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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (10): 2546-2559.doi: 10.3724/SP.J.1006.2022.14148

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Biochemical characteristics and expression differences of three members of CCRs in ramie (Boehmeria nivea)

TANG Ying-Hong1,3,4(), LIU Fang2, CHEN Jian-Rong2,*(), MAO Kai-Quan2, LI Hui1,3, WAN Hai-Qing1   

  1. 1College of Life and Environmental Science, Hunan University of Arts and Science, Changde 415000, Hunan, China
    2School of Biological and Environmental Engineering, Changsha University, Changsha 410022, Hunan, China
    3Changde Research Centre Agricultural Biological Macromolecule, Changde 415000, Hunan, China
    4Changde Research Centre for Artificial Intelligence and Biomedicine, Changde 415000, Hunan, China
  • Received:2021-08-15 Accepted:2022-01-05 Online:2022-10-12 Published:2022-02-22
  • Contact: CHEN Jian-Rong E-mail:yinghongtang2019@163.com;z20080849@ccsu.edu.cn
  • About author:First author contact:

    ** Contributed equally to this work

  • Supported by:
    Youth Fund Project of National Natural Science Foundation(31301374);Youth Fund Project of Hunan Natural Science Foundation(2021JJ40378);Doctoral Research Start Project of Hunan University of Arts and Science(19BSQD35)

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Abstract:

Cinnamoyl-CoA reductases (CCR) is a key enzyme in lignin biosynthesis. CCR mostly exists in the form of gene family in plants. In this study, four family members of CCR (BnCCR, BnCCR-1, BnCCR-2, and BnCCR-3) were screened and cloned by ramie transcriptome information. Sequence analysis revealed that the four BnCCRs can be divided into three categories. BnCCR belonged to bona fide CCR group, BnCCR-1 was a CCR like protein, while BnCCR-2 and BnCCR-3 sequences were different from bona fide CCR in NADPH binding domain, catalytic triad, and CCR substrate binding site, and formed a new group in ramie. Transmembrane structure analysis showed that BnCCR contained two transmembrane regions, and the other three BnCCRs didn’t contain transmembrane regions. Structural analysis indicated that the secondary and tertiary structure of the four BnCCRs were different, and the BnCCR-2 homologous modeling template was different from the other three BnCCRs. The qRT-PCR analysis demonstrated that BnCCR had higher expression levels in xylem at rapid growth stage, BnCCR-2 had the highest expression levels in phloem at rapid growth stage. In vitro enzyme activity analysis further found that BnCCR had typical bona fide CCR catalytic activity and substrate adaptability, while BnCCR-2 was different from the substrate adaptability of typical CCR, and had substrate specificity for cinnamoyl CoA and sinapoyl CoA. Therefore, BnCCR was involved in lignin biosynthesis, while BnCCR-2 was a new group with structure, tissue expression and in vitro biochemical function different from typical CCR. BnCCR-2 may not participate or not only participate in lignin biosynthesis, which provides a new reference for studying the evolution of CCR protein family.

Key words: ramie (Boehmeria nivea), cinnamoyl-CoA reductase, expression difference, catalytic specificity

Table 1

Primers for gene cloning and qRT-PCR used in this study"

引物用途
Primer function		 引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 产物长度
Amplication size (bp)		 退火温度
Tm (℃)
基因克隆
Gene cloning		 BnCCR-F
BnCCR-R		 AAGCGAAAATGCCAGTAGAC
TCAACCATACAAGTTACAACCG		 1343 55.0
BnCCR-1F
BnCCR-1R		 AATGGTTTAGGATTGAGAAAGTGG
GCTGAACAACAACTGCATCACAATA		 1195 63.0
BnCCR-2F
BnCCR-2R		 CTCTCATCACAATTTACAACCC
ATTCACTCTACGTGAGCCACT		 944 52.3
BnCCR-3F
BnCCR-3R		 ATGGACTGTGAAAAACCTGTCG
GATAAATCAACCCACCACAACAT		 979 50.9
荧光定量PCR
qRT-PCR		 BnCCR-P1
BnCCR-P2		 CCCGATGTTGTGGTTGATGAGTC
ACCAAATCCACGCCTTTCTCC		 137 60.0
BnCCR-1P1
BnCCR-1P2		 AAGACGAGACGCTGGTCAGTT
GACGCCGATTCCCTCATAA		 110 58.9
BnCCR-2P1
BnCCR-2P2		 GAATTTATGGCAGATGTGGAGGTA
AACAACGGCTGTGATTGAAGAAG		 111 61.2
BnCCR-3P1
BnCCR-3P2		 TGGATTGGGAGGTAAGAGGA
TGCTAGTGCTTGCCACAACT		 197 56.8
内参基因
Reference gene		 Actin I-P1
Actin I-P2		 CGTTGAACCCTAAGGC
ATCCAGCACGATACCAG		 137

