Cloning of Sucrose:Sucrose 1-Fructosyltransferase from Onion and Synthesis of Structurally Defined Fructan Molecules from Sucrose1

Sucrose (Suc):Suc 1-fructosyltransferase (1-SST) is the key enzyme in plant fructan biosynthesis, since it catalyzes de novo fructan synthesis from Suc. We have cloned 1-SST from onion (Allium cepa) by screening a cDNA library using acid invertase from tulip (Tulipa gesneriana) as a probe. Expression assays in tobacco (Nicotiana plumbaginifolia) protoplasts showed the formation of 1-kestose from Suc. In addition, an onion acid invertase clone was isolated from the same cDNA library. Protein extracts of tobacco protoplasts transformed with this clone showed extensive Suc-hydrolyzing activity. Conditions that induced fructan accumulation in onion leaves also induced 1-SST mRNA accumulation, whereas the acid invertase mRNA level decreased. Structurally different fructan molecules could be produced from Suc by a combined incubation of protein extract of protoplasts transformed with 1-SST and protein extract of protoplasts transformed with either the onion fructan:fructan 6G-fructosyltransferase or the barley Suc:fructan 6-fructosyltransferase.

Intracellular and intercellular compartmentation of carbohydrate metabolism in leaves of temperate gramineae

This review considers the importance of compartmentation in the regulation of carbohydrate metabolism in leaves. We draw particular attention to the role of the vacuole as a site for storage of soluble sugars based on sucrose, and discuss briefly their characteristic metabolism. We also point out inconsistencies between the observed properties of vacuoles and the behaviour in vitro of the enzymes of fructan biosynthesis that do not support the hypothesis that the vacuole is the site of synthesis as well as of storage. We also consider compartmentation of carbohydrate metabolism between different cell types, using mainly our studies on leaves of temperate C3 gramineae. Here we present evidence of significant differences in carbon metabolism between epidermis, mesophyll, bundle sheath and vasculature based upon both single-cell sampling and immunolocalisation. The implications of these differences for the control of metabolism in leaves are discussed.

Synthesis of fructans by fructosyltransferase from the tuberous roots of Viguiera discolor (Asteraceae)

Sucrose:sucrose fructosyltransferase (SST) and fructan:fructan fructosyl-transferase (FFT) activities from crude extracts of tuberous roots of Viguiera discolor growing in a preserved area of cerrado were analyzed in 1995-1996. SST activity was characterized by the synthesis of 1-kestose from sucrose and FFT activity by the production of nystose from 1-kestose. The highest fructan-synthesizing activity was observed during early dormancy (autumn), when both (SST and FFT) activities were high. The increase in synthetic activity seemed to start during the fruiting phase in the summer, when SST activity was higher than in spring. During winter and at the beginning of sprouting, both activities declined. The in vitro synthesis of high molecular mass fructans from sucrose by enzymatic preparations from tuberous roots collected in summer showed that long incubations of up to 288 h produced consistently longer polymers which resembled those found in vivo with respect to chromatographic profiles.

Phloem Transport of Fructans in the Crassulacean Acid Metabolism Species Agave deserti1

Four oligofructans (neokestose, 1-kestose, nystose, and an un-identified pentofructan) occurred in the vascular tissues and phloem sap of mature leaves of Agave deserti. Fructosyltransferases (responsible for fructan biosynthesis) also occurred in the vascular tissues. In contrast, oligofructans and fructosyltransferases were virtually absent from the chlorenchyma, suggesting that fructan biosynthesis was restricted to the vascular tissues. On a molar basis, these oligofructans accounted for 46% of the total soluble sugars in the vascular tissues (sucrose [Suc] for 26%) and for 19% in the phloem sap (fructose for 24% and Suc for 53%). The Suc concentration was 1.8 times higher in the cytosol of the chlorenchyma cells than in the phloem sap; the nystose concentration was 4.9 times higher and that of pentofructan was 3.2 times higher in the vascular tissues than in the phloem sap. To our knowledge, these results provide the first evidence that oligofructans are synthesized and transported in the phloem of higher plants. The polymer-trapping mechanism proposed for dicotyledonous C3 species may also be valid for oligofructan transport in monocotyledonous species, such as A. deserti, which may use a symplastic pathway for phloem loading of photosynthates in its mature leaves.

