Systemic Calcium Wave Propagation in Physcomitrella patens

Systemic Calcium Wave Propagation in Physcomitrella patens Abstract The adaptation to dehydration and rehydration cycles represents a key step in the evolution of photosynthetic organisms and requires the development of mechanisms by which to sense external stimuli and translate them into signaling components. In this study, we used genetically encoded fluorescent sensors to detect specific transient increases in the Ca2+ concentration in the moss Physcomitrella patens upon dehydration and rehydration treatment. Observation of the entire plant in a single time-series acquisition revealed that various cell types exhibited different sensitivities to osmotic stress and that Ca2+ waves originated from the basal part of the gametophore and were directionally propagated towards the top of the plant. Under similar conditions, the vascular plant Arabidopsis thaliana exhibited Ca2+ waves that propagated at a higher speed than those of P. patens. Our results suggest that systemic Ca2+ propagation occurs in plants even in the absence of vascular tissue, even though the rates can be different. Introduction Land plants form a monophyletic clade that evolved from freshwater charophyte macroalgae approximately 450 million years ago (Bowman 2013, de Vries et al. 2016). Land colonization by plants represents one of the most significant evolutionary events in the history of life on planet Earth and had a major impact on the Earth’s biosphere. During adaptation to their new environment, plants acquired the ability to survive cycles of dehydration and rehydration (Oliver et al. 2005), as well as exposure to stronger mechanical forces and more oxidative conditions (i.e. high light intensities, light enriched in UV wavelengths and high oxygen availability) (Kenrick and Crane 1997, Dahl et al. 2010). Because they diverged from their angiosperm ancestors soon after land colonization, bryophytes are a useful model system for the identification of early plant adaptations to the terrestrial environment. In particular, the moss Physcomitrella patens is used as a model plant for the study of evolution and development due to its evolutionary position and the availability of several molecular tools (Cove 2005, Rensing et al. 2008). The perception of external stimuli is pivotal for plant fitness, and these pathways probably involve calcium (Ca2+), a common second messenger that triggers cell responses in prokaryotes and eukaryotes during development, stress and long-distance signal transduction. Different external stimuli cause Ca2+ transient increases, waves and oscillations that are interpreted by cells as signals themselves (Whalley and Knight 2013, Choi et al. 2017). Ca2+ measurement in P. patens plants has been successfully performed using ratiometric dyes (Tucker et al. 2005, Qudeimat et al. 2008), the Ca2+-sensitive genetically encoded sensor aequorin from Aequorea victoria (Haley et al. 1995, Russell et al. 1996, Saidi et al. 2009) and GCaMP3 (Kleist et al. 2017). This allowed the assessment of the role of Ca2+ in the regulation of physiological and developmental processes at the level of protonemata cells in P. patens, including the UV-A response (Tucker et al. 2005) and thermal sensing mediated by cyclic nucleotide-gated calcium channels (CNGCs) (Finka et al. 2012). Moreover, two glutamate receptor-like ion channels (GLRs) were shown to be involved in the regulation of the cytoplasmic Ca2+ concentration in P. patens sperm cells. Changes in intracellular Ca2+ levels modulate sperm orientation towards the archegonium, thereby affecting the overall fertility of the plant (Ortiz-Ramírez et al. 2017). In this work, Cameleon YC3.60 (Nagai et al. 2004), a Förster resonance energy transfer (FRET)-based genetically encoded fluorescent sensor, was exploited to monitor the Ca2+ concentration in vivo with high spatiotemporal resolution inside different cell types and compartments of P. patens plants during osmotic stress. Results and Discussion The Cameleon YC3.60 sensor was targeted to the nucleoplasm, mitochondria and cytoplasm using specific N-terminal transit peptides or proteins previously utilized in Arabidopsis thaliana (Krebs et al. 2012, Loro et al. 2012, Costa et al. 2017). The presence of a clear homogeneous fluorescence signal in the expected compartment was verified by confocal laser scanning microscopy (Fig. 1a;Supplementary Fig. S1). The stability of fluorescent protein expression and the absence of major interference with cell physiology were verified by assessing plant growth, development and photosynthetic efficiency. Transgenic lines affected by the expression of the sensor were not used in further analyses (refer to Supplementary Fig. S1). Fig. 1 View largeDownload slide Expression of Yellow Cameleon 3.60 (YC3.60) and analysis of Ca2+ dynamics in Physcomitrella patens protonema cells. (a) The YC3.60 sensor was expressed under control of the constitutive 7113 promoter and was targeted to the cytoplasm. Chl and YC3.60 signals were independently acquired by confocal laser scanning microscopy and were then merged. Scale bar = 10 μm. (b, c) Changes in Ca2+ concentration in protonema cells in response to osmotic stress. CFP and cpVenus emissions were detected simultaneously after CFP excitation, and the cpVenus/CFP fluorescent ratio over that time is expressed as ΔR/R0. Osmotic stress was applied by continuous perfusion with solutions containing 400, 500 or 750 mM mannitol, which are represented by red, blue and black lines, respectively. The white and red bars on top of the panel indicate the absence and the presence, respectively, of mannitol in the perfusion solution. Data are shown as the mean of at least four independent plants. Fig. 1 View largeDownload slide Expression of Yellow Cameleon 3.60 (YC3.60) and analysis of Ca2+ dynamics in Physcomitrella patens protonema cells. (a) The YC3.60 sensor was expressed under control of the constitutive 7113 promoter and was targeted to the cytoplasm. Chl and YC3.60 signals were independently acquired by confocal laser scanning microscopy and were then merged. Scale bar = 10 μm. (b, c) Changes in Ca2+ concentration in protonema cells in response to osmotic stress. CFP and cpVenus emissions were detected simultaneously after CFP excitation, and the cpVenus/CFP fluorescent ratio over that time is expressed as ΔR/R0. Osmotic stress was applied by continuous perfusion with solutions containing 400, 500 or 750 mM mannitol, which are represented by red, blue and black lines, respectively. The white and red bars on top of the panel indicate the absence and the presence, respectively, of mannitol in the perfusion solution. Data are shown as the mean of at least four independent plants. Drought and salt stresses are major abiotic constraints affecting plant growth and productivity. The acquisition of tolerance to dynamic water availability is considered a keystone in land colonization (Rensing et al. 2008). Ca2+ concentration in response to salt and osmotic stress was assessed in vivo by imaging analyses of YC3.60 transgenic lines. Protonema cells were treated with various concentrations of NaCl, sorbitol and mannitol, and then the osmotic stress was released. Cyan fluorescent protein (CFP) and circularly permutated Venus (cpVenus) fluorescence signals were simultaneously acquired for the duration of the treatment to detect Ca2+-induced FRET transient efficiency change (Fig. 1b;Supplementary Fig. S2). Both the addition (hyperosmotic treatment) and removal of the osmoticum (hypo-osmotic treatment; mannitol was used) caused a detectable increase in Ca2+ concentration in various cell compartments, as shown by the clear change in ΔR/R0 (Fig. 1c;Supplementary Fig. S1). For all compartments, the transient increase in Ca2+ was more intense during the hypo-osmotic response than during the hyperosmotic response (Fig. 1c;Supplementary Fig. S1; Supplementary Table S1). Vicia