Seasonal pollen flow and progeny diversity in Amianthium muscaetoxicum: ecological potential for multiple mating in a self-incompatible,hermaphroditic perennial

Summary The ecological potential for multiple mating is high in Amianthium muscaetoxicum. The percentage of long-distance pollinations (20–100 m) is greater than reported for most insect-pollinated systems. Estimations of neighborhood area are at least an order of magnitude larger than any previously reported for plant species. Seasonal effects on fluorescent dust dispersal indicate that neighborhood areas change during the flowering season. The number of flowers marked with fluorescent dust on an inflorescence increases with increasing inflorescence size, and the proportions of available inflorescences that are marked decrease with distance from the source. Allozyme analysis indicates that heterozygosity levels are typical of outcrossing plants. The diversity of seed genotypes is increased by increasing the size of the floral display. The present investigation is the first to consider the effects of floral display on seed diversity and adds to existing data indicating that inflorescence size is important to fecundity and/or pollen donation in some systems.     

Ovule pre-emption and pollen limitation in a self-fertile perennial herb (Blandfordia grandiflora,Liliaceae)

The extent, frequency and causes of pollenlimited seed production were examined in partially selffertile populations of Blandfordia grandiflora for 2 years. Percentage seed set of open-pollinated plants (50–57%) did not differ within or between years, and was about 19% less than experimentally cross-pollinated plants (70–75%). Floral visits by honeybees did not differ through the flowering season and the number of pollen grains deposited on stigmas within 12 h of flowers opening exceeded the number of ovules per flower, indicating that the quality rather than the quantity of pollination limited seed set. Pollen limitation was caused by concurrent self- and cross-pollination and the subsequent abortion of some selfed ovules due to inbreeding depression. Natural seed set (55%) was intermediate between selfed (43%) and crossed (75%) flowers and was not increased when flowers that had been available to pollinators for 24 h were hand cross-pollinated, suggesting that ovules were already fertilized. Similarly, experimental pollination with both cross and self pollen within 24 h of flowers opening did not increase seed set relative to natural seed set, indicating that both cross- and self-fertilizations had occurred. In contrast, when selfing followed crossing by 48 h, or vice versa, seed set did not differ from crossed-only or selfed-only flowers, respectively, indicating that ovules were pre-empted by the first pollination. Collectively, these results indicate that under natural conditions self pollen pre-empts ovules, rendering them unavailable for cross-fertilization. This selfing reduces fecundity by 50%, as estimated from the natural production of cross seeds when selfing was prevented. Consequently, selection should favour floral traits, such as increased stigma-anther separation or protandry, that reduce interference between male and female functions that leads to selfing.     

Spatial genetic structure within two populations of a self-incompatible perennial,Chionographis japonica var.japonica (Liliaceae)

Intrapopulational spatial genetic structure was examined in two populations ofChionographis japonica var.japonica, a self-incompatible perennial, by spatial autocorrelation analysis of enzyme polymorphism. Although most spatial autocorrelation indices (Moran'sI) in the shortes distance class were significantly positive, most in the other distance classes did not significantly deviate from the values expected from random distributions of genotypes in both populations. This contrasts with a spatial genetic pattern previously reported for a population of the predominantly selfing congener,C. japonica var.kurohimensis, indicating that pollen-mediated gene flow highly impedes genetic substructuring within populations of outcrossingC. japonica var.japonica. Genetic similarity in very proximate distance found in outcrossingC. japonica var.japonica is probably due to restricted dispersal of seeds.     

Anther opening, pollen biology and stigma receptivity in the long blooming species, Parietaria judaica L. (Urticaceae)

Background and aimsThis paper is about pollen ecophysiology, anther opening, pollen dispersal and the timing of the male and female phases in Parietaria judaica (Urticaceae).MethodsEcophysiological (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 resultsPollen 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.ConclusionParietaria 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.     

