Heteranthery as a solution to the demand for pollen as food and for pollination – Legitimate flower visitors reject flowers without feeding anthers

• Heteranthery, the presence of feeding and pollinating anthers in the same flower, seems to mediate the evolutionary dilemma for plants to protect their gametes and yet provide food for pollinators. This study aims to elucidate the role of heteranthery in the buzz‐pollinated Senna reniformis.
• The fecundity of pollen from long‐, medium‐ and short‐sized anthers was determined by hand cross‐pollination experiments, and the quantity, size, ornamentation and viability of pollen of different anthers were compared. Rates of flower rejection by bees were measured in anther removal experiments to assess the preferences of flower visitors for feeding or pollinating anthers.
• Large bees, which were the effective pollinators of self‐incompatible S. reniformis, avoided flowers without short feeding anthers, but not those without medium or long anthers. Illegitimate small and medium‐sized bees were unresponsive to anther exclusion experiments. Long anthers deposited pollen on the back and short anthers on the venter of large bees. Pollen from long anthers had higher in vitro viability and higher fruit and seed set after cross‐pollination than pollen from other sized anthers.
• Short anthers produce feeding pollen to effective pollinators and long anthers are related to pollination of S. reniformis. Bee behaviour and size was found to directly influence the role of anthers in the ‘division of labour’. Only large bee pollinators that carry the pollinating pollen from long anthers in ‘safe sites’ associated short anthers with the presence of food. In the absence of these larger bee pollinators, the role of heteranthery in S. reniformis would be strongly compromised and its function would be lost.

     

Assessment of pollen reward and pollen availability in Solanum stramoniifolium and Solanum paniculatum for buzz‐pollinating carpenter bees

The two widespread tropical Solanum species S. paniculatum and S. stramoniifolium are highly dependent on the visits of large bees that pollinate the flowers while buzzing them. Both Solanum species do not offer nectar reward; the rewarding of bees is thus solely dependent on the availability of pollen. Flower visitors are unable to visually assess the amount of pollen, because the pollen is hidden in poricidal anthers. In this study we ask whether and how the amount of pollen determines the attractiveness of flowers for bees. The number of pollen grains in anthers of S. stramoniifolium was seven times higher than in S. paniculatum. By contrast, the handling time per five flowers for carpenter bees visiting S. paniculatum was 3.5 times shorter than of those visiting S. stramoniifolium. As a result foraging carpenter bees collected a similar number of pollen grains per unit time on flowers of both species. Experimental manipulation of pollen availability by gluing the anther pores showed that the carpenter bees were unable to detect the availability of pollen by means of chemical cues before landing and without buzzing. Our study shows that the efficiency of pollen collecting on S. paniculatum is based on large inflorescences with short between‐flower search times and short handling time of individual flowers, whereas that of S. stramoniifolium relies on a large amount of pollen per flower. Interestingly, large carpenter bees are able to adjust their foraging behaviour to drastically different strategies of pollen reward in otherwise very similar plant species.     

Floral micromorphology and microsporogenesis of the gynodioecious herb Glechoma longituba (Lamiaceae)

Gynodioecy, the phenomenon of having both hermaphrodite and female (i.e. male‐sterile) individuals within the same population, is an important intermediate step in the evolution of separate sexes in flowering plants. In this study, we investigated the floral micromorphology and microsporogenesis of the gynodioecious herb Glechoma longituba from four natural populations in Korea. The floral micromorphological characters of the different sex morphs were examined and compared using scanning electron microscopy (SEM), and the ultrastructure of microspores during microsporogenesis was studied. We also examined the development of anthers and pollen grains in the three sexual morphs (i.e. hermaphrodites, females, and gynomonoecious, i.e. individuals with a mixture of female and hermaphroditic flowers) by embryological investigation. The major difference in anther development between the three phenotypes was the early disintegration of the tapetal cells in the anthers of female flowers. While mature fertile pollen grains were found in both hermaphrodite and gynomonoecious phenotypes, females did not produce any pollen grains. In addition, both fertile and sterile pollen grains in gynomonoecious phenotypes were frequently observed. The results of the present study indicate that floral micromorphological characters were not distinct between sexual morphs of G. longituba, except for the structure of the inner cell surfaces of the anther. The observed tapetum abnormalities and degeneration of pollen grains in both gynomonoecious phenotypes and females may be the consequence of inbreeding depression in hermaphrodites.     