faba and A. thaliana guard cells, as well as A. thaliana leaves and roots, exhibited similar trends (Hayashi et al. 2006, Zhang et al. 2007, Loro et al. 2012), suggesting that the strong hypo-osmotic response is highly conserved. The detected Ca2+ transients exhibited different intensities and kinetics depending on the mannitol concentration used (400, 500 or 750 mM) and on the cellular compartments examined, revealing that Ca2+ dynamics are differentiated within the cell and also transduce information regarding stress severity (Fig. 1c; Supplementary Figs. S1, S3; Supplementary Table S1). Control experiments run in the absence of mannitol showed that the perfusion solution itself had no effect on Ca2+ dynamics (Supplementary Fig. S4). In vivo imaging analyses were implemented using lower magnification to observe more extended portions of the sample and different cell types simultaneously. It was possible to observe entire young P. patens gametophores, a significant advantage over A. thaliana, Oryza sativa and other plants of greater size in which the Cameleon sensor has already been expressed. Lines expressing YC3.60 in the cytoplasm were used preferentially because they showed more homogeneous and stable expression of the sensor in different cell types, especially old gametophores (Supplementary Fig. S5). Similar to previous observations in protonema, the exposure of entire young gametophores to 500 mM mannitol generated a concomitant increase in cpVenus fluorescence emission and a decrease in CFP fluorescence emission (Fig. 2b;Supplementary Videos S2, S3). The most intense Ca2+ transient was recorded again after the relief of the osmotic stress (Fig. 2b). The Ca2+ response, however, was not homogeneous throughout the plant but was more intense in the basal part of the gametophore than in the phyllids, demonstrating that different cell types have different sensitivities to the same stimulus (Fig. 2c, d;Supplementary Video S1). An independent quantitative analysis of the gametophore base and phyllids confirmed that Ca2+ responses are more intense in the former but are present in the latter, exhibiting a delay of approximately 1 min before reaching maximal signal intensity (Fig. 2d). Of note, our calibration of Ca2+ estimates that the maximum concentration of cytosolic Ca2+ at the base of the gametophore (634 ± 28 nM) was almost four times higher than in phyllids (152 ± 32) nM. Also in the case of gametophores, control experiments run in the absence of mannitol showed that the perfusion solution itself had no effect on Ca2+ response (Supplementary Fig. S6). Fig. 2 View largeDownload slide In vivo analysis of Ca2+ dynamics in response to osmotic stress throughout P. patens gametophores expressing CYT–YC3.60. (a) Representative snapshot of one gametophore; yellow boxes correspond to the regions of interests (ROIs 1–8) outlined to follow CFP and cpVenus fluorescence emission with time. (b) The signal is the result of the average signal emitted in ROIs 1–4 (left graph, base) and ROIs 5–8 (right graph, phyllid). (c) Snapshots of fluorescence ratio imaging acquisition during continuous perfusion with hyperosmotic (6 and 10 min) and hypo-osmotic (1, 13 and 28 min) solutions. False colors reflect the ratio of the cpVenus fluorescence emission to the CFP fluorescence emission at the specified time point. Scale bar = 500 μm. (d) Quantitative analysis of cpVenus/CFP fluorescent ratio measurements calculated as ΔR/R0 for the gametophore base and phyllids shown in (a–c). White and red bars on top of each graph indicate the presence and the absence of mannitol, respectively. The full acquisition cpVenus, CFP and cpVenus/CFP can be visualized as supplementary material (Supplementary Videos S1–S3). Fig. 2 View largeDownload slide In vivo analysis of Ca2+ dynamics in response to osmotic stress throughout P. patens gametophores expressing CYT–YC3.60. (a) Representative snapshot of one gametophore; yellow boxes correspond to the regions of interests (ROIs 1–8) outlined to follow CFP and cpVenus fluorescence emission with time. (b) The signal is the result of the average signal emitted in ROIs 1–4 (left graph, base) and ROIs 5–8 (right graph, phyllid). (c) Snapshots of fluorescence ratio imaging acquisition during continuous perfusion with hyperosmotic (6 and 10 min) and hypo-osmotic (1, 13 and 28 min) solutions. False colors reflect the ratio of the cpVenus fluorescence emission to the CFP fluorescence emission at the specified time point. Scale bar = 500 μm. (d) Quantitative analysis of cpVenus/CFP fluorescent ratio measurements calculated as ΔR/R0 for the gametophore base and phyllids shown in (a–c). White and red bars on top of each graph indicate the presence and the absence of mannitol, respectively. The full acquisition cpVenus, CFP and cpVenus/CFP can be visualized as supplementary material (Supplementary Videos S1–S3). The delay measured in phyllids was due to the directional propagation of Ca2+ transients from the base towards the tip of the plant (Fig. 3a–e;Supplementary Videos S4, S5). The directionality of propagation did not depend on the orientation of the plant with respect to the point of stress application. In Supplementary Video S4, the lower portion of the gametophore is closer to the entry point of the buffer (Fig. 3a), while in Supplementary Video S5 the orientation is reversed (Fig. 3b). In both cases, however, Ca2+ waves emanated from the lower part of the gametophore and propagated towards the phyllid. The dynamics of the osmo-induced Ca2+ increase were also analyzed independently at the base of the gametophore (Supplementary Fig. S7) and in phyllids (Fig. 3c–e). We also examined the same Ca2+ waves at the highest magnification available and found that only a few cells were in focus, thus precluding an analysis of long-distance transmission (Supplementary Fig. S8). In order to understand if a local mannitol stimulus could elicit Ca2+ propagation throughout gametophores, a local stress system was set up, in which gametophores were covered by a block of agarized perfusion solution and then subjected to the perfusion experiment (Supplementary Fig. S9). Even if the upper part of the plant was in less direct contact with mannitol solution, we still observed a retarded increase in cytoplasmic Ca2+ concentration in phyllids as compared with the base of the gametophore, suggesting the existence of a proper long-distance Ca2+ wave propagation in P. patens. Fig. 3 View largeDownload slide In vivo analysis of Ca2+ dynamics in response to osmotic stress in young Physcomitrella patens plantlets. (a, b) The response of two representative gametophores was in the same direction as or the direction opposing osmotic flow. From top to bottom, a sketch representing the direction of flow in the plant, and three snapshots of fluorescence ratio imaging acquisition corresponding to 0, 13.5 and 19 min. (c) Detail of one phyllid shown in (a). White dashed lines correspond to the regions of interest (ROIs 1–10) designated to follow CFP and cpVenus fluorescence emission during the indicated time. (d) Ca2+ concentration variation (ΔR/R0) during the hypo-osmotic phase. ROIs from 1 to 10 correspond to those drawn in (c). (e) Ratiometric false-color images from representative time series of a basal portion of a P. patens gametophore. The false colors represent the intensity of the cpVenus/CFP fluorescent ratio. The time (min) below each snapshot corresponds to the signal reported in (d). The full acquisitions are included as supplementary material (Supplementary Videos S4, S5). Scale bar = 250 μm. Ratio images are representative of n ≥ 3. Fig. 3 View largeDownload slide In vivo analysis of Ca2+ dynamics in response to osmotic stress in young Physcomitrella patens plantlets. (a, b) The response of two representative gametophores was in the same direction as or the direction opposing osmotic flow. From top to bottom, a sketch representing the direction of flow