Pollen Tube Growth Research Articles

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.

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.

Comparative Physiological, Transcriptomic, and Metabolomic Analyses of Acacia mangium Provide New Insights into Its Molecular Mechanism of Self-Incompatibility

Acacia mangium is well known as a valuable commercial tree species in the Acacia genus. A. mangium was recently found to be self-incompatible (SI), but its SI mechanism is not clear, which has hindered the progress of genetic improvement of A. mangium with strong resistance. To confirm the SI type of A. mangium, pollen germination was observed via fluorescence microscopy at 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h after self-pollination. We found over ninety percent of the pollen grains produced no pollen tube growth on the stigma/style. To further explore the SI molecular mechanism of A. mangium, tests of the transcriptome and metabolome were carried out after self-pollination. Observations of pollen germination after self-pollination using fluorescence microscopy suggested that the SI type of A. mangium is gametophytic self-incompatibility (GSI). A combined transcriptomic and metabolomic analysis showed that DEGs (differentially expressed genes) related to SI (6 S-glycoproteins, 93 F-box proteins, 69 26S proteasomes, 38 calcium-dependent protein kinases/calmodulin and 41 thioredoxin genes) were significantly enriched in six KEGG (sulfur metabolism, tyrosine metabolism, phenylalanine metabolism, butanoate metabolism, and valine, leucine, and isoleucine degradation). Further analysis of these six pathways revealed the enrichment of SI-related DEGs corresponding to succinate, methylmalonate, and 3-hydroxypropane. These three metabolites were significantly downregulated. The analysis of transcripts and metabolites suggested that transcripts of SI-related gene families (thioredoxin and F-box protein) were significantly upregulated under the regulation of transcription factors (TFs) after self-pollination, leading to a decrease in metabolites (such as succinate, methylmalonate, and 3-hydroxypropionate). We also further speculated that TFs (MYB, HB-HD-ZIP, AP2/ERF-ERF, and bZIP) and gene families (thioredoxin and F-box protein) were important factors related to the SI of A. mangium.

Histone Variant H3.3 Controls Arabidopsis Fertility by Regulating Male Gamete Development.

Reprograming of chromatin structures and changes in gene expression are critical for plant male gamete development, and epigenetic marks play an important role in these processes. Histone variant H3.3 is abundant in euchromatin and is largely associated with transcriptional activation. The precise function of H3.3 in gamete development remains unclear in plants. Here, we report that H3.3 is abundantly expressed in Arabidopsis anthers and its knockout mutant h3.3-1 is sterile due to male sterility. Transcriptome analysis of young inflorescence has identified 2348 genes downregulated in h3.3-1 mutant, among which 1087 target genes are directly bound by H3.3, especially at their 3' ends. As a group, this set of H3.3 targets is enriched in the reproduction-associated processes including male gamete generation, pollen sperm cell differentiation and pollen tube growth. The function of H3.3 in male gamete development is dependent on the Anti-Silencing Factor 1A/1B (ASF1A/1B)-Histone regulator A (HIRA)-mediated pathway. Our results suggest that ASF1A/1B-HIRA-mediated H3.3 deposition at its direct targets for transcription activation forms the regulatory networks responsible for male gamete development.

VAMP726 and VAMP725 regulate vesicle secretion and pollen tube growth in Arabidopsis.

VAMP726/VAMP725 and SYP131 can form a part of a SNARE complex to mediate vesicle secretion at the pollen tube apex. Secretory vesicle fusion with the plasma membrane of the pollen tube tip is a key step in pollen tube growth. Membrane fusion was mediated by SNAREs. However, little is known about the composition and function of the SNARE complex during pollen tube tip growth. In this study, we constructed a double mutant vamp725 vamp726 via CRISPR‒Cas9. Fluorescence labeling combined with microscopic observation, luciferase complementation imaging, co-immunoprecipitation and GST pull-down were applied in the study. We show that double mutation of the R-SNAREs VAMP726 and VAMP725 significantly inhibits pollen tube growth in Arabidopsis and slows vesicle exocytosis at the apex of the pollen tube. GFP-VAMP726 andVAMP725-GFP localize mainly to secretory vesicles and the plasma membrane at the apex of the pollen tube. In addition, fluorescence recovery after photobleaching (FRAP) experiments showed that mCherry-VAMP726 colocalizes with Qa-SNARESYP131 in the central region of the pollen tube apical plasma membrane. Furthermore, we found that VAMP726 and VAMP725 can interact with the SYP131. Based on these results, we suggest that VAMP726/VAMP725 and SYP131 can form a part of a SNARE complex to mediate vesicle secretion at the pollen tube apex, and vesicle secretion may mainly occur at the central region of the pollen tube apical plasma membrane.