Effect of flower orientation on the male and female traits of Myrtillocactus geometrizans (Cactaceae)

• Intra‐individual variation in the production and size of reproductive traits has been documented in columnar cacti, being higher in equator‐facing flowers. Such variation is attributed to the high amount of PAR intercepted by stems oriented towards the equator. Most studies focused on this phenomenon have documented the existence of intra‐individual variation on traits associated with the female function; however, its impact on traits associated with the male function has been neglected. We tested the hypothesis that equator‐facing flowers of Myrtillocactus geometrizans exhibit higher values on traits associated with both male and female functions than flowers facing against it.
• Number and size of anthers and ovaries, pollen:ovule ratio and number and quality of pollen grains (diameter, germinability, viability and pollen tube length) were estimated from reproductive structures facing north and south, and compared with t‐tests between orientations.
• Number of anthers per flower, number of pollen grains per anther and per floral bud; pollen size, viability and germinability; pollen tube length; ovary length and pollen:ovule ratio were significantly higher in reproductive structures oriented towards the south (i.e. equator).
• These findings suggest that intra‐individual variation in floral traits of M. geometrizans might be associated with different availability of resources in branches with contrasting orientation. Our results provide new evidence of the existence of a response to an orientation‐dependent extrinsic gradient. To our knowledge, this is the first study documenting the existence of intra‐individual variation on pollen quality and P:O ratio in Cactaceae species.

     

The mechanism of delayed selfing in Collinsia verna (Scrophulariaceae)

Collinsia verna, blue-eyed Mary, has floral attributes of an outcrossing species, yet most flowers readily self-pollinate under greenhouse conditions. Here we describe the mechanism of self-pollination in C. verna via changes in relative positions of the stigma and anthers and late timing of receptivity, resulting in delayed selfing. Each flower contains four anthers that dehisce sequentially over ∼1 wk. Pollen that is not collected by pollinators accumulates in the keel petal and retains high viability (>80% pollen germination) up to the time of corolla abscission. The stigmatic surface does not become receptive until after the third anther dehisces. This overlap in the sexual phases is concurrent with a change in herkogamy during floral development. In most flowers (70%), the stigma has moved to the front of the keel and is positioned near the anthers when the third anther dehisces. Under field conditions, fruiting success of plants within pollinator exclosures was ∼75% of the fruiting success in open-pollinated plants (33% fruiting success via autogamy vs. 44% fruiting success, respectively). Collinsia verna plants in pollinator exclosures exhibit variation in autogamy rates within natural populations (range 0–80%). In addition, only half of naturally pollinated, receptive flowers examined had pollen tubes growing in their styles. In contrast, shortly after corolla abscission, nearly all flowers examined (96%) had pollen tubes in their styles. Thus we find that in C. verna, autogamy occurs late in floral development, which has the potential to provide substantial reproductive assurance, and that individuals vary in their ability to set fruit through this mechanism. We suggest that delayed selfing mechanisms may be overlooked in other species and that variable pollinator availability may play a significant role in the maintenance of mixed mating in species with delayed selfing, such as C. verna.     

New acritarch species from Holocene sediments in central West Greenland

Anther development, microsporogenesis, and microgametogenesis were studied using both light and TEM microscopy in the six accessible subdioecious/cryptically dioecious species of Consolea (Cactaceae). Anther wall development, microsporogenesis, and microgametogenesis are uniform in staminate flowers of all six species, and are typical for Cactaceae. Breakdown of microsporogenesis in male‐sterile anthers occurs early, at the onset of meiosis, and results in anthers bearing no pollen grains. The abortive process follows a common pattern in all investigated species. The tapetum is the first layer to deviate from normal male‐fertile anther development. Tapetal cells in male‐sterile anthers elongate at an early stage and have abundant rER with atypical configurations. Ultimately, the tapetum becomes hypertrophied and non‐functional. Male‐sterility in pistillate flowers appears to be directly related to these anomalies. In addition, other anther layers and tissues are affected, and normal patterns of programmed cell death (PCD) are disrupted. The relationship between these patterns and the pattern of PCD in normal male‐fertile anthers is discussed. We hypothesize a single origin for the cryptically dioecious/subdioecious breeding system of Consolea based on the uniformity of the anther's abortive processes in pistillate flowers.     

