Bottom-up regulation of a tritrophic system by Beet yellows virus infection: consequences for aphid-parasitoid foraging behaviour and development.

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.

Ratio of sugar concentrations in the phloem sap and the cytosol of mesophyll cells in different tree species as an indicator of the phloem loading mechanism.

MAIN CONCLUSION: 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.

Phylogenetic and functional traits of ectomycorrhizal assemblages in top soil from different biogeographic regions and forest types.

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, ß-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.

Subcellular distribution of raffinose oligosaccharides and other metabolites in summer and winter leaves of Ajuga reptans (Lamiaceae).

MAIN CONCLUSION: In Ajuga reptans, raffinose oligosaccharides accumulated during winter. Stachyose, verbascose, and higher RFO oligomers were exclusively found in the vacuole whereas one-fourth of raffinose was localized in the stroma. The evergreen labiate Ajuga reptans L. can grow at low temperature. The carbohydrate metabolism changes during the cold phase, e.g., raffinose family oligosaccharides (RFOs) accumulate. Additionally, A. reptans translocates RFOs in the phloem. In the present study, subcellular concentrations of metabolites were studied in summer and winter leaves of A. reptans to gain further insight into regulatory instances involved in the cold acclimation process and into the function of RFOs. Subcellular metabolite concentrations were determined by non-aqueous fractionation. Volumes of the subcellular compartments of summer and winter leaves were analyzed by morphometric measurements. The metabolite content varied strongly between summer and winter leaves. Soluble metabolites increased up to tenfold during winter whereas the starch content was decreased. In winter leaves, the subcellular distribution showed a shift of carbohydrates from cytoplasm to vacuole and chloroplast. Despite this, the metabolite concentration was higher in all compartments in winter leaves compared to summer leaves because of the much higher total metabolite content in winter leaves. The different oligosaccharides did show different compartmentations. Stachyose, verbascose, and higher RFO oligomers were almost exclusively found in the vacuole whereas one-fourth of raffinose was localized in the stroma. Apparently, the subcellular distribution of the RFOs differs because they fulfill different functions in plant metabolism during winter. Raffinose might function in protecting chloroplast membranes during freezing, whereas higher RFO oligomers may exert protective effects on vacuolar membranes. In addition, the high content of RFOs in winter leaves may also result from reduced consumption of assimilates.

Characterization, localization, and seasonal changes of the sucrose transporter FeSUT1 in the phloem of Fraxinus excelsior.

Trees are generally assumed to be symplastic phloem loaders. A typical feature for most wooden species is an open minor vein structure with symplastic connections between mesophyll cells and phloem cells, which allow sucrose to move cell-to-cell through the plasmodesmata into the phloem. Fraxinus excelsior (Oleaceae) also translocates raffinose family oligosaccharides in addition to sucrose. Sucrose concentration was recently shown to be higher in the phloem sap than in the mesophyll cells. This suggests the involvement of apoplastic steps and the activity of sucrose transporters in addition to symplastic phloem-loading processes. In this study, the sucrose transporter FeSUT1 from F. excelsior was analysed. Heterologous expression in baker's yeast showed that FeSUT1 mediates the uptake of sucrose. Immunohistochemical analyses revealed that FeSUT1 was exclusively located in phloem cells of minor veins and in the transport phloem of F. excelsior. Further characterization identified these cells as sieve elements and possibly ordinary companion cells but not as intermediary cells. The localization and expression pattern point towards functions of FeSUT1 in phloem loading of sucrose as well as in sucrose retrieval. FeSUT1 is most likely responsible for the observed sucrose gradient between mesophyll and phloem. The elevated expression level of FeSUT1 indicated an increased apoplastic carbon export activity from the leaves during spring and late autumn. It is hypothesized that the importance of apoplastic loading is high under low-sucrose conditions and that the availability of two different phloem-loading mechanisms confers advantages for temperate woody species like F. excelsior.

Determinants of Acidobacteria activity inferred from the relative abundances of 16S rRNA transcripts in German grassland and forest soils.

