Abstract
Main conclusion
Two distinct cinnamoyl-coenzyme A reductases (CCRs) from Selaginella moellendorffii were evaluated, and of these, SmCCR2-1, which has both distinct sequence motifs and catalytic properties, was clustered into a new CCR subgroup.
Cinnamoyl-coenzyme A reductases (CCRs) have been reported in many land plants to have critical functions in monolignol biosynthesis. In this study, we performed a genome-wide screen and obtained two distinct SmCCRs from S. moellendorffii. Phylogenetic analysis indicated that SmCCR2 (both SmCCR2-1 and 2-2) and SmCCR3 together with PpaCCR belong to a distinct subgroup of genuine CCRs with variations in the NAD(P)H-binding motif. Enzymatic assays showed detectable activity by both SmCCR1 and SmCCR2-1 toward four hydroxycinnamoyl-CoA esters. SmCCR1, which clustered with reported CCRs from angiosperms and gymnosperms, exhibited specificity toward feruloyl-CoA, while SmCCR2-1 showed a preference for sinapoyl-CoA. Interestingly, the reaction temperature profiles for SmCCR1 and SmCCR2-1 are complementary. Homology models and molecular simulations suggest that the variations in NADPH-binding motifs, especially R(X)6K instead of R(X)5K, affect the NADP+ conformation. Notably, the signature motif NWYCY was replaced with NGYCL in SmCCR1 and with EWYCL in SmCCR2-1, while the signature residues H202 and R253, reported in a previous study, were conserved in SmCCR1 and SmCCR2-1 but varied in SmCCR-like genes. It is likely that NWYCY is not a reliable signature for CCRs in plants. The detectable activity of site-direct mutant S123T of SmCCR1 suggested that S123 which consists of catalytic triad is changeable. Possible evolution process for the emergence of two subgroups of genuine CCRs was also revealed. Altogether, these findings revise our understanding of CCRs with regard to divergence and active sites.
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Acknowledgements
This work was jointly supported by the Fundamental Research Funds for the Central Universities (BLYJ201504 to N.C.), National Natural Science Foundation (NSF 31300498 to Y.G.), High Technology Research and Development 863 Program (2011AA100203 to X. N. Jiang), (Grant Numbers J1103516, J1310005, ITR 13047) the Basic Science Basement Facility Buildup and Talent Training Program Project from National Natural Science Foundation of China (NSFC) and PCSIRT.
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425_2017_2678_MOESM3_ESM.tif
Online Resource 3 Expression profile of SmCCR and SmCCR-like genes in underground and aerial tissues. The left heatmap showed the expression profile of SmCCR and its homologs in root meristematic (MZ) and mixed elongation and differentiation (EDZ) zones with RPKM values and the right bars indicated the expression profile in aerial tissues of Selaginella moellendorffii (TIFF 774 kb)
425_2017_2678_MOESM4_ESM.tif
Online Resource 4 qRT-PCR analysis of transcript abundance of SmCCR1 and SmCCR2-1 in plants with different temperature treatment. Error bars represent STDEV of biological triplicates (TIFF 9871 kb)
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Chao, N., Li, S., Li, N. et al. Two distinct cinnamoyl-CoA reductases in Selaginella moellendorffii offer insight into the divergence of CCRs in plants. Planta 246, 33–43 (2017). https://doi.org/10.1007/s00425-017-2678-8
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DOI: https://doi.org/10.1007/s00425-017-2678-8