Cadmium induces cadmium-tolerant gene expression in the filamentous fungus Trichoderma harzianum.

The filamentous fungus Trichoderma harzianum, strain IMI 393899, was able to grow in the presence of the heavy metals cadmium and mercury. The main objective of this research was to study the molecular mechanisms underlying the tolerance of the fungus T. harzianum to cadmium. The suppression subtractive hybridization (SSH) method was used for the characterization of the genes of T. harzianum implicated in cadmium tolerance compared with those expressed in the response to the stress induced by mercury. Finally, the effects of cadmium exposure were also validated by measuring the expression levels of the putative genes coding for a glucose transporter, a plasma membrane ATPase, a Cd(2+)/Zn(2+) transporter protein and a two-component system sensor histidine kinase YcbA, by real-time-PCR. By using the aforementioned SSH strategy, it was possible to identify 108 differentially expressed genes of the strain IMI 393899 of T. harzianum grown in a mineral substrate with the addition of cadmium. The expressed sequence tags identified by SSH technique were encoding different genes that may be involved in different biological processes, including those associated to primary and secondary metabolism, intracellular transport, transcription factors, cell defence, signal transduction, DNA metabolism, cell growth and protein synthesis. Finally, the results show that in the mechanism of tolerance to cadmium a possible signal transduction pathway could activate a Cd(2+)/Zn(2+) transporter protein and/or a plasma membrane ATPase that could be involved in the compartmentalization of cadmium inside the cell.

Gene characterization, analysis of expression and in vitro synthesis of dihydroflavonol 4-reductase from [Citrus sinensis (L.) Osbeck].

Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) catalyzes the reduction of dihydroflavonols to leucoanthocyanins, a key "late" step in the biosynthesis of anthocyanins. In this study we showed that a strong reduction in DFR expression occurs in the non-red orange cultivar (Navel and Ovale) compared to that of the red orange (Tarocco) suggesting that the enzyme could be involved in the lack of production of anthocyanins. Therefore, we isolated and compared the cDNAs, the genomic clones, as well as the promoter regions of blood and blond orange dfrs. Our data revealed that the cDNA sequences of pigmented and non-pigmented orange DFRs were 100% homologous and contained a 1017 bp open reading frame which encodes a protein of 338 amino acid residues, corresponding to a molecular mass of 38010.76 Da, with a theoretical pI of 5.96. Moreover, we found that there were no significant differences in non-coding regions (introns and 5' upstream region) of dfr sequences. Southern blot analysis of genomic DNA indicated that dfr was present as a single copy gene in both cultivars. From these findings the low expression level of blond orange dfr, which might play a role in the phenotypic change from blood to blond orange, is thought to be the result of a likely mutation in a regulatory gene controlling the expression of dfr. In addition, here we reported the successful expression of orange DFR cDNAs leading to an active DFR enzyme which converts dihydroquercetin to leucoanthocyanidin, thus confirming the involvement of the isolated genes in the biosynthesis of anthocyanins. Moreover, as far as we know, this is the first report concerning the in vitro expression of DFR from fruit flesh whose biochemical properties might be very different from those of other plant organ DFRs.

Gene isolation, analysis of expression, and in vitro synthesis of glutathione S-transferase from orange fruit [Citrus sinensis L. (Osbeck)].

Glutathione S-transferases (GSTs) (EC 2.5.1.18) are ubiquitous enzymes that have a defined role in xenobiotic detoxification, but a deeper knowledge of their function in endogenous metabolism is still lacking. In this work, we isolated the cDNAs as well as the genomic clones of orange GSTs. Having considered gene organization and homology data, we suggest that the isolated GST gene is probably involved in the vacuolar import of anthocyanins. We also found that the blood and blond orange GSTs shared the same nucleotide sequences, but as expected, the GST expression in the nonpigmented orange cultivar [Citrus sinensis L. (Osbeck)] (Navel and Ovale) was strongly reduced as compared to that of the pigmented orange (Tarocco). Interestingly, in the crude extracts of pigmented orange fruits, the GST activity was reproducibly detected by providing either 1-chloro-2,4 dinitrobenzene (CDNB) or cyanidin-3-O-glucoside (C-3-G) as substrates; moreover, we have shown that cyanidin-3-O-glucoside acted as a powerful competitive inhibitor of 1-chloro-2,4 dinitrobenzene conjugation to reduced glutathione (GSH) in the pigmented orange, confirming that this molecule might easily bind to the active site of the enzyme and functions as a putative substrate. In addition, we have reported here the successful in vitro expression of orange GST cDNAs leading to a GST enzyme that is active against cyanidin-3-O-glucoside, thus suggesting the probable involvement of the isolated gene in the tagging of anthocyanins for vacuolar import. This last result will help to study the kinetic and structural properties of orange fruit GST avoiding time-consuming protein purification procedures.