Identification of products formed by a fructan:fructan fructosyltransferase activity from Lolium rigidum

The products formed by a fructan:fructan fructosyltransferase (FFT) activity purified from Lolium rigidum Gaudin were identified after gas chromatography-mass spectrometry of partially methylated alditol acetates, electrospray ionization-mass spectrometry and reversed-phase high-performance liquid chromatography. The FFT activity synthesized oligofructans up to degree of polymerization (DP) 6, but did not synthesize fructans of DP > 6 even when assayed with (1,1,1)-kestopentaose for up to 10 h. The FFT activity when assayed with 1-kestose or 6(G)-kestose synthesized fructan with fructosyl residues almost exclusively linked by beta-2,1-glycosidic linkages. When assayed with 1-kestose, the FFT activity synthesized tetrasaccharides and pentasaccharides with an internal glucosyl residue. The predominant tetrasaccharide was (1&6(G))-kestotetraose and the predominant pentasaccharide was (1&6(G),1)-kestopentaose. By comparison, tetrasaccharides and pentasaccharides extracted from L. rigidum also contained predominantly beta-2,1-glycosidic linked fructans with an internal glucosyl residue. The only exception was that one of the pentasaccharides contained beta-2,1- and beta-2,6-glycosidic linked fructosyl residues. This pentasaccharide was not synthesized by the FFT activity. The role of this FFT activity in formation of oligofructans in L. rigidum is discussed.

De Novo Assembly And Transcriptome Analysis Of The Rubber Tree (hevea Brasiliensis) And Snp Markers Development For Rubber Biosynthesis Pathways.

Hevea brasiliensis (Willd. Ex Adr. Juss.) Muell.-Arg. is the primary source of natural rubber that is native to the Amazon rainforest. The singular properties of natural rubber make it superior to and competitive with synthetic rubber for use in several applications. Here, we performed RNA sequencing (RNA-seq) of H. brasiliensis bark on the Illumina GAIIx platform, which generated 179,326,804 raw reads on the Illumina GAIIx platform. A total of 50,384 contigs that were over 400 bp in size were obtained and subjected to further analyses. A similarity search against the non-redundant (nr) protein database returned 32,018 (63%) positive BLASTx hits. The transcriptome analysis was annotated using the clusters of orthologous groups (COG), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam databases. A search for putative molecular marker was performed to identify simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). In total, 17,927 SSRs and 404,114 SNPs were detected. Finally, we selected sequences that were identified as belonging to the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, which are involved in rubber biosynthesis, to validate the SNP markers. A total of 78 SNPs were validated in 36 genotypes of H. brasiliensis. This new dataset represents a powerful information source for rubber tree bark genes and will be an important tool for the development of microsatellites and SNP markers for use in future genetic analyses such as genetic linkage mapping, quantitative trait loci identification, investigations of linkage disequilibrium and marker-assisted selection.

Analysis Of The Ergosterol Biosynthesis Pathway Cloning, Molecular Characterization And Phylogeny Of Lanosterol 14 α-demethylase (erg11) Gene Of Moniliophthora Perniciosa.

The phytopathogenic fungus Moniliophthora perniciosa (Stahel) Aime & Philips-Mora, causal agent of witches' broom disease of cocoa, causes countless damage to cocoa production in Brazil. Molecular studies have attempted to identify genes that play important roles in fungal survival and virulence. In this study, sequences deposited in the M. perniciosa Genome Sequencing Project database were analyzed to identify potential biological targets. For the first time, the ergosterol biosynthetic pathway in M. perniciosa was studied and the lanosterol 14α-demethylase gene (ERG11) that encodes the main enzyme of this pathway and is a target for fungicides was cloned, characterized molecularly and its phylogeny analyzed. ERG11 genomic DNA and cDNA were characterized and sequence analysis of the ERG11 protein identified highly conserved domains typical of this enzyme, such as SRS1, SRS4, EXXR and the heme-binding region (HBR). Comparison of the protein sequences and phylogenetic analysis revealed that the M. perniciosa enzyme was most closely related to that of Coprinopsis cinerea.

Isoprenoid biosynthesis in the erythrocytic stages of Plasmodium falciparum

The development of new drugs is one strategy for malaria control. Biochemical pathways localised in the apicoplast of the parasite, such as the synthesis of isoprenic precursors, are excellent targets because they are different or absent in the human host. Isoprenoids are a large and highly diverse group of natural products with many functions and their synthesis is essential for the parasite's survival. During the last few years, the genes, enzymes, intermediates and mechanisms of this biosynthetic route have been elucidated. In this review, we comment on some aspects of the methylerythritol phosphate pathway and discuss the presence of diverse isoprenic products such as dolichol, ubiquinone, carotenoids, menaquinone and isoprenylated proteins, which are biosynthesised during the intraerythrocytic stages of Plasmodium falciparum.

