Metabolic processes and carbon nutrient exchanges between host and pathogen sustain the disease development during sunflower infection by <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Interactions between the necrotrophic fungus Sclerotinia sclerotiorum and one of its hosts, Helianthus annuus L., were analyzed during fungal colonization of plant tissues. Metabolomic analysis, based on ¹³C- and ³¹P-NMR spectroscopy, was used to draw up the profiles of soluble metabolites of the two partners before interaction, and to trace the fate of metabolites specific of each partner during colonization. In sunflower cotyledons, the main soluble carbohydrates were glucose, fructose, sucrose and glutamate. In S. sclerotiorum extracts, glucose, trehalose and mannitol were the predominant soluble carbon stores. During infection, a decline in sugars and amino acids was observed in the plant and fungus total content. Sucrose and fructose, initially present almost exclusively in plant, were reduced by 85%. We used a biochemical approach to correlate the disappearance of sucrose with the expression and the activity of fungal invertase. The expression of two hexose transporters, Sshxt1 and Sshxt2, was enhanced during infection. A database search for hexose transporters homologues in the S. sclerotiorum genome revealed a multigenic sugar transport system. Furthermore, the composition of the pool of reserve sugars and polyols during infection was investigated. Whereas mannitol was produced in vitro and accumulated in planta, glycerol was exclusively produced in infected tissues and increased during colonization. The hypothesis that the induction of glycerol synthesis in S. sclerotiorum exerts a positive effect on osmotic protection of fungal cells and favors fungal growth in plant tissues is discussed. Taken together, our data revealed the importance of carbon–nutrient exchanges during the necrotrophic pathogenesis of S. sclerotiorum.     

Carbohydrates as regulatory factors on the rooting of <Emphasis Type="Italic">Eucalyptus saligna</Emphasis> Smith and <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> Labill

Comparisons between related species with different rooting capacities can provide insights into the mechanisms controlling adventitious root development. The availability of carbohydrates is often considered exclusively as an energetic requirement to drive root development; the major regulatory role in the process is often attributed to phytohormones, particularly auxin. The roles of light quantity (irradiance) and carbohydrate supply available to young aseptic donor-plants on the adventitious rooting response of Eucalyptus globulus (rooting recalcitrant) and Eucalyptus saligna (easy-to-root) were examined. The effects of the type of carbohydrate supply (sucrose or glucose) on the rooting response of cuttings was also evaluated. Light intensity supplied to mother-plants (30 or 60 mol m^–2 s^–1) had limited influence on the rooting response of both species, whereas dark periods were detrimental, particularly for E. globulus. In E. globulus, rooting was promoted by the absence of sucrose in donor-plant media. Presence of sucrose in donor plant medium promoted root number but did not affect rooting percentage of E. saligna. A positive effect of glucose on cutting rhizogenesis was found if this hexose was supplied during the root induction phase, followed by sucrose in the root formation step, especially for E. globulus. The same effect was not seen with fructose. The beneficial effect of glucose in the induction phase on root number was also evident under suboptimal auxin concentrations.     

Hormone and sugar effects on rice sucrose transporter <Emphasis Type="Italic">OsSUT1</Emphasis> expression in germinating embryos

The rice (Oryza sativa L.) OsSUT1 gene encodes a sucrose transporter protein. OsSUT1 was suggested to contribute to phloem loading of sucrose. OsSUT1 expression is highly induced in embryos after seeds were imbibed in water and peaked at 2 days after imbibition, but mRNA levels decline gradually afterwards. In this study, we demonstrated that phytohormones and sugars regulate OsSUT1 expression. Antagonism of abscisic acid and gibberellic acid appeared to play an important role in regulating OsSUT1 expression during embryo germination. In addition, our data showed a glucose and sucrose effect on OsSUT1 expression that represented a bi-phase process. Initially, glucose and sucrose functioned as negative regulators of OsSUT1 expression in germinating embryos after a 1-day treatment; however, when the treatment duration was extended to 5 days, OsSUT1 expression was significantly enhanced. Therefore, we hypothesized that the glucose and sucrose effect might occur in combination with other side effects, such as changes in hormone content or catabolism. Based on the effects that sugar analogs have on OsSUT1 expression, we suggest that the signal transduction for regulating glucose-responsive OsSUT1 expression in embryos occurs via a hexokinase-mediated pathway.     

