Unpollinated Pistils Research Articles

Morphological Changes to Fruit Development Induced by GA3 Application in Sweet Cherry (Prunus avium L.).

Cherry (Prunus avium) fruits are important sources of vitamins, minerals, and nutrients in the human diet; however, they contain a large stone, making them inconvenient to eat 'on the move' and process. The exogenous application of gibberellic acid (GA3) can induce parthenocarpy in a variety of fruits during development. Here, we showed that the application of GA3 to sweet cherry unpollinated pistils acted as a trigger for fruit set and permitted the normal formation of fruit up to a period of twenty-eight days, indicating that gibberellins are involved in the activation of the cell cycle in the ovary wall cells, leading to fruit initiation. However, after this period, fruit development ceased and developing fruit began to be excised from the branch by 35 days post treatment. This work also showed that additional signals are required for the continued development of fully mature parthenocarpic fruit in sweet cherry.

The origin and the genetic regulation of the self-compatibility mechanism in clementine (Citrus clementina Hort. ex Tan.).

Self-incompatibility (SI) is a genetic mechanism common in flowering plants to prevent self-fertilization. Among citrus species, several pummelo, mandarin, and mandarin-like accessions show SI behavior. In these species, SI is coupled with a variable degree of parthenocarpy ensuring the production of seedless fruits, a trait that is highly appreciated by consumers. In Citrus, recent evidences have shown the presence of a gametophytic SI system based on S-ribonucleases (S-RNases) ability to impair self-pollen tube growth in the upper/middle part of the style. In the present study, we combined PCR analysis and next-generation sequencing technologies, to define the presence of S7- and S11-Rnases in the S-genotype of the Citrus clementina (Hort. ex Tan.), the self-incompatible 'Comune' clementine and its self-compatible natural mutant 'Monreal'. The reference genome of 'Monreal' clementine is presented for the first time, providing more robust results on the genetic sequence of the newly discovered S7-RNase. SNP discovery analysis coupled with the annotation of the variants detected enabled the identification of 7,781 SNPs effecting 5,661 genes in 'Monreal' compared to the reference genome of C. clementina. Transcriptome analysis of unpollinated pistils at the mature stage from both clementine genotypes revealed the lack of expression of S7-RNase in 'Monreal' suggesting its involvement in the loss of the SI response. RNA-seq analysis followed by gene ontology studies enabled the identification of 2,680 differentially expressed genes (DEGs), a significant number of those is involved in oxidoreductase and transmembrane transport activity. Merging of DNA sequencing and RNA data led to the identification of 164 DEGs characterized by the presence of at least one SNP predicted to induce mutations with a high effect on their amino acid sequence. Among them, four candidate genes referring to two Agamous-like MADS-box proteins, to MYB111 and to MLO-like protein 12 were validated. Moreover, the transcription factor MYB111 appeared to contain a binding site for the 2.0-kb upstream sequences of the S7- and S11-RNase genes. These results provide useful information about the genetic bases of SI indicating that SNPs present in their sequence could be responsible for the differential expression and the regulation of S7-RNase and consequently of the SI mechanism.

Quantitative Phosphoproteomic Analysis Reveals Potential Regulatory Mechanisms of Early Fruit Enlargement in Pecan (Carya illinoinensis)

Pecan (Carya illinoinensis) is a popular tree nut. Its fruit development undergoes slow growth, rapid expansion, core hardening, and kernel maturation stages. However, little is known about how pecan initiates fruit development and enlargement after pollination. In this study, we performed the first large-scale identification of potential phosphorylation sites and proteins at early development of pecan fruit by a label-free phosphoproteomic quantification technique. A total of 2155 phosphosites were identified from 1953 phosphopeptides covering 1311 phosphoproteins in unpollinated pistils and fruits at 5 and 9 weeks after pollination. Of these, 699 nonredundant phosphoproteins were differentially phosphorylated (DP). Furthermore, the phosphorylation intensity of DP proteins in brassinolide (BR) and auxin signaling were analyzed, and the function of CiBZR1 was investigated. Ectopic expression of CiBZR1 resulted in BR response phenotypes with curled leaves and fruit, while enlarged seed size in Arabidopsis. Subcellular localization and transcriptional activation activity assay demonstrated that CiBZR1 distributed in both the nucleus and cytoplasm with transcriptional activity. When two phosphosites mutated, CiBZR1S201P,S205G moved to the nucleus completely, while the transcriptional activity remained unchanged. Taken together, our data reveal extensive phosphoproteins and lay a foundation to comprehensively dissect the potential post-translational regulation mechanism of early development of pecan fruit.