Identification and characterization of photo-thermo insensitive cowpea (Vigna unguiculata) genotypes for hot arid environment

High green pod yield, dual purpose type, earliness and photo-thermo insensitivity are the major breeding objectives of cowpea [Vigna unguiculata (L.) Walp.]. Photo-thermo sensitivity makes this crop vulnerable to photoperiod and temperature fluctuations particularly in hot arid regions, thereby affecting its yield potential drastically. Therefore, the present study was aimed to identify such elite genotypes with photo-thermo insensitivity, which can fit well across all seasons under hot arid environment. The experiments were conducted at ICAR-Central Institute for Arid Horticulture, Bikaner during rainy and summer seasons of 2019–20 and 2020–21. A wide range of variability among genotypes was observed with respect to various phenological and yield traits over the environments. Among the evaluated genotypes, AHCP-1-4-1 and AHCP-2-3 were found photo-thermo insensitive as these were able to flower and set pods at temperatures as low as 10oC and as high as 46°C. Pollen viability studies indicated that AHCP-1-4-1 had 83.72 and 88.24% pollen viability and AHCP-2-3 had 81.58 and 85.71% viable pollen at 11°C and 46°C, respectively and normal pollen tube growth at both the extremes of temperature. The identified genetic resources will contribute for developing photo-thermo insensitive cultivars and will improve the productivity and extend the availability of cowpea with quality pod yield across all seasons and locations of hot arid regions.

Floral Development on Vitis vinifera Is Associated with MADS-Box Transcription Factors through the Transcriptional Regulation of VviZIP3.

Several grapevine (Vitis vinifera L.) cultivars show a tendency to develop parthenocarpic seedless grapes, affecting fruit yield and quality. This reproductive disorder originates in defective ovule fertilization due to a failure in pollen tube growth. Zinc (Zn) is a crucial trace element, playing a vital role in various physiological and metabolic processes. It is particularly essential for the healthy growth of flowers and fruits. Insufficient zinc has been suggested as a potential reason for issues in this development process. This microelement is taken up through a mechanism that involves transporters, including the ZRT-IRT-like protein (ZIP) gene family, associated with the influx of Zn into the cell. In grapevines, 20 genes for ZIP-type transporters have been described. In this study, we analyzed the expression pattern of VviZIP3 during flower development and employ transgenic methods to assess its transcriptional regulation. Furthermore, through computational examination of the promoter region, we identified two CArG boxes, recognized as responsive elements to MADS transcription factors. These factors play a key role in shaping various components of a flower, such as pollen. Our investigation of the VviZIP3 promoter confirms the functionality of these CArG boxes. Overall, our results suggest that the increased expression of VviZIP3 during flowering is likely under the influence of MADS transcription factors.

Breeding system in Olea ferruginea Royle (Oleaceae): comparative performance of staminate and perfect flowers in reproductive assurance

ABSTRACT Olea ferruginea, a wild widely distributed species of the olive lineage, exhibits andromonoecy with protogynous perfect (hermaphrodite) and staminate (male) flowers. The present communication elucidates the differential performance of staminate and perfect flowers in reproductive assurance. The flowering chronology and function of perfect and staminate flowers in the breeding system were investigated to understand the breeding behavior of the species. In terms of breeding behavior, pollen from staminate and perfect flowers was used to establish their role in breeding system under different pollination treatments. Results indicate higher pollen viability, germination, and pollen tube growth of staminate than perfect flowers. Results from pollen counts and morphological investigations show slight differences in both types. The species is self-incompatible which is indicated by pollen germination, the average number of pollen tubes in the style, and the fruit set following self and cross-pollination. Hand cross-pollination improves natural fruit set, especially when pollen from staminate flowers was used. Although the relict role of attracting pollinators cannot be fully discarded, it is suggested that the main benefit supplied by the formation of staminate flowers in olives is to promote male fitness by raising pollen output at the population level.