Anther structure and pollen development in Melicoccus lepidopetalus (Sapindaceae): An evolutionary approach to dioecy in the family

Anther and pollen development in staminate and pistillate flowers of dioecious Melicoccus lepidopetalus (Sapindaceae) were examined by light and electron microscopy. Young anthers are similar in both types of flowers; they consist of epidermis, endothecium, two to four middle layers and a secretory tapetum. The microspore tetrads are tetrahedral. The mature anther in staminate flowers presents compressed epidermal cells and endothecium cells with fibrillar thickenings. A single locule is formed in the theca by dissolution of the septum and pollen grains are shed at two-celled stage. The mature anthers of pistillate flowers differ anatomically from those of staminate flowers. The epidermis is not compressed, the endothecium does not develop fibrillar thickenings, middle layers and tapetum are generally persisting, and the stomium is nonfunctional. Microspore degeneration begins after meiosis of microspore mother cells. At anthesis, uninucleate microspores and pollen grains with vegetative and generative nuclei with no cytokinesis are observed. Some pollen walls display an abnormal exine deposition, whereas others show a well formed exine, although both are devoid of intine. These results suggest that in the evolution towards unisexuality, the developmental differences of anther wall tissues and pollen grains between pistillate and staminate flowers might become more pronounced in a derived condition, such as dioecy.     

Anther culture of Sorghum bicolor (L.) Moench

The sequence of pollen development from the tetrad stage to the mature tricellular grain was studied in freshly harvested anthers of Sorghum bicolor. This pattern of development was then compared with that occurring during panicle pretreatment and subsequent anther incubation in vitro. It was found that during pretreatment at 7° C mitoses of the vegetative cell were induced in up to 30% of the pollen. During anther incubation procallus development was highly polarised with contributions from both the generative and vegetative cells. After pretreatment at 14 or 20° C the generative cell became detached from the pollen wall and it was not possible to determine whether subsequent development involved only the vegetative cell or both the vegetative and generative cells.Although retarded pollen grains were observed both in vivo and in vitro, and were occasionally seen to divide in culture, they did not appear to be the source of the procalluses produced.     

Down‐regulation of OsSPX1 caused semi‐male sterility,resulting in reduction of grain yield in rice

OsSPX1, a rice SPX domain gene, involved in the phosphate (Pi)‐sensing mechanism plays an essential role in the Pi‐signalling network through interaction with OsPHR2. In this study, we focused on the potential function of OsSPX1 during rice reproductive phase. Based on investigation of OsSPX1 antisense and sense transgenic rice lines in the paddy fields, we discovered that the down‐regulation of OsSPX1 caused reduction of seed‐setting rate and filled grain number. Through examination of anthers and pollens of the transgenic and wild‐type plants by microscopy, we found that the antisense of OsSPX1 gene led to semi‐male sterility, with lacking of mature pollen grains and phenotypes with a disordered surface of anthers and pollens. We further conducted rice whole‐genome GeneChip analysis to elucidate the possible molecular mechanism underlying why the down‐regulation of OsSPX1 caused deficiencies in anthers and pollens and lower seed‐setting rate in rice. The down‐regulation of OsSPX1 significantly affected expression of genes involved in carbohydrate metabolism and sugar transport, anther development, cell cycle, etc. These genes may be related to pollen fertility and male gametophyte development. Our study demonstrated that down‐regulation of OsSPX1 disrupted rice normal anther and pollen development by affecting carbohydrate metabolism and sugar transport, leading to semi‐male sterility, and ultimately resulted in low seed‐setting rate and grain yield.     

