Calystegine distribution in potato (Solanum tuberosum) tubers and plants

Potato plants contain calystegines in leaves, stems, flowers, fruits and roots. Calystegines A₃ and B₂ are the main constituents. Highest concentrations were measured in sprouts emerging from the tubers. In 3 mm long sprouts, 3.3 mg total calystegines per g fresh mass were detected. Dormant tubers directly after harvest contain less calystegines in all parts than sprouting tubers. Flowers and young leaves are the aerial plant tissues with the highest calystegine concentration, i.e. 150 μg total calystegines per g fresh mass. Calystegine levels did not rise when sprouts were wounded. Tropinone application to sprouts and aerial tissues lead to an accumulation of pseudotropine and not to tropine. That indicates that stereospecific tropinone reduction is active in potato.     

Calystegines in Calystegia sepium derive from the tropane alkaloid pathway.

Calystegines were measured in roots and aerial parts of Calystegia sepium. The accumulation appears developmentally regulated. Calystegine accumulation in hairy root cultures follows growth and reaches maximal values of 1,5 mg/g dry mass. 15N-Labelled tropinone was fed to root cultures and the incorporation of label into calystegines and further metabolites of the tropane alkaloid pathway was measured after 2, 4 and 6 days. Pseudotropine was completely labelled after 2 days, and calystegine A(3) was labelled faster than the calystegines of the B-group. 2,7-Dihydroxynortropane also incorporated 15N from tropinone and is suggested to be a by-product of the tropane alkaloid pathway leading to calystegines.     

Powdery mildew infection of wheat leaves changes host solute transport and invertase activity

The effect of powdery mildew ( Blumeria graminis ) infection of wheat leaves on solute transport and invertase activity of the host tissue has been examined. Sugars (glucose, sucrose, maltose) and amino acids (glutamine, histidine) were taken up by leaf pieces, and radioactivity was transferred to the fungal mycelium. Infection had a marked effect on sugar uptake, particularly for glucose which was taken up into infected tissue at considerably higher rates than into uninfected tissues. In contrast, amino acid uptake rates into infected tissues were lower when compared with those into uninfected tissue. The increase in glucose uptake could be correlated with a change in sugar transporter gene expression as a wheat homologue of the monosaccharide carrier AtSTP4 was shown to increase in infected tissue. Efflux analysis showed a higher leakage of preloaded glucose from infected leaves in comparison with uninfected tissue and transfer to the mycelium was greater for glucose than for the other solutes measured. All types of invertase, measured enzymatically, showed an increase in infected tissue, with the highest proportional increase observed for cell-wall invertase. A partial-length complementary DNA, TaINV2 , was isolated for a putative cell-wall invertase; expression studies indicated that levels for this or related sequences increased substantially 3 days after infection.     

Slow-growth phenotype of transgenic tomato expressing apoplastic invertase

The growth of transgenic tomato (Lycopersicon esculentum) plants that express in their apoplast yeast invertase under the control of the cauliflower mosaic virus 35S promoter is severely inhibited. The higher the level of invertase, the greater the inhibition of growth. A second phenotypic characteristic of these transgenic plants is the development of yellow and necrotic spots on the leaves, and leaf curling. Again the severity of the symptoms is correlated with the level of invertase. These symptoms do not develop in shaded leaves indicating the need for photosynthesis. Keeping the plants in the dark for a prolonged period (24 hours) results in the disappearance of leaf starch from the control plants, but not from the plants with apoplastic invertase. These results are consistent with the interpretation that apoplastic invertase prevents photosynthate export from source leaves and that phloem loading includes an apoplastic step.     

Biosynthesis of calystegines: 15N NMR and kinetics of formation in root cultures of Calystegia sepium

Calystegines are nortropane alkaloids bearing between three and five hydroxyl groups in various positions. [15N]Tropinone was administered to root cultures of Calystegia sepium and the incorporation into calystegines was followed. Increase of label in calystegines was measured by one-dimensional 15N NMR and inverse-detected 2D NMR techniques. The results show that tropinone and pseudotropine are metabolites in the biosynthetic pathway of calystegines. The velocity of calystegine accumulation was followed kinetically by transfer of root cultures from 15N-enriched medium to 14N-medium and analysis by GC-MS. A constant calystegine formation with no interference by excretion or degradation was observed. A biosynthetic rate for individual calystegines at each time point was calculated, the maximum was 0.4 mg/day/g of biomass. This allowed the velocity of individual biosynthetic steps to be estimated.     