Compounds composition of pollen tubes of Scots pine (Pinus sylvestris L.).

The aim of this research was to study the composition of pollen tubes of Scots pine (Pinus sylvestris L.). Pollen cultivation on deionized distilled water excluded the potential influence of the cultivation medium on the pollen tube growth and development. The fluorescent study indicated a gradual distribution of chemical compounds along the length of the tube. It was shown that the protoplast apical zone and the parietal layer near the tube's tip are most likely actively involved in the ion transport regulation in the growing pollen tube. The callose synthesis in the tip of matured pine tube completed the first stage of its active growth. Significant differences and pH gradients at the nucleus region and the parietal layer of the tube wall indicate that H+ gradient is the direct driving force of vesicle transport and can regulate the growth of pollen tubes. The distribution of amino acids, RNA, proteins and lipids was uniform throughout the length of the pine pollen tube. The content of amino acids, RNA, DNA and proteins slightly increased near the cell nucleus and drastically increased in the apical zone. At the very tip of the tube, a slight increase in the concentration of polysaccharides and a significant decrease in the content of amino acids, RNA, DNA, proteins and lipids were detected.

Reproductive systems and hybridization of Lymania species (Bromeliaceae) endemic to Northeast Brazil threatened with extinction

Reproductive systems among the Bromeliaceae family are very diverse at the species level, including allogamous, autogamous, cleistogamous, agamospermic or mixed plants. Hybridization allows the creation of new genotypes with different combinations of shapes and colors to meet the ornamental plant market's demand for novelty. Knowing the reproductive strategies of the species makes it possible to obtain these new materials with ornamental potential, in addition to being essential to define better conservation strategies. Little is known about the reproductive biology of Lymania species. In addition, at least 60% of these species are on the Brazilian National List of Species Threatened with Extinction. The purpose of this study was to identify the reproductive systems, the possible interspecific hybridizations, and the possible reproductive barriers of nine Lymania species based on the germination of pollen grains and the corresponding growth of the pollen tube in the pistil by using fluorescence microscopy with ultraviolet filtering. Natural and controlled pollinations of the nine species were carried out. The reproductive strategy was defined according to the autogamy index (AI), self-incompatibility index (SII), and reproductive efficiency index (REI). All species were self-incompatible except for L. smithii, which was autogamous. Agamospermy was not observed in any of the studied species. Forty-one hybridization combinations between species were performed, resulting in a 54% success rate, with incongruity in only 12 combinations. The main incongruence phenomena identified after interspecific crosses were: poor germination of pollen grains on the stigma surface; inhibition of pollen tube growth along the style; coiling of pollen tubes along the style; pollen tubes inside the ovary but without penetration into the micropyle; and reticulate deposition of callose in the pollen tube, among others. We suggest that self-incompatibility of the species is gametophytic, preventing self-fertilization. Results showed that it is possible to perform hybridization with some species. However, morphological, genetic, and molecular studies are needed to confirm the potential of these species. Seeds generated in the different reproductive systems, as well as in the hybridizations, had germination rates above 94%. The results presented here make important contributions to the conservation of species, seed generation, and hybridization of Bromeliaceae.

Embryo Rescue in Plant Breeding.

