Toward the identification of a phytocannabinoid-like compound in the flowers of a South African medicinal plant (Leonotis leonurus).

OBJECTIVE: Current global trends on natural therapeutics suggest an increasing market interest toward the use and discovery of new plant-derived therapeutic compounds, often referred to as traditional medicine (TM). The Cannabis industry is currently one such focal area receiving attention, owing to the occurrence of phytocannabinoids (pCBs) which have shown promise in health-promotion and disease prevention. However, the occurrence of pCBs in other plant species are often overlooked and rarely studied. Leonotis leonurus (L.) R. Br. is endemic to South Africa with a rich history of use in TM practices amongst indigenous people and, has been recorded to induce mild psychoactive effects akin to Cannabis. While the leaves have been well-reported to contain therapeutic phytochemicals, little information exists on the flowers. Consequently, as part of a larger research venture, we targeted the flowers of L. leonurus for the identification of potential pCB or pCB-like compounds. RESULTS: Flower extracts were separated and analyzed using high performance thin layer chromatography (HPTLC). A single pCB candidate was isolated from HPTLC plates and, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), we could successfully group this compound as a fatty amide and tentatively identified as 7,10,13,16-Docosatetraenoylethanolamine (adrenoyl-EA), a known bioactive compound.

Upregulation of pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity in strawberry.

Pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) is a cytosolic enzyme catalyzing the first committed step in glycolysis by reversibly phosphorylating fructose-6-phosphate to fructose-1,6-bisphosphate. The position of PFP in glycolytic and gluconeogenic metabolism, as well as activity patterns in ripening strawberry, suggest that the enzyme may influence carbohydrate allocation to sugars and organic acids. Fructose-2,6-bisphosphate activates and tightly regulates PFP activity in plants and has hampered attempts to increase PFP activity through overexpression. Heterologous expression of a homodimeric isoform from Giardia lamblia, not regulated by fructose-2,6-bisphosphate, was therefore employed to ensure in vivo increases in PFP activity. The coding sequence was placed into a constitutive expression cassette under control of the cauliflower mosaic virus 35S promoter and introduced into strawberry by Agrobacterium tumefaciens-mediated transformation. Heterologous expression of PFP resulted in an up to eightfold increase in total activity in ripe berries collected over two consecutive growing seasons. Total sugar and organic acid content of transgenic berries harvested during the first season were not affected when compared to the wild type, however, fructose content increased at the expense of sucrose. In the second season, total sugar content and composition remained unchanged while the citrate content increased slightly. Considering that PFP catalyses a reversible reaction, PFP activity appears to shift between gluconeogenic and glycolytic metabolism, depending on the metabolic status of the cell.

Differential expression of three purple acid phosphatases from potato.

Three cDNAs encoding purple acid phosphatase (PAP) were cloned from potato (Solanum tuberosum L. cv. Désirée) and expression of the corresponding genes was characterised. StPAP1 encodes a low-molecular weight PAP clustering with mammalian, cyanobacterial, and other plant PAPs. It was highly expressed in stem and root and its expression did not change in response to phosphorus (P) deprivation. StPAP2 and StPAP3 code for high-molecular weight PAPs typical for plants. Corresponding gene expression was shown to be responsive to the level of P supply, with transcripts of StPAP2 and StPAP3 being most abundant in P-deprived roots or both stem and roots, respectively. Root colonisation by arbuscular mycorrhizal fungi had no effect on the expression of any of the three PAP genes. StPAP1 mRNA is easily detectable along the root axis, including root hairs, but is barely detectable in root tips. In contrast, both StPAP2 and StPAP3 transcripts are abundant along the root axis, but absent in root hairs, and are most abundant in the root tip. All three PAPs described contain a predicted N-terminal secretion signal and could play a role in extracellular P scavenging, P mobilisation from the rhizosphere, or cell wall regeneration.

The Arabidopsis sex1 mutant is defective in the R1 protein, a general regulator of starch degradation in plants, and not in the chloroplast hexose transporter.

