Pollen Tube Guidance Research Articles

The Role of Female and Male Genes in Regulating Pollen Tube Guidance in Flowering Plants

The Role of Female and Male Genes in Regulating Pollen Tube Guidance in Flowering Plants

Deep imaging reveals dynamics and signaling in one-to-one pollen tube guidance

In the pistil of flowering plants, each ovule usually associates with a single pollen tube for fertilization. This one-to-one pollen tube guidance, which contributes to polyspermy blocking and efficient seed production, is largely different from animal chemotaxis of many sperms to one egg. However, the functional mechanisms underlying the directional cues and polytubey blocks in the depths of the pistil remain unknown. Here, we develop a two-photon live imaging method to directly observe pollen tube guidance in the pistil of Arabidopsis thaliana, clarifying signaling and cellular behaviors in the one-to-one guidance. Ovules are suggested to emit multiple signals for pollen tubes, including an integument-dependent directional signal that reaches the inner surface of the septum and adhesion signals for emerged pollen tubes on the septum. Not only FERONIA in the septum but ovular gametophytic FERONIA and LORELEI, as well as FERONIA- and LORELEI-independent repulsion signal, are involved in polytubey blocks on the ovular funiculus. However, these funicular blocks are not strictly maintained in the first 45 min, explaining previous reports of polyspermy in flowering plants.

Arabidopsis class A S-acyl transferases modify the pollen receptors LIP1 and PRK1 to regulate pollen tube guidance.

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.

Transcriptome Analysis Provides Insights into Korean Pine Tree Aging and Response to Shading

Age controls a tree’s responses to environmental cues and shading influences tree growth and physiology. These are basic principles of “Afforestation under canopy”, an approach that is widely used in the regeneration of Korean pine forests. Studying the underlying mechanism helps us to understand tree adaptation and utilize it in forest management. In this study, we investigated the transcriptomic changes in the uppermost main stems of the Korean pine tree (Pinus koraiensis, Sieb. et Zucc.) at different ages (5, 7, 10, 14, and 17 years) and in different growth conditions (open-grown and shade-grown trees) using RNA-Seq. In total, 434,005,837 reads were produced and assembled into 111,786 unigenes. After pairwise comparisons, 568 differentially expressed unigenes (DEUs) were identified. The greatest number of DEUs was identified in the comparison between 5-year-old open-grown trees and 17-year-old shade-grown trees, while no DEUs were identified in 15 pairwise comparisons. Among these 568 DEUs, 45 were assigned to gene ontology (GO) terms associated with response to environmental changes, including “response to stress” (26) and “response to light and temperature” (19); 12 were assigned to GO terms associated with sexual reproduction, such as “sexual reproduction”, “specification of floral organ identity”, “pollen tube guidance”, and “fruit ripening”; 15 were heat shock protein genes and showed decreased expression patterns with age; and one, annotated as Pinus tabuliformis DEFICIENS-AGAMOUS-LIKE 1, showed an increased expression pattern with age, independent of the reproductive state or growth conditions of Korean pine trees. Altogether, these findings not only demonstrate the molecular aspects of the developmental and physiological effects of age and shading on Korean pine trees, but also improve our understanding of the basic principles of “Afforestation under canopy”.

Identification and characterization of circular RNAs involved in the fertility stability of cotton CMS-D2 restorer line under heat stress

BackgroundAs 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.ResultsIn 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.ConclusionsThrough 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.

Two aspartic proteases, BnaAP36s and BnaAP39s, regulate pollen tube guidance in Brassica napus.

The online version contains supplementary material available at 10.1007/s11032-023-01377-1.

Pollen Tube Content Facilitates and Increases the Potential of Endosperm Proliferation Irrespective of Fertilization in <i>Arabidopsis thaliana</i>

Sexual reproduction in angiosperms is a complex and precise process of regulation, which includes the pollen tube guidance, double fertilization, and the seed development process. The previous report identified that for seed development, there is a period of preparation for fertilization (between pollen tube guidance and double fertilization) that is called pollen-tube-dependent ovule enlargement morphology (POEM). It has been shown that the pollen tube content (PTC) plays a crucial role in the enlargement of ovules and the initiation of seed coat formation. However, we did not investigate the potential of endosperm proliferation in autonomous mutants at a later stage. Here, we investigated this phenomenon using vanillin staining and transparent experiments to examine the manner, in which the PTC affects the potential of endosperm formation. Interestingly, the PTC increased the number of endosperm nuclei without fertilization equally and synchronously in mea and fis2 ovules. This finding might help improve the study of apomixes and our understanding of how the molecular mechanisms that regulate this phenomenon will contribute to plant reproductive science in the future.

