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Formin is an important player in promoting apical actin polymerization in pollen tubes, but the mechanism regulating its activity remains unknown. We here identify REN1, a Rho GTPase-activating protein, as a negative regulator of formins in Arabidopsis pollen tubes. Specifically, we found that depletion of REN1 promotes apical actin polymerization and increases the amount of filamentous actin in pollen tubes. Interestingly, the effect of REN1 loss of function phenocopies the effect of formin gain of function, as it causes the formation of supernumerary membrane-derived actin bundles, which leads to tube swelling and membrane deformation. Importantly, inhibition of formins suppresses the phenotypic defects in ren1 mutant pollen tubes. We further demonstrate that REN1 physically interacts with the Arabidopsis formin protein AtFH5, predominantly with the C terminus, and inhibits the ability of AtFH5 to nucleate and assemble actin in vitro. Depletion of AtFH5 partially suppresses the phenotype in ren1 mutant pollen tubes, demonstrating that REN1 regulates apical actin polymerization at least partially through inhibiting AtFH5. We thus uncover a novel mechanism regulating formins and actin polymerization in plants. [Display omitted] • Depletion of REN1 promotes apical actin polymerization in pollen tubes • REN1 inhibits the ability of AtFH5 to nucleate actin assembly in vitro • Depletion of AtFH5 partially suppresses the phenotype in ren1 mutant pollen tubes • AtFH5 interacts with REN1 predominantly through its C terminus Xu et al. demonstrate that REN1, a Rho GTPase-activating protein, regulates apical actin polymerization through inhibiting Arabidopsis formin protein AtFH5 in pollen tubes. This study uncovers a novel mechanism regulating formins and actin polymerization in plants. [ABSTRACT FROM AUTHOR]

Background and Aims Pollination failure occurs from insufficient pollen quantity or quality. However, the relative contributions of pollen quantity vs. quality to overall pollen limitation, and how this is affected by the co-flowering context, remain unknown for most plant populations. Here, we studied patterns of pollen deposition and pollen tube formation across populations of four predominately outcrossing species in the genus Clarkia to evaluate how the richness of co-flowering congeners affects the contribution of pollen quantity and quality to pollen limitation. Methods We partition variation in pollen deposition and pollen tube production across individuals, populations and species to identify the main sources of variation in components of reproductive success. We further quantify the relative contribution of pollen quantity and quality limitation to the reproductive success of the four Clarkia species using piecewise regression analyses. Finally, we evaluate how variation in the number of co-flowering Clarkia species in the community affects the strength of pollen quality and quality limitation. Results Across all contexts, pollen deposition and the proportion of pollen tubes produced varied greatly among individuals, populations and species, and these were not always correlated. For instance, C. xantiana received the smallest pollen loads yet produced the highest proportion of pollen tubes, while C. speciosa exhibited the opposite pattern. Yet, co-flowering richness had variable effects on the strength of pollen quantity and quality limitation among populations. Specifically, breakpoint values, which are an indicator of overall pollen limitation, were two-fold higher in the four-species community compared with one- and two-species communities for two Clarkia species, suggesting that pollen limitation can increase with increasing richness of co-flowering congeners. Conclusions Our results reveal a complex interplay between the quantity and quality of pollen limitation and co-flowering context that may have different evolutionary outcomes across species and populations. [ABSTRACT FROM AUTHOR]

Summary: Style penetration by pollen tubes is essential for reproductive success, a process requiring canonical Rab5s in Arabidopsis. However, functional loss of Arabidopsis Vps9a, the gene encoding for guanine nucleotide exchange factor (GEF) of Rab5s, did not affect male transmission, implying the presence of a compensation program or redundancy.By combining genetic, cytological, and molecular approaches, we report that Arabidopsis Vps9b is a pollen‐preferential gene, redundantly mediating pollen tube penetration of style with Vps9a.Vps9b is functionally interchangeable with Vps9a, whose functional distinction results from distinct expression profiles.Functional loss of Vps9a and Vps9b results in the mis‐targeting of Rab5‐dependent tonoplast proteins, defective vacuolar biogenesis, disturbed distribution of post‐Golgi vesicles, increased cellular turgor, cytosolic acidification, and disrupted organization of actin microfilaments (MF) in pollen tubes, which collectively lead to the failure of pollen tubes to grow through style. [ABSTRACT FROM AUTHOR]

Both the pollen tube and hyphae of filamentous pathogens penetrate the outer layer of the host and then grow within host tissues. Early epidermal responses are decisive for the outcome of these two-cell interaction processes. We identified a single cell type, the papilla in the stigma of Arabidospis, as a tool to conduct a comprehensive comparative analysis on how an epidermal cell responds to the invasion of an unwanted pathogen or a welcome pollen tube. We showed that Phytophtora parasitica , a root oomycete, effectively breaches the stigmatic cell wall and develops as a biotroph within the papilla cytoplasm. These invasive features resemble the behaviour exhibited by the pathogen within its natural host cell, but diverge from the manner in which the pollen tube progresses, being engulfed within the papilla cell wall. Quantitative analysis revealed that both invaders trigger reorganization of the stigmatic endomembrane system and the actin cytoskeleton. While some remodelling processes are shared between the two interactions, others appear more specific towards the respective invader. These findings underscore the remarkable ability of an epidermal cell to differentiate between two types of invaders, thereby enabling it to trigger the most suitable response during the onset of invasion. [ABSTRACT FROM AUTHOR]

Unlike early land plants, flowering plants have evolved a pollen tube that transports a pair of non-motile sperm cells to the female gametophyte. This process, known as siphonogamy, was first observed in gymnosperms and later became prevalent in angiosperms. However, the precise molecular mechanisms underlying the male–female interactions remain enigmatic. From the landing of the pollen grain on the stigma to gamete fusion, the male part needs to pass various tests: how does the stigma distinguish between compatible and incompatible pollen? what mechanisms guide the pollen tube towards the ovule? what factors trigger pollen tube rupture? how is polyspermy prevented? and how does the sperm cell ultimately reach the egg? Successful male–female communication is essential for surmounting these challenges, with cysteine-rich peptides (CRPs) playing a pivotal role in this dialogue. In this review, we summarize the characteristics of four distinct classes of CRPs, systematically review recent progress in the role of CRPs in four crucial stages of pollination and fertilization, consider potential applications of this knowledge in crop breeding, and conclude by suggesting avenues for future research. [ABSTRACT FROM AUTHOR]

Heterostyly is a specialized reproductive system associated with self‐ and intramorph incompatibility. It reinforces the intermorph breeding system in heterostylous plant species. However, distyly has not been comprehensively studied in Nyctanthes arbor‐tristis. The present study demonstrates the floral traits, breeding system, fruit and seed set, and morph ratio in distylous N. arbor‐tristis. The length of style and filament showed reciprocal herkogamy between long style (L‐morph) and short style (S‐morph). Flower size, corolla tube size, stamen and anther length, ovary length, and pollen diameter were larger in S‐morph. However, the style was longer in L‐morph. Our results demonstrated strongly incompatible self‐ and intramorph pollination combinations. The intermorph pollination was compatible, showing normal pollen tube growth throughout the style and ovary. The embryo formation was also higher in intermorph interaction. Moreover, the fruit setting was higher when the intermorph plants grew in the vicinity of natural conditions. Collectively, all data indicate that N. arbor‐tristis is a distylous species with a self‐incompatible breeding system, resulting in less seed formation in nature. [ABSTRACT FROM AUTHOR]

ABSTRACT Actin depolymerizing factors (ADFs), like other actin‐binding proteins (ABPs), are modified by phosphorylation to regulate the dynamics of the actin filaments, thereby functioning in various processes throughout the plant lifecycle. In this study, we found that the Arabidopsis thaliana cytoplasmic kinase AGC1.7 interacts with ADF7 in vitro and in vivo. AGC1.7 phosphorylates ADF7 at its Ser‐6, Ser‐103 and Ser‐104 residues in vitro, while replacing these residues with alanine promotes ADF7‐mediated actin depolymerization in vitro. Expression of the phosphorylation‐mimetic mutant protein ADF7S6/103/104D driven by the pollen‐specific LAT52 promoter fully rescues the defects in germination rate, silique length and seeds per silique in both adf7‐2 and agc1.5 agc1.7 (agcdm) mutants. Our data establish a model whereby AGC1.7‐mediated ADF7 phosphorylation plays an important role in pollen germination and pollen tube growth. [ABSTRACT FROM AUTHOR]

