). In contrast, there are rare known reports linking BrFLCS to flowering time. These observations suggest that BrFLCs function redundantly to repress flowering.

There have been several studies that have reported that specific sequence variations within BrFLCs are associated with flowering time differences. In vegetable-type B. rapa, a SNP at the 5' splice site in the sixth intron of BrFLCl contributed to flowering time variation. The presence of G instead of A at this site led to aberrant splicing, loss of function of BrFLCl, and early flowering across 121 B. rapa accessions (Yuan et al., 2009). Notably, this polymorphism was also presented in oil-type B. rapa, but it did not correlate with flowering time variations among oil-type B. rapa accessions (Wu et al., 2012). In oil-type B. rapa accessions, a 57-bp insertion/deletion (InDei) across exon 4 and intron 4 of BrFLC2 resulted in a non-functional gene that confers early flowering to accessions carrying this allele (Wu et al., 2012). Screening of 159 B. rapa accessions revealed that this InDei was not present in vegetable-type B. rapa subspecies, thus suggesting that this allele arose after the divergence of oil-type and vegetable-type B. rapa (Wu et al., 2012). A SNP (G/А) at the 5' splice site in the third intron of BrFLCS was significantly correlated with flowering time variation among a germplasm collection of 301 B. rapa accessions including turnip, mizuna, yellow sarson, Chinese cabbage and caixin (Xi et al., 2018). Accessions with a G allele express full-length cDNA, whereas the A allele leads to abnormal splicing and loss of function of BrFLCS. BrFLCS expression is typically much lower than that of BrFLCl and BrFLC2, which may explain why the regulatory effect of BrFLCS on flowering is weaker than that of BrFLCl (Xi et al., 2018). Notably, the Chiifu-402-41 accession used for reference genome assembly carries the nonfunctional BrFLCS allele. In turnip, the frequency of the functional BrFLCS allele was much higher than in other subspecies, thus suggesting that this allele may have been selected during B. rapa domestication (Xi et al., 2018).

A recent study showed that BrFLCl is a key variation source during spring Chinese cabbage selection (Su et al., 2018). Spring Chinese cabbage is sown in the early spring, and the seedlings have to experience low temperatures; therefore, bolting resistance is critical to prevent flowering before leafy head formation. Phenotypic and genomic analyses using 194 lines from spring-, summer-, and autumn-type Chinese cabbage identified 15 polymorphisms at the BrFLCl locus that have been associated with bolting time after vernalization (Su et al., 2018). These polymorphisms were classified into two main haplotype (BrFLClH1/H1, BrFLClH2/H2) groups and one minor haplotype (BrFLClH3/H3) group, among which BrFLClH1/H1 was carried by 39% of autumn accessions and 96.9% of spring accessions. This suggests that this haplotype group underwent extensive selection during spring Chinese cabbage breeding (Su et al., 2018).

3.2. FRI acts as an activator of FLC transcription

FRI is a major determinant of natural variation in Arabidopsis (Johanson et al., 2000). Many early flowering Arabidopsis accessions carry loss-of-function fri alleles (Johanson et al., 2000). FRI could promote FLC expression to confer vernalization requirements and a winter annual habit in Arabidopsis (Kim and Sung, 2014a). It interacts with the nuclear cap-binding complex and activates FLC transcription by increasing the proportion of FLC mRNA that possesses a 5' cap (Geraldo et al., 2009). FRI also participates in the FRI-C complex, which includes the SWR1 complex and SDG8, to facilitate the recruitment of modifying factors to FLC locus. This resulted in enhanced FLC transcription by increasing active chromatin modifications, such as НЗКЗбтеЗ, H3K4me3, and H3/H4 acetylation (Whittaker and Dean, 2017). In

B. rapa, there has been rare evidence implicating FRI in flowering time variation thus far (Akter et al., 2020a). This may be due to the redundant function of the two FRI paralogs in B. rapa, BrFRIa and BrFRIb (Table 1), both of which are considered activators of BrFLCs (Takada et al., 2019; Akter et al., 2020a). However, a recent study indicated that there might be functional divergence between BrFRIa and BrFRIb. Although BrFRIa and BrFRIb have similar expression patterns in different tissues, BrFRIa formed a stronger association with FLC chromatin to promote FLC expression, and transgenic Arabidopsis plants overexpressing BrFRIa flower much later than those overexpressing BrrFRIb (Zheng et al., 2021). BrFRIa and Arabidopsis AtFRI contain two coiled-coil domains that are required for their biological functions. Moreover, the first coiled-coil domain of BrFRIa contains most of the 37 conserved amino acids in the central domain of AtFRI (Zheng et al., 2021). In contrast, BrFRIb has only one coiled-coil domain homologous to those of BrFRIa and the central domain of AtFRI. The differences in protein sequences between BrFRIa and BrFRIb likely result in different protein activity. It is unclear whether this functional divergence is present in other B. rapa varieties. The

