Characterization of novel Brown midrib 6 mutations affecting lignin biosynthesis in sorghum

The presence of lignin reduces the quality of lignocellulosic biomass for forage materials and feedstock for biofuels. In C4 grasses,the brown midrib phenotype has been linked to mutations to genes in the monolignol biosynthesis pathway. For example,the Bmr6 gene in sorghum(Sorghum bicolor) has been previously shown to encode cinnamyl alcohol dehydrogenase(CAD),which catalyzes the final step of the monolignol biosynthesis pathway. Mutations in this gene have been shown to reduce the abundance of lignin,enhance digestibility,and improve saccharification efficiencies and ethanol yields. Nine sorghum lines harboring five different bmr6 alleles were identified in an EMS-mutagenized TILLING population. DNA sequencing of Bmr6 revealed that the majority of the mutations impacted evolutionarily conserved amino acids while three-dimensional structural modeling predicted that all of these alleles interfered with the enzyme's ability to bind with its NADPH cofactor. All of the new alleles reduced in vitro CAD activity levels and enhanced glucose yields following saccharification. Further,many of these lines were associated with higher reductions in acid detergent lignin compared to lines harboring the previously characterized bmr6-ref allele. These bmr6 lines represent new breeding tools for manipulating biomass composition to enhance forage and feedstock quality.     

Molecular characterization of allelic variation in spontaneous brown midrib mutants of sorghum (Sorghum bicolor (L.) Moench)

Modification of lignin composition and content are important to enhance the saccharification potential of lignocellulosic biomass. Brown midrib (bmr) mutants with altered lignin and enhanced glucose yields are a valuable resource for modification of the lignin biosynthetic pathway in sorghum (Sorghum bicolor (L.) Moench). Of the 38 bmr mutants reported in sorghum, some have been classified into four independent groups, namely bmr2, bmr6, bmr12 and bmr19, based on the allelic test, and a few have been characterized at the molecular level. The bmr2, bmr6 and bmr12 groups have mutations that impair 4-coumarate:coenzyme A ligase (4CL), cinnamyl alcohol dehydrogenase (CAD2) and caffeic O-methyltransferase (COMT), respectively. The molecular basis of bmr19 is unknown. In the present study, four spontaneous bmr mutants of sorghum were analyzed for allelic variation at two candidate gene loci. cDNAs of CAD2 and COMT genes were cloned and sequenced from these mutants. Sequence analysis revealed that two of these mutants, IS23789 and IS23253, share a new allele of CAD2. These mutants have a G-to-C transversion at position 3699 of the genomic sequence that leads to glycine-to-arginine (G191R) substitution in the CAD2 protein sequence. This mutation lies in the highly conserved glycine-rich motif ¹⁸⁸G(X)GGV(L)G¹⁹³ that participates in the binding of the pyrophosphate group of NADP⁺ cofactor and hence might impair the activity of CAD2. Phloroglucinol staining of midribs of these mutants also showed a dark wine-red color that is characteristic of the bmr6 group. These two mutants can be distinguished by an intron length polymorphic marker developed based on the COMT gene sequence in this study. Mutant IS23549, which has also been assigned to the bmr6 group, was found to have another new allele with alanine-to-valine (A164V) substitution in CAD2. Alanine-164 is highly conserved among MDR proteins in plants and hence may be necessary for the activity of the enzyme. In mutant IS11861, there was no mutation that led to a change in amino acid in CAD2, while a threonine-to-serine (T302S) substitution was found in COMT. This single nucleotide polymorphism (SNP) at position 2645 in the COMT gene was converted into a cleaved amplified polymorphic sequence marker that can be used for its identification. In addition, additional SNP- and/or indel-based markers were developed, which can be used for exploiting these alleles in the molecular breeding of sorghum for dedicated bioenergy feedstock.     

