Genome-Wide Identification of BrCMF Genes in Brassica rapa and Their Expression Analysis under Abiotic Stresses

CCT MOTIF FAMILY (CMF) genes belong to the CCT gene family and have been shown to play a role in diverse processes, such as flowering time and yield regulation, as well as responses to abiotic stresses. CMF genes have not yet been identified in Brassica rapa. A total of 25 BrCMF genes were identified in this study, and these genes were distributed across eight chromosomes. Collinearity analysis revealed that B. rapa and Arabidopsis thaliana share many homologous genes, suggesting that these genes have similar functions. According to sequencing analysis of promoters, several elements are involved in regulating the expression of genes that mediate responses to abiotic stresses. Analysis of the tissue-specific expression of BrCMF14 revealed that it is highly expressed in several organs. The expression of BrCMF22 was significantly downregulated under salt stress, while the expression of BrCMF5, BrCMF7, and BrCMF21 was also significantly reduced under cold stress. The expression of BrCMF14 and BrCMF5 was significantly increased under drought stress, and the expression of BrCMF7 was upregulated. Furthermore, protein–protein interaction network analysis revealed that A. thaliana homologs of BrCMF interacted with genes involved in the abiotic stress response. In conclusion, BrCMF5, BrCMF7, BrCMF14, BrCMF21, and BrCMF22 appear to play a role in responses to abiotic stresses. The results of this study will aid future investigations of CCT genes in B. rapa.


Introduction
CMF genes comprise a subfamily of the CCT gene family and have diverse functions.The CCT gene family contains conserved CCT motifs (CONSTANS, CO-like, and TOC1), and they were first described from the A. thaliana CONSTANS gene [1]; with these genes having been shown to be involved in flowering regulation and the response to photoperiod [2].Several studies have indicated that the CCT gene family can be divided into four subfamilies based on conserved domains: (1) the CONSTANS-like (COL) subfamily, which contains one or two zinc finger B-boxes (BBOX) and a CCT domain; (2) the Pseudo-response regulator (PRR) subfamily, which contains the CCT domain and a response regulator domain (REC) [3]; (3) CCT MOTIF FAMILY (CMF) subfamily, which contains only one conserved CCT domain [4]; and (4) the ZINC-FINGER PROTEIN EXPRESSED IN INFLORESCENCE MERISTEM (ZIM) subfamily, which is referred to as CMF in some studies, which contains the Tify, CCT, and ZnF_GATA domains [5][6][7].COL proteins are primarily involved in the regulation of flowering time and the biological clock.PRR proteins are primarily involved in circadian rhythm regulation, and ZIM proteins are involved in the elongation of the petiole and hypocotyl cells [6].The activities of the proteins encoded by CMF genes are Plants 2024, 13, 1118 3 of 18 Medicago truncatula (18) [10], Glycine max (19) [12], Cajanus cajan L. (12) [15], and Populus trichocarpa (14) [16].These genes were named according to their chromosomal distribution.ATCMF IDs, homologous gene names, chromosome positions, pI, MW, protein length (aa), subcellular localization, and their names (and alternate names) are listed in Table 1.The pI ranged from 4.45 (BrCMF11) to 9.67 (BrCMF24), the MW ranged from 17,412.59Da (BrCMF2) to 60,150.03Da (BrCMF24), and the protein length ranged from 153 aa (BrCMF2) to 535 aa (BrCMF24).The protein length was linearly positively correlated with the MW.All the predicted proteins were predicted to be located in the nucleus, among which a few were also predicted to be located in the cytosol, mitochondria, and chloroplast.CMF genes, which are mostly known for their role in regulating flowering time, might play key roles in the nucleus.Basic information of BrCMFs is shown in the above table, and the italics in parentheses in the A. thaliana column are the alternate names of the genes."MW" is molecular weight and "pI" is isoelectric point.

Chromosomal Localization, Synteny, and Phylogenetic Analysis of BrCMFs
BrCMF genes were unevenly distributed across eight chromosomes of B. rapa.Br-CMF25 was the only gene that was not mapped to one of the 10 chromosomes, and it was located on Scaffold000164, an unknown chromosome, and showed no collinearity with any Plants 2024, 13, 1118 4 of 18 other genes.The greatest number of BrCMF genes was located on Chromosome 10 (6), and the lowest number of BrCMF genes were located on Chromosome 1 (1).Chromosomes 2, 3, 4, 5, 8, and 9 contained four, four, two, two, three, and two BrCMF genes, respectively; no BrCMF genes were detected on Chromosomes 6 and 7 (Figure 1A).

