Manipulation of Guaiacyl and Syringyl Monomer Biosynthesis in an Arabidopsis Cinnamyl Alcohol Dehydrogenase Mutant Results in Atypical Lignin Biosynthesis and Modified Cell Wall Structure

Modifying lignin composition and structure is a key strategy to increase plant cell wall digestibility for biofuel production. Disruption of the genes encoding both cinnamyl alcohol dehydrogenases (CADs), including CADC and CADD, in Arabidopsis thaliana results in the atypical incorporation of hydroxycinnamaldehydes into lignin. Another strategy to change lignin composition is downregulation or overexpression of ferulate 5-hydroxylase (F5H), which results in lignins enriched in guaiacyl or syringyl units, respectively. Here, we combined these approaches to generate plants enriched in coniferaldehyde-derived lignin units or lignins derived primarily from sinapaldehyde. The cadc cadd and ferulic acid hydroxylase1 (fah1) cadc cadd plants are similar in growth to wild-type plants even though their lignin compositions are drastically altered. In contrast, disruption of CAD in the F5H-overexpressing background results in dwarfism. The dwarfed phenotype observed in these plants does not appear to be related to collapsed xylem, a hallmark of many other lignin-deficient dwarf mutants. cadc cadd, fah1 cadc cadd, and cadd F5H-overexpressing plants have increased enzyme-catalyzed cell wall digestibility. Given that these CAD-deficient plants have similar total lignin contents and only differ in the amounts of hydroxycinnamaldehyde monomer incorporation, these results suggest that hydroxycinnamaldehyde content is a more important determinant of digestibility than lignin content.     

Engineering Monolignol p-Coumarate Conjugates into Poplar and Arabidopsis Lignins

