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Chitosan is a partially deacetylated linear polysaccharide composed of β‐1,4‐linked units of d‐glucosamine and N‐acetyl glucosamine. As well as a structural component of fungal cell walls, chitosan is a potent antifungal agent. However, the mode of action of chitosan is poorly understood. Here, we report that chitosan is effective for control of rice blast disease. Chitosan application impairs growth of the blast fungus Magnaporthe oryzae and has a pronounced effect on appressorium‐mediated plant infection. Chitosan inhibits septin‐mediated F‐actin remodelling at the appressorium pore, thereby preventing repolarization of the infection cell. Chitosan causes plasma membrane permeabilization of M. oryzae and affects NADPH oxidase‐dependent synthesis of reactive oxygen species, essential for septin ring formation and fungal pathogenicity. We further show that toxicity of chitosan to M. oryzae requires the protein kinase C‐dependent cell wall integrity pathway, the Mps1 mitogen‐activated protein kinase and the Nox1 NADPH oxidase. A conditionally lethal, analogue (PP1)‐sensitive mutant of Pkc1 is partially remediated for growth in the presence of chitosan, while ∆nox1 mutants increase their glucan : chitin cell wall ratio, rendering them resistant to chitosan. Taken together, our data show that chitosan is a potent fungicide which requires the cell integrity pathway, disrupts plasma membrane function and inhibits septin‐mediated plant infection. This work was supported by AGL 2015 66833-R grant from the Spanish Ministry of Economy and Competitiveness and European H2020 Project MUSA-727624 and an EMBO Short-term Fellowship to FL-M. Work in NJT’s laboratory is supported by the Gatsby Charitable Foundation.

The chloroplast has recently emerged as pivotal to co-ordinating plant defence responses and as a target of plant pathogens. Beyond its central position in oxygenic photosynthesis and primary metabolism - key targets in the complex virulence strategies of diverse pathogens - the chloroplast integrates, decodes and responds to environmental signals. The capacity of chloroplasts to synthesize phytohormones and a diverse range of secondary metabolites, combined with retrograde and reactive oxygen signalling, provides exquisite flexibility to both perceive and respond to biotic stresses. These processes also represent a plethora of opportunities for pathogens to evolve strategies to directly or indirectly target 'chloroplast immunity'. This review covers the contribution of the chloroplast to pathogen associated molecular pattern and effector triggered immunity as well as systemic acquired immunity. We address phytohormone modulation of immunity and surmise how chloroplast-derived reactive oxygen species underpin chloroplast immunity through indirect evidence inferred from genetic modification of core chloroplast components and direct pathogen targeting of the chloroplast. We assess the impact of transcriptional reprogramming of nuclear-encoded chloroplast genes during disease and defence and look at future research challenges.

Rice blast is a devastating disease caused by the fungal pathogen Magnaporthe oryzae that threatens rice production around the world. The fungus produces a specialized infection cell, called the appressorium, that enables penetration through the plant cell wall in response to surface signals from the rice leaf. The underlying biology of plant infection, including the regulation of appressorium formation, is not completely understood. Here we report the identification of a network of temporally coregulated transcription factors that act downstream of the Pmk1 mitogen-activated protein kinase pathway to regulate gene expression during appressorium-mediated plant infection. We show that this tiered regulatory mechanism involves Pmk1-dependent phosphorylation of the Hox7 homeobox transcription factor, which regulates genes associated with induction of major physiological changes required for appressorium development—including cell-cycle control, autophagic cell death, turgor generation and melanin biosynthesis—as well as controlling a additional set of virulence-associated transcription factor–encoding genes. Pmk1-dependent phosphorylation of Mst12 then regulates gene functions involved in septin-dependent cytoskeletal re-organization, polarized exocytosis and effector gene expression, which are necessary for plant tissue invasion. Identification of this regulatory cascade provides new potential targets for disease intervention. Comparative RNA-seq, ChIP–seq and quantitative phosphoproteomics reveal how the blast fungus uses the Pmk1 MAP kinase to regulate a network of transcription factors that orchestrate the complex transcriptome changes necessary for infecting rice plants.

