CgPBA1 may be involved in nuclear degradation during secretory cavity formation by programmed cell death in Citrus grandis 'Tomentosa' fruits.

Caspase-3 is the crucial executor caspase of apoptosis in mammalian cells, which is essential for chromatin condensation and DNA fragmentation. Although plants have no caspase-3 homologs, PBA1 acts as a plant caspase-3-like enzyme in plant programmed cell death (PCD). PCD occurs during the formation of secretory cavities in Citrus fruits; hence, secretory cavities could be utilized as a new cell biology model for investigating the regulatory mechanisms of plant PCD. To further study the association between PBA1 and PCD during secretory cavity development in Citrus fruits, CgPBA1 was identified in the fruit of Citrus grandis 'Tomentosa'. The temporal and spatial expression of CgPBA1 during secretory cavity development were analyzed using quantitative real-time PCR and in situ hybridization, and the morphological changes in the apoptotic cell nuclei were observed using TUNEL assay and ultra-thin section technology. The results revealed that the full-length cDNA of CgPBA1 contains a 711 bp ORF that encodes a putative protein containing 236 amino acid with a proteasome-ß-6 functional domain that belongs to the Ntn hydrolase super family. CgPBA1 was predominantly expressed in the secretory cavities; its expression changes coincided with the morphological changes and DNA fragmentation in apoptotic cell nuclei. The green fluorescent fusion protein of CgPBA1 is also located in the nucleus of tobacco epidermal cells. Based on previous research and the findings of the present study, we speculate that CgPBA1 is a highly functional conserved protein in plants, and it might be involved in nuclear degradation during PCD for secretory cavity formation in C. grandis 'Tomentosa' fruits.

Reduced expression of CsPH8, a P-type ATPase gene, is the major factor leading to the low citrate accumulation in citrus leaves.

Citrate is an important intermediate product for the biosynthesis of several metabolites in plants. As two important organs of the citrus plant, fruits and leaves have their own metabolites characteristics; among them, citrate is normally high in fruit juice sacs (JS) and low in leaves. In this study, citrate content and transcript levels of citrate synthesis, transport, storage, and utilization related genes were compared between leaves and fruit JS of Citrus reticulata cv. 'Huagan No. 2', C. grandis cv. 'Hirado Buntan', and C. sinensis cv. 'Anliu'. Results indicated that the citrate content in fruit JS was significantly higher than in leaves of each cultivar. Only the relative mRNA levels of a P-type proton pump gene, CsPH8, was significantly lower in leaves than in fruit JS of three citrus cultivars, while other genes related to citrate biosynthesis, transport, storage, and utilization were highly expressed in leaves as compared to fruit JS. Furthermore, CsPH8 transient and stable transformation in leaves indicated that the change in citrate content is highly consistent with the change of CsPH8 transcript levels. Taken together, our results strongly suggest that the low accumulation of citrate in citrus leaves is mainly due to the low expression level of CsPH8; additionally, the high level of expression of citrate-utilizing genes would prevent citrate accumulation in the leaf organ.

Citrus CitERF6 Contributes to Citric Acid Degradation via Upregulation of CitAclα1, Encoding ATP-Citrate Lyase Subunit α.

Citric acid is the most abundant organic acid in citrus fruit, and the acetyl-CoA pathway potentially plays an important role in citric acid degradation, which occurs during fruit ripening. Analysis of transcripts during fruit development of key genes in the acetyl-CoA pathway and transient overexpression assay in citrus leaves indicated that CitAclα1 could be a potential target gene involved in citrate degradation. In order to understand more about CitAclα1, 23 transcription factors coexpressed with CitAclα1 in citrus fruit were identified by RNA-seq. Using dual-luciferase assays, CitERF6 was shown to trans-activate the promoter of CitAclα1 and electrophoretic mobility shift assays (EMSAs) showed that CitERF6 directly bound to a 5'-CAACA-3' motif in the CitAclα1 promoter. Furthermore, citric acid content was significantly reduced when CitERF6 was overexpressed in transgenic tobacco leaves. Taken together, these results indicate an important role for CitERF6 in transcriptional regulation of CitAclα1 and control of citrate degradation.