Response of fungal endophyte communities within Andropogon gerardii (Big bluestem) to nutrient addition and herbivore exclusion

Fungal endophytes may alter plant responses to the environment, but how does the environment affect the communities of fungal symbionts within plants? We examined the impact of nutrient addition and herbivore exclusion on endophyte communities of the prairie grass Andropogon gerardii in a full factorial field experiment. Fungi were cultured from stems, young leaves, and mature leaves, ITS sequences obtained, and endophyte incidence, community richness, and composition analyzed. Results indicate that in plots where nutrient addition and herbivore exclusion treatments had been applied separately, fungal endophyte incidence, community composition or evenness did not differ, but that greater species richness was observed in plots with nutrient addition and herbivore exclusion treatments applied in combination, compared to other treatments. Further, although fungal community composition was significantly different in stem and leaf tissues, OTU richness was greater in all endophyte communities in nutrient addition plus herbivore exclusion treatments, regardless of tissue type. Our results indicate the distinct fungal endophyte communities found in different plant tissues respond similarly to environmental factors.     

Inhibition of hydrolytic enzyme activities and plant pathogen growth by invertase inhibitors

The invertase inhibitory protein isolated from Cyphomandra betacea Sendt and Solanum tuberosum inhibited the invertase activity from different species, genera and even plant family. Furthermore, proteinaceous inhibitors are not invertase specific; fungal, bacterial and higher plant enzymes including polygalacturonase, pectinase, pectin lyase, alpha-L-arabinofuranosidase and beta-glucosidase are also shown to be inhibited. Both inhibitors exhibited an in vitro antibacterial action against phytopathogenics strains of Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae and Erwinia carotovora var carotovora.     

Involvement of cell-wall invertase in low-temperature hardening of tobacco plants

Specific features of low-temperature hardening (6 days at 8°C) of cold-sensitive tobacco plants (Nicotiana tabacum, cv. Samsun) related to changes in the cell-wall invertase activity were studied. During cold hardening, oppositely directed changes in this enzyme activity occurred in tobacco leaves and roots. In the leaves, cell-wall invertase was activated (approximately by 30%), the content of sugars increased (approximately by 25%), and the content of sucrose, the main transport form of sugars, in the apoplast reduced by three times; all these changes indicate that assimilate outflow from leaves to roots was inhibited. In contrast, in the root system, enzyme activity was decreased almost twice and the content of sugars in them was essentially unchanged. It is suggested that a strategy of low-temperature adaptation of cold-sensitive tobacco plants aimed at creating the high cold tolerance of aboveground parts, even at the expense of the root system, which, under conditions of native vegetation, is not practically exposed to damaging low temperatures.     

Variation of breeding system, floral rewards, and reproductive success in clonal Calystegia species (Convolvulaceae)

The reproductive biology of four Japanese Calystegia species (Convolvulaceae) was studied to examine the effects of clonality and population structure on reproductive success. Calystegia soldanella, C. hederacea, and C. japonica are self-incompatible, while C. sepium is self-compatible but needs pollinator services for self-pollination. The showy, bisexual flowers of Calystegia offer pollen and nectar that attract many kinds of insects such as bees and syrphid flies. Clones of C. soldanella often formed mats just above the high tide line on beaches and produced a great number of seeds. Calystegia hederacea and C. japonica were distributed as patches of separate clones and often failed to transfer pollen grains among clones. Fruit and seed sets in C. hederacea and C. japonica were not limited by pollinators but by the number of compatible pollen grains. Although C. sepium clones were also distributed in patches, high and stable fecundity was achieved by self-compatibility and pollinator attraction. We suggest that self-compatibility in C. sepium has evolved under fertilization limitation caused by geitonogamy or facilitated selfing.     

