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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 β-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.

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.

Nectar is an important floral resource in the establishment of plant-pollinator interactions. Recent studies have shown that nectariferous tissues are independent of the ABC model of floral development and that ecological interactions can modify their expression. In this sense, it would be interesting to study generalist species in relation to nectar production and nectary morpho-anatomy to verify the strategies used to attract different pollinator groups. We recorded nectar production dynamics in Aechmea vanhoutteana (Bromeliaceae) from a morpho-functional and ultrastructural perspective. We observed different species of hummingbirds, bees and butterflies visiting flowers of A.vanhoutteana, and their frequency varied throughout floral anthesis. The nectar volume and quantity of sugar also varied significantly during anthesis, and this spatial and temporal variability seems to be related to an increase in bee visits, representing an important aspect of the reproductive strategy of this species, since bees can fly longer distances than the observed hummingbirds (although both have territorial behaviours). Thus, it can be suggested that anatomical and physiological nectar traits may be related to pollen flow, an important aspect of the reproductive strategy of A. vanhoutteana, suggesting plant resource allocation for optimizing reproduction through nectar production.

Floral nectar is the primary reward directly consumed by floral visitors and its chemical composition affects their behaviour and fidelity. In turn, floral visitors are expected to alter floral nectar composition directly or indirectly through the introduction of external contaminants, such as pollen grains and microorganisms. To understand the effect of exogenous factors on nectar chemistry, we investigated the modifications of nectar sugar and amino acid composition in relation to floral visitors in the perennial herb Gentiana lutea. We quantified nectar contamination by floral visitors through visitor exclusion experiments in the field, estimating pollen and yeast concentration in visited flowers and in uncontaminated nectar at two different flower stages (flower bud and older open flowers). We then assessed changes in the composition and concentration of sugars and both protein and non-protein amino acid in the nectar. We found clear differences in the chemical composition of flower nectar in relation to flower stage and floral visitation. Nectar sugar and amino acid concentrations were significantly higher in older flowers than in flower buds. In addition, nectar sugar concentration was significantly higher in older flowers where visitors were excluded than in visited flowers. On the contrary, amino acid concentration was comparable between visited and non-visited older flowers. We also found that pollen contamination enriched the amino acid profile of the host nectar both quantitatively and qualitatively. Finally, nectar exposed to floral visitors showed a higher number of yeast cells than unvisited flowers although too low to cause the observed changes in the glucose:fructose ratio, suggesting the presence of fructose-related bacteria. Our results show that the chemical composition of floral nectar is altered by several endogenous and exogenous factors in a complex process of ecological relations, and suggest that the modulation of floral visitation through nectar is not solely related to the plant itself.

Floral nectar is a chemically complex aqueous solution within which several secondary metabolites have been identified that affect attractiveness for pollinators. Understanding preferences and aversions to nectar quality in flower visitors is crucial since this may influence the patterns of insect floral visitation with consequences on the plant fitness. We hypothesise that nectar chemical variation through different floral sexual phases may affect the number of insect visits that each phase receives. The study was realized on a population of Echium vulgare L. growing in a natural area close to Bologna. Nectar was collected from functionally male and female flowers to investigate its chemical composition through the HPLC technique. A total of 200min of behavioural observations on foraging insects was also carried out. Variation in nectar traits has been detected for the amino acid spectrum. The proportion of protein amino acids appeared to be significantly higher in male-phase flowers. This may explain the significantly higher number of visits on male flowers than expected observed for all bee taxa (except Hoplitis adunca females). Functionally male flowers presented higher concentrations of phenylalanine, whilst proline was highly represented in functionally female flowers. Since a recent study demonstrated that hymenopterans can oxidize proline at a high rate for ATP production, we can hypothesise that the quality of nectar offered by the two sexually distinct floral phases targets different insect behavioural traits and likely ensures an optimal pattern of visit among flower sexes, which are unequally distributed within and among individuals in the population.

Nectar production dynamics can show inter- and intraspecific variation, which can be associated with environmental and ecological factors and with the ultrastructural diversity of the floral nectary. In this context, we recorded nectar production dynamics from a morphofunctional perspective using the hummingbird-pollinated Billbergia distachia (Bromeliaceae). The scale-throated hermit Phaethornis eurynome was the only floral visitor observed, indicating a specialized pollination system. Nectar production showed significant differences between day and night, and the periods of major pollinator activity and nectar secretion were synchronous. The ultrastructural features of the nectary showed some evidence of nectar reabsorption in flowers at night, and it can be inferred that this process may be a key factor in the nocturnal pause in nectar production. In this way, nectary morphoanatomy, nectar traits and an energy-saving mechanism through nectar reabsorption contribute to the well-established relationship between B. distachia and P. eurynome.

