Genome-wide identification and expression profiling of the COBRA-like genes reveal likely roles in stem strength in rapeseed (Brassica napus L.).

The COBRA-like (COBL) genes play key roles in cell anisotropic expansion and the orientation of microfibrils. Mutations in these genes cause the brittle stem and induce pathogen responsive phenotypes in Arabidopsis and several crop plants. In this study, an in silico genome-wide analysis was performed to identify the COBL family members in Brassica. We identified 44, 20 and 23 COBL genes in B. napus and its diploid progenitor species B. rapa and B. oleracea, respectively. All the predicted COBL genes were phylogenetically clustered into two groups: the AtCOB group and the AtCOBL7 group. The conserved chromosome locations of COBLs in Arabidopsis and Brassica, together with clustering, indicated that the expansion of the COBL gene family in B. napus was primarily attributable to whole-genome triplication. Among the BnaCOBLs, 22 contained all the conserved motifs and derived from 9 of 12 subgroups. RNA-seq analysis was used to determine the tissue preferential expression patterns of various subgroups. BnaCOBL9, BnaCOBL35 and BnaCOBL41 were highly expressed in stem with high-breaking resistance, which implies these AtCOB subgroup members may be involved in stem development and stem breaking resistance of rapeseed. Our results of this study may help to elucidate the molecular properties of the COBRA gene family and provide informative clues for high stem-breaking resistance studies.

Introduction

Plant morphogenesis is dependent on the regulation of cell division and expansion. Most plant cells grow anisotropically through internal and isotropic turgor pressure yield from cell walls [1]. The plant cell wall is a dynamic, complex fibrillar network. After the plant cell expands to its final shape and the primary cell wall is formed, the secondary cell wall is formed and thickens between the primary cell wall and plasma membrane [2, 3]. The COBRA gene, which encodes a glycosylphosphatidylinositol (GPI) anchored protein [1, 4, 5], regulates microfibril deposition on the cell surface at the rapid elongation stage to guarantee a normal anisotropic expansion of the cell wall during plant morphogenesis.

The COBRA gene belongs to the COBRA-like (COBL) gene family. COBL proteins often contain an N-terminal secretion signal, a COBRA domain, potential N-glycosylation sites, a CCVS (Cys-rich) motif, and an ω-attachment site for GPI modification along with a hydrophobic C-terminal [1, 5]. Some of these proteins contain a predicted cellulose-binding site (CBM). The analysis of the cob allele indicated that COBLs can further affect the cellulose crystallinity status and cellulose content of the secondary cell wall [1].

The COBL family is conserved in monocots and eudicots [2, 5]. In Arabidopsis (Arabidopsis thaliana), there are 12 COBLs (AtCOBLs), which can be divided into two groups based on their protein sequences [1], one showing strong similarity to COBRA while the other exhibiting high similarity to AtCOBL7. There are 11, 11, and 10 COBLs that have been identified in the monocots rice (Oryza sativa ssp. japonica) [6], maize (Zea mays) [2], and sorghum (Sorghum bicolor) [7], respectively. Additionally, 17, 18, 24, and 33 COBLs have been reported in the eudicots tomato (Solanum lycopersicum) [8], Populus (Populus L.) [9], soybean (Glycine max) [10], and cotton (Gossypium spp.) [11], respectively. It appears that the family members increased to a certain extent in eudicots but remained almost constant in monocots. This phenomenon of expansion is presumed to be derived from whole-genome duplication [10, 11] and segmental duplication [5]. The phylogenetic relationship was similar to that of Arabidopsis among various reported species [10]. In the COBRA group, the AtCOB orthologous subgroup was predicted to be a sister clade of the AtCOBL4 subgroup and derived more recently after the division between monocots and eudicots [5].

The COBLs members have been found to mediate diverse physiological and developmental processes such as stem strength [6], pollen tube growth [12], pathogen resistance [13], and root-hair growth [4]. Silencing a COBL member, such as BRITTLE CULM1 (OsBC1) in rice, Brittle stalk 2 (ZmBk2) in maize, BRITTLE CULM1 (SbBC1) in sorghum, and TmBr1 in diploid wheat, caused plants to exhibit the brittle phenotype [6, 7, 14, 15]. Cuticle lacking, abnormal shape, and irregular size distribution were observed in the epidermal cells of a tomato mutant in which the SlCOBRA-like gene was repressed. These phenotypes resulted in extensive nonuniform cracking on the surface of the immature green fruits of these plants [8]. Mutations in AtCOBL10 were observed to cause gametophytic male sterility due to reduced pollen tube growth and compromised directional sensing in the female transmitting tract [12, 16, 17].

Rapeseed (Brassica napus L. AACC, 2n = 38) which supplies approximately 13–16% of vegetable oil worldwide [18], is an allotetraploid species that was formed approximately 7,500–12,500 years ago by a spontaneous cross of the diploid progenitors B. rapa (AA, 2n = 20) and B. oleracea (CC, 2n = 18) [19]. In this study, we identified COBL genes at the genome-wide level and performed a comprehensive in silico analysis including characterization of phylogeny, gene structure, conserved motifs, and chromosomal collinearity in rapeseed and its progenitors. We also evaluated the expression patterns of these genes in various tissues as well as stems with different stem breaking resistance (SBR) by transcriptome sequencing. Our results may help to further characterize the functions of COBL family, and provide clues for stem strength in rapeseed.

Materials and methods

Genome-wide identification of COBLs in Brassica napus and its both progenitor species

The B. napus (cv. ZhongShuang11, ZS11) genome sequence was downloaded from the BnPIR database (http://cbi.hzau.edu.cn/bnapus/index.php) [18, 20]. The genome sequences, CDSs and annotation files of B. rapa (v3.0) and B. oleracea (HDEM) were retrieved from the Brassica Database (BRAD, http://brassicadb.cn). The Arabidopsis COBL protein sequences were obtained from TAIR (http://www.arabidopsis.org) [5], and used as the query to identify COBL homologs in B. napus, B. rapa and B. oleracea by BLASTP [21], with the e-value being 1E-10. After redundant sequences and incomplete sequences were removed, the remaining protein sequences were submitted to SMART tools and the NCBI Conserved Domain Search Database to confirm the presence of previously characterized domains in the candidate sequences; sequences without COBRA domains were excluded from the downstream analysis [22].

The physicochemical parameters of BnaCOBL proteins, including the molecular weights (in kDa) and isoelectric points (pIs), were calculated by ExPASy [23]. The subcellular location of COBL proteins were predicted by Cell-PLoc v2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/).