Microarray Analysis of Gene Expression Involved in Anther Development in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

In flowering plants, anthers bear male gametophytes whose development is regulated by the elaborate coordination of many genes. In addition, both gibberellic acid (GA₃) and jasmonic acid (JA) play important roles in anther development and pollen fertility. To facilitate the analysis of anther development genes and how GA₃ and JA regulate anther development, we performed microarray experiments using a 10-K cDNA microarray with probes derived from seedlings, meiotic anthers, mature anthers and GA₃- or JA-treated suspension cells of rice. The expression level change of 2155 genes was significantly (by 2-fold or greater) detected in anthers compared with seedlings. Forty-seven genes, representing genes with potential function in cell cycle and cell structure regulation, hormone response, photosynthesis, stress resistance and metabolism, were differentially expressed in meiotic and mature anthers. Moreover, 314 genes responded to either GA₃ or JA treatment, and 24 GA₃- and 82 JA-responsive genes showed significant changes in expression between meiosis and the mature anther stages. RT-PCR demonstrated that gene y656d05 was not only highly expressed in meiotic anthers but also induced by GA₃. Strong RNA signals of y656d05 were detected in pollen mother cells and tapetum in in situ hybridization. Further characterization of these candidate genes can contribute to the understanding of the molecular mechanism of anther development and the involvement of JA and GA₃ signals in the control of anther development in rice.     

Developmental regulation of K accumulation in pollen, anthers, and papillae: are anther dehiscence, papillae hydration, and pollen swelling leading to pollination and fertilization in barley (Hordeum vulgare L.) regulated by changes in K concentration?

The presence of potassium (K) in pollen, anthers and papillaefrom barley (Hordeum vulgare L.) flowers with different levelsof developmental stages starting from boot stage to fully matureflower, was studied by using the K-sensitive fluorescent dyePBFI (potassium-binding benzofuran isophthalate) and confocallaser scanning microscopy. The presence of heavy K fluorescencewas detected only at the aperture area of the mature pollen.Similarly, the presence of K increased with the progressionfrom immature to mature anther and papillae. In addition, ahigher concentration of K was observed only at the stomium area(the place of anther dehiscence) of mature anthers. Keepingin view the role of K as an active osmoticum and the consistentand synchronized appearance of K in mature pollen, anthers,and papillae, it was concluded that K may regulate anther dehiscence,pollen imbibition, and papillae hydration leading to pollinationand fertilization. Key words: Anther, confocal microscopy, papillae, PBFI, pollen, potassium     

POLLINATION AND THE FUNCTION OF FLORAL PARTS IN CHAMAECRISTA FASCICULATA (FABACEAE)

Chamaecrista fasciculata has a rigid upper petal that curves over nine of the ten anthers. The single anther is deflexed, as is the style. An experimental pollination study was designed to test the hypotheses that: 1) the rigid petal acts as flight guide to ensure pollination; and 2) the nine grouped anthers serve as fodder anthers, whereas the single deflexed anther functions in pollination. The rigid petal was removed from 97 flowers. Only 5% of the manipulated flowers set fruit in comparison to a fruit set of 47% for the control group. The results of the study support the flight guide hypothesis. Pollen from both sets of anthers is viable and germinates on receptive stigmas. A pollen flow experiment using powdered metals, backscatter scanning electron microscopy, and X-ray microanalysis revealed that there is no difference in the frequency of pollen distribution from either set of anthers. Therefore, this study refutes the fodder/pollinating anther hypothesis.     

