AMEBaS: Automatic Midline Extraction and Background Subtraction of Ratiometric Fluorescence Time-Lapses of Polarized Single Cells.

Cell polarity is a macroscopic phenomenon established by a collection of spatially concentrated molecules and structures that culminate in the emergence of specialized domains at the subcellular level. It is associated with developing asymmetric morphological structures that underlie key biological functions such as cell division, growth, and migration. In addition, the disruption of cell polarity has been linked to tissue-related disorders such as cancer and gastric dysplasia. Current methods to evaluate the spatiotemporal dynamics of fluorescent reporters in individual polarized cells often involve manual steps to trace a midline along the cells' major axis, which is time consuming and prone to strong biases. Furthermore, although ratiometric analysis can correct the uneven distribution of reporter molecules using two fluorescence channels, background subtraction techniques are frequently arbitrary and lack statistical support. This manuscript introduces a novel computational pipeline to automate and quantify the spatiotemporal behavior of single cells using a model of cell polarity: pollen tube/root hair growth and cytosolic ion dynamics. A three-step algorithm was developed to process ratiometric images and extract a quantitative representation of intracellular dynamics and growth. The first step segments the cell from the background, producing a binary mask through a thresholding technique in the pixel intensity space. The second step traces a path through the midline of the cell through a skeletonization operation. Finally, the third step provides the processed data as a ratiometric timelapse and yields a ratiometric kymograph (i.e., a 1D spatial profile through time). Data from ratiometric images acquired with genetically encoded fluorescent reporters from growing pollen tubes were used to benchmark the method. This pipeline allows for faster, less biased, and more accurate representation of the spatiotemporal dynamics along the midline of polarized cells, thus advancing the quantitative toolkit available to investigate cell polarity. The AMEBaS Python source code is available at: https://github.com/badain/amebas.git.

Electrifying rhythms in plant cells.

Physiological oscillations (or rhythms) pervade all spatiotemporal scales of biological organization, either because they perform critical functions or simply because they can arise spontaneously and may be difficult to prevent. Regardless of the case, they reflect regulatory relationships between control points of a given system and offer insights as read-outs of the concerted regulation of a myriad of biological processes. Here we review recent advances in understanding ultradian oscillations (period < 24h) in plant cells, with a special focus on single-cell oscillations. Ion channels are at the center stage due to their involvement in electrical/excitabile phenomena associated with oscillations and cell-cell communication. We highlight the importance of quantitative approaches to measure oscillations in appropriate physiological conditions, which are essential strategies to deal with the complexity of biological rhythms. Future development of optogenetics techniques in plants will further boost research on the role of membrane potential in oscillations and waves across multiple cell types.

Spatiotemporal Quantification of Cytosolic pH in Arabidopsis Pollen Tubes.

Ion-specific probes and fluorescent indicators have been key in establishing the role of ion signaling, namely calcium, protons, and anions, in plant development, providing a robust approach for monitoring spatiotemporal changes in intracellular ion dynamics. The integration of protons/pH in signaling mechanisms is especially important as reports of their biological functions continue to expand; however, attaining quantitative estimates with high spatiotemporal resolution in single cells poses a major research challenge. Here, we detail the use of the genetically encoded pH-sensitive pHluorin reporter expressed in Arabidopsis thaliana pollen tubes to assess cytosolic measurements with calibration to provide actual pH values. This technique enabled us to identify critical phenotypes and establish the importance of tip-focused pH gradient for pollen tube growth, although it can be adapted to other experimental systems.

Structure of the Arabidopsis thaliana glutamate receptor-like channel GLR3.4.

