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Context. Reproductive success in dioecious plant species may be limited by severe pollen limitation owing to their separate sexes and pollination barriers. Aims. Dodonaea viscosa subsp. angustifolia (Jacq.) (Sapindaceae) is a dioecious and wind-pollinated species that has a long flowering period. This study sought to determine the relationship between its reproductive behaviour and pollen availability during different flowering phases. Methods. Extra-stigmatic pollen germination and reproductive performance during different phases of flowering were investigated and correlated with pistil elongation under natural conditions. Results. The species offers whole stigmatic and stylar surfaces for pollen to land and germinate under natural conditions. During pollen-limiting conditions, the length of the pistil increases significantly to enhance pollen capture. Depending on where on the pistil pollen lands, the timing of pollen tube arrival at the ovary varies. Conclusions. Dodonaea viscosa subsp. angustifolia is the first reported wild species in the Sapindaceae where pistil elongation is regulated by pollination conditions and extra-stigmatic pollen germination ensures reproduction during phases of differing pollen availability. Our results indicated that the flexibility of female function and reproductive behaviour in Dodonaea viscosa subsp. angustifolia adds to the evolutionary possibilities to overcome pollination constraints. Implications. This unique strategy for increasing female fitness through pollen presentation in D. viscosa subsp. angustifolia could be explored in other subspecies of D. viscosa on the Australian subcontinent. [ABSTRACT FROM AUTHOR]

Climate change and global warming represent the main threats for many agricultural crops. Tomato is one of the most extensively grown and consumed horticultural products and can survive in a wide range of climatic conditions. However, high temperatures negatively affect both vegetative growth and reproductive processes, resulting in losses of yield and fruit quality traits. Researchers have employed different parameters to evaluate the heat stress tolerance, including evaluation of leaf- (stomatal conductance, net photosynthetic rate, Fv/Fm), flower- (inflorescence number, flower number, stigma exertion), pollen-related traits (pollen germination and viability, pollen tube growth) and fruit yield per plant. Moreover, several authors have gone even further, trying to understand the plants molecular response mechanisms to this stress. The present review focused on the tomato molecular response to heat stress during the reproductive stage, since the increase of temperatures above the optimum usually occurs late in the growing tomato season. Reproductive- related traits directly affects the final yield and are regulated by several genes such as transcriptional factors, heat shock proteins, genes related to flower, flowering, pollen and fruit set, and epigenetic mechanisms involving DNA methylation, histone modification, chromatin remodelling and non-coding RNAs. We provided a detailed list of these genes and their function under high temperature conditions in defining the final yield with the aim to summarize the recent findings and pose the attention on candidate genes that could prompt on the selection and constitution of new thermotolerant tomato plant genotypes able to face this abiotic challenge. [ABSTRACT FROM AUTHOR]

Background: As a vital type of noncoding RNAs, circular RNAs (circRNAs) play important roles in plant growth and development and stress response. However, little is known about the biological roles of circRNAs in regulating the stability of male fertility restoration for cytoplasmic male sterility (CMS) conditioned by Gossypium harknessii cytoplasm (CMS-D2) cotton under high-temperature (HT) stress. Results: In this study, RNA-sequencing and bioinformatics analysis were performed on pollen grains of isonuclear alloplasmic near-isogenic restorer lines NH [N(Rf1rf1)] and SH [S(Rf1rf1)] with obvious differences in fertility stability under HT stress at two environments. A total of 967 circRNAs were identified, with 250 differentially expressed under HT stress. We confirmed the back-splicing sites of eight selected circRNAs using divergent primers and Sanger sequencing. Tissue-specific expression patterns of five differentially expressed circRNAs (DECs) were also verified by RT-PCR and qRT-PCR. Functional enrichment and metabolic pathway analysis revealed that the parental genes of DECs were significantly enriched in fertility-related biological processes such as pollen tube guidance and cell wall organization, as well as the Pentose and glucuronate interconversions, Steroid biosynthesis, and N-Glycan biosynthesis pathways. Moreover, we also constructed a putative circRNA-mediated competing endogenous RNA (ceRNA) network consisting of 21 DECs, eight predicted circRNA-binding miRNAs, and their corresponding 22 mRNA targets, especially the two ceRNA modules circRNA346-miR159a-MYB33 and circRNA484-miR319e-MYB33, which might play important biological roles in regulating pollen fertility stability of cotton CMS-D2 restorer line under HT stress. Conclusions: Through systematic analysis of the abundance, characteristics and expression patterns of circRNAs, as well as the potential functions of their parent genes, our findings suggested that circRNAs and their mediated ceRNA networks acted vital biological roles in cotton pollen development, and might be also essential regulators for fertility stability of CMS-D2 restorer line under heat stress. This study will open a new door for further unlocking complex regulatory mechanisms underpinning the fertility restoration stability for CMS-D2 in cotton. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. Significance Statement: Phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K) and its product phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2], a membrane lipid known to serve as a signal for cell polarization, dynamically localize to the future germination site on the plasma membrane of Arabidopsis pollen. Comprehensive genetic analysis reveals that Arabidopsis PIP5K4–6, which belong to a conserved clade in the molecular phylogenetic tree of angiosperm PIP5Ks, redundantly play an essential role in pollen germination, through the establishment of germination polarity. [ABSTRACT FROM AUTHOR]

Maize is a dynamic cereal of world’s agriculture community. It is grown in spring and autumn seasons in Pakistan and both pistillate and staminate flowers experience severe temperature stress during spring sowing (February sowing) at reproductive phase, which ultimately results in poor seed setting because of increased silk dryness and pollen desiccation. In-vivo haploid induction was performed by using 2nd generation tropically adapted maize haploid inducer allocated from CIMMYT. Indigenously developed maize double haploid (DH) lines using local segregating population as source were screened out against high temperature stress. DH lines were evaluated and selected by using various indicators cell membrane thermostability (CMT), leaf temperature (LT), pollen production potential (PPP), pollen germination %age (PG%), pollen tube length (PTL), grain yield per plant (GY/P), plant height (PH), ear height (EH), days to tasseling (DT), days to silking (DS), number of grains per row (NGPR) and number of rows per cob (NRPC). Significant differences were noted at allelic level among the DH lines for high temperature tolerance related indicators, which provided wide range of option for selection, opportunity to improve tolerance level against high temperature stress and usage of these DH lines in different future maize breeding programs. Correlation coefficient analysis depicted PH, NGPR, HGW, PPP, PTL, and PG as suitable selection criteria for GY/P. The most effective physiological benchmarks for evaluating germplasm resistance to high temperature stress are CMT, PG%, and PTL. According to principal component analysis (PCA) DH lines viz. 5 (PBG-5), 11 (PBG-11), 15 (PBG-15), 19 (PBG-19), 24(PBG-24), 30(PBG-30), 33(PBG-33), 39 (PBG-39), 46 (PBG-46) and 52 (PBG-52) showed high level of diversity and performed well under the normal and high temperature stress conditions and DH lines viz. 10 (PBG-10), 13 (PBG-13), 26 (PBG-26), 37 (PBG-37) and 51 (PBG-51) performed poor under the normal and high temperature stress conditions. To develop maize varieties that can thrive in high-temperature conditions, it is necessary to cultivate DH lines that exhibit strong adaptability when exposed to induced stress. These lines could serve as a valuable genetic resource, as they likely contain a diverse array of genes that contribute to high temperature tolerance. [ABSTRACT FROM AUTHOR]

