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Biomolecular condensates are membraneless organelle-like structures that can concentrate molecules and often form through liquid-liquid phase separation. Biomolecular condensate assembly is tightly regulated by developmental and environmental cues. Although research on biomolecular condensates has intensified in the past 10 years, our current understanding of the molecular mechanisms and components underlying their formation remains in its infancy, especially in plants. However, recent studies have shown that the formation of biomolecular condensates may be central to plant acclimation to stress conditions. Here, we describe the mechanism, regulation, and properties of stress-related condensates in plants, focusing on stress granules and processing bodies, two of the most well-characterized biomolecular condensates. In this regard, we showcase the proteomes of stress granules and processing bodies, in an attempt to suggest methods for elucidating the composition and function of biomolecular condensates. Finally, we discuss how biomolecular condensates modulate stress responses and how they might be used as targets for biotechnological efforts to improve stress tolerance.

Several approaches have been proposed to sequence the genome of Vitis vinifera L. (grapevine), utilizing low-resolution second-generation platforms that lead to partial coverage and low-quality reads. Nevertheless, the characterization of the grapevine genetic resources could be better addressed through extensive and high-resolution genome sequencing. MinION is a third-generation sequencer preferred by many laboratories due to its relatively low cost, ease of use, and small size. To exploit MinION at its full potential, it requires high-quality DNA which is difficult to obtain in grapevine. Here we establish a workflow for DNA for MinION sequencing long reads from grapevine. This protocol was tested with undeveloped leaves, apex, helices, petioles, and 4th leaves samples. In addition, a circulomic size selection approach was tested to provide an unparalleled size of DNA reads. Our results suggest that longer reads of grapevine DNA associate with the use of fresh undeveloped tissue and adjusted conditions for the purification of cell nuclei. Short read elimination protocol increased N50 read length with a reasonable compromise for overall data yield. Our workflow presents a significant advancement for long-read quality DNA isolation for grapevine, providing also technical considerations for high-quality DNA preparation compatible with MinION from plants.

The use of proximity-dependent biotin labelling (PDL) approaches coupled with mass spectrometry recently greatly advanced the identification of protein-protein interactions and study of protein complexation. PDL is based on the expression of a promiscuous biotin ligase (PBL), e.g. BirA* or a peroxidase fused to a bait protein of interest. In the presence of biotin as substrate, PBL enables covalent biotin labelling of proteins in the vicinity of the PBL-fused bait in vivo , allowing the subsequent capture and identification of interacting and neighbouring proteins without the need for the protein complex to remain intact during purification. To date, PDL has not been extensively used in plants. Here we present the results of a systematic multi-lab study applying a variety of PDL approaches in several plant systems under various conditions and bait proteins. We show that TurboID is the most promiscuous variant for PDL in plants and establish protocols for its efficient application. We demonstrate the applicability of TurboID in capturing membrane protein interactomes using the Lotus japonicus symbiotically active receptor kinases RLKs NOD FACTOR RECEPTOR 5 (NFR5) and LRR-RLK SYMBIOTIC RECEPTOR-KINASE (SYMRK) as test-cases. Furthermore, we benchmark the efficiency of various PBLs using the octameric endocytic TPLATE complex and compare PDL with one-step AP-MS approaches. Our results indicate that different PDL approaches in plants may differ in signal-to-noise ratio and robustness. We present a straightforward strategy to identify both non-biotinylated as well as biotinylated proteins in plants in a single experimental setup. Finally, we provide initial evidence that this technique has potential to infer structural information of protein complexes. Our methods, tools and adjustable pipelines provide a useful resource for the plant research community.

Hormonal pathways often converge on transcriptional repressors that can be degraded by the proteasome to initiate a response. We wish to draw attention to developments in a less-explored proteolytic branch called 'limited proteolysis' that, in addition to the classical proteolytic pathways, seems to regulate auxin and ethylene signaling.

In plants, auxin transport and development are tightly coupled, just as hormone and growth responses are intimately linked in multicellular systems. Here we provide insights into uncoupling this tight control by specifically targeting the expression of TINY ROOT HAIR 1 (TRH1), a member of plant high-affinity potassium (K+)/K+ uptake/K+ transporter (HAK/KUP/KT) transporters that facilitate K+ uptake by co-transporting protons, in Arabidopsis root cell files. Use of this system pinpointed specific root developmental responses to acropetal versus basipetal auxin transport. Loss of TRH1 function shows TRHs and defective root gravitropism, associated with auxin imbalance in the root apex. Cell file-specific expression of TRH1 in the central cylinder rescued trh1 root agravitropism, whereas positional TRH1 expression in peripheral cell layers, including epidermis and cortex, restored trh1 defects. Applying a system-level approach, the role of RAP2.11 and ROOT HAIR DEFECTIVE-LIKE 5 transcription factors (TFs) in root hair development was verified. Furthermore, ERF53 and WRKY51 TFs were overrepresented upon restoration of root gravitropism supporting involvement in gravitropic control. Auxin has a central role in shaping root system architecture by regulating multiple developmental processes. We reveal that TRH1 jointly modulates intracellular ionic gradients and cell-to-cell polar auxin transport to drive root epidermal cell differentiation and gravitropic response. Our results indicate the developmental importance of HAK/KUP/KT proton-coupled K+ transporters.

For most Gram-negative bacteria, pathogenicity largely depends on the type-III secretion system that delivers virulence effectors into eukaryotic host cells. The subcellular targets for the majority of these effectors remain unknown. Xanthomonas campestris, the causal agent of black rot disease of crucifers such as Brassica spp., radish, and turnip, delivers XopP, a highly conserved core-effector protein produced by X. campestris, which is essential for virulence. Here, we show that XopP inhibits the function of the host–plant exocyst complex by direct targeting of Exo70B, a subunit of the exocyst complex, which plays a significant role in plant immunity. XopP interferes with exocyst-dependent exocytosis and can do this without activating a plant NOD-like receptor that guards Exo70B in Arabidopsis. In this way, Xanthomonas efficiently inhibits the host’s pathogen-associated molecular pattern (PAMP)-triggered immunity by blocking exocytosis of pathogenesis-related protein-1A, callose deposition, and localization of the FLAGELLIN SENSITIVE2 (FLS2) immune receptor to the plasma membrane, thus promoting successful infection. Inhibition of exocyst function without activating the related defenses represents an effective virulence strategy, indicating the ability of pathogens to adapt to host defenses by avoiding host immunity responses.

