LARP6C orchestrates posttranscriptional reprogramming of gene expression during hydration to promote pollen tube guidance.

Increasing evidence suggests that posttranscriptional regulation is a key player in the transition between mature pollen and the progamic phase (from pollination to fertilization). Nonetheless, the actors in this messenger RNA (mRNA)-based gene expression reprogramming are poorly understood. We demonstrate that the evolutionarily conserved RNA-binding protein LARP6C is necessary for the transition from dry pollen to pollen tubes and the guided growth of pollen tubes towards the ovule in Arabidopsis thaliana. In dry pollen, LARP6C binds to transcripts encoding proteins that function in lipid synthesis and homeostasis, vesicular trafficking, and polarized cell growth. LARP6C also forms cytoplasmic granules that contain the poly(A) binding protein and possibly represent storage sites for translationally silent mRNAs. In pollen tubes, the loss of LARP6C negatively affects the quantities and distribution of storage lipids, as well as vesicular trafficking. In Nicotiana benthamiana leaf cells and in planta, analysis of reporter mRNAs designed from the LARP6C target MGD2 provided evidence that LARP6C can shift from a repressor to an activator of translation when the pollen grain enters the progamic phase. We propose that LARP6C orchestrates the timely posttranscriptional regulation of a subset of mRNAs in pollen during the transition from the quiescent to active state and along the progamic phase to promote male fertilization in plants.

Persistent directional growth capability in Arabidopsis thaliana pollen tubes after nuclear elimination from the apex.

During the double fertilization process, pollen tubes deliver two sperm cells to an ovule containing the female gametes. In the pollen tube, the vegetative nucleus and sperm cells move together to the apical region where the vegetative nucleus is thought to play a crucial role in controlling the direction and growth of the pollen tube. Here, we report the generation of pollen tubes in Arabidopsis thaliana whose vegetative nucleus and sperm cells are isolated and sealed by callose plugs in the basal region due to apical transport defects induced by mutations in the WPP domain-interacting tail-anchored proteins (WITs) and sperm cell-specific expression of a dominant mutant of the CALLOSE SYNTHASE 3 protein. Through pollen-tube guidance assays, we show that the physiologically anuclear mutant pollen tubes maintain the ability to grow and enter ovules. Our findings provide insight into the sperm cell delivery mechanism and illustrate the independence of the tip-localized vegetative nucleus from directional growth control of the pollen tube.

Quantitative cell biology of tip growth in moss.

KEY MESSAGE: Here we review, from a quantitative point of view, the cell biology of protonemal tip growth in the model moss Physcomitrium patens. We focus on the role of the cytoskeleton, vesicle trafficking, and cell wall mechanics, including reviewing some of the existing mathematical models of tip growth. We provide a primer for existing cell biological tools that can be applied to the future study of tip growth in moss. Polarized cell growth is a ubiquitous process throughout the plant kingdom in which the cell elongates in a self-similar manner. This process is important for nutrient uptake by root hairs, fertilization by pollen, and gametophyte development by the protonemata of bryophytes and ferns. In this review, we will focus on the tip growth of moss cells, emphasizing the role of cytoskeletal organization, cytoplasmic zonation, vesicle trafficking, cell wall composition, and dynamics. We compare some of the existing knowledge on tip growth in protonemata against what is known in pollen tubes and root hairs, which are better-studied tip growing cells. To fully understand how plant cells grow requires that we deepen our knowledge in a variety of forms of plant cell growth. We focus this review on the model plant Physcomitrium patens, which uses tip growth as the dominant form of growth at its protonemal stage. Because mosses and vascular plants shared a common ancestor more than 450 million years ago, we anticipate that both similarities and differences between tip growing plant cells will provide mechanistic information of tip growth as well as of plant cell growth in general. Towards this mechanistic understanding, we will also review some of the existing mathematical models of plant tip growth and their applicability to investigate protonemal morphogenesis. We attempt to integrate the conclusions and data across cell biology and physical modeling to our current state of knowledge of polarized cell growth in P. patens and highlight future directions in the field.

Two Expansin Genes, AtEXPA4 and AtEXPB5, Are Redundantly Required for Pollen Tube Growth and AtEXPA4 Is Involved in Primary Root Elongation in Arabidopsis thaliana.

