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Hemp (Cannabis sativaL.) is an extraordinarily versatile crop, with applications ranging from medicinal compounds to seed oil and fibre products.Cannabis sativais a short-day plant, and its flowering is tightly controlled by photoperiod. However, substantial genetic variation exists for photoperiod sensitivity inC. sativa,and photoperiod-insensitive (“autoflower”) cultivars are available.Using a bi-parental mapping population and bulked segregant analysis, we identifiedAutoflower2, a 0.5 Mbp locus significantly associated with photoperiod-insensitive flowering in hemp.Autoflower2contains an ortholog of the central flowering time regulatorFLOWERING LOCUS T(FT) fromArabidopsis thalianawhich we termedCsFT1. Extensive sequence divergence between alleles ofCsFT1was identified between photoperiod-sensitive and insensitive cultivars ofC. sativa, including a duplication ofCsFT1and sequence differences especially in introns. Genotyping of several mapping populations and a diversity panel confirmed a strong correlation betweenCsFT1alleles and photoperiod response as well as affirming that at least two independent loci for this agriculturally important trait,Autoflower1andAutoflower2, exist in theC. sativagene pool.This study reveals the multiple independent origins of photoperiod insensitivity inC. sativa,supporting the likelihood of a complex domestication history in this species. By integrating the genetic relaxation of photoperiod sensitivity into novelC. sativacultivars, expansion to higher latitudes will be permitted, thus allowing the full potential of this versatile crop to be reached.

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.

Societal Impact Statement Solutions to many of society's major challenges currently relate to plants, for example, feeding a growing global population, reducing crop water use, minimising pesticide use, and mitigating climate change, to name just a few. Solving global challenges requires high‐calibre plant science graduates with a fundamental understanding of plant evolution and the impacts of past climate change on extinction and speciation events, along with the knowledge required for sustainable global plant production, especially for food, under current and future climate change. Equipping these graduates for the future is a responsibility of university educators who must adopt innovative education methods to achieve this challenging goal. Summary The concept of land plant evolution is difficult for botany and biology undergraduates to grasp because it spans half a billion years, a timeline that cannot be easily visualised. We established the University College Dublin (UCD) Evolution of Land Plants Garden to improve student understanding, knowledge, and engagement in a purpose‐built ‘living’ outdoor classroom in which they can ‘walk’ through land plant history. We focus on the concept, configuration, and planting, how the garden has improved student learning, and how it could be adapted for other learning outcomes. The garden is divided into five sections depicting key innovations in land plant evolution, beginning with ‘Before land plants’, followed by ‘Cuticle’ where the Bryophyta appear, ‘Vascular tissue’ with the Pteridophyta; ‘Seed’ with the gymnosperms; and ‘Flower’ with the angiosperms. Analysis of quantitative student feedback and performance indicates that students' understanding of the subject was improved significantly by ‘walking through’ a living evolutionary history in practical classes, supported by lectures, laboratory work, and online quizzes. We present a template that could be adopted widely to engage students with plant evolution, in the context of decreasing engagement with plant sciences among young people.

Hemp (Cannabis sativa) is a highly versatile multi-purpose crop with a multitude of applications, from textiles, biofuel and building material to high-value food products for consumer markets. Furthermore, non-hallucinogenic cannabinoids like cannabidiol (CBD), which can be extracted from female hemp flowers, are potentially valuable pharmacological compounds. In addition, hemp has high carbon sequestration potential due to its rapid growth rate. Therefore, the hemp industry is gaining more traction and breeding hemp cultivars adapted to local climate conditions or bred for specific applications is becoming increasingly important.Here, we present a method for rapid generation cycling (speed breeding) for hemp. The speed breeding protocol makes use of the photoperiod sensitivity of Cannabis. It encompasses vegetative growth of the plants for two weeks under continuous light, followed by flower induction, pollination and seed development for four weeks under short-day conditions and a seed ripening phase under continuous light and water stress. With the protocol introduced here, a generation time of under nine weeks (61 days) from seed to seed can be achieved. Our method furthermore synchronises flowering time of different hemp cultivars, thus facilitating crosses between cultivars. The extremely short generation time will enable hemp researchers and breeders to perform crosses in a time-efficient way and generate new hemp cultivars with defined genetic characteristics in a shorter amount of time.

