Peter V. Bozhkov

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Programmed cell death (PCD) is an integral part of plant development and of responses to abiotic stress or pathogens. Although the morphology of plant PCD is, in some cases, well characterised and molecular mechanisms controlling plant PCD are beginning to emerge, there is still confusion about the classification of PCD in plants. Here we suggest a classification based on morphological criteria. According to this classification, the use of the term 'apoptosis' is not justified in plants, but at least two classes of PCD can be distinguished: vacuolar cell death and necrosis. During vacuolar cell death, the cell contents are removed by a combination of autophagy-like process and release of hydrolases from collapsed lytic vacuoles. Necrosis is characterised by early rupture of the plasma membrane, shrinkage of the protoplast and absence of vacuolar cell death features. Vacuolar cell death is common during tissue and organ formation and elimination, whereas necrosis is typically found under abiotic stress. Some examples of plant PCD cannot be ascribed to either major class and are therefore classified as separate modalities. These are PCD associated with the hypersensitive response to biotrophic pathogens, which can express features of both necrosis and vacuolar cell death, PCD in starchy cereal endosperm and during self-incompatibility. The present classification is not static, but will be subject to further revision, especially when specific biochemical pathways are better defined.

Metacaspases are cysteine-dependent proteases found in protozoa, fungi and plants and are distantly related to metazoan caspases. Although metacaspases share structural properties with those of caspases, they lack Asp specificity and cleave their targets after Arg or Lys residues. Studies performed over the past 10 years have demonstrated that metacaspases are multifunctional proteases essential for normal physiology of non-metazoan organisms. This article provides a comprehensive overview of the metacaspase function and molecular regulation during programmed cell death, stress and cell proliferation, as well as an analysis of the first metacaspase-mediated proteolytic pathway. To prevent further misapplication of caspase-specific molecular probes for measuring and inhibiting metacaspase activity, we provide a list of probes suitable for metacaspases.

Programmed cell death (PCD) is indispensable for eukaryotic development. In animals, PCD is executed by the caspase family of cysteine proteases. Plants do not have close homologues of caspases but possess a phylogenetically distant family of cysteine proteases named metacaspases. The cellular function of metacaspases in PCD is unknown. Here we show that during plant embryogenesis, metacaspase mcII-Pa translocates from the cytoplasm to nuclei in terminally differentiated cells that are destined for elimination, where it colocalizes with the nuclear pore complex and chromatin, causing nuclear envelope disassembly and DNA fragmentation. The cell-death function of mcII-Pa relies on its cysteine-dependent arginine-specific proteolytic activity. Accordingly, mutation of catalytic cysteine abrogates the proteolytic activity of mcII-Pa and blocks nuclear degradation. These results establish metacaspase as an executioner of PCD during embryo patterning and provide a functional link between PCD and embryogenesis in plants. Although mcII-Pa and metazoan caspases have different substrate specificity, they serve a common function during development, demonstrating the evolutionary parallelism of PCD pathways in plants and animals.

In the animal life cycle, the earliest manifestations of programmed cell death (PCD) can already be seen during embryogenesis. The aim of this work was to determine if PCD is also involved in the elimination of certain cells during plant embryogenesis. We used a model system of Norway spruce somatic embryogenesis, which represents a multistep developmental pathway with two broad phases. The first phase is represented by proliferating proembryogenic masses (PEMs). The second phase encompasses development of somatic embryos, which arise from PEMs and proceed through the same sequence of stages as described for their zygotic counterparts. Here we demonstrate two successive waves of PCD, which are implicated in the transition from PEMs to somatic embryos and in correct embryonic pattern formation, respectively. The first wave of PCD is responsible for the degradation of PEMs when they give rise to somatic embryos. We show that PCD in PEM cells and embryo formation are closely interlinked processes, both stimulated upon withdrawal or partial depletion of auxins and cytokinins. The second wave of PCD eliminates terminally differentiated embryo-suspensor cells during early embryogeny. During the dismantling phase of PCD, PEM and embryo-suspensor cells exhibit progressive autolysis, resulting in the formation of a large central vacuole. Autolytic degradation of the cytoplasm is accompanied by lobing and budding-like segmentation of the nucleus. Nuclear DNA undergoes fragmentation into both large fragments of about 50 kb and multiples of approximately 180 bp. The tonoplast rupture is delayed until lysis of the cytoplasm and organelles, including the nucleus, is almost complete. The protoplasm then disappears, leaving a cellular corpse represented by only the cell wall. This pathway of cell dismantling suggests overlapping of apoptotic and autophagic types of PCD during somatic embryogenesis in Norway spruce.

Several coniferous species can be propagated via somatic embryogenesis. This is a useful method for clonal propagation, but it can also be used for studying how embryo development is regulated in conifers. However, in conifers it is not known to what extent somatic and zygotic embryos develop similarly, because there has been little research on the origin and development of somatic embryos. A time-lapse tracking technique has been set up, and the development of more than 2000 single cells and few-celled aggregates isolated from embryogenic suspension cultures of Norway spruce (Picea abies L. Karst.) and embedded in thin layers of agarose has been traced. Experiments have shown that somatic embryos develop from proembryogenic masses which pass through a series of three characteristic stages distinguished by cellular organization and cell number (stages I, II and III) to transdifferentiate to somatic embryos. Microscopic inspection of different types of structures has revealed that proembryogenic masses are characterized by high interclonal variation of shape and cellular constitution. In contrast, somatic embryos are morphologically conservative structures, possessing a distinct protoderm-like cell layer as well as embryonal tube cells and suspensor. The lack of staining of the arabinogalactan protein epitope recognized by the monoclonal antibody JIM13 was shown to be an efficient marker for distinguishing proembryogenic masses from somatic embryos. The vast majority of cells in proembryogenic masses expressed this epitope and none of cells in the early somatic embryos. The conditions that promote cell proliferation (i.e. the presence of exogenous auxin and cytokinin), inhibit somatic embryo formation; instead, continuous multiplication of stage I proembryogenic masses by unequal division of embryogenic cells with dense cytoplasm is the prevailing process. Once somatic embryos have formed, their further development to mature forms requires abscisic acid and shares a common histodifferentiation pattern with zygotic embryos. Although the earliest stages of somatic embryo development comparable to proembryogeny could not be characterized, the subsequent developmental processes correspond closely to what occurs in the course of early and late zygotic embryogeny. A model for somatic embryogenesis pathways in Picea abies is presented.