Backcrossing between Sikitita and its male parent Arbequina, offers the possibility to check the suitability of different self-incompatibility models proposed for olive. To determine Sikitita's response to self- and cross-pollination treatments, including pollination with its father Arbequina, we compared the parameters following pollen–pistil interaction, the resulting initial and final fruit set, and the paternity of the seeds produced under different crosses. The results showed that Sikitita behaves as a self-incompatible cultivar due to the inhibition of pollen tube growth in the pistil of self-pollinated flowers. This incompatibility reaction led to a significant reduction of self-fertilization and fruit set. Seed paternity analyses confirmed the self-incompatibility response of Sikitita. A similar incompatibility response was observed in Sikitita flowers when hand-pollinated with pollen of Arbequina and Koroneiki. On the contrary, cross-pollination with Arbosana gave excellent results, with analyses showing that pollen of Arbosana is largely preferred by Sikitita to father its seeds more than the pollen of other cultivars presented in the orchard. The backcross failure of Sikitita with Arbequina pollen suggests that the self-incompatibility system in olives is not of the gametophytic type. In contrast, pollination tests fit features of previously reported sporophytic self-incompatibility systems. However, some amendments are proposed, among them the incompatibility groups for Sikitita and Koroneiki. [ABSTRACT FROM AUTHOR]

Elevated temperatures impair pollen performance and reproductive success, resulting in lower crop yields. The tomato (Solanum lycopersicum) anthocyanin reduced (are) mutant harbors a mutation in FLAVANONE 3-HYDROXYLASE (F3H), resulting in impaired flavonol antioxidant biosynthesis. The are mutant has reduced pollen performance and seed set relative to the VF36 parental line, phenotypes that are accentuated at elevated temperatures. Transformation of are with the wild-type F3H gene, or chemical complementation with flavonols, prevented temperature-dependent reactive oxygen species (ROS) accumulation in pollen and restored the reduced viability, germination, and tube elongation of are to VF36 levels. Overexpression of F3H in VF36 prevented temperature-driven ROS increases and impaired pollen performance, revealing that flavonol biosynthesis promotes thermotolerance. Although stigmas of are had reduced flavonol and elevated ROS levels, the growth of are pollen tubes was similarly impaired in both are and VF36 pistils. RNA-seq was performed at optimal and stress temperatures in are, VF36, and the F3H overexpression line at multiple timepoints across pollen tube elongation. The number of differentially expressed genes increased over time under elevated temperatures in all genotypes, with the greatest number in are. These findings suggest potential agricultural interventions to combat the negative effects of heat-induced ROS in pollen that lead to reproductive failure. Flavonol antioxidants reduce the negative impacts of high temperature on pollen performance by alleviating heat-induced reactive oxygen species levels and mediating transcriptome changes in pollen. [ABSTRACT FROM AUTHOR]

Self-incompatibility (SI) poses a significant reproductive barrier, severely impacting the yield, quality, and economic value of Camellia oleifera. In this study, methyl jasmonate (MeJA) was employed as an exogenous stimulus to alleviate SI in C. oleifera. The research findings revealed that an exogenous dose of 1000 μmol·L−1 MeJA enhanced the germination and tube growth of C. oleifera self-pollen and greatly improved ovule penetration (18.75%) and fertilization (15.81%), ultimately increasing fruit setting (18.67%). It was discovered by transcriptome analysis that the key genes (CAD, C4H) involved in the lignin production process exhibited elevated expression levels in self-pistils treated with MeJA. Further analysis showed that the lignin concentration in the MeJA-treated pistils was 31.70% higher compared with the control group. As verified by pollen germination assays in vitro, lignin in the appropriate concentration range could promote pollen tube growth. Gene expression network analysis indicated that transcription factor bHLH may be pivotal in regulating lignin biosynthesis in response to MeJA, which in turn affects pollen tubes. Further transient knockdown of bHLH (Co_33962) confirmed its important role in C. oleifera pollen tube growth. In summary, the application of MeJA resulted in the stimulation of self-pollen tube elongation and enhanced fruit setting in C. oleifera, which could be associated with the differential change in genes related to lignin synthesis and the increased lignin content. [ABSTRACT FROM AUTHOR]

Key message: Embryo abortion at the heart-shaped stage is the main reason for the failure of interspecific hybridization of hydrangea, and salicylic acid plays a key role during embryo abortion. Difficulties in obtaining seeds from interspecific hybridization between Hydrangea macrophylla and H. arborescens had severely restricted the process of breeding new hydrangea varieties. To clarify the cause of reproductive barriers, an interspecific hybridization was made between H. macrophylla 'Endless Summer' (female parent) and H. arborescens 'Annabelle' (male parent). The results showed that both parents’ floral organs developed normally, 'Annabelle' had high pollen viability (84.83% at 8 h after incubation), and the pollen tube could enter into the ovule of 'Endless Summer' at 72 h after pollination. Therefore, the pre-fertilization barrier was not the main reason for the failure of interspecific hybridization. However, observation of the embryo development by paraffin sections showed that the embryo was aborted at the heart-shaped stage. In addition, salicylic acid (SA) content was significantly higher (fourfold, P < 0.01) at 21 days after pollination (DAP) as compared to that of 17 DAP, which means SA may be closely correlated with embryo development. A total of 957 metabolites were detected, among which 78 were significantly different. During the embryo abortion, phenylpropanoids and polyketides were significantly down-regulated, while organic oxygen compounds were significantly up-regulated. Further analysis indicated that the metabolic pathway was enriched in the shikimic acid biosynthesis pathway, which suggests that more SA was synthesized. Taken together, it can be reasonably speculated that SA plays a key role leading to embryo abortion underlying the interspecific hybridization between Hydrangea macrophylla and H. arborescens. The result is helpful to direct the breeding of hydrangea through distant hybridization. [ABSTRACT FROM AUTHOR]

Summary: Reproduction in angiosperms relies on the precise growth of pollen tubes, facilitating the delivery of sperm cells to the ovule for double fertilization. LATERAL ORGAN BOUNDARIES DOMAIN10 (LBD10), a plant‐specific transcription factor, plays a pivotal role in Arabidopsis pollen development.Here, we uncovered LBD10's function in sustaining pollen tube growth and integrity.The lbd10 mutant exhibited elevated levels of reactive oxygen species (ROS) and hydrogen peroxide (H2O2) in both pollen grains and tubes, leading to compromised pollen tube growth. The inhibition of ROS synthesis and scavenging of excess ROS with an antioxidant treatment each alleviated these defects in lbd10. The lbd10 mutant displayed reduced flavonol accumulation in both pollen grains and tubes. All the altered phenotypes of lbd10 were complemented by expressing LBD10 under its native promoter. Exogenous application of flavonoids recused the defects in pollen tube growth and integrity in lbd10, along with reducing the excess levels of ROS and H2O2. LBD10 directly binds the promoters of key flavonol biosynthesis genes in chromatin and promotes reporter gene expression in Arabidopsis mesophyll protoplasts.Our findings indicate that LBD10 modulates ROS homeostasis by transcriptionally activating genes crucial for flavonol biosynthesis, thereby maintaining pollen tube growth and integrity. [ABSTRACT FROM AUTHOR]

• The pompom weed is likely pollinated by Apis mellifera and Hypolimnas misippus. • Gametophytic apomixis with autonomous endosperm development in triploids. • The co-occurrence of apomixis and polyploidy may be facilitating its invasiveness. • There is high pollen grain viability despite apomictic reproduction. • Low genetic diversity within and among the sampled triploid populations. Polyploidy and apomixis enable successful plant invasions into new regions. Campuloclinium macrocephalum (pompom weed) is a successful alien invasive species in South Africa that has been shown to comprise multiple ploidy levels. Despite the identification of triploid and tetraploid cytotypes in South African populations, the primary reproductive strategy was unknown. We investigated the reproductive strategy used by triploid populations of the pompom weed. We assessed whether insects contributed to pollen transfer using pollinator exclusion experiments, estimated genetic diversity, evaluated pollen tube growth in the styles, and estimated male fertility using pollen grain size and viability. We found evidence that strongly suggests that the pompom weed can reproduce via gametophytic apomixis with autonomous endosperm development. Pollinator exclusion experiments showed that while insects may positively influence reproductive success, the pompom weed can set lower quantities of seed in the absence of pollinators and that overall seed viability did not differ between open and excluded pollinator treatments. Further, while individuals are capable of uniparental reproduction, seed set appears to be independent of fertilization because we did not observe pollen tubes growing down the styles and/or reaching the ovules, despite high pollen viability. The sampled triploid populations also showed low genetic diversity, which may be a result low recombination frequencies. The low genetic variation should result in all of the sampled populations being equally susceptible to the biocontrol agents; however, this is not being observed in the sampled populations. Only one out of the four sampled populations showed a significant reduction in seed set and germination percentages due to the damage from Puccinia eupatorii (rust fungus) and Liothrips tractabilis (thrips). Nevertheless, the combination of an apomictic reproductive strategy and polyploidy is likely contributing to making pompom weed a formidable invader in South Africa. [ABSTRACT FROM AUTHOR]