Lignin acylation, the decoration of hydroxyls on lignin structural units with acyl groups, is common in many plant species. Monocot lignins are decorated with p-coumarates by the polymerization of monolignol p-coumarate conjugates. The acyltransferase involved in the formation of these conjugates has been identified in a number of model monocot species, but the effect of monolignol p-coumarate conjugates on lignification and plant growth and development has not yet been examined in plants that do not inherently possess p-coumarates on their lignins. The rice (Oryza sativa) p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE gene was introduced into two eudicots, Arabidopsis (Arabidopsis thaliana) and poplar (Populus alba × grandidentata), and a series of analytical methods was used to show the incorporation of the ensuing monolignol p-coumarate conjugates into the lignin of these plants. In poplar, specifically, the addition of these conjugates did not occur at the expense of the naturally incorporated monolignol p-hydroxybenzoates. Plants expressing the p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE transgene can therefore produce monolignol p-coumarate conjugates essentially without competing with the formation of other acylated monolignols and without drastically impacting normal monolignol production.Lignification of plant cell walls prototypically involves the polymerization of the monolignols (MLs), p-coumaryl alcohol, coniferyl alcohol (CA), and sinapyl alcohol (SA), predominantly by stepwise radical coupling of each monomer to the phenolic end of the growing polymer (Sarkanen and Ludwig, 1971; Boerjan et al., 2003; Ralph et al., 2004). The contribution of various MLs to the lignins depends on plant species, cell type, plant tissue, and tissue age. Although the majority of the lignin polymer is derived from these three MLs, the lignification process has a high degree of metabolic plasticity (Boerjan et al., 2003; Ralph et al., 2004; Ralph, 2007; Vanholme et al., 2012). Of particular interest are ML conjugates in which the ester group can be acetate (Ac; Sarkanen et al., 1967; Ralph, 1996; Ralph and Lu, 1998; Del Río et al., 2007; del Río et al., 2008; Martínez et al., 2008), p-hydroxybenzoate (pBz; Venverloo, 1971; Monties and Lapierre, 1981; Landucci et al., 1992; Tomimura, 1992a, 1992b; Hibino et al., 1994; Sun et al., 1999; Kuroda et al., 2001; Lu et al., 2004, 2015; Morreel et al., 2004; Rencoret et al., 2013), p-coumarate (pCA; Monties and Lapierre, 1981; Ralph et al., 1994; Crestini and Argyropoulos, 1997; del Río et al., 2008, 2012a, 2012b; Withers et al., 2012; Rencoret et al., 2013; Petrik et al., 2014), or ferulate (FA; Grabber et al., 2008; Ralph, 2010; Wilkerson et al., 2014). In all cases, the MLs are acylated before polymerization as proven by the presence in the lignins of unique β-β coupling products that only arise when one or both of the MLs are acylated, preventing the formation of the typical resinols from internal trapping of the quinone methide intermediates by the γ-OH (Lu and Ralph, 2002, 2008; Del Río et al., 2007; Lu et al., 2015).The BAHD acyltransferase, FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT), was recently identified in Angelica sinensis and transformed into poplar (Populus alba × grandidentata), which naturally incorporates other acylated MLs, namely ML-pBz conjugates, into its lignin (Wilkerson et al., 2014). Plants that incorporate ML-FAs into their lignins have the potential to be particularly important economically, because their lignin backbones are permeated with readily cleavable ester bonds, facilitating lignin breakdown and removal under alkaline pretreatment conditions. Determining the extent to which ML-FAs are incorporated into the lignin polymer is, however, extremely difficult because of the diversity of products generated during the polymerization events, which is described in the supplemental information in Wilkerson et al., 2014.There is currently only one technique, derivatization followed by reductive cleavage (DFRC), that can release diagnostic chemical marker compounds from lignins containing ML-FAs (Lu and Ralph, 2014; Wilkerson et al., 2014). The DFRC method selectively cleaves β-ethers while leaving ester linkages intact. This technique was recently used to show that ML-FA conjugates are fully incorporated into the lignin of the FMT poplar (Wilkerson et al., 2014), but the extent of incorporation, the spatial distribution, the exact mechanism of delivery to the developing cell wall, and the efficiency of incorporation remain largely unknown.The