Genome-Wide Identiﬁcation and Analysis of the Plant Cysteine Oxidase (PCO) Gene Family in Brassica napus and Its Role in Abiotic Stress Response

: Plant Cysteine Oxidase (PCO) is a plant O 2 -sensing enzyme catalyzing the oxidation of cysteine to Cys-sulﬁnic acid at the N-termini of target proteins. To better understand the Brassica napus PCO gene family, PCO genes in B. napus and related species were analyzed. In this study, 20, 7 and 8 PCO genes were identiﬁed in Brassica napus , Brassica rapa and Brassica oleracea , respectively. According to phylogenetic analysis, the PCOs were divided into ﬁve groups: PCO1 , PCO2 , PCO3 , PCO4 and PCO5 . Gene organization and motif distribution analysis suggested that the PCO gene family was relatively conserved during evolution. According to the public expression data, PCO genes were expressed in different tissues at different developmental stages. Moreover, qRT-PCR data showed that most of the Bna/Bra/BoPCO5 members were expressed in leaves, roots, ﬂowers and siliques, suggesting an important role in both vegetative and reproductive development. Expression of BnaPCO was induced by various abiotic stress, especially waterlogging stress, which was consistent with the result of cis-element analysis. In this study, the PCO gene family of Brassicaceae was analyzed for the ﬁrst time, which contributes to a comprehensive understanding of the origin and evolution of PCO genes in Brassicaceae and the function of BnaPCO in abiotic stress responses.


Introduction
Cysteine (Cys) oxidation is an essential post-translational modification (PTM), which controls protein half-life and protein function [1]. It is reported that ROS-mediated hormone signaling can be transduced by cysteine oxidation in plant, which is associated with root growth, pollen tube elongation and various abiotic stress responses [2]. Plant Cysteine Oxidases (PCOs) are the enzymes catalyzing N-terminal cysteinyl residues to sulfinic acid to promote proteasomal degradation in plants [3]. It has been shown that Plant Cysteine Oxidases use molecular oxygen atoms to catalyze dioxygenation of Nt-Cys to Cys-sulfinic acid in ERF-VIIs (ETHYLENE RESPONSE FACTOR group VIIs), as well as other substrates VRN2 and ZPR2 [4,5] for subsequent Nt-arginylation [6,7]. Meanwhile, ERF-VIIs are known as the regulators of hypoxia-regulated transcriptional reprogramming to adapt the environmental change [8][9][10]. PCO activity is sensitive to physiologically relevant fluctuations in O 2 availability [11], thus these enzymes can act as plant O 2 sensors with a key role in regulating ERF-VII stability. In other words, as an enzyme, Plant Cysteine Oxidases (PCOs) are a direct link between environmental stimuli and molecular physiological outcomes [12].
In Arabidopsis, AtPCOs catalyze the oxidation of cysteine to Cys-sulfinic acid at the N-termini of target proteins, a reaction that co-translational methionine cleavage exposes the N-terminal Cys for oxidation [6,7,11]. Oxidized N-terminal Cys residues are substrates for arginyl transferase enzymes, with the arising arginylated N-termini recognized by as well as total oil production in China. Rapeseed is mainly distributed in the Yangtze River basin in China and waterlogging, caused by the rainy and humid climate in this region, is a common abiotic stress affecting rapeseed production. The oxygen availability in waterlogging soil is greatly limited and the yield of rapeseed could decrease by 17-42% due to waterlogging stress in China [23]. Research on genetics, evolution and stress response of rapeseed is a good way to improve the quality and yield of rapeseed [24][25][26][27][28]. While AtPCOs are expected to play important roles in stress responses, detailed genome-wide analysis of the PCO gene family in Brassicaceae has not been performed. In this study, we investigated the significant role of PCO genes in Brassicaceae to provide evidence for resistance improvement of rapeseed. As a result, we identified 35 PCO genes of Brassica napus, Brassica rapa and Brassica oleracea, respectively, and characterized the gene structures, chromosomal locations, evolutionary relationships and expression patterns in different tissues and under different abiotic stress treatments with public data analysis. Moreover, we confirmed the BnaPCO gene expression patterns in different tissues and under waterlogging stress by qRT-PCR analysis. Therefore, this study provides a comprehensive understanding of PCOs in development and abiotic stress responses in Brassica napus.

