Effect of amino acid enriched diets on hemolymph amino acid composition in honey bees.

Amino acids (AAs) are an abundant class of nectar solutes, and they are involved in the nectar attractiveness to flower visitors. Among the various AAs, proline is the most abundant proteogenic AA, and γ-amino butyric acid (GABA) and ß-alanine are the two most abundant non-proteogenic AAs. These three AAs are known to affect insect physiology, being involved in flight metabolism and neurotransmission. The aim of this study was to investigate the effects of artificial diets enriched with either ß-alanine, GABA, or proline on consumption, survival, and hemolymph composition in honey bees belonging to two different ages and with different metabolism (i.e., newly emerged and foragers). Differences in feed intake among diets were not observed, while a diet enriched with ß-alanine improved the survival rate of newly emerged honey bees compared to the control group. Variations in the hemolymph AA concentrations occurred only in newly emerged honey bees, according to the diet and the time of hemolymph sampling. A greater susceptibility of young honey bees to enriched diets than older honey bees was observed. The variations in the concentrations of hemolymph AAs reflect either the accumulation of dietary AAs or the existence of metabolic pathways that may lead to the conversion of dietary AAs into different ones. This investigation could be an initial contribution to studying the complex dynamics that regulate hemolymph AA composition and its effect on honey bee physiology.

To each their own! Nectar plasticity within a flower mediates distinct ecological interactions.

Nuptial and extranuptial nectaries are involved in interactions with different animal functional groups. Nectar traits involved in pollination mutualisms are well known. However, we know little about those traits involved in other mutualisms, such as ant-plant interactions, especially when both types of nectaries are in the same plant organ, the flower. Here we investigated if when two types of nectaries are exploited by distinct functional groups of floral visitors, even being within the same plant organ, the nectar secreted presents distinct features that fit animal requirements. We compared nectar secretion dynamics, floral visitors and nectar chemical composition of both nuptial and extranuptial nectaries in natural populations of the liana Amphilophium mansoanum (Bignoniaceae). For that we characterized nectar sugar, amino acid and specialized metabolite composition by high-performance liquid chromatography. Nuptial nectaries were visited by three medium- and large-sized bee species and extranuptial nectaries were visited mainly by ants, but also by cockroaches, wasps and flies. Nuptial and extranuptial nectar differed regarding volume, concentration, milligrams of sugars per flower and secretion dynamics. Nuptial nectar was sucrose-dominated, with high amounts of γ-aminobutyric acid and ß-aminobutyric acid and with theophylline-like alkaloid, which were all exclusive of nuptial nectar. Whereas extranuptial nectar was hexose-rich, had a richer and less variable amino acid chemical profile, with high amounts of serine and alanine amino acids and with higher amounts of the specialized metabolite tyramine. The nectar traits from nuptial and extranuptial nectaries differ in energy amount and nutritional value, as well as in neuroactive specialized metabolites. These differences seem to match floral visitors' requirements, since they exclusively consume one of the two nectar types and may be exerting selective pressures on the composition of the respective resources of interest.

Secondary Metabolites in Nectar-Mediated Plant-Pollinator Relationships.

In recent years, our understanding of the complex chemistry of floral nectar and its ecological implications for plant-pollinator relationships has certainly increased. Nectar is no longer considered merely a reward for pollinators but rather a plant interface for complex interactions with insects and other organisms. A particular class of compounds, i.e., nectar secondary compounds (NSCs), has contributed to this new perspective, framing nectar in a more comprehensive ecological context. The aim of this review is to draft an overview of our current knowledge of NSCs, including emerging aspects such as non-protein amino acids and biogenic amines, whose presence in nectar was highlighted quite recently. After considering the implications of the different classes of NSCs in the pollination scenario, we discuss hypotheses regarding the evolution of such complex nectar profiles and provide cues for future research on plant-pollinator relationships.

Composition of Sexual Fluids in Cycas revoluta Ovules During Pollination and Fertilization.

