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Honeydew honey is produced by bees from excretions of plant-feeding insects, such as aphids and scale insects. Honeydew on conifers, like fir (Abies alba) or spruce (Picea abies), is produced by different species of the genera Cinara and Physokermes. This means that honeydew honey can stem from different botanical as well as zoological origins, but so far it is not possible to clearly distinguish the different types of honeys. In the attempt to identify distinguishing markers, 19 sugars, 25 amino acids and 9 inorganic ions were quantified in three groups of honeydew honey (fir/Cinara, spruce/Cinara and spruce/Physokermes) with 20 honey samples each. It could be demonstrated that the contents of isomaltose, raffinose, erlose, two undefined oligosaccharides, several amino acids, sulfate, and phosphate differed significantly between the three groups of honey. Furthermore, multivariate analyses resulted in a separation of spruce/Physokermes honey from spruce- or fir/Cinara honey due to its higher contents of phosphate, sulfate, erlose and two undefined oligosaccharides. Moreover, the amino acid composition and the isomaltose as well as the raffinose contents proved useful in the distinction between fir/Cinara and spruce/Cinara honey. In sum, the contents of sugars, amino acids, and inorganic ions in German fir and spruce honeys provide useful information about the botanical and zoological origin of honeydew honeys.

In temperate woody species, carbon transport from source to sink tissues is a striking physiological process, particularly considering seasonal changes. The functions of different tissues can also alternate across the seasons. In this regard, phloem loading and sugar distribution are important aspects of carbon partitioning, and sucrose uptake transporters (SUTs) play a key role in these processes. Therefore, the influence of seasons and different light-dark conditions on the expression of SUTs from 3-year-old Fagus sylvatica L., Quercus robur L. and Picea abies (L.) Karst. trees were analyzed. In addition, tissue-specific sugar and starch contents under these different environmental conditions were determined. Putative SUTs were identified in the gymnosperms (Picea abies, Ginkgo biloba L.), here for the first time, and also in the angiosperms (Q. robur, F. sylvatica). The identified SUT sequences of the different tree species cluster into three types, similar to other SUTs from herbaceous and tree species. Furthermore, the sequences from angiosperm and those from gymnosperm species form distinct clusters within the three types of SUTs. In F. sylvatica, Q. robur and P. abies, the expression levels of the different SUTs during seasons showed marked variations. Because of the high expression levels of type I SUTs in bark, wood and leaves during active growing phases in spring and summer, it can be assumed that they are involved in phloem loading, sucrose retrieval and possibly in further physiological processes. The expression patterns also indicate a flexible expression in all tissues depending on physiological requirements and environmental conditions. Compared with type I SUTs, the seasonal variations of type II SUT expression were less pronounced, whereas the seasonal variations of the type III SUT expression patterns were partly reverse. In addition to the seasonal regulation, the expressions of the different SUTs were also regulated by light in a diurnal manner.

Sucrose concentration in phloem sap was several times higher than in the cytosol of mesophyll cells. The results suggest that phloem loading involves active steps in the analyzed tree species. Phloem loading in source leaves is a key step for carbon partitioning and passive symplastic loading has been proposed for several tree species. However, experimental evidence to prove the potential for sucrose diffusion from mesophyll to phloem is rare. Here, we analyzed three tree species (two angiosperms, Fagus sylvatica, Magnolia kobus, and one gymnosperm, Gnetum gnemon) to investigate the proposed phloem loading mechanism. For this purpose, the minor vein structure and the sugar concentrations in phloem sap as well as in the subcellular compartments of mesophyll cells were investigated. The analyzed tree species belong to the open type minor vein subcategory. The sucrose concentration in the cytosol of mesophyll cells ranged between 75 and 165 mM and was almost equal to the vacuolar concentration. Phloem sap could be collected from F. sylvatica and M. kobus and the concentration of sucrose in phloem sap was about five- and 11-fold higher, respectively, than in the cytosol of mesophyll cells. Sugar exudation of cut leaves was decreased by p-chloromercuribenzenesulfonic acid, an inhibitor of sucrose-proton transporter. The results suggest that phloem loading of sucrose in the analyzed tree species involves active steps, and apoplastic phloem loading seems more likely.

In general, honey bees (Apis mellifera L.) feed on honey produced from collected nectar. In the absence of nectar, during certain times of the year or in monocultural landscapes, honey bees forage on honeydew. Honeydew is excreted by different herbivores of the order Hemiptera that consume phloem sap of plant species. In comparison to nectar, honeydew is composed of a higher variety of sugars and additional sugars with higher molecular weight, like the trisaccharide melezitose that can be a major constituent of honeydew. However, melezitose-containing honey is known to cause malnutrition in overwintering honey bees. Following the hypothesis that melezitose may be the cause for the so called ‘honeydew flow disease’, three independent feeding experiments with caged bees were conducted in consecutive years. Bees fed with melezitose showed increased food uptake, higher gut weights and elevated mortality compared to bees fed a control diet. Moreover, severe disease symptoms, such as swollen abdomen, abdomen tipping and impaired movement were observed in melezitose-fed bees. 16S-amplicon sequencing indicated that the melezitose diet changed the species composition of the lactic acid bacteria community within the gut microbiota. Based on these results, we conclude that melezitose cannot be easily digested by the host and may accumulate in the hindgut. Within cages or during winter, when there is no opportunity for excretion, the accumulated melezitose can cause severe intestinal symptoms and death of the bees, probably as result of poor melezitose metabolism capabilities in the intestinal microbiota. These findings confirm the causal relation between the trisaccharide melezitose and the honeydew flow disease and indicate a possible mechanism of pathogenesis.