Multiple alignments and phylogenetic analysis of COBLs from Brassica and Arabidopsis

All the predicted COBL protein sequences of B. napus, B. rapa, and B. oleracea, and the AtCOBLs protein sequences were aligned by Multiple Sequence Comparison by Log-Expectation (MUSCLE) [24]. The phylogenetic tree was generated in IQ-tree [25] software using the maximum likelihood (ML) method with 10,000 bootstrap replicates. The “figtree” (http://tree.bio.ed.ac.uk/software/figtree/) was used to draw the phylogenetic tree of COBL protein in four genomes.

Chromosomal locations and syntenic analyses of COBLs in Brassica napus and its both progenitor species

The chromosomal positions of the BnaCOBLs were obtained from the genome annotation file of ZS11. The start and end locations of each BnaCOBL were drawn on chromosomes using MapChart [26]. The synteny relationships between the BnaCOBLs and COBLs in B. rapa, and B. oleracea were evaluated using the McScanX [27] and drawn by TBtools [28].

Prediction of gene structures, conserved motifs, and cis-acting regulatory elements of BnaCOBLs

The gene structures (exon-intron) of BnaCOBLs were retrieved from the genome annotation file. The COBRA domain and potential N-glycosylation sites were predicted by GenomeNet Bioinformatics Tools (https://www.genome.jp/) and the NetNGlyc 1.0 server (http://www.cbs.dtu.dk/services/NetNGlyc/) [29]. The signal peptide, CCVS Cys-rich domain, and potential ω-sites for GPI modification were predicted with Signal 5.0 [30] and the GPI Prediction Server Version 3.0 [31]. Hydrophilicity analysis was performed by ExPASy-ProtScale (https://web.expasy.org/protscale/) [32, 33]. TBtools was used to draw the structural map of BnaCOBLs. To further analyze the COBRA domains of BnaCOBLs, the multi-sequence alignments were carried out by MEGA v7.0 [34] and the results were displayed by GeneDoc (http://www.cris.com/~Ketchup/ genedoc.shtml).

To analyze the putative cis-regulatory elements (CAREs) of BnaCOBLs, the promoter regions were defined as the 1.5-kb region upstream of the ATG start codon of each gene (i.e., the 1.5-kb downstream sequences were chosen if a gene was found to map on the opposite strand relative to the sequence strand deposited in the ZS11 genome). These sequences were used to detect the CAREs with the online database PlantCARE [35]. Next, considering the characters of plant core promoter regions, we checked the common promoter elements TATA-box and CAAT-box near the start codon (<500bp), the core promoter elements (i.e., TATA-box, CAAT-box) on the opposite strands of the corresponding genes were filtered out of the results because the core promoter regions are direction-sensitive [36]. We classified all the elements into core promoter elements, responsive elements, the temporal and spatial specific or unannotated elements according to their functional annotation.

Expression analysis of BnaCOBLs in various tissues

The RNA-seq data obtained from 12 tissues of the rapeseed cultivar ZS11, which was described in a previous study [37], were downloaded from National Center for Biotechnology Information (NCBI) (ID: PRJNA394926) to assess the tissue expression preference of different COBL family members of rapeseed.

For further evaluation of the expression profiles of BnaCOBLs in rapeseed stem, we selected previously reported [38] transcriptome expression data of four stem samples: FH (High stem breaking resistance (SBR) during Flowering), FL (Low-SBR during Flowering), SH (High-SBR during Silique development), and SL (Low-SBR during Silique development). The high SBR sample had averaged SBR of 115.49N; while the low SBR sample had averaged SBR of 31.69N [38]. The raw data were downloaded from the Short Read Archive (SRA) database of NCBI under the accession number SRP142441.

The NGSQCToolkit [39] was used to clean the raw data. The RSEM [40] and STAR [41] softwares were used to map the clean reads to the reference genome of ZS11 and calculate the transcripts per million (TPM) values of each gene, and the heat map of expression of BnaCOBL genes was drawn by TBtools.

Plant materials and qRT-PCR analysis

The seed of ZS11, a semi-winter rapeseed cultivar, was kindly provided by Oil Crops Research Institute, Chinese Academy of Agricultural Sciences and sown on the experimental farm of Hunan Agricultural University, Changsha. Three individual plants were harvested at the initial flowering stage. Their stems were cut into two parts, the upper (adjacent to inflorescence) and the lower (the first elongated internode). Fully expanded leaves were used as leaf samples whereas the taproot and the lateral roots were collected separately after being cleaned up.

Quantitative real-time RT–PCR (qRT–PCR) was performed to determine gene expression level. Total RNA was extracted from all sample tissues separately using an RNAqueous kit (Thermo Fisher, AM1912). The yield of RNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), and the integrity of the RNA was evaluated using agarose gel electrophoresis and staining with ethidium bromide. Each RT reaction consisted of 0.5 μg RNA, 2 μl of 5X TransScript All-in-One SuperMix for qPCR and 0.5 μl of gDNA Remover in a total volume of 10 μl. Reactions were performed in a GeneAmp® PCR System 9700 (Applied Biosystems, USA) for 15 min at 42°C and 5 s at 85°C. The 10-μl RT reaction mix was subsequently diluted tenfold in nuclease-free water. Real-time PCR was performed using LightCycler® 480 II Real-time PCR Instrument (Roche, Swiss) with 10 μl PCR reaction mixture that included 1 μl of cDNA, 5 μl of 2X PerfectStartTM Green qPCR SuperMix, 0.2 μl of forward primer, 0.2 μl of reverse primer and 3.6 μl of nuclease-free water. Reactions were incubated in a 384-well optical plate (Roche, Swiss) at 94°C for 30 s followed by 45 cycles of 94°C for 5 s and 60°C for 30 s. Each sample was repeated three times. The expression levels of mRNAs were normalized to BnaActin and were calculated using the comparative cycle threshold (Ct) method [42]. The primers were designed at the specific nucleotide among the CDSs of five BnaCOBLs and checked through electronic PCR on the CDSs of these genes. These primer sequences are listed in the S1 Table.

Results

Identification of the COBL genes in Brassica napus and its diploid progenitors

A total of 62 putative COBLs were identified in B. napus through a BLASTP search using 12 Arabidopsis COBL protein sequences as query. These sequences were submitted to SMART and the NCBI CDD (Conserved Domains Database) to confirm the existence of COBRA domains. Finally, 44 candidate COBLs were identified and designated as BnaCOBL1-44 in rapeseed, and their basic information is listed in the S2 Table. Among these proteins, BnaCOBL38 was determined to be the largest with 699 amino acids (aa), whereas BnaCOBL19 was the smallest with 200 aa. The molecular weights and isoelectric points of the BnaCOBLs ranged from 22.06 to 77.68 kDa and 5.25 to 10.09 (S2 Table), respectively. The BnaCOBLs were predicted to localize at the cell membrane (30), extracellular (12), and endoplasmic reticulum (2).

