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UDP-glycosyltransferases (UGTs) play a crucial role in the glycosylation of secondary metabolites in plants, which is of significant importance for growth and response to biotic or abiotic stress. Despite the wide identification of UGT family members in various species, limited information is available regarding this family in citrus. In this study, we identified 87 UGT genes from the Citrus sinensis genome and classified them into 14 groups. We characterized their gene structures and motif compositions, providing insights into the molecular basis underlying discrepant functions of UGT genes within each evolutionary branch. Tandem duplication events were found to be the main driving force behind UGT gene expansion. Additionally, we identified numerous cis-acting elements in the promoter region of UGT genes, including those responsive to light, growth factors, phytohormones, and stress conditions. Notably, light-responsive elements were found with a frequency of 100 %. We elucidated the expression pattern of UGTs during fruit development in Citrus aurantium using RNA-seq and quantitative real-time PCR (qRT-PCR), revealing that 10 key UGT genes are closely associated with biosynthesis of bitter flavanone neohesperidosides (FNHs). Furthermore, we identified Ca1,2RhaT as a flavonoid 1-2 rhamnosyltransferase (1,2RhaT) involved in FNHs biosynthesis for the first time. Isolation and functional characterization of the gene Ca1,2RhaT from Citrus aurantium in vitro and in vivo indicated that Ca1,2RhaT encoded a citrus 1,2RhaT and possessed rhamnosyl transfer activities. This work provides comprehensive information on the UGT family while offering new insights into understanding molecular mechanisms regulating specific accumulation patterns of FNHs or non-bitter flavanone rutinosides (FRTs) in citrus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

As one of the subtropical and tropical fruit trees, Citrus sinensis is sensitive to cold stress. However, most transcription factors (TFs) that regulate cold tolerance in citrus have not yet been reported. A phytochrome-interacting transcription factor (PIF) gene (CsPIF8) in citrus was significantly upregulated under cold stress. Overexpression of CsPIF8 increased cold tolerance in transgenic tomato plants and grapefruit callus, whereas virus-induced gene silencing-mediated suppression of PIF8 increased cold sensitivity in seedlings of Poncirus trifoliata. Superoxide dismutase (SOD) reduces the superoxide anion (O 2 - ) level to enhance cold tolerance in plants. Chromatin immunoprecipitation combined with high-throughput sequencing, yeast one hybrid, electrophoretic mobility shift and dual luciferase assays showed that CsPIF8 directly bound the E-box (CANNTG) of CsSOD promoter and activated the promoter of CsSOD. Furthermore, the expression level of CsSOD and CsSOD activity were significantly increased, whereas the level of O 2 - was significantly reduced in the transgenic lines. The Poncirus trifoliata seedlings with VIGS-mediated suppression of PIF8 exhibited the opposite effects. These results have shown that CsPIF8 improved cold tolerance in citrus through regulating the expression level of SOD and SOD activity. These findings may provide novel insights into the regulation of PIF8 in the response to cold stress in citrus., Competing Interests: Declaration of Competing Interest The authors have a patent pending related to this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Uronic acid-rich plant materials such as pectin are potential renewable feedstocks for the chemical industry. Uronic acid oxidase activity was first reported in extracts of citrus leaves, and was subsequently found to be widely distributed in plants, with the highest activity detected in extracts of the pectin-rich citrus peel. Herein we report the identification of the primary sequence of uronic acid oxidase from extracts of the peel of Citrus sinensis, by partial purification and protein mass spectrometry. Activity of the enzyme, a member of the berberine bridge family, was confirmed by recombinant expression in Pichia pastoris. Biochemical characterization of the recombinant enzyme is reported. Our findings facilitate further investigation of the biological function of uronic acid oxidation in the economically important orange fruit, and also provide a basis for the development of a catalyst for bioconversion of uronic acids., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Citrus fruit production occupies a place of considerable importance in the economy of the world including Pakistan. Tristeza disease caused by Citrus Tristeza Virus (CTV) exists in various forms that may or may not cause symptoms in the plants. The bioactive compounds and antioxidants are naturally present in plants and provide a defense mechanism that is generally accelerated in response to a stress. The objective of the present study was to target and analyze the citrus plants that were CTV positive to observe the changes in the enzymatic and non-enzymatic antioxidants of citrus (Sweet Oranges only). It was observed that in response to CTV infection, both the non-enzymatic antioxidants (total flavonoid, ascorbic acid, phenolic acid) and enzymatic antioxidants (catalase, superoxide dismutase and peroxidase) activities showed an increasing trend overall. The profiling of antioxidants in response to a viral infection may help in the discovery of new biomarkers that can be used as a monitoring tool in disease management.

Linalyl diphosphate (LPP) is the postulated intermediate in the enzymatic cyclization of monoterpenes catalyzed by terpene synthases. LPP is considered an obligate intermediate due to the conformationally restrictive trans -C2-C3 double bond of the substrate, geranyl diphosphate (GPP), which precludes the proper positioning of carbons C1 and C6 to enable cyclization. However, because of the complexity of potential carbocation-mediated rearrangements in these enzymatic reactions, it has proven difficult to directly demonstrate the formation of LPP despite significant efforts. Here we synthesized a fluorinated substrate analog, 8,9-difluorogeranyl diphosphate (DFGPP), which is designed to allow initial ionization/isomerization and form the fluorinated equivalent of LPP (DFLPP) while preventing the subsequent ionization/cyclization to produce the α-terpinyl cation. Steady-state kinetic studies with the model enzyme (+)-limonene synthase (LS) under catalytic conditions show that the cyclization of DFGPP is completely blocked and a single linear product, difluoromyrcene, is produced. When crystals of apo-LS are soaked with DFGPP under conditions limiting turnover of the enzyme, we show, using X-ray crystallography, that DFLPP is produced in the enzyme active site and trapped in the crystals. Clear electron density is observed in the active site of the enzyme, but it cannot be appropriately fit with a model for the DFGPP substrate analog, whereas it can accommodate an extended conformation of DFLPP. This result supports the current model for monoterpene cyclization by providing direct evidence of LPP as an intermediate.

Limonoids are natural products made by plants belonging to the Meliaceae (Mahogany) and Rutaceae (Citrus) families. They are well known for their insecticidal activity, contribution to bitterness in citrus fruits, and potential pharmaceutical properties. The best known limonoid insecticide is azadirachtin, produced by the neem tree ( Azadirachta indica ). Despite intensive investigation of limonoids over the last half century, the route of limonoid biosynthesis remains unknown. Limonoids are classified as tetranortriterpenes because the prototypical 26-carbon limonoid scaffold is postulated to be formed from a 30-carbon triterpene scaffold by loss of 4 carbons with associated furan ring formation, by an as yet unknown mechanism. Here we have mined genome and transcriptome sequence resources for 3 diverse limonoid-producing species ( A. indica , Melia azedarach , and Citrus sinensis ) to elucidate the early steps in limonoid biosynthesis. We identify an oxidosqualene cyclase able to produce the potential 30-carbon triterpene scaffold precursor tirucalla-7,24-dien-3β-ol from each of the 3 species. We further identify coexpressed cytochrome P450 enzymes from M. azedarach (MaCYP71CD2 and MaCYP71BQ5) and C. sinensis (CsCYP71CD1 and CsCYP71BQ4) that are capable of 3 oxidations of tirucalla-7,24-dien-3β-ol, resulting in spontaneous hemiacetal ring formation and the production of the protolimonoid melianol. Our work reports the characterization of protolimonoid biosynthetic enzymes from different plant species and supports the notion of pathway conservation between both plant families. It further paves the way for engineering crop plants with enhanced insect resistance and producing high-value limonoids for pharmaceutical and other applications by expression in heterologous hosts., Competing Interests: Conflict of interest statement: Authors involved in patent filing are H.H., M.J.S., and A.O., (Copyright © 2019 the Author(s). Published by PNAS.)