Table 2

Primers of construction of prokaryotic expression vector used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 蛋白分子量
Molecular weight (kD)		 退火温度
Tm (℃)
BnCCR-BamH I
BnCCR-Hind III		 CGCGGATCCATGCCAGTAGACAGCGCTTTGCCAG
CCCAAGCTTCTAAGATTGGATCTTGATGGAATCTTCTTGGG		 37 70.7
BnCCR-1 BamH I
BnCCR-1 Sma I		 CGCGGATCCATGATGGGGGAGAGAGTGTGTGTG
TCCCCCGGGTTAGCTTAGGGGTAGGACGCCGA		 35 66.4
BnCCR-2 BamH I
BnCCR-2 Hind III		 CGCGGATCCATGGCACCAACAGTCTGTGTAATG
CCCAAGCTTTCACTCTACGTGAGCCACTCTTTC		 33 61.6
BnCCR-3 Sac I
BnCCR-3 Sal I		 CGAGCTCATGGACTGTGAAAAACCTGTCG
ACGCGTCGACCTAGCAACAAGCAGTGCTCTCA		 34 59.2

Table S1

Unigene annotation of CCR gene"

基因 ID
Gene ID		 基因长度
Gene length (bp)		 Nr ID Nr得分
Nr score		 Nr 注释
Nr annotation
CL21616 1530 gi|316939060|gb|ADU64758.1| 610 肉桂酰辅酶A还原酶(巴西橡胶树)
Cinnamoyl-CoA reductase (Hevea brasiliensis)
CL14670 1357 gi|270315112|gb|ACZ74588.1| 352 肉桂酰辅酶A还原酶-类似蛋白1 (柳枝稷)
Cinnamoyl CoA reductase-like 1 (Panicum virgatum)
CL3762 1221 gi|229368454|gb|ACQ59093.1| 403 肉桂酰辅酶A还原酶1 (陆地棉)
Cinnamoyl-CoA reductase 1 (Gossypium hirsutum)
CL15530 1120 gi|21553548|gb|AAM62641.1| 424 肉桂酰辅酶A还原酶-类似蛋白(拟南芥)
Cinnamoyl-CoA reductase-like (Arabidopsis thaliana)

Fig. 1

Sequence alignment of BnCCRs gene Conserved sites involved in NADP(H) binding motifs are marked with triangles and the NADP+ specificity motifs are marked with full triangles. Sites involved in catalytic process are marked with full circles. Signature sites are marked with stars."

Fig. 2

Phylogenetic analysis of BnCCRs amino acid sequences Red solid dots indicate four genes of cinnamoyl CoA reductase in Boehmeria nivea: BnCCR, BnCCR-1, BnCCR-2, and BnCCR-3."

Fig. 2

Transmembrane helices analysis of BnCCRs protein based on TMHMM Red represents the transmembrane domain; the blue line represents the cytoplasmic loop; the red line represents the extracellular ocean."

Fig. 3

Deduced structural prediction of BnCCRs protein Extended strand is indicated by yellow ribbon; Alpha helix is indicated by pink ribbon; Beta Turn and Random coil is indicated by white ribbon. The R38(X)5K44 motif is indicated by red stick amino acid residues; the conserved sites S123-Y157-K161 of the short chain dehydrogenase superfamily is indicated by blue, earthy yellow, and purple stick amino acid residues, respectively. The important substrate binding site H202 of CCR is indicated by green stick amino acid residue; NAP in BnCCR is shown in wathet ball."

Fig. 4

Relative expression patterns of BnCCRs genes R: rapid growth period; M: maturity stage; L: late maturity."

Fig. 5

Purification of recombinant BnCCRs proteins M: protein 170 molecular marker; CK: control; 1: bacteria; 2: supernatant protein; 3: the target protein."