The composition of fluids that mediate fertilization in cycads is described for the first time. Using tandem mass spectrometry, proteomes of two stages of fluid production, megagametophyte fluid and archegonial chamber fluid production, are compared in Cycas revoluta. These were compared with the proteome of another sexual fluid produced by ovules, the pollination drop proteins. Cycad ovules produce complex liquids immediately prior fertilization. Compared with the pollination drops that mainly had few proteins in classes involved in defense and carbohydrate modification, megagametophyte fluid and archegonial chamber fluid had larger proteomes with many more protein classes, e.g. proteins involved in programmed cell death. Using high-performance liquid chromatography, megagametophyte fluid and archegonial chamber fluid were shown to have elevated concentrations of smaller molecular weight molecules including glucose, pectin and glutamic acid. Compared to megagametophyte fluid, archegonial chamber fluid had elevated pH as well as higher osmolality. Supplementary Information: The online version contains supplementary material available at 10.1007/s12229-021-09271-1.

Evolutionary and Ecological Considerations on Nectar-Mediated Tripartite Interactions in Angiosperms and Their Relevance in the Mediterranean Basin.

The Mediterranean basin hosts a high diversity of plants and bees, and it is considered one of the world's biodiversity hotspots. Insect pollination, i.e., pollen transfer from male reproductive structures to conspecific female ones, was classically thought to be a mutualistic relationship that links these two groups of organisms, giving rise to an admirable and complex network of interactions. Although nectar is often involved in mediating these interactions, relatively little is known about modifications in its chemical traits during the evolution of plants. Here, we examine how the current sucrose-dominated floral nectar of most Mediterranean plants could have arisen in the course of evolution of angiosperms. The transition from hexose-rich to sucrose-rich nectar secretion was probably triggered by increasing temperature and aridity during the Cretaceous period, when most angiosperms were radiating. This transition may have opened new ecological niches for new groups of insects that were co-diversifying with angiosperms and for specific nectar-dwelling yeasts that originated later (i.e., Metschnikowiaceae). Our hypothesis embeds recent discoveries in nectar biology, such as the involvement of nectar microbiota and nectar secondary metabolites in shaping interactions with pollinators, and it suggests a complex, multifaceted ecological and evolutionary scenario that we are just beginning to discover.

Effects of Few-Layer Graphene on the Sexual Reproduction of Seed Plants: An In Vivo Study with Cucurbita pepo L.

Products containing graphene-related materials (GRMs) are becoming quite common, raising concerns for environmental safety. GRMs have varying effects on plants, but their impact on the sexual reproduction process is largely unknown. In this study, the effects of few-layer graphene (FLG) and a similarly layered phyllosilicate, muscovite mica (MICA), were tested in vivo on the reproductive structures, i.e., pollen and stigma, of Cucurbita pepo L. ssp. pepo 'greyzini' (summer squash, zucchini). Pollen was exposed to FLG or MICA, after careful physical-chemical characterization, at concentrations of 0.5 and 2 mg of nanomaterial (NM) per g of pollen for up to six hours. Following this, pollen viability was tested. Stigmas were exposed to FLG or MICA for three hours and then analyzed by environmental scanning electron microscopy to verify possible alterations to their surface. Stigmas were then hand-pollinated to verify the effects of the two NMs on pollen adhesion and in vivo pollen germination. FLG and MICA altered neither pollen viability nor the stigmatic surface. However, both NMs equivalently decreased pollen adhesion and in vivo germination compared with untreated stigmas. These effects deserve further attention as they could impact on production of fruits and seeds. Importantly, it was shown that FLG is as safe as a naturally occurring nanomaterial.

Beyond graphene oxide acidity: Novel insights into graphene related materials effects on the sexual reproduction of seed plants.

Graphene related materials (GRMs) are currently being used in products and devices of everyday life and this strongly increases the possibility of their ultimate release into the environment as waste items. GRMs have several effects on plants, and graphene oxide (GO) in particular, can affect pollen germination and tube growth due to its acidic properties. Despite the socio-economic importance of sexual reproduction in seed plants, the effect of GRMs on this process is still largely unknown. Here, Corylus avellana L. (common Hazel) pollen was germinated in-vitro with and without 1-100 µg mL-1 few-layer graphene (FLG), GO and reduced GO (rGO) to identify GRMs effects alternative to the acidification damage caused by GO. At 100 µg mL-1 both FLG and GO decreased pollen germination, however only GO negatively affected pollen tube growth. Furthermore, GO adsorbed about 10 % of the initial Ca2+ from germination media accounting for a further decrease in germination of 13 % at the pH created by GO. In addition, both FLG and GO altered the normal tip-focused reactive oxygen species (ROS) distribution along the pollen tube. The results provided here help to understand GRMs effect on the sexual reproduction of seed plants and to address future in-vivo studies.