Embryo rescue (ER) techniques are among the oldest and most successful in vitro tissue culture protocols used with plant species. ER refers to a series of methods that promote the development of an immature or lethal embryo into a viable plant. Intraspecific, interspecific, or intergeneric crosses allow the introgression of important alleles of agricultural interest from wild species, such as resistance or tolerance to abiotic and biotic stresses or morphological traits in crops. However, pre-zygotic and post-zygotic reproductive barriers often present challenges in achieving successful hybridization. Pre-zygotic barriers manifest as incompatibility reactions that hinder pollen germination, pollen tube growth, or penetration into the ovule occurring in various tissues, such as the stigma, style, or ovary. To overcome these barriers, several strategies are employed, including cut-style or graft-on-style techniques, the utilization of mixed pollen from distinct species, placenta pollination, and in vitro ovule pollination. On the other hand, post-zygotic barriers act at different tissues and stages ranging from early embryo development to the subsequent growth and reproduction of the offspring. Many crosses among different genera result in embryo abortion due to the failure of endosperm development. In such cases, ER techniques are needed to rescue these hybrids. ER holds great promise for not only facilitating successful crosses but also for obtaining haploids, doubled haploids, and manipulating the ploidy levels for chromosome engineering by monosomic and disomic addition as well substitution lines. Furthermore, ER can be used to shorten the reproductive cycle and for the propagation of rare plants. Additionally, it has been repeatedly used to study the stages of embryonic development, especially in embryo-lethal mutants. The most widely used ER procedure is the culture of immature embryos taken and placed directly on culture media. In certain cases, the in vitro culture of ovule, ovaries or placentas enables the successful development of young embryos from the zygote stage to maturity.

SD-RLK28 positively regulates pollen hydration on stigmas as a PCP-Bβ receptor in Arabidopsis thaliana.

Pollen hydration on dry stigmas is strictly regulated by pollen-stigma interactions in Brassicaceae. Although several related molecular events have been described, the molecular mechanism underlying pollen hydration remains elusive. Multiple B-class pollen coat proteins (PCP-Bs) are involved in pollen hydration. Here, by analyzing the interactions of two PCP-Bs with three Arabidopsis thaliana stigmas strongly expressing S-domain receptor kinase (SD-RLK), we determined that SD-RLK28 directly interacts with PCP-Bβ. We investigated pollen hydration, pollen germination, pollen tube growth, and stigma receptivity in the sd-rlk28 and pcp-bβ mutants. PCP-Bβ acts in the pollen to regulate pollen hydration on stigmas. Loss of SD-RLK28 had no effect on pollen viability, and sd-rlk28 plants had normal life cycles without obvious defects. However, pollen hydration on sd-rlk28 stigmas was impaired and pollen tube growth in sd-rlk28 pistils was delayed. The defect in pollen hydration on sd-rlk28 stigmas was independent of changes in reactive oxygen species levels in stigmas. These results indicate that SD-RLK28 functions in the stigma as a PCP-Bβ receptor to positively regulate pollen hydration on dry stigmas.

Heat-dependent postpollination limitations on maize pollen tube growth and kernel sterility.

Heat stress has a negative impact on pollen development in maize (Zea mays L.) but the postpollination events that determine kernel sterility are less well characterised. The impact of short-term (hours) heat exposure during postpollination was therefore assessed in silks and ovaries. The temperatures inside the kernels housed within the husks was significantly lower than the imposed heat stress. This protected the ovaries and possibly the later phase of pollen tube growth from the adverse effects of heat stress. Failure of maize kernel fertilization was observed within 6 h of heat stress exposure postpollination. This was accompanied by a significant restriction of early pollen tube growth rather than pollen germination. Limitations on early pollen tube growth were therefore a major factor contributing to heat stress-induced kernel sterility. Exposure to heat stress altered the sugar composition of silks, suggesting that hexose supply contributed to the limitations on pollen tube growth. Moreover, the activities of sucrose metabolising enzymes, the expression of sucrose degradation and trehalose biosynthesis genes were decreased following heat stress. Significant increases in reactive oxygen species, abscisic acid and auxin levels accompanied by altered expression of phytohormone-related genes may also be important in the heat-induced suppression of pollen tube growth.

Differential sensitivity of ADF isovariants to a pH gradient promotes pollen tube growth.

The actin cytoskeleton is one of the targets of the pH gradient in tip-growing cells, but how cytosolic pH regulates the actin cytoskeleton remains largely unknown. We here demonstrate that Arabidopsis ADF7 and ADF10 function optimally at different pH levels when disassembling actin filaments. This differential pH sensitivity allows ADF7 and ADF10 to respond to the cytosolic pH gradient to regulate actin dynamics in pollen tubes. ADF7 is an unusual actin-depolymerizing factor with a low optimum pH in in vitro actin depolymerization assays. ADF7 plays a dominant role in promoting actin turnover at the pollen tube apex. ADF10 has a typically high optimum pH in in vitro assays and plays a dominant role in regulating the turnover and organization of subapical actin filaments. Thus, functional specification and cooperation of ADF isovariants with different pH sensitivities enable the coordination of the actin cytoskeleton with the cytosolic pH gradient to support pollen tube growth.