in the plant, and three snapshots of fluorescence ratio imaging acquisition corresponding to 0, 13.5 and 19 min. (c) Detail of one phyllid shown in (a). White dashed lines correspond to the regions of interest (ROIs 1–10) designated to follow CFP and cpVenus fluorescence emission during the indicated time. (d) Ca2+ concentration variation (ΔR/R0) during the hypo-osmotic phase. ROIs from 1 to 10 correspond to those drawn in (c). (e) Ratiometric false-color images from representative time series of a basal portion of a P. patens gametophore. The false colors represent the intensity of the cpVenus/CFP fluorescent ratio. The time (min) below each snapshot corresponds to the signal reported in (d). The full acquisitions are included as supplementary material (Supplementary Videos S4, S5). Scale bar = 250 μm. Ratio images are representative of n ≥ 3. Ca2+ waves are known to be involved in systemic acquired acclimation response to abiotic stress in A. thaliana ( Choi et al. 2014, Choi et al. 2017). Four-day-old A. thaliana plantlets expressing CYT–YC3.60 (Loro et al. 2012) (Supplementary Fig. S10; Supplementary Video S7) were treated with the same protocol used for P. patens for a direct comparison of their local and systemic Ca2+ responses. In this case, a Ca2+ wave was recorded and could be observed following the time needed to reach ΔRmax/R0 in four independent regions of interest (ROIs) from the root tip to the shoot (Supplementary Fig. S10). To better compare the Ca2+ propagation rates of the model plants A. thaliana and P. patens, ROIs of 400 μm were drawn and kymographs of the corresponding FRET signals were extrapolated (Fig. 4). In A. thaliana, Ca2+ waves moved at approximately 58.5 ± 18.9 μm s–1. In P. patens, both at the base of the gametophore and in phyllids, the speed of propagation was instead 4.5 ± 3.8 μm s–1, a value compatible with what has been measured using Fura-2-dextran ratio images in protonema apical cells in response to UV-A light (3.4–8.4 μm s–1) (Tucker et al. 2005). It is interesting to note that with our set-up, we managed to measure cell–cell communication in entire gametophores rather than simply the communication between two adjacent cells. The fast propagation measured in A. thaliana (Fig. 4; Supplementary Fig. S10) is in agreement with what has been reported previously for different tissues and stressors. Ca2+ waves traveled at 60–290 μm s–1 along the leaf veins of A. thaliana after exposure of the roots of plants expressing a bioluminescence resonance energy transfer (BRET)-based green fluorescent protein (GFP)–aequorin reporter to salt stress (Xiong et al. 2014). The response was fastest (400 μm s–1) when measured through root tissues at lower salt concentrations (Choi et al. 2014). Thus, the main component of Ca2+ wave propagation in P. patens is slower than that measured in A. thaliana roots and leaves. In order to understand whether extracellular Ca2+ was involved in Ca2+ wave propagation, we performed experiments by perfusing plants with a low Ca2+ concentration and lanthanum (III) chloride, a blocker of non-selective cation channels and stretch-activated Ca2+-permeable channels (Supplementary Fig. S11). The application of lanthanum has effectively reduced the amplitude of the Ca2+ increase in response to osmotic stress relaxation. On the other hand, we could still detect the propagation of a long-distance Ca2+ wave (Supplementary Fig. S11), suggesting that intracellular Ca2+ storage is involved in this process or that there are Ca2+ channels/transporters at the level of the plasma membrane that are insensitive to lanthanum. Moreover the rate of propagation is similar to calcium-induced calcium release measured in single cells (Jaffe 2010). Further work may elucidate whether Ca2+ waves serve a signaling function in the moss system and to uncover the molecular system responsible for the propagation of Ca2+ waves. Different models have been proposed for the interaction of propagating Ca2+ signals with electric signals and reactive oxygen species (ROS) waves through symplastic or apoplastic pathways (Choi et al. 2014, Choi et al. 2017). Vasculature seems to be fundamental for the propagation of Ca2+ waves when the signal is triggered by wounding or herbivore attack (Mousavi et al. 2013, Kiep et al. 2015). Interestingly, the aphid Myzus persicae elicits elevated cytosolic Ca2+ concentrations around piercing sites in the epidermal and mesophyll cells of A. thaliana (Vincent et al. 2017). This phloem-independent radial propagation proceeds at a speed of approximately 6 μm s–1 over an area of approximately 110 μm2 (Vincent et al. 2017). In the case of roots, transmission is likely to be supported by the activity of Ca2+ pumps and channels and their regulators within cortical and endodermal cells (Choi et al. 2016). Differences in the speed of Ca2+ propagation could be due to the obvious lack in P. patens of a proper vascular tissue. However, it is possible that different systems control Ca2+ homeostasis in the two model species. This second hypothesis is supported by genomic surveys highlighting the existence of transport systems differing in composition and complexity compared with other members of the Viridiplantae lineage (Edel and Kudla 2015). For example, GLRs have been identified as potential mediators of systemic electric signals in A. thaliana (Mousavi et al. 2013, Salvador-Recatalà 2016). P. patens encodes a small family of GLRs consisting of only two members (Edel and Kudla 2015, De Bortoli et al. 2016) that are thought to be involved in plant reproduction rather than gametophytic cell–cell signal transduction (Ortiz-Ramírez et al. 2017). Fig. 4 View largeDownload slide Kymographs illustrating Ca2+ wave dynamics in Arabidopsis thaliana and Physcomitrella patens lines expressing the cytoplasmic sensor (CYT–YC3.60). Ratiometric data (cpVenus/CFP fluorescent ratio) were extracted from a region of interest (ROI) of 400 μm parallel to the plant axis. For A. thaliana, the last segment of root before the hypocotyl was considered (Supplementary Fig. S10), while for P. patens, gametophores and phyllids were analyzed independently. The arrows indicate the tipward direction followed to draw the ROIs. The white and red bars represent the absence and the presence, respectively, of mannitol in the perfusion solution. Fig. 4 View largeDownload slide Kymographs illustrating Ca2+ wave dynamics in Arabidopsis thaliana and Physcomitrella patens lines expressing the cytoplasmic sensor (CYT–YC3.60). Ratiometric data (cpVenus/CFP fluorescent ratio) were extracted from a region of interest (ROI) of 400 μm parallel to the plant axis. For A. thaliana, the last segment of root before the hypocotyl was considered (Supplementary Fig. S10), while for P. patens, gametophores and phyllids were analyzed independently. The arrows indicate the tipward direction followed to draw the ROIs. The white and red bars represent the absence and the presence, respectively, of mannitol in the perfusion solution. The present work reveals the existence of long-distance transmission of Ca2+ waves in response to an osmoticum in the body of P. patens. The ability to propagate systemic Ca2+ waves, an important component for osmotic stress perception and osmotolerance in general, was present during land colonization. Materials and Methods Plant material and growth conditions Protonemal tissue of P. patens (the Gransden wild-type strain and YC3.60-expressing lines) were grown on minimum PpNO3 medium under controlled conditions: 24°C, a 16 h light/8 h dark photoperiod and a light intensity of 50 µmol photons m–2 s–1. Ten-day-old plants were used for protonemal tissue imaging, and gametophores were observed between 1 and 2 months of growth. Arabidopsis thaliana plants expressing cytoplasmic Cameleon (CYT–YC3.60) were used in this study (Krebs et al. 2012). Seeds were surface-sterilized and sown on half-strength Murashige and Skoog medium (MS including