Characterization of a Flavonoid 3'/5'/7-O-Methyltransferase from Citrus reticulata and Evaluation of the In Vitro Cytotoxicity of Its Methylated Products.

O-methylation of flavonoids is an important modification reaction that occurs in plants. O-methylation contributes to the structural diversity of flavonoids, which have several biological and pharmacological functions. In this study, an O-methyltransferase gene (CrOMT2) was isolated from the fruit peel of Citrus reticulata, which encoding a multifunctional O-methyltransferase and could effectively catalyze the methylation of 3'-, 5'-, and 7-OH of flavonoids with vicinal hydroxyl substitutions. Substrate preference assays indicated that this recombinant enzyme favored polymethoxylated flavones (PMF)-type substrates in vitro, thereby providing biochemical evidence for the potential role of the enzyme in plants. Additionally, the cytotoxicity of the methylated products from the enzymatic catalytic reaction was evaluated in vitro using human gastric cell lines SGC-7901 and BGC-823. The results showed that the in vitro cytotoxicity of the flavonoids with the unsaturated C2-C3 bond was increased after being methylated at position 3'. These combined results provide biochemical insight regarding CrOMT2 in vitro and indicate the in vitro cytotoxicity of the products methylated by its catalytic reaction.

Changes in Metabolisms of Antioxidant and Cell Wall in Three Pummelo Cultivars during Postharvest Storage.

The juice sacs of pummelo fruit is susceptible to softening during storage at 25 °C, which causes quality deterioration and flavor loss during postharvest pummelo storage. This study investigated the changes in metabolisms of antioxidant and cell wall in juice sacs of three pummelo cultivars-Hongroumiyou (HR), Bairoumiyou (BR) and Huangroumiyou (HuR)-during postharvest storage. The results revealed that, with the extension of storage, the juice sacs of three pummelo cultivars exhibited a decrease in total antioxidant capacity (TAC), DPPH and ABTS radical scavenging activity; a decline in total phenols (TP) content and an increase firstly then a decrease in total ascorbic acid (TAA) content; and a decrease in lipoxygenase (LOX) activity and a rise initially, but a decline in activities of ascorbate peroxidase (APX) and glutathione peroxidase (GPX). Additionally, increased water-soluble pectin (WSP), but declined propectin, ionic-soluble pectin (ISP) and chelator-soluble pectin (CSP); as well as an increase from 0 d to 60 d then followed by a decline in activities of pectinesterase (PE), polygalacturonase (PG) and pectate lyase (PL) were observed. These results suggested that the metabolisms of antioxidant and cell wall could result in softening and senescence of pummelo fruit.

Cit1,2RhaT and two novel CitdGlcTs participate in flavor-related flavonoid metabolism during citrus fruit development.

Neohesperidosides are disaccharides that are present in some flavonoids and impart a bitter taste, which can significantly affect the commercial value of citrus fruits. In this study, we identified three flavonoid-7-O-di-glucosyltransferase (dGlcT) genes closely related to 1,2-rhamnosyltransferase (1,2RhaT) in citrus genomes. However, only 1,2RhaT was directly linked to the accumulation of neohesperidoside, as demonstrated by association analysis of 50 accessions and co-segregation analysis of an F1 population derived from Citrus reticulata × Poncirus trifoliata. In transgenic tobacco BY2 cells, over-expression of CitdGlcTs resulted in flavonoid-7-O-glucosides being catalysed into bitterless flavonoid-7-O-di-glucosides, whereas over-expression of Cit1,2RhaT converted the same substrate into bitter-tasting flavonoid-7-O-neohesperidoside. Unlike 1,2RhaT, during citrus fruit development the dGlcTs showed an opposite expression pattern to CHS and CHI, two genes encoding rate-limiting enzymes of flavonoid biosynthesis. An uncoupled availability of dGlcTs and substrates might result in trace accumulation of flavonoid-7-O-di-glucosides in the fruit of C. maxima (pummelo). Past human selection of the deletion and functional mutation of 1,2RhaT has led step-by-step to the evolution of the flavor-related metabolic network in citrus. Our research provides the basis for potentially improving the taste in citrus fruit through manipulation of the network by knocking-out 1,2RhaT or by enhancing the expression of dGlcT using genetic transformation.