Poly(A) tail shortening is a critical step in messenger RNA (mRNA) decay and control of gene expression. The carbon catabolite repressor 4 (CCR4)-associated factor 1 (CAF1) component of the CCR4-NOT deadenylase complex plays an essential role in mRNA deadenylation in most eukaryotes. However, while CAF1 has been extensively investigated in yeast and animals, its role in plants remains largely unknown. Here, we show that the Citrus sinensis CAF1 (CsCAF1) is a magnesium-dependent deadenylase implicated in resistance against the citrus canker bacteria Xanthomonas citri. CsCAF1 interacted with proteins of the CCR4-NOT complex, including CsVIP2, a NOT2 homologue, translin-associated factor X (CsTRAX) and the poly(A)-binding proteins CsPABPN and CsPABPC. CsCAF1 also interacted with PthA4, the main X. citri effector required for citrus canker elicitation. We also present evidence suggesting that PthA4 inhibits CsCAF1 deadenylase activity in vitro and stabilizes the mRNA encoded by the citrus canker susceptibility gene CsLOB1, which is transcriptionally activated by PthA4 during canker formation. Moreover, we show that an inhibitor of CsCAF1 deadenylase activity significantly enhanced canker development, despite causing a reduction in PthA4-dependent CsLOB1 transcription. These results thus link CsCAF1 with canker development and PthA4-dependent transcription in citrus plants., (© 2019 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

The discovery of molecules that can inhibit the action of phytopathogens is essential to find alternative to current pesticides. Pectin methylesterases (PME), enzymes that fine-tune the degree of methylesterification of plant cell wall pectins, play a key role in the pathogenicity of fungi or bacteria. Here we report the synthesis of new lactoside derivatives and their analysis as potential PME inhibitors using three plants and one fungal PME. Because of its structure, abundance and reduced cost, lactose was chosen as a case study. Lactoside derivatives were obtained by TEMPO-mediated oxidation of methyl lactoside, followed by an esterification procedure. Three derivatives were synthesized: sodium (methyl-lactosid)uronate, methyl (methyl-lactosid)uronate and butyl (methyl-lactosid)uronate. The inhibition of the plant and pathogen enzyme activities by lactoside derivatives was measured in vitro, showing the importance of the substitution on lactose: methyl (methyl-lactosid)uronate was more efficient than butyl (methyl-lactosid)uronate. These results were confirmed by docking analysis showing the difference in the interaction between lactoside derivatives and PME proteins. In conclusion, this study identified novel inhibitors of pectin remodeling enzymes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Apart from non-enzymatic browning, polyphenol oxidase (PPO) also plays a role in the browning reaction of orange ( Citrus sinensis Osbeck) juice, and needs to be inactivated during the processing. In this study, the protein with high PPO activity was purified from orange ( Citrus sinensis Osbeck) and inactivated by ultrasonic processing. Fluorescence spectroscopy, circular dichroism (CD) and Dynamic light scattering (DLS) were used to investigate the ultrasonic effect on PPO activity and structural changes on purified PPO. DLS analysis illustrated that ultrasonic processing leads to initial dissociation and final aggregation of the protein. Fluorescence spectroscopy analysis showed the decrease in fluorescence intensity leading to the exposure of Trp residues to the polar environment, thereby causing the disruption of the tertiary structure after ultrasonic processing. Loss of α-helix conformation leading to the reorganization of secondary structure was triggered after the ultrasonic processing, according to CD analysis. Ultrasonic processing could induce aggregation and modification in the tertiary and secondary structure of a protein containing high PPO activity in orange ( Citrus sinensis Osbeck), thereby causing inactivation of the enzyme.

In plants, the final step of monolignols polymerization is catalyzed by laccase, a key enzyme in lignin biosynthesis. Laccase has been shown a multifunctional enzyme that plays many important roles. As information is not available on the laccase gene family in Citrus sinensis, genome-wide analysis has been carried out in this study using C. sinensis genome. Using bioinformatics approaches, 24 laccase genes (CsLAC1~CsLAC24) were identified from C. sinensis. Most CsLACs were found in C. sinensis chromosome 6, 7 and 8, while no CsLACs were found in chromosome 4, 5 and 9. In most CsLACs, four conserved signature sequences and three typical Cu-oxidase domains were observed. However, the CsLAC-encoding genes displayed distinct intron-exon patterns and relatively low sequence similarity. Phylogenetic clustering analysis indicated that the CsLACs were divided into seven groups, suggesting potential distinct functions and evolution. Putative signal sequences, subcellular location and glycosylation sites were predicted in the CsLACs. Moreover, sixteen CsLAC transcripts, which coding genes were clustering in chromosomes, were found to be potential targets of csi-miR397. Cis-regulatory elements and expression analyses indicated the possible involvement of some CsLAC members in diverse stresses and growth/development processes, respectively. These results may provide valuable clues for further studies on the functions of the CsLACs in citrus growth and adaptation to stress., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This work reports the molecular cloning and heterologous expression of the genes coding for α and β subunits of pyrophosphate-dependent phosphofructokinase (PPi-PFK) from orange. When expressed individually, both recombinant subunits were produced as highly purified monomeric proteins able to phosphorylate fructose-6-phosphate at the expenses of PPi (specific activity of 0.075 and 0.017 units. mg -1 for α and β subunits, respectively). On the other hand, co-expression rendered a α 3 β 3 hexamer with specific activity three orders of magnitude higher than the single subunits. All the conformations of the enzyme were characterized with respect to its kinetic properties and sensitivity to the regulator fructose-2,6-bisphosphate. A thorough review of current knowledge on the matter indicates that this is the first report of the recombinant production of active plant PPi-PFK and the characterization of its different conformations. This is a main contribution for future studies focused to better understand the enzyme properties and how it accomplishes its relevant role in plant metabolism., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Boron (B) deficiency and toxicity are the major factors that affect plant growth and yield. The present study revealed the effect of B deficiency and toxicity on plant growth, morphology, physiology, and cell structure. A hydroponic culture experiment was conducted with five B levels, B deficient (B0), sufficient (B20, B10, B40) and toxic (B100). Our results show that both B deficient as well as excess level inhibit plant growth. In B deficiency, the major visible symptoms were appeared in roots, while B excess burned the leaf margin of older leaves. The antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) decreased at B deficiency and also decreased up to some extent at B excess, while in sufficient treatments, the higher antioxidant enzymes were found at B20. In addition, the MDA concentration decreased at B deficiency and increased with B concentration. Moreover, the photosynthetic rate, transpiration rate, stomatal conductance, leaf gas exchange and intercellular CO 2 were reduced at both B deficiency as well as excess and higher at sufficient B20 treatment significantly. The chlorophyll and carotenoid content increased at B20 treatment, while decreased at B deficiency and excess. The middle lamellae of cell wall were found thick at B excess and normal at B20. The current study revealed that B deficiency as well as excess concentration affect plant growth and various morpho-physiological processes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Terpenes make up the largest and most diverse class of natural compounds and have important commercial and medical applications. Limonene is a cyclic monoterpene (C 10 ) present in nature as two enantiomers, (+) and (-), which are produced by different enzymes. The mechanism of production of the (-)-enantiomer has been studied in great detail, but to understand how enantiomeric selectivity is achieved in this class of enzymes, it is important to develop a thorough biochemical description of enzymes that generate (+)-limonene, as well. Here we report the first cloning and biochemical characterization of a (+)-limonene synthase from navel orange (Citrus sinensis). The enzyme obeys classical Michaelis-Menten kinetics and produces exclusively the (+)-enantiomer. We have determined the crystal structure of the apoprotein in an "open" conformation at 2.3 Å resolution. Comparison with the structure of (-)-limonene synthase (Mentha spicata), which is representative of a fully closed conformation (Protein Data Bank entry 2ONG ), reveals that the short H-α1 helix moves nearly 5 Å inward upon substrate binding, and a conserved Tyr flips to point its hydroxyl group into the active site.

