Tree species differ in their ability to use light efficiently, affecting carbon gain, establishment and survival in highly heterogeneous environments. This efficiency relies on the maintenance of the photosynthetic induction state, regulated by structural, biochemical, photochemical and stomatal processes that vary along the leaf economics spectrum (LES). Slow return species, such as shade-tolerant species (often late successional), are thought to sustain higher photosynthetic induction state, while quick return species, like light-demanding species (often early-successional) would have lower shade acclimation and shade-tolerant species lower acclimation to high light. Yet, results often deviate from these predictions. Moreover, most LES traits reflect steady state performance, not dynamic responses. Here, we investigated photosynthetic induction responses in four widely distributed Brazilian tree species representing contrasting successional groups and LES positions, grown under 10% light, 50% light and full sun. We quantified induction dynamics in terms of CO2 assimilation, stomatal conductance, electron transport rate, as well as chlorophyll content, and leaf mass per area (LMA). Acclimation to distinct light environments was assessed using a shade adjustment coefficient and a novel metric based on principal component analysis, relative plasticity (RP). Relative plasticity suggests an asymmetrical bell-shaped relationship with LES position: the slow return Hymenaea courbaril L. showed low plasticity and little change in resource allocation (LMA), photosynthetic rates or induction times; the fast-return Schinus terebinthifolia Raddi displayed moderate plasticity but unexpectedly high shade acclimation, showing high induction state and CO2 assimilation rates; and the intermediate strategists Cecropia pachystachya Trécul and Handroanthus impetiginosus (Mart. ex DC.) Mattos exhibited the highest plasticity, with coordinated increases in LMA, CO2 assimilation, stomatal conductance and photosynthetic induction under increasing light conditions. These findings highlight the importance of integrating photosynthetic dynamics into ecophysiological frameworks for species selection in reforestation, particularly in heterogeneous light environments, where adaptive flexibility can play a critical role on the resilience of an ecosystem.

Abstract Root hairs (RH) are excellent model systems for studying cell size and polarity since they elongate several hundred-fold their original size. Their tip growth is regulated by both intrinsic and environmental signals and is associated with the existence of a highly controlled cytoplasmic tip Ca²⁺ gradient, whose disruption impairs RH development. The molecular mechanisms underlying the Ca 2+ homeostasis fine tuning and the Ca 2+ organellar contributions to the cytoplasmic pool remain unclear. In the model plant Arabidopsis thaliana , many efflux routes are present, including those that employ Ca 2+ -pumps from the Autoinhibited Ca 2+ -ATPase (ACA) family. Here, we identified that the ER localized ACA2, and to a lower extent ACA7, are crucial ACAs required to control RH growth. By using genetically encoded Ca 2+ biosensors we showed that Ca 2+ -dynamics are compromised in the aca2-2 mutant, having lower cytosolic Ca 2+ concentration [Ca 2+ ] cyt and growth rate, showing an altered homeostatic calcium setpoint compared to Col-0. Accordingly, the ACA2 mutation changed the dynamics of [Ca 2+ ] cyt oscillations coupled to growth rate, inducing longer periods and more regular oscillations in the dominant high-frequency range (around 22 s), and slower oscillations (around 1 min) in the low-frequency range. Finally, expression of ACA2 with changes in four putative Ca 2+ binding residues (ACA2ΔCa 2+ ) failed to rescue the RH growth phenotype in the aca2-2 mutant. Collectively, our findings indicate that ER-localized ACA2 and possibly ACA7 are crucial for modulating cytoplasmic Ca 2+ signals, possibly composing a critical part of a negative feedback loop, and their absence leads to impairments in RH cell elongation.