Identification and Characterization of Four Missense Mutations in Brown midrib 12 (Bmr12), the Caffeic O-Methyltranferase (COMT) of Sorghum

Modifying lignin content and composition are targets to improve bioenergy crops for cellulosic conversion to biofuels. In sorghum and other C4 grasses, the brown midrib mutants have been shown to reduce lignin content and alter its composition. Bmr12 encodes the sorghum caffeic O-methyltransferase, which catalyzes the penultimate step in monolignol biosynthesis. From an EMS-mutagenized TILLING population, four bmr12 mutants were isolated. DNA sequencing identified the four missense mutations in the Bmr12 coding region, which changed evolutionarily conserved amino acids Ala71Val, Pro150Leu, Gly225Asp, and Gly325Ser. The previously characterized bmr12 mutants all contain premature stop codons. These newly identified mutants, along with the previously characterized bmr12-ref, represent the first allelic series of bmr12 mutants available in the same genetic background. The impacts of these newly identified mutations on protein accumulation, enzyme activity, Klason lignin content, lignin subunit composition, and saccharification yield were determined. Gly225Asp mutant greatly reduced protein accumulation, and Pro150Leu and Gly325Ser greatly impaired enzyme activity compared to wild type (WT). All four mutants significantly reduced Klason lignin content and altered lignin composition resulting in a significantly reduced S/G ratio relative to WT, but the overall impact of these mutations was less severe than bmr12-ref. Except for Gly325Ser, which is a hypomorphic mutant, all mutants increased the saccharification yield relative to WT. These mutants represent new tools to decrease lignin content and S/G ratio, possibly leading toward the ability to tailor lignin content and composition in the bioenergy grass sorghum.     

4-Coumarate:coenzyme A ligase in black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis>) catalyses the conversion of sinapate to sinapoyl-CoA

4-Coumarate:coenzyme A (CoA) ligase (4CL, EC 6.2.1.12) in crude enzyme preparation from the developing xylem of black locust (Robinia pseudoacacia) converted sinapate to sinapoyl CoA. The sinapate-converting activity was not inhibited by other cinnamate derivatives, such as p-coumarate, caffeate or ferulate, in the mixed-substrate assay. The crude extract prepared from the developing xylem was separated by anion-exchange chromatography into three different 4CL isoforms. The isoform 4CL1 had a strong substrate preference for p-coumarate, but lacked the activity for ferulate and sinapate. On the other hand, 4CL2 and 4CL3 displayed activity toward sinapate and also possessed high activity toward caffeate as well as p-coumarate. The crude extract from the shoots exhibited a very similar substrate preference to that of the developing xylem; therefore, 4CL2 may be a major isoform in both crude enzyme preparations. These results support the hypothesis that sinapate-converting 4CL isoform is constitutively expressed in lignin-forming cells.     

Identification of 4-coumarate:coenzyme A ligase (4CL) substrate recognition domains

4-coumarate:CoA ligase (4CL), the last enzyme of the general phenylpropanoid pathway, provides precursors for the biosynthesis of a large variety of plant natural products. 4 CL catalyzes the formation of CoA thiol esters of 4-coumarate and other hydroxycinnamates in a two step reaction involving the formation of an adenylate intermediate. 4 CL shares conserved peptide motifs with diverse adenylate-forming enzymes such as firefly luciferases, non-ribosomal peptide synthetases, and acyl:CoA synthetases. Amino acid residues involved in 4 CL catalytic activities have been identified, but domains involved in determining substrate specificity remain unknown. To address this question, we took advantage of the difference in substrate usage between the Arabidopsis thaliana 4 CL isoforms At4CL1 and At4CL2. While both enzymes convert 4-coumarate, only At4CL1 is also capable of converting ferulate. Employing a domain swapping approach, we identified two adjacent domains involved in substrate recognition. Both substrate binding domain I (sbd I) and sbd II of At4CL1 alone were sufficient to confer ferulate utilization ability upon chimeric proteins otherwise consisting of At4CL2 sequences. In contrast, sbd I and sbd II of At4CL2 together were required to abolish ferulate utilization in the context of At4CL1. Sbd I corresponds to a region previously identified as the substrate binding domain of the adenylation subunit of bacterial peptide synthetases, while sbd II centers on a conserved domain of so far unknown function in adenylate-forming enzymes (GEI/LxIxG). At4CL1 and At4CL2 differ in nine amino acids within sbd I and four within sbd II, suggesting that these play roles in substrate recognition.     