Research into the influence of stress factors, such as drought, different temperatures and/or varied light conditions, on plants due to climate changes is becoming increasingly important. Epiphytes, like many species of the Bromeliaceae, are particularly affected by this, but little is known about impacts on nectar composition and nectary metabolism. We investigated the influence of drought, different temperatures and light-dark regimes on nectar and nectaries of the epiphytic bromeliad species, Aechmea fasciata, and also the influence of drought with the terrestrial bromeliad, Billbergia nutans. The content of sugars, amino acids and ions in nectar and nectaries was analysed using HPLC. In addition, the starch content and the activities of different invertases in nectaries were determined. Compositions of nectar and nectaries were hardly influenced, neither by light nor dark, nor by different temperatures. In contrast, drought revealed changes in nectar volumes and nectar sugar compositions in the epiphytic bromeliad as well as in the terrestrial bromeliad. In both species, the sucrose-to-hexose ratio in nectar decreased considerably during the drought period. These changes in nectar sugar composition do not correlate with changes in the nectaries. The total sugar, amino acid and ion concentrations remained constant in nectar as well as in nectaries during the drought period. Changes in nectar composition or in the production of floral pollinator rewards are likely to affect plant-pollinator interactions. It remains questionable how far the adaptations of the bromeliads to drought and diverse light or temperature conditions are still sufficient.

Effects of plants on herbivores can cascade up the food web and modulate the abundance of higher trophic levels. In agro-ecosystems, plant viruses can affect the interactions between crops, crop pests, and natural enemies. Little is known, however, about the effects of viruses on higher trophic levels, including parasitoids and their ability for pest regulation. We tested the hypothesis that a plant virus affects parasitoid foraging behaviour through cascading effects on higher trophic levels. We predicted that the semi-persistent Beet yellows virus (BYV) would influence plant (Beta vulgaris) quality, as well as aphid host (Aphis fabae) quality for a parasitoid Lysiphlebus fabarum. We determined amino acid and sugar content in healthy and infected plants (first trophic level), lipid content and body size of aphids (second trophic level) fed on both plants, as well as foraging behaviour and body size of parasitoids (third trophic level) that developed on aphids fed on both plants. Our results showed that virus infection increased sugars and decreased total amino acid content in B. vulgaris. We further observed an increase in aphid size without modification in host aphid quality (i.e., lipid content), and a slight effect on parasitoid behaviour through an increased number of antennal contacts with host aphids. Although the BYV virus clearly affected the first two trophic levels, it did not affect development or emergence of parasitoids. As the parasitoid L. fabarum does not seem to be affected by the virus, we discuss the possibility of using it for the development of targeted biological control against aphids.

Nectar composition varies between species, depending on flowering time and pollinator type, among others. Various models of the biochemical and molecular mechanisms underlying nectar production and secretion have been proposed. To gain insights into these mechanisms, day- and night-flowering tobacco (Nicotiana) species with high or low proportions of hexoses in the nectar were analyzed. Nectar and nectaries were simultaneously collected, throughout the day and night. Soluble sugars and starch were determined and the activity and expression level of cell wall invertase (CW-INVs) were measured in nectaries. Nectaries and nectar of the five Nicotiana species contained different amounts of sucrose, glucose, and fructose. CW-INV activity was detected in the nectaries of all Nicotiana species and is probably involved in the hydrolysis of sucrose in the nectary tissue and during nectar secretion. The larger differences in the sucrose-to-hexose-ratio between nectaries and nectar in diurnal species compared to nocturnal species can be explained by higher sucrose cleavage within the nectaries in night-flowering species, and during secretion in day-flowering species. However, cell wall invertase alone cannot be responsible for the differences in sugar concentrations. Within the nectaries of the Nicotiana species, a portion of the sugars is transiently stored as starch. In general, night-flowering species showed higher starch contents in the nectaries compared to day-flowering species. Moreover, in night flowering species, the starch content decreased during the first half of the dark period, when nectar production peaks. The sucrose concentrations in the cytoplasm of nectarial cells were extrapolated from nectary sucrose contents. In day-flowering species, the sucrose concentration in the nectary cytoplasm was about twice as high as in nectar, whereas in night-flowering species the situation was the opposite, which implies different secretion mechanisms. The secreted nectar sugars remained stable for the complete flower opening period, which indicates that post-secretory modification is unlikely. On the basis of these results, we present an adapted model of the mechanisms underlying the secretion of nectar sugars in day- and night-flowering Nicotiana.