vitamins; Duchefa) supplemented with 0.1% (w/v) sucrose and 0.05% (w/v) MES, adjusted to pH 5.8 with KOH, and solidified with 0.8% (w/v) plant agar (Duchefa). After stratification at 4°C in the dark for 2–3 d, the seeds were transferred to a growth chamber with a 16/8 h light cycle (70 μmol photons m–2 s–1) at 24°C. Constructs Standard plasmid construction methods were used to obtain pMAK1-7113::YC3.60 plasmids. All PCRs were carried out using high-fidelity KAPA HiFi PCR Kits (Kapa Biosystems), according to the manufacturer’s instructions. PCR products were cloned into a pJET1.2/blunt vector (Thermo Fisher Scientific) and were sequenced after transfer to the expression vector pMAK1 (http://www.nibb.ac.jp/evodevo/PHYSCOmanual/17Overdriveplasmid/pMAK1.htm) under control of the constitutive promoter 7113 (Mitsuhara et al. 1996, Gerotto et al. 2012). The primers used to amplify the sequences and add signal sequences for mitochondrial or nucleoplasmic subcellular localization are as follows: MIT/NUC-for (5'-CTCGAGAGAGGACAGCCCAAGCTTATG-3') and MIT/NUC-rev (5'-GGGCCCTTTCAGCGTACCGAATTCTTA-3'). The PCR product was cloned with XhoI and ApaI. The primers used to amplify the sequences with cytoplasmic subcellular localization were: CYT-for (5'-GGGCCCATGCTGCAGAACGAGCTTGCTCTT-3') and CYT-rev (5'-GAATTCGTGCCAAGCTTCGATTGATG-3'). The PCR product was cloned with ApaI and EcoRI. Plant expression vectors were used as templates to perform the PCRs (Krebs et al. 2012, Loro et al. 2012, Costa et al. 2017). The Tnos terminator sequence in the pMAK1 plasmid was used to control the expression of the mitochondrion- and nucleoplasm-localized versions of YC3.60. The cytoplasm-localized version of YC3.60 was amplified together with the rbcs terminator, which was substituted for the Tnos terminator sequence in the pMAK1 plasmid. YC3.60 is targeted to the nucleoplasm by its fusion with the nuclear-localized Xenopus laevis nucleophosmin, to the mitochondrial matrix by four repeats of the mitochondrial targeting sequence from subunit VIII of human Cyt c oxidase (MIT) and to the cytoplasm by a nuclear export signal (CYT) of rabbit heat-stable protein kinase inhibitor α. Chl fluorescence measurements and growth tests To test plant performance, 10-day-old plants grown on PpNO3 were transferred to new plates as multiple independent spots of 2 mm diameter. In vivo Chl fluorescence was measured using Fluorcam 800MF (Photon Sytems Instruments) to evaluate the Fv/Fm parameter as (Fm –F0)/Fm. Pictures of the same plates were taken regularly, and plant growth was evaluated by measuring the size of different plant spots using ImageJ software (http://rsb.info.nih.gov/ij/) after background exclusion was performed with the ‘TRESHOLD COLOUR’ PLUGIN (http://www.mecourse.com/landinig/software/software.html). Confocal microscopy Laser scanning confocal microscopy analyses were performed using a Leica SP5 imaging system (Leica Microsytems). Images were acquired using a ×40 objective (HCX PL APO CS 40/1.25–0.75 oil) with different degrees of digital zoom. cpVenus was excited by the 514 nm line of the argon laser and the emission was collected between 525 and 540 nm. Chl fluorescence emission was collected simultaneously between 660 and 750 nm. Images were merged using ImageJ software (http://rsb.info.nih.gov/ij/). FRET-based calcium imaging For FRET measurements, plant material was removed from the plate and placed in an open-top chamber overlaid by cotton soaked in imaging solution (5 mM KCl, 10 mM MES and 10 mM CaCl2; adjusted to pH 5.8 with Trizma® base). Osmotic stress was achieved by adding imaging solutions containing different concentrations of mannitol or NaCl, using a continuous perfusion system. After 8 min, the hyperosmotic solution was replaced by the initial imaging solution to restore the baseline condition. We ensured that the increase in Ca2+ concentration was the result of osmotic stress and not mechanical stimulation by performing experiments with and without osmoticum (Supplementary Figs. S4, S6). Images were collected every 5 s using an inverted fluorescence microscope (Nikon Ti-E or Leica DMI6000) as described by Loro et al. (2012, 2016). Excitation was provided using a fluorescent lamp equipped with a 436/20 nm filter, and emission signals were filtered at 483/32 nm for CFP and at 542/27 nm for cpVenus with a dichroic mirror (510 nm). Experiments examining Ca2+ propagation throughout entire plants were carried out with a Nikon Ti-E microscope using a ×4 CFI 4 0.13 NA (numerical aperture), whereas for protonema cells, a ×20 CFI Plan APO VC, 0.75 NA dry objective was used (Loro et al. 2012, Loro et al. 2016). False-color images were obtained using the ImageJ ‘RATIO PLUS PLUGIN’ (Palmer and Tsien 2006). Fluorescence intensity was determined over ROIs corresponding to a single cell or organelle (in the case of nuclei) or to different plant tissues. The mean cpVenus and CFP signals of each ROI were used for ratio (R) calculation. Background subtraction was performed independently for both channels before calculating the ratio. To calculate the ΔR/R0, we used the following formula (R – R0)/R0). The ΔR/R0 was plotted vs. time. In situ calibration was performed by raising Ca2+ to saturating levels for YC3.60. This was attempted by permeabilizing plants for 5 min with 200 µM digitonin dissolved in a solution that we called Intracellular Like Medium (ILM: 100 mM K-gluconate, 1 mM MgCl2, 10 mM HEPES, pH 7.5 adjusted with Trizma®) in the presence of 5 mM EGTA to chelate Ca2+. Plants were then transferred to the imaging chamber where they were continuously perfused with ILM solution supplemented with 5 mM EGTA and imaged (acquiring CFP and cpVenus wavelengths) every 5 s for 6 min to measure the Rmin. To measure the Rmax, the plants were then perfused for 5 min with a modified ILM solution in which 5 mM EGTA was replaced with 10 mM CaCl2 and imaged. Plants were then perfused again with the ILM supplemented with 5 mM EGTA and imaged for 6–7 min. To make the Rmin and Rmax calculations, we averaged the traces of four independent experiments performed with two independent transgenic lines used herein. Rmax was 15.93 ± 0.09, Rmin was 5.11 ± 0.02 and the corresponding dynamic range was 3 and 11. In order to calculate the Ca2+ concentration, we considered the in vitro Kd and the Hill coefficient (n) of the YC3.60 reported in Nagai et al. 2004 corresponding to 250 nM and 1.7, respectively. We then used the equation previously published for the calculation of Ca2+ concentration in Palmer and Tsien (2006): [Ca2+] = {Kd^n×[(R – Rmin)/(Rmax – R)]}^1/n. Supplementary Data Supplementary data are available at PCP online. Acknowledgments This study was supported by the European Research Council [ERC starting grant BIOLEAP no. 309485 to T.M. and A.A.]; the Università degli Studi di Milano [PIANO DI SVILUPPO DI ATENEO 2016 to A.C.]; the Università degli Studi di Padova, Dipartimento di Biologia [BIRD173749/17 to A.A. ]. Disclosures There are no conflicts of interest to declare. Author contributions T.M., A.A. and A.C. planned and designed the research. A.A., A.C. and M.S. performed most of the experiments and analyzed the data. M.S., S.G. and M.Z. performed imaging analyses of protonemata cells. T.M. and A.A wrote the manuscript, which all authors revised and approved. References Bowman J.L. ( 2013 ) Walkabout on the long branches of plant evolution . Curr. Opin. 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Abbreviations Abbreviations CFP cyan fluorescent protein cpVenus circularly permutated Venus FRET Förster resonance energy transfer GFP green fluorescent protein GLR glutamate receptor-like ion channel NA numerical aperture ROI region of interest © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

Systemic Calcium Wave Propagation in Physcomitrella patens

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com
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0032-0781