Live Imaging of Arabidopsis Pollen Tube Reception and Double Fertilization Using the Semi-In Vitro Cum Septum Method.

In flowering plants, the growth and guidance of the pollen tube (male gametophyte) within the pistil and the reception of the pollen tube by the female gametophyte are essential for double fertilization and subsequent seed development. The interactions between male and female gametophytes during pollen tube reception culminate in pollen tube rupture and the release of two sperm cells to effect double fertilization. As pollen tube growth and double fertilization are deeply hidden within the tissues of the flower, this process is difficult to observe in vivo. A semi-in vitro (SIV) method for the live-cell imaging of fertilization in the model plant Arabidopsis thaliana has been developed and implemented in several investigations. These studies have helped to elucidate the fundamental features of how the fertilization process occurs in flowering plants and which cellular and molecular changes occur during the interaction of the male and female gametophytes. However, because these live cell imaging experiments involve the excision of individual ovules, they are limited to a low number of observations per imaging session, making this approach tedious and very time-consuming. Among other technical difficulties, a failure of the pollen tubes to fertilize the ovules in vitro isoften reported, which severely confounds such analyses. Here, a detailed video protocol for the imaging of pollen tube reception and fertilization in an automated and high-throughput manner is provided, allowing for up to 40 observations of pollen tube reception and rupture per imaging session. Coupled with the use of genetically encoded biosensors and marker lines, this method enables the generation of large sample sizes with a reduced time investment. Nuances and critical points of the technique, including flower staging, dissection, medium preparation, and imaging, are clearly detailed in video format to facilitate future research on the dynamics of pollen tube guidance, reception, and double fertilization.

Pollen tube emergence is mediated by ovary-expressed ALCATRAZ in cucumber

Pollen tube guidance within female tissues of flowering plants can be divided into preovular guidance, ovular guidance and a connecting stage called pollen tube emergence. As yet, no female factor has been identified to positively regulate this transition process. In this study, we show that an ovary-expressed bHLH transcription factor Cucumis sativus ALCATRAZ (CsALC) functions in pollen tube emergence in cucumber. CsALC knockout mutants showed diminished pollen tube emergence, extremely reduced entry into ovules, and a 95% reduction in female fertility. Further examination showed two rapid alkalinization factors CsRALF4 and CsRALF19 were less expressed in Csalc ovaries compared to WT. Besides the loss of male fertility derived from precocious pollen tube rupture as in Arabidopsis, Csralf4 Csralf19 double mutants exhibited a 60% decrease in female fertility due to reduced pollen tube distribution and decreased ovule targeting efficiency. In brief, CsALC regulates female fertility and promotes CsRALF4/19 expression in the ovary during pollen tube guidance in cucumber.

High temperatures during late floral bud stages decrease fertilization in strawberry (Fragaria × ananassa): pollen-pistil interaction and anatomical evidences

High temperature (HT) effects on pistil tissues and female gametophyte have been scarcely investigated in crops species. HT in strawberry can induce fruit malformations due to poor pollen performance in pistils, reducing the fertilization level. In this study, Fragaria × ananassa cvs. Earlibrite and Fortuna were exposed to ambient temperature (AT) or HT at late flower bud development over the duration of 3 or 5 days. To evaluate the capacity of heated apical and basal pistils to support and guide pollen tubes, we examined the performance of unheated pollen grains and their path along the pistil, as well as the anatomy of reproductive tissues. HT significantly induced a decrease in the number of adhered and germinated pollen grains and of pollen tubes in the style and the ovule micropyle. Microscopic observations revealed loss of stigmatic papillae turgidity and fertilization failures due to abnormal pollen tube paths. The latter finding was related to the high incidence of immature female gametophytes in apical pistils and unviable female gametophytes in basal pistils. Facultative apomixis is also described. This is the first report of impaired pistil functions when strawberry buds are exposed to HT, as revealed by in vivo poor pollen performance 24 h after pollination.