In plants, as in animals, programmed cell death (PCD) is a key process responsible for the elimination of unneeded structures and for overall shape remodeling during development [1]; however, the molecular mechanisms remain poorly understood. Despite the absence of canonical caspases in plants, dying plant cells show an increased proteolytic caspase-like activity [2]. Moreover, the cell death can be suppressed using synthetic [2] or natural [3] caspase inhibitors. This raises the question of whether plants have specific cysteine proteases with a role similar to metazoan caspases in the execution of PCD. Metacaspases are the best candidates to perform this role, because they contain a caspase-specific catalytic diad of histidine and cysteine as well as conserved caspase-like secondary structure [4,5]. Here we show the first experimental evidence for metacaspase function in the activation and/or execution of PCD in plants, and also demonstrate the fundamental requirement of plant metacaspase for embryogenesis.

Although animals eliminate apoptotic cells using macrophages, plants use cell corpses throughout development and disassemble cells in a cell-autonomous manner by vacuolar cell death. During vacuolar cell death, lytic vacuoles gradually engulf and digest the cytoplasmic content. On the other hand, acute stress triggers an alternative cell death, necrosis, which is characterized by mitochondrial dysfunction, early rupture of the plasma membrane, and disordered cell disassembly. How both types of cell death are regulated remains obscure. In this paper, we show that vacuolar death in the embryo suspensor of Norway spruce requires autophagy. In turn, activation of autophagy lies downstream of metacaspase mcII-Pa, a key protease essential for suspensor cell death. Genetic suppression of the metacaspase–autophagy pathway induced a switch from vacuolar to necrotic death, resulting in failure of suspensor differentiation and embryonic arrest. Our results establish metacaspase-dependent autophagy as a bona fide mechanism that is responsible for cell disassembly during vacuolar cell death and for inhibition of necrosis.

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.

Somatic embryogenesis (SE) is a process of differentiation of cells into a plant bypassing the fusion of gametes. As such, it represents a very powerful tool in biotechnology for propagation of species with a long reproductive cycle or low seed set and production of genetically modified plants with improved traits. SE is also a versatile model to study cellular and molecular mechanisms of plant embryo patterning. The morphology and molecular regulation of SE resemble those of zygotic embryogenesis and begin with establishment of apical–basal asymmetry. The apical domain, the embryo proper, proliferates and eventually gives rise to the plantlet, while the basal part, the embryo suspensor, is terminally differentiated and gradually removed via vacuolar programmed cell death (PCD). This PCD is essential for normal development of the apical domain. Emerging evidence demonstrates that signalling events in the apical and basal domains share homologous components. Here we provide an overview of the main pathways controlling the life and death events during SE.

•Autophagy can suppress, initiate, or execute cell death depending on the biological context. •Autophagy is an initiator of localized, hypersensitive response-associated cell death upon pathogen infection. •Autophagy executes ‘formative’ vacuolar cell death and prevents ‘destructive’ necrosis in terminally differentiated cells. •Homeostatic and anti-aging functions of autophagy complicate the dissection of its distinct roles in cell death. Autophagy plays multiple, often antagonistic roles in plants. In particular, cytoprotective functions of autophagy are well balanced by cell death functions to compensate for the absence of apoptosis culminating in phagocytic clearance of dead cells. If autophagy is indeed required for plant programmed cell death (PCD), then what place does it occupy in the PCD pathways? Recent studies have examined the effects of impaired autophagy on pathogen-induced hypersensitive response (HR) and developmental PCD. While HR death was efficiently suppressed, inhibition of autophagy induced a switch from vacuolar PCD essential for development to necrosis. We therefore propose a dual role for autophagy in plant PCD: as an effector of HR PCD lying upstream of the ‘point-of-no-return’, and also as a downstream mechanism for clearance of terminally differentiated cells during developmental PCD. Autophagy plays multiple, often antagonistic roles in plants. In particular, cytoprotective functions of autophagy are well balanced by cell death functions to compensate for the absence of apoptosis culminating in phagocytic clearance of dead cells. If autophagy is indeed required for plant programmed cell death (PCD), then what place does it occupy in the PCD pathways? Recent studies have examined the effects of impaired autophagy on pathogen-induced hypersensitive response (HR) and developmental PCD. While HR death was efficiently suppressed, inhibition of autophagy induced a switch from vacuolar PCD essential for development to necrosis. We therefore propose a dual role for autophagy in plant PCD: as an effector of HR PCD lying upstream of the ‘point-of-no-return’, and also as a downstream mechanism for clearance of terminally differentiated cells during developmental PCD. the dynamic process of autophagosome formation, delivery of autophagic substrates to the lysosome (or vacuole), and degradation of autophagic substrates inside the lysosome (or vacuole). a cytosolic membrane-bound compartment denoted by a limiting double membrane. from Greek, ‘eat oneself’; a major catabolic process in eukaryotic cells wherein a portion of the cytoplasm is engulfed by a specific membrane, delivered to lysosome (in animals) or vacuole (in fungi or plants), and is finally digested by hydrolytic enzymes. a group of proteins from different families, required for progression through specific stages of the autophagy pathway. a complex, high-amplitude resistance response induced by intracellular immune receptors of the nucleotide-binding leucine-rich (NB-LRR) protein family upon recognition of pathogen-secreted effector proteins or their activities on host cellular targets. ETI is typically associated with the development of HR and SAR. the digestion within a cell of a material taken in by phagocytosis from the cellular environment. At the final stages of apoptotic cell death, apoptotic bodies are removed by heterophagy. a rapid and locally restricted PCD reaction as a result of ETI. HR cell death is often but not always dispensable for ETI-mediated limitation of pathogen proliferation and spread. a group of cysteine-dependent proteases belonging to the C14 family that are found in protozoa, fungi, and plants. Two types of metacaspases are distinguished: (i) type I metacaspases have a N-terminal prodomain containing a proline-rich repeat motif and, in plant members, also a zinc-finger motif, (ii) type II metacaspases lack such a prodomain but harbor a linker region between the putative large (p20) and small (p10) caspase-like subunits. Unlike aspartate-specific caspases, metacaspases possess arginine/lysine substrate cleavage specificity. one of the two major types of cell death in plants, characterized by mitochondrial dysfunction, energetic catastrophe, and early rupture of plasma membrane. Necrosis is typically found under abiotic stress, whereas pathogen-induced HR cell death displays features of both necrosis and vacuolar cell death. Most morphological and biochemical characteristics of necrosis are conserved between animals and plants. a part of the plant embryo that functions as a conduit of nutrients and hormones to the embryo proper. The suspensor becomes obsolete at late stages of embryogenesis and undergoes slow degradation by vacuolar cell death. a broad-spectrum resistance response in uninfected tissue following local activation of ETI. SAR is usually characterized by salicylic acid (SA)- and NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1)-dependent induction of pathogenesis-related (PR) proteins. one of the two major types of cell death in plants, wherein the content of dying cell is gradually engulfed by growing lytic vacuoles without loss of protoplast turgor, and culminating in vacuolar collapse. Vacuolar cell death is commonly observed during plant development, for example in the embryo-suspensor and xylem elements.