Arabidopsis H3K36 methylase SDG8 associates with the FRI-C complex and is necessary for activating FLC transcription. Only one copy of SDG8 is present in the B. rapa genome (Table 1). The loss of function of BrSDGB led to decreased expression of all four of the BrFLCs and resulted in early bolting in Chinese cabbage (Fu et al., 2020). Concomitant knockdown or knockout of B. napus BnaSDG8.A/C could also promote flowering (Jiang et al., 2018), thus indicating a conserved function of SDG8 orthologs in FLC regulation. 3.3.

The autonomous pathway and COOLAIRs suppress FLC transcription The

autonomous pathway suppresses FLC expression independent and antagonistic to FRI activity (Kim and Sung, 2014a). In Arabidopsis, the autonomous pathway is comprised of FLOWERING CONTROL LOCUS A (FCA), FPA, FY, FLOWERING LOCUS D (FLD), FLOWERING LOCUS KH DOMAIN (FLK), LUMINIDEPENDENS (LD), and FVE (Whittaker and Dean, 2017). Loss of function of any one of these components delayed flowering in Arabidopsis. In B. rapa, GWAS analysis identified FPA and FY orthologs as candidate genes for flowering time variation in derived B. rapa lines (Kaur et al., 2021).

The components of the autonomous pathway encode proteins involved in RNA metabolism. They influence FLC expression by processing a collection of cold-induced, long non-coding RNAs (COOLAIRs) (Kim and Sung, 2014a). COOLAIRs are transcribed in the antisense direction, starting downstream of the poly-A site of FLC (Kim and Sung, 2014a). RNA processing and chromatin activities mediate chromatin silencing to repress FLC expression (Whittaker and Dean, 2017). COOLAIR-like transcripts identified in different species of Brassicaceae exhibit similar secondary structures, despite low sequence conservation, thus indicating functional similarity (Hawkes et al., 2016). In B. rapa, five COOLAIR-like transcripts were identified from the BrFLC2 locus (referred to as BrFLC2as), and the expression of these COOLAIRs increased during vernalization (Fig. 2) (Li et al., 2016). BrFLC2as are conserved in the rapid-cycling crop yellow sarson (var. trilocularis), medium-cycling crop Bre (ssp. pekinensis var. bre), and the slow-cycling crop Wantai (ssp. pekinensis var. wantai). BrFLC2as816 overexpression in Bre downregulated BrFLCl, BrFLC2, and BrFLC3 and abolished the requirement for vernalization, indicating that BrFLC2as816 can also regulate the expression of BrFLCl and BrFLC3 (Li et al., 2016). It is unclear whether COOLAIRs from BrFLC2 can function in trans to regulate other BrFLCs or if an independent mechanism leads to decreased expression of BrFLCl and BrFLC3. Surprisingly, no COOLAIR-like transcripts from the other three BrFLCs have been detected (Li et al., 2016; Shea et al., 2019).

In addition to COOLAIRs, the vernalization-mediated epigenetic silencing of FLC in Arabidopsis requires another long intronic noncoding RNA called COLD-ASSISTED INTRONIC NONCODING RNA (COLD AIR) (Kim and Sung, 2014a). COLD AIR is thought to take part in the recruitment of Polycomb repressive complex 2 (PRC2) to the FLC locus for chromatin modification. Intriguingly, no COLDAIR transcripts have been identified as being derived from BrFLCs (Li et al., 2016). The alternative mechanism that mediates PRC2 recruitment and FLC silencing in В. rapa remains elusive.