Similarly, we also identified 20 BraCOBLs and 23 BolCOBLs in B. rapa and B. oleracea, both progenitor species of B. napus, respectively. Their gene symbols and chromosomal locations are listed in S2 Table. There were approximately two times as many COBLs in B. rapa and B. oleracea as in Arabidopsis. The sum of COBLs in the diploid progenitors was almost equal to the quantity of BnaCOBLs.

Phylogenetic analysis of the COBL genes from B. napus, B. rapa, B. oleracea and Arabidopsis

To unravel the evolutionary relationships among the COBL genes from B. napus, B. rapa, B. oleracea and Arabidopsis, a phylogenetic tree was constructed based on whole protein sequences using ML method. As shown in Fig 1, all the COBL members were clustered into two groups, which corresponded with the AtCOB group and the AtCOBL7 group in Arabidopsis [5]. The AtCOB group (Group Ⅰ) contained AtCOB, AtCOBL1-6, 12 BraCOBLs, 15 BolCOBLs, and 25 BnaCOBLs, while the AtCOBL7 (Group Ⅱ) consisted of AtCOBL7-11, 8 BraCOBLs, 8 BolCOBLs, and 19 BnaCOBLs. Group Ⅰ contained more COBLs than Group Ⅱ in the four species analyzed.

Fig 1

Phylogenetic analysis of COBL proteins in Brassica napus, B. rapa, B. oleracea, and Arabidopsis.

All protein sequences were aligned by MUSCLE software. The phylogenetic tree was constructed by IQ-tree by ML method with 10,000 bootstrap replicates. These proteins were clustered into two groups. The red, blue, and green font represent the COBLs of Arabidopsis, B. rapa, and B. oleracea, respectively. The percentages of bootstrap numbers for the nodes are displayed on the branches.

Based on the bootstrap values and the topology of the phylogenetic tree, these proteins were further divided into 12 subgroups (Table 1). Each subgroup had COBLs from four species, except the BnaCOBL5/44 subgroup, which lacks COBLs from Arabidopsis. The subgroups AtCOBL1, AtCOBL7, AtCOBL8, and AtCOBL9 each retained two BnaCOBLs, while six and eight BnaCOBLs were retained in the subgroups AtCOBL2/3 and AtCOBL11, respectively. The other subgroups had three or five BnaCOBLs. These results indicated an unequal evolution among orthologous subgroups of BnaCOBLs when derived from corresponding AtCOBLs. The subgroup AtCOB was closer to AtCOBL5 in Group Ⅰ. while the subgroup AtCOBL10 was closer to AtCOBL11 in Group Ⅱ. This distribution was similar to that in Arabidopsis. Based on the triploidy and allotetraploidization events in the evolutionary history of rapeseed, each subgroup of this phylogenetic topology represented a class of orthologous COBLs in Brassica species derived from the corresponding AtCOBL.

Table 1

Domains of BnaCOBL proteins in rapeseed.

Gene Name	Chr.	Subgroup	CCVS1	N-terminal secretion signal cleavage site	ω-site2		Hydrophobic C-terminal
					Position	p-value
BnaCOBL9	A03	AtCOB	233	TEA-YD	N431	2.3*e-07	yes
BnaCOBL35	C07	AtCOB	233	TEA-YD	N431	2.4*e-06	yes
BnaCOBL41	C09	AtCOB	233	TEA-YD	N431	2.2*e-07	yes
BnaCOBL32	C05	AtCOBL1	231	ADA-YD	N428	3.7*e-04	yes
BnaCOBL33	C05	AtCOBL1	232	ADA-YD	A428	2.9*e-04	yes
BnaCOBL27	C03	AtCOBL2/3		-	A187	-	no
BnaCOBL6	A02	AtCOBL2/3	223	TEA-YD	N415	2.8*e-05	yes
BnaCOBL13	A06	AtCOBL2/3	223	TEA-YD	N412	2.5*e-07	yes
BnaCOBL25	C02	AtCOBL2/3	223	TEA-YD	N415	2.1*e-04	yes
BnaCOBL30	C05	AtCOBL2/3		-	G296	-	no
BnaCOBL34	C07	AtCOBL2/3	223	TEA-YD	N412	1.6*e-07	yes
BnaCOBL7	A03	AtCOBL4		ASA-YD	W526	-	yes
BnaCOBL19	A10	AtCOBL4		TSA-YD	G170	-	no
BnaCOBL24	C02	AtCOBL4		SSA-YD	G215	-	no
BnaCOBL26	C03	AtCOBL4		ASA-YD	M204	-	no
BnaCOBL10	A03	AtCOBL5		SEA-LT	M184	-	no
BnaCOBL18	A09	AtCOBL5		TEA-YD	G382	-	no
BnaCOBL36	C07	AtCOBL5		SEA-LT	S209	-	no
BnaCOBL42	C09	AtCOBL5		-	T183	-	no
BnaCOBL12	A06	AtCOBL6	222	SHG-YD	S270	-	no
BnaCOBL16	A08	AtCOBL6	219	THG-FD	S412	1.9*e-07	yes
BnaCOBL29	C05	AtCOBL6	221	SHG-YD	R548	-	no
BnaCOBL39	C08	AtCOBL6	218	THG-FD	S411	1.1*e-07	yes
BnaCOBL5	A02	-		SLG-RY	M186	-	no
BnaCOBL44	C09	-		-	M499	-	no
BnaCOBL2	A01	AtCOBL7	420	TTS-QS	S635	2.3*e-05	yes
BnaCOBL21	C01	AtCOBL7	419	TAS-QS	N635	3.0*e-05	yes
BnaCOBL4	A01	AtCOBL8	427	TSS-QP	S642	6.6*e-06	yes
BnaCOBL23	C01	AtCOBL8	423	TSS-QQ	N638	9.3*e-06	yes
BnaCOBL17	A09	AtCOBL9	421	SLS-QL	G638	9.8*e-05	yes
BnaCOBL40	C09	AtCOBL9	421	SLS-QL	S638	2.3*e-05	yes
BnaCOBL3	A01	AtCOBL10	433	CNG-QD	S646	1.6*e-05	yes
BnaCOBL8	A03	AtCOBL10	432	CNG-QD	S645	1.3*e-05	yes
BnaCOBL11	A05	AtCOBL10		-	G373	-	no
BnaCOBL22	C01	AtCOBL10	433	CNG-QD	S646	-	yes
BnaCOBL28	C03	AtCOBL10	422	CNG-QD	S635	9.2*e-06	yes
BnaCOBL1	A01	AtCOBL11	423	SFA-QD	S635	2.4*e-05	yes
BnaCOBL14	A07	AtCOBL11		-	A237	-	no
BnaCOBL15	A08	AtCOBL11	428	SLA-QD	Y641	-	yes
BnaCOBL20	C01	AtCOBL11	425	SRA-QD	S637	3.0*e-05	yes
BnaCOBL31	C05	AtCOBL11		-	S198		yes
BnaCOBL37	C08	AtCOBL11		-	E188	-	no
BnaCOBL38	C08	AtCOBL11	458	-	S670	-	yes
BnaCOBL43	C09	AtCOBL11		-	S198		yes

1The start site of the CCVS domain.

