RESUMO
Lignin is a phenolic polymer in plants that rigidifies the cell walls of water-conducting tracheary elements and support-providing fibers and stone cells. Different mechanisms have been suggested for the transport of lignin precursors to the site of lignification in the cell wall. Extracellular vesicle (EV)-enriched samples isolated from a lignin-forming cell suspension culture of Norway spruce (Picea abies L. Karst.) contained both phenolic metabolites and enzymes related to lignin biosynthesis. Metabolomic analysis revealed mono-, di- and oligolignols in the EV isolates, as well as carbohydrates and amino acids. In addition, salicylic acid (SA) and some proteins involved in SA signaling were detected in the EV-enriched samples. A proteomic analysis detected several laccases, peroxidases, ß-glucosidases, putative dirigent proteins, and cell wall-modifying enzymes such as glycosyl hydrolases, transglucosylase/hydrolases, and expansins in EVs. Our findings suggest that EVs are involved in transporting enzymes required for lignin polymerization in Norway spruce, and that radical coupling of monolignols can occur in these vesicles.
RESUMO
A comparative transcriptomic study and a single-cell metabolome analysis were combined to determine whether parenchymal ray cells contribute to the biosynthesis of monolignols in the lignifying xylem of Norway spruce (Picea abies). Ray parenchymal cells may function in the lignification of upright tracheids by supplying monolignols. To test this hypothesis, parenchymal ray cells and upright tracheids were dissected with laser-capture microdissection from tangential cryosections of developing xylem of spruce trees. The transcriptome analysis revealed that among the genes involved in processes typical for vascular tissues, genes encoding cell wall biogenesis-related enzymes were highly expressed in both developing tracheids and ray cells. Interestingly, most of the shikimate and monolignol biosynthesis pathway-related genes were equally expressed in both cell types. Nonetheless, 1,073 differentially expressed genes were detected between developing ray cells and tracheids, among which a set of genes expressed only in ray cells was identified. In situ single cell metabolomics of semi-intact plants by picoliter pressure probe-electrospray ionization-mass spectrometry detected monolignols and their glycoconjugates in both cell types, indicating that the biosynthetic route for monolignols is active in both upright tracheids and parenchymal ray cells. The data strongly support the hypothesis that in developing xylem, ray cells produce monolignols that contribute to lignification of tracheid cell walls.
Assuntos
Lignina/metabolismo , Picea/citologia , Picea/metabolismo , Xilema/citologia , Xilema/metabolismo , Vias Biossintéticas/genética , Parede Celular/metabolismo , Bases de Dados Genéticas , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Genes de Plantas , Metaboloma , Picea/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Xilema/anatomia & histologiaRESUMO
Both the mechanisms of monolignol transport and the transported form of monolignols in developing xylem of trees are unknown. We tested the hypothesis of an active, plasma membrane-localized transport of monolignol monomers, dimers, and/or glucosidic forms with membrane vesicles prepared from developing xylem and lignin-forming tissue-cultured cells of Norway spruce (Picea abies L. Karst.), as well as from control materials, comprising non-lignifying Norway spruce phloem and tobacco (Nicotiana tabacum L.) BY-2 cells. Xylem and BY-2 vesicles transported both coniferin and p-coumaryl alcohol glucoside, but inhibitor assays suggested that this transport was through the tonoplast. Membrane vesicles prepared from lignin-forming spruce cells showed coniferin transport, but the Km value for coniferin was much higher than those of xylem and BY-2 cells. Liquid chromatography-mass spectrometry analysis of membrane proteins isolated from spruce developing xylem, phloem, and lignin-forming cultured cells revealed multiple transporters. These were compared with a transporter gene set obtained by a correlation analysis with a selected set of spruce monolignol biosynthesis genes. Biochemical membrane vesicle assays showed no support for ABC-transporter-mediated monolignol transport but point to a role for secondary active transporters (such as MFS or MATE transporters). In contrast, proteomic and co-expression analyses suggested a role for ABC transporters and MFS transporters.