Male sterility, protogyny, and pollen-pistil interference in Plantago maritima (Plantaginaceae), a wind-pollinated, self-incompatible perennial

Evolution and maintenance of male sterility in seed plants can be explained by the maternal inheritance of mitochondria, which encode the trait, and by adaptive functions that enhance female fecundity in male-sterile compared to hermaphrodite individuals. Protogyny and male sterility can independently decrease the negative effect of pollen-pistil interference in self-incompatible species. In Plantago maritima, which possesses both traits, protogyny increases seed set in hermaphrodite individuals. This is shown both by a significantly positive association between seed set and retarded dehiscence of the anthers and by a more than 50% reduction in seed set following self-pollination. Male sterility does not seem to increase seed set further, as female and hermaphrodite plants do not differ significantly in mean seed set per capsule. Bagging experiments demonstrate strong self-incompatibility in the study populations. Hence, in P. maritima male sterility seems neither to prevent selfing nor to reduce the effect of pollen-pistil interference. Females had significantly larger stigmas than hermaphrodites, but seed set varied negatively with stigma length among females, indicating that the evolution of unisexuality in P. maritima is not due to prefertilization sex allocation. I therefore conclude that the genetical system of nucleocytoplasmic determination of gender is the main cause for maintenance of male sterility in P. maritima.     

Asymmetry in pollen flow promotes gender specialization in morphs of the distylous neotropical herb <Emphasis Type="Italic">Arcytophyllum lavarum</Emphasis> (Rubiaceae)

Distyly is a floral polymorphism, characterized by a reciprocal positioning between stigmas and anthers in different flowers, where two floral morphs, long-styled (pin) and short-styled (thrum) occur within the population. Distyly is suggested as one of the routes leading to the evolution of separate sexes in plants. In this evolutionary pathway, pollinators may disrupt the complementarity of pollen transfer between morphs. Consequently, the floral morphs gradually specialize as either male or female. A key process required for gender specialization in distylous plants is a deviation of the realized functional gender (i.e. the proportion of genes transmitted to the next generation via pollen donation and seed production) from the potential functional gender (i.e. the expected contribution of male and female function to reproductive success from the number of ovules or pollen grains produced by each morph). I selected the distylous herb Arcytophyllum lavarum (Rubiaceae) to determine if asymmetry in pollen flow promotes differences in seed production, pollen donation and a discrepancy between the potential and the realized functional genders in pin and thrum floral morphs. Pollen flow in A. lavarum is asymmetric and the pin morph is more efficient at performing cross-pollination than the thrum morph. Conversely, the thrum morph produced two times more seeds than the pin morph. Male and female contributions to the potential functional gender were equivalent in both morphs. However, the pin morph transmitted more genes through pollen donation and the thrum morph more through seed production than expected from their potential functional genders. These results support the hypothesis that if pollinators consistently promote asymmetric pollen flow between morphs over generations, it is possible that gender specialization may evolve to the extreme of dioecism from an original distylous condition.     

Ultrastructure of the dimorphic pollen and stigmas of the cleistogamous species,Collomia grandiflora (Polemoniaceae)

Summary An ultrastructural study of the pollen and stigma of the dimorphic flowers in the cleistogamous speciesCollomia grandiflora reveals significant differences in cytoplasmic features in the pollen and wall features in the papillae. Both pollen types contain lipid and starch reserves, but the smaller CL (cleistogamous) pollen shows a much greater abundance of starch compared to the CH (chasmogamous) pollen. In addition to the papular size and shape differences between the two stigma types, there is a more extensive cuticular stretching and wall microfibrillar loosening over the CH papillar tip. There is no apparent pellicle on the cuticle surface of either type of papilla, only scattered lipidic deposits. It is proposed that these structural differences may contribute to the cross incompatibility between the two floral morphs.     