Fig. 6

Chromatogram of recombinant BnCCR catalysis with five mixed substrates"

Fig. 7

Chromatogram of recombinant BnCCR-2 catalysis with five mixed substrates"

Table 3

Kinetic analysis of recombinant BnCCR and BnCCR-2"

底物
Substrate		 Km (μmol L-1) Vmax (nkat mg-1 protein) Kcat (s-1) Kcat/Km (mmol L-1 s-1)
BnCCR BnCCR-2 BnCCR BnCCR-2 BnCCR BnCCR-2 BnCCR BnCCR-2
肉桂酰CoA
Cinnamoyl CoA		 183.77 168.01 3.82 8.06 0.15 0.29 0.83 1.71
芥子酰CoA
Sinapoyl CoA		 391.62 197.68 7.68 4.35 0.31 0.16 0.78 0.79
对香豆酰CoA
p-coumaroyl CoA		 123.90 ND 4.55 ND 0.18 ND 1.47 ND
阿魏酰CoA
Feruloyl CoA		 806.38 ND 13.01 ND 0.52 ND 0.65 ND
咖啡酰CoA
Caffeoyl CoA		 ND ND ND ND ND ND ND ND

Fig. S2

Chromatogram of recombinant BnCCR catalysis with four substrates respectively"

Fig. S3

Chromatogram of recombinant BnCCR-2 catalysis with two substrates"

Fig. S4

Michaelis-Menten curve of recombinant BnCCR and BnCCR-2 catalysis with different substrates"