Assuntos
Picea , Lignina , Noruega , Proteômica , XilemaRESUMO
Lignans are bioactive compounds that are especially abundant in the Norway spruce (Picea abies L. Karst.) knotwood. By combining a variety of chromatographic, spectroscopic and imaging techniques, we were able to quantify, qualify and localise the easily extractable lignans in the xylem tissue. The knotwood samples contained 15 different lignans according to the gas chromatography-mass spectrometry analysis. They comprised 16% of the knotwood dry weight and 82% of the acetone extract. The main lignans were found to be hydroxymatairesinols HMR1 and HMR2. Cryosectioned and resin-embedded ultrathin sections of the knotwood were analysed with scanning transmission X-ray microscopy (STXM). Cryosectioning was found to retain only lignan residues inside the cell lumina. In the resin-embedded samples, lignan was interpreted to be unevenly distributed inside the cell lumina, and partially confined in deposits which were either readily present in the lumina or formed when OsO4 used in staining reacted with the lignans. Furthermore, the multi-technique characterisation enabled us to obtain information on the chemical composition of the structural components of knotwood. A simple spectral analysis of the STXM data gave consistent results with the gas chromatographic methods about the relative amounts of cell wall components (lignin and polysaccharides). The STXM analysis also indicated that a torus of a bordered pit contained aromatic compounds, possibly lignin.
Assuntos
Lignanas/análise , Microscopia Eletrônica de Transmissão e Varredura/métodos , Picea/química , Espectrometria por Raios X/métodos , Microtomografia por Raio-X/métodos , Lignanas/químicaRESUMO
The question of whether lignin is covalently linked to carbohydrates in native wood, forming what is referred to as lignin-carbohydrate complexes (LCCs), still lacks unequivocal proof. This is mainly due to the need to isolate lignin from woody materials prior to analysis, under conditions leading to partial chemical modification of the native wood polymers. Thus, the correlation between the structure of the isolated LCCs and LCCs in situ remains open. As a way to circumvent the problematic isolation, biomimicking lignin polymerization in vivo and in vitro is an interesting option. Herein, we report the detection of lignin-carbohydrate bonds in the extracellular lignin formed by tissue-cultured Norway spruce cells, and in modified biomimetic lignin synthesis (dehydrogenation polymers). Semi-quantitative 2D heteronuclear singular quantum coherence (HSQC)-, 31P -, and 13C-NMR spectroscopy were applied as analytical tools. Combining results from these systems, four types of lignin-carbohydrate bonds were detected; benzyl ether, benzyl ester, γ-ester, and phenyl glycoside linkages, providing direct evidence of lignin-carbohydrate bond formation in biomimicked lignin polymerization. Based on our findings, we propose a sequence for lignin-carbohydrate bond formation in plant cell walls.
Assuntos
Biomimética/métodos , Lignina/química , Polímeros/química , Espectroscopia de Ressonância MagnéticaRESUMO
Apoplastic events such as monolignol oxidation and lignin polymerization are difficult to study in intact trees. To investigate the role of apoplastic hydrogen peroxide (H2O2) in gymnosperm phenolic metabolism, an extracellular lignin-forming cell culture of Norway spruce (Picea abies) was used as a research model. Scavenging of apoplastic H2O2 by potassium iodide repressed lignin formation, in line with peroxidases activating monolignols for lignin polymerization. Time-course analyses coupled to candidate substrate-product pair network propagation revealed differential accumulation of low-molecular-weight phenolics, including (glycosylated) oligolignols, (glycosylated) flavonoids, and proanthocyanidins, in lignin-forming and H2O2-scavenging cultures and supported that monolignols are oxidatively coupled not only in the cell wall but also in the cytoplasm, where they are coupled to other monolignols and proanthocyanidins. Dilignol glycoconjugates with reduced structures were found in the culture medium, suggesting that cells are able to transport glycosylated dilignols to the apoplast. Transcriptomic analyses revealed that scavenging of apoplastic H2O2 resulted in remodulation of the transcriptome, with reduced carbon flux into the shikimate pathway propagating down to monolignol biosynthesis. Aggregated coexpression network analysis identified candidate enzymes and transcription factors for monolignol oxidation and apoplastic H2O2 production in addition to potential H2O2 receptors. The results presented indicate that the redox state of the apoplast has a profound influence on cellular metabolism.
