Roberto Bollini

Expression of the Stilbene Synthase (StSy) Gene from Grapevine in Transgenic White Poplar Results in High Accumulation of the Antioxidant Resveratrol Glucosides

When present, stilbene synthase leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. White poplar (Populus alba L.) was transformed with a construct containing a cDNA insert encoding stilbene synthase from grapevine (Vitis vinifera L.), under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a chimeric kanamycin resistance gene. Southern blot hybridization analysis demonstrated the presence and integration of exogenous DNA sequences in the poplar genome. Expression of the stilbene synthase-encoding gene in different transgenic lines was confirmed by Western blot and Northern analyses. Compared to the controls, in the transgenic plants two new compounds were detected and were identified as the trans- and cis- isomers of resvera trol-3-glucoside (piceid) by high-pressure liquid chromatography (HPLC), UV spectrophotometry, electrospray mass spectrometry (HPLC-ESI-MS) and enzymatic hydrolysis. Since poplaris a good biomass producer and piceids are accumulated in substantial amounts (up to 615.2 μg/g leaf fresh weight), the transgenic plants represent a potential alternative source for the production of these compounds with high pharmacological value. Despite the presence of piceid, in our experimental conditions no increased resistance against the pathogen Melampsora pulcherrima, which causes rust disease, was observed when in vitro bioassays were performed.

A defective ABC transporter of the MRP family, responsible for the bean lpa1 mutation, affects the regulation of the phytic acid pathway, reduces seed myo‐inositol and alters ABA sensitivity

• We previously identified the lpa1 (low phytic acid) 280-10 line that carries a mutation conferring a 90% reduction in phytic acid (InsP(6) ) content. In contrast to other lpa mutants, lpa1(280-10) does not display negative pleiotropic effects. In the present paper, we have identified the mutated gene and analysed its impact on the phytic acid pathway. • Here, we mapped the lpa1(280-10) mutation by bulk analysis on a segregating F(2) population, an then, by comparison with the soybean genome, we identified and sequenced a candidate gene. The InsP(6) pathway was analysed by gene expression and quantification of metabolites. • The mutated Pvmrp1(280-10) cosegregates with the lpa1(280-10) mutation, and the expression level of several genes of the InsP(6) pathway are reduced in the lpa1(280-10) mutant as well as the inositol and raffinosaccharide content. PvMrp2, a very similar paralogue of PvMrp1 was also mapped and sequenced. • The lpa1 mutation in beans is likely the result of a defective Mrp1 gene (orthologous to the lpa genes AtMRP5 and ZmMRP4), while its Mrp2 paralog is not able to complement the mutant phenotype in the seed. This mutation appears to down-regulate the InsP(6) pathway at the transcriptional level, as well as altering inositol-related metabolism and affecting ABA sensitivity.

Lectin-related resistance factors against bruchids evolved through a number of duplication events

Abstract. Abundant lectin-related proteins found in common beans (Phaseolus vulgaris L.) have been shown to confer resistance against the larvae of a number of bruchid species. Genes encoding for these proteins are members of the lectin multigene family, the most representative components being arcelins, phytohemagglutinins and α-amylase inhibitors. Arcelins have been described in seven variants, some of which are resistance factors against the Mexican bean weevil (Zabrotes subfasciatus), a major bean predator. In this study the isolation and sequencing of arcelin genes from wild P. vulgaris genotypes, containing Arc3 and Arc7 variants, is reported, and similarities and evolutionary relationships among the seven known arcelins are described. The evolutionary analysis shows that arcelins 3 and 4 cluster together and are the most-ancient variants. A duplication event gave rise to two additional clusters, one comprising arcelins 1, 2 and 6 and separated from the cluster of arcelins 5 and 7. A multiple number of arcelin genes were found in arcelin 3 and 4 genotypes indicating that more than one type of arcelin gene may be present in the same locus. Some of these sequences are reminiscent of ancient duplication events in arcelin evolution demonstrating that arcelins have evolved through multiple duplications. A further aim of this paper was to better understand and describe the evolution of the entire lectin multigene family. Beside arcelins, a number of other types of sequences, such as putative lectins and sequences not easily classifiable, were found in genotypes containing Arc3 and Arc4. These results, together with the evolutionary analysis, indicate that lectin loci are quite complex and confirm their origin by multiple duplication events.