Transcriptomic analysis of pollen-pistil interactions in almond (Prunus dulcis) identifies candidate genes for components of gametophytic self-incompatibility

The cultivated almond exhibits self-incompatibility of the gametophytic type regulated by the S-locus, which is expressed in both the pistil (S-RNase) and pollen (SFB protein). Although almond cultivars are mostly self-incompatible, some cultivars have been found to be self-compatible. For a long time, self-compatibility was unequivocally associated only with the presence of the Sf haplotype. However, recent studies reported the existence of self-incompatible almond cultivars carrying the Sf genotype. This finding suggests the involvement of new, hitherto undiscovered components involved in the almond self-incompatibility system. The aim of this study was to clarify the transcription pattern of the S-genes and to look for additional components of the gametophytic self-incompatibility system in almond. Transcriptome analysis of un-pollinated pistils and incompatible and compatible pollinations of self-compatible and self-incompatible almonds carrying the Sf haplotype was performed using high-throughput RNA sequencing technologies. Among the unigenes, 1357 were shown to be differentially expressed, and gene ontology annotation revealed that they are mostly involved in metabolic processes and binding molecular functions. The expression trend of fourteen representative genes, some of which are putatively involved in the self-(in)compatible response, was confirmed by RT-qPCR. This transcriptomic analysis provides candidate genes for almond components of gametophytic self-incompatibility and could be used as reference for subsequent comparative transcriptomic analyses of pollen and pistil.

Low genetic diversity and functional constraint of miRNA genes participating pollen-pistil interaction in rice.

In this study, we sequenced and analyzed the expression and evolution of rice miRNA genes participating pollen-pistil interaction that is crucial to rice yield. Pollen-pistil interaction is an essential reproductive process for all flowering plants. While microRNAs (miRNAs) are important noncoding small RNAs that regulate mRNA levels in eukaryotic cells, there is little knowledge about which miRNAs involved in the early stages of pollen-pistil interaction in rice and how they evolve under this conserved process. In this study, we sequenced the small RNAs in rice from unpollinated pistil (R0), pistil from 5min and 15min after pollination, respectively, to identify known and novel miRNAs that are involved in this process. By comparing the corresponding mRNA-seq dataset, we identified a group of miRNAs with strong negative expression pattern with their target genes. Further investigation of all miRNA loci (MIRNAs) across 1083 public rice accessions revealed significantly reduced genetic diversity in MIRNAs with strong negative expression of their targets when comparing to those with little or no impact on targets during pollen-pistil interaction. Annotation of targets suggested that those MIRNAs with strong impact on targets were pronounced in cell wall related processes such as xylan metabolism. Additionally, plant conserved miRNAs, such as those with functions in gibberellic acid, auxin and nitrate signaling, were also with strong negative expression of their targets. Overall, our analyses identified key miRNAs participating pollen-pistil interaction and their evolutionary patterns in rice, which can facilitate the understanding of molecular mechanisms associated with seed setting.

Evidence for post-zygotic self-incompatibility in Handroanthus impetiginosus (Bignoniaceae).