The outburst of plastic pollution in terrestrial ecosystems poses a potential threat to agriculture and food safety. Studies have already provided evidence for the uptake of plastic microparticles by several plant species, accompanied by numerous developmental effects, using fluorescence labelling techniques. Here, we introduce the implementation of confocal Raman spectroscopy, a label-free method, for the effective detection of microplastics (MPs) accumulation in the roots of a common edible root vegetable plant, Raphanus sativus, after treatment with acrylonitrile butadiene styrene (ABS) powder. We also demonstrate the concomitant occurrence of phenotypic defects in the polymer-treated plants. We anticipate that this work can provide new insights not only into the extent of the impact this widespread phenomenon has on crop plants but also on the methodological requirements to address it.

The outburst of plastic pollution in terrestrial ecosystems poses a potential threat to agriculture and food safety. Studies have already provided evidence for the uptake of plastic microparticles by several plant species, accompanied by numerous developmental effects, using fluorescence labelling techniques. Here, we introduce the implementation of confocal Raman spectroscopy, a label-free method, for the effective detection of microplastics (MPs) accumulation in the roots of a common edible root vegetable plant, Raphanus sativus, after treatment with acrylonitrile butadiene styrene (ABS) powder. We also demonstrate the concomitant occurrence of phenotypic defects in the polymer-treated plants. We anticipate that this work can provide new insights not only into the extent of the impact this widespread phenomenon has on crop plants but also on the methodological requirements to address it.

Background: There have been several attempts to sequence the genome of Vitis vinifera L. (grapevine), utilizing low-resolution second-generation platforms. Nevertheless, the characterization of the grapevine genetic resources and its adaptation to vulnerable conditions could be better addressed through extensive and high-resolution genome sequencing.MinION is a third-generation sequencer preferred by many laboratories due to its relatively low cost, ease of use and small size. Even though this long-read technology has been rapidly improving, to reach its full potential requires high-quality DNA.Results: Here we establish a workflow for DNA extraction suitable for MinION sequencing long reads from grapevine. This protocol was tested with leaf samples from different positions on annual growing branches of grapevine, Purified nuclei from fresh young leaves that led to high quality, long DNA fragments, suitable for long-read sequencing were successfully generated. It is evident that longer reads in grapevine associate with both fresh tissue and adjusted conditions used for nuclei purification.Conclusions: We propose that this workflow presents a significant advancement for long-read quality DNA isolation for grapevine and likely other plant species.

Tudor staphylococcal nuclease (TSN; also known as Tudor‐SN, p100, or SND1) is a multifunctional, evolutionarily conserved regulator of gene expression, exhibiting cytoprotective activity in animals and plants and oncogenic activity in mammals. During stress, TSN stably associates with stress granules (SGs), in a poorly understood process. Here, we show that in the model plant Arabidopsis thaliana, TSN is an intrinsically disordered protein (IDP) acting as a scaffold for a large pool of other IDPs, enriched for conserved stress granule components as well as novel or plant‐specific SG‐localized proteins. While approximately 30% of TSN interactors are recruited to stress granules de novo upon stress perception, 70% form a protein–protein interaction network present before the onset of stress. Finally, we demonstrate that TSN and stress granule formation promote heat‐induced activation of the evolutionarily conserved energy‐sensing SNF1‐related protein kinase 1 (SnRK1), the plant orthologue of mammalian AMP‐activated protein kinase (AMPK). Our results establish TSN as a docking platform for stress granule proteins, with an important role in stress signalling., Proteomic analysis of Arabidopsis stress granules identifies a role of intrinsically disordered Tudor staphylococcal nuclease (TSN) as docking platform for stress‐specific and stress‐independent components.

Fluorescent labelling of proteins without compromising their activity is crucial for determining their spatiotemporal localization while retaining their functionality. Spot-tag is a 12-amino acid peptide recognized by a single-domain nanobody. Here we introduce the spot-tag as a labelling strategy for proteins in fixed and living plant cells, using as an example the microtubule motor centromeric protein E-related Kinesin 7.3. Spot-tagging of ectopically introduced Kinesin 7.3 does not interfere with microtubules and spot staining results in a close-grained fluorophore labelling revealing a localization pattern that resembles “beads-on-a-string”. We anticipate that our protocol will apply to many more demanding protein cellular targets, offsetting activity perturbations and low photon quantum yields imposed by other protein-tagging approaches.

Metacaspases and paracaspases are proteases that were first identified as containing a caspase-like structural fold (Uren et al., 2000). Like caspases, metacaspases and paracaspases are multifunctional proteins regulating diverse biological phenomena, such as aging, immunity, proteostasis, and programmed cell death. The broad phylogenetic distribution of metacaspases and paracaspases across all kingdoms of life and large variation of their biochemical and structural features complicate classification and annotation of the rapidly growing number of identified homologs. Establishment of an adequate classification and unified nomenclature of metacaspases and paracaspases is especially important to avoid frequent confusion of these proteases with caspases—a tenacious misnomer that unfortunately does not appear to decline with time. This Letter represents a consensus opinion of researchers studying different aspects of caspases, metacaspases, and paracaspases in various organisms, ranging from microbes to plants and animals., This work was supported by the Knut and Alice Wallenberg Foundation.

SUMMARYAdaptation to stress depends on the modulation of gene expression. Regulation of mRNA stability and degradation in stress granules (SGs), - cytoplasmic membraneless organelles composed of messenger ribonucleoprotein (mRNP) complexes, - plays an important role in fine-tuning of gene expression. In addition, SG formation can modulate stress signaling pathways by protein sequestration. Molecular composition, structure, and function of SGs in plants remain obscure. Recently, we established Tudor Staphylococcal Nuclease (TSN or Tudor-SN; also known as SND1) as integral component of SGs in Arabidopsis thaliana. Here, we combined purification of TSN interactome with cell biology, reverse genetics and bioinformatics to study composition and function of SGs in plants. We found that under both normal (in the absence of stress) and stress conditions TSN interactome is enriched in the homologues of known mammalian and yeast SG proteins, in addition to novel or plant-specific SG components. We estimate that upon stress perception, approximately half of TSN interactors are recruited to SGs de novo, in a stress-dependent manner, while another half represent a dense protein-protein interaction network pre-formed before onset of stress. Almost all TSN-interacting proteins are moderately or highly disordered and approximately 20% of them are predisposed for liquid-liquid phase separation (LLPS). This suggests that plant SGs, similarly to mammalian and yeast counterparts, are multicomponent viscous liquid droplets. Finally, we have discovered that evolutionary conserved SNF1-related protein kinase 1 (SnRK1) interacts with TSN in heat-induced SGs and that SnRK1 activation critically depends on the presence of TSN and formation of SGs. Altogether, our results establish TSN as a docking platform for SG-associated proteins and important stress signal mediator in plants.