Contribution of processing events to the molecular heterogeneity of four banding types of phaseolin, the major storage protein of Phaseolus vulgaris L.

SummaryThe origin of the molecular heterogeneity of phaseolin was investigated by studying, both in vivo and in vitro, the synthesis and processing of four different banding types of phaseolin in five cultivars of Phaseolus vulgaris L. The results demonstrate: I) Newly-synthesized (unprocessed) phaseolin in all cultivars is composed of three major components. These differ between cultivars, both in charge and Mr. II) The processing of these precursors is highly conserved and consists of the co-translational cleavage of a signal peptide, two glycosylation steps in the endoplasmic reticulum and a further modification inside the protein bodies to give the mature form. III) Some of the molecular heterogeneity of each phaseolin banding type is due to a different extent of glycosylation of its polypeptide components.

In vivo endoproteolytically cleaved phaseolin is stable and accumulates in developing Phaseolus lunatus L. seeds

Phaseolin is the most abundant storage protein of bean seeds. To modify its amino-acidic composition by protein engineering, for the improvement of its nutritional value, regions which could be modified without detrimental effects on structural features of the protein must be identified. Data presented here, on the characterisation of the major storage protein of lima bean (Phaseolus lunatus L.) seeds, a phaseolin-like glycoprotein, provide good indications on one of such region. Phaseolus lunatus phaseolin consists of four major oligomers containing two subunit classes. Polypeptides of one class show a molecular mass ranging from 38.5 kDa to 32 kDa, while the molecular mass of polypeptides belonging to the other class ranges from 27 kDa to 21 kDa. The subunits originate from the cleavage of precursor forms, with molecular masses of 58 kDa and 54 kDa, which are still present - in residual amounts - in the nature protein. Comparison of their N-terminal sequences with those of the subunits demonstrate that cleavage occurs in a region of the molecule that instead remains uncleaved in phaseolins of the other species. Since this region can accommodate such a drastic modification, we suggest it could be a good candidate for in vitro manipulation.

Variation and genomic polymorphism of lectin-related proteins in Lima bean (Phaseolus lunatus L.) seeds

Variation of the lectin and the two lectin-related proteins, AIL (α-amylase inhibitor-like) and ARL (arcelin-like) was examined in wild and cultivated accessions of Lima bean (Phaseolus lunatus L.) using electrophoresis of total seed proteins, immunoblot and RFLP analysis of lectin-related genes. Results confirm that divergence of the two major Lima bean gene pools, Andean and Mesoamerican, also apply to this protein family. All three members of the family are present in both gene pools, with differences in size, abundance and composition between gene pools, giving the possibility to distinguish Andean from Mesoamerican lectin pattern types. Both patterns show some variants, such as lack of lectin or its presence as an abundant protein. The observed variation reflects, at least in part, into genomic polymorphism. The presence of arcelin- and α-amylase-related proteins in Lima bean could represent a tool to increase our knowledge in the evolution of the lectin family in Phaseolus species.

Arcelin in wild bean (Phaseolus vulgaris L.) seeds: sequence of arcelin 6 shows it is a member of the arcelins 1 and 2 subfamily

Arcelins are lectin-related proteins detected only in wild beans collected in Mexico and their presence has been related to resistance against the bean weevils. Six arcelin variants have been described and the gene sequence of four of them was determined. We have isolated and sequenced a cDNA clone encoding arcelin 6. Sequence data indicated that this protein is closely related to arcelin 1 and arcelin 2. The cluster dendrogram produced with the multiple alignment of the cDNA clones coding for arcelins showed that arcelins can be divided into three subgroups: i) arcelin 1, arcelin 2 and arcelin 6, ii) arcelin 4 and iii) arcelin 5a and arcelin 5b. Biochemical data indicate that arcelin 3 belongs to the same subgroup of arcelin 4. Southern blot analysis of genomic DNA yielded similar restriction patterns among members of each subgroup, confirming the results obtained with the multiple alignment. Furthermore, the hybridisation patterns were specific for each arcelin variant. On the basis of these evidences, we suggest that the lectin locus could be a useful tool for understanding evolutionary relationships in common bean and in the genus Phaseolus.