Changes in concentrations of soluble carbohydrates during germination of <Emphasis Type="Italic">Amaranthus caudatus</Emphasis> L. seeds in relation to ethylene,gibberellin A<Subscript>3</Subscript> and methyl jasmonate

Unimbibed Amaranthus caudatus seeds were found to contain stachyose, raffinose, verbascose, sucrose, galactinol, myo-inositol, glucose and fructose, while no galactose, maltose and maltotriose was detected. During imbibition, seed concentrations of verbascose, stachyose, raffinose, galactinol, myo-inositol (temporary) and fructose (transient) were observed to decrease; concentrations of galactose and maltose remained fairly constant, while those of sucrose, glucose and maltotriose increased, the increase in sucrose concentration was only temporary. Effects of gibberellin A₃ (GA₃) at 3 × 10⁻⁴ M and ethephon at 3 × 10⁻⁴ M alone or in the presence of methyl jasmonate (Me-JA) at 10⁻³ M on concentrations of soluble sugars during germination of A. caudatus seeds were examined. Me-JA was found to inhibit seed germination and fresh weight of the seeds, but did not affect sucrose, myo-inositol, galactose and maltose concentrations during imbibition for up to 20 h. The exogenously applied GA₃ was observed to enhance germination, stachyose breakdown and glucose concentration after 20 h of incubation. Ethephon stimulated seed germination as well as utilisation of stachyose, galactinol (both after 14 and 20 h) and raffinose (after 14 h of incubation). Although the stimulatory effect of either GA₃ or ethephon on seed germination was blocked by Me-JA; these stimulators increased mobilisation of raffinose and stachyose, but only ethephon enhanced both glucose and fructose after 14 and/or 20 h of incubation in the presence of Me-JA. The maltose concentration was increased by both GA₃ and ethephon alone and in the presence of Me-JA. Of the growth regulators studied, ethephon alone and/or in combination with Me-JA significantly increased the concentrations of glucose, fructose, galactose, maltose and maltotriose. The differences in sugar metabolism appear to be linked to ethylene or GA₃ applied simultaneously with Me-JA.     

Different effects of galactose and mannose on cell proliferation and intracellular soluble sugar levels in <Emphasis Type="Italic">Vigna angularis</Emphasis> suspension cultures

Plant cells utilize various sugars as carbon sources for growth, respiration and biosynthesis of cellular components. Suspension-cultured cells of azuki bean (Vigna angularis) proliferated actively in liquid growth medium containing 1% (w/v) sucrose, glucose, fructose, arabinose or xylose, but did not proliferate in medium containing galactose or mannose. These two latter sugars thus appeared distinct from other sugars used as growth substrates. Galactose strongly inhibited cell growth even in the presence of sucrose but mannose did not, suggesting a substantial difference in their effects on cell metabolism. Analysis of intracellular soluble-sugar fractions revealed that galactose, but not mannose, caused a conspicuous decrease in the cellular level of sucrose with no apparent effects on the levels of glucose or fructose. Such a galactose-specific decrease in sucrose levels also occurred in cells that had been cultured together with glucose in place of sucrose, suggesting that galactose inhibits the biosynthesis, rather than uptake, of sucrose in the cells. By contrast, mannose seemed to be metabolically inert in the presence of sucrose. From these results, we conclude that sucrose metabolism is important for the heterotrophic growth of cells in plant suspension-cultures.     

Phosphate starvation responses are mediated by sugar signaling in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Phosphate (Pi) is one of the least available plant nutrients in soils. It is associated with dynamic changes in carbon fluxes and several crucial processes that regulate plant growth and development. Pi levels regulate the expression of large number of genes including those involved in photosynthesis and carbon metabolism. Herein we show that sugar is required for Pi starvation responses including changes in root architecture and expression of phosphate starvation induced (PSI) genes in Arabidopsis. Active photosynthesis or the supplementation of sugar in the medium was essential for the expression of PSI genes under Pi limiting conditions. Expression of these genes was not only induced by sucrose but also detected, albeit at reduced levels, with other metabolizable sugars. Non-metabolizable sugar analogs did not induce the expression of PSI genes. Although sugar input appears to be downstream of initial Pi sensing, it is absolutely required for the completion of the PSI signaling pathway. Altered expression of PSI genes in the hexokinase signaling mutant gin2 indicates that hexokinase-dependent signaling is involved in this process. The study provides evidence for requirement of sugars in PSI signaling and evokes a role for hexokinase in some components of Pi response mechanism.     