Rhizoctonia solani (Rs) is a soil-borne pathogen with a broad host range. This pathogen incites a wide range of disease symptoms. Knowledge regarding its infection process is fragmented, a typical feature for basidiomycetes. In this study, we aimed at identifying potential fungal effectors and their function. From a group of 11 predicted single gene effectors, a rare lipoprotein A (RsRlpA), from a strain attacking sugar beet was analyzed. The RsRlpA gene was highly induced upon early-stage infection of sugar beet seedlings, and heterologous expression in Cercospora beticola demonstrated involvement in virulence. It was also able to suppress the hypersensitive response (HR) induced by the Avr4/Cf4 complex in transgenic Nicotiana benthamiana plants and functioned as an active protease inhibitor able to suppress Reactive Oxygen Species (ROS) burst. This effector contains a double-psi beta-barrel (DPBB) fold domain, and a conserved serine at position 120 in the DPBB fold domain was found to be crucial for HR suppression. Overall, R. solani seems to be capable of inducing an initial biotrophic stage upon infection, suppressing basal immune responses, followed by a switch to necrotrophic growth. However, regulatory mechanisms between the different lifestyles are still unknown.

Significance Animal NLRs form wheel-like structures called inflammasomes upon perception of pathogen-associated molecules. The induced proximity of the signaling domains at the center of the wheel is hypothesized to recruit caspases for the first step of immune signal transduction. We expressed a plant-animal NLR fusion to demonstrate that induced proximity of TIR signaling domains from plant NLRs is sufficient to activate plant immune signaling. This demonstrates that a signaling-competent inflammasome can be formed from known, minimal components. The intrinsic NADase activity of plant TIRs is necessary for immune signaling, but fusions to a bacterial or a mammalian TIR domain with NADase activity, which also lead to accumulation of NAD+ hydrolysis products (e.g. cyclic ADP-ribose), were unable to activate immune signaling., Plant and animal intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors detect pathogen-derived molecules and activate defense. Plant NLRs can be divided into several classes based upon their N-terminal signaling domains, including TIR (Toll-like, Interleukin-1 receptor, Resistance protein)- and CC (coiled-coil)-NLRs. Upon ligand detection, mammalian NAIP and NLRC4 NLRs oligomerize, forming an inflammasome that induces proximity of its N-terminal signaling domains. Recently, a plant CC-NLR was revealed to form an inflammasome-like hetero-oligomer. To further investigate plant NLR signaling mechanisms, we fused the N-terminal TIR domain of several plant NLRs to the N terminus of NLRC4. Inflammasome-dependent induced proximity of the TIR domain in planta initiated defense signaling. Thus, induced proximity of a plant TIR domain imposed by oligomerization of a mammalian inflammasome is sufficient to activate authentic plant defense. Ligand detection and inflammasome formation is maintained when the known components of the NLRC4 inflammasome is transferred across kingdoms, indicating that NLRC4 complex can robustly function without any additional mammalian proteins. Additionally, we found NADase activity of a plant TIR domain is necessary for plant defense activation, but NADase activity of a mammalian or a bacterial TIR is not sufficient to activate defense in plants.

Contents 'Summary' I. 'Introduction' II. 'DEK1: towards identification of protease substrates' III. 'Separases: when proteolytic modules attain nonproteolytic functions' IV. 'The peculiar case of a nonredundant subtilisin' V. 'Towards a solution to the protease redundancy problem' VI. 'Matters arising and closing remarks' 'Acknowledgements' References Summary Proteases are integral components of proteome remodelling networks that regulate turnover of proteins and expand their functional diversity. Accumulating evidence highlights the importance of proteases as being central hubs of developmental programs. Yet the molecular pathways that many proteases act on, their natural substrates and their putative nonproteolytic functions remain largely elusive. Here, we discuss recent findings on proteases with functions that converge into plant development regulation, such as DEFECTIVE KERNEL 1 (DEK1), separase and subtilisins, to highlight conspicuous but unexplored aspects of protease biology. We also suggest an exploratory framework for addressing protease functions.

Nitrogen (N) is a core component of fertilizers used in modern agriculture to increase yields and thus to help feed a growing global population. However, this comes at a cost to the environment, through run-off of excess N as a result of poor N-use efficiency (NUE) by crops. An obvious remedy to this problem would therefore be the improvement of NUE, which requires advancing our understanding on N homeostasis, sensing, and uptake. Proteolytic pathways are linked to N homeostasis as they recycle proteins that contain N and carbon; however, emerging data suggest that their functions extend beyond this simple recycling. Here, we highlight roles of proteolytic pathways in non-symbiotic and symbiotic N uptake and in systemic N sensing. We also offer a novel view in which we suggest that proteolytic pathways have roles in N homeostasis that differ from their accepted function in recycling.

Significance Plants and animals carry intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors. How NLR receptors activate defense on perceiving pathogen molecules is poorly understood, especially in plants. Some NLRs function in pairs, with one NLR carrying a domain that mimics a pathogen effector target. Effector action on this domain activates the second “helper” NLR. In the Arabidopsis RPS4 and RRS1 pair, RRS1 carries a WRKY transcription factor domain targeted by bacterial effectors AvrRps4 and PopP2. We monitored conformational changes in RPS4–RRS1 during activation and developed a “molecular padlock” to reversibly restrict such changes. This revealed domains within RRS1 required to keep the RRS1–RPS4 complex inactive prior to effector detection, and specific domain–domain interactions whose disruption or modification contributes to defense activation., Plant intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors often function in pairs to detect pathogen effectors and activate defense. The Arabidopsis RRS1-R–RPS4 NLR pair recognizes the bacterial effectors AvrRps4 and PopP2 via an integrated WRKY transcription factor domain in RRS1-R that mimics the effector’s authentic targets. How the complex activates defense upon effector recognition is unknown. Deletion of the WRKY domain results in an RRS1 allele that triggers constitutive RPS4-dependent defense activation, suggesting that in the absence of effector, the WRKY domain contributes to maintaining the complex in an inactive state. We show the WRKY domain interacts with the adjacent domain 4, and that the inactive state of RRS1 is maintained by WRKY–domain 4 interactions before ligand detection. AvrRps4 interaction with the WRKY domain disrupts WRKY–domain 4 association, thus derepressing the complex. PopP2-triggered activation is less easily explained by such disruption and involves the longer C-terminal extension of RRS1-R. Furthermore, some mutations in RPS4 and RRS1 compromise PopP2 but not AvrRps4 recognition, suggesting that AvrRps4 and PopP2 derepress the complex differently. Consistent with this, a “reversibly closed” conformation of RRS1-R, engineered in a method exploiting the high affinity of colicin E9 and Im9 domains, reversibly loses AvrRps4, but not PopP2 responsiveness. Following RRS1 derepression, interactions between domain 4 and the RPS4 C-terminal domain likely contribute to activation. Simultaneous relief of autoinhibition and activation may contribute to defense activation in many immune receptors.