Drought Stress Affects the Response of Italian Local Tomato (Solanum lycopersicum L.) Varieties in a Genotype-Dependent Manner.

Drought stress is one of the most severe conditions for plants, especially in the face of the emerging problem of global warming. This issue is important when considering economically relevant crops, including the tomato. For these plants, a promising solution is the valorization of local agrobiodiversity as a source of genetic variability. In this paper we investigated how six Italian tomato varieties react to a prolonged period of water depletion. We used a multidisciplinary approach, from genetics to plant physiology and cytology, to provide a detailed overview of the response of plants to stress. The varieties analyzed, each characterized by a specific genetic profile, showed a genotype-specific response with the variety 'Fragola' being the most resistant and the variety 'Pisanello' the most susceptible. For all the parameters evaluated, 'Fragola' performed in a manner comparable to that of control plants. On the contrary, 'Pisanello' appeared to be more affected and showed an increase in the number of stomata and a drastic increase in antioxidants, a symptom of acute oxidative stress. Our work suggests the existence of a valuable reservoir of genetic biodiversity with more drought-tolerant tomato genotypes opening the way to further exploitation and use of local germplasm in breeding programs.

Nutraceutical Characteristics of Ancient Malus x domestica Borkh. Fruits Recovered across Siena in Tuscany.

Background: A diet rich in fruits and vegetables contributes to lowering the risk of chronic diseases. The fruits of Malus x domestica are a rich dietary source of bioactive compounds, namely vitamins and antioxidants, with recognized action on human health protection. Tuscany is known for its rich plant biodiversity, especially represented by ancient varieties of fruit trees. Particularly noteworthy are the many ancient Tuscan varieties of apple trees. Methods: Sugar quantification via HPLC and spectrophotometric assays to quantify the antioxidant power and total polyphenol content revealed interesting differences in 17 old varieties of Malus x domestica Borkh. recovered in Siena (Tuscany). Results: The quantification of antioxidants, polyphenols, and the main free sugars revealed that their content in the old fruits was often superior to the widespread commercial counterparts ('Red Delicious' and 'Golden Delicious'). Such differences were, in certain cases, dramatic, with 8-fold higher values. Differences were also present for sugars and fibers (pectin). Most ancient fruits displayed low values of glucose and high contents of xylitol and pectin. Conclusions: The results reported here suggest the possible use of ancient apple varieties from Siena for nutraceutical purposes and draw attention to the valorization of local old varieties.

Effects of Non-Protein Amino Acids in Nectar on Bee Survival and Behavior.

Nectar mediates complex interactions between plants and animals. Recent research has focused on nectar secondary compounds that may play a role in regulating some of these interactions. These compounds may affect the behavior of nectar feeders by interacting with their neurobiology. Non-protein amino acids (NPAAs) can constitute a large portion of the amino acid content of floral nectar, but their ecological function has, to date, not been investigated. In this study, we tested the effects of diets with low and high concentrations of γ-amino butyric acid (GABA) and ß-alanine on the survival and behavior of Bombus terrestris and Apis mellifera. The most apparent effect on longevity was observed for B. terrestris workers that fed on high concentration of GABA, with longevity increased. By contrast, neither of the two NPAAs (at either concentration) had an affect on A. mellifera longevity. At the low NPAA concentration, only B. terrestris workers showed a difference in consumption, consuming more ß-alanine solution than the other two solutions. By contrast, at the high NPAA concentration, only A. mellifera workers showed a difference in consumption, consuming more ß-alanine solution. The effects of the NPAAs on behavior differed between the two species, with B. terrestris appearing more sensitive to the NPAAs than A. mellifera. After consuming NPAAs, B. terrestris showed changes in three (walking, flying, stationary) of the four behaviors recorded, although the effects varied with concentration and compound. In contrast, honey bees only showed a change in feeding behavior, with consumption of both NPAAs (at low concentrations) resulting in a decrease. Thus, pollinator intake of NPAAs may have important behavioral/ecological implications.

Spatiotemporal variation in the pollination systems of a supergeneralist plant: is Angelica sylvestris (Apiaceae) locally adapted to its most effective pollinators?