16S rRNA genes and transcripts of Acidobacteria were investigated in 57 grassland and forest soils of three different geographic regions. Acidobacteria contributed 9-31% of bacterial 16S rRNA genes whereas the relative abundances of the respective transcripts were 4-16%. The specific cellular 16S rRNA content (determined as molar ratio of rRNA : rRNA genes) ranged between 3 and 80, indicating a low in situ growth rate. Correlations with flagellate numbers, vascular plant diversity and soil respiration suggest that biotic interactions are important determinants of Acidobacteria 16S rRNA transcript abundances in soils. While the phylogenetic composition of Acidobacteria differed significantly between grassland and forest soils, high throughput denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism fingerprinting detected 16S rRNA transcripts of most phylotypes in situ. Partial least squares regression suggested that chemical soil conditions such as pH, total nitrogen, C : N ratio, ammonia concentrations and total phosphorus affect the composition of this active fraction of Acidobacteria. Transcript abundance for individual Acidobacteria phylotypes was found to correlate with particular physicochemical (pH, temperature, nitrogen or phosphorus) and, most notably, biological parameters (respiration rates, abundances of ciliates or amoebae, vascular plant diversity), providing culture-independent evidence for a distinct niche specialization of different Acidobacteria even from the same subdivision.

Apoplastic and symplastic phloem loading in Quercus robur and Fraxinus excelsior.

Whereas most of the research on phloem loading is performed on herbaceous plants, less is known about phloem loading strategies in trees. In this study, the phloem loading mechanisms of Quercus robur and Fraxinus excelsior were analysed. The following features were examined: the minor vein structure, the sugar concentrations in phloem sap by the laser-aphid-stylet technique, the distribution of photoassimilates in the mesophyll cells by non-aqueous fractionation, gradients of sugar concentrations and osmotic pressure, and the expression of sucrose transporters. The minor vein configurations of Q. robur and F. excelsior belong to the open type. Quercus robur contained companion cells in the minor veins whereas F. excelsior showed intermediary cells in addition to ordinary companion cells. The main carbon transport form in Q. robur was sucrose (~1M). In F. excelsior high amounts of raffinose and stachyose were also transported. However, in both tree species, the osmolality of phloem sap was higher than the osmolality of the mesophyll cells. The concentration gradients between phloem sap and the cytoplasm of mesophyll cells for sucrose were 16-fold and 14-fold for Q. robur and F. excelsior, respectively. Independent of the type of translocated sugars, sucrose transporter cDNAs were cloned from both species. The results indicate that phloem loading of sucrose and other metabolites must involve active loading steps in both tree species. Quercus robur seems to be an apoplastic phloem loader while F. excelsior shows indications of being a symplastic or mixed symplastic-apoplastic phloem loader.

Origin and Function of Amino Acids in Nectar and Nectaries of Pitcairnia Species with Particular Emphasis on Alanine and Glutamine.

Floral nectar contains sugars and numerous other compounds, including amino acids, but little is known about their function and origin in nectar. Therefore, the amino acid, sugar, and inorganic ion concentrations, as well as the activity of alanine aminotransferase (AlaAT) and glutamine synthetase (GS) in nectar, nectaries, and leaves were analyzed in 30 Pitcairnia species. These data were compared with various floral traits, the pollinator type, and the phylogenetic relationships of the species to find possible causes for the high amino acid concentrations in the nectar of some species. The highest concentrations of amino acids (especially alanine) in nectar were found in species with reddish flowers. Furthermore, the concentration of amino acids in nectar and nectaries is determined through analyzing flower color/pollination type rather than phylogenetic relations. This study provides new insights into the origin of amino acids in nectar. The presence of almost all amino acids in nectar is mainly due to their transport in the phloem to the nectaries, with the exception of alanine, which is partially produced in nectaries. In addition, active regulatory mechanisms are required in nectaries that retain most of the amino acids and allow the selective secretion of specific amino acids, such as alanine.

Sugar concentrations and expression of SUTs suggest active phloem loading in tall trees of Fagus sylvatica and Quercus robur.

Phloem loading and sugar distribution are key steps for carbon partitioning in herbaceous and woody species. Although the phloem loading mechanisms in herbs are well studied, less is known for trees. It was shown for saplings of Fagus sylvatica L. and Quercus robur L. that the sucrose concentration in the phloem sap was higher than in the mesophyll cells, which suggests that phloem loading of sucrose involves active steps. However, the question remains whether this also applies for tall trees. To approach this question, tissue-specific sugar and starch contents of small and tall trees of F. sylvatica and Q. robur as well as the sugar concentration in the subcellular compartments of mesophyll cells were examined. Moreover, sucrose uptake transporters (SUTs) were analyzed by heterology expression in yeast and the tissue-specific expressions of SUTs were investigated. Sugar content in leaves of the canopy (11 and 26 m height) was up to 25% higher compared with that of leaves of small trees of F. sylvatica and Q. robur (2 m height). The sucrose concentration in the cytosol of mesophyll cells from tall trees was between 120 and 240 mM and about 4- to 8-fold lower than the sucrose concentration in the phloem sap of saplings. The analyzed SUT sequences of both tree species cluster into three types, similar to SUTs from other plant species. Heterologous expression in yeast confirmed that all analyzed SUTs are functional sucrose transporters. Moreover, all SUTs were expressed in leaves, bark and wood of the canopy and the expression levels in small and tall trees were similar. The results show that the phloem loading in leaves of tall trees of F. sylvatica and Q. robur probably involves active steps, because there is an uphill concentration gradient for sucrose. SUTs may be involved in phloem loading.