2The amino acid and location of the ω-site (GPI attachment cleavage site). A site represented in italics means that the confidence of this prediction did not reach the threshold.

Chromosomal locations of COBLs and syntenic analyses between Brassica napus and its progenitor

The BnaCOBLs were unevenly distributed on 16 of 19 chromosomes (except for A04, C04 and C06) of rapeseed, with one to five members on each chromosome (Fig 2 and S2 Table). The BnaCOBLs were asymmetrically distributed in subgenomes: 19 were detected in the A subgenome, and 25 were detected in the C subgenome. However, the locations of BnaCOBLs on chromosome A01, A02, and A09 were much the same as the locations of their homologous C01, C02, and C09. Even the BnaCOBLs on A01 and BnaCOBLs at the homologous region on C01 were determined to belong to the same subgroups. This kind of gene pairs were also observed on some other homologous chromosomes.

Fig 2

Chromosomal locations of BnaCOBLs.

The blue and the green bar represent A- and C-subgenome chromosomes, respectively. The gene name is presented to the right of each bar, while the chromosome name is to the left. To the left of the A/C subgenome is a 10-Mb bar. The clusters are shown with a red frame.

Chalhoub et al [19] reported that 80.0% of genes in B. napus (cv. Damor) were orthologous to the genes of B. rapa and B. oleracea. Based on protein sequence identity and phylogenetic topology, we identified 18 and 21 orthologous gene pairs (S3 Table) between the subgenomes of rapeseed and their respective ancestral genomes. The locations of these orthologous pairs of COBLs showed high similarity between B. napus and B. rapa or B. oleracea, respectively (Fig 3A). We found two BraCOBLs (BraCOBL5 and BraCOBL13) and three BolCOBLs (BolCOBL6, BolCOBL16, and BolCOBL17) have lost their orthologous gene pair in B. napus. On the other hand, the five BnaCOBLs (BnaCOBL14, BnaCOBL27, BnaCOBL31, BnaCOBL43, and BnaCOBL44) did not detect orthologs in either B. rapa or B. oleracea.

Fig 3

Synteny of COBLs between B. napus and B. rapa or B. oleracea.

(a) Genome-wide synteny analysis for COBLs between B. napus and B. rapa or B. oleracea. The bars in red, purple, and green bars represent the chromosomes of B. napus, B. rapa, and B. oleracea, respectively. The homologous pairs of COBLs between B. napus and B. rapa or B. oleracea were connected with blue lines, (b) The syntenic relationships of gene clusters among B. napus, B. rapa, B. oleracea, and Arabidopsis. The blue square bracket marked the X block of Arabidopsis. The homologous COBLs of different genomes are connected by lines of the same color. A 10-Mb ruler is located in the lower-left corner.

The BnaCOBL gene clusters [43-45] containing two or three BnaCOBLs appear on the chromosome A03, C05, C07, C08, and C09 (Fig 2). The cluster on A03, C07, and C09 exhibited the same order on the “X block” [46] of B. rapa, B. oleracea and Arabidopsis (Fig 3B). The cluster on C08 was detected in B. oleracea but not in Arabidopsis. The cluster on C05 was detected only in B. napus, and this cluster may have been formed by segmental duplication in B. napus according to sequence and annotation of the ZS11 genome. These results suggested that the COBL gene family in rapeseed changed little during the allotetraploidization event from B. rapa and B. oleracea.

Structure and conserved domains of BnaCOBLs

We characterized gene structure and motif domains of the BnaCOBL genes. These genes had 2–12 exons (Fig 4). The number of exons varies between two groups. In Group Ⅰ, 17 of 25 members possessed over 6 exons, whereas all members in Group Ⅱ were determined to have only two to four exons. However, the average length of proteins was observed to be longer in Group II than in Group Ⅰ.

Fig 4

Domain compositions and the gene structure of BnaCOBLs.

The dotted rectangles represent the skipped and missed exons according to how the orthologous genes are spliced in B. rapa, B. oleracea, and Arabidopsis. The dashed lines modify the genomic sequences.

All the BnaCOBLs were observed to have the COBRA domain (Fig 4). The COBRA domain of the Group Ⅰ members is close to the N-terminal secretion signal peptide, while that of the Group Ⅱ members is in the middle of the protein sequences. But there are exceptions to this rule. The multiple-sequence alinements showed eight BnaCOBLs whose COBRA domain is largely defective (S1 Fig). For example, BnaCOBL31, BnaCOBL43 and BnaCOBL37 only retained 29 to 58 amino acids at C-terminal. The COBRA domain showed significant divergence between 12 subgroups and high conservation within the subgroup although it had almost the same in one-third amino acid residues among family members. One or more potential N-glycosylation sites were distributed to all BnaCOBLs. Thirty-four of these proteins were identified beginning with an N-terminal secretion signal. The CCVS (Cys-rich) motif of 27 BnaCOBLs was observed to have seven to ten amino acids away from the C-terminal of the COBRA domain. Half of the family members (Fig 4 and Table 1) were determined to have all the conserved domains, so did the ω-sites follow by a hydrophobic C-terminal domain.

Compared to AtCOBL4, BC1, BK2, the orthologous BnaCOBLs lost the C-terminal motif of COBRA domain, CCVS, and ω-site (S2 Fig) because of the exon skipping (BnaCOBL7 and BnaCOBL26) or the intron-retention (BnaCOBL19 and BnaCOBL24). These similar alternative splicing events were also identified in other rapeseed sequenced genomes so that no complete BnaCOBL7 can be found in the rapeseed pan-genome. The three members of AtCOB subgroup were conserved among all nine rapeseed genomes, which is shown in S2 Fig. In this subgroup, only two orthologous COBLs of B. rapa and B. oleracea were defective and also disappeared from B. napus.