Motif Analysis, Gene Structure, and Conserved Domain Analysis of BrCMFs
Using TBtools [23], we were able to obtain a conserved domain map of BrCMF genes, and the conserved domain diagram of the 25 predicted BrCMF genes is shown in Figure 2B.Each BrCMF has a conserved CCT domain.BrCMF18, BrCMF24, and BrCMF15 possess other conserved motifs in addition to the CCT domain.BrCMF18 contains a BBOX domain, which indicates that this gene is a member of the COL subfamily.Both the COL and CMF subfamilies contain CCT domains, and the COL family contains one to two BBOX domains, while CMF family members do not contain BBOX domains.Therefore, we predict that BrCMF18's domain has been evolutionarily acquired, given that the BBOX domain in COL genes might have been lost, and this led to the origin of the CMF family [4,9].
The similarity in the expression patterns of genes in organs was high among highly homologous genes.For example, the phylogenetic tree indicated that BrCMF15 and BrCMF19 were closely related to AtCMF3 (FITNESS), suggesting that these two genes might have functionally diversified in B.rapa.In addition, the expression patterns of these two genes were similar in various organs.BrCMF2, BrCMF6, and BrCMF24, which are homologous to AtCMF11, were expressed at low levels.Significant differences in the expression of BrCMF22, BrCMF1, and BrCMF9 were observed, presumably because AtCMF9 (CIL) was more closely related to BrCMF9 and BrCMF1 than to BrCMF22.This explains the similarity in the expression patterns of BrCMF9 and BrCMF1, as well as the contrasting expression patterns of BrCMF22 in different organs.

Motif Analysis, Gene Structure, and Conserved Domain Analysis of BrCMFs
Using TBtools [23], we were able to obtain a conserved domain map of BrCMF genes, and the conserved domain diagram of the 25 predicted BrCMF genes is shown in Figure 2B.Each BrCMF has a conserved CCT domain.BrCMF18, BrCMF24, and BrCMF15 possess other conserved motifs in addition to the CCT domain.BrCMF18 contains a BBOX domain, which indicates that this gene is a member of the COL subfamily.Both the COL and CMF subfamilies contain CCT domains, and the COL family contains one to two BBOX domains, while CMF family members do not contain BBOX domains.Therefore, we predict that BrCMF18's domain has been evolutionarily acquired, given that the BBOX domain in COL genes might have been lost, and this led to the origin of the CMF family [4,9].To further clarify the function of genes, we characterized the structure of BrCMF genes (Figure 2C).The coding sequence (CDS) represents the functional region of the gene containing exons, and lines indicate the functional region containing introns.The structure of 25 predicted genes varies greatly: BrCMF15 and BrCMF24 contain the most introns, while BrCMF18, BrCMF13, and BrCMF25 do not contain introns.Further evidence of the functional differentiation and specificity of the gene families is revealed by these structural differences.

Cis-Acting Element Analysis of BrCMFs
We identified 24 cis-acting elements in a 2000 bp 5′ region of BrCMF genes; cis-acting elements in the promoter of the BrCMF25 gene were not characterized.The functions of cis-acting elements can be divided into three categories: growth and development, phytohormone response, and stress response.The number and distribution of various cis-acting elements for each gene are shown in Figure 3.
In the first category (growth and development), all genes contain light-responsive- The motif diagram of BrCMF genes revealed that all genes have Motif 1 and Motif 2, and BrCMF22 has the most motifs (6).BrCMF1, BrCMF4, BrCMF14, BrCMF5, BrCMF7, BrCMF21, BrCMF20, BrCMF16, BrCMF12, and BrCMF11 each have five motifs; BrCMF18, BrCMF15, BrCMF23, BrCMF3, BrCMF10, and BrCMF17 only have one or two motifs.There was a close relationship between motif distributions and gene homology.Specifically, highly homologous genes tended to have similar motif distributions, indicating that similar genes have similar functions.
To further clarify the function of genes, we characterized the structure of BrCMF genes (Figure 2C).The coding sequence (CDS) represents the functional region of the gene containing exons, and lines indicate the functional region containing introns.The structure of 25 predicted genes varies greatly: BrCMF15 and BrCMF24 contain the most introns, while BrCMF18, BrCMF13, and BrCMF25 do not contain introns.Further evidence of the functional differentiation and specificity of the gene families is revealed by these structural differences.