Glutamate receptor-like channels (GLRs) play vital roles in various physiological processes in plants, such as wound response, stomatal aperture control, seed germination, root development, innate immune response, pollen tube growth, and morphogenesis. Despite the importance of GLRs, knowledge about their molecular organization is limited. Here we use X-ray crystallography and single-particle cryo-EM to solve structures of the Arabidopsis thaliana GLR3.4. Our structures reveal the tetrameric assembly of GLR3.4 subunits into a three-layer domain architecture, reminiscent of animal ionotropic glutamate receptors (iGluRs). However, the non-swapped arrangement between layers of GLR3.4 domains, binding of glutathione through S-glutathionylation of cysteine C205 inside the amino-terminal domain clamshell, unique symmetry, inter-domain interfaces, and ligand specificity distinguish GLR3.4 from representatives of the iGluR family and suggest distinct features of the GLR gating mechanism. Our work elaborates on the principles of GLR architecture and symmetry and provides a molecular template for deciphering GLR-dependent signaling mechanisms in plants.

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.

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.

Plasma membrane H+-ATPases sustain pollen tube growth and fertilization.

Pollen tubes are highly polarized tip-growing cells that depend on cytosolic pH gradients for signaling and growth. Autoinhibited plasma membrane proton (H+) ATPases (AHAs) have been proposed to energize pollen tube growth and underlie cell polarity, however, mechanistic evidence for this is lacking. Here we report that the combined loss of AHA6, AHA8, and AHA9 in Arabidopsis thaliana delays pollen germination and causes pollen tube growth defects, leading to drastically reduced fertility. Pollen tubes of aha mutants had reduced extracellular proton (H+) and anion fluxes, reduced cytosolic pH, reduced tip-to-shank proton gradients, and defects in actin organization. Furthermore, mutant pollen tubes had less negative membrane potentials, substantiating a mechanistic role for AHAs in pollen tube growth through plasma membrane hyperpolarization. Our findings define AHAs as energy transducers that sustain the ionic circuit defining the spatial and temporal profiles of cytosolic pH, thereby controlling downstream pH-dependent mechanisms essential for pollen tube elongation, and thus plant fertility.

Arabidopsis DIACYLGLYCEROL KINASE4 is involved in nitric oxide-dependent pollen tube guidance and fertilization.

Nitric oxide (NO) is a key signaling molecule that regulates diverse biological processes in both animals and plants, including important roles in male gamete physiology. In plants, NO is generated in pollen tubes (PTs) and affects intracellular responses through the modulation of Ca2+ signaling, actin organization, vesicle trafficking and cell wall deposition, bearing consequences in pollen-stigma interactions and PT guidance. In contrast, the NO-responsive proteins that mediate these responses remain elusive. Here, we show that PTs of Arabidopsis thaliana mutants impaired in the pollen-specific DIACYLGLYCEROL KINASE4 (DGK4) grow slower and become partially insensitive to NO-dependent growth inhibition and re-orientation responses. Recombinant DGK4 protein yields NO-responsive spectral and catalytic changes in vitro that are compatible with a role in NO perception and signaling in PTs. In addition to the expected phosphatidic acid-producing kinase activity, DGK4 recombinant protein also revealed guanylyl cyclase activity, as inferred by sequence analysis. Our results are compatible with a role for the fast-diffusible NO gas in signaling and cell-cell communication via the modulation of DGK4 activity during the progamic phase of angiosperm reproduction.

Molecular and electrophysiological characterization of anion transport in Arabidopsis thaliana pollen reveals regulatory roles for pH, Ca2+ and GABA.

We investigated the molecular basis and physiological implications of anion transport during pollen tube (PT) growth in Arabidopsis thaliana (Col-0). Patch-clamp whole-cell configuration analysis of pollen grain protoplasts revealed three subpopulations of anionic currents differentially regulated by cytoplasmic calcium ([Ca2+ ]cyt ). We investigated the pollen-expressed proteins AtSLAH3, AtALMT12, AtTMEM16 and AtCCC as the putative anion transporters responsible for these currents. AtCCC-GFP was observed at the shank and AtSLAH3-GFP at the tip and shank of the PT plasma membrane. Both are likely to carry the majority of anion current at negative potentials, as extracellular anionic fluxes measured at the tip of PTs with an anion vibrating probe were significantly lower in slah3-/- and ccc-/- mutants, but unaffected in almt12-/- and tmem16-/- . We further characterised the effect of pH and GABA by patch clamp. Strong regulation by extracellular pH was observed in the wild-type, but not in tmem16-/- . Our results are compatible with AtTMEM16 functioning as an anion/H+ cotransporter and therefore, as a putative pH sensor. GABA presence: (1) inhibited the overall currents, an effect that is abrogated in the almt12-/- and (2) reduced the current in AtALMT12 transfected COS-7 cells, strongly suggesting the direct interaction of GABA with AtALMT12. Our data show that AtSLAH3 and AtCCC activity is sufficient to explain the major component of extracellular anion fluxes, and unveils a possible regulatory system linking PT growth modulation by pH, GABA, and [Ca2+ ]cyt through anionic transporters.