The stereochemical course of monoterpene synthase reactions is thought to be determined early in the reaction sequence by selective binding of distinct conformations of the geranyl diphosphate (GPP) substrate. We explore here formation of early Michaelis complexes of the (+)-limonene synthase [(+)-LS] from Citrus sinensis using monofluorinated substrate analogues 2-fluoro-GPP (FGPP) and 2-fluoroneryl diphosphate (FNPP). Both are competitive inhibitors for (+)-LS with K I values of 2.4 ± 0.5 and 39.5 ± 5.2 μM, respectively. The K I values are similar to the K M for the respective nonfluorinated substrates, indicating that fluorine does not significantly perturb binding of the ligand to the enzyme. FGPP and FNPP are also substrates, but with dramatically reduced rates (k cat values of 0.00054 ± 0.00005 and 0.00024 ± 0.00002 s -1 , respectively). These data are consistent with a stepwise mechanism for (+)-LS involving ionization of the allylic GPP substrate to generate a resonance-stabilized carbenium ion in the rate-limiting step. Crystals of apo-(+)-LS were soaked with FGPP and FNPP to obtain X-ray structures at 2.4 and 2.2 Å resolution, respectively. The fluorinated analogues are found anchored in the active site through extensive interactions involving the diphosphate, three metal ions, and three active-site Asp residues. Electron density for the carbon chains extends deep into a hydrophobic pocket, while the enzyme remains mostly in the open conformation observed for the apoprotein. While FNPP was found in multiple conformations, FGPP, importantly, was in a single, relatively well-defined, left-handed screw conformation, consistent with predictions for the mechanism of stereoselectivity in the monoterpene synthases.

Citrus fruits are characterized by a complex mixture of volatiles making up their characteristic aromas, being the d-limonene the most abundant one. However, its role on citrus fruit and juice odor is controversial. Transgenic oranges engineered for alterations in the presence or concentration of few related chemical groups enable asking precise questions about their contribution to overall odor, either positive or negative, as perceived by the human nose. Here, either down- or up-regulation of a d-limonene synthase allowed us to infer that a decrease of as much as 51 times in d-limonene and an increase of as much as 3.2 times in linalool in juice were neutral for odor perception while an increase of only 3 times in ethyl esters stimulated the preference of 66% of the judges. The ability to address these questions presents exciting opportunities to understand the basic principles of selection of food., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Polyamine oxidase (PAO) is a key enzyme catalyzing polyamine catabolism leading to H2O2 production. We previously demonstrated that Citrus sinensis contains six putative PAO genes, but their functions are not well understood. In this work, we reported functional elucidation of CsPAO4 in polyamine catabolism and salt stress response. CsPAO4 was localized to the apoplast and used both spermidine (Spd) and spermine (Spm) as substrates for terminal catabolism. Transgenic plants overexpressing CsPAO4 displayed prominent increase in PAO activity, concurrent with marked decrease of Spm and Spd and elevation of H2O2. Seeds of transgenic lines displayed better germination when compared with wild type (WT) under salt stress. However, both vegetative growth and root elongation of the transgenic lines were prominently inhibited under salt stress, accompanied by higher level of H2O2 and more conspicuous programmed cell death (PCD). Exogenous supply of catalase (CAT), a H2O2 scavenger, partially recovered the vegetative growth and root elongation. In addition, spermine inhibited root growth of transgenic plants. Taken together, these data demonstrated that CsPAO4 accounts for production of H2O2 causing oxidative damages under salt stress and that down-regulation of a PAO gene involved in polyamine terminal catabolism may be an alternative approach for improving salt stress tolerance.

Aroma is a vital characteristic that determines the quality and commercial value of citrus fruits, and characteristic volatiles have been analyzed in different citrus species. In sweet orange, Citrus sinensis, the sesquiterpene (+)-valencene is a key volatile compound in the fruit peel. Valencene synthesis is catalyzed by the terpene synthase CsTPS1, but the transcriptional mechanisms controlling its gene expression are unknown. Here, the AP2/ERF (APETALA2/ethylene response factor) transcription factor, CitAP2.10, is characterized as a regulator of (+)-valencene synthesis. The expression pattern of CitAP2.10 was positively correlated with (+)-valencene content and CsTPS1 expression. Dual-luciferase assays indicated that CitAP2.10 could trans-activate the CsTPS1 promoter. Ethylene enhanced expression of CitAP2.10 and this effect was abolished by the ethylene antagonist 1-methylcyclopropene. The role and function of CitAP2.10 in (+)-valencene biosynthesis were confirmed using the Arabidopsis homolog (AtWRI1), which also transiently activated the CsTPS1 promoter. Furthermore, transient over-expression of CitAP2.10 triggered (+)-valencene biosynthesis in sweet orange fruit. These results indicate that CitAP2.10 regulates (+)-valencene synthesis via induction of CsTPS1 mRNA accumulation., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The O-methylation of various secondary metabolites is mainly catalyzed by S-adenosyl-l-methionine (SAM)-dependent O-methyltransferase (OMT) proteins that are encoded by the O-methyltransferase gene family. Citrus fruits are a rich source of O-methylated flavonoids that have a broad spectrum of biological activities, including anti-inflammatory, anticarcinogenic, and antiatherogenic properties. However, little is known about this gene family and its members that are involved in the O-methylation of flavonoids and their regulation in Citrus. In this study, 58 OMT genes were identified from the entire Citrus sinensis genome and compared with those from 3 other representative dicot plants. A comprehensive analysis was performed, including functional/substrate predictions, identification of chromosomal locations, phylogenetic relationships, gene structures, and conserved motifs. Distribution mapping revealed that the 58 OMT genes were unevenly distributed on the 9 citrus chromosomes. Phylogenetic analysis of 164 OMT proteins from C.sinensis, Arabidopsis thaliana, Populus trichocarpa, and Vitis vinifera showed that these proteins were categorized into group I (COMT subfamily) and group II (CCoAOMT subfamily), which were further divided into 10 and 2 subgroups, respectively. Finally, digital gene expression and quantitative real-time polymerase chain reaction analyses revealed that citrus OMT genes had distinct temporal and spatial expression patterns in different tissues and developmental stages. Interestingly, 18 and 11 of the 27 genes predicted to be involved in O-methylation of flavonoids had higher expression in the peel and pulp during fruit development, respectively. The citrus OMT gene family identified in this study might help in the selection of appropriate candidate genes and facilitate functional studies in Citrus., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Flavonoids, which comprise a large family of secondary plant metabolites, have received increased attention in recent years due to their wide range of features beneficial to human health. One of the most abundant flavonoid skeletons in citrus species is the flavanone naringenin, which is accumulated as glycosides containing terminal rhamnose (Rha) after serial glycosylation steps. The linkage type of Rha residues is a determining factor in the bitterness of the citrus fruit. Such Rha residues are attached by either an α1,2- or an α1,6-rhamnosyltransferase (1,2RhaT or 1,6RhaT). Although the genes encoding these RhaTs from pummelo (Citrus maxima) and orange (Citrus sinensis) have been functionally characterized, the details of the biochemical characterization, including the substrate preference, remain elusive due to the lack of availability of the UDP-Rha required as substrate. In this study, an efficient UDP-Rha in vivo production system using the engineered fission yeast expressing Arabidopsis thaliana rhamnose synthase 2 (AtRHM2) gene was constructed. The in vitro RhaT assay using the constructed UDP-Rha revealed that recombinant RhaT proteins (Cm1,2RhaT; Cs1,6RhaT; or Cm1,6RhaT), which were heterologously produced in fission yeast, catalyzed the rhamnosyl transfer to naringenin-7-O-glucoside as an acceptor. The substrate preference analysis showed that Cm1,2RhaT had glycosyl transfer activity toward UDP-xylose as well as UDP-Rha. On the other hand, Cs1,6RhaT and Cm1,6RhaT showed rhamnosyltransfer activity toward quercetin-3-O-glucoside in addition to naringenin-7-O-glucoside, indicating weak specificity toward acceptor substrates. Finally, naringin and narirutin from naringenin-7-O-glucoside were produced using the engineered fission yeast expressing the AtRHM2 and the Cm1,2RhaT or the Cs1,6RhaT genes as a whole-cell-biocatalyst.

