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1.  Defensin-Like ZmES4 Mediates Pollen Tube Burst in Maize via Opening of the Potassium Channel KZM1 
PLoS Biology  2010;8(6):e1000388.
Species-preferential osmotic pollen tube burst and sperm discharge in maize involve induced opening of the pollen tube-expressed potassium channel KZM1 by the egg apparatus-derived defensin-like protein ZmES4.
In contrast to animals and lower plant species, sperm cells of flowering plants are non-motile and are transported to the female gametes via the pollen tube, i.e. the male gametophyte. Upon arrival at the female gametophyte two sperm cells are discharged into the receptive synergid cell to execute double fertilization. The first players involved in inter-gametophyte signaling to attract pollen tubes and to arrest their growth have been recently identified. In contrast the physiological mechanisms leading to pollen tube burst and thus sperm discharge remained elusive. Here, we describe the role of polymorphic defensin-like cysteine-rich proteins ZmES1-4 (Zea mays embryo sac) from maize, leading to pollen tube growth arrest, burst, and explosive sperm release. ZmES1-4 genes are exclusively expressed in the cells of the female gametophyte. ZmES4-GFP fusion proteins accumulate in vesicles at the secretory zone of mature synergid cells and are released during the fertilization process. Using RNAi knock-down and synthetic ZmES4 proteins, we found that ZmES4 induces pollen tube burst in a species-preferential manner. Pollen tube plasma membrane depolarization, which occurs immediately after ZmES4 application, as well as channel blocker experiments point to a role of K+-influx in the pollen tube rupture mechanism. Finally, we discovered the intrinsic rectifying K+ channel KZM1 as a direct target of ZmES4. Following ZmES4 application, KZM1 opens at physiological membrane potentials and closes after wash-out. In conclusion, we suggest that vesicles containing ZmES4 are released from the synergid cells upon male-female gametophyte signaling. Subsequent interaction between ZmES4 and KZM1 results in channel opening and K+ influx. We further suggest that K+ influx leads to water uptake and culminates in osmotic tube burst. The species-preferential activity of polymorphic ZmES4 indicates that the mechanism described represents a pre-zygotic hybridization barrier and may be a component of reproductive isolation in plants.
Author Summary
Sperm cells of animals and lower plants are mobile and can swim to the oocyte or egg cell. In contrast, flowering plants generate immobile sperm encased in a pollen coat to protect them from drying out and are transported via the pollen tube cell towards the egg apparatus to achieve double fertilization. Upon arrival the pollen tube tip bursts to deliver two sperm cells, one fusing with the egg cell to generate the embryo and the other fusing with the central cell to generate the endosperm. Here, we report the mechanisms leading to pollen tube burst and sperm discharge in maize. We found that before fertilization the defensin-like protein ZmES1-4 is stored in the secretory zone of the egg apparatus cells and that pollen tubes cannot discharge sperm in ZmES1-4 knock-down plants. Application of chemically synthesized ZmES4 leads to pollen tube burst within seconds in maize, but not in other plant species, suggesting this mechanism may be species specific. Finally, we identified the pollen tube-expressed potassium channel KZM1 as a target of ZmES4, which opens after ZmES4 treatment and probably leads to K+ influx and sperm release after osmotic burst.
doi:10.1371/journal.pbio.1000388
PMCID: PMC2879413  PMID: 20532241
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.  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
4.  The large-scale investigation of gene expression in Leymus chinensis stigmas provides a valuable resource for understanding the mechanisms of poaceae self-incompatibility 
BMC Genomics  2014;15(1):399.
Background
Many Poaceae species show a gametophytic self-incompatibility (GSI) system, which is controlled by at least two independent and multiallelic loci, S and Z. Until currently, the gene products for S and Z were unknown. Grass SI plant stigmas discriminate between pollen grains that land on its surface and support compatible pollen tube growth and penetration into the stigma, whereas recognizing incompatible pollen and thus inhibiting pollination behaviors. Leymus chinensis (Trin.) Tzvel. (sheepgrass) is a Poaceae SI species. A comprehensive analysis of sheepgrass stigma transcriptome may provide valuable information for understanding the mechanism of pollen-stigma interactions and grass SI.