萱草花粉类动力蛋白生物物理学及药理学性质

利用FPLC技术从萱草花粉中鉴定并纯化了动力蛋白,研究了它的酶学性质及部分生物化学性质。结果如下:纯化的类动力蛋白分子量为100kD,等电点pI=6·15和6·80。在280nm波长激发下,最大的荧光发射波长是346nm。荧光光谱分析结合紫外吸收光谱及导数光谱分析推断它含有色氨酸和酪氨酸残基。药理学性质研究表明巯基可能在酶的活性中心起重要作用。     

PREM-1, a putative maize retroelement has LTR (long terminal repeat) sequences that are preferentially transcribed in pollen

A family of highly dispersed repetitive elements, designated PREM-1, which are transcribed primarily during pollen development, has been identified in maize. Sequence data from six PREM-1-containing genomic clones suggest that the PREM-1 sequences are the LTRs of a family of putative retroelements. PREM-1 LTRs are estimated to be present in about 10 000 to 40 000 copies in the maize genome. Although related sequences have been detected in sorghum and crab grass, highly homologous sequences appear to be specific to the genus Zea (maize and teosinte). A diverse group of RNAs that contain portions of the PREM-1 sequence at their 3 ends are transcribed in pollen; highest levels appear in early uninucleate microspores. The PREM-1-containing cDNAs do not appear to code for protein products since stop codons are present in all three reading frames. The possible significance of expression of retroelements in the male gametophyte, in terms of transposition of DNA, is discussed.     

Feeding friend and foe: ample pollen mitigates the effects of pollen theft for a gynodioecious plant,Polemonium foliosissimum (Polemoniaceae)

Background and AimsMost angiosperms rely on pollinators to transport pollen and effect fertilization. While some floral visitors are effective pollinators, others act as thieves, consuming pollen but effecting little pollination in return. The importance of pollen theft in male and female reproductive success has received little attention. Here, we examined if pollen consumption by flies altered pollen receipt and exacerbated pollen limitation for a bumblebee-pollinated plant, Polemonium foliosissimum (Polemoniaceae).MethodsTo examine the effect of pollen-thieving flies, we took a three-pronged approach. First, we used single-visit observations to quantify pollen removal and pollen deposition by flies and bumblebees. Second, we manipulated pollen in the neighbourhood around focal plants in two years to test whether pollen reduction reduced pollen receipt. Third, we combined pollen reduction with hand-pollination to test whether pollen thieving exacerbated pollen limitation. Polemonium foliosissimum is gynodioecious in most populations in the Elk Mountains of central Colorado, USA. Thus, we also tested whether pollen theft affected hermaphrodites and females differently.ResultsFlies removed significantly more pollen and deposited less pollen per visit than did bumblebees. Reduction of pollen in the neighbourhood around focal plants reduced pollen receipt in both years but only nearly significantly so in 2015. In 2016, plants were significantly pollen-limited; hand-pollination significantly increased seeds per fruit for both hermaphrodites and females. However, the reduction of pollen around focal plants did not exacerbate pollen limitation for either hermaphrodites or females.ConclusionsOur results suggest that plants tolerate significant consumption of pollen by thieves and pollinators by producing ample pollen to feed both and fertilize available ovules. Our results demonstrate that pollen limitation in P. foliosissimum is driven by lack of effective pollinators rather than lack of pollen. Teasing out these effects elucidates the relative importance of drivers of reproductive success and thus the expected response to selection by different floral visitors.     

In vitro germination and transient GFP expression of American chestnut (Castanea dentata) pollen

The development of the male reproductive structures of American chestnut (Castanea dentata) is described to advance our understanding of its reproductive behavior. This information has been vital in the development of a strategy to collect pollen grains from male catkins suitable for in vitro germination and transformation experiments. Cutting male catkins into small segments and rolling them over a culture plate resulted in evenly dispersed and large amounts of pollen with minimal unwanted accessory floral parts. To optimize pollen viability, the effect of various storage conditions on in vitro germination was examined. Our results showed that initial storage at 4°C for 2 weeks significantly increased percent germination as compared to freshly collected pollen and those stored directly at −20°C or −80°C. This also means that for long-term storage of American chestnut pollen, the catkins should first be kept at 4°C for a couple of weeks and then at −80°C. The use of pollen grains with high viability is necessary for the transformation of American chestnut pollen. To optimize pollen transformation via particle bombardment, the effects of target distance, target pressure, and pollen developmental stage were examined. Statistical analysis showed that bombardment of ungerminated pollen at 1,100 psi resulted in the highest percent transient GFP expression (4.1%).     