Environmental factors affect Acidobacterial communities below the subgroup level in grassland and forest soils.

In soil, Acidobacteria constitute on average 20% of all bacteria, are highly diverse, and are physiologically active in situ. However, their individual functions and interactions with higher taxa in soil are still unknown. Here, potential effects of land use, soil properties, plant diversity, and soil nanofauna on acidobacterial community composition were studied by cultivation-independent methods in grassland and forest soils from three different regions in Germany. The analysis of 16S rRNA gene clone libraries representing all studied soils revealed that grassland soils were dominated by subgroup Gp6 and forest soils by subgroup Gp1 Acidobacteria. The analysis of a large number of sites (n = 57) by 16S rRNA gene fingerprinting methods (terminal restriction fragment length polymorphism [T-RFLP] and denaturing gradient gel electrophoresis [DGGE]) showed that Acidobacteria diversities differed between grassland and forest soils but also among the three different regions. Edaphic properties, such as pH, organic carbon, total nitrogen, C/N ratio, phosphorus, nitrate, ammonium, soil moisture, soil temperature, and soil respiration, had an impact on community composition as assessed by fingerprinting. However, interrelations with environmental parameters among subgroup terminal restriction fragments (T-RFs) differed significantly, e.g., different Gp1 T-RFs correlated positively or negatively with nitrogen content. Novel significant correlations of Acidobacteria subpopulations (i.e., individual populations within subgroups) with soil nanofauna and vascular plant diversity were revealed only by analysis of clone sequences. Thus, for detecting novel interrelations of environmental parameters with Acidobacteria, individual populations within subgroups have to be considered.

Review primary and secondary metabolites in phloem sap collected with aphid stylectomy.

Phloem plays a central role in assimilate transport as well as in the transport of several secondary compounds. In order to study the chemical composition of phloem sap, different methods have been used for its collection, including stem incisions, EDTA-facilitated exudation or aphid stylectomy. Each collection method has several advantages and disadvantages and, unfortunately, the reported metabolite profiles and concentrations depend on the method used for exudate collection. This review therefore primarily focusses on sugars, amino acids, inorganic ions and further transported compounds like organic acids, nucleotides, phytohormons, defense signals, and lipophilic substances in the phloem sap obtained by aphid stylectomy to facilitate comparability of the data.

Comparative analyses of the metabolite and ion concentrations in nectar, nectaries, and leaves of 36 bromeliads with different photosynthesis and pollinator types.

Floral nectar contains mainly sugars as well as smaller amounts of amino acids and further compounds. The nectar composition varies between different plant species and it is related to the pollination type of the plant. In addition to this, other factors can influence the composition. Nectar is produced in and secreted from nectaries. A few models exist to explain the origin of nectar for dicotyl plant species, a complete elucidation of the processes, however, has not yet been achieved. This is particularly true for monocots or plant species with CAM photosynthesis. To get closer to such an elucidation, nectar, nectaries, and leaves of 36 bromeliad species were analyzed for sugars, starch, amino acids, and inorganic ions. The species studied include different photosynthesis types (CAM/C3), different pollination types (trochilophilous/chiropterophilous), or different live forms. The main sugars in nectar and nectaries were glucose, fructose, and sucrose, the total sugar concentration was about twofold higher in nectar than in nectaries, which suggests that sugars are actively transported from the nectaries into the nectar. The composition of amino acids in nectar is already determined in the nectaries, but the concentration is much lower in nectar than in nectaries, which suggests selective retention of amino acids during nectar secretion. The same applies to inorganic ions. Statistical analyses showed that the photosynthesis type and the pollination type can explain more data variation in nectar than in nectaries and leaves. Furthermore, the pollinator type has a stronger influence on the nectar or nectary composition than the photosynthesis type. Trochilophilous C3 plants showed significant correlations between the nitrate concentration in leaves and the amino acid concentration in nectaries and nectar. It can be assumed that the more nitrate is taken up, the more amino acids are synthesized in leaves and transported to the nectaries and nectar. However, chiropterophilous C3 plants show no such correlation, which means that the secretion of amino acids into the nectar is regulated by further factors. The results help understand the physiological properties that influence nectaries and nectar as well as the manner of metabolite and ion secretion from nectaries to nectar.