Rhizoctonia (in Greek root-killer) species and particularly R. solani attacks a broad range of plant species and crops. It belongs to Basidiomycota and is a soil-borne pathogen causing mainly damping-off in seedlings, although it can infect plants in any stage. It does not form any kind of asexual spores while the sexual form (Thanatephorus cucumeris) is extremely rare. However, it forms microsclerotia, able to survive in soil and plant debris for a long period and under harsh environmental conditions. Despite the severity of this disease, many aspects in R. solani infection biology still remain to be elucidated. In this study, we investigated the role of two effector candidates, derived from a R. solani AG2-2IIIB strain isolated from infected sugar beet plants. Gene expression analysis showed that genes encoding for a LysM effector and a RlpA-like protein were induced upon early infection stages, while their heterologous expression in Cercospora beticola contributed to virulence. Furthermore, the RsLysM effector showed chitin binding affinity, suppressing chitin-triggered immunity but could not protect hyphae from hydrolysis. The RlpA-like effector is homologous to a papain-like inhibitor and to a barwin-like endoglucanase, while our data showed that it was able to suppress hypersensitive response in Nicotiana bethamiana leaves. Overall, this study provides us with valuable information on R. solani infection biology towards understanding the molecular mechanisms in this pathosystem.

Plant intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors often function in pairs to detect pathogen effectors and activate defense. The Arabidopsis RRS1-R-RPS4 NLR pair recognizes the bacterial effectors AvrRps4 and PopP2 via an integrated WRKY transcription factor domain in RRS1-R that mimics the effector's authentic targets. How the complex activates defense upon effector recognition is unknown. Deletion of the WRKY domain results in an RRS1 allele that triggers constitutive RPS4-dependent defense activation, suggesting that in the absence of effector, the WRKY domain contributes to maintaining the complex in an inactive state. We show the WRKY domain interacts with the adjacent domain 4, and that the inactive state of RRS1 is maintained by WRKY-domain 4 interactions before ligand detection. AvrRps4 interaction with the WRKY domain disrupts WRKY-domain 4 association, thus derepressing the complex. PopP2-triggered activation is less easily explained by such disruption and involves the longer C-terminal extension of RRS1-R. Furthermore, some mutations in RPS4 and RRS1 compromise PopP2 but not AvrRps4 recognition, suggesting that AvrRps4 and PopP2 derepress the complex differently. Consistent with this, a "reversibly closed" conformation of RRS1-R, engineered in a method exploiting the high affinity of colicin E9 and Im9 domains, reversibly loses AvrRps4, but not PopP2 responsiveness. Following RRS1 derepression, interactions between domain 4 and the RPS4 C-terminal domain likely contribute to activation. Simultaneous relief of autoinhibition and activation may contribute to defense activation in many immune receptors.

Genome editing by CRISPR is now routinely used in plant biology for unravelling gene functions and improving agronomical traits. CRISPR opens up the possibility of genome manipulations which would have been unthinkable a few years ago. In this perspective, we discuss and suggest CRISPR-mediated approaches for steering plant development, also highlighting potential challenges.

Autophagy-related proteins Atg5 and Atg7 are rate-limiting components of autophagic flux in Arabidopsis. Overexpression of ATG5 or ATG7 genes stimulates Atg8 lipidation, autophagosome formation, and autophagic flux, leading to improved plant fitness., Autophagy is a major catabolic process whereby autophagosomes deliver cytoplasmic content to the lytic compartment for recycling. Autophagosome formation requires two ubiquitin-like systems conjugating Atg12 with Atg5, and Atg8 with lipid phosphatidylethanolamine (PE), respectively. Genetic suppression of these systems causes autophagy-deficient phenotypes with reduced fitness and longevity. We show that Atg5 and the E1-like enzyme, Atg7, are rate-limiting components of Atg8–PE conjugation in Arabidopsis. Overexpression of ATG5 or ATG7 stimulates Atg8 lipidation, autophagosome formation, and autophagic flux. It also induces transcriptional changes opposite to those observed in atg5 and atg7 mutants, favoring stress resistance and growth. As a result, ATG5- or ATG7-overexpressing plants exhibit increased resistance to necrotrophic pathogens and oxidative stress, delayed aging and enhanced growth, seed set, and seed oil content. This work provides an experimental paradigm and mechanistic insight into genetic stimulation of autophagy in planta and shows its efficiency for improving plant productivity.

Digestive proteolysis executed by the proteasome plays an important role in plant development. Yet, the role of limited proteolysis in this process is still obscured due to the absence of studies. Previously, we showed that limited proteolysis by the caspase-related protease separase (EXTRA SPINDLE POLES [ESP]) modulates development in plants through the cleavage of unknown substrates. Here we used a modified version of the positional proteomics method COmbined FRActional DIagonal Chromatography (COFRADIC) to survey the proteolytic landscape of wild-type and separase mutant RADIALLY SWOLLEN 4 (rsw4) root tip cells, as an attempt to identify targets of separase. We have discovered that proteins involved in the establishment of pH homeostasis and sensing, and lipid signalling in wild-type cells, suggesting novel potential roles for separase. We also observed significant accumulation of the protease PRX34 in rsw4 which negatively impacts growth. Furthermore, we observed an increased acetylation of N-termini of rsw4 proteins which usually comprise degrons identified by the ubiquitin-proteasome system, suggesting that separase intersects with additional proteolytic networks. Our results hint to potential pathways by which separase could regulate development suggesting also novel proteolytic functions.

