Yukiko Tsuji

Discovery of pinoresinol reductase genes in sphingomonads

Bacterial genes for the degradation of major dilignols produced in lignifying xylem are expected to be useful tools for the structural modification of lignin in plants. For this purpose, we isolated pinZ involved in the conversion of pinoresinol from Sphingobium sp. strain SYK-6. pinZ showed 43-77% identity at amino acid level with bacterial NmrA-like proteins of unknown function, a subgroup of atypical short chain dehydrogenases/reductases, but revealed only 15-21% identity with plant pinoresinol/lariciresinol reductases. PinZ completely converted racemic pinoresinol to lariciresinol, showing a specific activity of 46±3 U/mg in the presence of NADPH at 30°C. In contrast, the activity for lariciresinol was negligible. This substrate preference is similar to a pinoresinol reductase, AtPrR1, of Arabidopsis thaliana; however, the specific activity of PinZ toward (±)-pinoresinol was significantly higher than that of AtPrR1. The role of pinZ and a pinZ ortholog of Novosphingobium aromaticivorans DSM 12444 were also characterized.

Successful expression of a novel bacterial gene for pinoresinol reductase and its effect on lignan biosynthesis in transgenic Arabidopsis thaliana

Pinoresinol reductase and pinoresinol/lariciresinol reductase play important roles in an early step of lignan biosynthesis in plants. The activities of both enzymes have also been detected in bacteria. In this study, pinZ, which was first isolated as a gene for bacterial pinoresinol reductase, was constitutively expressed in Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. Higher reductive activity toward pinoresinol was detected in the resultant transgenic plants but not in wild-type plant. Principal component analysis of data from untargeted metabolome analyses of stem, root, and leaf extracts of the wild-type and two independent transgenic lines indicate that pinZ expression caused dynamic metabolic changes in stems, but not in roots and leaves. The metabolome data also suggest that expression of pinZ influenced the metabolisms of lignan and glucosinolates but not so much of neolignans such as guaiacylglycerol-8-O-4′-feruloyl ethers. In-depth quantitative analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS) indicated that amounts of pinoresinol and its glucoside form were markedly reduced in the transgenic plant, whereas the amounts of glucoside form of secoisolariciresinol in transgenic roots, leaves, and stems increased. The detected levels of lariciresinol in the transgenic plant following β-glucosidase treatment also tended to be higher than those in the wild-type plant. Our findings indicate that overexpression of pinZ induces change in lignan compositions and has a major effect not only on lignan biosynthesis but also on biosynthesis of other primary and secondary metabolites.

Tissue and cell-specific transcriptomes in cotton reveal the subtleties of gene regulation underlying the diversity of plant secondary cell walls

BackgroundKnowledge of plant secondary cell wall (SCW) regulation and deposition is mainly based on the Arabidopsis model of a ‘typical’ lignocellulosic SCW. However, SCWs in other plants can vary from this. The SCW of mature cotton seed fibres is highly cellulosic and lacks lignification whereas xylem SCWs are lignocellulosic. We used cotton as a model to study different SCWs and the expression of the genes involved in their formation via RNA deep sequencing and chemical analysis of stem and seed fibre.ResultsTranscriptome comparisons from cotton xylem and pith as well as from a developmental series of seed fibres revealed tissue-specific and developmentally regulated expression of several NAC transcription factors some of which are likely to be important as top tier regulators of SCW formation in xylem and/or seed fibre. A so far undescribed hierarchy was identified between the top tier NAC transcription factors SND1-like and NST1/2 in cotton. Key SCW MYB transcription factors, homologs of Arabidopsis MYB46/83, were practically absent in cotton stem xylem. Lack of expression of other lignin-specific MYBs in seed fibre relative to xylem could account for the lack of lignin deposition in seed fibre. Expression of a MYB103 homolog correlated with temporal expression of SCW CesAs and cellulose synthesis in seed fibres. FLAs were highly expressed and may be important structural components of seed fibre SCWs. Finally, we made the unexpected observation that cell walls in the pith of cotton stems contained lignin and had a higher S:G ratio than in xylem, despite that tissue’s lacking many of the gene transcripts normally associated with lignin biosynthesis.ConclusionsOur study in cotton confirmed some features of the currently accepted gene regulatory cascade for ‘typical’ plant SCWs, but also revealed substantial differences, especially with key downstream NACs and MYBs. The lignocellulosic SCW of cotton xylem appears to be achieved differently from that in Arabidopsis. Pith cell walls in cotton stems are compositionally very different from that reported for other plant species, including Arabidopsis. The current definition of a ‘typical’ primary or secondary cell wall might not be applicable to all cell types in all plant species.

FORMATION AND CHARACTERIZATION OF TRANSFORMED WOODY PLANTS INHIBITING LIGNIN BIOSYNTHESIS

ABSTRACT We have tried to form a super tree with new biotechnological and genetic engineering techniques. First subject of our research is to form a lower lignin content tree by controlling the lignin biosynthesis genes using the antisense RNA method. We have succeeded in isolating and sequencing the phenylalanine ammonia-lyase (PAL), O-methyltransferase (OMT) and peroxidase (PO) genes from hybrid aspen (Populus kitakamiensis), and also isolating the promoter region of these genes. These results show that the genes of PAL, OMT and PO involved in lignification are palg2a, homtl and prxA3a, respectively. We have been able to construct the system that includes transducing a foreign gene to the hybrid aspen by use of Ti-plasmid and infecting with Agrobacterium tumefaciens by the leaf desk method. In this paper, we focus on the peroxidase gene. First, transgenic poplars were made with the prxAl of a peroxidase gene and CaMV35S promoter by the antisense RNA method. They could not grow to young plants, because the promoter can not control its expression in situ. Therefore, a new vector having the original peroxidase (prxA3a) promoter and the antisense prxA3a gene involved in lignification was constructed. The transformants with this vector can grow as well as non-transformants. The transgenic poplars have lower total peroxidase activity (10-25%) than that of the control. From the result of peroxidase isozyme analysis by isoelectric focusing a peroxidase band (pI 3.8) involved in lignification disappeared in the transgenic plants. Lignin content in transgenic plant decreases 40-80% compared with control, on the basis of the results by the potassium permanganate oxidation method. The amount of glucose determined by the alditol acetate method in transformants increases 5-10% compared with non-transformants (control). These results show it is possible to form transgenic poplars having lower lignin content and higher glucose content which indicates the cellulose content.

