Identification of key enzymes responsible for protolimonoid biosynthesis in plants: Opening the door to azadirachtin production.

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-3ß-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-3ß-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.

Identification and Characterization of Citrus Peel Uronic Acid Oxidase.

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.

Ultrasonic Processing Induced Activity and Structural Changes of Polyphenol Oxidase in Orange (Citrus sinensis Osbeck).

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.

Functional and Structural Characterization of a (+)-Limonene Synthase from Citrus sinensis.

Terpenes make up the largest and most diverse class of natural compounds and have important commercial and medical applications. Limonene is a cyclic monoterpene (C10) 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.

Structural Characterization of Early Michaelis Complexes in the Reaction Catalyzed by (+)-Limonene Synthase from Citrus sinensis Using Fluorinated Substrate Analogues.

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 KI values of 2.4 ± 0.5 and 39.5 ± 5.2 µM, respectively. The KI values are similar to the KM 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 (kcat 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.

Deficiency and toxicity of boron: Alterations in growth, oxidative damage and uptake by citrange orange plants.

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 CO2 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.

CitAP2.10 activation of the terpene synthase CsTPS1 is associated with the synthesis of (+)-valencene in 'Newhall' orange.

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.

Substrate preference of citrus naringenin rhamnosyltransferases and their application to flavonoid glycoside production in fission yeast.

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.

Characteristics of ß-glucosidase from oranges during maturation and its relationship with changes in bound volatile compounds.

BACKGROUND: The hydrolysis of glycosidically bound volatile compounds can release potential aromas in oranges during maturation. ß-Glucosidase is the key enzyme that influences the hydrolysis of bound volatiles. In this study the changes in ß-glucosidase and bound volatile compounds in Jincheng oranges during maturation were investigated. The relationship between ß-glucosidase activity and bound volatiles was analyzed. RESULTS: The optimal temperature and pH of ß-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 ß-glucosidase was strongly inhibited by Zn(2+), Fe(2+), Cu(2+), Ag(+), Hg(2+) and Fe(3+). ß-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 ß-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 ß-glucosidase.

The molecular and enzymatic basis of bitter/non-bitter flavor of citrus fruit: evolution of branch-forming rhamnosyltransferases under domestication.

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.

Characterization of the glutathione S-transferase gene family through ESTs and expression analyses within common and pigmented cultivars of Citrus sinensis (L.) Osbeck.

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.

The sucrose synthase-1 promoter from Citrus sinensis directs expression of the ß-glucuronidase reporter gene in phloem tissue and in response to wounding in transgenic plants.

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 ß-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.

Light controls phospholipase A2alpha and beta gene expression in Citrus sinensis.

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.

Characterization of three terpenoid glycosyltransferase genes in 'Valencia' sweet orange (Citrus sinensis L. Osbeck).

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 phytochrome-interacting transcription factor CsPIF8 contributes to cold tolerance in citrus by regulating superoxide dismutase expression.

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 (O2-) 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 O2- 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.

Cloning, characterization and localization of CHS gene from blood orange, Citrus sinensis (L.) Osbeck cv. Ruby.

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.

Lactose derivatives as potential inhibitors of pectin methylesterases.

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.

Genome-wide identification and characterization of laccase gene family in Citrus sinensis.

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.

Heterologous expression and kinetic characterization of the α, ß and αß blend of the PPi-dependent phosphofructokinase from Citrus sinensis.

This work reports the molecular cloning and heterologous expression of the genes coding for α and ß 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 ß subunits, respectively). On the other hand, co-expression rendered a α3ß3 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.

Direct Evidence of an Enzyme-Generated LPP Intermediate in (+)-Limonene Synthase Using a Fluorinated GPP Substrate Analog.

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.