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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.
Methods
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.
Conclusions
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.
doi:10.1093/aob/mcp276
PMCID: PMC2814751  PMID: 19939980
Annona cherimola; Annonaceae; embryo sac; endosperm; Magnoliid; ovule; pollen–pistil interaction; pollen tube
2.  Genome-scale analysis and comparison of gene expression profiles in developing and germinated pollen in Oryza sativa 
BMC Genomics  2010;11:338.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2164-11-338
PMCID: PMC2895629  PMID: 20507633
3.  Developmental evolution of flowering plant pollen tube cell walls: callose synthase (CalS) gene expression patterns 
EvoDevo  2011;2:14.
Background
A number of innovations underlie the origin of rapid reproductive cycles in angiosperms. A critical early step involved the modification of an ancestrally short and slow-growing pollen tube for faster and longer distance transport of sperm to egg. Associated with this shift are the predominantly callose (1,3-β-glucan) walls and septae (callose plugs) of angiosperm pollen tubes. Callose synthesis is mediated by callose synthase (CalS). Of 12 CalS gene family members in Arabidopsis, only one (CalS5) has been directly linked to pollen tube callose. CalS5 orthologues are present in several monocot and eudicot genomes, but little is known about the evolutionary origin of CalS5 or what its ancestral function may have been.
Results
We investigated expression of CalS in pollen and pollen tubes of selected non-flowering seed plants (gymnosperms) and angiosperms within lineages that diverged below the monocot/eudicot node. First, we determined the nearly full length coding sequence of a CalS5 orthologue from Cabomba caroliniana (CcCalS5) (Nymphaeales). Semi-quantitative RT-PCR demonstrated low CcCalS5 expression within several vegetative tissues, but strong expression in mature pollen. CalS transcripts were detected in pollen tubes of several species within Nymphaeales and Austrobaileyales, and comparative analyses with a phylogenetically diverse group of sequenced genomes indicated homology to CalS5. We also report in silico evidence of a putative CalS5 orthologue from Amborella. Among gymnosperms, CalS5 transcripts were recovered from germinating pollen of Gnetum and Ginkgo, but a novel CalS paralog was instead amplified from germinating pollen of Pinus taeda.
Conclusion
The finding that CalS5 is the predominant callose synthase in pollen tubes of both early-diverging and model system angiosperms is an indicator of the homology of their novel callosic pollen tube walls and callose plugs. The data suggest that CalS5 had transient expression and pollen-specific functions in early seed plants and was then recruited to novel expression patterns and functions within pollen tube walls in an ancestor of extant angiosperms.
doi:10.1186/2041-9139-2-14
PMCID: PMC3146827  PMID: 21722365
4.  The Pollination of Trimenia moorei (Trimeniaceae): Floral Volatiles, Insect/Wind Pollen Vectors and Stigmatic Self‐incompatibility in a Basal Angiosperm 
Annals of Botany  2003;92(3):445-458.
Trimenia moorei (Oliv.) Philipson is an andromonoecious liane with >0·40 of the total flower buds maturing as bisexual flowers. Male and bisexual flowers are strongly scented with pollen, anther sacs and receptacle scars testing positively for volatile emissions. Scent analyses detect over 20 components. The major fatty acid derivative is 8‐heptadecene, and 2‐phenylethanol dominates the benzenoids. While hover‐flies in the genera Melangyna and Triglyphus contact the stigma with their probosces, the stigma secretes no free‐flowing, edible fluids. Copious pollen is the only edible reward consumed by hover‐flies (Syprhidae), sawflies (Pergidae) and bees in the families Apidae, Colletidae and Halictidae. All these insects carried pollen of T. moorei on their heads, legs and thoraces and female bees in the genera Apis, Exoneura, Leioproctus and Lasioglossum stored pollen on their hind legs. Pollen traps also indicate that pollen is shed directly into the air, permitting wind pollination. When bisexual flower buds are bagged (isolated from insect foragers) on the liane then subjected to a series of hand‐pollination experiments after perianth segments open, the structural analyses of pollen–carpel interactions indicate that T. moorei has a trichome‐rich dry‐type stigma with an early‐acting self‐incompatibility (SI) system. Bicellular pollen grains deposited on stigmas belonging to the same plant germinate but fail to penetrate intercellular spaces, while grains deposited following cross‐pollination reach the ovule within 24 h. Fluorescence analyses of 76 carpels collected at random from unbagged (open‐pollinated) flowers on five plants indicates that at least 64 % of carpels are cross‐pollinated in situ. Trimenia moorei is the first species within the ANITA group, and second within reilictual‐basal angiosperm lineages, to exhibit stigmatic SI in combination with dry‐type stigma and bicellular pollen, a condition once considered to be atypical for angiosperms as a whole but now known to be present in numerous taxa.
