Federico Pomar

Induction of shikimate dehydrogenase and peroxidase in pepper (Capsicum annuum L.) seedlings in response to copper stress and its relation to lignification

Pepper (Capsicum annuum L.) plants growing in a nutrient solution with excess copper, showed an increase in shikimate dehydrogenase (SKDH, EC 1.1.1.25) and peroxidase (EC 1.11.1.7) activities in the hypocotyl. In the roots, peroxidase was also induced, but SKDH activity per organ was depleted rather than enhanced. Copper stress caused stunting in the plants, reflected by a decrease in the fresh weight of all the organs. In the hypocotyl, the induction of both enzymatic activities was associated with the accumulation of soluble phenolics and lignin. The two SKDH isozymes present in the control hypocotyls (SKDH-3 and SKDH-4) increased in a similar proportion after copper stress. In the case of peroxidases, two new isozymes (PRX-A2 and PRX-A4) were detected in copper-stressed hypocotyls, and the other two isoperoxidases, PRX-B and PRX-A3, were enhanced c. 10 and three times, respectively, with respect to the control. The application of the chelator EDTA was able to counteract all the stress effects of the metal cited above. The role of these enzymes in phenolic metabolism under heavy metal stress is discussed.

The Ve-mediated resistance response of the tomato to Verticillium dahliae involves H2O2, peroxidase and lignins and drives PAL gene expression

BackgroundVerticillium dahliae is a fungal pathogen that infects a wide range of hosts. The only known genes for resistance to Verticillium in the Solanaceae are found in the tomato (Solanum lycopersicum) Ve locus, formed by two linked genes, Ve1 and Ve2. To characterize the resistance response mediated by the tomato Ve gene, we inoculated two nearly isogenic tomato lines, LA3030 (ve/ve) and LA3038 (Ve/Ve), with V. dahliae.ResultsWe found induction of H2O2 production in roots of inoculated plants, followed by an increase in peroxidase activity only in roots of inoculated resistant plants. Phenylalanine-ammonia lyase (PAL) activity was also increased in resistant roots 2 hours after inoculation, while induction of PAL activity in susceptible roots was not seen until 48 hours after inoculation. Phenylpropanoid metabolism was also affected, with increases in ferulic acid, p-coumaric acid, vanillin and p-hydroxybenzaldehyde contents in resistant roots after inoculation. Six tomato PAL cDNA sequences (PAL1 - PAL6) were found in the SolGenes tomato EST database. RT-PCR analysis showed that these genes were expressed in all organs of the plant, albeit at different levels. Real-time RT-PCR indicated distinct patterns of expression of the different PAL genes in V. dahliae-inoculated roots. Phylogenetic analysis of 48 partial PAL cDNAs corresponding to 19 plant species grouped angiosperm PAL sequences into four clusters, suggesting functional differences among the six tomato genes, with PAL2 and PAL6 presumably involved in lignification, and the remaining PAL genes implicated in other biological processes.An increase in the synthesis of lignins was found 16 and 28 days after inoculation in both lines; this increase was greater and faster to develop in the resistant line. In both resistant and susceptible inoculated plants, an increase in the ratio of guaiacyl/syringyl units was detected 16 days after inoculation, resulting from the lowered amount of syringyl units in the lignins of inoculated plants.ConclusionsThe interaction between the tomato and V. dahliae triggered a number of short- and long-term defensive mechanisms. Differences were found between compatible and incompatible interactions, including onset of H2O2 production and activities of peroxidase and PAL, and phenylpropanoid metabolism and synthesis of lignins.

Peroxidase: a multifunctional enzyme in grapevines

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.

Bioinformatic and functional characterization of the basic peroxidase 72 from Arabidopsis thaliana involved in lignin biosynthesis.