The expression of a potato (Solanum tuberosum) S-RNase gene in Nicotiana tabacum pollen

Self-incompatibility in the Solanaceae is controlled by a single multiallelic genetic locus, the S locus. The stylar gene products of the S locus are abundant glycoproteins with ribonuclease activity, secreted in the transmitting tract tissue of the pistil. To investigate the structural and functional integrity and possible phenotypic effects of expression of the S-gene product in the male gametophyte, N. tabacum plants were transformed with a construct containing the genomic S ₂ -RNase coding sequence from S. tuberosum under the control of the promoter of the pollen-specific LAT52 gene from tomato. The expression pattern of the S ₂ RNase in the male gametophyte at both the protein and RNA level was found to be identical to that already reported for expression of the -glucuronidase (GUS) gene directed by the LAT52 promoter in transgenic tomato and tobacco. The S ₂ -RNase gene fusion led to a tissue-specific and developmentally regulated accumulation of the S ₂ polypeptide in pollen of transgenic tobacco plants. The transgenic protein product was of the same size and charge as the potato stylar product, had ribonuclease activity, and was glycosylated. The transgenic plants, however, did not show any morphological variations in their flower organs, and their fertility was not influenced by the accumulation of the S ₂ -RNase protein in pollen.     

Floral display, pollinator visitation and reproductive success in the dioecious perennial herb Wurmbea dioica (Liliaceae)

Floral traits that increase attractiveness to pollinators are predicted to evolve through selection on male function rather than on female function. To determine the importance of male-biased selection in dioecious Wurmbea dioica, we examined sexual dimorphism in flower size and number and the effects of these traits on pollinator visitation and reproductive success of male and female plants. Males produced more and larger flowers than did females. Bees and butterflies responded to this dimorphism and visited males more frequently than females, although flies did not differentiate between the sexes. Within sexes, insect pollinators made more visits to and visited more flowers on plants with many flowers. However, visits per flower did not vary with flower number, indicating that visitation was proportional to the number of flowers per plant. When flower number was experimentally held constant, visitation increased with flower size under sunny but not overcast conditions. Flower size but not number affected pollen removal per flower in males and deposition in females. In males, pollen removal increased with flower size 3 days after flowers opened, but not after 6 days when 98% of pollen was removed. Males with larger flowers therefore, may have higher fitness not because pollen removal is more complete, but because pollen is removed more rapidly providing opportunities to pre-empt ovules. In females, pollen deposition increased with flower size 3 days but not 6 days after flowers opened. At both times, deposition exceeded ovule production by four-fold or more, and for 2 years seed production was not limited by pollen. Flower size had no effect on seed production per plant and was negatively related to percent seed set, implying a tradeoff between allocation to attraction and reproductive success. This indicates that larger flower size in females is unlikely to increase fitness. In both sexes, gamete production was positively correlated with flower size. In males, greater pollen production would increase the advantage of large flowers, but in females more ovules may represent a resource cost. Selection to increase flower size and number in W. dioica has probably occurred through male rather than female function. Received: 15 June 1997 / Accepted: 12 February 1998     

Global warming and the earlier start of the Japanese-cedar (Cryptomeria japonica) pollen season in Toyama, Japan