biological role of pCA in lignin has been highly speculative. It is hypothesized that the pCA moieties may function as a radical sensitizer (Takahama and Oniki, 1996, 1997; Takahama et al., 1996; Ralph et al., 2004; Hatfield et al., 2008; Ralph, 2010). Peroxidases and/or laccases readily oxidize pCA to a radical but are poor oxidizers for SA. Free radicals of pCA readily undergo radical transfer to SA, which in turn, forms a homodimer or couples to the end of a growing polymer chain. Conjugating pCA to an ML, like SA, to form SA-pCA, the most prevalent ML-pCA conjugate in grasses, creates a compound with a built-in radical sensitizer that can participate in the polymerization event. The prevalence of these conjugates in potential biofuel crops and the impact that these ester-linked conjugates have on the lignin polymer during pretreatment and downstream fermentation processes have driven the search to find the genes and their enzymes responsible for acylating MLs in monocots (Withers et al., 2012; Marita et al., 2014; Petrik et al., 2014; Wilkerson et al., 2014).In rice (Oryza sativa), enzymes have been characterized that function specifically in the addition of pCA onto hemicelluloses (Bartley et al., 2013) or lignin (Withers et al., 2012; Petrik et al., 2014). The p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (PMT) was identified as one of many grass-specific BAHD acyltransferases produced by rice and found to coexpress with many ML biosynthetic enzymes (Withers et al., 2012). The enzyme preferentially forms a γ-ester through its specificity toward p-coumaroyl-CoA and an ML, and has kinetic efficiency with p-coumaryl alcohol > SA > CA. In most grasses, the PMT enzyme predominantly produces SA-pCA conjugates that are then incorporated into the lignin polymer (Petrik et al., 2014).To test the role of PMT during cell wall lignification, genetic manipulation of PMT genes has been performed in Brachypodium distachyon and maize (Zea mays), two model monocots. The suppression and overexpression of a BdPMT revealed the PMT to be involved only in the acylation of MLs before polymerization and not in the acylation of hemicelluloses (Petrik et al., 2014). RNA interference-mediated suppression of BdPMT resulted in decreased incorporation of ML-pCA conjugates into the cell wall without adversely affecting growth, height, or digestibility of the mature plants. Even deleterious mutations in the BdPMT gene, which resulted in a complete absence of pCA-acylating B. distachyon lignins, did not affect plant growth or development (Petrik et al., 2014). The arabinose-bound FA and pCA levels remained virtually unchanged in the PMT-misregulated plants, illustrating the specificity of the PMT enzyme for the p-coumaroyl-CoA substrate and its ML acylation. The PMT enzyme identified in maize (pCAT = ZmPMT) also displayed the highest catalytic efficiency with p-coumaroyl-CoA and SA as substrates (Marita et al., 2014). RNAi-mediated suppression of ZmPMT also resulted in decreased production of the ML conjugates. The effect on the lignin polymer when introducing PMT into plants that do not normally express a homologous enzyme is, however, unknown.pCAs, because they favor radical transfer over radical coupling, are overwhelmingly seen as free-phenolic pendant entities on the lignin polymer (Ralph et al., 1994; Ralph, 2010). As a result, the pCA itself can be completely quantified by simple saponification. The units to which the pCA is attached are, like their normal ML-derived counterparts, not fully releasable from lignin as identifiable monomers (during degradative reactions), but the pCA’s terminal location makes p-coumaroylated units more readily releasable and detectable than if they participated in lignification (as FAs do). Examining the effect of PMT and its resulting conjugates on lignification in plants that do not naturally produce such conjugates will contribute to our understanding of the role of PMT in lignification in general.In this study, we aimed to assess the ability of the model eudicot plants Arabidopsis (Arabidopsis thaliana) and poplar, neither of which naturally produces ML-pCA conjugates, to express a PMT gene and incorporate these novel conjugates into their cell wall lignins. We also investigated the effect that the introduction of PMT has on the native levels of ML-pBz conjugates in poplar lignin. Various analytical techniques were optimized and used to examine the cell walls of the transgenic plants for pCA conjugates and determine whether they were specifically incorporated into the lignin polymer in the cell wall.     

