On the mechanisms of nectar secretion: revisited

Background and Scope

Models of nectar formation and exudation in multilayered nectaries with modified stomata or permeable cuticle are evaluated. In the current symplasmic model the pre-nectar moves from terminal phloem through the symplasm into the apoplasm (cell walls and intercellular spaces) with nectar formation by either granulocrine or eccrine secretion and its diffusion outwards. It is concluded, however, that no secretory granules are actually produced by the endoplasmic reticulum, and that secretory Golgi vesicles are not involved in the transport of nectar sugar. Therefore, the concept of granulocrine secretion of nectar should be discarded. The specific function of the endomembrane system in nectary cells remains unknown. According to the apoplasmic model, the pre-nectar moves from the terminal phloem in the apoplasm and, on the way, is transformed from phloem sap into nectar. However, viewed ultrastructurally, the unloading (terminal) phloem of nectaries appears to be less active than that of the leaf minor veins, and is therefore not actively involved in the secretion of pre-nectar components into the apoplasm. This invalidates the apoplasmic model. Neither model provides an explanation for the origin of the driving force for nectar discharge.

Proposal

A new model is proposed in which nectar moves by a pressure-driven mass flow in the nectary apoplasm while pre-nectar sugars diffuse from the sieve tubes through the symplasm to the secretory cells, where nectar is formed and sugars cross the plasma membrane by active transport (‘eccrine secretion’). The pressure originates as the result of water influx in the apoplasm from the symplasm along the sugar concentration gradient. It follows from this model that there can be no combinations of apoplasmic and symplasmic pre-nectar movements. The mass-flow mechanism of nectar exudation appears to be universal and applicable to all nectaries irrespective of their type, morphology and anatomy, presence or absence of modified stomata, and their own vascular system.     

Holocrine secretion and cytoplasmic content of Helleborus foetidus L. (Ranunculaceae) nectar

We used electron microscopy to investigate the fine structure of nectary secretions of Helleborus foetidus. During the secretion period, epidermal cells of nectaries discharge the whole contents of the cytoplasm into the nectary cavity. The external wall of the cell breaks, releasing the cytoplasm as a dense aggregate that later disperses in the nectary cavity. Cell components, such as chromatin, plastids, mitochondria, lipid droplets and membranes, were found in the nectar of H. foetidus, evincing the complex nature of the secreted material. These results confirm that nectar secretion in H. foetidus is of the holocrine type.     

Flowers respond to pollinator sound within minutes by increasing nectar sugar concentration

Can plants sense natural airborne sounds and respond to them rapidly? We show that Oenothera drummondii flowers, exposed to playback sound of a flying bee or to synthetic sound signals at similar frequencies, produce sweeter nectar within 3 min, potentially increasing the chances of cross pollination. We found that the flowers vibrated mechanically in response to these sounds, suggesting a plausible mechanism where the flower serves as an auditory sensory organ. Both the vibration and the nectar response were frequency‐specific: the flowers responded and vibrated to pollinator sounds, but not to higher frequency sound. Our results document for the first time that plants can rapidly respond to pollinator sounds in an ecologically relevant way. Potential implications include plant resource allocation, the evolution of flower shape and the evolution of pollinators sound. Finally, our results suggest that plants may be affected by other sounds as well, including anthropogenic ones.     

Changes in floral nectar are unlikely adaptive responses to pollinator flight sound

Under noiseless experimental conditions, sugar concentration of secreted floral nectar may increase after flower exposure to nearby sounds of pollinator flight (Veits et al. 2019). However, we reject the argument that this represents adaptive plant behaviour, and consider that the appealing analogy between a flower and human ear is unjustified.     

A regulatory invertase from sugar cane leaf-sheaths

A soluble β-fructofuranosidase was isolated from sugar cane leaf-sheaths. The enzyme attacks sucrose with an activation energy of 5700 cal/mol above 30° and 17 000 cal/mol below 30°. The enzyme was inhibited by the reaction products. Glucose is a simple non-competitive inhibitor, but fructose is a competitive inhibitor. Kinetic studies using double reciprocal plots and replots of 1/K_i, slope vs inhibitor concentration showed that fructose binds to two interacting sites of the enzyme. Per cent residual activity plotted against inhibitor concentration, and Hill plots confirmed the regulatory properties of the invertase. n was found to be close to 2, the number of binding sites established with the double reciprocal method. The tissue and cellular levels of sucrose, fructose and glucose were measured. Fructose was found at inhibitory concentrations confirming that the activity of the enzyme is probably modulated by the hexose pool of the leaf-sheaths.     