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1471-9053
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10.1093/pcp/pcy104
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Abstract

Abstract The adaptation to dehydration and rehydration cycles represents a key step in the evolution of photosynthetic organisms and requires the development of mechanisms by which to sense external stimuli and translate them into signaling components. In this study, we used genetically encoded fluorescent sensors to detect specific transient increases in the Ca2+ concentration in the moss Physcomitrella patens upon dehydration and rehydration treatment. Observation of the entire plant in a single time-series acquisition revealed that various cell types exhibited different sensitivities to osmotic stress and that Ca2+ waves originated from the basal part of the gametophore and were directionally propagated towards the top of the plant. Under similar conditions, the vascular plant Arabidopsis thaliana exhibited Ca2+ waves that propagated at a higher speed than those of P. patens. Our results suggest that systemic Ca2+ propagation occurs in plants even in the absence of vascular tissue, even though the rates can be different. Introduction Land plants form a monophyletic clade that evolved from freshwater charophyte macroalgae approximately 450 million years ago (Bowman 2013, de Vries et al. 2016). Land colonization by plants represents one of the most significant evolutionary events in the history of life on planet Earth and had a major impact on the Earth’s biosphere. During adaptation to their new environment, plants acquired the ability to survive cycles of dehydration and rehydration (Oliver et al. 2005), as well as exposure to stronger mechanical forces and more oxidative conditions (i.e. high light intensities, light enriched in UV wavelengths and high oxygen availability) (Kenrick and Crane 1997, Dahl et al. 2010). Because they diverged from their angiosperm ancestors soon after land colonization, bryophytes are a useful model system for the identification of early plant adaptations to the terrestrial environment. In particular, the moss Physcomitrella patens is used as a model plant for the study of evolution and development due to its evolutionary position and the availability of several molecular tools (Cove 2005, Rensing et al. 2008). The perception of external stimuli is pivotal for plant fitness, and these pathways probably involve calcium (Ca2+), a common second messenger that triggers cell responses in prokaryotes and eukaryotes during development, stress and long-distance signal transduction. Different external stimuli cause Ca2+ transient increases, waves and oscillations that are interpreted by cells as signals themselves (Whalley and Knight 2013, Choi et al. 2017). Ca2+ measurement in P. patens plants has been successfully performed using ratiometric dyes (Tucker et al. 2005, Qudeimat et al. 2008), the Ca2+-sensitive genetically encoded sensor aequorin from Aequorea victoria (Haley et al. 1995, Russell et al. 1996, Saidi et al. 2009) and GCaMP3 (Kleist et al. 2017). This allowed the assessment of the role of Ca2+ in the regulation of physiological and developmental processes at the level of protonemata cells in P. patens, including the UV-A response (Tucker et al. 2005) and thermal sensing mediated by cyclic nucleotide-gated calcium channels (CNGCs) (Finka et al. 2012). Moreover, two glutamate receptor-like ion channels (GLRs) were shown to be involved in the regulation of the cytoplasmic Ca2+ concentration in P. patens sperm cells. Changes in intracellular Ca2+ levels modulate sperm orientation towards the archegonium, thereby affecting the overall fertility of the plant (Ortiz-Ramírez et al. 2017). In this work, Cameleon YC3.60 (Nagai et al. 2004), a Förster resonance energy transfer (FRET)-based genetically encoded fluorescent sensor, was exploited to monitor the Ca2+ concentration in vivo with high spatiotemporal resolution inside different cell types and compartments of P. patens plants during osmotic stress. Results and Discussion The Cameleon YC3.60 sensor was targeted to the nucleoplasm, mitochondria and cytoplasm using specific N-terminal transit peptides or proteins previously utilized in Arabidopsis thaliana (Krebs et al. 2012, Loro et al. 2012, Costa et al. 2017). The presence of a clear homogeneous fluorescence signal in the expected compartment was verified by confocal laser scanning microscopy (Fig. 1a;Supplementary Fig. S1). The stability of fluorescent protein expression and the absence of major interference with cell physiology were verified by assessing plant growth, development and photosynthetic efficiency. Transgenic lines affected by the expression of the sensor were not used in further analyses (refer to Supplementary Fig. S1). Fig. 1 View largeDownload slide Expression of Yellow Cameleon 3.60 (YC3.60) and analysis of Ca2+ dynamics in Physcomitrella patens protonema cells. (a) The YC3.60 sensor was expressed under control of the constitutive 7113 promoter and was targeted to the cytoplasm. Chl and YC3.60 signals were independently acquired by confocal laser scanning microscopy and were then merged. Scale bar = 10 μm. (b, c) Changes in Ca2+ concentration in protonema cells in response to osmotic stress. CFP and cpVenus emissions were detected simultaneously after CFP excitation, and the cpVenus/CFP fluorescent ratio over that time is expressed as ΔR/R0. Osmotic stress was applied by continuous perfusion with solutions containing 400, 500 or 750 mM mannitol, which are represented by red, blue and black lines, respectively. The white and red bars on top of the panel indicate the absence and the presence, respectively, of mannitol in the perfusion solution. Data are shown as the mean of at least four independent plants. Fig. 1 View largeDownload slide Expression of Yellow Cameleon 3.60 (YC3.60) and analysis of Ca2+ dynamics in Physcomitrella patens protonema cells. (a) The YC3.60 sensor was expressed under control of the constitutive 7113 promoter and was targeted to the cytoplasm. Chl and YC3.60 signals were independently acquired by confocal laser scanning microscopy and were then merged. Scale bar = 10 μm. (b, c) Changes in Ca2+ concentration in protonema cells in response to osmotic stress. CFP and cpVenus emissions were detected simultaneously after CFP excitation, and the cpVenus/CFP fluorescent ratio over that time is expressed as ΔR/R0. Osmotic stress was applied by continuous perfusion with solutions containing 400, 500 or 750 mM mannitol, which are represented by red, blue and black lines, respectively. The white and red bars on top of the panel indicate the absence and the presence, respectively, of mannitol in the perfusion solution. Data are shown as the mean of at least four independent plants. Drought and salt stresses are major abiotic constraints affecting plant growth and productivity. The acquisition of tolerance to dynamic water availability is considered a keystone in land colonization (Rensing et al. 2008). Ca2+ concentration in response to salt and osmotic stress was assessed in vivo by imaging analyses of YC3.60 transgenic lines. Protonema cells were treated with various concentrations of NaCl, sorbitol and mannitol, and then the osmotic stress was released. Cyan fluorescent protein (CFP) and circularly permutated Venus (cpVenus) fluorescence signals were simultaneously acquired for the duration of the treatment to detect Ca2+-induced FRET transient efficiency change (Fig. 1b;Supplementary Fig. S2). Both the addition (hyperosmotic treatment) and removal of the osmoticum (hypo-osmotic treatment; mannitol was used) caused a detectable increase in Ca2+ concentration in various cell compartments, as shown by the clear change in ΔR/R0 (Fig. 1c;Supplementary Fig. S1). For all compartments, the transient increase in Ca2+ was more intense during the hypo-osmotic response than during the hyperosmotic