CORNICHON sorting and regulation of GLR channels underlie pollen tube Ca2+ homeostasis.

Compared to animals, evolution of plant calcium (Ca2+) physiology has led to a loss of proteins for influx and small ligand-operated control of cytosolic Ca2+, leaving many Ca2+ mechanisms unaccounted for. Here, we show a mechanism for sorting and activation of glutamate receptor-like channels (GLRs) by CORNICHON HOMOLOG (CNIH) proteins. Single mutants of pollen-expressed Arabidopsis thaliana GLRs (AtGLRs) showed growth and Ca2+ flux phenotypes expected for plasma membrane Ca2+ channels. However, higher-order mutants of AtGLR3.3 revealed phenotypes contradicting this assumption. These discrepancies could be explained by subcellular AtGLR localization, and we explored the implication of AtCNIHs in this sorting. We found that AtGLRs interact with AtCNIH pairs, yielding specific intracellular localizations. AtCNIHs further trigger AtGLR activity in mammalian cells without any ligand. These results reveal a regulatory mechanism underlying Ca2+ homeostasis by sorting and activation of AtGLRs by AtCNIHs.

Oscillatory signatures underlie growth regimes in Arabidopsis pollen tubes: computational methods to estimate tip location, periodicity, and synchronization in growing cells.

Oscillations in pollen tubes have been reported for many cellular processes, including growth, extracellular ion fluxes, and cytosolic ion concentrations. However, there is a shortage of quantitative methods to measure and characterize the different dynamic regimes observed. Herein, a suite of open-source computational methods and original algorithms were integrated into an automated analysis pipeline that we employed to characterize specific oscillatory signatures in pollen tubes of Arabidopsis thaliana (Col-0). Importantly, it enabled us to detect and quantify a Ca2+ spiking behaviour upon growth arrest and synchronized oscillations involving growth, extracellular H+ fluxes, and cytosolic Ca2+, providing the basis for novel hypotheses. Our computational approach includes a new tip detection method with subpixel resolution using linear regression, showing improved ability to detect oscillations when compared to currently available methods. We named this data analysis pipeline 'Computational Heuristics for Understanding Kymographs and aNalysis of Oscillations Relying on Regression and Improved Statistics', or CHUKNORRIS. It can integrate diverse data types (imaging, electrophysiology), extract quantitative and time-explicit estimates of oscillatory characteristics from isolated time series (period and amplitude) or pairs (phase relationships and delays), and evaluate their synchronization state. Here, its performance is tested with ratiometric and single channel kymographs, ion flux data, and growth rate analysis.

Pollen tube growth regulation by free anions depends on the interaction between the anion channel SLAH3 and calcium-dependent protein kinases CPK2 and CPK20.