Circadian rhythms have been shown to play an important role in plant-pathogen interactions between plants and fungi. These protocols describe the methodology used to determine the function and characteristics of the diurnal and circadian behavior in the hemibiotrophic fungal pathogen, Magnaporthe oryzae. Here we describe methods to determine how conidiation, conidial development, and pathogenicity may be altered in M. oryzae as a result of differing diurnal or circadian environmental conditions.

Circadian rhythms have been shown to play an important role in plant-pathogen interactions between plants and fungi. These protocols describe the methodology used to determine the function and characteristics of the diurnal and circadian behavior in the hemibiotrophic fungal pathogen, Magnaporthe oryzae. Here we describe methods to determine how conidiation, conidial development, and pathogenicity may be altered in M. oryzae as a result of differing diurnal or circadian environmental conditions.

SummaryChitosan is a partially deacetylated linear polysaccharide composed of β-1,4-linked units of D-glucosamine and N-acetyl glucosamine. As well as acting as a structural component of fungal cell walls, chitosan can be applied as a potent antifungal agent. However, the mode-of-action of chitosan in fungal pathogens is poorly understood.Here, we report that chitosan is effective for control of rice blast disease. Chitosan application impairs growth of the blast fungusMagnaporthe oryzaeand has a pronounced effect on appressorium-mediated plant infection. Chitosan inhibits septin-mediated F-actin re-modelling at the appressorium pore, thereby preventing re-polarisation of the infection cell and rice leaf cuticle penetration.We found that chitosan causes plasma membrane permeabilization ofM. oryzaeand affects NADPH oxidase-dependent synthesis of reactive oxygen species, essential for septin ring formation and fungal pathogenicity. Our data further show that the toxicity of chitosan toM. oryzaerequires the protein kinase C-dependent cell wall integrity pathway and the Nox1 NADPH oxidase. A conditionally lethal, analogue (PP1)-sensitive mutant of Pkc1 is partially remediated for growth in the presence of chitosan and PP1, while Δnox1mutants increase their glucan/chitin cell wall ratio, rendering them resistant to chitosan.Taken together, our data show that chitosan is a potent fungicide for control of the rice blast fungus which involves the cell wall integrity pathway, disrupts plasma membrane and inhibits septin-mediated plant infection.

Rice blast is a pervasive and devastating disease that threatens rice production across the world. In spite of its importance to global food security, however, the underlying biology of plant infection by the blast fungus Magnaporthe oryzae remains poorly understood. In particular, it is unclear how the fungus elaborates a specialised infection cell, the appressorium, in response to surface signals from the rice leaf. Here, we report the identification of a network of temporally co-regulated transcription factors that act downstream of the Pmk1 mitogen-activated protein kinase pathway to regulate gene expression during appressorium-mediated plant infection. We have functionally characterised this network of transcription factors and demonstrated the operation of a hierarchical transcriptional control system. We show that this tiered regulatory mechanism involves Pmk1-dependent phosphorylation of the Hox7 homeobox transcription factor, which represses hyphal-associated gene expression and simultaneously induces major physiological changes required for appressorium development, including cell cycle arrest, autophagic cell death, turgor generation and melanin biosynthesis. Mst12 then regulates gene functions involved in septin-dependent cytoskeletal re-organisation, polarised exocytosis and effector gene expression necessary for plant tissue invasion.

Rice blast is a devastating disease caused by the fungal pathogen Magnaporthe oryzae that threatens rice production around the world. The fungus produces a specialized infection cell, called the appressorium, that enables penetration through the plant cell wall in response to surface signals from the rice leaf. The underlying biology of plant infection, including the regulation of appressorium formation, is not completely understood. Here we report the identification of a network of temporally coregulated transcription factors that act downstream of the Pmk1 mitogen-activated protein kinase pathway to regulate gene expression during appressorium-mediated plant infection. We show that this tiered regulatory mechanism involves Pmk1-dependent phosphorylation of the Hox7 homeobox transcription factor, which regulates genes associated with induction of major physiological changes required for appressorium development-including cell-cycle control, autophagic cell death, turgor generation and melanin biosynthesis-as well as controlling a additional set of virulence-associated transcription factor-encoding genes. Pmk1-dependent phosphorylation of Mst12 then regulates gene functions involved in septin-dependent cytoskeletal re-organization, polarized exocytosis and effector gene expression, which are necessary for plant tissue invasion. Identification of this regulatory cascade provides new potential targets for disease intervention.