A candidate-gene approach to clone the sorghum <Emphasis Type="Italic">Brown midrib </Emphasis>gene encoding caffeic acid <Emphasis Type="Italic">O</Emphasis>-methyltransferase

The brown midrib (bmr) mutants of sorghum have brown vascular tissue in the leaves and stem as a result of changes in lignin composition. The bmr mutants were generated via chemical mutagenesis with diethyl sulfate (DES) and resemble the brown midrib (bm) mutants of maize. The maize and sorghum brown midrib mutants are of particular value for the comparison of lignin biosynthesis across different, yet evolutionarily related, species. Although the sorghum brown midrib mutants were first described in 1978, none of the Brown midrib genes have been cloned. We have used a candidate-gene approach to clone the first Brown midrib gene from sorghum. Based on chemical analyses of the allelic mutants bmr12, bmr18 and bmr26, we hypothesized that these mutants had reduced activity of the lignin biosynthetic enzyme caffeic acid O-methyltransferase (COMT). After a northern analysis revealed strongly reduced expression of the COMT gene, the gene was cloned from the mutants and the corresponding wild types using PCR. In all three mutants, point mutations resulting in premature stop codons were identified: bmr12, bmr18 and bmr26 are therefore mutant alleles of the gene encoding COMT. RT-PCR indicated that all three mutants express the mutant allele, but at much lower levels relative to the wild-type controls. Molecular markers were developed for each of the three mutant alleles to facilitate the use of these mutant alleles in genetic studies and breeding programs.     

Cinnamate:coenzyme A ligase from the filamentous bacterium streptomyces coelicolor A3(2)

4-Coumarate:coenzyme A ligase (4CL) plays a key role in phenylpropanoid metabolism, providing precursors for a large variety of important plant secondary metabolites, such as lignin, flavonoids, and phytoalexins. Although 4CLs have been believed to be specific to plants, a gene encoding a 4CL-like enzyme which shows more than 40% identity in amino acid sequence to plant 4CLs was found in the genome of the gram-positive, filamentous bacterium Streptomyces coelicolor A3(2). The recombinant enzyme, produced in Escherichia coli with a histidine tag at its N-terminal end, showed distinct 4CL activity. The optimum pH and temperature of the reaction were pH 8.0 and 30 degrees C, respectively. The K(m) value for 4-coumarate and k(cat) were determined as 131 +/- 4 micro M and 0.202 +/- 0.007 s(-1), respectively. The K(m) value was comparable to those of plant 4CLs. The substrate specificity of this enzyme was, however, distinctly different from those of plant 4CLs. The enzyme efficiently converted cinnamate (K(m), 190 +/- 2 micro M; k(cat), 0.475 +/- 0.012 s(-1)), which is a very poor substrate for plant 4CLs. Furthermore, the enzyme showed only low activity toward caffeate and no activity toward ferulate, both of which are generally good substrates for plant 4CLs. The enzyme was therefore named ScCCL for S. coelicolor A3(2) cinnamate CoA ligase. To determine the amino acid residues providing the unique substrate specificity of ScCCL, eight ScCCL mutant enzymes having a mutation(s) at amino acid residues that probably line up along the substrate-binding pocket were generated. Mutant A294G used caffeate as a substrate more efficiently than ScCCL, and mutant A294G/A318G used ferulate, which ScCCL could not use as a substrate, suggesting that Ala(294) and Ala(318) are involved in substrate recognition. Furthermore, the catalytic activities of A294G and A294G/A318G toward cinnamate and 4-coumarate were greatly enhanced compared with those of the wild-type enzyme.     

Structure and evolution of 4-coumarate:coenzyme A ligase (4CL) gene families

The phenylpropanoid enzyme 4-coumarate:coenzyme A ligase (4CL) plays a key role in general phenylpropanoid metabolism. 4CL is related to a larger class of prokaryotic and eukaryotic adenylate-forming enzymes and shares several conserved peptide motifs with these enzymes. In order to better characterize the nature of 4CL gene families in poplar, parsley, and tobacco, we used degenerate primers to amplify 4CL sequences from these species. In each species additional, divergent 4CL genes were found. Complete cDNA clones for the two new poplar 4CL genes were obtained, allowing examination of their expression patterns and determination of the substrate utilization profile of a xylem-specific isoform. Phylogenetic analysis of these genes and gene fragments confirmed previous results showing that 4CL proteins fall into two evolutionarily ancient subgroups . A comparative phylogenetic analysis of enzymes in the adenylate-forming superfamily showed that 4CLs, luciferases, and acetate CoA ligases each form distinct clades within the superfamily. According to this analysis, four Arabidopsis 4CL-like genes identified from the Arabidopsis Genome Project are only distantly related to bona fide 4CLs or are more closely related to fatty acid CoA ligases, suggesting that the three Arabidopsis 4CL genes previously characterized represent the extent of the 4CL gene family in this species.     