Late-acting self-incompatibility (LSI) has been defined as a genetically controlled self-sterility mechanism that prevents seed set by selfing, despite normal pollen tube growth and ovule penetration in self-pollinated pistils. In species of the Bignoniaceae with LSI, such as Handroanthus impetiginosus, the selfed pistils are characterized by a marked delay in ovule penetration, fertilization, and endosperm initiation, followed by uniform pistil abscission. This highlights the contentious possibility of a post-zygotic self-incompatibility system. However, previous studies were unable to confirm fusion of the sperm and egg cell nuclei in selfed ovules. In the present study, the cytology of the embryo sac, double fertilization, and pistil longevity was investigated in H. impetiginosus using comparative nuclei microspectrofluorometry of DAPI-stained sections of self- vs. unpollinated pistils. Differences in both pistil longevity and ovary size between self- and unpollinated flowers at the time of pistil abscission were significant. Zygotes with double the DNA content in their nuclei relative to unfertilized egg cell nuclei were verified in selfed ovules from the first day after pollination onward, and G1 karyogamy appeared to have occurred. Our cytological analysis clearly indicates that ovules of self-pollinated pistils in H. impetiginosus are fertilized before pistil abscission but no embryogenesis initiation occurs, which strongly supports the idea of a post-zygotic self-incompatibility mechanism.

Identification of the ligand of Pru p 3, a peach LTP.

Pru p 3, a peach LTP, is located in pollinated flower styles and secreting downy hairs, transporting a derivative of camptothecin bound to phytosphingosine. Pru p 3 may inhibit a second pollination and may keep away herbivores until seed maturation. The allergen Pru p 3, a peach lipid transfer protein, has been well studied. However, its physiological function remains to be elucidated. Our results showed that Pru p 3 usually carries a lipid ligand that play an essential role in its function in plants. Using ESI-qToF, we observed that the ligand was a derivative of camptothecin binding to phytosphingosine, wich that is inserted into the hydrophobic tunnel of the protein. In addition, the described ligand displayed topoisomerase I activity inhibition and self-fluorescence, both recognized as camptothecin properties. During flower development, the highest expression of Pru p 3 was detected in the styles of pollinated flowers, in contrast to its non-expression in unpollinated pistils, where expression decreased after anthesis. During ripening, the expression of Pru p 3 were observed mainly in peel but not in pulp. In this sense, Pru p 3 protein was also localized in trichomes covering the fruit epidermis.

Pollen tube access to the ovule is mediated by glycoprotein secretion on the obturator of apple (Malus × domestica, Borkh).

Within the ovary, the obturator bridges the pathway of the pollen tube from the style to the ovule. Despite its widespread presence among flowering plants, its function has only been studied in a handful of species, and the molecules involved in pollen tube-obturator cross-talk have not been explored hitherto. This work evaluates the involvement of glucans and glycoproteins on pollen tube growth in the obturator of apple flowers ( Malus × domestica) . Pollen tube kinetics were sequentially examined in the pistil and related to changes occurring on the obturator using histochemistry and inmunocytochemistry. To discriminate between changes in the obturator induced by pollen tubes from those developmentally regulated, both pollinated and unpollinated pistils were examined. Pollen tube growth rates were slow in the stigma, faster in the style and slow again in the ovary. The arrival of pollen tubes at the obturator was concomitant with the secretion of proteins, saccharides and glycoprotein epitopes belonging to extensins and arabinogalactan proteins (AGPs). While some of these secretions - extensins and AGPs labelled by JIM13 - were developmentally regulated, others - AGPs labelled by JIM8 - were elicited by the presence of pollen tubes. Following pollen tube passage, all these glycoproteins were depleted. The results show a timely secretion of glycoproteins on the obturator surface concomitant with pollen tube arrival at this structure. The fact that their secretion is depleted following pollen tube passage strongly suggests their role in regulating pollen tube access to the ovule. Remarkably, both the regulation of the secretion of the different glycoproteins, as well as their association with the performance of pollen tubes exhibit similarities with those observed in the stigma, in line with their common developmental origin.

