RESUMEN

Cell wall-bound hydroxycinnamic acids and the composition of lignin were studied in relation to the digestibility of a collection of 91 maize silages in wethers. Total lignin and guaiacyl content showed the highest correlation coefficients with digestibility. Using the above-mentioned chemical parameters, eight equations were also developed to predict digestibility. The prediction of organic matter digestibility produced a high adjusted R2 value (0.487) using total lignin, guaiacyl, esterified ferulic acid and esterified p-coumaric acid content as predictors. The prediction of in vivo dry matter digestibility produced a higher adjusted R2 value (0.516) using the same variables as predictors. Cell wall digestibility depends on a multiplicity of factors and it is not possible to attribute a causal effect on in vivo digestibility to any single factor. However, total lignin, guaiacyl and p-coumaric acid content emerge as good predictors of digestibility.

Asunto(s)

Ácidos Cumáricos/química , Digestión/fisiología , Lignina/química , Ovinos/fisiología , Ensilaje/análisis , Zea mays/química , Alimentación Animal/análisis , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Dieta/veterinaria , Masculino

RESUMEN

Class III peroxidases (Prxs) are plant enzymes capable of using H(2)O(2) to oxidize a range of plant secondary metabolites, notably phenolic compounds. These enzymes are localized in the cell wall or in the vacuole, which is a target for secondary metabolite accumulation, but very little is known about the function of vacuolar Prxs. Here, the physiological role of the main leaf vacuolar Prx of the medicinal plant Catharanthus roseus, CrPrx1, was further investigated namely by studying its capacity to oxidize co-localized phenolic substrates at the expense of H(2)O(2). LC-PAD-MS analysis of the phenols from isolated leaf vacuoles detected the presence of three caffeoylquinic acids and four flavonoids in this organelle. These phenols or similar compounds were shown to be good CrPrx1 substrates, and the CrPrx1-mediated oxidation of 5-O-caffeoylquinic acid was shown to form a co-operative regenerating cycle with ascorbic acid. Interestingly, more than 90% of total leaf Prx activity was localized in the vacuoles, associated to discrete spots of the tonoplast. Prx activity inside the vacuoles was estimated to be 1809 nkat ml(-1), which, together with the determined concentrations for the putative vacuolar phenolic substrates, indicate a very high H(2)O(2) scavenging capacity, up to 9 mM s(-1). Accordingly, high light conditions, known to increase H(2)O(2) production, induced both phenols and Prx levels. Therefore, it is proposed that the vacuolar couple Prx/secondary metabolites represent an important sink/buffer of H(2)O(2) in green plant cells.

Asunto(s)

Catharanthus/enzimología , Peróxido de Hidrógeno/metabolismo , Peroxidasa/metabolismo , Fenoles/metabolismo , Plantas Medicinales/enzimología , Vacuolas/enzimología , Ácido Ascórbico/metabolismo , Catharanthus/efectos de la radiación , Catharanthus/ultraestructura , Isoenzimas/metabolismo , Luz , Espectrometría de Masas , Células del Mesófilo/citología , Células del Mesófilo/enzimología , Células del Mesófilo/efectos de la radiación , Células del Mesófilo/ultraestructura , Oxidación-Reducción/efectos de la radiación , Fenoles/química , Fenoles/aislamiento & purificación , Extractos Vegetales , Hojas de la Planta/enzimología , Hojas de la Planta/efectos de la radiación , Hojas de la Planta/ultraestructura , Plantas Medicinales/efectos de la radiación , Plantas Medicinales/ultraestructura , Protoplastos/metabolismo , Espectrofotometría Ultravioleta , Especificidad por Sustrato/efectos de la radiación , Factores de Tiempo , Vacuolas/efectos de la radiación , Vacuolas/ultraestructura

RESUMEN

Suspension cultured cells of Capsicum chinense secrete proteins to the culture medium in both control conditions and under methyl jasmonate treatment. The exogenous application of methyl jasmonate induced the accumulation of putative pathogenesis-related proteins, class I chitinase, leucin-rich repeat protein, NtPRp27-like protein and pectinesterase which were also found in suspension cultured cells of C. annuum elicited with methyl jasmonate. However, a germin-like protein, which has never been described in methyl jasmonate-elicited C. chinense suspension cultured cells, was found. The different effects described as being the result of exogenous application of signalling molecules like methyl jasmonate on the expression of germin-like protein suggest that germin-like proteins may play a variety of roles in protecting plants against pathogen attacks and different stresses. Further studies will be necessary to characterize the differential expression of these pathogenesis-related proteins and to throw light on the complexity of their regulation.

