Flux modeling for monolignol biosynthesis

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The glucosyltransferase UGT72E2 is responsible for monolignol 4-O-glucoside production in Arabidopsis thaliana

The phenylpropanoid pathway in plants leads to the synthesis of a wide range of soluble secondary metabolites, many of which accumulate as glycosides. In Arabidopsis, a small cluster of three closely related genes, UGT72E1-E3, encode glycosyltransferases shown to glucosylate several phenylpropanoids in vitro, including monolignols, hydroxycinnamic acids and hydroxycinnamic aldehydes. The role of these genes in planta has now been investigated through genetically downregulating the expression of individual genes or silencing the entire cluster. Analysis of these transgenic Arabidopsis plants showed that the levels of coniferyl and sinapyl alcohol 4-O-glucosides that accumulate in light-grown roots were significantly reduced. A 50% reduction in both glucosides was observed in plants in which UGT72E2 was downregulated, whereas silencing the three genes led to a 90% reduction, suggesting some redundancy of function within the cluster. The gene encoding UGT72E2 was constitutively overexpressed in transgenic Arabidopsis to determine whether increased glucosylation of monolignols could influence flux through the soluble phenylpropanoid pathway. Elevated expression of UGT72E2 led to increased accumulation of monolignol glucosides in root tissues and also the appearance of these glucosides in leaves. In particular, coniferyl alcohol 4-O-glucoside accumulated to massive amounts (10 micromol g(-1) FW) in root tissues of these plants. Increased glucosylation of other phenylpropanoids also occurred in plants overexpressing this glycosyltransferase. Significantly changing the pattern of glycosides in the leaves also led to a pronounced change in accumulation of the hydroxycinnamic ester sinapoyl malate. The data demonstrate the plasticity of phenylpropanoid metabolism and the important role that glucosylation of secondary metabolites can play in cellular homeostasis.     

AtABCG29 is a monolignol transporter involved in lignin biosynthesis

Lignin is the defining constituent of wood and the second most abundant natural polymer on earth. Lignin is produced by the oxidative coupling of three monolignols: p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. Monolignols are synthesized via the phenylpropanoid pathway and eventually polymerized in the cell wall by peroxidases and laccases. However, the mechanism whereby monolignols are transported from the cytosol to the cell wall has remained elusive. Here we report the discovery that AtABCG29, an ATP-binding cassette transporter, acts as a p-coumaryl alcohol transporter. Expression of AtABCG29 promoter-driven reporter genes and a Citrine-AtABCG29 fusion construct revealed that AtABCG29 is targeted to the plasma membrane of the root endodermis and vascular tissue. Moreover, yeasts expressing AtABCG29 exhibited an increased tolerance to p-coumaryl alcohol by excreting this monolignol. Vesicles isolated from yeasts expressing AtABCG29 exhibited a p-coumaryl alcohol transport activity. Loss-of-function Arabidopsis mutants contained less lignin subunits and were more sensitive to p-coumaryl alcohol. Changes in secondary metabolite profiles in abcg29 underline the importance of regulating p-coumaryl alcohol levels in the cytosol. This is the first identification of a monolignol transporter, closing a crucial gap in our understanding of lignin biosynthesis, which could open new directions for lignin engineering.     

Brittle culm15 encodes a membrane-associated chitinase-like protein required for cellulose biosynthesis in rice

Plant chitinases, a class of glycosyl hydrolases, participate in various aspects of normal plant growth and development, including cell wall metabolism and disease resistance. The rice (Oryza sativa) genome encodes 37 putative chitinases and chitinase-like proteins. However, none of them has been characterized at the genetic level. In this study, we report the isolation of a brittle culm mutant, bc15, and the map-based cloning of the BC15/OsCTL1 (for chitinase-like1) gene affected in the mutant. The gene encodes the rice chitinase-like protein BC15/OsCTL1. Mutation of BC15/OsCTL1 causes reduced cellulose content and mechanical strength without obvious alterations in plant growth. Bioinformatic analyses indicated that BC15/OsCTL1 is a class II chitinase-like protein that is devoid of both an amino-terminal cysteine-rich domain and the chitinase activity motif H-E-T-T but possesses an amino-terminal transmembrane domain. Biochemical assays demonstrated that BC15/OsCTL1 is a Golgi-localized type II membrane protein that lacks classical chitinase activity. Quantitative real-time polymerase chain reaction and β-glucuronidase activity analyses indicated that BC15/OsCTL1 is ubiquitously expressed. Investigation of the global expression profile of wild-type and bc15 plants, using Illumina RNA sequencing, further suggested a possible mechanism by which BC15/OsCTL1 mediates cellulose biosynthesis and cell wall remodeling. Our findings provide genetic evidence of a role for plant chitinases in cellulose biosynthesis in rice, which appears to differ from their roles as revealed by analysis of Arabidopsis (Arabidopsis thaliana).     