Chromosomal Distribution and Duplication of BnaPCOs
It was uncovered that B. napus (AnAnCnCn, 2n = 38) originated from the hybridization event between B. rapa (AnAn, n = 10) and B. oleracea (CnCn, n = 9) approximately 7500 years ago [20][21][22], and Brassicaceae species underwent a whole-genome triplication event [20]. As shown in Figure 2a, three genes in B. rapa were oriented from At2G42670, which was consistent with the duplication theory. According to the evolution theory, there are 15, 15 and 30 PCOs in B. rapa, B. oleracea and B. napus expanded from the 5 AtPCOs. However, only 7, 8 and 20 genes were identified in B. rapa, B. oleracea and B.napus, respectively (Table S1). As shown in Figure 2, 7 BraPCOs were scattered on 6 of the 10 B. rapa chromosomes, 8 BoPCOs were scattered on 5 of the 9 B. oleracea chromosomes, 20 BnaPCOs were scattered on 11 of the 19 B. napus chromosomes. The numbers of BnaPCOs on the An-subgenome (8 genes) and the Cn-subgenome (12 genes) showed a biased trend, with more genes on the Cn-subgenome. It was indicated that some PCOs may have been lost during evolution.
represents the protein from Arabidopsis.

Chromosomal Distribution and Duplication of BnaPCOs
It was uncovered that B. napus (AnAnCnCn, 2n = 38) originated from the hybridization event between B. rapa (AnAn, n = 10) and B. oleracea (CnCn, n = 9) approximately 7500 years ago [20][21][22], and Brassicaceae species underwent a whole-genome triplication event [20]. As shown in Figure 2a, three genes in B. rapa were oriented from At2G42670, which was consistent with the duplication theory. According to the evolution theory, there are 15, 15 and 30 PCOs in B. rapa, B. oleracea and B. napus expanded from the 5 AtPCOs. However, only 7, 8 and 20 genes were identified in B. rapa, B. oleracea and B.napus, respectively (Table S1). As shown in Figure 2, 7 BraPCOs were scattered on 6 of the 10 B. rapa chromosomes, 8 BoPCOs were scattered on 5 of the 9 B. oleracea chromosomes, 20 BnaPCOs were scattered on 11 of the 19 B. napus chromosomes. The numbers of BnaPCOs on the Ansubgenome (8 genes) and the Cn-subgenome (12 genes) showed a biased trend, with more genes on the Cn-subgenome. It was indicated that some PCOs may have been lost during evolution. Previous study reported that the An and Cn subgenomes of B. napus were collinear with the diploid B. rapa (Ar) and B. oleracea (Co) genomes, and most of the An-Ar and Cn-Co homologous pairs showed similar chromosomal locations [20]. However, the C genome of B. napus had more homologous genes corresponding to theBoPCO3 gene (Bo5G025500), and the A genome had more homologous genes corresponding to the BraPCO4 gene (BraA05G003220), while the homologous genes of PCO1, PCO2 and PCO5 groups were relatively conserved (Table 1). It was inferred that the PCO sequences were mutated or duplicated in the homologous evolution process. Previous study reported that the An and Cn subgenomes of B. napus were collinear with the diploid B. rapa (Ar) and B. oleracea (Co) genomes, and most of the An-Ar and Cn-Co homologous pairs showed similar chromosomal locations [20]. However, the C genome of B. napus had more homologous genes corresponding to the BoPCO3 gene (Bo5G025500), and the A genome had more homologous genes corresponding to the BraPCO4 gene (BraA05G003220), while the homologous genes of PCO1, PCO2 and PCO5 groups were relatively conserved (Table 1). It was inferred that the PCO sequences were mutated or duplicated in the homologous evolution process.