Cis-Acting Element Analysis of BrCMFs
We identified 24 cis-acting elements in a 2000 bp 5 ′ region of BrCMF genes; cis-acting elements in the promoter of the BrCMF25 gene were not characterized.The functions of cis-acting elements can be divided into three categories: growth and development, phytohormone response, and stress response.The number and distribution of various cis-acting elements for each gene are shown in Figure 3.
Plants 2024, 13, x FOR PEER REVIEW 8 of 18 respectively.Most BrCMF genes contained three-four cis-acting elements, and BrCMF4 did not contain phytohormone-responsive cis-acting elements.The promoters of 10, 11, 21, and 16 BrCMF genes had defense and stress-responsiveness, drought-inducibility, anaerobic induction, and low-temperature-responsiveness elements, respectively.Most genes contain two-three types cis-acting elements in the Stress response group, suggesting that some of these genes play an important role in stress responses.

Tissue Specific Expression of BrCMFs
To identify the potential roles of BrCMFs in abiotic stress responses, we analyzed tissue-specific expression data for callus, flower, leaf, root, silique, and stem tissues of B. rapa from the BRAD (http://brassicadb.cn/,accessed on 22 November 2022) and made expression heat maps (Figure 4, Table S1) to enhance our understanding of their potential functions.In most tissues, the expression of the genes was low; however, the expression of eight genes (BrCMF5, BrCMF7, BrCMF10, BrCMF14, BrCMF15, BrCMF19, BrCMF21, and BrCMF22) was high in some tissues and low in others.Genes with similar sequences tended to have similar high expression levels.BrCMF14 and BrCMF22 are highly expressed in multiple tissues, while the expression of BrCMF14 was high in callus, flower, root, silique, and stem tissues, and the expression of BrCMF22 was high in flower, leaf, and stem tissues, suggesting that these two genes play roles in several life activities.Both BrCMF19 and BrCMF15 were highly expressed in leaves, and BrCMF10 was highly expressed in roots, suggesting that they play key roles in diverse life activities.The homologous genes BrCMF5, BrCMF7, and BrCMF21 were highly expressed in silique and stem tissue, and BrCMF7 was highly expressed in flower and leaf tissue.In the first category (growth and development), all genes contain light-responsiveness elements, which is consistent with the role of CCT genes in regulating photoperiodic flowering.Many circadian rhythm-related elements were detected in BrCMF1, BrCMF3, BrCMF7, BrCMF11, and BrCMF14.In addition, some elements involved in the seed-specific regulation of endosperm expression and zein metabolism regulation were observed.
In the phytohormone response group, genes contained five different types of elements: ABA-responsiveness, auxin-responsiveness, gibberellin-responsiveness, MeJAresponsiveness, and salicylic acid-responsiveness elements.Most of these genes contained elements that are MeJA-responsive, and BrCMF8 contained the most cis-acting elements (10).ABA and gibberellin-responsive elements were present in 16 and 14 BrCMF genes, respectively.Most BrCMF genes contained three-four cis-acting elements, and BrCMF4 did not contain phytohormone-responsive cis-acting elements.
The promoters of 10, 11, 21, and 16 BrCMF genes had defense and stress-responsiveness, drought-inducibility, anaerobic induction, and low-temperature-responsiveness elements, respectively.Most genes contain two-three types cis-acting elements in the Stress response group, suggesting that some of these genes play an important role in stress responses.

Tissue Specific Expression of BrCMFs
To identify the potential roles of BrCMFs in abiotic stress responses, we analyzed tissue-specific expression data for callus, flower, leaf, root, silique, and stem tissues of B. rapa from the BRAD (http://brassicadb.cn/,accessed on 22 November 2022) and made expression heat maps (Figure 4, Table S1) to enhance our understanding of their potential functions.In most tissues, the expression of the genes was low; however, the expression of eight genes (BrCMF5, BrCMF7, BrCMF10, BrCMF14, BrCMF15, BrCMF19, BrCMF21, and BrCMF22) was high in some tissues and low in others.Genes with similar sequences tended to have similar high expression levels.BrCMF14 and BrCMF22 are highly expressed in multiple tissues, while the expression of BrCMF14 was high in callus, flower, root, silique, and stem tissues, and the expression of BrCMF22 was high in flower, leaf, and stem tissues, suggesting that these two genes play roles in several life activities.Both BrCMF19 and BrCMF15 were highly expressed in leaves, and BrCMF10 was highly expressed in roots, suggesting that they play key roles in diverse life activities.The homologous genes BrCMF5, BrCMF7, and BrCMF21 were highly expressed in silique and stem tissue, and BrCMF7 was highly expressed in flower and leaf tissue.