Starch is the major storage carbohydrate in higher plants and of considerable importance for the human diet and for numerous technical applications. In addition, starch can be accumulated transiently in chloroplasts as a temporary deposit of carbohydrates during ongoing photosynthesis. This transitory starch has to be mobilized during the subsequent dark period. Mutants defective in starch mobilization are characterized by high starch contents in leaves after prolonged periods of darkness and therefore are termed starch excess (sex) mutants. Here we describe the molecular characterization of the Arabidopsis sex1 mutant that has been proposed to be defective in the export of glucose resulting from hydrolytic starch breakdown. The mutated gene in sex1 was cloned using a map-based cloning approach. By complementation of the mutant, immunological analysis, and analysis of starch phosphorylation, we show that sex1 is defective in the Arabidopsis homolog of the R1 protein and not in the hexose transporter. We propose that the SEX1 protein (R1) functions as an overall regulator of starch mobilization by controlling the phosphate content of starch.

Undernutrition and childhood infections: a prospective study of childhood infections in relation to growth in the Sudan.

UNLABELLED: The relationships between both diarrhoea and respiratory infections and linear and ponderal growth were prospectively examined among 28 753 Sudanese pre-school children. Childhood infections were significantly and inversely associated with attained height and attained weight and gain in height and weight over a 6-mo period. They were significantly and positively associated also with stunting after adjusting for age, gender, socio-economic status, dietary variables and previous morbidity. Attained height was on average 17 mm lower (95% CI [-19 -15]) for children with diarrhoea and 11 mm lower (95% CI [-3 -9]) for children with complicated cough than for those without these symptoms. The association between morbidity and attained weight was significant for diarrhoea and complicated cough, but the differences between children with and without symptoms were negligible. The risk of being stunted 6 mo later was 1.38 times (95% CI [1.20 1.59]), 1.29 times (95% CI [0.97 1.72]) and 1.32 times (95% CI [1.13 1.54]) greater among normally-nourished children with diarrhoea, febrile diarrhoea and fever, respectively, than among children without these symptoms. The difference in attained height between children with diarrhoea or complicated cough and those without symptoms increased with age, and was larger among the non-breastfed children compared with breastfed children. CONCLUSION: The results underline the need to reduce child morbidity to prevent the impairment of growth and development.

Antisense inhibition of plastidial phosphoglucomutase provides compelling evidence that potato tuber amyloplasts import carbon from the cytosol in the form of glucose-6-phosphate.

The aim of this work was to establish whether plastidial phosphoglucomutase is involved in the starch biosynthetic pathway of potato tubers and thereby to determine the form in which carbon is imported into the potato amyloplast. For this purpose, we cloned the plastidial isoform of potato PGM (StpPGM), and using an antisense approach generated transgenic potato plants that exhibited decreased expression of the StpPGM gene and contained significantly reduced total phosphoglucomutase activity. We confirmed that this loss in activity was due specifically to a reduction in plastidial PGM activity. Potato lines with decreased activities of plastidial PGM exhibited no major changes in either whole-plant or tuber morphology. However, tubers from these lines exhibited a dramatic (up to 40%) decrease in the accumulation of starch, and significant increases in the levels of sucrose and hexose phosphates. As tubers from these lines exhibited no changes in the maximal catalytic activities of other key enzymes of carbohydrate metabolism, we conclude that plastidial PGM forms part of the starch biosynthetic pathway of the potato tuber, and that glucose-6-phosphate is the major precursor taken up by amyloplasts in order to support starch synthesis.

Understanding and influencing starch biochemistry.

Starch is one of the most important products synthesized by plants that is used in industrial processes. If it were possible to increase production or modify starches in vivo, using combinations or either genetically altered or mutant plants, it may make them cheaper for use by industry, or open up new markets for the modified starches. The conversion of sucrose to starch in storage organs is, therefore, discussed. In particular the roles of the different enzymes directly involved in synthesizing the starch molecules on altering starch structure are reviewed, as well as the different models for the production of the fine structure of amylopectin. In addition, the process of starch phosphorylation, which is also important in determining the physical properties of starches, is reviewed. It is hoped that detailed knowledge of these processes will lead to the rational design of tailored starches. Starch degradation is also an important process, for example, in the cold-sweetening of potato tubers, but outside of cereal endosperm little is known about the processes involved. The enzymes thought to be involved and the evidence for this are discussed.