Transcriptomic Insight into the Pollen Tube Growth of Olea europaea L. subsp. europaea Reveals Reprogramming and Pollen-Specific Genes Including New Transcription Factors.

The pollen tube is a key innovation of land plants that is essential for successful fertilisation. Its development and growth have been profusely studied in model organisms, but in spite of the economic impact of olive trees, little is known regarding the genome-wide events underlying pollen hydration and growth in this species. To fill this gap, triplicate mRNA samples at 0, 1, 3, and 6 h of in vitro germination of olive cultivar Picual pollen were analysed by RNA-seq. A bioinformatics R workflow called RSeqFlow was developed contemplating the best practices described in the literature, covering from expression data filtering to differential expression and clustering, to finally propose hub genes. The resulting olive pollen transcriptome consisted of 22,418 reliable transcripts, where 5364 were differentially expressed, out of which 173 have no orthologue in plants and up to 3 of them might be pollen-specific transcription factors. Functional enrichment revealed a deep transcriptional reprogramming in mature olive pollen that is also dependent on protein stability and turnover to allow pollen tube emergence, with many hub genes related to heat shock proteins and F-box-containing proteins. Reprogramming extends to the first 3 h of growth, including processes consistent with studies performed in other plant species, such as global down-regulation of biosynthetic processes, vesicle/organelle trafficking and cytoskeleton remodelling. In the last stages, growth should be maintained from persistent transcripts. Mature pollen is equipped with transcripts to successfully cope with adverse environments, even though the in vitro growth seems to induce several stress responses. Finally, pollen-specific transcription factors were proposed as probable drivers of pollen germination in olive trees, which also shows an overall increased number of pollen-specific gene isoforms relative to other plants.

Role of Phenylpropanoids and Flavonoids in Plant Defense Mechanism

Phenylpropanoids and flavonoids are specialized metabolites that play a crucial role in plant defenses against biotic and abiotic stresses. Secondary metabolites in plants, such as phenylpropanoids, flavonoids, lignins, monolignols, phenolic acids, stilbenes and coumarins, play a significant role in biotic and abiotic stress responses and interactions with their environment. Under stress-free conditions, flavonoids influence pollen tube growth, seed maturation, dormancy, and the longevity of plant reproductive organs and seeds. Under adverse abiotic conditions, flavonoids can mediate defense responses, such as the accumulation of flavonoids in leaves and glandular trichomes. Metabolomics has identified specialized PPP metabolites correlated with plant resistance to fungi and oomycetes. However, the correlation can be in opposite directions depending on the pathogen and a systemic approach is required to achieve increased resistance without decreasing resistance to another pathogen. The PAL gene is located upstream in the PPP pathway and phenylpropanoids and flavonoids have antifungal activity against Fusarium oxysporum, Rhizoctonia solani and Alternaria alternata. Phenylpropanoids have been studied in vitro to develop biological alternatives to synthetic phytoprotectants but the host's defense system is the most effective mode of action. Post-inoculation transcriptomic shifts reveal that some genes encoding enzymes in the PPP are rapidly activated, while others are induced later in the infection response. Insects are the most problematic macroscopic pests on cultivated plants and plants' natural defense mechanisms against herbivores include releasing volatile organic compounds, attracting enemies and producing phenylpropanoids and flavonoids. Increasing the content of flavonoids in infested plants can reduce the population of insects that cause damage to plants and spread viruses. Plant-environment interaction, mediated by secondary metabolites like phenylpropanoids and flavonoids, leads to diversity in gene structures. Systemic biology approach will be realized through sequencing, structural elucidation, and analytical tools. Model plants and crops can be used for exploration.

Cytogenetic Study and Pollen Viability of Phalaenopsis Queen Beer 'Mantefon'.