Nramp5 is a major transporter responsible for manganese and cadmium uptake in rice

Paddy rice (Oryza sativa) is able to accumulate high concentrations of Mn without showing toxicity; however, the molecular mechanisms underlying Mn uptake are unknown. Here, we report that a member of the Nramp (for the Natural Resistance-Associated Macrophage Protein) family, Nramp5, is involved in Mn uptake and subsequently the accumulation of high concentrations of Mn in rice. Nramp5 was constitutively expressed in the roots and encodes a plasma membrane-localized protein. Nramp5 was polarly localized at the distal side of both exodermis and endodermis cells. Knockout of Nramp5 resulted in a significant reduction in growth and grain yield, especially when grown at low Mn concentrations. This growth reduction could be partially rescued by supplying high concentrations of Mn but not by the addition of Fe. Mineral analysis showed that the concentration of Mn and Cd in both the roots and shoots was lower in the knockout line than in wild-type rice. A short-term uptake experiment revealed that the knockout line lost the ability to take up Mn and Cd. Taken together, Nramp5 is a major transporter of Mn and Cd and is responsible for the transport of Mn and Cd from the external solution to root cells.     

Granule-bound starch synthase I is responsible for biosynthesis of extra-long unit chains of amylopectin in rice

A rice Wx gene encoding a granule-bound starch synthase I (GBSSI) was introduced into the null-mutant waxy (wx) rice, and its effect on endosperm starches was examined. The apparent amylose content was increased from undetectable amounts for the non-transgenic wx cultivars to 21.6-22.2% of starch weight for the transgenic lines. The increase was in part due to a significant amount of extra-long unit chains (ELCs) of amylopectin (7.5-8.4% of amylopectin weight), that were absent in the non-transgenic wx cultivars. Thus, actual amylose content was calculated to be 14.9-16.0% for the transgenic lines. Only slight differences were found in chain-length distribution for the chains other than ELCs, indicating that the major effect of the Wx transgene on amylopectin structure was ELC formation. ELCs isolated from debranched amylopectin exhibited structures distinct from amylose. Structures of amylose from the transgenic lines were slightly different from those of cv. Labelle (Wx(a)) in terms of a higher degree of branching and size distribution. The amylose and ELC content of starches of the transgenic lines resulted in the elevation of pasting temperature, a 50% decrease in peak viscosity, a large decrease in breakdown and an increase in setback. As yet undetermined factors other than the GBSSI activity are thought to be involved in the control of formation and/or the amount of ELCs. Structural analysis of the Wx gene suggested that the presence of a tyrosine residue at position 224 of GBSSI correlates with the formation of large amounts of ELCs in cultivars carrying Wx(a).     

C5orf42 is the major gene responsible for OFD syndrome type VI

Oral-facial-digital syndrome type VI (OFD VI) is a recessive ciliopathy defined by two diagnostic criteria: molar tooth sign (MTS) and one or more of the following: (1) tongue hamartoma (s) and/or additional frenula and/or upper lip notch; (2) mesoaxial polydactyly of one or more hands or feet; (3) hypothalamic hamartoma. Because of the MTS, OFD VI belongs to the “Joubert syndrome related disorders”. Its genetic aetiology remains largely unknown although mutations in the TMEM216 gene, responsible for Joubert (JBS2) and Meckel-Gruber (MKS2) syndromes, have been reported in two OFD VI patients. To explore the molecular cause(s) of OFD VI syndrome, we used an exome sequencing strategy in six unrelated families followed by Sanger sequencing. We identified a total of 14 novel mutations in the C5orf42 gene in 9/11 families with positive OFD VI diagnostic criteria including a severe fetal case with microphthalmia, cerebellar hypoplasia, corpus callosum agenesis, polydactyly and skeletal dysplasia. C5orf42 mutations have already been reported in Joubert syndrome confirming that OFD VI and JBS are allelic disorders, thus enhancing our knowledge of the complex, highly heterogeneous nature of ciliopathies.     