Background and Aims: In terms of pollination systems, umbellifers (plants of the carrot family, Apiaceae) are regarded as generalists, since their (usually dichogamous) flowers are visited by a wide range of insects representing several taxonomic orders. However, recent analyses of insect effectiveness revealed that these plants may be pollinated effectively by a narrow assemblage of insect visitors. Of particular interest was whether populations of an umbellifer species varied in pollinator assemblages and whether this could lead to local specialization of the pollination system. We also explored whether variation in pollinator assemblages was associated with variation in floral traits, and whether this variation influences reproductive output. Methods: The focus was on Angelica sylvestris, a common European species visited by a taxonomically diverse insect assemblage. In three populations, located along an ~700-km transect, over three growth seasons insect visitors were identified, their effectiveness was assessed by surveying pollen loads present on the insect body, insect activity on umbels, nectar and scent composition was studied, and transplantation experiments were performed. Key Results: The populations investigated in this study differed in their nectar and scent profiles and, despite the similar taxonomic composition of insect visitor assemblages, were effectively pollinated by disparate pollinator morphogroups, i.e. flies and beetles. Although this suggested local adaptations to the most effective pollinators, analyses of body pollen loads and behaviour on umbels demonstrated functional equivalency of the visitor morphogroups, which is probably related to the fact that A. sylvestris bears few ovules per flower. The transplantation experiments confirmed that reproductive success was not related to the source of experimental plants and that the insects do not exhibit preferences towards local genotypes. Conclusions: Angelica sylvestris is morphologically well adapted to ecological generalization, and there is little evidence that the surveyed populations represent distinct pollination ecotypes. Most likely, the observed variation in floral characters can be interpreted as 'adaptive wandering'. Specialization in this family seems possible only under very special circumstances, for example when the pollinator community comprises insect visitor groups that clearly differ in their pollination capacity (e.g. due to differences in their functional morphology) and/or have different perceptional biases (e.g. for colour or scent). However, the barrier to the evolution of morphological adaptations resulting in the fine-tuning of the flower towards particular pollinator types may arise from the architectural constraints on the floral bauplan that make umbellifers so uniform in their floral displays and so successful in attracting large numbers of pollinators.

An Unexplored Side of Regeneration Niche: Seed Quantity and Quality Are Determined by the Effect of Temperature on Pollen Performance.

In 1977, Peter Grubb introduced the regeneration niche concept, which assumes that a plant species cannot persist if the environmental conditions are only suitable for adult plant growth and survival, but not for seed production, dispersal, germination, and seedling establishment. During the last decade, this concept has received considerable research attention as it helps to better understand community assembly, population dynamics, and plant responses to environmental changes. Yet, in its present form, it focuses too much on the post-fertilization stages of plant sexual reproduction, neglecting the fact that the environment can operate as a constraint at many points in the chain of processes necessary for successful regeneration. In this review, we draw the attention of the plant ecology research community to the pre-fertilization stages of plant sexual reproduction, an almost ignored but important aspect of the regeneration niche, and their potential consequences for successful seed production. Particularly, we focus on how temperature affects pollen performance and determines plant reproduction success by playing an important role in the temporal and spatial variations in seed quality and quantity. We also review the pollen adaptations to temperature stresses at different levels of plant organization and discuss the plasticity of the performance of pollen under changing temperature conditions. The reviewed literature demonstrates that pre-fertilization stages of seed production, particularly the extreme sensitivity of male gametophyte performance to temperature, are the key determinants of a species' regeneration niche. Thus, we suggest that previous views stating that the regeneration niche begins with the production of seeds should be modified to include the preceding stages. Lastly, we identify several gaps in pollen-related studies revealing a framework of opportunities for future research, particularly how these findings could be used in the field of plant biology and ecology.

Nectar in Plant-Insect Mutualistic Relationships: From Food Reward to Partner Manipulation.