Gene Structures and Motif Analysis of PCOs in B. napus
In order to study the homology domain and conservation degree of the BnaPCOs, MEME [29] and TBtools [30] were used to predict and visualize their conserved domain and gene organization, respectively. 8 motifs were predicted by MEME (https://memesuite.org/meme/tools/meme, accessed on 10 April 2023) ( Figure S1). As shown in Figure 3, Motif 2 and 5 were the most conserved, with 38 of the 40 genes containing these two motifs. All of the PCO4 and PCO5 proteins contained Motif 8, whereas none of the other genes contained Motif 8. All PCO5 proteins contained eight motifs, of which the Motif 7 was located at the C terminal. However, all PCO2 proteins contained seven motifs in addition to the Motif 8, with the Motif 7 located at the N terminal. Two of the four short abnormal proteins, BnaC05G0471900ZS and BnaC09G0521600ZS, lacked Motif 1-3-4-6-7-8, while BnaC04G0074400ZS and BnaC05G0283800ZS lacked Motif 1-2-4-5-6-8. Further protein analysis exhibited that BnaC05G0471900ZS and BnaC09G0521600ZS aligned C-terminal of BnaPCO3 protein and BnaC04G0074400ZS and BnaC05G0283800ZS aligned N-terminal of BnaPCO3 protein ( Figure S8). MEME [29] and TBtools [30] were used to predict and visualize their conserved domain and gene organization, respectively. 8 motifs were predicted by MEME (https://memesuite.org/meme/tools/meme , accessed on 10 April 2023) ( Figure S1). As shown in Figure  3, Motif 2 and 5 were the most conserved, with 38 of the 40 genes containing these two motifs. All of the PCO4 and PCO5 proteins contained Motif 8, whereas none of the other genes contained Motif 8. All PCO5 proteins contained eight motifs, of which the Motif 7 was located at the C terminal. However, all PCO2 proteins contained seven motifs in addition to the Motif 8, with the Motif 7 located at the N terminal. Two of the four short abnormal proteins, BnaC05G0471900ZS and BnaC09G0521600ZS, lacked Motif 1-3-4-6-7-8, while BnaC04G0074400ZS and BnaC05G0283800ZS lacked Motif 1-2-4-5-6-8. Further protein analysis exhibited that BnaC05G0471900ZS and BnaC09G0521600ZS aligned Cterminal of BnaPCO3 protein and BnaC04G0074400ZS and BnaC05G0283800ZS aligned N-terminal of BnaPCO3 protein ( Figure S8). It is reported that some introns play an essential role in transcriptional regulation [31]. We also investigated the distribution of introns and exons to study the diversity of gene structure. Five exons and four introns were distributed on most of PCO genes. Additionally, the intron phases of BnaPCOs were highly conserved in the same group, implicating the evolutionary similarity between these members. It is reported that some introns play an essential role in transcriptional regulation [31]. We also investigated the distribution of introns and exons to study the diversity of gene structure. Five exons and four introns were distributed on most of PCO genes. Additionally, the intron phases of BnaPCOs were highly conserved in the same group, implicating the evolutionary similarity between these members.

Cis-Element Analysis of BnaPCOs
Cis-elements regulate the initiation and efficiency of gene transcription by binding to transcription factors [32]. We analyzed the cis-elements of 20 PCOs promoters with PlantCARE [33]. Cis-elements of plant growth and development, hormone response and abiotic stress response were identified in BnaPCOs promoter region (Table S2). The identified environmental stress-related elements included anaerobic induction, circadian control, defense and stress responsive, drought induction, light response, low temperature response, meristem expression, and wound response. Among them, the most common elements were associated with light response and anaerobic induction, indicating that the growth and development of plants regulated by BnaPCOs was affected by light and oxygen. PCOs was known as a sensor of oxygen, which was consistent with the cis-elements result. However, the light response of PCOs needed further study.
In addition, there were more anaerobic-induction elements on PCO2s promoter region compared with other genes and there was no anaerobic-induction elements on the promoter of BnaC05G0471900ZS (belongs to BnaPCO3) ( Figure 4). The cis-elements in the upstream promoter region of genes are closely related to the expression and function of downstream genes [34]. Therefore, according to the anaerobic-induction elements result, it indicated that the expression of PCO2s may be highly induced by hypoxia and it also implies the diversification of biological functions of PCO genes in B. napus. and development of plants regulated by BnaPCOs was affected by light and oxygen. PCOs was known as a sensor of oxygen, which was consistent with the cis-elements result. However, the light response of PCOs needed further study.
In addition, there were more anaerobic-induction elements on PCO2s promoter region compared with other genes and there was no anaerobic-induction elements on the promoter of BnaC05G0471900ZS (belongs to BnaPCO3) (Figure 4). The cis-elements in the upstream promoter region of genes are closely related to the expression and function of downstream genes [34]. Therefore, according to the anaerobic-induction elements result, it indicated that the expression of PCO2s may be highly induced by hypoxia and it also implies the diversification of biological functions of PCO genes in B. napus.