Undernutrition in relation to childhood infections: a prospective study in the Sudan.

OBJECTIVE: The aim of the study was to examine the relationships between nutritional status and diarrhoea and respiratory infections. DESIGN: Prospective cohort study within the framework of a randomized double-blind placebo-controlled intervention trial. SETTING: In rural communities in the Khartoum and Gezira regions, in Northern Sudan. SUBJECTS: 28,753 Sudanese pre-school children between 6 months and 6 y old. METHODS: Relative risks of subsequent diarrhoea and respiratory infections in relation to nutritional status measured by anthropometry (Z-scores of height-for-age (H/A), weight-for-height (W/H), and weight-for-age (W/A), which reflect stunting, wasting and underweight, respectively) were estimated using odds ratios from logistic regression adjusting for various covariates. RESULTS: H/A, W/H and W/A were significantly and inversely associated with subsequent diarrhoea and febrile diarrhoea (P for trend <0.001) with risks being 2.00 times higher (95% confidence interval, CI (1.64, 2.43)) among children with W/A Z-scores below -4 Z, and 1.75 times higher (95% CI (1.56, 1.96)) among those with a W/A Z-score between -4 and -3 Z compared with children having a W/A Z-score > or =1. Age, gender, region of residence and seasonality modified these associations. Also, febrile cough was inversely associated with W/A and W/H (P<0.03), with risks ranging from 1.41 times higher (95% CI (1.02, 1.97)) to 1.21 times higher (95% CI (1.04, 1.41)) in the group of underweight children with W/A Z-scores below -4 and between -2 and -1 Z, all compared with normally nourished children (> or =-1 Z). CONCLUSIONS: The reduction of severe but also mild and moderate undernutrition is necessary through nutrition, health and socio-economic improvement in order to prevent morbidity.

Introduction of polyphosphate as a novel phosphate pool in the chloroplast of transgenic potato plants modifies carbohydrate partitioning.

Potato plants (Solanum tuberosum L., cv. Désirée) were transformed with the polyphosphate kinase gene from Escherichia coli fused to the leader sequence of the ferredoxin oxidoreductase gene (FNR) from Spinacea oleracea under the control of the leaf specific St-LS1 promoter to introduce a novel phosphate pool in the chloroplasts of green tissues. Transgenic plants (cpPPK) in tissue culture developed necrotic lesions in older leaves and showed earlier leaf senescence while greenhouse plants showed no noticeable phenotype. Leaves of cpPPK plants contained less starch but higher concentrations of soluble sugars. The presence of polyphosphate in cpPPK leaves was demonstrated by toluidine blue staining and unambiguously verified and quantified by in vitro 31P-NMR of extracts. Polyphosphate accumulated during leaf development from 0.06 in juvenile leaves to 0.83 mg P g-1 DW in old leaves and had an average chain length of 18 residues in mature leaves. In situ 31P-NMR on small leaf pieces perfused with well-oxygenated medium showed only 0.036 mg P g-1 DW polyphosphate that was, however, greatly increased upon treatment with 50 mM ammonium sulfate at pH 7.3. This phenomenon along with a yield of 0.47 mg P g-1 DW polyphosphate from an extract of the same leaf material suggests that 93% of the polyphosphate pool is immobile. This conclusion is substantiated by the observation that no differences in polyphosphate pool sizes could be discerned between darkened and illuminated leaves, leaves treated with methylviologen or anaerobis and control leaves, treatments causing a change in the pool of ATP available for polyPi synthesis. Results are discussed in the context of the chelating properties of polyphosphates for cations and its consequences for the partitioning of photoassimilate between starch and soluble sugars.

Antisense inhibition of the GDP-mannose pyrophosphorylase reduces the ascorbate content in transgenic plants leading to developmental changes during senescence.