Several hemipteran species feed on the phloem sap of plants and produce large amounts of honeydew that is collected by bees to produce honeydew honey. Therefore, it is important to know whether it is predominantly the hemipteran species or the host plant to influence the honeydew composition. This is particularly relevant for those botanical and zoological species from which the majority of honeydew honey originates. To investigate this issue, honeydew from two Cinara species located on Abies alba as well as from two Cinara and two Physokermes species located on Picea abies were collected. Phloem exudates of the host plants were also analyzed. Honeydew of all species contained different proportions of hexoses, sucrose, melezitose, erlose, and further di- and trisaccharides, whereas the phloem exudates of the host trees contained no trisaccharides. Moreover, the proportions of sugars differed significantly between hemipteran species feeding on the same tree species. Sucrose hydrolysis and oligosaccharide formation was shown in whole-body homogenates of aphids. The type of the produced oligosaccharides in the aphid-extracts correlated with the oligosaccharide composition in the honeydew of the different aphid species. The total contents of amino acids and inorganic ions in the honeydew were much lower than the sugar content. Glutamine and glutamate were predominant amino acids in the honeydew of all six hemipteran species and also in the phloem exudates of both tree species. Potassium was the dominant inorganic ion in all honeydew samples and also in the phloem exudate. Statistical analyses reveal that the sugar composition of honeydew is determined more by the hemipteran species than by the host plant. Consequently, it can be assumed that the sugar composition of honeydew honey is also more influenced by the hemipteran species than by the host tree.

Floral nectar contains mainly sugars but also amino acids, organic acids, inorganic ions and secondary compounds to attract pollinators. The genus Nicotiana exhibits great diversity among species in floral morphology, flowering time, nectar compositions, and predominant pollinators. We studied nectar samples of 20 Nicotiana species, composed equally of day- and night-flowering plants and attracting different groups of pollinators (e.g. hummingbirds, moths or bats) to investigate whether sugars, amino acids, organic acids and inorganic ions are influenced by pollinator preferences. Glucose, fructose and sucrose were the only sugars found in the nectar of all examined species. Sugar concentration of the nectar of day-flowering species was 20% higher and amino acid concentration was 2-3-fold higher compared to the nectar of night-flowering species. The sucrose-to-hexose ratio was significantly higher in night-flowering species and the relative share of sucrose based on the total sugar correlated with the flower tube length in the nocturnal species. Flowers of different tobacco species contained varying volumes of nectar which led to about 150-fold higher amounts of total sugar per flower in bat- or sunbird-pollinated species than in bee-pollinated or autogamous species. This difference was even higher for total amino acids per flower (up to 1000-fold). As a consequence, some Nicotiana species invest large amounts of organic nitrogen for certain pollinators. Higher concentrations of inorganic ions, predominantly anions, were found in nectar of night-flowering species. Therefore, higher anion concentrations were also associated with pollinator types active at night. Malate, the main organic acid, was present in all nectar samples but the concentration was not correlated with pollinator type. In conclusion, statistical analyses revealed that pollinator types have a stronger effect on nectar composition than phylogenetic relations. In this context, nectar sugars and amino acids are more strongly correlated with the preferences of predominant pollinators than organic acids and inorganic ions.

Ectomycorrhizal (EM) fungal taxonomic, phylogenetic, and trait diversity (exploration types) were analyzed in beech and conifer forests along a north-to-south gradient in three biogeographic regions in Germany. The taxonomic community structures of the ectomycorrhizal assemblages in top soil were influenced by stand density and forest type, by biogeographic environmental factors (soil physical properties, temperature, and precipitation), and by nitrogen forms (amino acids, ammonium, and nitrate). While α-diversity did not differ between forest types, β-diversity increased, leading to higher γ-diversity on the landscape level when both forest types were present. The highest taxonomic diversity of EM was found in forests in cool, moist climate on clay and silty soils and the lowest in the forests in warm, dry climate on sandy soils. In the region with higher taxonomic diversity, phylogenetic clustering was found, but not trait clustering. In the warm region, trait clustering occurred despite neutral phylogenetic effects. These results suggest that different forest types and favorable environmental conditions in forests promote high EM species richness in top soil presumably with both high functional diversity and phylogenetic redundancy, while stressful environmental conditions lead to lower species richness and functional redundancy.

A first comprehensive dataset of nectar sugar composition and concentration in Bromeliaceae is presented, covering 111 species belonging to all three subfamilies. Based on this dataset, we examined the relationship between nectar traits and pollination syndromes in the family. Sugars in samples were assayed by high pressure liquid chromatography. All sampled species were grouped into three broad categories (trochilophilous, chiropterophilous, or lepidopterophilous) according to their main pollination mode. Significant differences between the different pollination syndromes were found in nectar sugar composition as well as concentration. For a total of four genera (Guzmania, Pitcairnia, Tillandsia and Vriesea), a comparison of nectar composition showed significant differences between trochilophilous and chiropterophilous species. Data presented here indicate that the characteristics of nectar in Bromeliaceae are predominantly determined by putative adaptations of nectar sugars to preferences of the pollinators rather than by phylogenetic relations.

Nectar composition and concentration were analyzed for 75 samples of 70 species of Acanthaceae representing all major intrafamiliar groups. Analyses of variance were conducted to test for significant differences between pollination syndromes and between taxonomical or phylogenetic groups (∼genera). The available data indicate that the characteristics of nectar in Acanthaceae are predominantly determined by adaptation to needs of the pollinators rather than by phylogenetic constraints.