The growth of plant cells is inseparable from relaxation and expansion of cell walls. Expansins are a class of cell wall binding proteins, which play important roles in the relaxation of cell walls. Although there are many members in expansin gene family, the functions of most expansin genes in plant growth and development are still poorly understood. In this study, the functions of two expansin genes, AtEXPA4 and AtEXPB5 were characterized in Arabidopsis thaliana. AtEXPA4 and AtEXPB5 displayed consistent expression patterns in mature pollen grains and pollen tubes, but AtEXPA4 also showed a high expression level in primary roots. Two single mutants, atexpa4 and atexpb5, showed normal reproductive development, whereas atexpa4atexpb5 double mutant was defective in pollen tube growth. Moreover, AtEXPA4 overexpression enhanced primary root elongation, on the contrary, knocking out AtEXPA4 made the growth of primary root slower. Our results indicated that AtEXPA4 and AtEXPB5 were redundantly involved in pollen tube growth and AtEXPA4 was required for primary root elongation.

The Tip-Localized Phosphatidylserine Established by Arabidopsis ALA3 Is Crucial for Rab GTPase-Mediated Vesicle Trafficking and Pollen Tube Growth.

RabA4 subfamily proteins, the key regulators of intracellular transport, are vital for tip growth of plant polar cells, but their unique distribution in the apical zone and role in vesicle targeting and trafficking in the tips remain poorly understood. Here, we found that loss of Arabidopsis (Arabidopsis thaliana) AMINOPHOSPHOLIPID ATPASE 3 (ALA3) function resulted in a marked decrease in YFP-RabA4b/ RFP-RabA4d- and FM4-64-labeled vesicles from the inverted-cone zone of the pollen tube tip, misdistribution of certain intramembrane compartment markers, and an obvious increase in pollen tube width. Additionally, we revealed that phosphatidylserine (PS) was abundant in the inverted-cone zone of the apical pollen tube in wild-type Arabidopsis and was mainly colocalized with the trans-Golgi network/early endosome, certain post-Golgi compartments, and the plasma membrane. Loss of ALA3 function resulted in loss of polar localization of apical PS and significantly decreased PS distribution, suggesting that ALA3 is a key regulator for establishing and maintaining the polar localization of apical PS in pollen tubes. We further demonstrated that certain Rab GTPases colocalized with PS in vivo and bound to PS in vitro. Moreover, ALA3 and RabA4d collectively regulated pollen tube growth genetically. Thus, we propose that the tip-localized PS established by ALA3 is crucial for Rab GTPase-mediated vesicle targeting/trafficking and polar growth of pollen tubes in Arabidopsis.

LINC-complex mediated positioning of the vegetative nucleus is involved in calcium and ROS signaling in Arabidopsis pollen tubes.

Nuclear movement and positioning play a role in developmental processes throughout life. Nuclear movement and positioning are mediated primarily by linker of nucleoskeleton and cytoskeleton (LINC) complexes. LINC complexes are comprised of the inner nuclear membrane SUN proteins and the outer nuclear membrane (ONM) KASH proteins. In Arabidopsis pollen tubes, the vegetative nucleus (VN) maintains a fixed distance from the pollen tube tip during growth, and the VN precedes the sperm cells (SCs). In pollen tubes of wit12 and wifi, mutants deficient in the ONM component of a plant LINC complex, the SCs precede the VN during pollen tube growth and the fixed VN distance from the tip is lost. Subsequently, pollen tubes frequently fail to burst upon reception. In this study, we sought to determine if the pollen tube reception defect observed in wit12 and wifi is due to decreased sensitivity to reactive oxygen species (ROS). Here, we show that wit12 and wifi are hyposensitive to exogenous H2O2, and that this hyposensitivity is correlated with decreased proximity of the VN to the pollen tube tip. Additionally, we report the first instance of nuclear Ca2+ peaks in growing pollen tubes, which are disrupted in the wit12 mutant. In the wit12 mutant, nuclear Ca2+ peaks are reduced in response to exogenous ROS, but these peaks are not correlated with pollen tube burst. This study finds that VN proximity to the pollen tube tip is required for both response to exogenous ROS, as well as internal nuclear Ca2+ fluctuations.

Restricted Pollination for Tracing Individual Pollen Tubes in a Pistil.

As one of the essential steps to complete sexual reproduction, a pollen tube is precisely guided to an embryo sac to deliver the sperm cells. This ovule targeting by a pollen tube is one of the essential steps in pollen tube guidance. To assess the ovule targeting ability of the pollen tube from a certain mutant line, comparative analysis of pollen tube behaviors between wild-type and mutant genotypes is important. Here, we provide a protocol that traces all pollen tubes germinated from the quartet tetrad in a pistil by restricted pollination and aniline blue staining. By this analysis, statistical comparison between wild-type and the mutant pollen tube functions under the same in vivo condition is possible.