Increasingly frequent heat waves threaten the reproduction of flowering plants; compromising the future persistence, adaptive capacity, and dispersal of wild plant populations, and also the yield of fruit-bearing crop plants. Heat damages the development of sensitive floral organs and gametes, which inhibits pollen germination, pollen tube growth, and fertilization. However, the role of heat has not been integrated into the framework of pollen quantity and quality limitation and how heat influences the success of cross and self-pollination. We exposed developing flowers to either controlled temperature (25 °C:20 °C) or extreme heat (35 °C:20 °C) over 72 h. We then hand-pollinated them with either crossed or self-derived pollen from the same temperature treatment to determine the direct and interactive effects of simulated heatwaves on pollen tube growth and resulting seed set. We also collected anthers from virgin flowers to measure heat impacts on pollen production. Under cooler control temperatures pollen tube survival of self-derived pollen was approximately 27% lower than that of crossed pollen. Pollen tube survival in heat-treated cross-pollinated and heat-treated self-pollinated flowers were 71% and 77% lower compared to flowers cross-pollinated at control temperatures. These differences in pollen tube survival rate between heat-treated cross-pollinated and heat-treated self-pollinated flowers were insignificant. Furthermore, extreme heat reduced seed set by 87%, regardless of pollen origin, and also reduced pollen production during flower development by approximately 20%. Our results suggest flowers that develop during heatwaves are likely to experience exacerbated pollen quantity and quality limitation driven by changes in pollen production and pollen vigour. Heatwave-induced pollen limitation will likely reduce crop yields in agricultural systems, and depress mating and reproduction in wild plant species, the latter of which may hinder the adaptive capacity of plants to a rapidly changing world. [ABSTRACT FROM AUTHOR]

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Main conclusion: Four cultivars of Paeonia lactiflora pollen have a different viability after cryopreservation, and that the difference of pollen viability is related to calcium ions and cell wall deposition. Cryopreservation is a vital technique for preserving germplasm resources, offering extensive application prospects. Understanding the factors influencing pollen viability after cryopreservation is crucial for the permanent preservation and exchange of pollen resources. This study investigated pollen from four Paeonia lactiflora cultivars with varying viability after cryopreservation, aiming to determine whether calcium ions (Ca2+) and cell wall deposition affect these viability changes. The results showed that Ca2+-ATPase activity and cytoplasmic Ca2+ of all four cultivars exhibited an increasing trend after cryopreservation; the calmodulin (CaM) content varied with cultivars. Correlation analysis showed that fresh pollen viability was significantly negatively correlated with cytoplasmic Ca2+ content and positively correlated with Ca2+-ATPase activity, while pollen viability after cryopreservation exhibited a significantly negative correlation with cytoplasmic Ca2+ content and a positive correlation with CaM content. The pollen cell wall of the cultivar ‘Zi Feng Chao Yang’ (ZFCY), which showed increased viability after cryopreservation, contained significantly higher levels of low-temperature tolerance-related phospholipids and proteins compared to other cultivars. Additionally, all cultivars maintained a clear Ca2+ gradient at the tips of pollen tubes after cryopreservation, without significant callose accumulation. These findings suggest that differences in Ca2+ signaling and cell wall components deposition influence changes in pollen viability after cryopreservation, and the Ca2+ gradient and callose at the tip of pollen tubes are not responsible for preventing pollen tube growth. [ABSTRACT FROM AUTHOR]

The complex processes of pollen germination and pollen tube elongation are important events in the sexual reproduction of flowering plants and are essential to the success of seed formation. However, unfavourable environmental and climatic conditions affect pollen performance negatively. Integrating the application of various biostimulants represents one of the most innovative and promising strategies to improve crop productivity. Biostimulants are known to sustainably improve pollen viability, germination and elongation. However, there is limited information on the effects of biostimulants on pollen grains. The present review investigates the potential of biostimulants (such as seaweed extracts, smoke–water, polyamines, amino acids, melatonin, carbohydrate-based biostimulants and inorganic biostimulants) as signaling molecules that can improve crucial stages of pollen growth in a range of crops, both in optimal and suboptimal environments. Furthermore, this review investigates the stages of pollen development, including uninucleate microspores and different stages of mature pollen, following various biostimulant treatment during the flowering period. Regulation of biostimulant-induced pollen germination and elongation has also been discussed along with recent developments regarding the mechanism of action of biostimulants in various growth aspects of pollen. This review not only summarizes what is known currently, but it also lays the groundwork for future investigations to clarify the complex physiological and molecular processes involved in pollen performance as well as the regulatory systems that are impacted by biostimulants. [ABSTRACT FROM AUTHOR]

Rhododendron resources are abundant in China, and hybridization breeding technology is the primary method for cultivating Rhododendron varieties. In order to optimize the utilization of wild Rhododendron resources for distant hybridization, this study took three horticultural varieties of Rhododendron subgenus Tsutsusi and the Rhododendron decorum Franch. of the subgenus Hymenanthes as the research objects, and the cross-compatibility between subgenera was analyzed from the aspects of pollen tube growth and ovary and seed development. At the same time, the statistics of ovary swelling rate and fruit bearing rate, numbers of capsule seeds, 1000 seed weight of hybrid seeds, germination rate, green seedling rate, and other indexes were analyzed to comprehensively evaluate hybrid fertility. The results showed that there was obvious pre-fertilization and post-fertilization barriers existing in the hybridization of Rhododendron × hybridum hort. and R. decorum. During the growth of pollen tubes, a large amount of callus appeared, which led to the entanglement, distortion, and abnormal development of the pollen tubes; only part of the pollen tubes entered into the ovary or ovule. The pre-fertilization barriers can be overcome by early pollination and delayed pollination. According to the observation of the ovary paraffin section, 45 d after pollination, the seed was shriveled and developed abnormally. The comprehensive evaluation of hybrid fertility showed that 'Sima' × R. decorum was fertile, 'Yin Taohong' × R. decorum was a weakly fertile, and 'Little Taohong' × R. decorum was sterile. This study provided a scientific basis for intergeneric hybridization breeding between the subgenus Tsutsusi and the subgenus Hymenanthes. [ABSTRACT FROM AUTHOR]

Flowering plants require normal pollen germination and growth to be fertilized, but studies on the mechanism regulating pollen tube growth in Fraxinus mandshurica are limited. Here, we used transcriptomic data to study the oxidative phosphorylation pathway during pollen tube growth in Fraxinus mandshurica. Our study identified 8,734 differentially expressed genes during the stages S1 to S3 of pollen tube growth. Significant enrichment of the oxidative phosphorylation pathway, amino acid synthesis, protein processing in the ER, carbon metabolism, pyruvate metabolism, citrate cycle (TCA cycle), and glycolysis/gluconeogenesis were examined using the Kyoto Encyclopedia of Genes and Genomes, and 58 genes linked to ROS synthesis and scavenging during the S1–S3 stages were identified. Also, H2DCFDA staining confirmed ROS formation in the pollen and the pollen tubes, and treatment with copper (II) chloride (CuCl2) and diphenyleneiodonium (DPI) was shown to reduce ROS in the pollen tube. Reduction in ROS content caused decreased pollen germination and pollen tube length. Furthermore, FmRbohH (respiratory burst oxidase homolog H) expression was detected in the pollen and pollen tube, and an antisense oligodeoxynucleotide assay demonstrated reduced ROS and pollen tube growth in Fraxinus mandshurica. This study shed more light on the RbohH gene functions during pollen tube growth. [ABSTRACT FROM AUTHOR]

Background: The dramatic temperature fluctuations spurred by global warming and the accompanying extreme weather events inhibit mango growth and threaten mango productivity. Particularly, mango flowering is highly sensitive to temperature changes. The mango fruit setting rate was significantly positively correlated with pollen activity, and pollen activity was regulated by different metabolites. Methods: In this study, the in vitro pollen of two mango varieties ('Renong No.1' and 'Jinhuang'), in which sensitivity to temperature differed significantly, were subjected to different temperature stresses (15 °C, 25 °C and 35 °C), and their metabolomics were analyzed. Results: The present results showed that 775 differential metabolites were screened by liquid chromatography–mass spectrometry and divided into 12 categories. The two varieties had significant differences in metabolite expression under different temperature stresses and the effect of low temperature on 'Renong No.1' mainly focused on amino acid metabolism, while the effect on 'Jinhuang' was mainly related to glycolysis. However, under the 35 °C temperature stress, 'Renong No.1' responded by redistributing riboflavin and betaine in vivo and the most obvious metabolic pathway of 'Jinhuang' enrichment was pyrimidine metabolism, which had undergone complex main body formation and extensive regulatory processes. The changes of metabolites of different varieties under low temperature and high temperature stress were different. Among them, flavonoids or flavonoid derivatives were included in class A (216 metabolites), C (163 metabolites) and D (233 metabolites) metabolites, indicating that flavonoid metabolites had an obvious regulatory effect on mango pollen metabolism under different temperature stress. Conclusions: The present results provide valuable information for reproductive biology studies and breeding in mango, in particular, the selection and breeding of the most suitable varieties for different production areas. [ABSTRACT FROM AUTHOR]