3.4. Chromatin modifications play an important role in regulating FLC expression during vernalization

During vernalization, FLC expression is repressed. Importantly, plants can "remember" the cold they have experienced. This results in reduced FLC expression even after plants are subjected to warmer temperatures. The mechanism behind "cell memory" induced by long-term cold treatment is mediated by chromatin modifications that lead to gene silencing. In Arabidopsis, histone modifications during vernalization have been extensively studied (Kim and Sung, 2014a; Berry and Dean, 2015; Whittaker and Dean, 2017). Upon cold exposure, the transcription of COOLAIR is increased, coinciding with decreased transcription of FLC. Meanwhile, histone deacetylase HDA19 is recruited to the FLC locus to reduce FLC transcription. During cold exposure, chromatin modifications in the FLC sequence lead to epigenetic silencing. The histone modifications prior to vernalization, including H3K4me3, H2B ubiquitination, histone acetylation, and НЗКЗбтеЗ, are replaced with repressive modifications, such as H3K27me3. H3K27me3 is initially deposited in a small, specific region called the nucleation region, which contains about three nucleosomes. This process is mediated by the PHD-PRC2 complex, which comprises several Polycomb family proteins and PHD finger proteins. Upon returning to warm temperatures, H3K27me3 spreads across the FLC locus to establish stable silencing that is heritable across cell divisions. After flowering, epigenetic modifications are erased from the FLC locus during embryo development to reset FLC expression in the next generation, a process that involves the H3K27me3 demethylase ELF6 (Kim and Sung, 2014a; Berry and Dean, 2015; Whittaker and Dean, 2017).

In pre-vernalized B. rapa plants, BrFLCs are enriched with H3K4me3 modifications (Méhraj et al., 2021). Upon cold exposure, H3K27me3 accumulates in the nucleation region of the four BrFLC paralogs, which results in decreased expression of BrFLC (Akter et al., 2020b). Upon returning to warm temperatures, H3K27me3 modifications spread across the four BrFLC paralogs, which stabilize the repression of BrFLCs (Kawanabe et al., 2016; Akter et al., 2019; Takada et al., 2019) (Fig. 2). These observations suggest that the regulatory mechanism of chromatin modifications during vernalization is largely similar between B. rapa and Arabidopsis.

Loss of function of key components in the Arabidopsis vernalization pathway, such as VRNI, VRN2, VIN3, and VINS, eliminated the requirement for vernalization to promote flowering (Amasino, 2010). Vernalization-induced VINS encodes a PHD finger protein that interacts with VRN5 to increase H3K27me3 accumulation at the FLC locus by enhancing PRC2 histone methyltransferase activity in Arabidopsis (Whittaker and Dean, 2017). The B. rapa genome encodes two VIN3 paralogs that are induced during vernalization (Table 1) (Dai et al., 2020). Recent studies have identified BrVINS.l as a QTL associated with flowering time in B. rapa (Liu et al., 2016; Wang et al., 2018). Polymorphisms in the BrVINS.l promoter region are associated with vernalization variations in Chinese cabbage (Su et al., 2018). Phenotypic and genomic analysed of 194 lines from spring-, summer-, and autumn-type Chinese cabbage identified five haplotype groups of BrVINS.l, among which BrVIN3.1H1/H1 substantially contributed to bolting time. Analysis of promoter activity confirmed that polymorphisms in the BrVINS.l promoter confer different transcriptional responses of BrVINS.l to vernalization (Su et al., 2018). Therefore, it is possible to produce vernalization-resistant varieties by modifying the sequence of the BrVINS.l promoter with gene editing. The BrVIN3.1 protein localized to both BrFLCl and BrFLC2 loci, but it accumulated to higher levels at the BrFLCl locus, thus suggesting that BrFLCl is the main target of BrVIN3.1 (Su et al., 2018). Both BrVINS.lH1/H1 and BrFLClH1/H1 were intensively selected during Chinese cabbage breeding (Su et al., 2018). Because all four of the BrFLCs are repressed by vernalization, it would be interesting to investigate whether the other BrVINS paralogs are responsible for regulating the expression of other BrFLCs. In addition, VRNs and VINs maybe good candidates for breeding vernalization-resistant B. rapa varieties suitable for planting in

CURLY LEAF (CLF) is a major component of the PRC2 complex (Kim and Sung, 2014b; Xiao and Wagner, 2015). In Arabidopsis, loss of function of CLF decreased global H3K27me3 levels, leading to multiple developmental defects and early flowering (Shu et al., 2019). CLF regulates the expression of FLC, FT, and several floral homeotic genes, including AG and AGLs (Liu et al., 2018; Shu et al., 2020). In B. rapa, mutations in the BrCLF gene led to enhanced expression of BrFLCs, BrFT, BrAGs, and BrAGL19s, and early flowering (Huang et al., 2020; Tan et al., 2021).

In Arabidopsis, five H3K27 demethylases participate in several developmental processes and function in different pathways to influence flowering, among which ELF6 is required for epigenetic reprogramming of the FLC locus during sexual reproduction (Crevillen et al., 2014). ELF6 is highly expressed in flowers and embryos. The elf mutant failed to fully reset FLC expression in the next generation, which then resulted in early flowering (Crevillen et al., 2014). The B. rapa genome contains only one copy of ELF6 (Table 1). Loss of function of BrELF6 increased H3K27me3 levels at the BrFLCl, -2, and -3 loci and led to early flowering, thus indicating a conserved function for BrELF6 in flowering (Poza-Viejo et al., 2022). In addition, BrELF6 has been identified as one of the candidate genes contributing to flowering time variations in B. rapa (Kaur et al., 2021).