Four o'clock pollination biology: nectaries,nectar and flower visitors in Nyctaginaceae from southern South America

Floral nectary structure and nectar sugar composition were investigated in relation to other floral traits and flower visitors in contrasting species of Nyctaginaceae from southern South America, representing four tribes (Bougainvilleeae, Colignonieae, Nyctagineae, Pisoneae). Our comparative data will aid in the understanding of plant–pollinator interactions and in the development of hypotheses on the origin of floral and reproductive characters in this family. The nectaries are located on the inner side of the staminal tube. The nectariferous tissue is composed of an epidermis and three to ten layers of secretory parenchymal cells, supplied indirectly by the filament vascular bundles. Stomata appear to be associated with nectar secretion. For the first time in Nyctaginaceae, nectary ultrastructure is described in Boerhavia diffusa var. leiocarpa. Nectary parenchyma cells are densely cytoplasmic and contain numerous starch grains. Plasmodesmata connect the nectariferous cells. Flowers of Nyctaginaceae secrete a small volume of nectar of variable concentration (10–47%). Nectar is dominated by hexoses, but Mirabilis jalapa showed a balanced proportion of sucrose and hexoses. Hymenoptera are the most common visitors for most species; nocturnal Lepidoptera are the most common visitors for M. jalapa and Bougainvillea stipitata. We found relatively low variation in the nectary characteristics of Nyctaginaceae compared with broad variation in flower structure, shape, colour and nectar traits. © 2013 The Linnean Society of London     

Honeybees prefer warmer nectar and less viscous nectar,regardless of sugar concentration

The internal temperature of flowers may be higher than air temperature, and warmer nectar could offer energetic advantages for honeybee thermoregulation, as well as being easier to drink owing to its lower viscosity. We investigated the responses of Apis mellifera scutellata (10 colonies) to warmed 10% w/w sucrose solutions, maintained at 20–35°C, independent of low air temperatures, and to 20% w/w sucrose solutions with the viscosity increased by the addition of the inert polysaccharide Tylose (up to the equivalent of 34.5% sucrose). Honeybee crop loads increased with nectar temperature, as did the total consumption of sucrose solutions over 2 h by all bees visiting the feeders. In addition, the preference of marked honeybees shifted towards higher nectar temperatures with successive feeder visits. Crop loads were inversely proportional to the viscosity of the artificial nectar, as was the total consumption of sucrose solutions over 2 h. Marked honeybees avoided higher nectar viscosities with successive feeder visits. Bees thus showed strong preferences for both warmer and less viscous nectar, independent of changes in its sugar concentration. Bees may benefit from foraging on nectars that are warmer than air temperature for two reasons that are not mutually exclusive: reduced thermoregulatory costs and faster ingestion times due to the lower viscosity.     

Inter- and intra-floral heterogeneity of nectar production in Helleborus foetidus L. (Ranunculaceae)

Three southern Spanish populations of Helleborusfoetidus L. (Ranunculaceae) were sampled for nectar content in the absence of nectarivorous flower visitors. Nectar volume was measured in individual nectaries of flowers at the same stage in the anthesis cycle. Total nectar content per flower was extremely variable between plants within populations and between flowers within plants, but much less so between populations. Average sugar content per flower was roughly similar in the three populations sampled. High variances in nectar abundance occurred also among nectaries within the same flower. Heterogeneity in pollinator rewards thus occurs simultaneously at several spatial scales, namely between nectaries, between flowers and between plants. This implies a strong component of uncertainty to foraging pollinators, which may therefore tend to avoid Helleborus flowers.     

Reproductive biology, variability of nectar features and pollination of Combretum fruticosum (Combretaceae) in Argentina

Flower morphology, nectary structure, nectar features (chemical composition, secretion pattern, standing crop, removal effects) and flower visitors are analysed in an Argentinian population of Combretum fruticosum. The variability of these data was examined throughout the flower lifetime. Nectar is hexose dominant. Its chemical composition and concentration are constant for all flowering stageS. Nectar volume varies as a function of flower age due to a combination of nectar secretion, cessation and resorption periods. The overall sugar production is decreased by nectar removal. The plant is self-incompatible and xenogamouS. Only 16.2% of the flowers set seedS. Inflorescences with green flowers were exclusively visited by two hummingbird and three perching bird species which transfer the pollen. A clear link was observed among nectar production pattern, standing crop of nectar, and visitors' behaviour.     