SUMMARY: Being a bona fide actin bundler, Arabidopsis villin5 (VLN5) plays a crucial role in regulating actin stability and organization within pollen tubes. Despite its significance, the precise mechanism through which VLN5 bundles actin filaments has remained elusive. Through meticulous deletion analysis, we have unveiled that the link between gelsolin repeat 6 (G6) and the headpiece domain (VHP), rather than VHP itself, is indispensable for VLN5‐mediated actin bundling. Further refinement of this region has pinpointed a critical sequence spanning from Val763 to Ser823, essential for VLN5's actin‐bundling activity. Notably, the absence of Val763‐Ser823 in VLN5 results in decreased filamentous decoration within pollen tubes and a diminished ability to rescue actin bundling defects in vln2vln5 mutant pollen tubes compared to intact VLN5. Moreover, our findings highlight that the Val763‐Ser823 sequence harbors a binding site for F‐actin, suggesting that this linker‐based F‐actin binding site, in conjunction with the F‐actin binding site localized in G1–G6, enables a single VLN5 to concurrently bind to two adjacent actin filaments. Therefore, our study unveils a novel mechanism by which VLN5 bundles actin filaments. [ABSTRACT FROM AUTHOR]

Protein S-acylation catalyzed by protein S-acyl transferases (PATs) is a reversible lipid modification regulating protein targeting, stability, and interaction profiles. PATs are encoded by large gene families in plants, and many proteins including receptor-like cytoplasmic kinases (RLCKs) and receptor-like kinases (RLKs) are subject to S-acylation. However, few PATs have been assigned substrates, and few S-acylated proteins have known upstream enzymes. We report that Arabidopsis (Arabidopsis thaliana) class A PATs redundantly mediate pollen tube guidance and participate in the S-acylation of POLLEN RECEPTOR KINASE1 (PRK1) and LOST IN POLLEN TUBE GUIDANCE1 (LIP1), a critical RLK or RLCK for pollen tube guidance, respectively. PAT1, PAT2, PAT3, PAT4, and PAT8, collectively named PENTAPAT for simplicity, are enriched in pollen and show similar subcellular distribution. Functional loss of PENTAPAT reduces seed set due to male gametophytic defects. Specifically, pentapat pollen tubes are compromised in directional growth. We determine that PRK1 and LIP1 interact with PENTAPAT, and their S-acylation is reduced in pentapat pollen. The plasma membrane (PM) association of LIP1 is reduced in pentapat pollen, whereas point mutations reducing PRK1 S-acylation affect its affinity with its interacting proteins. Our results suggest a key role of S-acylation in pollen tube guidance through modulating PM receptor complexes. Five Arabidopsis class A protein S-acyl transferases mediate pollen tube guidance by S-acylating two pollen receptors. [ABSTRACT FROM AUTHOR]

Previous studies on the mountain plant Ranunculus kuepferi concluded that apomictic self-compatible tetraploids have experienced a niche shift toward a colder climate during the Holocene, which suggests a fitness advantage over the sexual, self-sterile diploid parents under cold and stressful high-mountain conditions. However, there is still a lack of information on whether reproductive development would be advantageous for tetraploids. Here, we report on microsporogenesis, megagametogenesis, the dynamics of flower and seed development, and the consequences for reproductive success in a common garden experiment along a 1000 m climatic elevation gradient and in natural populations. Flower buds were initiated in the year preceding anthesis and passed winter in a pre-meiotic stage. Flower morphology differed in the known cytotype-specific way in that tetraploid flowers produced about twice as many carpels and fewer petals, stamens, and pollen grains than diploid flowers. Tetraploids developed precociously aposporous embryo sacs and showed a high rate of developmental disturbances. Sexual seed formation prevailed in diploids and pseudogamous apomixis in tetraploids. Along the elevation gradient, stigma pollen load, pollen performance, and seed output decreased. Combinations of reproductive traits, namely, bypass of meiosis irregularities and uniparental reproduction, might have promoted the vast expansion of apomictic R. kuepferi lines across the European Alps. [ABSTRACT FROM AUTHOR]

This document is an editorial for a special issue of the journal Biomolecules titled "Phytohormones 2022-2023." It emphasizes the significance of plant hormones in regulating plant growth and development, as well as their potential as biotechnological tools. The special issue covers various aspects of plant hormones, including their role in pollen germination, self-incompatibility, nitrogen use efficiency, stress resistance, and the accumulation of biomass and secondary metabolites. The articles in the special issue explore topics such as the presence of ABA in stigma exudates, programmed cell death as a self-incompatibility factor, the interaction between phytohormones and nutrients in woody plants, and the mechanisms underlying delayed recovery of gas exchange after drought relief. Other articles discuss the growth-inhibitory effect of increased planting density and its reduction by ABA-degrading bacteria, the capacity of methyl jasmonate to increase resistance to cadmium stress, the role of lipoxygenase3 in rice plants' defense response to blast disease, and the influence of CKs and ABA on the growth, development, and virulence of the pathogenic fungus Stagonospora nodorum. Additionally, the effects of auxin and jasmonic acid on the formation of adventitious roots and the production of secondary metabolites in Acmella radicans and Salvia bulleyana are explored. The article also discusses the use of auxin indole-3-acetic acid and cytok [Extracted from the article]

Zinc (Zn) is an essential element for plants. Numerous proteins in different cellular compartments require Zn for their structure and function. Zn can be toxic when it accumulates in high levels in the cytoplasm. Therefore, Zn homeostasis at tissue, cell, and organelle levels is vital for plant growth. A part of the metal tolerance protein (MTP) / Cation Diffusion Facilitator (CDF) transporters functions as Zn transporters, exporting Zn from the cytosol to various membrane compartments. In Arabidopsis thaliana, MTP1, MTP2, MTP3, MTP4, MTP5, and MTP12 are classified as Zn transporters (Zn-CDF). In this study, we systematically analyzed the localization of GFP-fused Zn-CDFs in the leaf epidermal cells of Nicotiana benthamiana. As previously reported, MTP1 and MTP3 were localized to tonoplast, MTP2 to endoplasmic reticulum, and MTP5 to Golgi. In addition, we identified the localization of MTP4 to trans-Golgi Network (TGN). Since MTP4 is specifically expressed in pollen, we analyzed the localization of MTP4-GFP in the Arabidopsis pollen tubes and confirmed that it is in the TGN. We also showed the Zn transport capability of MTP4 in yeast cells. We then analyzed the phenotype of an mtp4 T-DNA insertion mutant under both limited and excess Zn conditions. We found that their growth and fertility were not largely different from the wild-type. Our study has paved the way for investigating the possible roles of MTP4 in metallating proteins in the secretory pathway or in exporting excess Zn through exocytosis. In addition, our system of GFP-fused MTPs will help study the mechanisms for targeting transporters to specific membrane compartments. [ABSTRACT FROM AUTHOR]

Key message: Two pollen-preferential thaumatin-like proteins show both common and distinctive expression profiles. Precocious expression of one of them drastically disturbs timely deposition and dissolution of callose during microsporogenesis, leading to microspore death. Thaumatin-like proteins (TLPs), members of the pathogenesis-related protein family 5 (PR-5), are involved in plant defenses against biotic and abiotic stresses through antifungal activity and enhanced tolerance. Accordingly, studies on TLPs have focused on their responses to various pathogens and stresses and on engineering agronomically valuable crops that can be cultivated in suboptimal environments. On the other hand, the role of TLP members in plant development and their genetic regulation remains largely unexplored. Recently, we reported that the generative cell internalization after pollen mitosis I, an essential pollen patterning step for the nonmotile sperm cell delivery through a pollen tube, depends on STICKY GENERATIVE CELL which suppresses callose deposition in the nascent generative cell and interacts with a germline cell preferential GCTLP1 in Arabidopsis. Here, we additionally identified GCTLP2 which is similarly expressed in the germline cells. We generated various transgenic lines and examined their expressions and phenotypes to elucidate GCTLP functions during pollen development. Expression profiles suggest two GCTLP proteins may have common but also distinctive roles during pollen development. Importantly, ectopic expression analyses show that precocious expression of GCTLP2 severely disturbs the timely deposition and degradation of callose during microsporogenesis which is essential to produce viable microspores. Therefore, our study broadens the knowledge of TLP function and callose regulation for successful pollen development in Arabidopsis. [ABSTRACT FROM AUTHOR]

Key message: GPI anchor addition is important for JAGGER localization and in vivo function. Loss of correct GPI anchor addition in JAGGER, negatively affects its localization and function. In flowering plants, successful double fertilization requires the correct delivery of two sperm cells to the female gametophyte inside the ovule. The delivery of a single pair of sperm cells is achieved by the entrance of a single pollen tube into one female gametophyte. To prevent polyspermy, Arabidopsis ovules avoid the attraction of multiple pollen tubes to one ovule–polytubey block. In Arabidopsisjagger mutants, a significant number of ovules attract more than one pollen tube to an ovule due to an impairment in synergid degeneration. JAGGER encodes a putative arabinogalactan protein which is predicted to be anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Here, we show that JAGGER fused to citrine yellow fluorescent protein (JAGGER-cYFP) is functional and localizes mostly to the periphery of ovule integuments and transmitting tract cells. We further investigated the importance of GPI-anchor addition domains for JAGGER localization and function. Different JAGGER proteins with deletions in predicted ω-site regions and GPI attachment signal domain, expected to compromise the addition of the GPI anchor, led to disruption of JAGGER localization in the cell periphery. All JAGGER proteins with disrupted localization were also not able to rescue the polytubey phenotype, pointing to the importance of GPI-anchor addition to in vivo function of the JAGGER protein. [ABSTRACT FROM AUTHOR]