4. Photoperiod regulation of flowering time

Studies in Arabidopsis have shown that the photoperiod functions through the CO transcription factor to regulate FT expression (Amasino, 2010; Golembeski and Imaizumi, 2015; Freytes et al., 2021). The transcriptional and post-transcriptional regulation of CO by integrated signals from the circadian clock and light quality is an output of the photoperiod, and it is critical for sensing optimal long day conditions (Amasino, 2010; Golembeski and Imaizumi, 2015). Upon sensing the light of dawn, the core clock component CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) activate the DOF domain transcription factor CYCLING DOF FACTOR (CDF), which represses CO transcription. During the afternoon and evening, CDF expression is repressed by the clock component PSUEDO RESPONSE REGULATORS (PRR5/7/9). This clock-mediated regulation of CO transcription results in a daily oscillation of its mRNA level, with a minimum in the morning and a maximum at night under long days. Accumulation of the CDF protein is regulated by a blue light-induced complex composed of GIGANTEA (GI), FLAVIN BINDING, KELCH REPEAT, and F-BOX 1 (FKF1). This complex mediates the proteasome-dependent degradation of CDF to release CO repression. Meanwhile, CO protein accumulation is regulated by light quality mediated by photoreceptors, the ONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1)SUPPRESSOR OF PHYA (SPA) complex, and HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1). In the dark, CO is degraded by the COP1-SPA complex. In the morning, the absorption of red light by the phytochrome photoreceptor PHYTOCHROME B (PHYB) and HOS1 promotes the degradation of CO. In the afternoon, CO is stabilized by the absorption of far-red light by PHYA and blue light by FKF1. Under long days, CO protein accumulation coincides with increased CO transcripts in the afternoon to promote FT activation and floral transition (Amasino, 2010; Golembeski and Imaizumi, 2015; Freytes et al., 2021).

The B. rapa genome contains one CO ortholog that exhibits a similar daily expression oscillation to its Arabidopsis ortholog (Table 1) (Wang et al., 2011; Kim et al., 2019). Moreover, the cisregulatory regions responsible for the expression pattern of CO are conserved among Brassicaceae species, thus suggesting that the clock-mediated regulation of CO expression is likely conserved between B. rapa and Arabidopsis (Ledger et al., 2001). However, it is unknown whether the BrCO protein is regulated by light quality. In Arabidopsis, natural variations in the CO locus have been associated with flowering time (Rosas et al., 2014). Although several studies have shown that CO homologs in B. napus are co-localized with the QTL region associated with flowering time (Osterberg et al., 2002; Xu et al., 2016; Li et al., 2018), the role of BrCO on flowering still needs to be fully elucidated.

Most genes encoding the B. rapa core clock components, CCA1 and PRR gene families, exhibit preferential retention during diploidization following whole genome triplication (Lou et al., 2012). The B. rapa genome contains a single copy of the CCA1 gene (Table 1) (Lou et al., 2012), which exhibits a similar daily pattern of expression peaking at dawn as Arabidopsis CCA1 (Kim et al., 2019). Sequence variation (InDeis) in the fourth intron of BrCCAl has been associated with flowering time (Yi et al., 2017). Interestingly, vernalization led to DNA demethylation and enhanced expression of genes encoding two subunits of the clock regulator casein kinase II (CK2), BrCKA2, and BrCKB4, thus resulting in a shortened period of BrCCAl and allowing the vernalized plant to perceive long days (Duan et al., 2017). This indicates that there may be interactions between vernalization and photoperiod in flowering regulation. The other clock genes in B. rapa have between two and three paralogs in Arabidopsis (Table 1), with at least one copy of each gene exhibiting a similar oscillation pattern as its Arabidopsis ortholog (Kim et al., 2019). Little is currently known about the role of BrPRRs in flowering time regulation, although BnPRR7 has been found to co-localize with the QTL region associated with flowering time in B. napus (Jian et al., 2019).

