Reproductive Performance of the Alpine Plant Species Ranunculus kuepferi in a Climatic Elevation Gradient: Apomictic Tetraploids Do Not Show a General Fitness Advantage over Sexual Diploids.

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.

Quantifying Protein Dynamics by Kymograph Analysis.

Time-lapse imaging of the subcellular localization and dynamic behavior of proteins is critical to understand their biological functions in cells. With the advent of various methodologies and computational tools, the precise tracking and quantification of protein spatiotemporal dynamics have become feasible. Kymograph analysis, in particular, has been extensively adopted for the quantitative assessment of proteins, vesicles, and organelle movements. However, conventional kymograph analysis, which is based on a single linear trajectory, may not comprehensively capture the complexity of proteins that alter their course during intracellular transport and activity. In this chapter, we introduced an advanced protocol for whole-cell kymograph analysis that allows for three-dimensional (3D) tracking of protein dynamics. This method was validated through the analysis of tip-focused endocytosis and exocytosis processes in growing tobacco pollen tubes by employing both the advanced whole-cell and classical kymograph methods. In addition, we enhanced this method by integrating pseudo-colored kymographs that enables the direct visualization of changes in protein fluorescence intensity with fluorescence recovery after photobleaching to advance our understanding of protein localization and dynamics. This comprehensive method offers a novel insight into the intricate dynamics of protein activity within the cellular context.

Co-flowering richness has variable effects on pollen quantity and quality limitation in four Clarkia species.

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 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 times larger 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 quantity and quality of pollen limitation and co-flowering context that may have different evolutionary outcomes across species and populations.

Arabidopsis pollen prolyl-hydroxylases P4H4/6 are relevant for correct hydroxylation and secretion of LRX11 in pollen tubes.

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.

Comprehensive Embryological Analyses of Common Buckwheat (Fagopyrym esculentum Moench).

Knowledge of detailed reproductive biology of cultivated species is important as requirements for fruit and seed production allow the development of effective management strategies and a sustainable use. Embryological processes of common buckwheat (Fagopyrum esculentum Moench) are difficult to interpret due to the influence of genetic determinants, i.e., dimorphic heterostyly resulting in the production of long- and short-styled flowers, and environmental predisposition, i.e., sensitivity of ovules to thermal stress. Furthermore, the situation is complicated by overproduction of flowers and depletion of resources as the plant ages. Herein we provide protocols that allow to visualize both basic and more specific embryological features and also disturbances in sexual reproduction of common buckwheat resulting from external and internal factors. All stages of plant material fixation, preparation, staining, and observation are described and explained in detail. Technical tips and pictures of properly prepared microscopic sections are also provided.

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

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

How pollen tubes fight for food: the impact of sucrose carriers and invertases of Arabidopsis thaliana on pollen development and pollen tube growth.

Pollen tubes of higher plants grow very rapidly until they reach the ovules to fertilize the female gametes. This growth process is energy demanding, however, the nutrition strategies of pollen are largely unexplored. Here, we studied the function of sucrose transporters and invertases during pollen germination and pollen tube growth. RT-PCR analyses, reporter lines and knockout mutants were used to study gene expression and protein function in pollen. The genome of Arabidopsis thaliana contains eight genes that encode functional sucrose/H+ symporters. Apart from AtSUC2, which is companion cell specific, all other AtSUC genes are expressed in pollen tubes. AtSUC1 is present in developing pollen and seems to be the most important sucrose transporter during the fertilization process. Pollen of an Atsuc1 knockout plant contain less sucrose and have defects in pollen germination and pollen tube growth. The loss of other sucrose carriers affects neither pollen germination nor pollen tube growth. A multiple knockout line Atsuc1Atsuc3Atsuc8Atsuc9 shows a phenotype that is comparable to the Atsuc1 mutant line. Loss of AtSUC1 can`t be complemented by AtSUC9, suggesting a special function of AtSUC1. Besides sucrose carriers, pollen tubes also synthesize monosaccharide carriers of the AtSTP family as well as invertases. We could show that AtcwINV2 and AtcwINV4 are expressed in pollen, AtcwINV1 in the transmitting tissue and AtcwINV5 in the funiculi of the ovary. The vacuolar invertase AtVI2 is also expressed in pollen, and a knockout of AtVI2 leads to a severe reduction in pollen germination. Our data indicate that AtSUC1 mediated sucrose accumulation during late stages of pollen development and cleavage of vacuolar sucrose into monosaccharides is important for the process of pollen germination.

