Ivana Puglisi

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.

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.

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.

The glutathione S-transferase gene superfamily: an in silico approach to study the post translational regulation

The use of plants to reclaim contaminated soils and groundwater, known as phytoremediation, is a promising biotechnological strategy which has gained a lot of attention in the last few years. Plants have evolved sophisticated detoxification systems against the toxin chemicals: following the uptake, the compounds are activated so that certain functional groups can conjugate hydrophilic molecules, such as thiols. The resulting conjugates are recognized by the tonoplast transporters and sequestered into the vacuoles. The xenobiotic conjugation with glutathione is mediated by enzymes which belong to the superfamily of glutathione S-transferases (GSTs) catalyzing the nucleophylic attack of the sulphur of glutathione on the electrophilic groups of the cytotoxic substrates therefore playing a crucial role in their degradation. This study was designed to identify the putative correlation between structural and functional characteristics of plant GST classes belonging to different plant species. Consequently, the protein sequences of the expressed GSTs have been retrieved from UniGene, classified and then analyzed in order to assess the evolutionary trend and to predict secondary structure. Moreover, the fingerprint analysis was performed with SCAN Prosite in the attempt to correlate meaningful signature profile and biological information. The results evidenced that all the soluble GSTs have a tendency to assume the α-helix secondary structure followed by random coil and β-sheet. The fingerprint analysis revealed that specific signature profiles related mainly to protein phosphorylation are in the GST classes of all considered species thus suggesting that they might be subjected to reversible activation by phosphorylation-mediated regulation. This approach provides the knowledge of the relationship between presence of conserved signature profile and biological function in the view of future selection of GSTs which might be employed in either mutagenesis or genetic engineering studies.

Two previously unknown Phytophthora species associated with brown rot of Pomelo (Citrus grandis) fruits in Vietnam

Two distinct Phytophthora taxa were found to be associated with brown rot of pomelo (Citrus grandis), a new disease of this ancestral Citrus species, in the Vinh Long province, Mekong River Delta area, southern Vietnam. On the basis of morphological characters and using the ITS1-5.8S-ITS2 region of the rDNA and the cytochrome oxidase subunit 1 (COI) as barcode genes, one of the two taxa was provisionally named as Phytophthora sp. prodigiosa, being closely related to but distinct from P. insolita, a species in Phytophthora Clade 9, while the other one, was closely related to but distinct from the Clade 2 species P. meadii and was informally designated as Phytophthora sp. mekongensis. Isolates of P. sp. prodigiosa and P. sp. mekongensis were also obtained from necrotic fibrous roots of Volkamer lemon (C. volkameriana) rootstocks grafted with ‘King’ mandarin (Citrus nobilis) and from trees of pomelo, respectively, in other provinces of the Mekong River Delta, indicating a widespread occurrence of both Phytophthora species in this citrus-growing area. Koch’s postulates were fulfilled via pathogenicity tests on fruits of various Citrus species, including pomelo, grapefruit (Citrus x paradisi), sweet orange (Citrus x sinensis) and bergamot (Citrus x bergamia) as well as on the rootstock of 2-year-old trees of pomelo and sweet orange on ‘Carrizo’ citrange (C. sinensis ‘Washington Navel’ x Poncirus trifoliata). This is the first report of a Phytophthora species from Clade 2 other than P. citricola and P. citrophthora as causal agent of fruit brown rot of Citrus worldwide and the first report of P. insolita complex in Vietnam. Results indicate that likely Vietnam is still an unexplored reservoir of Phytophthora diversity.

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.

Biostimulant activity of humic-like substances from agro-industrial waste on Chlorella vulgaris and Scenedesmus quadricauda

ABSTRACT The use of microalgae in a number of sectors, including biodiesel, feed and food production, is proving to be of great interest. An evaluation was made of the possible biostimulant effects on Chlorella vulgaris and Scenedesmus quadricauda of humic-like substances (HLs) extracted from agro-industrial wastes. These included digestate from the waste of an agro-livestock farm (D-HL), oil extraction residues from rape (B-HL, Brassica napus L.) and tomato residues (T-HL). The microalgal growth medium (BG11) was supplemented with HLs to evaluate their effect on biomass yield as well as carbohydrate, chlorophylls a and b, lipid and fatty acid contents. Our results showed that the HLs used in the test are effective biostimulants of C. vulgaris and S. quadricauda. The biostimulant effect seems to depend on the type of extract used for cultivating the microalgae, the concentration and the species treated. Among the extracts applied to the growth medium, D-HL and T-HL seem to have a significant effect on microalgal biomass and lipid production. Although B-HL showed no significant effect on the biomass and lipid content of C. vulgaris and S. quadricauda, its presence in the growth medium increased the saturated:unsaturated fatty acid ratio (SFA/UFA) and stimulated the sugar metabolism of the microalgae by increasing their carbohydrate and chlorophyll content.

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 Cd2+/Zn2+ 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 Cd2+/Zn2+ transporter protein and/or a plasma membrane ATPase that could be involved in the compartmentalization of cadmium inside the cell.

Novel bioprocess for the cultivation of microalgae in hydroponic growing system of tomato plants

Hydroponic growing systems and the application of natural biostimulant substances are becoming very attractive option for crop cultivation due to their economic relevance since they allow reduction in the use of fertilizer and increase the yield. In order to perform a hydroponic co-cultivation system of microalgae (Chlorella vulgaris or Scenedesmus quadricauda) and tomato plants, grown in Hoagland nutrient solution, their mutual effect and the influence of a natural biostimulant obtained by alkaline extraction from a digestate of agro-livestock residues (DHL) were evaluated. The results showed that the co-cultivation system positively affected the growth of both tomato plants and microalgae. The best option, aimed to a mutual benefit for both cultures, resulted to be the co-cultivation system of tomato plants and S. quadricauda in the presence of DHL, positively affecting the growth of tomato plants along with a great increase in microalgal biomass.