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Background and Aims Plants underpin life on Earth and are essential to human existence. Alarmingly, almost 40% of plant species are under threat of extinction, with plants that are not directly useful to humans being particularly vulnerable. Plant diversity and its untapped resources require urgent protection to safeguard our future, but conservation initiatives are biased towards mammals and birds. Plant awareness disparity, formerly known as plant blindness, describes our tendency to ignore plant life and has been suggested to play a crucial role in the bias against funding and support for plant conservation programmes. Previous studies indicate that nature documentaries can generate shifts in audience awareness of animal species by providing vicarious connections to nature. Here, we investigated whether the plant-focused popular BBC show Green Planet had a similar effect for plants and stimulated audience engagement for information after the broadcast. Methods Online searches for further information were considered a form of engagement for evaluation of the interest of the audience in plants portrayed in Green Planet episodes. The big data activities (Google search engine and Wikipedia pageviews trends) related to the plants mentioned in Green Planet episodes were examined over the period covering the broadcast of the show in UK. Key Results Analyses indicate that Green Planet generated increased awareness and stimulated audience engagement for further information about plants featured in the show, with audience reaction driven by the screen time. Conclusions Natural history films can promote plant awareness, and culturomic tools can be used to assess their impact on the general public, potentially also to inform plant conservation strategies. These are promising findings as we strive to increase public awareness of the value of plant life.

Natural selection has shaped a wide range of lifespans across mammals, with a few long-lived species showing negligible signs of ageing. Approaches used to elucidate the genetic mechanisms underlying mammalian longevity usually involve phylogenetic selection tests on candidate genes, analyses of differential gene expression between age cohorts or species, and measuring age-related epigenetic changes. However, the link between gene duplication and evolution of mammalian longevity has not been widely investigated. Here, we explored the association between gene duplication and mammalian lifespan by analysing 287 human longevity-associated genes across 37 placental mammals. We estimated that the expansion rate of these genes is eight times higher than their contraction rate across these 37 species. Using phylogenetic approaches, we identified 43 genes whose duplication levels are significantly correlated with longevity quotients (FDR < 0.05). In particular, strong correlation observed for four genes (CREBBP,PIK3R1,HELLS,FOXM1) appears to be driven mainly by their high duplication levels in two ageing extremists, the naked mole rat (Heterocephalus glaber) and the greater mouse-eared bat (Myotis myotis). Further sequence and expression analyses suggest that the genePIK3R1may have undergone a convergent duplication event, whereby the similar region of its coding sequence was independently duplicated multiple times in both of these long-lived species. Collectively, this study identified several candidate genes whose duplications may underlie the extreme longevity in mammals, and highlighted the potential role of gene duplication in the evolution of mammalian long lifespans.

Programmed cell death (PCD) facilitates targeted elimination of redundant, damaged, or infected cells via genetically controlled pathways. In plants, PCD is often an essential component of normal development and can also mediate responses to abiotic and biotic stress stimuli. However, studying the transcriptional regulation of this fundamental process is hindered by difficulties in sampling small groups of cells undergoing PCD that are often buried within the bulk of living plant tissue. We addressed this challenge by using RNA sequencing (RNA-Seq) ofArabidopsis thalianasuspension cells, a system that allows precise monitoring of PCD activation and progression. The use of three PCD-inducing treatments (salicylic acid, heat and critical dilution), in combination with three cell death modulators (3- methyladenine, lanthanum chloride and conditioned medium), allowed isolation of candidate ‘core’ and stimuli-specific PCD genes, inference of underlying gene regulatory networks and identification of putative transcriptional regulators. This analysis underscored cell cycle disturbance and the repression of both pro-survival stress responses and mitochondrial retrograde signalling as key elements of the PCD-associated transcriptional signature in plants. Further, phenotyping of twentyArabidopsisT-DNA insertion mutants in selected candidate genes confirmed a role for several in PCD and stress tolerance regulation, and validated the potential of these generated resources to identify novel genes involved in plant PCD pathways and/or stress tolerance in plants.

