Self-(in)compatibility in Tunisian apple accessions [Malus domestica. Borkh]: S-genotypes identification and pollen tube growth analysis.

MAIN CONCLUSION: Self-incompatibility studies have revealed a potential use of Tunisian apple resources for crop improvement and modern breeding programs and a likely correlation between the pollen tube growth and flowering period. Apples [Malus domestica. Borkh] exhibit an S-RNase-based gametophytic self-incompatibility (GSI) system. Four primer combinations were used to S-genotype eighteen Tunisian local apple accessions and twelve introduced accessions that served as references. Within the Tunisian local accessions, S2, S3, S7, and S28 S-alleles were the most frequent and were assigned to 14 S-genotypes; among them, S7S28, S3S7, S2S5, and S2S3 were the most abundant. PCA plot showed that population structuring was affected by the S-alleles frequencies and revealed a modern origin of the Tunisian varieties rather than being ancient ones. Nonetheless, the results obtained with 17 SSR markers showed a separate grouping of local Tunisian accessions that calls into question the hypothesis discussed. Pollination experiments showed that the pollen started to germinate within 24 h of pollination but 48 h after pollination in the "El Fessi" accession. The first pollen tubes arrived in the styles within 36 h of pollination in two early flowering accessions known as "Arbi" and "Bokri", and after 72 h of pollination in late flowering "El Fessi" and 48 h after pollination in remaining accessions. The first pollen tube arrests were observed in accessions "Arbi" and "Bokri" within 84 h of pollination, within 108 h of pollination in "El Fessi" and within 108 h of pollination in remaining accessions. In the apple accession called "Boutabgaya," the pollen tubes reached the base of the style within 120 h of pollination without being aborted. Nevertheless, the self-compatible nature of "Boutabgaya" needs more studies to be confirmed. However, our results revealed the malfunction of the female component of the GSI in this accession. To conclude, this work paved the path for further studies to enhance the insight (i) into the relation between the flowering period and the pollen tube growth, (ii) self-compatible nature of "Boutabgaya", and (iii) the origin of the Tunisian apple.

Redundant role of OsCNGC4 and OsCNGC5 encoding cyclic nucleotide-gated channels in rice pollen germination and tube growth.

In staple crops, such as rice (Oryza sativa L.), pollen plays a crucial role in seed production. However, the molecular mechanisms underlying rice pollen germination and tube growth remain underexplored. Notably, we recently uncovered the redundant expression and mutual interaction of two rice genes encoding cyclic nucleotide-gated channels (CNGCs), OsCNGC4 and OsCNGC5, in mature pollen. Building on these findings, the current study focused on clarifying the functional roles of these two genes in pollen germination and tube growth. To overcome functional redundancy, we produced gene-edited rice plants with mutations in both genes using the CRISPR-Cas9 system. The resulting homozygous OsCNGC4 and OsCNGC5 gene-edited mutants (oscngc4/5) exhibited significantly lower pollen germination rates than the wild type (WT), along with severely reduced fertility. Transcriptome analysis of the double oscngc4/5 mutant revealed downregulation of genes related to receptor kinases, transporters, and cell wall metabolism. To identify the direct regulators of OsCNGC4, which form a heterodimer with OsCNGC5, we screened a yeast two-hybrid library containing rice cDNAs from mature anthers. Subsequently, we identified two calmodulin isoforms (CaM1-1 and CaM1-2), NETWORKED 2 A (NET2A), and proline-rich extension-like receptor kinase 13 (PERK13) proteins as interactors of OsCNGC4, suggesting its roles in regulating Ca2+ channel activity and F-actin organization. Overall, our results suggest that OsCNGC4 and OsCNGC5 may play critical roles in pollen germination and elongation by regulating the Ca2+ gradient in growing pollen tubes.

Distribution of exchangeable Ca2+ during the process of Larix decidua Mill. pollination and germination.