Contents 958 I. 958 II. 959 III. 960 IV. 962 V. 962 962 References 963 SUMMARY: Proteases can either digest target proteins or perform the so-called 'limited proteolysis' by cleaving polypeptide chains at specific site(s). Autophagy and the ubiquitin-proteasome system (UPS) are two main mechanisms carrying out digestive proteolysis. While the net outcome of digestive proteolysis is the loss of function of protein substrates, limited proteolysis can additionally lead to gain or switch of function. Recent evidence of crosstalk between autophagy, UPS and limited proteolysis indicates that these pathways are parts of the same proteolytic nexus. Here, we focus on three emerging themes within this area: limited proteolysis as a mechanism modulating autophagy; interplay between autophagy and UPS, including autophagic degradation of proteasomes (proteophagy); and specificity of protein degradation during bulk autophagy.

Factors regulating dynamics of chromatin structure have direct impact on expression of genetic information. Cohesin is a multi-subunit protein complex crucial for pairing sister chromatids during cell division, DNA repair and regulation of gene transcription and silencing. In non-plant species cohesin is loaded on chromatin by the Scc2/Scc4 (NIBPL/MAU2) complex. Here we identify AtSCC4, the Arabidopsis homolog of Scc4, and show that it forms a functional complex with AtSCC2, the homolog of Scc2. We demonstrate that AtSCC2 and AtSCC4 act in the same pathway and that both proteins are indispensable for cell fate determination during early stages of embryo development. Mutant embryos lacking either of these proteins develop only up to the globular stage, and show suspensor over-proliferation phenotype preceded by ectopic auxin maxima distribution. We further establish a new assay to reveal the AtSCC4-dependent dynamics of cohesin loading on chromatin in vivo. Our findings define Scc2/Scc4 complex as an evolutionary conserved machinery controlling cohesin loading and chromatin structure maintenance and provide new insight into plant-specific role of this complex in controlling cell fate during embryogenesis.

Summary The WUSCHEL-RELATED HOMEOBOX 8/9 is important for correct orientation of the cell division plane and for cell fate determination during embryo pattern formation in the gymnosperm Norway spruce., Proper embryo development is crucial as that is when the primary body axes are established. In Arabidopsis, AtWOX8 and AtWOX9, members of the WUSCHEL-RELATED HOMEOBOX (WOX) gene family, are critical for embryo development. In Norway spruce, PaWOX8/9, which is expressed in embryos, is the homologue of AtWOX8 and AtWOX9. In this work, it is shown that the transcript abundance of PaWOX8/9 is high during early and late embryogeny and that it decreases when the maturation phase starts. To address the function of PaWOX8/9 during embryo development, RNAi lines were established to down-regulate the transcript level of PaWOX8/9, using both constitutive and inducible promoters. Embryos in the PaWOX8/9 RNAi lines show an aberrant morphology caused by disturbed orientation of the cell division plane at the basal part of the embryonal mass during early and late embryogeny. In addition, the transcript level of several key cell-cycle-regulating genes, for example, PaE2FAB-LIKE and PaCYCLIN B-LIKE, are affected in the PaWOX8/9 RNAi lines. Taken together, our results suggest that PaWOX8/9 may perform an evolutionarily conserved function as a regulator of the establishment of the apical–basal embryo pattern.

Vesicle trafficking plays an important role in cell division, establishment of cell polarity, and translation of environmental cues to developmental responses. However, the molecular mechanisms regulating vesicle trafficking remain poorly understood. Here, we report that the evolutionarily conserved caspase-related protease separase (EXTRA SPINDLE POLES [ESP]) is required for the establishment of cell polarity and cytokinesis in Arabidopsis thaliana. At the cellular level, separase colocalizes with microtubules and RabA2a (for RAS GENES FROM RAT BRAINA2a) GTPase-positive structures. Separase facilitates polar targeting of the auxin efflux carrier PIN-FORMED2 (PIN2) to the rootward side of the root cortex cells. Plants with the radially swollen4 (rsw4) allele with compromised separase activity, in addition to mitotic failure, display isotropic cell growth, perturbation of auxin gradient formation, slower gravitropic response in roots, and cytokinetic failure. Measurements of the dynamics of vesicle markers on the cell plate revealed an overall reduction of the delivery rates of KNOLLE and RabA2a GTPase in separase-deficient roots. Furthermore, dissociation of the clathrin light chain, a protein that plays major role in the formation of coated vesicles, was slower in rsw4 than in the control. Our results demonstrate that separase is a key regulator of vesicle trafficking, which is indispensable for cytokinesis and the establishment of cell polarity.

Factors regulating dynamics of chromatin structure have direct impact on expression of genetic information. Cohesin is a multi-subunit protein complex that is crucial for pairing sister chromatids during cell division, DNA repair and regulation of gene transcription and silencing. In non-plant species, cohesin is loaded on chromatin by the Scc2-Scc4 complex (also known as the NIBPL-MAU2 complex). Here, we identify the

The apoplastic polyamine oxidase (PAO) catalyzes the oxidation of the higher polyamines spermidine and spermine, contributing to hydrogen peroxide (H2O2) accumulation. However, it is yet unclear whether apoplastic PAO is part of a network that coordinates the accumulation of reactive oxygen species (ROS) under salinity or if it acts independently. Here, we unravel that NADPH oxidase and apoplastic PAO cooperate to control the accumulation of H2O2 and superoxides (O2·-) in tobacco (Nicotiana tabacum). To examine to what extent apoplastic PAO constitutes part of a ROS-generating network, we examined ROS accumulation in guard cells of plants overexpressing or down-regulating apoplastic PAO (lines S2.2 and A2, respectively) or down-regulating NADPH oxidase (line AS-NtRbohD/F). The H2O2-specific probe benzene sulfonyl-H2O2 showed that, under salinity, H2O2 increased in S2.2 and decreased in A2 compared with the wild type. Surprisingly, the O2·--specific probe benzene sulfonyl-So showed that O2·- levels correlated positively with that of apoplastic PAO (i.e. showed high and low levels in S2.2 and A2, respectively). By using AS-NtRbohD/F lines and a pharmacological approach, we could show that H2O2 and O2·- accumulation at the onset of salinity stress was dependent on NADPH oxidase, indicating that NADPH oxidase is upstream of apoplastic PAO. Our results suggest that NADPH oxidase and the apoplastic PAO form a feed-forward ROS amplification loop, which impinges on oxidative state and culminates in the execution of programmed cell death. We propose that the PAO/NADPH oxidase loop is a central hub in the plethora of responses controlling salt stress tolerance, with potential functions extending beyond stress tolerance.