Made available in DSpace on 2020-12-12T02:47:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Cactaceae exhibit highly modified spines that are considered to be extrafloral nectaries (EFNs). Despite their ecological and taxonomical relevance in this family, little is known on their structure and function. We have described the anatomy, ontogenesis, and ultrastructure of the secretory glochids in two Opuntioideae species. Young cladodes of Brasiliopuntia brasiliensis (Willd.) A. Berger and Nopalea cochenillifera (L.) Salm-Dyck were processed for light and electron microscopy studies. The composition of the secretions was analyzed by high performance liquid chromatography. The secretory glochids were soft, massive, and barbed, as well as translucent. Hyaline droplets on the secretory glochid apex were collected by aggressive ants. The secretory glochids originated from the areolar meristem, beginning as small protuberances formed by protoderm and ground meristem. Mature secretory glochids consisted of a central multiseriate axis of ground cells covered by uniseriate epidermis with a continuous cuticle, and exhibited three regions: (i) dilated vascularized base with parenchyma cells exhibiting features associated to nectar secretion; (ii) elongated median region with juxtaposed fusiform non-lignified parenchyma cells; and (iii) tapered apical portion with immature fibers loosely arranged cells. The exudate was sucrose-dominant with a similar amino acid profile in both species. Our results shed light on the secretory activity of glochids in Cactaceae and their role in cactus–ant interactions. UNESP – São Paulo State University Institute of Biosciences Department of Botany University of Siena Department of Life Sciences UNESP – São Paulo State University Institute of Biosciences Department of Botany CAPES: 001 FAPESP: 2017 / 14891-5 CNPq: 303981/ 2018-0 CNPq: 304396/2015-0

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.

To investigate the effects of two nectar nonprotein amino acids, β-alanine and γ-aminobutyric acid (GABA), on Osmia bicornis survival and locomotion, two groups of caged bees were fed with sugar syrup enriched with β-alanine and GABA, respectively. A further control group was fed with sugar syrup. Five behavioural categories were chosen according to the principle of parsimony and intrinsic unitary consistency from start to end, and recorded by scan sampling: two states (remaining under paper or in tubes) and three events (walking on net, feeding from flower and flying). We also analysed the amino acid content of haemolymph sampled from an additional 45 bees fed the same diets (15 per diet type). Bees fed with β-alanine had a significantly shorter survival time than those fed with the control and GABA diets. The GABA diet induced higher levels of locomotion than β-alanine. The former nonprotein amino acid was only detected in the haemolymph of bees fed GABA. The results suggest that insects consuming nonprotein amino-acid-rich diets absorb and transfer these substances to the haemolymph and that nonprotein amino acids affect survival and locomotion. Ecological consequences are discussed in the framework of plant reproductive biology.

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.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.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.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 β-alanine, that are known to influence insect feeding behaviour and physiology.

Made available in DSpace on 2018-12-11T17:30:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-02-01 Zeyheria tuberculosa, a Bignoniaceae tree that occurs in a broad latitudinal range in Brazilian forests, is a threatened species of seasonal tropical forest. It is crucial to enhance the knowledge on this species’ floral biology, since interactions related to reproduction of plants are critical for the long term maintenance of their populations. Thus, we investigated the sexual reproduction and floral biology of Z. tuberculosa, including nectar features, aiming to understand the ecological processes associated to this mutualistic plant-animal interaction in the current modified landscape. For this, we performed experiments under natural conditions and in the laboratory. All the evaluated plants were self-incompatible. Zeyheria tuberculosa was pollinated by Bombus pauloensis, a medium-sized bee, and offered hexose-rich nectar as resource. Flowers were also visited by small-sized bee species, Trigona spinipes, that acted as pollen thief, and Pseudoaugochlora pandora and P. graminae that acted as nectar thieves. These three bee groups exhibited temporal separation in the peak of visits throughout the day. The peak of pollinator visits occurred firstly, which may allow the pollinator to access nectar and pollen loads before they are exploited by thieves. However, bee pollinator visits were scarce, and this fact, associated to the self-incompatibility of Z. tuberculosa, may explain the low fruit set registered in the studied plants. It could be of special concern for this threatened species maintenance in the medium and long term, which depends on the offspring originated by sexual reproduction. Graduation Program in Biological Sciences (Botany) São Paulo State University (Unesp) Institute of Biosciences, Caixa Postal 510 Department of Life Sciences University of Siena, Via P. A. Mattioli 4 Department of Botany São Paulo State University (Unesp) Institute of Biosciences, Caixa Postal 510 Graduation Program in Biological Sciences (Botany) São Paulo State University (Unesp) Institute of Biosciences, Caixa Postal 510 Department of Botany São Paulo State University (Unesp) Institute of Biosciences, Caixa Postal 510

International audience; 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.