Key message: The VAMP721, VAMP722, SYP121, SYP122 and SNAP33 SNAREs are required in the Arabidopsis stigma for pollen hydration, further supporting a role for vesicle trafficking in the stigma's pollen responses. In the Brassicaceae, the process of accepting compatible pollen is a key step in successful reproduction and highly regulated following interactions between the pollen and the stigma. Central to this is the initiation of secretion in the stigma, which is proposed to provide resources to the pollen for hydration and germination and pollen tube growth. Previously, the eight exocyst subunit genes were shown to be required in the Arabidopsis stigma to support these pollen responses. One of the roles of the exocyst is to tether secretory vesicles at the plasma membrane for membrane fusion by the SNARE complex to enable vesicle cargo release. Here, we investigate the role of Arabidopsis SNARE genes in the stigma for pollen responses. Using a combination of different knockout and knockdown SNARE mutant lines, we show that VAMP721, VAMP722, SYP121, SYP122 and SNAP33 are involved in this process. Significant disruptions in pollen hydration were observed following pollination of wildtype pollen on the mutant SNARE stigmas. Overall, these results place the Arabidopsis SNARE complex as a contributor in the stigma for pollen responses and reaffirm the significance of secretion in the stigma to support the pollen–stigma interactions. [ABSTRACT FROM AUTHOR]

Pollen grains are central to sexual plant reproduction and their viability and longevity/storage are critical for plant physiology, ecology, plant breeding, and many plant product industries. Our goal is to present progress in assessing pollen viability/longevity along with recent advances in our understanding of the intrinsic and environmental factors that determine pollen performance: the capacity of the pollen grain to be stored, germinate, produce a pollen tube, and fertilize the ovule. We review current methods to measure pollen viability, with an eye toward advancing basic research and biotechnological applications. Importantly, we review recent advances in our understanding of how basic aspects of pollen/stigma development, pollen molecular composition, and intra- and intercellular signaling systems interact with the environment to determine pollen performance. Our goal is to point to key questions for future research, especially given that climate change will directly impact pollen viability/longevity. We find that the viability and longevity of pollen are highly sensitive to environmental conditions that affect complex interactions between maternal and paternal tissues and internal pollen physiological events. As pollen viability and longevity are critical factors for food security and adaptation to climate change, we highlight the need to develop further basic research for better understanding the complex molecular mechanisms that modulate pollen viability and applied research on developing new methods to maintain or improve pollen viability and longevity. [ABSTRACT FROM AUTHOR]

Prezygotic isolation is important for successful fertilization in rice, significantly affecting yield. This study focused on F 5:6 generation plants derived from inter-subspecific crosses (Nipponbare × KDML105) with low (LS) and high seed-setting rates (HS), in which normal pollen fertility was observed. However, LS plants showed a reduced number of pollen grains adhering to the stigma and fewer pollen tubes reaching the ovules at 4‒5 h post-pollination, compared with HS plants. Bulked segregant RNA-Seq analysis of pollinated pistils from the HS and LS groups revealed 249 and 473 differentially expressed genes (DEGs), respectively. Kyoto Encyclopedia of Genes and Genomes analysis of the HS and LS- specific DEGs indicated enrichment in metabolic pathways, pentose and glucuronate interconversions, and flavonoid biosynthesis. Several of these DEGs exhibited co-expression with pollen development genes and formed extensive clusters of co-expression networks. Compared with LS pistils, enzyme genes controlling pectin degradation, such as OsPME35 and OsPLL9 , showed similar expression patterns, with higher levels in HS pistils pre-pollination. Os02g0467600 , similar to cinnamate 4-hydroxylase gene (CYP73), involved in flavonoid biosynthesis, displayed higher expression in HS pistils post-pollination. Our findings suggest that OsPME35 , OsPLL9 , and Os02g0467600 contribute to prezygotic isolation by potentially modifying the stigma cell wall (OsPME35 and OsPLL9) and controlling later processes such as pollen-stigma adhesion (Os02g0467600) genes. Furthermore, several DEGs specific to HS and LS were co-localized with QTLs and functional genes associated with spikelet fertility. These findings provide valuable insights for further research on rice spikelet fertility, ultimately contributing to the development of high-yielding rice varieties. [ABSTRACT FROM AUTHOR]

Supra-optimal temperature adversely affects plant growth and development. Among different growth phases, the reproductive stage of plants is highly sensitive to high temperature stress. The heat tolerance of plants is reliant on a complex interplay of various factors, including the activation of heat stress responses, downstream signaling pathways, and biosynthesis of plant hormones. This intricate network of mechanisms results in the production of several secondary metabolites, which play a crucial role in enhancing the plant's ability to cope with high temperature stress. Many of these secondary metabolites are crucial for the reproductive performance of plants, as they are involved in the attraction of pollinators, pollen germination, pollen tube growth, pollen viability, pollen fertility, and fertilization. Reproductive tissues of plants are highly vulnerable to heat stress, but increased biosynthesis of secondary metabolites (flavonoids, polyamines, glutathione, etc.) induces reproductive thermotolerance through reactive oxygen species homeostasis, protecting the cellular membranes, improving pollen fertility, reducing anther abortion, regulating cellular signaling and activation of genes/transcription factors associated with metabolic pathway network. The identification and engineering of some genes controlling the secondary metabolic pathways network have proved effective in inducing terminal heat stress tolerance in transgenic plants. This review highlights the effect of heat stress on plant secondary metabolism and the role of different plant secondary metabolites in contributing to heat stress tolerance in reproductive tissues. The engineering of genes involved in plant secondary metabolic pathway and their effect on heat stress tolerance has also been discussed. [ABSTRACT FROM AUTHOR]

Glycoside hydrolases (GHs), enzymes that break down glycosidic bonds in carbohydrates and between carbohydrates and non-carbohydrates, are prevalent in plants, animals, microorganisms, and other organisms. The tomato is a significant crop that contains the GH17 gene family. However, its role in tomatoes has yet to be fully investigated. In this study, we identified 43 GH17 genes from the tomato genome, distributed unevenly across 12 chromosomes. We further analyzed their gene structure, phylogenetic relationships, promoter elements, and expression patterns. The promoter element analysis indicated their potential roles in response to biotic and abiotic stresses as well as phytohormone effects on growth and development. The expression studies across different tomato tissues revealed that 10 genes were specifically expressed in floral organs, with SlA6 prominently expressed early during bud formation. By using CRISPR/Cas9 gene-editing technology, SlA6 knockout plants were generated. Phenotypic characterization showed that pollen viability, pollen tube germination, fruit weight, and seed number were significantly reduced in the Sla6 mutant, but the soluble solids content (TSS) was significantly higher in the Sla6 mutant, suggesting that SlA6 affects pollen development and fruit quality. [ABSTRACT FROM AUTHOR]

Key message: The Arabidopsis KASH protein SINE3 is involved in male and female gametophyte development, likely affecting the first post-meiotic mitosis in both cases, and is required for full seed set. Linker of nucleoskeleton and cytoskeleton (LINC) complexes are protein complexes spanning the inner and outer membranes of the nuclear envelope (NE) and are key players in nuclear movement and positioning. Through their roles in nuclear movement and cytoskeletal reorganization, plant LINC complexes affect processes as diverse as pollen tube rupture and stomatal development and function. KASH proteins are the outer nuclear membrane component of the LINC complex, with conserved C-termini but divergent N-terminal cytoplasmic domains. Of the known Arabidopsis KASH proteins, SUN-INTERACTING NUCLEAR ENVELOPE PROTEIN 3 (SINE3) has not been functionally characterized. Here, we show that SINE3 is expressed at all stages of male and female gametophyte development. It is located at the NE in male and female gametophytes. Loss of SINE3 results in a female-derived seed set defect, with sine3 mutant ovules arresting at stage FG1. Pollen viability is also significantly reduced, with microspores arresting prior to pollen mitosis I. In addition, sine3 mutants have a minor male meiosis defect, with some tetrads containing more than four spores. Together, these results demonstrate that the KASH protein SINE3 plays a crucial role in male and female gametophyte development, likely affecting the first post-meiotic nuclear division in both cases. [ABSTRACT FROM AUTHOR]