Results
The transcript abundance profiles of mature stigmas, mature ovaries and leaves were examined using high-throughput next generation sequencing technology. A comparative transcriptomic analysis of these tissues identified 1,025 specifically or preferentially expressed genes in sheepgrass stigmas. These genes contained a significant proportion of genes predicted to function in cell-cell communication and signal transduction. We identified 111 putative transcription factors (TFs) genes and the most abundant groups were MYB, C2H2, C3H, FAR1, MADS. Comparative analysis of the sheepgrass, rice and Arabidopsis stigma-specific or preferential datasets showed broad similarities and some differences in the proportion of genes in the Gene Ontology (GO) functional categories. Potential SI candidate genes identified in other grasses were also detected in the sheepgrass stigma-specific or preferential dataset. Quantitative real-time PCR experiments validated the expression pattern of stigma preferential genes including homologous grass SI candidate genes.
Conclusions
This study represents the first large-scale investigation of gene expression in the stigmas of an SI grass species. We uncovered many notable genes that are potentially involved in pollen-stigma interactions and SI mechanisms, including genes encoding receptor-like protein kinases (RLK), CBL (calcineurin B-like proteins) interacting protein kinases, calcium-dependent protein kinase, expansins, pectinesterase, peroxidases and various transcription factors. The availability of a pool of stigma-specific or preferential genes for L. chinensis offers an opportunity to elucidate the mechanisms of SI in Poaceae.
Electronic supplementary material
The online version of this article (doi: 10.1186/1471-2164-15-399) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-399
PMCID: PMC4045969  PMID: 24886329
5.  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.
doi:10.1371/journal.pgen.1003933
PMCID: PMC3836714  PMID: 24278028
6.  Penetration of the Stigma and Style Elicits a Novel Transcriptome in Pollen Tubes, Pointing to Genes Critical for Growth in a Pistil 
PLoS Genetics  2009;5(8):e1000621.
Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. Using microarray analysis in Arabidopsis, we show that pollen tubes that have grown through stigma and style tissues of a pistil have a distinct gene expression profile and express a substantially larger fraction of the Arabidopsis genome than pollen grains or pollen tubes grown in vitro. Genes involved in signal transduction, transcription, and pollen tube growth are overrepresented in the subset of the Arabidopsis genome that is enriched in pistil-interacted pollen tubes, suggesting the possibility of a regulatory network that orchestrates gene expression as pollen tubes migrate through the pistil. Reverse genetic analysis of genes induced during pollen tube growth identified seven that had not previously been implicated in pollen tube growth. Two genes are required for pollen tube navigation through the pistil, and five genes are required for optimal pollen tube elongation in vitro. Our studies form the foundation for functional genomic analysis of the interactions between the pollen tube and the pistil, which is an excellent system for elucidation of novel modes of cell–cell interaction.
Author Summary
For successful reproduction in flowering plants, a single-celled pollen tube must rapidly extend through female pistil tissue, locate female gametes, and deliver sperm. Pollen tubes undergo a dramatic transformation while growing in the pistil; they grow faster compared to tubes grown in vitro and become competent to perceive and respond to navigation cues secreted by the pistil. The genes expressed by pollen tubes in response to growth in the pistil have not been characterized. We used a surgical procedure to obtain large quantities of uncontaminated pollen tubes that grew through the pistil and defined their transcriptome by microarray analysis. Importantly, we identify a set of genes that are specifically expressed in pollen tubes in response to their growth in the pistil and are not expressed during other stages of pollen or plant development. We analyzed mutants in 33 pollen tube–expressed genes using a sensitive series of pollen function assays and demonstrate that seven of these genes are critical for pollen tube growth; two specifically disrupt growth in the pistil. By identifying pollen tube genes induced by the pistil and describing a mutant analysis scheme to understand their function, we lay the foundation for functional genomic analysis of pollen–pistil interactions.
doi:10.1371/journal.pgen.1000621
PMCID: PMC2726614  PMID: 19714218
7.  A Species-Specific Cluster of Defensin-Like Genes Encodes Diffusible Pollen Tube Attractants in Arabidopsis 
PLoS Biology  2012;10(12):e1001449.
AtLURE1 defensin-like peptides, which show species-specific evolution, are essential in Arabidopsis for attracting pollen tubes and can function in the breakdown of reproductive isolation barriers.