It has been known for centuries that floral and extra-floral nectar secreted by plants attracts and rewards animals. Extra-floral nectar is involved in so-called indirect defense by attracting animals (generally ants) that prey on herbivores, or by discouraging herbivores from feeding on the plant. Floral nectar is presented inside the flower close to the reproductive organs and rewards animals that perform pollination while visiting the flower. In both cases nectar is a source of carbon and nitrogen compounds that feed animals, the most abundant solutes being sugars and amino acids. Plant-animal relationships involving the two types of nectar have therefore been used for a long time as text-book examples of symmetric mutualism: services provided by animals to plants in exchange for food provided by plants to animals. Cheating (or deception or exploitation), namely obtaining the reward/service without returning any counterpart, is however, well-known in mutualistic relationships, since the interacting partners have conflicting interests and selection may favor cheating strategies. A more subtle way of exploiting mutualism was recently highlighted. It implies the evolution of strategies to maximize the benefits obtained by one partner while still providing the reward/service to the other partner. Several substances other than sugars and amino acids have been found in nectar and some affect the foraging behavior of insects and potentially increase the benefits to the plant. Such substances can be considered plant cues to exploit mutualism. Recent evidence motivated some authors to use the term "manipulation" of animals by plants in nectar-mediated mutualistic relationships. This review highlights the recent background of the "manipulation" hypothesis, discussing it in the framework of new ecological and evolutionary scenarios in plant-animal interactions, as a stimulus for future research.

Phylogenetic and functional signals in gymnosperm ovular secretions.

BACKGROUND AND AIMS: Gymnosperms are either wind-pollinated (anemophilous) or both wind- and insect-pollinated (ambophilous). Regardless of pollination mode, ovular secretions play a key role in pollen capture, germination and growth; they are likely also involved in pollinator reward. Little is known about the broad-scale diversity of ovular secretions across gymnosperms, and how these may relate to various reproductive functions. This study analyses the sugar and amino acid profiles of ovular secretions across a range of ambophilous (cycads and Gnetales) and anemophilous gymnosperms (conifers) to place them in an evolutionary context of their possible functions during reproduction. METHODS: Ovular secretions from 13 species representing all five main lineages of extant gymnosperms were sampled. High-performance liquid chromatography techniques were used to measure sugar and amino acid content. Multivariate statistics were applied to assess whether there are significant differences in the chemical profiles of anemophilous and ambophilous species. Data were compared with published chemical profiles of angiosperm nectar. Chemical profiles were placed in the context of phylogenetic relationships. KEY RESULTS: Total sugar concentrations were significantly higher in ovular secretions of ambophilous species than wind-pollinated taxa such as Pinaceae and Cupressophyta. Ambophilous species had lower amounts of total amino acids, and a higher proportion of non-protein amino acids compared with anemophilous lineages, and were also comparable to angiosperm nectar. Results suggest that early gymnosperms likely had ovular secretion profiles that were a mosaic of those associated with modern anemophilous and ambophilous species. Ginkgo, thought to be anemophilous, had a profile typical of ambophilous taxa, suggesting that insect pollination either exists in Gingko, but is undocumented, or that its ancestral populations were insect-pollinated. CONCLUSIONS: Chemical profiles of ovular secretions of ambophilous gymnosperms show a clear signal of pollinator-driven selection, including higher levels of carbohydrates than anemophilous taxa, lower levels of amino acids, and the presence of specific amino acids, such as ß-alanine, that are known to influence insect feeding behaviour and physiology.

It is a matter of timing: asynchrony during pollen development and its consequences on pollen performance in angiosperms-a review.

Functional pollen is needed to successfully complete fertilization. Pollen is formed inside the anthers following a specific sequence of developmental stages, from microsporocyte meiosis to pollen release, that concerns microsporocytes/microspores and anther wall tissues. The processes involved may not be synchronous within a flower, an anther, and even a microsporangium. Asynchrony has been barely analyzed, and its biological consequences have not been yet assessed. In this review, different processes of pollen development and lifetime, stressing on the possible consequences of their differential timing on pollen performance, are summarized. Development is usually synchronized until microsporocyte meiosis I (occasionally until meiosis II). Afterwards, a period of mostly asynchronous events extends up to anther opening as regards: (1) meiosis II (sometimes); (2) microspore vacuolization and later reduction of vacuoles; (3) amylogenesis, amylolysis, and carbohydrate inter-conversion; (4) the first haploid mitosis; and (5) intine formation. Asynchrony would promote metabolic differences among developing microspores and therefore physiologically heterogeneous pollen grains within a single microsporangium. Asynchrony would increase the effect of competition for resources during development and pollen tube growth and also for water during (re)hydration on the stigma. The differences generated by developmental asynchronies may have an adaptive role since more efficient pollen grains would be selected with regard to homeostasis, desiccation tolerance, resilience, speed of (re)hydration, and germination. The performance of each pollen grain which landed onto the stigma will be the result of a series of selective steps determined by its development, physiological state at maturity, and successive environmental constrains.