Key message: ARID-HMG DNA binding protein, AtHMGB15, regulates pollen development and pollen germination in Arabidopsis. Previous studies have shown that ARID-HMG DNA binding protein, AtHMGB15 regulate pollen development and pollen germination in Arabidopsis. Here, we performed transcriptome and cytological studies to understand the role of AtHMGB15 in regulating pollen wall morphology and the pollen tube germination rate. Our result showed abnormal vacuolization in the tapetal cells during anther maturation and prolonged PCD in AtHMGB15 loss-of-function mutant. The tapetum has the ability to perform both secretory and biosynthetic activities critical for pollen maturation and pollen viability. Interestingly, expression of PCD executer genes CEP1, MC9 and RNS3 were significant down-regulation of in athmgb15-4. The growth of pollen tubes is regulated by the actin cytoskeleton dynamics. To address the defect in pollen tube growth of athmgb15, we monitored the actin network in growing pollen tubes of wildtype and athmgb15-4 using Rhodamine-phalloidin fluorescence. Our results indicate a highly fragmented actin distribution in athmgb15-4 pollen tubes with a lesser number of long actin fibers and significantly low f-actin concentration at the apex. q-RTPCR further indicates significant downy-regulation of actin regulatory proteins VLN2 and PRF4. Collectively, our results suggest that AtHMGB15 being a nuclear architectural protein orchestrates high-order chromatin organization to promote the transcription of genes responsible for pollen development and pollen germination. [ABSTRACT FROM AUTHOR]

Key message: Total PME activity in reproductive tissues was related to haplotypes at maize cross incompatibility loci, suggesting that these loci function by controlling PME activity. In maize, the pollination outcome depends on the haplotypes of the interacting male gametophyte (germinated pollen) and female sporophyte (silk) at several cross-incompatibility loci. Functional alleles (-S haplotypes) of the cross-incompatibility loci Ga1 and Ga2, both encode two pectin methylesterases (PMEs), one that is expressed in silk and the other in pollen. We examined total PME activity in reproductive tissues containing functional and null haplotypes at the Ga1 or Ga2 loci. In pollinated silks, there was a correlation between total PME activity and the -S haplotype pollen in both Ga1 and Ga2 systems. We did not detect a significant relationship between PME activity and pollination outcome of either system. We re-examined previously reported active site amino acid substitutions in PMEs encoded by cross incompatibility loci. We observed that different active site substitutions are present in the pollen and silk PMEs of cross incompatibility loci and these differences are conserved across Ga1, Ga2 and Tcb-1. This work establishes a relationship between total PME activity and the haplotypes of the Ga1 locus in pollinated silks. [ABSTRACT FROM AUTHOR]

Cryopreservation of pollen stands out as a significant strategy in conserving species diversity, serving as a tool for genetic enhancement. This study investigated crucial factors for pollen cryopreservation, revealing unprecedented results linked to dehydration conditions and periods, encompassing peculiarities of the evaluated species. Data on the cryopreservation of wild passion fruit species are presented, outlining an innovative and efficient protocol to influence future research on pollen cryopreservation, reinforcing genetic improvement, and establishing a cryobank for pollen from wild species of the genus. Pollen grains from eight Passiflora species were evaluated, subjected to dehydration in a desiccator with silica gel and an oven at 35 °C at intervals of 0, 20, 40, or 60 min. The dehydrated material was subsequently cryopreserved for 24 h, followed by in vitro germination tests and measurements of pollen tube length to determine the optimal dehydration time for each species. Furthermore, controlled pollination was performed using pollen cryopreserved for 24 h. This pollen exhibited germination rates ranging from 71% (P. edmundoi) to 28% (P. misera), while fresh pollen showed germination rates from 89% (P. edmundoi) to 37% (P. organensis). Field tests involving controlled pollination using cryopreserved pollen resulted in fruiting rates from 50% (P. edmundoi) to 40% (P. gibertii), producing seeds with germination percentages ranging from 67 to 99%. These in vitro and in vivo results highlight the effectiveness of cryopreservation as a technique to conserve sets of haploid genes within the cryogenic bank. [ABSTRACT FROM AUTHOR]

This article explores the molecular mechanisms underlying pollen tube growth in pear trees. The researchers focused on the role of flavonoids, a group of plant secondary metabolites, in regulating pollen development and growth. They identified a specific gene, PbrF3H, that is involved in flavonoid biosynthesis and found that it is regulated by a transcription factor called PbrMYB186. Knockdown experiments showed that both PbrF3H and PbrMYB186 are essential for flavonoid production and pollen tube growth. The study provides insights into the regulatory network of flavonoids in pear pollen tubes. [Extracted from the article]

Gymnosperms originated in the Middle Devonian and have experienced a long evolutionary history with pulses of speciation and extinction, which resulted in the four morphologically distinct extant groups, i.e., cycads, Ginkgo, conifers and gnetophytes. For over a century, the systematic relationships within the extant gymnosperms have been debated because different authors emphasized different characters. Recent phylogenomic studies of gymnosperms have given a consistent topology, which aligns well with extant gymnosperms classified into three classes, five subclasses, eight orders, and 13 families. Here, we review the historical opinions of systematics of gymnosperms with special reference to several problematic taxa and reconsider the evolution of some key morphological characters previously emphasized by taxonomists within a phylogenomic context. We conclude that (1) cycads contain two families, i.e., the Cycadaceae and the Zamiaceae; (2) Ginkgo is sister to cycads but not to conifers, with the similarities between Ginkgo and conifers being the result of parallel evolution including a monopodial growth pattern, pycnoxylic wood in long shoots, and the compound female cones, and the reproductive similarities between Ginkgo and cycads are either synapomorphic or plesiomorphic, e.g., the boat-shaped pollen, the branched pollen tube, and the flagellate sperms; (3) conifers are paraphyletic with gnetophytes nested within them, thus gnetophytes are derived conifers, and our newly delimited coniferophytes are equivalent to the Pinopsida and include three subclasses, i.e., Pinidae, Gnetidae, and Cupressidae; (4) fleshy cones of conifers originated multiple times, the Podocarpaceae are sister to the Araucariaceae, the Cephalotaxaceae and the Taxaceae comprise a small clade, which is sister to the Cupressaceae; (5) the Cephalotaxaceae are distinct from the Taxaceae, because the former family possesses typical female cones and the fleshy part of the seed is derived from the fleshiness of integument, while the latter family has reduced female cones and preserves no traces of the seed scale complexes. [ABSTRACT FROM AUTHOR]

• Specialised plant pollination systems can suffer reduced fertility if they receive insufficient pollinator service. • The Cape endemic Cyanella alba possess specialised floral adaptations – mirror-image flowers and dimorphic anthers – usually associated with buzz pollination by bees. • Experimental pollinations demonstrated that plants possess ovarian self-incompatibility and depend on pollinators for cross-pollination. • Chronic pollen limitation of seed set owing to the near absence of visitation by buzz pollinating bees was evident in several natural populations. • Land use and climate change may explain the paucity of buzz pollination with implications for ecology and evolution of plant populations. Plants with specialised pollination systems may be susceptible to reduced fertility if they are incapable of autonomous self-pollination and are visited infrequently by pollinators capable of promoting effective cross-pollination. Here, we investigate this question by examining the reproductive ecology of the Cape endemic Cyanella alba subsp. flavescens (Tecophilaeaceae), which has mirror-image flowers (enantiostyly) and dimorphic anthers (heteranthery). These traits are usually associated with buzz pollination mediated by bees that extract pollen from the poricidal anthers of flowers through vibration. We quantified insect visitation to flowers in three populations over two years in the vicinity of the Biedouw Valley, Western Cape, South Africa to determine whether plants were visited by buzz-pollinating bees. We also recorded visits by pollinators to other co-flowering species at each site and constructed a network on insect visitation. We conducted controlled hand pollinations and bagging experiments to determine whether plants were self-compatible and capable of autonomous self-pollination, and compared natural levels of fruit and seed set with those obtained from hand cross-pollination to evaluate evidence for pollen limitation of seed fertility. Despite 74 h of field observations, only two actively buzz-pollinating bees were recorded visiting flowers, although they were observed visiting other species in the community. The most frequent visitors to C. alba were opportunistic pollen-consuming beetles although we obtained no direct evidence that they were effective in promoting cross-pollination. Plants in each population exhibited chronic pollen limitation of seed set. Controlled pollinations demonstrated that plants were moderately self-sterile and incapable of autonomous self-pollination. Although fruit set was high following self-pollination, few seeds were evident within developing fruit. Observations of pollen-tube growth following controlled hand self- and cross-pollination revealed abundant pollen tubes at the base of styles in both treatments. Thus, self-sterility in C. alba probably results from late-acting ovarian self-incompatibility, and possibly also the abortion of developing seed due to inbreeding depression. We discuss the possible causes of scarce buzz pollination in C. alba and consider its ecological and evolutionary consequences. [ABSTRACT FROM AUTHOR]

