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1.  The progamic phase of an early-divergent angiosperm, Annona cherimola (Annonaceae) 
Annals of Botany  2009;105(2):221-231.
Background and Aims
Recent studies of reproductive biology in ancient angiosperm lineages are beginning to shed light on the early evolution of flowering plants, but comparative studies are restricted by fragmented and meagre species representation in these angiosperm clades. In the present study, the progamic phase, from pollination to fertilization, is characterized in Annona cherimola, which is a member of the Annonaceae, the largest extant family among early-divergent angiosperms. Beside interest due to its phylogenetic position, this species is also an ancient crop with a clear niche for expansion in subtropical climates.
The kinetics of the reproductive process was established following controlled pollinations and sequential fixation. Gynoecium anatomy, pollen tube pathway, embryo sac and early post-fertilization events were characterized histochemically.
Key Results
A plesiomorphic gynoecium with a semi-open carpel shows a continuous secretory papillar surface along the carpel margins, which run from the stigma down to the obturator in the ovary. The pollen grains germinate in the stigma and compete in the stigma-style interface to reach the narrow secretory area that lines the margins of the semi-open stylar canal and is able to host just one to three pollen tubes. The embryo sac has eight nuclei and is well provisioned with large starch grains that are used during early cellular endosperm development.
A plesiomorphic simple gynoecium hosts a simple pollen–pistil interaction, based on a support–control system of pollen tube growth. Support is provided through basipetal secretory activity in the cells that line the pollen tube pathway. Spatial constraints, favouring pollen tube competition, are mediated by a dramatic reduction in the secretory surface available for pollen tube growth at the stigma–style interface. This extramural pollen tube competition contrasts with the intrastylar competition predominant in more recently derived lineages of angiosperms.
PMCID: PMC2814751  PMID: 19939980
Annona cherimola; Annonaceae; embryo sac; endosperm; Magnoliid; ovule; pollen–pistil interaction; pollen tube
2.  Establishment of the male germline and sperm cell movement during pollen germination and tube growth in maize 
Plant Signaling & Behavior  2010;5(7):885-889.
Two sperm cells are required to achieve double fertilization in flowering plants (angiosperms). In contrast to animals and lower plants such as mosses and ferns, sperm cells of flowering plants (angiosperms) are immobile and are transported to the female gametes (egg and central cell) via the pollen tube. The two sperm cells arise from the generative pollen cell either within the pollen grain or after germination inside the pollen tube. While pollen tube growth and sperm behavior has been intensively investigated in model plant species such as tobacco and lily, little is know about sperm dynamics and behavior during pollen germination, tube growth and sperm release in grasses. In the March issue of Journal of Experimental Botany, we have reported about the sporophytic and gametophytic control of pollen tube germination, growth and guidance in maize.1 Five progamic phases were distinguished involving various prezygotic crossing barriers before sperm cell delivery inside the female gametophyte takes place. Using live cell imaging and a generative cell-specific promoter driving α-tubulin-YFP expression in the male germline, we report here the formation of the male germline inside the pollen grain and the sperm behaviour during pollen germination and their movement dynamics during tube growth in maize.
PMCID: PMC3014542  PMID: 20505353
male gametophyte; generative cell; sperm; pollen tube; tubulin; fertilization; maize
3.  How does timing, duration and severity of heat stress influence pollen-pistil interactions in angiosperms? 
Plant Signaling & Behavior  2011;6(7):930-933.
Reproductive development in sexual plants is substantially more sensitive to high temperature stress than vegetative development, resulting in negative implications for food and fiber production under the moderate temperature increases projected to result from global climate change. High temperature exposure either during early pollen development or during the progamic phase of pollen development will negatively impact pollen performance and reproductive output; both phases of pollen development are considered exceptionally sensitive to moderate heat stress. However, moderately elevated temperatures either before or during the progamic phase can limit fertilization by negatively impacting important pollen pistil interactions required for successful pollen tube growth toward the ovules. This mini-review identifies the impacts of heat stress on pollen-pistil interactions and sexual reproduction in angiosperms. A special emphasis is placed on the biochemical response of the pistil to moderately high temperature and the resultant influence on in vivo pollen performance and fertilization.
PMCID: PMC3257764  PMID: 21628998
pollen-pistil interaction; carbohydrates; heat stress; fertilization; pollen tube growth; climate change
4.  Sporophytic control of pollen tube growth and guidance in maize 
Journal of Experimental Botany  2009;61(3):673-682.
Pollen tube germination, growth, and guidance (progamic phase) culminating in sperm discharge is a multi-stage process including complex interactions between the male gametophyte as well as sporophytic tissues and the female gametophyte (embryo sac), respectively. Inter- and intra-specific crossing barriers in maize and Tripsacum have been studied and a precise description of progamic pollen tube development in maize is reported here. It was found that pollen germination and initial tube growth are rather unspecific, but an early, first crossing barrier was detected before arrival at the transmitting tract. Pollination of maize silks with Tripsacum pollen and incompatible pollination of Ga1s/Ga1s-maize silks with ga1-maize pollen revealed another two incompatibility barriers, namely transmitting tract mistargeting and insufficient growth support. Attraction and growth support by the transmitting tract seem to play key roles for progamic pollen tube growth. After leaving transmitting tracts, pollen tubes have to navigate across the ovule in the ovular cavity. Pollination of an embryo sac-less maize RNAi-line allowed the role of the female gametophyte for pollen tube guidance to be determined in maize. It was found that female gametophyte controlled guidance is restricted to a small region around the micropyle, approximately 50–100 μm in diameter. This area is comparable to the area of influence of previously described ZmEA1-based short-range female gametophyte signalling. In conclusion, the progamic phase is almost completely under sporophytic control in maize.