Transcriptome Analysis of BrCMFs
We compared the expression levels of 24 BrCMF genes under cold, drought, and salt stress using transcriptome data (Figure 5A-C, Table S2).The expression of most genes was low under salt, drought, and cold stress.For example, the expression levels of BrCMF8, BrCMF12, BrCMF13, BrCMF18, and BrCMF19 were always 0 under three stress conditions, corresponding to no height of the columns; however, significant changes were observed in the expression of some genes.Salt treatment decreased the expression of BrCMF5, BrCMF21, and BrCMF22 and increased the expression of BrCMF7 and BrCMF15.Following exposure to drought stress, the expression of several genes was significantly downregulated and the expression of BrCMF7 was upregulated, which suggests that BrCMF7 plays a special role in the response to drought stress.During cold treatment, the expression of BrCMF5, BrCMF7, and BrCMF21 was downregulated; however, the expression of BrCMF15 and BrCMF22 was upregulated.Changes in the expression of BrCMF5, BrCMF7, BrCMF15, BrCMF21, and BrCMF22 strongly suggest that they play an important role in the response to abiotic stress.
To confirm our hypothesis, we conducted RT-qPCR analyses of key genes that were inferred to have tissue-specific expression patterns according to RNA-seq analysis, and the screened genes showed a significant downregulation trend within 12 h under both salt and cold stress.The expression levels of BrCMF22, BrCMF5, BrCMF7, and BrCMF21 inferred by RNA-seq and RT-qPCR analyses were consistent, and they were hypothesized  S1).Colors in the graph indicate the level of expression, light colors represent low expression levels, while red represents high expression levels.The data were all log-transformed to enhance contrast.

Transcriptome Analysis of BrCMFs
We compared the expression levels of 24 BrCMF genes under cold, drought, and salt stress using transcriptome data (Figure 5A-C, Table S2).The expression of most genes was low under salt, drought, and cold stress.For example, the expression levels of BrCMF8, BrCMF12, BrCMF13, BrCMF18, and BrCMF19 were always 0 under three stress conditions, corresponding to no height of the columns; however, significant changes were observed in the expression of some genes.Salt treatment decreased the expression of BrCMF5, BrCMF21, and BrCMF22 and increased the expression of BrCMF7 and BrCMF15.Following exposure to drought stress, the expression of several genes was significantly downregulated and the expression of BrCMF7 was upregulated, which suggests that BrCMF7 plays a special role in the response to drought stress.During cold treatment, the expression of BrCMF5, BrCMF7, and BrCMF21 was downregulated; however, the expression of BrCMF15 and BrCMF22 was upregulated.Changes in the expression of BrCMF5, BrCMF7, BrCMF15, BrCMF21, and BrCMF22 strongly suggest that they play an important role in the response to abiotic stress.
Plants 2024, 13, 1118 9 of 18 promote reactive oxygen species (ROS) scavenging and contribute to the positive feedbac regulation of melatonin (MT) to enhance the cold tolerance of plants [25,26], but BrCMF1 lacks MeJA-response elements.This is consistent with the high expression of the othe genes according to the qPCR analysis.S2 and S3.

Protein-Protein Interaction (PPI) Network Analysis of BrCMFs
The STRING tool was used to predict interactions between proteins and A. thalian  S2 and S3.