Tudor Staphylococcal Nuclease (TSN or Tudor-SN; also known as SND1) is an evolutionarily conserved protein involved in the transcriptional and posttranscriptional regulation of gene expression in animals. Although TSN was found to be indispensable for normal plant development and stress tolerance, the molecular mechanisms underlying these functions remain elusive. Here, we show that Arabidopsis thaliana TSN is essential for the integrity and function of cytoplasmic messenger ribonucleoprotein (mRNP) complexes called stress granules (SGs) and processing bodies (PBs), sites of posttranscriptional gene regulation during stress. TSN associates with SGs following their microtubule-dependent assembly and plays a scaffolding role in both SGs and PBs. The enzymatically active tandem repeat of four SN domains is crucial for targeting TSN to the cytoplasmic mRNA complexes and is sufficient for the cytoprotective function of TSN during stress. Furthermore, our work connects the cytoprotective function of TSN with its positive role in stress-induced mRNA decapping. While stress led to a pronounced increase in the accumulation of uncapped mRNAs in wild-type plants, this increase was abrogated in TSN knockout plants. Taken together, our results establish TSN as a key enzymatic component of the catabolic machinery responsible for the processing of mRNAs in the cytoplasmic mRNP complexes during stress.

Autophagy and the ubiquitin-proteasome system (UPS) are two major protein degradation pathways implicated in the response to microbial infections in eukaryotes. In animals, the contribution of autophagy and the UPS to antibacterial immunity is well documented and several bacteria have evolved measures to target and exploit these systems to the benefit of infection. In plants, the UPS has been established as a hub for immune responses and is targeted by bacteria to enhance virulence. However, the role of autophagy during plant-bacterial interactions is less understood. Here, we have identified both pro- and antibacterial functions of autophagy mechanisms upon infection of Arabidopsis thaliana with virulent Pseudomonas syringae pv tomato DC3000 (Pst). We show that Pst activates autophagy in a type III effector (T3E)-dependent manner and stimulates the autophagic removal of proteasomes (proteaphagy) to support bacterial proliferation. We further identify the T3E Hrp outer protein M1 (HopM1) as a principle mediator of autophagy-inducing activities during infection. In contrast to the probacterial effects of Pst-induced proteaphagy, NEIGHBOR OF BRCA1-dependent selective autophagy counteracts disease progression and limits the formation of HopM1-mediated water-soaked lesions. Together, we demonstrate that distinct autophagy pathways contribute to host immunity and bacterial pathogenesis during Pst infection and provide evidence for an intimate crosstalk between proteasome and autophagy in plant-bacterial interactions.

Autophagy is a eukaryotic catabolic pathway essential for growth and development. In plants, it is activated in response to environmental cues or developmental stimuli. However, in contrast to other eukaryotic systems, we know relatively little regarding the molecular players involved in autophagy and the regulation of this complex pathway. In the framework of the COST (European Cooperation in Science and Technology) action TRANSAUTOPHAGY (2016-2020), we decided to review our current knowledge of autophagy responses in higher plants, with emphasis on knowledge gaps. We also assess here the potential of translating the acquired knowledge to improve crop plant growth and development in a context of growing social and environmental challenges for agriculture in the near future.