Cyanobacteria can form nitrogen-fixing symbioses with a broad range of plant species. Unlike other plant-bacteria symbioses, little is understood about the immunological responses induced by plant cyanobionts (symbiotic cyanobacteria). Here, we demonstrated that the model plant-symbiotic cyanobacteria, Nostoc punctiforme is capable of protecting against programmed cell death (PCD) when induced in Arabidopsis thaliana suspension cell cultures. We also profiled the early transcriptomic changes that were induced in response to conditioned medium (CM) from N. punctiforme cell cultures. Interestingly, the reduction in PCD was preceded by the induction of genes associated with defence and immunity, the most striking of which were a number of WRKY-family transcription factors. Down-regulated genes included those involved in the regulation of cell growth and differentiation. This work is the first to show that a cyanobiont can affect plant PCD and provides a useful transcriptome resource for studying early plant cell responses to symbiotic cyanobacteria.

Sphingolipids, a class of amino-alcohol-based lipids, are well characterized in eukaryotes and in some anaerobic bacteria. However, the only sphingolipids so far identified in cyanobacteria are two ceramides (i.e.

Cannabis sativa is most prominent for its psychoactive secondary compound tetrahydrocannabinol, or THC. However, THC is only one of many phytocannabinoids found in this (in)famous medicinal plant. The stepwise legalization of Cannabis in many countries has opened opportunities for its medicinal and commercial use, sparking scientific interest in the genetics and biochemistry of phytocannabinoid synthesis. Advances in plant biology and genomics help to accelerate research in the Cannabis field, which is still lagging behind other comparable high-value crops. Here, we discuss the intriguing genetics and evolutionary history of phytocannabinoid synthases, and also show that an increased understanding of Cannabis developmental genetics and morphology are of critical importance to leverage the full potential of phytocannabinoid production.

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.

Cadmium (Cd) toxicity induces oxidative burst and leads to programmed cell death (PCD) in plant cells. The role of salicylic acid-induced protein kinase (SIPK) signaling pathway in Cd-induced oxidative stress was investigated in suspension-cultured tobacco (Nicotiana tabacum L. cv. Barley 21). The cells were pretreated with 40 μM PD98059 (inhibitor of MAPKK) and then exposed to 50 μM Cd for 24 h. The percentages of cell viability, apoptosis, necrosis, and the content of reactive oxygen species (ROS) were monitored by flow cytometry. Expression of PCD related gene (Hsr203J) and the contents of certain signaling molecules were measured as well. The results showed that Cd increased the expression of SIPK, Hsr203J, and CAT genes, the activities of catalase and caspase-3-like enzymes. Addition of PD98059 inhibitor reduced the expression of Hsr203J and CAT genes, decreased CAT activity, but increased ROS and SA contents, and caspase-3-like activity and apoptosis rate. The highest apoptosis level was accompanied by the highest level of Hsr203J gene expression. From the results it can be suggested that upon treatment of tobacco cells with Cd, internal SA content increased and induced the SIPK signaling pathway, thereby inhibited the antioxidant system and led to PCD.

Cannabis sativa is an extraordinarily versatile species. Hemp and its cousin marijuana, both C. sativa, have been used for millennia as a source of fibre, oil and for medicinal, spiritual and recreational purposes. Because the consumption of Cannabis can have psychoactive effects, the plant has been widely banned throughout the last century. In the past decade, evidence of its medicinal properties did lead to the relaxation of legislation in many countries around the world. Consequently, the genetics and development of Cannabis as well as Cannabis-derived products are the subject of renewed attention.Here, we review the biology of C. sativa, including recent insights from taxonomy, morphology and genomics, with an emphasis on the genetics of cannabinoid synthesis. Because the female Cannabis flower is of special interest as the site of cannabinoid synthesis, we explore flower development, flowering time well as the species’ unique sex determination system in detail. Furthermore, we outline the tremendous medicinal, engineering, and environmental opportunities that Cannabis bears. Together, the picture emerges that our understanding of Cannabis biology currently progresses at an unusual speed. A future challenge will be to preserve the multi-purpose nature of Cannabis, and to harness its medicinal properties and sustainability advantages simultaneously.