Cell theory has officially reached 350 years of age as the first use of the word 'cell' in a biological context can be traced to a description of plant material by Robert Hooke in his historic publication 'Micrographia: or some physiological definitions of minute bodies'. The 2015 Royal Microscopical Society Botanical Microscopy meeting was a celebration of the streams of investigation initiated by Hooke to understand at the subcellular scale how plant cell function and form arises. Much of the work presented, and Honorary Fellowships awarded, reflected the advanced application of bioimaging informatics to extract quantitative data from micrographs that reveal dynamic molecular processes driving cell growth and physiology. The field has progressed from collecting many pixels in multiple modes to associating these measurements with objects or features that are meaningful biologically. The additional complexity involves object identification that draws on a different type of expertise from computer science and statistics that is often impenetrable to biologists. There are many useful tools and approaches being developed, but we now need more interdisciplinary exchange to use them effectively. In this review we show how this quiet revolution has provided tools available to any personal computer user. We also discuss the oft-neglected issue of quantifying algorithm robustness and the exciting possibilities offered through the integration of physiological information generated by biosensors with object detection and tracking.

Magnaporthe oryzae is the causal agent of rice blast disease, the most devastating disease of cultivated rice (Oryza sativa) and a continuing threat to global food security. To cause disease, the fungus elaborates a specialized infection cell called an appressorium, which breaches the cuticle of the rice leaf, allowing the fungus entry to plant tissue. Here, we show that the exocyst complex localizes to the tips of growing hyphae during vegetative growth, ahead of the Spitzenkörper, and is required for polarized exocytosis. However, during infection-related development, the exocyst specifically assembles in the appressorium at the point of plant infection. The exocyst components Sec3, Sec5, Sec6, Sec8, and Sec15, and exocyst complex proteins Exo70 and Exo84 localize specifically in a ring formation at the appressorium pore. Targeted gene deletion, or conditional mutation, of genes encoding exocyst components leads to impaired plant infection. We demonstrate that organization of the exocyst complex at the appressorium pore is a septin-dependent process, which also requires regulated synthesis of reactive oxygen species by the NoxR-dependent Nox2 NADPH oxidase complex. We conclude that septin-mediated assembly of the exocyst is necessary for appressorium repolarization and host cell invasion.

The cuticle is a ubiquitous, predominantly waxy layer on the aerial parts of higher plants that fulfils a number of essential physiological roles, including regulating evapotranspiration, light reflection, and heat tolerance, control of development, and providing an essential barrier between the organism and environmental agents such as chemicals or some pathogens. The structure and composition of the cuticle are closely associated but are typically investigated separately using a combination of structural imaging and biochemical analysis of extracted waxes. Recently, techniques that combine stain-free imaging and biochemical analysis, including Fourier transform infrared spectroscopy microscopy and coherent anti-Stokes Raman spectroscopy microscopy, have been used to investigate the cuticle, but the detection sensitivity is severely limited by the background signals from plant pigments. We present a new method for label-free, in vivo structural and biochemical analysis of plant cuticles based on stimulated Raman scattering (SRS) microscopy. As a proof of principle, we used SRS microscopy to analyze the cuticles from a variety of plants at different times in development. We demonstrate that the SRS virtually eliminates the background interference compared with coherent anti-Stokes Raman spectroscopy imaging and results in label-free, chemically specific confocal images of cuticle architecture with simultaneous characterization of cuticle composition. This innovative use of the SRS spectroscopy may find applications in agrochemical research and development or in studies of wax deposition during leaf development and, as such, represents an important step in the study of higher plant cuticles.

This chapter describes the steps needed to inoculate host plants with a fungus of interest, and subsequently to visualize the infection using confocal microscopy. As an exemplar, we consider the interaction between wheat and the Septoria leaf blotch fungus, Zymoseptoria tritici. This method is easiest when a GFP- or other fluorophore-tagged strain of the studied fungus is available, but notes are also provided which describe possible staining techniques which may be employed if fluorescent fungus is unavailable in your system.