Protein–Protein and Protein–Membrane Associations in the Lignin Pathway

Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein–protein, and protein–membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.     

A Missense Mutation in the Glucosamine-6-Phosphate N-Acetyltransferase–Encoding Gene Causes Temperature-Dependent Growth Defects and Ectopic Lignin Deposition in Arabidopsis

To study the regulatory mechanisms underlying lignin biosynthesis, we isolated and characterized lignescens (lig), a previously undescribed temperature-sensitive mutant of Arabidopsis thaliana that exhibits ectopic lignin deposition and growth defects under high-temperature conditions. The lig mutation was identified as a single base transition in GNA1 encoding glucosamine-6-phosphate N-acetyltransferase (GNA), a critical enzyme of UDP-N-acetylglucosamine (UDP-GlcNAc) biosynthesis. lig harbors a glycine-to-serine substitution at residue 68 (G68S) of GNA1. Enzyme activity assays of the mutant protein (GNA1^G68S) showed its thermolability relative to the wild-type protein. The lig mutant exposed to the restrictive temperature contained a significantly smaller amount of UDP-GlcNAc than did the wild type. The growth defects and ectopic lignification of lig were suppressed by the addition of UDP-GlcNAc. Since UDP-GlcNAc is an initial sugar donor of N-glycan synthesis and impaired N-glycan synthesis is known to induce the unfolded protein response (UPR), we examined possible relationships between N-glycan synthesis, UPR, and the lig phenotype. N-glycans were reduced and LUMINAL BINDING PROTEIN3, a typical UPR gene, was expressed in lig at the restrictive temperature. Furthermore, treatment with UPR-inducing reagents phenocopied the lig mutant. Our data collectively suggest that impairment of N-glycan synthesis due to a shortage of UDP-GlcNAc leads to ectopic lignin accumulation, mostly through the UPR.     

Role of Aminoalcoholphosphotransferases 1 and 2 in Phospholipid Homeostasis in Arabidopsis

Aminoalcoholphosphotransferase (AAPT) catalyzes the synthesis of phosphatidylcholine (PC) and phosphotidylethanolamine (PE), which are the most prevalent membrane phospholipids in all eukaryotic cells. Here, we show that suppression of AAPTs results in extensive membrane phospholipid remodeling in Arabidopsis thaliana. Double knockout (KO) mutants that are hemizygous for either aapt1 or aapt2 display impaired pollen and seed development, leading to embryotic lethality of the double KO plants, whereas aapt1 or aapt2 single KO plants show no overt phenotypic alterations. The growth rate and seed yield of AAPT RNA interference (RNAi) plants are greatly reduced. Lipid profiling shows decreased total galactolipid and phospholipid content in aapt1-containing mutants, including aapt1, aapt1/aapt1 aapt2/AAPT2, aapt1/AAPT1 aapt2/aapt2, and AAPT RNAi plants. The level of PC in leaves was unchanged, whereas that of PE was reduced in all AAPT-deficient plants, except aapt2 KO. However, the acyl species of PC was altered, with increased levels of C34 species and decreased C36 species. Conversely, the levels of PE and phosphatidylinositol were decreased in C34 species. In seeds, all AAPT-deficient plants, including aapt2 KO, displayed a decrease in PE. The data show that AAPT1 and AAPT2 are essential to plant vegetative growth and reproduction and have overlapping functions but that AAPT1 contributes more than AAPT2 to PC production in vegetative tissues. The opposite changes in molecular species between PC and PE and unchanged PC level indicate the existence of additional pathways that maintain homeostatic levels of PC, which are crucial for the survival and proper development of plants.     

An Evolutionarily Conserved Signaling Mechanism Mediates Far-Red Light Responses in Land Plants

Phytochromes are plant photoreceptors important for development and adaptation to the environment. Phytochrome A (PHYA) is essential for the far-red (FR) high-irradiance responses (HIRs), which are of particular ecological relevance as they enable plants to establish under shade conditions. PHYA and HIRs have been considered unique to seed plants because the divergence of seed plants and cryptogams (e.g., ferns and mosses) preceded the evolution of PHYA. Seed plant phytochromes translocate into the nucleus and regulate gene expression. By contrast, there has been little evidence of a nuclear localization and function of cryptogam phytochromes. Here, we identified responses to FR light in cryptogams, which are highly reminiscent of PHYA signaling in seed plants. In the moss Physcomitrella patens and the fern Adiantum capillus-veneris, phytochromes accumulate in the nucleus in response to light. Although P. patens phytochromes evolved independently of PHYA, we have found that one clade of P. patens phytochromes exhibits the molecular properties of PHYA. We suggest that HIR-like responses had evolved in the last common ancestor of modern seed plants and cryptogams and that HIR signaling is more ancient than PHYA. Thus, other phytochromes in seed plants may have lost the capacity to mediate HIRs during evolution, rather than that PHYA acquired it.     