Expression Profiling of PCO Genes in Different Tissues
Based on Arabidopsis eFP Browser data (http://bar.utoronto.ca/efp/cgibin/efpWeb.cgi, accessed on 28 March 2023) and RNA-seq data (B. rapa: GSE43245, B. oleracea: GSE42891 and B. napus: BnIR database) (Table S3), the PCO genes were expressed in different vegetative and reproductive organs of the four species at different developmental stages ( Figure 5). qRT-PCR was performed to verify the expression pattern in Arabidopsis, B. rapa, B. oleracea and B. napus (Figures 6, 7 and S9). In general, the expression pattern of PCO differed between groups ( Figure 5). Almost all PCOs were weakly expressed in pollen, indicating that PCO expression was down-regulated in sperm cells, presumably due to chromosomal structure or histone modifications. On the other hand, most

Expression Profiling of PCO Genes in Different Tissues
Based on Arabidopsis eFP Browser data (http://bar.utoronto.ca/efp/cgi-bin/efpWeb. cgi, accessed on 28 March 2023) and RNA-seq data (B. rapa: GSE43245, B. oleracea: GSE42891 and B. napus: BnIR database) (Table S3), the PCO genes were expressed in different vegetative and reproductive organs of the four species at different developmental stages ( Figure 5). qRT-PCR was performed to verify the expression pattern in Arabidopsis, B. rapa, B. oleracea and B. napus (Figure 6, Figure 7 and Figure S9). In general, the expression pattern of PCO differed between groups ( Figure 5). Almost all PCOs were weakly expressed in pollen, indicating that PCO expression was down-regulated in sperm cells, presumably due to chromosomal structure or histone modifications. On the other hand, most of PCO5s were expressed in different tissues, indicating that it involved in both vegetative and reproductive development. Compared with the public data, qRT-PCR analysis of BnaPCOs showed the similar results (Figure 7). of PCO5s were expressed in different tissues, indicating that it involved in both vegetative and reproductive development. Compared with the public data, qRT-PCR analysis of BnaPCOs showed the similar results (Figure 7).       There were six PCO3 genes in B. napus, and genes (BnaA06G0127100 and BnaC05G0155300) with higher sequence similarity to ancestral genes (BraA6G014250 and Bo5G025500) had higher expression levels, whereas genes with lower sequence similarity There were six PCO3 genes in B. napus, and genes (BnaA06G0127100 and BnaC05G0155300) with higher sequence similarity to ancestral genes (BraA6G014250 and Bo5G025500) had higher expression levels, whereas genes with lower sequence similarity to ancestral genes had lower expression levels (Figures 7 and S4). This suggested that genes with higher sequence similarity to the ancestral genes may play a major role, while the new genes may be pseudogenes that have been amplified during evolution or may be silent under normal conditions and be as a backup for their homolog genes under special conditions.

Expression Profiling of PCO Genes under Abiotic Stress Treatment
To reveal the roles of PCOs in stress response in B. napus, the expression patterns upon various abiotic treatment were investigated ( Figure 8, Table S4). In general, the expression of most PCO genes in leaves did not change significantly under various abiotic stress treatments (Figure 8a). In leaves, PCO3 gene expression was strongly induced under freezing stress, and PCO5 gene expression was increased under salt and osmotic stress as well. However, the response of PCO genes to stress was much stronger in roots than in leaves (Figure 8b). In roots, the expression of PCO1 and PCO2 were strongly induced by drought, and the expression of PCO1, PCO2, PCO3 and PCO4 were extremely downregulated by freezing and cold stress. However, PCO5 gene expression in roots changed weakly under the abiotic stress treatments compared with other PCO genes, which was consistent with the hypothesis that PCO5 worked as a fundamental gene. Though there were similar motifs of drought-inducible and low-temperature responsive on PCOs promoter region, the gene expression pattern was various under drought, freezing or cold stress treatment. It implied that there were other regulators along with cis-elements to regulate PCO gene expression. It was interesting that PCO3 was up-regulated in leaves and down-regulated in roots after freezing treatment. In other words, PCO3 showed opposite response patterns in leaves and roots under freezing stress treatment. Though there were similar motifs of drought-inducible and low-temperature responsive on PCOs promoter region, the gene expression pattern was various under drought, freezing or cold stress treatment. It implied that there were other regulators along with ciselements to regulate PCO gene expression. It was interesting that PCO3 was up-regulated in leaves and down-regulated in roots after freezing treatment. In other words, PCO3 showed opposite response patterns in leaves and roots under freezing stress treatment. This suggested that PCO3 may play different roles in leaves and roots under freezing stress, and further studies were needed to clarify this.