Rice No Pollen 1 (NP1) is required for anther cuticle formation and pollen exine patterning

Angiosperm male reproductive organs (anthers and pollen grains) have complex and interesting morphological features, but mechanisms that underlie their patterning are poorly understood. Here we report the isolation and characterization of a male sterile mutant of No Pollen 1 (NP1) in rice (Oryza sativa). The np1‐4 mutant exhibited smaller anthers with a smooth cuticle surface, abnormal Ubisch bodies, and aborted pollen grains covered with irregular exine. Wild‐type exine has two continuous layers; but np1‐4 exine showed a discontinuous structure with large granules of varying size. Chemical analysis revealed reduction in most of the cutin monomers in np1‐4 anthers, and less cuticular wax. Map‐based cloning suggested that NP1 encodes a putative glucose‐methanol‐choline oxidoreductase; and expression analyses found NP1 preferentially expressed in the tapetal layer from stage 8 to stage 10 of anther development. Additionally, the expression of several genes involved in biosynthesis and in the transport of lipid monomers of sporopollenin and cutin was decreased in np1‐4 mutant anthers. Taken together, these observations suggest that NP1 is required for anther cuticle formation, and for patterning of Ubisch bodies and the exine. We propose that products of NP1 are likely important metabolites in the development of Ubisch bodies and pollen exine, necessary for polymerization, assembly, or both.     

LEVER ACTION ANTHERS AND THE FORCIBLE SHEDDING OF POLLEN IN TORENIA (SCROPHULARIACEAE)

The anthers of Torenia fournieri were found to shed pollen forcibly by lever action. Anther structure was modified for this function by a flangelike outgrowth of the lateral pollen sac wall forming a lever. When pressed, this lever causes an infolding of the thinner, subadjacent, pollen sac wall forcing pollen from the stomium. A force of 1–1.5 g pressing against the four levers of an anther pair resulted in the forcible shedding of 2,000–3,000 pollen grains in two parallel rows. During the 2-day anthesis the flowers shift from functioning as males to hermaphroditic outcrossers, and yet only have a pollen:ovule ratio of 98.6, a ratio more indicative of facultative autogamy. The outermost pair of anthers functions on the first day of anthesis, while the second, inner pair functions on both days. In each flower, the 2-day anther pair produces approximately twice as many pollen grains as the 1 -day anther pair, a pollen production highly correlated with the length of their functional lives. This difference in pollen production is apparent in the larger size of the 2-day anthers, a size difference that first appears with the initiation of the anther primordia.     

Breeding system and bumblebee drone pollination of an explosively pollen‐releasing plant,Meliosma tenuis (Sabiaceae)

• Explosive pollen release is a mechanism used by some angiosperms that serves to attach pollen to a pollinator's body. It is usually adopted by species with zygomorphic tubular flowers and pollinated by birds and bees. The tree genus Meliosma (Sabiaceae, Proteales) has unique disc‐like flowers that are externally actinomorphic, but internally zygomorphic, and release pollen explosively.
• To elucidate the adaptive significance of explosive pollen release, we observed flowering behaviour, the breeding system and pollinator visits to flowers of the Japanese species Meliosma tenuis in a temperate forest. Flowers bloomed in June and were nectariferous and protandrous. Explosive pollen release was triggered by slight tactile stimuli to anther filaments or staminodes in male‐stage flowers.
• Because pollen cannot come into contact with the pistils enclosed by staminodes, M. tenuis is functionally protandrous. Artificial pollination treatments revealed that M. tenuis is allogamous. The dominant flower visitors were nectar‐seeking drones of the bumblebee species Bombus ardens (Apidae). The drones’ behaviour, pollen attachment on their bodies and fruit set of visit‐restricted flowers suggest that they are the only agent triggering the explosive pollen release mechanism, and are the main pollinator of M. tenuis.
• The finding that bumblebee workers rarely visit these flowers suggests that the explosive pollen release has another function, namely to discourage pollen‐harvesting bumblebee workers.

     

Could learning of pollen odours by honey bees (Apis mellifera) play a role in their foraging behaviour?