Polyamines (PAs) are nitrogenous molecules that are indispensable for cell viability and with an agreed-on role in the modulation of stress responses. Tobacco plants with downregulated SAMDC (AS-SAMDC) exhibit reduced PAs synthesis but normal levels of PA catabolism. We used AS-SAMDC to increase our understanding on the role of PAs in stress responses. Surprisingly, at control conditions AS-SAMDC plants showed increased biomass and altered developmental characteristics, such as increased height and leaf number. On the contrary, during salt stress AS-SAMDC plants showed reduced vigor when compared to the WT. During salt stress, the AS-SAMDC plants although showing compensatory readjustments of the antioxidant machinery and of photosynthetic apparatus, they failed to sustain their vigor. AS-SAMDC sensitivity was accompanied by inability to effectively control H2O2 levels and concentrations of monovalent and divalent cations. In accordance with these findings, we suggest that PAs may regulate the trade-off between growth and tolerance responses.

The caspase‐related protease separase (EXTRA SPINDLE POLES, ESP) plays a major role in chromatid disjunction and cell expansion in Arabidopsis thaliana. Whether the expansion phenotypes are linked to defects in cell division in Arabidopsis ESP mutants remains elusive. Here we present the identification, cloning and characterization of the gymnosperm Norway spruce (Picea abies, Pa) ESP. We used the P. abies somatic embryo system and a combination of reverse genetics and microscopy to explore the roles of Pa ESP during embryogenesis. Pa ESP was expressed in the proliferating embryonal mass, while it was absent in the suspensor cells. Pa ESP associated with kinetochore microtubules in metaphase and then with anaphase spindle midzone. During cytokinesis, it localized on the phragmoplast microtubules and on the cell plate. Pa ESP deficiency perturbed anisotropic expansion and reduced mitotic divisions in cotyledonary embryos. Furthermore, whilst Pa ESP can rescue the chromatid nondisjunction phenotype of Arabidopsis ESP mutants, it cannot rescue anisotropic cell expansion. Our data demonstrate that the roles of ESP in daughter chromatid separation and cell expansion are conserved between gymnosperms and angiosperms. However, the mechanisms of ESP‐mediated regulation of cell expansion seem to be lineage‐specific., This work was supported by grants from the VR Swedish Research Council (to P.N.M. and P.V.B.), Pehrssons Fund (to P.V.B.), the Swedish Foundation for Strategic Research (to P.V.B.), the Olle Engkvist Foundation (to P.V.B.), the Knut and Alice Wallenberg Foundation (to P.V.B.), the August T. Larsson Foundation (to A.P.S. and P.V.B.), Hatch Grant WNP00826 (to A.P.S.), and a Spanish Ministry of Science and Innovation grant (AGL2010‐15684 to M.F.S). V.S‐V. was recipient of a FPI fellowship from the Spanish Ministry of Science and Innovation (BES‐2008‐003592).

Microtubules play an essential role in breaking cellular symmetry. We have previously shown that separase associates with microtubules and regulates microtubule-dependent establishment of cell polarity in Arabidopsis. However, separase lacks microtubule-binding activity, raising questions about mechanisms underlying this phenomenon. Here we report that the N-terminal non-catalytic domain of separase binds to the C-terminal tail domain of three homologs of the centromeric protein CENP-E Kinesin 7 (Kin7). Conformational changes of Kin7 induced upon binding to separase facilitate recruitment of Kin7/separase complex (KISC) onto microtubules. KISC operates independently of proteolytic activity of separase in promoting microtubule rescue and pauses, as well as in suppressing catastrophes. Genetic complementation experiments in conditional separase mutant rsw4 background demonstrate the importance of KISC for the establishment of cell polarity and for plant development. Our study establishes a mechanism governing microtubule dynamics via the separase-dependent activation of CENP-E-related kinesins., This work was supported by grants from the Swedish Research Council (to P.N.M. and P.V.B.), Knut and Alice Wallenberg Foundation (to P.V.B.), the Swedish Foundation for Strategic Research (to P.V.B.), August T. Larsson Foundation (to A.S. and P.V.B.), Olle Engkvist Foundation (to P.V.B.), Pehrssons Fund (to P.V.B.), and NIFA hatch project WNP00826 (to A.S.).

""Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine catabolism. In Arabidopsis thaliana, five PAOs (AtPAO1-5) are present with cytosolic or peroxisomal localization. Here, we present a detailed study of the expression pattern of AtPAO1, AtPAO2, AtPAO3 and AtPAO5 during seedling and flower growth and development through analysis of promoter activity in AtPAO::β-glucuronidase (GUS) transgenic Arabidopsis plants. The results reveal distinct expression patterns for each studied member of the AtPAO gene family. AtPAO1 is mostly expressed in the transition region between the meristematic and the elongation zone of roots and anther tapetum, AtPAO2 in the quiescent center, columella initials and pollen, AtPAO3 in columella, guard cells and pollen, and AtPAO5 in the vascular system of roots and hypocotyls. Furthermore, treatment with the plant hormone abscisic acid induced expression of AtPAO1 in root tip and AtPAO2 in guard cells. These data suggest distinct physiological role(s) for each member of the AtPAO gene family. . ""

Spermidine (Spd) treatment inhibited root cell elongation, promoted deposition of phenolics in cell walls of rhizodermis, xylem elements, and vascular parenchyma, and resulted in a higher number of cells resting in G1 and G2 phases in the maize (Zea mays) primary root apex. Furthermore, Spd treatment induced nuclear condensation and DNA fragmentation as well as precocious differentiation and cell death in both early metaxylem and late metaxylem precursors. Treatment with either N-prenylagmatine, a selective inhibitor of polyamine oxidase (PAO) enzyme activity, or N,N 1-dimethylthiourea, a hydrogen peroxide (H2O2) scavenger, reverted Spd-induced autofluorescence intensification, DNA fragmentation, inhibition of root cell elongation, as well as reduction of percentage of nuclei in S phase. Transmission electron microscopy showed that N-prenylagmatine inhibited the differentiation of the secondary wall of early and late metaxylem elements, and xylem parenchymal cells. Moreover, although root growth and xylem differentiation in antisense PAO tobacco (Nicotiana tabacum) plants were unaltered, overexpression of maize PAO (S-ZmPAO) as well as down-regulation of the gene encoding S-adenosyl-l-methionine decarboxylase via RNAi in tobacco plants promoted vascular cell differentiation and induced programmed cell death in root cap cells. Furthermore, following Spd treatment in maize and ZmPAO overexpression in tobacco, the in vivo H2O2 production was enhanced in xylem tissues. Overall, our results suggest that, after Spd supply or PAO overexpression, H2O2 derived from polyamine catabolism behaves as a signal for secondary wall deposition and for induction of developmental programmed cell death.