Nectar sugars and bird visitation define a floral niche for basidiomycetous yeast on the Canary Islands.

BACKGROUND: Studies on the diversity of yeasts in floral nectar were first carried out in the late 19th century. A narrow group of fermenting, osmophilous ascomycetes were regarded as exclusive specialists able to populate this unique and species poor environment. More recently, it became apparent that microorganisms might play an important role in the process of plant pollination. Despite the importance of these nectar dwelling yeasts, knowledge of the factors that drive their diversity and species composition is scarce. RESULTS: In this study, we linked the frequencies of yeast species in floral nectars from various host plants on the Canary Islands to nectar traits and flower visitors. We estimated the structuring impact of pollination syndromes (nectar volume, sugar concentration and sugar composition) on yeast diversity.The observed total yeast diversity was consistent with former studies, however, the present survey yielded additional basidiomycetous yeasts in unexpectedly high numbers. Our results show these basidiomycetes are significantly associated with ornithophilous flowers. Specialized ascomycetes inhabit sucrose-dominant nectars, but are surprisingly rare in nectar dominated by monosaccharides. CONCLUSIONS: There are two conclusions from this study: (i) a shift of floral visitors towards ornithophily alters the likelihood of yeast inoculation in flowers, and (ii) low concentrated hexose-dominant nectar promotes colonization of flowers by basidiomycetes. In the studied floral system, basidiomycete yeasts are acknowledged as regular members of nectar. This challenges the current understanding that nectar is an ecological niche solely occupied by ascomycetous yeasts.

Post-pollination prefertilization drops affect germination rates of heterospecific pollen in larch and Douglas-fir.

Pollen of larch (Larix × marschlinsii) and Douglas-fir (Pseudotsuga menziesii) was used in homospecific and heterospecific crosses. Germination of heterospecific pollen in ovulo was reduced in post-pollination prefertilization drops. This provides evidence of selection against foreign pollen by open-pollinated exposed ovules in these two sister taxa, which share the same type of pollination mechanism. Of the other prezygotic stages in pollen-ovule interactions, uptake of pollen by stigmatic hairs did not show any selection. Pollen tube penetration of the nucellus was similar for hetero- and homospecific pollen tubes, but heterospecific tubes only delivered gametes in one cross. To test for differences in the post-pollination prefertilization drops of each species, drops were gathered and analysed. Glucose and fructose were present in similar amounts in Douglas-fir and larch, while sucrose was found in larch only. Other carbohydrates such as xylose and melezitose were species-specific. In P. menziesii, sucrose is absent due to its conversion to glucose and fructose by apoplastic invertases. In contrast, Larix × marschlinsii drops have sucrose because they lack apoplastic invertases. The presence of invertase activity shows that the composition of gymnosperm post-pollination prefertilization drops is not static but dynamic. Drops of these two species also differed in their calcium concentrations.

Water status and associated processes mark critical stages in pollen development and functioning.

BACKGROUND: The male gametophyte developmental programme can be divided into five phases which differ in relation to the environment and pollen hydration state: (1) pollen develops inside the anther immersed in locular fluid, which conveys substances from the mother plant--the microsporogenesis phase; (2) locular fluid disappears by reabsorption and/or evaporation before the anther opens and the maturing pollen grains undergo dehydration--the dehydration phase; (3) the anther opens and pollen may be dispersed immediately, or be held by, for example, pollenkitt (as occurs in almost all entomophilous species) for later dispersion--the presentation phase; (4) pollen is dispersed by different agents, remaining exposed to the environment for different periods--the dispersal phase; and (5) pollen lands on a stigma and, in the case of a compatible stigma and suitable conditions, undergoes rehydration and starts germination--the pollen-stigma interaction phase. SCOPE: This review highlights the issue of pollen water status and indicates the various mechanisms used by pollen grains during their five developmental phases to adjust to changes in water content and maintain internal stability. CONCLUSIONS: Pollen water status is co-ordinated through structural, physiological and molecular mechanisms. The structural components participating in regulation of the pollen water level, during both dehydration and rehydration, include the exine (the outer wall of the pollen grain) and the vacuole. Recent data suggest the involvement of water channels in pollen water transport and the existence of several molecular mechanisms for pollen osmoregulation and to protect cellular components (proteins and membranes) under water stress. It is suggested that pollen grains will use these mechanisms, which have a developmental role, to cope with environmental stress conditions.

