RESUMO
Some nucleotide-binding and leucine-rich repeat receptors (NLRs) indirectly detect pathogen effectors by monitoring their host targets. In Arabidopsis thaliana, RIN4 is targeted by multiple sequence-unrelated effectors and activates immune responses mediated by RPM1 and RPS2. These effectors trigger cell death in Nicotiana benthamiana, but the corresponding NLRs have yet not been identified. To identify N. benthamiana NLRs (NbNLRs) that recognize Arabidopsis RIN4-targeting effectors, we conducted a rapid reverse genetic screen using an NbNLR VIGS library. We identified that the N. benthamiana homolog of Ptr1 (Pseudomonas tomato race 1) recognizes the Pseudomonas effectors AvrRpt2, AvrRpm1, and AvrB. We demonstrated that recognition of the Xanthomonas effector AvrBsT and the Pseudomonas effector HopZ5 is conferred independently by the N. benthamiana homolog of Ptr1 and ZAR1. Interestingly, the recognition of HopZ5 and AvrBsT is contributed unequally by Ptr1 and ZAR1 in N. benthamiana and Capsicum annuum. In addition, we showed that the RLCK XII family protein JIM2 is required for the NbZAR1-dependent recognition of AvrBsT and HopZ5. The recognition of sequence-unrelated effectors by NbPtr1 and NbZAR1 provides an additional example of convergently evolved effector recognition. Identification of key components involved in Ptr1 and ZAR1-mediated immunity could reveal unique mechanisms of expanded effector recognition.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas/metabolismo , Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Pseudomonas , Receptores Imunológicos/metabolismo , Proteínas de Bactérias/metabolismo , Pseudomonas syringae/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Arabidopsis/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
Cassava (Manihot esculenta) is a starchy root crop that supports over a billion people in tropical and subtropical regions of the world. This staple, however, produces the neurotoxin cyanide and requires processing for safe consumption. Excessive consumption of insufficiently processed cassava, in combination with protein-poor diets, can have neurodegenerative impacts. This problem is further exacerbated by drought conditions which increase this toxin in the plant. To reduce cyanide levels in cassava, we used CRISPR-mediated mutagenesis to disrupt the cytochrome P450 genes CYP79D1 and CYP79D2 whose protein products catalyze the first step in cyanogenic glucoside biosynthesis. Knockout of both genes eliminated cyanide in leaves and storage roots of cassava accession 60444; the West African, farmer-preferred cultivar TME 419; and the improved variety TMS 91/02324. Although knockout of CYP79D2 alone resulted in significant reduction of cyanide, mutagenesis of CYP79D1 did not, indicating these paralogs have diverged in their function. The congruence of results across accessions indicates that our approach could readily be extended to other preferred or improved cultivars. This work demonstrates cassava genome editing for enhanced food safety and reduced processing burden, against the backdrop of a changing climate.
RESUMO
Plants are resistant to most pathogens because of an immune system that perceives invading microbes and activates defense. A large repertoire of innate immune receptors mediates specific direct or indirect recognition of pathogen-derived molecules. Disease is often a consequence of insufficient immune surveillance, and the transfer of immune receptor genes from resistant plants to susceptible crop varieties is an effective strategy for combating disease outbreaks. We discuss approaches for identifying intracellular and cell surface immune receptors, with particular focus on recently developed and emerging methodologies. We also review considerations for the transfer of immune receptor genes into crop species, including additional host factors that may be required for immune receptor function. Together, these concepts lay out a broadly applicable playbook for developing crop varieties with durable disease resistance.