Spermidine (Spd) has been correlated with various physiological and developmental processes in plants, including pollen tube growth. In this work, we show that Spd induces an increase in the cytosolic Ca(2+) concentration that accompanies pollen tube growth. Using the whole-cell patch clamp and outside-out single-channel patch clamp configurations, we show that exogenous Spd induces a hyperpolarization-activated Ca(2+) current: the addition of Spd cannot induce the channel open probability increase in excised outside-out patches, indicating that the effect of Spd in the induction of Ca(2+) currents is exerted via a second messenger. This messenger is hydrogen peroxide (H₂O₂), and is generated during Spd oxidation, a reaction mediated by polyamine oxidase (PAO). These reactive oxygen species trigger the opening of the hyperpolarization-activated Ca(2+) -permeable channels in pollen. To provide further evidence that PAO is in fact responsible for the effect of Spd on the Ca(2+) -permeable channels, two Arabidopsis mutants lacking expression of the peroxisomal-encoding AtPAO3 gene, were isolated and characterized. Pollen from these mutants was unable to induce the opening of the Ca(2+) -permeable channels in the presence of Spd, resulting in reduced pollen tube growth and seed number. However, a high Spd concentration triggers a Ca(2+) influx beyond the optimal, which has a deleterious effect. These findings strongly suggest that the Spd-derived H₂O₂ signals Ca(2+) influx, thereby regulating pollen tube growth.

International audience; In higher plants the glutamate dehydrogenase (GDH) enzyme catalyzes the reversible amination of 2-oxoglutarate to form glutamate, using ammonium as a substrate. For a better understanding of the physiological function of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate, we used transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the two genes encoding the enzyme. An in vivo real time (15)N-nuclear magnetic resonance (NMR) spectroscopy approach allowed the demonstration that, when the two GDH genes were overexpressed individually or simultaneously, the transgenic plant leaves did not synthesize glutamate in the presence of ammonium when glutamine synthetase (GS) was inhibited. In contrast we confirmed that the primary function of GDH is to deaminate Glu. When the two GDH unlabeled substrates ammonium and Glu were provided simultaneously with either [(15)N]Glu or (15)NH(4)(+) respectively, we found that the ammonium released from the deamination of Glu was reassimilated by the enzyme GS, suggesting the occurrence of a futile cycle recycling both ammonium and Glu. Taken together, these results strongly suggest that the GDH enzyme, in conjunction with NADH-GOGAT, contributes to the control of leaf Glu homeostasis, an amino acid that plays a central signaling and metabolic role at the interface of the carbon and nitrogen assimilatory pathways. Thus, in vivo NMR spectroscopy appears to be an attractive technique to follow the flux of metabolites in both normal and genetically modified plants.

Polyamines have long been implicated in plant growth and development, as well as adaptation to abiotic and biotic stress. As a general rule of thumb the higher the polyamine titers the better. However, their molecular roles in plant stress responses still remain obscure. It has been postulated that they could act through their catabolism, which generates molecules which may act as secondary messengers signalling networks of numerous developmental and stress adaptation processes. Recently it was shown that plant and mammalian polyamine catabolism share critical features, giving new insight in plant polyamine catabolism. In this review, the advances in genes and proteins of polyamine catabolism in plants is presented and compared to other models.

In contrast to animals, where polyamine (PA) catabolism efficiently converts spermine (Spm) to putrescine (Put), plants have been considered to possess a PA catabolic pathway producing 1,3-diaminopropane, Δ 1-pyrroline, the corresponding aldehyde, and hydrogen peroxide but unable to back-convert Spm to Put. Arabidopsis (Arabidopsis thaliana) genome contains at least five putative PA oxidase (PAO) members with yet-unknown localization and physiological role(s). AtPAO1 was recently identified as an enzyme similar to the mammalian Spm oxidase, which converts Spm to spermidine (Spd). In this work, we have performed in silico analysis of the five Arabidopsis genes and have identified PAO3 (AtPAO3) as a nontypical PAO, in terms of homology, compared to other known PAOs. We have expressed the gene AtPAO3 and have purified a protein corresponding to it using the inducible heterologous expression system of Escherichia coli. AtPAO3 catalyzed the sequential conversion/oxidation of Spm to Spd, and of Spd to Put, thus exhibiting functional homology to the mammalian PAOs. The best substrate for this pathway was Spd, whereas the N 1-acetyl-derivatives of Spm and Spd were oxidized less efficiently. On the other hand, no activity was detected when diamines (agmatine, cadaverine, and Put) were used as substrates. Moreover, although AtPAO3 does not exhibit significant similarity to the other known PAOs, it is efficiently inhibited by guazatine, a potent PAO inhibitor. AtPAO3 contains a peroxisomal targeting motif at the C terminus, and it targets green fluorescence protein to peroxisomes when fused at the N terminus but not at the C terminus. These results reveal that AtPAO3 is a peroxisomal protein and that the C terminus of the protein contains the sorting information. The overall data reinforce the view that plants and mammals possess a similar PA oxidation system, concerning both the subcellular localization and the mode of its action.