This chapter describes the steps needed to inoculate host plants with a fungus of interest, and subsequently to visualize the infection using confocal microscopy. As an exemplar, we consider the interaction between wheat and the Septoria leaf blotch fungus, Zymoseptoria tritici. This method is easiest when a GFP- or other fluorophore-tagged strain of the studied fungus is available, but notes are also provided which describe possible staining techniques which may be employed if fluorescent fungus is unavailable in your system.

Microalgae are widely viewed as a promising and sustainable source of renewable chemicals and biofuels. Botryococcus braunii synthesizes and secretes significant amounts of long‐chain (C30‐C40) hydrocarbons that can be subsequently converted into gasoline, diesel, and aviation fuel. B. braunii cultures are not axenic and the effects of co‐cultured microorganisms on B. braunii growth and hydrocarbon yield are important, but sometimes contradictory. To understand the composition of the B. braunii microbial consortium, we used high throughput Illumina sequencing of metagenomic DNA to profile the microbiota within a well established, stable B. braunii culture and characterized the demographic changes in the microcosm following modification to the culture conditions. DNA sequences attributed to B. braunii were present in equal quantities in all treatments, whereas sequences assigned to the associated microbial community were dramatically altered. Bacterial species least affected by treatments, and more robustly associated with the algal cells, included members of Rhizobiales, comprising Bradyrhizobium and Methylobacterium, and representatives of Dyadobacter, Achromobacter and Asticcacaulis. The presence of bacterial species identified by metagenomics was confirmed by additional 16S rDNA analysis of bacterial isolates. Our study demonstrates the advantages of high throughput sequencing and robust metagenomic analyses to define microcosms and further our understanding of microbial ecology.

The use of citizen science to obtain annotations from multiple annotators has been shown to be an effective method for annotating datasets in which computational methods alone are not feasible. The way in which the annotations are obtained is an important consideration which affects the quality of the resulting consensus annotation. In this paper, we examine three separate approaches to obtaining consensus scores for instances rather than merely binary classifications. To obtain a consensus score, annotators were asked to make annotations in one of three paradigms: classification, scoring and ranking. A web-based citizen science experiment is described which implements the three approaches as crowdsourced annotation tasks. The tasks are evaluated in relation to the accuracy and agreement among the participants using both simulated and real-world data from the experiment. The results show a clear difference in performance between the three tasks, with the ranking task obtaining the highest accuracy and agreement among the participants. We show how a simple evolutionary optimiser may be used to improve the performance by reweighting the importance of annotators.

The growing world population puts ever-increasing demands on the agricultural and agrochemical industries to increase agricultural yields. This can only be achieved by investing in fundamental plant and agrochemical research and in the development of improved analytical tools to support research in these areas. There is currently a lack of analytical tools that provide noninvasive structural and chemical analysis of plant tissues at the cellular scale. Imaging techniques such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy provide label-free chemically specific image contrast based on vibrational spectroscopy. Over the past decade, these techniques have been shown to offer clear advantages for a vast range of biomedical research applications. The intrinsic vibrational contrast provides label-free quantitative functional analysis, it does not suffer from photobleaching, and it allows near real-time imaging in 3D with submicrometer spatial resolution. However, due to the susceptibility of current detection schemes to optical absorption and fluorescence from pigments (such as chlorophyll), the plant science and agrochemical research communities have not been able to benefit from these techniques and their application in plant research has remained virtually unexplored. In this paper, we explore the effect of chlorophyll fluorescence and absorption in CARS and SRS microscopy. We show that with the latter it is possible to use phase-sensitive detection to separate the vibrational signal from the (electronic) absorption processes. Finally, we demonstrate the potential of SRS for a range of in planta applications by presenting in situ chemical analysis of plant cell wall components, epicuticular waxes, and the deposition of agrochemical formulations onto the leaf surface.

University-based scientists hold the collective responsibility for educating the next generation of citizens, scientists and voters, but the degree to which they are individually trained and rewarded for this pursuit is variable. This F1000Research channel has its origin in a Society for Experimental Biology Conference held in Prague, 2015 and brings together researchers who excel at undergraduate education or the scholarship of teaching and learning to discuss challenges and best practices in contemporary higher science education.