Background: The hydrolysis of glycosidically bound volatile compounds can release potential aromas in oranges during maturation. β-Glucosidase is the key enzyme that influences the hydrolysis of bound volatiles. In this study the changes in β-glucosidase and bound volatile compounds in Jincheng oranges during maturation were investigated. The relationship between β-glucosidase activity and bound volatiles was analyzed., Results: The optimal temperature and pH of β-glucosidase from Jincheng oranges were 40 °C and 5-6 respectively. Its Km and Vmax values were 0.61 mmol L(-1) and 0.009 U mg(-1) respectively. The activity of β-glucosidase was strongly inhibited by Zn(2+), Fe(2+), Cu(2+), Ag(+), Hg(2+) and Fe(3+). β-Glucosidase activity in pulp increased gradually during maturation, while that in peel first increased and then decreased in November. In total, 12 and 14 bound volatiles were found in pulp and peel respectively of this orange during maturation., Conclusion: The concentration of bound volatiles in pulp and peel decreased with the rise in β-glucosidase activity in pulp and peel during maturation. This indicated that bound volatiles in Jincheng oranges were released during maturation owing to the increase in β-glucosidase., (© 2014 Society of Chemical Industry.)

The glutathione transferases (GSTs) are members of a superfamily of enzymes with pivotal role in the detoxification of both xenobiotic and endogenous compounds. In this work, the generation and characterization of transgenic tobacco plants over-expressing tau glutathione transferases from Citrus sinensis (CsGSTU1 and CsGSTU2) and several cross-mutate forms of these genes are reported. Putative transformed plants were verified for the presence of the transgenes and the relative quantification of transgene copy number was evaluated by Taqman real time PCR. The analysis of gene expression revealed that transformed plants exhibit high levels of CsGSTU transcription suggesting that the insertion of the transgenes occurred in transcriptional active regions of the tobacco genome. In planta studies demonstrate that transformed tobacco plants gain tolerance against fluorodifen. Simultaneously, the wild type CsGSTU genes were in vitro expressed and their kinetic properties were determined using fluorodifen as substrate. The results show that CsGSTU2 follows a Michaelis-Menten hyperbolic kinetic, whereas CsGSTU1 generates a sigmoid plot typical of the regulatory enzymes, thus suggesting that when working at sub-lethal fluorodifen concentrations CsGSTU2 can counteract the herbicide injury more efficiently than the CsGSTU1. Moreover, the transgenic tobacco plant over-expressing CsGSTs exhibited both drought and salinity stress tolerance. However, as we show that CsGSTUs do not function as glutathione peroxidase in vitro, the protective effect against salt and drought stress is not due to a direct scavenging activity of the oxidative stress byproducts. The transgenic tobacco plants, which are described in the present study, can be helpful for phytoremediation of residual xenobiotics in the environment and overall the over-expression of CsGSTUs can be helpful to develop genetically modified crops with high resistance to abiotic stresses., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Polyamine oxidases (PAOs) are FAD-dependent enzymes associated with polyamine catabolism. In plants, increasing evidences support that PAO genes play essential roles in abiotic and biotic stresses response. In this study, six putative PAO genes (CsPAO1-CsPAO6) were unraveled in sweet orange (Citrus sinensis) using the released citrus genome sequences. A total of 203 putative cis-regulatory elements involved in hormone and stress response were predicted in 1.5-kb promoter regions at the upstream of CsPAOs. The CsPAOs can be divided into four major groups, with similar organizations with their counterparts of Arabidopsis thaliana. Transcripts of CsPAOs were detected in leaf, stem, cotyledon, and root, with the highest levels detected in the roots. The CsPAOs displayed various responses to exogenous treatments with polyamines and ABA and were differentially altered by abiotic stresses, including cold, salt, and mannitol. Overexpression of CsPAO3 in tobacco demonstrated that spermidine and spermine were decreased in the transgenic line, while putrescine was significantly enhanced, implying a potential role of this gene in polyamine back conversion. These data provide valuable knowledge for understanding the roles of the PAO genes in the future., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The presence of lipases was observed in three byproducts of orange juice processing: peel, core and frit. The enzymes were characterised biochemically over a wide pH range from neutral (6-7) to alkaline (8-9). The optimal temperature for the activity of these byproducts showed wide range at 20°C to 70°C, indicating fairly high thermostability. The activities were monitored on p-NP-butyrate, p-NP-laurate and p-NP-palmitate. For the first time, lipase activity was detected in these residues, reaching 68.5 lipase U/g for the crude extract from fractions called frit., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Boron (B) deficiency is a widespread problem in many crops, including Citrus. The effects of B-deficiency on gas exchange, carbohydrates, organic acids, amino acids, total soluble proteins and phenolics, and the activities of key enzymes involved in organic acid and amino acid metabolism in 'Xuegan' [Citrus sinensis (L.) Osbeck] leaves and roots were investigated. Boron-deficient leaves displayed excessive accumulation of nonstructural carbohydrates and much lower CO2 assimilation, demonstrating feedback inhibition of photosynthesis. Dark respiration, concentrations of most organic acids [i.e., malate, citrate, oxaloacetate (OAA), pyruvate and phosphoenolpyruvate] and activities of enzymes [i.e., phosphoenolpyruvate carboxylase (PEPC), NAD-malate dehydrogenase, NAD-malic enzyme (NAD-ME), NADP-ME, pyruvate kinase (PK), phosphoenolpyruvate phosphatase (PEPP), citrate synthase (CS), aconitase (ACO), NADP-isocitrate dehydrogenase (NADP-IDH) and hexokinase] involved in glycolysis, the tricarboxylic acid (TCA) cycle and the anapleurotic reaction were higher in B-deficient leaves than in controls. Also, total free amino acid (TFAA) concentration and related enzyme [i.e., NADH-dependent glutamate 2-oxoglutarate aminotransferase (NADH-GOGAT) and glutamate OAA transaminase (GOT)] activities were enhanced in B-deficient leaves. By contrast, respiration, concentrations of nonstructural carbohydrates and three organic acids (malate, citrate and pyruvate), and activities of most enzymes [i.e., PEPC, NADP-ME, PK, PEPP, CS, ACO, NAD-isocitrate dehydrogenase, NADP-IDH and hexokinase] involved in glycolysis, the TCA cycle and the anapleurotic reaction, as well as concentration of TFAA and activities of related enzymes (i.e., nitrate reductase, NADH-GOGAT, glutamate pyruvate transaminase and glutamine synthetase) were lower in B-deficient roots than in controls. Interestingly, leaf and root concentration of total phenolics increased, whereas that of total soluble protein decreased, in response to B-deficiency. In conclusion, respiration, organic acid (i.e., glycolysis and the TCA cycle) metabolism, the anapleurotic pathway and amino acid biosynthesis were upregulated in B-deficient leaves with excessive accumulation of carbohydrates to 'consume' the excessive carbon available, but downregulated in B-deficient roots