[1] Zhao Q. Lignification: flexibility, biosynthesis and regulation. Trends Plant Sci, 2016, 21: 713-721.
doi: 10.1016/j.tplants.2016.04.006
[2] 郭亚玉, 许会敏, 赵媛媛, 吴鸿洋, 林金星. 植物木质化过程及其调控的研究进展. 中国科学: 生命科学, 2020, 50: 111-122.
Guo Y Y, Xu H M, Zhao Y Y, Wu H X, Lin J X. Plant lignification and its regulation. Sci Sin (Vitae), 2020, 50: 111-122 (in Chinese with English abatract).
doi: 10.2298/SOS1801111K
[3] Day A, Ruel K, Neutelings G, Cronier D, David H, Hawkins S, Chabbert B. Lignification in the flax stem: evidence for an unusual lignin in bast fibers. Planta, 2005, 222: 234-245.
doi: 10.1007/s00425-005-1537-1
[4] 袁有美, 唐映红, 刘芳, 陈建荣. 免耕种植水土保持用苎麻木质纤维主要成分分析. 中国农学通报, 2015, 31(31): 242-246.
Yuan Y M, Tang Y H, Liu F, Chen J R. Analysis of fiber main ingredients in no-till planting ramie. Chin Agric Sci Bull, 2015, 31(31): 242-246. (in Chinese with English abstract)
[5] 徐益, 张力岚, 祁建民, 张列梅, 张立武. 主要麻类作物基因组学与遗传改良: 现状与展望. 作物学报, 2021, 47: 997-1019.
doi: 10.3724/SP.J.1006.2021.04121
Xu Y, Zhang L L, Qi J M, Zhang L M, Zhang L W. Genomics and genetic improvement in main bast fiber crops: advances and perspectives. Acta Agron Sin, 2021, 47: 997-1019. (in Chinese with English abstract)
[6] Sonawane P, Vishwakarma R K, Khan B M. Biochemical characterization of recombinant cinnamoyl CoA reductase 1 (Ll- CCRH1) from Leucaena leucocephala. Int J Biol Macromol, 2013, 58: 154-159.
doi: 10.1016/j.ijbiomac.2013.03.050
[7] Barakat A, Yassin N B M, Park J S, Choi A, Herr J, Carlson J E. Comparative and phylogenomic analyses of cinnamoyl-CoA reductase and cinnamoyl-CoA-reductase-like gene family in land plants. Plant Sci, 2011, 181: 249-257.
doi: 10.1016/j.plantsci.2011.05.012
[8] Labeeuw L, Martone P T, Boucher Y, Case R J. Ancient origin of the biosynthesis of lignin precursors. Biol Direct, 2015, 10: 23.
[9] Escamilla-Trevino L L, Shen H, Uppalapati S R, Ray T, Tang Y, Hernandez T, Yin Y, Xu Y, Dixon R A. Switchgrass (Panicum virgatum) possesses a divergent family of cinnamoyl CoA reductases with distinct biochemical properties. New Phytol, 2010, 185: 143-155.
doi: 10.1111/j.1469-8137.2009.03018.x pmid: 19761442
[10] Zhou R, Jackson L, Shadle G, Nakashima J, Temple S, Chen F, Dixon R A. Distinct cinnamoyl CoA reductases involved in parallel routes to lignin in Medicago truncatula. Proc Natl Acad Sci USA, 2010, 107: 17803-17808.
doi: 10.1073/pnas.1012900107
[11] Chao N, Li N, Qi Q, Li S, Lyu T, Jiang X N, Gai Y. Characterization of the cinnamoyl-CoA reductase (CCR) gene family in Populus tomentosa reveals the enzymatic active sites and evolution of CCR. Planta, 2017, 245: 61-75.
doi: 10.1007/s00425-016-2591-6 pmid: 27580618
[12] Baltas M, Lapeyre C, Bedos-Belval F, Maturano M, Saint-Aguet P, Roussel L, Duran H, Grima-Pettenati J. Kinetic and inhibition studies of cinnamoyl-CoA reductase 1 from Arabidopsis thaliana. Plant Physiol Biochem, 2005, 43: 746-753.
doi: 10.1016/j.plaphy.2005.06.003
[13] Sonawane P, Patel K, Vishwakarma R K, Srivastava S, Singh S, Gaikwad S, Khan B M. Probing the active site of cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala. Int J Biol Macromol, 2013, 60: 33-38.
doi: 10.1016/j.ijbiomac.2013.05.005
[14] Bomati E K, Noel J P. Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase. Plant Cell, 2005, 17: 1598-1611.
pmid: 15829607
[15] Ma Q H. Characterization of a cinnamoyl-CoA reductase that is associated with stem development in wheat. J Exp Bot, 2007, 58: 2011-2021.
doi: 10.1093/jxb/erm064
[16] Lauvergeat V, Lacomme C, Lacombe E, Lasserre E, Roby D, Grima-Pettenati J. Two cinnamoyl-CoA reductase (CCR) genes from Arabidopsis thaliana are differentially expressed during development and in response to infection with pathogenic bacteria. Phytochemistry, 2001, 57: 1187-1195.
pmid: 11430991
[17] Goffner D, Campbell M M, Campargue C, Clastre M, Borderies G, Boudet A, Boudet A M. Purification and characterization of cinnamoyl-coenzyme A: NADP oxidoreductase in Eucalyptus gunnii. Plant Physiol, 1994, 106: 625-632.
pmid: 12232355
[18] Pinçon G, Maury S, Hoffmann L, Geoffroy P, Lapierre C, Pollet B, Legrand M. Repression of O-methyltransferase genes in transgenic tobacco affects lignin synthesis and plant growth. Phytochemistry, 2001, 57: 1167-1176.
pmid: 11430989
[19] 李雪平, 彭镇华, 高志民, 胡陶. 抑制COMT基因表达对转基因烟草木质素合成的影响. 分子植物育种, 2012, 10: 689-692.