Lignins result from the oxidative polymerization of three hydroxycinnamyl (p-coumaryl, coniferyl, and sinapyl) alcohols in a reaction mediated by peroxidases. The most important of these is the cationic peroxidase from Zinnia elegans (ZePrx), an enzyme considered to be responsible for the last step of lignification in this plant. Bibliographical evidence indicates that the arabidopsis peroxidase 72 (AtPrx72), which is homolog to ZePrx, could have an important role in lignification. For this reason, we performed a bioinformatic, histochemical, photosynthetic, and phenotypical and lignin composition analysis of an arabidopsis knock-out mutant of AtPrx72 with the aim of characterizing the effects that occurred due to the absence of expression of this peroxidase from the aspects of plant physiology such as vascular development, lignification, and photosynthesis. In silico analyses indicated a high homology between AtPrx72 and ZePrx, cell wall localization and probably optimal levels of translation of AtPrx72. The histochemical study revealed a low content in syringyl units and a decrease in the amount of lignin in the atprx72 mutant plants compared to WT. The atprx72 mutant plants grew more slowly than WT plants, with both smaller rosette and principal stem, and with fewer branches and siliques than the WT plants. Lastly, chlorophyll a fluorescence revealed a significant decrease in ΦPSII and qL in atprx72 mutant plants that could be related to changes in carbon partitioning and/or utilization of redox equivalents in arabidopsis metabolism. The results suggest an important role of AtPrx72 in lignin biosynthesis. In addition, knock-out plants were able to respond and adapt to an insufficiency of lignification.

Structural motifs of syringyl peroxidases predate not only the gymnosperm–angiosperm divergence but also the radiation of tracheophytes

•  The most distinctive variation in the monomer composition of lignins in vascular land plants is that found between the two main groups of seed plants. Thus, while gymnosperm lignins are typically composed of guaiacyl (G) units, angiosperm lignins are largely composed of similar levels of G and syringyl (S) units. •  However, and contrary to what might be expected, peroxidases isolated from basal (Cycadales and Ginkgoales) and differentially evolved (Coniferales and Gnetales) gymnosperms are also able to oxidize S moieties, and this ability is independent of the presence or absence of S-type units in their lignins. •  The results obtained led us to look at the protein database to search for homologies between gymnosperm peroxidases and true eudicot S-peroxidases, such as the Zinnia elegans peroxidase. •  The findings showed that certain structural motifs characteristic of eudicot S-peroxidases (certain amino acid sequences and β-sheet secondary structures) predate the gymnosperm-angiosperm divergence and the radiation of tracheophytes, since they are found not only in peroxidases from basal gymnosperms, ferns and lycopods, but also in peroxidases from the moss Physcomitrella patens (Bryopsida) and the liverwort Marchantia polymorpha (Marchantiopsida), which, as typical of bryophytes, do not have xylem tissue nor lignins.

The presence of sinapyl lignin in Ginkgo biloba cell cultures changes our views of the evolution of lignin biosynthesis

Suspension cell cultures (SCCs) from one of the oldest seed plants, Ginkgo biloba, show unpredictable alterations in the nature of the lignins, such as is the recruitment of sinapyl alcohol for lignin biosynthesis, compared with the woody tissues of the same species, which lack syringyl (S) lignins. These results show that, in this gymnosperm, the genes involved in sinapyl alcohol biosynthesis are latent and that their regulatory regions respond, by initiating gene expression, to the developmental signals and the environmental clues, which condition its in vitro culture. G. biloba SCCs not only synthesize S lignins but also their extracellular proteome contains both class III peroxidases capable of oxidizing sinapyl alcohol and enzymes involved in H2O2 production, observation which suggests that the peroxidase branch for the oxidative coupling of sinapyl alcohol units into lignins is operative. The incomplete knowledge of the G. biloba peroxidase-encoding genes led us to purify, characterize and partially sequence the peroxidase responsible for monolignol oxidation. When the major peroxidase from G. biloba SCCs (GbPrx) was purified to homogeneity, it showed absorption maxima in the visible region at 414 (Soret band), and at 543 and 570 nm, which calls to mind those shown by low-spin ferric peroxidases. However, the results also showed that the paraperoxidase-like character of GbPrx is not an obstacle for oxidizing the three monolignols compared with high-spin ferric peroxidases. Taken together, these results mean that the time at which the evolutionary gain of the segment of the route that leads to the biosynthesis of S lignins took place in seed plants needs to be revised.