Genes directly involved in male/female and host/parasite interactions are believed to be under positive selection. The flowering plant Arabidopsis thaliana has more than 300 defensin-like (DEFL) genes, which are likely to be involved in both natural immunity and cell-to-cell communication including pollen–pistil interactions. However, little is known of the relationship between the molecular evolution of DEFL genes and their functions. Here, we identified a recently evolved cluster of DEFL genes in A. thaliana and demonstrated that these DEFL (cysteine-rich peptide [CRP810_1]) peptides, named AtLURE1 peptides, are pollen tube attractants guiding pollen tubes to the ovular micropyle. The AtLURE1 genes formed the sole species-specific cluster among DEFL genes compared to its close relative, A. lyrata. No evidence for positive selection was detected in AtLURE1 genes and their orthologs, implying neutral evolution of AtLURE1 genes. AtLURE1 peptides were specifically expressed in egg-accompanying synergid cells and secreted toward the funicular surface through the micropyle. Genetic analyses showed that gametophytic mutants defective in micropylar guidance (myb98, magatama3, and central cell guidance) do not express AtLURE1 peptides. Downregulation of the expression of these peptides impaired precise pollen tube attraction to the micropylar opening of some populations of ovules. Recombinant AtLURE1 peptides attracted A. thaliana pollen tubes at a higher frequency compared to A. lyrata pollen tubes, suggesting that these peptides are species-preferential attractants in micropylar guidance. In support of this idea, the heterologous expression of a single AtLURE1 peptide in the synergid cell of Torenia fournieri was sufficient to guide A. thaliana pollen tubes to the T. fournieri embryo sac and to permit entry into it. Our results suggest the unique evolution of AtLURE1 genes, which are directly involved in male–female interaction among the DEFL multigene family, and furthermore suggest that these peptides are sufficient to overcome interspecific barriers in gametophytic attraction and penetration.
Author Summary
Defensin-like (DEFL) peptides commonly function as effector peptides and are involved in male-female and host-parasite interactions in eukaryotes. In higher plants, DEFL genes belong to a large multigene family and are highly variable between species. However, little is known about the relationship between the molecular evolution of DEFL genes and their functions. By comparing multiply duplicated DEFL genes between A. thaliana and its close relative A. lyrata, we have now identified pollen tube attractant peptides called AtLURE1 peptides, in A. thaliana. We find that AtLURE1 genes form a species-specific gene cluster and that the AtLURE1 peptides these genes encode are specifically expressed in the synergid (egg-accompanying) cells and are secreted along the path down which the pollen tube elongates to reach the female gametophyte. AtLURE1 peptides attract pollen tubes in a species-preferential manner and their downregulation impairs pollen tube guidance. Interestingly, the genetic introduction of a single AtLURE1 gene from A. thaliana into another plant, T. fournieri, is sufficient to breakdown reproductive isolation barriers in pollen tube guidance and penetration. These results suggest that AtLURE1 peptides, which show species-specific evolution, are key molecules that attract pollen tubes from a plant's own species to the embryo sac to enable successful reproduction.
doi:10.1371/journal.pbio.1001449
PMCID: PMC3525529  PMID: 23271953
8.  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.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2164-13-294
PMCID: PMC3416702  PMID: 22748054
9.  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
10.  Involvement of MLPK Pathway in Intraspecies Unilateral Incompatibility Regulated by a Single Locus With Stigma and Pollen Factors 
G3: Genes|Genomes|Genetics  2013;3(4):719-726.
Plants have evolved many systems to prevent undesirable fertilization. Among these, incompatibility is a well-organized system in which pollen germination or pollen tube growth is inhibited in pistils. We previously found that a novel one-way pollen–stigma incompatibility response [unilateral incompatibility (UI)] occurred between two self-incompatible Brassica rapa plants, a Turkish line, and a Japanese cultivated hybrid variety, “Osome.” Pollen from the Turkish line is rejected on the stigma of the Osome line, but the reverse cross is compatible; such a UI phenotype closely resembles self-incompatibility (SI). The pollen factor of this UI has been genetically explained by a single locus which is different from the S-locus. In this study, we performed further genetic analyses of this intraspecies UI and showed that the stigma factor was also controlled by a single locus, and we named the loci corresponding to the stigma and pollen factors of the intraspecies UI, stigmatic unilateral incompatibility (SUI), and pollen unilateral incompatibility (PUI) loci, respectively. Interestingly, segregation analyses of SUI and PUI indicated that they are closely linked to each other and behave as a single unit. To investigate the effect of an SI-related gene, MLPK in this UI, we produced segregation lines for SUI and mlpk. A distorted segregation ratio of SUI phenotype in an mlpk background indicated involvement of MLPK in SUI, suggesting the existence of an MLPK-dependent novel pollen–stigma recognition mechanism.