Sporophytic apomixis in polyembryonic Handroanthus serratifolius (Vahl) S.O. Grose (Bignoniaceae) characterizes the species as an agamic polyploid complex

Neopolyploidy has been associated with gametophytic apomixis and breakdown of gametophytic self-incompatibility. Nevertheless, Bignoniaceae presents agamic polyploid complexes with neopolyploidy associated to sporophytic apomixis. Apomictic populations are commonly polyploid, polyembryonic and self-fertile, while diploids are mostly late-acting self-incompatible (LSI) and monoembryonic. Contrastingly, Handroanthus serratifolius shows hexaploid monoembryonic and polyembryonic populations, although breeding system has been studied only for monoembryonic individuals, which are LSI. Our aim here was to investigate breeding system and early embryology in polyembryonic individuals of H. serratifolius to define if they form an agamic polyploid complex. Experimental pollinations and histological analyzes of ovules and young seeds were carried out. Megasporogenesis and megagametogenesis occurred as in other sexual species of Bignoniaceae. The polyembryonic individuals were self-fertile and double fertilization was observed both after self and cross-pollinated pistils. Adventitious embryos originated from the hypostasis and integument of the ovule, indicating sporophytic apomixis. Adventitious embryo precursor cells occurred in all pistils, including unpollinated ones. But unpollinated pistils aborted possibly due to absence of endosperm, and pollination was required for fruit-set (pseudogamy). It is possible that the self-fertility in polyembryonic individuals ensues as the initial endosperm of self-fertilized ovules supply early adventitious embryos development, and these embryos would later prevent the abortion of selfed pistils. The sporophytic apomixis in polyembryonic populations and the occurrence of sexual monoembryonic populations of H. serratifolius allows us to consider the species part of an agamic polyploid complex. But in contrast with other Handroanthus agamic complexes, both apomictic and sexual LSI plants were hexaploid.

Pollination triggers female gametophyte development in immature Nicotiana tabacum flowers

In Nicotiana tabacum, female gametophytes are not fully developed at anthesis, but flower buds pollinated 12 h before anthesis produce mature embryo sacs. We investigated several pollination-associated parameters in N. tabacum flower buds to determine the developmental timing of important events in preparation for successful fertilization. First, we performed hand pollinations in flowers from stages 4 to 11 to study at which developmental stage pollination would produce fruits. A Peroxtesmo test was performed to correlate peroxidase activity on the stigma surface, indicative of stigma receptivity, with fruit set. Pollen tube growth and female gametophyte development were microscopically analyzed in pistils of different developmental stages. Fruits were obtained only after pollinations of flower buds at late stage 7 and older; fruit weight and seed germination capacity increased as the developmental stage of the pollinated flower approached anthesis. Despite positive peroxidase activity and pollen tube growth, pistils at stages 5 and 6 were unable to produce fruits. At late stage 7, female gametophytes were undergoing first mitotic division. After 24 h, female gametophytes of unpollinated pistils were still in the end of the first division, whereas those of pollinated pistils showed egg cells. RT-qPCR assay showed that the expression of the NtEC1 gene, a marker of egg cell development, is considerably higher in pollinated late stage 7 ovaries compared with unpollinated ovaries. To test whether ethylene is the signal eliciting female gametophyte maturation, the expression of ACC synthase was examined in unpollinated and pollinated stage 6 and late stage 7 stigmas/styles. Pollination induced NtACS expression in stage 6 pistils, which are unable to produce fruits. Our results show that pollination is a stimulus capable of triggering female gametophyte development in immature tobacco flowers and suggests the existence of a yet undefined signal sensed by the pistil.

ITRAQ-based quantitative proteomic analysis of pistils and anthers from self-incompatible and self-compatible almonds with the S f haplotype

