Spermine and spermidine inhibit or induce programmed cell death in Arabidopsis thaliana in vitro and in vivo in a dose-dependent manner.

Polyamines are ubiquitous biomolecules with a number of established functions in eukaryotic cells. In plant cells, polyamines have previously been linked to abiotic and biotic stress tolerance, as well as to the modulation of programmed cell death (PCD), with contrasting reports on their pro-PCD and pro-survival effects. Here, we used two well-established platforms for the study of plant PCD, Arabidopsis thaliana suspension cultures cells and the root hair assay, to examine the roles of the polyamines spermine and spermidine in the regulation of PCD. Using these systems for precise quantification of cell death rates, we demonstrate that both polyamines can trigger PCD when applied exogenously at higher doses, whereas at lower concentrations they inhibit PCD induced by both biotic and abiotic stimuli. Furthermore, we show that concentrations of polyamines resulting in inhibition of PCD generated a transient ROS burst in our experimental system, and activated the expression of oxidative stress- and pathogen response-associated genes. Finally, we examined PCD responses in existing Arabidopsis polyamine synthesis mutants, and identified a subtle PCD phenotype in Arabidopsis seedlings deficient in thermo-spermine. The presented data show that polyamines can have a role in PCD regulation; however, that role is dose-dependent and consequently they may act as either inhibitors, or inducers, of PCD in Arabidopsis.

A FLOWERING LOCUS T ortholog is associated with photoperiod-insensitive flowering in hemp (Cannabis sativa L.).

Hemp (Cannabis sativa L.) is an extraordinarily versatile crop, with applications ranging from medicinal compounds to seed oil and fibre products. Cannabis sativa is a short-day plant, and its flowering is highly controlled by photoperiod. However, substantial genetic variation exists for photoperiod sensitivity in C. sativa, and photoperiod-insensitive ("autoflower") cultivars are available. Using a bi-parental mapping population and bulked segregant analysis, we identified Autoflower2, a 0.5 Mbp locus significantly associated with photoperiod-insensitive flowering in hemp. Autoflower2 contains an ortholog of the central flowering time regulator FLOWERING LOCUS T (FT) from Arabidopsis thaliana which we termed CsFT1. We identified extensive sequence divergence between alleles of CsFT1 from photoperiod-sensitive and insensitive cultivars of C. sativa, including a duplication of CsFT1 and sequence differences, especially in introns. Furthermore, we observed higher expression of one of the CsFT1 copies found in the photoperiod-insensitive cultivar. Genotyping of several mapping populations and a diversity panel confirmed a correlation between CsFT1 alleles and photoperiod response, affirming that at least two independent loci involved in the photoperiodic control of flowering, Autoflower1 and Autoflower2, exist in the C. sativa gene pool. This study reveals the multiple independent origins of photoperiod insensitivity in C. sativa, supporting the likelihood of a complex domestication history in this species. By integrating the genetic relaxation of photoperiod sensitivity into novel C. sativa cultivars, expansion to higher latitudes will be permitted, thus allowing the full potential of this versatile crop to be reached.

Roadmap for the next decade of plant programmed cell death research.

Programmed cell death (PCD) is fundamentally important for plant development, abiotic stress responses and immunity, but our understanding of its regulation remains fragmented. Building a stronger research community is required to accelerate progress in this area through knowledge exchange and constructive debate. In this Viewpoint, we aim to initiate a collective effort to integrate data across a diverse set of experimental models to facilitate characterisation of the fundamental mechanisms underlying plant PCD and ultimately aid the development of a new plant cell death classification system in the future. We also put forward our vision for the next decade of plant PCD research stemming from discussions held during the 31st New Phytologist workshop, 'The Life and Death Decisions of Plant Cells' that took place at University College Dublin in Ireland (14-15 June 2023). We convey the key areas of significant progress and possible future research directions identified, including resolving the spatiotemporal control of cell death, isolation of its molecular and genetic regulators, and harnessing technical advances for studying PCD events in plants. Further, we review the breadth of potential impacts of plant PCD research and highlight the promising new applications of findings from this dynamically evolving field.

Arabidopsis cell suspension culture and RNA sequencing reveal regulatory networks underlying plant-programmed cell death.