To confirm our hypothesis, we conducted RT-qPCR analyses of key genes that were inferred to have tissue-specific expression patterns according to RNA-seq analysis, and the screened genes showed a significant downregulation trend within 12 h under both salt and cold stress.The expression levels of BrCMF22, BrCMF5, BrCMF7, and BrCMF21 inferred by RNA-seq and RT-qPCR analyses were consistent, and they were hypothesized to be involved in salt and cold stress.BrCMF15 showed a significant upregulation trend in both the transcriptome and RT-qPCR after 7 days of cold treatment (Figure 5C,E, Tables S2 and S3), and it was hypothesized that BrCMF15 might function under prolonged cold stress.Expression patterns (transcriptome and RT-qPCR) were consistent for the majority of genes, with differences in a few gene changes possibly being due to experimental or sampling differences, as has occurred in previous studies [24].Under drought stress, the expression levels of BrCMF14 and BrCMF5 were significantly downregulated more than 3-fold and 10-fold, respectively, and the expression of BrCMF7 was significantly upregulated more than 3-fold (Figure 5D, Table S3).These results were consistent with the transcriptome data (Figure 5B, Table S2), confirming that these genes might be involved in drought-stress-related functions.The expression level of BrCMF15 significantly increased after 4 h of drought treatment, and, as the treatment time increased, the expression level of BrCMF15 was sharply downregulated.After 6 h of drought stress, the expression level in RT-qPCR experiments was significantly downregulated (Figure 5D, Table S3), which is consistent with the transcriptome data (Figure 5B, Table S2).It proves the correctness of the experimental results.The relative expression of BrCMFs was significantly downregulated under short duration cold stress, and the expression of BrCMF15 of these genes was lowest under such conditions (Figure 5E, Table S3).Studies have shown that MeJA could promote reactive oxygen species (ROS) scavenging and contribute to the positive feedback regulation of melatonin (MT) to enhance the cold tolerance of plants [25,26], but BrCMF15 lacks MeJA-response elements.This is consistent with the high expression of the other genes according to the qPCR analysis.
At1G07050 (AtCMF3) interacted with AT5G23240 and AT5G42900 (COR27)-related proteins; AT5G23240 plays a role in root development by mediating protein folding and preventing the aggregation of proteins in chloroplasts during salt stress.The COR27 gene plays a role in the response to cold stress by mediating the effects of cold signals on the biological clock, enhancing freezing tolerance, and entraining circadian rhythms [27].CIL (AtCMF9) interacts with EMB1138, which encodes plastid-specific enzymes involved in ABA biosynthesis; this plays an important role in maintaining ABA levels and mediating salt stress responses.AT4G27900 interacted with AT5G53420 and was associated with PRR7 and ARP6.ARP6 is responsible for mediating ambient temperature responses.Moreover, PRR7 is encoded by a CCT gene and has been shown to modulate drought and ABA responses as well as the expression of genes under cold stress [28].The target gene of PRR7 is regulated by ABA and contains ABA-responsive elements in its upstream region [29], while PRR7 is involved in plant growth and development, regulation of the biological clock, and the photoperiodic flowering response.
PRR7 and ARP6.ARP6 is responsible for mediating ambient temperature responses.Moreover, PRR7 is encoded by a CCT gene and has been shown to modulate drought and ABA responses as well as the expression of genes under cold stress [28].The target gene of PRR7 is regulated by ABA and contains ABA-responsive elements in its upstream region [29], while PRR7 is involved in plant growth and development, regulation of the biological clock, and the photoperiodic flowering response.