The female germ unit is essential for pollen tube funicular guidance in Arabidopsis thaliana.

In angiosperm, two immotile sperm cells are delivered to the female gametes for fertilization by a pollen tube, which perceives guidance cues from ovules at least at two critical sites, micropyle for short-distance guidance and funiculus for comparably longer distance guidance. Compared with the great progress in understanding pollen tube micropylar guidance, little is known about the signaling for funicular guidance. Here, we show that funiculus plays an important role in pollen tube guidance and report that female gametophyte (FG) plays a critical role in funicular guidance by analysis of a 3-dehydroquinate synthase (DHQS) mutant. Loss function of DHQS in FG interrupts pollen tube funicular guidance, suggesting that the guiding signal is generated from FG. We show the evidence that the capacity of funicular guidance is established during FG functional specification after the establishment of cell identity. Specific expression of DHQS in the synergid cells, central cells, or egg cells can rescue funicular guidance defect in dhqs/+, indicating all the female germ unit cells are involved in the funicular guidance. The finding reveals that the attracting signal of pollen tube funicular guidance was generated at a site and stage manner and provides novel clue to locate and search for the signal.

Receptor Kinases: A Sophisticate Protein Network to Maintain Plant life Continuity. An Update

Abstract To govern the plant growth, defense from pathogen attack, to tolerate the harsh conditions and reproduction, plant employ very sophisticated and highly effective machinery in the form of receptor kinases (RKs) which make them adoptable in the diverse land environment. Different classes of RKs including receptor like cytoplasmic kinases (RLCKs) unique in their functionality are of paramount importance in transmembrane signaling; and have emerged as a major class of signaling proteins, which regulate the normal plant life continuity. For their central importance in vital functions of plants, RKs have brought overwhelming intention of researchers across the globe and have been considered as most flashpoint of research to investigate, by the plant biologists. We intended to provide an overview of current progress in understanding the associations of RKs with plant cellular responses to regulate the downstream signaling nodes to orchestrate a complex array of defense against microbial pathogens; to face any drought condition and how RKs are associated with signal peptides to coordinate the plant growth, pollen tube guidance and embryonic pattering. Following the rising attention of scientific community to explore the physiology and anatomy, these central players of plant life regulation in depth, we believe that this systematic review will provide an update on research about RKs and will be a source of blooming growth of research for beneficial knowledge to deal the diseases of plants.

F-actin regulates the polarized secretion of pollen tube attractants in Arabidopsis synergid cells.

Pollen tube attraction is a key event of sexual reproduction in flowering plants. In the ovule, two synergid cells neighboring the egg cell control pollen tube arrival via the active secretion of attractant peptides such as AtLURE1 and XIUQIU from the filiform apparatus (FA) facing toward the micropyle. Distinctive cell polarity together with longitudinal F-actin and microtubules are hallmarks of the synergid cell in various species, though the functions of these cellular structures are unclear. In this study, we used genetic and pharmacological approaches to indicate the roles of cytoskeletal components in FA formation and pollen tube guidance in Arabidopsis thaliana. Genetic inhibition of microtubule formation reduced invaginations of the plasma membrane but did not abolish micropylar AtLURE1.2 accumulation. By contrast, the expression of a dominant-negative form of ACTIN8 induced disorganization of the FA and loss of polar AtLURE1.2 distribution toward the FA. Interestingly, after pollen tube reception, F-actin became unclear for a few hours in the persistent synergid cell, which may be involved in pausing and resuming pollen tube attraction during early polytubey block. Our data suggest that F-actin plays a central role in maintaining cell polarity and in mediating male-female communication in the synergid cell.

New insights on the expression patterns of specific Arabinogalactan proteins in reproductive tissues of Arabidopsis thaliana.