Investigation of ROP GTPase Activity and Cytoskeleton Dynamics During Tip Growth in Root Hairs and Pollen Tubes.

Pollen tubes and root hairs are typical tip-growing cells and are employed as model systems to study plant cell polarity. Previous studies have shown that the Rho family ROP GTPase plays a critical role in the regulation of pollen tube and root hair growth. Periodically, activated ROP GTPase coordinates with the tip-focused calcium gradient, to regulate actin dynamics and vesicle trafficking. Moreover, microtubules are also involved in organelle movement and growth directionality. Here, we describe methods for analyzing the spatiotemporal localization and activity of ROP, cortical microtubule organization, and F-actin dynamics in pollen tubes and/or root hairs.

Poly(A) polymerase 1 contributes to competence acquisition of pollen tubes growing through the style in Arabidopsis thaliana.

Polyadenylation of mRNAs is critical for their export from the nucleus, stability, and efficient translation. The Arabidopsis thaliana genome encodes three isoforms of canonical nuclear poly(A) polymerase (PAPS) that redundantly polyadenylate the bulk of pre-mRNAs. However, previous studies have indicated that subsets of pre-mRNAs are preferentially polyadenylated by either PAPS1 or the other two isoforms. Such functional specialization raises the possibility of an additional level of gene-expression control in plants. Here we test this notion by studying the function of PAPS1 in pollen-tube growth and guidance. Pollen tubes growing through female tissue acquire the competence to find ovules efficiently and upregulate PAPS1 expression at the transcriptional, but not detectably at the protein level compared with in vitro grown pollen tubes. Using the temperature-sensitive paps1-1 allele we show that PAPS1 activity during pollen-tube growth is required for full acquisition of competence, resulting in inefficient fertilization by paps1-1 mutant pollen tubes. While these mutant pollen tubes grow almost at the wild-type rate, they are compromised in locating the micropyles of ovules. Previously identified competence-associated genes are less expressed in paps1-1 mutant than in wild-type pollen tubes. Estimating the poly(A) tail lengths of transcripts suggests that polyadenylation by PAPS1 is associated with reduced transcript abundance. Our results therefore suggest that PAPS1 plays a key role in the acquisition of competence and underline the importance of functional specialization between PAPS isoforms throughout different developmental stages.

Depletion plays a pivotal role in self-incompatibility, revealing a link between cellular energy status, cytosolic acidification and actin remodelling in pollen tubes.

Self-incompatibility (SI) involves specific interactions during pollination to reject incompatible ('self') pollen, preventing inbreeding in angiosperms. A key event observed in pollen undergoing the Papaver rhoeas SI response is the formation of punctate F-actin foci. Pollen tube growth is heavily energy-dependent, yet ATP levels in pollen tubes have not been directly measured during SI. Here we used transgenic Arabidopsis lines expressing the Papaver pollen S-determinant to investigate a possible link between ATP levels, cytosolic pH ([pH]cyt ) and alterations to the actin cytoskeleton. We identify for the first time that SI triggers a rapid and significant ATP depletion in pollen tubes. Artificial depletion of ATP triggered cytosolic acidification and formation of actin aggregates. We also identify in vivo, evidence for a threshold [pH]cyt of 5.8 for actin foci formation. Imaging revealed that SI stimulates acidic cytosolic patches adjacent to the plasma membrane. In conclusion, this study provides evidence that ATP depletion plays a pivotal role in SI upstream of programmed cell death and reveals a link between the cellular energy status, cytosolic acidification and alterations to the actin cytoskeleton in regulating Papaver SI in pollen tubes.

Mechanics and hydraulics of pollen tube growth.