Almond [Prunus dulcis (Miller) D.A. Webb] exhibits self-incompatibility of the gametophytic type, which is controlled by a locus named S with expression in pistils (S-RNase) and in pollen (SFB protein). Recent studies support the involvement of other unidentified components (named modifier factors) in the incompatibility system of this species. A few proteomic studies have tried to identify modifier factors in Prunus species using comparative proteomics of pollinated pistils. However, there are no studies in which the pollen and un-pollinated pistils from self-incompatible and self-compatible Prunus species are compared. In the present work, IEF and RT-PCR analyses of pistils from two almond selections with the S f haplotype but different incompatibility phenotypes revealed that lack of ribonuclease activity in the self-compatible individual is not due to the absence of Sf-RNase transcripts. To identify the proteins differentially expressed in the pistils and anthers of these selections, which could be candidates for modifier factors, isobaric tags for relative and absolute quantitation and 2D-nano-liquid chromatography–electrospray ionisation tandem mass spectrometry were carried out. After a BLAST search against the UniProtKB/SwissProt database, 23 proteins were identified as differentially expressed in pistils and 17 proteins were identified as differentially expressed in anthers. Grouping these proteins into functional categories revealed that most of the pistil proteins had a metabolic function, followed by proteins with a stress resistance and defence function. In the case of anthers, metabolic proteins together with stress resistance and defence proteins were also predominant, although this time in equal proportion. Moreover, the differentially expressed proteins were associated with particular genes, which were located in the Prunus persica Whole Genome version 1.0. Some of the proteins identified here have been linked to pollen development, to pollen dynamics or to pollen–pistil interactions. These results not only provide proteomic profiles of differential expression in mature pistils and anthers, but could also serve as a reference for other comparative proteomic studies of pollinated pistils in almond and in other species with the same incompatibility system.

Exogenous steroid hormones stimulate full development of autonomous endosperm in Arabidopsis thaliana

<p>Most flowering plants, including important crops, require double fertilization to form an embryo and endosperm, which nourishes it. Independence from fertilization is a feature of apomictic plants that produce seeds, from which the plants that are clones of the mother plant arise. The phenomenon of apomixis occurs in some sexual plants under specific circumstances. Since the launch of a fertilization-independent mechanism is considered a useful tool for plant breeding, there have been efforts to artificially induce apomixis. We have been able to produce fertilization-independent endosperm in vitro in <em>Arabidopsis</em> over the last few years. This paper demonstrates the methods of improving the quality of the endosperm obtained using plant and mammalian steroid hormones. Additionally, it shows the study on the autonomous endosperm (AE) formation mechanism in vitro.</p><p>This paper examines the effect of exogenous steroid hormones on unfertilized egg and central cell divisions in culture of unpollinated pistils of <em>Arabidopsis</em> Col-0 wild-type and <em>fie-1</em> mutant. All media with hormones used (estrone, androsterone, progesterone, and epibrassinolide) stimulated central cell divisions and fertilization-independent endosperm development. The stages of AE development followed the pattern of <em>Arabidopsis thaliana</em> wild type after fertilization. Subsequent stages of AE were observed from 2-nuclear up to cellular with the most advanced occurring on medium with 24-epibrassinolide and progesterone. The significant influence of mammalian sex hormones on speed of AE development and differentiation was noticed. Using restriction analysis, the changes in methylation of <em>FIE</em> gene was established under in vitro condition. The authors of this paper showed that <em>Arabidopsis thaliana</em> has a high potency to fertilization-independent development.</p>

Glycoprotein composition along the pistil of Malus x domestica and the modulation of pollen tube growth

BackgroundThe characteristics of pollen tube growth are not constant, but display distinct patterns of growth within the different tissues of the pistil. In the stigma, the growth rate is slow and autotrophic, whereas in the style, it is rapid and heterotrophic. Very little is known about the interactions between these distinct maternal tissues and the traversing pollen tube and the role of this interaction on the observed metabolism. In this work we characterise pollen tube growth in the apple flower and look for differences in glycoprotein epitope localization between two different maternal tissues, the stigma and the style.ResultsWhile immunocytochemically-detected arabinogalactan proteins were present at high levels in the stigma, they were not detected in the transmitting tissue of the style, where extensins were abundant. Whereas extensins remained at high levels in unpollinated pistils, they were no longer present in the style following pollen tube passage. Similarily, while abundant in unpollinated styles, insoluble polysaccharides such as β-glucans, were depleted in pollinated pistils.ConclusionsThe switch from autotropic to heterotrophic pollen tube growth correlates spatially with a change of glycoprotein epitopes between the stigma and the style. The depletion of extensins and polysaccharides following pollen tube passage in the style suggest a possible contribution to the acceleration of heterotrophic pollen tube growth, which would imply an active contribution of female tissues on prezygotic male–female crosstalk.