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.

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 β-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 β-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 β-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.

Generalist flowers are visited by a broad variety of insects that function as pollinators, occasional visitors and as pollen and/or nectar robbers. Moreover, among legitimate pollinators the pollination efficiency can be different. Nectar greatly affects visitor behaviour and fidelity to a certain species, influencing plant reproductive effort. In this study we have investigated a generalist system (Gentiana lutea L.), examining the role of flower visitors and quantifying the contribution of each pollinating taxon in three natural populations. In order to verify the level of generalization, we introduce an index of Pollinator Performance (PoP), based on insect visitation rate and cross-pollen transport efficiency. Our results confirm the high degree of pollinator-generalization of the study species. Nevertheless, flower visitors show various degree of pollinating performance, mainly defined by their sedentary versus dynamic behaviour. Sedentary insects enhance geitonogamous pollen transfer, which results in reduced seed set and pollen limitation. In particular, an unusual sluggish behaviour was observed in bumblebees feeding on nectar. The hexose-rich abundant nectar offered by G. lutea flowers is remarkably rich in proline and β-alanine amino acids: this composition presumably influences feeding choice and insect dynamism, likely exerting a narcotic effect on pollinators. The consequences on plant fitness are discussed in an evolutionary perspective.

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 &lsquo, greyzini&rsquo, (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.

The production of graphene based materials (GBMs) is steadily increasing but the effects of the possible release of GBMs in the environment are far from being understood. Graphene oxide (GO) is among the most active GBMs and it causes widely varying effects on the vegetative body of seed plants. However, nothing is known yet about its potential effects on the reproductive process. This study addresses the effects of GO on pollen germination and pollen tube elongation in the model species Nicotiana tabacum and in the non-model species Corylus avellana. In vitro germination experiments were conducted without or with GO (control and treated samples, respectively) at concentrations of 25, 50 and 100 μg mL−1. Pollen germination and tube elongation were affected at GO concentrations ≥50 μg mL−1, decreasing by 20% and 19% in N. tabacum and by 68% and 58% in C. avellana, respectively. GO did not affect the viability of N. tabacum pollen, but doubled the frequency of bent tubes. Microscopy observations of pollen tubes exposed to a cell-permeant, dual-excitation ratiometric pH indicator revealed that GO affected the intracellular pH homeostasis. Further germination experiments on C. avellana conducted by inverting the pH conditions of the control and treated (100 μg GO mL−1) samples demonstrated that the main factor influencing the pollen performance is the acidic properties of GO. This might affect the reproductive process of numerous seed plants thus being relevant from an environmental point of view.

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.

The present study examines the cytological, physiological, chemical and ecological characteristics of pollen and nectar offered by male and female flowers of the dioecious plant Laurus nobilis . The various phases of floral phenology and the insect pollinators were observed. We used cytological methods to determine anther, pollen and nectary structure. Nectar sugar composition was evaluated by HPLC. Pollen viability in time was compared with cytoplasmic and intine water content. Pollen presentation was found to be reversible by opening and closing of anther valves, determined by hydration of the mechanical layer of the anther. Pollen, covered by pollenkitt, was presented for dispersal for 3 consecutive days and during this time the intine and cytoplasm lost water and pollen viability diminished. At germination exine ruptured together with the outermost layer of the intine. Nectaries of male flowers were observed on the anther filament and on staminodes of female flowers. The nectar consisted almost entirely of sucrose and was more concentrated in male flowers. Secreted through stomata, nectar was presented in a thin layer. In the study area, the main pollinators (about half the total number of all visits) were hymenopterans. Pollen is of the recalcitrant type due to its high water content (>30%) but its viability is long-lasting because the intine is thick and stores water, keeping the cytoplasm of the vegetative cell hydrated and viable, and because anther valves may close under adverse conditions, protecting the pollen. Insects are attracted by male and female flowers similarly, males offer nectar and pollen, whilst females only nectar.

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 &lsquo, Fragola&rsquo, being the most resistant and the variety &lsquo, Pisanello&rsquo, the most susceptible. For all the parameters evaluated, &lsquo, performed in a manner comparable to that of control plants. On the contrary, &lsquo, 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.