with less accumulation of carbohydrates to maintain the net carbon balance., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Response surface methodology was used to evaluate the optimal high pressure processing treatment (300-500 MPa, 5-15 min) combined with Stevia rebaudiana (Stevia) addition (0-2.5% (w/v)) to guarantee food safety while maintaining maximum retention of nutritional properties. A fruit extract matrix was selected and Listeria monocytogenes inactivation was followed from the food safety point of view while polyphenoloxidase (PPO) and peroxidase (POD) activities, total phenolic content (TPC) and antioxidant capacity (TEAC and ORAC) were studied from the food quality point of view. A combination of treatments achieved higher levels of inactivation of L. monocytogenes and of the oxidative enzymes, succeeding in completely inactivating POD and also increasing the levels of TPC, TEAC and ORAC. A treatment of 453 MPa for 5 min with a 2.5% (w/v) of Stevia succeeded in inactivating over 5 log cycles of L. monocytogenes and maximizing inactivation of PPO and POD, with the greatest retention of bioactive components., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Six low-methoxy pectins with different degrees of methylesterification and amidation, and molecular weights were used to prepare gels with similar moduli of elasticity by varying the concentrations of pectin and calcium phosphate. Five aroma compounds were added to the gels and their sensory textural properties, release and perception of aromas were investigated. Sensory firmness, springiness, adhesiveness, chewiness and cohesiveness differed according to the gel type, even though the moduli of elasticity were not significantly different (p<0.05). Release and perception of aromas also displayed significant difference according to the gel type (p<0.05). Low-methoxy amidated pectin exhibited the lowest release and perception for all the aroma compounds, while pectin-methylesterase-treated pectin gels exhibited relatively higher aroma release and perception. These results showed that the structural properties of pectins and gelling factors that increase the non-polar character of the gel matrices could decrease the release and perception of aromas in pectin gel systems., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Glutathione S-transferases (GSTs) represent a ubiquitous gene family encoding detoxification enzymes able to recognize reactive electrophilic xenobiotic molecules as well as compounds of endogenous origin. Anthocyanin pigments require GSTs for their transport into the vacuole since their cytoplasmic retention is toxic to the cell. Anthocyanin accumulation in Citrus sinensis (L.) Osbeck fruit flesh determines different phenotypes affecting the typical pigmentation of Sicilian blood oranges. In this paper we describe: i) the characterization of the GST gene family in C. sinensis through a systematic EST analysis; ii) the validation of the EST assembly by exploiting the genome sequences of C. sinensis and C. clementina and their genome annotations; iii) GST gene expression profiling in six tissues/organs and in two different sweet orange cultivars, Cadenera (common) and Moro (pigmented)., Results: We identified 61 GST transcripts, described the full- or partial-length nature of the sequences and assigned to each sequence the GST class membership exploiting a comparative approach and the classification scheme proposed for plant species. A total of 23 full-length sequences were defined. Fifty-four of the 61 transcripts were successfully aligned to the C. sinensis and C. clementina genomes. Tissue specific expression profiling demonstrated that the expression of some GST transcripts was 'tissue-affected' and cultivar specific. A comparative analysis of C. sinensis GSTs with those from other plant species was also considered. Data from the current analysis are accessible at http://biosrv.cab.unina.it/citrusGST/, with the aim to provide a reference resource for C. sinensis GSTs., Conclusions: This study aimed at the characterization of the GST gene family in C. sinensis. Based on expression patterns from two different cultivars and on sequence-comparative analyses, we also highlighted that two sequences, a Phi class GST and a Mapeg class GST, could be involved in the conjugation of anthocyanin pigments and in their transport into the vacuole, specifically in fruit flesh of the pigmented cultivar.

Despite the longstanding importance of the thermally tolerant pectin methylesterase (TT-PME) activity in citrus juice processing and product quality, the unequivocal identification of the protein and its corresponding gene has remained elusive. TT-PME was purified from sweet orange [ Citrus sinensis (L.) Osbeck] finisher pulp (8.0 mg/1.3 kg tissue) with an improved purification scheme that provided 20-fold increased enzyme yield over previous results. Structural characterization of electrophoretically pure TT-PME by MALDI-TOF MS determined molecular masses of approximately 47900 and 53000 Da for two principal glycoisoforms. De novo sequences generated from tryptic peptides by MALDI-TOF/TOF MS matched multiple anonymous Citrus EST cDNA accessions. The complete tt-pme cDNA (1710 base pair) was cloned from a fruit mRNA library using RT- and RLM-RACE PCR. Citrus TT-PME is a novel isoform that showed higher sequence identity with the multiply glycosylated kiwifruit PME than to previously described Citrus thermally labile PME isoforms.

Domestication and breeding of citrus species/varieties for flavor and other characteristics, based on the ancestral species pummelo, mandarin and citron, has been an ongoing process for thousands of years. Bitterness, a desirable flavor characteristic in the fruit of some citrus species (pummelo and grapefruit) and undesirable in others (oranges and mandarins), has been under positive or negative selection during the breeding process of new species/varieties. Bitterness in citrus fruit is determined by the composition of branched-chain flavanone glycosides, the predominant flavonoids in citrus. The flavor-determining biosynthetic step is catalyzed by two branch-forming rhamnosyltransferases that utilize flavanone-7-O-glucose as substrate. The 1,2-rhamnosytransferase (encoded by Cm1,2RhaT) leads to the bitter flavanone-7-O-neohesperidosides whereas the 1,6-rhamnosytransferase leads to the tastelessflavanone-7-O-rutinosides. Here, we describe the functional characterization of Cs1,6RhaT, a 1,6-rhamnosyltransferase-encoding gene directing biosynthesis of the tasteless flavanone rutinosides common to the non-bitter citrus species. Cs1,6RhaT was found to be a substrate-promiscuous enzyme catalyzing branched-chain rhamnosylation of flavonoids glucosylated at positions 3 or 7. In vivo substrates include flavanones, flavones, flavonols and anthocyanins. Cs1,6RhaT enzyme levels were shown to peak in young fruit and leaves, and gradually subside during development. Phylogenetic analysis of Cm1,2RhaT and Cs1,6RhaT demonstrated that they both belong to the branch-forming glycosyltransferase cluster, but are distantly related and probably originated separately before speciation of the citrus genome. Genomic data from citrus, supported by a study of Cs1,6RhaT protein levels in various citrus species, suggest that inheritance, expression levels and mutations of branch-forming rhamnosyltransferases underlie the development of bitter or non-bitter species/varieties under domestication., (© 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.)