GDP-mannose pyrophosphorylase (GMPase, EC 2.7.7.22) catalyses the synthesis of GDP-D-mannose and represents the first committed step in the formation of all guanosin-containing sugar nucleotides found in plants which are precursors for cell wall biosynthesis and, probably more important, the synthesis of ascorbate. A full-length cDNA encoding GMPase from S. tuberosum was isolated. Transgenic potato plants were generated in which the GMPase cDNA was introduced in antisense orientation to the 35S promoter. Transformants with reduced GMPase activity were selected. Transgenic plants were indistinguishable from the wild-type when held under tissue culture conditions, however, a major change was seen 10 weeks after transfer into soil. Transgenic plants showed dark spots on leaf veins and stems with this phenotype developing from the bottom to the top of the plant. In case of the line with the strongest reduction, all aerial parts finally dried out after 3 months in soil, in contrast to the wild-type plants which did not start to senesce at this time. This coincides with a reduction of ascorbate contents in the transgenic plants, which is in agreement with the recently proposed pathway of ascorbate biosynthesis. Furthermore, leaf cell walls of the transgenic potato plants had mannose contents that were reduced to 30-50% of the wild-type levels, whereas the composition of tuber cell walls was unchanged. The glycosylation pattern of proteins was unaffected by GMPase inhibition, as studied by affinoblot analysis.

The influence of alterations in ADP-glucose pyrophosphorylase activities on starch structure and composition in potato tubers.

In order to examine whether alterations in the supply of precursor molecules into the starch biosynthetic pathway affected various characteristics of the starch, starch was isolated from potato (Solanum tuberosum L.) tubers containing reduced amounts of the enzyme ADP-glucose pyrophosphorylase (AGPase). It was found that although the type of crystalline polymorph in the starch was not altered, the amylose content was severely reduced. In addition, amylopectin from the transgenic plants accumulated more relatively short chains than that from control plants and the sizes of starch granules were reduced. The starch granules from the transgenic plants contained a greater amount of granule-bound starch synthase enzyme, which led to an increase in the maximum activity of the enzyme per unit starch tested. The K(m) for ADP-glucose was, at most, only slightly altered in the transgenic lines. Potato plants containing reduced AGPase activity were also transformed with a bacterial gene coding for AGPase to test whether this enzyme can incorporate phosphate monoesters into amylopectin. A slight increase in phosphate contents in the starch in comparison with the untransformed control was found, but not in comparison with starch from the line with reduced AGPase activity into which the bacterial gene was transformed.Key words: ADP-glucose pyrophosphorylase. Amylopectin structure. Amylose. Solanum (starch. tuber). Starch granule size. Starch phosphorylation

Cloning and functional analysis of a cDNA encoding a starch synthase from potato (Solanum tuberosum L.) that is predominantly expressed in leaf tissue.

Three isoforms of starch synthase (SS) were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. A cDNA encoding SSI from rice was used as a probe to clone a corresponding cDNA from potato. The deduced amino acid sequence identified the protein as an SS from potato with an M(r) of 70.6 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SSI. An analysis of the expression pattern of the gene indicated that SSI is predominantly expressed in sink and source leaves, and, to a lower extent in tubers. In several independent transgenic potato lines, where the expression of SSI was repressed using the antisense approach, the activity of a specific SS isoform was reduced to non-detectable levels as determined through activity staining after native gel electrophoresis. The reduction in the amount of this isoform of SS did not lead to any detectable changes in starch structure, probably due to the fact that this isoform only represents a minor activity in potato tubers.

Production of modified polymeric carbohydrates.

The cloning of a gene responsible for the phosphorylation of glucans has made it possible to genetically engineer the phosphorylation level of starches in higher plants. Through the manipulation of starch synthase activity, it is now also possible to genetically tailor the chain-length distribution in the amylopectin. Both findings will lead to the development of novel starches utilized as a renewable resource. The production of fructans on a large scale can also be envisioned for the near future.

Simultaneous antisense inhibition of two starch-synthase isoforms in potato tubers leads to accumulation of grossly modified amylopectin.