Assuntos
Doenças das Plantas , Imunidade Vegetal , Produtos Agrícolas/genética , Resistência à Doença/genética , Doenças das Plantas/genética , Imunidade Vegetal/genéticaRESUMO
Xanthomonas species, Pseudomonas syringae and Ralstonia species are bacterial plant pathogens that cause significant yield loss in many crop species. Generating disease-resistant crop varieties can provide a more sustainable solution to control yield loss compared to chemical methods. Plant immune receptors encoded by nucleotide-binding, leucine-rich repeat (NLR) genes typically confer resistance to pathogens that produce a cognate elicitor, often an effector protein secreted by the pathogen to promote virulence. The diverse sequence and presence/absence variation of pathogen effector proteins within and between pathogen species usually limits the utility of a single NLR gene to protecting a plant from a single pathogen species or particular strains. The NLR protein Recognition of XopQ 1 (Roq1) was recently identified from the plant Nicotiana benthamiana and mediates perception of the effector proteins XopQ and HopQ1 from Xanthomonas and P. syringae respectively. Unlike most recognized effectors, alleles of XopQ/HopQ1 are highly conserved and present in most plant pathogenic strains of Xanthomonas and P. syringae. A homolog of XopQ/HopQ1, named RipB, is present in most Ralstonia strains. We found that Roq1 confers immunity to Xanthomonas, P. syringae, and Ralstonia when expressed in tomato. Strong resistance to Xanthomonas perforans was observed in three seasons of field trials with both natural and artificial inoculation. The Roq1 gene can therefore be used to provide safe, economical, and effective control of these pathogens in tomato and other crop species and reduce or eliminate the need for traditional chemical controls.
RESUMO
Photosynthetic organisms experience wide fluctuations in light intensity and regulate light harvesting accordingly to prevent damage from excess energy. The antenna quenching component qH is a sustained form of energy dissipation that protects the photosynthetic apparatus under stress conditions. This photoprotective mechanism requires the plastid lipocalin LCNP and is prevented by SUPPRESSOR OF QUENCHING1 (SOQ1) under non-stress conditions. However, the molecular mechanism of qH relaxation has yet to be resolved. Here, we isolated and characterized RELAXATION OF QH1 (ROQH1), an atypical short-chain dehydrogenase-reductase that functions as a qH-relaxation factor in Arabidopsis. The ROQH1 gene belongs to the GreenCut2 inventory specific to photosynthetic organisms, and the ROQH1 protein localizes to the chloroplast stroma lamellae membrane. After a cold and high-light treatment, qH does not relax in roqh1 mutants and qH does not occur in leaves overexpressing ROQH1. When the soq1 and roqh1 mutations are combined, qH can neither be prevented nor relaxed and soq1 roqh1 displays constitutive qH and light-limited growth. We propose that LCNP and ROQH1 perform dosage-dependent, antagonistic functions to protect the photosynthetic apparatus and maintain light-harvesting efficiency in plants.
Assuntos
Arabidopsis/genética , Estiolamento/genética , Arabidopsis/enzimologiaRESUMO
The immune pathway responsible for perception of the Xanthomonas perforans effector XopJ4 was identified in the plant Nicotiana benthamiana. This pathogen causes significant yield loss in commercial tomato cultivation. Genetic mapping and viral-induced gene silencing were used to identify immune signaling components of the XopJ4 perception pathway in N. benthamiana. Transient complementation assays were performed to determine the functionality of gene variants and co-immunoprecipitation assays were used to gain insight into the molecular mechanism of the pathway. Two N. benthamiana ethyl methanesulfonate (EMS) mutants deficient for XopJ4 perception were identified as having loss-of-function mutations in the gene encoding the nucleotide binding, leucine-rich repeat (NLR) protein NbZAR1. Silencing of a receptor-like cytoplasmic kinase family XII gene, subsequently named XOPJ4 IMMUNITY 2 (JIM2), blocks perception of XopJ4. This study demonstrates the feasibility of conducting mutant screens in N. benthamiana to investigate the genetic basis of the plant immune system and other processes. The identification of NbZAR1 and JIM2 as mediating XopJ4 perception in N. benthamiana supports the model of ZAR1 being involved in the perception of many different pathogen effector proteins with specificity dictated by associated receptor-like cytoplasmic kinases.