Metacaspases and paracaspases are proteases that were first identified as containing a caspase-like structural fold (Uren et al., 2000Uren A.G. O’Rourke K. Aravind L.A. Pisabarro M.T. Seshagiri S. Koonin E.V. Dixit V.M. Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma.Mol. Cell. 2000; 6: 961-967Abstract Full Text Full Text PDF PubMed Google Scholar). 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. The current classification of proteases provided by the MEROPS database clusters caspases, metacaspases, and paracaspases to the same family, C14, within the CD clan (https://www.ebi.ac.uk/merops/). All members of the C14 family are annotated to possess aspartate P1 cleavage specificity, and the family is further split into two subfamilies: C14A (caspases) and C14B (metacaspases and paracaspases). Importantly, the MEROPS approach of grouping proteases into families or subfamilies is based on statistically significant similarities of the amino acid sequence within the peptidase domain or part thereof, without considering their biochemical properties (Rawlings et al., 2018Rawlings N.D. Barrett A.J. Thomas P.D. Huang X. Bateman A. Finn R.D. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database.Nucleic Acids Res. 2018; 46: D624-D632Crossref PubMed Scopus (756) Google Scholar). Being valuable for high-throughput protease classification, this approach, however, has substantial drawbacks if implemented without further adjustment. Indeed, in contradiction with the MEROPS description, none of the metacaspases or paracaspases characterized so far cleave after an aspartate residue. Instead, paracaspases are arginine specific (Coornaert et al., 2008Coornaert B. Baens M. Heyninck K. Bekaert T. Haegman M. Staal J. Sun L. Chen Z.J. Marynen P. Beyaert R. T cell antigen receptor stimulation induces MALT1 paracaspase-mediated cleavage of the NF-kappaB inhibitor A20.Nat. Immunol. 2008; 9: 263-271Crossref PubMed Scopus (342) Google Scholar, Hachmann et al., 2012Hachmann J. Snipas S.J. van Raam B.J. Cancino E.M. Houlihan E.J. Poreba M. Kasperkiewicz P. Drag M. Salvesen G.S. Mechanism and specificity of the human paracaspase MALT1.Biochem. J. 2012; 443: 287-295Crossref PubMed Scopus (66) Google Scholar, Rebeaud et al., 2008Rebeaud F. Hailfinger S. Posevitz-Fejfar A. Tapernoux M. Moser R. Rueda D. Gaide O. Guzzardi M. Iancu E.M. Rufer N. et al.The proteolytic activity of the paracaspase MALT1 is key in T cell activation.Nat. Immunol. 2008; 9: 272-281Crossref PubMed Scopus (238) Google Scholar), whereas metacaspases can cleave after either arginine or lysine (Figure S1A; Sundström et al., 2009Sundström J.F. Vaculova A. Smertenko A.P. Savenkov E.I. Golovko A. Minina E. Tiwari B.S. Rodriguez-Nieto S. Zamyatnin Jr., A.A. Välineva T. et al.Tudor staphylococcal nuclease is an evolutionarily conserved component of the programmed cell death degradome.Nat. Cell Biol. 2009; 11: 1347-1354Crossref PubMed Scopus (164) Google Scholar, Vercammen et al., 2004Vercammen D. van de Cotte B. De Jaeger G. Eeckhout D. Casteels P. Vandepoele K. Vandenberghe I. Van Beeumen J. Inzé D. Van Breusegem F. Type II metacaspases Atmc4 and Atmc9 of Arabidopsis thaliana cleave substrates after arginine and lysine.J. Biol. Chem. 2004; 279: 45329-45336Crossref PubMed Scopus (263) Google Scholar). Such fundamental differences in the proteolytic specificity between caspases, metacaspases, and paracaspases imply distinct repertoires of new proteoforms that they generate and point to the complex diversification and coevolution of their substrates and downstream pathways. One unfortunate consequence of the current classification is the misuse of caspase-specific probes for studying metacaspases and paracaspases that is commonly found in the literature and leads to false conclusions. Apart from substrate specificity, caspases, metacaspases, and paracaspases feature other fundamental differences (Figure S1A). For example, active metacaspases are monomers and their activation usually requires millimolar concentrations of calcium (Hander et al., 2019Hander T. Fernández-Fernández Á.D. Kumpf R.P. Willems P. Schatowitz H. Rombaut D. Staes A. Nolf J. Pottie R. Yao P. et al.Damage on plants activates Ca2+-dependent metacaspases for release of immunomodulatory peptides.Science. 2019; 363: 1-10Crossref Scopus (113) Google Scholar, McLuskey et al., 2012McLuskey K. Rudolf J. Proto W.R. Isaacs N.W. Coombs G.H. Moss C.X. Mottram J.C. Crystal structure of a Trypanosoma brucei metacaspase.Proc. Natl. Acad. Sci. USA. 2012; 109: 7469-7474Crossref PubMed Scopus (65) Google Scholar, Wong et al., 2012Wong A.H.H. Yan C. Shi Y. Crystal structure of the yeast metacaspase Yca1.J. Biol. Chem. 2012; 287: 29251-29259Crossref PubMed Scopus (70) Google Scholar). In contrast, active caspases and paracaspases are calcium-independent dimers (Hachmann et al., 2012Hachmann J. Snipas S.J. van Raam B.J. Cancino E.M. Houlihan E.J. Poreba M. Kasperkiewicz P. Drag M. Salvesen G.S. Mechanism and specificity of the human paracaspase MALT1.Biochem. J. 2012; 443: 287-295Crossref PubMed Scopus (66) Google Scholar, Wiesmann et al., 2012Wiesmann C. Leder L. Blank J. Bernardi A. Melkko S. Decock A. D’Arcy A. Villard F. Erbel P. Hughes N. et al.Structural determinants of MALT1 protease activity.J. Mol. Biol. 2012; 419: 4-21Crossref PubMed Scopus (64) Google Scholar, Yu et al., 2011Yu J.W. Jeffrey P.D. Ha J.Y. Yang X. Shi Y. Crystal structure of the mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) paracaspase region.Proc. Natl. Acad. Sci. USA. 2011; 108: 21004-21009Crossref PubMed Scopus (65) Google Scholar). This indicates that upstream pathways regulating activation of caspases, metacaspases, and paracaspases are likewise different. In the past two decades we have learned about important differences between caspases, metacaspases, and paracaspases. Thus, simple extrapolation of features typical for caspases to all other members of the C14 family is not justified anymore. Instead caspases, metacaspases, and paracaspases should be separated into three corresponding groups within the family and each group should be properly annotated by having its key biochemical and structural characteristics provided. We kindly request curators of the MEROPS database to make corresponding changes. Since the structure and substrate specificity of prokaryotic caspase-like proteases named “orthocaspases” remain largely unknown (Klemenčič et al., 2015Klemenčič M. Novinec M. Dolinar M. Orthocaspases are proteolytically active prokaryotic caspase homologues: the case of Microcystis aeruginosa.Mol. Microbiol. 2015; 98: 142-150Crossref PubMed Scopus (34) Google Scholar), we leave their classification and nomenclature open until their structural and biochemical properties have been clarified. The name “caspase” stands for “cysteine-dependent aspartate-specific protease.” Thus, the names “metacaspase” and “paracaspase” imply the wrong substrate specificity for these proteases. However, since these names have been used for two decades, we propose to keep them, provided that caspases, metacaspases, and paracaspases are recognized as three separate groups within the C14 family. Based on domain composition and arrangement, metacaspases and paracaspases are further subdivided into three and two types, respectively (Figure S1A). For the sake of consistency, we propose to maintain a common nomenclature for the different types of metacaspases and paracaspases using Latin numerals (e.g., type I metacaspases). As for the conserved protein structures, they will be referred to as the p20-like region, the p10-like region, the linker region, and the N-terminal pro-domain, matching the nomenclature of caspases (Figure S1A; Alnemri et al., 1996Alnemri E.S. Livingston D.J. Nicholson D.W. Salvesen G. Thornberry N.A. Wong W.W. Yuan J. Human ICE/CED-3 protease nomenclature.Cell. 1996; 87: 171Abstract Full Text Full Text PDF PubMed Scopus (2139) Google Scholar). The p20, p10, and linker regions have been previously defined for the caspase group of the C14 family (Fuentes-Prior and Salvesen, 2004Fuentes-Prior P. Salvesen G.S. The protein structures that shape caspase activity, specificity, activation and inhibition.Biochem. J. 2004; 384: 201-232Crossref PubMed Scopus (687) Google Scholar) and can be easily identified in metacaspases and paracaspase homologs based on a hidden Markov model (HMM) alignment with the C14 peptidase domain (Figure S1B). Notably, although not always clearly stated in the literature, most known members of the C14 family contain the linker region. Furthermore, type II metacaspases are distinguished by a long linker between the p20 and p10 regions and an additional linker within the p10 region (Figure S1A), which are frequently referred to as a single linker. We suggest the consideration of the active form of metacaspases or paracaspases as a monomer if it is a cleaved or intact polypeptide chain derived from a single translational event, and a dimer if it comprises uncut or processed products of two translational events. We propose to establish a unified nomenclature of metacaspases and paracaspases in order to (i) facilitate the comparison of orthologs from different organisms and (ii) make it suitable for annotating homologs of species with partially sequenced genomes. Thus, we suggest using simple root symbols such as MCA for metacaspases and PCA for paracaspases. When naming individual family members, these root symbols will be preceded by the abbreviated Latin name of the species and followed by a hyphen, a Latin number representing the type, and then a small alpha character indicating in alphabetical order the number of the homolog of this type in a given genome (Figure S1C). Proenzymes that require proteolytic processing for activation could be annotated with the prefix “pro-”, e.g. pro-AtMCA-Ia for the metacaspase 1 of type I from A. thaliana. Spliceoforms should be indicated by a decimal number (e.g. AtMCA-Ia.1). Please note that these conventions do not consider the letter case, which should conform to gene and protein nomenclature established for a given model organism or taxonomic group. Importantly, this nomenclature should be used synonymously for metacaspases and paracaspase homologs with well-established names, e.g., human MALT1/HsPCA-Ia and A. thaliana AtMC1/AtMCA-Ia or AtMC4/AtMCA-IIa. We encourage all researchers to adopt these recommendations. The new classification and unified nomenclature of metacaspases and paracaspases will facilitate a more comprehensive exchange of relevant findings within the scientific community and help to bridge already existing knowledge with newly discovered homologs, thus promoting mechanistic understanding of these ancient, evolutionarily conserved proteases. This work was supported by the Knut and Alice Wallenberg Foundation. We apologize to colleagues whose work has not been cited due to space limitations. 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Initially found to be critically involved in inflammation and apoptosis, caspases have since then been implicated in the regulation of various signaling pathways in animals. How caspases and caspase-mediated processes evolved is a topic of great interest and hot debate. In fact, caspases are just the tip of the iceberg, representing a relatively small group of mostly animal-specific enzymes within a broad family of structurally related cysteine proteases (family C14 of CD clan) found in all kingdoms of life. Apart from caspases, this family encompasses para- and metacaspases, and all three groups of proteases exhibit significant variation in biochemistry and function in vivo. Notably, metacaspases are present in all eukaryotic lineages with a remarkable absence in animals. Thus, metacaspases and caspases must have adapted to operate under distinct cellular and physiological settings. Here we discuss biochemical properties and biological functions of metacaspases in comparison to caspases, with a major focus on the regulation of developmental aspects in plants versus animals.