Progress in analysing the cellular functions of many structural proteins has accelerated through the use of confocal microscopy together with transient gene expression. Several methods for transient expression have been developed in the past few years, but their application has seen limited success beyond a few tractable species and tissues. We have developed a simple and efficient method to visualize fluorescent proteins in Arabidopsis root epidermis using co-cultivation of seedlings with Agrobacterium rhizogenes. The method is equally suitable for transient gene expression in other species, including Thellungiella, and can be combined with supporting molecular and biochemical analyses. The method promises significant advantages for study of membrane dynamics, cellular development and polar growth in root hairs without interference in the development of the plant. Since the method targets specifically the root epidermis, it also offers a powerful tool to approach issues of root-rhizosphere interactions, such as ion transport and nutrient acquisition. As a proof of principle, we carried out transfections with fluorescent markers for the plasma membrane (NpPMA2-GFP, Nicotiana plumbaginifolia L. Plasma Membrane H(+)-ATPase 2), the endoplasmic reticulum (YFP-HDEL), and the Golgi apparatus (sialyl transferase-GFP) to trace their distribution in growing Arabidopsis root hairs and epidermis. The results demonstrate that, in Arabidopsis root hairs, movement of the Golgi is faster than previously reported for tobacco leaf epidermal cells, consistent with the high secretory dynamics of the tip growing cell; they show a pattern to the endoplasmic reticulum within the cytoplasm that is more diffuse than found in tobacco leaf epidermis, and they confirm previous findings of a polarized distribution of the endoplasmic reticulum at the tip of growing root hairs.

Cell theory has officially reached 350 years of age as the first use of the word 'cell' in a biological context can be traced to a description of plant material by Robert Hooke in his historic publication 'Micrographia: or some physiological definitions of minute bodies'. The 2015 Royal Microscopical Society Botanical Microscopy meeting was a celebration of the streams of investigation initiated by Hooke to understand at the subcellular scale how plant cell function and form arises. Much of the work presented, and Honorary Fellowships awarded, reflected the advanced application of bioimaging informatics to extract quantitative data from micrographs that reveal dynamic molecular processes driving cell growth and physiology. The field has progressed from collecting many pixels in multiple modes to associating these measurements with objects or features that are meaningful biologically. The additional complexity involves object identification that draws on a different type of expertise from computer science and statistics that is often impenetrable to biologists. There are many useful tools and approaches being developed, but we now need more interdisciplinary exchange to use them effectively. In this review we show how this quiet revolution has provided tools available to any personal computer user. We also discuss the oft-neglected issue of quantifying algorithm robustness and the exciting possibilities offered through the integration of physiological information generated by biosensors with object detection and tracking.

Transcriptional reprogramming is integral to effective plant defense. Pathogen effectors act transcriptionally and posttranscriptionally to suppress defense responses. A major challenge to understanding disease and defense responses is discriminating between transcriptional reprogramming associated with microbial-associated molecular pattern (MAMP)-triggered immunity (MTI) and that orchestrated by effectors. A high-resolution time course of genome-wide expression changes following challenge with Pseudomonas syringae pv tomato DC3000 and the nonpathogenic mutant strain DC3000hrpA allowed us to establish causal links between the activities of pathogen effectors and suppression of MTI and infer with high confidence a range of processes specifically targeted by effectors. Analysis of this information-rich data set with a range of computational tools provided insights into the earliest transcriptional events triggered by effector delivery, regulatory mechanisms recruited, and biological processes targeted. We show that the majority of genes contributing to disease or defense are induced within 6 h postinfection, significantly before pathogen multiplication. Suppression of chloroplast-associated genes is a rapid MAMP-triggered defense response, and suppression of genes involved in chromatin assembly and induction of ubiquitin-related genes coincide with pathogen-induced abscisic acid accumulation. Specific combinations of promoter motifs are engaged in fine-tuning the MTI response and active transcriptional suppression at specific promoter configurations by P. syringae.