Assuntos
Proteínas de Bactérias/metabolismo , Nicotiana/genética , Doenças das Plantas/imunologia , Imunidade Vegetal , Proteínas de Plantas/metabolismo , Transdução de Sinais , Xanthomonas/fisiologia , Proteínas de Bactérias/genética , Solanum lycopersicum/genética , Solanum lycopersicum/imunologia , Solanum lycopersicum/microbiologia , Proteínas NLR/genética , Proteínas NLR/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Nicotiana/imunologia , Nicotiana/microbiologiaRESUMO
Effector-triggered immunity (ETI) in plants involves a large family of nucleotide-binding leucine-rich repeat (NLR) immune receptors, including Toll/IL-1 receptor-NLRs (TNLs) and coiled-coil NLRs (CNLs). Although various NLR immune receptors are known, a mechanistic understanding of NLR function in ETI remains unclear. The TNL Recognition of XopQ 1 (Roq1) recognizes the effectors XopQ and HopQ1 from Xanthomonas and Pseudomonas, respectively, which activates resistance to Xanthomonas euvesicatoria and Xanthomonas gardneri in an Enhanced Disease Susceptibility 1 (EDS1)-dependent way in Nicotiana benthamiana In this study, we found that the N. benthamiana N requirement gene 1 (NRG1), a CNL protein required for the tobacco TNL protein N-mediated resistance to tobacco mosaic virus, is also essential for immune signaling [including hypersensitive response (HR)] triggered by the TNLs Roq1 and Recognition of Peronospora parasitica 1 (RPP1), but not by the CNLs Bs2 and Rps2, suggesting that NRG1 may be a conserved key component in TNL signaling pathways. Besides EDS1, Roq1 and NRG1 are necessary for resistance to Xanthomonas and Pseudomonas in N. benthamiana NRG1 functions downstream of Roq1 and EDS1 and physically associates with EDS1 in mediating XopQ-Roq1-triggered immunity. Moreover, RNA sequencing analysis showed that XopQ-triggered gene-expression profile changes in N. benthamiana were almost entirely mediated by Roq1 and EDS1 and were largely regulated by NRG1. Overall, our study demonstrates that NRG1 is a key component that acts downstream of EDS1 to mediate various TNL signaling pathways, including Roq1 and RPP1-mediated HR, resistance to Xanthomonas and Pseudomonas, and XopQ-regulated transcriptional changes in N. benthamiana.
Assuntos
Nicotiana/genética , Nicotiana/metabolismo , Subpopulações de Linfócitos B/metabolismo , Proteínas de Ligação a DNA , Proteínas de Repetições Ricas em Leucina , Proteínas NLR/metabolismo , Neuregulina-1/genética , Neuregulina-1/fisiologia , Doenças das Plantas , Imunidade Vegetal , Proteínas de Plantas/genética , Domínios Proteicos , Proteínas/genética , Pseudomonas , Transdução de Sinais , Transcriptoma , XanthomonasRESUMO
Light utilization is finely tuned in photosynthetic organisms to prevent cellular damage. The dissipation of excess absorbed light energy, a process termed nonphotochemical quenching (NPQ), plays an important role in photoprotection. Little is known about the sustained or slowly reversible form(s) of NPQ and whether they are photoprotective, in part due to the lack of mutants. The Arabidopsis thaliana suppressor of quenching1 (soq1) mutant exhibits enhanced sustained NPQ, which we term qH. To identify molecular players involved in qH, we screened for suppressors of soq1 and isolated mutants affecting either chlorophyllide a oxygenase or the chloroplastic lipocalin, now renamed plastid lipocalin (LCNP). Analysis of the mutants confirmed that qH is localized to the peripheral antenna (LHCII) of photosystem II and demonstrated that LCNP is required for qH, either directly (by forming NPQ sites) or indirectly (by modifying the LHCII membrane environment). qH operates under stress conditions such as cold and high light and is photoprotective, as it reduces lipid peroxidation levels. We propose that, under stress conditions, LCNP protects the thylakoid membrane by enabling sustained NPQ in LHCII, thereby preventing singlet oxygen stress.