doi:10.1093/aob/mcg157
PMCID: PMC4257518  PMID: 12930730
Andromonoecy; dry stigma; insect pollination; self‐incompatibility; transmitting tissue; Trimeniaceae; volatiles; wind‐pollination
5.  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
6.  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.
Methods
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.
Conclusions
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.
doi:10.1093/aob/mcq198
PMCID: PMC2990668  PMID: 21047886
Autogamy; basal angiosperm; delayed self-pollination; Hydatellaceae; Nymphaeales; wind pollination; reproductive assurance; reproductive timing; stigma receptivity; Trithuria submersa
7.  Molecular cloning and transcriptional activity of a new Petunia calreticulin gene involved in pistil transmitting tract maturation, progamic phase, and double fertilization 
Planta  2013;239(2):437-454.
Calreticulin (CRT) is a highly conserved and ubiquitously expressed Ca2+-binding protein in multicellular eukaryotes. As an endoplasmic reticulum-resident protein, CRT plays a key role in many cellular processes including Ca2+ storage and release, protein synthesis, and molecular chaperoning in both animals and plants. CRT has long been suggested to play a role in plant sexual reproduction. To begin to address this possibility, we cloned and characterized the full-length cDNA of a new CRT gene (PhCRT) from Petunia. The deduced amino acid sequence of PhCRT shares homology with other known plant CRTs, and phylogenetic analysis indicates that the PhCRT cDNA clone belongs to the CRT1/CRT2 subclass. Northern blot analysis and fluorescent in situ hybridization were used to assess PhCRT gene expression in different parts of the pistil before pollination, during subsequent stages of the progamic phase, and at fertilization. The highest level of PhCRT mRNA was detected in the stigma–style part of the unpollinated pistil 1 day before anthesis and during the early stage of the progamic phase, when pollen is germinated and tubes outgrow on the stigma. In the ovary, PhCRT mRNA was most abundant after pollination and reached maximum at the late stage of the progamic phase, when pollen tubes grow into the ovules and fertilization occurs. PhCRT mRNA transcripts were seen to accumulate predominantly in transmitting tract cells of maturing and receptive stigma, in germinated pollen/growing tubes, and at the micropylar region of the ovule, where the female gametophyte is located. From these results, we suggest that PhCRT gene expression is up-regulated during secretory activity of the pistil transmitting tract cells, pollen germination and outgrowth of the tubes, and then during gamete fusion and early embryogenesis.
doi:10.1007/s00425-013-1971-4
PMCID: PMC3902078  PMID: 24213153
Calcium homeostasis; Calreticulin; Chaperone activity; Gene expression; Nucleolus; Pollen–pistil interactions
8.  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.
doi:10.1093/jxb/erp330
PMCID: PMC2814102  PMID: 19926683
Female gametophyte; maize; pollen tube guidance; prezygotic barriers; transmitting tract; Tripsacum
9.  The Causes of Self‐sterility in Natural Populations of the Relictual Angiosperm, Illicium floridanum (Illiciaceae) 
Annals of Botany  2004;94(1):43-50.
• Background and Aims Illicium floridanum, a species belonging to the basal extant angiosperm taxon Illiciaceae, reportedly exhibits self‐incompatibility (SI). To date, the site and timing of SI within the carpel of this species remains unidentified. Thus, the objective of this research was to determine the cellular and temporal aspects of SI in I. floridanum.