The involvement of Ca2+ ions in angiosperms sexual processes is well established, while in gymnosperms, such knowledge remains limited and is still a topic of discussion. In this study, we focused on Larix decidua, using Alizarin-red S staining and the pyroantimonate method to examine the tissue and subcellular distribution of free and loosely bound Ca2+ ions at different stages of the male gametophyte's development and its interaction with the ovule. Our findings show that in larch, both the germination of pollen grains and the growth of pollen tubes occur in an environment rich in Ca2+. These ions play a crucial role in the adhesion of the pollen grain to the stigmatic tip and its subsequent movement to the micropylar canal. There is a significant presence of free and loosely bound Ca2+ ions in both the fluid of the micropylar canal and the extracellular matrix of the nucellus. As the pollen tube extends through the nucellus, we observed a notable accumulation of Ca2+ ions just above the entry to the mature archegonium, a region likely crucial for the male gametophyte's directional growth. Meanwhile, the localized presence of free and loosely bound Ca2+ ions within the egg cell cytoplasm may inhibit the pollen tubes growth and rupture, playing an important role in fertilization.

Defective pollen tube tip growth induces neo-polyploid infertility.

Genome duplication (generating polyploids) is an engine of novelty in eukaryotic evolution and a promising crop improvement tool. Yet newly formed polyploids often have low fertility. Here we report that a severe fertility-compromising defect in pollen tube tip growth arises in new polyploids of Arabidopsis arenosa. Pollen tubes of newly polyploid A. arenosa grow slowly, have aberrant anatomy and disrupted physiology, often burst prematurely, and have altered gene expression. These phenotypes recover in evolved polyploids. We also show that gametophytic (pollen tube) genotypes of two tip-growth genes under selection in natural tetraploid A. arenosa are strongly associated with pollen tube performance in the tetraploid. Our work establishes pollen tube tip growth as an important fertility challenge for neo-polyploid plants and provides insights into a naturally evolved multigenic solution.

Identification of self-incompatibility alleles in Quince (Cydonia oblonga Mill.).

The Quince (Cydonia oblonga Mill.), typically known for its self-compatibility, surprisingly presents a degree of self-incompatibility. This research focused on exploring the diversity within the self-incompatibility gene locus (S) in various C. oblonga genotypes. Through meticulous DNA sequencing, the study sought to unearth potential novel S alleles. In the process of genotyping the S gene across multiple quince genotypes, not only were the previously documented S1 and S2 alleles identified, but this investigation also uncovered two previously unrecognized alleles, termed S4 and S5. These alleles, particularly S4, emerged as the most prevalent among the tested genotypes. To corroborate the findings derived from DNA sequencing, the study employed pollen tube growth germination assays. These assays elucidated a higher pollen germination rate in the Ardabil2 genotype in contrast to Behta. Additionally, the study involved assessing pollen tube growth in both Ardabil2 and Behta through cross-pollination techniques, meticulously tracking the development of pollen tubes at various stages. Remarkably, the outcomes demonstrated that the Behta genotype possesses self-incompatibility, whereas the Ardabil2 genotype showcases a notable degree of self-compatibility. This groundbreaking discovery of new S alleles in quince not only affirms the species' self-compatibility but also sheds light on the complexities of allelic diversity and its impact on self-incompatibility. Such insights are invaluable for enhancing the yield of quince orchards through strategic breeding programs.

The PPO family in Nicotiana tabacum is an important regulator to participate in pollination.