5-hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of monolignol biosynthesis in angiosperms

S-Adenosyl-L-methionine-dependent caffeate O-methyltransferase (COMT, EC 2.1.1.6) has traditionally been thought to catalyze the methylation of caffeate and 5- hydroxyferulate for the biosynthesis of syringyl monolignol, a lignin constituent of angiosperm wood that enables efficient lignin degradation for cellulose production. However, recent recognition that coniferyl aldehyde prevents 5-hydroxyferulate biosynthesis in lignifying tissue, and that the hydroxylated form of coniferyl aldehyde, 5-hydroxyconiferyl aldehyde, is an alternative COMT substrate, demands a re-evaluation of the role of COMT during monolignol biosynthesis. Based on recombinant aspen (Populus tremuloides) COMT enzyme kinetics coupled with mass spectrometry analysis, this study establishes for the first time that COMT is in fact a 5-hydroxyconiferyl aldehyde O-methyltransferase (AldOMT), and that 5-hydroxyconiferyl aldehyde is both the preferred AldOMT substrate and an inhibitor of caffeate and 5-hydroxyferulate methylation, as measured by K(m) and K(i) values. 5-Hydroxyconiferyl aldehyde also inhibited the caffeate and 5-hydroxyferulate methylation activities of xylem proteins from various angiosperm tree species. The evidence that syringyl monolignol biosynthesis is independent of caffeate and 5-hydroxyferulate methylation supports our previous discovery that coniferyl aldehyde prevents ferulate 5-hydroxylation and at the same time ensures a coniferyl aldehyde 5-hydroxylase (CAld5H)-mediated biosynthesis of 5-hydroxyconiferyl aldehyde. Together, our results provide conclusive evidence for the presence of a CAld5H/AldOMT-catalyzed coniferyl aldehyde 5-hydroxylation/methylation pathway that directs syringyl monolignol biosynthesis in angiosperms.     

C2 domain is responsible for targeting rice phosphoinositide specific phospholipase C

Phosphoinositide-specific phospholipase C (PLC) is involved in Ca²⁺ mediated signalling events that lead to altered cellular status. Using various sequence-analysis methods, we identified two conserved motifs in known PLC sequences. The identified motifs are located in the C2 domain of plant PLCs and are not found in any other protein. These motifs are specifically found in the Ca²⁺ binding loops and form adjoining beta strands. Further, we identified certain conserved residues that are highly distinct from corresponding residues of animal PLCs. The motifs reported here could be used to annotate plant-specific phospholipase C sequences. Furthermore, we demonstrated that the C2 domain alone is capable of targeting PLC to the membrane in response to a Ca²⁺ signal. We also showed that the binding event results from a change in the hydrophobicity of the C2 domain upon Ca²⁺ binding. Bioinformatic analyses revealed that all PLCs from Arabidopsis and rice lack a transmembrane domain, myristoylation and GPI-anchor protein modifications. Our bioinformatic study indicates that plant PLCs are located in the cytoplasm, the nucleus and the mitochondria. Our results suggest that there are no distinct isoforms of plant PLCs, as have been proposed to exist in the soluble and membrane associated fractions. The same isoform could potentially be present in both subcellular fractions, depending on the calcium level of the cytosol. Overall, these data suggest that the C2 domain of PLC plays a vital role in calcium signalling.     

Mathematical modeling of monolignol biosynthesis in Populus xylem

Recalcitrance of lignocellulosic biomass to sugar release is a central issue in the production of biofuel as an economically viable energy source. Among all contributing factors, variations in lignin content and its syringyl-guaiacyl monomer composition have been directly linked with the yield of fermentable sugars. While recent advances in genomics and metabolite profiling have significantly broadened our understanding of lignin biosynthesis, its regulation at the pathway level is yet poorly understood. During the past decade, computational and mathematical methods of systems biology have become effective tools for deciphering the structure and regulation of complex metabolic networks. As increasing amounts of data from various organizational levels are being published, the application of these methods to studying lignin biosynthesis appears to be very beneficial for the future development of genetically engineered crops with reduced recalcitrance. Here, we use techniques from flux balance analysis and nonlinear dynamic modeling to construct a mathematical model of monolignol biosynthesis in Populus xylem. Various types of experimental data from the literature are used to identify the statistically most significant parameters and to estimate their values through an ensemble approach. The thus generated ensemble of models yields results that are quantitatively consistent with several transgenic experiments, including two experiments not used in the model construction. Additional model results not only reveal probable substrate saturation at steps leading to the synthesis of sinapyl alcohol, but also suggest that the ratio of syringyl to guaiacyl monomers might not be affected by genetic modulations prior to the reactions involving coniferaldehyde. This latter model prediction is directly supported by data from transgenic experiments. Finally, we demonstrate the applicability of the model in metabolic engineering, where the pathway is to be optimized toward a higher yield of xylose through modification of the relative amounts of the two major monolignols. The results generated by our preliminary model of in vivo lignin biosynthesis are encouraging and demonstrate that mathematical modeling is poised to become an effective and predictive complement to traditional biotechnological and transgenic approaches, not just in microorganisms but also in plants.     