Discussion
CMF, COL, and PRR genes are subfamilies of the CCT gene family, and their roles in flowering time regulation have been studied.The PRR subfamily and the COL subfamily of the B. rapa CCT gene family have been identified [30,31], but whole-genome identification and analysis of BrCMF genes have not yet been carried out in B. rapa.We identified 25 BrCMF genes on eight chromosomes in B. rapa, which is more than the number of CMF genes identified in previously studied plants of this family.Previous studies have demonstrated that Brassicaceae underwent three whole genome replication events (WGT) approximately 9-15 million years ago, which greatly increased the number of genomes of Brassicaceae [32].The higher number of BrCMFs compared to other species also reflects the WGT event in the B.rapa.Multiple BrCMF genes in B. rapa can jointly regulate the same physiological process.
The BrCMF genes differed greatly in sequence length as well as structure, and the intron-exon content varied considerably, indicating that members of the CMF family show high functional specificity and diversity.The phylogenetic trees of the CMF gene family in B. rapa, A. thaliana, and O. sativa have revealed that CMF genes can be divided into four Plants 2024, 13, 1118 13 of 18 groups based on their degree of developmental relatedness.The divergence of OsCMFs occurred earlier than that of BrCMFs, suggesting that the differentiation of BrCMFs occurred later than that of OsCMF in the monocotyledonous plant O. sativa [4].Only two pairs of OsCMF and BrCMF genes showed high covariance, whereas more than 30 pairs of AtCMF and BrCMF genes showed high covariance.This 15-fold increase in the latter suggests that B. rapa is more closely related to A. thaliana and that they share homologous genes.
Plant hormones play a key role in regulating the stress resistance of plants [33].ABA is a typical plant hormone associated with abiotic stress.It plays an important role in the responses to external stress signals to promote the expression of resistance-related genes [34][35][36] and regulate stomatal closure to limit water loss [37], which helps plants adapt to drought stress [37,38] and salt stress [37,39,40].MtCMFs have been shown to regulate drought and salt stress either through ABA-dependent or ABA-independent mechanisms [10].By regulating osmotic ions or transmitting signals via ABA [41], methyl jasmonate (MeJA) plays an important role [42] in regulating resistance to salt stress, drought stress [43,44], and low-temperature stress [26,45].Gibberellin (GA) regulates the response to cold stress by inhibiting protein degradation via the activation of DELLA protein [46], and the accumulation of DELLA protein can reduce ROS levels, promote stress tolerance [47], and mediate ET signal transduction to promote salt tolerance [48].The promoter regions of some BrCMF genes contain cis-acting elements, which is consistent with our findings; these genes are involved in abiotic stress resistance.
Both RNA-seq and RT-qPCR experiments revealed consistent changes in multiple genes, indicating that several genes within the BrCMF family are associated with different abiotic stress responses.BrCMF22 is thought to respond to salt stress; BrCMF14, BrCMF5, and BrCMF7 may regulate the response to drought stress; and BrCMF21, BrCMF5, and BrCMF7 may respond to cold stress.
The expression of BrCMF22 was high in flower and leaf tissues, and the promoter region is enriched with reactive oxygen species-inducing response elements associated with abiotic stress resistance and hormone-associated cis-acting elements.Additionally, there are anywhere from 1-8 response elements for ABA, jasmonic acid, and gibberellin, which are phytohormones that modulate salt stress signaling.The expression of some CMF genes was significantly downregulated under salt stress, such as SiCCT31 in S. italica [11] and AetCCT16 and AetCCT20 in A. tauschii [14].The expression of BrCMF22 was also significantly downregulated under salt treatment according to RNA-seq and RT-qPCR analyses, as indicated by a study showing that the Arabidopsis homologous gene AtCMF9 interacts with EMB1138 salt stress-related proteins.This indicates that BrCMF22 regulates the salt stress response through hormone-protein interactions.
The results suggest that BrCMF14, BrCMF5, and BrCMF7 may play a role in the regulation of drought stress.The transcriptome data indicated that BrCMF14 was highly expressed in several organs, including roots, flowers, and fruits.Both transcriptome datasets indicated that drought stress reduced the expression level of this gene, and this was consistent with the expression patterns of CMF family genes in A. tauschii [14].ABA and MeJA response elements in the promoter region of this gene were identified, and the Arabidopsis homolog AtCMF10 interacts with PRR7, which regulates drought and ABA.Therefore, BrCMF14 might play a regulatory role in drought stress by interacting with the PRR7 interaction protein and the ABA response element.In the roots and other organs, both homologous genes, BrCMF5 and BrCMF7, were highly expressed.However, the expression of BrCMF5 was significantly downregulated after drought treatment, whereas the expression of BrCMF7 was significantly upregulated, which is consistent with the fact that both genes contain defense and stress-responsive elements, as well as MeJA-responsive elements.The expression of GmCMF06 and GmCMF07 [12] in G. max and SiCCT3 [11] in S. italica was also significantly upregulated under drought stress.In addition to interacting with PRR7, the Arabidopsis homologous gene AtCMF13 performs a similar function under drought conditions.BrCMF7 contains an ABA response element, and PRR7 is regulated by ABA to regulate the response to drought stress [29], which might explain the upregulation of PRR7 expression after drought treatment compared with BrCMF5.
The B. rapa genes BrCMF21, BrCMF5, and BrCMF7, which are homologous to AtCMF13, might be involved in the response to cold stress.RNA-seq and RT-qPCR analysis of these three genes revealed that cold treatment significantly reduced their expression levels, and the homologous gene AtCMF13 interacted with the ambient temperature response protein APP6.Therefore, we hypothesized that BrCMF21, BrCMF5, and BrCMF7 might be involved in the regulation of cold stress.There are two low-temperature-responsive cis-acting elements in BrCMF5 and BrCMF7, as well as two−six MeJA-responsive elements in each of the three genes, and both BrCMF21 and BrCMF5 contain gibberellin elements, which impair cold tolerance by degrading the DELLA protein.Furthermore, these cis-acting elements suggest that these genes may also be involved in the response to cold stress, which is consistent with our findings.