The investigation and improvement of nitrogen efficiency in oilseed rape (Brassica napus L.) are important issues in rapeseed breeding. The objective of this study was to modify ammonium assimilation in transgenic rapeseed plants through the expression of the Escherichia coli asparagine synthetase (AsnA, E.C. 6.3.1.1) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and to study its influence on amino acid composition in leaves and on seed traits related to nitrogen efficiency. In regenerated transgenic plants, the 37kDa AsnA protein was detected by Western blot analysis, but was lacking in untransformed control plants of cv. Drakkar. In the transformants, in vitro asparagine synthetase activities ranged from 105 to 185 nmol asparagine mg -1 protein h -1 , whereas, in untransformed control plants, only negligible asparagine synthetase activities of up to 5 nmol asparagine mg -1 protein h -1 were found. Despite these significant activities, no changes in the amino acid composition in the leaves or in the phloem of transgenic plants were detectable. In a pot experiment, two transgenic lines expressing the prokaryotic asparagine synthetase clearly performed inferiorly to control plants at limiting nitrogen (N) fertilizer supply. Although the seed N content was increased, the seed yield and the seed N yield were reduced, which was interpreted as an increased nitrate assimilation leading, at limiting N supply, to a reduced seed yield and seed N yield. At high N fertilizer supply, the differences were less pronounced for one transgenic line, whereas the other showed a higher seed N yield and an improved nitrogen harvest index. The results show that the expression of the E. coil asnA gene in oilseed rape could be of advantage at high N supply, but not at limiting N fertilizer supply.

SUMMARY The obligately biotrophic rust fungi are dependent on nutrient supply from their host plants. A cDNA library of infection structures of the rust fungus Uromyces fabae was used to identify a gene (UfAAT3) that encodes a protein with a high degree of sequence similarity to fungal amino acid permeases. The expression profile revealed by RT-PCR shows an up-regulation very early during rust development, with the highest level in haustoria and infected leaves. Heterologous expression of UfAAT3p in Xenopus oocytes revealed an amino acid permease energized by co-transport with protons and exhibiting a broad substrate specificity. Compared to the previously described U. fabae amino acid transporter (AAT1), which represented the highest transport activities for lysine and histidine, electrophysiological measurements with cRNA of UfAAT3-injected oocytes showed substrate preferences for leucine and the sulphur containing amino acids methionine and cysteine. The unique contribution of the amino acid permeases and their substrate affinities might be connected with the availability of amino acids in the leaf tissue. Thus, in order to compare the substrate profiles of AAT1p and UfAAT3p with the natural environment of U. fabae we analysed the amino acid concentration in the apoplastic space, in addition to that in extracts of Vicia faba leaves. The predominant free amino acids were asparagine, alanine, glutamine and glutamate. However, most amino acids were present at low concentrations (between 0.02 and 0.16 mm), including the preferred substrates of the U. fabae amino acid permeases AAT1p and UfAAT3p.

A sucrose (Suc) transporter cDNA has been cloned from Alonsoa meridionalis, a member of the Scrophulariaceae. This plant species has an open minor vein configuration and translocates mainly raffinose and stachyose in addition to Suc in the phloem (C. Knop, O. Voitsekhovskaja, G. Lohaus [2001] Planta 213: 80–91). These are typical properties of symplastic phloem loaders. For functional characterization, AmSUT1 cDNA was expressed in bakers' yeast (Saccharomyces cerevisiae). Substrate and inhibitor specificities, energy dependence, and K m value of the protein agree well with the properties measured for other Suc transporters of apoplastic phloem loaders. A polyclonal antiserum against the 17 N-terminal amino acids of the A. meridionalis Suc transporter AmSUT1 was used to determine the cellular localization of the AmSUT1 protein. Using fluorescence labeling on sections from A. meridionalis leaves and stems, AmSUT1 was localized exclusively in phloem cells. Further histological characterization identified these cells as companion cells and sieve elements. p-Chloromercuribenzenesulfonic acid affected the sugar exudation of cut leaves in such a way that the exudation rates of Suc and hexoses decreased, whereas those of raffinose and stachyose increased. The data presented indicate that phloem loading of Suc and retrieval of Suc in A. meridionalis are at least partly mediated by the activity of AmSUT1 in addition to symplastic phloem loading.

In order to study differences between sugar transport in oligosaccharide-translocating and sucrose-translocating species, two members of the Scrophulariaceae, Asarina barclaiana Pennell and Alonsoa meridionalis O. Kuntze, were analysed regarding minor-vein anatomy, sugar concentrations in leaves and phloem sap, and expression of sucrose transporters. The minor veins of Asarina barclaiana possess mainly transfer cells and modified intermediary cells and those of Alonsoa meridionalis have intermediary cells and ordinary companion cells. Phloem sap from these plants was collected by the laser-aphid-stylet technique. The main carbon transport forms in Asarina were sucrose and in Alonsoa raffinose and stachyose. The sum of the carbohydrate concentrations in the phloem sap of both species was as high as that in apoplastic phloem loaders. In Asarina the ratio of the sucrose concentration in the phloem to that in the cytosol of source cells was about 35 and the corresponding ratio in Alonsoa was about two. Sucrose transporter cDNAs were isolated from leaves of both species. By means of semi-quantitative reverse transcription-polymerase chain reaction, sucrose transporter mRNA was detected in different organs and also in the phloem sap. This is the first time that sucrose transporters have been found in oligosaccharide-translocating species and that the mRNA of these sucrose transporters has been localized directly in the phloem sap. Taken together, our observations indicate that Asarina is an apoplastic phloem loader, while the results for Alonsoa are ambiguous: some properties are typical of the symplastic phloem-loading mechanism, but probably a sucrose transporter is involved in loading and/or retrieval of sucrose into the phloem.

