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1.
Molecules ; 25(5)2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-32150921

RESUMO

A large-scale glycol lignin (GL) production process (50 kg wood meal per batch) based on acid-catalyzed polyethylene glycol (PEG) solvolysis of Japanese cedar (JC) was developed at the Forestry and Forest Products Research Institute (FFPRI), Tsukuba, Japan. JC wood meal with various particle size distributions (JC-S < JC-M < JC-L) (average meal size, JC-S (0.4 mm) < JC-M (0.8 mm) < JC-L (1.6 mm)) and liquid PEG with various molecular masses are used as starting materials to produce PEG-modified lignin derivatives, namely, GLs, with various physicochemical and thermal properties. Because GLs are considered a potential feedstock for industrial applications, the effect of heat treatment on GL properties is an important issue for GL-based material production. In this study, GLs obtained from PEG400 solvolysis of JC-S, JC-M, and JC-L were subjected to heating in a constant-temperature drying oven at temperatures ranging from 100 to 220 °C for 1 h. All heat-treated GL series were thermally stable, as determined from the Klason lignin content, TMA, and TGA analyses. SEC analysis suggests the possibility of condensation among lignin fragments during heat treatment. ATR-FTIR spectroscopy, thioacidolysis, and 2D HSQC NMR demonstrated that a structural rearrangement occurs in the heat-treated GL400 samples, in which the content of α-PEG-ß-O-4 linkages decreases along with the proportional enrichments of ß-5 and ß-ß linkages, particularly at treatment temperatures above 160 °C.

2.
New Phytol ; 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31909485

RESUMO

Lignin is a major component of cell wall biomass and decisively affects biomass utilisation. Engineering of lignin biosynthesis is extensively studied, while lignin modification often causes growth defects. We developed a strategy for cell-type-specific modification of lignin to achieve improvements in cell wall property without growth penalty. We targeted a lignin-related transcription factor, LTF1, for modification of lignin biosynthesis. LTF1 can be engineered to a nonphosphorylation form which is introduced into Populus under the control of either a vessel-specific or fibre-specific promoter. The transgenics with lignin suppression in vessels showed severe dwarfism and thin-walled vessels, while the transgenics with lignin suppression in fibres displayed vigorous growth with normal vessels under phytotron, glasshouse and field conditions. In-depth lignin structural analyses revealed that such cell-type-specific downregulation of lignin biosynthesis led to the alteration of overall lignin composition in xylem tissues reflecting the population of distinctive lignin polymers produced in vessel and fibre cells. This study demonstrates that fibre-specific suppression of lignin biosynthesis resulted in the improvement of wood biomass quality and saccharification efficiency and presents an effective strategy to precisely regulate lignin biosynthesis with desired growth performance.

3.
Plant Biotechnol (Tokyo) ; 36(2): 113-118, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31768112

RESUMO

Podophyllotoxin is a starting material of the semisynthetic anticancer medicines etoposide, teniposide, and etopophos. The major plant source of podophyllotoxin is rhizomes of Podophyllum hexandrum, which is a Himalayan endangered species; therefore, alternative sources of podophyllotoxin or bioproduction systems have been pursued to avoid exploiting this limited natural resource. In this paper, we report de novo transcriptome analysis of Thujopsis dolablata var. hondae, which accumulates the podophyllotoxin derivatives (deoxypodophyllotoxin and ß-peltatin A methyl ether) in its needles. We analyzed transcriptomes of the T. dolablata var. hondae young needles to obtain the sequences that putatively encode O-methyltransferases, cytochrome P450s, and a 2-oxoglutarate dependent dioxygenase because these protein families are responsible for podophyllotoxin-related compound formation in P. hexandrum. The resulting transcriptomes contained considerable numbers of coding sequences classified into the three protein families. Our results are a genetic basis for identifying genes involved in the biosynthesis of podophyllotoxin and related compounds and also for future metabolic engineering of podophyllotoxin in heterologous hosts.