Lectin Gene Sequences and Species Relationships among Cultivated Legumes

Lectins are a class of defence proteins of non-immune origin that bind carbohydrate in a reversible fashion. In some cultivated legume species, lectin protein coding genes were PCR amplified using primers designed on the basis of conserved N- and C-terminal amino acid sequences of the common bean (one-chain) or pea (two-chains) lectins. Amplification products of the expected length were obtained in Lathyrus sativus L., Vicia faba L. var. faba, Phaseolus coccineus L., and Vigna unguiculata (L.) Walp. No amplification product or agglutinating activity against blood cells, and/or cross-reaction with specific antibodies were detected in Lupinus albus L. and Cicer arietinum L. Finally, the new isolated nucleotide sequences, together with other legume lectin sequences already present in the EMBL Database, were used for evolutionary analysis. This last indicated the existence of two main clusters; one grouping all the species belonging to the Phaseoleae tribe and the other one grouping Lens culinaris Medik., Pisum sativum L., L. sativus, and V. faba, members of the Vicieae tribe. Results were congruent with the taxonomic classification and suggested that the lectin genes divergence in legume followed species evolution.

Evolutionary analysis of the APA genes in the Phaseolus genus: wild and cultivated bean species as sources of lectin-related resistance factors?

The APA (Arcelin/Phytohemagglutinin/α-Amylase inhibitor) gene family is composed of various members, present in Phaseolus species and coding for lectin and lectin-related seed proteins having the double role of storage and defense proteins. Here members of the APA family have been identified by immunological, functional, and molecular analyses and representative genes were sequenced in nine wild species of Phaseolus. All taxa possessed at least one member of the true lectin gene. No arcelin type sequences have been isolated from the species examined. Among the wild species studied, only P. costaricensis contained an α-amylase inhibitor (α-AI). In addition P. augusti, P. maculatus, P. microcarpus, and P. oligospermus showed the presence of the lectin-related α-amylase inhibitor-like (AIL) genes and α-AI activity. Data from Southern blot analysis indicated the presence of only one lectin gene in P. parvulus and P. filiformis, while an extensive gene duplication of the APA locus was found in the other Phaseolus species. Phylogenetic analysis carried out on the nucleotide sequences showed the existence of two main clusters and clearly indicated that lectin-related genes originated from a paralogous duplication event preceding the development of the ancestor to the Phaseolus genus. The finding of detectable α-AI activity in species containing AIL genes suggests that exploiting APA genes variability in the Phaseolus genus may represent a valuable tool to find new members that may have acquired insecticidal activities.

Antioxidant effect of a fermented powder of Lady Joy bean in primary rat hepatocytes

Abstract The role and beneficial effects of plant and food extracts against various diseases induced by oxidative stress have received much attention in recent years. Legumes are rich in bioactive compounds, and some studies suggest a correlation between their consumption and a reduced incidence of diseases. Primary cultures of rat hepatocytes were used to investigate whether and how an extract obtained from a fermented powder of bean named Lady Joy (Phaseolus vulgaris L.) is able to regulate antioxidant and detoxifying enzymes through the NRF2 pathway, inhibit NF-kB activation, and reduce H2O2-induced endoplasmic reticulum (ER) stress. All of the antioxidant and detoxifying enzymes studied were significantly up-regulated by Lady Joy treatment. Western blot showed that Nrf2 was activated by Lady Joy treatment. Also, cells treated with this fermented bean were partially protected against NF-kB activation resulting from H2O2 stress. As a link between oxidative stress and ER dysfunction is hypothesized, we verified whether Lady Joy was able to protect cells from H2O2-induced ER stress, by studying the response of the proteins CHOP, BiP and caspase 12. The results of this study show that Lady Joy can induce the Nrf2 pathway, inhibit NF-kB, and protect ER from stress induced by H2O2.