Cis-acting regulatory elements in the promoter region of BnaCOBLs

The control over gene transcription via upstream cis-acting regulatory elements (CAREs) is the most prominent mechanism governing gene expression regulation [47]. The analysis of CAREs may help elucidate the expression levels of BnaCOBLs in specific tissues and conditions [48]. To predict putative cis-elements in the BnaCOBLs, DNA sequences 1500 bp upstream of the start codon (ATG) were searched for in the PlantCARE database to identify the CAREs associated with plant growth, development, and stress response. Eighty-five CAREs were found in all BnaCOBLs. All promoter regions of BnaCOBLs contained CAAT-box which is the major determinant of promoter efficiency. Three or more TATA-boxes were found in all the genes except two, BnaCOBL10 and BnaCOBL36 which had the TATA-less type of promoters [49].

We also analyzed the phytohormones and environment responsive elements (Fig 5 and S4 Table). The stress-related CAREs (S4 Table) were the most common and identified among all the BnaCOBLs. These stress-related CAREs included MYB, MYC, ARE and as-1, which correspond to abiotic and biotic stress. The most frequent stress CAREs were the MYCs, a dehydration-responsive element. The BnaCOBLs were probably regulated by methyl jasmonate (MeJA), ethylene (ETH), and abscisic acid (ABA) since these phytohormones-responsive CAREs were detected frequently in putative promoter regions. We found 22 light-responsive elements located in all BnaCOBLs. These discoveries indicated that BnaCOBLs could be regulated by stress-related, phytohormone-responsive, and light-induced transcription factors.

Fig 5

Cis-acting regulatory elements in the promoter region of BnaCOBLs.

Different colors represent cis-regulatory elements with different predicted functions. In this figure, MYB represents MYB, MBS, MBSI, the MYB-like sequence, the Myb-binding site, and the MYB recognition site; ABA responsiveness represents ABRE, ABRE3a, and ABRE4; MeJA responsiveness represents the TGACG-motif and the CGTCA-motif; GA responsiveness represents the P-box, the GARE-motif, and the ATC-box; Auxin represents the TGA element and the AuxRR core, and stress responsiveness and light responsiveness covered 11 and 22 cis-regulatory elements respectively.

Tissue specificity of expression of BnaCOBLs in rapeseed

The function of COBLs has been reported in root, flower, stem, and fruit skin of Arabidopsis, rice, maize, and tomato. We collected the RNA-seq data from the Sequence Read Archive (SRA) to examine the tissue-specific expression of the 44 BnaCOBLs. These tissues include stem, leaf, root, flower, stamen, ovule, pistil, silique, sepal, pericarp, blossomy pistil, and wilting pistil. The TPM values are listed in the S5 Table.

The expression profiling (Fig 6) in the various tissues demonstrated that BnaCOBLs participated in biological processes in all examined tissues, especially the COBLs of the subgroups the AtCOB, AtCOBL7, and AtCOBL8, whereas the COBLs of the subgroups AtCOBL1, AtCOBL10, and AtCOBL11 were specifically expressed in floral organs. Even individual genes, such as BnaCOBL6 and BnaCOBL25, were expressed in the ovule. The expression levels in tissues were mostly conserved in the intra orthologous subgroups but were different in inter orthologous subgroups. The eight members (BnaCOBL5, BnaCOBL11, BnaCOBL14 BnaCOBL31, BnaCOBL37, BnaCOBL42, BnaCOBL43, and BnaCOBL44) were found not to be expressed in any tissues. These expression characteristics of BnaCOBLs implied that specific subgroup members have different functions in different organs.

Fig 6

Heatmap of the expression levels of BnaCOBLs.

Blue represents little or no expression, and red represents a high level of expression. The expression patterns of BnaCOBLs in various tissues and in the stem with different stem breaking resistance (SBR) are presented. The subgroups are noted to the left of the gene names.

Considering the reported brittle culm mutants and the importance of stem stress resistance, we further compared the expression levels of BnaCOBLs in stems with different SBR levels. All three AtCOB subgroup members BnaCOBL9, BnaCOBL35, and BnaCOBL41 were most active in the stem compared with other subgroups (Fig 6) and expressed at higher levels in the High-SBR one. Among these three genes, BnaCOBL41 was expressed highest. In contrast, the AtCOBL4 subgroup members BnaCOBL7, BnaCOBL26, BnaCOBL19, and BnaCOBL24, which were found to be involved in stem breaking in cereal crops, were weakly expressed in the High-SBR stem.

To confirm these results, we selected the above three AtCOB subgroup members BnaCOBL9, BnaCOBL35, and BnaCOBL41, and two AtCOBL4 subgroup genes BnaCOBL19, and BnaCOBL24 to quantify their expression with qRT-PCR in taproots, lateral roots, flower buds, leaves, upper and lower stems of rapeseed (ZS11) at the flowering stage. The amplification curves showed that the average expression level of the AtCOB subgroup genes was higher in the stem than that of the AtCOBL4 members (Fig 7).

Fig 7

qRT-PCR analyses of five selected BnaCOBLs.

A relatively high expression level was observed for the three AtCOB subgroup genes in the stem, root, and leaf, with the BnaCOBL41 gene being expressed more strongly in the root, while the other two genes were expressed at higher levels in stems. The AtCOBL4 subgroup genes were expressed at higher levels than other genes in these tissues. The stem adjacent to an inflorescence is defined as the “upper stem”; the first elongated node is defined as the “lower stem”. Single and double asterisks represent differences from the control sample at the 5% and 1% significance levels, respectively. Error bars represent the standard deviations of three independent measurements.

Furthermore, the results of qRT-PCR on various parts of stems implied that the genes BnaCOBL9, BnaCOBL35, and BnaCOBL41 have different functions not only at different developmental stages but also in different internodes.

Discussion

In this study, we identified 44 COBRA-like genes in rapeseed and analyzed their phylogenetic relationships, chromosome locations, domain composition, and putative cis-elements. Together with the tissue specific expression patterns, these characters were differentiated by subgroups which were orthologs from different COBLs of Arabidopsis.

The COBRA family has been reported in many species in the plant kingdom, even in the moss Physcomitrella patens [5]. This family has already emerged in the ancestor of Arabidopsis. In Arabidopsis, there are 12 AtCOBLs, and segmental duplication contributed to AtCOBLs, as two pairs of duplication had been identified (AtCOBL2 and AtCOBL3, AtCOBL1 and AtCOBL4) [5].

We identified 44 BnaCOBLs, 20 BraCOBLs, and 23 BolCOBLs in the genomes of the allotetraploid Brassica napus and its diploid progenitor species B. rapa and B. oleracea, respectively (Fig 1). Brassica evolved from a Brassiceae lineage-specific whole genome triplication (WGT) [19] after diverged from a common ancestor with Arabidopsis about 20 million years ago [50, 51]. After WGT the number of BraCOBLs and BolCOBLs almost doubled compared to the number of AtCOBLs. However, the number of BnaCOBLs was close to the sum of the number of BraCOBLs and BolCOBLs after allopolyploidization. Furthermore, BnaCOBLs are highly syntenic to [52-55] and conserved in gene clusters of BraCOBLs and BolCOBLs (Fig 1). We propose that whole-genome triplication event contributed to the expansion of BnaCOBLs.