In order to investigate the relationship between the amino acid concentration in the phloem sap of leaves and the protein content in seeds, two Brassica napus genotypes and one B. carinata genotype with low, medium and high seed protein contents were analyzed. Phloem sap was collected from the B. napus winter rapeseed breeding line DSV15 with 19% protein of dry weight in the seeds, the spring cultivar 'Duplo' with 25% protein in the seeds and from the B. carinata line BRA1151/90 with 39% protein in the seeds by using the aphid-stylet technique. The total amino acid contents measured in the phloem varied considerably among the three genotypes analysed, and correlated positively with their respective seed protein contents. The total amino acid-to-sucrose ratio was lowest in B. napus line DSV15 which had the lowest seed protein content and highest in the B. carinata line BRA1151/90 which had the highest seed protein content. The amino-N translocation in the phloem during the light period was about 2-fold higher in the B. carinata line BRA1151/90 than in the B. napus lines Dulpo and DSV15. Predominant amino acids in the phloem were glutamine and glutamate, followed by serine, aspartate, and threonine. The amino acid patterns in the leaves resembled those in the phloem, although their absolute concentrations were higher in the phloem than in the cytosol of mesophyll tissue. Furthermore, the concentration gradient of amino acids between the cytosol of mesophyll cells and the phloem was higher in the B. carinata line BRA1151/90 than in the B. napus lines Duplo and DSV15. These results lead to the conclusion that the phloem translocation of amino-N and the phloem loading process of amino acids are decisive factors for the protein content in the seeds of Brassica species.

Strategies for avoiding ion accumulation in leaves of plants grown at high concentration of NaCl (100 mol m(-3)) in the rooting media, i.e. retranslocation via the phloem and leaching from the leaf surface, were quantified for fully developed leaves of maize plants cultivated hydroponically with or without salt, and with or without sprinkling (to induce leaching). Phloem sap, apoplastic fluid, xylem sap, solutes from leaf and root tissues, and the leachate were analysed for carbohydrates, amino acids, malate, and inorganic ions. In spite of a reduced growth rate Na(+) and Cl(-) concentrations in the leaf apoplast remained relatively low (about 4-5 mol m(-3)) under salt treatment. Concentrations of Na(+) and Cl(-) in the phloem sap of salt-treated maize did not exceed 12 and 32 mol m(-3), respectively, and thus remained lower than described for other species. However, phloem transport rates of these ions were higher than reported for other species. The relatively high translocation rate of ions found in maize may be due to the higher carbon translocation rate observed for C(4) plants as opposed to C(3) plants. Approximately 13-36% of the Na(+) and Cl(-) imported into the leaves through the xylem were exported by the phloem. It is concluded that phloem transport plays an important role in controlling the NaCl content of the leaf in maize. Surprisingly, leaching by artificial rain did not affect plant growth. Ion concentrations in the leachate were lower than reported for other plants but increased with NaCl treatment.

Infection with phloem limited Abutilon Mosaic Virus caused localized carbohydrate accumulation (high levels of starch, sucrose, and hexoses) in leaves of Abutilon striatum during early symptom development. In mature leaves with attenuated symptoms, tissues showing faint vein-clearing had markedly higher carbohydrate contents than uniformly green areas of the same leaf. A similar pattern of carbohydrate accumulation was found in pale-green mosaics in mature leaves with overt symptoms when compared to green-islands of the same leaf, but overnight carbohydrate loses were comparable to controls. Because leaves with attenuated symptoms showed no further symptom development whereas the pale-green mosaics became yellow and eventually necrotic in leaves with overt symptoms, it seems unlikely that carbohydrate accumulation following impaired translocation was responsible for symptom expression. High carbohydrate status in leaves with attenuated symptoms had little effect on nonphotochemical quenching during early stages of photosynthetic induction. In leaves with overt symptoms, areas of high carbohydrate status with pale-green mosaics showed markedly slower nonphotochemical quenching. Early symptom areas of young leaves, and advanced symptom areas of mature leaves had low starch contents but were otherwise similar to controls in carbohydrate status. Impaired nonphotochemical quenching in these tissues tended to reflect the state of symptom development, rather than carbohydrate status. Plants with overt symptoms grew about half as fast as plants with attenuated symptoms.