Starch is one of the major components of cereals, tubers, and fruits. Genes encoding granule-bound starch synthase (GBSS), which is responsible for amylose synthesis, have been extensively studied in cereals but little is known about them in fruits. Due to their low copy gene number, GBSS genes have been used to study plant phylogenetic and evolutionary relationships. In this study, GBSS genes have been isolated and characterized in three fruit trees, including apple, peach, and orange. Moreover, a comprehensive evolutionary study of GBSS genes has also been conducted between both monocots and eudicots. Results have revealed that genomic structures of GBSS genes in plants are conserved, suggesting they all have evolved from a common ancestor. In addition, the GBSS gene in an ancestral angiosperm must have undergone genome duplication ∼251 million years ago (MYA) to generate two families, GBSSI and GBSSII. Both GBSSI and GBSSII are found in monocots; however, GBSSI is absent in eudicots. The ancestral GBSSII must have undergone further divergence when monocots and eudicots split ∼165 MYA. This is consistent with expression profiles of GBSS genes, wherein these profiles are more similar to those of GBSSII in eudicots than to those of GBSSI genes in monocots. In dicots, GBSSII must have undergone further divergence when rosids and asterids split from each other ∼126 MYA. Taken together, these findings suggest that it is GBSSII rather than GBSSI of monocots that have orthologous relationships with GBSS genes of eudicots. Moreover, diversification of GBSS genes is mainly associated with genome-wide duplication events throughout the evolutionary course of history of monocots and eudicots.

Interest in phloem-specific promoters for the engineering of transgenic plants has been increasing in recent years. In this study we isolated two similar, but distinct, alleles of the Citrus sinensis sucrose synthase-1 promoter (CsSUS1p) and inserted them upstream of the β-glucuronidase (GUS) gene to test their ability to drive expression in the phloem of transgenic Arabidopsis thaliana and Nicotiana tabacum. Although both promoter variants were capable of conferring localized GUS expression in the phloem, the CsSUS1p-2 allele also generated a significant level of expression in non-target tissues. Unexpectedly, GUS expression was also instigated in a minority of CsSUS1p::GUS lines in response to wounding in the leaves of transgenic Arabidopsis. Deletion analysis of the CsSUS1p suggested that a fragment comprising nucleotides -410 to -268 relative to the translational start site contained elements required for phloem-specific expression while nucleotides -268 to -103 contained elements necessary for wound-specific expression. Interestingly, the main difference between the two CsSUS1p alleles was the presence of a 94-bp insertion in allele 2. Fusion of this indel to a minimal promoter and GUS reporter gene indicated that it contained stamen and carpel-specific enhancer elements. This finding of highly specific and separable regulatory units within the CsSUS1p suggests that this promoter may have a potential application in the generation of constructs for the use in the development of transgenic plants resistant to a wide variety of target pests.

The multiple forms of the enzyme pectin methylesterase (PME) present in citrus fruit tissues vary in activity toward juice cloud-associated pectin substrates and, thus, in their impact on juice cloud stability and product quality. Because the proteins responsible for individual PME activities are rarely identified by structural properties or correlated to specific PME genes, matrix-assisted laser desorption-ionization with tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) was investigated as a direct means to unequivocally identify the thermolabile (TL-) PME isoforms isolated from sweet orange [ Citrus sinensis (L.) Osbeck] fruit tissue. Affinity-purified TL-PME preparations were separated by SDS-PAGE prior to trypsin digestion and analyzed by MS for peptide mass fingerprinting. The two major PME isoforms accumulated in citrus fruit matched existing accessions in the SwissProt database. Although similar in size by SDS-PAGE, isoform-specific peptide ion signatures easily distinguished the two PMEs.

Three putative terpenoid UDP-glycosyltransferase (UGT) genes, designated CsUGT1, CsUGT2, and CsUGT3, were isolated and characterized in 'Valencia' sweet orange (Citrus sinensis L. Osbeck). CsUGT1 consisted of 1493 nucleotides with an open reading frame encoding 492 amino acids, CsUGT2 consisted of 1727 nucleotides encoding 504 amino acids, and CsUGT3 consisted of 1705 nucleotides encoding 468 amino acids. CsUGT3 had a 145 bp intron at 730-874, whereas CsUGT1 and CsUGT2 had none. The three deduced glycosyltransferase proteins had a highly conserved plant secondary product glycosyltransferase motif in the C terminus. Phylogenetic analysis showed that CsUGT1 and CsUGT3 were classified into group L of glycosyltransferase family 1, and CsUGT2 was classified into group D. Through Southern blotting analysis, CsUGT1 was found to have two copies in the sweet orange genome, whereas CsUGT2 and CsUGT3 had at least seven and nine copies, respectively. CsUGT1, CsUGT2, and CsUGT3 were constitutively expressed in leaf, flower, and fruit tissues. The results facilitate further investigation of the function of terpenoid glycosyltransferases in citrus and the biosynthesis of terpenoid glycosides in vitro.

The low-molecular weight secretory phospholipase A2alpha (CssPLA2alpha) and beta (CsPLA2beta) cloned in this study exhibited diurnal rhythmicity in leaf tissue of Citrus sinensis. Only CssPLA2alpha displayed distinct diurnal patterns in fruit tissues. CssPLA2alpha and CsPLA2beta diurnal expression exhibited periods of approximately 24 h; CssPLA2alpha amplitude averaged 990-fold in the leaf blades from field-grown trees, whereas CsPLA2beta amplitude averaged 6.4-fold. Diurnal oscillation of CssPLA2alpha and CsPLA2beta gene expression in the growth chamber experiments was markedly dampened 24 h after transfer to continuous light or dark conditions. CssPLA2alpha and CsPLA2beta expressions were redundantly mediated by blue, green, red and red/far-red light, but blue light was a major factor affecting CssPLA2alpha and CsPLA2beta expression. Total and low molecular weight CsPLA2 enzyme activity closely followed diurnal changes in CssPLA2alpha transcript expression in leaf blades of seedlings treated with low intensity blue light (24 micromol m(-2) s(-1)). Compared with CssPLA2alpha basal expression, CsPLA2beta expression was at least 10-fold higher. Diurnal fluctuation and light regulation of PLA2 gene expression and enzyme activity in citrus leaf and fruit tissues suggests that accompanying diurnal changes in lipophilic second messengers participate in the regulation of physiological processes associated with phospholipase A2 action.