Structural Studies of Cinnamoyl-CoA Reductase and Cinnamyl-Alcohol Dehydrogenase,Key Enzymes of Monolignol Biosynthesis

The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates.     

Tricin,a Flavonoid Monomer in Monocot Lignification

Tricin was recently discovered in lignin preparations from wheat (Triticum aestivum) straw and subsequently in all monocot samples examined. To provide proof that tricin is involved in lignification and establish the mechanism by which it incorporates into the lignin polymer, the 4′-O-β-coupling products of tricin with the monolignols (p-coumaryl, coniferyl, and sinapyl alcohols) were synthesized along with the trimer that would result from its 4′-O-β-coupling with sinapyl alcohol and then coniferyl alcohol. Tricin was also found to cross couple with monolignols to form tricin-(4′-O-β)-linked dimers in biomimetic oxidations using peroxidase/hydrogen peroxide or silver (I) oxide. Nuclear magnetic resonance characterization of gel permeation chromatography-fractionated acetylated maize (Zea mays) lignin revealed that the tricin moieties are found in even the highest molecular weight fractions, ether linked to lignin units, demonstrating that tricin is indeed incorporated into the lignin polymer. These findings suggest that tricin is fully compatible with lignification reactions, is an authentic lignin monomer, and, because it can only start a lignin chain, functions as a nucleation site for lignification in monocots. This initiation role helps resolve a long-standing dilemma that monocot lignin chains do not appear to be initiated by monolignol homodehydrodimerization as they are in dicots that have similar syringyl-guaiacyl compositions. The term flavonolignin is recommended for the racemic oligomers and polymers of monolignols that start from tricin (or incorporate other flavonoids) in the cell wall, in analogy with the existing term flavonolignan that is used for the low-molecular mass compounds composed of flavonoid and lignan moieties.Lignin, a complex phenylpropanoid polymer in the plant cell wall, is predominantly deposited in the cell walls of secondary-thickened cells (Vanholme et al., 2010). It is synthesized via oxidative radical coupling reactions from three prototypical monolignols, p-coumaryl, coniferyl, and sinapyl alcohols, differentiated by their degree of methoxylation ortho to the phenolic hydroxyl group. Considered within the context of the entire polymer, the main structural features of lignin can be defined in terms of its p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, derived respectively from these three monolignols (Ralph, 2010). Several novel monomers, all deriving from the monolignol biosynthetic pathway, have been found to incorporate into lignin in wild-type and transgenic plants. For example, monolignol acetate, p-hydroxybenzoate, and p-coumarate ester conjugates have all been shown to incorporate into lignin polymers and are the source of naturally acylated lignins (Ralph et al., 2004; Lu and Ralph, 2008); lignins derived solely from caffeyl alcohol were found in the seed coats of both monocot and dicot plants (Chen et al., 2012a, 2012b); lignins derived solely from 5-hydroxyconiferyl alcohol were found in a cactus (for example, in a member of the genera Astrophytum) seed coat (Chen et al., 2012a); a Medicago truncatula transgenic deficient in cinnamyl alcohol dehydrogenase exhibited a lignin that was overwhelmingly derived from hydroxycinnamaldehydes (instead of their usual hydroxycinnamyl alcohol analogs; Zhao et al., 2013); and iso-sinapyl alcohol was implicated as a monomer in caffeic acid O-methyltransferase down-regulated switchgrass (Panicum virgatum; Tschaplinski et al., 2012). These findings imply that plants are quite flexible in being able to use a variety of monomers during lignification to form the heterogenous lignin polymer. Most recently, and as addressed more fully here, the flavonoid tricin has been implicated as a monomer in monocot lignins (del Río et al., 2012). To our knowledge, tricin is the first monomer from outside the monolignol biosynthetic pathway to be implicated in lignification.Tricin [5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one], a member of the flavonoid family, is recognized as a valuable human health compound due to its antioxidant, antiaging, anticancer, and cardioprotective potentials (Ogo et al., 2013). Tricin and its derivatives can be solvent extracted from monocot samples such as wheat (Triticum aestivum), oat bran (Avena sativa), bamboo (Leleba oldhami), sugarcane (Saccharum officinarum), and maize (Zea mays). Extracted compounds can take the form of tricin itself, 7-O-glycosylated tricin, or the flavonolignan in which tricin is 4′-O-etherified by putative coupling with coniferyl alcohol (Ju et al., 1998; Bouaziz et al., 2002; Wenzig et al., 2005; Duarte-Almeida et al., 2007; Van Hoyweghen et al., 2010; Nakano et al., 2011; Bottcher et al., 2013; Moheb et al., 2013).In 2012, we reported, to our knowledge, the first evidence that tricin was incorporated into lignin, as implicated by two previously unassigned correlation peaks at δ_C/δ_H 94.1/6.56 and 98.8/6.20 in a heteronuclear single-quantum coherence (HSQC) NMR spectrum from the whole cell wall and an isolated milled wood lignin of (unacetylated) wheat straw (del Río et al., 2012). The same evidence has now been found in the HSQC spectrum of wheat straw lignin isolated via different methods (Yelle et al., 2013; Zeng et al., 2013). Additional studies have verified the presence of tricin in lignin fractions from a variety of monocots, including bamboo (You et al., 2013), coconut coir (Cocos nucifera; Rencoret et al., 2013), maize, and others examined in our laboratories. The implication that tricin is the first phenolic from outside the monolignol biosynthetic pathway found to be integrated into the polymer has prompted further study with the aim of identifying and mechanistically delineating the role of tricin in lignin and its biosynthetic incorporation pathway.Tricin, unlike the monolignols that derive from the shikimate biosynthetic pathway (Sarkanen and Ludwig, 1971), is derived from a combination of the shikimate and acetate/malonate-derived polyketide pathways (Winkel-Shirley, 2001), as shown in Supplemental Figure S1. After p-coumaroyl-CoA is synthesized from p-coumaric acid by 4-coumarate:CoA ligase, it branches from the monolignol biosynthetic route to be transformed via chalcone synthase and chalcone isomerase into naringenin, the central precursor of most flavonoids. Naringenin is subsequently converted into apigenin by flavone synthase. Further hydroxylation at C-3′ and C-5′ followed by O-methylation furnishes tricin (Koes et al., 1994; Winkel-Shirley, 2001). The incorporation of tricin into lignin, therefore, suggests that an additional biosynthetic pathway, namely the polyketide pathway, may be associated with cell wall lignification in monocots.The revelation that tricin is incorporated into the lignin polymer was precipitated by closer study of signals found within the NMR spectra of various monocot samples. Before this discovery, tricin had not been noted in any lignin fractions, and although it is reasonable to anticipate compatibility based on its chemical structure, there is no direct and reliable evidence to date showing that tricin is able to react with monolignols through radical coupling; therefore, the efficiency and selectivity of the coupling reactions between tricin and various monolignols were also unknown. Synthetic model compounds that would facilitate the elucidation of the role of tricin within plant cell walls are desirable as aids to be used in a mechanistic study of flavonolignin generation. (We coin the term flavonolignin to describe the racemic oligomers and polymers of monolignols that start from tricin [or other flavonoids] in the cell wall, in analogy with the existing term flavonolignan that is used for the low-molecular mass compounds composed of flavonoid and lignan moieties that are presumably made in the cytoplasm [Begum et al., 2010; Niculaes et al., 2014; Dima et al., 2015]).The overall objective of this study is to demonstrate that tricin incorporates into the lignin polymer of monocots, with maize/corn stover as the representative experimental material. To this end, we have synthesized tricin and various model compounds in which tricin is conjugated to monolignols in the manner expected for the lignification process. Next, we verified whether these synthetic compounds could be made from their assumed precursors under the biomimetic radical conditions anticipated for lignification. Subsequently, NMR data generated from these synthetic and biomimetic coupling products were compared with NMR data from native maize stover lignin, including high-M_r fractions. We conclude that tricin is a monomer in monocot lignification and that, because little syringaresinol is found in maize lignin, tricin is functioning as a nucleation site that initiates lignin polymer chains.     