Asunto(s)

Acetatos/farmacología , Capsicum/efectos de los fármacos , Capsicum/metabolismo , Ciclopentanos/farmacología , Oxilipinas/farmacología , Proteínas de Plantas/metabolismo , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos

RESUMEN

Capsicum annuum suspension cell cultures were used to evaluate the effect of cyclodextrins and methyl jasmonate as elicitors of defence responses. The induced defence responses included the accumulation of sesquiterpenes and phytosterols and the activation of pathogenesis-related proteins, leading to reinforcement and modification of the cell wall architecture during elicitation and protection cells against biotic stress. The results showed that the addition of both cyclodextrins and methyl jasmonate induced the biosynthesis of two sesquiterpenes, aromadendrene and solavetivone. This response was clearly synergistic since the increase in the levels of these compounds was much greater in the presence of both elicitors than when they were used separately. The biosynthesis of phytosterols was also induced in the combined treatment, as the result of an additive effect. Likewise, the exogenous application of methyl jasmonate induced the accumulation of pathogenesis-related proteins. The analysis of the extracellular proteome showed the presence of amino acid sequences homologous to PR1 and 4, NtPRp27-like proteins and class I chitinases, peroxidases and the hydrolytic enzymes LEXYL1 and 2, arabinosidases, pectinases, nectarin IV and leucin-rich repeat protein, which suggests that methyl jasmonate plays a role in mediating defence-related gene product expression in C. annuum. Apart from these methyl jamonate-induced proteins, other PR proteins were found in both the control and elicited cell cultures of C. annuum. These included class IV chitinases, beta-1,3-glucanases, thaumatin-like proteins and peroxidases, suggesting that their expression is mainly constitutive since they are involved in growth, development and defence processes.

Asunto(s)

Capsicum/citología , Capsicum/metabolismo , Espacio Extracelular/metabolismo , Fitosteroles/biosíntesis , Proteínas de Plantas/metabolismo , Sesquiterpenos/metabolismo , Acetatos/farmacología , Secuencia de Aminoácidos , Capsicum/efectos de los fármacos , Células Cultivadas , Ciclopentanos/farmacología , Electroforesis en Gel de Poliacrilamida , Espacio Extracelular/efectos de los fármacos , Espectrometría de Masas , Datos de Secuencia Molecular , Oxilipinas/farmacología , Péptidos/química , Proteínas de Plantas/química , Proteoma/metabolismo , Sesquiterpenos/química , beta-Ciclodextrinas/farmacología

RESUMEN

Hypocotyl formation during the epigeal germination of seedlings is under strict hormonal regulation. In a 3 d old Zinnia elegans seedling system, gibberellic acid (GA(3)) exerts an opposite effect to that exerted by light on hypocotyl photomorphogenesis because GA(3) promotes an etiolated-like growth with an inhibition of radial (secondary) growth. For this reason, the effect of GA(3) on the basic peroxidase isoenzyme from Z. elegans (ZePrx), an enzyme involved in hypocotyl lignin biosynthesis, was studied. The results showed that GA(3) reduces ZePrx activity, similarly to the way in which it reduces seedling secondary growth. This hormonal response is supported by the analysis of the ZePrx promoter, which contains four types of GA(3)-responsive cis-elements: the W Box/O2S; the Pyr Box; the GARE; and the Amy Box. Taken together, these results suggest that ZePrx is directly regulated by GA(3), with this effect matching the inhibitory effect of GA on the hypocotyl secondary growth.

Asunto(s)

Asteraceae/efectos de los fármacos , Asteraceae/metabolismo , Giberelinas/farmacología , Isoenzimas/metabolismo , Peroxidasa/metabolismo , Plantones/efectos de los fármacos , Plantones/metabolismo , Asteraceae/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Plantones/crecimiento & desarrollo