Summary 1. Studies from the last decade clearly demonstrate that nectar is much more than just a simple alimentary reward for pollinators. Considerable progress has been made in recent years with regard to the chemical ecology of nectar, demonstrating unequivocally that its chemistry is strongly involved in the interactions between plants and a larger variety of organisms - from bacteria to fungi and animals - than previously thought. As far as plant-pollinator relationships are concerned, attention has recently been directed towards secondary compounds, mainly alkaloids that are able to induce a wide range of behavioural and physiological responses inoral visi- tors. Nonetheless the study of some other secondary compounds, such as non-protein amino acids (NPAAs), has been neglected. The roles of these substances in nectar are still unknown although several ecological and physiological functions have been attributed to a number of NAAs in both animals and plants in several ecolog- ical and physiological contexts. 2. The existence of NPAAs inoral nectar wasrst reported in early surveys of nectar composition dating from the 1970s. Since that time, only a very few papers have mentioned the presence of these compounds in nectar, but without any hypothesis relating to their ecological role and their putative effect onower visitors. This appears sur- prising considering that interaction with other organisms is a recognized function of NPAAs in plants. 3. Here, after exploring the multiple roles of NPAAs at the community level and focusing on the more recent advances in nectar chemistry and ecology, I review the small body of literature relating to the presence of NPAAs in nectar before outlining some ecological roles in the light of complex, nectar-mediated, plant-animal interactions that have been recognized recently for other nectar secondary compounds based also on data and information obtained for other biological systems (from arthropods to humans). 4. Synthesis. By integrating data obtained from both literature andeld observations, I have arrived at the pro- posal that non-protein amino acids in nectar may contribute to the plant-insect network of interactions in a number of ways: by affecting the physiology of the nervous system of the insect, regulating nectar intake through phagostimulation and promoting muscle function duringight. Conrmation of these hypotheses is needed in order to reinforce the concept that plants are able to affect the foraging behaviour of insects through nectar chemistry, thereby eventually optimizing their pollination effectiveness.

Pollen hydration status at dispersal depends on many parameters and on this basis two main groups of grains may be distinguished: those with a low water content are usually named orthodox or desiccation-resistant, while those with a higher water content at dispersal are known as recalcitrant or desiccation-sensitive due to their reduced mechanisms to keep water constant. On exposure, the latter lose water and die quickly. Although the end of flower receptivity may occur in different ways, in species possessing recalcitrant pollen it often consists in corolla closure. In addition to data available from current literature, also a new set of experiments was performed: the viability of pollen of eight entomophilous species having nectar as pollinator reward, desiccation-sensitive pollen and corolla closure at the end of receptivity, irrespective of pollination, was tested from the onset of anthesis until flower closure using a fluorochromatic reaction. Pollen viability fell sharply in all species, albeit at different rates, depending on initial water content, and may be also on types of carbohydrate reserves and inherent enzymes interconverting them. On the basis of experimental data and the literature, the authors speculate on the effects of corolla closure: it avoids dispersal of pollen with reduced viability, it avoids collection of unconsumed nectar while facilitating its reabsorption by the nectary parenchyma and its utilization for other purposes, and it avoids contamination by moulds and bacterial spores that could penetrate the plant via the nectar and nectary parenchyma.

A population of Buglossoides purpuroca- erulea (L.) I.M. Johnst. (Boraginaceae-Lithosper- meae) located in Lecceto (Siena Province, Tuscany, central Italy) has been studied to compare floral nectar attributes and forager species between sun-exposed and shaded plants. Flower anthesis and maturity of sexual organs were also investigated. Average flower anthesis lasted 3-4 days. Stigma receptivity and anther dehiscence occurred on the first day. Nectar production also began on the first day and maximum production occurred on second-third day. Signifi- cantly greater volumes and total sugars were recorded in individuals exposed to the sun. Nectar HPLC analysis showed a similar hexose-dominant sugar profile for all the individuals with percentages of sucrose, glucose and fructose around 5, 48 and 47 %, respectively. Protein amino acids represent the 90 % of the overall free amino acids profile. Significant differences between relative percentages of serine and proline were found between sun-exposed and shaded individuals. Empis pennipes and Bombilyus major were the most frequent insect visitors to shaded and sun-exposed individuals, respectively. The hexose dominance of the nectar of B. purpurocaerulea ,a n exception among the Mediterranean Lithospermeae, may be related to the habitat where this plant generally grows, i.e. the forest-edge, and to pollination mainly performed by dipterans.