Localization of membrane-associated calcium in unpollinated and pollinated pistil of Petunia hybrida Hort.

In the pistil of <em>Petunia hybrida</em>, the transmitting tract and the ovules are the sites which give Ca<sup>2+</sup>-CTC fluorescence. In unpollinated pistil the level of membrane-associated Ca<sup>2+</sup> decreases from the stigma to the base of the style. The renewed strong rise of Ca<sup>2+</sup>-CTC fluorescence appears on the placenta surface and in the ovule integuments. Following pollination, when the pollen tubes have grown through the pistil, the pattern of membrane-associated Ca<sup>2+</sup> on the path stigma - ovary is reversed. The highest fluorescence is found in the base of the style. In pollinated ovules the Ca<sup>2+</sup>-CTC fluorescence increases markedly. In the transmitting cells membrane-associated Ca<sup>2+</sup> occurs mainly in the polar regions of the cell. During cell degeneration following pollination the cytoplasmic clusters show Ca<sup>2+</sup>-CTC fluorescence. The used <em>P. hybrida</em> cultivar is self-fertile. The selection of pollen tubes occurs mainly in the upper part of the style. The rejected pollen tubes show a steady high level of membrane-associated calcium.

Effect of estrone on somatic and female gametophyte cell division and differentiation in Arabidospis thaliana cultured in vitro

The aim of the study was to determine the effect of the mammalian female sex hormone estrone on differentiation of somatic tissues and on induction of autonomous endosperm in culture of female gametophyte cells of Arabidopsis thaliana ecotype Columbia (Col-0). In culture, estrone-stimulated development of autonomous endosperm (AE) occurred in 14.7% of unpollinated pistils. The AE represented development stages similar to those of young endosperm after fertilization and AE of fis mutants in vivo. In the majority of ovules the AE was in a few-nucleate young stage. Some ovules showed more advanced stages of AE development, with nuclei and cytoplasm forming characteristic nuclear cytoplasmic domains (NCDs). Sporadically, AE was divided into regions characteristic for Arabidopsis endosperm formed after fertilization. Direct organogenesis (caulogenesis, rhizogenesis), callus proliferation and formation of trichome-like structures were observed during in vitro culture of hypocotyls and cotyledons of 3-day-old seedlings cultured on medium supplemented with estrone for 28 days. Histological analysis showed adventitious root formation and changes in explant anatomy caused by estrone.

Molecular Cloning, Expression Analysis and Localization of Exo70A1 Related to Self Incompatibility in Non-Heading Chinese Cabbage (Brassica campestris ssp. chinensis)

The exocyst is a conserved protein complex, and required for vesicles tethering, fusion and polarized exocytosis. Exo70A1, the exocyst subunit, is essential for assembly of the exocyst complex. To better understand potential roles of Exo70A1 in non-heading Chinese cabbage (Brassica campestris ssp. chinensis), we obtained the full-length cDNA of Exo70A1 gene, which consisted of 1917 bp and encoded a protein of 638 amino acids. BlastX showed BcExo70A1 shared 94.9% identity with Brassica oleracea var. acephala (AEI26267.1), and clustered into a same group with other homologues in B. oleracea var. acephala and Brassica napus. Subcellular localization analysis showed BcExo70A1 was localized to punctate structures in cytosol of onion epithelial cells. Results showed that BcExo70A1 was widely presented in stamens, young stems, petals, unpollinated pistils, roots and leaves of self compatible and incompatible plants. The transcripts of BcExo70A1 in non-heading Chinese cabbage declined during initial 1.5 h after incompatible pollination, while an opposite trend was presented after compatible pollination. Our study reveals that BcExo70A1 could play essential roles in plant growth and development, and is related to the rejection of self pollen in non-heading Chinese cabbage.

Molecular cloning and transcriptional activity of a new Petunia calreticulin gene involved in pistil transmitting tract maturation, progamic phase, and double fertilization

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.