Effect of overexpression of citrus 9-cis-epoxycarotenoid dioxygenase 3 (CsNCED3) on the physiological response to drought stress in transgenic tobacco.

9-cis-epoxycarotenoid dioxygenase (NCED) encodes a key enzyme in abscisic acid (ABA) biosynthesis. Little is known regarding the regulation of stress response by NCEDs at physiological levels. In the present study, we generated transgenic tobacco overexpressing an NCED3 ortholog from citrus (CsNCED3) and investigated its relevance in the regulation of drought stress tolerance. Wild-type (WT) and transgenic plants were grown under greenhouse conditions and subjected to drought stress for 10 days. Leaf predawn water potential (Ψwleaf), stomatal conductance (gs), net photosynthetic rate (A), transpiration rate (E), instantaneous (A/E) and intrinsic (A/gs) water use efficiency (WUE), and in situ hydrogen peroxide (H2O2) and abscisic acid (ABA) production were determined in leaves of irrigated and drought-stressed plants. The Ψwleaf decreased throughout the drought stress period in both WT and transgenic plants, but was restored after re-watering. No significant differences were observed in gs between WT and transgenic plants under normal conditions. However, the transgenic plants showed a decreased (P ≤ 0.01) gs on the 4th day of drought stress, which remained lower (P ≤ 0.001) than the WT until the end of the drought stress. The A and E levels in the transgenic plants were similar to those in WT; therefore, they exhibited increased A/gs under drought conditions. No significant differences in A, E, and gs values were observed between the WT and transgenic plants after re-watering. The transgenic plants had lower H2O2 and higher ABA than the WT under drought conditions. Our results support the involvement of CsNCED3 in drought avoidance.

Molecular cloning and characterization of a flavonoid-O-methyltransferase with broad substrate specificity and regioselectivity from Citrus depressa.

BACKGROUND: Flavonoids are secondary metabolites that play significant roles in plant cells. In particular, polymethoxy flavonoids (PMFs), including nobiletin, have been reported to exhibit various health-supporting properties such as anticancer, anti-inflammatory, and anti-pathogenic properties. However, it is difficult to utilize PMFs for medicinal and dietary use because plant cells contain small amounts of these compounds. Biosynthesis of PMFs in plant cells is carried out by the methylation of hydroxyl groups of flavonoids by O-methyltransferases (FOMT), and many kinds of FOMTs with different levels of substrate specificity and regioselectivity are cooperatively involved in this biosynthesis. RESULTS: In this study, we isolated five genes encoding FOMT (CdFOMT1, 3, 4, 5, and 6) from Citrus depressa, which is known to accumulate nobiletin in the peels of its fruits. The genes encoded Mg(2+)-independent O-methyltransferases and showed high amino acid sequence similarity (60-95 %) with higher plant flavonoid O-methyltransferases. One of these genes is CdFOMT5, which was successfully expressed as a soluble homodimer enzyme in Escherichia coli. The molecular mass of the recombinant CdFOMT5 subunit was 42.0 kDa including a 6× histidine tag. The enzyme exhibited O-methyltransferase activity for quercetin, naringenin, (-)-epicatechin, and equol using S-adenosyl-L-methionine (SAM) as a methyl donor, and its optimal pH and temperature were pH 7.0 and 45 °C, respectively. The recombinant CdFOMT5 demonstrated methylation activity for the 3-, 5-, 6-, and 7-hydroxyl groups of flavones, and 3,3',5,7-tetra-O-methylated quercetin was synthesized from quercetin as a final product of the whole cell reaction system. Thus, CdFOMT5 is a O-methyltransferase possessing a broad range of substrate specificity and regioselectivity for flavonoids. CONCLUSIONS: Five FOMT genes were isolated from C. depressa, and their nucleotide sequences were determined. CdFOMT5 was successfully expressed in E. coli cells, and the enzymatic properties of the recombinant protein were characterized. Recombinant CdFOMT5 indicated O-methyltransferase activity for many flavonoids and a broad regioselectivity for quercetin as a substrate. Whole-cell biocatalysis using CdFOMT5 expressed in E. coli cells was performed using quercetin as a substrate, and 3,3',5,7-tetramethylated quercetin was obtained as the final product.