RESUMEN

Catharanthus roseus produces low levels of two dimeric terpenoid indole alkaloids, vinblastine and vincristine, which are widely used in cancer chemotherapy. The dimerization reaction leading to alpha-3',4'-anhydrovinblastine is a key regulatory step for the production of the anticancer alkaloids in planta and has potential application in the industrial production of two semisynthetic derivatives also used as anticancer drugs. In this work, we report the cloning, characterization, and subcellular localization of an enzyme with anhydrovinblastine synthase activity identified as the major class III peroxidase present in C. roseus leaves and named CrPrx1. The deduced amino acid sequence corresponds to a polypeptide of 363 amino acids including an N-terminal signal peptide showing the secretory nature of CrPrx1. CrPrx1 has a two-intron structure and is present as a single gene copy. Phylogenetic analysis indicates that CrPrx1 belongs to an evolutionary branch of vacuolar class III peroxidases whose members seem to have been recruited for different functions during evolution. Expression of a green fluorescent protein-CrPrx1 fusion confirmed the vacuolar localization of this peroxidase, the exact subcellular localization of the alkaloid monomeric precursors and dimeric products. Expression data further supports the role of CrPrx1 in alpha-3',4'-anhydrovinblastine biosynthesis, indicating the potential of CrPrx1 as a target to increase alkaloid levels in the plant.

Asunto(s)

Alcaloides/metabolismo , Alcaloides/farmacología , Antineoplásicos Fitogénicos/metabolismo , Catharanthus/enzimología , Peroxidasas/genética , Peroxidasas/metabolismo , Vacuolas/enzimología , Secuencia de Aminoácidos , Antineoplásicos Fitogénicos/farmacología , Secuencia de Bases , Catharanthus/genética , Clonación Molecular , Regulación de la Expresión Génica de las Plantas , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo

RESUMEN

Lignins are cell wall heteropolymers that arise from the peroxidase-mediated coupling of p-coumaryl, coniferyl and sinapyl alcohols. In gymnosperms, they are derived from coniferyl alcohol, whereas in angiosperms, lignins are derived from coniferyl and sinapyl alcohols. Thus, although it is frequently assumed that the chemical complexity of lignins has increased during plant evolution, it is frequently forgotten that pteridophytes have lignins that are derived from sinapyl alcohol. Until recently, most peroxidases characterized in flowering plants only oxidized coniferyl alcohol. However, recent reports have described the molecular characterization of peroxidases capable of oxidizing sinapyl alcohol (syringyl peroxidases). Current molecular studies propose that the structural motifs of syringyl peroxidases predate the radiation of tracheophytes, which suggests that syringyl peroxidases existed before the appearance of syringyl lignins.

Asunto(s)

Lignina/metabolismo , Peroxidasas/química , Peroxidasas/metabolismo , Syringa/enzimología , Secuencia de Aminoácidos , Catálisis , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Plantas/metabolismo , Conformación Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

The major basic peroxidase (ZePrx) from Zinnia elegans suspension cell cultures was purified and cloned. The purification resolved ZePrxs in two isoforms (ZePrx33.44 and ZePrx34.70), whose co-translational and post-translational modifications are characterized. Based on the N-terminal sequence obtained by Edman degradation of mature ZePxs, it may be expected that the immature polypeptides of ZePrxs contain a signal peptide (N-terminal pro-peptide) of 30 amino acids, which directs the polypeptide chains to the ER membrane. These immature polypeptides are co-translationally processed by proteolytic cleavage, and modeling studies of digestions suggested that the processing of the N-terminal pro-peptide of ZePrxs is performed by a peptidase from the SB clan (S8 family, subfamily A) of serine-type proteases. When the post-translational modifications of ZePrxs were characterized by trypsin digestion, and tryptic peptides were analyzed by reverse phase nano liquid chromatography (RP-nanoLC) coupled to MALDI-TOF MS, it was seen that, despite the presence in the primary structure of the protein of several (disulphide bridges, N-glycosylation, phosphorylation and N-myristoylation) potential post-translational modification sites, ZePrxs are only post-translationated modified by the formation of N-terminal pyroglutamate residues, disulphide bridges and N-glycosylation. Glycans of ZePrxs belong to three main types and conduce to the existence of at least ten different molecular isoforms. The first glycans belong to both low and high mannose-type glycans, with the growing structure Man(3-9)(GlcNAc)(2). Low mannose-type glycans, Man(3-4)(GlcNAc)(2), coexist with the truncated (paucimannosidic-type) glycan, Man(3)Xyl(1)Fuc(1)(GlcNAc)(2), in the G(3) and G(4 )sub-isoforms of ZePrx33.44. In ZePrx34.70, on the other hand, the complex-type biantennary glycan, Man(3)Xyl(1)Fuc(3)(GlcNAc)(5), and the truncated (paucimannosidic-type) glycan, Man(3)Xyl(1)Fuc(1)(GlcNAc)(2), appear to fill the two putative sites for N-glycosylation. Since the two N-glycosylation sites in ZePrxs are located in an immediately upstream loop region of helix F'' (close to the proximal histidine) and in helix F'' itself, and are flanked by positive-charged amino acids that produce an unusual positive-net surface electrostatic charge pattern, it may be expected that glycans not only affect reaction dynamics but may well participate in protein/cell wall interactions. These results emphasize the complexity of the ZePrx proteome and the difficulties involved in establishing any fine structure-function relationship.