S-Nitrosylation of Ascorbate Peroxidase Is Part of Programmed Cell Death Signaling in Tobacco Bright Yellow-2 Cells

Nitric oxide (NO) is a small redox molecule that acts as a signal in different physiological and stress-related processes in plants. Recent evidence suggests that the biological activity of NO is also mediated by S-nitrosylation, a well-known redox-based posttranslational protein modification. Here, we show that during programmed cell death (PCD), induced by both heat shock (HS) or hydrogen peroxide (H₂O₂) in tobacco (Nicotiana tabacum) Bright Yellow-2 cells, an increase in S-nitrosylating agents occurred. NO increased in both experimentally induced PCDs, although with different intensities. In H₂O₂-treated cells, the increase in NO was lower than in cells exposed to HS. However, a simultaneous increase in S-nitrosoglutathione (GSNO), another NO source for S-nitrosylation, occurred in H₂O₂-treated cells, while a decrease in this metabolite was evident after HS. Consistently, different levels of activity and expression of GSNO reductase, the enzyme responsible for GSNO removal, were found in cells subjected to the two different PCD-inducing stimuli: low in H₂O₂-treated cells and high in the heat-shocked ones. Irrespective of the type of S-nitrosylating agent, S-nitrosylated proteins formed upon exposure to both of the PCD-inducing stimuli. Interestingly, cytosolic ascorbate peroxidase (cAPX), a key enzyme controlling H₂O₂ levels in plants, was found to be S-nitrosylated at the onset of both PCDs. In vivo and in vitro experiments showed that S-nitrosylation of cAPX was responsible for the rapid decrease in its activity. The possibility that S-nitrosylation induces cAPX ubiquitination and degradation and acts as part of the signaling pathway leading to PCD is discussed.Nitric oxide (NO) is a gaseous and diffusible redox molecule that acts as a signaling compound in both animal and plant systems (Pacher et al., 2007; Besson-Bard et al., 2008). In plants, NO has been found to play a key role in several physiological processes, such as germination, lateral root development, flowering, senescence, stomatal closure, and growth of pollen tubes (Beligni and Lamattina, 2000; Neill et al., 2002; Correa-Aragunde et al., 2004; He et al., 2004; Prado et al., 2004; Carimi et al., 2005). In addition, NO has been reported to be involved in plant responses to both biotic and abiotic stresses (Leitner et al., 2009; Siddiqui et al., 2011) and in the signaling pathways leading to programmed cell death (PCD; Delledonne et al., 1998; de Pinto et al., 2006; De Michele et al., 2009; Lin et al., 2012; Serrano et al., 2012).The cellular environment may greatly influence the chemical reactivity of NO, giving rise to different biologically active NO-derived compounds, collectively named reactive nitrogen species, which amplify and differentiate its ability to activate physiological and stress-related processes. Many of the biological properties of NO are due to its high affinity with transition metals of metalloproteins as well as its reactivity with reactive oxygen species (ROS; Hill et al., 2010). However, recent evidence suggests that protein S-nitrosylation, due to the addition of NO to reactive Cys thiols, may act as a key mechanism of NO signaling in plants (Wang et al., 2006; Astier et al., 2011). NO is also able to react with reduced glutathione (GSH), the most abundant cellular thiol, thus producing S-nitrosoglutathione (GSNO), which also acts as an endogenous trans-nitrosylating agent. GSNO is also considered as a NO store and donor and, as it is more stable than NO, acts as a long-distance NO transporter through the floematic flux (Malik et al., 2011). S-Nitrosoglutathione reductase (GSNOR), which is an enzyme conserved from bacteria to humans, has been suggested to play a role in regulating S-nitrosothiols (SNO) and the turnover of S-nitrosylated proteins in plants (Liu et al., 2001; Rusterucci et al., 2007).A number of proteins involved in metabolism, stress responses, and redox homeostasis have been identified as potential targets for S-nitrosylation in Arabidopsis (Arabidopsis thaliana; Lindermayr et al., 2005). During the hypersensitive response (HR), 16 proteins were identified to be S-nitrosylated in the seedlings of the same species (Romero-Puertas et al., 2008); in Citrus species, S-nitrosylation of about 50 proteins occurred in the NO-mediated resistance to high salinity (Tanou et al., 2009).However, while the number of candidate proteins for S-nitrosylation is increasing, the functional significance of protein S-nitrosylation has been explained only in a few cases, such as for nonsymbiotic hemoglobin (Perazzolli et al., 2004), glyceraldehyde 3-phosphate dehydrogenase (Lindermayr et al., 2005; Wawer et al., 2010), Met adenosyltransferase (Lindermayr et al., 2006), and metacaspase9 (Belenghi et al., 2007). Of particular interest are the cases in which S-nitrosylation involves enzymes controlling ROS homeostasis. For instance, it has been reported that S-nitrosylation of peroxiredoxin IIE regulates the antioxidant function of this enzyme and might contribute to the HR (Romero-Puertas et al., 2007). It has also been shown that in the immunity response, S-nitrosylation of NADPH oxidase inactivates the enzyme, thus reducing ROS production and controlling HR development (Yun et al., 2011).Recently, S-nitrosylation has also been shown to be involved in PCD of nitric oxide excess1 (noe1) rice (Oryza sativa) plants, which are mutated in the OsCATC gene coding for catalase (Lin et al., 2012). In these plants, which show PCD-like phenotypes under high-light conditions, glyceraldehyde 3-phosphate dehydrogenase and thioredoxin are S-nitrosylated. This suggests that the NO-dependent regulation of these proteins is involved in plant PCD, similar to what occurs in animal apoptosis (Sumbayev, 2003; Hara et al., 2005; Lin et al., 2012). The increase in hydrogen peroxide (H₂O₂) after exposure to high light in noe1 plants is responsible for the production of NO required for leaf cell death induction (Lin et al., 2012). There is a strict relationship between H₂O₂ and NO in PCD activation (Delledonne et al., 2001; de Pinto et al., 2002); however, the mechanism of this interplay is largely still unknown (for review, see Zaninotto et al., 2006; Zhao, 2007; Yoshioka et al., 2011). NO can induce ROS production and vice versa, and their reciprocal modulation in terms of intensity and timing seems to be crucial in determining PCD activation and in controlling HR development (Delledonne et al., 2001; Zhao, 2007; Yun et al., 2011).In previous papers, we demonstrated that heat shock (HS) at 55°C and treatment with 50 mm H₂O₂ promote PCD in tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells (Vacca et al., 2004; de Pinto et al., 2006; Locato et al., 2008). In both experimental conditions, NO production and decrease in cytosolic ascorbate peroxidase (cAPX) were observed as early events in the PCD pathway, and cAPX decrease has been suggested to contribute to determining the redox environment required for PCD (de Pinto et al., 2006; Locato et al., 2008).In this study, the production of nitrosylating agents (NO and GSNO) in the first hours of PCD induction by HS or H₂O₂ treatment in tobacco BY-2 cells and their role in PCD were studied. The possibility that S-nitrosylation could be a first step in regulating cAPX activity and turnover as part of the signaling pathway leading to PCD was also investigated.     