A chimaeric antisense construct was used to reduce the activities of the two major starch-synthase isoforms in potato tubers simultaneously. A range of reductions in total starch-synthase activities were found in the resulting transgenic plants, up to a maximum of 90% inhibition. The reduction in starch-synthase activity had a profound effect on the starch granules, which became extremely distorted in appearance compared with the control lines. Analysis of the starch indicated that the amounts produced in the tubers, and the amylose content of the starch, were not affected by the reduction in activity. In order to understand why the starch granules were distorted, amylopectin was isolated and the constituent chain lengths analysed. This indicated that the amylopectin was very different to that of the control. It contained more chains of fewer than 15 glucose units in length, and fewer of between 15 and 80 glucose units. In addition, the amylopectin contained more very long chains. Amylopectin from plants repressed in just one of the activities of the two starch-synthase isoforms, which we have reported upon previously, were also analysed. Using a technique different to that used previously we show that both isoforms also affect the amylopectin, but in a way that is different to when both isoforms are repressed together.

Inhibition of a starch-granule-bound protein leads to modified starch and repression of cold sweetening.

We have cloned a gene involved in starch metabolism that was identified by the ability of its product to bind to potato starch granules. Reduction in the protein level of transgenic potatoes leads to a reduction in the phosphate content of the starch. The complementary result is obtained when the protein is expressed in Escherichia coli, as this leads to an increased phosphate content of the glycogen. It is possible that this protein is responsible for the incorporation of phosphate into starch-like glucans, a process that is not understood at the biochemical level. The reduced phosphate content in potato starch has some secondary effects on its degradability, as the respective plants show a starch excess phenotype in leaves and a reduction in cold-sweetening in tubers.

cDNA sequence and heterologous expression of monomeric spinach pullulanase: multiple isomeric forms arise from the same polypeptide.

The spinach pullulanase gene was cloned and sequenced using peptide sequences of the purified enzyme as a starting point and employing PCR techniques and cDNA library screening. Its open reading frame codes for a protein of 964 amino acids which represents a precursor of the pullulanase. The N-terminal transit peptide consists of 65 amino acids, and the mature protein, comprising 899 amino acids, has a calculated molecular mass of 99kDa. Pullulanase is a member of the alpha-amylase family. In addition to a characteristic catalytic (beta/alpha)8-barrel domain, it contains a domain, F, that is specific for branching and debranching enzymes. Pullulanase cDNA was expressed in Escherichia coli, and the purified protein was compared with the enzyme from spinach leaves. Identity of the two proteins was confirmed in terms of catalytic properties, N-terminal amino acid sequences and molecular masses. The pullulanase produced by E. coli showed the same microheterogeneity as the spinach leaf enzyme: it could be resolved into two substrate-induced forms by electrophoresis in amylopectin-containing polyacrylamide gels, and, in the absence of substrate, into several free forms (charge isomers) by isoelectric focusing or chromatofocusing. Rechromatofocusing of single free forms resulted in the originally observed pattern of molecular forms. However, heterogeneity of the protein disappeared on isoelectric focusing under completely denaturing conditions when only one protein band was observed. Post-translational modifications such as glycosylation and phosphorylation could be excluded as potential explanations for the protein heterogeneity. Therefore the microheterogeneity of spinach leaf pullulanase results from neither genetic variation nor post-translational modifications, but is a property of the single unmodified gene product. The different interconvertible forms of the pullulanase represent protein populations of different tertiary structure of the same polypeptide.

Homodimers and heterodimers of Pho1-type phosphorylase isoforms in Solanum tuberosum L. as revealed by sequence-specific antibodies.