PMCID: PMC2814102  PMID: 19926683
Female gametophyte; maize; pollen tube guidance; prezygotic barriers; transmitting tract; Tripsacum
5.  De novo post-pollen mitosis II tobacco pollen tube transcriptome 
Plant Signaling & Behavior  2012;7(8):918-921.
In our previous study we applied the Agilent 44K tobacco gene chip to introduce and analyze the tobacco male gametophyte transcriptome in mature pollen and 4h pollen tubes. Here we extended our analysis post-pollen mitosis II (PMII) by including a new data set obtained from more advanced stage of the ongoing progamic phase – pollen tubes cultivated in vitro for 24 h. Pollen mitosis II marks key events in the control of male gametophyte development, the production of two sperm cells. In bicellular species covering cca 70% of angiosperms including Nicotiana tabacum, PMII takes place after pollen germination in growing pollen tube. We showed the stable and even slightly increasing complexity of tobacco male gametophyte transcriptome over long period of progamic phase–24 h of pollen tube growth. We also demonstrated the ongoing transcription activity and specific transcript accumulation in post-PMII pollen tubes cultivated in vitro. In all, we have identified 320 genes (2.2%) that were newly transcribed at least after 4h of pollen tube cultivation in vitro. Further, 699 genes (4.8%) showed over 5-fold increased accumulation after the 24h of cultivation.
PMCID: PMC3474685  PMID: 22827945
de novo pollen tube transcriptome; male gametophyte development; pollen tube growth; transcriptomics
6.  Pollination function transferred: modified tepals of Albuca (Hyacinthaceae) serve as secondary stigmas 
Annals of Botany  2012;110(3):565-572.
Background and Aims
The stigma, a structure which serves as a site for pollen receipt and germination, has been assumed to have evolved once, as a modification of carpels, in early angiosperms. Here it is shown that a functional stigma has evolved secondarily from modified tepals in some Albuca species (Hyacinthaceae).
Deposition of pollen on Albuca floral organs by bees was recorded. Pollen germination and fruit set was measured in flowers that had pollen deposited solely on their tepals or had their tepal tips experimentally isolated or removed after pollination.
Key Results
Leafcutter bees deposit pollen onto the papillate apices of the inner tepals of Albuca flowers. Pollen germinates in tepal-derived fluid secreted 2 or 3 d after anthesis and pollen tubes subsequently penetrate the style during flower wilting. Application of cross-pollen to the inner tepal apices of A. setosa flowers led to high fruit set. No fruits were produced in pollinated flowers in which the inner tepals were mechanically isolated or removed.
Pollen capture by tepals in the Albuca clade probably evolved in response to selection for floral morphology that maximizes the accuracy of pollen transfer. These findings show how pollination function can be transferred among floral organs, and shed light on how the original angiosperm stigma developed from sporophylls.
PMCID: PMC3400444  PMID: 22652419
Hyacinthaceae; Ornithogaloideae; pollen; pollen germination; pollen receipt; pollen tube; pollination; sexual interference
7.  High-Throughput Sequencing of Small RNAs from Pollen and Silk and Characterization of miRNAs as Candidate Factors Involved in Pollen-Silk Interactions in Maize 
PLoS ONE  2013;8(8):e72852.
In angiosperms, successful pollen-pistil interactions are the prerequisite and guarantee of subsequent fertilization and seed production. Recent profile analyses have helped elucidate molecular mechanisms underlying these processes at both transcriptomic and proteomic levels, but the involvement of miRNAs in pollen-pistil interactions is still speculative. In this study, we sequenced four small RNA libraries derived from mature pollen, in vitro germinated pollen, mature silks, and pollinated silks of maize (Zea mays L.). We identified 161 known miRNAs belonging to 27 families and 82 novel miRNAs. Of these, 40 conserved and 16 novel miRNAs showed different expression levels between mature and germinated pollen, and 30 conserved and eight novel miRNAs were differentially expressed between mature and pollinated silks. As candidates for factors associated with pollen-silk (pistil) interactions, expression patterns of the two sets of differentially expressed miRNAs were confirmed by stem-loop real-time RT-PCR. Transcript levels of 22 predicted target genes were also validated using real-time RT-PCR; most of these exhibited expression patterns contrasting with those of their corresponding miRNAs. In addition, GO analysis of target genes of differentially expressed miRNAs revealed that functional categories related to auxin signal transduction and gene expression regulation were overrepresented. These results suggest that miRNA-mediated auxin signal transduction and transcriptional regulation have roles in pollen-silk interactions. The results of our study provide novel information for understanding miRNA regulatory roles in pollen-pistil interactions.
PMCID: PMC3749131  PMID: 23991159
8.  Comprehensive Cell-specific Protein Analysis in Early and Late Pollen Development from Diploid Microsporocytes to Pollen Tube Growth* 
Pollen development in angiosperms is one of the most important processes controlling plant reproduction and thus productivity. At the same time, pollen development is highly sensitive to environmental fluctuations, including temperature, drought, and nutrition. Therefore, pollen biology is a major focus in applied studies and breeding approaches for improving plant productivity in a globally changing climate. The most accessible developmental stages of pollen are the mature pollen and the pollen tubes, and these are thus most frequently analyzed. To reveal a complete quantitative proteome map, we additionally addressed the very early stages, analyzing eight stages of tobacco pollen development: diploid microsporocytes, meiosis, tetrads, microspores, polarized microspores, bipolar pollen, desiccated pollen, and pollen tubes. A protocol for the isolation of the early stages was established. Proteins were extracted and analyzed by means of a new gel LC-MS fractionation protocol. In total, 3817 protein groups were identified. Quantitative analysis was performed based on peptide count. Exceedingly stage-specific differential protein regulation was observed during the conversion from the sporophytic to the gametophytic proteome. A map of highly specialized functionality for the different stages could be revealed from the metabolic activity and pronounced differentiation of proteasomal and ribosomal protein complex composition up to protective mechanisms such as high levels of heat shock proteins in the very early stages of development.