Assuntos
Peróxido de Hidrogênio/metabolismo , Fenóis/metabolismo , Picea/metabolismo , Antioxidantes/metabolismo , Espaço Extracelular/metabolismo , Sequestradores de Radicais Livres/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Genes de Plantas , Lignina/metabolismo , Anotação de Sequência Molecular , Estresse Oxidativo , Picea/genética , Análise de Componente Principal , Transdução de Sinais , Especificidade por Substrato , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
Maize (Zea mays L.) suspension-cultured cells habituated to a cellulose biosynthesis inhibitor 2,6-dichlorobenzonitrile (DCB) have a modified cell wall, in which the reduction in the cellulose content is compensated by a network of highly cross-linked feruloylated arabinoxylans and the deposition of lignin-like polymers. For both arabinoxylan cross-linking and lignin polymerization, class III peroxidases (POXs) have been demonstrated to have a prominent role. For the first time, a comparative study of POX activity and isoforms in control and cellulose-impaired cells has been addressed, also taking into account their cellular distribution in different compartments. Proteins from the spent medium (SM), soluble cellular (SC), ionically (ICW) and covalently bound cell wall protein fractions were assayed for total and specific peroxidase activity by using coniferyl and sinapyl alcohol and ferulic acid as substrates. The isoPOX profile was obtained by isoelectric focusing. POX activity was higher in DCB-habituated than in non-habituated cells in all protein fractions at all cell culture stages. For all substrates assayed, SC and ICW fractions showed higher activity at the early log growth phase than at the late log phase. However, the highest POX activity in the spent medium was found at the late log phase. According to the isoPOX profiles, the highest diversity of isoPOXs was detected in the ICW and SM protein fractions. The latter fraction contained isoPOXs with higher activity in DCB-habituated cells. Some of the isoPOXs detected could be involved in cross-linking of arabinoxylans and in the lignin-like polymer formation in DCB-habituated cells.
Assuntos
Peroxidases/metabolismo , Zea mays/metabolismo , Celulose/metabolismo , Lignina/metabolismo , Nitrilas/metabolismoRESUMO
A proportion of the plant's l-ascorbate (vitamin C) occurs in the apoplast, where it and its metabolites may act as pro-oxidants and anti-oxidants. One ascorbate metabolite is 2,3-diketogulonate (DKG), preparations of which can non-enzymically generate H2O2 and delay peroxidase action on aromatic substrates. As DKG itself generates several by-products, we characterised these and their ability to generate H2O2 and delay peroxidase action. DKG preparations rapidly produced a by-product, compound (1), with λmax 271 and 251 nm at neutral and acidic pH respectively. On HPLC, (1) co-eluted with the major H2O2-generating and peroxidase-delaying principle. Compound (1) was slowly destroyed by ascorbate oxidase, and was less stable at pH 6 than at pH 1. Electrophoresis of an HPLC-enriched preparation of (1) suggested a strongly acidic (pKa ≈ 2.3) compound. Mass spectrometry suggested that un-ionised (1) has the formula C6H6O5, i.e. it is a reduction product of DKG (C6H8O7). In conclusion, compound (1) is the major H2O2-generating, peroxidase-delaying principle formed non-enzymically from DKG in the pathway ascorbate â dehydroascorbic acid â DKG â (1). We hypothesise that (1) generates apoplastic H2O2 (and consequently hydroxyl radicals) and delays cell-wall crosslinking - both these effects favouring wall loosening, and possibly playing a role in pathogen defence.
Assuntos
Ácido 2,3-Dicetogulônico/química , Peróxido de Hidrogênio/química , Peroxidase do Rábano Silvestre/químicaRESUMO
MAIN CONCLUSION: Externally added coniferyl alcohol at high concentrations reduces the growth of Nicotiana cells and seedlings. Coniferyl alcohol is metabolized by BY-2 cells to several compounds. Coniferyl alcohol (CA) is a common monolignol and a building block of lignin. The toxicity of monolignol alcohols has been stated in the literature, but there are only few studies suggesting that this is true. We investigated the physiological effects of CA on living plant cells in more detail. Tobacco (Nicotiana tabacum) Bright yellow-2 cells (BY-2) and Nicotiana benthamiana seedlings both showed concentration-dependent growth retardation in response to 0.5-5 mM CA treatment. In some cases, CA addition caused cell death in BY-2 cultures, but this response was dependent on the growth stage of the cells. Based on LC-MS/MS analysis, BY-2 cells did not accumulate the externally supplemented CA, but metabolized it to ferulic acid, ferulic acid glycoside, coniferin, and to some other phenolic compounds. In addition to growth inhibition, CA caused the formation of a lignin-like compound detected by phloroglucinol staining in N. benthamiana roots and occasionally in BY-2 cells. To prevent this, we added potassium iodide (KI, at 5 mM) to overcome the peroxidase-mediated CA polymerization to lignin. KI had, however, toxic effects on its own: in N. benthamiana seedlings, it caused reduction in growth; in BY-2 cells, reduction in growth and cell viability. Surprisingly, CA restored the growth of KI-treated BY-2 cells and N. benthamiana seedlings. Our results suggest that CA at high concentrations is toxic to plant cells.