Polyphenol oxidases (PPOs) are type-3 copper enzymes and are involved in many biological processes. However, the potential functions of PPOs in pollination are not fully understood. In this work, we have screened 13 PPO members in Nicotiana. tabacum (named NtPPO1-13, NtPPOs) to explore their characteristics and functions in pollination. The results show that NtPPOs are closely related to PPOs in Solanaceae and share conserved domains except NtPPO4. Generally, NtPPOs are diversely expressed in different tissues and are distributed in pistil and male gametes. Specifically, NtPPO9 and NtPPO10 are highly expressed in the pistil and mature anther. In addition, the expression levels and enzyme activities of NtPPOs are increased after N. tabacum self-pollination. Knockdown of NtPPOs would affect pollen growth after pollination, and the purines and flavonoid compounds are accumulated in self-pollinated pistil. Altogether, our findings demonstrate that NtPPOs potentially play a role in the pollen tube growth after pollination through purines and flavonoid compounds, and will provide new insights into the role of PPOs in plant reproduction.

EGG CELL 1 contributes to egg-cell-dependent preferential fertilization in Arabidopsis.

During double fertilization in angiosperms, the pollen tube delivers two sperm cells into an embryo sac; one sperm cell fuses with an egg cell, and the other sperm cell fuses with the central cell. It has long been proposed that the preference for fusion with one or another female gamete cell depends on the sperm cells and occurs during gamete recognition. However, up to now, sperm-dependent preferential fertilization has not been demonstrated, and results on preferred fusion with either female gamete have remained conflicting. To investigate this topic, we generated Arabidopsis thaliana mutants that produce single sperm-like cells or whose egg cells are eliminated; we found that although the three different types of sperm-like cell are functionally equivalent in their ability to fertilize the egg and the central cell, each type of sperm-like cell fuses predominantly with the egg cell. This indicates that it is the egg cell that controls its preferential fertilization. We also found that sperm-activating small secreted EGG CELL 1 proteins are involved in the regulation of egg-cell-dependent preferential fertilization, revealing another important role for this protein family during double fertilization.

Arabidopsis leucine-rich repeat malectin receptor-like kinases regulate pollen-stigma interactions.

Flowering plants contain tightly controlled pollen-pistil interactions required for promoting intraspecific fertilization and preventing interspecific hybridizations. In Arabidopsis (Arabidopsis thaliana), several receptor kinases (RKs) are known to regulate the later stages of intraspecific pollen tube growth and ovular reception in the pistil, but less is known about RK regulation of the earlier stages. The Arabidopsis RECEPTOR-LIKE KINASE IN FLOWERS1 (RKF1)/RKF1-LIKE (RKFL) 1-3 cluster of 4 leucine-rich repeat malectin (LRR-MAL) RKs was previously found to function in the stigma to promote intraspecific pollen hydration. In this study, we tested additional combinations of up to 7 Arabidopsis LRR-MAL RK knockout mutants, including RKF1, RKFL1-3, LysM RLK1-INTERACTING KINASE1, REMORIN-INTERACTING RECEPTOR1, and NEMATODE-INDUCED LRR-RLK2. These LRR-MAL RKs were discovered to function in the female stigma to support intraspecific Arabidopsis pollen tube growth and to establish a prezygotic interspecific barrier against Capsella rubella pollen. Thus, this study uncovered additional biological functions for this poorly understood group of RKs in regulating the early stages of Arabidopsis sexual reproduction.

PbRbohH/J mediates ROS generation to regulate the growth of pollen tube in pear.

Respiratory burst oxidase homolog (Rboh) family genes play crucial functions in development and growth. However, comprehensive and systematic investigation of Rboh family members in Rosaceae and their specific functions during pear pollen development are still limited. In the study, 63 Rboh genes were identified from eight Rosaceae genomes (Malus domestica, Pyrus bretschneideri, Pyrus communis, Prunus persica, Rubus occidentalis, Fragaria vesca, Prunus mume and Prunus avium) and divided into seven main subfamilies (I-VII) according to phylogenetic and structural features. Different modes of gene duplication led to the expansion of Rboh family, with purifying selection playing a vital role in the evolution of Rboh genes. In addition, RNA sequencing and qRT-PCR results indicated that PbRbohH and PbRbohJ were specifically high-expressed in pear pollen. Subsequently, subcellular localization revealed that PbRbohH/J distributed at the plasma membrane. Furthermore, by pharmacological analysis and antisense oligodeoxynucleotide assay, PbRbohH/J were demonstrated to mediate the formation of reactive oxygen species (ROS) to manage pollen tube growth. In conclusion, our results provide useful insights into the functions, expression patterns, evolutionary history of the Rboh genes in pear and other Rosaceae species.