PMCID: PMC3879621  PMID: 24078888
9.  Proteomic analyses of apoplastic proteins from germinating Arabidopsis thaliana pollen 
Biochimica et biophysica acta  2011;1814(12):1964-1973.
Pollen grains play important roles in the reproductive processes of flowering plants. The roles of apoplastic proteins in pollen germination and in pollen tube growth are comparatively less well understood. To investigate the functions of apoplastic proteins in pollen germination, the global apoplastic proteins of mature and germinated Arabidopsis thaliana pollen grains were prepared for differential analyses by using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE) saturation labeling techniques. One hundred and three proteins differentially expressed (p value ≤ 0.01) in pollen germinated for 6h compare with un-germination mature pollen, and 98 spots, which represented 71 proteins, were identified by LC-MS/MS. By bioinformatics analysis, 50 proteins were identified as secreted proteins. These proteins were mainly involved in cell wall modification and remodeling, protein metabolism and signal transduction. Three of the differentially expressed proteins were randomly selected to determine their subcellular localizations by transiently expressing YFP fusion proteins. The results of subcellular localization were identical with the bioinformatics prediction. Based on these data, we proposed a model for apoplastic proteins functioning in pollen germination and pollen tube growth. These results will lead to a better understanding of the mechanisms of pollen germination and pollen tube growth.
PMCID: PMC3214693  PMID: 21798377
Arabidopsis thaliana; pollen germination; apoplast; 2-D DIGE; proteomic
10.  MYB97, MYB101 and MYB120 Function as Male Factors That Control Pollen Tube-Synergid Interaction in Arabidopsis thaliana Fertilization 
PLoS Genetics  2013;9(11):e1003933.
Pollen tube reception involves a pollen tube-synergid interaction that controls the discharge of sperm cells into the embryo sac during plant fertilization. Despite its importance in the sexual reproduction of plants, little is known about the role of gene regulation in this process. We report here that the pollen-expressed transcription factors MYB97, MYB101 and MYB120 probably control genes whose encoded proteins play important roles in Arabidopsis thaliana pollen tube reception. They share a high amino acid sequence identity and are expressed mainly in mature pollen grains and pollen tubes. None of the single or double mutants of these three genes exhibited any visible defective phenotype. Although the myb97 myb101 myb120 triple mutant was not defective in pollen development, pollen germination, pollen tube growth or tube guidance, the pollen tubes of the triple mutants exhibited uncontrolled growth and failed to discharge their sperm cells after entering the embryo sac. In addition, the myb97 myb101 myb120 triple mutation significantly affected the expression of a group of pollen-expressed genes in mature pollen grains. All these results indicate that MYB97, MYB101 and MYB120 participate in pollen tube reception, possibly by controlling the expression of downstream genes.
Author Summary
Pollen tube reception is an important step of fertilization and is controlled by interactions between the pollen tube and synergid. Components of both the pollen tube and synergid are believed to be involved in the process. Several proteins associated with this process have been identified in synergid cells. However, very little is known about the components contributed by the pollen tube. This work identified a group of Arabidopsis pollen-expressed MYB transcription factors, among which at least three members are involved in pollen tube reception. The myb97 myb101 myb120 triple mutation caused overgrowth of the pollen tube into the embryo sac and disrupted sperm cell discharge, leading to failed fertilization. This study provides novel evidence demonstrating that male factors are involved in pollen tube reception.
PMCID: PMC3836714  PMID: 24278028
11.  Pollen dispersal and gene flow within and into a population of the alpine monocarpic plant Campanula thyrsoides 
Annals of Botany  2012;110(7):1479-1488.
Background and Aims
Gene flow by seed and pollen largely shapes the genetic structure within and among plant populations. Seed dispersal is often strongly spatially restricted, making gene flow primarily dependent on pollen dispersal within and into populations. To understand distance-dependent pollination success, pollen dispersal and gene flow were studied within and into a population of the alpine monocarpic perennial Campanula thyrsoides.
A paternity analysis was performed on sampled seed families using microsatellites, genotyping 22 flowering adults and 331 germinated offspring to estimate gene flow, and pollen analogues were used to estimate pollen dispersal. The focal population was situated among 23 genetically differentiated populations on a subalpine mountain plateau (<10 km2) in central Switzerland.
Key Results
Paternity analysis assigned 110 offspring (33·2 %) to a specific pollen donor (i.e. ‘father’) in the focal population. Mean pollination distance was 17·4 m for these offspring, and the pollen dispersal curve based on positive LOD scores of all 331 offspring was strongly decreasing with distance. The paternal contribution from 20–35 offspring (6·0–10·5 %) originated outside the population, probably from nearby populations on the plateau. Multiple potential fathers were assigned to each of 186 offspring (56·2 %). The pollination distance to ‘mother’ plants was negatively affected by the mothers' degree of spatial isolation in the population. Variability in male mating success was not related to the degree of isolation of father plants.
Pollen dispersal patterns within the C. thyrsoides population are affected by spatial positioning of flowering individuals and pollen dispersal may therefore contribute to the course of evolution of populations of this species. Pollen dispersal into the population was high but apparently not strong enough to prevent the previously described substantial among-population differentiation on the plateau, which may be due to the monocarpic perenniality of this species.