4.
Sci Rep ; 9(1): 17153, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31748605

RESUMO

Lignin is a complex phenylpropanoid polymer deposited in plant cell walls. Lignin has long been recognized as an important limiting factor for the polysaccharide-oriented biomass utilizations. To mitigate lignin-associated biomass recalcitrance, numerous mutants and transgenic plants that produce lignocellulose with reduced lignin contents and/or lignins with altered chemical structures have been produced and characterised. However, it is not fully understood how altered lignin chemistry affects the supramolecular structure of lignocellulose, and consequently, its utilization properties. Herein, we conducted comprehensive chemical and supramolecular structural analyses of lignocellulose produced by a rice cad2 mutant deficient in CINNAMYL ALCOHOL DEHYDROGENASE (CAD), which encodes a key enzyme in lignin biosynthesis. By using a solution-state two-dimensional NMR approach and complementary chemical methods, we elucidated the structural details of the altered lignins enriched with unusual hydroxycinnamaldehyde-derived substructures produced by the cad2 mutant. In parallel, polysaccharide assembly and the molecular mobility of lignocellulose were investigated by solid-state 13C MAS NMR, nuclear magnetic relaxation, X-ray diffraction, and Simon's staining analyses. Possible links between CAD-associated lignin modifications (in terms of total content and chemical structures) and changes to the lignocellulose supramolecular structure are discussed in the context of the improved biomass saccharification efficiency of the cad2 rice mutant.

5.
Sci Rep ; 9(1): 11597, 2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31406182

RESUMO

Lignin is a phenylpropanoid polymer produced in the secondary cell walls of vascular plants. Although most eudicot and gymnosperm species generate lignins solely via polymerization of p-hydroxycinnamyl alcohols (monolignols), grasses additionally use a flavone, tricin, as a natural lignin monomer to generate tricin-incorporated lignin polymers in cell walls. We previously found that disruption of a rice 5-HYDROXYCONIFERALDEHYDE O-METHYLTRANSFERASE (OsCAldOMT1) reduced extractable tricin-type metabolites in rice vegetative tissues. This same enzyme has also been implicated in the biosynthesis of sinapyl alcohol, a monolignol that constitutes syringyl lignin polymer units. Here, we further demonstrate through in-depth cell wall structural analyses that OsCAldOMT1-deficient rice plants produce altered lignins largely depleted in both syringyl and tricin units. We also show that recombinant OsCAldOMT1 displayed comparable substrate specificities towards both 5-hydroxyconiferaldehyde and selgin intermediates in the monolignol and tricin biosynthetic pathways, respectively. These data establish OsCAldOMT1 as a bifunctional O-methyltransferase predominantly involved in the two parallel metabolic pathways both dedicated to the biosynthesis of tricin-lignins in rice cell walls. Given that cell wall digestibility was greatly enhanced in the OsCAldOMT1-deficient rice plants, genetic manipulation of CAldOMTs conserved in grasses may serve as a potent strategy to improve biorefinery applications of grass biomass.

6.
Mol Plant ; 12(10): 1325-1337, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31145998

RESUMO

Lignin is specifically deposited in plant secondary cell walls, and initiation of lignin biosynthesis is regulated by a variety of developmental and environmental signals. However, the mechanisms governing the regulation of lignin biosynthesis remain to be elucidated. In this study, we identified a lignin biosynthesis-associated transcription factor (LTF) from Populus, LTF1, which binds the promoter of a key lignin biosynthetic gene encoding 4-coumarate-CoA ligase (4CL). We showed that LTF1 in its unphosphorylated state functions as a regulator restraining lignin biosynthesis. When LTF1 becomes phosphorylated by PdMPK6 in response to external stimuli such as wounding, it undergoes degradation through a proteasome pathway, resulting in activation of lignification. Expression of a phosphorylation-null mutant version of LTF1 led to stable protein accumulation and persistent attenuation of lignification in wood cells. Taken together, our study reveals a mechanism whereby LTF1 phosphorylation acts as a sensory switch to regulate lignin biosynthesis in response to environmental stimuli. The discovery of novel modulators and mechanisms modifying lignin biosynthesis has important implications for improving the utilization of cell-wall biomass.