doi:10.1534/g3.113.005892
PMCID: PMC3618358  PMID: 23550141
Brassica rapa L; unilateral incompatibility; self-incompatibility; pollen-stigma recognition
11.  Distinct short-range ovule signals attract or repel Arabidopsis thaliana pollen tubes in vitro 
BMC Plant Biology  2006;6:7.
Background
Pollen tubes deliver sperm after navigating through flower tissues in response to attractive and repulsive cues. Genetic analyses in maize and Arabidopsis thaliana and cell ablation studies in Torenia fournieri have shown that the female gametophyte (the 7-celled haploid embryo sac within an ovule) and surrounding diploid tissues are essential for guiding pollen tubes to ovules. The variety and inaccessibility of these cells and tissues has made it challenging to characterize the sources of guidance signals and the dynamic responses they elicit in the pollen tubes.
Results
Here we developed an in vitro assay to study pollen tube guidance to excised A. thaliana ovules. Using this assay we discerned the temporal and spatial regulation and species-specificity of late stage guidance signals and characterized the dynamics of pollen tube responses. We established that unfertilized A. thaliana ovules emit diffusible, developmentally regulated, species-specific attractants, and demonstrated that ovules penetrated by pollen tubes rapidly release diffusible pollen tube repellents.
Conclusion
These results demonstrate that in vitro pollen tube guidance to excised A. thaliana ovules efficiently recapitulates much of in vivo pollen tube behaviour during the final stages of pollen tube growth. This assay will aid in confirming the roles of candidate guidance molecules, exploring the phenotypes of A. thaliana pollen tube guidance mutants and characterizing interspecies pollination interactions.
doi:10.1186/1471-2229-6-7
PMCID: PMC1489931  PMID: 16595022
12.  Pollination and late-acting self-incompatibility in Cyrtanthus breviflorus (Amaryllidaceae): implications for seed production 
Annals of Botany  2010;106(4):547-555.
Background and Aims
Animal pollination is typically an uncertain process that interacts with self-incompatibility status to determine reproductive success. Seed set is often pollen-limited, but species with late-acting self-incompatibility (SI) may be particularly vulnerable, if self-pollen deposition results in ovule discounting. Pollination is examined and the occurrence of late-acting SI and ovule discounting assessed in Cyrtanthus breviflorus.
Methods
The pollination system was characterized by observing floral visitors and assessing nectar production and spectral reflectance of flowers. To assess late-acting SI and ovule discounting, growth of self- and cross-pollen tubes, and seed set following open pollination or hand pollination with varying proportions of self- and cross-pollen, were examined.
Key Results
Native honeybees Apis mellifera scutellata pollinated flowers as they actively collected pollen. Most flowers (≥70 %) did not contain nectar, while the rest produced minute volumes of dilute nectar. The flowers which are yellow to humans are visually conspicuous to bees with a strong contrast between UV-reflecting tepals and UV-absorbing anthers and pollen. Plants were self-incompatible, but self-rejection was late-acting and both self- and cross-pollen tubes penetrated ovules. Seed set of open-pollinated flowers was pollen-limited, despite pollen deposition exceeding ovule number by 6-fold. Open-pollinated seed set was similar to that of the cross + self-pollen treatment, but was less than that of the cross-pollen-only treatment.
Conclusions
Flowers of C. breviflorus are pollinated primarily by pollen-collecting bees and possess a late-acting SI system, previously unknown in this clade of the Amaryllidaceae. Pollinators of C. breviflorus deposit mixtures of cross- and self-pollen and, because SI is late-acting, self-pollen disables ovules, reducing female fertility. This study thus contributes to growing evidence that seed production in plants with late-acting SI systems is frequently limited by pollen quality, even when pollinators are abundant.
doi:10.1093/aob/mcq149
PMCID: PMC2944973  PMID: 20647225
Amarydillaceae; Cyrtanthus breviflorus; honeybee pollination; late-acting self-incompatibility; ovule discounting; pollen limitation; pollen quantity and quality
13.  A Group-1 Grass Pollen Allergen Influences the Outcome of Pollen Competition in Maize 
PLoS ONE  2007;2(1):e154.