The male and female gametes of higher plants are immobile, but compatible gametes can recognize, attach, and fuse to fulfill fertilization and start embryogenesis after sperm cells are released from the pollen tube. The two fusions of egg and central cells with two sperm cells are controlled by accurate regulation mechanisms that ensure one-to-one gamete fusion. Many of the molecules involved in this process remain unknown, especially the egg cell proteins that are responsible for sperm-egg recognition, attachment, and fusion. The cytoplasm of sperm cells can trigger egg activation without the fusion of male and female gamete nuclei, suggesting that a gene controlling egg division is suppressed in the absence of fertilization. Fertilization in higher plants induces structural, physiological, and molecular biological changes in the fused egg, which are collectively known as egg activation. This review focuses on the early changes that occur in the fused egg of higher plants before fusion of the nuclei of male and female gametes. [ABSTRACT FROM AUTHOR]

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 3ʹ 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. [ABSTRACT FROM AUTHOR]

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Introduction: The effects of temperature increases on the growth of tomato fields are among the obvious results of global warming and are considered an important issue that should be investigated. To maintain and develop the cultivation systems of this crop, a proper understanding of the heat tolerance mechanisms and physiological responses in tomatoes should be achieved. The primary objective of this research is to discover the impact of heat stress on the germination and growth of pollen grains in research tomato germplasms. The researchers' knowledge about the response of different tomato cultivars to abiotic stresses is limited and only the effects of enzymes involved in the response process, heat shock proteins and some hormones have been investigated. The process of detecting heat stress-sensitive stages and their enhancement is facilitated by having a correct understanding of physiological processes. Materials and methods: The seeds of heat-resistant (LA2661 and LA2662) and -sensitive (LA3911) research cultivars of tomato were used to evaluate the effects of increasing day and night temperatures. The obtained seedlings were grown under optimal temperature conditions (24°C day/18°C night), and after observing the first flower primordium, were incubated in growth chambers to apply daytime heat stress treatments, including temperatures of 28°C, 32°C and 36°C day/18°C night and night stress treatments including temperatures of 28°C, 32°C, and 36°C at night/ 24°C day for 7 days. Pollen grains were then evaluated for their survival, germination, and growth. Results: The findings of the daytime heat stress tests show that the percentage of survival and germination of pollen grains and growth of pollen tubes of cultivars LA2661, LA2662 and LA3911 decreased as daytime temperature rose from 24°C to 36°C. This reduction is more noticeable for the sensitive cultivar LA3911. Degraded pollen grains increased in the LA3911 cultivar due to heat stress. The survival percentage of pollen grains in all three studied cultivars decreased due to the application of heat stress at night. The resistant cultivars LA2661 and LA2662 had a higher germination percentage compared to the sensitive cultivar LA3911. Pollen grains germination decreased by 50% as a result of increasing the night temperature from 18°C to 36°C. Pollen tube length was reduced in both cultivars and night treatments. Conclusion: The effects of heat stress in the early stages of flowering when flowers are visible are high, and reproductive stages are very sensitive to high temperatures and affect fertility and processes after insemination, and finally, they lead to yield loss. The daytime temperature increase relative to the natural temperature range (22°C to 24°C) during growth severely impacts the number of pollen grains released from tomato flowers. The number of non-living pollen grains is higher at 36°C day and 32°C and 36°C night temperatures compared to optimal temperature conditions. It appears that the increase in nighttime temperature results in more severe consequences than the increase in daytime temperature. [ABSTRACT FROM AUTHOR]

Under natural conditions, most Hibiscus syriacus L. individuals form very few mature seeds or the mature seeds that do form are of poor quality. As a result, seed yield is poor and seeds have low natural germinability. These phenomena strongly hinder utilization of the excellent germplasm resources of H. syriacus. The study has shown that pollen activity and stigma receptivity were high on the day of anthesis, and the pistils and stamens were fertile. Pollen release and stigma receptivity were synchronous. But in styles following self and cross-pollination, pollen tube abnormalities (distortion and twisting of the pollen tubes) and callose deposition were observed. Cross-pollinated pollen tubes elongated faster and fewer pollen tube abnormalities were observed compared with self-pollinated pollen tubes. And during embryo development, abnormalities during the heart-shaped embryo stage led to embryo abortion. Imbalance in antioxidant enzyme activities and low contents of auxin and cytokinin during early stages of embryo development may affect embryo development. Therefore, a low frequency of outcrossing and mid-development embryo abortion may be important developmental causes of H. syriacus seed abortion. Nutrient deficiencies, imbalance in antioxidant enzyme activities, and a high content of abscisic acid at advanced stages of seed development may be physiological causes of seed abortion. [ABSTRACT FROM AUTHOR]

Key message: 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 and VAMP725-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-SNARE SYP131 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. [ABSTRACT FROM AUTHOR]

Pollen, as the male gametophyte of plants, plays a critical role in plant sexual reproduction. Investigating pollen-specific promoters not only helps to understand the regulatory mechanisms of pollen development but also provides tissue-specific promoters for plant genetic engineering. Previously, wheat promoter PSG076 was found to confer pollen-specific activity in tobacco. To determine the tissue-specific activity of PSG076 in wheat, a 1.4-kb promoter fragment was fused with the β-glucuronidase (GUS) reporter gene and stably introduced into wheat via particle bombardment. Histochemical analysis in T1 progeny plants showed that the activity of PSG076 promoter was detected specifically in mature pollen and pollen tube. No GUS activity was found in other floral and vegetable tissues. Weak GUS staining was also visible in pollen at 45 days post anthesis (DPA). Further analysis showed that GUS activity was found weakly in middle tricellular pollen grains and increased rapidly as pollen matured. These results indicated that PSG076 promoter has strong activity specifically at late pollen development stage and can be utilized in genetic engineering investigations for creating male sterility in wheat and other plant species for hybrid seed production. [ABSTRACT FROM AUTHOR]

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. Summary statement: Just 6 h of postpollination heat stress exposure was solely responsible for failure in maize. Heat stress altered sugar composition, increased ABA and auxin and higher reactive oxygen species accumulation in silks, negatively impacts pollen tube growth. [ABSTRACT FROM AUTHOR]