Intergeneric and interspecific hybridization has been employed for the breeding of Phalaenopsis to transfer desirable traits between species, producing novel phenotypes with improved size, color, form, and flower-bearing ability. These characteristics are often enhanced; however, many of these hybrids are triploids and have reduced or complete sterility, for example, Phalaenopsis Queen Beer 'Mantefon', an important novelty-type cultivar in Asia, particularly in China, Japan, and Republic of Korea. Despite the increasing demand for the crop for ornamental purposes, little is known about its cytogenetics, which is essential for breeding and, consequently, crop improvement. In this study, karyotyping using fluorescence in situ hybridization, meiotic chromosome behavior analysis, pollen staining, and in vitro viability germination tests were performed to understand the cause of hybrid sterility and pollen abnormality in Phalaenopsis Queen Beer 'Mantefon' from a cytogenetic perspective. Viability tests revealed pollen infertility at all flower developmental stages, confirmed by the absence of pollen tube growth. Aberrant chromosomal behavior was observed in pollen mother cells (PMCs), frequently forming univalents, chromosomal bridges, and laggards during the entire meiotic process. PMCs were also divided irregularly into sporads with varying numbers of micronuclei, which may be responsible for pollen sterility in this cultivar. Altogether, the cytogenetic analyses provided insights into the pollen development of Phalaenopsis Queen Beer 'Mantefon' and the conceivable causes of its infertility.

Identification and analysis of CdS-RNase in Camellia drupifera: A key determinant of late-acting self-incompatibility

Plant self-incompatibility (SI) is of great significance in reproductive biology and is also the main factor affecting yield in agricultural production. Late-acting self-incompatibility (LSI) is one of the main types of plant SI, but the mechanism of LSI is not systematically analyzed. Tea oil Camellia, a strictly outcrossing plant, is a well-known woody oil cash crop. SI is one of the main reasons for the low yield of tea oil Camellia. In this study, we found that C. drupifera, one of the main species of tea oil Camellia, shows LSI, in which SI occurs in the style, and pollen tube growth in the ovary ceases at 96 h after selfing. The CdS-RNase gene of Camellia drupifera was successfully cloned, and the gene showed a length of 922 bp and encoded 238 amino acids. CdS-RNase had typical S-RNase characteristics. The functional validation results revealed that CdS-RNase was a key female factor for SI in C. drupifera, indicating that S-RNase plays an important role in the plant LSI mechanism. CdS-RNase localized to the cell membrane, was specifically expressed in the style and transferred to the ovary, and inhibited pollen tube growth in the style and ovary. There was an "enhancer factor" for the expression of CdS-RNase in selfing styles. Between 24 h and 48 h after selfing, CdS-RNase expression was significantly upregulated 28.3 times, which was more than 2.5 times higher than the outcross expression level and more than 5 times lower than the cross expression level. A total of 28 CdS-RNase proteins were identified, including 6 CdSm-RNases, and outcrossing among different CdS-RNase genotypes had a significant effect on the fruit setting rate. It may be that mismatches among CdS-RNase genotypes and self-inbreeding are the main reasons for the low yield of C. drupifera. These results provide a basis for the systematic analysis of the LSI mechanism and for solving the SI problem of tea oil Camellia.

Effects of species and varietal differences on intergeneric cross-compatibility between apple and pear

Hybridization across species and genus has been utilized to improve agriculturally important traits in crops. These interspecific and intergeneric hybridizations are often inhibited by reproductive barriers at the prezygotic and postzygotic stages. In this study, we used intergeneric hybridization between apple and pear to investigate the effects of species and varietal differences on pollen tube growth, seed germination, and seedling survival in a wide range of germplasms. In apple pistils pollinated by pears, inferior pollen tube growth was often observed for European pear cultivars, whereas hybrid seedlings derived from a cross between apple and European pear showed higher survival rate. In contrast, Japanese pear and Chinese pear pollination resulted in higher fruit setting which likely resulted from a higher rate of compatible pistils but a lower rate of seedling survival. Our results suggest the presence of both prezygotic and postzygotic barriers that occur independently in different crossing combinations. We also developed a Cleaved Amplified Polymorphic Sequence marker for detecting intergeneric hybrids derived from a wide range of crosses between apples and pears. The results of the present study will help in the development of intergeneric hybrids between apple and pear for introducing valuable traits from a diverse gene pool.

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

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 14months. 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.