Elucidation of the Structure and Reaction Mechanism of Sorghum Hydroxycinnamoyltransferase and Its Structural Relationship to Other Coenzyme A-Dependent Transferases and Synthases

Hydroxycinnamoyltransferase (HCT) from sorghum (Sorghum bicolor) participates in an early step of the phenylpropanoid pathway, exchanging coenzyme A (CoA) esterified to p-coumaric acid with shikimic or quinic acid as intermediates in the biosynthesis of the monolignols coniferyl alcohol and sinapyl alcohol. In order to elucidate the mode of action of this enzyme, we have determined the crystal structures of SbHCT in its apo-form and ternary complex with shikimate and p-coumaroyl-CoA, which was converted to its product during crystal soaking. The structure revealed the roles of threonine-36, serine-38, tyrosine-40, histidine-162, arginine-371, and threonine-384 in catalysis and specificity. Based on the exact chemistry of p-coumaroyl-CoA and shikimic acid in the active site and an analysis of kinetic and thermodynamic data of the wild type and mutants, we propose a role for histidine-162 and threonine-36 in the catalytic mechanism of HCT. Considering the calorimetric data, substrate binding of SbHCT should occur sequentially, with p-coumaroyl-CoA binding prior to the acyl acceptor molecule. While some HCTs can use both shikimate and quinate as an acyl acceptor, SbHCT displays low activity toward quinate. Comparison of the structure of sorghum HCT with the HCT involved in chlorogenic acid synthesis in coffee (Coffea canephora) revealed many shared features. Taken together, these observations explain how CoA-dependent transferases with similar structural features can participate in different biochemical pathways across species.Lignin is a major structural and protective component of plant cell walls. Lignin exists as a polymer of mainly three hydroxycinnamyl alcohols and related compounds, referred to as monolignols. The most common monolignols are coniferyl, sinapyl, and p-coumaryl alcohol (Ralph et al., 2004; Vanholme et al., 2010). After polymerization, structures derived from those compounds are referred to as guaiacyl, syringyl, and p-hydroxyphenyl subunits, respectively. The specific composition of lignin subunits varies among species, tissues, and developmental stages. Gymnosperm trees produce lignin that is primarily made of guaiacyl subunits, angiosperm trees contain guaiacyl and syringyl subunits, whereas grasses contain guaiacyl and syringyl subunits with small amounts (approximately 5%) of p-hydroxyphenyl residues. This observed variation in subunit composition across species may reflect the heterogeneity in substrate specificity and kinetic parameters among various monolignol biosynthetic enzymes (Weng et al., 2008).Biosynthesis of the monolignols occurs via the phenylpropanoid pathway using Phe precursors (Vanholme et al., 2010). Phe ammonia lyase, cinnamate-4-hydroxylase, and 4-coumarate coenzyme A (CoA) ligase (4CL) generate p-coumaroyl-CoA from Phe (Vanholme et al., 2010). Grasses can bypass cinnamate-4-hydroxylase by using Tyr as a substrate for Phe ammonia lyase (Neish, 1961; Rösler et al., 1997). The hydroxycinnamoyltransferase (HCT) enzymes exchange the CoA functionality esterified to p-coumaric acid with shikimic or quinic acid to allow for the subsequent conversion of the p-coumaroyl moiety to a caffeoyl moiety by p-coumarate-3′-hydroxylase (C3′H). The hydroxycinnamoyl-CoA shikimate hydroxycinnamoyltransferases (HSTs) exhibit preference for shikimate, whereas the hydroxycinnamoyl-CoA quinate hydroxycinnamoyltransferases prefer quinate as a substrate (Sander and Petersen, 2011). Subsequent reactions ultimately lead to coniferyl and sinapyl alcohol via reduction of the γ-carbon on the propane side chain and substitution of the C3 and C5 positions of the phenol ring (Boerjan et al., 2003).Sorghum (Sorghum bicolor) is an attractive bioenergy crop with typical dry biomass yields between 20 and 25 Mg ha⁻¹ and yields as high as 40 Mg ha⁻¹ possible under optimal conditions (Venuto and Kindiger, 2008). Moreover, sorghum utilizes nitrogen-based fertilizer more efficiently than maize (Zea mays) and sugarcane (Saccharum officinarum), leading to less groundwater contamination and lower CO₂ emission (Propheter and Staggenborg, 2010; Wortmann and Regassa, 2011). Overall, sorghum has a higher sugar yield potential per land area and requires less water for growth than maize, allowing it to grow in a more diverse range of environments (Saballos, 2008). The sorghum genome sequence has been released (Paterson et al., 2009), and Targeting Induced Local Lesions in Genomes populations exist (Xin et al., 2008) in which various cell wall mutants have been identified (Sattler et al., 2012; Vermerris and Saballos, 2012).A detailed understanding of the catalytic mechanism of phenylpropanoid-related enzymes will enable the targeted modification of lignin subunit composition. The presence of lignin poses a major obstacle to the production of biofuels and chemicals from lignocellulosic biomass, because of its ability to hinder the activity of enzymes required to degrade cellulose to sugars that can be fermented for ethanol production (Yang and Wyman, 2004; Berlin et al., 2006). Genetic modification of plant cell wall composition, especially lignin content and subunit composition, has been shown to improve biomass conversion to fermentable sugars (Chen and Dixon, 2007; Vermerris et al., 2007; Jung et al., 2012). In particular, HCT silencing in Arabidopsis (Arabidopsis thaliana) causes an accumulation of p-hydroxyphenyl residues in the lignin and decreased content of guaiacyl and syringyl residues, leading to a dwarf phenotype (Li et al., 2010). Down-regulation of HCT has also been shown to result in decreased plant growth in alfalfa (Medicago sativa; Shadle et al., 2007). Concomitantly, ruminant digestibility and the yield of fermentable sugars following enzymatic saccharification increased (Chen and Dixon, 2007; Shadle et al., 2007). Reduced HCT activity may alter cell wall polymer interactions and allow better access of cellulolytic enzymes to the cellulose. Therefore, it has the potential to reduce the energy and processing costs associated with the conversion of biomass to fuels and chemicals. However fine-tuning will be necessary to limit the negative impacts on plant growth, which will require a detailed understanding of the catalytic mechanism of HCT.Given the difference in lignin subunit composition among different species and the prominence of grasses among dedicated bioenergy crops, we have focused on elucidating the crystal structure and activity of monolignol-related enzymes of sorghum, starting with the HST-like HCT. HCT belongs to the BAHD superfamily of plant-specific acyl-CoA-dependent acyltransferases (Ma et al., 2005; D’Auria, 2006). However, the BAHD superfamily has functionally and structurally diverse members that frequently possess little (as low as 10%) sequence identity among them (St-Pierre and Luca, 2000). Recent studies led to the crystal structure of the HST-like HCT from robusta coffee (Coffea canephora), an angiosperm dicot with a binding pocket elucidated by molecular docking and mutagenesis (Lallemand et al., 2012). In this report, we present the three-dimensional structures of HCT in its apo-form and ternary complex, supplemented by mutagenic studies to elucidate its reaction mechanism and structural relationship to other members in this growing functional class.     