Expression Profiling of PCO Genes under Waterlogging Stress
Waterlogging removes air from soil leading to a blockage of gas exchange between the soil and the atmosphere [38,39]. Meanwhile, the diffusion rate of oxygen in water is only one tenth of that in air. As a result, oxygen availability in flooded soils is greatly limited, leading to suppression of root respiration. As mentioned before, PCO is an oxygen sensor in plant [11]. Therefore, it is speculated that PCO plays an important role in the hypoxic response induced by waterlogging stress. In order to elucidate the potential function of BnaPCO in response to waterlogging stress, RNA-seq and qRT-PCR assay were performed with leaves and roots after waterlogging stress in B.napus. Transcriptome data and qRT-PCR data (Figures 9 and 10, Table S5) showed that compared with CK, the expression of PCOs was significantly induced by waterlogging stress. Compared to other genes, the expression of PCO2 gene pairs (BnaA04G0111100ZS and BnaC04G0395100ZS) were strongly induced both in leaves and roots, while the expression of PCO4 gene pairs (BnaA05G0034400ZS and BnaC03G0244400ZS) were particularly strongly expressed in leaves. In addition, gene expression of PCO3 (BnaC05G0471900ZS and BnaC09G0521600ZS) were barely detectable in the transcriptome data, which was consistent with the previous result ( Figures 5 and 7). According to the expression results, the function of PCO was conserved under hypoxic stress. This suggested that PCO3 may play different roles in leaves and roots under freezing stress, and further studies were needed to clarify this.

Expression Profiling of PCO Genes under Waterlogging Stress
Waterlogging removes air from soil leading to a blockage of gas exchange between the soil and the atmosphere [38,39]. Meanwhile, the diffusion rate of oxygen in water is only one tenth of that in air. As a result, oxygen availability in flooded soils is greatly limited, leading to suppression of root respiration. As mentioned before, PCO is an oxygen sensor in plant [11]. Therefore, it is speculated that PCO plays an important role in the hypoxic response induced by waterlogging stress. In order to elucidate the potential function of BnaPCO in response to waterlogging stress, RNA-seq and qRT-PCR assay were performed with leaves and roots after waterlogging stress in B.napus. Transcriptome data and qRT-PCR data (Figures 9 and 10, Table S5) showed that compared with CK, the expression of PCOs was significantly induced by waterlogging stress. Compared to other genes, the expression of PCO2 gene pairs (BnaA04G0111100ZS and BnaC04G0395100ZS) were strongly induced both in leaves and roots, while the expression of PCO4 gene pairs (BnaA05G0034400ZS and BnaC03G0244400ZS) were particularly strongly expressed in leaves. In addition, gene expression of PCO3 (BnaC05G0471900ZS and BnaC09G0521600ZS) were barely detectable in the transcriptome data, which was consistent with the previous result ( Figures 5 and 7). According to the expression results, the function of PCO was conserved under hypoxic stress.  BnaPCO2 (e,f), BnaPCO3 ((g-l)), BnaPCO4 ((m-p)) and BnaPCO5 ((q-t)) genes, respectively. CK represents normal conditions. W represents waterlogging stress treatment. The mRNA level (relative to BnaActin7) of each gene in CK-Root or CK-Leaf was set to 1. Bars indicate ± SD (n = 3).