Microbe associated molecular pattern (MAMP) receptors in plants recognize MAMPs and activate basal defences; however a complete understanding of the molecular and physiological mechanisms conferring immunity remains elusive. Pathogens suppress active defence in plants through the combined action of effector proteins. Here we show that the chloroplast is a key component of early immune responses. MAMP perception triggers the rapid, large-scale suppression of nuclear encoded chloroplast-targeted genes (NECGs). Virulent Pseudomonas syringae effectors reprogramme NECG expression in Arabidopsis, target the chloroplast and inhibit photosynthetic CO2 assimilation through disruption of photosystem II. This activity prevents a chloroplastic reactive oxygen burst. These physiological changes precede bacterial multiplication and coincide with pathogen-induced abscisic acid (ABA) accumulation. MAMP pretreatment protects chloroplasts from effector manipulation, whereas application of ABA or the inhibitor of photosynthetic electron transport, DCMU, abolishes the MAMP-induced chloroplastic reactive oxygen burst, and enhances growth of a P. syringae hrpA mutant that fails to secrete effectors.

Plant leaves are optically complex, which makes them difficult to image by light microscopy. Careful sample preparation is therefore required to enable researchers to maximise the information gained from advances in fluorescent protein labelling, cell dyes and innovations in microscope technologies and techniques. We have previously shown that mounting leaves in the non-toxic, non-fluorescent perfluorocarbon (PFC), perfluorodecalin (PFD) enhances the optical properties of the leaf with minimal impact on physiology. Here, we assess the use of the perfluorocarbons PFD, and perfluoroperhydrophenanthrene (PP11) for in vivo plant leaf imaging using 4 advanced modes of microscopy: laser scanning confocal microscopy (LSCM), Two-photon fluorescence (TPF) microscopy, second harmonic generation (SHG) microscopy and stimulated Raman scattering (SRS) microscopy. For every mode of imaging tested, we observed an improved signal when leaves were mounted in PFD or in PP11, compared to mounting the samples in water. Using an image analysis technique based on autocorrelation to quantitatively assess LSCM image deterioration with depth, we show that PP11 outperformed PFD as a mounting medium by enabling the acquisition of clearer images deeper into the tissue. In addition, we show that SRS microscopy can be used to image perfluorocarbons directly in the mesophyll and thereby easily delimit the negative space within a leaf, which may have important implications for studies of leaf development. Direct comparison of on and off resonance SRS micrographs show that PFCs do not to form intracellular aggregates in live plants. We conclude that the application of PFCs as mounting media substantially increases advanced microscopy image quality of living mesophyll and leaf vascular bundle cells.

The study of plant cell physiology is currently experiencing a profound transformation. Novel techniques allow dynamic in vivo imaging with subcellular resolution, covering a rapidly growing range of plant cell physiology. Several basic biological questions that have been inaccessible by the traditional combination of biochemical, physiological and cell biological approaches now see major progress. Instead of grinding up tissues, destroying their organisation, or describing cell- and tissue structure, without a measure for its function, novel imaging approaches can provide the critical link between localisation, function and dynamics. Thanks to a fast growing collection of available fluorescent protein variants and sensors, along with innovative new microscopy technologies and quantitative analysis tools, a wide range of plant biology can now be studied in vivo, including cell morphology & migration, protein localization, topology & movement, protein-protein interaction, organelle dynamics, as well as ion, ROS & redox dynamics. Within the cell, genetic targeting of fluorescent protein probes to different organelles and subcellular locations has started to reveal the stringently compartmentalized nature of cell physiology and its sophisticated spatiotemporal regulation in response to environmental stimuli. Most importantly, such cellular processes can be monitored in their natural 3D context, even in complex tissues and organs – a condition not easily met in studies on mammalian cells. Recent new insights into plant cell physiology by functional imaging have been largely driven by technological developments, such as the design of novel sensors, innovative microscopy & imaging techniques and the quantitative analysis of complex image data. Rapid further advances are expected which will require close interdisciplinary interaction of plant biologists with chemists, physicists, mathematicians and computer scientists. High-throughput approaches will become increasingly important, to fill genomic data with ‘life’ on the scale of cell physiology. If the vast body of information generated in the -omics era is to generate actual mechanistic understanding of how the live plant cell works, functional imaging has enormous potential to adopt the role of a versatile standard tool across plant biology and crop breeding. We welcome original research papers, methodological papers, reviews and mini reviews, with particular attention to contributions in which novel imaging techniques enhance our understanding of plant cell physiology and permits to answer questions that cannot be easily addressed with other techniques.