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 plantspecific 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.

Abstract 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, 2 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.

Plant embryogenesis is intimately associated with programmed cell death. The mechanisms of initiation and control of programmed cell death during plant embryo development are not known. Proteolytic activity associated with caspase-like proteins is paramount for control of programmed cell death in animals and yeasts. Caspase family of proteases has unique strong preference for cleavage of the target proteins next to asparagine residue. In this work, we have used synthetic peptide substrates containing caspase recognition sites and corresponding specific inhibitors to analyse the role of caspase-like activity in the regulation of programmed cell death during plant embryogenesis. We demonstrate that VEIDase is a principal caspase-like activity implicated in plant embryogenesis. This activity increases at the early stages of embryo development that coincide with massive cell death during shape remodeling. The VEIDase activity exhibits high sensitivity to pH, ionic strength and Zn(2+) concentration. Altogether, biochemical assays show that VEIDase plant caspase-like activity resembles that of both mammalian caspase-6 and yeast metacaspase, YCA1. In vivo, VEIDase activity is localised specifically in the embryonic cells during both the commitment and in the beginning of the execution phase of programmed cell death. Inhibition of VEIDase prevents normal embryo development via blocking the embryo-suspensor differentiation. Our data indicate that the VEIDase activity is an integral part in the control of plant developmental cell death programme, and that this activity is essential for the embryo pattern formation.

Cell and tissue patterning in plant embryo development is well documented. Moreover, it has recently been shown that successful embryogenesis is reliant on programmed cell death (PCD). The cytoskeleton governs cell morphogenesis. However, surprisingly little is known about the role of the cytoskeleton in plant embryogenesis and associated PCD. We have used the gymnosperm, Picea abies, somatic embryogenesis model system to address this question. Formation of the apical-basal embryonic pattern in P. abies proceeds through the establishment of three major cell types: the meristematic cells of the embryonal mass on one pole and the terminally differentiated suspensor cells on the other, separated by the embryonal tube cells. The organisation of microtubules and F-actin changes successively from the embryonal mass towards the distal end of the embryo suspensor. The microtubule arrays appear normal in the embryonal mass cells, but the microtubule network is partially disorganised in the embryonal tube cells and the microtubules disrupted in the suspensor cells. In the same embryos, the microtubule-associated protein, MAP-65, is bound only to organised microtubules. In contrast, in a developmentally arrested cell line, which is incapable of normal embryonic pattern formation, MAP-65 does not bind the cortical microtubules and we suggest that this is a criterion for proembryogenic masses (PEMs) to passage into early embryogeny. In embryos, the organisation of F-actin gradually changes from a fine network in the embryonal mass cells to thick cables in the suspensor cells in which the microtubule network is completely degraded. F-actin de-polymerisation drugs abolish normal embryonic pattern formation and associated PCD in the suspensor, strongly suggesting that the actin network is vital in this PCD pathway.