Influence of light,dark, temperature and drought on metabolite and ion composition in nectar and nectaries of an epiphytic bromeliad species (Aechmea fasciata)

• 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.

     

Sugar and amino acid composition of ant‐attended nectar and honeydew sources from an Australian rainforest

Abstract Ants (Hymenoptera: Formicidae) consume a broad spectrum of liquid food sources including nectar and honeydew, which play a key role in their diet especially in tropical forests. This study compares carbohydrates and amino acids from a representative spectrum of liquid sources used by ants in the canopy and understorey of a tropical rainforest in northern Queensland, Australia. Eighteen floral nectars, 16 extrafloral nectars, two wound sap and four homopteran honeydew sources were analysed using high performance liquid chromatography. Wounds comprised flower abscission scars on Normanbya normanbyi L. H. Bailey and bitemarks on Cardwellia sublimis F. Muell. where ants were actively involved in wounding. Discriminant analysis was performed to model differences between food sources in sugar and amino acid concentration and composition. All characteristics varied significantly among plant species. Honeydew contained a broader spectrum of sugars (including melezitose, raffinose, melibiose, lactose and maltose) than nectar (sucrose, glucose, fructose), but certain extrafloral nectars had similar amino acid profiles and, like honeydew sources, were often monopolized by ants. Most common amino acids across the sources were proline, alanine and threonine among 17 α‐amino acids identified. Interspecific variability concealed characteristic differences in sugar and amino acid parameters between nectar, honeydew and wound sap across all plants, but these types differed significantly when found on the same plant. Among all sources studied, only a few flower nectars were naturally not consumed by ants and they were significantly less attended than sugar controls in feeding trials. These nectars did not differ in sugars and amino acids from ant‐attended flower nectars, suggesting the activity of repellents. Apart from these exceptions, variability in amino acids and carbohydrates is proposed to play a key role in ant preferences and nutrition.     

Pollinator limitation and inbreeding depression in orchid species with and without nectar rewards

Many orchids produce no nectar rewards. Foraging pollinators should visit more flowers per inflorescence in species with nectar, which could increase geitonogamous self-fertilization. If a history of selfing decreases genetic load, then nectar-producing orchids should harbour lower inbreeding depression than nectarless species. Here, I tested this hypothesis by quantifying inbreeding depression and pollinator limitation in populations of three closely related orchid species, one of which provides nectar. I also compared inbreeding depression for nectarless and nectar-producing species of orchids using published studies. All field populations expressed pollinator limitation, but the nectar-providing species was intermediate to the two nectarless species. All populations expressed inbreeding depression, and levels increased in later life-history stages. There was no tendency for nectarless species to express higher inbreeding depression either in experiments or published studies. Nectarless orchids may not express higher levels of inbreeding depression because pollinators fail to visit more flowers in nectar-bearing species, because such visitations do not result in greater selfing, and/or because higher selfing may be ineffective in purging the mutations that cause load.     

Nectary and gender‐biased nectar production in dichogamous Chamaenerion angustifolium (L.) Scop. (Onagraceae)

In dichogamous plants, nectar characteristics (i.e. nectar amount and its composition) can differ between sexual phases. In the present study, we investigated the structural organization of the floral nectary, nectar production and carbohydrate composition in the protandrous Chamaenerion angustifolium (L.) Scop. (Onagraceae). The receptacular nectary consisted of an epidermis with numerous nectarostomata, several layers of photosynthetic secretory parenchyma, and subsecretory parenchyma. Nectariferous tissue was not directly vascularized and starch grains were rarely observed in the secretory cells, occurring exclusively in the guard cells of modified stomata. The nectar was released via nectarostomata. The floral nectar was hexose rich (32.8/39.1/28.1% glucose/fructose/sucrose) and the total concentration was constant throughout the anthesis (47% on average). However, contrasting patterns in nectar amount and carbohydrate composition between the floral sexual phases were observed. On average, female‐phased flowers produced 1.4‐fold more nectar than male‐phased flowers, and although the nectar was sucrose rich during the male phase, it was hexose rich during the female phase, suggesting sucrose hydrolysis.     