PMCID: PMC3489145  PMID: 22718086
Gene flow; pollen dispersal; Campanula thyrsoides; European Alps; male mating success; monocarpic perenniality; paternity analysis; pollen analogues; pollination distance
12.  Floral traits mediate the vulnerability of aloes to pollen theft and inefficient pollination by bees 
Annals of Botany  2012;109(4):761-772.
Background and Aims
Pollen-collecting bees are among the most important pollinators globally, but are also the most common pollen thieves and can significantly reduce plant reproduction. The pollination efficiency of pollen collectors depends on the frequency of their visits to female(-phase) flowers, contact with stigmas and deposition of pollen of sufficient quantity and quality to fertilize ovules. Here we investigate the relative importance of these components, and the hypothesis that floral and inflorescence characteristics mediate the pollination role of pollen collection by bees.
For ten Aloe species that differ extensively in floral and inflorescence traits, we experimentally excluded potential bird pollinators to quantify the contributions of insect visitors to pollen removal, pollen deposition and seed production. We measured corolla width and depth to determine nectar accessibility, and the phenology of anther dehiscence and stigma receptivity to quantify herkogamy and dichogamy. Further, we compiled all published bird-exclusion studies of aloes, and compared insect pollination success with floral morphology.
Key Results
Species varied from exclusively insect pollinated, to exclusively bird pollinated but subject to extensive pollen theft by insects. Nectar inaccessibility and strong dichogamy inhibited pollination by pollen-collecting bees by discouraging visits to female-phase (i.e. pollenless) flowers. For species with large inflorescences of pollen-rich flowers, pollen collectors successfully deposited pollen, but of such low quality (probably self-pollen) that they made almost no contribution to seed set. Indeed, considering all published bird-exclusion studies (17 species in total), insect pollination efficiency varied significantly with floral shape.
Species-specific floral and inflorescence characteristics, especially nectar accessibility and dichogamy, control the efficiency of pollen-collecting bees as pollinators of aloes.
PMCID: PMC3286288  PMID: 22278414
Pollen theft; pollination efficiency; dichogamy; floral morphology; Aloe; Alooideae; Xanthorrhoeaceae; Asphodeloideae
13.  Cell Type-Specific Transcriptome of Brassicaceae Stigmatic Papilla Cells From a Combination of Laser Microdissection and RNA Sequencing 
Plant and Cell Physiology  2013;54(11):1894-1906.
Pollination is an early and critical step in plant reproduction, leading to successful fertilization. It consists of many sequential processes, including adhesion of pollen grains onto the surface of stigmatic papilla cells, foot formation to strengthen pollen–stigma interaction, pollen hydration and germination, and pollen tube elongation and penetration. We have focused on an examination of the expressed genes in papilla cells, to increase understanding of the molecular systems of pollination. From three representative species of Brassicaceae (Arabidopsis thaliana, A. halleri and Brassica rapa), stigmatic papilla cells were isolated precisely by laser microdissection, and cell type-specific gene expression in papilla cells was determined by RNA sequencing. As a result, 17,240, 19,260 and 21,026 unigenes were defined in papilla cells of A. thaliana, A. halleri and B. rapa, respectively, and, among these, 12,311 genes were common to all three species. Among the17,240 genes predicted in A. thaliana, one-third were papilla specific while approximately half of the genes were detected in all tissues examined. Bioinformatics analysis revealed that genes related to a wide range of reproduction and development functions are expressed in papilla cells, particularly metabolism, transcription and membrane-mediated information exchange. These results reflect the conserved features of general cellular function and also the specific reproductive role of papilla cells, highlighting a complex cellular system regulated by a diverse range of molecules in these cells. This study provides fundamental biological knowledge to dissect the molecular mechanisms of pollination in papilla cells and will shed light on our understanding of plant reproduction mechanisms.
PMCID: PMC3814185  PMID: 24058146
Arabidopsis halleri; Arabidopsis thaliana; Brassica rapa; Laser microdissection; Papilla cell; RNA sequencing
14.  Why fast-growing pollen tubes give rise to vigorous progeny: the test of a new mechanism 
A positive correlation between the speed of pollen tube growth and the quality of the resulting progeny in several species of flowering plants has traditionally been explained as being caused by an overlap in gene expression between gametophytes and sporophytes. We experimentally manipulated the pollen tube growth rates of pollen donors, such that the genotype controlling the rate was uncoupled from the phenotype, to test the alternative hypothesis that the correlation arises because ovules fertilized early are better provisioned by the maternal plant than later-fertilized ovules. Crosses using Silene vulgaris individuals bearing distinctive genetic markers revealed a correlation between the order of fertilization by pollen grains and vigour of the resulting sporophytes, which was independent of the speed of growth of the pollen tubes. Seeds sired by donors with relatively fast-growing pollen germinated earlier and grew larger than those sired by slow-growing pollen when pollen from the two donors was applied simultaneously. Reversing the order of arrival in the ovary by placing slow-growing pollen on the styles earlier and closer to the ovary led to reverse results. These results suggest that differential provisioning by the maternal plant can lead to differences in progeny vigour following pollen competition.
PMCID: PMC1689050
15.  Reproductive ecology of the basal angiosperm Trithuria submersa (Hydatellaceae) 
Annals of Botany  2010;106(6):909-920.