Cyanobacteria such as Nostoc spp. can form nitrogen-fixing symbioses with a broad range of plant species. Unlike other plant-bacteria symbioses, little is understood about the immunological and developmental signalling events induced by Nostoc cyanobionts (symbiotic cyanobacteria). Here, we used suspension cell cultures to elucidate the early molecular mechanisms underpinning the association between cyanobionts and plants by studying the effects of conditioned medium (CM) from Nostoc punctiforme cultures on plant programmed cell death (PCD), a typical immune response activated during incompatible interactions. We showed that N. punctiforme-CM could suppress PCD induced by a temperature stress. Interestingly, this was preceded by significant transcriptional reprogramming, as evidenced by the differential regulation of a network of defence-associated genes, as well as genes implicated in regulating cell growth and differentiation. This work is the first to show that cyanobionts can regulate PCD in plants and provides a valuable transcriptome resource for the early immunological and developmental signalling events elicited by Nostoc cyanobionts.

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.

High-throughput screening of an ethyl methanesulfonate-generated mutant collection of Cucurbita pepo using the ethylene triple-response test resulted in the identification of two semi-dominant ethylene-insensitive mutants: etr1a and etr2b. Both mutations altered sex determination mechanisms, promoting conversion of female into bisexual or hermaphrodite flowers, and monoecy into andromonoecy, thereby delaying the transition to female flowering and reducing the number of pistillate flowers per plant. The mutations also altered the growth rate and maturity of petals and carpels in pistillate flowers, lengthening the time required for flowers to reach anthesis, as well as stimulating the growth rate of ovaries and the parthenocarpic development of fruits. Whole-genome sequencing allowed identification of the causal mutation of the phenotypes as two missense mutations in the coding region of CpETR1A and CpETR2B, each one corresponding to one of the duplicates of ethylene receptor genes highly homologous to Arabidopsis ETR1 and ETR2. The phenotypes of homozygous and heterozygous single- and double-mutant plants indicated that the two ethylene receptors cooperate in the control of the ethylene response. The level of ethylene insensitivity, which was determined by the strength of each mutant allele and the dose of wild-type and mutant etr1a and etr2b alleles, correlated with the degree of phenotypic changes in the mutants., Ethylene receptor mutants reveal the involvement of ethylene perception in the control of sex expression, sex determination, and fruit set in Cucurbita pepo.

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

Schilling et al. introduce and discuss Cannabis.

Programmed cell death (PCD) is a form of cellular suicide that serves to eliminate damaged or unwanted cells. In plants PCD operates from embryogenesis to the death of the whole plant and is an integral component of both plant defence and development. Studying the regulatory mechanisms of PCD in vivo can be difficult as often cells undergoing PCD are buried beneath living tissue or it is only possible to observe the cells post mortem. For many of the hallmark characteristics there is only a finite window of opportunity to detect their presence and even then, the presence of these markers is only enough to give a qualitative and not a quantitative measure of PCD. To examine the subtle role environmental factors may have on altering PCD activation thresholds it is necessary to quantitatively assess rates and timing of PCD events.

Peroxidase and hydrogen peroxide both play important roles in the final stages of the lignification pathway. Peroxidase, in the presence of H2O2 catalyses the oxidation of monolignols to give lignin. In order to examine this process we looked at lignification in transgenic tobacco plants expressing a barley peroxidase gene, HvPrx8, either alone or in combination with a wheat germin gene, g.f 2.8, which encodes oxalate oxidase, thereby providing a source of H2O2. Elevated activity of the antioxidant ascorbate peroxidase was found in plants expressing oxalate peroxidase and was greatly increased by co-expression with the barley peroxidase, although the latter had no effect when expressed alone. An increase was observed in the oxidation of the lignin monomer, syringaldazine in cell lines over-expressing barley peroxidase, while a decrease was observed in double transformants. Plants over-expressing barley peroxidase have elevated levels of lignin deposition compared to that of wild type tobacco plants. Over-expression of the individual enzymes was also shown to enhance heat-induced programmed cell death (PCD) in cell suspension cultures, an effect which was greatly reduced in the double-expressing lines.