Key message: Functional loss of Arabidopsis Sar1b with that of either Sar1a or Sar1c inhibits mitosis of functional megaspores, leading to defective embryo sac formation and reduced fertility. Vesicular trafficking among diverse endomembrane compartments is critical for eukaryotic cells. Anterograde trafficking from endoplasmic reticulum (ER) to the Golgi apparatus is mediated by coat protein complex II (COPII) vesicles. Among five cytosolic components of COPII, secretion-associated Ras-related GTPase 1 (Sar1) mediates the assembly and disassembly of the COPII coat. Five genes in Arabidopsis encode Sar1 isoforms, whose different cargo specificities and redundancy were both reported. We show here that Arabidopsis Sar1a, Sar1b, and Sar1c mediate the development of female gametophytes (FGs), in which Sar1b plays a major role, whereas Sar1a and Sar1c play a minor role. We determined that female transmission of sar1a;sar1b or sar1c;sar1b was significantly reduced due to defective mitosis of functional megaspores. Half of ovules in sar1a;sar1b/+ or sar1c;sar1b/+ plants failed to attract pollen tubes, leading to fertilization failure. The homozygous sar1a;sar1b or sar1c;sar1b double mutant was obtained by introducing either UBQ10:GFP-Sar1b or UBQ10:GFP-Sar1c, supporting their redundant function in FG development. [ABSTRACT FROM AUTHOR]

Due to self-incompatibility (SI) prevents self-fertilization, natural or artificial cross-pollination has been conducted in many orchards to stabilize fruit yield. However, it is still puzzled which routes of self S-RNase arresting pollen tube growth. Herein, 17 COBRA genes were isolated from pear genome. Of these genes, the pollen-specifically expressed PbCOB.A.1 and PbCOB.A.2 positively mediates pollen tube growth. The promoters of PbCOB.A.1 and/or PbCOB.A.2 were bound and activated by PbABF.E.2 (an ABRE-binding factor) and PbC2H2.K16.2 (a C2H2-type zinc finger protein). Notably, the expressions of PbCOB.A.1, PbCOB.A.2, and PbC2H2.K16.2 were repressed by self S-RNase, suggesting that self S-RNase reduces the expression of PbCOB.A.1 and PbCOB.A.2 by decreasing the expression of their upstream factors, such as PbC2H2.K16.2, to arrest pollen tube growth. PbCOB.A.1 or PbCOB.A.2 accelerates the growth of pollen tubes treated by self S-RNase, but can hardly affect level of reactive oxygen species and deploymerization of actin cytoskeleton in pollen tubes and cannot physically interact with any reported proteins involved in SI. These results indicate that PbCOB.A.1 and PbCOB.A.2 may not relieve S-RNase toxicity in incompatible pollen tube. The information provides a new route to elucidate the arresting pollen tube growth during SI reaction. [ABSTRACT FROM AUTHOR]

The in vitro growth of the pollen tube (PT), an object of comprehensive and intensive research, is a model for studying the mechanisms of sexual reproduction in higher plants. We have studied the potential role of reactive oxygen species (ROS) in the in vitro germination and growth maintenance of the petunia (Petunia hybrida E. Vilm.) male gametophyte. The exogenous treatment with H2O2 influences the PT germination and polar growth in vitro. The addition of H2O2 to culture medium increases both the percentage of pollen grain germination and the PT length in the case of long cultivation, but inhibits both processes during the first hour of cultivation. This suggests that endogenous ROS play a decisive role in the early stages of pollen germination, with the sensitivity to endogenous ROS emerging later over the course of their growth. The addition of diphenylene iodonium chloride (DPI), a NADPH oxidase inhibitor, considerably decreases both the germination and the growth of the petunia male gametophyte at low concentrations (0.1 μM), and completely arrests the growth at high concentrations (1 μM). ROS are necessary for polar growth of the petunia male gametophyte; they are secreted in the early stages of pollen grain activation and are further localized to the initiation of the PT, mainly in the PT apical part, during polar growth, as confirmed with the help of intravital fluorescence microscopy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This document is an interview with Debora Gasperini, a researcher in plant science. Gasperini explains that her interest in research began at a young age, inspired by her love for the natural world and engaging science classes. She chose to study molecular biology and became fascinated with plants due to their unique communication and resilience. Gasperini is motivated by the thrill of discovery and problem-solving, and she considers scientists like Rita Levi-Montalcini and Katherine Esau as role models for their passion and perseverance. She also discusses her favorite papers from the journal New Phytologist, which covers a wide range of plant science topics. Gasperini appreciates the adaptability and enduring nature of plants, which continue to inspire her research. [Extracted from the article]

Within flowers, the style channel receives pollen and transmits male gametes inside elongating pollen tubes to ovules. The styles of maize/corn are called silks. Fertilization-stage silks possess complex microbiomes, which may partially derive from pollen. These microbiomes lack functional analysis. We hypothesize that fertilization-stage silk microbiomes promote host fertilization to ensure their own vertical transmission. We further hypothesize that these microbes encode traits to survive stresses within the silk (water/nitrogen limitation) and pollen (dehydration/aluminum) habitats. Here, bacteria cultured from fertilization-stage silks of 14 North American maize genotypes underwent genome mining and functional testing, which revealed osmoprotection, nitrogen-fixation, and aluminum-tolerance traits. Bacteria contained auxin biosynthesis genes, and testing confirmed indole compound secretion, which is relevant, since pollen delivers auxin to silks to stimulate egg cell maturation. Some isolates encoded biosynthetic/transport compounds known to regulate pollen tube guidance/growth. The isolates encoded ACC deaminase, which degrades the precursor for ethylene that otherwise accelerates silk senescence. The findings suggest that members of the microbiome of fertilization-stage silks encode adaptations to survive the stress conditions of silk/pollen and have the potential to express signaling compounds known to impact reproduction. Overall, whereas these microbial traits have traditionally been assumed to primarily promote vegetative plant growth, this study proposes they may also play selfish roles during host reproduction.

In vitro pollen germination is considered the most efficient method to assess pollen viability. The pollen germination frequency and pollen tube length, which are key indicators of pollen viability, should be accurately measured during in vitro culture. In this study, a Mask R-CNN model trained using microscopic images of tree peony (Paeonia suffruticosa) pollen has been proposed to rapidly detect the pollen germination rate and pollen tube length. To reduce the workload during image acquisition, images of synthesized crossed pollen tubes were added to the training dataset, significantly improving the model accuracy in recognizing crossed pollen tubes. At an Intersection over Union threshold of 50%, a mean average precision of 0.949 was achieved. The performance of the model was verified using 120 testing images. The R 2 value of the linear regression model using detected pollen germination frequency against the ground truth was 0.909 and that using average pollen tube length was 0.958. Further, the model was successfully applied to two other plant species, indicating a good generalizability and potential to be applied widely. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Annual Medicago species contribute significantly to improved fodder production in Algeria. The leguminous plant model is Medicago truncatula Gaertner. Because of its high protein content, this plant is essential for natural soil fertilization as well as good quality nutrition for animals and humans. However, abiotic stresses such as salinity are the leading cause of land degradation and crop productivity limitations worldwide, and they have an impact on legume physiology and metabolism. We investigated the pollen tube growth behavior in two contrasting Medicago truncatula ecotypes under salt stress (Tru 42, tolerant, and Tru 242, sensitive) with different NaCl concentrations (68, 102, and 137 mM) because pollen tube growth mechanisms can be affected by environmental stresses such as water and salt stress. According to the data, the Tru242 ecotype has a pollen tube elongation of 1.41 to 8.45 um, whereas the high pollen tube elongation of the Tru 42 ecotype is between 2.8 and 18.83 um. It is crucial to comprehend the physiological reactions of male gametophytes in order to reproduce, as salinity stress can hinder their ability to do so. It is intriguing to include the tolerant genotype in a selection program for leguminous breeding, as the analysis revealed that the tolerant ecotype has better pollen tube elongation than the sensitive one. in fact, there is not enough knowledge about pollen tolerance mechanism to salt stress of Medicago truncatula or other Medicago species. [ABSTRACT FROM AUTHOR]