Discussion
The Plant Cysteine Oxidase family (PCO) is a set of plant O2-sensing enzymes, which catalyze the O2-dependent step [11]. In Arabidopsis, PCO has five members, PCO1, PCO2, PCO3, PCO4 and PCO5. In this study, 20, 8 and 7 PCO genes in B. napus, B. oleracea and B. rapa were identified, respectively (Figure 1). In B. napus, the number of PCO genes in the An subgenome (8) was almost the same as that in the diploid ancestors B. rapa (7) ( Table  1). This showed that the An subgenome PCO genes were relatively preserved after the whole-genome duplication event in B. napus. However, PCO3 genes in the Cn subgenome (BnaC04G0074400ZS, BnaC05G0155300ZS, BnaC05G0283800ZS, BnaC05G0471900ZS and BnaC09G0521600ZS) were mutated from the diploid ancestor B. oleracea (Bo5G025500). It suggested that the Cn subgenome was much flexible during the evolution, compared with the An subgenome [40]. PCO sequence alignment (Figures S2-S6) revealed that most PCOs Figure 10. BnaPCO gene expression after waterlogging stress treatment for 6 h and 12 h by qRT-PCR assays. Genes with purple, yellow, green, orange and blue background represent BnaPCO1 ((a-d)), BnaPCO2 (e,f), BnaPCO3 ((g-l)), BnaPCO4 ((m-p)) and BnaPCO5 ((q-t)) genes, respectively. CK represents normal conditions. W represents waterlogging stress treatment. The mRNA level (relative to BnaActin7) of each gene in CK-Root or CK-Leaf was set to 1. Bars indicate ± SD (n = 3).

Discussion
The Plant Cysteine Oxidase family (PCO) is a set of plant O 2 -sensing enzymes, which catalyze the O 2 -dependent step [11]. In Arabidopsis, PCO has five members, PCO1, PCO2, PCO3, PCO4 and PCO5. In this study, 20, 8 and 7 PCO genes in B. napus, B. oleracea and B. rapa were identified, respectively (Figure 1). In B. napus, the number of PCO genes in the An subgenome (8) was almost the same as that in the diploid ancestors B. rapa (7) ( Table 1). This showed that the An subgenome PCO genes were relatively preserved after the whole-genome duplication event in B. napus. However, PCO3 genes in the Cn subgenome (BnaC04G0074400ZS, BnaC05G0155300ZS, BnaC05G0283800ZS, BnaC05G0471900ZS and BnaC09G0521600ZS) were mutated from the diploid ancestor B. oleracea (Bo5G025500). It suggested that the Cn subgenome was much flexible during the evolution, compared with the An subgenome [40]. PCO sequence alignment (Figures S2-S6) revealed that most PCOs were conserved in Brassicaceae, indicating that these duplicated PCO genes can still retain the function of their ancestors in these species.
The gene expression pattern of duplicated genes with similar functions may change during the formation of allopolyploids, which takes several typical patterns, including transgressive up/down-regulation, unequal parental contributions, and silencing [40,41]. Although PCO3 was expanded in B. napus, half of them were slightly expressed in different tissues or abiotic stress treatment. According to the results of PCO3 alignment ( Figure  S4), BnaA06G0127100ZS was derived from BraA06G014250, BnaC05G0155300ZS was derived from Bo5G025500, and the remaining four genes may be generated by mutation or amplification of DNA fragments during the evolution of polyploid. The expression patterns of BnaA06G0127100ZS and BnaC05G0155300ZS maintained their expression patterns in two diploid progenitors. In addition, the expression of BnaC04G0074400ZS and BnaC05G0283800ZS responded to abiotic stress (Figures 8-10). It indicated that the newly generated genes may contribute to phenotypic differences between allopolyploids and their parental species under abiotic stress conditions.
It has been studied that transcription factors (TFs) bind to cis-regulatory DNA sequences at the 5' upstream end of genes to activate or repress gene expression [42,43]. In general, genes containing stress response elements in their promoter region are likely to be regulated by stress related TFs [25,31,39,44,45]. It is reported that HSFB2b directly binds to GmC4H and GmCHS3 to regulate the gene expression in response to salt stress, since there are HSEs (Heat Shock Elements) in the promoter regions of the GmC4H and GmCHS3 [46]. It was showed that hormone-responsive elements and environmental stress-related elements were distributed on the BnaPCO promoters (Table S2). Combined with the expression data with abiotic stress treatment, BnaPCO expression was regulated by various stress responses, especially drought, freezing, cold and waterlogging stress (Figures 8-10). However, the expression level was different under different stress conditions. For instance, there were seven anaerobic-induction cis-elements on the promoter region of BnaC04G0395100ZS, but the degree of waterlogging induced expression was much different between leaves and roots (Figures 9 and 10). As chromosome structure, histone modification, DNA methylation, transcriptional factors, cis-elements and other regulators work together to regulate gene expression [43], more studies are needed to explore the regulation mechanism of PCO expression under abiotic stress.
Oxygen homeostasis is critical for crop development, and hypoxia in plants is typically a consequence of reduced O 2 diffusion under conditions of waterlogging or submergence [24,[47][48][49]. The quality and yield of rapeseed are seriously affected by waterlogging stress in China [28,49]. It is reported that the response to hypoxia in rice, Arabidopsis and barley is mediated by the group VII ETHYLENE RESPONSE FACTORs (ERF-VIIs) [4,[8][9][10]19]. Moreover, the PCOs directly link O 2 availability to ERF-VII stability and anaerobic adaptation, leading to the suggestion that they act as plant O 2 sensors [6,7,11]. There were multiple anaerobic-induction cis-elements distributed on the BnaPCO promoters, and BnaPCO expression were induced by waterlogging stress after 6 h treatment both in leaves and roots. According to the expression results (Figures 9 and 10), it suggested that BnaPCO was a vital component, connecting environmental stimulus with cellular and physiological response and BnaPCO2 could be a potential target for improving waterlogging stress tolerance. Furthermore, H164 and D176 were in the AtPCO4 active site [12] and it was conserved in BnaPCO4 as shown in Figure S7. It implied that the function of BnaPCO in catalyzing ERF-VIIs could be conserved and targeting PCOs will be an effective way to improve the rapeseed tolerance to waterlogging stress by manipulating their O 2 sensitivity and/or substrate specificity.