The expression profiling demonstrated expression patterns of BnaCOBLs in twelve tissues (Fig 6). As the stem with reinforced mechanical strength showed higher resistance to lodging and pathogen attack [13, 38]. We concentrated on their expression levels in stems with different breaking resistance and found all three AtCOB subgroup members BnaCOBL9, BnaCOBL35, and BnaCOBL41 were expressed at higher levels in the High-SBR stem than in the Low-SBR one. BnaCOBL9 is located near the lodging coefficient QTL on A03, and BnaCOBL41 is located 300 kb upstream of the breaking force QTL on C09 in rapeseed [56]. Both BnaCOBL9 and BnaCOBL35 are reported to be hub genes with some CesA in a co-expression module, which was predicted to be relevant to cellulose biosynthesis [38]. We postulate that the AtCOB subgroup BnaCOBLs may play a role in the formation of stem strength in rapeseed.

Contrary to the expectation, the cloned AtCOBL4 subgroup COBL genes such as BC1 in rice, BK2 in maize, which were shown to be associated with the stem-breaking resistance in the grass family [57], were weakly expressed in High-SBR stems of rapeseed, which indicates AtCOBL4 subgroup members are not involved in the formation of stem strength in rapeseed. None of all AtCOBL4 subgroup members maintained all core motifs of COBLs (Fig 4), whereas members in this subgroup of B. rapa and B. oleracea were complete (S2 Fig). This structural change brought about alternative splicing variants, that is, exon skipping (BnaCOBL7 and BnaCOBL26) or intron-retention (BnaCOBL19 and BnaCOBL24). These splicing variants were confirmed in the reported rapeseed genomes [18]. Whether do structural change and alternative splicing cause neofunctionalization and/or subfunctionalization of AtCOBL4 subgroup members in rapeseed is worth more studies.

Sequence alignment of the COBRA domain of COBL proteins in Arabidopsis, rice, corn and rapeseed.

BC1_ rice_Japo (AAQ56120.1) and BC1_rice_Indi (AAQ56121.1) represent the Brittle Culm1 protein in Oryza sativa subsp. Indica and Oryza sativa subsp. Japonica respectively. BK2 (ABJ99754.1) encodes brittle_stalk-2 protein in corn.

(TIF)

Click here for additional data file.

Sequence alignment of AtCOB and AtCOBL4 orthologous subgroup proteins in Arabidopsis, B. rapa, B. oleracea and B. napus.

The blue square brackets contain the COBL gene across four B. rapa genomes; The green square brackets contain the COBL gene across three B. oleracea genomes; The purple-red square brackets contain the COBL gene across nine B. napus genomes. Conservative domains are in the rectangle. "M1", " M2", "M3" and " M5" are the crucial sites that were verified by mutants to BC1 of rice; “M4” point at a transposon insertion site in BK2 of corn.

(SVG)

Click here for additional data file.

Primer sequences designed for qRT-PCR of selected BnaCOBLs.

(XLSX)

Click here for additional data file.

The basic information concerning COBLs in B. napus, B. rapa, and B. oleracea.

(XLSX)

Click here for additional data file.

Orthologous COBL gene pairs between B. napus with B. rapa and B. oleracea.

(XLSX)

Click here for additional data file.

Cis-acting regulatory elements related to stress, light and phytohormone responsiveness in the promoter region of BnaCOBL genes.

(XLSX)

Click here for additional data file.

TPM values of BnaCOBLs in different tissues and stems with distinct SBR.

(XLSX)

Click here for additional data file.

21 May 2021

PONE-D-21-09114

Genome-wide identification of the COBRA-like gene family in rapeseed (Brassica napus L.)

PLOS ONE

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Reviewer #1: Partly

Reviewer #2: Yes

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Reviewer #2: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, the authors conducted a genome-wide analysis of the COBRA-like gene family in rapeseed. They also performed phylogenetic analysis, cis-element, and gene expression analysis. However, I do have some concerns before its final acceptance.

1. In phylogenetic analysis COBLs, authors had done analysis using protein sequences from Brassica and A. thaliana only. They can draw a better picture/results if they use more species for phylogenetic analysis.

2. Why authors randomly selected the five genes out of 44 for Q-RT PCR analysis? In my opinion, there should be a criterion/logic for selection. Like, (a) selection based on their subclass division; phylogenetic based, (b) selection of genes based on their in-silico expression results.

3. “BnaCOBLs were involved in stem lodging resistance” based on these experiments, it overestimated results. In my opinion, some more experiments should be conducted to further functional validations of results to meet the journal standards. Functional validation of genes using transgenic approach/VIGS/CRISPAR.

4. It is also suggested that authors should improve the discussion section. They have to discuss their results and compare them with some earlier and recently published papers in more depth and clarity.

5. Please provide the Pfam ID used for COBL gene identification.

6. Detail of BnaActin gene used in this study. i.e., NCBI ID, or any other database ID should be provided.

7. How many biological replicates of each tissue were used for qRT-PCR analysis, since it is not clear from the martial and methods section. Please clear?

8. How they named COBL genes? Please provide details.

Reviewer #2: In this paper, using the method of biological information, 44 COBRA-like genes were identified in rapeseed. Then, the phylogenetic relationships, the chromosome location, the domain composition, and the putative cis element distribution of BnaCOBLs were determined. Different expression levels in stems with distinct lodging resistance suggested that there was some association between BnaCOBLs and stem lodging resistance. The logic of the article is very clear, and the scientific issues of concern are also well explained. But there are still several issues that need attention:

(1) In line 127, when determining the promoter, you should not only use the length as the standard, but should combine some basic characteristics of the promoter, such as the -10 region and the -35 region. Because the promoter may exist in the last gene, or the two genes mentioned in your article share the promoter. The sequence of the promoter directly affects the results of the subsequent analysis. For example, the speculation of gene function in line 445 may be biased.

(2) Line 117. Why you choosed 10 motifs and motif width of 8-50 amino-acid residues as this papaer's parameters. The basis should be pointed out here.

(3) In the discussion section, when the article directly quoted the content of the article results, the corresponding picture number was not marked. I think it will be easier for readers to read this article by marking the corresponding picture numbers.