Brachypodium distachyon is emerging as the model species for temperate monocotyledonous grasses of the Pooideae, and the genome of the B. distachyon community inbred line Bd21 has recently been sequenced. Here, we report the development of a procedure for the efficient establishment of a cell suspension culture derived from calli. We show that embryogenic potential is maintained in 3-month-old cultures as the cells were positively labelled by the monoclonal antibody, JIM8 which recognizes a carbohydrate epitope often present in arabinogalactan proteins found in the cell walls of cells in embryogenic cultures. Additionally, we were able to regenerate plants from these cell suspension cultures. The cell suspension culture we have established can also be used in studies on plant programmed cell death (PCD). Our results clearly demonstrate that B. distachyon cells can undergo apoptosis-like PCD (AL-PCD) as visualised by the characteristic retraction of the protoplast from the cell wall. As B. distachyon is genetically related to important temperate cereal grass crops like wheat and barley, the ability to utilise cell suspension cultures of B. distachyon to dissect the underlying mechanisms PCD will have important implications for understanding developmental processes in economically important cereal crops.

In this commentary we argue that the recent renaming of all types of plant programmed cell death (PCD) into two categories of vacuolar cell death and necrosis is premature and does not fully take into account different forms of cell death that may operate in plant cells. Specifically, we believe that the condensed protoplast morphology associated with many instances of PCD may come about due to an active cell death-associated cellular retraction rather than simple rupture of the plasma membrane. We argue that it is important to be able to distinguish between cells that have died having undergone this protoplast retraction and those which have died without protoplast retraction. In our opinion this differentiation is essential as the control of these two types of death may differ in several respects.

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.

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.

Chloroplasts produce reactive oxygen species (ROS) during cellular stress. ROS are known to act as regulators of programmed cell death (PCD) in plant and animal cells, so it is possible that chloroplasts have a role in regulating PCD in green tissue. Arabidopsis thaliana cell suspension cultures are model systems in which to test this, as here it is shown that their cells contain well-developed, functional chloroplasts when grown in the light, but not when grown in the dark. Heat treatment at 55 °C induced apoptotic-like (AL)-PCD in the cultures, but light-grown cultures responded with significantly less AL-PCD than dark-grown cultures. Chloroplast-free light-grown cultures were established using norflurazon, spectinomycin, and lincomycin and these cultures responded to heat treatment with increased AL-PCD, demonstrating that chloroplasts affect AL-PCD induction in light-grown cultures. Antioxidant treatment of light-grown cultures also resulted in increased AL-PCD induction, suggesting that chloroplast-produced ROS may be involved in AL-PCD regulation. Cycloheximide treatment of light-grown cultures prolonged cell viability and attenuated AL-PCD induction; however, this effect was less pronounced in dark-grown cultures, and did not occur in antioxidant-treated light-grown cultures. This suggests that a complex interplay between light, chloroplasts, ROS, and nuclear protein synthesis occurs during plant AL-PCD. The results of this study highlight the importance of taking into account the time-point at which cells are observed and whether the cells are light-grown and chloroplast-containing or not, for any study on plant AL-PCD, as it appears that chloroplasts can play a significant role in AL-PCD regulation.

Programmed cell death (PCD) in plants is a crucial component of development and defence mechanisms. In animals, different types of cell death (apoptosis, autophagy, and necrosis) have been distinguished morphologically and discussed in these morphological terms. PCD is largely used to describe the processes of apoptosis and autophagy (although some use PCD and apoptosis interchangeably) while necrosis is generally described as a chaotic and uncontrolled mode of death. In plants, the term PCD is widely used to describe most instances of death observed. At present, there is a vast array of plant cell culture models and developmental systems being studied by different research groups and it is clear from what is described in this mass of literature that, as with animals, there does not appear to be just one type of PCD in plants. It is fundamentally important to be able to distinguish between different types of cell death for several reasons. For example, it is clear that, in cell culture systems, the window of time in which 'PCD' is studied by different groups varies hugely and this can have profound effects on the interpretation of data and complicates attempts to compare different researcher's data. In addition, different types of PCD will probably have different regulators and modes of death. For this reason, in plant cell cultures an apoptotic-like PCD (AL-PCD) has been identified that is fairly rapid and results in a distinct corpse morphology which is visible 4-6 h after release of cytochrome c and other apoptogenic proteins. This type of morphology, distinct from autophagy and from necrosis, has also been observed in examples of plant development. In this review, our model system and how it is used to distinguish specifically between AL-PCD and necrosis will be discussed. The different types of PCD observed in plants will also be discussed and the importance of distinguishing between different forms of cell death will be highlighted.