The B. rapa genome contains a single copy of GI that exhibits a similar expression pattern as Arabidopsis GI (Kim et al., 2019). BrGI was identified as a major QTL associated with the circadian period in B. rapa using a recombinant inbred line (RIL) population developed from a cross between the oilseed R500 and the rapid cycling IMB211 (Xie et al., 2015). An amino acid substitution (S264A) resulting from a nucleotide polymorphism between the two GI alleles underlay variations in the circadian period. The BrGIR500 encoding an A amino acid caused a short circadian period in the RIL population (Xie et al., 2015). Interestingly, this amino acid substitution did not affect flowering time, as both BrGIR500 and BrGIIMB211 fully rescued the photoperiodic flowering defect of Arabidopsis gi-201 mutants (Xie et al., 2015). Further study has shown that loss of function of BrGI caused a defective circadian rhythm and delayed flowering, thus indicating that BrGI plays a critical role in maintaining the circadian rhythm and regulating flowering (Xie et al., 2015).

In Arabidopsis, ZEITLUPE (ZTL), LOV KELCH PROTEIN2 (LKP2), FLAVIN BINDING, KELCH REPEAT, and FKF1 act partially redundantly in the turnover of clock proteins. They participate in an SCF E3 ligase complex to target substrate proteins for proteasomal degradation (Nelson et al., 2000; Somers et al., 2000; Baudry et al., 2010). Surprisingly, the B. rapa genome has lost all of the copies of ZTL and FKF1, but it has retained three tightly linked copies of LKP2 (Table 1) (Lou et al., 2012). The expression pattern of LKP2a was identical to Arabidopsis FKF1, while the other two homologs exhibited no circadian oscillation (Kim et al., 2019). It would be interesting to investigate whether these BrLKP2s can fulfill the functions of FKF1 to control flowering in B. rapa.

5. The role of gibberellin (GA) in flowering time regulation

In Arabidopsis, the phytohormone GA plays an essential role in promoting flowering. Disruption of GA biosynthesis genes or loss of function of GA-signaling components affects flowering (Bao et al., 2020). As the central components of the GA signaling pathway, DELLA proteins (DELLAs) negatively regulate flowering by suppressing the transcriptional activity of FT activators, such as CO and PHYTOCHROME INTERACTING FACTOR 4 (PIF4), while stabilizing FT transcriptional repressors, such as MYC3 and FLC (Bao et al., 2020). Under long day conditions conducive to flowering, GA production in leaves promotes DELLA degradation, thus resulting in increased FT expression. In the shoot apex, GA can promote SOCI and LFY expression under short days (Bao et al., 2020). Exogenous GA3 (one of the major bioactive GAs) promoted flowering time primarily by upregulating SOCI (Moon et al., 2003). The expression of several key genes involved in GA metabolism and transport, such as GA3oxl, GA3ox2, GA2Oox2, GA2ox2 and GA2ox8, is influenced by the photoperiodic and thermosensory pathways, which maintain GA homeostasis to ensure floral transition under suitable conditions (Bao et al., 2020).

Exogenous GA3 application can also promote flowering in B. rapa (Guan et al., 2021). GA3 treatment decreased the expression of the DELLA, BrFLC, and BrSVP genes while increasing BrSOCl expression in flowering Chinese cabbage (Guan et al., 2021). However, there are few reports on gene mutations or QTLs associated with GA signaling or homeostasis affecting flowering time in B. rapa. Interestingly, the Jumonji H3K27me3 demethylase BrREF6 likely regulates flowering time by affecting the transcription of genes involved in GA metabolism (Poza-Viejo et al., 2022). Disruption of BrREF6 affected GA metabolism and delayed flowering in B. rapa yellow sarson R-o-18, which could be rescued by GA3 application (Poza-Viejo et al., 2022). Studies in Arabidopsis suggest that REF6 can regulate FLC, FT, and SOCI expression (Noh et al., 2004). However, the expression of BrFLC3, the major functional FLC paralog in R-o-18, was unaffected in the braA.re/6 mutant. In contrast, the functional FT paralog BrFTa is downregulated (Poza-Viejo et al., 2022). As GA promotes FT expression, BrREF6 may promote BrFT expression through the regulation of GA homeostasis.

6. The thermosensory pathway regulates flowering time

In fact, flowering time varies significantly among Arabidopsis natural accessions in response to temperature changes (Lempe et al., 2005). Flowering time in most Arabidopsis accessions is accelerated by higher temperatures (23-27 °C) and delayed by lower temperatures (16-23 °C) (Song et al., 2013). This response to short-term temperature changes is mediated by the thermosensory pathway, which regulates FT transcription through several components (Song et al., 2013; Golembeski and Imaizumi, 2015). Under low ambient temperatures, the E3 ligase HOS1 downregulates FT expression, either by promoting CO degradation or by repressing FLC transcription through associations with FLC repressors (Golembeski and Imaizumi, 2015). FT expression is also repressed by a complex consisting of SVP, FLC, and FLM that binds the FT promoter. Warm temperatures induced alternative splicing of the FLM transcript, thus resulting in a protein that negatively affects the activity of the SVP-FLM complex (Golembeski and Imaizumi, 2015). This leads to the derepression of FT, followed by a transition to flowering. Warm temperatures also reduced H2A.Z accumulation at the FT promoter, which allowed PIF4 to bind and promote FT activation under short day conditions (Golembeski and Imaizumi, 2015).