7.
New Phytol ; 223(1): 204-219, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30883799

RESUMO

In rice (Oryza sativa), OsF2H and OsFNSII direct flavanones to independent pathways that form soluble flavone C-glycosides and tricin-type metabolites (both soluble and lignin-bound), respectively. Production of soluble tricin metabolites requires CYP75B4 as a chrysoeriol 5'-hydroxylase. Meanwhile, the close homologue CYP75B3 is a canonical flavonoid 3'-hydroxylase (F3'H). However, their precise roles in the biosynthesis of soluble flavone C-glycosides and tricin-lignins in cell walls remain unknown. We examined CYP75B3 and CYP75B4 expression in vegetative tissues, analyzed extractable flavonoid profiles, cell wall structure and digestibility of their mutants, and investigated catalytic activities of CYP75B4 orthologues in grasses. CYP75B3 and CYP75B4 showed co-expression patterns with OsF2H and OsFNSII, respectively. CYP75B3 is the sole F3'H in flavone C-glycosides biosynthesis, whereas CYP75B4 alone provides sufficient 3',5'-hydroxylation for tricin-lignin deposition. CYP75B4 mutation results in production of apigenin-incorporated lignin and enhancement of cell wall digestibility. Moreover, tricin pathway-specific 3',5'-hydroxylation activities are conserved in sorghum CYP75B97 and switchgrass CYP75B11. CYP75B3 and CYP75B4 represent two different pathway-specific enzymes recruited together with OsF2H and OsFNSII, respectively. Interestingly, the OsF2H-CYP75B3 and OsFNSII-CYP75B4 pairs appear to be conserved in grasses. Finally, manipulation of tricin biosynthesis through CYP75B4 orthologues can be a promising strategy to improve digestibility of grass biomass for biofuel and biomaterial production.

8.
Plant J ; 98(6): 975-987, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30773774

RESUMO

Breeding approaches to enrich lignins in biomass could be beneficial to improving the biorefinery process because lignins increase biomass heating value and represent a potent source of valuable aromatic chemicals. However, despite the fact that grasses are promising lignocellulose feedstocks, limited information is yet available for molecular-breeding approaches to upregulate lignin biosynthesis in grass species. In this study, we generated lignin-enriched transgenic rice (Oryza sativa), a model grass species, via targeted mutagenesis of the transcriptional repressor OsMYB108 using CRISPR/Cas9-mediated genome editing. The OsMYB108-knockout rice mutants displayed increased expressions of lignin biosynthetic genes and enhanced lignin deposition in culm cell walls. Chemical and two-dimensional nuclear magnetic resonance (NMR) analyses revealed that the mutant cell walls were preferentially enriched in γ-p-coumaroylated and tricin lignin units, both of which are typical and unique components in grass lignins. NMR analysis also showed that the relative abundances of major lignin linkage types were altered in the OsMYB108 mutants.

9.
Plant Physiol ; 179(4): 1796-1809, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30670602

RESUMO

Striga species are parasitic weeds that seriously constrain the productivity of food staples, including cereals and legumes, in Sub-Saharan Africa and Asia. In eastern and central Africa, Striga spp. infest as much as 40 million hectares of smallholder farmland causing total crop failure during severe infestation. As the molecular mechanisms underlying resistance are yet to be elucidated, we undertook a comparative metabolome study using the Striga-resistant rice (Oryza sativa) cultivar 'Nipponbare' and the susceptible cultivar 'Koshihikari'. We found that a number of metabolites accumulated preferentially in the Striga-resistant cultivar upon Striga hermonthica infection. Most apparent was increased deposition of lignin, a phenylpropanoid polymer mainly composed of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) aromatic units, around the site of interaction in Nipponbare. The increased deposition of lignin was accompanied by induction of the expression of corresponding enzyme-encoding genes in the phenylpropanoid pathway. In addition, perturbing normal lignin composition by knocking down or overexpressing the genes that regulate lignin composition, i.e. p-COUMARATE 3-HYDROXYLASE or FERULATE 5-HYDROXYLASE, enhanced susceptibility of Nipponbare to S hermonthica infection. These results demonstrate that enhanced lignin deposition and maintenance of the structural integrity of lignin polymers deposited at the infection site are crucial for postattachment resistance against S hermonthica.