SUMMARY: The successful interaction between pollen and stigma is a critical process for plant sexual reproduction, involving a series of intricate molecular and physiological events. After self‐compatible pollination, a significant reduction in reactive oxygen species (ROS) production has been observed in stigmas, which is essential for pollen grain rehydration and subsequent pollen tube growth. Several scavenging enzymes tightly regulate ROS homeostasis. However, the potential role of these ROS‐scavenging enzymes in the pollen‐stigma interaction in Brassica napus remains unclear. Here, we showed that the activity of ascorbate peroxidase (APX), an enzyme that plays a crucial role in the detoxification of hydrogen peroxide (H2O2), was modulated depending on the compatibility of pollination in B. napus. We then identified stigma‐expressed APX1s and generated pentuple mutants of APX1s using CRISPR/Cas9 technology. After compatible pollination, the BnaAPX1 pentuple mutants accumulated higher levels of H2O2 in the stigma, while the overexpression of BnaA09.APX1 resulted in lower levels of H2O2. Furthermore, the knockout of BnaAPX1 delayed the compatible response‐mediated pollen rehydration and germination, which was consistent with the effects of a specific APX inhibitor, ρ‐Aminophenol, on compatible pollination. In contrast, the overexpression of BnaA09.APX1 accelerated pollen rehydration and germination after both compatible and incompatible pollinations. However, delaying and promoting pollen rehydration and germination did not affect the seed set after compatible and incompatible pollination in APX1 pentuple mutants and overexpression lines, respectively. Our results demonstrate the fundamental role of BnaAPX1 in pollen rehydration and germination by regulating ROS homeostasis during the pollen‐stigma interaction in B. napus. Significance Statement: Our research found that self‐incompatibility in Brassica napus requires the accumulation of reactive oxygen species (ROS) levels in the stigma. We identified the importance of BnaAPX1 in regulating ROS homeostasis, which is critical for pollen rehydration and germination in B. napus. [ABSTRACT FROM AUTHOR]

SUMMARY: Redox changes of pyridine nucleotides in cellular compartments are highly dynamic and their equilibria are under the influence of various reducing and oxidizing reactions. To obtain spatiotemporal data on pyridine nucleotides in living plant cells, typical biochemical approaches require cell destruction. To date, genetically encoded fluorescent biosensors are considered to be the best option to bridge the existing technology gap, as they provide a fast, accurate, and real‐time readout. However, the existing pyridine nucleotides genetically encoded fluorescent biosensors are either sensitive to pH change or slow in dissociation rate. Herein, we employed the biosensors which generate readouts that are pH stable for in planta measurement of NADH/NAD+ ratio and NADPH level. We generated transgenic Arabidopsis lines that express these biosensors in plastid stroma and cytosol of whole plants and pollen tubes under the control of CaMV 35S and LAT52 promoters, respectively. These transgenic biosensor lines allow us to monitor real‐time dynamic changes in NADH/NAD+ ratio and NADPH level in the plastids and cytosol of various plant tissues, including pollen tubes, root hairs, and mesophyll cells, using a variety of fluorescent instruments. We anticipate that these valuable transgenic lines may allow improvements in plant redox biology studies. Significant Statement: Monitoring dynamic changes in pyridine nucleotides (e.g., NADPH and NADH/NAD+ ratio) in plants at the subcellular level is a major obstacle in plant bioenergetics research. Genetically encoded fluorescent biosensors are considered to be the best option so far because they provide fast, accurate, and real‐time readouts. Here, we present a detailed study on the application of pyridine nucleotide biosensors, and we anticipate that these transgenic lines will be useful in plant redox biology studies. [ABSTRACT FROM AUTHOR]

Fertilization barriers often hinder the introgression of desired traits of interest in interspecific hybridization. In present study, the fertilization barriers in interspecific crosses of Cucurbita moschata (Punjab Nawab and Punjab Sardar) and Cucurbita pepo (PMK-1) were investigated. The results revealed that pollen grains were viable and stigmas were receptive at the time of pollination in all the genotypes. The maximum pollen germination (%) was observed in Punjab Nawab, while maximum pollen size, pollen tube length and style length were recorded in Punjab Sardar. Additionally, fluorescence microscopic images showed that the shorter pollen tubes of PMK-1 did not grow enough in the longer style of Punjab Sardar, hence the interspecific cross of Punjab Sardar × PMK-1 was incompatible. While in the reciprocal cross (PMK-1 × Punjab Sardar), formation of callose plugs in the pollen tubes of Punjab Sardar and its slow growth might had contributed to the failure of fertilization. However, in the interspecific cross of Punjab Nawab × PMK-1 and vice-versa, the pollen grains successfully germinated on stigma and pollen tubes reached the style but embryo was not able to attain a particular shape, therefore, the cross was partially incompatible. The fruit setting (%), fruit diameter, fruit weight, seed number, seed weight and seed germination (%) was lesser in interspecific crosses as compared to self-pollinated genotypes. Furthermore, cluster analysis revealed that partially compatible and incompatible crosses are present in different clusters and partially compatible cross showed strong positive correlation with fruit characters. Collectively, pre-and post-fertilization barriers hampered the interspecific hybridization between C. pepo and C. moschata genotypes. These findings would help the breeders to overcome such barriers and introgress the hull-less seed trait of C. pepo and disease resistant trait of C. moschata. [ABSTRACT FROM AUTHOR]

Camellia drupifera is an important woody oil plant in South China, renowned for its seed oil that is rich in unsaturated fatty acids and possesses significant antioxidant, anti-cancer, and immune-enhancing properties. The low fruit-setting rate of C. drupifera is influenced by multiple factors, including flowering stage climate, flowering habits, pollination biology, soil conditions, and self-incompatibility. Among these, large-scale pure forest plantations are the primary cause of the low fruit-setting rate. Although previous studies have explored the impact of self-incompatibility on fruit-setting in C. drupifera, research on the dynamic changes of endogenous substances during the flowering stage in pure forest environments remains limited. Research findings indicate that tannase activity is relatively high in the pistils of C. drupifera, creating a favorable environment for pollen tube growth. Plant hormones such as indole-3-acetic acid (IAA), cytokinin (CTK), gibberellin (GA), and ethylene (ETH) regulate the development and aging of floral organs through complex interactions. Specifically, high levels of IAA in the pistil promote pollen tube growth, while changes in ETH and ABA are closely related to the aging of floral organs. Under oxidative stress conditions, high levels of H2O2 in the pistil may contribute to self-incompatibility. The activity of superoxide dismutase (SOD) in the floral organs during the flowering stage is significantly higher compared to peroxidase (POD) and catalase (CAT), highlighting the critical role of SOD in regulating oxidative stress during this stage. This study provides new insights into the changes in endogenous substances in the floral organs of C. drupifera during the flowering stage. It offers theoretical references for understanding its sexual reproduction process and for the application of plant growth regulators to improve fruit setting. [ABSTRACT FROM AUTHOR]

Background: The increasing ambient temperature significantly impacts plant growth, development, and reproduction. Uncovering the temperature-regulating mechanisms in plants is of high importance, for increasing our fundamental understanding of plant thermomorphogenesis, for its potential in applied science, and for aiding plant breeders in improving plant thermoresilience. Thermomorphogenesis, the developmental response to warm temperatures, has been primarily studied in seedlings and in the regulation of flowering time. PHYTOCHROME B and PHYTOCHROME-INTERACTING FACTORs (PIFs), particularly PIF4, are key components of this response. However, the thermoresponse of other adult vegetative tissues and reproductive structures has not been systematically evaluated, especially concerning the involvement of phyB and PIFs. Results: We screened the temperature responses of the wild type and several phyB-PIF4 pathway Arabidopsis mutant lines in combined and integrative phenotyping platforms for root growth in soil, shoot, inflorescence, and seed. Our findings demonstrate that phyB-PIF4 is generally involved in the relay of temperature signals throughout plant development, including the reproductive stage. Furthermore, we identified correlative responses to high ambient temperature between shoot and root tissues. This integrative and automated phenotyping was complemented by monitoring the changes in transcript levels in reproductive organs. Transcriptomic profiling of the pistils from plants grown under high ambient temperature identified key elements that may provide insight into the molecular mechanisms behind temperature-induced reduced fertilization rate. These include a downregulation of auxin metabolism, upregulation of genes involved auxin signalling, miRNA156 and miRNA160 pathways, and pollen tube attractants. Conclusions: Our findings demonstrate that phyB-PIF4 involvement in the interpretation of temperature signals is pervasive throughout plant development, including processes directly linked to reproduction. [ABSTRACT FROM AUTHOR]