• Methods Following controlled application of cross‐ and self‐pollen in natural populations of I. floridanum, embryo sac development and temporal aspects of stigma receptivity, as well as pollen tube growth, fertilization, and embryo and endosperm development, were investigated with the aid of light and fluorescence microscopy.
• Key Results Flowers of I. floridanum exhibited complete dichogamy whereby stigmas only supported cross‐ and self‐pollen tube growth prior to anther dehiscence. In contrast to earlier reports of SI in this species, a prezygotic SI resulting in rejection of self‐pollen tube growth at the stigma was absent and there were no significant differences between cross‐ versus self‐pollen germination and pollen tube growth within the style and ovary during the first 5 d after pollination. Structural development of the four‐celled embryo sac was not differentially influenced by pollen type as noted to occur in other angiosperms with late‐acting ovarian SI. The ovule micropyle and embryo sac were penetrated equally by cross‐ and self‐pollen tubes. In addition, there were no statistically significant differences in cross‐ versus self‐fertilization. A resting zygote and multicellular endosperm at a variety of developmental stages was present by 30 d after application of cross‐ or self‐pollen.
• Conclusions In the clear absence of a prezygotic SI that was previously reported to result in differential self‐pollen tube growth at the stigma, self‐ sterility in I. floridanum is likely due to early‐acting inbreeding depression, although late‐acting post‐zygotic ovarian SI cannot be ruled out.
doi:10.1093/aob/mch113
PMCID: PMC4242369  PMID: 15155377
Illicium floridanum; self‐incompatibility; self‐sterility; complete dichogamy; early‐acting inbreeding depression; Illiciaceae; Austrobaileyales
10.  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.
doi:10.4161/psb.20745
PMCID: PMC3474685  PMID: 22827945
de novo pollen tube transcriptome; male gametophyte development; pollen tube growth; transcriptomics
11.  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).
Methods
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.
Conclusions
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.
doi:10.1093/aob/mcs114
PMCID: PMC3400444  PMID: 22652419
Hyacinthaceae; Ornithogaloideae; pollen; pollen germination; pollen receipt; pollen tube; pollination; sexual interference
12.  Ultrastructure of stomatal development in early-divergent angiosperms reveals contrasting patterning and pre-patterning 
Annals of Botany  2013;112(6):1031-1043.
Background and Aims
Angiosperm stomata consistently possess a pair of guard cells, but differ between taxa in the patterning and developmental origin of neighbour cells. Developmental studies of phylogenetically pivotal taxa are essential as comparative yardsticks for understanding the evolution of stomatal development.
Methods
We present a novel ultrastructural study of developing stomata in leaves of Amborella (Amborellales), Nymphaea and Cabomba (Nymphaeales), and Austrobaileya and Schisandra (Austrobaileyales), representing the three earliest-divergent lineages of extant angiosperms (the ANITA-grade).
Key Results
Alternative developmental pathways occur in early-divergent angiosperms, resulting partly from differences in pre-patterning and partly from the presence or absence of highly polarized (asymmetric) mitoses in the stomatal cell lineage. Amplifying divisions are absent from ANITA-grade taxa, indicating that ostensible similarities with the stomatal patterning of Arabidopsis are superficial. In Amborella, ‘squared’ pre-patterning occurs in intercostal regions, with groups of four protodermal cells typically arranged in a rectangle; most guard-mother cells are formed by asymmetric division of a precursor cell (the mesoperigenous condition) and are typically triangular or trapezoidal. In contrast, water-lily stomata are always perigenous (lacking asymmetric divisions). Austrobaileya has occasional ‘giant’ stomata.