Assuntos
Interações Hospedeiro-Parasita/genética , Lignina/química , Oryza/genética , Striga/fisiologia , Lignina/genética , Oryza/parasitologia , Doenças das Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/parasitologia
10.
Plant J ; 97(3): 543-554, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30375064

RESUMO

The aromatic composition of lignin is an important trait that greatly affects the usability of lignocellulosic biomass. We previously identified a rice (Oryza sativa) gene encoding coniferaldehyde 5-hydroxylase (OsCAld5H1), which was effective in modulating syringyl (S)/guaiacyl (G) lignin composition ratio in rice, a model grass species. Previously characterized OsCAld5H1-knockdown rice lines, which were produced via an RNA-interference approach, showed augmented G lignin units yet contained considerable amounts of residual S lignin units. In this study, to further investigate the effect of suppression of OsCAld5H1 on rice lignin structure, we generated loss-of-function mutants of OsCAld5H1 using the CRISPR/Cas9-mediated genome editing system. Homozygous OsCAld5H1-knockout lines harboring anticipated frame-shift mutations in OsCAld5H1 were successfully obtained. A series of wet-chemical and two-dimensional NMR analyses on cell walls demonstrated that although lignins in the mutant were predictably enriched in G units all the tested mutant lines produced considerable numbers of S units. Intriguingly, lignin γ-p-coumaroylation analysis by the derivatization followed by reductive cleavage method revealed that enrichment of G units in lignins of the mutants was limited to the non-γ-p-coumaroylated units, whereas grass-specific γ-p-coumaroylated lignin units were almost unaffected. Gene expression analysis indicated that no homologous genes of OsCAld5H1 were overexpressed in the mutants. These data suggested that CAld5H is mainly involved in the production of non-γ-p-coumaroylated S lignin units, common in both eudicots and grasses, but not in the production of grass-specific γ-p-coumaroylated S units in rice.


Assuntos
Acroleína/análogos & derivados , Lignina/metabolismo , Oxigenases de Função Mista/metabolismo , Oryza/genética , Acroleína/metabolismo , Biomassa , Sistemas CRISPR-Cas , Parede Celular/metabolismo , Mutação com Perda de Função , Oxigenases de Função Mista/genética , Oryza/enzimologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Propionatos/metabolismo
11.
Plant Cell Physiol ; 59(11): 2278-2287, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30085233

RESUMO

Plant specialized metabolites are often found as lineage-specific diastereomeric isomers. For example, Sesamum alatum accumulates the specialized metabolite (+)-2-episesalatin, a furofuran-type lignan with a characteristic diastereomeric configuration rarely found in other Sesamum spp. However, little is known regarding how diastereomeric specificity in lignan biosynthesis is implemented in planta. Here, we show that S. alatum CYP81Q3, a P450 orthologous to S. indicum CYP81Q1, specifically catalyzes methylenedioxy bridge (MDB) formation in (+)-epipinoresinol to produce (+)-pluviatilol. Both (+)-epipinoresinol and (+)-pluviatilol are putative intermediates of (+)-2-episesalatin based on their diastereomeric configurations. On the other hand, CYP81Q3 accepts neither (+)- nor (-)-pinoresinol as a substrate. This diastereomeric selectivity of CYP81Q3 is in clear contrast to that of CYP81Q1, which specifically converts (+)-pinoresinol to (+)-sesamin via (+)-piperitol by the sequential formation of two MDBs but does not accept (+)-epipinoresinol as a substrate. Moreover, (+)-pinoresinol does not interfere with the conversion of (+)-epipinoresinol to (+)-pluviatilol by CYP81Q3. Amino acid substitution and CO difference spectral analyses show that polymorphic residues between CYP81Q1 and CYP81Q3 proximal to their putative substrate pockets are crucial for the functional diversity and stability of these two enzymes. Our data provide clues to understanding how the lineage-specific functional differentiation of respective biosynthetic enzymes substantiates the stereoisomeric diversity of lignan structures.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Lignanas/metabolismo , Proteínas de Plantas/metabolismo , Sesamum/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Redes e Vias Metabólicas , Filogenia , Proteínas de Plantas/genética , Sementes/metabolismo , Estereoisomerismo , Especificidade por Substrato
12.
Plant J ; 2018 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-29890017