Arabidopsis mutants sup and /im showed temperatureinsensitive flowering (Lee et al., 2013). The B. rapa genome encodes a single orthologous copy of AtSVP (Table 1) (Wang et al., 2011). Overexpression of BrSVP in Arabidopsis decreased FT expression and delayed flowering, and BcSVP expression driven by Arabidopsis SVP promoter can rescue the phenotype of Arabidopsis sup mutant, suggesting a conserved function of BrSVP in mediating temperature-regulated flowering (Lee et al., 2007). However, the mechanism of flowering time regulation in response to lower temperatures in B. rapa remains elusive.

A study on B. rapa yellow sarson R-o-18 demonstrated that the effect of warm temperatures (28 °C) on B. rapa flowering time is different from that of Arabidopsis (Del Olmo et al., 2019). R-o-18 plants growing at 28 °C bolted later than those growing at 21 °C. In contrast to Arabidopsis (Col-0), warm temperatures led to high H2.AZ accumulation at the BrFT locus and low BrFTa expression in R-o-18 (Del Olmo et al., 2019). In cauliflower (B. oleraceae), flowering could also be delayed by warm temperatures (Verhage et al., 2017). Apparently, plants within the same genus and species have evolved different temperature requirements for flowering as an adaptation to their local environment.

Recent studies have shown that controlling the photoperiod and temperature can promote flowering and ripening, thus accelerating the breeding process. This so-called "speed breeding" method has been proven to be effective in different crops, such as wheat, rice, barley, soybean, oilseeds, sunflower, apple, banana and so on (Samantara et al., 2022). It is also worthwhile to explore suitable control conditions for the rapid breeding of B. rapa to improve breeding efficiency.

7. Floral integrators

The major floral integrators in Arabidopsis include FT, SOCI, AGL24, and SPL (Golembeski and Imaizumi, 2015). These components act as "hubs" in the flowering network by integrating environmental and developmental signals that are sensed by different upstream pathways. Highlighted below are a few studies on В. rapa that have shed some light on the role of BrFTs and BrSOCls in flowering.

7.1. BrFTs

The florigenic protein FT is a small protein belonging to the PEBP family. It is produced in leaves and travels to the shoot apex, where it initiates meristematic reprogramming and the formation of reproductive structures (Turek et al., 2008; Song et al., 2013). The central regulator of the vernalization pathway, FLC, represses FT transcription by binding to the CArG box in the FT promoter (Deng et al., 2011). FLC expression is also regulated by GA signaling and the thermosensory pathway (Amasino, 2010; Song et al., 2013). Activation of FT transcription in response to photoperiod and light is primarily mediated by CO, which can bind to СО-responsive elements (COREs) in the FT promoter. This activation accelerates flowering under optimal long day conditions (Turek et al., 2008; Song et al., 2013). CO activity is also affected by GA and low temperatures (Turek et al., 2008; Song et al., 2013). Additionally, PIF4 functions in the thermosensory pathway and photoperiod pathway to promote FT transcription (Golembeski and Imaizumi, 2015). FT expression is also affected by chromatin status, which involves components of the PRC and the mediator complex (Golembeski and Imaizumi, 2015). These factors together form a complex regulatory network that controls flowering time (Golembeski and Imaizumi, 2015).

The B. rapa genome contains two syntenic orthologs of FT (Table 1) (Del Olmo et al., 2019). BrFTa is highly expressed in B. rapa leaves, and loss of function of BrFTa resulted in extremely delayed flowering in yellow sarson R-o-18 (Del Olmo et al., 2019). BrFTb was identified as a major QTL associated with flowering time with a recombinant inbred line (RIL) population from a cross between two vernalization-independent lines, namely, yellow sarson R-o-18 and caixin L58 (Zhang et al., 2015). Yellow sarson R0-18 carries a loss-of-function allele for BrFTb with an LTRtransposon insertion in the second intron, which leads to late flowering time (Zhang et al., 2015).