Worldwide, 400 million people suffer from hay fever and seasonal asthma. The major causative agents of these allergies are pollen specific proteins called the group-1 grass pollen allergens. Although details of their antigenicity have been studied for 40 years with an eye towards immunotherapy, their function in the plant has drawn scant attention. Zea m 1 constitutes a class of abundant grass pollen allergens coded for by several genes that loosen the walls of grass cells, including the maize stigma and style. We have examined the impact of a transposon insertion into one of these genes (EXPB1, the most abundant isoform of Zea m 1) on the production of Zea m 1 protein, pollen viability, and pollen tube growth, both in vitro and in vivo. We also examined the effect of the insertional mutation on the competitive ability of the pollen by experimentally varying the sizes of the pollen load deposited onto stigmas using pollen from heterozygous plants and then screening the progeny for the presence of the transposon using PCR. We found that the insertional mutation reduced the levels of Zea m 1 in maize pollen, but had no effect on pollen viability, in vitro pollen tube growth or the proportion of progeny sired when small pollen loads are deposited onto stigmas. However, when large pollen loads are deposited onto the stigmas, the transposon mutation is vastly underrepresented in the progeny, indicating that this major pollen allergen has a large effect on pollen tube growth rates in vivo, and plays an important role in determining the outcome of the pollen-pollen competition for access to the ovules. We propose that the extraordinary abundance (4% of the extractable protein in maize pollen) of this major pollen allergen is the result of selection for a trait that functions primarily in providing differential access to ovules.
doi:10.1371/journal.pone.0000154
PMCID: PMC1764715  PMID: 17225858
14.  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.
doi:10.1093/jxb/ert093
PMCID: PMC3654424  PMID: 23676884
pollen tube; pollen-predominant; profilin; short open reading frame; Zea mays; Zm908p11.
15.  The beginning of a seed: regulatory mechanisms of double fertilization 
The launch of seed development in flowering plants (angiosperms) is initiated by the process of double fertilization: two male gametes (sperm cells) fuse with two female gametes (egg and central cell) to form the precursor cells of the two major seed components, the embryo and endosperm, respectively. The immobile sperm cells are delivered by the pollen tube toward the ovule harboring the female gametophyte by species-specific pollen tube guidance and attraction mechanisms. After pollen tube burst inside the female gametophyte, the two sperm cells fuse with the egg and central cell initiating seed development. The fertilized central cell forms the endosperm while the fertilized egg cell, the zygote, will form the actual embryo and suspensor. The latter structure connects the embryo with the sporophytic maternal tissues of the developing seed. The underlying mechanisms of double fertilization are tightly regulated to ensure delivery of functional sperm cells and the formation of both, a functional zygote and endosperm. In this review we will discuss the current state of knowledge about the processes of directed pollen tube growth and its communication with the synergid cells resulting in pollen tube burst, the interaction of the four gametes leading to cell fusion and finally discuss mechanisms how flowering plants prevent multiple sperm cell entry (polyspermy) to maximize their reproductive success.
doi:10.3389/fpls.2014.00452
PMCID: PMC4160995  PMID: 25309552
pollen tube; ovule; gamete interaction; cell fusion; signaling; fertilization; polyspermy
16.  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
17.  Pollen Lipidomics: Lipid Profiling Exposes a Notable Diversity in 22 Allergenic Pollen and Potential Biomarkers of the Allergic Immune Response 
PLoS ONE  2013;8(2):e57566.
Background/Aim
Pollen grains are the male gametophytes that deliver sperm cells to female gametophytes during sexual reproduction of higher plants. Pollen is a major source of aeroallergens and environmental antigens. The pollen coat harbors a plethora of lipids that are required for pollen hydration, germination, and penetration of the stigma by pollen tubes. In addition to proteins, pollen displays a wide array of lipids that interact with the human immune system. Prior searches for pollen allergens have focused on the identification of intracellular allergenic proteins, but have largely overlooked much of the extracellular pollen matrix, a region where the majority of lipid molecules reside. Lipid antigens have attracted attention for their potent immunoregulatory effects. By being in close proximity to allergenic proteins on the pollen surface when they interact with host cells, lipids could modify the antigenic properties of proteins.