Conclusions
Similar mature stomatal phenotypes can result from contrasting morphogenetic factors, although the results suggest that paracytic stomata are invariably the product of at least one asymmetric division. Loss of asymmetric divisions in stomatal development could be a significant factor in land plant evolution, with implications for the diversity of key structural and physiological pathways.
doi:10.1093/aob/mct169
PMCID: PMC3783234  PMID: 23969762
Epidermal pre-patterning; meristemoids; mesogenous stomata; perigenous stomata; stomatal development; ANITA; early-divergent angiosperms
13.  Using maize as a model to study pollen tube growth and guidance, cross-incompatibility and sperm delivery in grasses 
Annals of Botany  2011;108(4):727-737.
Background
In contrast to animals and lower plants such as mosses and ferns, sperm cells of flowering plants (angiosperms) are immobile and require transportation to the female gametes via the vegetative pollen tube cell to achieve double fertilization. The path of the pollen tube towards the female gametophyte (embryo sac) has been intensively studied in many intra- and interspecific crossing experiments with the aim of increasing the gene pool of crop plants for greater yield, improved biotic and abiotic stress resistance, and for introducing new agronomic traits. Many attempts to hybridize different species or genotypes failed due to the difficulty for the pollen tubes in reaching the female gametophyte. Detailed studies showed that these processes are controlled by various self-incompatible (intraspecific) and cross-incompatible (interspecific) hybridization mechanisms.
Scope
Understanding the molecular mechanisms of crossing barriers is therefore of great interest in plant reproduction, evolution and breeding research. In particular, pre-zygotic hybridization barriers related to pollen tube germination, growth, guidance and sperm delivery, which are considered the major hybridization controls in nature and thus also contribute to species isolation and speciation, have been intensively investigated. Despite this general interest, surprisingly little is known about these processes in the most important agronomic plant family, the Gramineae, Poaceae or grasses. Small polymorphic proteins and their receptors, degradation of sterility locus proteins and general compounds such as calcium, γ-aminobutyric acid or nitric oxide have been shown to be involved in progamic pollen germination, adhesion, tube growth and guidance, as well as sperm release. Most advances have been made in the Brassicaceae, Papaveraceae, Linderniaceae and Solanaceae families including their well-understood self-incompatibility (SI) systems. Grass species evolved similar mechanisms to control the penetration and growth of self-pollen to promote intraspecific outcrossing and to prevent fertilization by alien sperm cells. However, in the Poaceae, the underlying molecular mechanisms are still largely unknown.
Conclusions
We propose to develop maize (Zea mays) as a model to investigate the above-described processes to understand the associated intra- and interspecific crossing barriers in grasses. Many genetic, cellular and biotechnological tools including the completion of a reference genome (inbred line B73) have been established in the last decade and many more maize inbred genomes are expected to be available soon. Moreover, a cellular marker line database as well as large transposon insertion collections and improved Agrobacterium transformation protocols are now available. Additionally, the processes described above are well studied at the morphological level and a number of mutants have been described already, awaiting disclosure of the relevant genes. The identification of the first key players in pollen tube growth, guidance and burst show maize to be an excellent grass model to investigate these processes in more detail. Here we provide an overview of our current understanding of these processes in Poaceae with a focus on maize, and also include relevant discoveries in eudicot model species.
doi:10.1093/aob/mcr017
PMCID: PMC3170146  PMID: 21345919
Maize; male germline; sperm cell; interspecific crosses; self- and cross-incompatibility; pollen tube growth and guidance; fertilization; reproductive isolation
14.  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.
doi:10.4161/psb.6.7.15315
PMCID: PMC3257764  PMID: 21628998
pollen-pistil interaction; carbohydrates; heat stress; fertilization; pollen tube growth; climate change
15.  Asymmetrical conspecific seed-siring advantage between Silene latifolia and S. dioica 
Annals of Botany  2010;105(4):595-605.
Background and Aims
Silene dioica and S. latifolia experience only limited introgression despite overlapping flowering phenologies, geographical distributions, and some pollinator sharing. Conspecific pollen precedence and other reproductive barriers operating between pollination and seed germination may limit hybridization. This study investigates whether barriers at this stage contribute to reproductive isolation between these species and, if so, which mechanisms are responsible.