Background and Aims
Trithuria, the sole genus in the family Hydatellaceae, is an important group for understanding early angiosperm evolution because of its sister relationship to the ancient lineage, Nymphaeales (water lilies). Although also aquatic, Trithuria differs from water lilies in that all species are extremely small, and most have an annual life form and grow in seasonal wetlands. Very little is known about their reproductive ecology. This paper reports on reproductive timing, mode of pollination and characteristics of the breeding system of Trithuria submersa in Western Australia.
Mass collections of open-pollinated plants from different ecological settings were used to characterize the reproductive developmental sequence and natural pollen reception. Hand-pollination, caging and emasculation experiments were used to measure outcross + geitonogamous pollen reception versus autonomous self-pollination in two populations over two field seasons.
Key Results
Natural outcross or geitonogamous pollination was by wind, not by water or insects, but pollen reception was extremely low. Pollen production was very low and pollen release was non-synchronous within populations. The pollen to ovule (P/O) ratio was 23·9, compared with 1569·1 in dioecious Trithuria austinensis. Stigmas became receptive before male phase and remained so until anthers dehisced and autonomous self-pollination occurred. Natural pollen loads are composed primarily of self pollen. Self- and open-pollinated plants had equivalent seed set (both >70 %). Self-pollinated plants produced seed within 17 d.
Autonomous self-pollination and self-fertilization are predominant in T. submersa. The low P/O ratio is not an artefact of small plant size and is inconsistent with long-term pollination by wind. It indicates that T. submersa has evolved a primarily autogamous breeding system. Selfing, along with the effect of small plant size on the speed of reproduction, has enabled T. submersa to colonize marginal ephemeral wetlands in the face of unpredictable pollination.
PMCID: PMC2990668  PMID: 21047886
Autogamy; basal angiosperm; delayed self-pollination; Hydatellaceae; Nymphaeales; wind pollination; reproductive assurance; reproductive timing; stigma receptivity; Trithuria submersa
16.  Pollen tube growth and guidance: roles of small, secreted proteins 
Annals of Botany  2011;108(4):627-636.
Pollination is a crucial step in angiosperm (flowering plant) reproduction. Highly orchestrated pollen–pistil interactions and signalling events enable plant species to avoid inbreeding and outcrossing as a species-specific barrier. In compatible pollination, pollen tubes carrying two sperm cells grow through the pistil transmitting tract and are precisely guided to the ovules, discharging the sperm cells to the embryo sac for fertilization.
In Lilium longiflorum pollination, growing pollen tubes utilize two critical mechanisms, adhesion and chemotropism, for directional growth to the ovules. Among several molecular factors discovered in the past decade, two small, secreted cysteine-rich proteins have been shown to play major roles in pollen tube adhesion and reorientation bioassays: stigma/style cysteine-rich adhesin (SCA, approx. 9·3 kDa) and chemocyanin (approx. 9·8 kDa). SCA, a lipid transfer protein (LTP) secreted from the stylar transmitting tract epidermis, functions in lily pollen tube tip growth as well as in forming the adhesive pectin matrix at the growing pollen tube wall back from the tip. Lily chemocyanin is a plantacyanin family member and acts as a directional cue for reorienting pollen tubes. Recent consecutive studies revealed that Arabidopsis thaliana homologues for SCA and chemocyanin play pivotal roles in tip polarity and directionality of pollen tube growth, respectively. This review outlines the biological roles of various secreted proteins in angiosperm pollination, focusing on plant LTPs and chemocyanin.
PMCID: PMC3170145  PMID: 21307038
Angiosperm fertilization; Arabidopsis thaliana; chemocyanin; cysteine-rich peptides (CRPs); Lilium longiflorum; lipid transfer proteins (LTPs); plantacyanins; pollen tube tip growth; stigma/style cysteine-rich adhesin (SCA)
17.  Zm908p11, encoded by a short open reading frame (sORF) gene, functions in pollen tube growth as a profilin ligand in maize 
Journal of Experimental Botany  2013;64(8):2359-2372.
Double fertilization of flowering plants depends on the targeted transportation of sperm to the embryo sac by the pollen tube. Currently, little is known about the underlying molecular mechanisms that regulate pollen germination and pollen tube growth in maize (Zea mays). Here, a maize pollen-predominant gene Zm908, with several putative short open reading frames (sORFs), was isolated and characterized. The longest ORF of Zm908 encodes a small protein of 97 amino acids. This was designated as Zm908p11 and is distributed throughout the maize pollen tube. Western blot detected the small peptide in mature pollen. Quantitative reverse transcription–PCR and northern blot analysis revealed that Zm908p11 was expressed predominantly in mature pollen grains. Ectopic overexpression of full-length Zm908 and Zm908p11 in tobacco resulted in defective pollen, while transgenic tobacco plants with a site-specific mutation or a frameshift mutation of Zm908p11 showed normal pollen development. Overexpression of Zm908p11 in maize decreased pollen germination efficiency. Maize pollen cDNA library screening and protein–protein interaction assays demonstrated that Zm908p11 interacts with maize profilin 1 (ZmPRO1). A microarray analysis identified 273 up-regulated and 203 down-regulated genes in the overexpressing transgenic Zm908p11 pollen. Taken together, these results indicate that Zm908 functions as Zm908p11, and binds to profilins as a novel ligand, with a required role during pollen tube growth in maize. Accordingly, a model is proposed for the role of Zm908p11 during pollen tube growth in maize.
PMCID: PMC3654424  PMID: 23676884
pollen tube; pollen-predominant; profilin; short open reading frame; Zea mays; Zm908p11.
18.  Wind of change: new insights on the ecology and evolution of pollination and mating in wind-pollinated plants 
Annals of Botany  2009;103(9):1515-1527.