Polarization of the zygote defines the body axis during plant development. In Arabidopsis (Arabidopsis thaliana), the zygote becomes polarized and elongates in the longitudinal direction, ultimately forming the apical–basal axis of the mature plant. Despite its importance, the mechanism for this elongation remains poorly understood. Based on live-cell imaging of the zygote, we developed new image analysis methods, referred to as coordinate normalization, that appropriately fix and align positions in an image, preventing fluctuation across a temporal sequence of images. Using these methods, we discovered that the zygote elongates only at its apical tip region, similar to tip-growing cells such as pollen tubes and root hairs. We also investigated the spatiotemporal dynamics of the apical tip contour of the zygote and observed that the zygote tip retains its isotropic, hemispherical apical shape during cell elongation. By looking at the elliptical fitting of the contour over time, we further discovered that the apical cell tip becomes thinner at first and then thickens, with a transient increase in growth speed that is followed by the first cell division. We performed the same series of analyses using root hairs and established that both the hemispherical tip shape and the changes in growth rate associated with changes in tip size are specific to the zygote. In summary, the Arabidopsis zygote undergoes directional elongation as a tip-growing cell, but its tip retains an unusual isotropic shape, and the manner of growth changes with the developmental stage. [ABSTRACT FROM AUTHOR]

Styles transmit pollen-derived sperm nuclei from pollen to ovules, but also transmit environmental pathogens. The microbiomes of styles are likely important for reproduction/disease, yet few studies exist. Whether style microbiome compositions are spatially responsive to pathogens is unknown. The maize pathogen Fusarium graminearum enters developing grain through the style (silk). We hypothesized that F. graminearum treatment shifts the cultured transmitting silk microbiome (TSM) compared to healthy silks in a distance-dependent manner. Another objective of the study was to culture microbes for future application. Bacteria were cultured from husk-covered silks of 14 F. graminearum-treated diverse maize genotypes, proximal (tip) and distal (base) to the F. graminearum inoculation site. Long-read 16S sequences from 398 isolates spanned 35 genera, 71 species, and 238 OTUs. More bacteria were cultured from F. graminearum-inoculated tips (271 isolates) versus base (127 isolates); healthy silks were balanced. F. graminearum caused a collapse in diversity of ~20–25% across multiple taxonomic levels. Some species were cultured exclusively or, more often, from F. graminearum-treated silks (e.g., Delftia acidovorans, Klebsiella aerogenes, K. grimontii, Pantoea ananatis, Stenotrophomonas pavanii). Overall, the results suggest that F. graminearum alters the TSM in a distance-dependent manner. Many isolates matched taxa that were previously identified using V4-MiSeq (core and F. graminearum-induced), but long-read sequencing clarified the taxonomy and uncovered greater diversity than was initially predicted (e.g., within Pantoea). These isolates represent the first comprehensive cultured collection from pathogen-treated maize silks to facilitate biocontrol efforts and microbial marker-assisted breeding. [ABSTRACT FROM AUTHOR]

SUMMARY: Ovules are female reproductive organs of angiosperms, consisting of sporophytic integuments surrounding female gametophytes, that is, embryo sacs. Synchronization between integument growth and embryo sac development requires intracellular communication. However, signaling routes through which cells of the two generations communicate are unclear. We report that symplastic signals through plasmodesmata (PDs) of integuments are critical for the development of female gametophytes. Genetic interferences of PD biogenesis either by functional loss of CHOLINE TRANSPORTER‐LIKE1 (CTL1) or by integument‐specific expression of a mutated CALLOSE SYNTHASE 3 (cals3m) compromised PD formation in integuments and reduced fertility. Close examination of pINO:cals3m or ctl1 ovules indicated that female gametophytic development was either arrested at various stages after the formation of functional megaspores. In both cases, defective ovules could not attract pollen tubes, leading to the failure of fertilization. Results presented here demonstrate a key role of the symplastic route in sporophytic control of female gametophytic development. Significance Statement: By examining a mutant defective in plasmadesmata (PDs) formation and by genetically interfering with PD formation specifically in integuments, this study demonstrates a key role of PD in integument‐mediated female gametophytic development, implying the involvement of syplasmic transport in this process. [ABSTRACT FROM AUTHOR]

The pollen viability of Siberian spruce (Picea obovata Ledeb.) has been studied on the border of the city Krasnoyarsk, as well as under unpolluted growth conditions. This study has discovered the variability and correlation between the functional characteristics of pollens, as well as a low rate of development anomalies. The viability of pollen growth in forest ecosystems of the middle mountains of the Eastern Sayans (the northeastern border of the Krasnoyarskie Stolby national park) have decreased under pollution press. The great variety of pollen viability characteristics of the Siberian spruce pollen makes the high degree of flexibility and adaptability of species reproductive processes evident. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Pollen tubes have dynamic tubular vacuoles. Functional loss of AP‐3, a regulator of one vacuolar trafficking route, reduces pollen tube growth. However, the role of canonical Rab5 GTPases that are responsible for two other vacuolar trafficking routes in Arabidopsis pollen tubes is obscure.By using genomic editing, confocal microscopy, pollen tube growth assays, and transmission electron microscopy, we demonstrate that functional loss of canonical Rab5s in Arabidopsis, RHA1 and ARA7, causes the failure of pollen tubes to grow through style and thus impairs male transmission.Functional loss of canonical Rab5s compromises vacuolar trafficking of tonoplast proteins, vacuolar biogenesis, and turgor regulation. However, rha1;ara7 pollen tubes are comparable to those of wild‐type in growing through narrow passages by microfluidic assays.We demonstrate that functional loss of canonical Rab5s compromises endocytic and secretory trafficking at the plasma membrane (PM), whereas the targeting of PM‐associated ATPases is largely unaffected. Despite that, rha1;ara7 pollen tubes contain a reduced cytosolic pH and disrupted actin microfilaments, correlating with the mis‐targeting of vacuolar ATPases (VHA). These results imply a key role of vacuoles in maintaining cytoplasmic proton homeostasis and in pollen tube penetrative growth through style. [ABSTRACT FROM AUTHOR]