Purification, characterization and kinetic properties of pepper fruit acidic peroxidase

Abstract A soluble acidic peroxidase (EC 1.11.1.7) was purified about 300-fold from the pericarp of pepper ( Capsicum annuum L.) fruits by ammonium sulphate fractionation followed by chromatography in columns of Sephadex G-100, Q-Sepharose and Superose 12 PC 3.2/30. The purified enzyme has a pI of 3.8 and a M r , determined by gel filtration, of 50 k. The enzyme was stable in a pH range from pH 6 to 9 and was resistant to high temperature. The ability of the acidic peroxidase to oxidize capsaicin was studied. The oxidation follows the accepted model for peroxidase oxidations, in which compound I (CoI) and compound II (CoII) appear to be the main intermediates in the catalytic cycle. Kinetic constants for H 2 O 2 [ K 1 (CoI formation constant) = 41 μM −1 sec −1 ] and for capsaicin [ K 3 (CoII reduction constant) = 3.5 μM −1 sec −1 ] suggest that the acidic peroxidase has a higher H 2 O 2 reactivity than other peroxidases, and that capsaicin is a good substrate for CoII reduction.

Characterization of the last step of lignin biosynthesis in Zinnia elegans suspension cell cultures

The last step of lignin biosynthesis in Zinnia elegans suspension cell cultures (SCCs) catalyzed by peroxidase (ZePrx) has been characterized. The k 3 values shown by ZePrx for the three monolignols revealed that sinapyl alcohol was the best substrate, and were proportional to their oxido/reduction potentials, signifying that these reactions are driven exclusively by redox thermodynamic forces. Feeding experiments demonstrate that cell wall lignification in SCCs is controlled by the rate of supply of H2O2. The results also showed that sites for monolignol β‐O‐4 cross‐coupling in cell walls may be saturated, suggesting that the growth of the lineal lignin macromolecule is not infinite.

The suppression of AtPrx52 affects fibers but not xylem lignification in Arabidopsis by altering the proportion of syringyl units

Lignins result from the oxidative polymerization of three hydroxycinnamyl (p-coumaryl, coniferyl and sinapyl) alcohols in a reaction mediated by peroxidases (EC 1.11.1.7) and laccases (EC 1.10.3.2), yielding H, G and S units, respectively. Although both acidic and basic peroxidases can oxidize p-coumaryl and coniferyl alcohol, only basic peroxidases are able to oxidize sinapyl alcohol. The AtPrx52 from Arabidopsis is a basic peroxidase that has been reported to be highly homologous to the basic peroxidase of Zinnia elegans, the only peroxidase which has been unequivocally linked to lignin formation. Here, we show how the suppression of AtPrx52 causes a change in lignin composition, mainly at the level of stem interfascicular fibers. Quantification of lignins in two different atprx52 knock-out mutants revealed a decrease of lignin amount compared with wild type. The S/G ratio, obtained by both nitrobenzene oxidation and thioacidolysis, indicated a decrease in S units in the atprx52 mutants. As deduced from Wiesner and mainly Mäule staining, this reduction in S unit content appears to be restricted to the interfascicular fibers. Moreover, quantitative polymerase chain reaction analysis in atprx52 plants showed a general downregulation of genes involved in lignin biosynthetic pathway, as well as genes related to secondary cell wall. On the other hand, other routes from phenylpropanoid metabolism were induced. Taken together, our results indicate that AtPrx52 is involved in the synthesis of S units in interfascicular fibers at late stages of the lignification process.

Peroxidase 4 is involved in syringyl lignin formation in Arabidopsis thaliana.

Syringyl lignins result from the oxidative polymerization of sinapyl alcohol in a reaction mediated by syringyl (basic) peroxidases. Several peroxidases have been identified in the genome of Arabidopsis thaliana as close homologues to ZePrx, the best characterized basic peroxidase so far, but none of these has been directly involved in lignification. We have used a knock-out mutant of AtPrx4, the closest homologue to ZePrx, to study the involvement of this basic peroxidase in the physiology of the plant under both long- and short-day light conditions. Our results suggest that AtPrx4 is involved in cell wall lignification, especially in syringyl monomer formation. The disruption of AtPrx4 causes a decrease in syringyl units proportion, but only when light conditions are optimal. Moreover, the effect of AtPrx4 disruption is age-dependent, and it is only significant when the elongation process of the stem has ceased and lignification becomes active. In conclusion, AtPrx4 emerges as a basic peroxidase regulated by day length with an important role in lignification.