Methods
Pollen-tube lengths for pollen of both species in styles of both species were compared. Additionally, both species were pollinated with majority S. latifolia and majority S. dioica pollen mixes; then seed set, seed germination rates and hybridity of the resulting seedlings were determined using species-specific molecular markers.
Key Results
The longest pollen tubes were significantly longer for conspecific than heterospecific pollen in both species, indicating conspecific pollen precedence. Seed set but not seed germination was lower for flowers pollinated with pure heterospecific versus pure conspecific pollen. Mixed-species pollinations resulted in disproportionately high representation of nonhybrid offspring for pollinations of S. latifolia but not S. dioica flowers.
Conclusions
The finding of conspecific pollen precedence for pollen-tube growth but not seed siring in S. dioica flowers may be explained by variation in pollen-tube growth rates, either at different locations in the style or between leading and trailing pollen tubes. Additionally, this study finds a barrier to hybridization operating between pollination and seed germination against S. dioica but not S. latifolia pollen. The results are consistent with the underlying cause of this barrier being attrition of S. dioica pollen tubes or reduced success of heterospecifically fertilized ovules, rather than time-variant mechanisms. Post-pollination, pre-germination barriers to hybridization thus play a partial role in limiting introgression between these species.
doi:10.1093/aob/mcq013
PMCID: PMC2850797  PMID: 20147372
Conspecific pollen precedence; hybridization; pollen tubes; reproductive isolation; Silene dioica; Silene latifolia
16.  The PTI1-like kinase ZmPti1a from maize (Zea mays L.) co-localizes with callose at the plasma membrane of pollen and facilitates a competitive advantage to the male gametophyte 
BMC Plant Biology  2006;6:22.
Background
The tomato kinase Pto confers resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato in a gene for gene manner. Upon recognition of specific avirulence factors the Pto kinase activates multiple signal transduction pathways culminating in induction of pathogen defense. The soluble cytoplasmic serine/threonine kinase Pti1 is one target of Pto phosphorylation and is involved in the hypersensitive response (HR) reaction. However, a clear role of Pti1 in plant pathogen resistance is uncertain. So far, no Pti1 homologues from monocotyledonous species have been studied.
Results
Here we report the identification and molecular analysis of four Pti1-like kinases from maize (ZmPti1a, -b, -c, -d). These kinase genes showed tissue-specific expression and their corresponding proteins were targeted to different cellular compartments. Sequence similarity, expression pattern and cellular localization of ZmPti1b suggested that this gene is a putative orthologue of Pti1 from tomato. In contrast, ZmPti1a was specifically expressed in pollen and sequestered to the plasma membrane, evidently owing to N-terminal modification by myristoylation and/or S-acylation. The ZmPti1a:GFP fusion protein was not evenly distributed at the pollen plasma membrane but accumulated as an annulus-like structure which co-localized with callose (1,3-β-glucan) deposition. In addition, co-localization of ZmPti1a and callose was observed during stages of pollen mitosis I and pollen tube germination. Maize plants in which ZmPti1a expression was silenced by RNA interference (RNAi) produced pollen with decreased competitive ability. Hence, our data provide evidence that ZmPti1a plays an important part in a signalling pathway that accelerates pollen performance and male fitness.
Conclusion
ZmPti1a from maize is involved in pollen-specific processes during the progamic phase of reproduction, probably in crucial signalling processes associated with regions of callose deposition. Pollen-sporophyte interactions and pathogen induced HR show certain similarities. For example, HR has been shown to be associated with cell wall reinforcement through callose deposition. Hence, it is hypothesized that Pti1 kinases from maize act as general components in evolutionary conserved signalling processes associated with callose, however during different developmental programs and in different tissue types.
doi:10.1186/1471-2229-6-22
PMCID: PMC1609167  PMID: 17022830
17.  Comprehensive analysis of tobacco pollen transcriptome unveils common pathways in polar cell expansion and underlying heterochronic shift during spermatogenesis 
BMC Plant Biology  2012;12:24.