Background: In a genetic breeding program, understanding the viability of pollen germination and pollen tube growth is an essential factor for artificial hybridization. Currently, little is known about the influence of sucrose, boric acid concentrations and calcium chloride on pollen viability in alfalfa. This study is valuable for providing information about pollen germination requirements to support further improvement of artificial pollination and breeding programs in alfalfa. Methods: The single-factor experimental and an orthogonal design method (L16 [4]3) was used to compare the effectiveness of key factors (e.g., concentrations of sucrose, boric acid and calcium chloride) to determine the pollen vigor of Medicago varia Martin. 'Caoyuan No.1'and M. ruthenica L. Trautv. 'zhilixing' and to optimize the culture medium for in vitro pollen germination. Four different varieties were chosen to compare the diurnal variation of pollen vigor. Result: The four factors that influenced pollen germination in Medicago varia Martin. 'Caoyuan No.1' and M. ruthenica L. Trautv. 'zhilixing' in order of significance are as follows: sucrose > H3BO3 > CaCl2. These variables influenced pollen tube growth in the following order: sucrose > H3BO3 > CaCl2. The optimal culture medium for promoting pollen vigor of Medicago varia Martin. 'Caoyuan No.1' was 10% sucrose, 300 mon/L H3BO3 and 100 mon/L CaCl2; that of M. ruthenica L. Trautv. 'zhilixing' was 10% sucrose, 200 mon/L H3BO3 and 50 mon/L CaCl2. Among several species of alfalfa, 'Caoyuan No.1' shows the highest pollen vigor and the daily pollen activity curves of all varieties showed a single peak type. [ABSTRACT FROM AUTHOR]

Summary: In angiosperms, the timely delivery of sperm cell nuclei by pollen tube (PT) to the ovule is vital for double fertilization. Penetration of PT into maternal stigma tissue is a critical step for sperm cell nuclei delivery, yet little is known about the process. Here, a male‐specific and sporophytic mutant xt6, where PTs are able to germinate but unable to penetrate the stigma tissue, is reported in Oryza sativa. Through genetic study, the causative gene was identified as Chalcone synthase (OsCHS1), encoding the first enzyme in flavonoid biosynthesis. Indeed, flavonols were undetected in mutant pollen grains and PTs, indicating that the mutation abolished flavonoid biosynthesis. Nevertheless, the phenotype cannot be rescued by exogenous application of quercetin and kaempferol as reported in maize and petunia, suggesting a different mechanism exists in rice. Further analysis showed that loss of OsCHS1 function disrupted the homeostasis of flavonoid and triterpenoid metabolism and led to the accumulation of triterpenoid, which inhibits significantly α‐amylase activity, amyloplast hydrolysis and monosaccharide content in xt6, these ultimately impaired tricarboxylic acid (TCA) cycle, reduced ATP content and lowered the turgor pressure as well. Our findings reveal a new mechanism that OsCHS1 modulates starch hydrolysis and glycometabolism through modulating the metabolic homeostasis of flavonoids and triterpenoids which affects α‐amylase activity to maintain PT penetration in rice, which contributes to a better understanding of the function of CHS1 in crop fertility and breeding. [ABSTRACT FROM AUTHOR]

During angiosperm sexual reproduction, pollen tubes must penetrate through multiple cell types in the pistil to mediate successful fertilization. Although this process is highly choreographed and requires complex chemical and mechanical signaling to guide the pollen tube to its destination, aspects of our understanding of pollen tube penetration through the pistil are incomplete. Our previous work demonstrated that disruption of the Arabidopsis thaliana O-FUCOSYLTRANSFERASE1 (OFT1) gene resulted in decreased pollen tube penetration through the stigma–style interface. Here, we demonstrate that second site mutations of Arabidopsis GALACTURONOSYLTRANSFERASE 14 (GAUT14) effectively suppress the phenotype of oft1 mutants, partially restoring silique length, seed set, pollen transmission, and pollen tube penetration deficiencies in navigating the female reproductive tract. These results suggest that disruption of pectic homogalacturonan (HG) synthesis can alleviate the penetrative defects associated with the oft1 mutant and may implicate pectic HG deposition in the process of pollen tube penetration through the stigma–style interface in Arabidopsis. These results also support a model in which OFT1 function directly or indirectly modifies structural features associated with the cell wall, with the loss of oft1 leading to an imbalance in the wall composition that can be compensated for by a reduction in pectic HG deposition. [ABSTRACT FROM AUTHOR]

Pollen tube growth is essential for successful double fertilization, which is critical for grain yield in crop plants. Rapid alkalinization factors (RALFs) function as ligands for signal transduction during fertilization. However, functional studies on RALF in monocot plants are lacking. Herein, we functionally characterized two pollen‐specific RALFs in rice (Oryza sativa) using multiple clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR‐associated protein 9‐induced loss‐of‐function mutants, peptide treatment, expression analyses, and tag reporter lines. Among the 41 RALF members in rice, OsRALF17 was specifically expressed at the highest level in pollen and pollen tubes. Exogenously applied OsRALF17 or OsRALF19 peptide inhibited pollen tube germination and elongation at high concentrations but enhanced tube elongation at low concentrations, indicating growth regulation. Double mutants of OsRALF17 and OsRALF19 (ralf17/19) exhibited almost full male sterility with defects in pollen hydration, germination, and tube elongation, which was partially recovered by exogenous treatment with OsRALF17 peptide. This study revealed that two partially functionally redundant OsRALF17 and OsRALF19 bind to Oryza sativa male‐gene transfer defective 2 (OsMTD2) and transmit reactive oxygen species signals for pollen tube germination and integrity maintenance in rice. Transcriptomic analysis confirmed their common downstream genes, in osmtd2 and ralf17/19. This study provides new insights into the role of RALF, expanding our knowledge of the biological role of RALF in regulating rice fertilization. [ABSTRACT FROM AUTHOR]

Self-incompatibility (SI) is a widespread genetically determined system in flowering plants that prevents self-fertilization to promote gene flow and limit inbreeding. S-RNase–based SI is characterized by the arrest of pollen tube growth through the pistil. Arrested pollen tubes show disrupted polarized growth and swollen tips, but the underlying molecular mechanism is largely unknown. Here, we demonstrate that the swelling at the tips of incompatible pollen tubes in pear (Pyrus bretschneideri [Pbr]) is mediated by the SI-induced acetylation of the soluble inorganic pyrophosphatase (PPA) PbrPPA5. Acetylation at Lys-42 of PbrPPA5 by the acetyltransferase GCN5-related N-acetyltransferase 1 (GNAT1) drives accumulation of PbrPPA5 in the nucleus, where it binds to the transcription factor PbrbZIP77, forming a transcriptional repression complex that inhibits the expression of the pectin methylesterase (PME) gene PbrPME44. The function of PbrPPA5 as a transcriptional repressor does not require its PPA activity. Downregulating PbrPME44 resulted in increased levels of methyl-esterified pectins in growing pollen tubes, leading to swelling at their tips. These observations suggest a mechanism for PbrPPA5-driven swelling at the tips of pollen tubes during the SI response. The targets of PbrPPA5 include genes encoding cell wall–modifying enzymes, which are essential for building a continuous sustainable mechanical structure for pollen tube growth. In the self-incompatibility response, acetylation of PbrPPA5 enhances the repression of PbrPME44, resulting in the accumulation of methyl-esterified pectins of the pollen tube cell wall and tip swelling. [ABSTRACT FROM AUTHOR]

