title: Phosphorylation is an on/off switch for 5-hydroxyconiferaldehyde O-methyltransferase activity in poplar monolignol biosynthesis.
abstract: Although phosphorylation has long been known to be an important regulatory modification of proteins, no unequivocal evidence has been presented to show functional control by phosphorylation for the plant monolignol biosynthetic pathway. Here, we present the discovery of phosphorylation-mediated on/off regulation of enzyme activity for 5-hydroxyconiferaldehyde O-methyltransferase 2 (PtrAldOMT2), an enzyme central to monolignol biosynthesis for lignification in stem-differentiating xylem (SDX) of Populus trichocarpa. Phosphorylation turned off the PtrAldOMT2 activity, as demonstrated in vitro by using purified phosphorylated and unphosphorylated recombinant PtrAldOMT2. Protein extracts of P. trichocarpa SDX, which contains endogenous kinases, also phosphorylated recombinant PtrAldOMT2 and turned off the recombinant protein activity. Similarly, ATP/Mn(2+)-activated phosphorylation of SDX protein extracts reduced the endogenous SDX PtrAldOMT2 activity by ∼ 60%, and dephosphorylation fully restored the activity. Global shotgun proteomic analysis of phosphopeptide-enriched P. trichocarpa SDX protein fractions identified PtrAldOMT2 monophosphorylation at Ser(123) or Ser(125) in vivo. Phosphorylation-site mutagenesis verified the PtrAldOMT2 phosphorylation at Ser(123) or Ser(125) and confirmed the functional importance of these phosphorylation sites for O-methyltransferase activity. The PtrAldOMT2 Ser(123) phosphorylation site is conserved across 93% of AldOMTs from 46 diverse plant species, and 98% of the AldOMTs have either Ser(123) or Ser(125). PtrAldOMT2 is a homodimeric cytosolic enzyme expressed more abundantly in syringyl lignin-rich fiber cells than in guaiacyl lignin-rich vessel cells. The reversible phosphorylation of PtrAldOMT2 is likely to have an important role in regulating syringyl monolignol biosynthesis of P. trichocarpa.
authors.affiliation: State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China; Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC 27695;
authors.forename: Jack P
authors.initials: JP
authors.lastname: Wang
authors.affiliation: Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC 27695;
authors.forename: Ling
authors.initials: L
authors.lastname: Chuang
authors.affiliation: W. M. Keck FT-ICR MS Laboratory for Human Health Research, Department of Chemistry, North Carolina State University, Raleigh, NC 27695.
authors.forename: Philip L
authors.initials: PL
authors.lastname: Loziuk
authors.affiliation: Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC 27695;
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China; Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC 27695;
authors.forename: Ying-Chung
authors.initials: YC
authors.lastname: Lin
authors.affiliation: Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC 27695;
authors.forename: Rui
authors.initials: R
authors.lastname: Shi
authors.affiliation: State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China;
authors.forename: Guan-Zheng
authors.initials: GZ
authors.lastname: Qu
authors.affiliation: W. M. Keck FT-ICR MS Laboratory for Human Health Research, Department of Chemistry, North Carolina State University, Raleigh, NC 27695.
authors.forename: David C
authors.initials: DC
authors.lastname: Muddiman
authors.affiliation: Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC 27695; vchiang@ncsu.edu ron_sederoff@ncsu.edu.
authors.forename: Ronald R
authors.initials: RR
authors.lastname: Sederoff
authors.affiliation: State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China; Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC 27695; vchiang@ncsu.edu ron_sederoff@ncsu.edu.
authors.forename: Vincent L
authors.initials: VL
authors.lastname: Chiang
mesh_terms: D000171:Acrolein / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D002396:Catechols / Q000378:metabolism*; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008031:Lignin / Q000096:biosynthesis*; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D032107:Populus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D011523:Protoplasts / Q000201:enzymology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D053719:Tandem Mass Spectrometry
chemical_list: C405696:5-hydroxyconiferyl aldehyde; D002396:Catechols; D007527:Isoenzymes; D010750:Phosphoproteins; D010940:Plant Proteins; C434469:syringyl monolignol; D049452:Green Fluorescent Proteins; D000171:Acrolein; D008031:Lignin; D008780:Methyltransferases
keywords: AldOMT; COMT; lignin; phosphoproteomics; phosphoregulation