Background
Many flowering plants produce bicellular pollen. The two cells of the pollen grain are destined for separate fates in the male gametophyte, which provides a unique opportunity to study genetic interactions that govern guided single-cell polar expansion of the growing pollen tube and the coordinated control of germ cell division and sperm cell fate specification. We applied the Agilent 44 K tobacco gene chip to conduct the first transcriptomic analysis of the tobacco male gametophyte. In addition, we performed a comparative study of the Arabidopsis root-hair trichoblast transcriptome to evaluate genetic factors and common pathways involved in polarized cell-tip expansion.
Results
Progression of pollen grains from freshly dehisced anthers to pollen tubes 4 h after germination is accompanied with > 5,161 (14.9%) gametophyte-specific expressed probes active in at least one of the developmental stages. In contrast, > 18,821 (54.4%) probes were preferentially expressed in the sporophyte. Our comparative approach identified a subset of 104 pollen tube-expressed genes that overlap with root-hair trichoblasts. Reverse genetic analysis of selected candidates demonstrated that Cu/Zn superoxide dismutase 1 (CSD1), a WD-40 containing protein (BP130384), and Replication factor C1 (NtRFC1) are among the central regulators of pollen-tube tip growth. Extension of our analysis beyond the second haploid mitosis enabled identification of an opposing-dynamic accumulation of core regulators of cell proliferation and cell fate determinants in accordance with the progression of the germ cell cycle.
Conclusions
The current study provides a foundation to isolate conserved regulators of cell tip expansion and those that are unique for pollen tube growth to the female gametophyte. A transcriptomic data set is presented as a benchmark for future functional studies using developing pollen as a model. Our results demonstrated previously unknown functions of certain genes in pollen-tube tip growth. In addition, we highlighted the molecular dynamics of core cell-cycle regulators in the male gametophyte and postulated the first genetic model to account for the differential timing of spermatogenesis among angiosperms and its coordination with female gametogenesis.
doi:10.1186/1471-2229-12-24
PMCID: PMC3305590  PMID: 22340370
18.  A Tale of Two Morphs: Modeling Pollen Transfer, Magic Traits, and Reproductive Isolation in Parapatry 
PLoS ONE  2014;9(9):e106512.
The evolution of the flower is commonly thought to have spurred angiosperm diversification. Similarly, particular floral traits might have promoted diversification within specific angiosperm clades. We hypothesize that traits promoting the precise positional transfer of pollen between flowers might promote diversification. In particular, precise pollen transfer might produce partial reproductive isolation that facilitates adaptive divergence between parapatric populations differing in their reproductive-organ positions. We investigate this hypothesis with an individual-based model of pollen transfer dynamics associated with heterostyly, a floral syndrome that depends on precise pollen transfer. Our model shows that precise pollen transfer can cause sexual selection leading to divergence in reproductive-organ positions between populations served by different pollinators, pleiotropically causing an increase in reproductive isolation through a “magic trait” mechanism. Furthermore, this increased reproductive isolation facilitates adaptive divergence between the populations in an unlinked, ecologically selected trait. In a different pollination scenario, however, precise pollen transfer causes a decrease in adaptive divergence by promoting asymmetric gene flow. Our results highlight the idea that magic traits are not “magic” in isolation; in particular, the effect size of magic traits in speciation depends on the external environment, and also on other traits that modify the strength of the magic trait's influence on non-random mating. Overall, we show that the evolutionary consequences of pollen transfer dynamics can depend strongly on the available pollinator fauna and on the morphological fit between flowers and pollinators. Furthermore, our results illustrate the potential importance of even weak reproductive isolating barriers in facilitating adaptive divergence.
doi:10.1371/journal.pone.0106512
PMCID: PMC4161326  PMID: 25211280
19.  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.
Methods
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.
Conclusions
Species-specific floral and inflorescence characteristics, especially nectar accessibility and dichogamy, control the efficiency of pollen-collecting bees as pollinators of aloes.
doi:10.1093/aob/mcr324
PMCID: PMC3286288  PMID: 22278414
Pollen theft; pollination efficiency; dichogamy; floral morphology; Aloe; Alooideae; Xanthorrhoeaceae; Asphodeloideae
20.  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.
doi:10.1371/journal.pgen.1004384
PMCID: PMC4022456  PMID: 24830428
21.  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.