Ectopic expression of soybean leghemoglobin in chloroplasts impairs gibberellin biosynthesis and induces dwarfism in transgenic potato plants

We have characterized potato (Solanum tuberosum L.) plants expressing a soybean leghemoglobin that is targeted to plastids. Transgenic plants displayed a dwarf phenotype caused by short internode length, and exhibited increased tuberization in vitro. Under in vivo conditions that do not promote tuberization, plants showed smaller parenchymal cells than control plants. Analysis of gibberellin (GA) concentrations indicated that the transgenic plants have a substantial reduction (approximately 10-fold) of bioactive GA(1) concentration in shoots. Application of GA(3) to the shoot apex of the transformed plants completely restored the wild type phenotype suggesting that GA-biosynthesis rather than signal transduction was limiting. Since the first stage of the GA-biosynthetic pathway is located in the plastid, these results suggest that an early step in the pathway may be affected by the presence of the leghemoglobin.

Lysine biosynthesis and nitrogen metabolism in quinoa (Chenopodium quinoa): Study of enzymes and nitrogen-containing compounds

Aspartate kinase (AK, EC 2.7.2.4), homoserine dehydrogenase (HSDH, EC 1.1.1.3) and dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) were isolated and partially purified from immature Chenopodium quinoa Willd seeds. Enzyme activities were studied in the presence of the aspartate-derived amino acids lysine, threonine and methionine and also the lysine analogue S-2-aminoethyl-L-cysteine (AEC), at 1 mM and 5 mM. The results confirmed the existence of, at least, two AK isoenzymes, one inhibited by lysine and the other inhibited by threonine, the latter being predominant in quinoa seeds. HSDH activity was also shown to be partially inhibited by threonine, whereas some of the activity was resistant to the inhibitory effect, indicating the presence of two isoenzymes, one resistant and another sensitive to threonine inhibition. Only one DHDPS isoenzyme highly sensitive to lysine inhibition was detected. The results suggest that the high concentration of lysine observed in quinoa seeds is possibly due to a combined effect of increased lysine, synthesis and accumulation in the soluble form and/or as protein lysine. Nitrogen assimilation was also investigated and based on nitrate content, nitrate reductase activity, amino acid distribution and ureide content, the leaves were identified as the predominant site of nitrate reduction in this plant species. The amino acid profile analysis in leaves and roots also indicated an important role of soluble glutamine as a nitrogen transporting compound. (c) 2007 Elsevier Masson SAS. All rights reserved.

Influence of ethylene on carotenoid biosynthesis during papaya postharvesting ripening

The carotenoid composition was evaluated during ripening of papaya cv. `Golden` under untreated (control) conditions and treated with ethylene and 1-methylcyclopropene (1-MCP). At the end of the experiments, the total carotenoid content in the control group (2194.4 mu g/100 g) was twice as high as that found in ethylene (1018.1 mu g/100 g) and 1-MCP (654.5 mu g/100 g) gas-treated samples. Separation of 21 carotenoids by HPLC connected to photodiode array and mass spectrometry detectors showed that no minor carotenoids seemed to be particularly favoured by the treatments. Lycopene was the major carotenoid in all untreated and gas-treated samples, ranging from 461.5 to 1321.6 mu g/100 g at the end of the experiments. According to the proposed biosynthetic pathway, lycopene is the central compound, since it is the most abundant carotenoid indicating a high stimulation of its upstream steps during ripening, and it is the source for the synthesis of other derivative compounds, such as beta-cryptoxanthin. The influence of both gas treatments on the carotenoid biosynthetic pathway was considered. (C) 2011 Elsevier Inc. All rights reserved.

Increased expression of fructan 1-exohydrolase in rhizophores of Vernonia herbacea during sprouting and exposure to low temperature

Rhizophores of Vernonia herbacea, an Asteraceae found in the Brazilian Cerrado, store high amounts of fructans that vary in composition over the phenological cycle. Fructan 1-exohydrolase (1-FEH) activity is detectable during the sprouting phase, mainly in the proximal regions of rhizophores, of plants induced to sprout by defoliation and/or cold storage. We found an increase in 1-FEH gene expression during natural and induced sprouting and further enhancement through low-temperature treatment. Furthermore, a comparative analysis of 1-FEH gene expression in different regions of the rhizophores during the transition from dormancy to sprouting is presented. Transcripts were detected mainly in the proximal region, coinciding with high 1-FEH activity and a high concentration of free fructose. Low temperature promoted the accumulation of fructans of a low degree of polymerization (DP) and enhanced 1-FEH activity and gene expression. It is hypothesized that a set of 1-FEH proteins acts in two different ways during fructan mobilization: (1) by hydrolyzing fructo-oligosaccharides and -polysaccharides in sprouting plants (naturally or induced) for carbon supply and (2) by hydrolyzing preferably fructo-polysaccharides under low temperature to maintain the oligosaccharide pool for plant cold acclimation. (C) 2010 Elsevier GmbH. All rights reserved.