Successful embryonic development in plants, as in animals, requires a strict coordination of cell proliferation, cell differentiation, and cell-death programs. The role of cell death is especially critical for the establishment of polarity at early stages of plant embryogenesis, when the differentiation of the temporary structure, the suspensor, is followed by its programmed elimination. Here, we review the emerging knowledge of this and other functions of programmed cell death during plant embryogenesis, as revealed by developmental analyses of Arabidopsis embryo-specific mutants and gymnosperm (spruce and pine) model embryonic systems. Cell biological studies in these model systems have helped to identify and order the cellular processes occurring during self-destruction of the embryonic cells. While metazoan embryos can recruit both apoptotic and autophagic cell deaths, the ultimate choice depending on the developmental task and conditions, plant embryos use autophagic cell disassembly as a single universal cell-death pathway. Dysregulation of this pathway leads to aberrant or arrested embryo development. We address the role of distinct cellular components in the execution of the autophagic cell death, and outline an overall mechanistic view of how cells are eliminated during plant embryonic pattern formation. Finally, we discuss the possible roles of some of the candidate plant cell-death proteins in the regulation of developmental cell death.

The bimolecular fluorescence complementation (BiFC) phenomenon has been successfully applied for in vivo protein-protein interaction studies and protein tagging analysis. Here we report a novel BiFC-based technique for investigation of integral membrane protein topology in living plant cells. This technique relies on the formation of a fluorescent complex between a non-fluorescent fragment of the yellow fluorescent protein (YFP) targeted into a specific cellular compartment and a counterpart fragment attached to the integral membrane protein N- or C-terminus or inserted into the internal loop(s). We employed this technique for topological studies of beet yellows virus-encoded p6 membrane-embedded movement protein, a protein with known topology, and the potato mop-top virus-encoded integral membrane TGBp2 protein with predicted topology. The results confirm that p6 is a type III integral transmembrane protein. Using a novel method, the central hydrophilic region of TGBp2 was localized into the ER lumen, whereas the N- and C-termini localized to the cytosol. We conclude that the BiFC-based reporter system for membrane protein topology analysis is a relatively fast and efficient method that can be used for high-throughput analysis of proteins integrated into the endoplasmic reticulum in living plant cells.

During plant embryogenesis, once the suspensor organ of the plant embryo has fulfilled its role, it is removed by programmed cell death (PCD). The pro-death cathepsin protease NtCP14 initiates this PCD, but is inhibited by the cystatin NtCYS until the suspensor function is fulfilled.

Tudor staphylococcal nuclease (TSN, also known as Tudor-SN, SND1 or p100) is an evolutionarily conserved protein with invariant domain composition, represented by tandem repeat of staphylococcal nuclease domains and a tudor domain. Conservation along significant evolutionary distance, from protozoa to plants and animals, suggests important physiological functions for TSN. It is known that TSN is critically involved in virtually all pathways of gene expression, ranging from transcription to RNA silencing. Owing to its high protein-protein binding affinity coexistent with enzymatic activity, TSN can exert its biochemical function by acting as both a scaffolding molecule of large multiprotein complexes and/or as a nuclease. TSN is indispensible for normal development and stress resistance, whereas its increased expression is closely associated with various types of cancer. Thus, TSN is an attractive target for anti-cancer therapy and a potent tumor marker. Considering ever increasing interest to further understand a multitude of TSN-mediated processes and a mechanistic role of TSN in these processes, here we took an attempt to summarize and update the available information about this intriguing multifunctional protein.

Agriculture faces enormous challenges including the need to substantially increase productivity, reduce environmental footprint, and deliver renewable alternatives that are being addressed by developing new oil crops for the future. The efforts include domestication of Lepidium spp. using genomics-aided breeding as a cold hardy perennial high-yielding oil crop that provides substantial environmental benefits, expands the geography for oil crops, and improves farmers’ economy. In addition, genetic engineering in Crambe abyssinica may lead to a dedicated industrial oil crop to replace fossil oil. Redirection of photosynthates from starch to oil in plant tubers and cereal endosperm also provides a path for enhancing oil production to meet the growing demands for food, fuel, and biomaterials. Insect pheromone components are produced in seed oil plants in a cost-effective and environmentally friendly pest management replacing synthetically produced pheromones. Autophagy is explored for increasing crop fitness and oil accumulation using genetic engineering in Arabidopsis.

Development of multiple embryos from a single zygote, the phenomenon called monozygotic polyembryony, is a widespread reproductive strategy found in higher plants and especially in gymnosperms. The enigma of plant monozygotic polyembryony is that only one embryo in a polyembryonic seed usually survives while the others are eliminated at an early stage. Here we report that programmed cell death (PCD) is the major mechanism responsible for elimination of subordinate embryos in a polyembryonic seed. Using post-fertilized pine (Pinus sylvestris) ovules, we show that once the dominant embryo is selected and, subsequently, the entire female gametophyte is affected by PCD, the cells of subordinate embryos initiate an autolytic self-destruction program. The progression of embryonic PCD follows a rigid basal-apical pattern, first killing the most basally situated cells, adjacent to the suspensor, and then proceeding towards the apical region until all cells in the embryonal mass are doomed. Our data demonstrate that during polyembryony, PCD serves to halt competition among monozygotic embryos in order to ensure survival of one embryo.