Methodology/Principal Findings
We performed a comparative pollen lipid profiling of 22 commonly allergenic plant species by the use of gas chromatography-mass spectroscopy, followed by detailed data mining and statistical analysis. Three experiments compared pollen lipid profiles. We built a database library of the pollen lipids by matching acquired pollen-lipid mass spectra and retention times with the NIST/EPA/NIH mass-spectral library. We detected, identified, and relatively quantified more than 106 lipid molecular species including fatty acids, n-alkanes, fatty alcohols, and sterols. Pollen-derived lipids stimulation up-regulate cytokines expression of dendritic and natural killer T cells co-culture.
Conclusions/Significance
Here we report on a lipidomic analysis of pollen lipids that can serve as a database for identifying potential lipid antigens and/or novel candidate molecules involved in allergy. The database provides a resource that facilitates studies on the role of lipids in the immunopathogenesis of allergy. Pollen lipids vary greatly among allergenic species and contain many molecules that have stimulatory or regulatory effects on immune responses.
doi:10.1371/journal.pone.0057566
PMCID: PMC3585183  PMID: 23469025
18.  Breeding systems, hybridization and continuing evolution in Avon Gorge Sorbus 
Annals of Botany  2013;111(4):563-575.
Background and Aims
Interspecific hybridization and polyploidy are key processes in plant evolution and are responsible for ongoing genetic diversification in the genus Sorbus (Rosaceae). The Avon Gorge, Bristol, UK, is a world ‘hotspot’ for Sorbus diversity and home to diploid sexual species and polyploid apomictic species. This research investigated how mating system variation, hybridization and polyploidy interact to generate this biological diversity.
Methods
Mating systems of diploid, triploid and tetraploid Sorbus taxa were analysed using pollen tube growth and seed set assays from controlled pollinations, and parent–offspring genotyping of progeny from open and manual pollinations.
Key Results
Diploid Sorbus are outcrossing and self-incompatible (SI). Triploid taxa are pseudogamous apomicts and genetically invariable, but because they also display self-incompatibility, apomictic seed set requires pollen from other Sorbus taxa – a phenomenon which offers direct opportunities for hybridization. In contrast tetraploid taxa are pseudogamous but self-compatible, so do not have the same obligate requirement for intertaxon pollination.
Conclusions
The mating inter-relationships among Avon Gorge Sorbus taxa are complex and are the driving force for hybridization and ongoing genetic diversification. In particular, the presence of self-incompatibility in triploid pseudogamous apomicts imposes a requirement for interspecific cross-pollination, thereby facilitating continuing diversification and evolution through rare sexual hybridization events. This is the first report of naturally occurring pseudogamous apomictic SI plant populations, and we suggest that interspecific pollination, in combination with a relaxed endosperm balance requirement, is the most likely route to the persistence of these populations. We propose that Avon Gorge Sorbus represents a model system for studying the establishment and persistence of SI apomicts in natural populations.
doi:10.1093/aob/mct013
PMCID: PMC3605949  PMID: 23408832
Hybridization; evolution; polyploidy; apomixis; pseudogamy; self-incompatibility; Sorbus
19.  Three MYB transcription factors control pollen tube differentiation required for sperm release 
Current biology : CB  2013;23(13):1209-1214.
Summary
In flowering plants, immotile sperm cells develop within the pollen grain and are delivered to female gametes by a pollen tube [1, 2]. Upon arrival at the female gametophyte, the pollen tube stops growing and releases sperm cells for successful fertilization [3]. Several female signaling components essential for pollen tube reception have been identified [4–11]); however, male components remain unknown. We show that the expression of three closely related MYB transcription factors is induced in pollen tubes by growth in the pistil. Pollen tubes lacking these three transcriptional regulators fail to stop growing in synergids, specialized cells flanking the egg cell that attract pollen tubes [12–16] and degenerate upon pollen tube arrival [17, 18]. myb triple mutant pollen tubes also fail to release their sperm cargo. We define a suite of pollen tube-expressed genes regulated by these critical MYBs and identify transporters, carbohydrate active enzymes, and small peptides as candidate molecular mediators of pollen-female interactions necessary for flowering plant reproduction. Our data indicate that de novo transcription in the pollen tube nucleus during growth in the pistil leads to pollen tube differentiation required for release of sperm cells.
doi:10.1016/j.cub.2013.05.021
PMCID: PMC4009620  PMID: 23791732
20.  Unilateral Incompatibility in Capsicum (Solanaceae): Occurrence and Taxonomic Distribution 
Annals of Botany  2004;94(2):289-295.