In order to understand the metabolic processes governing the protein content in maize grains, we compared metabolic parameters of the two maize strains Illinois High Protein (IHP) and Illinois Low Protein (ILP), which differ largely in their relative starch and protein content. The activity of nitrate reductase (NR) and the transcription level of asparagine synthetase, two enzymes that play a central role in the flow of N into organic compounds, were also investigated. It was shown that IHP plants contained a higher activity of NR than ILP plants, which was in part due to an increased level of NR activity in the roots. The root-to-shoot ratios in the ILP and IHP strains were 0.41 and 0.54, respectively. The total amino nitrogen contained in the amino acids of the leaves, phloem, xylem and grain was much higher in IHP plants than in ILP plants. The enhanced contents of amino nitrogen in IHP plants were mainly due to an increase in the asparagine level, which was 13 times higher in the xylem of IHP plants than in that of ILP plants. In the roots the asparagine synthetase was expressed constitutively. We conclude from these results that IHP maize differs from ILP maize in having a high capacity for delivering asparagine as a product of root metabolism in addition to the amino acids supplied by nitrate assimilation of the leaves.

Background: Floral nectar contains sugars and amino acids to attract pollinators. In addition, nectar also contains different secondary compounds, but little is understood about their origin or function. Does nectar composition reflect phloem composition, or is nectar synthesized and/or modified in nectaries? Studies where both, the nectar as well as the phloem sap taken from the same plant species were analyzed in parallel are rare. Therefore, phloem sap and nectar from different plant species (Maurandya barclayana, Lophospermum erubescens, and Brassica napus) were compared. Methodology and Principal Findings: Nectar was collected with microcapillary tubes and phloem sap with the laser-aphidstylet technique. The nectar of all three plant species contained high amounts of sugars with different percentages of glucose, fructose, and sucrose, whereas phloem sap sugars consisted almost exclusively of sucrose. One possible reason for this could be the activity of invertases in the nectaries. The total concentration of amino acids was much lower in nectars than in phloem sap, indicating selective retention of nitrogenous solutes during nectar formation. Nectar amino acid concentrations were negatively correlated with the nectar volumes per flower of the different plant species. Both members of the tribe Antirrhineae (Plantaginaceae) M. barclayana and L. erubescens synthesized the iridoid glycoside antirrhinoside. High amounts of antirrhinoside were found in the phloem sap and lower amounts in the nectar of both plant species. Conclusions/Significance: The parallel analyses of nectar and phloem sap have shown that all metabolites which were found in nectar were also detectable in phloem sap with the exception of hexoses. Otherwise, the composition of both aqueous solutions was not the same. The concentration of several metabolites was lower in nectar than in phloem sap indicating selective retention of some metabolites. Furthermore, the existence of antirrhinoside in nectar could be based on passive secretion from the phloem. peerReviewed

The interactions between the assimilation and transport of nitrogen and carbon were investigated in barley and spinach leaves. Both plants were fumigated with NH 3 (1 mg m -3 ) and the content of amino acids, sucrose and carbon intermediates of amino acid metabolism were analysed in the leaves, apoplast and phloem sap. The following changes took place in the C- and N-metabolism of barley leaves during 5 h of fumigation with NH 3 . (a) The contents of amino acids, especially glutamine, largely increased and the contents of sucrose, 2-oxoglutarate, phosphoenolpyruvate, and glycerate-3-phosphate declined. (b) A decrease in the phophoenolpyruvate content was accompanied by an increased activity of phosphoenolpyruvate carboxylase. (c) The altered cytosolic concentrations of amino acids and sucrose during NH 3 fumigation correlated with similar changes in the apoplast and phloem sap. The altered percentage of each amino acid relative to the total amino acid concentration in the cytosol, caused by NH 3 fumigation, is reflected in the apoplast and the phloem sap. The results indicate that the concentrations of amino acids in the cytosol determine their concentrations in the phloem.

The concentrations of sucrose, amino acids, nitrate and malate in the apoplastic compartment of illuminated leaves of barley and spinach were determined and compared with the corresponding concentrations in the cytosolic compartment of mesophyll cells and in the phloem sap, as measured previously with plants grown under identical conditions. The concentrations of sucrose and amino acids in the apoplast are found to be much lower than in the cytosol and in the phloem sap, indicating that not only the uptake into the phloem of sucrose, but also of amino acids, requires transport against a concentration gradient. The gradient of sucrose and amino acids between the cytosol and the apoplast was maintained when phloem transport had been blocked by cold girdling. Apparently, the efflux of sucrose and amino acids from the source cells to the apoplast is regulated in such a way that it meets the requirements of phloem transport. The percentages of the single amino acids as part of the total amino acids are quite similar in the cytosol, apoplast and phloem sap. The ratio of sucrose to the total amino acids in the cytosol is similar to that in the apoplast but about five times higher in the phloem sap. It appears from these results that the preferential extraction of sucrose over amino acids from the source cells to the phloem is due to the uptake from the apoplast into the phloem.