Synergid cell calcium oscillations refine understanding of FERONIA/LORELEI signaling during interspecific hybridization.

KEY MESSAGE: Pollen tubes from closely related species and mutants lacking pollen tube MYB transcription factors are able to initiate FER/LRE-dependent synergid cell calcium oscillations. Reproductive isolation leads to the evolution of new species; however, the molecular mechanisms that maintain reproductive barriers between sympatric species are not well defined. In flowering plants, sperm cells are immotile and are delivered to female gametes by the pollen grain. After landing on the stigmatic surface, the pollen grain germinates a polarized extension, the pollen tube, into floral tissue. After growing via polar extension to the female gametes and shuttling its cargo of sperm cells through its cytoplasm, the pollen tube signals its arrival and identity to synergid cells that flank the egg. If signaling is successful, the pollen tube and receptive synergid cell burst, and sperm cells are released for fusion with female gametes. To better understand cell-cell recognition during reproduction and how reproductive barriers are maintained between closely related species, pollen tube-initiated synergid cell calcium ion dynamics were examined during interspecific crosses. It was observed that interspecific pollen tubes successfully trigger synergid cell calcium oscillations-a hallmark of reproductive success-but signaling fails downstream of key signaling genes and sperm are not released. This work further defines pollen tube-synergid cell signaling as a critical block to interspecific hybridization and suggests that the FERONIA/LORELEI signaling mechanism plays multiple parallel roles during pollen tube reception.

Evolution of the thermostability of actin-depolymerizing factors enhances the adaptation of pollen germination to high temperature.

Double fertilization in many flowering plants (angiosperms) often occurs during the hot summer season, but the mechanisms that enable angiosperms to adapt specifically to high temperatures are largely unknown. The actin cytoskeleton is essential for pollen germination and the polarized growth of pollen tubes, yet how this process responds to high temperatures remains unclear. Here, we reveal that the high thermal stability of 11 Arabidopsis (Arabidopsis thaliana) actin-depolymerizing factors (ADFs) is significantly different: ADFs that specifically accumulate in tip-growing cells (pollen and root hairs) exhibit high thermal stability. Through ancestral protein reconstruction, we found that subclass II ADFs (expressed specifically in pollen) have undergone a dynamic wave-like evolution of the retention, loss, and regeneration of thermostable sites. Additionally, the sites of AtADF7 with high thermal stability are conserved in ADFs specific to angiosperm pollen. Moreover, the high thermal stability of ADFs is required to regulate actin dynamics and turnover at high temperatures to promote pollen germination. Collectively, these findings suggest strategies for the adaptation of sexual reproduction to high temperature in angiosperms at the cell biology level.

Reproductive biology of the "Brazilian pine" (Araucaria angustifolia-Araucariaceae): the pollen tube growth and the seed cone development.