The rich literature that characterizes the field of pollination biology has focused largely on animal-pollinated plants. At least 10 % of angiosperms are wind pollinated, and this mode of pollination has evolved on multiple occasions among unrelated lineages, and hence this discrepancy in research interest is surprising. Here, the evolution and functional ecology of pollination and mating in wind-pollinated plants are discussed, a theoretical framework for modelling the selection of wind pollination is outlined, and pollen capture and the occurrence of pollen limitation in diverse wind-pollinated herbs are investigated experimentally.
Scope and Conclusions
Wind pollination may commonly evolve to provide reproductive assurance when pollinators are scarce. Evidence is presented that pollen limitation in wind-pollinated plants may not be as common as it is in animal-pollinated species. The studies of pollen capture in wind-pollinated herbs demonstrate that pollen transfer efficiency is not substantially lower than in animal-pollinated plants as is often assumed. These findings challenge the explanation that the evolution of few ovules in wind-pollinated flowers is associated with low pollen loads. Floral and inflorescence architecture is crucial to pollination and mating because of the aerodynamics of wind pollination. Evidence is provided for the importance of plant height, floral position, and stamen and stigma characteristics in promoting effective pollen dispersal and capture. Finally, it is proposed that geitonogamous selfing may alleviate pollen limitation in many wind-pollinated plants with unisexual flowers.
PMCID: PMC2701749  PMID: 19218583
Wind pollination; reproductive assurance; pollen limitation; geitonogamy; sex allocation; inflorescence architecture; mating systems
19.  Sterile flowers increase pollinator attraction and promote female success in the Mediterranean herb Leopoldia comosa 
Annals of Botany  2012;111(1):103-111.
Background and Aims
Large floral displays have opposing consequences for animal-pollinated angiosperms: they attract more pollinators but also enable elevated among-flower self-pollination (geitonogamy). The presence of sterile flowers as pollinator signals may enhance attraction while allowing displays of fewer open fertile flowers, limiting geitonogamy. The simultaneous contributions of fertile and non-fertile display components to pollinator attraction and reproductive output remain undetermined.
The simultaneous effects of the presence of sterile flowers and fertile-flower display size in two populations of Leopoldia comosa were experimentally assessed. Pollinator behaviour, pollen removal and deposition, and fruit and seed production were compared between intact plants and plants with sterile flowers removed.
Key Results
The presence of sterile flowers almost tripled pollinator attraction, supplementing the positive effect of the number of fertile flowers on the number of bees approaching inflorescences. Although attracted bees visited more flowers on larger inflorescences, the number visited did not additionally depend on the presence of sterile flowers. The presence of sterile flowers improved all aspects of plant performance, the magnitude of plant benefit being context dependent. During weather favourable to pollinators, the presence of sterile flowers increased pollen deposition on stigmas of young flowers, but this difference was not evident in older flowers, probably because of autonomous self-pollination in poorly visited flowers. Total pollen receipt per stigma decreased with increasing fertile display size. In the population with more pollinators, the presence of sterile flowers increased fruit number but not seed set or mass, whereas in the other population sterile flowers enhanced seeds per fruit, but not fruit production. These contrasts are consistent with dissimilar cross-pollination and autonomous self-pollination, coupled with the strong predispersal inbreeding depression exhibited by L. comosa populations.
Sterile flowers enrich pollination quality by promoting pollen export and import, while limiting the mating costs of geitonogamy associated with large fertile displays.
PMCID: PMC3523656  PMID: 23131298
Anthophora; cross-pollination; geitonogamy; fertile floral display; mating cost; Leopoldia comosa; non-fertile flowers; outcrossing; pollen deposition; pollen quality; pollen removal; sterile flowers
20.  Floral symmetry affects speciation rates in angiosperms. 
Despite much recent activity in the field of pollination biology, the extent to which animal pollinators drive the formation of new angiosperm species remains unresolved. One problem has been identifying floral adaptations that promote reproductive isolation. The evolution of a bilaterally symmetrical corolla restricts the direction of approach and movement of pollinators on and between flowers. Restricting pollinators to approaching a flower from a single direction facilitates specific placement of pollen on the pollinator. When coupled with pollinator constancy, precise pollen placement can increase the probability that pollen grains reach a compatible stigma. This has the potential to generate reproductive isolation between species, because mutations that cause changes in the placement of pollen on the pollinator may decrease gene flow between incipient species. I predict that animal-pollinated lineages that possess bilaterally symmetrical flowers should have higher speciation rates than lineages possessing radially symmetrical flowers. Using sister-group comparisons I demonstrate that bilaterally symmetric lineages tend to be more species rich than their radially symmetrical sister lineages. This study supports an important role for pollinator-mediated speciation and demonstrates that floral morphology plays a key role in angiosperm speciation.
PMCID: PMC1691633  PMID: 15156918
21.  Phosphorylation of a WRKY Transcription Factor by MAPKs Is Required for Pollen Development and Function in Arabidopsis 
PLoS Genetics  2014;10(5):e1004384.
Plant male gametogenesis involves complex and dynamic changes in gene expression. At present, little is known about the transcription factors involved in this process and how their activities are regulated. Here, we show that a pollen-specific transcription factor, WRKY34, and its close homolog, WRKY2, are required for male gametogenesis in Arabidopsis thaliana. When overexpressed using LAT52, a strong pollen-specific promoter, epitope-tagged WRKY34 is temporally phosphorylated by MPK3 and MPK6, two mitogen-activated protein kinases (MAPKs, or MPKs), at early stages in pollen development. During pollen maturation, WRKY34 is dephosphorylated and degraded. Native promoter-driven WRKY34-YFP fusion also follows the same expression pattern at the protein level. WRKY34 functions redundantly with WRKY2 in pollen development, germination, and pollen tube growth. Loss of MPK3/MPK6 phosphorylation sites in WRKY34 compromises the function of WRKY34 in vivo. Epistasis interaction analysis confirmed that MPK6 belongs to the same genetic pathway of WRKY34 and WRKY2. Our study demonstrates the importance of temporal post-translational regulation of WRKY transcription factors in the control of developmental phase transitions in plants.