All kingdoms of life have evolved tip-growing cells able to mine their environment or deliver cargo to remote targets. The basic cellular processes supporting these functions are understood in increasing detail, but the multiple interactions between them lead to complex responses that require quantitative models to be disentangled. Here, I review the equations that capture the fundamental interactions between wall mechanics and cell hydraulics starting with a detailed presentation of James Lockhart's seminal model. The homeostatic feedbacks needed to maintain a steady tip velocity are then shown to offer a credible explanation for the pulsatile growth observed in some tip-growing cells. Turgor pressure emerges as a central variable whose role in the morphogenetic process has been a source of controversy for more than 50 yr. I argue that recasting Lockhart's work as a process of chemical stress relaxation can clarify how cells control tip growth and help us internalise the important but passive role played by turgor pressure in the morphogenetic process.

Proline-rich extensin-like receptor kinases PERK5 and PERK12 are involved in pollen tube growth.

Proline-rich extensin-like receptor kinases (PERKs) belong to the hydroxyproline-rich glycoprotein (HRGP) superfamily known to be involved in many plant developmental processes. Here, we characterized two pollen-expressed PERKs from Arabidopsis thaliana, PERK5 and PERK12. Pollen tube growth was impaired in single and double perk5-1 perk12-1 loss of function mutants, with an impact on seed production. When the segregation was analysed, a male gametophytic defect was found, indicating that perk5-1 and perk12-1 mutants carry deficient pollen transmission. Furthermore, perk5-1 perk12-1 displayed an excessive accumulation of pectins and cellulose at the cell wall of the pollen tubes. Our results indicate that PERK5 and PERK12 are necessary for proper pollen tube growth, highlighting their role in cell wall assembly and reactive oxygen species homeostasis.

Microfluidics-Based Bioassays and Imaging of Plant Cells.

Many plant processes occur in the context of and in interaction with a surrounding matrix such as soil (e.g. root growth and root-microbe interactions) or surrounding tissues (e.g. pollen tube growth through the pistil), making it difficult to study them with high-resolution optical microscopy. Over the past decade, microfabrication techniques have been developed to produce experimental systems that allow researchers to examine cell behavior in microstructured environments that mimic geometrical, physical and/or chemical aspects of the natural growth matrices and that cannot be generated using traditional agar plate assays. These microfabricated environments offer considerable design flexibility as well as the transparency required for high-resolution, light-based microscopy. In addition, microfluidic platforms have been used for various types of bioassays, including cellular force assays, chemoattraction assays and electrotropism assays. Here, we review the recent use of microfluidic devices to study plant cells and organs, including plant roots, root hairs, moss protonemata and pollen tubes. The increasing adoption of microfabrication techniques by the plant science community may transform our approaches to investigating how individual plant cells sense and respond to changes in the physical and chemical environment.

Measuring Extracellular Proton and Anionic Fluxes in Arabidopsis Pollen Tubes.

The ion-selective vibrating probe has been used to detect and quantify the magnitude and direction of transmembrane fluxes of several ions in a wide range of biological systems. Inherently non-invasive, vibrating probes have been essential to access relevant electrophysiological parameters related to apical growth and morphogenesis in pollen tubes, a highly specialized cell where spatiotemporal tuning of ion dynamics is fundamental. Of relevance, crucial processes to the cell physiology of pollen tubes associated with protons and anions have been elucidated using vibrating probes, allowing the identification of diverse molecular players underlying and regulating their extracellular fluxes. The use of Arabidopsis thaliana as a genetic model system posed new challenges given their relatively small dimensions and difficult manipulation in vitro. Here, we describe protocol optimizations that made the use of the ion-selective vibrating probe in Arabidopsis pollen tubes feasible, ensuring consistent and reproducible data. Quantitative methods like this enabled characterizing phenotypes of ion transporter mutants, which are not directly detectable by evident morphological and reproductive defects, providing valuable insights into molecular and cellular mechanisms. The protocol for quantifying extracellular proton and anionic fluxes detailed here can be adjusted to other systems and species, while the sample preparation can be applied to correlated techniques, facilitating the research of pollen tube growth and development.