Functional aspects of floral nectar secretion of Ananas ananassoides, an ornithophilous bromeliad from the Brazilian savanna.

BACKGROUND AND AIMS: Several members of Bromeliaceae show adaptations for hummingbird pollination in the Neotropics; however, the relationships between floral structure, nectar production, pollination and pollinators are poorly understood. The main goal of this study was to analyse the functional aspects of nectar secretion related to interaction with pollinators by evaluating floral biology, cellular and sub-cellular anatomy of the septal nectary and nectar composition of Ananas ananassoides, including an experimental approach to nectar dynamics. METHODS: Observations on floral anthesis and visitors were conducted in a population of A. ananassoides in the Brazilian savanna. Nectary samples were processed using standard methods for light and transmission electron microscopy. The main metabolites in nectary tissue were detected via histochemistry. Sugar composition was analysed by high-performance liquid chromatography (HPLC). The accumulated nectar was determined from bagged flowers ('unvisited'), and floral response to repeated nectar removal was evaluated in an experimental design simulating multiple visits by pollinators to the same flowers ('visited') over the course of anthesis. KEY RESULTS: The hummingbirds Hylocharis chrysura and Thalurania glaucopis were the most frequent pollinators. The interlocular septal nectary, composed of three lenticular canals, extends from the ovary base to the style base. It consists of a secretory epithelium and nectary parenchyma rich in starch grains, which are hydrolysed during nectar secretion. The median volume of nectar in recently opened 'unvisited' flowers was 27·0 µL, with a mean (sucrose-dominated) sugar concentration of 30·5 %. Anthesis lasts approx. 11 h, and nectar secretion begins before sunrise. In 'visited' flowers (experimentally emptied every hour) the nectar total production per flower was significantly higher than in the 'unvisited' flowers (control) in terms of volume (t = 4·94, P = 0·0001) and mass of sugar (t = 2·95, P = 0·007), and the concentration was significantly lower (t = 8·04, P = 0·0001). CONCLUSIONS: The data suggest that the total production of floral nectar in A. ananassoides is linked to the pollinators' activity and that the rapid renewal of nectar is related to the nectary morphological features.

Nectar and pollination drops: how different are they?

BACKGROUND: Pollination drops and nectars (floral nectars) are secretions related to plant reproduction. The pollination drop is the landing site for the majority of gymnosperm pollen, whereas nectar of angiosperm flowers represents a common nutritional resource for a large variety of pollinators. Extrafloral nectars also are known from all vascular plants, although among the gymnosperms they are restricted to the Gnetales. Extrafloral nectars are not generally involved in reproduction but serve as 'reward' for ants defending plants against herbivores (indirect defence). SCOPE: Although very different in their task, nectars and pollination drops share some features, e.g. basic chemical composition and eventual consumption by animals. This has led some authors to call these secretions collectively nectar. Modern techniques that permit chemical analysis and protein characterization have very recently added important information about these sugary secretions that appear to be much more than a 'reward' for pollinating (floral nectar) and defending animals (extrafloral nectar) or a landing site for pollen (pollination drop). CONCLUSIONS: Nectar and pollination drops contain sugars as the main components, but the total concentration and the relative proportions are different. They also contain amino acids, of which proline is frequently the most abundant. Proteomic studies have revealed the presence of common functional classes of proteins such as invertases and defence-related proteins in nectar (floral and extrafloral) and pollination drops. Invertases allow for dynamic rearrangement of sugar composition following secretion. Defence-related proteins provide protection from invasion by fungi and bacteria. Currently, only few species have been studied in any depth. The chemical composition of the pollination drop must be investigated in a larger number of species if eventual phylogenetic relationships are to be revealed. Much more information can be provided from further proteomic studies of both nectar and pollination drop that will contribute to the study of plant reproduction and evolution.