Nanotechnology provides tremendous potential in agriculture, mitigating climate change impact and improving abiotic stress management strategy. Chitosan nanoparticles (NCS) were synthesised using the ion gelation method and characterised for size (75.5 nm in particle size analyser), shape (spherical under scanning electron microscopy) and stability (132.2 mV zeta potential). Further, salicylic acid was incorporated into NCS to craft salicylic acid-functionalised chitosan nanoparticles (SA-NCS) and illustrated for size (517 nm), shape (spherical) and stability (197.1 mV). The influence of the exogenous application of SA-NCS (0.08%) was studied at the reproductive stage of three genotypes of cotton (Gossypium hirsutum): (1) heat-tolerant Solar-651 BGII; (2) moderately heat-tolerant Solar-701 BGII; and (3) heat-susceptible Solar-805 BGII, exposed to different temperature regimes: (1) H1 (optimal), 32/20 ± 2°C; (2) H2 (sub-optimal), 38/24 ± 2°C; H3 (supra-optimal), 45/30 ± 2°C. Heat stress significantly reduces carbon-fixing Rubisco, enzymes related to sucrose metabolism and pollen tube length. Considering three genotypes and reproductive stages (sepal and anther tissues), activities of Rubisco (sepals), invertase (sepals), sucrose phosphate synthase (anthers), sucrose content (sepals) and pollen tube length were elevated under high-temperature regimes, signifying better source to sink transposition of sucrose influenced by SA-NCS. The study provides new insights into SA-NCS to improve source–sink imbalance and restore sucrose metabolism for better growth of reproductive structure under heat stress in cotton. Nanotechnology offers enormous potential in agriculture, including alleviating the effects of climate change and enhancing abiotic stress management. Salicylic acid-functionalised chitosan nanoparticles (SA-NCS) were synthesised and their applications illustrated slow release of SA from SA-NCS, which may effectively protect developing anthers against heat stress in cotton. The study provides new insights into SA-NCS to improve source-sink imbalance and enlighten to restore sucrose metabolism for better growth of reproductive structure under heat stress in cotton (Gossypium hirsutum L.). [ABSTRACT FROM AUTHOR]

Societal Impact Statement: Lateral gene transfer (LGT) refers to the transmission of genetic material without sexual reproduction. LGT is widespread in a number of plant species, including grasses. But how these genes of foreign origin got there is presently unknown. In this review, we show that transformation techniques used to genetically modify organisms could occur in the wild and be responsible for the frequently observed grass‐to‐grass LGTs. The distinction between natural evolutionary processes and genetic engineering might be arbitrary, and its validity will be further debated as agricultural biotechnology becomes more widely used and examples of "natural genetic engineering" through LGT increase. Summary: Lateral gene transfer (LGT) is the transmission of genetic material among species without sexual reproduction. LGT was initially thought to be restricted to prokaryotes, but it has since been reported in a wide range of eukaryotes, including plants. Grasses seem to be particularly prone to LGT and frequently exchange genes among species. However, the mechanism(s) facilitating these transfers in this economically and ecologically important group of plants are debated. Here, we review vector‐mediated, direct tissue‐to‐tissue contact, wide‐crossing and reproductive contamination LGT mechanisms and discuss the likelihood of each in light of recent studies. Of particular relevance are transformation approaches that require minimal human intervention to transfer DNA among grasses in the lab that could mimic the mechanisms facilitating grass‐to‐grass LGT in the wild. These approaches include relatively simple techniques, such as pollen tube pathway‐mediated transformation, that take advantage of the permeability of the reproductive process to introduce alien genetic material from a third individual into an embryo. This process could be easily mirrored in the wild where pollen from one species lands on the stigma of another, acting as a source of alien DNA that can ultimately contaminate the reproductive process. This contamination is likely to be prevalent in wind pollinated species such as grasses, where the rates of illegitimate pollination will be high. In conclusion, plant transformation methods requiring minimal intervention are likely paralleled in the wild where they act as the mechanism underpinning LGT between distantly related grass species. [ABSTRACT FROM AUTHOR]

Arabinogalactan proteins (AGPs) are widely distributed in the plant kingdom and play a vital role during the process of plant sexual reproduction. In this study, we performed a comprehensive identification of the PbrAGPs expressed in pear pollen and further explored their influences on pollen tube growth. Among the 187 PbrAGPs that were found to be expressed in pear pollen tubes, 38 PbrAGPs were specifically expressed in pollen according to the RNA-seq data. The PbrAGPs were divided into two groups of highly expressed and specifically expressed in pear pollen. We further tested their expression patterns using RT-PCR and RT-qPCR. Most of the PbrAGPs were expressed in multiple tissues and their expression levels were consistent with reads per kilobase per million map reads (RPKM) values during pollen tube growth, implying that PbrAGPs might be involved in the regulation of pear pollen tube growth. We also constructed phylogenetic trees to identify the functional genes in pear pollen tube growth. Therefore, 19 PbrAGPs (PbrAGP1 to PbrAGP19) were selected to test their influences on pollen tube growth. Recombinant proteins of the 19 PbrAGP-His were purified and used to treat pear pollen, and 11 of the PbrAGP-His recombinant proteins could promote pear pollen tube growth. Additionally, pollen tube growth was inhibited when the expression levels of PbrAGP1 and PbrAGP5 were knocked down using an antisense oligonucleotide assay. PbrAGP1 and PbrAGP5 were localized in the plasma membrane and might not alter the distribution of pectin in the pollen tube. In summary, this study identified the PbrAGPs expressed in pear pollen and lays the foundation for further exploring their functions in pollen tube growth.

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. [ABSTRACT FROM AUTHOR]

The current pace of crop plant optimization is insufficient to meet future demands and there is an urgent need for novel breeding strategies. It was previously shown that plants tolerate the generation of triparental polyspermy-derived plants and that polyspermy can bypass hybridization barriers. Polyspermy thus has the potential to harness previously incompatible climate-adapted wild varieties for plant breeding. However, factors that influence polyspermy frequencies were not previously known. The endopeptidases ECS1 and ECS2 have been reported to prevent the attraction of supernumerary pollen tubes by cleaving the pollen tube attractant LURE1. Here, we show that these genes have an earlier function that is manifested by incomplete double fertilization in plants defective for both genes. In addition to supernumerary pollen tube attraction, ecs1 ecs2 mutants exhibit a delay in synergid disintegration, are susceptible to heterofertilization, and segregate haploid plants that lack a paternal genome contribution. Our results thus uncover ECS1 and ECS2 as the first female factors triggering the induction of maternal haploids. Capitalizing on a high-throughput polyspermy assay, we in addition show that the double mutant exhibits an increase in polyspermy frequencies. As both haploid induction and polyspermy are valuable breeding aims, our results open new avenues for accelerated generation of climate-adapted cultivars. [ABSTRACT FROM AUTHOR]