Higher plants possess two types of glucan phosphorylase (EC 2.4.1.1). One isozyme type, designated as Pho1, is located in the plastid whereas the other type, Pho2, is restricted to the cytosol. For Solanum tuberosum L. two Pho1 type phosphorylases have been sequenced [Nakano, K. & Fukui, T. (1986) J. Biol. Chem. 261, 8230-8236; Sonnewald, U., Basner, A., Greve, B. & Steup, M. (1995) Plant Mol. Biol. 27, 567-576]. Both proteins (referred to as Pho1a and Pho1b, respectively) are highly similar (81-84% amino acid identity over most parts of the two sequences) with the exception of the N-terminal transit peptide and the large insertion located between the N- and the C-terminal domains. In this communication antibodies that bind specifically to either Pho1a or Pho1b were used to study both isoforms at the protein level. The antibodies were applied to both potato tuber and leaf extracts following either denaturing or non-denaturing electrophoresis. Pho1a but not Pho1b was immunochemically detectable in tuber extracts whereas leaf extracts contained both the Pho1a and Pho1b protein. During denaturing electrophoresis the two antigens comigrated. When the leaf Pho1 isoforms were separated by affinity electrophoresis three bands of activity were resolved; all of them were recognized by the anti-Pho1a antibodies, but only two of these reacted with the anti-Pho1b antibodies. The isoform binding exclusively to the anti-Pho1a antibodies comigrated with the Pho1 isozyme from potato tubers. Immunoprecipitation experiments performed with anti-Pho1a antibodies removed the entire Pho1 phosphorylase activity from both tuber and leaf extracts. Addition of anti-Pho1b antibodies to tuber extracts did not affect the enzyme pattern, whereas in leaf extracts one isoform remained unchanged but the two other bands were strongly retarded. This indicates that the Pho1a protein is present in all three forms and Pho1b is associated with Pho1a. Association of Pho1a and Pho1b was further demonstrated by cross-linking experiments using bis(sulfosuccinimidyl)suberate as linker. Immunoprecipitation experiments were also performed using extracts of transformed Escherichia coli cells that expressed either Pho1a or Pho1b or both simultaneously. Under these conditions a homodimeric Pho1b phosphorylase was observed that had a lower electrophoretic mobility than the heterodimer from leaves. In leaves of transgenic potato plants antisense inhibition of the Pho1a gene affected the formation of (Pho1a)2 more strongly than that of the heterodimer. Thus, in leaves, Pho1a exists both as a homodimer, (Pho1a)2 and as heterodimer, (Pho1a-Pho1b); a part of it appears to be covalently modified. Pho1b, in the homodimeric form, is often below the limit of detection. In tubers the homodimer, (Pho1a)2, is the only detectable Pho1-type enzyme. To our knowledge this is the first report on a heterodimeric structure of plant phosphorylase.

Morphology and tuber formation of in-vitro-grown potato plants harboring the yeast invertase gene and/or the rolC gene.

Growth and tuber formation of transgenic potato plants (Solanum tuberosum cv. Désirée) harboring the yeast invertase gene and the rolC gene individually or in combination under the transcriptional control of the patatin promoter were investigated under different conditions in vitro. Plants expressing only the invertase gene were morphologically similar to control plants. rolC transgenic plants had an increased tiller number, improved root growth, and a higher total biomass. Tuber formation and growth were altered by the introduced transgenes. The sucrose requirement to induce tubers was shifted to lower or higher concentrations for invertase- or rolC-expressing clones, respectively. In addition, rolC plants formed tubers of altered morphology. A comparison with soil-grown plants showed that morphological parameters can be predicted to some extent from in vitro studies, while for reliable prescreening of parameters concerning tuber formation and growth, an optimization of currently used protocols is necessary.

Starches from A to C. Chlamydomonas reinhardtii as a model microbial system to investigate the biosynthesis of the plant amylopectin crystal.

Wide-angle powder x-ray diffraction analysis was carried out on starch extracted from wild-type and mutant Chlamydomonas reinhardtii cells. Strains containing no defective starch synthases as well as mutants carrying a disrupted granule-bound starch synthase structural gene displayed the A type of diffraction pattern with a high degree of crystallinity. Mutants carrying a defect for the major soluble starch synthase (SSS), SSS II, were characterized by a switch to the B type of diffraction pattern with very low crystallinity. Mutant strains carrying SSS I as the only glucan elongation enzyme regained some of their crystallinity but switched to the C type of diffraction pattern. Differential scanning calorimetry analysis correlated tightly with the x-ray diffraction results. Together with the electron microscopy analyses, these results establish C. reinhardtii as a microbial model system displaying all aspects of cereal starch synthesis and structure. We further show that SSS II is the major enzyme involved in the synthesis of crystalline structures in starch and demonstrate that SSS I alone builds a new type of amylopectin structure.