Phylogenetic Analysis, Chromosomal Locations and Syntenic Relationship
ClustalW was used to align the multiple sequences of all PCO proteins (from Arabidopsis thaliana, B. napus, B. rapa and B. oleracea), and MEGA 11 was used to build a phylogenetic tree using the neighbor-joining (NJ) phylogenetic technique with 1000 bootstrap replicates. Arabidopsis_thaliana.TAIR10.dna (genome) and Arabidopsis_thaliana.TAIR10.gff3 (annotation information) were downloaded from the public database in https://www. arabidopsis.org/ (accessed on 10 January 2023). The genome and annotation information of B. rapa_Chiifu_V3.5, B._oleracea.BOL, and B. napus ZS11 were downloaded from the public database in https://yanglab.hzau.edu.cn/BnIR (accessed on 15 January 2023) [51]. TBtools version 1.116 was used to examine the gene chromosomal localization and syntenic relationship with the genome data, gff3 files and multiple synteny plot tool kit following the software instruction [30].

Analysis of Gene Structure, Motif Composition and Cis-Element
The motifs of BnaPCO proteins were predicted using the MEME v5.5.2 [29]. The number of motifs should not exceed 8. The distribution of motifs occurs zero or one time in each sequence. In order to investigate the structural characteristics of the BnaPCOs, the gff3 file (ZS11.annotation.gff3) was downloaded from the B. napus database (http://cbi.hzau.edu. cn/cgi-bin/rape/download_ext, accessed on 15 January 2023), which has the annotation information of the B. napus genome. The position information of introns and exons is obtained from the gff3 file. Meanwhile, the motif information was submitted to TBtools to graphically display gene structures and motif distributions.
To identify the cis-element of BnaPCOs, TBtools was used to obtain the 2000 bp sequences in front of the genomic CDS. Then, the PlantCARE [33] was used to predict the cis-elements on these promoters. Thus, the number and types of different cis-acting elements in BnaPCOs were classified and visualized with TBtools.