Sugar loading is not required for phloem sap flow in maize plants.

Phloem transport of photoassimilates from leaves to non-photosynthetic organs, such as the root and shoot apices and reproductive organs, is crucial to plant growth and yield. For nearly 90 years, evidence has been generally consistent with the theory of a pressure-flow mechanism of phloem transport. Central to this hypothesis is the loading of osmolytes, principally sugars, into the phloem to generate the osmotic pressure that propels bulk flow. Here we used genetic and light manipulations to test whether sugar import into the phloem is required as the driving force for phloem sap flow. Using carbon-11 radiotracer, we show that a maize sucrose transporter1 (sut1) loss-of-function mutant has severely reduced export of carbon from photosynthetic leaves (only ~4% of the wild type level). Yet, the mutant remarkably maintains phloem pressure at ~100% and sap flow speeds at ~50-75% of those of wild type. Potassium (K+) abundance in the phloem was elevated in sut1 mutant leaves. Fluid dynamic modelling supports the conclusion that increased K+ loading compensated for decreased sucrose loading to maintain phloem pressure, and thereby maintained phloem transport via the pressure-flow mechanism. Furthermore, these results suggest that sap flow and transport of other phloem-mobile nutrients and signalling molecules could be regulated independently of sugar loading into the phloem, potentially influencing carbon-nutrient homoeostasis and the distribution of signalling molecules in plants encountering different environmental conditions.

Leaf Epidermis: The Ambiguous Symplastic Domain.

The ability to develop secondary (post-cytokinetic) plasmodesmata (PD) is an important evolutionary advantage that helps in creating symplastic domains within the plant body. Developmental regulation of secondary PD formation is not completely understood. In flowering plants, secondary PD occur exclusively between cells from different lineages, e.g., at the L1/L2 interface within shoot apices, or between leaf epidermis (L1-derivative), and mesophyll (L2-derivative). However, the highest numbers of secondary PD occur in the minor veins of leaf between bundle sheath cells and phloem companion cells in a group of plant species designated "symplastic" phloem loaders, as opposed to "apoplastic" loaders. This poses a question of whether secondary PD formation is upregulated in general in symplastic loaders. Distribution of PD in leaves and in shoot apices of two symplastic phloem loaders, Alonsoa meridionalis and Asarina barclaiana, was compared with that in two apoplastic loaders, Solanum tuberosum (potato) and Hordeum vulgare (barley), using immunolabeling of the PD-specific proteins and transmission electron microscopy (TEM), respectively. Single-cell sampling was performed to correlate sugar allocation between leaf epidermis and mesophyll to PD abundance. Although the distribution of PD in the leaf lamina (except within the vascular tissues) and in the meristem layers was similar in all species examined, far fewer PD were found at the epidermis/epidermis and mesophyll/epidermis boundaries in apoplastic loaders compared to symplastic loaders. In the latter, the leaf epidermis accumulated sugar, suggesting sugar import from the mesophyll via PD. Thus, leaf epidermis and mesophyll might represent a single symplastic domain in Alonsoa meridionalis and Asarina barclaiana.

Silencing of Vlaro2 for chorismate synthase revealed that the phytopathogen Verticillium longisporum induces the cross-pathway control in the xylem.

The first leaky auxotrophic mutant for aromatic amino acids of the near-diploid fungal plant pathogen Verticillium longisporum (VL) has been generated. VL enters its host Brassica napus through the roots and colonizes the xylem vessels. The xylem contains little nutrients including low concentrations of amino acids. We isolated the gene Vlaro2 encoding chorismate synthase by complementation of the corresponding yeast mutant strain. Chorismate synthase produces the first branch point intermediate of aromatic amino acid biosynthesis. A novel RNA-mediated gene silencing method reduced gene expression of both isogenes by 80% and resulted in a bradytrophic mutant, which is a leaky auxotroph due to impaired expression of chorismate synthase. In contrast to the wild type, silencing resulted in increased expression of the cross-pathway regulatory gene VlcpcA (similar to cpcA/GCN4) during saprotrophic life. The mutant fungus is still able to infect the host plant B. napus and the model Arabidopsis thaliana with reduced efficiency. VlcpcA expression is increased in planta in the mutant and the wild-type fungus. We assume that xylem colonization requires induction of the cross-pathway control, presumably because the fungus has to overcome imbalanced amino acid supply in the xylem.