Identification of CMF Genes in the B. rapa Genome
Based on previous research [4] and a search in the Phytozome database (Phytozome (doe.gov), 15 AtCMFs were obtained.The B. rapa genome data file was extracted from BRAD (http://brassicadb.cn/,accessed on 17 November 2022), and the A. thaliana genome data file was extracted from TAIR (http://www.Arabidopsis.org/,accessed on 16 November 2022).These genomes and TBtools (v1.120) [23] were then used to screen predicted genes based on their conserved domains, and genes without CCT domains were eliminated, with a total of 25 CMF genes being identified.Chromosome location information was retrieved from the genome annotation files in the B. rapa genome database.The length (aa), molecular weight (Da), and isoelectric point (pI) of the proteins encoded by the CMF genes were obtained from ExPASy [49] (https://web.expasy.org/protparam/,accessed on 21 November 2022).Subcellular localization data were obtained from PSORT (WoLF PSORT: Protein Subcellular Localization Prediction (hgc.jp)).

Chromosomal Localization, Synteny, and Phylogenetic Analysis
The locations of CMF genes on the B. rapa chromosome were determined from the B. rapa gff3 genome annotation information using TBtools (v1.120) software [23], while the chromosome density was calculated using the Gene Density Profile plug-in in TBtools (v1.120) with default settings.BrCMFs were named according to their chromosomal location.
Using the Dual Systeny Plot plug-in, Advanced Circos plug-in, and Table row extract or filter plug-in in the TBtools (v1.120) software, collinearity relationships between duplicate genes within B. rapa and between species were analyzed and visualized using data from B. rapa, O. sativa, and A. thaliana.
Phylogenetic trees of the complete amino acid sequences for BrCMFs, AtCMFs, and Os-CMFs were constructed using the maximum likelihood method in MEGA X [50] with default parameters, while branch support was evaluated using 1000 bootstrap replicates.We used iTOL (https://itol.embl.de/accessed on 27 November 2022) to make the phylogenetic tree.

Motif analysis, Gene Structure, and Conservative Domain Analysis
The Simple MEME Wrapper in TBtools (v1.120) was used to analyze conserved motifs using default parameter settings, with the exception of the Number of Motifs, which was set to 10. Conserved motif files were obtained from Batch CD-Search in NCBI (https: //www.ncbi.nlm.nih.gov/,accessed on 22 November 2022) and visualized using the Visualize Pfam Domain Pattern tool (from Pfam Search) in TBtools.

Cis-Acting Element Analysis
The GXF Sequences Extract tool in TBtools (v1.120) was used to extract BrCMF promoter sequences 2000 bp upstream of the CDS, and the PlantCARE database [51] (PlantCARE, a database of plant promoters and their cis-acting regulatory elements (https: //www.ugent.be/)accessed on 22 November 2022) was used for predictive analysis with default parameters.The results were visualized using TBtools (v1.120).
4.6.Stress Treatments, Total RNA Extraction, Transcriptome and RT-qPCR Analysis Seeds were sown in an MS-modified medium (containing vitamins, sucrose, and agar) (PM10121-307, Coolaber, Beijing, China) in a plant incubator.Six-leaved seedlings with similar growth statuses were obtained, and the seedlings were placed in a hydroponic system under salt stress (150 mM NaCl), drought stress (15% PEG6000), and cold stress (4 • C), while control (CK) seedlings were treated with dd H 2 O and grown at 25 • C/15 • C, with a light/dark cycle of 16/8 h.Materials that had been treated with salt stress for 12 h, drought stress for 6 h, and cold stress for 7 d were used for transcriptome analysis, BrCMF genes were subjected to Paired-end (PE) sequencing using Next-Generation Sequencing (NGS) based on the Illumina HiSeq sequencing platform by BioMarker Technologies (Beijing, China), and three biological replicates were collected for each sample.RT-qPCR experiments were conducted using samples that had been exposed to salt and drought stress treatment for 4 h, 6 h, and 12 h and exposed to cold stress treatment for 4 h, 6 h, 12 h, and 7 days.A minimum of three biological replicates were performed for each treatment, and samples were stored at −80 • C during storage.
Total RNA was extracted using a FastPure ® Cell/Tissue Total RNA Isolation Kit V2 (Vazyme Biotech Co., Ltd., Nanjing, China).Primer sequences were designed using the qPrimerDB-qPCR Primer Database (https://biodb.swu.edu.cn/qprimerdb/,accessed on 19-20 November 2022).RT-qPCR primer sequences are shown in Table S4.TransScript ® Uni All-in-One First-Strand cDNA Synthesis SuperMix was used for the reverse transcription of RNA samples for RT-qPCR analysis.RT-qPCR was performed using TransStart ® Green qPCR SuperMix (TransGen Biotech, Beijing, China) on a qTOWER3 qPCR machine, with BrACTIN2 as the internal reference gene, while the reaction was performed in three technical replicates.The data were analyzed using the 2 −∆∆CT method [52] and plotted using Excel 2020.

PPI Network Analysis
PPI network analysis was conducted using STRING (https://cn.string-db.org/,accessed on 23 November 2022) with default parameters, and Cytoscape v3.9.1 [53] was used to construct the interaction network.

Conclusions
A total of 25 BrCMF genes were identified in B. rapa, and some of these genes were involved in the regulation of abiotic stresses responses.BrCMF22 might play a role in the response to salt stress, BrCMF14, BrCMF5, and BrCMF7 might play a role in the response to drought stress, and BrCMF21, BrCMF5, and BrCMF7 might play a role in the response to cold stress.Additional research is needed to clarify the specific mechanism underlying the observed responses.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants13081118/s1. Table S1: Tissue-specific expression data; Table S2: The expression of BrCMF genes after abiotic stress treatments in transcriptome; Table S3: The RT-qPCR data; Table S4: List of primers used for RT-qPCR.