Semi-In Vivo Assay for Pollen Tube Attraction.

In flowering plants, each pollen tube delivers two sperm cells into the ovule to complete double fertilization. During the process, pollen tubes need to be navigated into the ovule, where accurate and complex pre-ovule guidance and ovule guidance are required. In recent years, different methods have been established to study those genes involved in the regulation of pollen tube guidance. Semi-in vivo ovule targeting mimics in vivo pollen tube micropylar guidance, and the semi-in vivo ovule targeting assay has been used to investigate function of genes involved in micropylar guidance. Moreover, the ovule targeting assay is the best way to do live cell imaging, which facilitates observation of pollen tube reception, synergid cell degeneration, and semi-in vivo gamete fusion. Meanwhile, semi-in vivo pollen tube attraction assay is another useful method to directly determine whether a certain molecule has pollen tube attraction activity.

Internally Controlled Methods to Quantify Pollen Tube Growth and Penetration Defects in Arabidopsis thaliana.

Double-fertilization in angiosperms requires precise communication between the male gametophyte (pollen), the female tissues, and the associated female gametophyte (embryo sac) to facilitate efficient fertilization. Numerous small molecules, proteins, and peptides have been shown to impact double-fertilization through the disruption of pollen germination, pollen tube growth, pollen tube guidance, or pollen tube penetration of the female tissues. The genetic basis of signaling events that lead to successful double-fertilization has been greatly facilitated by studies in the model organism Arabidopsis thaliana, which possesses a relatively simple reproductive physiology and a widely available T-DNA mutant seed collection. In this chapter, we detail methods for determining the effects of single gene loss-of-function mutations on pollen behavior through the creation of an internally controlled fluorescent hemizygous complement line. By transforming a single copy of the disrupted gene back into the homozygous mutant background, a precise endogenous control is generated due to the fact that pollen containing equal ratios of mutant and complemented pollen can be collected from a single flower. Using this experimental design, we describe multiple assays that can be performed in series to assess mutant pollen defects in germination, pollen tube elongation rate, and pistil penetration, which can be easily quantified alongside a "near-wildtype" complemented counterpart.

Arabidopsis thaliana Pollen Tube Culture for Multi-omics Studies.

Pollen tubes have been key models to study plant cell wall elongation. Arabidopsis thaliana, although small, is a nice model, easy to grow and with a large set of studies to simplify result integration and interpretation. Pollen tubes may be used for gene expression essays, but also for biochemical characterization of the cell wall composition. However, pollen tube culture methods though seemingly straightforward have often a multitude of small technical details crucial for success, quickly deterring the more inexperienced and setting back experiments for months at the time. Here we propose a detailed method to set up easily a pollen tube culture routine in any lab, with a minimal set of equipment, to isolate and process pollen tubes for gene expression and/or cell wall biochemistry studies.

Obtaining Mutant Pollen for Phenotypic Analysis and Pollen Tube Dual Staining.

Mutant phenotype observation is the most useful and important method to study which biological process a gene-of-interest is involved in. In flowering plants, excessive pollen grains land and germinate on the stigma, then pollen tubes grow through the transmitting tract to reach the ovules, eventually enter the micropyle to complete double fertilization. First, for mutants whose homozygotes could not be obtained due to pollen tube defects, it is difficult to observe the defect phenotype since the pollen grains of different genotypes are mixed together. Here, we provide a detailed protocol to pick out mutant pollen grains from the heterozygous mutant plants in Arabidopsis thaliana. By using this method, we could obtain sufficient mutant pollen grains for phenotypic analysis. Second, it is difficult to compare the pollen/pollen tube behavior of two different genotypes/species in vivo in a same pistil. Here, we develop a new dual staining method which combines GUS staining with aniline blue staining. By using this method, we can analyze the competence of the two different pollen tubes in the same pistil.

Analyzing Intracellular Gradients in Pollen Tubes.

Conspicuous intracellular gradients manifest and/or drive intracellular polarity in pollen tubes. However, quantifying these gradients raises multiple technical challenges. Here we present a sensible computational protocol to analyze gradients in growing pollen tubes and to filter nonrepresentative time points. As an example, we use imaging data from pollen tubes expressing a genetically encoded ratiometric Ca2+ probe, Yellow CaMeleon 3.6, from which a kymograph is extracted. The tip of the pollen tube is detected with CHUKNORRIS, our previously published methodology, allowing the reconstruction of the intracellular gradient through time. Statistically confounding time points, such as growth arrest where gradients are highly oscillatory, are filtered out and a mean spatial profile is estimated with a local polynomial regression method. Finally, we estimate the gradient slope by the linear portion of the decay in mean fluorescence, offering a quantitative method to detect phenotypes of gradient steepness, location, intensity, and variability. The data manipulation protocol proposed can be achieved in a simple and efficient manner using the statistical programming language R, opening paths to perform high-throughput spatiotemporal phenotyping of intracellular gradients in apically growing cells.