The use of citizen science to obtain annotations from multiple annotators has been shown to be an effective method for annotating datasets in which computational methods alone are not feasible. The way in which the annotations are obtained is an important consideration which affects the quality of the resulting consensus estimates. In this paper, we examine three separate approaches to obtaining scores for instances rather than merely classifications. To obtain a consensus score annotators were asked to make annotations in one of three paradigms: classification, scoring and ranking. A web-based citizen science experiment is described which implements the three approaches as crowdsourced annotation tasks. The tasks are evaluated in relation to the accuracy and agreement among the participants using both simulated and real-world data from the experiment. The results show a clear difference in performance between the three tasks, with the ranking task obtaining the highest accuracy and agreement among the participants. We show how a simple evolutionary optimiser may be used to improve the performance by reweighting the importance of annotators.

Exposure of photosynthetic cells of leaf tissues of Arabidopsis thaliana (Arabidopsis) to high light intensities (HL) may provoke a rapid rise in hydrogen peroxide (H2O2) levels in chloroplasts and subcellular compartments, such as peroxisomes, associated with photosynthetic metabolism. It has been hypothesized that when H2O2 is contained at or near its site of production then it plays an important role in signaling to induce acclimation to HL. However, should this discrete containment fail and H2O2 levels exceed the capacity of antioxidant systems to scavenge them, then oxidative stress ensues which triggers cell death. To test this hypothesis, the spatiotemporal accumulation of H2O2 needs to be quantified in different subcellular compartments. In this chapter, preliminary experiments are presented on the use of Arabidopsis seedlings transformed with a nuclear-encoded cytosol-located yellow fluorescent protein-based sensor for H2O2, called HyPer. HyPer allows ratiometric determination of its fluorescence at two excitation wavelengths, which frees quantification of H2O2 from the variable levels of HyPer in vivo. HyPer fluorescence was shown to have the potential to provide the necessary spatial, temporal, and quantitative resolution to study HL responses of seedlings using confocal microscopy. Chlorophyll fluorescence imaging was used to quantify photoinhibition of photosynthesis induced by HL treatment of seedlings on the microscope staging. However, several technical issues remain, the most challenging of which is the silencing of HyPer expression beyond the seedling stage. This limited our pilot studies to cotyledon epidermal cells, which while not photosynthetic, nevertheless responded to HL with 45% increase in cytosolic H2O2.

Exposure of photosynthetic cells of leaf tissues of Arabidopsis thaliana (Arabidopsis) to high light intensities (HL) may provoke a rapid rise in hydrogen peroxide (H2O2) levels in chloroplasts and subcellular compartments, such as peroxisomes, associated with photosynthetic metabolism. It has been hypothesized that when H2O2 is contained at or near its site of production then it plays an important role in signaling to induce acclimation to HL. However, should this discrete containment fail and H2O2 levels exceed the capacity of antioxidant systems to scavenge them, then oxidative stress ensues which triggers cell death. To test this hypothesis, the spatiotemporal accumulation of H2O2 needs to be quantified in different subcellular compartments. In this chapter, preliminary experiments are presented on the use of Arabidopsis seedlings transformed with a nuclear-encoded cytosol-located yellow fluorescent protein-based sensor for H2O2, called HyPer. HyPer allows ratiometric determination of its fluorescence at two excitation wavelengths, which frees quantification of H2O2 from the variable levels of HyPer in vivo. HyPer fluorescence was shown to have the potential to provide the necessary spatial, temporal, and quantitative resolution to study HL responses of seedlings using confocal microscopy. Chlorophyll fluorescence imaging was used to quantify photoinhibition of photosynthesis induced by HL treatment of seedlings on the microscope staging. However, several technical issues remain, the most challenging of which is the silencing of HyPer expression beyond the seedling stage. This limited our pilot studies to cotyledon epidermal cells, which while not photosynthetic, nevertheless responded to HL with 45% increase in cytosolic H2O2.

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.