The co–occurrence of drought and heat stress in the field is especially frequent and the plant response to a combination of these abiotic stresses is singular, remaining largely unknown. Understanding physiological responses to a combined stress is of particular importance in face of the imminent global climate changes, which is predicted to entail not only higher temperatures, but also increase in drought, imposing restrictions to the productivity of economically important species as E. globulus . This forest species is critically affected by changes in temperature and water availability, since its high productivity implies in high rates of water use. In order to evaluate the photosynthetic responses of Eucalyptus globulus to these environmental conditions, young plants were grown at 25° C and 35° C under different irrigation regimes: full irrigation – FI, moderate drought – MD, and severe drought – SD. Plants grown at 25° C FI and MD were also subjected to a short–term exposure to 35° C. Several ecophysiological variables, derived mainly from leaf gas exchange and chlorophyll a fluorescence measurements, were compared in order to test the hypotheses that: i) E. globulus show different responses to short or long–term exposure to 35° C; ii) the effect of both stresses combined will not be simply the sum of each condition applied separately. Our results showed that plants respond differently to water deficit according to the thermal regime. The combination of water deficit and a long–term exposure to 35° C caused a down–acclimation of the photosynthetic capacity, while plants under the short–term treatment showed a higher performance, even when compared to the 25° C control condition, supporting the hypothesis i . Thus, the short–term exposure to 35° C induced an increase in tolerance to water deficit. Since the response to a combination of heat and drought differed from the superposition of individual responses, as observed mainly at 35° C, the hypothesis ii was also supported by our results. ### Competing Interest Statement The authors have declared no competing interest. Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, 2019/26129-6

Pressurized cells with strong walls make up the hydrostatic skeleton of plants. Assembly and expansion of such stressed walls depend on a family of secreted RAPID ALKALINIZATION FACTOR (RALF) peptides, which bind both a membrane receptor complex and wall-localized LEUCINE-RICH REPEAT EXTENSIN (LRXs) in a mutually exclusive way. Here we show that, in root hairs, the RALF22 peptide has a dual structural and signalling role in cell expansion. Together with LRX1, it directs the compaction of charged pectin polymers at the root hair tip into periodic circumferential rings. Free RALF22 induces the formation of a complex with LORELEI-LIKE-GPI-ANCHORED PROTEIN 1 and FERONIA, triggering adaptive cellular responses. These findings show how a peptide simultaneously functions as a structural component organizing cell wall architecture and as a feedback signalling molecule that regulates this process depending on its interaction partners. This mechanism may also underlie wall assembly and expansion in other plant cell types. The authors show that RALF22 has a dual role in cell wall assembly in root hairs: as a structural component organizing cell wall architecture and as a feedback signalling molecule that regulates this process depending on its interaction partners.

Summary Pressurized cells with strong walls make up the hydrostatic skeleton of plants. Assembly and expansion of such stressed walls depend on a family of secreted RAPID ALKALINIZATION FACTOR (RALF) peptides which, curiously, bind both a membrane receptor complex and wall-localized LEUCINE-RICH REPEAT EXTENSINs (LRXs) in a mutually exclusive way. Here we show that, in root hairs, the RALF22 peptide has a dual structural and signaling role in cell expansion. Together with LRX1, it directs the compaction of charged pectin polymers at the root hair tip into periodic circumferential rings. Free RALF22 induces the formation of a complex with LORELEI-LIKE-GPI-ANCHORED PROTEIN 1 (LLG1) and FERONIA (FER), triggering adaptive cellular responses. These findings show how a peptide simultaneously functions as a structural component organizing cell wall architecture and as a signaling molecule that regulates this process. This mechanism may also underlie wall assembly and expansion in other plant cell types.

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

Polar growth and guidance of pollen tubes (PTs) is regulated by chemical and electrical signaling cues. Central regulators controlling cellular protrusion of PTs tip-focused Ca2+ and H+ cytosolic gradients and ion fluxes depend, but also feedback, on membrane voltage (V-m). However, the functional relationship between V-m, Ca2+, and H+ dynamics is largely unclear. We carried out parallel live-cell Ca2+ and H+-imaging together with V-m recordings followed by cross-correlation analysis to study their interconnection in tobacco PTs. Upon electrode impalement at the tip or shank, PT growth ceased but sustained oscillatory behavior of all three signals continued and remained mostly in phase, although shifted in time. The Ca2+ oscillations were largely restricted to the apex, temporally leading the H+ increases by & SIM;18 s, whereas H+ tip-focused peaks spread into the shank. Voltage recordings in the shank (50-80 & mu;m behind the tip) revealed transient bell-shaped depolarizations that correlated strongly with H+ but not Ca2+ oscillations. In contrast, V-m oscillations readout at the tip correlated equally with tip Ca2+- and H+-signals, having action potential-like appearances that were approximately five times faster compared with those in the shank. Our multiparameter readout approach identified distinct relationships of chemical and electric signals along PTs, where at the tip changes in both Ca2+ and H+ correlated with V-m dynamics, whereas at the shank only H+ correlated with V-m. We propose that spatially confined chemo-electric coupling support the manifestation of propagating voltage and pH waves along nongrowing PTs, which are preceded by short-lasting but high amplitude tip Ca2+ spikes.