KEY MESSAGE: In Araucaria angustifolia, the seed scale is part of the ovule, the female gametophyte presents a monosporic origin and arises from a coenocytic tetrad, and the pollen tube presents a single axis. The seed cone of conifers has many informative features, and its ontogenetic data may help interpret relationships among function, development patterns, and homology among seed plants. We reported the seed cone development, from pollination to pre-fertilization, including seed scale, ovule ontogeny, and pollen tube growth in Araucaria angustifolia. The study was performed using light microscopy, scanning electron microscopy, and X-ray microcomputed tomography (µCT). During the pollination period, the ovule arises right after the seed scale has emerged. From that event to the pre-fertilization period takes about 14 months. Megasporogenesis occurs three weeks after ovule formation, producing a coenocytic tetrad. At the same time as the female gametophyte's first nuclear division begins, the pollen tube grows through the seed scale adaxial face. Until maturity, the megagametophyte goes through the free nuclei stage, cellularization stage, and cellular growth stage. Along its development, many pollen tubes develop in the nucellar tissue extending straight toward the female gametophyte. Our observations show that the seed scale came out of the same primordia of the ovule, agreeing with past studies that this structure is part of the ovule itself. The formation of a female gametophyte with a monosporic origin that arises from a coenocytic tetrad was described for the first time in conifers, and the three-dimensional reconstruction of the ovule revealed the presence of pollen tubes with only one axis and no branches, highlighting a new pattern of pollen tube growth in Araucariaceae.

Signalling between the sexes during pollen tube reception.

Plant reproduction is a complex, highly-coordinated process in which a single, male germ cell grows through the maternal reproductive tissues to reach and fertilise the egg cell. Focussing on Arabidopsis thaliana, we review signalling between male and female partners which is important throughout the pollen tube journey, especially during pollen tube reception at the ovule. Numerous receptor kinases and their coreceptors are implicated in signal perception in both the pollen tube and synergid cells at the ovule entrance, and several specific peptide and carbohydrate ligands for these receptors have recently been identified. Clarifying the interplay between these signals and the downstream responses they instigate presents a challenge for future research and may help to illuminate broader principles of plant cell-cell communication.

Comparative genomic analysis of the RabGAP gene family in seven Rosaceae species, and functional identification of PbrRabGAP10 in controlling pollen tube growth by mediating cellulose deposition in pear.

Rab GTPase-activating proteins (RabGAPs), serving as crucial signaling switches, play essential roles in several physiological processes related to plant growth and development. However, despite their importance, information regarding the RabGAP gene family and their biological functions remains unknown in the Rosaceae. In this study, we identified a total of 127 RabGAP genes in seven Rosaceae species, which were divided into five subfamilies. Our findings indicate that whole genome duplication (WGD) events or dispersed duplication events largely contributed to the expansion of RabGAP family members within Rosaceae species. Through tissue-specific expression analyses, we revealed that the PbrRabGAP genes exhibited distinct expression patterns in different pear tissues. Furthermore, by examining the expression pattern during pollen development and employing an antisense oligonucleotide approach, we demonstrated that PbrRabGAP10, located in the cytoplasm, mediates the imbalance of cellulose distribution, thus regulating pollen tube elongation. In conclusion, the present study offers an overview of the RabGAP family in Rosaceae genomes and serves as the basis for further functional studies.

Redundant function of the Arabidopsis phosphatidylinositol 4-phosphate 5-kinase genes PIP5K4-6 is essential for pollen germination.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is a key enzyme producing the signaling lipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 ] in eukaryotes. Although PIP5K genes are reported to be involved in pollen tube germination and growth, the essential roles of PIP5K in these processes remain unclear. Here, we performed a comprehensive genetic analysis of the Arabidopsis thaliana PIP5K4, PIP5K5, and PIP5K6 genes and revealed that their redundant function is essential for pollen germination. Pollen with the pip5k4pip5k5pip5k6 triple mutation was sterile, while pollen germination efficiency and pollen tube growth were reduced in the pip5k6 single mutant and further reduced in the pip5k4pip5k6 and pip5k5pip5k6 double mutants. YFP-fusion proteins, PIP5K4-YFP, PIP5K5-YFP, and PIP5K6-YFP, which could rescue the sterility of the triple mutant pollen, preferentially localized to the tricolpate aperture area and the future germination site on the plasma membrane prior to germination. Triple mutant pollen grains under the germination condition, in which spatiotemporal localization of the PtdIns(4,5)P2 fluorescent marker protein 2xmCHERRY-2xPHPLC as seen in the wild type was abolished, exhibited swelling and rupture of the pollen wall, but neither the conspicuous protruding site nor site-specific deposition of cell wall materials for germination. These data indicate that PIP5K4-6 and their product PtdIns(4,5)P2 are essential for pollen germination, possibly through the establishment of the germination polarity in a pollen grain.