Background
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.
doi:10.1093/aob/mcp035
PMCID: PMC2701749  PMID: 19218583
Wind pollination; reproductive assurance; pollen limitation; geitonogamy; sex allocation; inflorescence architecture; mating systems
22.  Comparison of ultrastructure, pollen tube growth pattern and starch content in developing and abortive ovaries during the progamic phase in hazel 
HIGHLIGHTS In an abortive ovary of hazel, an integument seldom differentiated and a mature embryo sac never developed.In an abortive ovary of hazel, pollen tube growth was arrested at the style base about 40 days after blooming. Thus, fertilization of the ovule was precluded.Ovary abortion in the four hybrid cultivars was indicated to be associated with insufficient resource availability to support fruit set by all flowers, whereas ovary abortion in C. heterophylla was at least partly determined by pollen availability.
In Northeast China, a high frequency of ovary abortion contributes to serious losses in yield of hazelnut. The development of pistillate inflorescences and fruit clusters of four large-fruited hybrid hazel cultivars and the small-fruited Corylus heterophylla were used to study ovary abortion and its possible causes during the progamic phase in hazel. The average number of pistillate (ANP) flowers per inflorescence and average number of fruit (ANF) per cluster of four hybrid hazel cultivars were 7.6–8.5 and 2.4–3.0 respectively; in C. heterophylla, its ANP and ANF was 5.8–6.2 and 3.5, respectively. The total drop varied from 50 to 67%. Ovary abortion in hazel initiated from about 30 days after blooming. The percentage of abortive ovaries (PAO) in the four hybrid hazel cultivars ranged from 63 to 72%, and was significantly higher than that of C. heterophylla (29–42%). Only the abortive ovary ratio of C. heterophylla was significantly reduced after artificial pollination. Fruit number per cluster was positively and negatively correlated with yield and nut mass, respectively. In abortive ovaries, the diameter remained less than 2 mm during the entire fruit development, an integument seldom differentiated and a mature embryo sac never developed. In addition, pollen tube growth was arrested at the style base about 40 days after blooming. Thus, fertilization of the ovule was precluded. Compared with abortive ovary, starch content in developing ovary of four hybrid hazel cultivars and C. heterophylla were significantly higher. This study suggests that abortive ovary was incapable to finish fertilization process due to the absence of mature embryo sac and arrested pollen tubes, and this is likely associate with insufficient resource availability to support fruit set by all flowers in four hybrid hazel cultivars, whereas ovary abortion in C. heterophylla is at least partly determined by pollen availability.
doi:10.3389/fpls.2014.00528
PMCID: PMC4186277  PMID: 25339969
hazelnut; abortive ovary; pistillate inflorescence; fruit cluster; delayed fertilization
23.  Secondary Evolution of a Self-Incompatibility Locus in the Brassicaceae Genus Leavenworthia 
PLoS Biology  2013;11(5):e1001560.
Self-incompatibility enables plants to avoid inbreeding by self-pollination. Here we report that the genetic locus encoding self-pollen recognition has evolved twice in the Brassicaceae family, challenging the notion that loss of self-incompatibility is irreversible.