Author Summary
Pollen development, or male gametogenesis, is a process by which a haploid uninucleate microspore undergoes cell division and specification to form a mature pollen grain containing two sperm cells. The highly defined cell linage makes pollen development an ideal model to understand the regulation of plant cellular development. Pollen development has multiple phases and involves dynamic changes in gene expression, which highlights the importance of transcription factors and their regulatory pathway(s). In this report, we demonstrate that WRKY34 and WRKY2, two closely related WRKY transcription factors in Arabidopsis, play important roles in pollen development. WRKY34 is phosphorylated by MPK3/MPK6, two functionally redundant mitogen-activated protein kinases (MAPKs or MPKs), at early stages in pollen development. Utilizing a combination of genetic, biochemical, and cytological tools, we determined that this MAPK-WRKY signaling module functions at the early stage of pollen development. Loss of function of this pathway reduces pollen viability, and the surviving pollen has poor germination and reduced pollen tube growth, all of which reduce the transmission rate of the mutant pollen. This study discovers a novel stage-specific signaling pathway in pollen development.
PMCID: PMC4022456  PMID: 24830428
22.  Identification of genes specifically or preferentially expressed in maize silk reveals similarity and diversity in transcript abundance of different dry stigmas 
BMC Genomics  2012;13:294.
In plants, pollination is a critical step in reproduction. During pollination, constant communication between male pollen and the female stigma is required for pollen adhesion, germination, and tube growth. The detailed mechanisms of stigma-mediated reproductive processes, however, remain largely unknown. Maize (Zea mays L.), one of the world’s most important crops, has been extensively used as a model species to study molecular mechanisms of pollen and stigma interaction. A comprehensive analysis of maize silk transcriptome may provide valuable information for investigating stigma functionality. A comparative analysis of expression profiles between maize silk and dry stigmas of other species might reveal conserved and diverse mechanisms that underlie stigma-mediated reproductive processes in various plant species.
Transcript abundance profiles of mature silk, mature pollen, mature ovary, and seedling were investigated using RNA-seq. By comparing the transcriptomes of these tissues, we identified 1,427 genes specifically or preferentially expressed in maize silk. Bioinformatic analyses of these genes revealed many genes with known functions in plant reproduction as well as novel candidate genes that encode amino acid transporters, peptide and oligopeptide transporters, and cysteine-rich receptor-like kinases. In addition, comparison of gene sets specifically or preferentially expressed in stigmas of maize, rice (Oryza sativa L.), and Arabidopsis (Arabidopsis thaliana [L.] Heynh.) identified a number of homologous genes involved either in pollen adhesion, hydration, and germination or in initial growth and penetration of pollen tubes into the stigma surface. The comparison also indicated that maize shares a more similar profile and larger number of conserved genes with rice than with Arabidopsis, and that amino acid and lipid transport-related genes are distinctively overrepresented in maize.
Many of the novel genes uncovered in this study are potentially involved in stigma-mediated reproductive processes, including genes encoding amino acid transporters, peptide and oligopeptide transporters, and cysteine-rich receptor-like kinases. The data also suggest that dry stigmas share similar mechanisms at early stages of pollen-stigma interaction. Compared with Arabidopsis, maize and rice appear to have more conserved functional mechanisms. Genes involved in amino acid and lipid transport may be responsible for mechanisms in the reproductive process that are unique to maize silk.
PMCID: PMC3416702  PMID: 22748054
23.  Genome-scale analysis and comparison of gene expression profiles in developing and germinated pollen in Oryza sativa 
BMC Genomics  2010;11:338.
Pollen development from the microspore involves a series of coordinated cellular events, and the resulting mature pollen has a specialized function to quickly germinate, produce a polar-growth pollen tube derived from the vegetative cell, and deliver two sperm cells into the embryo sac for double fertilization. The gene expression profiles of developing and germinated pollen have been characterised by use of the eudicot model plant Arabidopsis. Rice, one of the most important cereal crops, has been used as an excellent monocot model. A comprehensive analysis of transcriptome profiles of developing and germinated pollen in rice is important to understand the conserved and diverse mechanism underlying pollen development and germination in eudicots and monocots.
We used Affymetrix GeneChip® Rice Genome Array to comprehensively analyzed the dynamic changes in the transcriptomes of rice pollen at five sequential developmental stages from microspores to germinated pollen. Among the 51,279 transcripts on the array, we found 25,062 pollen-preferential transcripts, among which 2,203 were development stage-enriched. The diversity of transcripts decreased greatly from microspores to mature and germinated pollen, whereas the number of stage-enriched transcripts displayed a "U-type" change, with the lowest at the bicellular pollen stage; and a transition of overrepresented stage-enriched transcript groups associated with different functional categories, which indicates a shift in gene expression program at the bicellular pollen stage. About 54% of the now-annotated rice F-box protein genes were expressed preferentially in pollen. The transcriptome profile of germinated pollen was significantly and positively correlated with that of mature pollen. Analysis of expression profiles and coexpressed features of the pollen-preferential transcripts related to cell cycle, transcription, the ubiquitin/26S proteasome system, phytohormone signalling, the kinase system and defense/stress response revealed five expression patterns, which are compatible with changes in major cellular events during pollen development and germination. A comparison of pollen transcriptomes between rice and Arabidopsis revealed that 56.6% of the rice pollen preferential genes had homologs in Arabidopsis genome, but 63.4% of these homologs were expressed, with a small proportion being expressed preferentially, in Arabidopsis pollen. Rice and Arabidopsis pollen had non-conservative transcription factors each.
