Fuencisla Merino

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.

O-4-Linked coniferyl and sinapyl aldehydes in lignifying cell walls are the main targets of the Wiesner (phloroglucinol-HCl) reaction.

Summary. The nature and specificity of the Wiesner test (phloroglucinol-HCl reagent) for the aromatic aldehyde fraction contained in lignins is studied. Phloroglucinol reacted in ethanol-hydrochloric acid with coniferyl aldehyde, sinapyl aldehyde, vanillin, and syringaldehyde to yield either pink pigments (in the case of hydroxycinnamyl aldehydes) or red-brown pigments (in the case of hydroxybenzaldehydes). However, coniferyl alcohol, sinapyl alcohol, and highly condensed dehydrogenation polymers derived from these cinnamyl alcohols and aldehydes did not react with phloroglucinol in ethanol-hydrochloric acid. The differences in the reactivity of phloroglucinol with hydroxycinnamyl aldehydes and their dehydrogenation polymers may be explained by the fact that, in the latter, the unsubstituted (α,β-unsaturated) cinnamaldehyde functional group, which is responsible for the dye reaction, is lost due to lateral chain cross-linking reactions involving the β carbon. Fourier transform infrared spectroscopy and thioacidolysis analyses of phloroglucinol-positive lignifying plant cell walls belonging to the plant species Zinnia elegans L., Capsicum annuum var. annuum, Populus alba L., and Pinus halepensis L. demonstrated the presence of 4-O-linked hydroxycinnamyl aldehyde end groups and 4-O-linked 4-hydroxy-3-methoxy-benzaldehyde (vanillin) end groups in lignins. However, given the relatively low abundance of 4-O-linked vanillin in lignifying cell walls and the low extinction coefficient of its red-brown phloroglucinol adduct, it is unlikely that vanillin contributes to a great extent to the phloroglucinol-positive stain reaction. These results suggest that the phloroglucinol-HCl pink stain of lignifying xylem cell walls actually reveals the 4-O-linked hydroxycinnamyl aldehyde structures contained in lignins. Histochemical studies showed that these aldehyde structures are assembled, as in the case of coniferyl aldehyde, during the early stages of xylem cell wall lignification.

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.

Distribution of lignin monomers and the evolution of lignification among lower plants.

Through application of chemical, biochemical and histochemical analyses, we provide new data on the absence/presence of syringyl lignins in the algal species Mastocarpus stellatus, Cystoseira baccata and Ulva rigida, the bryophytes Physcomitrella patens and Marchantia polymorpha, the lycophytes Selaginella martensii, Isoetes fluitans and Isoetes histrix, the sphenophyte Equisetum telmateia, the ferns Ceratopteris thalictroides, Ceratopteris cornuta, Pteridium aquilinum, Phyllitis scolopendrium and Dryopteris affinis, and the angiosperm Posidonia oceanica. Lignins, and especially syringyl lignins, are distributed from non-vascular basal land plants, such as liverworts, to lycopods and ferns. This distribution, along with the already reported presence of syringyl lignins in ginkgoopsids, suggests that syringyl lignin is a primitive character in land plant evolution. Here, we discuss whether the pathway for sinapyl alcohol recruitment was iterative during the evolution of land plants or, alternatively, was incorporated into the earliest land plants and subsequently repressed in several basal liverworts, lycopods, equisetopsids and ferns. This last hypothesis, which is supported by recent studies of transcriptional regulation of the biosynthesis of lignins, implies that lignification originated as a developmental enabler in the peripheral tissues of protracheophytes and would only later have been co-opted for the strengthening of tracheids in eutracheophytes.

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.

Differential activation of defense-related genes in susceptible and resistant pepper cultivars infected with Phytophthora capsici

This study investigated the expression pattern of genes encoding for a basic PR-1 protein, a basic beta-1,3-glucanase, a peroxidase, and a sesquiterpene cyclase involved in defense responses in three pepper cultivars with different levels of resistance to Phytophthora capsici. All genes were up-regulated in infected stems of the pepper cultivars, with expression being detected 8h post-inoculation. mRNA levels of these genes increased markedly by 24h post-inoculation, and maximal induction levels were observed for the PR-1 and sesquiterpene cyclase genes. PR-1, peroxidase, and sesquiterpene genes were always expressed at higher levels in resistant cultivars than in the susceptible cultivar, although up-regulation was observed in both, suggesting that the differences between these pepper genotypes in susceptibility and resistance are a matter of the timing and magnitude of the defense response.

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.

Wound-induced shikimate dehydrogenase and peroxidase related to lignification in pepper (Capsicum annuum L.) leaves

Summary Wounding on non-excised Capsicum annuum leaves, maintained on the whole growing plant, causes an increase in shikimate dehydrogenase (SKDH, EC 1.1.1.25) and peroxidase (EC 1.11.1.7) activity. The observed induction of both enzymatic activities was related to lignin accumulation, and in the case of peroxidase, it was located in the vicinity of the wounds. However, wounding did not cause changes in soluble phenolic compounds. PAGE analysis showed that the shikimate dehydrogenase pattern was the same in both control and wounded leaves, but each isozyme was enhanced to a different degree: SKDH-1 = SKDH-2 > SKDH-5 > SKDH-4 > SKDH-3. The relationship between the degree of induction and both subcellular location and biosynthetic function is proposed. In the case of peroxidases, one new acidic isozyme (PRX-A2, pI 4.5) was detected by isoelectric focusing in wounded leaves, and another one (PRX-A4, pI 3.7) was enhanced more than 8 times with respect to control leaves. The possible role of these isozymes in lignin biosynthesis and wound healing is discussed.

New Insights into Capsicum spp Relatedness and the Diversification Process of Capsicum annuum in Spain

The successful exploitation of germplasm banks, harbouring plant genetic resources indispensable for plant breeding, will depend on our ability to characterize their genetic diversity. The Vegetable Germplasm Bank of Zaragoza (BGHZ) (Spain) holds an important Capsicum annuum collection, where most of the Spanish pepper variability is represented, as well as several accessions of other domesticated and non-domesticated Capsicum spp from all over the five continents. In the present work, a total of 51 C. annuum landraces (mainly from Spain) and 51 accessions from nine Capsicum species maintained at the BGHZ were evaluated using 39 microsatellite (SSR) markers spanning the whole genome. The 39 polymorphic markers allowed the detection of 381 alleles, with an average of 9.8 alleles per locus. A sizeable proportion of alleles (41.2%) were recorded as specific alleles and the majority of these were present at very low frequencies (rare alleles). Multivariate and model-based analyses partitioned the collection in seven clusters comprising the ten different Capsicum spp analysed: C. annuum, C. chinense, C. frutescens, C. pubescens, C. bacatum, C. chacoense and C. eximium. The data clearly showed the close relationships between C. chinense and C. frutescens. C. cardenasii and C. eximium were indistinguishable as a single, morphologically variable species. Moreover, C. chacoense was placed between C. baccatum and C. pubescens complexes. The C. annuum group was structured into three main clusters, mostly according to the pepper fruit shape, size and potential pungency. Results suggest that the diversification of C. annuum in Spain may occur from a rather limited gene pool, still represented by few landraces with ancestral traits. This ancient population would suffer from local selection at the distinct geographical regions of Spain, giving way to pungent and elongated fruited peppers in the South and Center, while sweet blocky and triangular types in Northern Spain.