Unlocking a 'lock-key' mechanism governing pollen-pistil interactions.

Pollen-pistil interactions ensure genetic diversity and shape the reproductive success of plants. Lan et al. recently revealed that the interaction among various receptor-like kinases, cell-wall proteins, and stigmatic RALF peptides (sRALFs) or pollen RALF peptides (pRALFs) on the stigma surface govern the penetration of pollen tubes in members of the Brassicaceae.

The RALF signaling pathway regulates cell wall integrity during pollen tube growth in maize.

Autocrine signaling pathways regulated by RAPID ALKALINIZATION FACTORs (RALFs) control cell wall integrity during pollen tube germination and growth in Arabidopsis (Arabidopsis thaliana). To investigate the role of pollen-specific RALFs in another plant species, we combined gene expression data with phylogenetic and biochemical studies to identify candidate orthologs in maize (Zea mays). We show that Clade IB ZmRALF2/3 mutations, but not Clade III ZmRALF1/5 mutations, cause cell wall instability in the sub-apical region of the growing pollen tube. ZmRALF2/3 are mainly located in the cell wall and are partially able to complement the pollen germination defect of their Arabidopsis orthologs AtRALF4/19. Mutations in ZmRALF2/3 compromise pectin distribution patterns leading to altered cell wall organization and thickness culminating in pollen tube burst. Clade IB, but not Clade III ZmRALFs, strongly interact as ligands with the pollen-specific Catharanthus roseus RLK1-like (CrRLK1L) receptor kinases Z. mays FERONIA-like (ZmFERL) 4/7/9, LORELEI-like glycosylphosphatidylinositol-anchor (LLG) proteins Z. mays LLG 1 and 2 (ZmLLG1/2), and Z. mays pollen extension-like (PEX) cell wall proteins ZmPEX2/4. Notably, ZmFERL4 outcompetes ZmLLG2 and ZmPEX2 outcompetes ZmFERL4 for ZmRALF2 binding. Based on these data, we suggest that Clade IB RALFs act in a dual role as cell wall components and extracellular sensors to regulate cell wall integrity and thickness during pollen tube growth in maize and probably other plants.

The fertility tracks of pollen tube in the ovary of Solanum nigrum by three-dimensional reconstruction.

In this paper, we present an enhanced method for automatically capturing a large number of consecutive paraffin sections using a microscope. Leveraging these microstructural images, we employed three-dimensional visualisation and reconstruction techniques to investigate the dispersal growth process of pollen tube bundles upon entering the ovary of Solanum nigrum. Additionally, we explored their behaviour within different ovules and examined the relationship between the germination rate of seeds and the fertilisation process. Our findings reveal that despite the abundance of Solanum nigrum seeds, only a fraction of them is capable of successful germination. The germination rate of seeds is closely related to whether fertilisation of the ovules and pollen tubes is completed. Due to the limited number of pollen tubes entering the ovary, only a portion of the ovules can be fertilised. The proportion of fertilised ovules positively correlates with the germination rate of the seeds. Through three-dimensional reconstruction, we observed a phenomenon of proximity during the pollination process, wherein ovules closer to the pollen tube bundles are more likely to be fertilised. Furthermore, fertilised ovules exhibited significant changes in morphology and embryo sac structure. The number of fertilised ovules directly impacts the germination rate of wild Solanum nigrum seeds. Although all Solanum nigrum ovules have the potential to develop into seeds, most seeds originating from unfertilised ovules are unable to germinate normally, resulting in an incomplete germination rate of seeds and preventing it from reaching 100%.