Assuntos
Nicotiana/citologia , Fenóis/farmacologia , Plântula/efeitos dos fármacos , Nicotiana/efeitos dos fármacosRESUMO
Secondarily thickened cell walls of water-conducting vessels and tracheids and support-giving sclerenchyma cells contain lignin that makes the cell walls water impermeable and strong. To what extent laccases and peroxidases contribute to lignin biosynthesis in muro is under active evaluation. We performed an in silico study of Norway spruce (Picea abies (L.) Karst.) laccases utilizing available genomic data. As many as 292 laccase encoding sequences (genes, gene fragments, and pseudogenes) were detected in the spruce genome. Out of the 112 genes annotated as laccases, 79 are expressed at some level. We isolated five full-length laccase cDNAs from developing xylem and an extracellular lignin-forming cell culture of spruce. In addition, we purified and biochemically characterized one culture medium laccase from the lignin-forming cell culture. This laccase has an acidic pH optimum (pH 3.8-4.2) for coniferyl alcohol oxidation. It has a high affinity to coniferyl alcohol with an apparent Km value of 3.5 µM; however, the laccase has a lower catalytic efficiency (V(max)/K(m)) for coniferyl alcohol oxidation compared with some purified culture medium peroxidases. The properties are discussed in the context of the information already known about laccases/coniferyl alcohol oxidases of coniferous plants.
Assuntos
Lacase/metabolismo , Lignina/biossíntese , Picea/enzimologia , Técnicas de Cultura de Tecidos/métodos , Álcoois/metabolismo , Clonagem Molecular , Meios de Cultura , Eletroforese em Gel de Poliacrilamida , Genes de Plantas , Concentração de Íons de Hidrogênio , Cinética , Lacase/genética , Lacase/isolamento & purificação , Oxirredução , Picea/genética , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Análise de Sequência de ProteínaRESUMO
There are no earlier reports with successful isolation of plasma membranes from lignin-forming tissues of conifers. A method to isolate cellular membranes from extracellular lignin-producing tissue-cultured cells and developing xylem of Norway spruce was optimized. Modifications to the homogenization buffer were needed to obtain membranes from these phenolics-rich tissues. Membranes were separated by aqueous polymer two-phase partitioning. Chlorophyll a determination, marker enzyme assays and western blot analyses using antibodies for each membrane type showed that mitochondrial, chloroplastic and to a certain extent also ER and Golgi membranes were efficiently diminished from the upper phase, but tonoplast and plasma membranes distributed evenly between the upper and lower phases. Redox enzymes present in the partially purified membrane fractions were assayed in order to reveal the origin of H(2)O(2) needed for lignification. The membranes of spruce contained enzymes able to generate superoxide in the presence of NAD(P)H. Besides members of the flavodoxin and flavodoxin-like family proteins, cytochrome b5, cytochrome P450 and several stress responsive proteins were identified by nitroblue tetrazolium staining of isoelectric focusing gels and by mass spectrometry. Naphthoquinones juglone and menadione increased superoxide production in activity-stained gels. Some juglone-activated enzymes were preferentially using NADH. With NADH, menadione activated only some of the enzymes that juglone did, whereas with NADPH the activation patterns were identical. Duroquinone, a benzoquinone, did not affect superoxide production. Superoxide dismutase, ascorbate peroxidase, catalase and an acidic class III peroxidase isoenzyme were detected in partially purified spruce membranes. The possible locations and functions of these enzymes are discussed.