Monosaccharides promote flowering in <Emphasis Type="Italic">Kniphofia leucocephala</Emphasis> in vitro

An investigation into the role of carbohydrates in flowering of the endangered species Kniphofia leucocephala Baijnath. (Asphodelaceae) in vitro revealed that a carbohydrate source is essential for the induction of inflorescences. Both the concentration and type of sugar influenced the percentage of flowering, with 60 g l⁻¹ fructose and 10 μM N⁶-benzyladenine (BA) inducing the best flowering response. A high percentage of flowering was also observed with 60 g l⁻¹ glucose and 10 μM BA. The optimal concentration of sucrose for flower induction was 30 g l⁻¹, beyond which the flowering percentage declined, but could be partially restored by increasing the BA concentration. Although overall plant growth declined at high sugar concentrations, there does not appear to be any significant correlations between various growth parameters and flowering percentage, suggesting that sugars play a direct role in floral transition in vitro.     

Effects of sugars and growth regulators on <Emphasis Type="Italic">in vitro</Emphasis> growth of <Emphasis Type="Italic">Dactylorhiza</Emphasis> species

The influence of sugars and growth regulators on shoot and root growth of Dactylorhiza species was studied under in vitro conditions. The seedling development was stimulated with the application of glucose and sucrose at concentration of 10 g dm⁻³ each. The improvement of shoot growth rate and shoot length was enhanced by cytokinins N ⁶-(2-isopentenyl)adenine or N ⁶-benzyladenine and their combination with auxin indolebutyric acid (IBA). The root growth rate and root length of seedlings increased in the presence of IBA and α-naphthaleneacetic acid. Individual Dactylorhiza species showed statistically significant differences in shoot and root development depending on sugar and growth regulator combinations.     

Influence of the carbon source on growth and rosmarinic acid production in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei were characterized with respect to growth and rosmarinic acid formation in media with different sugars and various sugar concentrations. Sucrose is the sugar with the highest stimulating effect on growth and rosmarinic acid accumulation, followed by glucose and fructose. The sugar alcohol mannitol cannot be metabolized by the plant cells. Sucrose is cleaved into glucose and fructose by the Coleus cells. Sucrose concentrations from 1 to 5% have an increasing positive effect on growth and rosmarinic acid synthesis in the cell cultures with a maximum rosmarinic acid content of 12% of the dry weight in medium with 5% sucrose; in medium with 6% sucrose rosmarinic acid accumulation obviously did not reach its highest level in the culture period of 14 days. A very high yield of rosmarinic acid (2 mg ml^-1 suspension) could also be achieved by maintaining a sucrose concentration of 2% during the whole culture period. The start of rosmarinic acid synthesis by the cell cultures seems to be regulated by the growth limitation when a nutrient, e.g. phosphate is depleted from the medium. The rate of rosmarinic acid accumulation is related to the amount of carbon left in the medium when growth ceases.Abbreviations RA rosmarinic acid     

Enhanced Biotransformation Capacity of <Emphasis Type="Italic">Rhodiola rosea</Emphasis> Callus Cultures for Glycosid Production

Rhodiola rosea is a promising medicinal plant that produces various glycosides. Recently we developed a successful method for cultivating it in liquid cultures of compact callus aggregates. In a previous study we reported the successful production of the glycosides of R. rosea by biotransformation of cinnamyl alcohol and tyrosol. In the present study we investigated the possibility of further increasing the yields of the biotransformation products by addition of glucose to the culture medium aside from sucrose, which was used earlier as carbon source. Surprisingly, glucose addition doubled the yield of cinnamyl alcohol glycosides. Rosavin was not produced at all when only sucrose was used. When glucose was added the accumulation dynamics of rosin and a recently described derivative glycoside (designed as compound 321) were similar. Both increased during the first days and then remained constant, while other glycoside compounds increased continuously throughout the cultivation. Rosavin reached its maximum concentration after nine days. In contrast to the beneficial effect on cinnamyl alcohol related glycosides the addition of glucose did not affect the accumulation of the tyrosol derivative salidroside.     