Asunto(s)

Asteraceae/enzimología , Peroxidasa/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Asteraceae/genética , ADN Complementario/genética , Activación Enzimática/efectos de los fármacos , Glicosilación , Interacciones Hidrofóbicas e Hidrofílicas , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Peroxidasa/química , Peroxidasa/genética , Polisacáridos/metabolismo , Biosíntesis de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Electricidad Estática , Homología Estructural de Proteína , Compuestos de Sulfhidrilo/farmacología

RESUMEN

Comparison of the stability of five plant peroxidases (horseradish, royal palm tree leaf, soybean, and cationic and anionic peanut peroxidases) was carried out under acidic conditions favorable for synthesis of polyelectrolyte complexes of polyaniline (PANI). It demonstrates that palm tree peroxidase has the highest stability. Using this peroxidase as a catalyst, the enzymatic synthesis of polyelectrolyte complexes of PANI and poly(2-acrylamido-3-methyl-1-propanesulfonic acid) (PAMPS) was developed. The template polymerization of aniline was carried out in aqueous buffer at pH 2.8. Varying the concentrations of aniline, PAMPS, and hydrogen peroxide as reagents, favorable conditions for production of PANI were determined. UV-vis-NIR absorption and EPR demonstrated that PAMPS and PANI formed the electroactive complex similar to PANI doped traditionally using low molecular weight sulfonic acids. The effect of pH on conformational variability of the complex was evaluated by UV-vis spectroscopy. Atomic force microscopy showed that a size of the particles of the PANI-PAMPS complexes varied between 10 and 25 nm, depending on a concentration of PAMPS in the complex. The dc conductivity of the complexes depends also on the content of PAMPS, the higher conductivity being for the complexes containing the lower content of the polymeric template.

Asunto(s)

Compuestos de Anilina/síntesis química , Peroxidasa/metabolismo , Polímeros/síntesis química , Ácidos Sulfónicos/síntesis química , Árboles/enzimología , Compuestos de Anilina/análisis , Catálisis/efectos de los fármacos , Electrólitos , Concentración de Iones de Hidrógeno , Polímeros/análisis , Ácidos Sulfónicos/análisis

RESUMEN

Peroxidases are heme-containing enzymes that catalyse the one-electron oxidation of several substrates at the expense of H2O2. They are probably encoded by a large multigene family in grapevines, and therefore show a high degree of polymorphism. Grapevine peroxidases are glycoproteins of high thermal stability, whose molecular weight usually ranges from 35 to 45 kDa. Their visible spectrum shows absorption bands characteristic of high-spin class III peroxidases. Grapevine peroxidases are capable of accepting a wide range of natural compounds as substrates, such as the cell wall protein extensin, plant growth regulators such as IAA, and phenolics such as benzoic acids, stilbenes, flavonols, cinnamyl alcohols and anthocyanins. They are located in cell walls and vacuoles. These locations are in accordance with their key role in determining the final cell wall architecture, especially regarding lignin deposition and extensin insolubilization, and the turnover of vacuolar phenolic metabolites, a task that also forms part of the molecular program of disease resistance. Although peroxidase is a constitutive enzyme in grapevines, its levels are strongly modulated during plant cell development and in response to both biotic and abiotic environmental factors. To gain an insight into the metabolic regulation of peroxidase, several authors have studied how grapevine peroxidase and H2O2 levels change in response to a changing environment. Nevertheless, the results obtained are not always easy to interpret. Despite such difficulties, the response of the peroxidase-H2O2 system to both UV-C radiation and Trichoderma viride elicitors is worthy of study. Both UV-C and T. viride elicitors induce specific changes in peroxidase isoenzyme / H2O2 levels, which result in specific changes in grapevine physiology and metabolism. In the case of T. viride-elicited grapevine cells, they show a particular mechanism for H2O2 production, in which NADPH oxidase-like activities are apparently not involved. However, they offer a unique system whereby the metabolic regulation of peroxidase by H2O2, with all its cross-talks and downstream signals, may be elegantly dissected.