Autophagy maintains cellular homeostasis and its dysfunction has been implicated in aging. Bats are the longest-lived mammals for their size, but the molecular mechanisms underlying their extended healthspan are not well understood. Here, drawing on >8 years of mark-recapture field studies, we report the first longitudinal analysis of autophagy regulation in bats. Mining of published population level aging blood transcriptomes (M. myotis, mouse and human) highlighted a unique increase of autophagy related transcripts with age in bats, but not in other mammals. This bat-specific increase in autophagy transcripts was recapitulated by the western blot determination of the autophagy marker, LC3II/I ratio, in skin primary fibroblasts (Myotis myotis, Pipistrellus kuhlii, mouse), that also showed an increase with age in both bat species. Further phylogenomic selection pressure analyses across eutherian mammals (n=70 taxa; 274 genes) uncovered 10 autophagy-associated genes under selective pressure in bat lineages. These molecular adaptations potentially mediate the exceptional age-related increase of autophagy signalling in bats, which may contribute to their longer healthspans.

Both auxin signalling and programmed cell death (PCD) are essential components of a normally functioning plant. Auxin underpins plant growth and development, as well as regulating plant defences against environmental stresses. PCD, a genetically controlled pathway for selective elimination of redundant, damaged, or infected cells, is also a key element of many developmental processes and stress response mechanisms in plants. An increasing body of evidence suggests that auxin signalling and PCD regulation are often connected. While generally auxin appears to suppress cell death, it has also been shown to promote PCD events, most likely via stimulation of ethylene biosynthesis. Intriguingly, certain cells undergoing PCD have also been suggested to control the distribution of auxin in plant tissues, by either releasing a burst of auxin or creating an anatomical barrier to auxin transport and distribution. These recent findings indicate novel roles of localized PCD events in the context of plant development such as control of root architecture, or tissue regeneration following injury, and suggest exciting possibilities for incorporation of this knowledge into crop improvement strategies.

The optimum assessment structure measures student knowledge accurately and without bias. In this study, the performance of the first-year undergraduate science students from the University College Dublin was evaluated to test the gender equality of the assessment structure in place. Results of male and female students taking three life science modules were analysed, for two academic years, with assessment structure based on a combination of three types of evaluation: continuous assessment and multiple choice questions (MCQ) exam scored with/without negative marking. We found no significant gender effect associated with performance in continuous assessment, or MCQ exams scored without negative marking. However, a significant bias against females was consistently observed for the same cohort of students in the MCQ exams with negative marking of 0.25 points. This bias was at least partially linked to a gender difference in willingness to guess and preliminary data suggest that it disappears after removal of negative marking from the MCQ exams. Our results support the view of a diverse assessment structure being fairer to the students. Moreover, caution is advised while using negative marking, and regular reviews of assessment strategy should be implemented by higher education institutions to ensure gender-bias free evaluation of students’ performance. European Research Council

Programmed cell death (PCD) is a genetically controlled pathway that plants can use to selectively eliminate redundant or damaged cells. In addition to its fundamental role in plant development, PCD can often be activated as an essential defense response when dealing with biotic and abiotic stresses. For example, localized, tightly controlled PCD can promote plant survival by restricting pathogen growth, driving the development of morphological traits for stress tolerance such as aerenchyma, or triggering systemic pro-survival responses. Relatively little is known about the molecular control of this essential process in plants, especially in comparison to well-described cell death models in animals. However, the networks orchestrating transcriptional regulation of plant PCD are emerging. Transcription factors (TFs) regulate the clusters of stimuli inducible genes and play a fundamental role in plant responses, such as PCD, to abiotic and biotic stresses. Here, we discuss the roles of different classes of transcription factors, including members of NAC, ERF and WRKY families, in cell fate regulation in response to environmental stresses. The role of TFs in stress-induced mitochondrial retrograde signaling is also reviewed in the context of life-and-death decisions of the plant cell and future research directions for further elucidation of TF-mediated control of stress-induced PCD events are proposed. An increased understanding of these complex signaling networks will inform and facilitate future breeding strategies to increase crop tolerance to disease and/or abiotic stresses.

Bats are unique among mammals given their ability to fly, apparent tolerance of deadly viruses and extraordinary longevity. We propose that these traits are linked and driven by adaptations of the innate immune system. To explore this hypothesis we challenged macrophages from the greater mouse-eared bat, Myotis myotis and the house mouse, Mus musculus with Toll Like Receptors (TLRs) ligands, lipopolysaccharides, LPS and polyinosinic-polycytidylic acid, Poly(I:C). Macrophages from both species presented a high level of mRNA induction of inferon ß ( INF-ß) , tumor necrosis factor ( TNF) and interleukin-1ß (Il- ß) . However, in bat macrophages, this antiviral, proinflammatory response was balanced by a sustained high-level transcription of the anti-inflammatory cytokine Il-10, which was not observed in mouse, potentially resulting from adaptive regulation in bats. Additionally, phylogenomic selection tests across the basal divergences in mammals (n = 39) uncovered bat-specific adaptations in six genes involved in antiviral and proinflammatory signalling. Based on this pilot study, we put forward a hypothesis that bats may have evolved unique anti-inflamma tory responses to neutralize proinflammatory stimuli resulting from flight. This in turn may drive their extraordinary longevity and viral tolerance by limiting inflammation driven ageing and infection-induced immunopathology. Further data from other individuals and bat species are required to advance this intriguing hypothesis. European Research Council University College Dublin http://www.bioone.org/loi/acta