Anthocyanins accumulation and related gene expression in red orange fruit induced by low temperature storage.

The aim of this work was to study the impact of moderately long storage periods at 4 degrees C upon red orange [Citrus sinensis (L.) Osbeck] anthocyanins production and the expression of structural genes involved in their biosynthesis such as phehylalanine ammonia lyase (PAL), chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), and UDP-glucose flavonoid glucosyl transferase (UFGT). Our results showed that low temperature-induced anthocyanins accumulation in red orange juice vesicles after 75 days reached values eight times higher than those kept at 25 degrees C. Furthermore, real-time polymerase chain reaction showed that expression of PAL, CHS, DFR, and UFGT was strongly induced during low temperature exposure since levels of all transcripts increased at least 40-fold with respect to control samples. Interestingly, in orange fruits subjected to a brief exposure at low temperature (45 days) and subsequently kept at 25 degrees C, the anthocyanins content dropped although samples still maintained higher levels of these pigments than those registered in control oranges. Concordantly, the expression of chs, dfr, and ufgt declined upon return to control conditions, but it was always much higher in samples subjected to brief cold induction than in the control samples. On the contrary, the amount of PAL transcripts became negligible immediately after the temperature change from 4 to 25 degrees C, thus indicating that "early" and "late" genes, respectively, implicated in the first and in the last steps leading to the anthocyanins, might be affected by different regulation mechanisms.

Characterization of "lettucine", a serine-like protease from Lactuca sativa leaves, as a novel enzyme for milk clotting.

In this work we focused on the characterization of a novel plant rennet purified from lettuce leaves (Lactuca sativa L. cv Romana). The lettuce protease, lettucine, showed trypsin-like, SV8-like, and caseinolytic activities. Although the enzyme did not recognize peptides having hydrophobic amino acid residues in the P(1) position of the target bond, it did show milk-clotting activity, suggesting that different bonds rather than the Phe(105)-Met(106) of the kappa-casein might be cleaved, still inducing milk-clotting. The enzyme exhibited proteolytic activity toward alpha-casein, beta-casein, kappa-casein, and milks with different fat contents, with the highest activity observed with partially skimmed milk, total casein, and alpha- and kappa-casein. SDS-PAGE studies showed that lettucine cleaved alpha-casein, beta-casein, and kappa-casein. In particular, we showed that alpha-casein breakdown occurred even though total casein or milks were supplied, suggesting that the lettuce enzyme is able to operate a significant disorganization of the casein's micellar structure. Moreover, the proteolytic activity of the enzyme analyzed under various technological parameters, such as temperature and pH, indicated that the lettuce enzyme is highly consistent with the milk-clotting process.

Short cold storage enhances the anthocyanin contents and level of transcripts related to their biosynthesis in blood oranges.

The health benefits associated with the consumption of anthocyanin-containing foods are extensively documented. Mature fruits of blood oranges and their hybrids are characterized by the presence of these bioactive pigments, the abundance of which can be enhanced by storing fruit at cooling nonfreezing temperature. In this work the effects of short low-temperature exposure (4 °C × 15 days) upon orange anthocyanin content and the expression of structural genes belonging to the pigment biosynthesis pathway were investigated. The results highlight that anthocyanin levels of fruit exposed to cold sharply increase, reaching, after 6 days of storage, a value 8 times higher than that observed in the time zero samples, thus suggesting that fruit with enhanced health-related attributes might be obtained at this storage stage. The analysis of gene expression shows that the amount of transcripts of all considered genes (CM1, PAL, CHS, DFR, ANS, UFGT, and GST) sharply increased after 3-6 days of cold storage, confirming previous data showing that the biosynthesis of anthocyanins is a cold-regulated pathway. By comparing the expression of selected genes (PAL, DFR, and UFGT) between blood and common oranges, it turns out that those genes strictly involved in anthocyanin biosynthesis are not cold responsive in common oranges. Moreover, the data highlight that the EST encoding the transcription factor NAC domain protein is selectively induced by cold in blood oranges but not in common oranges, thus proposing it as a candidate gene specifically involved in blood orange response to cold exposure.