Nicotinate O-Glucosylation Is an Evolutionarily Metabolic Trait Important for Seed Germination under Stress Conditions in Arabidopsis thaliana

The glycosylation of nicotinate (NA), a key intermediate of the NAD salvage pathway, occurs widely in land plants. However, the physiological function of NA glycosylation is not well understood in planta, and no gene encoding NA glycosyltransferase has been reported to date. NA glycosylation in Arabidopsis thaliana occurs at either the N- or the O-position of the NA molecule, and O-glucosylation appears to be unique to the Brassicaceae. Using gene-enzyme correlations focused on Family 1 glycosyltransferases (GTs; EC 2.4), we identified and characterized three Arabidopsis GTs, which are likely involved in NA glycosylation. These include one NAOGT (UGT74F2; previously identified as a salicylic acid glycosyltransferases) and two NANGTs (UGT76C4 and UGT76C5). Arabidopsis mutants of UGT74F2 accumulate higher levels of free NA, but not salicylic acid, than that of the wild type, and this inversely correlated with seed germination rates under various abiotic stresses. The germination defect of the ugt74f2-1 mutant could be fully complemented by overexpression of UGT74F2. These observations, together with comprehensive chemical analysis, suggest that NA glycosylation may function to protect plant cells from the toxicity of NA overaccumulation during seed germination. Combined with phylogenetic analysis, our results suggest that NAOGTs arose recently in the Brassicaceae family and may provide a fitness benefit. The multifunctionality of UGT74F2 in Arabidopsis is also investigated and discussed.