Very few principles have been unraveled that explain the relationship between soil properties and soil biota across large spatial scales and different land-use types. Here, we seek these general relationships using data from 52 differently managed grassland and forest soils in three study regions spanning a latitudinal gradient in Germany. We hypothesize that, after extraction of variation that is explained by location and land-use type, soil properties still explain significant proportions of variation in the abundance and diversity of soil biota. If the relationships between predictors and soil organisms were analyzed individually for each predictor group, soil properties explained the highest amount of variation in soil biota abundance and diversity, followed by land-use type and sampling location. After extraction of variation that originated from location or land-use, abiotic soil properties explained significant amounts of variation in fungal, meso- and macrofauna, but not in yeast or bacterial biomass or diversity. Nitrate or nitrogen concentration and fungal biomass were positively related, but nitrate concentration was negatively related to the abundances of Collembola and mites and to the myriapod species richness across a range of forest and grassland soils. The species richness of earthworms was positively correlated with clay content of soils independent of sample location and land-use type. Our study indicates that after accounting for heterogeneity resulting from large scale differences among sampling locations and land-use types, soil properties still explain significant proportions of variation in fungal and soil fauna abundance or diversity. However, soil biota was also related to processes that act at larger spatial scales and bacteria or soil yeasts only showed weak relationships to soil properties. We therefore argue that more general relationships between soil properties and soil biota can only be derived from future studies that consider larger spatial scales and different land-use types. peerReviewed

Summary Malate is a characteristic metabolite in the photosynthesis of C4 and CAM plants. Furthermore, changes in the intracellular concentration of this organic acid provide part of the osmotic motor for guard cells. Since alterations in the malate concentration influence the photosynthetic capacity on one side and stomatal action on the other, it was studied whether the extracellular malate level represents an indicator of changes in the ambient CO2 concentration and a key regulator of ion transport in guard cells. Here it is demonstrated that alterations in the ambient CO2 level modify the extracellular malate concentration of Vicia faba leaves. Elevated external malate caused stomatal closure in a concentration-dependent manner (Kmmal = 0.3 mM). Slight variations in the external malate concentration strongly regulate the voltage-dependent properties of GCAC1, an anion-release channel in the plasma membrane of guard cells. Superfusion of guard cell protoplasts with malate levels in the physiological range (Kmmal = 0.4 mM) caused the voltage gate to shift towards the resting potential of the cell-activating GCAC1. Single-channel conductance was dependent on the extracellular chloride concentration (KmCl = 3 mM). In the absence of extracellular chloride the plasma membrane lacked anion conductance until the addition of malate induced channel opening. Isophthalate was a powerful agonist in both malate-induced processes, channel regulation and stomatal closure, indicating that modulation of GCAC1 is a key step in stomatal action. It was thus concluded that feedback regulation of volume and turgor with respect to the ambient CO2 concentration via malate-sensitive anion channels may provide a CO2 sensor to guard cells.

Amino acid and sucrose contents were analyzed in the chloroplastic, cytosolic, and vacuolar compartments and in the phloem sap of illuminated spinach leaves (Spinacia oleracea L.). The determination of subcellular metabolite distribution was carried out by nonaqueous fractionation of frozen and lyophilized leaf material using a novel three-compartment calculation method. The phloem sap was collected by aphid stylets which had been severed by a laser beam. Subcellular analysis revealed that the amino acids found in leaves are located mainly in the chloroplast stroma and in the cytosol, the sum of their concentrations amounting to 151 and 121 millimolar, respectively, whereas the amino acid concentrations in the vacuole are one order of magnitude lower. The amino acid concentrations in the phloem sap are found to be not very different from the cytosolic concentrations, whereas the sieve tube concentration of sucrose is found to be one order of magnitude higher than in the cytosol. It is concluded that the phloem loading results in a preferential extraction of sucrose from the source cells.

Sugar and sugar alcohol concentrations were analyzed in subcellular compartments of mesophyll cells, in the apoplast, and in the phloem sap of leaves of Plantago major (common plantain), Plantago maritima (sea plantain), Prunus persica (peach) and Apium graveolens (celery). In addition to sucrose, common plantain, sea plantain, and peach also translocated substantial amounts of sorbitol, whereas celery translocated mannitol as well. Sucrose was always present in vacuole and cytosol of mesophyll cells, whereas sorbitol and mannitol were found in vacuole, stroma, and cytosol in all cases except for sea plantain. The concentration of sorbitol, mannitol and sucrose in phloem sap was 2- to 40-fold higher than that in the cytosol of mesophyll cells. Apoplastic carbohydrate concentrations in all species tested were in the low millimolar range versus high millimolar concentrations in symplastic compartments. Therefore, the concentration ratios between the apoplast and the phloem were very strong, ranging between 20- to 100-fold for sorbitol and mannitol, and between 200- and 2000-fold for sucrose. The woody species, peach, showed the smallest concentration ratios between the cytosol of mesophyll cells and the phloem as well as between the apoplast and the phloem, suggesting a mixture of apoplastic and symplastic phloem loading, in contrast to the herbal plant species (common plantain, sea plantain, celery) which likely exhibit an active loading mode for sorbitol and mannitol as well as sucrose from the apoplast into the phloem. peerReviewed

