Shinya Kajita

Structural Characterization of Modified Lignin in Transgenic Tobacco Plants in Which the Activity of 4-Coumarate:Coenzyme A Ligase Is Depressed.

Transgenic tobacco (Nicotiana tabacum L.) plants in which the activity of 4-coumarate:coenzyme A ligase is very low contain a novel lignin in their xylem. Details of changes in hydroxycinnamic acids bound to cell walls and in the structure of the novel lignin were identified by base hydrolysis, alkaline nitrobenzene oxidation, pyrolysis-gas chromatography, and 13C-nuclear magnetic resonance analysis. In the brownish tissue of the transgenic plants, the levels of three hydroxycinnamic acids, p-coumaric, ferulic, and sinapic, which were bound to cell walls, were apparently increased as a result of down-regulation of the expression of the gene for 4-coumarate:coenzyme A ligase. Some of these hydroxycinnamic acids were linked to cell walls via ester and ether linkages. The accumulation of hydroxycinnamic acids also induced an increase in the level of condensed units in the novel lignin of the brownish tissue. Our data indicate that the behavior of some of the incorporated hydroxycinnamic acids resembles lignin monomers in the brownish tissue, and their accumulation results in dramatic changes in the biosynthesis of lignin in transgenic plants.

Down-regulation of an anionic peroxidase in transgenic aspen and its effect on lignin characteristics

It is generally accepted that peroxidases catalyze the final step in the biosynthesis of lignin. In this study, to examine how expression of prxA3a, a gene for an anionic peroxidase, might be related to lignification in plant tissues, we produced transgenic tobacco plants that harbored a gene for β-glucuronidase (GUS) fused to the prxA3a promoter. Histochemical staining for GUS activity indicated that the prxA3a promoter was active mainly in the lignifying cells of stem tissues. Further, to examine the effects of suppressing the expression of prxA3a, we transferred an antisense prxA3a gene construct into the original host, hybrid aspen (Populus sieboldii ×P. gradidentata), under the control of the original promoter of the prxA3a gene. Eleven transformed aspens were obtained and characterized, and the stable integration of the antisense construct was confirmed by PCR and Southern blotting analysis in all these lines. Assays of enzymatic activity showed that both total peroxidase activity and acidic peroxidase activity were lower in most transgenic lines than in the control plants. In addition, the reduction of peroxidase activity was associated with lower lignin content and modified lignin composition. Transgenic lines with the highest reduction of peroxidase activity displayed a higher syringyl/vanillin (S/V) ratio and a lower S+V yield, mainly because of a decreased amount of V units. Thus, our results indicate that prxA3a is involved in the lignification of xylem tissue and that the down-regulation of anionic peroxidase alters both lignin content and composition in hybrid aspen.

Multiple classes of transcription factors regulate the expression of VASCULAR-RELATED NAC-DOMAIN7, a master switch of xylem vessel differentiation.

The secondary cell walls of xylem cells, including vessel elements, provide mechanical strength and contribute to the conduction of water and minerals. VASCULAR-RELATED NAC-DOMAIN7 (VND7) is a NAC-domain transcription factor that regulates the expression of genes required for xylem vessel element formation. Transient expression assays using 68 transcription factors that are expressed during xylem vessel differentiation showed that 14 transcription factors, including VND1-VND7, are putative positive regulators of VND7 expression. Electrophoretic mobility shift assays revealed that all seven VND proteins bound to the VND7 promoter region at its SMBE/TERE motif, indicating that VND7 is a direct target of all of the VND transcription factors. Overexpression of VND1-VND5, GATA12 and ANAC075, newly identified transcription factors that function upstream of VND7, resulted in ectopic xylem vessel element formation. These data suggest that VND7 transcription is a regulatory target of multiple classes of transcription factors.

Genetic engineering of woody plants: current and future targets in a stressful environment

Abiotic stress is a major factor in limiting plant growth and productivity. Environmental degradation, such as drought and salinity stresses, will become more severe and widespread in the world. To overcome severe environmental stress, plant biotechnologies, such as genetic engineering in woody plants, need to be implemented. The adaptation of plants to environmental stress is controlled by cascades of molecular networks including cross-talk with other stress signaling mechanisms. The present review focuses on recent studies concerning genetic engineering in woody plants for the improvement of the abiotic stress responses. Furthermore, it highlights the recent advances in the understanding of molecular responses to stress. The review also summarizes the basis of a molecular mechanism for cell wall biosynthesis and the plant hormone responses to regulate tree growth and biomass in woody plants. This would facilitate better understanding of the control programs of biomass production under stressful conditions.

Agrobacterium-mediated transformation of poplar using a disarmed binary vector and the overexpression of a specific member of a family of poplar peroxidase genes in transgenic poplar cell

Abstract An efficient method was established for transformation of the poplar hybrid Populus kitakamiensis (Populus sieboldii × Populus gradidentata) using a binary disarmed strain of Agrobacterium tumefaciens LBA4404 and Ti-binary vectors. The frequency of transformation of poplar leaf segments reached as high as 60%. In transgenic poplar plants, the gene for β-glucuronidase (gus) was expressed at high levels under the control of the cauliflower mosaic virus 35S (CaMV35S) promoter. Poplars possess a number of peroxidase isozymes whose pattern of expression is tissue-specific, developmentally regulated and influenced by environmental factors. We altered the expressin of a peroxidase isozyme by introducing an identified genomic gene for a peroxidase (prxA1) under the control of the CaMV35S promoter. Transgenic poplars obtained by introducing the chimeric peroxidase gene (CaMV35S promoter-prxA1) were shown to have an increase in total peroxidase activity that was accounted for by the specific overproduction of the peroxidase isozyme (PrxA1). From this study, the anionic peroxidase isozyme encoded by the identified genomic gene, prxA1, was demonstrated to be the anionic peroxidase isozyme with a pI of 4.4 among various isozymes of poplar peroxidase. On the basis of this assignment, we characterized the tissue-specific and UV-light-inducible regulation of expression of this isozyme.