In each BrFT promoter, there is a CArG box, which can be bound by all four of the BrFLCs in vitro (Ma et al., 2021). The expression of both of the BrFTs is upregulated during vernalization (Jung et al., 2018). The remaining question is whether the different BrFLC paralogs have similar effects on the repression of BrFT paralogs in planta. Similar to AtFT, BrFTa expression has a circadian oscillation that peaks at the end of the day in yellow sarson R-o-18 (Del Olmo et al., 2019). However, BrFTa expression in response to warm temperature treatment differs from that of AtFT (Del Olmo et al., 2019) (discussed in Section 5). A systematic analysis of the cis-elements in the BrFT promoter and the expression pattern of BrFTs under different environmental conditions would assist in illustrating the differences in flowering time signaling mechanisms between B. rapa and Arabidopsis.

7.2. BrSOCls

SOC1/AGL20 encodes a MADS box transcription factor that interacts with AGL24 to promote differentiation of primordia into floral meristems via upregulating LFY. SOCI expression is directly regulated by FT and FLC (Romera-Branchat et al., 2020). After FT moves from the leaves to the SAM, it forms a complex with the bZIP transcription factor FD/ATBZIP14, which binds to a G-box (CACGTG) element in the SOCI promoter to activate SOCI expression. The FLC-SVP complex also represses the expression of SOCI by directly binding the SOCI promoter or by repressing FT and FD transcription (Amasino, 2010).

The B. rapa genome possesses three SOCI genes (Table 1) (Franks et al., 2015). CRISPR/Cas9-mediated simultaneous knockout of BrSOCl-1 and BrSOCl-2 resulted in delayed flowering time, thus suggesting that BrSOCl-1 and BrSOCl-2 function redundantly in flowering time regulation (Jung et al., 2021). Additionally, BrSOCl-1 overexpression in B. napus caused early flowering, suggesting a conserved function of S0C1-1 among Brassica species (Hongét al., 2012). Comparative transcriptomics of two Chinese cabbage inbred lines, namely, '4004' (early bolting) and '50' (late bolting), revealed that in the early bolting line, three BrSOCls were upregulated after vernalization, whereas in the latebolting line, the expression of BrSOCls expression was low regardless of vernalization (Jung et al., 2021). In a natural population of B. rapa, BrSOCl-1 and BrSOCl-2 expression tends to be higher in early-flowering accessions as compared to lateflowering accessions. BrSOCl-3 expression showed no significant difference between early and late flowering accessions, thus suggesting that BrSOCl-1 and BrSOCl-2 are important for flowering time (Franks et al., 2015). Unexpectedly, no correlation was found between variations in the promoter region of BrSOCl-1/ BrSOCl-2 and the flowering time difference, thus indicating that variations in the upstream components contribute to differences in two BrSOCls expression and flowering time in the population (Franks et al., 2015).

The promoter regions of BrSOCl-1, BrSOCl-2, and BrSOCl-3 contain two, two, and one FLC-binding motifs, respectively. An in vitro binding assay confirmed the binding of the BrSOCl-1 promoter by BrFLCl/5, the BrSOCl-2 promoter by BrFLCl/2/3, and the BrSOCl-3 promoter by all four of the BrFLCs (Ma et al., 2021). The different binding patterns may indicate that the BrFLC and BrSOCl paralogs experience functional diversification among B. rapa varieties, thus resulting in specific regulation modules under certain conditions. The divergent role of BrSOCs in flowering time control remains to be elucidated.