Programmed cell death (PCD) facilitates selective, genetically controlled elimination of redundant, damaged, or infected cells. In plants, PCD is often an essential component of normal development and can mediate responses to abiotic and biotic stress stimuli. However, studying the transcriptional regulation of PCD is hindered by difficulties in sampling small groups of dying cells that are often buried within the bulk of living plant tissue. We addressed this challenge by using RNA sequencing and Arabidopsis thaliana suspension cells, a model system that allows precise monitoring of PCD rates. 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), enabled isolation of candidate core- and stimuli-specific PCD genes, inference of underlying regulatory networks and identification of putative transcriptional regulators of PCD in plants. This analysis underscored a disturbance of the cell cycle and mitochondrial retrograde signaling, and repression of pro-survival stress responses, as key elements of the PCD-associated transcriptional signature. Further, phenotyping of Arabidopsis T-DNA insertion mutants in selected candidate genes validated the potential of generated resources to identify novel genes involved in plant PCD pathways and/or stress tolerance.

The boundary of life and death: changes in mitochondrial and cytosolic proteomes associated with programmed cell death of Arabidopsis thaliana suspension culture cells.

Introduction: Despite the critical role of programmed cell death (PCD) in plant development and defense responses, its regulation is not fully understood. It has been proposed that mitochondria may be important in the control of the early stages of plant PCD, but the details of this regulation are currently unknown. Methods: We used Arabidopsis thaliana cell suspension culture, a model system that enables induction and precise monitoring of PCD rates, as well as chemical manipulation of this process to generate a quantitative profile of the alterations in mitochondrial and cytosolic proteomes associated with early stages of plant PCD induced by heat stress. The cells were subjected to PCD-inducing heat levels (10 min, 54°C), with/without the calcium channel inhibitor and PCD blocker LaCl3. The stress treatment was followed by separation of cytosolic and mitochondrial fractions and mass spectrometry-based proteome analysis. Results: Heat stress induced rapid and extensive changes in protein abundance in both fractions, with release of mitochondrial proteins into the cytosol upon PCD induction. In our system, LaCl3 appeared to act downstream of cell death initiation signal, as it did not affect the release of mitochondrial proteins, but instead partially inhibited changes occurring in the cytosolic fraction, including upregulation of proteins with hydrolytic activity. Discussion: We characterized changes in protein abundance and localization associated with the early stages of heat stress-induced PCD. Collectively, the generated data provide new insights into the regulation of cell death and survival decisions in plant cells.

A simple and cost-effective method for studying anoxia tolerance in plants.

Premise: We developed a novel, cost-effective protocol that facilitates testing anoxia tolerance in plants without access to specialized equipment. Methods and Results: Arabidopsis thaliana and barley (Hordeum vulgare) seedlings were treated in airtight 2-L Kilner jars. An anoxic atmosphere was generated using Oxoid AnaeroGen 2.5-L sachets placed on in-house, custom-built wire stands. The performed experiments confirmed a higher sensitivity to low oxygen stress previously observed in anac017 A. thaliana mutants and the positive effect of exogenous sucrose on anoxia tolerance reported by previous studies in A. thaliana. Barley seedlings displayed typical responses to anoxia treatment, including shoot growth cessation and the induction of marker genes for anaerobic metabolism and ethylene biosynthesis in root tissue. Conclusions: The results validate the novel method as an inexpensive, simple alternative for testing anoxia tolerance in plants, where access to an anaerobic workstation is not possible. The novel protocol requires minimum investment and is easily adaptable.

A protocol for rapid generation cycling (speed breeding) of hemp (Cannabis sativa) for research and agriculture.

Hemp (Cannabis sativa) is a highly versatile 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 associated with 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 the rapid generation cycling (speed breeding) of hemp. The speed breeding protocol makes use of the photoperiod sensitivity of Cannabis. It encompasses vegetative growth of the plants for 2 weeks under continuous light, followed by 4 weeks under short-day conditions, during which flower induction, pollination and seed development proceed, and finally a seed ripening phase under continuous light and water stress. With the protocol described here, a generation time of under 9 weeks (61 days) from seed to seed can be achieved. Furthermore, our method synchronises the 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 over a short period of time.

Genetic and lipidomic analyses suggest that Nostoc punctiforme, a plant-symbiotic cyanobacterium, does not produce sphingolipids.