To determine the driving forces for symplastic sugar flux between mesophyll and phloem, gradients of sugar concentrations and osmotic pressure were studied in leaf tissues of two Scrophulariaceae species, Alonsoa meridionalis and Asarina barclaiana. A. meridionalis has a typical symplastic configuration of minor-vein phloem, i.e. intermediary companion cells with highly developed plasmodesmal connections to bundle-sheath cells. In A. barclaiana, two types of companion cells, modified intermediary cells and transfer cells, were found in minor-vein phloem, giving this species the potential to have a complex phloem-loading mode. We identified all phloem-transported carbohydrates in both species and analyzed the levels of carbohydrates in chloroplasts, vacuoles, and cytoplasm of mesophyll cells by nonaqueous fractionation. Osmotic pressure was measured in single epidermal and mesophyll cells and in whole leaves and compared with calculated values for phloem sap. In A. meridionalis, a 2-fold concentration gradient for sucrose between mesophyll and phloem was found. In A. barclaiana, the major transported carbohydrates, sucrose and antirrhinoside, were present in the phloem in 22- and 6-fold higher concentrations, respectively, than in the cytoplasm of mesophyll cells. The data show that diffusion of sugars along their concentration gradients is unlikely to be the major mechanism for symplastic phloem loading if this were to occur in these species. We conclude that in both A. meridionalis and A. barclaiana, apoplastic phloem loading is an indispensable mechanism and that symplastic entrance of solutes into the phloem may occur by mass flow. The conditions favoring symplastic mass flow into the phloem are discussed.

We have identified and characterized a novel NADP+-dependent D-arabitol dehydrogenase and the corresponding gene from the rust fungus Uromyces fabae, a biotrophic plant pathogen on broad bean (Vicia faba). The new enzyme was termed ARD1p (D-arabitol dehydrogenase 1). It recognizes D-arabitol and mannitol as substrates in the forward reaction, and D-xylulose, D-ribulose and D-fructose as substrates in the reverse reaction. Co-factor specificity was restricted to NADP(H). Kinetic data for the major substrates and co-factors are presented. A detailed analysis of the organization and expression pattern of the ARD1 gene are also given. Immunocytological data indicate a localization of the gene product predominantly in haustoria, the feeding structures of these fungi. Analyses of metabolite levels during pathogenesis indicate that the D-arabitol concentration rises dramatically as infection progresses, and D-arabitol was shown in an in vitro system to be capable of quenching reactive oxygen species involved in host plant defence reactions. ARD1p may therefore play an important role in carbohydrate metabolism and in establishing and/or maintaining the biotrophic interaction in U. fabae.

Non-aqueous fractionation of frozen, lyophilized leaves and morphometric analysis were used to determine metabolite concentrations in the cytosolic, chloroplastic and vacuolar fraction of leaves. In barley, potato, spinach and tobacco the concentrations of amino acids and sucrose are very high in the cytosol, but low in the vacuole. In contrast, hexoses are almost exclusively located in the vacuole in these species. The concentration of metabolites in the apoplast was measured in the solution obtained following vacuum infiltration of leaves. To complement these measurements, the concentrations of sucrose and amino acids in the phloem sap were determined in samples of exudate obtained from the severed stylets of aphids feeding on the plants. Comparison of the concentrations of sucrose and amino acids in the cytosol, the apoplast and the phloem sap suggests that the efflux of these compounds from the cytosol to the apoplast is restricted, and that sucrose is transported into the sieve tubes in preference to amino acids. Differences in the phloem concentrations of sucrose and amino acids in sugar beet and fodder beet correlate with differences in the composition of the storage products in the tap roots of these two cultivars. This suggests that phloem loading may have a major influence on the quality of harvest products accumulated by non-photosynthetic storage tissues in plants.

Rates of CO2 fixation during the light period and the rates of CO2 release during the night period were measured using mature leaves from 39- to 49-d-old spinach (Spinacia oleracea L., US Hybrid 424; grown in 9 h light, 15 h darkness, daily) and mature leaves from 21-d-old barley (Hordeum vulgare L., cv. Apex; grown in 14 h light, 10 h darkness, daily). At certain times during the light and dark periods leaves were harvested for assay of their contents of soluble carbohydrates, starch, malate and the various amino acids. Evaluation of the results of these measurements shows that in spinach and barley leaves 46% and 26%, respectively, of the carbon assimilated during the light period is deposited in the leaves for export during the night period. Taking into account the carbon consumption in the source leaves by dark respiration, it is evaluated that rates of assimilate export during the light period from spinach and barley leaves [38 and 42 μatom C · (mg Chl)−1 · h−1] are reduced in the dark period to 16 μatom C · (mg Chl)−1 · h−1 in both species. The calculated C/N ratios of the photoassimilates exported during the dark period were 0.029 and 0.015 for spinach and barley leaves, respectively.

A comparison of barley (Hordeum vulgare L.) leaves was made between the cytosolic content of amino acids and sucrose as determined by subcellular fractionation and the corresponding concentration in phloem sap, which was collected continuously for up to 6 days from severed aphid stylets. Because amino acids were found to be almost absent from the vacuoles, and because the amino acid patterns in the stroma and cytosol are similar, whole leaf contents could be taken as a measure of cytosolic amino acid levels for a comparison of data during a diurnal cycle. The results show that the pattern of amino acids in the phloem sap was very similar to the pattern in the cytosol. Therefore, we concluded that the overall process of transfer of amino acids from the cytosol of the source cells into the sieve tubes, although carrier mediated, may be a passive process and that the translocation of amino acids via the sieve tubes requires the mass flow of sucrose driven by the active sucrose transport involved by the phloem loading.