Tobacco suspension cultures were subjected to a range of heat stresses and used to compare morphological aspects of programmed cell death (PCD) and necrosis. Cells undergoing PCD were found to display characteristic death morphology, caused by cytoplasmic retraction of the protoplast, and to have cleaved DNA. We evaluated if the morphological characteristics of PCD could be used to monitor changes in cell death induction thresholds in transgenic cell cultures with high levels of peroxidase activity. Again, using a heat shock assay, we show that tobacco cell cultures with elevated levels of peroxidase have higher cell death induction threshold levels than wild type tobacco cell cultures. Thus, assessing PCD associated morphological changes can report on the effect of altering peroxidase genes on cell death activation in tobacco. This study demonstrates that PCD morphology could routinely be used to monitor the effects of introduced genes on programmed cell death induction thresholds in plants.

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 (PCD) is a fundamental process that occurs in plants during development, the hypersensitive response and situations involving abiotic or biotic stress. One form of PCD is characterised morphologically by the retraction of the protoplast from the cell wall and has been observed in many examples of the aforementioned processes. The mitochondria play a significant role in plant PCD, and research has begun to provide us with answers about the role of the chloroplast in plant PCD. In this chapter we discuss what happens when the mitochondria and chloroplast sense and respond to stress or developmental signals and whether they can play a coordinated role in determining whether a plant cell will live or die.

Five populations of Glyceria fluitans (L.) R. Br. from metal-contaminated and non-contaminated sites across Europe were investigated for innate zinc tolerance. The plants were grown hydroponically in zinc-amended nutrient solutions. Growth and survival of plants from all five populations occurred at all levels of elevated zinc treatments (2, 300, 600 and 1000 μmol L −1 ZnSO 4 ·7H 2 O). There were only slight differences in growth between the populations from contaminated and non-contaminated sites. Uptake of zinc did differ between populations, but this did not affect tolerance. The findings support the theory that wetland angiosperm species tend to be tolerant to exposure to high levels of metals, regardless of their origin.

The key role for mitochondria in mammalian apoptosis, a form of programmed cell death (PCD), is well established, but a similar role for plant mitochondria is just emerging. In order to unravel the molecular mechanisms linking plant mitochondria to the downstream events of PCD, we have developed an Arabidopsis cell-free system that can be used to monitor biochemical and morphological changes in isolated nuclei that are associated with PCD. Using this system, two activities that resulted in nuclear DNA degradation could be distinguished, both of which were facilitated by the addition of mitochondria. One activity mediated the generation of 30 kb DNA fragments within 3 h and chromatin condensation within 6 h, when nuclei were incubated with mitochondria alone. The second activity required cytosolic extract in addition to mitochondria and resulted in oligonucleosome-sized DNA cleavage after >12 h. Submitochondrial fractionation and pharmacological studies suggested the presence of an Mg2+-dependent nuclease activity in the intermembrane space, which is responsible for the former in vitro activity. The evolutionary conservation of the role of mitochondria in PCD in animals and plants is discussed.

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.

15 páginas. -- The definitive version is available at http://jxb.oxfordjournals.org, Light-grown Arabidopsis thaliana cell suspension culture (ACSC) were subjected to mild photooxidative damage with Rose Bengal (RB) with the aim of gaining a better understanding of singlet oxygen-mediated defence responses in plants. Additionally, ACSC were treated with H2O2 at concentrations that induced comparable levels of protein oxidation damage. Under low to medium light conditions, both RB and H2O2 treatments activated transcriptional defence responses and inhibited photosynthetic activity, but they differed in that programmed cell death (PCD) was only observed in cells treated with RB. When dark-grown ACSC were subjected to RB in the light, PCD was suppressed, indicating that the singlet oxygen-mediated signalling pathway in ACSC requires functional chloroplasts. Analysis of up-regulated transcripts in light-grown ACSC, treated with RB in the light, showed that both singlet oxygen-responsive transcripts and transcripts with a key role in hormone-activated PCD (i.e. ethylene and jasmonic acid) were present. A co-regulation analysis proved that ACSC treated with RB exhibited higher correlation with the conditional fluorescence (flu) mutant than with other singlet oxygen-producing mutants or wild-type plants subjected to high light. However, there was no evidence for the up-regulation of EDS1, suggesting that activation of PCD was not associated with the EXECUTER- and EDS1-dependent signalling pathway described in the flu mutant. Indigo Carmine and Methylene Violet, two photosensitizers unable to enter chloroplasts, did not activate transcriptional defence responses in ACSC; however, whether this was due to their location or to their inherently low singlet oxygen quantum efficiencies was not determined., This work is funded by the Junta de Castilla y León (ref: CSI002A10-2). CTD was funded by the Irish Research Council for Science, Engineering and Technology.