Major constituents of the plant cell walls are structural proteins that belong to the hydroxyproline-rich glycoprotein (HRGP) family. Leucine-rich repeat extensin (LRX) proteins contain a leucine-rich domain and a C-terminal domain with repetitive Ser–Pro3–5 motifs that are potentially to be O -glycosylated. It has been demonstrated that pollen-specific LRX8–LRX11 from Arabidopsis thaliana are necessary to maintain the integrity of the pollen tube cell wall during polarized growth. In HRGPs, including classical extensins (EXTs), and probably in LRXs, proline residues are converted to hydroxyproline by prolyl-4-hydroxylases (P4Hs), thus defining novel O -glycosylation sites. In this context, we aimed to determine whether hydroxylation and subsequent O -glycosylation of Arabidopsis pollen LRXs are necessary for their proper function and cell wall localization in pollen tubes. We hypothesized that pollen-expressed P4H4 and P4H6 catalyze the hydroxylation of the proline units present in Ser–Pro3–5 motifs of LRX8–LRX11. Here, we show that the p4h4-1 p4h6-1 double mutant exhibits a reduction in pollen germination rates and a slight reduction in pollen tube length. Pollen germination is also inhibited by P4H inhibitors, suggesting that prolyl hydroxylation is required for pollen tube development. Plants expressing pLRX11::LRX11-GFP in the p4h4-1 p4h6-1 background show partial re-localization of LRX11–green fluorescent protein (GFP) from the pollen tube tip apoplast to the cytoplasm. Finally, immunoprecipitation-tandem mass spectrometry analysis revealed a decrease in oxidized prolines (hydroxyprolines) in LRX11–GFP in the p4h4-1 p4h6-1 background compared with lrx11 plants expressing pLRX11::LRX11-GFP. Taken together, these results suggest that P4H4 and P4H6 are required for pollen germination and for proper hydroxylation of LRX11 necessary for its localization in the cell wall of pollen tubes. [ABSTRACT FROM AUTHOR]

The escalation in the intensity, frequency, and duration of high-temperature (HT) stress is currently unparalleled, which aggravates the challenges for crop production. Yet, the stage-dependent responses of reproductive organs to HT stress at the morphological, physiological, and molecular levels remain inadequately explored in pivotal staple crops. This review synthesized current knowledge regarding the mechanisms by which HT stress induces abnormalities and aberrations in reproductive growth and development, as well as by which it alters the morphology and function of florets, flowering patterns, and the processes of pollination and fertilization in maize (Zea mays L.). We identified the stage-specific sensitivities to HT stress and accurately defined the sensitive period from a time scale of days to hours. The microspore tetrad phase of pollen development and anthesis (especially shortly after pollination) are most sensitive to HT stress, and even brief temperature spikes during these stages can lead to significant kernel loss. The impetuses behind the heat-induced impairments in seed set are closely related to carbon, reactive oxygen species, phytohormone signals, ion (e.g. Ca2+) homeostasis, plasma membrane structure and function, and others. Recent advances in understanding the genetic mechanisms underlying HT stress responses during maize sexual reproduction have been systematically summarized. [ABSTRACT FROM AUTHOR]

SUMMARY: Low phytic acid (lpa) crop is considered as an effective strategy to improve crop nutritional quality, but a substantial decrease in phytic acid (PA) usually has negative effect on agronomic performance and its response to environment adversities. Myo‐inositol‐3‐phosphate synthase (MIPS) is the rate‐limiting enzyme in PA biosynthesis pathway, and regarded as the prime target for engineering lpa crop. In this paper, the rice MIPS gene (RINO2) knockout mutants and its wild type were performed to investigate the genotype‐dependent alteration in the heat injury‐induced spikelet fertility and its underlying mechanism for rice plants being imposed to heat stress at anthesis. Results indicated that RINO2 knockout significantly enhanced the susceptibility of rice spikelet fertility to heat injury, due to the severely exacerbated obstacles in pollen germination and pollen tube growth in pistil for RINO2 knockout under high temperature (HT) at anthesis. The loss of RINO2 function caused a marked reduction in inositol and phosphatidylinositol derivative concentrations in the HT‐stressed pollen grains, which resulted in the strikingly lower content of phosphatidylinositol 4,5‐diphosphate (PI (4,5) P2) in germinating pollen grain and pollen tube. The insufficient supply of PI (4,5) P2 in the HT‐stressed pollen grains disrupted normal Ca2+ gradient in the apical region of pollen tubes and actin filament cytoskeleton in growing pollen tubes. The severely repressed biosynthesis of PI (4,5) P2 was among the regulatory switch steps leading to the impaired pollen germination and deformed pollen tube growth for the HT‐stressed pollens of RINO2 knockout mutants. Significance Statement: For engineering lpa rice, the knockout of RINO2 significantly enhanced susceptibility of spikelet fertility to heat injury at anthesis, which was mostly ascribed to the heat injury‐induced obstacle in pollen germination and pollen tube growth. High temperature significantly reduced the concentrations of inositol and PI (4,5) P2 concentration in pollen grains, this occurrence was strongly responsible for the disorder Ca2+ gradient and actin filament cytoskeleton in growing pollen tubes. These findings provide new understanding for the regulatory mechanism by which the loss of RINO2 function negatively affects pollen germination and pollen tube growth in response to HT exposure at anthesis. [ABSTRACT FROM AUTHOR]

The objective of this study was to characterize the reproductive compatibility among cultivars and selections of Japanese plum through controlled pollination, both in vivo and in situ, to obtain a profile of reproductively compatible cultivars. In situ and in vivo self-pollinations were carried out in an F1 population of Japanese plum, and in vivo pollinations were conducted on the pollen-grain receptor cultivars: Laetitia, Zafira and Fortune, and the pollen-grain donor cultivars: Piamontesa, Selection Plum 86-13, Harry Pickstone, Black Amber, Bruce, SC-17, Laetitia, SC-15, SC 7, Zafira, Amarelinha, América, Reubennel and Fortune, to verify the percentage of germination of pollen grains and growth of pollen tubes in the pistils of the recipient plants. The in situ and in vivo assays for the F1 population confirm the self-compatibility of genotypes 13 and 35. The Laetitia cultivar is self-incompatible; however, it is compatible with the Selection 'Plum 86-13'. 'Zafira' is self-incompatible. 'Fortune' is compatible with 'Amarelinha', 'SC-7', 'America', 'Harry Pickstone' and 'Reubennel', but it is self-incompatible. [ABSTRACT FROM AUTHOR]

In nature, the ability to avoid self-fertilization has evolved to prevent the deleterious effects of inbreeding. However, under cultivation conditions, self-incompatibility can reduce the pollination efficiency of Coffea canephora. The objective of this study was to characterize the self-incompatibility expression of the most cultivated genotypes in Western Amazonia, to improve the management of this coffee plant. In vitro pollinations were conducted among 45 genotypes, and the development of pollen tubes was evaluated using fluorescence microscopy. Pollination efficiency was evaluated considering the allelic variability within a breeding population from an ideal condition of maximum genetic variability. Based on the compatibility response, the genotypes were organized into six groups: group I (24.4%), group II (31.1%), group III (24.4%), group IV (2.2%), group V (2.2%), and group VI (15.6%). The lower frequencies of groups IV, V, and VI were associated with the lower frequency of the rarest allelic forms in this breeding population (p = 0.36, q = 0.26, r = 0.29, and s = 0.10). The correspondence between allelic and genotypic frequencies indicates that this population is in Hardy–Weinberg equilibrium (HWE) for this trait. Considering the cultivation of 2 to 10 clones, the population studied showed intermediate pollination efficiency between an ideal HWE population with p = q = r = s = 0.25 and a population with the rarest allelic forms (p = 0.48, q = 0.32, r = 0.19, s = 0.01). Efficiency estimates were stabilized from the cultivation of five clones, indicating that cultivating a minimum number of clones should be considered. Theoretically, maximum pollination efficiency is achieved by representing all alleles in equal proportions, whereas in practice, farmers should ensure the cultivation of plants from different compatibility groups, without significant imbalances. [ABSTRACT FROM AUTHOR]

Trendy high-density olive fields are often monovarietal orchards, mostly using the cultivar Arbequina. However, Arbequina shows a strong self-incompatibility response, and its yields depend on wind cross-pollination, which is not always available. With the aim of finding suitable self-compatible cultivars that can replace Arbequina, we evaluated pollen–pistil interaction, fruit set and seed paternity in Arbosana under different pollination treatments: self-pollination, open-pollination and three cross-pollination treatments: × Arbequina, × Sikitita and × Koroneiki. All these cultivars are low-vigor cultivars suitable for high-density orchards, making them potential pollinizers for Arbosana. The results show that Arbosana is also self-incompatible with a strong reduction in fruit set due to a lower fertilization level caused by a strong inhibition of pollen tube growth in self-pollinated flowers. Seed-paternity analyses confirmed the self-incompatibility response of Arbosana and suggest that some fruit obtained in bagged shoots under self-pollination were, in fact, a product of cross-fertilization. In conclusion, we recommend against the use of Arbosana in large monovarietal orchards. On the contrary, good results were obtained under cross-pollination with Sikitita, Arbequina and Koroneiki pollen, allowing us to recommend them as pollinizers for Arbosana in appropriate pollination designs. This is the first time Arbosana self-incompatibility has been reported. [ABSTRACT FROM AUTHOR]