Silicone Chambers for Pollen Tube Imaging in Microstructured In Vitro Environments.

Live cell imaging at high resolution of pollen tubes growing in vitro requires an experimental setup that maintains the elongated cells in a single optical plane and allows for controlled exchange of growth medium. As a low-cost alternative to lithography-based microfluidics, we developed a silicone-based spacer system that allows introducing spatial features and flexible design. These growth chambers can be cleaned and reused repeatedly.

Imaging and Analysis of the Content of Callose, Pectin, and Cellulose in the Cell Wall of Arabidopsis Pollen Tubes Grown In Vitro.

To achieve fertilization, pollen tubes have to protect and properly deliver sperm cells through the pistil to the ovules. Pollen tube growth is a representative example of polarized growth where new components of the cell wall and plasma membrane are continuously deposited at the tip of the growing cell. The integrity of the cell wall is of fundamental importance to maintain apical growth. For this reason, pollen tube growth has become an excellent model to study the role of polysaccharides and structural cell wall proteins involved in polar cell expansion. However, quantification of structural polysaccharides at the pollen tube cell wall has been challenging due to technical complexity and the difficulty of finding specific dyes. Here, we propose simple methods for imaging and quantification of callose, pectin , and cellulose using specific dyes such as Aniline Blue, Propidium Iodide, and Pontamine Fast Scarlet 4B.

Characterization of Growth Behavior and the Resulting Forces Applied by Pollen Tubes in a 3D Matrix.

The question of how pollen tubes orient themselves on their way to the egg cell is a major focus of plant reproduction research. The role of physical guidance through the tissues of the pistil in relation to the mechanical perception and growth adaptation of the pollen tubes has not been sufficiently investigated. In order to advance research on the mechanical perception of pollen tubes and their force application during invasive growth, we present simple methods for the observation and mechanical characterization of pollen tubes in vitro, which can be established with little effort in any biological laboratory with standard equipment. Pollen grains are germinated in a hydrogel containing agarose and their growth is recorded in 3D using brightfield microscopy. Using suitable analysis software, parameters such as growth rate and pollen tube diameter can then be determined to estimate the exerted penetration force.

Flow Chamber Assay to Image the Response of FRET-Based Nanosensors in Pollen Tubes to Changes in Medium Composition.

Pollen tubes growing in the transmitting tract are presented with an extracellular matrix rich in a variety of substances. The expression of a multitude of genes for transport proteins in the pollen tube indicates that pollen tubes take up at least some of the components provided by the transmitting tract, for example nutrients, ions, or signaling molecules. FRET (Förster resonance energy transfer)-based nanosensors are perfectly suited to study the uptake of these molecules into pollen tubes. They are genetically encoded and can easily be expressed in Arabidopsis pollen tubes. Furthermore, the method is noninvasive and nanosensors for a wide range of substances are available. This chapter will describe the design of plasmids required to generate stable Arabidopsis lines with a pollen tube-specific expression of nanosensor constructs. We also present a method to germinate Arabidopsis pollen tubes in a flow chamber slide that allows the perfusion of the pollen tubes with liquid medium supplemented with the substrate of the nanosensor. Simultaneous evaluation of the FRET efficiency of the nanosensor by confocal microscopy reveals whether the substance is taken up by the pollen tubes. Together with the great number of available nanosensors this method can generate a detailed picture of the substances that are taken up during pollen tubes growth.

Isolation of male and female gametes, zygotes and proembryos of leek (Allium tuberosum Roxb).

The isolation of male and female gametes is an effective method to study the fertilization mechanisms of higher plants. An osmotic shock method was used to rupture pollen grains of Allium tuberosum Roxb and release the pollen contents, including generative cells, which were mass collected. The pollinated styles were cut following 3 h of in vivo growth, and cultured in medium for 6-8 h, during which time pollen tubes grew out of the cut end of the style. After pollen tubes were transferred into a solution containing 6% mannitol, tubes burst and released pairs of sperm cells. Ovules of A. tuberosum were incubated in an enzyme solution for 30 min, and then dissected to remove the integuments. Following transfer to a dissecting solution free of enzymes, each nucellus was cut in the middle, and squeezed gently on the micropylar end, resulting in the liberation of the egg, zygote and proembryo from ovules at selected stages. These cells can be used to explore fertilization and embryonic development using molecular biological methods for each cell type and development stage.