response (Fig. 1c;Supplementary Fig. S1; Supplementary Table S1). Vicia faba and A. thaliana guard cells, as well as A. thaliana leaves and roots, exhibited similar trends (Hayashi et al. 2006, Zhang et al. 2007, Loro et al. 2012), suggesting that the strong hypo-osmotic response is highly conserved. The detected Ca2+ transients exhibited different intensities and kinetics depending on the mannitol concentration used (400, 500 or 750 mM) and on the cellular compartments examined, revealing that Ca2+ dynamics are differentiated within the cell and also transduce information regarding stress severity (Fig. 1c; Supplementary Figs. S1, S3; Supplementary Table S1). Control experiments run in the absence of mannitol showed that the perfusion solution itself had no effect on Ca2+ dynamics (Supplementary Fig. S4). In vivo imaging analyses were implemented using lower magnification to observe more extended portions of the sample and different cell types simultaneously. It was possible to observe entire young P. patens gametophores, a significant advantage over A. thaliana, Oryza sativa and other plants of greater size in which the Cameleon sensor has already been expressed. Lines expressing YC3.60 in the cytoplasm were used preferentially because they showed more homogeneous and stable expression of the sensor in different cell types, especially old gametophores (Supplementary Fig. S5). Similar to previous observations in protonema, the exposure of entire young gametophores to 500 mM mannitol generated a concomitant increase in cpVenus fluorescence emission and a decrease in CFP fluorescence emission (Fig. 2b;Supplementary Videos S2, S3). The most intense Ca2+ transient was recorded again after the relief of the osmotic stress (Fig. 2b). The Ca2+ response, however, was not homogeneous throughout the plant but was more intense in the basal part of the gametophore than in the phyllids, demonstrating that different cell types have different sensitivities to the same stimulus (Fig. 2c, d;Supplementary Video S1). An independent quantitative analysis of the gametophore base and phyllids confirmed that Ca2+ responses are more intense in the former but are present in the latter, exhibiting a delay of approximately 1 min before reaching maximal signal intensity (Fig. 2d). Of note, our calibration of Ca2+ estimates that the maximum concentration of cytosolic Ca2+ at the base of the gametophore (634 ± 28 nM) was almost four times higher than in phyllids (152 ± 32) nM. Also in the case of gametophores, control experiments run in the absence of mannitol showed that the perfusion solution itself had no effect on Ca2+ response (Supplementary Fig. S6). Fig. 2 View largeDownload slide In vivo analysis of Ca2+ dynamics in response to osmotic stress throughout P. patens gametophores expressing CYT–YC3.60. (a) Representative snapshot of one gametophore; yellow boxes correspond to the regions of interests (ROIs 1–8) outlined to follow CFP and cpVenus fluorescence emission with time. (b) The signal is the result of the average signal emitted in ROIs 1–4 (left graph, base) and ROIs 5–8 (right graph, phyllid). (c) Snapshots of fluorescence ratio imaging acquisition during continuous perfusion with hyperosmotic (6 and 10 min) and hypo-osmotic (1, 13 and 28 min) solutions. False colors reflect the ratio of the cpVenus fluorescence emission to the CFP fluorescence emission at the specified time point. Scale bar = 500 μm. (d) Quantitative analysis of cpVenus/CFP fluorescent ratio measurements calculated as ΔR/R0 for the gametophore base and phyllids shown in (a–c). White and red bars on top of each graph indicate the presence and the absence of mannitol, respectively. The full acquisition cpVenus, CFP and cpVenus/CFP can be visualized as supplementary material (Supplementary Videos S1–S3). Fig. 2 View largeDownload slide In vivo analysis of Ca2+ dynamics in response to osmotic stress throughout P. patens gametophores expressing CYT–YC3.60. (a) Representative snapshot of one gametophore; yellow boxes correspond to the regions of interests (ROIs 1–8) outlined to follow CFP and cpVenus fluorescence emission with time. (b) The signal is the result of the average signal emitted in ROIs 1–4 (left graph, base) and ROIs 5–8 (right graph, phyllid). (c) Snapshots of fluorescence ratio imaging acquisition during continuous perfusion with hyperosmotic (6 and 10 min) and hypo-osmotic (1, 13 and 28 min) solutions. False colors reflect the ratio of the cpVenus fluorescence emission to the CFP fluorescence emission at the specified time point. Scale bar = 500 μm. (d) Quantitative analysis of cpVenus/CFP fluorescent ratio measurements calculated as ΔR/R0 for the gametophore base and phyllids shown in (a–c). White and red bars on top of each graph indicate the presence and the absence of mannitol, respectively. The full acquisition cpVenus, CFP and cpVenus/CFP can be visualized as supplementary material (Supplementary Videos S1–S3). The delay measured in phyllids was due to the directional propagation of Ca2+ transients from the base towards the tip of the plant (Fig. 3a–e;Supplementary Videos S4, S5). The directionality of propagation did not depend on the orientation of the plant with respect to the point of stress application. In Supplementary Video S4, the lower portion of the gametophore is closer to the entry point of the buffer (Fig. 3a), while in Supplementary Video S5 the orientation is reversed (Fig. 3b). In both cases, however, Ca2+ waves emanated from the lower part of the gametophore and propagated towards the phyllid. The dynamics of the osmo-induced Ca2+ increase were also analyzed independently at the base of the gametophore (Supplementary Fig. S7) and in phyllids (Fig. 3c–e). We also examined the same Ca2+ waves at the highest magnification available and found that only a few cells were in focus, thus precluding an analysis of long-distance transmission (Supplementary Fig. S8). In order to understand if a local mannitol stimulus could elicit Ca2+ propagation throughout gametophores, a local stress system was set up, in which gametophores were covered by a block of agarized perfusion solution and then subjected to the perfusion experiment (Supplementary Fig. S9). Even if the upper part of the plant was in less direct contact with mannitol solution, we still observed a retarded increase in cytoplasmic Ca2+ concentration in phyllids as compared with the base of the gametophore, suggesting the existence of a proper long-distance Ca2+ wave propagation in P. patens. Fig. 3 View largeDownload slide In vivo analysis of Ca2+ dynamics in response to osmotic stress in young Physcomitrella patens plantlets. (a, b) The response of two representative gametophores was in the same direction as or the direction opposing osmotic flow. From top to bottom, a sketch representing the direction of flow in the plant, and three snapshots of fluorescence ratio imaging acquisition corresponding to 0, 13.5 and 19 min. (c) Detail of one phyllid shown in (a). White dashed lines correspond to the regions of interest (ROIs 1–10) designated to follow CFP and cpVenus fluorescence emission during the indicated time. (d) Ca2+ concentration variation (ΔR/R0) during the hypo-osmotic phase. ROIs from 1 to 10 correspond to those drawn in (c). (e) Ratiometric false-color images from representative time series of a basal portion of a P. patens gametophore. The false colors represent the intensity of the cpVenus/CFP fluorescent ratio. The time (min) below each snapshot corresponds to the signal reported in (d). The full acquisitions are included as supplementary material (Supplementary Videos S4, S5). Scale bar = 250 μm. Ratio images are representative of n ≥ 3. Fig. 3 View largeDownload slide In vivo analysis of Ca2+ dynamics in response to osmotic stress in young Physcomitrella patens plantlets. (a, b) The response of two representative gametophores was in the same direction as or the direction opposing osmotic