RESUMO

p-Coumaroyl ester 3-hydroxylase (C3'H) is a key enzyme involved in the biosynthesis of lignin, a phenylpropanoid polymer that is the major constituent of secondary cell walls in vascular plants. Although the crucial role of C3'H in lignification and its manipulation to upgrade lignocellulose have been investigated in eudicots, limited information is available in monocotyledonous grass species, despite their potential as biomass feedstocks. Here we address the pronounced impacts of C3'H deficiency on the structure and properties of grass cell walls. C3'H-knockdown lines generated via RNA interference (RNAi)-mediated gene silencing, with about 0.5% of the residual expression levels, reached maturity and set seeds. In contrast, C3'H-knockout rice mutants generated via CRISPR/Cas9-mediated mutagenesis were severely dwarfed and sterile. Cell wall analysis of the mature C3'H-knockdown RNAi lines revealed that their lignins were largely enriched in p-hydroxyphenyl (H) units while being substantially reduced in the normally dominant guaiacyl (G) and syringyl (S) units. Interestingly, however, the enrichment of H units was limited to within the non-acylated lignin units, with grass-specific γ-p-coumaroylated lignin units remaining apparently unchanged. Suppression of C3'H also resulted in relative augmentation in tricin residues in lignin as well as a substantial reduction in wall cross-linking ferulates. Collectively, our data demonstrate that C3'H expression is an important determinant not only of lignin content and composition but also of the degree of cell wall cross-linking. We also demonstrated that C3'H-suppressed rice displays enhanced biomass saccharification.

13.
Biosci Biotechnol Biochem ; 82(7): 1143-1152, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29558856

RESUMO

A comprehensive understanding of the structure and properties of gramineous lignocelluloses is needed to facilitate their uses in biorefinery. In this study, lignocelluloses from fractionated internode tissues of two taxonomically close species, Erianthus arundinaceus and sugarcane (Saccharum spp.), were characterized. Our analyses determined that syringyl (S) lignins were predominant over guaiacyl (G) or p-hydroxyphenyl (H) lignins in sugarcane tissues; on the other hand, S lignin levels were similar to those of G lignin in Erianthus tissues. In addition, tricin units were detected in sugarcane tissues, but not in Erianthus tissues. Distributions of lignin inter-monomeric linkage types were also different in Erianthus and sugarcane tissues. Alkaline treatment removed lignins from sugarcane tissues more efficiently than Erianthus tissues, resulting in a higher enzymatic digestibility of sugarcane tissues compared with Erianthus tissues. Our data indicate that Erianthus biomass displayed resistance to alkaline delignification and enzymatic digestion.


Assuntos
Álcalis/química , Biomassa , Enzimas/metabolismo , Lignina/química , Polissacarídeos/metabolismo , Saccharum/química , Saccharum/classificação , Saccharum/enzimologia , Especificidade da Espécie
14.
New Phytol ; 218(2): 710-723, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29498051