Atmospheric pollen surveys were conducted in Toyama City, Japan over a 21-year period (1983–2003). Airborne pollen was collected by two methods, the gravimetric method and the volumetric method. The gravimetric method indicated that the start of the Cryptomeria japonica pollen season, as indicated by pollen dispersion, has advanced from day 73 (from January 1) in 1983 to day 47 in 2003. Measurements taken using the volumetric method confirmed this trend. There was a significant correlation between the start dates obtained by both methods. Meteorological data indicated that the most noticeable elevation in temperature during the experimental period occurred in February – an increase of 2.1°C. Significant correlations existed between the mean temperatures and the start dates of the pollen season. These results support the steadily increasing number of reports indicating a global warming trend. The temperature change in February in affecting the start dates of the C. japonica pollen season is particularly relevant in the context of human health. Further studies will be needed to clarify the effects of the global warming trend on the pollen season and human health in more detail.     

棉铃虫激活增强子结合蛋白AP-4基因的原核表达、多克隆抗体制备、表达谱分析及功能鉴定

【目的】激活增强子结合蛋白4(activating enhancer binding protein 4, AP-4)是近年来备受关注的一类功能广泛的转录因子,参与Wnt/β-catenin信号通路的调控。本研究旨在研究棉铃虫Helicoverpa armigera HaAP-4基因的原核表达并制备多克隆抗体,明确HaAP-4基因的时空表达谱,初步探究HaAP-4对棉铃虫胆固醇载体蛋白2(sterol carrier protein-2, SCP-2)基因(HaSCP-2)表达的调控作用。【方法】通过PCR扩增棉铃虫HaAP-4基因片段,将其克隆至pET-28a原核表达载体,转化至大肠杆菌Escherichia coli BL21,经IPTG诱导表达,采用镍柱纯化重组蛋白,免疫新西兰兔制备多克隆抗体。运用RT-qPCR检测HaAP-4基因在棉铃虫不同发育阶段(卵、幼虫、预蛹、蛹和成虫)以及5龄幼虫和预蛹不同组织(中肠、脂肪体、头和表皮)中的表达水平。设计并合成HaAP-4 siRNA,转染棉铃虫Ha细胞,通过RT-qPCR检测和分析使用HaAP-4 siRNA进行RNA干扰后Ha细胞中HaAP-4和HaSCP-2基因 mRNA的表达情况。【结果】成功构建pET-28a-HaAP-4重组表达质粒,在大肠杆菌细胞中获得高效表达的可溶性蛋白,目的蛋白大小约为55 kD,经纯化获得了纯度较高的重组蛋白,免疫新西兰兔成功制备了多克隆抗体,通过Western blotting检测显示抗体具有较好的特异性,ELISA分析表明抗体效价较高。发育阶段特异性表达结果显示,HaAP-4基因在棉铃虫不同发育阶段均有表达,其中在卵期、5龄幼虫期和预蛹期的表达量较高。组织表达结果表明,HaAP-4基因在5龄幼虫和预蛹不同组织中均有表达,且在中肠和头部具有高表达,而在表皮和脂肪体中表达量较低。RNA干扰实验发现,HaAP-4基因的敲降对HaSCP-2基因转录有显著影响,HaSCP-2基因mRNA表达水平下降了约55%。【结论】利用pET-28a原核表达系统能有效地在体外表达可溶性的HaAP-4,并制备了较好的多克隆抗体。RNAi实验结果提示,在中肠内高表达的HaAP-4基因能促进HaSCP-2基因的转录表达,在棉铃虫脂质生理代谢过程中发挥作用。本研究为进一步深入阐明棉铃虫HaAP-4的潜在功能打下了基础。     