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.

It is unclear whether West Nile virus (WNV) circulates endemically in Portugal. Despite the country’s adequate climate for transmission, Portugal has only reported four human WNV infections so far. We performed a review of WNV-related data (1966–2020), explored mosquito (2016–2019) and land type distributions (1992–2019), and used climate data (1981–2019) to estimate WNV transmission suitability in Portugal. Serological and molecular evidence of WNV circulation from animals and vectors was largely restricted to the south. Land type and climate-driven transmission suitability distributions, but not the distribution of WNV-capable vectors, were compatible with the North-South divide present in serological and molecular evidence of WNV circulation. Our study offers a comprehensive, data-informed perspective and review on the past epidemiology, surveillance and climate-driven transmission suitability of WNV in Portugal, highlighting the south as a subregion of importance. Given the recent WNV outbreaks across Europe, our results support a timely change towards local, active surveillance.

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.

Background: West Nile virus (WNV) is a mosquito-borne pathogen with a globally expanding geographical range. Many European countries regularly report outbreaks affecting both human and equine populations, with an increase in epidemic activity in the past decade. Portugal, on the far west of Europe, has not yet experienced large outbreaks of WNV, despite its suitable Mediterranean climate. Objectives: To provide a comprehensive, data-driven perspective on the past epidemiology, surveillance and transmission suitability of WNV in Portugal. Methods: Literature and database reviews were performed, covering the period between 1966 and 2020 on existing evidence for WNV circulation. Climatic data was used to estimate the theoretical transmission potential of WNV, and to analyse long-term climate trends between 1981 and 2019. Results: Existing data show that WNV-capable mosquito species are spread across the country, while WNV molecular and serological evidence is vastly concentrated in the south. Our analyses suggest that two different climate types, characteristic of the north and south of Portugal, contribute to quantitative differences in WNV theoretical transmission potential. This supports the north-south divide observed from previous molecular and serological evidence. Due to long-term changes in local climate, suitability for WNV transmission is shown to have been slowly increasing in the country. Discussion: Data and theoretical analyses support the notion that the south of Portugal is more suitable for WNV transmission, while not rejecting the possibility of transmission in the north. Mosquito monitoring has been effective, but surveillance of key sentinel species remains passive, creating important knowledge gaps for epidemic control and public health. To date, evidence supports WNV circulation, but it remains unclear whether it is endemic or sporadically transmitted. This study supports a timely change towards WNV active surveillance, to avoid and mitigate potential public health emergencies as those recently observed in other European countries.

Whereas the role of calcium ions (Ca2+) in plant signaling is well studied, the physiological significance of pH-changes remains largely undefined. Here we developed CapHensor, an optimized dual-reporter for simultaneous Ca2+ and pH ratio-imaging and studied signaling events in pollen tubes (PTs), guard cells (GCs), and mesophyll cells (MCs). Monitoring spatio-temporal relationships between membrane voltage, Ca2+- and pH-dynamics revealed interconnections previously not described. In tobacco PTs, we demonstrated Ca2+-dynamics lag behind pH-dynamics during oscillatory growth, and pH correlates more with growth than Ca2+. In GCs, we demonstrated abscisic acid (ABA) to initiate stomatal closure via rapid cytosolic alkalization followed by Ca2+ elevation. Preventing the alkalization blocked GC ABA-responses and even opened stomata in the presence of ABA, disclosing an important pH-dependent GC signaling node. In MCs, a flg22-induced membrane depolarization preceded Ca2+-increases and cytosolic acidification by c. 2 min, suggesting a Ca2+/pH-independent early pathogen signaling step. Imaging Ca2+ and pH resolved similar cytosol and nuclear signals and demonstrated flg22, but not ABA and hydrogen peroxide to initiate rapid membrane voltage-, Ca2+- and pH-responses. We propose close interrelation in Ca2+- and pH-signaling that is cell type- and stimulus-specific and the pH having crucial roles in regulating PT growth and stomata movement.