The Boraginales are now universally accepted as monophyletic and firmly placed in Lamiidae. However, a consensus about familial classification has remained elusive, with some advocating recognition of a single, widely variable family, and others proposing recognition of several distinct families. A consensus classification is proposed here, based on recent molecular phylogenetic studies, morphological characters, and taking nomenclatural stability into consideration. We suggest the recognition of eleven, morphologically well-defined and clearly monophyletic families, namely the Boraginaceae s.str., Codonaceae, Coldeniaceae fam. nov., Cordiaceae, Ehretiaceae, Heliotropiaceae, Hoplestigmataceae, Hydrophyllaceae, Lennoaceae, Namaceae, and Wellstediaceae. Descriptions, synonomy, a taxonomic key, and a list of genera for these eleven families are provided, including the new family Coldeniaceae (monogeneric) and Namaceae (segregated from Hydrophyllaceae and comprising Nama, Eriodictyon, Turricula, and Wigandia), the latter necessitating a revised circumscription of a more morphologically coherent Hydrophyllaceae.

The floral nectaries of Helleborus foetidus L. were studied using scanning and transmission electron microscopy, as well as light microscopy. Nectaries are tubular and consist of an external epidermis, a photosynthesizing parenchyma, large branches of vascular tissue, a nectar-producing parenchyma, and an internal epidermis. The external epidermis is characterized by thick outer walls and a thin cuticle. Cells of the photosynthesizing parenchyma are characterized by chloroamyloplasts. The nectar-producing parenchyma consists of small cells with lobed nucleus, several small vacuoles, and numerous undifferentiated elongated plastids. These cells contain lipid bodies, Golgi membranes, and rough endoplasmic reticulum and are connected by numerous plasmodesmata. Parenchyma cells around sieve elements contain some amyloplasts. The outer cell wall of the internal epidermis displays a central thinned area containing a depression. The cuticle is very thick with amorphous structure, especially evident in its proximal part. Secretion takes place only in the inner basal region of the nectary cup. The surface of the internal epidermis is devoid of secreting structures. Nectar is released by fracture of the cuticle and underlying cell wall; however, epidermal cell death is not apoptotic. This holocrine secretion is coupled with a more common merocrine secretion through which nectar accumulates in subcuticular spaces. This combined secretion mode produces an enriched “nectar soup” crucial for interactions with pollinators and yeasts during winter flowering.

Nectar is considered a primary alimentary reward for a large variety of pollinators. Recent studies demonstrated that nectar may have other functions in addition to attracting pollinators. Mainly other two functions have been recognized: defense against microbial invasion and post-secretion modification of sugar profile. The floral nectar of Cucurbita pepo L. has been analyzed to confirm the presence of defense proteins, namely xylosidases that were identified by means of a proteomic approach in a previous study. An enzyme assay was also performed that revealed low invertase activity for which the optimal pH was determined. This invertase activity is not able to modify the sugar profile significantly during the short period of anthesis (6 h). The amino acids complement of the nectar of both sexes was also determined. Sixteen of the 20 protein amino acids have been detected. Proline comprises more than 30% of total amino acid content in male and female flowers. Three non-protein amino acids (taurine, β-alanine, and GABA) represent almost one third of the total amino acid content, and of these, GABA is the most abundant (16%). Several biological activities were attributed to these amino acids and further studies are needed to assess their presumed involvement in managing the foraging behavior of insects. More recent studies, including our own, demonstrate that the various functions of nectar are much more complex than previously thought.

The structure of perigonal nectaries, nectar production and carbohydrate composition were compared at various stages in the lifespan of the flower of Fritillaria meleagris L. The six nectaries each occupied a groove that is located 2–4 mm above the tepal base. The average nectary measured 11.0 mm long and 1.0–1.2 mm wide. The structure of nectaries situated on both inner and outer tepal whorls was identical, and at anthesis they were equally accessible to potential pollinators. However, secretion from nectaries associated with inner tepals tended to exceed that produced by nectaries located on the outer tepals. On average, regardless of flower stage, one flower secreted 10.87 ± 12.98 mg of nectar (mean and SD; N = 182). The nectar concentration ranged between 3 and 75%, with average concentration of sugars exceeding 50%. Both nectar production and concentration were dependent on the stage of anthesis, with the highest scores being recorded during full anthesis (21.75 ± 16.08 mg; 70.5%, mass and concentration, respectively) and the lowest at the end of anthesis (1.32 ± 2.69 mg; 16.9%, mass and concentration, respectively). A decline in both mass of nectar secreted and nectar concentration during the final stage of anthesis indicates nectar resorption. Nectar was composed of sucrose, glucose and fructose in approx. equal quantities, and its composition did not change significantly during subsequent stages of flowering. The nectaries comprised a single-layered secretory epidermis and several layers of subepidermal parenchyma. The nectariferous cells did not accumulate starch during any of the investigated stages. The nectary was supplied with one large and several smaller vascular bundles comprising xylem and phloem. Transport of assimilates and nectar secretion by protoplasts of secretory cells (and probably also nectar resorption) were facilitated by cell wall ingrowths present on the tangential walls of epidermal cells and subepidermal parenchyma. Epidermal cells lacked stomata. Nectar passed across the cell wall and through the cuticle which was clearly perforated with pores.