Sugar, amino acid and inorganic ion profiling of the honeydew from different hemipteran species feeding on Abies alba and Picea abies.

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.

The trisaccharide melezitose impacts honey bees and their intestinal microbiota.

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.

Evidence for functional heterogeneity of sieve element-companion cell complexes in minor vein phloem of Alonsoa meridionalis.

Two modes of phloem loading have been proposed, apoplastic and symplastic, depending on the structure of sieve element-companion cell complexes (SE-CCCs) in minor vein phloem. Species are usually classified as either apoplastic or symplastic loaders although the cytology of SE-CCCs in minor veins of the majority of plants indicates that both mechanisms can be simultaneously involved in phloem loading. The functions of structurally different SE-CCCs in minor veins of the stachyose-translocating plant Alonsoa meridionalis were examined. A stachyose synthase gene, AmSTS1, was expressed in intermediary cells but not in the ordinary companion cell of the same vein. In contrast, sucrose transporter AmSUT1 protein was present in ordinary companion cells but not in the neighbouring intermediary cells. These data reveal the principles of phloem sap formation in A. meridionalis and, probably, in many other dicots. The two types of SE-CCCs within one and the same minor vein load different carbohydrates, using contrasting mechanisms for their delivery into the phloem. Lateral sieve pores in the minor vein phloem lead to mixing of the carbohydrates soon after loading. While symplastic and apoplastic pathways can function simultaneously during phloem loading, they are separated at the level of different SE-CCCs combined in phloem endings.

Measurement of Subcellular Metabolite Concentrations in Relation to Phloem Loading.

The key step of carbon export from green leaves is the loading of sugars into the phloem. To fully understand and quantify this process, it is essential to know the concentration of sugars in the different compartments of the cells along the phloem loading pathway. However, determining subcellular metabolite concentrations has been technically challenging. This paper describes a technique to measure metabolite levels in the chloroplast, the cytosol, and the vacuole of mesophyll cells with high accuracy. The nonaqueous fractionation (NAF) technique is arguably the method of choice to analyze the subcellular metabolite distributions as it minimizes the risk of metabolite interconversions or redistribution during the process. The principle of NAF is the separation of small subcellular particles, which are obtained by homogenization, lyophilization, and sonication, in a nonaqueous density gradient. Due to the varying composition-dependent density of the fragments, their segregation reflects compartmental distributions throughout the gradient. By determining marker enzymes for chloroplast stroma, cytosol, and vacuole in gradient fractions the proportions of each subcellular compartment in each gradient fraction can be analyzed. The measured distribution of marker enzymes and of metabolites in each fraction of the gradient can be used to calculate the subcellular distribution of the metabolites.

What Do Nectarivorous Bats Like? Nectar Composition in Bromeliaceae With Special Emphasis on Bat-Pollinated Species.

Floral nectar is the most important reward for pollinators and an integral component of the pollination syndrome. Nectar research has mainly focused on sugars or amino acids, whereas more comprehensive studies on the nectar composition of closely related plant species with different pollination types are rather limited. Nectar composition as well as concentrations of sugars, amino acids, inorganic ions, and organic acids were analyzed for 147 species of Bromeliaceae. This plant family shows a high diversity in terms of floral morphology, flowering time, and predominant pollination types (trochilophilous, trochilophilous/entomophilous, psychophilous, sphingophilous, chiropterophilous). Based on the analyses, we examined the relationship between nectar traits and pollination type in this family. Nectar of all analyzed species contained high amounts of sugars with different proportions of glucose, fructose, and sucrose. The total concentrations of amino acids, inorganic cations, and anions, or organic acids were much lower. The analyses revealed that the sugar composition, the concentrations of inorganic cations and anions as well as the concentration of malate in nectar of bat-pollinated species differed significantly from nectar of species with other pollination types. Flowers of bat-pollinated species contained a higher volume of nectar, which results in a total of about 25-fold higher amounts of sugar in bat-pollinated species than in insect-pollinated species. This difference was even higher for amino acids, inorganic anions and cations, and organic acids (between 50 and 100-fold). In general, bat-pollinated plant species invest large amounts of organic and inorganic compounds for their pollinators. Furthermore, statistical analyses reveal that the characteristics of nectar in Bromeliaceae are more strongly determined by the pollinator type rather than by taxonomic groups or phylogenetic relations. However, a considerable part of the variance cannot be explained by either of the variables, which means that additional factors must be responsible for the differences in the nectar composition.