Li X P, Peng Z H, Gao Z M, Hu T. The Effects of depressing expression of COMT on lignin synthesis of transgenic tobacco. Mol Plant Breed, 2012, 10: 689-692. (in Chinese with English abstract)
[20] Li Y, Kim J I, Pysh L, Chapple C. Four isoforms of Arabidopsis thaliana 4-coumarate: CoA ligase (4CL) have overlapping yet distinct roles in phenylpropanoid metabolism. Plant Physiol, 2015, 169: 2409-2421.
[21] Wang Q, Dai X R, Pang H Y, Cheng Y X, Huang X, Li H, Yan X J, Lu F C, Wei H R, Sederoff R R, Li Q Z. BEL1-like homeodomain protein BLH6a is a negative regulator of CAl5H2 in sinapyl alcohol monolignol biosynthesis in poplar. Front Plant Sci, 2021, 12: 1-14.
[22] Chen J R, Liu F, Tang Y H, Yuan Y M, Guo Q Q. Transcriptome sequencing and profiling of expressed genes in phloem and xylem of ramie (Boehmeria nivea L. Gaud). PLoS One, 2014, 10: e110623.
[23] 唐映红, 陈建荣, 刘芳, 袁有美, 郭清泉, 昌洪涛. 苎麻肉桂酰辅酶A还原酶基因cDNA序列的克隆与分析. 作物学报, 2015, 41: 1324-1332.
doi: 10.3724/SP.J.1006.2015.01324
Tang Y H, Chen J R, Liu F, Yuan Y M, Guo Q Q, Chang H T. cDNA Cloning and analysis of Cinnamoyl-CoA reductase gene from Boehmeria nivea. Acta Agron Sin, 2015, 41: 1324-1332. (in Chinese with English abstract)
doi: 10.3724/SP.J.1006.2015.01324
[24] Tang Y H, Liu F, Mao K Q, Xing H C, Chen J R, Guo Q Q. Cloning and characterization of the key 4-coumarate CoA ligase genes in Boehmeria nivea. South Afr J Bot, 2018, 116: 123-130.
doi: 10.1016/j.sajb.2018.02.398
[25] Tang Y H, Liu F, Xing H C, Mao K Q, Chen G, Guo Q Q, Chen J R. Correlation analysis of lignin accumulation and expression of key genes involved in lignin biosynthesis of ramie (Boehmeria nivea). Genes, 2019, 10: 389.
[26] Luan M B, Jian J B, Chen P, Chen J H, Chen J H, Gao Q, Gao G, Zhou J H, Chen K M, Guang X M, Chen J K, Zhang Q Q, Wang X F, Fang L, Sun Z M, Bai M Z, Fang X D, Zhao S C, Xiong H P, Yu C M, Zhu A G. Draft genome sequence of ramie, Boehmeria nivea (L.) Gaudich. Mol Ecol Resour, 2018, 18: 639-645.
doi: 10.1111/1755-0998.12766
[27] Chao N, Li S, Li N, Qi Q, Jiang W T, Jiang X N, Gai Y. Two distinct cinnamoyl-CoA reductases in Selaginella moellendorffii offer insight into the divergence of CCRs in plants. Planta, 2017, 246: 33-43.
doi: 10.1007/s00425-017-2678-8 pmid: 28321576
[28] Pan H, Zhou R, Louie G V, Mühlemann J K, Bomati E K, Bowman M E, Dudareva N, Dixon R A, Noel J P, Wang X Q. Structural studies of cinnamoyl-CoA reductase and cinnamyl-alcohol dehydrogenase, key enzymes of monolignol biosynthesis. Plant Cell, 2014, 26: 3709-3727.
doi: 10.1105/tpc.114.127399
[29] Sattler S A, Walker A M, Vermerris W, Sattler S E, Kang C. Structural and biochemical characterization of Cinnamoyl-CoA reductases. Plant Physiol, 2017, 173: 1031-1044.
doi: 10.1104/pp.16.01671 pmid: 27956488
[30] Chao N, Jiang W T, Wang X C, Jiang X N, Gai Y. Novel motif is capable of determining CCR and CCR-like proteins based on the divergence of CCRs in plants. Tree Physiol, 2019, 39: 2019-2026.
doi: 10.1093/treephys/tpz098 pmid: 31748812
[31] Jones L, Ennos A R, Turner S R. Cloning and characterization of irregular xylem4 (irx4) a severely lignin-deficient mutant of Arabidopsis. Plant J, 2001, 26: 205-216.
pmid: 11389761
[32] 宋恩慧, 蔡诚, 魏国, 高慧, 项艳. RNA干涉培育低木质素杨树. 安徽林业科技, 2012, 38(2): 58-62.
Song E H, Cai C, Wei G, Gao H, Xiang Y. Research on RNA- interference breeding of low- lignin poplar varieties. Anhui For Sci Technol, 2012, 38(2): 58-62 (in Chinese with English abatract).
[33] Wadenbäck J, von Arnold S, Egertsdotter U, Walter M H, Grima-Pettenati J, Goffner D, Gellerstedt G, Gullion T, Clapham D. Lignin biosynthesis in transgenic Norway spruce plants harboring an antisense construct for cinnamoyl CoA reductase (CCR). Transgenic Res, 2008, 17: 379-392.
pmid: 17610137
[34] Kawasaki T, Koita H, Nakatsubo T, Hasegawa K, Wakabayashi K, Takahashi H, Umemura K, Umezawa T, Shimamoto K. Cinnamoyl-CoA reductase, a key enzyme in lignin biosynthesis, is an effector of small GTPase Rac in defense signaling in rice. Proc Natl Acad Sci USA, 2006, 103: 230-235.
doi: 10.1073/pnas.0509875103
[1] TANG Ying-Hong, CHEN Jian-Rong, LIU Fang, YUAN You-Mei, GUO Qing-Quan, CHANG Hong-Tao. cDNA Cloning and Analysis of Cinnamoyl-CoA Reductase Gene from Boehmeria nivea [J]. Acta Agron Sin, 2015, 41(09): 1324-1332.
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