A polyketide synthase gene, ACRTS2, is responsible for biosynthesis of host-selective ACR-toxin in the rough lemon pathotype of Alternaria alternata

The rough lemon pathotype of Alternaria alternata produces host-selective ACR-toxin and causes Alternaria leaf spot disease of rough lemon (Citrus jambhiri). The structure of ACR-toxin I (MW = 496) consists of a polyketide with an α-dihydropyrone ring in a 19-carbon polyalcohol. Genes responsible for toxin production were localized to a 1.5-Mb chromosome in the genome of the rough lemon pathotype. Sequence analysis of this chromosome revealed an 8,338-bp open reading frame, ACRTS2, that was present only in the genomes of ACR-toxin-producing isolates. ACRTS2 is predicted to encode a putative polyketide synthase of 2,513 amino acids and belongs to the fungal reducing type I polyketide synthases. Typical polyketide functional domains were identified in the predicted amino acid sequence, including β-ketoacyl synthase, acyl transferase, methyl transferase, dehydratase, β-ketoreductase, and phosphopantetheine attachment site domains. Combined use of homologous recombination-mediated gene disruption and RNA silencing allowed examination of the functional role of multiple paralogs in ACR-toxin production. ACRTS2 was found to be essential for ACR-toxin production and pathogenicity of the rough lemon pathotype of A. alternata.     

Inactivation of an aminoaldehyde dehydrogenase is responsible for fragrance in rice

Rice (Oryza sativa) has two betaine aldehyde dehydrogenase homologs, BAD1 and BAD2, encoded on chromosome four and chromosome eight respectively. BAD2 is responsible for the characteristic aroma of fragrant rice. Complementary DNA clones of both BAD1 and BAD2 were isolated and expressed in E. coli. BAD2 had optimum activity at pH 10, little to no affinity towards N-acetyl-gamma-aminobutyraldehyde (NAGABald) with a Km of approximately 10 mM and moderate affinity towards gamma-guanidinobutyraldehyde (GGBald) and betaine aldehyde (bet-ald) with Km values of approximately 260 microM and 63 microM respectively. A lower Km of approximately 9 microM was observed with gamma-aminobutyraldehyde (GABald), suggesting BAD2 has a higher affinity towards this substate in vivo. The enzyme encoded on chromosome four, BAD1, had optimum activity at pH 9.5, showed little to no affinity towards bet-ald with a Km of 3 mM and had moderate affinity towards GGBald, NAGABald and GABald with Km values of approximately 545, 420 and 497 microM respectively. BAD1 had a half life roughly double that of BAD2. We discuss the implications of these findings on the pathway of fragrance generation in Basmati and Jasmine rice and the potential of rice to accumulate the osmoprotectant glycine betaine.     

Cloning of a gene responsible for the biosynthesis of glutathione in Escherichia coli B

A gene (gshI) responsible for gamma-glutamylcysteine synthetase (GSH-I) activity was cloned to construct an Escherichia coli B strain having high glutathione synthesizing activity. For this purpose, two E. coli B mutants (strains C912 and RC912) were used. C912 was deficient in GSH-I activity. RC912, a revertant of C912, had a GSH-I activity that was desensitized to feedback inhibition of reduced glutathione. To clone gshI, chromosomal DNAs of RC912 and plasmid vector pBR322 were digested with various restriction endonucleases and then ligated with T4 DNA ligase. The whole ligation mixture was used to transform C912, and the transformants were selected as tetramethylthiuramdisulfide-resistant colonies. Of about 20 resistant colonies, 2 or 3 became red when treated with nitroprusside and showed appreciably high GSH-I activities. The chimeric plasmid DNA, designated pBR322-gshI, was isolated from the strain having the highest GSH-I activity and transformed into RC912. The structure and molecular size of pBR322-gshI in RC912 were determined. The molecular size of this plasmid was 6.2 megadaltons, and the plasmid contained a 3.4-megadalton segment derived from RC912 chromosomal DNA, which included gshI gene. The GSH-I activity of RC912 cells containing pBR322-gshI was fourfold higher than that of RC912 cells without pBR322-gshI.     