Expression of genes of Trichoderma harzianum in response to the presence of cadmium in the substrate.

Trichoderma harzianum is a very important fungus for the reduction of the amount of heavy metals resulting from agricultural and industrial activities. This filamentous fungus has been shown to be tolerant towards several heavy metals (e.g. Cd, Pb, Zn, Ni and Mn), but the mechanism underlying this tolerance is not entirely understood. In this study, we confirmed the ability of T. harzianum to grow in the presence of cadmium and observed a significant stimulation of its growth in the presence of mercury. A molecular approach to investigate the cadmium tolerance mechanisms was carried out by the application of the suppression subtractive hybridisation (SSH) technique. To this end, we have used a particular strategy to discriminate cadmium-induced differentially expressed genes from those generally expressed upon heavy metal treatment. Several genes (109) were found to be overexpressed in the presence of cadmium, confirming the dramatic metabolic modification underlying the metal stress response of the fungus.

Identification of differentially expressed genes in response to mercury I and II stress in Trichoderma harzianum.

Filamentous fungi are very promising organisms in both the control and the reduction of the amount of heavy metal released by human and industrial activities. In particular, Trichoderma harzianum demonstrated to be tolerant towards different heavy metals, such as mercury and cadmium, even though the mechanism underlying this tolerance is not fully understood. By using a particular strategy of the suppression subtractive hybridization technique, we were able to identify in the strain IMI 393899 of T. harzianum eight different genes up-regulated in the presence of mercury II with respect to cadmium. Among the genes identified, a possible role in the tolerance mechanism could be envisaged for hydrophobin, due to its ability to dissolve hydrophobic molecules into aqueous media. We also show that IMI 393899 grows at the same rate of control culture in the presence of mercury I and that all eight genes isolated were also up-regulated in this condition.

Expression analysis in response to low temperature stress in blood oranges: implication of the flavonoid biosynthetic pathway.

The productivity and the geographical distribution of most commercially important Citrus varieties are markedly affected by environmental low temperatures. As gene engineering has been shown to be a favourable alternative to produce germplasm with improved cold tolerance, a broad group of cold regulated genes have been previously identified from several Citrus spp. By contrast, little information regarding the cold stress response of pigmented sweet orange varieties is available although they might provide a pivotal contribution to define the whole events occurring in cold exposed Citrus fruits. In our work, the transcriptome analysis based on subtractive hybridisation was performed in order to emphasise the overall induction in gene expression after the exposure of blood oranges [(Citrus sinensis) L. Osbeck Tarocco Sciara] to low temperature. The cold induction of several gene expressions was then validated by real-time RT-PCR. Overall, we observed the enhancement of transcripts involved in the defence mechanisms against oxidative damage, osmoregulating processes, lipid desaturation as well as many ESTs implicated in the primary and secondary metabolisms. In particular, the results show that cold stress induces transcriptomic modifications directed towards the increase of flavonoid biosynthesis, including those reactions involved in anthocyanin biosynthesis, as well as of the metabolic pathways supplying it. By comparing the blood orange response to cold stress with those of other plant sources, such as grapefruit, it seems to be similar to that of the chilling acclimated species. Interestingly, among the genes encoding for the regulatory proteins, several transcription factors have been identified for the first time as cold responsive genes in plants, indicating novel investigation lanes which should receive special attention in the future.

Different roles of functional residues in the hydrophobic binding site of two sweet orange tau glutathione S-transferases.