Isolation of a sesquiterpene synthase expressing in specialized epithelial cells surrounding the secretory cavities in rough lemon (Citrus jambhiri).

Volatile terpenoids such as monoterpenes and sesquiterpenes play multiple roles in plant responses and are synthesized by terpene synthases (TPSs). We have previously isolated a partial TPS gene, RlemTPS4, that responds to microbial attack in rough lemon. In this study, we isolated a full length RlemTPS4 cDNA from rough lemon. RlemTPS4 localized in the cytosol. The recombinant RlemTPS4 protein was obtained using a prokaryotic expression system and GC-MS analysis of the terpenes produced by the RlemTPS4 enzymatic reaction determined that RlemTPS4 produces some sesquiterpenes such as δ-elemene. The RlemTPS4 gene was specifically expressed in specialized epithelial cells surrounding the oil secretory cavities in rough lemon leaf tissue.

ICE1 of Poncirus trifoliata functions in cold tolerance by modulating polyamine levels through interacting with arginine decarboxylase.

ICE1 (Inducer of CBF Expression 1) encodes a MYC-like basic helix-loop-helix transcription factor that acts as a central regulator of cold response. In this study, we elucidated the function and underlying mechanisms of PtrICE1 from trifoliate orange [Poncirus trifoliata (L.) Raf.]. PtrICE1 was upregulated by cold, dehydration, and salt, with the greatest induction under cold conditions. PtrICE1 was localized in the nucleus and could bind to a MYC-recognizing sequence. Ectopic expression of PtrICE1 in tobacco and lemon conferred enhanced tolerance to cold stresses at either chilling or freezing temperatures. Yeast two-hybrid screening revealed that 21 proteins belonged to the PtrICE1 interactome, in which PtADC (arginine decarboxylase) was confirmed as a bona fide protein interacting with PtrICE1. Transcript levels of ADC genes in the transgenic lines were slightly elevated under normal growth condition but substantially increased under cold conditions, consistent with changes in free polyamine levels. By contrast, accumulation of the reactive oxygen species, H2O2 and O2 (-), was appreciably alleviated in the transgenic lines under cold stress. Higher activities of antioxidant enzymes, such as superoxide dismutase and catalase, were detected in the transgenic lines under cold conditions. Taken together, these results demonstrated that PtrICE1 plays a positive role in cold tolerance, which may be due to modulation of polyamine levels through interacting with the ADC gene.

Inactivation, aggregation, secondary and tertiary structural changes of germin-like protein in Satsuma mandarine with high polyphenol oxidase activity induced by ultrasonic processing.

The inhibition of Polyphenol oxidase (PPO) in plants has been widely researched for their important roles in browning reaction. A newly found germin-like protein (GLP) with high PPO activity in Satsuma mandarine was inactivated by low-frequency high-intensity ultrasonic (20 kHz) processing. The effects of ultrasound on PPO activity and structure of GLP were investigated using dynamic light scattering (DLS) analysis, transmission electron microscopy (TEM), circular dichroism (CD) spectral measurement and fluorescence spectral measurement. The lowest PPO activity achieved was 27.4% following ultrasonication for 30 min at 400 W. DLS analysis showed ultrasound caused both aggregation and dissociation of GLP particles. TEM images also demonstrated protein aggregation phenomena. CD spectra exhibited a certain number of loss in α-helix structure content. Fluorescence spectra showed remarkable increase in fluorescence intensity with tiny blue-shift following ultrasonication. In conclusion, ultrasound applied in this study induced structural changes of GLP and eventually inactivated PPO activity.

