Goffredo Petrone

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.

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.

Anthocyaninless Cultivars of Sweet Orange Lack to Express the UDP-Glucose Flavonoid 3-O-Glucosyl Transferase

In this study we isolated the partial cDNA clone of three enzymes active in the late steps of anthocyanins biosynthetic pathway such as DFR, ANS and UFGT from Citrus sinensis (L) Osbeck. The predicted aminoacids sequences shared very high identity values ranging from 77 % for DFR (Pyrus communis, Malus domestica), to 86 % for ANS (Vitis vinifera) with homologous available protein sequences. Only the UFGT sequence showed lower identity value (59 %), but this finding is in accordance with data obtained for other enzymes belonging to the plant glucosyl transferase superfamily. The expression of CHS, CHI, F3OH, DFR, ANS and UFGT was also investigated in both coloured and uncoloured oranges juice vesicles. Transcripts encoding the enzymes involved in the earlier reactions have been detected in either blood and blonde oranges by RT-PCR. Conversely, considerable difference emerged in the case of the later gene ufgt which was totally unexpressed in blonde oranges juice vesicles probably determining the lack of anthocyanins production in all blonde orange cultivars analysed.

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.

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 kcat values for CDNB, suggesting that Lys117 might positively influence the formation of the transition state during catalysis. No changes in the Km 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 Km. 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.

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.

Purification and partial characterization of an ATP-hydrolyzing serine protease from lettuce leaves

Abstract A novel ATP-hydrolyzing protease has been purified from lettuce leaves by combination of (NH 4 ) 2 SO 4 fractionation, gel filtration and anionic exchange chromatography. The purified enzyme is made up of a single subunit with an apparent molecular weight of 40 000, even though a possible higher molecular organization might occur in vivo induced by the presence of ATP or proteases substrate. The lettuce protease showed caseinolytic, trypsin-like and, to a smaller extent, peptidyl glutamyl hydrolase activities. It is a serine protease as both peptidase activities are highly sensitive to tosyl- l -lysinechloromethylketone (TLCK) and leupeptin. The trypsin-like activity of the enzyme was not affected by MgATP complex or ATP alone. Peptidyl glutamyl hydrolase activity, instead, and ATP hydrolysis were strictly correlated as incubation of the enzyme with MgATP, but not with ATP alone, stimulated the peptidase activity of the enzyme, while peptide substrate as well as TLCK enhanced ATPase activity. Moreover, the ATPase inhibitor vanadate, which also blocked the peptidyl glutamyl hydrolase activity, caused a strong activation of the trypsin-like activity of the enzyme. Therefore, these studies could indicate the existence of multiple functional states of the enzyme achieved in vivo by ATP hydrolysis. The cytosolic localization of the enzyme is finally discussed.

Different kinetic and regulatory properties of soluble and membrane-bound nitrate reductases in tomato leaves

Abstract In the present study we emphasized the differences between tomato ( Lycopersicon esculentum cv. Mill) leaves cytosolic (cytosolic nitrate reductase, cNR) and membrane-bound nitrate reductase (mNR) by comparing their activity during prolonged darkness, under salt stress conditions as well as their response to nitrate induction. Tomato leaves mNR remained more active in the dark than cNR and it was more easily recovered after transfer to light. Under saline stress both enzyme forms were inhibited but cNR showed values of residual activity markedly lower than mNR. As expected, cNR activity was strongly induced and strictly dependent by the presence of NO 3 − in the nutrient solution during plant growth. Conversely, mNR was only weakly stimulated by NO 3 − . Similar pH dependence but considerable different K m values for nitrate were observed too, thus suggesting a distinct physiological role for the two enzymes. We also tried to solubilize the membrane-bound enzyme by detergent treatment. Among the detergents tested, β-octylglucoside showed a stimulatory effect upon the enzyme as well as a stronger extracting power. The enzyme extracted from the microsomal fraction exhibited similar kinetic properties to the membrane-bound form.

Nitrogen metabolism and ion content of sweet pepper under salt and heat stress.

The effects of salinity and high temperature stress conditions which often occur during greenhouse cultivation have been investigated in sweet pepper (Capsicum annuum L.). The responses were evaluated by measuring growth,nitrate content and reduction, protein and chlorophyll contents, proteolytic activity, as well as Na + , K + , Ca 2 + and Cl - contents. Plants exposed to salt stress strongly reduced growth and reproductive activity. In addition, salinity caused higher rates of membrane-bound NR activities and higher levels of total chlorophyll. Na + and Cl - contents increased. On the contrary, high temperature did not affect growth and reproductive activity. In response to heat, pepper showed a significant increase in the NADH-dependent NR activity and increased nitrate content suggesting a possible enhancement in nitrate uptake during heat stress. A slight increase in chlorophyll content and a strong reduction in Na + and Cl - content were observed under the same conditions.

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.