RESUMO

Parasitic plants in the family Orobanchaceae are destructive weeds of agriculture worldwide. The haustorium, an essential parasitic organ used by these plants to penetrate host tissues, is induced by host-derived phenolic compounds called haustorium-inducing factors (HIFs). The origin of HIFs remains unknown, although the structures of lignin monomers resemble that of HIFs. Lignin is a natural phenylpropanoid polymer, commonly found in secondary cell walls of vascular plants. We therefore investigated the possibility that HIFs are derived from host lignin. Various lignin-related phenolics, quinones and lignin polymers, together with nonhost and host plants that have different lignin compositions, were tested for their haustorium-inducing activity in two Orobanchaceae species, a facultative parasite, Phtheirospermum japonicum, and an obligate parasite, Striga hermonthica. Lignin-related compounds induced haustoria in P. japonicum and S. hermonthica with different specificities. High concentrations of lignin polymers induced haustorium formation. Treatment with laccase, a lignin degradation enzyme, promoted haustorium formation at low concentrations. The distinct lignin compositions of the host and nonhost plants affected haustorium induction, correlating with the response of the different parasitic plants to specific types of lignin-related compounds. Our study provides valuable insights into the important roles of lignin biosynthesis and degradation in the production of HIFs.


Assuntos
Interações Hospedeiro-Parasita , Lignina/metabolismo , Orobanchaceae/anatomia & histologia , Striga/anatomia & histologia , Antocianinas/metabolismo , Arabidopsis/parasitologia , Vias Biossintéticas , Oryza/parasitologia , Plantas Geneticamente Modificadas , Quinonas/metabolismo
15.
Sci Rep ; 8(1): 1290, 2018 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-29358744

RESUMO

Termites represent one of the most efficient lignocellulose decomposers on earth. The mechanism by which termites overcome the recalcitrant lignin barrier to gain access to embedded polysaccharides for assimilation and energy remains largely unknown. In the present study, softwood, hardwood, and grass lignocellulose diets were fed to Coptotermes formosanus workers, and structural differences between the original lignocellulose diets and the resulting feces were examined by solution-state multidimensional nuclear magnetic resonance (NMR) techniques as well as by complementary wet-chemical methods. Overall, our data support the view that lignin polymers are partially decomposed during their passage through the termite gut digestive system, although polysaccharide decomposition clearly dominates the overall lignocellulose deconstruction process and the majority of lignin polymers remain intact in the digestive residues. High-resolution NMR structural data suggested preferential removal of syringyl aromatic units in hardwood lignins, but non-acylated guaiacyl units as well as tricin end-units in grass lignins. In addition, our data suggest that termites and/or their gut symbionts may favor degradation of C-C-bonded ß-5 and resinol-type ß-ß lignin inter-monomeric units over degradation of ether-bonded ß-O-4 units, which is in contrast to what has been observed in typical lignin biodegradation undertaken by wood-decaying fungi.


Assuntos
Trato Gastrointestinal/metabolismo , Isópteros/fisiologia , Lignina/química , Polissacarídeos/química , Madeira/metabolismo , Animais , Sequência de Carboidratos , Fezes/química , Hidrólise , Japão , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Polissacarídeos/classificação , Polissacarídeos/isolamento & purificação , Madeira/classificação
16.
J Insect Physiol ; 103: 57-63, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-29038014

RESUMO

We investigated the effects of lignins as diet components on the physiological activities of a lower termite, Coptotermes formosanus Shiraki. Artificial diets composed of polysaccharides with and without purified lignins (milled-wood lignins) from Japanese cedar (softwood), Japanese beech (hardwood), and rice (grass), were fed to C. formosanus workers. The survival and body mass of the workers as well as the presence of three symbiotic protists in the hindguts of the workers were then periodically examined. The survival rates of workers fed on diets containing lignins were, regardless of the lignocellulose diet sources, significantly higher than those of workers fed on only polysaccharides. In addition, it was clearly observed that all the tested lignins have positive effects on the maintenance of two major protists in the hindguts of C. formosanus workers, i.e., Pseudotrichonympha grassii and Holomastigotoides hartmanni. Overall, our data suggest that the presence of lignin is crucial to maintaining the physiological activities of C. formosanus workers during their lignocellulose decomposition. Our data also suggested that some components, possibly minerals and/or non-structural carbohydrates, in grass lignocellulose negatively affect the survival of C. formosanus workers as well as the present rate of the symbiotic protists in their hindguts.