Molecular cloning and characterization of a geranyl diphosphate-specific aromatic prenyltransferase from lemon.

Prenyl residues confer divergent biological activities such as antipathogenic and antiherbivorous activities on phenolic compounds, including flavonoids, coumarins, and xanthones. To date, about 1,000 prenylated phenolics have been isolated, with these compounds containing various prenyl residues. However, all currently described plant prenyltransferases (PTs) have been shown specific for dimethylallyl diphosphate as the prenyl donor, while most of the complementary DNAs encoding these genes have been isolated from the Leguminosae. In this study, we describe the identification of a novel PT gene from lemon (Citrus limon), ClPT1, belonging to the homogentisate PT family. This gene encodes a PT that differs from other known PTs, including flavonoid-specific PTs, in polypeptide sequence. This membrane-bound enzyme was specific for geranyl diphosphate as the prenyl donor and coumarin as the prenyl acceptor. Moreover, the gene product was targeted to plastid in plant cells. To our knowledge, this is the novel aromatic PT specific to geranyl diphosphate from citrus species.

Characterization of germin-like protein with polyphenol oxidase activity from Satsuma mandarine.

Polyphenol oxidases (PPOs) catalyzing the oxygen dependent oxidation of phenols to quinones are ubiquitously distributed in plants and are assumed to be involved in plant defense against pests and pathogens. A protein with high PPO activity was identified in Satsuma mandarine, extracted with Tris-HCl buffer, purified by salt precipitation and column chromatography, and characterized by mass spectrometry as germin-like protein (GLP), which belongs to pathogenesis related protein (PR) family. In the present study, the structure and enzymatic properties of GLP were characterized using spectroscopy methods. Based on native PAGE analysis, the molecular weight of GLP was estimated to be 108 kDa and GLP was identified as a pentamer containing five subunits of 22 kDa. The optimum pH and temperature for PPO catalyzing activity of GLP was 6.5 and 65°C, respectively. Kinetic constants were 0.0365 M and 0.0196 M with the substrates catechol and pyrogallol, respectively. The structural characterization of GLP provided better insights into the regions responsible for its PPO activity.

Influence of crop load on the expression patterns of starch metabolism genes in alternate-bearing citrus trees.

The fruit is the main sink organ in Citrus and captures almost all available photoassimilates during its development. Consequently, carbohydrate partitioning and starch content depend on the crop load of Citrus trees. Nevertheless, little is known about the mechanisms controlling the starch metabolism at the tree level in relation to presence of fruit. The aim of this study was to find the relation between the seasonal variation of expression and activity of the genes involved in carbon metabolism and the partition and allocation of carbohydrates in 'Salustiana' sweet orange trees with different crop loads. Metabolisable carbohydrates, and the expression and activity of the enzymes involved in sucrose and starch metabolism, including sucrose transport, were determined during the year in the roots and leaves of 40-year-old trees bearing heavy crop loads ('on' trees) and trees with almost no fruits ('off' trees). Fruit altered photoassimilate partitioning in trees. Sucrose content tended to be constant in roots and leaves, and surplus fixed carbon is channeled to starch production. Differences between 'on' and 'off' trees in starch content can be explained by differences in ADP-glucose pyrophosphorylase (AGPP) expression/activity and α-amylase activity which varies depending on crop load. The observed relation of AGPP and UGPP (UDP-glucose pyrophosphorylase) is noteworthy and indicates a direct link between sucrose and starch synthesis. Furthermore, different roles for sucrose transporter SUT1 and SUT2 have been proposed. Variation in soluble sugars content cannot explain the differences in gene expression between the 'on' and 'off' trees. A still unknown signal from fruit should be responsible for this control.

Altered sensitivity to ethylene in 'Tardivo', a late-ripening mutant of Clementine mandarin.