Assuntos
Lignina/metabolismo , Picea/metabolismo , Proteínas de Plantas/metabolismo , Catalase/metabolismo , Membrana Celular/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Peróxido de Hidrogênio/metabolismo , NAD/metabolismo , NADP/metabolismo , Oxirredução , Peroxidase/metabolismo , Peroxidases/metabolismo , Proteínas de Plantas/isolamento & purificação , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo , Xilema/metabolismoRESUMO
In order to develop more economic uses of lignin, greater knowledge regarding its native structure is required. This can inform the development of optimized extraction methods that preserve desired structural properties. Current extraction methods alter the polymeric structure of lignin, leading to a loss of valuable structural groups or the formation of new non-native ones. In this study, Norway spruce (Picea abies) tissue-cultured cells that produce lignin extracellularly in a suspension medium were employed. This system enables the investigation of unaltered native lignin, as no physicochemical extraction steps are required. For the first time, this culture was used to investigate the interactions between lignin and xylan, a secondary cell wall hemicellulose, and to study the importance of lignin-carbohydrate complexes (LCCs) on the polymerization and final structure of extracellular lignin (ECL). This has enabled us to study the impact of xylan on monolignol composition and structure of the final lignin polymer. We find that the addition of xylan to the solid culture medium accelerates cell growth and impacts the ratio of monolignols in the lignin. However, the presence of xylan in the lignin polymerization environment does not significantly alter the structural properties of lignin as analyzed by two-dimensional nuclear magnetic resonance (NMR) spectroscopy and size exclusion chromatography (SEC). Nevertheless, our data indicate that xylan can act as a nucleation point, leading to more rapid lignin polymerization, an important insight into biopolymer interactions during cell wall synthesis in wood. Lignin structure and interactions with a secondary cell wall hemicellulose were investigated in a model cell culture: we found that the polymerization and final structure of lignin are altered when the hemicellulose is present during cell growth and monolignol production. The physicochemical interactions between lignin and xylan partly define the extractability and utility of native lignin in high value applications, so this work has implications for lignin extraction as well as fundamental plant biology.
RESUMO
The blackening of cut carrots causes substantial economic losses to the food industry. Blackening was not observed in carrots that had been stored underground for less than a year, but the susceptibility to blackening increased with the age of the carrots that were stored underground for longer periods. Samples of black, border, and orange tissues from processed carrot batons and slices, prepared under industry standard conditions, were analyzed to identify the molecular and metabolic mechanisms underpinning processing-induced blackening. The black tissues showed substantial molecular and metabolic rewiring and large changes in the cell wall structure, with a decreased abundance of xyloglucan, pectins (homogalacturonan, rhamnogalacturonan-I, galactan and arabinan), and higher levels of lignin and other phenolic compounds when compared to orange tissues. Metabolite profiling analysis showed that there was a major shift from primary to secondary metabolism in the black tissues, which were depleted in sugars, amino acids, and tricarboxylic acid (TCA) cycle intermediates but were rich in phenolic compounds. These findings suggest that processing triggers a release from quiescence. Transcripts encoding proteins associated with secondary metabolism were less abundant in the black tissues, but there were no increases in transcripts associated with oxidative stress responses, programmed cell death, or senescence. We conclude that restraining quiescence release alters cell wall metabolism and composition, particularly regarding pectin composition, in a manner that increases susceptibility to blackening upon processing.
Assuntos
Daucus carota , Daucus carota/metabolismo , Células Vegetais , Lignina/metabolismo , Parede Celular/químicaRESUMO
Plant respiratory burst oxidase homologs (RBOHs) are plasma membrane-localized NADPH oxidases that generate superoxide anion radicals, which then dismutate to H2O2, into the apoplast using cytoplasmic NADPH as an electron donor. PaRBOH1 is the most highly expressed RBOH gene in developing xylem as well as in a lignin-forming cell culture of Norway spruce (Picea abies L. Karst.). Since no previous information about regulation of gymnosperm RBOHs exist, our aim was to resolve how PaRBOH1 is regulated with a focus on phosphorylation. The N-terminal part of PaRBOH1 was found to contain several putative phosphorylation sites and a four-times repeated motif with similarities to the Botrytis-induced kinase 1 target site in Arabidopsis AtRBOHD. Phosphorylation was indicated for six of the sites in in vitro kinase assays using 15 amino-acid-long peptides for each of the predicted phosphotarget site in the presence of protein extracts of developing xylem. Serine and threonine residues showing positive response in the peptide assays were individually mutated to alanine (kinase-inactive) or to aspartate (phosphomimic), and the wild type PaRBOH1 and the mutated constructs transfected to human kidney embryogenic (HEK293T) cells with a low endogenous level of extracellular ROS production. ROS-producing assays with HEK cells showed that Ca2+ and phosphorylation synergistically activate the enzyme and identified several serine and threonine residues that are likely to be phosphorylated including a novel phosphorylation site not characterized in other plant species. These were further investigated with a phosphoproteomic study. Results of Norway spruce, the first gymnosperm species studied in relation to RBOH regulation, show that regulation of RBOH activity is conserved among seed plants.