An enclosed-chamber labeling system for the safe <Superscript>14</Superscript>C-enrichment of phytochemicals in plant cell suspension cultures

Summary Various plant secondary products have been implicated in the promotion of good health or the prevention of disease in humans, but little is known about the way they are absorbed in the gut, or in which tissues they are deposited throughout the body. While these issues could be studied if the phytochemicals were isotopically labeled, generating labeled molecules often is problematic because many compounds of interest can be synthesized only in planta at present. In order to generale ¹⁴C-labeled phytochemicals of high radioactive enrichment, we developed an enclosed-chamber labeling system in which cell suspension cultures can be safely and efficiently grown when supplied with ¹⁴C-enriched precursors. The system is designed to hold culture flasks within a clear, polyacrylic compartment that is affixed to the top of a rotary shaker. The flow-through gas exchange nature of the system allows for O₂ replenishment and complete capture of respired ¹⁴CO₂ throughout the entire period of cell culture. Air is circulated internally with the aid of a small fan, and chamber air temperature is monitored continuously with an internal temperature probe and data logger. Production runs of 12–14 d with Vaccinium pahalae (ohelo berry) and Vitis vinifera (grape) suspension cultures, using [¹⁴C]sucrose as the carbon source, demonstrated a 20–23% efficiency of ¹⁴C incorporation into the flavonoid-rich fractions. Further studies with ohelo cell cultures showed that flavonoids were produced with either sucrose or glucose as the carbohydrate source, although flavonoid productivity (measured as anthocyanins) was higher with sucrose. This comprehensive chamber system should have broad applicability with numerous cell types and can be used to generate a wide array of labeled phytochemicals.     

Identification,cloning and characterization of <Emphasis Type="Italic">sis7</Emphasis> and <Emphasis Type="Italic">sis10</Emphasis> sugar-insensitive mutants of <Emphasis Type="Italic">Arabidopsis</Emphasis>

Background  

The levels of soluble sugars, such as glucose and sucrose, help regulate many plant metabolic, physiological and developmental processes. Genetic screens are helping identify some of the loci involved in plant sugar response and reveal extensive cross-talk between sugar and phytohormone response pathways.     

Evaluating potential sugar food sources from the olive grove agroecosystems for Prays oleae parasitoid Chelonus elaeaphilus

Chelonus elaeaphilus Silvestri (Hymenoptera: Braconidae) is a host-specific parasitoid of the olive moth, Prays oleae (Bernard), that can cause parasitism rates of up to 80% in Mediterranean olive groves. A laboratory study was carried out to assess the potential of sugars provided by wild plant species in olive grove agroecosystem to enhance the fitness of C. elaeaphilus. Insects were reared in a climate-controlled chamber at 25?±?2°C, 60?±?5% relative humidity (RH) with a photoperiod of 16:8 (L:D) h. Five naturally occurring wild plant nectar sugars (sucrose, fructose, glucose, maltose and mannose) were tested for their effect on insect longevity. The nectar sugar content of sucrose, fructose and glucose in 12 selected olive grove agroecosystem plant species was analysed and categorised on the basis of sugar ratios. Female insect longevity was increased when they were fed on both sucrose and glucose compared to either maltose or fructose, suggesting that sucrose-dominant nectars would bene?t this parasitoid. Sucrose was predominant in the nectar of five of the studied plant species (Silene gallica, Borago officinalis, Echium plantagineum, Lavandula stoechas and Lonicera hispânica). The results are discussed in terms of potential enhancement of the biological control of P. oleae.     

Expression of the <Emphasis Type="BoldItalic">Escherichia coli pntAB</Emphasis> genes encoding a membrane-bound transhydrogenase in <Emphasis Type="BoldItalic">Corynebacterium glutamicum</Emphasis> improves <Emphasis Type="SmallCaps">l</Emphasis>-lysine formation

A critical factor in the biotechnological production of l-lysine with Corynebacterium glutamicum is the sufficient supply of NADPH. The membrane-integral nicotinamide nucleotide transhydrogenase PntAB of Escherichia coli can use the electrochemical proton gradient across the cytoplasmic membrane to drive the reduction of NADP⁺ via the oxidation of NADH. As C. glutamicum does not possess such an enzyme, we expressed the E. coli pntAB genes in the genetically defined C. glutamicum lysine-producing strain DM1730, resulting in membrane-associated transhydrogenase activity of 0.7 U/mg protein. When cultivated in minimal medium with 10% (w/v) carbon source, the presence of transhydrogenase slightly reduced glucose consumption, whereas the consumption of fructose, glucose plus fructose, and, in particular, sucrose was stimulated. Biomass was increased by pntAB expression between 10 and 30% on all carbon sources tested. Most importantly, the lysine concentration was increased in the presence of transhydrogenase by ∼10% on glucose, ∼70% on fructose, ∼50% on glucose plus fructose, and even by ∼300% on sucrose. Thus, the presence of a proton-coupled transhydrogenase was shown to be an efficient way to improve lysine production by C. glutamicum. In contrast, pntAB expression had a negative effect on growth and glutamate production of C. glutamicum wild type.     