Analysis of Transgenic Poplar in Which the Expression of Peroxidase Gene is Suppressed

ABSTRACT Aims of our research are to produce a tree with a lower lignin content by controlling lignin biosynthesis genes using antisense RNA and to clear the relationship between lignification and the expression of peroxidase gene. The isolation and sequencing of some peroxidase genes from hybrid aspen ( Populus kitakamiensis ) and also the isolation of promoter region of these genes were achieved. From the results of northern hybridization and expression of these genes, it was concluded that the peroxidase gene involved in lignification was prxA3a . A new vector, containing the original peroxidase gene ( prxA3a ) promoter and the antisense prxA3a gene involved in lignification was constructed. Transgenic poplars formed had lower total peroxidase activity compared with that of the control. After peroxidase isozyme analysis by the isoelectric focusing, it was clear that the enzyme activity of a peroxidase band (pI 3.8) was suppressed in the transgenic poplars. The lignin content of the transgenic plants decreased 3-26% compared with that of the control, when the potassium permanganate oxidation method was used to determine the lignin content. From the microscopic observation with cross-sections after staining by color reactions, it was cleared that the peroxidase activity and the staining of maule and phloroglucinol color reactions were disappeared in the xylem region near cambial zone in transgenic poplar samples. The fact shows that the transformants in which the expression of the peroxidase gene was suppressed by antisense RNA had peroxidase activity decreased and lignin formation suppressed in early stage of secondary xylem formation, resulting that transgenic poplars with a lowered lignin content were formed.

Introduction of alkali-labile units into lignin in transgenic plants by genetic engineering

Background Lignin is one of major components of plant secondary cell wall. In plant cell wall, it is synthesized via radical coupling of precursors such as p-coumaryl, coniferyl, and sinapyl alcohols. In early stage of the lignification, 8-O-4’, 8-8’ and 8-5’ dimers are thought to be synthesized mainly from the precursors in the wall. A gramnegative bacterium, Shingobium sp. strain SYK-6 (hereafter refer to as SYK-6) is able to catabolize a wide variety of phenolic compounds including the lignin precursors by its unique enzymatic system. One of catabolic enzymes, LigD, catalyzes oxidation at alpha (benzyl) position of 8-O-4’ dimers and forms carbonyl group at the position (Figure 1). This oxidation is the first step of catabolic pathway of 8-O-4’ dimers in SYK-6. When we express LigD polypeptide in the cell wall of transgenic plants, the oxidative dimers will be expected to be generated and then incorporated into lignin polymer. In some past studies, it has been shown that the presence of carbonyl groups at the alpha position of aryl propane units in lignin greatly speeds up the rate of cleavage of beta-aryl ether linkages during kraft pulping condition [1,2]. In order to contribute to efficient and sustainable production of kraft pulp and the other biomass-derived products such as bioethanol, we introduced the ligD gene into Arabidopsis and hybrid aspen and tried to generate transgenic plants whose lignin can be easy to remove from hollocellulose fraction under alkaline conditions. Method Because of codon usage is significantly different between genes in plants and SYK-6, we chemically synthesized open reading frame (ORF) of the ligD gene for improving its expression in the transgenic plants. After addition of nucleotide sequence for apoplast-targeting signal peptide to the synthesized ligD ORF, it was introduced into Arabidopsis thaliana, tobacco BY-2 and hybrid aspen under the control of cauliflower mosaic virus 35S promoter. LigD expression in the transgenic plants was monitored by Western blot analysis and enzymatic activity with crude extract prepared from each transgenic line. Preliminary analysis of lignin structure by 2D-NMR and nitrobenzene oxidation was also performed.

RNAi-suppression of barley caffeic acid O-methyltransferase modifies lignin despite redundancy in the gene family

Summary Caffeic acid O‐methyltransferase (COMT), the lignin biosynthesis gene modified in many brown‐midrib high‐digestibility mutants of maize and sorghum, was targeted for downregulation in the small grain temperate cereal, barley (Hordeum vulgare), to improve straw properties. Phylogenetic and expression analyses identified the barley COMT orthologue(s) expressed in stems, defining a larger gene family than in brachypodium or rice with three COMT genes expressed in lignifying tissues. RNAi significantly reduced stem COMT protein and enzyme activity, and modestly reduced stem lignin content while dramatically changing lignin structure. Lignin syringyl‐to‐guaiacyl ratio was reduced by ~50%, the 5‐hydroxyguaiacyl (5‐OH‐G) unit incorporated into lignin at 10‐–15‐fold higher levels than normal, and the amount of p‐coumaric acid ester‐linked to cell walls was reduced by ~50%. No brown‐midrib phenotype was observed in any RNAi line despite significant COMT suppression and altered lignin. The novel COMT gene family structure in barley highlights the dynamic nature of grass genomes. Redundancy in barley COMTs may explain the absence of brown‐midrib mutants in barley and wheat. The barley COMT RNAi lines nevertheless have the potential to be exploited for bioenergy applications and as animal feed.