FERONIA controls pectin- and nitric oxide-mediated male-female interaction.

Species that propagate by sexual reproduction actively guard against the fertilization of an egg by multiple sperm (polyspermy). Flowering plants rely on pollen tubes to transport their immotile sperm to fertilize the female gametophytes inside ovules. In Arabidopsis, pollen tubes are guided by cysteine-rich chemoattractants to target the female gametophyte1,2. The FERONIA receptor kinase has a dual role in ensuring sperm delivery and blocking polyspermy3. It has previously been reported that FERONIA generates a female gametophyte environment that is required for sperm release4. Here we show that FERONIA controls several functionally linked conditions to prevent the penetration of female gametophytes by multiple pollen tubes in Arabidopsis. We demonstrate that FERONIA is crucial for maintaining de-esterified pectin at the filiform apparatus, a region of the cell wall at the entrance to the female gametophyte. Pollen tube arrival at the ovule triggers the accumulation of nitric oxide at the filiform apparatus in a process that is dependent on FERONIA and mediated by de-esterified pectin. Nitric oxide nitrosates both precursor and mature forms of the chemoattractant LURE11, respectively blocking its secretion and interaction with its receptor, to suppress pollen tube attraction. Our results elucidate a mechanism controlled by FERONIA in which the arrival of the first pollen tube alters ovular conditions to disengage pollen tube attraction and prevent the approach and penetration of the female gametophyte by late-arriving pollen tubes, thus averting polyspermy.

Extracellular ionic fluxes suggest the basis for cellular life at the 1/f ridge of extended criticality.

The criticality hypothesis states that a system may be poised in a critical state at the boundary between different types of dynamics. Previous studies have suggested that criticality has been evolutionarily selected, and examples have been found in cortical cell cultures and in the human nervous system. However, no one has yet reported a single- or multi-cell ensemble that was investigated ex vivo and found to be in the critical state. Here, the precise 1/f noise was found for pollen tube cells of optimum growth and for the physiological ("healthy") state of blood cells. We show that the multi-scale processes that arise from the so-called critical phenomena can be a fundamental property of a living cell. Our results reveal that cell life is conducted at the border between order and disorder, and that the dynamics themselves drive a system towards a critical state. Moreover, a temperature-driven re-entrant state transition, manifest in the form of a Lorentz resonance, was found in the fluctuation amplitude of the extracellular ionic fluxes for the ensemble of elongating pollen tubes of Nicotiana tabacum L. or Hyacintus orientalis L. Since this system is fine-tuned for rapid expansion to reach the ovule at a critical temperature which results in fertilisation, the core nature of criticality (long-range coherence) offers an explanation for its potential in cell growth. We suggest that the autonomous organisation of expansive growth is accomplished by self-organised criticality, which is an orchestrated instability that occurs in an evolving cell.

Aminooxyacetic acid (АОА), inhibitor of 1-aminocyclopropane-1-carboxilic acid (AСС) synthesis, suppresses self-incompatibility-induced programmed cell death in self-incompatible Petunia hybrida L. pollen tubes.

Self-incompatibility (SI) is genetically determined reproductive barrier preventing inbreeding and thereby providing the maintenance of plant species diversity. At present, active studies of molecular bases of SI mechanisms are underway. S-RNAse-based SI in Petunia hybrida L. is a self-/non-self recognition system that allows the pistil to reject self pollen and to accept non-self pollen for outcrossing. In the present work, using fluorescent methods including the TUNEL method allowed us to reveal the presence of markers of programmed cell death (PCD), such as DNA fragmentation, in growing in vivo petunia pollen tubes during the passage of the SI reaction. The results of statistical analysis reliably proved that PCD is the factor of S-RNAse-based SI. It was found that preliminary treatment before self-pollination of stigmas of petunia self-incompatible line with aminooxyacetic acid (AOA), inhibitor of ACC synthesis, led to stimulation of pollen tubes growth when the latter did not exhibit any hallmarks of PCD. These data argue in favor of assumption that ethylene controls the passage of PCD in incompatible pollen tubes in the course of S-RNAse-based SI functioning. The involvement of the hormonal regulation in SI mechanism in P. hybrida L. is the finding observed by us for the first time.