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.

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 (alpha,beta-unsaturated) cinnamaldehyde functional group, which is responsible for the dye reaction, is lost due to lateral chain cross-linking reactions involving the beta carbon. Fourier transform infrared spectroscopy and thioacidolysis analyses of phloroglucinol-positive lignifying plant cell walls belonging to the plant species Zinnia elegans L., Capsicum annuumvar. annuum, Populus albaL., and Pinus halepensisL. 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.

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 q L 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.

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Pepper fruits, of Capsicum annuum cv. Padron, undergo changes in content of capsaicinoids, lignin, and free phenolics during the maturation process. Although capsaicinoids increase with development, the maximal levels of free phenolics and lignin are observed during the early stages of development. A decrease of peroxidase activity was observed during maturation, and this was related with a decrease in other physiological parameters studied, namely chlorophylls and pH. Subcellular fractionation studies reveal that most peroxidase activity is localized in the soluble fraction throughout development. The changes in the peroxidase activity were accompanied by changes in the different isoenzymes. Acidic isoenzymes increased whereas the basic isoenzymes decreased over the same period, and the changes in these isoenzymes were related with capsaicin metabolism.

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.

Pepper (cv. Padrón) shows a spatial gradient in the content of phenolic compounds, and particularly of capsaicinoids, along the stem. These compounds were consistently more abundant in apical fruits than in fruits belonging to middle and basal segments. Analysis of the two principal capsaicinoids in fruits showed that the proportion of capsaicin was always higher than that of dihydrocapsaicin. Capsaicinoids were also found to be present in vegetative organs, such as stem and leaves. In this case, the proportion of individual capsaicinoids was different than that in fruits, and dihydrocapsaicin was found to be more abundant. To find out whether the capsaicinoids in vegetative organs came from the fruits, the floral buds were removed and fruit formation was prevented. Capsaicinoids were not detected in the stem and leaves of floral bud-deprived plants, suggesting that they did originate from the fruit.

Summary• Verticillium dahliae is a soilborne pathogen that causes vascular wilt in pepper ( Capsicum annuum var. annuum ). Here we study to what extent changes in the lignification response of peppers condition tolerance of wilt.• For this, the quantum yield ( Φ PSII ), the linear e lectron t ransport r ate (ETR), and the lignification response (monomer composition and crosslinking) were studied in three C. annuum cultivars differing in degree of tolerance.• The results showed that in tolerant cultivars (Padrón and Yolo Wonder), both Φ PSII and ETR showed significantly higher levels at saturating photosynthetically active radiation values. This was not, however, the case for cv. Luesia, which showed a significant decrease in Φ PSII , ETR and nonphotochemical quenching values, suggesting that photochemical processes are strongly damaged in this cultivar as a consequence of the disease. The analysis of stem lignins in tolerant cultivars revealed that they were mainly composed of p -hydroxyphenyl units, while levels of β -O-4-linked coniferyl and sinapyl alcohols were significantly lower.• It is concluded that through the observed changes in stem lignins (monomer composition and crosslinking) peppers retard, since they maintain leaf photosynthetic integrity, but do not stop (since wilt symptoms are not avoided) V. dahliae fungal hyphae penetration.