ISSR fingerprinting for the assessment of the bindweed biocontrol agent Stagonospora convolvuli LA39 after field release

AIMS: To develop a molecular identification method based on ISSR fingerprints to monitor the fungal leaf pathogen Stagonospora convolvuli LA39 used to biologically control bindweeds after a field release. METHODS AND RESULTS: The developed method proved to be suitable to clearly distinguish LA39 from resident Stagonospora spp. and was applied in two field experiments. First, the environmental persistence of LA39 was assessed in an overwintering experiment. LA39 could be re-isolated from infected bindweed 1 year after field application, but with very low frequency of occurrence. Secondly, LA39 was applied in an area with natural bindweed infestation and re-isolated from infected bindweed. The dispersal of LA39 during one season was poor (4-5 m). CONCLUSIONS: ISSR fingerprinting has been shown to be a valuable tool to monitor the environmental fate of S. convolvuli in the field. It is concluded that an LA39-based mycoherbicide will have minimal environmental impact caused by the restricted mobility, poor proliferation and poor persistence over seasons of LA39. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies about the dispersal and survival of biocontrol agents after field release as well as the development of methods needed for this purpose are indispensable for a comprehensive risk assessment for biocontrol agents.     

Isolation of a novel plant lectin with an unusual specificity from Calystegia sepium

A novel plant lectin has been isolated from the rhizomes of Calystegia sepium (hedge bindweed) and partially characterized. The lectin is a dimeric protein composed of two identical non-covalently linked subunits of 16kDa. Hapten inhibition studies indicate that the novel lectin is best inhibited by maltose and mannose and hence exhibits a sugar binding specificity that differs in some respects from that of all previously isolated plant lectins. Mitogenicity tests have shown that the Calystegia lectin is a powerful T-cell mitogen. Affinity purification of human, plant and fungal glycoproteins on immobilized C. sepium lectin demonstrates that this novel lectin can be used for the isolation of glycoconjugates from various sources. Moreover, it can be expected that by virtue of its distinct specificity, the new lectin will become an important tool in glycobiology. Abbreviations: Calsepa, lectin isolated from Calystegia sepium; ConA, concanavalin A; LPS, lipopolysaccharide; PBS, phosphate buffered saline (1.5 mMKH2PO4, 10 mM Na2HPO4, 3 mM KCl, 140 mM NaCl, pH 7.4) This revised version was published online in November 2006 with corrections to the Cover Date.     

Effects of drought on non-mycorrhizal and mycorrhizal maize: changes in the pools of non-structural carbohydrates, in the activities of invertase and trehalase, and in the pools of amino acids and imino acids

To study the response of non-mycorrhizal and mycorrhizal maize plants to drought, the changes in the pools of non-structural carbohydrates and amino acids were analysed in leaves and roots of two maize cvs. Plants well colonized by the arbuscular mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) (60% of root length infected) and comparable non-mycorrhizal plants were subjected to moderate drought stress by reducing the water supply. This stress induced a conspicuous increase in the trehalose pool in the mycorrhizal roots, probably because it was accumulated by the fungal symbiont. Furthermore, glucose and fructose were accumulated in leaves and roots of non-mycorrhizal plants but not in the mycorrhizal ones. Starch disappeared completely from the leaves of both mycorrhizal and non-mycorrhizal plants in response to drought. Activities of soluble acid invertase and trehalase were also measured. Acid invertase activity increased during drought in the leaves of both non-mycorrhizal and mycorrhizal plants whilst in the roots it was unaffected in non-mycorrhizal plants and decreased in the mycorrhizal ones. Without drought stress, trehalase activity was considerably higher in the leaves and roots of mycorrhizal plants than in those of non-mycorrhizal plants. It increased conspicuously during drought, primarily in the leaves of non-mycorrhizal plants. A drought-induced accumulation of amino acids as well as imino acids was found in roots and leaves of both mycorrhizal and non-mycorrhizal plants; leaves of mycorrhizal plants accumulated more imino acids than those of non-mycorrhizal ones. Our results show that drought stress and the presence of a mycorrhizal fungus have a considerable effect on carbon partitioning, imino acid and amino acid accumulation in maize plants.     