Micro-organisms behind the pollination scenes: microbial imprint on floral nectar sugar variation in a tropical plant community

Background and Aims

Variation in the composition of floral nectar reflects intrinsic plant characteristics as well as the action of extrinsic factors. Micro-organisms, particularly yeasts, represent one extrinsic factor that inhabit the nectar of animal-pollinated flowers worldwide. In this study a ‘microbial imprint hypothesis’ is formulated and tested, in which it is proposed that natural community-wide variation in nectar sugar composition will partly depend on the presence of yeasts in flowers.

Methods

Occurrence and density of yeasts were studied microscopically in single-flower nectar samples of 22 animal-pollinated species from coastal xeric and sub-humid tropical habitats of the Yucatán Peninsula, Mexico. Nectar sugar concentration and composition were concurrently determined on the same samples using high-performance liquid chromatography (HPLC) methods.

Key Results

Microscopical examination of nectar samples revealed the presence of yeasts in nearly all plant species (21 out of 22 species) and in about half of the samples examined (51·8 % of total, all species combined). Plant species and individuals differed significantly in nectar sugar concentration and composition, and also in the incidence of nectar yeasts. After statistically controlling for differences between plant species and individuals, nectar yeasts still accounted for a significant fraction of community-wide variance in all nectar sugar parameters considered. Significant yeast × species interactions on sugar parameters revealed that plant species differed in the nectar sugar correlates of variation in yeast incidence.

Conclusions

The results support the hypothesis that nectar yeasts impose a detectable imprint on community-wide variation in nectar sugar composition and concentration. Since nectar sugar features influence pollinator attraction and plant reproduction, future nectar studies should control for yeast presence and examine the extent to which microbial signatures on nectar characteristics ultimately have some influence on pollination services in plant communities.     

Heterologous expression of the apple hexose transporter MdHT2.2 altered sugar concentration with increasing cell wall invertase activity in tomato fruit

Sugar transporters are necessary to transfer hexose from cell wall spaces into parenchyma cells to boost hexose accumulation to high concentrations in fruit. Here, we have identified an apple hexose transporter (HTs), MdHT2.2, located in the plasma membrane, which is highly expressed in mature fruit. In a yeast system, the MdHT2.2 protein exhibited high ¹⁴C‐fructose and ¹⁴C‐glucose transport activity. In transgenic tomato heterologously expressing MdHT2.2, the levels of both fructose and glucose increased significantly in mature fruit, with sugar being unloaded via the apoplastic pathway, but the level of sucrose decreased significantly. Analysis of enzyme activity and the expression of genes related to sugar metabolism and transport revealed greatly up‐regulated expression of SlLIN5, a key gene encoding cell wall invertase (CWINV), as well as increased CWINV activity in tomatoes transformed with MdHT2.2. Moreover, the levels of fructose, glucose and sucrose recovered nearly to those of the wild type in the sllin5‐edited mutant of the MdHT2.2‐expressing lines. However, the overexpression of MdHT2.2 decreased hexose levels and increased sucrose levels in mature leaves and young fruit, suggesting that the response pathway for the apoplastic hexose signal differs among tomato tissues. The present study identifies a new HTs in apple that is able to take up fructose and glucose into cells and confirms that the apoplastic hexose levels regulated by HT controls CWINV activity to alter carbohydrate partitioning and sugar content.     

Comparative study of stability of soluble and cell wall invertase from Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae is the most significant source of enzyme invertase. It is mainly used in the food industry as a soluble or immobilized enzyme. The greatest amount of invertase is located in the periplasmic space in yeast. In this work, it was isolated into two forms of enzyme from yeast S. cerevisiae cell, soluble and cell wall invertase (CWI). Both forms of enzyme showed same temperature optimum (60°C), similar pH optimum, and kinetic parameters. The significant difference between these biocatalysts was observed in their thermal stability, stability in urea and methanol solution. At 60°C, CWI had 1.7 times longer half-life than soluble enzyme, while at 70°C CWI showed 8.7 times longer half-life than soluble enzyme. After 2-hr of incubation in 8?M urea solution, soluble invertase and CWI retained 10 and 60% of its initial activity, respectively. During 22?hr of incubation of both enzymes in 30 and 40% methanol, soluble invertase was completely inactivated, while CWI changed its activity within the experimental error. Therefore, soluble invertase and CWI have not shown any substantial difference, but CWI showed better thermal stability and stability in some of the typical protein-denaturing agents.