Assuntos
Isópteros/fisiologia , Lignina/fisiologia , Animais , Dieta , Parabasalídeos/fisiologia , Simbiose
17.
Plant Physiol ; 174(2): 972-985, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28385728

RESUMO

Lignin, a ubiquitous phenylpropanoid polymer in vascular plant cell walls, is derived primarily from oxidative couplings of monolignols (p-hydroxycinnamyl alcohols). It was discovered recently that a wide range of grasses, including cereals, utilize a member of the flavonoids, tricin (3',5'-dimethoxyflavone), as a natural comonomer with monolignols for cell wall lignification. Previously, we established that cytochrome P450 93G1 is a flavone synthase II (OsFNSII) indispensable for the biosynthesis of soluble tricin-derived metabolites in rice (Oryza sativa). Here, our tricin-deficient fnsII mutant was analyzed further with an emphasis on its cell wall structure and properties. The mutant is similar in growth to wild-type control plants with normal vascular morphology. Chemical and nuclear magnetic resonance structural analyses demonstrated that the mutant lignin is completely devoid of tricin, indicating that FNSII activity is essential for the deposition of tricin-bound lignin in rice cell walls. The mutant also showed substantially reduced lignin content with decreased syringyl/guaiacyl lignin unit composition. Interestingly, the loss of tricin in the mutant lignin appears to be partially compensated by incorporating naringenin, which is a preferred substrate of OsFNSII. The fnsII mutant was further revealed to have enhanced enzymatic saccharification efficiency, suggesting that the cell wall recalcitrance of grass biomass may be reduced through the manipulation of the flavonoid monomer supply for lignification.


Assuntos
Biomassa , Lignina/metabolismo , Oxigenases de Função Mista/metabolismo , Oryza/enzimologia , Vias Biossintéticas/genética , Parede Celular/metabolismo , Fertilidade/genética , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Genes de Plantas , Espectroscopia de Ressonância Magnética , Mutação/genética , Oryza/genética , Fenótipo
18.
Planta ; 246(2): 337-349, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28421330

RESUMO

MAIN CONCLUSION: Regulation of a gene encoding coniferaldehyde 5-hydroxylase leads to substantial alterations in lignin structure in rice cell walls, identifying a promising genetic engineering target for improving grass biomass utilization. The aromatic composition of lignin greatly affects utilization characteristics of lignocellulosic biomass and, therefore, has been one of the primary targets of cell wall engineering studies. Limited information is, however, available regarding lignin modifications in monocotyledonous grasses, despite the fact that grass lignocelluloses have a great potential for feedstocks of biofuel production and various biorefinery applications. Here, we report that manipulation of a gene encoding coniferaldehyde 5-hydroxylase (CAld5H, or ferulate 5-hydroxylase, F5H) leads to substantial alterations in syringyl (S)/guaiacyl (G) lignin aromatic composition in rice (Oryza sativa), a major model grass and commercially important crop. Among three CAld5H genes identified in rice, OsCAld5H1 (CYP84A5) appeared to be predominantly expressed in lignin-producing rice vegetative tissues. Down-regulation of OsCAld5H1 produced altered lignins largely enriched in G units, whereas up-regulation of OsCAld5H1 resulted in lignins enriched in S units, as revealed by a series of wet-chemical and NMR structural analyses. Our data collectively demonstrate that OsCAld5H1 expression is a major factor controlling S/G lignin composition in rice cell walls. Given that S/G lignin composition affects various biomass properties, we contemplate that manipulation of CAld5H gene expression represents a promising strategy to upgrade grass biomass for biorefinery applications.