**********

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Reviewer #1: No

Reviewer #2: Yes: Guanghui Xiao

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Submitted filename: WHQ comments.docx

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16 Aug 2021

Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, the authors conducted a genome-wide analysis of the COBRA-like gene family in rapeseed. They also performed phylogenetic analysis, cis-element, and gene expression analysis. However, I do have some concerns before its final acceptance.

1. In phylogenetic analysis COBLs, authors had done analysis using protein sequences from Brassica and A. thaliana only. They can draw a better picture/results if they use more species for phylogenetic analysis.

In this case, we revealed the evolutionary source and expansion of BnaCOBLs family mainly by the phylogenetic analysis, so we chose only A.thaliana and two diploid progenitor species B. rapa and B. oleracea. The COBL family had already discovered conserving in monocots and eudicots from other researches in rice, soybean and corn.

2. Why authors randomly selected the five genes out of 44 for Q-RT PCR analysis? In my opinion, there should be a criterion/logic for selection. Like, (a) selection based on their subclass division; phylogenetic based, (b) selection of genes based on their in-silico expression results.

We chose the five genes for Q-RT PCR analysis justly based on their phylogenetic subgroup division, and their in-silico expression. The BnaCOBL9, BnaCOBL35, and BnaCOBL41 belong to AtCOB subgroup; the BnaCOBL19 and BnaCOBL24 belong to AtCOBL4 subgroup. The members in both subgroups differently expressed in different levels of the stem breaking resistance.

3. “BnaCOBLs were involved in stem lodging resistance” based on these experiments, it overestimated results. In my opinion, some more experiments should be conducted to further functional validations of results to meet the journal standards. Functional validation of genes using transgenic approach/VIGS/CRISPAR.

Thanks for your suggestion, we also thought the evidence was not sufficient. We changed the conclusion.

4. It is also suggested that authors should improve the discussion section. They have to discuss their results and compare them with some earlier and recently published papers in more depth and clarity.

Yes, we have revised the discussion section in the manuscript.

5. Please provide the Pfam ID used for COBL gene identification.

The Pfam ID of COBRA-like gene family is PF04833

6. Detail of BnaActin gene used in this study. i.e., NCBI ID, or any other database ID should be provided.

The expression levels of mRNAs were normalized to BnaActin (BnaA10g06670D), which has been listed on S1 Table.

7. How many biological replicates of each tissue were used for qRT-PCR analysis, since it is not clear from the martial and methods section. Please clear?

We performed three biological replicates, which have been provided in line 158 of the manuscript.

8. How they named COBL genes? Please provide details.

COBLs is the abbreviation of COBRA-like genes. The number suffixes were added according to the chromosome location from Chromosome A01 to Chromosome C09. This could also be shown on Figure 2.

Reviewer #2: In this paper, using the method of biological information, 44 COBRA-like genes were identified in rapeseed. Then, the phylogenetic relationships, the chromosome location, the domain composition, and the putative cis-element distribution of BnaCOBLs were determined. Different expression levels in stems with distinct lodging resistance suggested that there was some association between BnaCOBLs and stem lodging resistance. The logic of the article is very clear, and the scientific issues of concern are also well explained. But there are still several issues that need attention :

(1) In line 127, when determining the promoter, you should not only use the length as the standard, but should combine some basic characteristics of the promoter, such as the -10 region and the -35 region. Because the promoter may exist in the last gene, or the two genes mentioned in your article share the promoter. The sequence of the promoter directly affects the results of the subsequent analysis. For example, the speculation of gene function in line 445 may be biased.

After further reviewing the literature on promoter research, we identified cis-elements in all 1500bp upstream of the ATG codon and did not filter out the elements on the opposite strand. Since the cis-element may exist in the genes on the upstream. The elements in core promoter regions nearing the TSS (transcript start site) are direction-sensitive, but the elements in the distal region are direction-insensitive.

We checked the TATA-box and CAAT-box in the proximal region (-500bp) of the start codon, only BnaCOBL10 and BnaCOBL36 loss their TATA-box. But the TATA-less type promoter takes account for 79.9% of all promters in Arabidopsis thaliana. So we temporarily identified the elements in the 1500bp upstream region of the start codon when batch analyzing.

(2) Line 117. Why you choosed 10 motifs and motif width of 8-50 amino-acid residues as this papaer's parameters. The basis should be pointed out here.

Once, we had predicted the conserved motifs of COBL family by MEME software with these parameters. Then we use the known COBRA domains of COBL family to evaluate BnaCOBLs instead. This made the protein structural analysis more meaningful.

(3) In the discussion section, when the article directly quoted the content of the article results, the corresponding picture number was not marked. I think it will be easier for readers to read this article by marking the corresponding picture numbers.

Yes, we have marked these signs in the revised discussion section.

Submitted filename: Responses to the Refrees Comments.docx

Click here for additional data file.

26 Aug 2021

PONE-D-21-09114R1

Genome-wide identification and expression profiling of the COBRA-like genes reveal likely roles in stem strength in rapeseed (Brassica napusL.)

PLOS ONE

Dear Dr. Liu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Oct 10 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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Kun Lu, Ph.D.

Academic Editor

PLOS ONE

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Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

Reviewer #4: Yes

**********

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Reviewer #4: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: Yang et al., used bioinformatics method to analyze the COBL gene family in rapeseed. The results are interesting, and may have potential guiding significance for lodging resistance improvement. The major comments are:

1. Whether the animal (like mouse or human) has COBL homeologues, if has, please introduce the gene function of COBL genes in Introduction.

2. The authors are better to give the uniform naming in B. napus, B. rapa and B. oleracea based on Fig. 1 result, and combine the Table S2 and S3 together. Otherwise the readers hard to compare the same homeologue in different species.

3. Based on the protein location predication result, the BnaCOBL10 and BnaCOBL42 were not on cell membrane, while these two genes were in the same subgroup (Fig. 2). The authors may discuss the possibility of the differentiation of gene function in Discussion.

4. The authors need to check the italics of the gene name fully (e.g. Line 37, 44, 56, 57, 79, 141, 223, 226, 229, 230 etc.)

Reviewer #4: In this manuscript, 44 COBRA-like genes were identified in rapeseed by sequence identity and conservative COBRA domain. The author characterized these genes on phylogenetic grouping, the chromosome location, the domains, and the putative cis-element distribution in promoters of BnaCOBLs. The expression profiles and the protein domains of AtCOB and AtCOBL4 subgroup imply these AtCOB subgroup members may be involved in stem development and stem breaking resistance of rapeseed, rather than members in AtCOBL4 subgroup, which are functional in crops of grass family. The logic of the article is clear, and the scientific issues of concern are also well explained. But I think authors should address following points in this manuscript before publishing in “ PLOS ONE ”:

1. The conclusion in line 79 to 80 seemed to be expanded according to the results and discusses.

2. As the topology of the phylogenetic tree (Fig 1), BnaCOBL5 and BnaCOBL44 did not belong to any subgroup orthologs from AtCOBLs. Where they evolved from? Maybe these two genes are not the members of COBL family in rapeseed.