Expression of a plant expansin is involved in the establishment of root knot nematode parasitism in tomato

A group of plant proteins, expansins, have been identified as wall-loosening factors and as facilitators of cell expansion in vivo. The root knot nematode Meloidogyne javanica establishes a permanent feeding site composed of giant cells surrounded by gall tissue. We used quantitative PCR and in situ localization to demonstrate the induction of a tomato (Lycopersicon esculentum cv. VF36) expansin (LeEXPA5) expression in gall cells adjacent to the nematode feeding cells. To further characterize the biological role of LeEXPA5 we have generated LeEXPA5-antisense transgenic roots. The ability of the nematode to establish a feeding site and complete its life cycle, the average root cell size and the rate of root elongation were determined for the transgenic roots, as well as the level of LeEXPA5 expression in non-infected and nematode-infected roots. Our results demonstrated that a decrease of LeEXPA5 expression reduces the ability of the nematode to complete its life cycle in transgenic roots. We suggest that a plant-originated expansin is necessary for a successful parasitic nematode–plant interaction.     

Regeneration of flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) plants from anther culture and somatic tissue with increased resistance to<Emphasis Type="Italic"> Fusarium oxysporum</Emphasis>

The aim of this study was to establish a protocol for the efficient production of flax plants of microspore origin. The results were compared to those obtained for plants regenerated from somatic explants from hypocotyls, cotyledons, leaves, stems and roots. All the plants obtained during the experiments were regenerated from callus that was grown for periods from a few weeks to a few months before the regeneration was achieved. Anther cultures were less effective in plant regeneration than somatic cell cultures. However, regenerants derived from anther cells showed valuable breeding features, including increased resistance to fungal wilt. The age of the donor plants and the season they grew in had a noticeable effect on their anther callusing and subsequent plant regeneration. Low temperature had a negative effect and dark pre-treatment a positive effect on callusing and plant regeneration. Different media were most effective for callus induction, shoot induction and rooting. For callus induction two carbon sources (2.5% sucrose and 2.5% glucose) were most effective; for shoots, only sucrose at lower concentration (2%) was effective. Rooting was most efficient in 1% sucrose and reduced (50%) mineral concentration in the medium. It was found that the length of in vitro cultivation significantly increases the ploidy and affects such features as regenerant morphological characteristics, petal colour, and resistance to Fusarium oxysporum-induced fungal wilt. The established plant regeneration system provides a basis for the creation of transgenic flax.Abbreviations BAP 6-Benzyl-aminopurine - IAA Indole-3-acetic acid - MS Murashige and Skoog medium - NAA -Naphthalene-acetic acidCommunicated by H. Lörz     

Effect of Different Sugars on Photosynthesis and Chlorophyll Fluorescence in Photoautotrophic Tomato Suspension Cell Cultures

The effects of metabolisable sugars sucrose and glucose along with non-metabolisable isomers of sucrose palatinose and turanose were tested. Rate of oxygen evolution (P), electron transport rate (ETR), and photochemical quenching (q_p) showed substantial decrease after 24 and 48 h by glucose and sucrose treatments, whereas there was no effect on all these parameters by the treatment with palatinose and turanose. Also the F_v/F_m ratio remained constant through the time of studies revealing that the maximal photochemical capacity of the cells was unchanged. Non-photochemical quenching (q_N) showed a decrease compared to the control values by all the treatments. Hence P and Chl fluorescence parameter were affected only by those sugars which are used in the metabolic pathways and not by sugar analogues.     

The importance of root carbohydrate abundance in ammonium uptake

The influence of carbohydrates on ammonium uptake and ammonium transporter (AMT1) expression was investigated in roots of field pea (Pisum arvense) and rutabaga (Brassica napus var. rapifera). Ammonium transport into field pea seedlings diminished markedly following cotyledon removal, which indicated that uptake of ammonium was under control of reserves stored in the cotyledons. Excision of cotyledons decreased also the level of some amino acids, glucose and total reducing sugars in field pea roots. To investigate the importance of the sugar supply for the regulation of ammonium uptake at low external NH ₄ ⁺ level, 1 mM glucose or sucrose was supplied for several hours to the field pea seedlings deprived cotyledons or to intact rutabaga plants. Supply of both sugars resulted in a substantial increase in ammonium uptake by both plant species and enhanced markedly the expression of AMT1 in rutabaga roots. The results indicate that sugars may regulate ammonium transport at the genetic level.