The gene carD encodes the aldehyde dehydrogenase responsible for neurosporaxanthin biosynthesis in Fusarium fujikuroi

Neurosporaxanthin (β-apo-4'-carotenoic acid) biosynthesis has been studied in detail in the fungus Fusarium fujikuroi. The genes and enzymes for this biosynthetic pathway are known until the last enzymatic step, the oxidation of the aldehyde group of its precursor, β-apo-4'-carotenal. On the basis of sequence homology to Neurospora crassa YLO-1, which mediates the formation of apo-4'-lycopenoic acid from the corresponding aldehyde substrate, we cloned the carD gene of F. fujikuroi and investigated the activity of the encoded enzyme. In vitro assays performed with heterologously expressed protein showed the formation of neurosporaxanthin and other apocarotenoid acids from the corresponding apocarotenals. To confirm this function in vivo, we generated an Escherichia coli strain producing β-apo-4'-carotenal, which was converted into neurosporaxanthin upon expression of carD. Moreover, the carD function was substantiated by its targeted disruption in a F. fujikuroi carotenoid-overproducing strain, which resulted in the loss of neurosporaxanthin and the accumulation of β-apo-4'-carotenal, its derivative β-apo-4'-carotenol, and minor amounts of other carotenoids. Intermediates accumulated in the ΔcarD mutant suggest that the reactions leading to neurosporaxanthin in Neurospora and Fusarium are different in their order. In contrast to ylo-1 in N. crassa, carD mRNA content is enhanced by light, but to a lesser extent than other enzymatic genes of the F. fujikuroi carotenoid pathway. Furthermore, carD mRNA levels were higher in carotenoid-overproducing mutants, supporting a functional role for CarD in F. fujikuroi carotenogenesis. With the genetic and biochemical characterization of CarD, the whole neurosporaxanthin biosynthetic pathway of F. fujikuroi has been established.     

Identification of a gene cluster responsible for the biosynthesis of cyclic lipopeptide verlamelin

Only limited studies are available on the molecular-level biosynthesis of cyclic lipopeptides (cyclic and hybrid molecules consisting of peptide and fatty acid moieties) in filamentous fungi. Here, we identified and characterized biosynthetic genes of the cyclic lipopeptides, known as verlamelins. Only four genes, coding for non-ribosomal peptide synthetase (NRPS), fatty acid hydroxylase, thioesterase, and AMP-dependent ligase, were found to be involved in verlamelin biosynthesis by the analysis of corresponding gene knockouts. Surprisingly, no gene(s) coding for fatty acid synthase or polyketide synthase was present in the cluster, while verlamelin A/B contained a 5-hydroxytetradecanoic acid moiety. Precursor feeding experiment indicated that both fatty acid hydroxylase and thioesterase are involved to supply 5-hydroxytetradecanoic acid. The results suggested that 5-hydroxytetradecanoic acid was supplied from primary metabolism via fatty acid hydroxylase and loaded onto NRPS. Elongation of the peptide and final cyclization were accomplished by NRPS. The knowledge obtained through this study should provide new insight into fungal lipopeptide biosynthesis.     

Clade classification of monolignol biosynthesis gene family members reveals target genes to decrease lignin in Lolium perenne

In monocots, lignin content has a strong impact on the digestibility of the cell wall fraction. Engineering lignin biosynthesis requires a profound knowledge of the role of paralogues in the multigene families that constitute the monolignol biosynthesis pathway. We applied a bioinformatics approach for genome‐wide identification of candidate genes in Lolium perenne that are likely to be involved in the biosynthesis of monolignols. More specifically, we performed functional subtyping of phylogenetic clades in four multigene families: 4CL, COMT, CAD and CCR. Essential residues were considered for functional clade delineation within these families. This classification was complemented with previously published experimental evidence on gene expression, gene function and enzymatic activity in closely related crops and model species. This allowed us to assign functions to novel identified L. perenne genes, and to assess functional redundancy among paralogues. We found that two 4CL paralogues, two COMT paralogues, three CCR paralogues and one CAD gene are prime targets for genetic studies to engineer developmentally regulated lignin in this species. Based on the delineation of sequence conservation between paralogues and a first analysis of allelic diversity, we discuss possibilities to further study the roles of these paralogues in lignin biosynthesis, including expression analysis, reverse genetics and forward genetics, such as association mapping. We propose criteria to prioritise paralogues within multigene families and certain SNPs within these genes for developing genotyping assays or increasing power in association mapping studies. Although L. perenne was the target of the analyses presented here, this functional subtyping of phylogenetic clades represents a valuable tool for studies investigating monolignol biosynthesis genes in other monocot species.     

Starch metabolism in the leaf sheaths and culm of rice

The levels of starch and dextrin, free sugars, soluble protein, and enzymes involved in starch metabolism—α-amylase, β-amylase, phosphorylase, Q-enzyme, R-enzyme, and ADP-glucose starch synthetases—were assayed in the leaf sheaths and culm of the rice plant (Oryza sativa L., variety IR8) during growth.