A combined application of abscisic acid (ABA) and high molecular mass osmoticum, polyethylene glycol (PEG) has become a routine method for stimulating somatic embryo maturation in some genera of Coniferales. The goals of the present study were to clarify how the PEG 4000‐attributed low osmotic potential (ψ s ) of the maturation medium affects the yield and morphology of mature somatic embryos as well as subsequent developmental processes during germination and ex vitro plantlet growth in different genotypes of Picea abies belonging to 3 full sib seed families. Despite high within‐ and among‐family variation, a stimulatory effect of 7.5% PEG (ψ s =−0.645 MPa) on somatic embryo maturation was recorded for 13 out of 17 cell lines ( F = 2.83, P = 0.1). PEG‐treated somatic embryos were more dehydrated than embryos matured in the absence of PEG. Subsequently, embryos were partially desiccated using a high relative humidity treatment (HRH‐treatment). The dynamics of embryo water content (WC) during HRH‐treatment differed between embryos developed on maturation medium for 5 or 7 weeks. These two patterns remained unchanged irrespective of the ψ s of the maturation medium. In 5‐week somatic embryos, the WC decreased to the lowest level (in the range 25‐35%) within the first 8 days of HRH‐treatment and was not further substantially changed. Seven‐week embryos also lost water within 8 to 16 days (decrease to 15‐25% WC), but this drop was followed by rehydration of embryonic tissues by 24th day of HRH‐treatment up to nearly the initial WC. Thus, 7‐week embryos experienced both desiccation and slow imbibition in the course of the 24‐day HRH‐treatment. This could account for their increased germinability compared to 5‐week somatic embryos found in the present study. Addition of 7.5% PEG to the maturation medium significantly inhibited somatic embryo germination for the vast majority of genotypes ( F = 7.35; P = 0.01). Moreover, even after ex vitro transfer, both radicle elongation and lateral root formation were substantially suppressed ( F = 3.8; P = 0.03) in those plantlets produced from PEG‐treated somatic embryos. Alterations both in the organization of the root meristem and in the structure of the root cap were found by histomorphological analysis of PEG‐treated somatic embryos. All those embryos possessed massive root caps with numerous intercellular spaces in the pericolumn tissue. Cells of the quiescent center exhibited clear symptoms of degradation manifested in shrinkage and collapse of the protoplasm. In addition, PEG‐treated embryos were of smaller size compared to embryos matured without osmoticum. When grown in artificial substrate (up to 5 months) the PEG‐induced inhibitory post‐effect gradually decreased. At this stage, the duration of maturation was the only factor separating plantlets into slow‐ and fast‐growing categories. Somatic embryos matured for 5 weeks produced plantlets twice the size of those produced by 7‐week embryos ( F = 37.8; P < 0.0001). This trend did not depend on ψ s of the maturation medium, nor on the genotype.

The biotechnology of somatic embryogenesis holds considerable promise for clonal propagation and breeding programs in forestry. To efficiently regulate the whole process of plant regeneration through somatic embryogenesis, it is of outmost importance to understand early developmental events when somatic embryos are just formed. In Norway spruce, somatic embryos transdifferentiate from proembryogenic masses (PEMs). This work describes the developmental dynamics (frequency distribution of PEMs and early somatic embryos) of the whole embryogenic suspension culture growing in the presence and absence of plant growth regulators (PGRs), auxin and cytokinin. The experiments have shown that PEM-to-somatic embryo transition is a key developmental switch that determines the yield and quality of mature somatic embryos and ultimately plant production. This switch was induced by the withdrawal of PGRs in cell suspension leading to a rapid accumulation of early somatic embryos (to a maximum of 75% of the entire population of suspension culture) and concomitant degradation of PEMs. The latter was evident from increased level of cell death measured through spectrophotometric Evans blue staining assay. Proembryogenic mass-to-embryo transition and concomitant activation of cell death were mediated by strong extracellular acidification. Therefore, buffering PGR-free culture medium at high (pH 5.8) or low (pH 4.5) levels of pH inhibited both PEM-to-embryo transition and cell death. The yield of mature somatic embryos on abscisic acid (ABA)-containing medium was increased up to 10-fold if the suspension culture had been pretreated for 1 to 9 days in unbuffered PGR-free medium. In this case a large proportion (75%) of the total number of mature embryos was formed within a short, 5-week, contact with ABA. The latter is practically important because prolonged contact with ABA suppresses the growth of somatic embryo plants. Based on these results, an improved method for regulating somatic embryogenesis was set up and tested for nine genotypes of Norway spruce. Over 800 plants regenerated from all tested genotypes demonstrated a good performance in the greenhouse and they were transferred to the field.

With the advent of agriculture, plants have been essential to the wellness of our society and the sustainability of our planet's ecosystem for the past 10 000 years. Their potential new use as renewable biofactories to transition our economy from fossil fuel-based resources ensures that advances in plant biology will be critical if we are to continue to sustain human development while minimizing impacts on global climate. Understanding fundamental processes that govern plant development, evolution and environmental responses is essential to usher in this new era of plant domestication for energy and novel products.

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.

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.

Abstract Plant peroxisomes maintain a plethora of key life processes including fatty acid β-oxidation, photorespiration, synthesis of hormones, and homeostasis of reactive oxygen species (ROS). Abundance of peroxisomes in cells is dynamic; however mechanisms controlling peroxisome proliferation remain poorly understood because measuring peroxisome abundance is technically challenging. Counting peroxisomes in individual cells of complex organs by electron or fluorescence microscopy is expensive and time consuming. Here we present a simple technique for quantifying peroxisome abundance using the small probe Nitro-BODIPY, which in vivo fluoresces selectively inside peroxisomes. The physiological relevance of our technique was demonstrated using salinity as a known inducer of peroxisome proliferation. While significant peroxisome proliferation was observed in wild-type Arabidopsis leaves following 5-hour exposure to NaCl, no proliferation was detected in the salt-susceptible mutants fry1-6, sos1-14, and sos1-15 . We also found that N-BODIPY detects aggregation of peroxisomes during final stages of programmed cell death and can be used as a marker of this stage. Furthermore, accumulation of peroxisomes in an autophagy-deficient Arabidopsis mutant atg5 correlated with N-BODIPY labeling. In conclusion, the technique reported here enables quantification of peroxisomes in plant material at various physiological settings. Its potential applications encompass identification of genes controlling peroxisome homeostasis and capturing stress-tolerant genotypes.