'Tardivo' mandarin is a mutant of 'Comune' Clementine with a delay in peel degreening and coloration, allowing late harvesting. In this work, we have explored if the late-harvesting phenotype of 'Tardivo' mandarin is related to altered perception and sensitivity to ethylene. The peel degreening rate was examined after a single ethephon treatment or during a continuous ethylene application in fruits at two maturation stages. In general, ethylene-induced peel degreening was considerably delayed and reduced in fruits of 'Tardivo', as well as the concomitant reduction of chlorophyll (Chl) and chloroplastic carotenoids, and the accumulation of chromoplastic carotenoids. Analysis of the expression of genes involved in Chl degradation, carotenoids, ABA, phenylpropanoids and ethylene biosynthesis revealed an impairment in the stimulation of most genes by ethylene in the peel of 'Tardivo' fruits with respect to 'Comune', especially after 5 days of ethylene application. Moreover, ethylene-induced expression of two ethylene receptor genes, ETR1 and ETR2, was also reduced in mutant fruits. Expression levels of two ethylene-responsive factors, ERF1 and ERF2, which were repressed by ethylene, were also impaired to a different extent, in fruits of both genotypes. Collectively, results suggested an altered sensitivity of the peel of 'Tardivo' to ethylene-induced physiological and molecular responses, including fruit degreening and coloration processes, which may be time-dependent since an early moderated reduction in the responses was followed by the latter inability to sustain ethylene action. These results support the involvement of ethylene in the regulation of at least some aspects of peel maturation in the non-climacteric citrus fruit.

Enzymatic formation of ß-citraurin from ß-cryptoxanthin and Zeaxanthin by carotenoid cleavage dioxygenase4 in the flavedo of citrus fruit.

In this study, the pathway of ß-citraurin biosynthesis, carotenoid contents and the expression of genes related to carotenoid metabolism were investigated in two varieties of Satsuma mandarin (Citrus unshiu), Yamashitabeni-wase, which accumulates ß-citraurin predominantly, and Miyagawa-wase, which does not accumulate ß-citraurin. The results suggested that CitCCD4 (for Carotenoid Cleavage Dioxygenase4) was a key gene contributing to the biosynthesis of ß-citraurin. In the flavedo of Yamashitabeni-wase, the expression of CitCCD4 increased rapidly from September, which was consistent with the accumulation of ß-citraurin. In the flavedo of Miyagawa-wase, the expression of CitCCD4 remained at an extremely low level during the ripening process, which was consistent with the absence of ß-citraurin. Functional analysis showed that the CitCCD4 enzyme exhibited substrate specificity. It cleaved ß-cryptoxanthin and zeaxanthin at the 7,8 or 7',8' position. But other carotenoids tested in this study (lycopene, α-carotene, ß-carotene, all-trans-violaxanthin, and 9-cis-violaxanthin) were not cleaved by the CitCCD4 enzyme. The cleavage of ß-cryptoxanthin and zeaxanthin by CitCCD4 led to the formation of ß-citraurin. Additionally, with ethylene and red light-emitting diode light treatments, the gene expression of CitCCD4 was up-regulated in the flavedo of Yamashitabeni-wase. These increases in the expression of CitCCD4 were consistent with the accumulation of ß-citraurin in the two treatments. These results might provide new strategies to improve the carotenoid contents and compositions of citrus fruits.

Metabolic responses to iron deficiency in roots of Carrizo citrange [Citrus sinensis (L.) Osbeck. x Poncirus trifoliata (L.) Raf].