Glutathione S-transferases (GSTs) catalyze the conjugation of glutathione to hydrophobic compounds, contributing to the metabolism of toxic chemicals. In this study, we show that two naturally occurring tau GSTs (GSTUs) exhibit distinctive kinetic parameters towards 1-chloro-2,4-dinitrobenzene (CDNB), although they differ only in three amino acids (Arg89, Glu117 and Ile172 in GSTU1 are replaced by Pro89, Lys117 and Val172 in GSTU2). In order to understand the effects of the single mismatched residues, several mutant GSTs were generated through site-directed mutagenesis. The analysis of the kinetic parameters of the mutants led to the conclusion that Glu117 provides a critical contribution to the maintenance of a high-affinity CDNB-binding site. However, the substitution E117K gives rise to mutants showing increased k(cat) values for CDNB, suggesting that Lys117 might positively influence the formation of the transition state during catalysis. No changes in the K(m) values towards glutathione were found between the naturally occurring GSTs and mutants, except for the mutant caused by the substitution R89P in GSTU1, which showed a sharp increase in K(m). Moreover, the analysis of enzyme reactivation after denaturation showed that this R89P substitution leads to a two-fold enhancement of the refolded enzyme yield, suggesting that the insertion of proline might induce critical structural modifications. In contrast, the substitution P89R in GSTU2 does not modify the reactivation yield and does not impair the affinity of the mutant for glutathione, suggesting that all three residues investigated in this work are fundamental in the creation of enzymes characterized by unique biochemical properties.

Covalent selection of the thiol proteome on activated thiol sepharose: a robust tool for redox proteomics.

Protein thiols contribute significantly to antioxidant defence and selective oxidation of cysteines is important in signal transduction even in sub-stress scenarios. However, cysteine is the second rarest residue in proteins and it can be difficult to target low-abundance thiol (-SH)-containing proteins in proteomic separations. Activated thiol sepharose (ATS) allows covalent selection of -SH-containing proteins which can then be recovered by reduction with mercaptoethanol or dithiothreitol. This is a robust method for enriching -SH-containing proteins. We have used ATS to estimate the percentage (by weight) of thiol-containing proteins in cell extracts from a range of biological sources: a bacterium, Escherichia coli; a fungus, Trichoderma harzianum; and a bivalve mollusc Mytilus edulis. -SH-containing proteins account for 2.52% (E. coli), 1.4% (T. harzianum) and 1.4% (M. edulis) of total protein. Exposure to pro-oxidants did not materially alter these values. On removal of low M(r) thiols such as glutathione, the values for M. edulis did not significantly change but those for T. harzianum increased threefold. The two-dimensional electrophoresis profiles of ATS-selected proteins for each organism were compared in control and pro-oxidant-exposed preparations. This revealed that some proteins present in controls were absent in pro-oxidant-treated extracts which we attribute to thiol oxidation. ATS has significant potential in enrichment for -SH-containing proteins in redox proteomics.

Gene isolation and expression analysis of two distinct sweet orange [Citrus sinensis L. (Osbeck)] tau-type glutathione transferases.

Glutathione S-transferases (GSTs) represent a multifunctional family of enzymes grouped into four main classes (tau, phi, theta, and zeta) conjugating endobiotic and xenobiotic compounds to glutathione. In plants, this is considered to be a crucial step in the detoxification process as the S-glutathionylated metabolites are tagged for vacuolar sequestration. In this work, we have isolated two glutathione S-transferases belonging to the tau class GSTs from sweet orange leaves. The cDNA clones contained a complete open reading frame of 651 bp encoding two 216 amino acid proteins. Homology search and sequence alignment showed that the deduced amino acid sequences shared high identity with GSTs from other plant sources, including several strictly conservative motifs and distinctive amino acid residues specific of the tau class GSTs. The genomic clones of both isoforms were also isolated and the analysis of the gene organization confirmed the membership of both enzymes to the tau class GSTs. The encoded proteins differ only for three amino acids: the triplet R89, E117 and I172 found in the isoform named GSTU1 is replaced by the triplet P89, K117 and V172 in the GSTU2 isoform. The successful in vitro expression of the proteins led to the functional active form of both enzymes which showed different specific activity against CDNB as substrate, the GSTU1 showing values three fold lower than that observed for the GSTU2 enzyme. The analysis of the gene expressions suggested that the GST isoforms show either different distribution between leaf and flesh, the isoforms being decidedly expressed in the leaf, or cultivar related specificity, the U2 being highly expressed in the leaves of red orange whereas the U1 in the blond orange leaves. Furthermore, we also showed that the expression of U1 gene was remarkably induced in response to cadmium sulphate, CDNB and cyhalothrin treatments as well as to cold stress. On the contrary, the U2 isoform was constitutively expressed probably playing some sort of "default scavenging" activity in vivo. Taken together these results suggested that GSTU1 is a stress responsive gene and can be considered as potential target that is genetically modified so as create novel germoplasm with enhanced stress tolerance.