In April and May 2010, a natural population of Cerinthe major (Boraginaceae-Lithospermeae) was investigated with regard to: floral morphology, phenology, sexual receptivity during anthesis, the production of nectar and its sugar and amino acid content. The pollinator array of this species was also investigated. Cerinthe major has showy, pentamerous, hermaphroditic flowers with a deep yellow corolla tube arranged on numerous scorpioid inflorescences. Nectar and pollen are the main floral rewards for pollinators. The arrangement of stamens limits access to nectar and promotes pollen loading onto pollinator bodies as they force their way towards the source of nectar. By limiting the direct exposure of nectar to the environment, the corolla tube and the arrangement of anther filaments seemingly protect nectar from evaporation resulting from high temperatures and low relative humidity during flower anthesis. The green, annular nectary located at the base of the ovary lobes is composed of a very thin epidermis enclosing a dense mass of parenchyma. The epidermis lacks stomata, and the thin cuticle has an irregular surface. The parenchyma cells do not store starch during the pre-secretory stage, suggesting that immediate photosynthesis is the most likely source of nectar carbohydrates. Generally, anthesis lasts 4–5 days; the gynoecium becomes fully receptive on the second day and this is synchronous with anther dehiscence. Since nectar production begins during the bud stage, a substantial volume is available for flower visitors at anthesis. Nectar production decreases drastically in senescent flowers and, if not consumed, the nectar can be re-absorbed. Analysis of sugars revealed a sucrose-dominant nectar (sucrose = 93.18 ± 1.35%). Proline, GABA, taurine, leucine, citrulline and alanine were the main amino acids present and are probably implicated in pollinator flight performance. The bees Bombus hortorum, Anthophora sp. and Eucera sp. were constant and frequent visitors of C. major flowers. Moreover, the manner in which they interacted with the flower in order to obtain nectar and pollen loads was sufficient evidence for their role as pollinators. Their different foraging behaviours suggest that each species may promote differences in pollen flow both within and among individuals of the population.

In most species, arrest of growth and a decrease in water content occur in seeds and pollen before they are dispersed. However, in a few cases, pollen and seeds may continue to develop (germinate). Examples are cleistogamy and vivipary. In all other cases, seeds and pollen are dispersed with a variable water content (2-70%), and consequently they respond differently to environmental relative humidity that affects dispersal and maintenance of viability in time. Seeds with low moisture content shed by the parent plant after maturation drying can generally desiccate further to moisture contents in the range of 1-5% without damage and have been termed 'orthodox'. Pollen that can withstand dehydration also was recently termed orthodox. Seeds and pollen that do not undergo maturation drying and are shed at relatively high moisture contents (30-70%) are termed 'recalcitrant'. Since recalcitrant seeds and pollen are highly susceptible to desiccation damage, they cannot be stored under conditions suitable for orthodox seeds and pollen. Hence, there are four types of plants with regard to tolerance of pollen and seeds to desiccation. Orthodoxy allows for dispersal over greater distances, longer survival, and greater resistance to low relative humidity. The advantage of recalcitrance is fast germination. Orthodoxy and recalcitrance are often related to environment rather than to systematics. It has been postulated that certain types of genes are involved during presentation and dispersal of pollen and seeds, since molecules (sucrose, polyalcohols, late embryogenic abundant proteins, antioxidants, etc.) that protect different cell compartments during biologically programmed drying have been detected in both.