Mycological studies on the angiosperm root parasiteCynomorium coccineum L. and two of its halophytic hosts

The fungal colonization of the angiosperm root parasiteCynomorium coccineum and the halophytic hostsLimonium delicatulum andArthrocnemum glaucum were investigated in a Mediterranean salt marsh in March 1992. The main fungal inhabitants on the leaves or shoot surface of the test plants wereAspergillus niger, Penicillium chrysogenum andCladosporium herbarum. The qualitative analysis of the fungal species associating the parasite, the hosts and the non-infected plants showed similar pattern. However, the total population exhibited quantitative differences coupled with the amount and the chemical composition of the exudates on plant surface and the quantity of transpired water. The fungal catch from the aerial shoot (inflorescence) of the parasite was higher than that collected from either the leaves or aerial shoots of non-infected or host plants. The fungal density on the leaves ofL. delicatulum was higher than those isolated from the aerial shoots ofA. glaucum. Infection byC. coccineum caused a marked drop in the total fungal population on leaves or shoot surfaces of the hosts as compared to the corresponding non-infected individuals. The stimulative effect of washings on spore germination of some isolated fungal species was matched with the density of fungi on the target plants.     

Biological Control of Hedge Bindweed (Calystegia sepium) with Stagonospora convolvuli Strain LA39 in Combination with Competition from Red Clover (Trifolium pratense)

In a maize cropping system where a living green cover suppresses many weeds, Calystegia sepium is able to escape control. In this paper we report the potential for biological control of C. sepium by using the bindweed pathogen Stagonospora convolvuli strain LA39 as a mycoherbicide in combination with competition by the green cover plant Trifolium pratense. In a greenhouse experiment, competition from shoots of T. pratense caused a strong reduction of the biomass of C. sepium, and combined competition from shoots and roots had the same effect. In a second, factorial greenhouse experiment, competition by T. pratense again reduced C. sepium biomass. However, S. convolvuli did not influence the number of leaves or the biomass of C. sepium in the greenhouse even though severe necrosis was observed on inoculated bindweed leaves. In contrast, in a 2-year field study, S. convolvuli caused severe disease and a strong reduction of C. sepium ground coverage in maize. Underseeding with T. pratense had no effect on disease severity, but T. pratense reduced ground coverage by C. sepium at one of eight samplings in the first year. In conclusion, S. convolvuli is useful in the field and, as shown in the greenhouse, a competitive green cover might improve biological control of C. sepium.     

The impact of reduced vacuolar invertase activity on the photosynthetic and carbohydrate metabolism of tomato

The impact of reduced vacuolar invertase activity on photosynthetic and carbohydrate metabolism was examined in tomato (Solanum lycopersicon L.). The introduction of a co-suppression construct (derived from tomato vacuolar invertase cDNA) produced plants containing a range of vacuolar invertase activities. In the leaves of most transgenic plants from line INV-B, vacuolar invertase activity was below the level of detection, whereas leaves from line INV-A and untransformed wild-type plants showed considerable variation. Apoplasmic invertase activity was not affected by the co-suppression construct. It has been suggested that, in leaves, vacuolar invertase activity regulates sucrose content and its availability for export, such that in plants with high vacuolar invertase activity a futile cycle of sucrose synthesis and degradation takes place. In INV-B plants with no detectable leaf vacuolar invertase activity, sucrose accumulated to much higher levels than in wild-type plants, and hexoses were barely detectable. There was a clear threshold relationship between invertase activity and sucrose content, and a linear relationship with hexose content. From these data the following conclusions can be drawn. (i) In INV-B plants sucrose enters the vacuole where it accumulates as hydrolysis cannot take place. (ii) There was not an excess of vacuolar invertase activity in the vacuole; the rate of sucrose hydrolysis depended upon the concentration of the enzyme. (iii) The rate of import of sucrose into the vacuole is also important in determining the rate of sucrose hydrolysis. The starch content of leaves was not significantly different in any of the plants examined. In tomato plants grown at high irradiance there was no impact of vacuolar invertase activity on the rate of photosynthesis or growth. The impact of the cosuppression construct on root vacuolar invertase activity and carbohydrate metabolism was less marked.Abbreviations CaMV Cauliflower Mosaic Virus - WT wild type     

