Identification of Grass-specific Enzyme That AcylatesMonolignols with p-Coumarate*SReceived for publication, July 25, 2011, and in revised form, January 19, 2012 Published, JBC Papers in Press, January 21, 2012, DOI 10.1074/jbc.M111.284497
Saunia Withers, Fachuang Lu**, Hoon Kim**, Yimin Zhu, John Ralph**, and Curtis G. Wilkerson1
From the Department of Plant Biology, Great Lakes Bioenergy Research Center, and the Department of Biochemistry andMolecular Biology, Michigan State University, East Lansing, Michigan 48824 and the Department of Biochemistry, Great LakesBioenergy Research Center, and the **Wisconsin Bioenergy Initiative, University of Wisconsin, Madison, Wisconsin 53706
Background: In grasses, monolignols can be enzymatically preacylated and incorporated into lignin with unknown effectson lignin function.Results: The grass-specific acyltransferase enzyme OsPMT, expressed in Escherichia coli, acylated monolignols withp-coumarate.Conclusion:OsPMT likely encodes an enzyme responsible for acylation of monolignols in grasses.Significance: The identification of enzymes responsible for monolignol acylation allows for the assessment of lignin acylation.
Lignin is amajor component of plant cellwalls that is essentialto their function. However, the strong bonds that bind the vari-ous subunits of lignin, and its cross-linking with other plant cellwall polymers, make it one of the most important factors in therecalcitrance of plant cell walls against polysaccharide utiliza-tion. Plantsmake lignin from a variety ofmonolignols includingp-coumaryl, coniferyl, and sinapyl alcohols to produce the threeprimary lignin units: p-hydroxyphenyl, guaiacyl, and syringyl,respectively, when incorporated into the lignin polymer. Ingrasses, these monolignols can be enzymatically preacylated byp-coumarates prior to their incorporation into lignin, and thesemonolignol conjugates can also be monomer precursors oflignin. Although monolignol p-coumarate-derived units maycomprise up to 40%of the lignin in somegrass tissues, thep-cou-marate moiety from such conjugates does not enter into theradical coupling (polymerization) reactions of lignification.With a greater understanding of monolignol p-coumarate con-jugates, grass lignins could be engineered to contain fewer pen-dent p-coumarate groups andmoremonolignol conjugates thatimprove lignin cleavage. We have cloned and expressed anenzyme from rice that has p-coumarate monolignol transferaseactivity and determined its kinetic parameters.
Lignin is an important polymer in plant cell walls, providingstructural support to cells, strength to stems, a waterproof lin-ing to the vascular system, and many other functions. Plantsmake lignin from a variety of monomers, but lignin is generallyconsidered to arise primarily from the three monolignols,p-coumaryl alcohol 1H, coniferyl alcohol 1G, and sinapyl alco-hol 1S (Fig. 1), which produce p-hydroxyphenyl (H), guaiacyl(G), and syringyl (S) units, respectively, when incorporated into
the lignin polymer. These monolignols are synthesized from abranch of the phenylpropanoid pathway, where p-coumaricacid is activated by 4-coumarate: CoA ligase (4CL) to formp-coumaroyl-CoA 2a (Fig. 2) (1). The CoA esters caffeoyl-CoA2b and feruloyl-CoA 2c are derived from p-coumaroyl-CoA 2a.Each of these CoA esters can then be reduced, first by cinnam-oyl-CoA reductase and then by cinnamyl alcohol dehydrogen-ase, to the monolignols 1. Additional pathways are possible; forexample, the 4CL enzyme can activate caffeic and ferulic acids(data not shown) to form their CoA esters (2, 3). Once themonolignols are synthesized, they are transported to the cellwall, oxidized, and polymerized into lignin via radical couplingreactions (4, 5).Lignins in nature are often found to be naturally partially
acylated. It is now reasonably well established that acylatedlignins arise from preacylatedmonomers rather than from acy-lation of the polymer (68). Studies indicate that monolignolscan be enzymatically preacylated by various acids including:acetates, which occur at low levels in hardwoods but at highlevels in palms, kenaf, abaca, and sisal (6, 911); p-hydroxyben-zoates in palms and Populus species (willow, aspen, poplar)(1218); and p-coumarates in both C3 and C4 grasses (1922),prior to their incorporation into lignin. The function of suchacylation is essentially unknown, but these monolignol conju-gates can also be monomer precursors of lignin (23). Theseacylated monolignols, or monolignol conjugates, are thenincorporated into the lignin polymer by polymerization andco-polymerization with the traditional monolignols, resultingin lignins that are (partially) acylated at their -positions (24).With p-coumarate levels being as high as 18% of corn lignin(20), acetylation of kenaf lignins being as high as 60% in kenaf(9), and apparently as high as 80% in agave (25, 26), total acyla-tion (by p-coumarate and acetate) as high as 85% in abaca (25),and asmonolignolp-coumarate-derived unitsmay comprise upto 40% of the lignin in some grass tissues, it is evident that thesemonolignol conjugates can comprise a very high fraction of themonomer pool for those lignins. This acylation is found pre-dominantly on syringyl units, suggesting that sinapyl alcoholrather than coniferyl alcohol is the primary monomer used for
* This work was supported by the Department of Energy Great Lakes Bioen-ergy Research Center Office of Science Grant DE-FC02-07ER64494.