8. Conclusions and perspectives

Based on the current findings, the main regulatory pathways governing flowering time are largely conserved between Arabidopsis and B. rapa. However, some notable differences have also been observed. Fig. 3 summarizes the major flowering time regulators identified in B. rapa. The transition to flowering in B. rapa is regulated by the environmental and developmental cues that are integrated to control the expression of floral integrators, BrFTs and BrSOCls, to regulate flowering (Hong et al., 2012; Zhang et al., 2015; Del Olmo et al., 2019; Jung et al., 2021). The mechanism of vernalization-regulated flowering is similar between B. rapa and Arabidopsis. The expression of BrFLCs is activated by the FRI-C complex and suppressed by COOLAIR-like transcripts (Li et al., 2016; Shea et al., 2019; Takada et al., 2019; Akter et al., 2020a). Chromatin modifications play a crucial role in BrFLC repression, which involves the function of BrVIN3, BrCLF, and BrELF6 (Kawanabe et al., 2016; Su et al., 2018; Akter et al., 2019, 2020b; Huang et al., 2020; Méhraj et al., 2021; Tan et al., 2021; Poza-Viejo et al., 2022). B. rapa clock genes have a similar daily oscillation expression pattern to their Arabidopsis orthologs. However, our understanding of the role of the circadian clock and photoperiod in B. rapa flowering time regulation remains limited; only BrCCAl and BrGI are involved in regulating flowering (Xie et al., 2015; Yi et al., 2017). GA promotes flowering in B. rapa, likely by repressing BrFLC and BrSVP. Under low temperatures, the expression of BrFTs is repressed by BrSVP (Lee et al., 2007), whereas warm temperatures delay flowering in B. rapa by reducing BraA.FT.A expression (Del Olmo et al., 2019). The main differences in the molecular mechanism of flowering time regulation between B. rapa and Arabidopsis are as follows. 1) In Arabidopsis, FKF1, ZTL, and LKP2 function partially redundantly in circadian clock regulation and the output to the photoperiodic flowering time pathway, resulting in CO repression by targeting proteasomedependent degradation of CDF (Nelson et al., 2000; Somers et al., 2000; Baudry et al., 2010). In B. rapa, neither ZTL nor FKFl genes are present (Lou et al., 2012). It is unclear whether the functions of the three BrLKP2 genes replace that of ZTL and FKFl in photoperiod-mediated flowering time regulation. 2) The H3K27me3 demethylase BraA.REF6 modulates B. rapa flowering time by influencing the expression of GA metabolic genes (PozaViejo et al., 2022), unlike Arabidopsis REF6, which regulates the expression of FLC, FT, and SOCI (Noh et al., 2004). Loss of function of BrREFG in B. rapa does not alter the expression of BrFLC genes (Poza-Viejo et al., 2022). 3) Under high ambient temperatures, B. rapa flowers late due to increased H2A.Z accumulation in the BrFT locus and decreased BrFT expression (Del Olmo et al., 2019). In contrast, high temperature accelerates flowering in Arabidopsis by reducing H2A.Z incorporation in the FT locus (Golembeski and Imaizumi, 2015).

Flowering time is one of the most important agronomic traits for breeding because it influences crop yield, quality, and adaptation. B. rapa crops have different growth habits and flowering traits that have been adapted to their culturing environment during longterm domestication, thus resulting from the accumulation of beneficial gene alleles in various cultivars that fit seasonal and geographical conditions. For example, the incorporation of elite alleles of BrVIN3.1 and BrFLC loci gave rise to bolting-resistant spring Chinese cabbage, which meets the annual demand for Chinese cabbage production (Su et al., 2018). The domestication process requires long-term artificial selection, but uncovering the molecular mechanisms and genetic variations of flowering time control of different B. rapa crops can help accelerate crop improvement. The identified key genes and QTL involved in flowering time regulation provide potential genetic targets for molecular breeding, which allow us to increase crop adaptability and plasticity by manipulating the flowering transition of different B. rapa varieties. This could not only help to improve crop yield and quality, but it could also guide us in designing robust cultivars to composite rapidly changing patterns of temperature and rainfall variation and ensure crop production in the face of global

climate change. Currently, our understanding of the regulatory mechanism of flowering time control in B. rapa is rather limited. One of the major challenges for future research is to use the learned knowledge in Arabidopsis and B. rapa as a starting framework to mine more functional flowering genes using the latest highthroughput muti-OMICS techniques, such as GWAS, single-cell transcriptomics, and cell-specific epigenomics. This would deepen our understanding of the regulation network and provide more potential target genes for breeding. With the development of new technologies, it has become more convenient and efficient to select or modify these target loci in crop improvement. First, high-throughput molecular markerassisted breeding and whole-genome selection technology can be used to efficiently aggregate target alleles scattered in different germplasm, which can accelerate the process of selection and crop improvement. Second, using transgenic technology or the newly developed genome-editing technology, we can deliver interested genes to the genome or accurately modify the target DNA, which allows us to make targeted, specific, and predictable changes to the flowering genes. Moreover, a comprehensive understanding of the crosstalk of flowering regulatory networks and the genetic basis of other traits in B. rapa should allow for the final aggregation of beneficial alleles of flowering traits together with other agronomic traits, such as yield, quality, and disease and stress resistance, thus resulting in superior

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Our work is supported by National Natural Science Foundation of China (Grant Nos. 32372733, 32172594), Natural Science Foundation of Hebei (Grant No. C2020204111), S & T Program of Hebei (Grant No. 21326344D), State Key Laboratory of North China Crop Improvement and Regulation (Grant No. NCCIR2023ZZ-1), and the Starting Grant from Hebei Agricultural University (Grant No. YJ201920).

Footnote

1 These authors contributed equally to this work.

* Corresponding authors.

E-mail addresses: [email protected]; [email protected]

Peer review under responsibility of Chinese Society of Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS)

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