Cloning and Phylogenetic Analysis of Brassica napus L. Caffeic Acid O-Methyltransferase 1 Gene Family and Its Expression Pattern under Drought Stress

Abstract

For many plants, regulating lignin content and composition to improve lodging resistance is a crucial issue. Caffeic acid O-methyltransferase (COMT) is a lignin monomer-specific enzyme that controls S subunit synthesis in plant vascular cell walls. Here, we identified 12 BnCOMT1 gene homologues, namely BnCOMT1-1 to BnCOMT1-12. Ten of 12 genes were composed of four highly conserved exons and three weakly conserved introns. The length of intron I, in particular, showed enormous diversification. Intron I of homologous BnCOMT1 genes showed high identity with counterpart genes in Brassica rapa and Brassica oleracea, and intron I from positional close genes in the same chromosome were relatively highly conserved. A phylogenetic analysis suggested that COMT genes experience considerable diversification and conservation in Brassicaceae species, and some COMT1 genes are unique in the Brassica genus. Our expression studies indicated that BnCOMT1 genes were differentially expressed in different tissues, with BnCOMT1-4, BnCOMT1-5, BnCOMT1-8, and BnCOMT1-10 exhibiting stem specificity. These four BnCOMT1 genes were expressed at all developmental periods (the bud, early flowering, late flowering and mature stages) and their expression level peaked in the early flowering stage in the stem. Drought stress augmented and accelerated lignin accumulation in high-lignin plants but delayed it in low-lignin plants. The expression levels of BnCOMT1s were generally reduced in water deficit condition. The desynchrony of the accumulation processes of total lignin and BnCOMT1s transcripts in most growth stages indicated that BnCOMT1s could be responsible for the synthesis of a specific subunit of lignin or that they participate in other pathways such as the melatonin biosynthesis pathway.

Fig 1. Sequence identity of intron I for BnCOMT1s.

Data on the upper right present the sequence identity regardless of the unmatched regions possibly emerged in either end of two aligned intron sequences, and data on the bottom left present the sequence identity of full-length intron sequences.

Fig 2. Protein sequence identity and similarity of BnCOMT1s.

Data on the upper right present the sequence identity, and data on the bottom left present the sequence similarity.

Fig 3. Domains and conserved sites prediction of BnCOMT1s.

The red bars stand for winged helix-turn-helix DNA-binding domain (IPR011991), blue bars for plant methyltransferase dimerisation domain (IPR012967), grey bars for S-adenosyl-L-methionine-dependent methyltransferase domain (IPR029063). The blue circles present conserved site (IPR022657). The blue triangles stand for S-adenosyl-L-methionine binding sites, and red ones for proton acceptor active sites.

Fig 4. Phylogenetic trees for COMT proteins in Brassica napus and other plants.

The tree was generated from an amino acid sequence alignment of 42BnCOMTs, 22BrCOMTs, 20BoCOMTs, 14AtCOMTs and another Arabidopsis protein with Methyltransferase_11 domain serving as outgroup species using MEGA 6.0 with the ClustalW program. The trees were constructed by ML methods (1000 bootstrap replicates). COMT proteins were clustered into five distinct groups (Groups A, B, C, D, and E). The tree is based on homologous groups showing evolutionary relationships with COMTs. Numbers next to the nodes are bootstrap values indicating frequencies of respective furcations found in 1000 replications of subset tree calculations. Only bootstrap values greater than 50% are denoted at the nodes.

Fig 5. ML phylogenetic tree for Methyltransf_2 domains in COMT proteins.

The phylogenetic tree derived by the ML method with bootstrap analysis (1000 replicates) from alignment of amino acid sequences of Methyltransferase domains predicted in COMT proteins from Arabidopsis, B. rapa, B. oleracea, B. napus, and outgroup using MEGA 6.0 program.

Fig 6. ML phylogenetic tree for intron I in Brassicaceae COMT1 genes.

The phylogenetic tree derived by the ML method with bootstrap analysis (1000 replicates) from alignment of gene sequences of intron I from three Brassicaceae species using MEGA 6.0 program.

Fig 7. Tissue specific expression characteristics of BnCOMT1s.

The tissue-specific expression levels of 10 of twelve BnCOMT1 genes (except BnCOMT1-1 and BnCOMT1-7) in eight different tissues (root, stem, leaf, bud, flower, 15D, 30D and 45D) were checked by real-time qRT-PCR.

Fig 8. Total lignin content and spatial expression characteristics of stem specific BnCOMT1s under drought stress.

H1, H2 were high-lignin content materials and L1, L2 were low materials. The grey color represents the drought treatment and blue represents the natural condition. BS, ES, LS, and MS were respectively short for budding stage, early flowering stage, late flowering stage and mature stage.