S This article contains supplemental Table I and Fig. 1.1 To whom correspondence should be addressed: Dept. of Plant Biology,
Great Lakes Bioenergy Research Center, Dept. of Biochemistry and Molec-ular Biology, Michigan State University, S124 Plant Biology, 178 Wilson Rd.,East Lansing, MI 48824. Tel.: 517-353-5554; Fax: 517-353-1926; E-mail:firstname.lastname@example.org.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 11, pp. 83478355, March 9, 2012 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
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acylation and then lignification. Approximately 10% of p-cou-marate can be found on guaiacyl units in maize (27, 28). p-Hy-droxybenzoates and acetates have been found almost exclu-sively on syringyl units (6, 7, 11, 24).p-Coumaroylation in grasses has been hypothesized as a way
to produce sinapyl alcohol and lignin syringyl unit radicals viaperoxidases that otherwise oxidize such substrates slowly (24).p-Coumarates are oxidized to the phenolic p-coumarate radicalrapidly by most peroxidases; however, the p-coumarate moietydoes not typically undergo radical coupling reactions in plantaand remains almost entirely as terminal units with an unsatu-rated side chain and a free phenolic group (20, 29). We nowknow thatwhen other phenolics are present, radical transfer (orradical exchange) from the p-coumarate radical to sinapyl alco-hol or syringyl units (producingmore stable radicals) is favored(30, 31). Lignification with sinapyl p-coumarate ester conju-gates produces an essentially normal lignin core simply withpendant p-coumarate groups; no new bonds in the backbone ofthe lignin polymer arise from the radical coupling reactions(although postcoupling rearomatization reactions of -coupled units must proceed via a different pathway than inter-nal trapping by the -OH when the -OH is acylated) (68),and the resulting lignin backbone is not significantly more eas-ily cleaved.The pathways, and the required enzymes and genes by which
monolignol conjugates are synthesized, are becoming impor-tant to delineate. First, as noted above, we have little knowledgeof the function of lignin acylation. The ability to down-regulateacylation in plants that have acylated lignins, and to possiblyintroduce acylated lignins into plants that do not, should helpshed some light on the roles and consequences of acylation. Forexample, with the gene responsible for the enzyme involvedin biosynthesizing monolignol p-coumarate conjugates, grasslignins could be engineered to contain fewer pendent p-couma-rate groups. Also, in a related project, our group has been inter-ested in introducingmonolignol ferulate conjugates, analogs ofmonolignol p-coumarates, into lignins in an attempt to intro-duce readily cleavable linkages into the backbone of the poly-
mer, facilitating lignin depolymerization in pulping or biomasspretreatments to reduce energy requirements (24, 32).Currently, we do not know which enzymes are responsible
for the biosynthesis of the monolignol conjugates. We expectcandidates for this type of activity to be part of the BAHD acyl-transferase family, which have been shown to acylate diversesubstrates (33), including the acetylation ofmonolignols for usein isoeugenol synthesis (34). We have also established thatp-coumaroylation in maize utilizes transferases that act onmonolignols and p-coumaroyl-CoA (35). Here, we report on agene from Oryza sativa that expresses a BAHD acyltransferasethat catalyzes the acylation ofmonolignols 1with p-coumarate,via p-coumaroyl-CoA 2a (Fig. 1). This gene is found only ingrasses and is co-expressed with genes involved in monolignolbiosynthesis in rice (supplemental Table I) (36). We thereforesuggest that this gene is implicated in the acylation of monoli-gnols by p-coumarate, producing the monolignol conjugates 3(p-coumaryl p-coumarate 3Ha, coniferyl p-coumarate 3Ga,and sinapyl p-coumarate 3Sa) that are involved in lignificationin grasses. Identifying the enzyme responsible for acylatinggrass lignins will help us to determine the function of this abun-dant modification.