† Background and Aims Nectar is a very complex mixture of substances. Some components (sugars and amino acids) are considered primary alimentary rewards for animals and have been investigated and characterized in numerous species for many years. In contrast, nectar proteins have been the subject of few studies and little is known of their function. Only very recently have detailed studies and characterization of nectar proteins been undertaken, and then for only a very few species. This current work represents a first step in the identification of a protein profile for the floral nectar of Cucurbita pepo. In this regard, the species studied is of particular interest in that it is monoecious with unisexual flowers and, consequently, it is possible that nectar proteins derived from male and female flowers may differ. † Methods Manually excised spots from two-dimensional (2-D) electrophoresis were subjected to in-gel protein digestion. The resulting peptides were sequenced using nanoscale LC‐ESI/MS-MS (liquid chromatography‐ electrospray ionization/tandem mass spectrometry). An MS/MS ions search was carried out in Swiss-Prot and NCBInr databases using MASCOT software. † Key Results Two-dimensional electrophoresis revealed a total of 24 spots and a different protein profile for male and female flower nectar. Four main proteins recognized by 2-D electrophoresis most closely resemble b-D-xylosidases from Arabidopsis thaliana and have some homology to a b-D-xylosidase from Medicago varia. They were present in similar quantities in male and female flowers and had the same molecular weight, but with slightly different isoelectric points. † Conclusions A putative function for xylosidases in floral nectar of C. pepo is proposed, namely that they may be involved in degrading the oligosaccharides released by the nectary cell walls in response to hydrolytic enzymes produced by invading micro-organisms. Several types of oligosaccharides have been reported to increase the pathogenic potential of micro-organisms. Thus, it is possible that such a mechanism may reduce the virulence of pathogens present in nectar.

Male flowers of Cucurbita pepo L. cv. Alberello di Sarzana produce nectar in the first hours of anthesis (5:00 a.m.–8:00 a.m.). However, nectar production ceases soon after 9:00 a.m. Removal of nectar had no effect on total nectar volume and sugar concentration. Nectar substitution experiments were performed using low (20 μL) and high (100 μL) nectar volumes in combination with low (10%) and high (60%) nectar sugar concentrations. Experiments were performed during the nectar production phase (5:00 a.m.) and after the cessation phase began (10:00 a.m.). In both phases, high-volume treatments tended to reverse the passage of water from the nectary to the nectar. Although reabsorption of water and sugar is more obvious after the commencement of the cessation phase, and especially during high-concentration treatments, it can also occur during the secretory phase, thereby demonstrating that the flowers are able to secrete and reabsorb water and sugar independently throughout the whole of anthesis. This is very...

The nectar–sugar profile (fructose, glucose and sucrose) of 14 species of Anchusa and five members of the allied genera Anchusella, Cynoglottis, Hormuzakia and Lycopsis (Boraginaceae: tribe Boragineae) was determined. Most of the species examined (c. 74%) produce sucrose-dominant nectar, whereas the remaining taxa produce sucrose-rich nectars. Little variation in nectar–sugar composition was found in some species, even when sampling was repeated in different years and/or localities. Average sucrose concentration was 57.75% (coefficient of variation 19.1%). The only floral morphological character that was correlated with the nectar–sugar profile is the length of the corolla tube, as taxa with relatively long floral tubes produce nectar with lower glucose concentrations. The flowering period is also related to sugar composition, as nectar of late-flowering species contains lower sucrose concentrations. However, small differences in sugar profiles do not reflect phylogenetic relationships based on molecular studies. It would appear that dry habitats and time of flowering are the main determinants of nectar–sugar composition in the genus Anchusa sensu lato. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 162, 616–627.

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.

† Background and Aims The pollination drop is a liquid secretion produced by the ovule and exposed outside the micropyle. In many gymnosperms, pollen lands on the surface of the pollination drop, rehydrates and enters the ovule as the drop retracts. The objective of this work was to study the formation of the pollination drop in Juniperus communis, its carbohydrate composition and the response to deposition of conspecific pollen, foreign pollen and other particulate material, in an attempt to clarify the mechanism of pollination drop retraction. † Methods Branches with female cones close to pollination drop secretion were collected. On the first day of pollination drop exposure, an eyelash mounted on a wooden stick with paraffin was used to collect pollen or silica gel particles, which were then deposited by contact with the drop. Volume changes in pollination drops were measured by using a stereomicroscope with a micrometer eyepiece 3 h after deposition. The volume of non-pollinated control drops was also recorded. On the first day of secretion, drops were also collected for sugar analysis by high-performance liquid chromatography. † Key Results The pollination drop persisted for about 12 d if not pollinated, and formed again after removal for up to four consecutive days. After pollination with viable conspecific pollen, the drop retracted quickly and did not form again. Partial withdrawal occurred after deposition of other biological and non-biological material. Fructose was the dominant sugar; glucose was also present but at a much lower percentage. † Conclusions Sugar analysis confirmed the general trend of fructose dominance in gymnosperm pollination drops. Complete pollination drop withdrawal appears to be triggered by a biochemical mechanism resulting from interaction between pollen and drop constituents. The results of particle deposition suggest the existence of a nonspecific, particle-size-dependent mechanism that induces partial pollination drop withdrawal. These results suggest that the non-specific response may decrease the probability of pollen landing on the drop, reducing pollination efficiency.