Primary structures and catalytic properties of isoenzymes encoded by the two 4-coumarate: CoA ligase genes in parsley

We have determined the primary structures of two 4-coumarate: CoA ligase (4CL) isoenzymes in parsley (Petroselinum crispum) by sequencing near full-length cDNAs corresponding to the two 4CL genes, Pc4CL-1 and Pc4CL-2, present in this plant. Comparison of the cDNA and genomic nucleotide sequences showed that each 4CL gene is organized in five exons separated by introns of varying lengths. The positions of introns are the same in both genes and 97-99% of the corresponding nucleotide sequences are identical. The two isoenzymes, which are nearly identical in their primary structures, were separated by ion-exchange chromatography, and were found to be indistinguishable with regard to substrate specificity. Assignment to Pc4CL-1 and Pc4CL-2 was achieved by comparison with catalytically active 4CL proteins, isolated from Escherichia coli cells which had been transformed with plasmids harboring the corresponding cDNAs.     

Chromosomal localization of parsley 4-coumarate: CoA ligase genes by in situ hybridization with a complementary DNA

A near full-length cDNA (1.9 kb) was used as probe forin situ hybridization to assign one of the two highly homologous 4-coumarate: CoA ligase genes in parsley (Petroselinum crispum) to the short arm of a submetacentric chromosome. The results suggest, but do not definitely prove, that the second gene is located on a metacentric chromosome and is thus unlinked from the other.Abbreviations CHS chalcone synthase - 4CL 4-coumarate:CoA ligase - Kb kilobases (kilobasepairs)     

Identification of a 4-coumarate:CoA ligase gene family in the moss, Physcomitrella patens

Since the early evolution of land plants from primitive green algae, phenylpropanoid compounds have played an important role. In the biosynthesis of phenylpropanoids, 4-coumarate:CoA ligase (4CL; EC 6.2.1.12) has a pivotal role at the divergence point from general phenylpropanoid metabolism to several major branch pathways. Although higher plant 4CLs have been extensively studied, little information is available on the enzymes from bryophytes. In Physcomitrella patens, we have identified a 4CL gene family consisting of four members, taking advantage of the available EST sequences and a draft sequence of the P. patens genome. The encoded proteins of three of the genes display similar substrate utilization profiles with highest catalytic efficiency towards 4-coumarate. Interestingly, the efficiency with cinnamate as substrate is in the same range as with caffeate and ferulate. The deduced proteins of the four genes share sequence identities between 78% and 86%. The intron/exon structures are pair wise similar. Pp4CL2 and Pp4CL3 each consists of four exons and three introns, whereas Pp4CL1 and Pp4CL4 are characterized each by five exons and four introns. Pp4CL1, Pp4CL2 and Pp4CL3 are expressed in both gametophore and protonema tissue of P. patens, unlike Pp4CL4 whose expression could not be demonstrated under the conditions employed. Phylogenetic analysis suggests an early evolutionary divergence of Pp4CL gene family members. Using Streptomyces coelicolor cinnamate:CoA ligase (ScCCL) as an outgroup, the P. patens 4CLs are clearly separated from the spermatophyte proteins, but are intercalated between the angiosperm 4CL class I and class II. A comparison of three P. patens subspecies from diverse geographical locations shows high sequence identities for the four 4CL isoforms.     