In angiosperms, pollen tube growth is critical for double fertilization and seed formation. Many of the factors involved in pollen tube tip growth are unknown. Here, we report the roles of pollen‐specific GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE‐LIKE (GDPD‐LIKE) genes in pollen tube tip growth. Arabidopsis thaliana GDPD‐LIKE6 (AtGDPDL6) and AtGDPDL7 were specifically expressed in mature pollen grains and pollen tubes and green fluorescent protein (GFP)‐AtGDPDL6 and GFP‐AtGDPDL7 fusion proteins were enriched at the plasma membrane at the apex of forming pollen tubes. Atgdpdl6 Atgdpdl7 double mutants displayed severe sterility that was rescued by genetic complementation with AtGDPDL6 or AtGDPDL7. This sterility was associated with defective male gametophytic transmission. Atgdpdl6 Atgdpdl7 pollen tubes burst immediately after initiation of pollen germination in vitro and in vivo, consistent with the thin and fragile walls in their tips. Cellulose deposition was greatly reduced along the mutant pollen tube tip walls, and the localization of pollen‐specific CELLULOSE SYNTHASE‐LIKE D1 (CSLD1) and CSLD4 was impaired to the apex of mutant pollen tubes. A rice pollen‐specific GDPD‐LIKE protein also contributed to pollen tube tip growth, suggesting that members of this family have conserved functions in angiosperms. Thus, pollen‐specific GDPD‐LIKEs mediate pollen tube tip growth, possibly by modulating cellulose deposition in pollen tube walls. [ABSTRACT FROM AUTHOR]

Main conclusion: The pistillate flowers of Lithocarpus dealbatus show two pollen tube (PT) arresting sites (the style-joining and micropyle) within the pistil during the postpollination-prezygotic stage. The PT, arrested at the pre-ovule stage, enhanced PT competition allowing the most compatible PTs to enter the ovary to ensure the highest fertilization success. During the shift from animal pollination to wind pollination, plants require a series of changes in reproductive traits. The mode of pollination is striking labile in Fagaceae. Lithocarpus is insect pollinated and is closely related to wind-pollinated Quercus. Little is known about the sexual reproduction of Lithocarpus. This study aimed to reveal the sexual reproduction of Lithocarpus dealbatus and to explore the evolutionary pattern of the key sexual reproduction traits to better understand their possible role in labile pollination. We found that after pollination, L. dealbatus PTs grew slowly in the style reaching style-joining in mid-January of the second year; then PT growth was arrested at style-joining for four months. Only two to three PTs resumed growth in mid-May to reach the micropyle, where PT growth ceased for one month before one PT resumed growth and passed through the micropyle to the embryo sac. Fagaceae showed a generalized mating system. Vast pollen production, small-sized pollen grains, long stigmatic receptive time, and reduced perianth were compatible with beetle pollination syndrome, representing the plesiomorphic status in Fagaceae. A large stigmatic surface and dry pollen grains linked to wind pollination might be independently derived several times in fagaceous lineages. Beetle pollination syndrome can cope with the uncertainty of pollinators to ensure conspecific pollen capture, which represents pre-adaptation status and has a selective advantage when conditions change, favouring wind pollination. The arrest of the PT at style-joining is a unique mechanism in later derived fagaceous lineages to enhance PT competition and promote outcrossing. [ABSTRACT FROM AUTHOR]

Apomixis is a unique reproductive process that produces fertile offspring without the combination of sperm and egg cells. This process perfectly reproduces maternal DNA, making it possible to fix heterosis during reproduction. Malus shizongensis is a newly discovered species that is closely related to Malus hupehensis Rehd. After de-male bagging, it was found that the fruit set rate reached 78.7%. Preliminary analysis indicated that M. shizongensis have apomictic reproductive characteristics. In this work, we employed paraffin sectioning and electron scanning microscopy to explore apomixis in M. shizongensis during the development of male–female gametes and embryo sacs. Stigma fluorescence assays showed that pollen germination was normal, but less pollen entered the ovaries. Additionally, analysis of anthers indicated the presence of dysplasia and paraffin sectioning revealed that the pollen mother cells were aborted due to abnormal disintegration of the tapetum layer. Taken together, our results indicate that the primary causes of apomixis in M. shizongensis are anther dysplasia and male gamete development failure, resulting in reduced pollen tube entry into ovaries and reduced reproduction of female gametes. In conclusion, this study provide a theoretical basis and technical supports for apple stock breeding and apple industry development. [ABSTRACT FROM AUTHOR]

Camellia yuhsienensis was used to cross with Camellia oleifera to improve the resistance of oil camellia anthracnose. However, unilateral cross-incompatibility (UCI) between C. oleifera and C. yuhsienensis was found during the breeding process. Five C.oleifera cultivars and four C. uhsienensis materials were tested to confirm the UCI between C. oleifera and C. yuhsienensis. 'Huashuo' (HS) and 'Youza 2' (YZ2) were used to represent these two species to characterize the UCI, including pollen tube growth, fertilization and fruit development. The results demonstrated that UCI was prevalent between C. oleifera and C. yuhsienensis. The asynchronous flowering period was a pre-pollination barrier that limited mating between these two species under natural conditions. Interspecific pollen tubes were observed through the styles of these two plants, though the growth rates differed considerably. At 96 hours after pollination, the pollen tube of YZ2 barely entered the ovule, but remained at the base of the style and became swollen. However, the HS pollen tube entered the ovule 48 hours after pollination, double fertilization was observed, and the fruit and seeds developed commonly. Relative to compatible combinations, most unfertilized ovules in incompatible combinations failed to grow, turned brown 150 days after pollination, and the fruits were smaller than expected with uneven enlargement. Investigations on both semi-in vivo and in vitro pollen tubes gave us new idea for thought: the HS style has a stronger inhibitory effect on the interspecific pollen tubes, while calcium alleviates the inhibitory of styles but failed to prevent the appearance of abnormal pollen tube morphology. This study provides useful information on interspecific hybridization between C. oleifera and C. yuhsienensis for understanding reproductive isolation mechanisms and breeding programs in genus Camellia. [ABSTRACT FROM AUTHOR]

Pollen tubes of higher plants grow very rapidly until they reach the ovules to fertilize the female gametes. This growth process is energy demanding, however, the nutrition strategies of pollen are largely unexplored. Here, we studied the function of sucrose transporters and invertases during pollen germination and pollen tube growth. RT-PCR analyses, reporter lines and knockout mutants were used to study gene expression and protein function in pollen. The genome of Arabidopsis thaliana contains eight genes that encode functional sucrose/H+ symporters. Apart from AtSUC2, which is companion cell specific, all other AtSUC genes are expressed in pollen tubes. AtSUC1 is present in developing pollen and seems to be the most important sucrose transporter during the fertilization process. Pollen of an Atsuc1 knockout plant contain less sucrose and have defects in pollen germination and pollen tube growth. The loss of other sucrose carriers affects neither pollen germination nor pollen tube growth. A multiple knockout line Atsuc1Atsuc3Atsuc8Atsuc9 shows a phenotype that is comparable to the Atsuc1 mutant line. Loss of AtSUC1 can't be complemented by AtSUC9, suggesting a special function of AtSUC1. Besides sucrose carriers, pollen tubes also synthesize monosaccharide carriers of the AtSTP family as well as invertases. We could show that AtcwINV2 and AtcwINV4 are expressed in pollen, AtcwINV1 in the transmitting tissue and AtcwINV5 in the funiculi of the ovary. The vacuolar invertase AtVI2 is also expressed in pollen, and a knockout of AtVI2 leads to a severe reduction in pollen germination. Our data indicate that AtSUC1 mediated sucrose accumulation during late stages of pollen development and cleavage of vacuolar sucrose into monosaccharides is important for the process of pollen germination. [ABSTRACT FROM AUTHOR]