Background and aims This paper is about pollen ecophysiology, anther opening, pollen dispersal and the timing of the male and female phases in Parietaria judaica (Urticaceae). Methods Ecophysiological (effects of different relative humidities (RHs) and osmotic relationships) and cytological methods (stigmatic receptivity, pollen viability, histology and histochemistry) were used to determine pollen and pollination features during the long blooming period of this species. Key results Pollen is dispersed by rapid uncurling of the filament and anther opening. The filament and anther lack cells with lignified wall thickenings, which are usually responsible for anther opening and ballistic pollen dispersal. Instead, dispersal is the result of the sudden movement of the filament. Pollen is of the partially hydrated type, i.e. with a water content greater than 30% at shedding, and readily loses water, and hence viability, at low RH. Pollen carbohydrate reserves differ with season. Starchless grains germinate quickly and are less subject to water loss. Flowers are protogynous, pollen release occurring only after complete cessation of the female phase within an inflorescence. Conclusion Parietaria has partially hydrated pollen which differs from typical pollen of this type because of its reduced size and the absence of callose. Because of its low water content at the time of shedding it survives better at higher RH. Dispersal and pollination are adapted to pollen features.

Many plant-derived chemicals may have an impact on the functioning of the animal brain. The mechanisms by which the psychoactive components of these various products have their effects have been widely described, but the question of why they have these effects has been almost totally ignored. Recent evidence suggests that plants may produce chemicals to manipulate their partner ants and to make reciprocation more beneficial. In the present review we propose that these plant-derived chemicals could have evolved in plants to attract and manipulate ant behaviour; this would place the plant–animal interaction in a different ecological context and open new ecological and neurobiological perspectives for drug seeking and use., Plant–ant interactions are generally considered as mutualisms, with both parties gaining benefits from the association. It has recently emerged that some of these mutualistic associations have, however, evolved towards other forms of relationships and, in particular, that plants may manipulate their partner ants to make reciprocation more beneficial, thereby stabilizing the mutualism. Focusing on plants bearing extrafloral nectaries, we review recent studies and address three key questions: (i) how can plants attract potential partners and maintain their services; (ii) are there compounds in extrafloral nectar that could mediate partner manipulation; and (iii) are ants susceptible to such compounds? After reviewing the current knowledge on plant–ant associations, we propose a possible scenario where plant-derived chemicals, such as secondary metabolites, known to have an impact on animal brain, could have evolved in plants to attract and manipulate ant behaviour. This new viewpoint would place plant–animal interaction in a different ecological context, opening new ecological and neurobiological perspectives of drug seeking and use.

Nectar is the most common floral pollinator reward. In dichogamous species, floral nectar production rates can differ between sexual phases. We studied the structure of nectaries located on the stylopodium and nectar production in protandrous umbellifer Angelica sylvestris. Our study species produced nectar in both floral sexual phases. Nectar sugar concentration was low (on average 22 ± 11 %, mean ± SD) and the nectar hexose rich and composed of sucrose, glucose, fructose and a small amount of amino acids, including β-alanine, a non-protein amino acid. Although nectar composition and sugar concentration varied little between floral sexual phases, nectar production showed a threefold reduction during the stigma receptive period. This is in contrast to other studies of Apiaceae that have reported female-biased nectar production, but in the direction predicted by plant sexual selection theory, suggesting that in pollen-unlimited species, floral rewards mainly enhance male reproductive success. The structure of the nectary was similar at the two sexual stages investigated, and composed of a secretory epidermis and several layers of nectariferous and subsecretory parenchyma. The nectary cells were small, had large nuclei, numerous small vacuoles and dense, intensely staining cytoplasm with abundant endoplasmic reticulum, mitochondria and secretory vesicles. They contained abundant resin-like material that may potentially act as defence against microbes. Starch was rarely observed in the nectary cells, occurring predominantly at the female stage and mainly in guard and parenchyma cells in close proximity to stomata, and in subsecretory parenchyma. The main route of nectar release in A. sylvestris seems to be via modified stomata.