Assuntos
Arabidopsis/metabolismo , Lipocalinas/metabolismo , Processos Fotoquímicos , Plastídeos/metabolismo , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/metabolismo , Clorofila/metabolismo , Temperatura Baixa , Genes de Plantas , Genes Supressores , Testes Genéticos , Luz , Mutação/genética , Oxigenases/metabolismo , Processos Fotoquímicos/efeitos da radiação , Plastídeos/efeitos da radiação , Tiorredoxinas/metabolismo , Sequenciamento Completo do GenomaRESUMO
Xanthomonas spp. are phytopathogenic bacteria that can cause disease on a wide variety of plant species resulting in significant impacts on crop yields. Limited genetic resistance is available in most crop species and current control methods are often inadequate, particularly when environmental conditions favor disease. The plant Nicotiana benthamiana has been shown to be resistant to Xanthomonas and Pseudomonas due to an immune response triggered by the bacterial effector proteins XopQ and HopQ1, respectively. We used a reverse genetic screen to identify Recognition of XopQ 1 (Roq1), a nucleotide-binding leucine-rich repeat (NLR) protein with a Toll-like interleukin-1 receptor (TIR) domain, which mediates XopQ recognition in N. benthamiana. Roq1 orthologs appear to be present only in the Nicotiana genus. Expression of Roq1 was found to be sufficient for XopQ recognition in both the closely-related Nicotiana sylvestris and the distantly-related beet plant (Beta vulgaris). Roq1 was found to co-immunoprecipitate with XopQ, suggesting a physical association between the two proteins. Roq1 is able to recognize XopQ alleles from various Xanthomonas species, as well as HopQ1 from Pseudomonas, demonstrating widespread potential application in protecting crop plants from these pathogens.
Assuntos
Resistência à Doença , Nicotiana/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Pseudomonas/metabolismo , Xanthomonas/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
A mutation in the ALTERED XYLOGLUCAN9 (AXY9) gene was found to be causative for the decreased xyloglucan acetylation phenotype of the axy9.1 mutant, which was identified in a forward genetic screen for Arabidopsis (Arabidopsis thaliana) mutants. The axy9.1 mutant also exhibits decreased O-acetylation of xylan, implying that the AXY9 protein has a broad role in polysaccharide acetylation. An axy9 insertional mutant exhibits severe growth defects and collapsed xylem, demonstrating the importance of wall polysaccharide O-acetylation for normal plant growth and development. Localization and topological experiments indicate that the active site of the AXY9 protein resides within the Golgi lumen. The AXY9 protein appears to be a component of the plant cell wall polysaccharide acetylation pathway, which also includes the REDUCED WALL ACETYLATION and TRICHOME BIREFRINGENCE-LIKE proteins. The AXY9 protein is distinct from the TRICHOME BIREFRINGENCE-LIKE proteins, reported to be polysaccharide acetyltransferases, but does share homology with them and other acetyltransferases, suggesting that the AXY9 protein may act to produce an acetylated intermediate that is part of the O-acetylation pathway.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Polissacarídeos/metabolismo , Acetilação , Acetiltransferases/genética , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Mapeamento Cromossômico , Genes Reporter , Genótipo , Glucanos/metabolismo , Complexo de Golgi/metabolismo , Modelos Biológicos , Mutagênese Insercional , Fenótipo , Filogenia , Caules de Planta/citologia , Caules de Planta/genética , Caules de Planta/metabolismo , Xilanos/metabolismoRESUMO
MAIN CONCLUSION: Putative XyG xylosyltransferases from Tropaeolum majus (nasturtium) and Solanum lycopersicum (tomato) homologous to characterized Arabidopsis genes were identified and shown to functionally complement Arabidopsis mutants lacking xyloglucan demonstrating they represent xyloglucan xylosyltransferases. Xyloglucan is a major hemicellulose in the plant cell wall and is important for the structural organization of the wall. The fine structure of xyloglucan can vary dependent on plant species and tissue type. Most vascular seed-bearing plants including Arabidopsis thaliana and nasturtium (Tropaeolum majus) have a xyloglucan structure, in which three out of four backbone glucosyl-residues are substituted with xylosyl-residues. In contrast, the xyloglucan found in plants of the Solanaceae family, which includes tomato (Solanum lycopersicum), is typically less xylosylated with only two of the four backbone glucosyl-residues substituted with xylosyl-residues. To investigate the genetics of xyloglucan xylosylation, candidate xyloglucan xylosyltransferase genes (XXTs) homologous to known A. thaliana XXTs were cloned from nasturtium and tomato. These candidate XXTs were expressed in the A. thaliana xxt1/2 double and xxt1/2/5 triple mutant, whose walls lack detectable xyloglucan. Expression of the orthologs of XXT5 resulted in no detectable xyloglucan in the transgenic A. thaliana plants, consistent with a lack of xyloglucan in the A. thaliana xxt1/2 double mutant. However, transformation of both the tomato and nasturtium orthologs of AtXXT1 and AtXXT2 resulted in the production of xyloglucan with a xylosylation pattern similar to wild type A. thaliana indicating that both SlXXT2 and TmXXT2 likely have xylosyltransferase activity. As the expression of the SlXXT2 did not result in xyloglucan with a decreased xylosylation frequency found in tomato, this gene is not responsible for the unique xylosylation pattern found in the solanaceous plants.