Membrane receptor-mediated mechano-transduction maintains cell integrity during pollen tube growth within the pistil.

Invasive or penetrative growth is critical for developmental and reproductive processes (e.g., pollen tube penetration of pistils) and disease progression (e.g., cancer metastasis and fungal hyphae invasion). The invading or penetrating cells experience drastic changes in mechanical pressure from the surroundings and must balance growth with cell integrity. Here, we show that Arabidopsis pollen tubes sense and/or respond to mechanical changes via a cell-surface receptor kinase Buddha's Paper Seal 1 (BUPS1) while emerging from compressing female tissues. BUPS1-defective pollen tubes fail to maintain cell integrity after emergence from these tissues. The mechano-transduction function of BUPS1 is established by using a microfluidic channel device mimicking the mechanical features of the in vivo growth path. BUPS1-based mechano-transduction activates Rho-like GTPase from Plant 1 (ROP1) GTPase to promote exocytosis that facilitates secretion of BUPS1's ligands for mechanical signal amplification and cell wall rigidification in pollen tubes. These findings uncover a membrane receptor-based mechano-transduction system for cells to cope with the physical challenges during invasive or penetrative growth.

Recent advances in understanding the biological roles of the plant nuclear envelope.

The functional organization of the plant nuclear envelope is gaining increasing attention through new connections made between nuclear envelope-associated proteins and important plant biological processes. Animal nuclear envelope proteins play roles in nuclear morphology, nuclear anchoring and movement, chromatin tethering and mechanical signaling. However, how these roles translate to functionality in a broader biological context is often not well understood. A surprising number of plant nuclear envelope-associated proteins are plant-unique, suggesting that separate functionalities evolved after the split of Opisthokonta and Streptophyta. Significant progress has now been made in discovering broader biological roles of plant nuclear envelope proteins, increasing the number of known plant nuclear envelope proteins, and connecting known proteins to chromatin organization, gene expression, and the regulation of nuclear calcium. The interaction of viruses with the plant nuclear envelope is another emerging theme. Here, we survey the recent developments in this still relatively new, yet rapidly advancing field.

Diversification of crown group Araucaria: the role of Araucaria famii sp. nov. in the mid-Cretaceous (Campanian) radiation of Araucariaceae in the Northern Hemisphere.

PREMISE: Exceptional anatomical preservation of a fossil araucarian seed cone from a marine carbonate concretion from Vancouver Island, British Columbia, Canada provides unusually complete evidence for cone structure including seeds, megagametophytes, microgametophytes, and embryos of an Upper Cretaceous (Campanian) species of Araucaria, providing important new insights into the structure and relationships of Cretaceous Northern Hemisphere Araucariaceae. METHODS: The cone was studied from serial thin sections prepared by the coal ball peel technique. Phylogenetic analysis using a modified morphological matrix with both discrete and continuous characters was performed using TNT version 1.5. RESULTS: The nearly spherical cone, 6 × 6 cm in diameter, has helically arranged cone-scale complexes, consisting of a large bract with an upturned tip and a small, fleshy ovuliferous scale. Vascularization of the cone-scale complex is single at its origin. Widely winged bracts, with a bulging base, contain numerous vascular bundles, interspersed with transfusion tissue, and a large number of resin canals. Seeds are ovoid, 1.2 cm long, 1.2 cm in diameter. Nucellus is free from the integument, except at its base, with a convoluted apex, containing possible pollen tubes. Megagametophytes and mature cellular embryos occur in several seeds. CONCLUSIONS: This small cone with attached, imbricate leaves, wide bracts, and unusually large seeds, most closely resembles those of Araucaria Section Eutacta. Width and continuity of secondary xylem in the cone axis, and intact cone-scale complexes indicate that this cone probably did not disarticulate readily at maturity. When added to a modified, previously published phylogenetic analysis, Araucaria famii sp. nov. enhances our understanding of the Cretaceous radiation of Northern Hemisphere Araucaria Section Eutacta.

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.

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.