alpha-Amylase activity was screened in the peel, as waste fruit, of 13 species and cultivars of Egyptian citrus. The species Citrus sinensis cv. Abosora had the highest activity. alpha-Amylase AI from Abosora peel was purified to homogeneity using anion and cation-exchange, and gel filtration chromatographies. Molecular weight of alpha-amylase AI was found to be 42 kDa. The hydrolysis properties of alpha-amylase AI toward different substrates indicated that corn starch is the best substrate. The alpha-amylase had the highest activity toward glycogen compared with amylopectin and dextrin. Potato starch had low affinity toward alpha-amylase AI but it did not hydrolyze beta-cyclodextrin and dextran. Apparent Km for alpha-amylase AI was 5 mg (0.5%) starch/ml. alpha-Amylase AI showed optimum activity at pH 5.6 and 40 degrees C. The enzyme was thermally stable up to 40 degrees C and inactivated at 70 degrees C. The effect of mono and divalent metal ions were tested for the alpha-amylase AI. Ba2+ was found to have activating effect, where as Li+ had negligible effect on activity. The other metals caused inhibition effect. Activity of the alpha-amylase AI was increased one and half in the presence of 4 mM Ca2+ and was found to be partially inactivated at 10 mM Ca2+. The reduction of starch viscosity indicated that the enzyme is endoamylase. The results suggested that, in addition to citrus peel is a rich source of pectins and flavanoids, alpha-amylase AI from orange peel could be involved in the development and ripening of citrus fruit and may be used for juice processing.

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.

The higher plant mitochondrial electron transport chain contains an alternative pathway that ends with the AOX (alternative oxidase). The AOX proteins are encoded by a small gene family composed of two discrete gene subfamilies. Aox1 is present in both monocot and eudicot plants, whereas Aox2 is only present in eudicot plants. We isolated a genomic clone from Citrus sinensis containing the Aox1a gene. The orange Aox1a consists of four exons interrupted by three introns and its promoter harbours diverse putative stress-specific regulatory motifs including pathogen response elements. The role of the Aox1a gene was evaluated during the compatible interaction between C. sinensis and Xanthomonas axonopodis pv. citri and no induction of the Aox1a at the transcriptional level was observed. On the other hand, Aox1a was studied in orange plants during non-host interactions with Pseudomonas syringae pv. tomato and Xanthomonas campestris pv. vesicatoria, which result in hypersensitive response. Both phytopathogens produced a strong induction of Aox1a, reaching a maximum at 8 h post-infiltration. Exogenous application of salicylic acid produced a slight increase in the steady-state level of Aox1a, whereas the application of fungi elicitors showed the highest induction. These results suggest that AOX1a plays a role during biotic stress in non-host plant pathogen interaction.

Chalcone synthase (CHS) is involved in the biosynthesis of anthocyanin. In this study, a full-length DNA of CHS gene (named as CsCHS-bo) was cloned from the blood orange, Citrus sinensis (L.) Osbeck cv. Ruby. The gene was 1,512 bp in size containing an open reading frame (1,176 bp) encoding 391 amino acids. Comparative and bioinformatic analyses revealed that the deduced protein of CsCHS-bo was highly homologous to CHS from other plant species. The protein of CsCHS-bo had four CHS-specific conserved motifs and a CHS-family signature sequence GFGPG. Phylogenetic analysis indicated that the protein of CsCHS-bo was in a subgroup with CHS of Ruta Palmatum. The CsCHS-bo was localized to the chromosomes 2p, 4p and 6p by an improved fluorescence in situ hybridization technique, indicating that at least three copies of CsCHS-bo were present in the genome.

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.

Glucosylation is a predominant flavonoid modification reaction affecting the solubility, stability, and subsequent bioavailability of these metabolites. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. In this work, a Citrus paradisi glucosyltransferase gene was identified, cloned, and introduced into the pET recombinant protein expression system utilizing primers designed against a predicted flavonoid glucosyltransferase gene (AY519364) from Citrus sinensis. The encoded C. paradisi protein is 51.2 kDa with a predicted pI of 6.27 and is 96% identical to the C. sinensis homologue. A number of compounds from various flavonoid subclasses were tested, and the enzyme glucosylated only the flavonol aglycones quercetin (K(m)(app)=67 microM; V(max)=20.45 pKat/microg), kaempferol (K(m)(app)=12 microM; V(max)=11.63 pKat/microg), and myricetin (K(m)(app)=33 microM; V(max)=12.21 pKat/microg) but did not glucosylate the anthocyanidin, cyanidin. Glucosylation occurred at the 3 hydroxyl position as confirmed by HPLC and TLC analyses with certified reference compounds. The optimum pH was 7.5 with a pronounced buffer effect noted for reactions performed in Tris-HCl buffer. The enzyme was inhibited by Cu(2+), Fe(2+), and Zn(2+) as well as UDP (K(i)(app)=69.5 microM), which is a product of the reaction. Treatment of the enzyme with a variety of amino acid modifying compounds suggests that cysteine, histidine, arginine, tryptophan, and tyrosine residues are important for activity. The thorough characterization of this C. paradisi flavonol 3-O-glucosyltransferase adds to the growing base of glucosyltransferase knowledge, and will be used to further investigate structure-function relationships.

The influence of high pressure carbon dioxide (HPCD) on the activity and structure of pectin methylesterase (PME) from orange was investigated. The pressures were 8-30 MPa, temperature 55 degrees C and time 10 min. HPCD caused significant inactivation on PME, the lowest residual activity was about 9.3% at 30 MPa. The SDS-PAGE electrophoretic behavior of HPCD-treated PME was not altered, while changes in the secondary and tertiary structures were found. The beta-structure fraction in the secondary structure decreased and the fluorescence intensity increased as HPCD pressures were elevated. After 7-day storage at 4 degrees C, no alteration of its activity and no reversion of its beta-structure fraction were observed, while its fluorescence intensity further decreased.

A specific indicator of freshness, allowing routine distinction between freshly squeezed orange juices (FSOJs) and FSOJ-like products, was to be identified. Using the Actijoule unit of a tubular heater at a flow rate of 60 L/h, FSOJs from Citrus sinensis (L.) Osbeck cv. Valencia Late were continuously heated on a pilot plant scale at six different temperatures (42-92 degrees C), followed by continuous cooling to ambient temperature and subsequent filling into sterilized glass jars. The cloud stability and residual activities of pectin methylesterase (PE) and peroxidase (POD) were monitored over the storage at 4 degrees C for up to 62 days, thus considering the storage conditions of FSOJs in retail markets. As shown by the viable microbial counts throughout storage, microbial activity was insignificant due to good sanitary practice, thus proving that the enzyme activities detected were of plant origin. The juices processed at temperatures > or =62 degrees C were characterized by minor residual activities. When exposed to temperatures <62 degrees C in the genuine acidic matrix of the juices, the heat stability of PE exceeded that of POD. Compared with the aforementioned samples, the juice processed at 52 degrees C with a residual PE activity of 33.8% was hardly inferior in terms of cloud stability within the first 14 days. After the juice was processed at 42 degrees C, rapid clarification occurred within the first 8 days, consistent with undetectable PE deactivation. Hence, only the range of approximately 50-60 degrees C is relevant in minimal heat-processing for the retention of cloud stability within the short turnover period of FSOJ-like products, with partial PE and POD deactivation being already sufficient to distinguish those juices from FSOJs. Irrespective of the previous thermal treatment, the total PE activity remained nearly constant during storage, whereas the POD activity rapidly declined to minor levels after 20 days. Consequently, as to the future analysis of samples with unknown processing history, PE was suggested as an indicator enzyme for the freshness of FSOJs, allowing their unambiguous distinction from minimally heat-processed juices.