Background Cross-reactivity to SARS-CoV-2 from exposure to endemic human coronaviruses (eHCoV) is gaining increasing attention as a possible driver of both protection against infection and COVID-19 severity. Here we explore the potential role of cross-reactivity induced by eHCoVs on age-specific COVID-19 severity in a mathematical model of eHCoV and SARS-CoV-2 transmission. Methods We use an individual-based model, calibrated to prior knowledge of eHCoV dynamics, to fully track individual histories of exposure to eHCoVs. We also model the emergent dynamics of SARS-CoV-2 and the risk of hospitalisation upon infection. Results We hypothesise that primary exposure with any eHCoV confers temporary cross-protection against severe SARS-CoV-2 infection, while life-long re-exposure to the same eHCoV diminishes cross-protection, and increases the potential for disease severity. We show numerically that our proposed mechanism can explain age patterns of COVID-19 hospitalisation in EU/EEA countries and the UK. We further show that some of the observed variation in health care capacity and testing efforts is compatible with country-specific differences in hospitalisation rates under this model. Conclusions This study provides a “proof of possibility” for certain biological and epidemiological mechanisms that could potentially drive COVID-19-related variation across age groups. Our findings call for further research on the role of cross-reactivity to eHCoVs and highlight data interpretation challenges arising from health care capacity and SARS-CoV-2 testing.

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.

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.

The interacting binary Eta Carinae remains one of the most enigmatic massive stars in our Galaxy despite over four centuries of observations. In this work, its light curve from the ultraviolet to the near-infrared is analysed using spatially resolved HST observations and intense monitoring at the La Plata Observatory, combined with previously published photometry. We have developed a method to separate the central stellar object in the ground-based images using HST photometry and applying it to the more numerous ground-based data, which supports the hypothesis that the central source is brightening faster than the almost-constant Homunculus. After detrending from long-term brightening, the light curve shows periodic orbital modulation (Delta V similar to 0.6 mag) attributed to the wind-wind collision cavity as it sweeps around the primary star and it shows variable projected area to our line-of-sight. Two quasi-periodic components with time-scales of 2-3 and 8-10 yr and low amplitude, Delta V < 0.2 mag, are superimposed on the brightening light curve, being the only stellar component of variability found, which indicates minimal stellar instability. Moreover, the light-curve analysis shows no evidence of 'shell ejections' at periastron. We propose that the long-term brightening of the stellar core is due to the dissipation of a dusty clump in front of the central star, which works like a natural coronagraph. Thus, the central stars appear to be more stable than previously thought since the dominant variability originates from a changing circumstellar medium. We predict that the brightening phase, due mainly to dust dissipation, will be completed around 2032 +/- 4 yr, when the star will be brighter than in the 1600s by up to Delta V similar to 1 mag.

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 receptorlike channels (GLRs) by CORNICHON HOMOLOG (CNIH) proteins. Single mutants of pollenexpressed 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.

Glutamate receptors are well characterized channels that mediate cell-to-cell communication during neurotransmission in animals, but their functional role in organisms without a nervous system remains unclear. In plants, genes of the GLUTAMATE RECEPTOR-LIKE (GLR) family have been implicated in defence against pathogens, reproduction, control of stomata aperture and light signal transduction. However, the large number of GLR genes present in angiosperm genomes (20 to 70) has prevented the observation of strong phenotypes in loss-of-function mutants. Here we show that in the basal land plant Physcomitrella patens, mutation of the GLR genes GLR1 and GLR2 causes failure of sperm cells to target the female reproductive organs. In addition, we show that GLR genes encode non-selective Ca2+-permeable channels that can regulate cytoplasmic Ca2+ and are needed to induce the expression of a BELL1-like transcription factor essential for zygote development. Our work reveals functions for GLR channels in sperm chemotaxis and transcriptional regulation. Sperm chemotaxis is essential for fertilization in both animals and early land plants such as bryophytes and pteridophytes. Therefore, our results suggest that ionotropic glutamate receptors may have been conserved throughout plant evolution to mediate cell-to-cell communication during sexual reproduction.

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.