Polyamines (PAs) exert a protective effect against stress challenges, but their molecular role in this remains speculative. In order to detect the signaling role of apoplastic PA-derived hydrogen peroxide (H2O2) under abiotic stress, we developed a series of tobacco (Nicotiana tabacum cv Xanthi) transgenic plants overexpressing or downregulating apoplastic polyamine oxidase (PAO; S-pao and A-pao plants, respectively) or downregulating S-adenosyl-l-methionine decarboxylase (samdc plants). Upon salt stress, plants secreted spermidine (Spd) into the apoplast, where it was oxidized by the apoplastic PAO, generating H2O2. A-pao plants accumulated less H2O2 and exhibited less programmed cell death (PCD) than did wild-type plants, in contrast with S-pao and samdc downregulating plants. Induction of either stress-responsive genes or PCD was dependent on the level of Spd-derived apoplastic H2O2. Thus, in wild-type and A-pao plants, stress-responsive genes were efficiently induced, although in the latter at a lower rate, while S-pao plants, with higher H2O2 levels, failed to accumulate stress-responsive mRNAs, inducing PCD instead. Furthermore, decreasing intracellular PAs, while keeping normal apoplastic Spd oxidation, as in samdc downregulating transgenic plants, caused enhanced salinity-induced PCD. These results reveal that salinity induces the exodus of Spd into the apoplast, where it is catabolized by PAO, producing H2O2. The accumulated H2O2 results in the induction of either tolerance responses or PCD, depending also on the levels of intracellular PAs.

To dissect differences in both lipid accumulation and composition and the role of these modifications during drought stress, four grapevine cultivars exhibiting differential tolerance to drought were subjected to water shortage. Tolerant cultivars, Kahli Kerkennah and Cardinal, exhibited higher leaf water potential (Ψw), and lower lipid peroxidation compared to the sensitive cultivars Guelb Sardouk and Superior Seedless during stress. Total lipid amounts increased during stress only in the leaves of the tolerant cultivars. Drought induced increases in the ratios digalactosyldiacylglycerol/monogalactosyldiacylglycerol and phosphatidylcholine/phoshatidylethanolamine of almost all the drought stressed cultivars. Moreover, the overall analysis of the composition of fatty acids revealed that a linolenic acid was prevalent in grapevine and the unsaturation level of lipids increased under water stress in all the cultivars. Specific adjustments in the lipid composition during stress could compromise stress tolerance.

Adaptation to stress entails a repertoire of molecular pathways that remodel the proteome, thereby promoting selective translation of pro-survival proteins. Yet, translation of other proteins, especially those which are harmful for stress adaptation is, on the contrary, transiently suppressed through mRNA decay or storage. Proteome remodeling under stress is intimately associated with the cytoplasmic ribonucleoprotein (RNP) complexes called stress granules (SGs) and processing bodies (PBs). The molecular composition and regulation of SGs and PBs in plants remain largely unknown. Recently, we identified the Arabidopsis Tudor Staphylococcal Nuclease (TSN, Tudor-SN or SND1) as a SG- and PB-associated protein required for mRNA decapping under stress conditions. Here we show that SGs localize in close proximity to PBs within plant cells that enable the exchange of molecular components. Furthermore, we provide a meta-analysis of mRNA degradome of TSN-deficient plants suggesting that TSN might inhibit the degradation of mRNAs which are involved in stress adaptation. Our results establish TSN as a versatile mRNA regulator during stress.

Summary In angiosperms, WUSCHEL‐RELATED HOMEOBOX 3 (WOX3) genes are required for the recruitment of founder cells from the lateral domains of shoot meristems that form lateral regions of leaves. However, the regulation of the formation of lateral organs in gymnosperms remains unknown.By using somatic embryos of Norway spruce (Picea abies) we have studied the expression and function of PaWOX3 during embryo development. The mRNA abundance of PaWOX3 was determined by quantitative real‐time PCR, and the spatial expression of PaWOX3 was analysed by histochemical β‐glucuronidase (GUS) assays and in situ mRNA hybridization. To investigate the function of PaWOX3, we analysed how downregulation of PaWOX3 in RNA interference lines affected embryo development and morphology. PaWOX3 was highly expressed in mature embryos at the base of each cotyledon close to the junction between the cotyledons, and in the lateral margins of cotyledons and needles, separating them into an adaxial and an abaxial side. Downregulation of the expression of PaWOX3 caused defects in lateral margin outgrowth in cotyledons and needles, and reduced root elongation.Our data suggest that the WOX3 function in margin outgrowth in lateral organs is conserved among the seed plants, whereas its function in root elongation may be unique to gymnosperms.

Summary Caloric restriction (CR) extends lifespan in various heterotrophic organisms ranging from yeasts to mammals, but whether a similar phenomenon occurs in plants remains unknown. Plants are autotrophs and use their photosynthetic machinery to convert light energy into the chemical energy of glucose and other organic compounds. As the rate of photosynthesis is proportional to the level of photosynthetically active radiation, the CR in plants can be modeled by lowering light intensity. Here, we report that low light intensity extends the lifespan in Arabidopsis through the mechanisms triggering autophagy, the major catabolic process that recycles damaged and potentially harmful cellular material. Knockout of autophagy-related genes results in the short lifespan and suppression of the lifespan-extending effect of the CR. Our data demonstrate that the autophagy-dependent mechanism of CR-induced lifespan extension is conserved between autotrophs and heterotrophs.

Factors regulating dynamics of chromatin structure have direct impact on expression of genetic information. Cohesin is a multi-subunit protein complex crucial for pairing sister chromatids during cell division, DNA repair and regulation of gene transcription and silencing. In non-plant species cohesin is loaded on chromatin by the Scc2/Scc4 (NIBPL/MAU2) complex. Here we identify AtSCC4, the Arabidopsis homolog of Scc4, and show that it forms a functional complex with AtSCC2, the homolog of Scc2. We demonstrate that AtSCC2 and AtSCC4 act in the same pathway and that both proteins are indispensable for cell fate determination during early stages of embryo development. Mutant embryos lacking either of these proteins develop only up to the globular stage, and show suspensor over-proliferation phenotype preceded by ectopic auxin maxima distribution. We further establish a new assay to reveal the AtSCC4-dependent dynamics of cohesin loading on chromatin in vivo . Our findings define Scc2/Scc4 complex as an evolutionary conserved machinery controlling cohesin loading and chromatin structure maintenance and provide new insight into plant-specific role of this complex in controlling cell fate during embryogenesis.

Polyamines (PAs) have been considered as important molecules for survival. However, evidence reinforces that PAs are also implicated, directly or indirectly, in pathways regulating programmed cell death (PCD). Direct correlation of PAs with cell death refers to their association with particular biological processes, and their physical contact with molecules or structures involved in cell death. Indirectly, PAs regulate PCD through their metabolic derivatives, such as catabolic and interconversion products. Cytotoxic products of PA metabolism are involved in PCD cascades, whereas it remains largely elusive how PAs directly control pathways leading to PCD. In this review, we present and compare advances in PA-dependent PCD in animals and plants.