Our results demonstrated that rice pollen expressed a set of reduced but specific transcripts in comparison with vegetative tissues, and the number of stage-enriched transcripts displayed a "U-type" change during pollen development, with the lowest at the bicellular pollen stage. These features are conserved in rice and Arabidopsis. The shift in gene expression program at the bicellular pollen stage may be important to the transition from earlier cell division to later pollen maturity. Pollen at maturity pre-synthesized transcripts needed for germination and early pollen tube growth. The transcription regulation associated with pollen development would have divergence between the two species. Our results also provide novel insights into the molecular program and key components of the regulatory network regulating pollen development and germination.
PMCID: PMC2895629  PMID: 20507633
24.  Tracking the development of the petaloid fertile stamen in Canna indica: insights into the origin of androecial petaloidy in the Zingiberales 
AoB Plants  2013;5:plt009.
The order Zingiberales comprises ∼2500 species of tropical to subtropical plants, including agriculturally (e.g. banana, ginger) and horticulturally (e.g. cannas, heliconias, bird-of-paradise) important plants. Throughout the evolution of this order, the stamens have been modified from the ancestral filamentous structures that produce pollen (seen in Banana flowers) to petal-like structures that no longer bear pollen sacs (seen in Canna flowers). This results in a reduction of pollen, but an effective increase in the overall size of the floral display and perhaps in the efficacy of specialized pollinators by converting stamens into ‘petals’. This study investigates the genetic mechanisms that are involved in making petal-like structures in place of pollen-producing stamens.
Flowers of the order Zingiberales demonstrate a remarkable trend of reduction in the number of fertile stamens; from five or six fertile, filamentous stamens bearing two thecae each in Musaceae and Strelitziaceae to just a single petaloid stamen bearing a single theca in Cannaceae and Marantaceae. As one progresses from ancestral to derived floral forms, 5–6 fertile stamens are replaced by 4–5 petaloid staminodes. In Cannaceae and Costaceae, all members of the androecial whorls exhibit petaloidy, including the fertile stamen. In Costaceae, a single fertile stamen develops two thecae embedded on a broad petaloid appendage, while in Cannaceae the single fertile stamen is further reduced to a single theca with a prominent, expanded petaloid appendage. Whether petaloidy of the fertile stamen is a synapomorphy of the entire ginger clade (including Cannaceae, Costaceae, Zingiberaceae and Marantaceae), or the result of independent convergent evolution in Cannaceae, Costaceae, and some Zingiberaceae, is unclear. We combine a developmental series of the formation of the petaloid fertile stamen in Canna indica with data on the expression of B- and C-class floral organ identity genes to elucidate the organogenetic identity of the petaloid stamen and staminodes. Our data indicate that the single fertile theca in C. indica and its petaloid appendage are derived from one-half of the primordium of a single stamen, with no contribution from the remaining part of the stamen (i.e. the second theca primordium) which aborts early in development. The petaloid appendage expands later, and develops from the position of the filament/connective of the developing theca. Floral identity gene expression shows that petal identity genes (i.e. B-class genes) are expressed in all floral organs studied while C-class gene AG-1 is expressed in an increasing gradient from sepals to gynoecium, and AG-2 is expressed in all floral organs except the petals. The canonical model for molecular specification of floral organ identity is not sufficient to explain petaloidy in the androecial whorl in Canna sp. Further studies understanding the regulation of gene networks are required.
PMCID: PMC3608240  PMID: 23539493
Canna; evo-devo; floral development; MADS-box genes; petaloid stamens; petaloidy; Zingiberales
25.  Generalist passerine pollination of a winter-flowering fruit tree in central China 
Annals of Botany  2011;109(2):379-384.
Background and Aims
Winter-flowering plants outside the tropics may experience a shortage of pollinator service, given that insect activity is largely limited by low temperature. Birds can be alternative pollinators for these plants, but experimental evidence for the pollination role of birds in winter-flowering plants is scarce.
Pollinator visitation to the loquat, Eriobotrya japonica (Rosaceae), was observed across the flowering season from November to January for two years in central China. Self- and cross-hand pollination was conducted in the field to investigate self-compatibility and pollen limitation. In addition, inflorescences were covered by bird cages and nylon mesh nets to exclude birds and all animal pollinators, respectively, to investigate the pollination role of birds in seed production.
Self-fertilization in the loquat yielded few seeds. In early winter insect visit frequency was relatively higher, while in late winter insect pollinators were absent and two passerine birds (Pycnonotus sinensis and Zosterops japonicus) became the major floral visitors. However, seed-set of open-pollinated flowers did not differ between early and late winter. Exclusion of bird visitation greatly reduced seed-set, indicating that passerine birds were important pollinators for the loquat in late winter. The whitish perigynous flowers reward passerines with relatively large volumes of dilute nectar. Our observation on the loquat and other Rosaceae species suggested that perigyny might be related to bird pollination but the association needs further study.
These findings suggest that floral traits and phenology would be favoured to attract bird pollinators in cold weather, in which insect activity is limited.
PMCID: PMC3268538  PMID: 22112440
Winter flowering; bird pollination; Pycnonotus sinensis; generalist pollination system; perigyny; self-incompatibility; ornithophily

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