Arabinogalactan proteins (AGPs) are hydroxyproline-rich glycoproteins containing a high proportion of carbohydrates, widely distributed in the plant kingdom and ubiquitously present in land plants. AGPs have long been suggested to play important roles in plant reproduction and there is already evidence that specific glycoproteins are essential for male and female gametophyte development, pollen tube growth and guidance, and successful fertilization. However, the functions of many of these proteins have yet to be uncovered, mainly due to the difficulty to study individual AGPs. In this work, we generated molecular tools to analyze the expression patterns of a subgroup of individual AGPs in different Arabidopsis tissues, focusing on reproductive processes. This study focused on six AGPs: four classical AGPs (AGP7, AGP25, AGP26, AGP27), one AG peptide (AGP24) and one chimeric AGP (AGP31). These AGPs were first selected based on their predicted expression patterns along the reproductive tissues from available RNA-seq data. Promoter analysis using β-glucuronidase fusions and qPCR in different Arabidopsis tissues allowed to confirm these predictions. AGP7 was mainly expressed in female reproductive tissues, more precisely in the style, funiculus, and integuments near the micropyle region. AGP25 was found to be expressed in the style, septum and ovules with higher expression in the chalaza and funiculus tissues. AGP26 was present in the ovules and pistil valves. AGP27 was expressed in the transmitting tissue, septum and funiculus during seed development. AGP24 was expressed in pollen grains, in mature embryo sacs, with highest expression at the chalazal pole and in the micropyle. AGP31 was expressed in the mature embryo sac with highest expression at the chalaza and, occasionally, in the micropyle. For all these AGPs a co-expression analysis was performed providing new hints on its possible functions. This work confirmed the detection in Arabidopsis male and female tissues of six AGPs never studied before regarding the reproductive process. These results provide novel evidence on the possible involvement of specific AGPs in plant reproduction, as strong candidates to participate in pollen-pistil interactions in an active way, which is significant for this field of study.

ALA3 regulates pollen tube growth and guidance by keeping PRKs in all the right places.

ALA3 regulates pollen tube growth and guidance by keeping PRKs in all the right places.

The phospholipid flippase ALA3 regulates pollen tube growth and guidance in Arabidopsis

Pollen tube guidance regulates the growth direction and ovule targeting of pollen tubes in pistils, which is crucial for the completion of sexual reproduction in flowering plants. The Arabidopsis (Arabidopsis thaliana) pollen-specific receptor kinase (PRK) family members PRK3 and PRK6 are specifically tip-localized and essential for pollen tube growth and guidance. However, the mechanisms controlling the polar localization of PRKs at the pollen tube tip are unclear. The Arabidopsis P4-ATPase ALA3 helps establish the polar localization of apical phosphatidylserine (PS) in pollen tubes. Here, we discovered that loss of ALA3 function caused pollen tube defects in growth and ovule targeting and significantly affected the polar localization pattern of PRK3 and PRK6. Both PRK3 and PRK6 contain two polybasic clusters in the intracellular juxtamembrane domain, and they bound to PS in vitro. PRK3 and PRK6 with polybasic cluster mutations showed reduced or abolished binding to PS and altered polar localization patterns, and they failed to effectively complement the pollen tube-related phenotypes of prk mutants. These results suggest that ALA3 influences the precise localization of PRK3, PRK6, and other PRKs by regulating the distribution of PS, which plays a key role in regulating pollen tube growth and guidance.

Quantification of Species-Preferential Micropylar Chemoattraction in Arabidopsis by Fluorescein Diacetate Staining of Pollen Tubes.

Sexual reproduction between males and females of the same species is essential for species maintenance. Ovular micropylar guidance, the last step of pollen tube guidance in angiosperms, contributes to species-preferential reproduction. Previous studies using semi-in vivo attraction assays showed that species-preferential attractant peptides are secreted from the ovule through its micropyle. However, conventional semi-in vivo assays usually depend on transgenic pollen tubes expressing a fluorescent protein to determine whether the tubes are attracted to the ovule to precisely penetrate the micropyle. Here, we found that fluorescein diacetate (FDA) staining was suitable for evaluating the micropylar guidance rate of non-transgenic pollen tubes in semi-in vivo conditions. Micropylar guidance was quantified for ovules and pollen tubes of Arabidopsis thaliana and Arabidopsis lyrata by combining FDA staining with modified semi-in vivo assays. Our results using the simple staining method showed that the ovules of each species secrete species-preferential attractants, and that pollen tubes respond more strongly to attractants of their own species compared with those of closely related species. LURE-type CRP810 attractant peptides were shown to be responsible for micropylar attraction of A. thaliana in the semi-in vivo assay. The POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 6 (PRK6) receptor for LURE1, as well as an unidentified receptor for other LURE-type attractants, are involved in the species-preferential response of these two Arabidopsis species.