诱导免疫哈密瓜植株的苯丙烷类代谢酶活力及新蛋白质的出现

用瓜类刺盘孢(Colletotrichum langenarium)对哈密瓜进行免疫诱导处理,诱导植株的提取物对瓜类疫霉具有抑制作用。在诱导免疫植株中,苯丙烷类代谢的3个酶(PAL,CA4H和4CL)的活性都比未经免疫诱导的植株增强,过氧化物酶及其同工酶的活性在诱导免疫植株中也明显增强。用10％聚丙烯酰胺凝胶电泳在诱导免疫植株中分离到一种新的蛋白质,这是一种酸性蛋白,等电点为pH5.0,其分子量在15.5kD左右,此蛋白不能直接抑制瓜类疫霉孢子萌发。     

Genome-wide analysis of a land plant-specific acyl:coenzyme A synthetase (ACS) gene family in Arabidopsis, poplar, rice and Physcomitrella

The plant enzyme 4-coumarate:coenzyme A ligase (4CL) is part of a family of adenylate-forming enzymes present in all organisms. Analysis of genome sequences shows the presence of '4CL-like' enzymes in plants and other organisms, but their evolutionary relationships and functions remain largely unknown. 4CL and 4CL-like genes were identified by BLAST searches in Arabidopsis, Populus, rice, Physcomitrella, Chlamydomonas and microbial genomes. Evolutionary relationships were inferred by phylogenetic analysis of aligned amino acid sequences. Expression patterns of a conserved set of Arabidopsis and poplar 4CL-like acyl-CoA synthetase (ACS) genes were assayed. The conserved ACS genes form a land plant-specific class. Angiosperm ACS genes grouped into five clades, each of which contained representatives in three fully sequenced genomes. Expression analysis revealed conserved developmental and stress-induced expression patterns of Arabidopsis and poplar genes in some clades. Evolution of plant ACS enzymes occurred early in land plants. Differential gene expansion of angiosperm ACS clades has occurred in some lineages. Evolutionary and gene expression data, combined with in vitro and limited in vivo protein function data, suggest that angiosperm ACS enzymes play conserved roles in octadecanoid and fatty acid metabolism, and play roles in organ development, for example in anthers.     

Stable and specific expression of 4-coumarate:coenzyme A ligase gene (4CL1) driven by the xylem-specific Pto4CL1 promoter in the transgenic tobacco

The ability of 4-coumarate:coenzyme A ligase promoter from Populus tomentosa (Pto4CL1p) to drive expression of the GUS reporter gene and 4-coumarate:coenzyme A ligase gene in tobacco has been studied using transgenic plants produced by Agrobacterium-mediated transformation. Intense GUS histochemical staining was detected in the xylem of stem in transgenic tobacco plants carrying the 1140 bp Pto4CL1p promoter. To further investigate the regulation function of the tissue-specific expression promoter, Pto4CL1p, a binary vector containing Pto4CL1p promoter fused with 4CL1 gene was transferred into tobacco. The activity of the 4CL1 enzyme doubled in the stems of transgenic tobacco but did not increase in the leaves. The content of lignin was increased 25% in the stem but there was no increase in the leaves of transgenic tobacco.     

The Origin and Biosynthesis of the Benzenoid Moiety of Ubiquinone (Coenzyme Q) in Arabidopsis

It is not known how plants make the benzenoid ring of ubiquinone, a vital respiratory cofactor. Here, we demonstrate that Arabidopsis thaliana uses for that purpose two separate biosynthetic branches stemming from phenylalanine and tyrosine. Gene network modeling and characterization of T-DNA mutants indicated that acyl-activating enzyme encoded by At4g19010 contributes to the biosynthesis of ubiquinone specifically from phenylalanine. CoA ligase assays verified that At4g19010 prefers para-coumarate, ferulate, and caffeate as substrates. Feeding experiments demonstrated that the at4g19010 knockout cannot use para-coumarate for ubiquinone biosynthesis and that the supply of 4-hydroxybenzoate, the side-chain shortened version of para-coumarate, can bypass this blockage. Furthermore, a trans-cinnamate 4-hydroxylase mutant, which is impaired in the conversion of trans-cinnamate into para-coumarate, displayed similar defects in ubiquinone biosynthesis to that of the at4g19010 knockout. Green fluorescent protein fusion experiments demonstrated that At4g19010 occurs in peroxisomes, resulting in an elaborate biosynthetic architecture where phenylpropanoid intermediates have to be transported from the cytosol to peroxisomes and then to mitochondria where ubiquinone is assembled. Collectively, these results demonstrate that At4g19010 activates the propyl side chain of para-coumarate for its subsequent β-oxidative shortening. Evidence is shown that the peroxisomal ABCD transporter (PXA1) plays a critical role in this branch.