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.

Cell-cell and extracellular cell matrix (ECM) interactions provide cells with information essential for controlling morphogenesis, cell-fate specification, and cell death. In animals, one of the major groups of enzymes that degrade the ECM is the matrix metalloproteinases (MMPs). Here, we report the characterization of the cucumber (Cucumis sativus L. cv Marketmore)Cs1-MMP gene encoding such an enzyme likely to play a role in plant ECM degradation. Cs1-MMP has all the hallmark motif characteristics of animal MMPs and is a pre-pro-enzyme having a signal peptide, propeptide, and zinc-binding catalytic domains. Cs1-MMP also displays functional similarities with animal MMPs. For example, it has a collagenase-like activity that can cleave synthetic peptides and type-I collagen, a major component of animal ECM. Cs1-MMP activity is completely inhibited by a hydroxamate-based inhibitor that binds at the active site of MMPs in a stereospecific manner. TheCs1-MMP gene is expressed de novo at the end stage of developmental senescence, prior to the appearance of DNA laddering in cucumber cotyledons leaf discs and male flowers. As the steady-state level of Cs1-MMP mRNA peaks late in senescence and the pro-enzyme must undergo maturation and activation, the protease is probably not involved in nutrient remobilization during senescence but may have another function. The physiological substrates for Cs1-MMP remain to be determined, but the enzyme represents a good candidate for plant ECM degradation and may be involved in programmed cell death (PCD). Our results suggest that PCD occurs only at the culmination of the senescence program or that the processes are distinct with PCD being triggered at the end of senescence.

In plants most instances of programmed cell death (PCD) occur in a number of related, or neighbouring, cells in specific tissues. However, recent research with plant cell cultures has demonstrated that PCD can be induced in single cells. The uniformity, accessibility and reduced complexity of cell cultures make them ideal research tools to investigate the regulation of PCD in plants. PCD has now been induced in cell cultures from a wide range of species including many of the so-called model species. We will discuss the establishment of cell cultures, the fractionation of single cells and isolation of protoplasts, and consider the characteristic features of PCD in cultured cells. We will review the wide range of methods to induce cell death in cell cultures ranging from abiotic stress, absence of survival signals, manipulation of signal pathway intermediates, through the induction of defence-related PCD and developmentally induced cell death.

In mammals mitochondria play a critical role in the activation of programmed cell death (PCD). One mechanism by which mitochondria can commit a cell to death is by translocating cytochrome c into the cytosol where it activates cell death caspases. However, release of cytochrome c does not appear to be a feature of caspase activation in nematodes or insects, similarly, there is no evidence for cytochrome c release during the caspase-independent PCD that can occur in Dictyostelium cells. In an attempt to understand the underlying regulation of PCD in plants we investigated if mitochondrial components were released into the cytosol when plant cells are induced to undergo PCD. PCD was triggered in cucumber cotyledons by subjecting them to a short 55 degrees C heat treatment. This heat treatment has previously been shown to trigger PCD in other plant species and cell death was confirmed in cucumber using morphological (cellular condensation) and molecular (DNA 'laddering') markers of PCD. We present evidence that, unlike Dictyostelium and invertebrate PCDs, cytochrome c release is an early event in plant PCD. The mitochondrial release of cytochrome c following a PCD-inducing stimulus in both plants and mammals suggests the pathways have been conserved during evolution, having been derived from ancestral unicellular death programmes.