Assuntos
Carboxiliases/metabolismo , Lignina/metabolismo , Oryza/enzimologia , Acroleína/análogos & derivados , Acroleína/química , Acroleína/metabolismo , Biocombustíveis , Biomassa , Vias Biossintéticas , Carboxiliases/genética , Parede Celular/metabolismo , Regulação para Baixo , Engenharia Genética , Lignina/química , Oryza/citologia , Oryza/genética , Oryza/crescimento & desenvolvimento , Filogenia , Folhas de Planta/anatomia & histologia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regulação para Cima
19.
Plant Biotechnol (Tokyo) ; 34(1): 7-15, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-31275003

RESUMO

Lignin encrusts lignocellulose polysaccharides, and has long been considered an obstacle for the efficient use of polysaccharides during processes such as pulping and bioethanol fermentation. However, lignin is also a potential feedstock for aromatic products and is an important by-product of polysaccharide utilization. Therefore, producing biomass plant species exhibiting enhanced lignin production is an important breeding objective. Herein, we describe the development of transgenic rice plants with increased lignin content. Five Arabidopsis thaliana (Arabidopsis) and one Oryza sativa (rice) MYB transcription factor genes that were implicated to be involved in lignin biosynthesis were transformed into rice (O. sativa L. ssp. japonica cv. Nipponbare). Among them, three Arabidopsis MYBs (AtMYB55, AtMYB61, and AtMYB63) in transgenic rice T1 lines resulted in culms with lignin content about 1.5-fold higher than that of control plants. Furthermore, lignin structures in AtMYB61-overexpressing rice plants were investigated by wet-chemistry and two-dimensional nuclear magnetic resonance spectroscopy approaches. Our data suggested that heterologous expression of AtMYB61 in rice increased lignin content mainly by enriching syringyl units as well as p-coumarate and tricin moieties in the lignin polymers. We contemplate that this strategy is also applicable to lignin upregulation in large-sized grass biomass plants, such as Sorghum, switchgrass, Miscanthus and Erianthus.

20.
Planta ; 242(3): 589-600, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26070439

RESUMO

MAIN CONCLUSION: A rice MYB transcription factor, OsMYB58/63, was found to directly upregulate the expression of a rice secondary wall-specific cellulose synthase gene, cellulose synthase A7 ( OsCesA7 ); in contrast, the Arabidopsis putative orthologs AtMYB58 and AtMYB63 have been shown to specifically activate lignin biosynthesis. Although indirect evidence has shown that grass plants are similar to but partially different from dicotyledonous ones in transcriptional regulation of lignocellulose biosynthesis, little is known about the differences. This study showed that a rice MYB transcription factor, OsMYB58/63, directly upregulated the expression of a rice secondary wall-specific cellulose synthase gene, cellulose synthase A7 (OsCesA7). Gene co-expression analysis showed that, in rice, OsMYB58/63 and several rice MYB genes were co-expressed with genes encoding lignocellulose biosynthetic enzymes. The expression levels of OsMYB55/61, OsMYB55/61-L, OsMYB58/63, and OsMYB42/85 were commonly found to be high in culm internodes and nodes. All four MYB transcription factors functioned as transcriptional activators in yeast cells. OsMYB58/63 most strongly transactivated the expression of OsCesA7 in rice protoplasts. Moreover, recombinant OsMYB58/63 protein was bound to two distinct cis-regulatory elements, AC-II and SMRE3, in the OsCesA7 promoter. This is in sharp contrast to the role of Arabidopsis orthologs, AtMYB58 and AtMYB63, which had been reported to specifically activate lignin biosynthesis. The promoter analysis revealed that AC elements, which are the binding sites for MYB58 and MYB63, were lacking in cellulose and xylan biosynthetic genes in Arabidopsis, but present in cellulose, xylan, and lignin biosynthetic genes in rice, implying that the difference of transcriptional regulation between rice and Arabidopsis is due to the distinct composition of promoters. Our results provide a new insight into transcriptional regulation in grass lignocellulose biosynthesis.


Assuntos
Parede Celular/enzimologia , Parede Celular/metabolismo , Glucosiltransferases/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Parede Celular/genética , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/genética , Oryza/enzimologia , Oryza/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética
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