An invertase inhibitory protein from Pteris deflexa link fronds

Plant invertases play important roles in sucrose metabolism. Cell wall invertase was reported to participate in phloem loading and unloading. Soluble invertases would be involved in hexose level regulation in mature tissues and in stored sucrose utilization within vacuoles. Invertase inhibitory proteins were described as one of the possible mechanisms for invertase activity regulation in some plant species; nevertheless, these proteins were found only in sink tissues, suggesting that this mechanism would not be relevant in the sucrose turnover of leaves. This report describes the purification of invertase from Pteris deflexa fronds and the occurrence of an invertase inhibitory protein in this fern organ, as well as its purification and invertase-inhibitor interactions. The Mr of the invertase and of its inhibitory protein were 90,000 and 18,000, respectively. SDS-PAGE in the presence of 2-mercaptoetanol gave two subunits for the enzyme (Mr=66,000 and 30,000) and only one for the inhibitor. The inhibitor protein is a glycoprotein (12% w/w of neutral sugars) that did not show agglutinating activity like some others, and also showed a high heat stability at pH 5.0. The optimum pH of invertase activity is 5.0, while invertase inhibitory protein caused maximal inhibition at the same pH value. Invertase-inhibitor complex formation occurs in an immediate manner and a protease activity was discarded. The inhibition is non-competitive (Ki=1.5 x 10(-6) M) without interactions among the binding sites. The complex is slightly dissociable and sucrose was able to partially reduce the inhibitory effect. Up to the present, invertase inhibitory proteins have been found solely in heterotrophic tissues. In this work we demonstrate that this protein is also present in an autotrophic tissue of a lower vascular plant.     

Acid invertase (sucrose hydrolysis) is not required for sucrose mobilization from the vacuole

The possible involvement of acid invertase (sucrose hydrolysis) as a prerequisite for sucrose mobilization from the vacuole of storage cells was investigated. Sugarcane ( Saccharum officinarum ) stalks, carrot ( Daucus carota ) roots and red beet ( Beta vulgaris ) hypocotyls were planted under greenhouse conditions and allowed to resume growth. The plants, however, were not permitted to become photosynthetically autotrophic by removing the new expanded leaves. Sucrose levels declined significantly in all three tissues without the development of acid invertase (EC 3.2.1.26) during the 21‐day experimental period. Acid invertase and thus sucrose hydrolysis within the vacuole was, therefore, not required for sucrose mobilization.     

Antisense repression of vacuolar and cell wall invertase in transgenic carrot alters early plant development and sucrose partitioning.

To unravel the functions of cell wall and vacuolar invertases in carrot, we used an antisense technique to generate transgenic carrot plants with reduced enzyme activity. Phenotypic alterations appeared at very early stages of development; indeed, the morphology of cotyledon-stage embryos was markedly changed. At the stage at which control plantlets had two to three leaves and one primary root, shoots of transgenic plantlets did not separate into individual leaves but consisted of stunted, interconnected green structures. When transgenic plantlets were grown on media containing a mixture of sucrose, glucose, and fructose rather than sucrose alone, the malformation was alleviated, and plantlets looked normal. Plantlets from hexose-containing media produced mature plants when transferred to soil. Plants expressing antisense mRNA for cell wall invertase had a bushy appearance due to the development of extra leaves, which accumulated elevated levels of sucrose and starch. Simultaneously, tap root development was markedly reduced, and the resulting smaller organs contained lower levels of carbohydrates. Compared with control plants, the dry weight leaf-to-root ratio of cell wall invertase antisense plants was shifted from 1:3 to 17:1. Plants expressing antisense mRNA for vacuolar invertase also had more leaves than did control plants, but tap roots developed normally, although they were smaller, and the leaf-to-root ratio was 1.5:1. Again, the carbohydrate content of leaves was elevated, and that of roots was reduced. Our data suggest that acid invertases play an important role in early plant development, most likely via control of sugar composition and metabolic fluxes. Later in plant development, both isoenzymes seem to have important functions in sucrose partitioning.