flow. From top to bottom, a sketch representing the direction of flow in the plant, and three snapshots of fluorescence ratio imaging acquisition corresponding to 0, 13.5 and 19 min. (c) Detail of one phyllid shown in (a). White dashed lines correspond to the regions of interest (ROIs 1–10) designated to follow CFP and cpVenus fluorescence emission during the indicated time. (d) Ca2+ concentration variation (ΔR/R0) during the hypo-osmotic phase. ROIs from 1 to 10 correspond to those drawn in (c). (e) Ratiometric false-color images from representative time series of a basal portion of a P. patens gametophore. The false colors represent the intensity of the cpVenus/CFP fluorescent ratio. The time (min) below each snapshot corresponds to the signal reported in (d). The full acquisitions are included as supplementary material (Supplementary Videos S4, S5). Scale bar = 250 μm. Ratio images are representative of n ≥ 3. Ca2+ waves are known to be involved in systemic acquired acclimation response to abiotic stress in A. thaliana ( Choi et al. 2014, Choi et al. 2017). Four-day-old A. thaliana plantlets expressing CYT–YC3.60 (Loro et al. 2012) (Supplementary Fig. S10; Supplementary Video S7) were treated with the same protocol used for P. patens for a direct comparison of their local and systemic Ca2+ responses. In this case, a Ca2+ wave was recorded and could be observed following the time needed to reach ΔRmax/R0 in four independent regions of interest (ROIs) from the root tip to the shoot (Supplementary Fig. S10). To better compare the Ca2+ propagation rates of the model plants A. thaliana and P. patens, ROIs of 400 μm were drawn and kymographs of the corresponding FRET signals were extrapolated (Fig. 4). In A. thaliana, Ca2+ waves moved at approximately 58.5 ± 18.9 μm s–1. In P. patens, both at the base of the gametophore and in phyllids, the speed of propagation was instead 4.5 ± 3.8 μm s–1, a value compatible with what has been measured using Fura-2-dextran ratio images in protonema apical cells in response to UV-A light (3.4–8.4 μm s–1) (Tucker et al. 2005). It is interesting to note that with our set-up, we managed to measure cell–cell communication in entire gametophores rather than simply the communication between two adjacent cells. The fast propagation measured in A. thaliana (Fig. 4; Supplementary Fig. S10) is in agreement with what has been reported previously for different tissues and stressors. Ca2+ waves traveled at 60–290 μm s–1 along the leaf veins of A. thaliana after exposure of the roots of plants expressing a bioluminescence resonance energy transfer (BRET)-based green fluorescent protein (GFP)–aequorin reporter to salt stress (Xiong et al. 2014). The response was fastest (400 μm s–1) when measured through root tissues at lower salt concentrations (Choi et al. 2014). Thus, the main component of Ca2+ wave propagation in P. patens is slower than that measured in A. thaliana roots and leaves. In order to understand whether extracellular Ca2+ was involved in Ca2+ wave propagation, we performed experiments by perfusing plants with a low Ca2+ concentration and lanthanum (III) chloride, a blocker of non-selective cation channels and stretch-activated Ca2+-permeable channels (Supplementary Fig. S11). The application of lanthanum has effectively reduced the amplitude of the Ca2+ increase in response to osmotic stress relaxation. On the other hand, we could still detect the propagation of a long-distance Ca2+ wave (Supplementary Fig. S11), suggesting that intracellular Ca2+ storage is involved in this process or that there are Ca2+ channels/transporters at the level of the plasma membrane that are insensitive to lanthanum. Moreover the rate of propagation is similar to calcium-induced calcium release measured in single cells (Jaffe 2010). Further work may elucidate whether Ca2+ waves serve a signaling function in the moss system and to uncover the molecular system responsible for the propagation of Ca2+ waves. Different models have been proposed for the interaction of propagating Ca2+ signals with electric signals and reactive oxygen species (ROS) waves through symplastic or apoplastic pathways (Choi et al. 2014, Choi et al. 2017). Vasculature seems to be fundamental for the propagation of Ca2+ waves when the signal is triggered by wounding or herbivore attack (Mousavi et al. 2013, Kiep et al. 2015). Interestingly, the aphid Myzus persicae elicits elevated cytosolic Ca2+ concentrations around piercing sites in the epidermal and mesophyll cells of A. thaliana (Vincent et al. 2017). This phloem-independent radial propagation proceeds at a speed of approximately 6 μm s–1 over an area of approximately 110 μm2 (Vincent et al. 2017). In the case of roots, transmission is likely to be supported by the activity of Ca2+ pumps and channels and their regulators within cortical and endodermal cells (Choi et al. 2016). Differences in the speed of Ca2+ propagation could be due to the obvious lack in P. patens of a proper vascular tissue. However, it is possible that different systems control Ca2+ homeostasis in the two model species. This second hypothesis is supported by genomic surveys highlighting the existence of transport systems differing in composition and complexity compared with other members of the Viridiplantae lineage (Edel and Kudla 2015). For example, GLRs have been identified as potential mediators of systemic electric signals in A. thaliana (Mousavi et al. 2013, Salvador-Recatalà 2016). P. patens encodes a small family of GLRs consisting of only two members (Edel and Kudla 2015, De Bortoli et al. 2016) that are thought to be involved in plant reproduction rather than gametophytic cell–cell signal transduction (Ortiz-Ramírez et al. 2017). Fig. 4 View largeDownload slide Kymographs illustrating Ca2+ wave dynamics in Arabidopsis thaliana and Physcomitrella patens lines expressing the cytoplasmic sensor (CYT–YC3.60). Ratiometric data (cpVenus/CFP fluorescent ratio) were extracted from a region of interest (ROI) of 400 μm parallel to the plant axis. For A. thaliana, the last segment of root before the hypocotyl was considered (Supplementary Fig. S10), while for P. patens, gametophores and phyllids were analyzed independently. The arrows indicate the tipward direction followed to draw the ROIs. The white and red bars represent the absence and the presence, respectively, of mannitol in the perfusion solution. Fig. 4 View largeDownload slide Kymographs illustrating Ca2+ wave dynamics in Arabidopsis thaliana and Physcomitrella patens lines expressing the cytoplasmic sensor (CYT–YC3.60). Ratiometric data (cpVenus/CFP fluorescent ratio) were extracted from a region of interest (ROI) of 400 μm parallel to the plant axis. For A. thaliana, the last segment of root before the hypocotyl was considered (Supplementary Fig. S10), while for P. patens, gametophores and phyllids were analyzed independently. The arrows indicate the tipward direction followed to draw the ROIs. The white and red bars represent the absence and the presence, respectively, of mannitol in the perfusion solution. The present work reveals the existence of long-distance transmission of Ca2+ waves in response to an osmoticum in the body of P. patens. The ability to propagate systemic Ca2+ waves, an important component for osmotic stress perception and osmotolerance in general, was present during land colonization. Materials and Methods Plant material and growth conditions Protonemal tissue of P. patens (the Gransden wild-type strain and YC3.60-expressing lines) were grown on minimum PpNO3 medium under controlled conditions: 24°C, a 16 h light/8 h dark photoperiod and a light intensity of 50 µmol photons m–2 s–1. Ten-day-old plants were used for protonemal tissue imaging, and gametophores were observed between 1 and 2 months of growth. Arabidopsis thaliana plants expressing cytoplasmic Cameleon (CYT–YC3.60) were used in this study (Krebs et al. 2012). Seeds were surface-sterilized and