Understanding leaf and fruit abscission is essential in order to develop strategies for controlling the process in fruit crops. Mechanisms involved in signalling leaf and fruit abscission upon induction by abscission agents were investigated in Citrus sinensis cv. 'Valencia'. Previous studies have suggested a role for phospholipid signalling; hence, two phospholipase D cDNA sequences, CsPLDalpha1 and CsPLDgamma1, were isolated and their role was examined. CsPLDalpha1 expression was reduced in leaves but unaltered in fruit peel tissue treated with an ethylene-releasing compound (ethephon), or a fruit-specific abscission agent, 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMNP). By contrast, CsPLDgamma1 expression was up-regulated within 6 h (leaves) and 24 h (fruit peel) after treatment with ethephon or CMNP, respectively. CsPLDalpha1 expression was diurnally regulated in leaf blade but not fruit peel. CsPLDgamma1 exhibited strong diurnal oscillation in expression in leaves and fruit peel with peak expression around midday. While diurnal fluctuation in CsPLDalpha1 expression appeared to be light-entrained in leaves, CsPLDgamma1 expression was regulated by light and the circadian clock. The diurnal expression of both genes was modulated by ethylene-signalling. The ethephon-induced leaf abscission and the ethephon- and CMNP-induced decrease in fruit detachment force were enhanced by application during rising diurnal expression of CsPLDgamma1. The results indicate differential regulation of CsPLDalpha1 and CsPLDgamma1 in leaves and fruit, and suggest possible roles for PLD-dependent signalling in regulating abscission responses in citrus.

Chlorophyll is a central player in harvesting light energy for photosynthesis, yet the rate-limiting steps of chlorophyll catabolism and the regulation of the catabolic enzymes remain unresolved. To study the role and regulation of chlorophyllase (Chlase), the first enzyme of the chlorophyll catabolic pathway, we expressed precursor and mature versions of citrus (Citrus sinensis) Chlase in two heterologous plant systems: (1) squash (Cucurbita pepo) plants using a viral vector expression system; and (2) transiently transformed tobacco (Nicotiana tabacum) protoplasts. Expression of full-length citrus Chlase resulted in limited chlorophyll breakdown in protoplasts and no visible leaf phenotype in whole plants, whereas expression of a Chlase version lacking the N-terminal 21 amino acids (ChlaseDeltaN), which corresponds to the mature protein, led to extensive chlorophyll breakdown in both tobacco protoplasts and squash leaves. ChlaseDeltaN-expressing squash leaves displayed a dramatic chlorotic phenotype in plants grown under low-intensity light, whereas under natural light a lesion-mimic phenotype occurred, which was demonstrated to follow the accumulation of chlorophyllide, a photodynamic chlorophyll breakdown product. Full-length and mature citrus Chlase versions were localized to the chloroplast membrane fraction in expressing tobacco protoplasts, where processing of the N-terminal 21 amino acids appears to occur. Results obtained in both plant systems suggest that Chlase functions as a rate-limiting enzyme in chlorophyll catabolism controlled via posttranslational regulation.

Xylella fastidiosa (Xf) is a fastidious bacterium that grows exclusively in the xylem of several important crop species, including grape and sweet orange (Citrus sinensis L. Osb.) causing Pierce disease and citrus variegated chlorosis (CVC), respectively. The aim of this work was to study the nitrogen metabolism of a highly susceptible variety of sweet orange cv. 'Pêra' (C. sinensis L. Osbeck) infected with Xf. Plants were artificially infected and maintained in the greenhouse until they have developed clear disease symptoms. The content of nitrogen compounds and enzymes of the nitrogen metabolism and proteases in the xylem sap and leaves of diseased (DP) and uninfected healthy (HP) plants was studied. The activity of nitrate reductase in leaves did not change in DP, however, the activity of glutamine synthetase was significantly higher in these leaves. Although amino acid concentration was slightly higher in the xylem sap of DP, the level dropped drastically in the leaves. The protein contents were lower in the sap and in leaves of DP. DP and HP showed the same amino acid profiles, but different proportions were observed among them, mainly for asparagine, glutamine, and arginine. The polyamine putrescine was found in high concentrations only in DP. Protease activity was higher in leaves of DP while, in the xylem sap, activity was detected only in DP. Bidimensional electrophoresis showed a marked change in the protein pattern in DP. Five differentially expressed proteins were identified (2 from HP and 3 from DP), but none showed similarity with the genomic (translated) and proteomic database of Xf, but do show similarity with the proteins thaumatin, mucin, peroxidase, ABC-transporter, and strictosidine synthase. These results showed that significant changes take place in the nitrogen metabolism of DP, probably as a response to the alterations in the absorption, assimilation and distribution of N in the plant.

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.

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.

There is now biochemical and genetic evidence that oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED) is the critical step in the regulation of abscisic acid (ABA) synthesis in higher plants. The peel of Citrus fruit accumulates large amounts of ABA during maturation. To understand the regulation of ABA biosynthesis in Citrus, two full-length cDNAs (CsNCED1 and CsNCED2) encoding NCEDs were isolated and characterized from the epicarp of orange fruits (Citrus sinensis L. Osbeck). Expression of the CsNCED1 gene increased in the epicarp during natural and ethylene-induced fruit maturation, and in water-stressed leaves, in a pattern consistent with the accumulation of ABA. The second gene, CsNCED2, was not detected in dehydrated leaves and, in fruits, exhibited a differential expression to that of CsNCED1. Taken together, these results suggests that CsNCED1 is likely to play a primary role in the biosynthesis of ABA in both leaves and fruits, while CsNCED2 appears to play a subsidiary role restricted to chromoplast-containing tissue. Furthermore, analysis of 9-cis-violaxanthin and 9'-cis-neoxanthin, as the two possible substrates for NCEDs, revealed that the former was the main carotenoid in the outer coloured part of the fruit peel as the fruit ripened or after ethylene treatment, whereas 9'-cis-neoxanthin was not detected or was in trace amounts. By contrast, turgid and dehydrated leaves contained 9'-cis-neoxanthin but 9-cis-violaxanthin was absent. Based on these results, it is suggested that 9-cis-violaxanthin may be the predominant substrate for NCED in the peel of Citrus fruits, whereas 9'-cis-neoxanthin would be the precursor of ABA in photosynthetic tissues.

Valencia pectinmethylesterase (PME) fractions, B-PME, containing 36 and 13 kDa protein bands and U-PME, containing a 36 and 27 kDa protein bands, were used to de-esterify original pectin (O-Pec) from 73% degree of esterification (%DE) to 63% (B-Pec) and 61% DE (U-Pec), respectively. Most O-Pec eluted from ion exchange chromatography at low salt concentration and a smaller component eluted at higher ionic strength. B-Pec and U-Pec eluted as one broad peak at higher ionic strength. PME modification did not change molecular weight: O-pectin (134,000 g/mol), U-Pec (133,850 g/mol), and B-Pec (132,250 g/mol). The NMR signal of GG and GGG increased after modification, whereas the signal of EE and EEE decreased. The negative zeta-potential increased with pH for all pectins. U-PME and B-PME created differently modified pectins that vary in degree and length of multiple attacks and fraction of the pectin population that was modified.

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.