Apical growth in pollen tubes (PTs) is associated with the presence of tip-focused ion gradients and fluxes, implying polar localization or regulation of the underlying transporters. The molecular identity and regulation of anion transporters in PTs is unknown. Here we report a negative gradient of cytosolic anion concentration focused on the tip, in negative correlation with the cytosolic Ca(2+) concentration. We hypothesized that a possible link between these two ions is based on the presence of Ca(2+)-dependent protein kinases (CPKs). We characterized anion channels and CPK transcripts in PTs and analyzed their localization. Yellow fluorescent protein (YFP) tagging of a homolog of SLOW ANION CHANNEL-ASSOCIATED1 (SLAH3:YFP) was widespread along PTs, but, in accordance with the anion efflux, CPK2/CPK20/CPK17/CPK34:YFP fluorescence was strictly localized at the tip plasma membrane. Expression of SLAH3 with either CPK2 or CPK20 (but not CPK17/CPK34) in Xenopus laevis oocytes elicited S-type anion channel currents. Interaction of SLAH3 with CPK2/CPK20 (but not CPK17/CPK34) was confirmed by Förster-resonance energy transfer fluorescence lifetime microscopy in Arabidopsis thaliana mesophyll protoplasts and bimolecular fluorescence complementation in living PTs. Compared with wild-type PTs, slah3-1 and slah3-2 as well as cpk2-1 cpk20-2 PTs had reduced anion currents. Double mutant cpk2-1 cpk20-2 and slah3-1 PTs had reduced extracellular anion fluxes at the tip. Our studies provide evidence for a Ca(2+)-dependent CPK2/CPK20 regulation of the anion channel SLAH3 to regulate PT growth.

Glutamate receptor-like genes form Ca2+ channels in pollen tubes and are regulated by pistil D-serine.

Elevations in cytosolic free calcium concentration ([Ca(2+)](cyt)) constitute a fundamental signal transduction mechanism in eukaryotic cells, but the molecular identity of Ca(2+) channels initiating this signal in plants is still under debate. Here, we show by pharmacology and loss-of-function mutants that in tobacco and Arabidopsis, glutamate receptor-like channels (GLRs) facilitate Ca(2+) influx across the plasma membrane, modulate apical [Ca(2+)](cyt) gradient, and consequently affect pollen tube growth and morphogenesis. Additionally, wild-type pollen tubes grown in pistils of knock-out mutants for serine-racemase (SR1) displayed growth defects consistent with a decrease in GLR activity. Our findings reveal a novel plant signaling mechanism between male gametophyte and pistil tissue similar to amino acid-mediated communication commonly observed in animal nervous systems.

Low temperature and defoliation affect fructan-metabolizing enzymes in different regions of the rhizophores of Vernonia herbacea.

In addition to the storage function, fructans in Asteraceae from floras with seasonal growth have been associated with drought and freezing tolerance. Vernonia herbacea, native of the Brazilian Cerrado, bears underground reserve organs, rhizophores, accumulating inulin-type fructans. The rhizophore is a cauline branched system with positive geotropic growth, with the apex (distal region) presenting younger tissues; sprouting of new shoots occurs by development of buds located on the opposite end (proximal region). Plants induced to sprouting by excision of the aerial organs present increased 1-fructan exohydrolase (1-FEH) activity in the proximal region, while plants at the vegetative stage present high 1-sucrose:sucrose fructosyltransferase (1-SST) in the distal region. The aim of the present study was to analyze how low temperature (5 degrees C) could affect fructan-metabolizing enzymes and fructan composition in the different regions of the rhizophores of intact and excised plants. 1-SST and 1-fructan:fructan fructosyltransferase (1-FFT) were higher in the distal region decreasing towards the proximal region in intact plants at the vegetative phase, and were drastically diminished when cold and/or excision were imposed. In contrast, 1-FEH increased in the proximal region of treated plants, mainly in excised plants subjected to cold. The ratio fructo-oligo to fructo-polysaccharides was significantly higher in plants exposed to low temperature (1.17 in intact plants and 1.64 in excised plants) than in plants exposed to natural temperature conditions (0.84 in intact vegetative plants and 0.58 in excised plants), suggesting that oligosaccharides are involved in the tolerance of plants to low temperature via 1-FEH, in addition to 1-FFT. Principal component analysis indicated different response mechanisms in fructan metabolism under defoliation and low temperature, which could be interpreted as part of the strategies to undergo unfavorable environmental conditions prevailing in the Cerrado during winter.