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., an acetylsphingomyelin and a cerebroside), both based on unbranched, long-chain base (LCB) sphingolipids in Scytonema julianum and Moorea producens , respectively. The first step in de novo sphingolipid biosynthesis is the condensation of l-serine with palmitoyl-CoA to produce 3-keto-diyhydrosphingosine (KDS). This reaction is catalyzed by serine palmitoyltransferase (SPT), which belongs to a small family of pyridoxal phosphate-dependent α-oxoamine synthase (AOS) enzymes. Based on sequence similarity to molecularly characterized bacterial SPT peptides, we identified a putative SPT (Npun_R3567) from the model nitrogen-fixing, plant-symbiotic cyanobacterium, Nostoc punctiforme strain PCC 73102 (ATCC 29133). Gene expression analysis revealed that Npun_R3567 is induced during late-stage diazotrophic growth in N. punctiforme . However, Npun_R3567 could not produce the SPT reaction product, 3-keto-diyhydrosphingosine (KDS), when heterologously expressed in Escherichia coli . This agreed with a sphingolipidomic analysis of N. punctiforme cells, which revealed that no LCBs or ceramides were present. To gain a better understanding of Npun_R3567, we inferred the phylogenetic position of Npun_R3567 relative to other bacterial AOS peptides. Rather than clustering with other bacterial SPTs, Npun_R3567 and the other cyanobacterial BioF homologues formed a separate, monophyletic group. Given that N. punctiforme does not appear to possess any other gene encoding an AOS enzyme, it is altogether unlikely that N. punctiforme is capable of synthesizing sphingolipids. In the context of cross-kingdom symbiosis signalling in which sphingolipids are emerging as important regulators, it appears unlikely that sphingolipids from N. punctiforme play a regulatory role during its symbiotic association with plants.

Evolution, genetics and biochemistry of plant cannabinoid synthesis: a challenge for biotechnology in the years ahead.

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.

Plant programmed cell death meets auxin signalling.

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.

Cyanobacteria-Derived Proline Increases Stress Tolerance in Arabidopsis thaliana Root Hairs by Suppressing Programmed Cell Death.

Nitrogen-fixing heterocystous cyanobacteria are used as biofertilizer inoculants for stimulating plant growth but can also alleviate plant stress by exometabolite secretion. However, only a small number of studies have focused on elucidating the identity of said bioactives because of the wide array of exuded compounds. Here, we used the root hair assay (RHA) as a rapid programmed cell death (PCD) screening tool for characterizing the bioactivity of cyanobacteria Nostoc muscorum conditioned medium (CM) on Arabidopsis thaliana root hair stress tolerance. We found that heat-stressed A. thaliana pre-treated with N. muscorum CM fractions exhibited significantly lower root hair PCD levels compared to untreated seedlings. Treatment with CM increased stress tolerance by suppressing PCD in root hairs but not necrosis, indicating the bioactive compound was specifically modulating the PCD pathway and not a general stress response. Based on documented N. muscorum exometabolites, we identified the stress-responsive proline as a compound of interest and strong evidence from the ninhydrin assay and HPLC indicate that proline is present in N. muscorum CM. To establish whether proline was capable of suppressing PCD, we conducted proline supplementation experiments. Our results showed that exogenous proline had a similar effect on root hairs as N. muscorum CM treatment, with comparable PCD suppression levels and insignificant necrosis changes. To verify proline as one of the biologically active compounds in N. muscorum CM, we used three mutant A. thaliana lines with proline transporter mutations (lht1, aap1 and atprot1-1::atprot2-3::atprot3-2). Compared with the wild-type seedlings, PCD-suppression in lht1and aap1 mutants was significantly reduced when supplied with low proline (1-5 µM) levels. Similarly, pre-treatment with N. muscorum CM resulted in elevated PCD levels in all three mutant lines compared to wild-type seedlings. Our results show that plant uptake of cyanobacteria-derived proline alters their root hair PCD sensitivity threshold. This offers evidence of a novel biofertilizer mechanism for reducing stress-induced PCD levels, independent of the existing mechanisms documented in the literature.

Stressed to Death: The Role of Transcription Factors in Plant Programmed Cell Death Induced by Abiotic and Biotic Stimuli.

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.

Cannabis sativa.

Schilling et al. introduce and discuss Cannabis.

The role of SIPK signaling pathway in antioxidant activity and programmed cell death of tobacco cells after exposure to cadmium.

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.

The retraction of the protoplast during PCD is an active, and interruptible, calcium-flux driven process.

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.

Methods to Study Plant Programmed Cell Death.

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.

Mitochondrial Markers of Programmed Cell Death in Arabidopsis thaliana.

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.