Figure 1 .
Figure 1.(A) The location of BrCMFs on A. thaliana chromosomes, with chromosomes indicated in yellow font, gene names in red font, and blue lines indicating collinearity relationships among genes.(B) Chromosomal collinearity relationships between B. rapa and A. thaliana and between B. rapa and O. sativa.Purple, blue, and orange indicate B. rapa, A. thaliana, and O. sativa, respectively, with red lines indicating collinearity relationships, while the gray lines represent orthologous gene blocks between Brassica rapa and other species.(C) The phylogenetic tree of CMF genes of three species: B. rapa, A. thaliana, and O. sativa.Yellow, green, blue, and purple correspond to Group 1, Group 2, Group 3, and Group 4, respectively.Other names of the genes are shown in parentheses.

Figure 2 .
Figure 2. (A) The conserved motifs of BrCMFs, (B) the domains of BrCMFs, and (C) gene structure.The evolutionary relationships among the 25 genes are shown on the left.

Figure 2 .
Figure 2. (A) The conserved motifs of BrCMFs, (B) the domains of BrCMFs, and (C) gene structure.The evolutionary relationships among the 25 genes are shown on the left.

Figure 3 .
Figure 3.A total of 24 BrCMFs contain various cis-acting elements, and these cis-acting elements were classified into three groups based on their functions, including growth and development, phytohormone response, and stress response, as indicated on the right side of the figure.The functions of the cis-acting elements are shown on the left side, along with the number of cis-acting elements in each gene, while blank cells indicate the absence of the cis-acting element.Each column corresponds to a gene, which is indicated at the bottom, and each number indicates the number of cisacting elements present in the 2000 bp 5' region of the corresponding gene.

Figure 3 .
Figure 3.A total of 24 BrCMFs contain various cis-acting elements, and these cis-acting elements were classified into three groups based on their functions, including growth and development, phytohormone response, and stress response, as indicated on the right side of the figure.The functions of the cis-acting elements are shown on the left side, along with the number of cis-acting elements in each gene, while blank cells indicate the absence of the cis-acting element.Each column corresponds to a gene, which is indicated at the bottom, and each number indicates the number of cis-acting elements present in the 2000 bp 5' region of the corresponding gene.

Figure 4 .
Figure 4. Heatmap of the tissue-specific expression patterns of BrCMF genes in various organs (TableS1).Colors in the graph indicate the level of expression, light colors represent low expression levels, while red represents high expression levels.The data were all log-transformed to enhance contrast.

Figure 4 .
Figure 4. Heatmap of the tissue-specific expression patterns of BrCMF genes in various organs (TableS1).Colors in the graph indicate the level of expression, light colors represent low expression levels, while red represents high expression levels.The data were all log-transformed to enhance contrast.

Figure 5 .
Figure 5. Transcriptome and RT-qPCR analysis reveals stress-induced changes in the expression o BrCMFs (BrACTIN2 was used as reference for estimated ∆CT in Figure 5A-C).(A) The expressio levels of BrCMFs transcriptome after 12 h of NaCl treatment.(B) The expression levels of BrCMF transcriptome after 6 h of PEG treatment.(C) The expression of BrCMFs transcriptome after 7 d o cold treatment.The height of column represents the expression level.(D) The expression levels o BrCMFs according to RT-qPCR analyses after 0 h (CK), 4 h, 6 h, and 12 h of NaCl and PEG trea ments.(E) The expression levels of BrCMFs according to RT-qPCR analyses after 0 h (CK), 4 h, 6 h 12 h and 7 d of cold treatments.The expression of BrCMF5 and BrCMF21 decreased to 0 after 7 day of cold stress.Error bars are indicated for each column.CK refers to 0 h treatment, 7 d refers to days.PEG refers to the PEG6000 treatment.Full details are given in TablesS2 and S3.

Figure 5 .
Figure 5. Transcriptome and RT-qPCR analysis reveals stress-induced changes in the expression of BrCMFs (BrACTIN2 was used as reference for estimated ∆CT in Figure 5A-C).(A) The expression levels of BrCMFs transcriptome after 12 h of NaCl treatment.(B) The expression levels of BrCMFs transcriptome after 6 h of PEG treatment.(C) The expression of BrCMFs transcriptome after 7 d of cold treatment.The height of column represents the expression level.(D) The expression levels of BrCMFs according to RT-qPCR analyses after 0 h (CK), 4 h, 6 h, and 12 h of NaCl and PEG treatments.(E) The expression levels of BrCMFs according to RT-qPCR analyses after 0 h (CK), 4 h, 6 h, 12 h and 7 d of cold treatments.The expression of BrCMF5 and BrCMF21 decreased to 0 after 7 days of cold stress.Error bars are indicated for each column.CK refers to 0 h treatment, 7 d refers to 7 days.PEG refers to the PEG6000 treatment.Full details are given in TablesS2 and S3.