sown on half-strength Murashige and Skoog medium (MS including vitamins; Duchefa) supplemented with 0.1% (w/v) sucrose and 0.05% (w/v) MES, adjusted to pH 5.8 with KOH, and solidified with 0.8% (w/v) plant agar (Duchefa). After stratification at 4°C in the dark for 2–3 d, the seeds were transferred to a growth chamber with a 16/8 h light cycle (70 μmol photons m–2 s–1) at 24°C. Constructs Standard plasmid construction methods were used to obtain pMAK1-7113::YC3.60 plasmids. All PCRs were carried out using high-fidelity KAPA HiFi PCR Kits (Kapa Biosystems), according to the manufacturer’s instructions. PCR products were cloned into a pJET1.2/blunt vector (Thermo Fisher Scientific) and were sequenced after transfer to the expression vector pMAK1 (http://www.nibb.ac.jp/evodevo/PHYSCOmanual/17Overdriveplasmid/pMAK1.htm) under control of the constitutive promoter 7113 (Mitsuhara et al. 1996, Gerotto et al. 2012). The primers used to amplify the sequences and add signal sequences for mitochondrial or nucleoplasmic subcellular localization are as follows: MIT/NUC-for (5'-CTCGAGAGAGGACAGCCCAAGCTTATG-3') and MIT/NUC-rev (5'-GGGCCCTTTCAGCGTACCGAATTCTTA-3'). The PCR product was cloned with XhoI and ApaI. The primers used to amplify the sequences with cytoplasmic subcellular localization were: CYT-for (5'-GGGCCCATGCTGCAGAACGAGCTTGCTCTT-3') and CYT-rev (5'-GAATTCGTGCCAAGCTTCGATTGATG-3'). The PCR product was cloned with ApaI and EcoRI. Plant expression vectors were used as templates to perform the PCRs (Krebs et al. 2012, Loro et al. 2012, Costa et al. 2017). The Tnos terminator sequence in the pMAK1 plasmid was used to control the expression of the mitochondrion- and nucleoplasm-localized versions of YC3.60. The cytoplasm-localized version of YC3.60 was amplified together with the rbcs terminator, which was substituted for the Tnos terminator sequence in the pMAK1 plasmid. YC3.60 is targeted to the nucleoplasm by its fusion with the nuclear-localized Xenopus laevis nucleophosmin, to the mitochondrial matrix by four repeats of the mitochondrial targeting sequence from subunit VIII of human Cyt c oxidase (MIT) and to the cytoplasm by a nuclear export signal (CYT) of rabbit heat-stable protein kinase inhibitor α. Chl fluorescence measurements and growth tests To test plant performance, 10-day-old plants grown on PpNO3 were transferred to new plates as multiple independent spots of 2 mm diameter. In vivo Chl fluorescence was measured using Fluorcam 800MF (Photon Sytems Instruments) to evaluate the Fv/Fm parameter as (Fm –F0)/Fm. Pictures of the same plates were taken regularly, and plant growth was evaluated by measuring the size of different plant spots using ImageJ software (http://rsb.info.nih.gov/ij/) after background exclusion was performed with the ‘TRESHOLD COLOUR’ PLUGIN (http://www.mecourse.com/landinig/software/software.html). Confocal microscopy Laser scanning confocal microscopy analyses were performed using a Leica SP5 imaging system (Leica Microsytems). Images were acquired using a ×40 objective (HCX PL APO CS 40/1.25–0.75 oil) with different degrees of digital zoom. cpVenus was excited by the 514 nm line of the argon laser and the emission was collected between 525 and 540 nm. Chl fluorescence emission was collected simultaneously between 660 and 750 nm. Images were merged using ImageJ software (http://rsb.info.nih.gov/ij/). FRET-based calcium imaging For FRET measurements, plant material was removed from the plate and placed in an open-top chamber overlaid by cotton soaked in imaging solution (5 mM KCl, 10 mM MES and 10 mM CaCl2; adjusted to pH 5.8 with Trizma® base). Osmotic stress was achieved by adding imaging solutions containing different concentrations of mannitol or NaCl, using a continuous perfusion system. After 8 min, the hyperosmotic solution was replaced by the initial imaging solution to restore the baseline condition. We ensured that the increase in Ca2+ concentration was the result of osmotic stress and not mechanical stimulation by performing experiments with and without osmoticum (Supplementary Figs. S4, S6). Images were collected every 5 s using an inverted fluorescence microscope (Nikon Ti-E or Leica DMI6000) as described by Loro et al. (2012, 2016). Excitation was provided using a fluorescent lamp equipped with a 436/20 nm filter, and emission signals were filtered at 483/32 nm for CFP and at 542/27 nm for cpVenus with a dichroic mirror (510 nm). Experiments examining Ca2+ propagation throughout entire plants were carried out with a Nikon Ti-E microscope using a ×4 CFI 4 0.13 NA (numerical aperture), whereas for protonema cells, a ×20 CFI Plan APO VC, 0.75 NA dry objective was used (Loro et al. 2012, Loro et al. 2016). False-color images were obtained using the ImageJ ‘RATIO PLUS PLUGIN’ (Palmer and Tsien 2006). Fluorescence intensity was determined over ROIs corresponding to a single cell or organelle (in the case of nuclei) or to different plant tissues. The mean cpVenus and CFP signals of each ROI were used for ratio (R) calculation. Background subtraction was performed independently for both channels before calculating the ratio. To calculate the ΔR/R0, we used the following formula (R – R0)/R0). The ΔR/R0 was plotted vs. time. In situ calibration was performed by raising Ca2+ to saturating levels for YC3.60. This was attempted by permeabilizing plants for 5 min with 200 µM digitonin dissolved in a solution that we called Intracellular Like Medium (ILM: 100 mM K-gluconate, 1 mM MgCl2, 10 mM HEPES, pH 7.5 adjusted with Trizma®) in the presence of 5 mM EGTA to chelate Ca2+. Plants were then transferred to the imaging chamber where they were continuously perfused with ILM solution supplemented with 5 mM EGTA and imaged (acquiring CFP and cpVenus wavelengths) every 5 s for 6 min to measure the Rmin. To measure the Rmax, the plants were then perfused for 5 min with a modified ILM solution in which 5 mM EGTA was replaced with 10 mM CaCl2 and imaged. Plants were then perfused again with the ILM supplemented with 5 mM EGTA and imaged for 6–7 min. To make the Rmin and Rmax calculations, we averaged the traces of four independent experiments performed with two independent transgenic lines used herein. Rmax was 15.93 ± 0.09, Rmin was 5.11 ± 0.02 and the corresponding dynamic range was 3 and 11. In order to calculate the Ca2+ concentration, we considered the in vitro Kd and the Hill coefficient (n) of the YC3.60 reported in Nagai et al. 2004 corresponding to 250 nM and 1.7, respectively. We then used the equation previously published for the calculation of Ca2+ concentration in Palmer and Tsien (2006): [Ca2+] = {Kd^n×[(R – Rmin)/(Rmax – R)]}^1/n. Supplementary Data Supplementary data are available at PCP online. Acknowledgments This study was supported by the European Research Council [ERC starting grant BIOLEAP no. 309485 to T.M. and A.A.]; the Università degli Studi di Milano [PIANO DI SVILUPPO DI ATENEO 2016 to A.C.]; the Università degli Studi di Padova, Dipartimento di Biologia [BIRD173749/17 to A.A. ]. Disclosures There are no conflicts of interest to declare. Author contributions T.M., A.A. and A.C. planned and designed the research. A.A., A.C. and M.S. performed most of the experiments and analyzed the data. M.S., S.G. and M.Z. performed imaging analyses of protonemata cells. T.M. and A.A wrote the manuscript, which all authors revised and approved. References Bowman J.L. ( 2013 ) Walkabout on the long branches of plant evolution . Curr. Opin. 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Abbreviations Abbreviations CFP cyan fluorescent protein cpVenus circularly permutated Venus FRET Förster resonance energy transfer GFP green fluorescent protein GLR glutamate receptor-like ion channel NA numerical aperture ROI region of interest © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Plant and Cell PhysiologyOxford University Press

Published: Jun 6, 2018

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