3. The alternative splicing of subgroup AtCOBL4 presented in the Result section should be supported by figures.

4. Why the members in AtCOBL4 subgroup with incomplete protein still are expressed in stems at a certain level?

5. In line 19 in the Abstract section, whether the expansion of COBL family in B.napus was attributable to ‘whole-genome duplication’ or ‘whole-genome triplication’ as written in line 423?

6. Some syntax and formatting errors such as, in line 243, ‘The kind of gene pair was’ should revise as ‘This kind of gene pairs were’ in this context; in Table 1, the ‘p-value’ in line where the ‘Gene Name’ is ‘BnaCOBL20’, does not line up with other elements in the same line.

7. Add some correlation descriptions between the genes (such as BnaCOBL9, BnaCOBL35 and BnaCOBL24 etc.) expression change and stem strength in results.

**********

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Reviewer #3: No

Reviewer #4: No

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28 Sep 2021

Dear Referees:

We thank the reviewers for their enthusiasm and interest in this research. We would like to acknowledge the referees for spending their time and effort sharing their views and providing constructive comments. These comments help us improve our manuscript. The detailed response is provided below following the referee’s specific comments.

Reviewer #3: Yang et al., used bioinformatics method to analyze the COBL gene family in rapeseed. The results are interesting, and may have potential guiding significance for lodging resistance improvement. The major comments are:

1. Whether the animal (like mouse or human) has COBL homeologues, if has, please introduce the gene function of COBL genes in Introduction.

The COBRA-like gene family (pfam04833) is the only member of the superfamily cl04787 which only be found in embryophyte from a search of the conserved domain database (Search on 1st Sep, 2021).

2. The authors are better to give the uniform naming in B. napus, B. rapa and B. oleracea based on Fig. 1 result, and combine the Table S2 and S3 together. Otherwise the readers hard to compare the same homeologue in different species.

As you recommended, we have renamed the COBL genes of B. rapa (20) and B. oleracea (23) as BraCOBL1-20 and BolCOBL1-23 based on their chromosome locations. We combined the Tables S2 and S3 together to form a new S2 Table.

3. Based on the protein location predication result, the BnaCOBL10 and BnaCOBL42 were not on cell membrane, while these two genes were in the same subgroup (Fig. 2). The authors may discuss the possibility of the differentiation of gene function in Discussion.

Both BnaCOBL10 and BnaCOBL42 are orthologous from AtCOBL5, that is the only member without the GPIω-attachment site in Arabidpsis. The GPIω-attachment site is important for COBL proteins to be anchored in plasma membrane. So the orthologs of AtCOBL5 subgroup in rapeseed are not predicted to be on the plasma membrane. The function of the gene AtCOBL5 in Arabidpsis is little known, so did the orthologs of AtCOBL5 subgroup in other plants.

4. The authors need to check the italics of the gene name fully (e.g. Line 37, 44, 56, 57, 79, 141, 223, 226, 229, 230 etc.)

The words with capital letters in lines listed by reviewer, are not the names of genes but the names of subgroups or gene families. It is not necessary to italic the names of subgroups or gene families.

Reviewer #4: In this manuscript, 44 COBRA-like genes were identified in rapeseed by sequence identity and conservative COBRA domain. The author characterized these genes on phylogenetic grouping, the chromosome location, the domains, and the putative cis-element distribution in promoters of BnaCOBLs. The expression profiles and the protein domains of AtCOB and AtCOBL4 subgroup imply these AtCOB subgroup members may be involved in stem development and stem breaking resistance of rapeseed, rather than members in AtCOBL4 subgroup, which are functional in crops of grass family. The logic of the article is clear, and the scientific issues of concern are also well explained. But I think authors should address following points in this manuscript before publishing in “PLOS ONE ”:

1. The conclusion in line 79 to 80 seemed to be expanded according to the results and discusses.

Based on our results we redrawn the conclusion.

2. As the topology of the phylogenetic tree (Fig 1), BnaCOBL5 and BnaCOBL44 did not belong to any subgroup orthologs from AtCOBLs. Where they evolved from? Maybe these two genes are not the members of COBL family in rapeseed.

The BnaCOBL5 and BnaCOBL44 contain the COBRA domain, which meets the criteria of a member of COBL family. In fact, analysis of identity indicated both genes evolve from AtCOBL2/3 orthologs.

3. The alternative splicing of subgroup AtCOBL4 presented in the Result section should be supported by figures.

As shown in Fig4.tif, we illustrated the skipped exons in rectangle with dotted line.

4. Why the members in AtCOBL4 subgroup with incomplete protein still are expressed in stems at a certain level?

This phenomenon is caused by the unchanged promoters of members in AtCOBL4 subgroup.

5. In line 19 in the Abstract section, whether the expansion of COBL family in B. napus was attributable to ‘whole-genome duplication’ or ‘whole-genome triplication’ as written in line 423?

It is ‘whole-genome triplication’ in line 19.

6. Some syntax and formatting errors such as, in line 243, ‘The kind of gene pair was’ should revise as ‘This kind of gene pairs were’ in this context; in Table 1, the ‘p-value’ in line where the ‘Gene Name’ is ‘BnaCOBL20’, does not line up with other elements in the same line.

We carefully checked and corrected the grammatical and formatting errors in the revised manuscript.

7. Add some correlation descriptions between the genes (such as BnaCOBL9, BnaCOBL35 and BnaCOBL24 etc.) expression change and stem strength in results.

We have described the expression change patterns in line 376 to 382.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

8 Nov 2021

Genome-wide identification and expression profiling of the COBRA-like  genes reveal likely roles in stem strength in rapeseed (Brassica napus L.)

PONE-D-21-09114R2

Dear Dr. Liu,

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Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

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Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #3: Yang et al. addressed all the comments, and revised the manuscript fully. The manuscript is matched the publication criteria of PLOS ONE right now.

Reviewer #4: This paper is focused on identification and expression profiling of the COBRA-like  genes reveal likely roles in stem strength in rapeseed, it is well organized and its presentation is good. I think it meets the current publication criteria of PLOS ONE.

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Reviewer #3: No

Reviewer #4: No

16 Nov 2021

PONE-D-21-09114R2

Genome-wide identification and expression profiling of the COBRA-like  genes reveal likely roles in stem strength in rapeseed (Brassica napus L.)

Dear Dr. Liu:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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