C-型咽侧体抑制素(AST-C)和促前胸腺激素释放激素(PTTH)基因在家蚕脑组织中的时空表达分析

【目的】咽侧体抑制素在昆虫体内具有重要的调控功能。本研究比较了家蚕Bombyx mori C-型咽侧体抑制素(allatostatin C,AST-C)与促前胸腺激素释放激素(prothoracicotropic hormone,PTTH)基因在不同发育阶段家蚕脑组织中转录表达的模式及其在脑组织的表达定位,以期为家蚕AST-C的功能研究提供重要的线索。【方法】利用脑组织芯片数据分析比较AST-C和PTTH基因在家蚕脑组织中的发育表达特征,RT-PCR验证其芯片数据,分析AST-C在不同发育阶段家蚕中枢神经系统中的表达模式,并用全组织包埋原位杂交技术对AST-C和PTTH在脑组织的表达进行定位。【结果】AST-C和PTTH在不同发育阶段家蚕脑组织中有相似的转录表达模式,且都在脑组织外侧一对神经分泌细胞中表达。【结论】AST-C可能与PTTH以相同的转录表达模式共同参与家蚕的变态发育调控。     

Lily pollen alkaline phytase is a histidine phosphatase similar to mammalian multiple inositol polyphosphate phosphatase (MINPP)

Phytic acid is the most abundant inositol phosphate in cells; it constitutes 1-5% of the dry weight of cereal grains and legumes. Phytases are the primary enzymes responsible for the hydrolysis of phytic acid and thus play important roles in inositol phosphate metabolism. A novel alkaline phytase in lily pollen (LlALP) was recently purified in our laboratory. In this paper, we describe the cloning and characterization of LlALP cDNA from lily pollen. Two isoforms of alkaline phytase cDNAs, LlAlp1 and LlAlp2, which are 1467 and 1533 bp long and encode proteins of 487 and 511 amino acids, respectively, were identified. The deduced amino acid sequences contains the signature heptapeptide of histidine phosphatases, -RHGXRXP-, but shares < 25% identity to fungal histidine acid phytases. Phylogenetic analysis reveals that LlALP is most closely related to multiple inositol polyphosphate phosphatase (MINPP) from humans (25%) and rats (23%). mRNA corresponding to LlAlp1 and LlAlp2 were expressed in leaves, stem, petals and pollen grains. The expression profiles of LlAlp isoforms in anthers indicated that mRNA corresponding to both isoforms were present at all stages of flower development. The expression of LlAlp2 cDNA in Escherichia coli revealed the accumulation of the active enzyme in inclusion bodies and confirmed that the cDNA encodes an alkaline phytase. In summary, plant alkaline phytase is a member of the histidine phosphatase family that includes MINPP and exhibits properties distinct from bacterial and fungal phytases.     

Between-year changes in functional gender expression of Palicourea padifolia (Rubiaceae), a distylous, hummingbird-pollinated shrub

BACKGROUND AND AIMS: Because distylous species have two hermaphroditic style-length floral morphs, they face two sex allocation problems: the equilibrium morph ratio and the optimal allocation to pollen and seed production in each floral morph. Gender specialization is expected among distylous species when floral morphs differ in reproductive output. However, spatio-temporal variability in female reproductive output between morphs needs to be investigated to fully understand patterns of sexual expression and gender specialization in distylous plants. Between-year variation in flower and fruit production of hummingbird-pollinated Palicourea padifolia (Rubiaceae) was examined, focusing on functional gender expression of long- and short-styled morphs and comparing their reproductive performance in five consecutive years (1998-2002). METHODS: Between-year variation in inflorescence, floral bud and fruit production was monitored and quantified. These traits were then used as parameters to determine functional gender differences between floral morphs through time. KEY RESULTS: Inflorescence production varied among years but no significant differences were found between floral morphs. Long-styled plants initiated more floral buds per inflorescence every year than short-styled plants, suggesting higher allocation to pollinator attraction and, potentially, an increase in male fitness through pollen donation. Although fruit production was similar between morphs, their functional gender shifted across years. CONCLUSIONS: The gender expression inconsistency across years is surprising because a number of floral characters and attributes that contribute to differently attracting and rewarding effective pollinators in P. padifolia suggest gender specialization. The evidence that morphs of distylous species might specialize in functional gender mostly comes from differences among populations in seed production and non-equilibrium morph ratios based on 1-year field population surveys. The results suggest that more sampling through time is needed to detect gender specialization among distylous species with a perennial habit.