Assuntos
Arabidopsis/enzimologia , Glucanos/metabolismo , Pentosiltransferases/metabolismo , Xilanos/metabolismo , Xilose/metabolismo , Arabidopsis/genética , Configuração de Carboidratos , Glucanos/química , Pentosiltransferases/classificação , Pentosiltransferases/genética , Filogenia , Xilanos/químicaRESUMO
Plant cells are surrounded by a cell wall that plays a key role in plant growth, structural integrity, and defense. The cell wall is a complex and diverse structure that is mainly composed of polysaccharides. The majority of noncellulosic cell wall polysaccharides are produced in the Golgi apparatus from nucleotide sugars that are predominantly synthesized in the cytosol. The transport of these nucleotide sugars from the cytosol into the Golgi lumen is a critical process for cell wall biosynthesis and is mediated by a family of nucleotide sugar transporters (NSTs). Numerous studies have sought to characterize substrate-specific transport by NSTs; however, the availability of certain substrates and a lack of robust methods have proven problematic. Consequently, we have developed a novel approach that combines reconstitution of NSTs into liposomes and the subsequent assessment of nucleotide sugar uptake by mass spectrometry. To address the limitation of substrate availability, we also developed a two-step reaction for the enzymatic synthesis of UDP-l-rhamnose (Rha) by expressing the two active domains of the Arabidopsis UDP-l-Rha synthase. The liposome approach and the newly synthesized substrates were used to analyze a clade of Arabidopsis NSTs, resulting in the identification and characterization of six bifunctional UDP-l-Rha/UDP-d-galactose (Gal) transporters (URGTs). Further analysis of loss-of-function and overexpression plants for two of these URGTs supported their roles in the transport of UDP-l-Rha and UDP-d-Gal for matrix polysaccharide biosynthesis.
Assuntos
Arabidopsis/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Família Multigênica , Ramnose/metabolismo , Uridina Difosfato Glucose/metabolismo , Arabidopsis/enzimologia , Transporte Biológico , Cinética , Dados de Sequência Molecular , Pectinas/metabolismo , Filogenia , Proteolipídeos/metabolismo , Frações Subcelulares/metabolismo , Fatores de TempoRESUMO
Xyloglucan (XyG) is a hemicellulose found in the cell walls of all land plants including early-divergent groups such as liverworts, hornworts and mosses. The basic structure of XyG, a xylosylated glucan, is similar in all of these plants but additional substituents can vary depending on plant family, tissue, and developmental stage. A comprehensive list of known XyG sidechain substituents is assembled including their occurrence within plant families, thereby providing insight into the evolutionary origin of the various sidechains. Recent advances in DNA sequencing have enabled comparative genomics approaches for the identification of XyG biosynthetic enzymes in Arabidopsis thaliana as well as in non-model plant species. Characterization of these biosynthetic genes not only allows the determination of their substrate specificity but also provides insights into the function of the various substituents in plant growth and development.
RESUMO
Xyloglucan (XyG) is the dominant hemicellulose present in the primary cell walls of dicotyledonous plants. Unlike Arabidopsis (Arabidopsis thaliana) XyG, which contains galactosyl and fucosyl substituents, tomato (Solanum lycopersicum) XyG contains arabinofuranosyl residues. To investigate the biological function of these differing substituents, we used a functional complementation approach. Candidate glycosyltransferases were identified from tomato by using comparative genomics with known XyG galactosyltransferase genes from Arabidopsis. These candidate genes were expressed in an Arabidopsis mutant lacking XyG galactosylation, and two of them resulted in the production of arabinosylated XyG, a structure not previously found in this plant species. These genes may therefore encode XyG arabinofuranosyltransferases. Moreover, the addition of arabinofuranosyl residues to the XyG of this Arabidopsis mutant rescued a growth and cell wall biomechanics phenotype, demonstrating that the function of XyG in plant growth, development, and mechanics has considerable flexibility in terms of the specific residues in the side chains. These experiments also highlight the potential of reengineering the sugar substituents on plant wall polysaccharides without compromising growth or viability.
