Deciphering the role of the phenylpropanoid metabolism in the tolerance of Capsicum annuum L. to Verticillium dahliae Kleb.

Verticillium dahliae is an economically relevant soilborne pathogen that causes vascular wilt in several crops, including pepper (Capsicum annuum). Fungal infection is usually visualized as a vascular browning, likely due to the onset of phenylpropanoid metabolism, which also seems to play a crucial role in the tolerance of some pepper varieties. In the current work, the potential function of distinct phenylpropanoid derivatives (suberin, lignin and phenolic compounds) in the pepper tolerance response against V. dahliae, was investigated. Histochemical and biochemical analyses ruled out suberin as a key player in the pepper-fungus interaction. However, changes observed in lignin composition and higher deposition of bound phenolics in infected stems seemed to contribute to the reinforcement of cell walls and the impairment of V. dahliae colonization. Most importantly, this is the first time that the accumulation of the hydroxycinnamic acid amide N-feruloyltyramine was reported in pepper stems in response to a vascular fungus. Fungitoxic activity for that hydroxycinnamate-tyramine conjugate was demonstrated as well.

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.

A proteomic approach to Physcomitrella patens rhizoid exudates.

The interaction between plants and the surrounding environment has been widely studied, specially the defence reactions and the plant-plant interactions. One of the most remarkable metabolic features of plant roots is the ability to secrete a vast array of compounds into the rhizosphere, not only of low molecular weight but also polysaccharides and proteins. Here, we took advantage of proteomics to study the rhizoid exudates of Physcomitrella patens at early and late development stages (7 and 28 days of culture in liquid medium). Samples were extracted, separated and detected with nanoLC-MALDI-TOF/TOF MS/MS, identifying 47 proteins at the development stage of 7 days, and 66 proteins at 28 days. Moreover, 21 proteins were common to the two analyzed periods. All the identified proteins were classified into 8 functional categories: response to stress, response to stimulus, oxido-reduction, cell wall modification, photosynthesis and carbohydrate metabolism, transport, DNA metabolic process and regulation/signalling. Our results show important differences in the protein expression profile along the development of P. patens, mainly at the level of regulation- and senescence-related proteins. Defence-related proteins, such as chitinases, thaumatins and peroxidases have a major role in the interaction of P. patens with the environment.

Purification and kinetic characterization of two peroxidases of Selaginella martensii Spring. involved in lignification.

Two cationic peroxidases from Selaginella martensii Spring. (SmaPrx2 and SmaPrx3) were purified using a three-step protocol which includes ammonium sulfate precipitation, adsorption chromatography on phenyl sepharose and cationic exchange chromatography on SP sepharose. The molecular mass for SmaPrx2 and SmaPrx3 was calculated to be 36.3 kDa and 45.6 kDa, respectively, according to MALDI-TOF/TOF. The isoelectric points were estimated in 9.2 and 9.5 for SmaPrx2 and SmaPrx3, respectively, according to isoelectrofocusing. Both enzymes show a typical peroxidase UV-visible spectrum with a Soret peak at 403 nm for SmaPrx2 and 404 nm for SmaPrx3. The specific activities showed against several substrates and the kinetic parameters suggest SmaPrx2 and SmaPrx3 have specific roles in cell wall formation and especially in lignin biosynthesis. Several peptides from tryptic digestion of both peroxidases were identified through MALDI-TOF MS/MS. The presence in these peptides of structural determinants typical of syringyl peroxidases indicates these proteins show no structural restrictions to oxidize syringyl moieties. These data, along with the in vitro capacity of using sinapyl alcohol as substrate and the low K(m) in the µM range suggest these two peroxidases may be responsible for the oxidation of syringyl monolignols that leads to syringyl lignins biosynthesis.

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

BACKGROUND: Verticillium 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. RESULTS: We 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. CONCLUSIONS: The 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.

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.

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 beta-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.

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 H(2)O(2). The results also showed that sites for monolignol beta-O-4 cross-coupling in cell walls may be saturated, suggesting that the growth of the lineal lignin macromolecule is not infinite.

Changes in stem lignins (monomer composition and crosslinking) and peroxidase are related with the maintenance of leaf photosynthetic integrity during Verticillium wilt in Capsicum annuum.

• 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 (ΦPSII ), the linear electron transport rate (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 ΦPSII 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 ΦPSII , 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 ß-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.

Capsaicinoids in vegetative organs of Capsicum annuum L. in relation to fruiting.

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.