The effects of iron (Fe) deficiency on the low-molecular-weight organic acid (LMWOA) metabolism have been investigated in Carrizo citrange (CC) [Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.] roots. Major LMWOAs found in roots, xylem sap and root exudates were citrate and malate and their concentrations increased with Fe deficiency. The activities of several enzymes involved in the LMWOA metabolism were also assessed in roots. In the cytosolic fraction, the activities of malate dehydrogenase (cMDH) and phosphoenolpyruvate carboxylase (PEPC) enzymes were 132 and 100% higher in Fe-deficient conditions, whereas the activity of pyruvate kinase was 31% lower and the activity of malic enzyme (ME) did not change. In the mitochondrial fraction, the activities of fumarase, MDH and citrate synthase enzymes were 158, 117 and 53% higher, respectively, in Fe-deficient extracts when compared with Fe-sufficient controls, whereas no significant differences between treatments were found for aconitase (ACO) activity. The expression of their corresponding genes in roots of Fe-deficient plants was higher than that measured in Fe-sufficient controls, except for ACO and ME. Also, dicarboxylate-tricarboxylate carrier (DTC) expression was significantly increased in Fe-deficient roots. In conclusion, Fe deficiency in CC seedlings causes a reprogramming of the carbon metabolism that involves an increase of anaplerotic fixation of carbon via PEPC and MDH activities in the cytosol and a shift of the Krebs cycle in the mitochondria towards a non-cyclic mode, as previously described in herbaceous species. In this scheme, DTC could play an important role shuttling both malate and reducing equivalents between the cytosol and the mitochondria. As a result of this metabolic switch malate and citrate concentrations in roots, xylem sap and root exudates increase.

Bicarbonate blocks iron translocation from cotyledons inducing iron stress responses in Citrus roots.

The effect of bicarbonate ion (HCO3(-)) on the mobilization of iron (Fe) reserves from cotyledons to roots during early growth of citrus seedlings and its influence on the components of the iron acquisition system were studied. Monoembryonic seeds of Citrus limon (L.) were germinated "in vitro" on two iron-deprived media, supplemented or not with 10mM HCO3(-) (-Fe+Bic and -Fe, respectively). After 21d of culture, Fe concentration in seedling organs was measured, as well as gene expression and enzymatic activities. Finally, the effect of Fe resupply on the above responses was tested in the presence and absence of HCO3(-) (+Fe+Bic or +Fe, respectively). -Fe+Bic seedlings exhibited lower Fe concentration in shoots and roots than -Fe ones but higher in cotyledons, associated to a significative inhibition of NRAMP3 expression. HCO3(-) upregulated Strategy I related genes (FRO1, FRO2, HA1 and IRT1) and FC-R and H(+)-ATPase activities in roots of Fe-starved seedlings. PEPC1 expression and PEPCase activity were also increased. When -Fe+Bic pre-treated seedlings were transferred to Fe-containing media for 15d, Fe content in shoots and roots increased, although to a lower extent in the +Fe+Bic medium. Consequently, the above-described root responses became markedly repressed, however, this effect was less pronounced in +Fe+Bic seedlings. In conclusion, it appears that HCO3(-) prevents Fe translocation from cotyledons to shoot and root, therefore reducing their Fe levels. This triggers Fe-stress responses in the root, enhancing the expression of genes related with Fe uptake and the corresponding enzymatic activities.

Purification and characterization of a thermostable soluble peroxidase from Citrus medica leaf.

A soluble and thermostable peroxidase enzyme (POD) was extracted from the leaf of Citrus medica. The enzyme was purified 15.10-fold with a total yield of 28.6% by ammonium sulfate precipitation followed by Sephadex G-100 gel filtration chromatography. The purified enzyme came as a single band on native polyacrylamide gel electrophoresis (PAGE) as well as sodium dodecyl sulfate (SDS) PAGE. The molecular mass of the enzyme was about 32 kD as determined by SDS-PAGE. The enzyme was optimally active at pH 6.0 and 50°C temperature. The enzyme was active in wide range of pH (5.0-8.0) and temperature (30-80°C). From the thermal inactivation studies in the range of 60-75°C, the half-life (t(1/2)) values of the enzyme ranged from 8 to 173 min. The inactivation energy (Ea) value of POD was estimated to be 21.7 kcal mol(-1). The Km values for guaiacol and H(2)O(2) were 8 mM and 1.8 mM, respectively. This enzyme was activated by some metals and reagents such as Ca(2+), Cu(2+), Mg(2+), Co(2+), ferulic acid, and indole acetic acid (IAA), while it was inhibited by Fe(2+), Zn(2+), Hg(2+), and Mn(2+), L-cysteine, L-proline, and protocatechuic acid.

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 ß-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.