Assuntos
Glucanos/química , Pentosiltransferases/genética , Proteínas de Plantas/genética , Solanum lycopersicum/enzimologia , Xilanos/química , Arabidopsis/genética , Parede Celular/metabolismo , Pentosiltransferases/metabolismo , Pentosiltransferases/fisiologia , Filogenia , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas/metabolismoRESUMO
One major component of plant cell walls is a diverse group of polysaccharides, the hemicelluloses. Hemicelluloses constitute roughly one-third of the wall biomass and encompass the heteromannans, xyloglucan, heteroxylans, and mixed-linkage glucan. The fine structure of these polysaccharides, particularly their substitution, varies depending on the plant species and tissue type. The hemicelluloses are used in numerous industrial applications such as food additives as well as in medicinal applications. Their abundance in lignocellulosic feedstocks should not be overlooked, if the utilization of this renewable resource for fuels and other commodity chemicals becomes a reality. Fortunately, our understanding of the biosynthesis of the various hemicelluloses in the plant has increased enormously in recent years mainly through genetic approaches. Taking advantage of this knowledge has led to plant mutants with altered hemicellulosic structures demonstrating the importance of the hemicelluloses in plant growth and development. However, while we are on a solid trajectory in identifying all necessary genes/proteins involved in hemicellulose biosynthesis, future research is required to combine these single components and assemble them to gain a holistic mechanistic understanding of the biosynthesis of this important class of plant cell wall polysaccharides.
Assuntos
Parede Celular/metabolismo , Glucanos/biossíntese , Mananas/biossíntese , Células Vegetais/metabolismo , Polissacarídeos/biossíntese , Xilanos/biossínteseRESUMO
Xylan is the second most abundant polysaccharide on Earth and represents an immense quantity of stored energy for biofuel production. Despite its importance, most of the enzymes that synthesize xylan have yet to be identified. Xylans have a backbone of ß-1,4-linked xylose residues with substitutions that include α-(1â2)-linked glucuronosyl, 4-O-methyl glucuronosyl, and α-1,2- and α-1,3-arabinofuranosyl residues. The substitutions are structurally diverse and vary by taxonomy, with grass xylan representing a unique composition distinct from dicots and other monocots. To date, no enzyme has yet been identified that is specific to grass xylan synthesis. We identified a xylose-deficient loss-of-function rice mutant in Os02g22380, a putative glycosyltransferase in a grass-specific subfamily of family GT61. We designate the mutant xax1 for xylosyl arabinosyl substitution of xylan 1. Enzymatic fingerprinting of xylan showed the specific absence in the mutant of a peak, which was isolated and determined by (1)H-NMR to be (ß-1,4-Xyl)(4) with a ß-Xylp-(1â2)-α-Araf-(1â3). Rice xax1 mutant plants are deficient in ferulic and coumaric acid, aromatic compounds known to be attached to arabinosyl residues in xylan substituted with xylosyl residues. The xax1 mutant plants exhibit an increased extractability of xylan and increased saccharification, probably reflecting a lower degree of diferulic cross-links. Activity assays with microsomes isolated from tobacco plants transiently expressing XAX1 demonstrated xylosyltransferase activity onto endogenous acceptors. Our results provide insight into grass xylan synthesis and how substitutions may be modified for increased saccharification for biofuel generation.