In flowering plants, pollination, pollen tube growth, and fertilization are regarded as the first hierarchical processes of producing offspring. However, their independent contributions to fruit set and development remain unclear. In this study, we examined the effect of three different types of pollen, intact pollen (IP), soft X-ray-treated pollen (XP) and dead pollen (DP), on pollen tube growth, fruit development and gene expression in "Micro-Tom" tomato. Normal germination and pollen tube growth were observed in flowers pollinated with IP; pollen tubes started to penetrate the ovary at 9 h after pollination, and full penetration was achieved after 24 h (IP24h), resulting in ~94% fruit set. At earlier time points (3 and 6 h after pollination; IP3h and IP6h, respectively), pollen tubes were still in the style, and no fruit set was observed. Flowers pollinated with XP followed by style removal after 24 h (XP24h) also demonstrated regular pollen tubes and produced parthenocarpic fruits with ~78% fruit set. As expected, DP could not germinate and failed to activate fruit formation. Histological analysis of the ovary at 2 days after anthesis (DAA) revealed that IP and XP comparably increased cell layers and cell size; however, mature fruits derived from XP were significantly smaller than those derived from IP. Furthermore, there was a high correlation between seed number and fruit size in fruit derived from IP, illustrating the crucial role of fertilization in the latter stages of fruit development. RNA-Seq analysis was carried out in ovaries derived from IP6h, IP24h, XP24h and DP24h in comparison with emasculated and unpollinated ovaries (E) at 2 DAA. The results revealed that 65 genes were differentially expressed (DE) in IP6h ovaries; these genes were closely associated with cell cycle dormancy release pathways. Conversely, 5062 and 4383 DE genes were obtained in IP24h and XP24h ovaries, respectively; top enriched terms were mostly associated with cell division and expansion in addition to the 'plant hormone signal transduction' pathway. These findings indicate that full penetration of pollen tubes can initiate fruit set and development independently of fertilization, most likely by activating the expression of genes regulating cell division and expansion. [ABSTRACT FROM AUTHOR]

Research into molecular mechanisms of self-incompatibility (SI) in plants can be observed in representatives of various families, including Solanaceae. Earlier studies of the mechanisms of S-RNase-based SI in petunia (Petunia hybrida E. Vilm.) demonstrate that programmed cell death (PCD) is an SI factor. These studies suggest that the phytohormon cytokinin (CK) is putative activator of caspase-like proteases (CLPs). In this work, data confirming this hypothesis were obtained in two model objects—petunia and tomato (six Solanaceae representatives). The exogenous zeatin treatment of tomato and petunia stigmas before a compatible pollination activates CLPs in the pollen tubes in vivo, as shown via the intravital imaging of CLP activities. CK at any concentration slows down the germination and growth of petunia and tomato male gametophytes both in vitro and in vivo; shifts the pH of the cytoplasm (PHc) to the acid region, thereby creating the optimal conditions for CLP to function and inhibiting the F-actin formation and/or destructing the cytoskeleton in pollen tubes to point foci during SI-induced PCD; and accumulates in style tissues during SI response. The activity of the ISOPENTENYLTRANSFERASE 5 (IPT5) gene at this moment exceeds its activity in a cross-compatible pollination, and the levels of expression of the CKX1 and CKX2 genes (CK OXIDASE/DEHYDROGENASE) are significantly lower in self-incompatible pollination. All this suggests that CK plays a decisive role in the mechanism underlying SI-induced PCD. [ABSTRACT FROM AUTHOR]

The purpose of this work was to study the effect of various components of the nutrient medium on the germination of pollen from a number of homostylous and heterostylous flax species. Four homostylous, including cultivated flax, and four heterostylous flax species were used as pollen sources. Artificial nutrient medium used for in vitro pollen germination included basic components - boric acid and calcium chloride in the concentrations of 200 mg/l, and tested ingredients - polyethylene glycol, sucrose, and tris (tris (hydroxymethyl) aminomethane). Pollen was germinated for 3-4 hours at the temperature of 25±1 °C in the dark. From 300 to 400 pollen grains in five replications were analyzed in each treatment. Pollen grains with not-burst (normal) and burst pollen tubes were recorded separately. Results. The media with PEG-2000 in concentrations of 20-30 % ensured germination of pollen from both homostylous and heterostylous Linum species up to 40-50 %. The addition of sucrose to a medium with PEG disadvantageously influenced pollen grain germination as well as the increase in the PEG molecular weight. Pollen germination on the medium containing PEG-20000 led to the increase in the number of pollen grains with burst tubes. Addition of tris did not influence pollen germination in L. usitatissimum or negatively affected ability of pollen grains to germinate in L. grandiflorum. The successful in vitro germination of pollen in species of the Linum genus allows to evaluate the quality of flax pollen quickly and efficiently and more successfully implement many genetic and breeding programs. This is especially important for such an important agricultural crop as cultivated flax for which artificial medium for pollen germination was not developed for now. [ABSTRACT FROM AUTHOR]

Pollen tube polar growth is a key physiological activity for angiosperms to complete double fertilization, which is highly dependent on the transport of polar substances mediated by secretory vesicles. The exocyst and Sec1/Munc18 (SM) proteins are involved in the regulation of the tethering and fusion of vesicles and plasma membranes, but the molecular mechanism by which they regulate pollen tube polar growth is still unclear. In this study, we found that loss of function of SEC1A, a member of the SM protein family in Arabidopsis thaliana, resulted in reducing pollen tube growth and a significant increase in pollen tube width. SEC1A was diffusely distributed in the pollen tube cytoplasm, and was more concentrated at the tip of the pollen tube. Through co‐immunoprecipitation‐mass spectrometry screening, protein interaction analysis and in vivo microscopy, we found that SEC1A interacted with the exocyst subunit SEC6, and they mutually affected the distribution and secretion rate at the tip of the pollen tube. Meanwhile, the functional loss of SEC1A and SEC6 significantly affected the distribution of the SNARE (soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor) complex member SYP125 at the tip of the pollen tube, and led to the disorder of pollen tube cell wall components. Genetic analysis revealed that the pollen tube‐related phenotype of the sec1a sec6 double mutant was significantly enhanced compared with their respective single mutants. Therefore, we speculated that SEC1A and SEC6 cooperatively regulate the fusion of secretory vesicles and plasma membranes in pollen tubes, thereby affecting the length and the width of pollen tubes. [ABSTRACT FROM AUTHOR]