A phospholipase A(2) from Bothrops asper snake venom activates neutrophils in culture: Expression of cyclooxygenase-2 and PGE(2) biosynthesis

In this study, the production of prostaglandin E(2) (PGE(2)) and up-regulation in cyclooxygenase (COX) pathway induced by a phospholipase A(2) (PLA(2)), myotoxin-III (MT-III), purified from Bothrops asper snake venom, in isolated neutrophils were investigated. The arachidonic acid (AA) production and the participation of intracellular PLA(2)s (cytosolic PLA(2) and Ca(2+)-independent PLA(2)) in these events were also evaluated. MT-III induced COX-2, but not COX-1 gene and protein expression in neutrophils and increased PGE(2) levels. Pretreatment of neutrophils with COX-2 and COX-1 inhibitors reduced PGE(2) production induced by MT-III. Arachidonyl trifluoromethyl ketone (AACOCF(3)), an intracellular PLA(2) inhibitor, but not bromoenol lactone (BEL), an iPLA(2) inhibitor, suppressed the MT-III-induced AA and PGE(2) release. In conclusion, MT-III directly stimulates neutrophils inducing COX-2 mRNA and protein expression followed by production of PGE(2). COX-2 isoform is preeminent over COX-1 for production of PGE(2) stimulated by MT-III. PGE(2) and AA release by MT-III probably is related to cPLA(2) activation. (c) 2010 Elsevier Ltd. All rights reserved.

Secretory phospholipases A(2) isolated from Bothrops asper and from Crotalus durissus terrificus snake venoms induce distinct mechanisms for biosynthesis of prostaglandins E-2 and D-2 and expression of cyclooxygenases

The effects of myotoxin III (MT-III), a phospholipase A(2) (sPLA(2)) from Bothrops asper snake venom, and crotoxin B (CB), a neurotoxic and myotoxic sPLA2 from the venom of Crotalus durissus terrificus, on cyclooxygenases (COXs) expression and biosynthesis of prostaglandins (PGs) were evaluated, together with the mechanisms involved in these effects. Upon intraperitoneal injection in mice, both sPLA(2)s promoted the synthesis of PGD(2) and PGE(2), with a different time-course. MT-III, but not CB, induced COX-2 expression by peritoneal leukocytes without modification on COX-1 constitutive expression, whereas CB increased the constitutive activity of COX-1. MT-III increased the enzymatic activity of COX-1 and COX-2. Similar effects were observed when these sPLA(2)s were incubated with isolated macrophages, evidencing a direct effect on these inflammatory cells. Moreover, both toxins elicited the release of arachidonic acid from macrophages in vitro. inhibition of cPLA(2) by AACOCF(3), but not of iPLA(2) by PACOCF(3) or BEL, significantly reduced PGD2, PGE2 and arachidonic acid (AA) release promoted by MT-III. These inhibitors did not affect MT-III-induced COX-2 expression. In contrast, cPLA2 inhibition did not modify the effects of CB, whereas iPLA2 inhibition reduced PGD2 and AA production induced by CB. These findings imply that distinct regulatory mechanisms leading to PGs` synthesis are triggered by these snake venom sPLA(2)s. Such differences are likely to explain the dissimilar patterns of inflammatory reaction elicited by these sPLA(2)s in vivo. (C) 2008 Elsevier Ltd. All rights reserved.

Iron bioavailability from ferric pyrophosphate in rats fed with fructan-containing yacon (Smallanthus sonchifolius) flour

The effects of inulin-type fructans (ITF)-containing yacon flour (YF) on Fe bioavailability from ferric pyrophosphate (FP) were evaluated in Fe-deficient rats using the Hb repletion efficiency (HRE) assay. Weanling male Wistar rats were fed a low-Fe diet (12 mg/kg) for 15 days followed by 2 weeks of Fe repletion with diets providing 35 mg Fe/kg as either ferrous sulphate (FS) or FP, supplemented with 7.5% ITF as either YF or Raftilose (RAF), a purified ITF. ITF increased caecal fermentation, whereas YF was more butyrogenic than RAF. ITF improved FIRE in FP-fed rats, and those fed YF had a higher relative biological value compared with those fed FP and RAF. Liver Fe was increased by ITF, but only YF led to values similar to those in the FS group. It is observed that ITF increased caecal fermentation and Fe bioavailability. These effects were more pronounced when YF was the ITF source. (C) 2010 Elsevier Ltd. All rights reserved.