The protoplast retracts during apoptosis-like programmed cell death (AL-PCD) and, if this retraction is an active component of AL-PCD, it should be used as a defining feature for this type of programmed cell death. We used an array of pharmacological and genetic tools to test if the rates of protoplast retraction in cells undergoing AL-PCD can be modulated. Disturbing calcium flux signalling, ATP synthesis and mitochondrial permeability transition all inhibited protoplast retraction and often also the execution of the death programme. Protoplast retraction can precede loss of plasma membrane integrity and cell death can be interrupted after the protoplast retraction had already occurred. Blocking calcium influx inhibited the protoplast retraction, reduced DNA fragmentation and delayed death induced by AL-PCD associated stresses. At higher levels of stress, where cell death occurs without protoplast retraction, blocking calcium flux had no effect on the death process. The results therefore strongly suggest that retraction of the protoplast is an active biological process dependent on an early Ca2+-mediated trigger rather than cellular disintegration due to plasma membrane damage. Therefore this morphologically distinct cell type is a quantifiable feature, and consequently, reporter of AL-PCD. Irish Research Council UCD School of Biology and Environmental Science postgraduate funding award

In Europe, 2 different diploid chromosome numbers, 2n = 54 and 2n = 56, have been described in the lesser horseshoe bat (Rhinolophushipposideros). The eastern form with 2n = 56 extends from the Czech Republic to Greece. To date, specimens with 54 chromosomes have been reported only from Spain and Germany. This study expands the distributional area of the western variant to Ireland. Strikingly, this distribution of European chromosomal variants is in contrast to the available molecular data that indicate little genetic differentiation of R. hipposideros populations spanning Northwestern to Central Europe. Further, we have developed an optimized protocol for establishing fibroblast cell cultures, suitable for karyotype analyses, from 3-mm wing membrane biopsies. This is a useful technique for cytogenetic studies of endangered bat species, as this non-lethal sampling method imposes only minimum stress to the animal without lasting adverse effects and is routinely used to sample tissue probes for molecular genetic studies in bats.

Telomeres do not shorten with age in longest-lived bats., Understanding aging is a grand challenge in biology. Exceptionally long-lived animals have mechanisms that underpin extreme longevity. Telomeres are protective nucleotide repeats on chromosome tips that shorten with cell division, potentially limiting life span. Bats are the longest-lived mammals for their size, but it is unknown whether their telomeres shorten. Using >60 years of cumulative mark-recapture field data, we show that telomeres shorten with age in Rhinolophus ferrumequinum and Miniopterus schreibersii, but not in the bat genus with greatest longevity, Myotis. As in humans, telomerase is not expressed in Myotis myotis blood or fibroblasts. Selection tests on telomere maintenance genes show that ATM and SETX, which repair and prevent DNA damage, potentially mediate telomere dynamics in Myotis bats. Twenty-one telomere maintenance genes are differentially expressed in Myotis, of which 14 are enriched for DNA repair, and 5 for alternative telomere-lengthening mechanisms. We demonstrate how telomeres, telomerase, and DNA repair genes have contributed to the evolution of exceptional longevity in Myotis bats, advancing our understanding of healthy aging.

Programmed cell death (PCD) is a critical component of plant development, defense against invading pathogens, and response to environmental stresses. In this chapter, we provide detailed technical methods for studying PCD associated with plant development or induced by abiotic stress. A root hair assay or electrolyte leakage assay are excellent techniques for the quantitative determination of PCD and/or cellular injury induced in response to abiotic stress, whereas the lace plant provides a unique model that facilitates the study of genetically regulated PCD during leaf development.