Abstract. The role of pollen odour cues in the foraging behaviour of honey bees (Apis mellifera L.) is poorly understood. Using classical conditioning of the proboscis extension response, in which bees learn to associate an odour with a sucrose reward, the present study tests whether odours of bee-collected pollen from the hive environment or odours of fresh pollen on the anthers of flowers could be used in pollen foraging. Honey bees efficiently learn odours from field-bean (Vicia faba) bee-collected pollen and oilseed-rape (Brassica napus) bee-collected pollen, hand-collected pollen, anthers and whole flowers, demonstrating that honey bees can learn pollen odours associatively in biologically realistic concentrations. Honey bees learn pollen odours of oilseed rape better than field bean and, although they generalize these two odours, they easily distinguish between them in discrimination tests, suggesting that pollen odours may be used in species recognition/discrimination. There is little evidence that honey bees can recognize whole flowers based on previous experience of bee-collected pollen odour. However, they generalize the odours of oilseed-rape anthers and whole flowers, suggesting that anther pollen in situ may play a more prominent role than bee-collected pollen in foraging behaviour.     

Hormonal control of floral abscission in zucchini squash (<Emphasis Type="Italic">Cucurbita pepo</Emphasis>)

In the zucchini squash, Cucurbita pepo, a well coordinated abscission of the female flower during fruit set is essential to obtain a fruit of commercial value. In Spain zucchini is mainly produced in greenhouses in Almería, where high temperatures during the spring-summer period provoke a cultivar-dependent defect in fruits known as the “sticky flower” syndrome. This disorder is characterised by an arrest in growth and maturation of floral organs, and a lack of female floral abscission, thus diminishing fruit shelf-life, commercial quality and value. The aim of the present work was to improve knowledge of the abscission process in C. pepo to better understand the fundamental causes of this disorder. The anatomical analysis of abscission shows a well defined male floral abscission zone (AZ), few hours after anthesis, which differs from the female zone which is not differentiated from the adjacent tissue until the abscission process has begun, and which occurs as a consequence of AZ cell enlargement and the dissolution of their cell walls. To evaluate the role of ethylene and auxins in the regulation of floral abscission in zucchini we performed several treatments, with: ethylene, added as 0.25% ethrel solution; AVG, the inhibitor of ethylene synthesis, at 100 μM; indol-3-acetic acid, 100 μM; and TIBA, the inhibitor of auxin polar transport, at 10 mM. These treatments show that ethylene is an accelerator of zucchini floral abscission, and also promotes abscission in isolated AZs of sticky flowers. On the other hand, IAA delays abscission of the female flowers, whilst the inhibitor of auxin polar transport promotes it. The activity of the cell wall hydrolytic enzymes, polygalacturonase and cellulase, sharply increased just before the shedding of zucchini floral organs (72 h after anthesis). Moreover, both enzyme activities were induced by ethylene, which partly explains the ethylene promoting effect.     

Patterns of pollen removal and deposition in Polemonium brandegeei (Polemoniaceae): the role of floral visitors,floral design and sexual interference

The arrangement, colour, shape and size of floral parts (collectively floral design) have evolved primarily to promote mating success via animal‐mediated pollen transfer. Although numerous studies have examined variation in pollinator assemblages, relatively few have examined patterns of pollen removal and deposition in the presence of fluctuating pollinators and ineffective floral visitors; therefore, net pollen removal and deposition by entire visitor assemblages are unclear. We studied the timing (diurnal or nocturnal) and effects of floral traits on pollen removal and deposition under a dynamic visitor assemblage of Polemonium brandegeei. We quantified pollen grains remaining in anthers (pollen removal) and deposited on stigmas (pollen deposition) of plants visited during either the day (07:30–20:00 h) or night (20:30–07:30 h) in natural populations over two flowering seasons. Pollen removal and deposition occurred both diurnally and nocturnally during our study. Increased diurnal removal and deposition coincided with peak floral visitations in 2006. This increase in pollen removal and deposition may reflect increased visits by pollen consumers, effective hawkmoth pollinators and increased self‐pollen deposition due to hot, dry weather. Nonlinear effects of style length significantly affected pollen removal, with less pollen remaining in flowers with intermediate style lengths. Pollen deposition was more complex, with herkogamy and anther height affecting deposition. Further, close proximity of stigmas and anthers increased the potential for sexual interference between pollen removal and deposition. Overall, flower visitations and pollen removal and deposition varied between years and populations, but sex organ placement consistently influenced the removal and deposition of pollen.