Identification of gene expression profile during fertilization in Brassica campestris subsp. chinensis

Fertilization is controlled by a complex gene regulatory network. To study the fertilization mechanism, we determined time courses of the four developmental stages of fertilization in Chinese cabbage pak-choi (Brassica campestris subsp. chinensis) by cytological observation. We then used the Arabidopsis ATH1 microarray to characterize the gene expression profiles of pollinated and unpollinated pistils in B. campestris subsp. chinensis. The result showed 44 up-regulated genes and 33 down-regulated genes in pollinated pistils compared with unpollinated pistils. Gene ontology analysis identified 20% of the up-regulated genes as belonging to the category of cell wall metabolism. We compared the up-regulated genes in pollinated pistils with previously identified pollen development related genes. Ten genes were found to be in common, which were termed as continuously expressed genes, in the two processes in the present article. Their expression patterns during pollen development and fertilization processes were then verified by RT-PCR. One of the continuously expressed genes, the homologous gene of At3g01270 in B. campestris subsp. chinensis, was confirmed as specifically expressed in microspores and pollinated pistils by using in situ hybridization. The potential biological functions of the other continuously expressed genes were also discussed.

Mating in the pseudogamic apomicticAnemopaegma acutifoliumDC: another case of pseudo‐self‐compatibility inBignoniaceae?

Self-compatibility in apomictic pseudogamic species is considered fundamental to assure reproduction by seeds in extreme situations, making apomictic species more advantageous than sexual ones in these scenarios. Anemopaegma acutifolium is a polyploidy, apomictic sporophytic species with no endosperm development in ovules of unpollinated pistils, which indicates obligate pseudogamy. Thus, the aim of the present work is to study the breeding system and post-pollination events to test if there is similar pseudogamous development irrespective of pollination treatment. We analysed fruit and seed set obtained in controlled experimental pollinations, as well as embryo number per seed, and the progress of ovule penetration, fertilisation and early endosperm development between self- and cross-pollinated pistils. We found that the species is self-fertile and that spontaneous selfing fruit set is also possible, although emasculated flowers never form fruits. Selfed pistils were as efficient as crossed ones for all parameters analysed, except for a delay in endosperm development observed in the former that may be an effect of the late-acting self-incompatibility. Therefore, the avoidance of selfed pistil abortion seems to be promoted by the presence of adventitious embryos and a normal endosperm. We conclude that A.acutifolium shows apomixis-related pseudo-self-compatibility, as in other self-fertile apomictic species of Bignoniaceae, which confer reproductive assurance and increases fruit-set and persistence ability in fast-changing tropical habitats.

Pollen–pistil interactions and early fruiting in parthenocarpic citrus

An intense pollen-pistil interaction precedes fertilization. This interaction is of particular relevance in agronomically important species where seeds or fruits are the edible part. Over time some agronomically species have been selected for the ability to produce fruit without seeds. While this phenomenon is critical for commercial production in some species, very little is known about the events behind the production of seedless fruit. In this work, the relationship between pollen-pistil interaction and the onset of fruiting was investigated in citrus mandarin. Pistils were sequentially examined in hand-pollinated flowers paying attention to pollen-tube behaviour, and to cytochemical changes along the pollen-tube pathway. To evaluate which of these changes were induced by pollination/fertilization and which were developmentally regulated, pollinated and unpollinated pistils were compared. Also the onset of fruiting was timed and changes in the ovary examined. Conspicuous changes occurred in the pistil along the pollen-tube pathway, which took place in a basipetal way encompassing the timing of pollen-tube growth. However, these changes appear to be developmentally regulated as they happened in the same way and at the same time in unpollinated flowers. Moreover, the onset of fruiting occurred prior to fertilization and the very same changes could be observed in unpollinated flowers. Pollen-pistil interaction in citrus showed similarities with unrelated species and families belonging to other taxa. The uncoupling of the reproductive and fruiting processes accounts for the parthenocarpic ability of unpollinated flowers to produce fruit in citrus. However, the maintenance of a functional reproductive process reflects the potential to produce seeded fruits, providing a basis for the understanding of the production of seeded or unseeded fruits and further understanding of the process of parthenocarpy in other species.