Assuntos
Parede Celular/química , Oryza/enzimologia , Pentosiltransferases/metabolismo , Xilanos/metabolismo , Xilose/metabolismo , Biocombustíveis , Espectroscopia de Ressonância Magnética , Microssomos , Oryza/metabolismo , Pentosiltransferases/genética , UDP Xilose-Proteína XilosiltransferaseRESUMO
A deep-sequencing approach was pursued utilizing 454 and Illumina sequencing methods to discover new genes involved in xyloglucan biosynthesis. cDNA sequences were generated from developing nasturtium (Tropaeolum majus) seeds, which produce large amounts of non-fucosylated xyloglucan as a seed storage polymer. In addition to known xyloglucan biosynthetic genes, a previously uncharacterized putative xyloglucan galactosyltransferase was identified. Analysis of an Arabidopsis thaliana mutant line defective in the corresponding ortholog (AT5G62220) revealed that this gene shows no redundancy with the previously characterized xyloglucan galactosyltransferase, MUR3, but is required for galactosyl-substitution of xyloglucan at a different position. The gene was termed XLT2 for Xyloglucan L-side chain galactosylTransferase position 2. It represents an enzyme in the same subclade of glycosyltransferase family 47 as MUR3. A double mutant defective in both MUR3 (mur3.1) and XLT2 led to an Arabidopsis plant with xyloglucan that consists essentially of only xylosylated glucosyl units, with no further substitutions.
Assuntos
Galactosiltransferases/metabolismo , Glucanos/biossíntese , Proteínas de Plantas/metabolismo , Sementes/crescimento & desenvolvimento , Tropaeolum/enzimologia , Tropaeolum/genética , Xilanos/biossíntese , Galactosiltransferases/genética , Sequenciamento de Nucleotídeos em Larga Escala , Dados de Sequência Molecular , Proteínas de Plantas/genética , RNA de Plantas/genética , Sementes/enzimologia , Sementes/genética , Sementes/metabolismo , Análise de Sequência de RNA , Tropaeolum/crescimento & desenvolvimento , Tropaeolum/metabolismoRESUMO
In an Arabidopsis thaliana forward genetic screen aimed at identifying mutants with altered structures of their hemicellulose xyloglucan (axy mutants) using oligosaccharide mass profiling, two nonallelic mutants (axy4-1 and axy4-2) that have a 20 to 35% reduction in xyloglucan O-acetylation were identified. Mapping of the mutation in axy4-1 identified AXY4, a type II transmembrane protein with a Trichome Birefringence-Like domain and a domain of unknown function (DUF231). Loss of AXY4 transcript results in a complete lack of O-acetyl substituents on xyloglucan in several tissues, except seeds. Seed xyloglucan is instead O-acetylated by the paralog AXY4like, as demonstrated by the analysis of the corresponding T-DNA insertional lines. Wall fractionation analysis of axy4 knockout mutants indicated that only a fraction containing xyloglucan is non-O-acetylated. Hence, AXY4/AXY4L is required for the O-acetylation of xyloglucan, and we propose that these proteins represent xyloglucan-specific O-acetyltransferases, although their donor and acceptor substrates have yet to be identified. An Arabidopsis ecotype, Ty-0, has reduced xyloglucan O-acetylation due to mutations in AXY4, demonstrating that O-acetylation of xyloglucan does not impact the plant's fitness in its natural environment. The relationship of AXY4 with another previously identified group of Arabidopsis proteins involved in general wall O-acetylation, reduced wall acetylation, is discussed.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Glucanos/metabolismo , Proteínas de Membrana/metabolismo , Xilanos/metabolismo , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Parede Celular/química , Parede Celular/metabolismo , DNA Bacteriano , Ecótipo , Técnicas de Inativação de Genes , Proteínas de Membrana/genética , Mutação , Filogenia , Polissacarídeos/metabolismo , Estrutura Terciária de Proteína , Sementes/genética , Sementes/metabolismoRESUMO
An Arabidopsis thaliana mutant with an altered structure of its hemicellulose xyloglucan (XyG; axy-8) identified by a forward genetic screen facilitating oligosaccharide mass profiling was characterized. axy8 exhibits increased XyG fucosylation and the occurrence of XyG fragments not present in the wild-type plant. AXY8 was identified to encode an α-fucosidase acting on XyG that was previously designated FUC95A. Green fluorescent protein fusion localization studies and analysis of nascent XyG in microsomal preparations demonstrated that this glycosylhydrolase acts mainly on XyG in the apoplast. Detailed structural analysis of XyG in axy8 gave unique insights into the role of the fucosidase in XyG metabolism in vivo. The genetic evidence indicates that the activity of glycosylhydrolases in the apoplast plays a major role in generating the heterogeneity of XyG side chains in the wall. Furthermore, without the dominant apoplastic glycosylhydrolases, the XyG structure in the wall is mainly composed of XXXG and XXFG subunits.