Plant Materials and Treatments
Zhongshuang 11 (ZS11, a semi-winter cultivar of Brassica napus is widely planted in Southern China and the genome sequence is available) seeds were germinated on filter paper, and the seedlings were then transplanted into pots with soil and nurtured in a growth chamber for four weeks (23 • C, 16 h light/8 h dark cycle, a relative humidity of 60%, 300 µmol m −2 s −1 light intensity). The waterlogging treatment was performed as previously described [27]. The pots of 4-week-old seedlings were placed in a 28 cm × 14 cm × 14 cm container filled with water and the water level was maintained at approximately 2 cm above the soil surface. Control plants (CK) remained well-watered throughout the experiment.

RNA-Seq and Heat Map Analysis of the PCO Transcriptome Data
Leaf and root samples of CK, 6 h treatment and 12 h-treatment were collected for RNA isolation and total RNA isolated using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's introductions. There were three biological replicates for each sample. Total amounts and integrity of RNA were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). A total of 1 µg total RNA per sample was used as input material for the lncRNA library preparation. Strand-specific libraries were generated using NEBNext ® UltraTM RNA Library Prep Kit for Illumina ® (NEB, Ipswich, MA, USA) following the manufacturer's recommendations and index codes were added to attribute sequences to each sample. RNA Sequencing was performed by the Illumina NovaSeq 6000 (Novogene, Beijing, China). Hisat2 v2.0.5 was used to map the reads to the reference genome and the gene expression level was determined by FPKM (number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced) calculation [52]. Differential expression analysis between CK and waterlogging treatment was performed using the edgeR R package. The p values were adjusted using the Benjamini and Hochberg method. Significantly differential expression genes were screened based on the following criteria: Corrected p-value < 0.05.
The expression patterns of BnaPCOs in different tissues and other abiotic stress treatment were obtained from the BnTIR (Brassica napus transcriptome information resource) database [37]. The public expression data of AtPCOs, BraPCOs and BoPCOs were obtained from Arabidopsis eFP Browser data, RNA-seq data of B. rapa (GSE43245) [35] and RNA-seq data of B. oleracea (GSE42891) [36]. All the expression data were standardized based on a log2 scale, and clustered and visualized with TBtools.

Quantitative Real-Time RT-PCR (qRT-PCR) Analysis
To verify the PCO gene expression in different tissues, samples of root, stem, leaf, flower, silique and seed from Arabidopsis and B. napus, and samples of root, stem and leaf from B. rapa and B. oleracea were collected. Leaf and root samples of B. napus under waterlogging stress treatment with 6 h and 12 h were collected as well. Total RNA was extracted using MolPure ® Plant RNA Kit according to manufacturer instructions (Yeasen, Shanghai, China). The first strand cDNA was synthesized by Hifair ® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (Yeasen, Shanghai, China). Then, the gene relative expression was detected by qRT-PCR assay using PerfectStart ® Green qPCR SuperMix (TransGen, Beijing, China) and a CFX96™ Real-Time PCR Detection System (BIO-RAD, Hercules, CA, USA). Gene expression was normalized to AtActin2, BraGAPDH, BoActin and BnaActin7, respectively [27,[53][54][55]. Relative gene expression values were calculated with the ∆∆Ct method. Experiments were performed with three biological replicates. Primers for qRT-PCR were listed in Table S6.

Conclusions
In this study, we identified 20, 7 and 8 PCO (Plant Cysteine Oxidase) proteins in B. napus, B. rapa and B. oleracea, respectively, by exploring the important role of PCO genes in Brassicaceae plants. Collinearity analysis shows that the PCO gene family was relatively conserved in evolution of B. rapa, B. oleracea, Arabidopsis, and B. napus. However, there were mutations or duplications of PCO3 and PCO4 during homologous evolution process. The cis-elements that regulate hormone response and response to abiotic stresses were found in the BnaPCO promoters. In addition, we found that the Bna/Bra/BoPCO genes were expressed differently in different tissues at different developmental stages. Remarkably, BnaPCO2s were significantly induced after waterlogging treatment, which was consistent with the cis-element analysis and previous studies. BnaPCO2 could be the potential target for waterlogging tolerance improvement. This study provides a foundation for further understanding the biology and stress response functions of PCO family genes in B. napus.

Data Availability Statement:
The clean reads of RNA-seq in this paper have been deposited in the National Genomics Data Center [56,57] (GSA: CRA010912). All relevant data are available from the corresponding author on request (mani@caas.cn).