RESUMO
Lignin, a phenolic polymer abundant in cell walls of certain cell types, has given challenges to scientists studying its structure or biosynthesis. In plants lignified tissues are distributed between other, non-lignified tissues. Characterization of native lignin in the cell wall has been difficult due to the highly cross-linked nature of the wall components. Model systems, like plant tissue cultures with tracheary element differentiation or extracellular lignin formation, have provided useful information related to lignin structure and several aspects of lignin formation. For example, many enzyme activities in the phenylpropanoid pathway have been first identified in tissue cultures. This review focuses on studies where the use of plant tissue cultures has been advantageous in structural and biosynthesis studies of lignin, and discusses the validity of tissue cultures as models for lignin biosynthesis.
Assuntos
Lignina/biossíntese , Plantas/metabolismo , Transporte Biológico , Parede Celular/metabolismo , Modelos BiológicosRESUMO
Plant tissue cultures are an efficient system to study cell wall biosynthesis in living cells in vivo. Tissue cultures also provide cells and culture medium from which enzymes and cell wall polymers can easily be separated for further studies. Tissue cultures with tracheary element differentiation or extracellular lignin formation have provided useful information related to several aspects of xylem and lignin formation. In this chapter, methods for nutrient medium preparation and callus culture initiation and its maintenance as well as those for protoplast isolation and viability observation are described. As a case study, we describe the establishment of a xylogenic culture of Zinnia elegans mesophyll cells.
Assuntos
Plantas/metabolismo , Técnicas de Cultura de Tecidos/métodos , Asteraceae/citologia , Diferenciação Celular , Divisão Celular , Parede Celular/metabolismo , Células Cultivadas , Germinação , Células do Mesofilo/citologia , Células do Mesofilo/metabolismo , Folhas de Planta/citologia , Protoplastos/metabolismo , Esterilização , Nicotiana/citologiaRESUMO
A cell culture of Picea abies (L.) Karst. was used for studies of H(2)O(2) generation during constitutive extracellular lignin formation and after elicitation by cell wall fragments of a pathogenic fungus, Heterobasidium parviporum. Stable, micromolar levels of H(2)O(2) were present in the culture medium during lignin formation. Elicitation induced a burst of H(2)O(2), peaking at ca. 90 min after elicitation. Of exogenous reducing substrates that may be responsible for the synthesis of H(2)O(2) from O(2), NADH stimulated H(2)O(2) production irrespective of elicitation. Cysteine (Cys) and glutathione (GSH) partially scavenged the constitutive H(2)O(2), but usually increased or prolonged elicitor-induced H(2)O(2) formation. Culture medium peroxidases were not able to generate H(2)O(2) in vitro with Cys or GSH as reductants. These thiols, however, generated H(2)O(2) non-enzymically at pH 4.5. [(35)S]Sulphate feeding to spruce cells showed that endogenous sulphur-containing compounds (including GSH, GSSG and cysteic acid) existed in the culture medium. The apoplastic levels of these were, however, undetectable by the monobromobimane method suggesting that their contribution to apoplastic H(2)O(2) formation is probably minor. Azide, an inhibitor of haem-containing enzymes, slightly inhibited constitutive H(2)O(2) generation but strongly delayed the elicitor-induced H(2)O(2) accumulation. Diphenylene iodonium, an inhibitor of flavin-containing enzymes, efficiently inhibited H(2)O(2) production irrespective of elicitation. Elicitation led to downregulation of the expression of several peroxidase genes, and peroxidase activity in the culture medium was slightly reduced. Expression of three other peroxidase genes and a respiratory burst oxidase homologue (rboh) gene were upregulated. These data suggest that both peroxidases and rboh may contribute to H(2)O(2) generation.
Assuntos
Apoptose/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Lignina/biossíntese , Picea/metabolismo , Células Cultivadas , Picea/citologiaRESUMO
Gaining membrane vesicles from different plant species and tissue types is crucial for membrane studies. Membrane vesicles can be used for further purification of individual membrane types, and, for example, in studies of membrane enzyme activities, transport assays, and in proteomic analysis. Membrane isolation from some species, such as conifers, has proved to be more difficult than that of angiosperm species. In this paper, we describe steps for isolating cellular membranes from developing xylem, phloem, and lignin-forming tissue-cultured cells of Norway spruce, followed by partial enrichment of plasma membranes by aqueous polymer two-phase partitioning and purity analyses. The methods used are partially similar to the ones used for mono- and dicotyledonous plants, but some steps require discreet optimization, probably due to a high content of phenolic compounds present in the tissues and cultured cells of Norway spruce.
Assuntos
Fracionamento Celular/métodos , Membrana Celular , Picea/citologia , Técnicas de Cultura de Células , Lignina/metabolismo , Floema/citologia , Xilema/citologiaRESUMO
Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.