In Europe, 2 different diploid chromosome numbers, 2n = 54 and 2n = 56, have been described in the lesser horseshoe bat (Rhinolophushipposideros). The eastern form with 2n = 56 extends from the Czech Republic to Greece. To date, specimens with 54 chromosomes have been reported only from Spain and Germany. This study expands the distributional area of the western variant to Ireland. Strikingly, this distribution of European chromosomal variants is in contrast to the available molecular data that indicate little genetic differentiation of R. hipposideros populations spanning Northwestern to Central Europe. Further, we have developed an optimized protocol for establishing fibroblast cell cultures, suitable for karyotype analyses, from 3-mm wing membrane biopsies. This is a useful technique for cytogenetic studies of endangered bat species, as this non-lethal sampling method imposes only minimum stress to the animal without lasting adverse effects and is routinely used to sample tissue probes for molecular genetic studies in bats.

Programmed cell death (PCD) is a critical component of plant development, defense against invading pathogens, and response to environmental stresses. In this chapter, we provide detailed technical methods for studying PCD associated with plant development or induced by abiotic stress. A root hair assay or electrolyte leakage assay are excellent techniques for the quantitative determination of PCD and/or cellular injury induced in response to abiotic stress, whereas the lace plant provides a unique model that facilitates the study of genetically regulated PCD during leaf development.

In plants, apoptosis-like programmed cell death (AL-PCD) is readily distinguished from other forms of programmed cell death (PCD) through a distinct morphology. Detection of cytochrome c release from mitochondria and changes in mitochondrial morphology are the earliest markers for detection of this form of PCD in plants. In this chapter we provide detailed technical methods for the visualization of both of these mitochondrial markers of AL-PCD in Arabidopsis.

Programmed cell death can be defined as an organized cellular destruction and can be activated throughout plant development, as a defense response against invading pathogens or during environmental stress. The root hair assay presented herein enables in vivo quantitative measurements of programmed cell death based on the morphological changes of dying root hairs. Application of this novel, simple technique eliminates the need for establishing cell suspension cultures, resulting in a significant reduction in time, cost, and labor input. Here, we present a detailed root hair assay protocol for the dicotyledonous model plant Arabidopsis thaliana, where results from germination to scoring of cell death can be obtained within 7 days. We also suggest and present a panel of cell death inducing treatments which can be used to study abiotic stress- and mycotoxin-induced programmed cell death in the root hair system in Arabidopsis. A root hair assay protocol for the monocotyledonous model species Brachypodium distachyon is also included.

Programmed cell death can be defined as an organized cellular destruction and can be activated throughout plant development, as a defense response against invading pathogens or during environmental stress. The root hair assay presented herein enables in vivo quantitative measurements of programmed cell death based on the morphological changes of dying root hairs. Application of this novel, simple technique eliminates the need for establishing cell suspension cultures, resulting in a significant reduction in time, cost, and labor input. Here, we present a detailed root hair assay protocol for the dicotyledonous model plant Arabidopsis thaliana, where results from germination to scoring of cell death can be obtained within 7 days. We also suggest and present a panel of cell death inducing treatments which can be used to study abiotic stress- and mycotoxin-induced programmed cell death in the root hair system in Arabidopsis. A root hair assay protocol for the monocotyledonous model species Brachypodium distachyon is also included.

The Fusarium mycotoxin deoxynivalenol (DON) can cause cell death in wheat (Triticum aestivum), but can also reduce the level of cell death caused by heat shock in Arabidopsis (Arabidopsis thaliana) cell cultures. We show that 10 μg mL−1 DON does not cause cell death in Arabidopsis cell cultures, and its ability to retard heat-induced cell death is light dependent. Under dark conditions, it actually promoted heat-induced cell death. Wheat cultivars differ in their ability to resist this toxin, and we investigated if the ability of wheat to mount defense responses was light dependent. We found no evidence that light affected the transcription of defense genes in DON-treated roots of seedlings of two wheat cultivars, namely cultivar CM82036 that is resistant to DON-induced bleaching of spikelet tissue and cultivar Remus that is not. However, DON treatment of roots led to genotype-dependent and light-enhanced defense transcript accumulation in coleoptiles. Wheat transcripts encoding a phenylalanine ammonia lyase (PAL) gene (previously associated with Fusarium resistance), non-expressor of pathogenesis-related genes-1 (NPR1) and a class III plant peroxidase (POX) were DON-upregulated in coleoptiles of wheat cultivar CM82036 but not of cultivar Remus, and DON-upregulation of these transcripts in cultivar CM82036 was light enhanced. Light and genotype-dependent differences in the DON/DON derivative content of coleoptiles were also observed. These results, coupled with previous findings regarding the effect of DON on plants, show that light either directly or indirectly influences the plant defense responses to DON. Science Foundation Ireland EU FP5 Project