• Background and aims Unilateral incompatibility (UI) occurs when pollinations between species are successful in one direction but not in the other. Self-incompatible (SI) species frequently show UI with genetically related, self-compatible (SC) species, as pollen of SI species is compatible on the SC pistil, but not vice versa. Many examples of unilateral incompatibility, and all those which have been studied most intensively, are found in the Solanaceae, particularly Lycopersicon, Solanum, Nicotiana and Petunia. The genus Capsicum is evolutionarily somewhat distant from Lycopersicon and Solanum and even further removed from Nicotiana and Petunia. Unilateral incompatibility has also been reported in Capsicum; however, this is the first comprehensive study of crosses between all readily available species in the genus.
• Methods All readily available (wild and domesticated) species in the genus are used as plant material, including the three genera from the Capsicum pubescens complex plus eight other species. Pollinations were made on pot-grown plants in a glasshouse. The number of pistils pollinated per cross varied (from five to 40 pistils per plant), depending on the numbers of flowers available. Pistils were collected 24 h after pollination and fixed for 3–24 h. After staining, pistils were mounted in a drop of stain, squashed gently under a cover slip and examined microscopically under ultra-violet light for pollen tube growth.
• Key results Unilateral incompatibility is confirmed in the C. pubescens complex. Its direction conforms to that predominant in the Solanaceae and other families, i.e. pistils of self-incompatible species, or self-compatible taxa closely related to self-incompatible species, inhibit pollen tubes of self-compatible species.
• Conclusions Unilateral incompatibility in Capsicum does not seem to have arisen to prevent introgression of self-compatibility into self-incompatible taxa, but as a by-product of divergence of the C. pubescens complex from the remainder of the genus.
doi:10.1093/aob/mch139
PMCID: PMC4242164  PMID: 15229125
Capsicum pubescens complex; chile pepper; incompatibility; incongruity; interspecific hybridization; pollen tube growth; S gene
21.  Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue 
Journal of Experimental Botany  2008;59(11):3187-3201.
After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen–stigma interactions that regulate pollen tube growth in Nicotiana.
doi:10.1093/jxb/ern175
PMCID: PMC2504342  PMID: 18689443
Kunitz proteinase inhibitor homologue; Nicotiana; pollen–pistil interactions; pollination
22.  Time-lapse imaging of self- and cross-pollinations in Brassica rapa 
Annals of Botany  2013;112(1):115-122.
Background and Aims
Pollination is an important process in the life cycle of plants and is the first step in bringing together the male and female gametophytes for plant reproduction. While pollination has been studied for many years, accurate knowledge of the morphological aspects of this process is still far from complete. This study therefore focuses on a morphological characterization of pollination, using time-series image analysis of self- and cross-pollinations in Brassica rapa.
Methods
Time-lapse imaging of pollen behaviour during self- and cross-pollinations was recorded for 90 min, at 1 min intervals, using a stereoscopic microscope. Using time-series digital images of pollination, characteristic features of pollen behaviours during self- and cross-pollinations were studied.
Key Results
Pollen exhibited various behaviours in both self- and cross-pollinations, and these were classified into six representative patterns: germination, expansion, contraction, sudden contraction, pulsation and no change. It is noteworthy that in ‘contraction’ pollen grains shrunk within a short period of 30–50 min, and in ‘pulsation’ repeated expansion and contraction occurred with an interval of 10 min, suggesting that a dehydration system is operating in pollination. All of the six patterns were observed on an individual stigma with both self- and cross-pollinations, and the difference between self- and cross-pollinations was in the ratios of the different behaviours. With regard to water transport to and from pollen grains, this occurred in multiple steps, before, during and after hydration. Thus, pollination is regulated by a combination of multiple components of hydration, rehydration and dehydration systems.