Isolation of a novel cell wall architecture mutant of rice with defective Arabidopsis COBL4 ortholog BC1 required for regulated deposition of secondary cell wall components

We recently reported that the cwa1 mutation disturbed the deposition and assembly of secondary cell wall materials in the cortical fiber of rice internodes. Genetic analysis revealed that cwa1 is allelic to bc1, which encodes glycosylphosphatidylinositol (GPI)-anchored COBRA-like protein with the highest homology to Arabidopsis COBRA-like 4 (COBL4) and maize Brittle Stalk 2 (Bk2). Our results suggested that CWA1/BC1 plays a role in assembling secondary cell wall materials at appropriate sites, enabling synthesis of highly ordered secondary cell wall structure with solid and flexible internodes in rice. The N-terminal amino acid sequence of CWA1/BC1, as well as its orthologs (COBL4, Bk2) and other BC1-like proteins in rice, shows weak similarity to a family II carbohydrate-binding module (CBM2) of several bacterial cellulases. To investigate the importance of the CBM-like sequence of CWA1/BC1 in the assembly of secondary cell wall materials, Trp residues in the CBM-like sequence, which is important for carbohydrate binding, were substituted for Val residues and introduced into the cwa1 mutant. CWA1/BC1 with the mutated sequence did not complement the abnormal secondary cell walls seen in the cwa1 mutant, indicating that the CBM-like sequence is essential for the proper function of CWA1/BC1, including assembly of secondary cell wall materials.The plant secondary cell wall is a highly ordered structure composed of various polysaccharides, phenolic components and proteins. Its coordinated regulation of a number of complex metabolic pathways and assembly has not been resolved. To understand the molecular mechanisms that regulate secondary cell wall synthesis, we isolated a novel rice mutant, cell wall architecture1 (cwa1), that exhibits an irregular thickening pattern in the secondary cell wall of sclerenchyma, as well as culm brittleness and reduced cellulose content in mature internodes. Light and transmission electron microscopy revealed that the cwa1 mutant plant has regions of local aggregation in the secondary cell walls of the cortical fibers in its internodes, showing uneven thickness. Ultraviolet microscopic observation indicated that localization of cell wall phenolic components was perturbed and that these components abundantly deposited at the aggregated cell wall regions in sclerenchyma. Therefore, regulation of deposition and assembly of secondary cell wall materials, i.e. phenolic components, appear to be disturbed by mutation of the cwa1 gene. Genetic analysis showed that cwa1 is allelic to brittle culm1 (bc1), which encodes the glycosylphosphatidylinositol-anchored COBRA-like protein specifically in plants. BC1 is known as a regulator that controls the culm mechanical strength and cellulose content in the secondary cell walls of sclerenchyma, but the precise function of BC1 has not been resolved. Our results suggest that CWA1/BC1 has an essential role in assembling cell wall constituents at their appropriate sites, thereby enabling synthesis of solid and flexible internodes in rice.

Characterization of the Third Glutathione S-Transferase Gene Involved in Enantioselective Cleavage of the β-Aryl Ether by Sphingobium sp. Strain SYK-6

The glutathione S-transferases, LigF and LigE, of Sphingobium sp. strain SYK-6 respectively play a role in cleavage of the β-aryl ether of (+)-(βS)-α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV) and (−)-(βR)-MPHPV. The ligP gene, which showed 59% similarity to ligE at the amino acid level, was isolated from SYK-6. LigP produced in Escherichia coli revealed enantioselectivity for (−)-(βR)-MPHPV, and ligE and ligP alone contributed to the degradation of (−)-(βR)-MPHPV in SYK-6.

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.

Effective Removal of Bisphenol a from Contaminated Areas by Recombinant Plant Producing Lignin Peroxidase

We have attempted to enhance the environmental decontamination functions of plants by introducing appropriate enzymatic activities from microorganisms. Lignin peroxidase is a well-known enzyme used for the degradation of some environmental pollutants. In the present study, we introduced an extracellular fungal enzyme, the lignin peroxidase of Trametes versicolor , into tobacco plants. Six transgenic plant, designated FLP-1, 2, 3, 4, 5 and 8, produced lignin peroxidase in a crude extract of the root. The FLP-1, FLP-2 and FLP-8 were able to remove 10μmol of bisphenol A g -1 dry weight from hydroponic culture. The efficiency of this removal was approximately 4-fold greater than that of control lines. Our results should stimulate efforts to develop plant-based technologies for the removal of environmental pollutants from contaminated environments.

Hybrid aspen with a transgene for fungal manganese peroxidase is a potential contributor to phytoremediation of the environment contaminated with bisphenol A

To assess the possible utility of a fungal gene for manganese-dependent peroxidase (MnP) produced by a transgenic plant in phytoremediation, we transformed hybrid aspen with a chimeric gene for MnP. Our gene construct allowed expression of the gene for MnP in plants and relatively high MnP activity was detected in the hydroponic medium in which roots of plants that expressed the transgene had been cultured. Some of our transgenic plants were able to remove bisphenol A from the medium more efficiently than wild-type plants. Our results demonstrate that, without any modification of the coding sequence, a chimeric gene for fungal MnP can be expressed in a woody plant, with secretion of active MnP from roots into the rhizosphere. Our strategy suggests new options using woody plants for phytoremediation.