The Evolution and Expression Profiles of EC1 Gene Family during Development in Cotton.

Fertilization is essential to sexual reproduction of flowering plants. EC1 (EGG CELL 1) proteins have a conserved cysteine spacer characteristic and play a crucial role in double fertilization process in many plant species. However, to date, the role of EC1 gene family in cotton is fully unknown. Hence, detailed bioinformatics analysis was explored to elucidate the biological mechanisms of EC1 gene family in cotton. In this study, we identified 66 genes in 10 plant species in which a total of 39 EC1 genes were detected from cotton genome. Phylogenetic analysis clustered the identified EC1 genes into three families (I-III) and all of them contain Prolamin-like domains. A good collinearity was observed in the synteny analysis of the orthologs from cotton genomes. Whole-genome duplication was determined to be one of the major impetuses for the expansion of the EC1 gene family during the process of evolution. qRT-PCR analysis showed that EC1 genes were highly expressed in reproductive tissues under multiple stresses, signifying their potential role in enhancing stress tolerance or responses. Additionally, gene interaction networks showed that EC1 genes may be involved in cell stress and response transcriptional regulator in the synergid cells and activate the expression of genes required for pollen tube guidance. Our results provide novel functional insights into the evolution and functional elucidation of EC1 gene family in cotton.

Arabidopsis SKU5 Similar 11 and 12 play crucial roles in pollen tube integrity, growth and guidance.

Pollen tube integrity, growth and guidance are crucial factors in plant sexual reproduction. Members of the plant Skewed5 (SKU5) Similar (SKS) family show strong similarity to multicopper oxidases (MCOs), but they lack conserved histidines in MCO active sites. The functions of most SKS family members are unknown. Here, we show that Arabidopsis pollen-expressed SKS11 and SKS12 play important roles in pollen tube integrity, growth and guidance. The sks11sks12 mutant exhibited significantly reduced male fertility. Most of the pollen from sks11sks12 plants burst when germinated, and the pollen tubes grew slowly and exhibited defective growth along the funiculus and micropyle. SKS11-GFP and SKS12-mCherry were detected at the cell wall in pollen tubes. The contents of several cell wall polysaccharides and arabinogalactans were decreased in the pollen tube cell walls of sks11sks12 plants. Staining with a reactive oxygen species (ROS)-sensitive dye and use of the H2 O2 sensor HyPer revealed that the ROS content in the pollen tubes of sks11sks12 plants was remarkably reduced. SKS11444His-Ala , in which the last conserved histidine was mutated, could restore the mutant phenotypes of sks11sks12. Thus, SKS11/12 are required for pollen tube integrity, growth and guidance possibly by regulating the ROS level and cell wall polysaccharide deposition or remodeling in pollen tubes.

Physalis floridana CRABS CLAW mediates neofunctionalization of GLOBOSA genes in carpel development.

Floral B-function MADS-box genes, such as GLOBOSA (GLO), function in corolla and stamen organ identity specification. The functions of these genes outside these floral whorls are rarely reported. DOLL1 is a GLO gene controlling corolla and androecium organ identity. In this study we found that, in Physalis floridana double-layered-lantern 1 (doll1) mutant pollinated with wild-type pollen, fruit set was extremely low, indicating that doll1 females are dysfunctional. Stigma and style structure, stigma receptivity, pollen tube guidance, and embryo sac development were also impaired in doll1. P. floridana CRABS CLAW (PFCRC), predominantly expressed in carpels, was repressed in doll1 native carpels. Loss-of-function of PFCRC altered carpel meristem determinacy, carpel closure, and ovule number, and the resultant 'pistil' consisted of multiple spirally-arranged dorsiventral carpels occasionally with 1-2 naked ovules on the margin and trichomes at each mutated carpel tip, implying an alteration of carpel organ identity. Regulatory and genetic interactions between B-class MADS-box genes and PFCRC were revealed in a context-dependent manner in floral development. Our work reveals a new role for the B-function genes in carpel and ovule development via regulating PFCRC, providing a new understanding of genetic regulatory networks between MADS-domain and CRC transcription factors in mediating carpel organ specification, functionality, and origin.