The activation of programmed cell death (PCD) is often a result of complex signalling pathways whose relationship and intersection are not well understood. We recently described a PCD root hair assay and proposed that it could be used to rapidly screen genetic or pharmacological modulators of PCD. To further assess the applicability of the root hair assay for studying multiple signalling pathways leading to PCD activation we have investigated the crosstalk between salicylic acid, autophagy and apoptosis-like PCD (AL-PCD) in Arabidopsis thaliana. The root hair assay was used to determine rates of AL-PCD induced by a panel of cell death inducing treatments in wild type plants treated with chemical modulators of salicylic acid synthesis or autophagy, and in genetic lines defective in autophagy or salicylic acid signalling. The assay demonstrated that PCD induced by exogenous salicylic acid or fumonisin B1 displayed a requirement for salicylic acid signalling and was partially dependent on the salicylic acid signal transducer NPR1. Autophagy deficiency resulted in an increase in the rates of AL-PCD induced by salicylic acid and fumonisin B1, but not by gibberellic acid or abiotic stress. The phenylalanine ammonia lyase-dependent salicylic acid synthesis pathway contributed only to death induced by salicylic acid and fumonisin B1. 3-Methyladenine, which is commonly used as an inhibitor of autophagy, appeared to influence PCD induction in all treatments suggesting a possible secondary, non-autophagic, effect on a core component of the plant PCD pathway. The results suggest that salicylic acid signalling is negatively regulated by autophagy during salicylic acid and mycotoxin-induced AL-PCD. However, this crosstalk does not appear to be directly involved in PCD induced by gibberellic acid or abiotic stress. This study demonstrates that the root hair assay is an effective tool for relatively rapid investigation of complex signalling pathways leading to the activation of PCD.

The Fusarium genus of fungi is responsible for commercially devastating crop diseases and the contamination of cereals with harmful mycotoxins. Fusarium mycotoxins aid infection, establishment, and spread of the fungus within the host plant. We investigated the effects of the Fusarium mycotoxin deoxynivalenol (DON) on the viability of Arabidopsis cells. Although it is known to trigger apoptosis in animal cells, DON treatment at low concentrations surprisingly did not kill these cells. On the contrary, we found that DON inhibited apoptosis-like programmed cell death (PCD) in Arabidopsis cells subjected to abiotic stress treatment in a manner independent of mitochondrial cytochrome c release. This suggested that Fusarium may utilise mycotoxins to suppress plant apoptosis-like PCD. To test this, we infected Arabidopsis cells with a wild type and a DON-minus mutant strain of F. graminearum and found that only the DON producing strain could inhibit death induced by heat treatment. These results indicate that mycotoxins may be capable of disarming plant apoptosis-like PCD and thereby suggest a novel way that some fungi can influence plant cell fate. Science Foundation Ireland

In Arabidopsis thaliana we demonstrate that dying root hairs provide an easy and rapid in vivo model for the morphological identification of apoptotic-like programmed cell death (AL-PCD) in plants. The model described here is transferable between species, can be used to investigate rates of AL-PCD in response to various treatments and to identify modulation of AL-PCD rates in mutant/transgenic plant lines facilitating rapid screening of mutant populations in order to identify genes involved in AL-PCD regulation.

Programmed cell death (PCD) describes a small number of processes that result in a highly controlled, and organised, form of cellular destruction, activated in every part of the plant, throughout its entire life cycle. For example, PCD is a critical component of many vegetative and reproductive developmental processes, senescence programmes, pathogen defence mechanisms and stress responses. Cell destruction can manifest as apoptotic-like, necrotic or autophagic cell death, and these processes are likely to overlap extensively, sharing several regulatory mechanisms. Several of the key PCD regulators and signals have now been revealed, for example, many cell organelles, including mitochondria, chloroplasts, Golgi apparatus, endoplasmic reticulum and vacuoles have been shown to have a role in controlling PCD activation. Following activation the actual dismantling of the cell appears to involve cell death proteases including those with caspase-like, or metacaspase, activity. This review will examine the current state of knowledge about the regulation of events during plant PCD. We will describe numerous examples of developmental or environmentally induced deaths and outline their potential as model systems for use in PCD research programmes. Similarly, a range of techniques and in vitro model systems that can be used to identify, and quantify, rates of plant PCD are reviewed. These model systems and techniques can be used to identify the underlying signals and events that drive and regulate PCD and ultimately reveal the steps necessary for the botanical dance of death.