Conclusions
Regulated hydration of pollen is a key process for both pollination and self-incompatibility, and this is achieved by a balanced complex of hydration, dehydration and nutrient supply to pollen grains from stigmatic papilla cells.
doi:10.1093/aob/mct102
PMCID: PMC3690991  PMID: 23644359
Brassicaceae; Brassica rapa; pollination; live imaging; pollen hydration; pollen–stigma interaction; self-incompatibility; time-lapse imaging
23.  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.
doi:10.1098/rspb.1998.0381
PMCID: PMC1689050
24.  Pollen-tube growth rates in Collinsia heterophylla (Plantaginaceae): one-donor crosses reveal heritability but no effect on sporophytic-offspring fitness 
Annals of Botany  2009;103(6):941-950.
Background and Aims
Evolutionary change in response to natural selection will occur only if a trait confers a selective advantage and there is heritable variation. Positive connections between pollen traits and fitness have been found, but few studies of heritability have been conducted, and they have yielded conflicting results. To understand better the evolutionary significance of pollen competition and its potential role in sexual selection, the heritability of pollen tube-growth rate and the relationship between this trait and sporophytic offspring fitness were investigated in Collinsia heterophylla.
Methods
Because the question being asked was if female function benefited from obtaining genetically superior fathers by enhancing pollen competition, one-donor (per flower) crosses were used in order to exclude confounding effects of post-fertilization competition/allocation caused by multiple paternity. Each recipient plant was crossed with an average of five pollen donors. Pollen-tube growth rate and sporophytic traits were measured in both generations.
Key Results
Pollen-tube growth rate in vitro differed among donors, and the differences were correlated with in vivo growth rate averaged over two to four maternal plants. Pollen-tube growth rate showed significant narrow-sense heritability and evolvability in a father–offspring regression. However, this pollen trait did not correlate significantly with sporophytic-offspring fitness.
Conclusions
These results suggest that pollen-tube growth rate can respond to selection via male function. The data presented here do not provide any support for the hypothesis that intense pollen competition enhances maternal plant fitness through increased paternity by higher-quality sporophytic fathers, although this advantage cannot be ruled out. These data are, however, consistent with the hypothesis that pollen competition is itself selectively advantageous, through both male and female function, by reducing the genetic load among successful gametophytic fathers (pollen), and reducing inbreeding depression associated with self–pollination in plants with mix-mating systems.
doi:10.1093/aob/mcp014
PMCID: PMC2707896  PMID: 19202136
Collinsia heterophylla; evolvability; female fitness; good genes; heritability; male fitness; mixed-mating system; Plantaginaceae; pollen competition; sexual selection
25.  Developmental onset of reproductive barriers and associated proteome changes in stigma/styles of Solanum pennellii  
Journal of Experimental Botany  2012;64(1):265-279.
Although self-incompatibility (SI) in plants has been studied extensively, far less is known about interspecific reproductive barriers. One interspecific barrier, known as unilateral incongruity or incompatibility (UI), occurs when species display unidirectional compatibility in interspecific crosses. In the wild tomato species Solanum pennellii, both SI and self-compatible (SC) populations express UI when crossed with domesticated tomato, offering a useful model system to dissect the molecular mechanisms involved in reproductive barriers. In this study, the timing of reproductive barrier establishment during pistil development was determined in SI and SC accessions of S. pennellii using a semi-in vivo system to track pollen-tube growth in developing styles. Both SI and UI barriers were absent in styles 5 days prior to flower opening, but were established by 2 days before flower opening, with partial barriers detected during a transition period 3–4 days before flower opening. The developmental expression dynamics of known SI factors, S-RNases and HT proteins, was also examined. The accumulation of HT-A protein coincided temporally and spatially with UI barriers in developing pistils. Proteomic analysis of stigma/styles from key developmental stages showed a switch in protein profiles from cell-division-associated proteins in immature stigma/styles to a set of proteins in mature stigma/styles that included S-RNases, HT-A protein and proteins associated with cell-wall loosening and defense responses, which could be involved in pollen–pistil interactions. Other prominent proteins in mature stigma/styles were those involved in lipid metabolism, consistent with the accumulation of lipid-rich material during pistil maturation.
doi:10.1093/jxb/ers324
PMCID: PMC3528032  PMID: 23166371
pistil development; reproductive barriers; self-incompatibility; Solanum pennellii; stigma/stylar proteins; unilateral incongruity/incompatibility

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