Self-incompatibility (SI) is the flowering plant reproductive system in which self pollen tube growth is inhibited, thereby preventing self-fertilization. SI has evolved independently in several different flowering plant lineages. In all Brassicaceae species in which the molecular basis of SI has been investigated in detail, the product of the S-locus receptor kinase (SRK) gene functions as receptor in the initial step of the self pollen-rejection pathway, while that of the S-locus cysteine-rich (SCR) gene functions as ligand. Here we examine the hypothesis that the S locus in the Brassicaceae genus Leavenworthia is paralogous with the S locus previously characterized in other members of the family. We also test the hypothesis that self-compatibility in this group is based on disruption of the pollen ligand-producing gene. Sequence analysis of the S-locus genes in Leavenworthia, phylogeny of S alleles, gene expression patterns, and comparative genomics analyses provide support for both hypotheses. Of special interest are two genes located in a non-S locus genomic region of Arabidopsis lyrata that exhibit domain structures, sequences, and phylogenetic histories similar to those of the S-locus genes in Leavenworthia, and that also share synteny with these genes. These A. lyrata genes resemble those comprising the A. lyrata S locus, but they do not function in self-recognition. Moreover, they appear to belong to a lineage that diverged from the ancestral Brassicaceae S-locus genes before allelic diversification at the S locus. We hypothesize that there has been neo-functionalization of these S-locus-like genes in the Leavenworthia lineage, resulting in evolution of a separate ligand-receptor system of SI. Our results also provide support for theoretical models that predict that the least constrained pathway to the evolution of self-compatibility is one involving loss of pollen gene function.
Author Summary
Self-incompatibility (SI) is a pollen recognition system that enables plants to avoid the inbreeding caused by self-pollination. It involves a pair of tightly linked genes known as the S locus. The product of one of these genes acts as the receptor and recognizes the pollen protein produced by the same plant, while the product of the other gene is the pollen protein that is recognized by the receptor. In this study, we have analyzed the gene sequence, genome organization, and gene evolutionary history of S loci in members of the Brassicaceae family, which includes plants of the genus Leavenworthia. From our analyses, we conclude that both genes that comprise the ancestral S locus in the Brassicaceae were lost in Leavenworthia. We show, however, that plants of this genus possess two other linked genes that exhibit patterns of polymorphism and expression that are characteristic of an S locus. These genes occupy the same genomic position in Leavenworthia as do two non-S-locus genes in the related species Arabidopsis lyrata, genes that are not known to function in self-recognition in this species. We suggest that these genes have evolved to assume the function of the pollen recognition system of SI in Leavenworthia—that is, that there has been de novo emergence of a distinct Brassicaceae S locus in this genus. We also present evidence that the breakdown of the SI system in two Leavenworthia races is due to independent mutations in the S-locus pollen gene, in accordance with theoretical predictions for the spread of S-locus disrupting mutations.
doi:10.1371/journal.pbio.1001560
PMCID: PMC3653793  PMID: 23690750
24.  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.
doi:10.1371/journal.pone.0072852
PMCID: PMC3749131  PMID: 23991159
25.  Stigma Development and Receptivity in Almond (Prunus dulcis) 
Annals of Botany  2006;97(1):57-63.
• Background and Aims Fertilization is essential in almond production, and pollination can be limiting in production areas. This study investigated stigma receptivity under defined developmental stages to clarify the relationship between stigma morphology, pollen germination, tube growth and fruit set.
• Methods Light and scanning electron microscopy were employed to examine stigma development at seven stages of flower development ranging from buds that were swollen to flowers in which petals were abscising. Flowers at different stages were hand pollinated and pollen germination and tube growth assessed. Artificial pollinations in the field were conducted to determine the effect of flower age on fruit set.
• Key Results Later stages of flower development exhibited greater stigma receptivity, i.e. higher percentages of pollen germination and more extensive tube growth occurred in older (those opened to the flat petal stage or exhibiting petal fall) than younger flowers. Enhanced stigma receptivity was associated with elongation of stigmatic papillae and increased amounts of stigmatic exudate that inundated papillae at later developmental stages. Field pollinations indicated that the stigma was still receptive and nut set was maintained in older flowers.
• Conclusions Stigma receptivity in almond does not become optimal until flowers are past the fully open stage. The stigma is still receptive and fruit set is maintained in flowers even at the stage when petals are abscising. Strategies to enhance pollination and crop yield, including the timing and placement of honey bees, should consider the effectiveness of developmentally advanced flowers.
doi:10.1093/aob/mcj013
PMCID: PMC2803376  PMID: 16287904
Almond; effective pollination period; Prunus dulcis; stigma receptivity; stigmatic exudate

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