Somatic embryogenesis of Norway spruce (Picea abies L.) is a versatile model system to study molecular mechanisms regulating embryo development because it proceeds through defined developmental stages corresponding to specific culture treatments. Normal embryonic development involves early differentiation of proembryogenic masses (PEMs) into somatic embryos, followed by early and late embryogeny leading to the formation of mature cotyledonary embryos. In some cell lines there is a developmental arrest at the PEM−somatic embryo transition. To learn more about the molecular mechanisms regulating embryogenesis, we compared the transcript profiles of two normal lines and one developmentally arrested line. Ribonucleic acid, extracted from these cell lines at successive developmental stages, was analyzed on DNA microarrays containing 2178 expressed sequence tags (ESTs) (corresponding to 2110 unique cDNAs) from loblolly pine (Pinus taeda L.). Hybridization between spruce and pine species on microarrays has been shown to be effective (van Zyl et al. 2002, Stasolla et al. 2003). In contrast to the developmentally arrested line, the early phases of normal embryo development are characterized by a precise pattern of gene expression, i.e., repression followed by induction. Comparison of transcript levels between successive stages of embryogenesis allowed us to identify several genes that showed unique expression responses during normal development. Several of these genes encode proteins involved in detoxification processes, methionine synthesis and utilization, and carbohydrate metabolism. The potential role of these genes in embryo development is discussed.

Abstract Zinc is a potent regulator of programmed cell death (PCD) in animals. While certain, cell-type-specific concentrations of intracellular free zinc are required to protect cells from death, zinc depletion commits cells to death in diverse systems. As in animals, PCD has a fundamental role in plant biology, but its molecular regulation is poorly understood. In particular, the involvement of zinc in the control of plant PCD remains unknown. Here, we used somatic embryos of Norway spruce (Picea abies) to investigate the role of zinc in developmental PCD, which is crucial for correct embryonic patterning. Staining of the early embryos with zinc-specific molecular probes (Zinquin-ethyl-ester and Dansylaminoethyl-cyclen) has revealed high accumulation of zinc in the proliferating cells of the embryonal masses and abrupt decrease of zinc content in the dying terminally differentiated suspensor cells. Exposure of early embryos to a membrane-permeable zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine led to embryonic lethality, as it induced ectopic cell death affecting embryonal masses. This cell death involved the loss of plasma membrane integrity, metacaspase-like proteolytic activity, and nuclear DNA fragmentation. To verify the anti-cell death effect of zinc, we incubated early embryos with increased concentrations of zinc sulfate. Zinc supplementation inhibited developmental PCD and led to suppression of terminal differentiation and elimination of the embryo suspensors, causing inhibition of embryo maturation. Our data demonstrate that perturbation of zinc homeostasis disrupts the balance between cell proliferation and PCD required for plant embryogenesis. This establishes zinc as an important cue governing cell fate decisions in plants.

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.

Genotypic instability is commonly observed in plants derived from tissue culture and is at least partly due to in vitro-induced stress. In this work, the issues of whether genetic instability induced by in vitro stress varies among families and if genetic instability influences the adaptation to in vitro conditions and embryo development have been addressed. By comparing the stability of four variable nuclear microsatellite loci in embryogenic cultures and zygotic embryos of Pinus sylvestris, a significant difference in genetic stability among families was found. In six out of 10 families analysed, the level of genetic stability was similar between somatic and zygotic embryos. However, for the rest of the families, the mutation rate was significantly higher during somatic embryogenesis. Families showing a low genetic stability during establishment of embryogenic cultures had a higher embryogenic potential than those which were genetically more stable. In contrast, embryo development was suppressed in genetically unstable families. The relatively high mutation rates found for some families might reflect the plasticity of the families to adapt to stress, which is important for widely distributed species such as Pinus sylvestris.

Vacuolar programmed cell death (PCD) is indispensable for plant development and is accompanied by a dramatic growth of lytic vacuoles, which gradually digest cytoplasmic content leading to self-clearance of dying cells. Our recent data demonstrate that vacuolar PCD critically requires autophagy and its upstream regulator, a caspase-fold protease metacaspase. Furthermore, both components lie downstream of the point of no return in the cell-death pathway. Here we consider the possibilities that i) autophagy could have both cytotoxic and cytoprotective roles in the vacuolar PCD, and ii) metacaspase could augment autophagic flux through targeting an as yet unknown autophagy repressor.

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.

Autophagy is a major catabolic process in eukaryotes with a key role in homeostasis, programmed cell death, and aging. In plants, autophagy is also known to regulate agronomically important traits such as stress resistance, longevity, vegetative biomass, and seed yield. Despite its significance, there is still a shortage of reliable tools modulating plant autophagy. Here, we describe the first robust pipeline for identification of specific plant autophagy-modulating compounds. Our screening protocol comprises four phases: (1) high-throughput screening of chemical compounds in cell cultures of tobacco (Nicotiana tabacum); (2) confirmation of the identified hits in planta using Arabidopsis (Arabidopsis thaliana); (3) further characterization of the effect using conventional molecular biology methods; and (4) verification of chemical specificity on autophagy in planta. The methods detailed here streamline the identification of specific plant autophagy modulators and aid in unraveling the molecular mechanisms of plant autophagy.

Abstract Background Animals and plants diverged over one billion years ago and evolved unique mechanisms for many cellular processes, including cell death. One of the most well-studied cell death programmes in animals, apoptosis, involves gradual cell dismantling and engulfment of cellular fragments, apoptotic bodies, through phagocytosis. However, rigid cell walls prevent plant cell fragmentation and thus apoptosis is not applicable for executing cell death in plants. Furthermore, plants are devoid of the key components of apoptotic machinery, including phagocytosis as well as caspases and Bcl-2 family proteins. Nevertheless, the concept of plant “apoptosis-like programmed cell death” (AL-PCD) is widespread. This is largely due to superficial morphological resemblances between plant cell death and apoptosis, and in particular between protoplast shrinkage in plant cells killed by various stimuli and animal cell volume decrease preceding fragmentation into apoptotic bodies. Results Here, we provide a comprehensive spatio-temporal analysis of cytological and biochemical events occurring in plant cells subjected to heat shock at 40–55 °C and 85 °C, the experimental conditions typically used to trigger AL-PCD and necrotic cell death, respectively. We show that cell death under both conditions was not accompanied by membrane blebbing or formation of apoptotic bodies, as would be expected during apoptosis. Instead, we observed instant and irreversible permeabilization of the plasma membrane and ATP depletion. These processes did not depend on mitochondrial functionality or the presence of Ca 2+ and could not be prevented by an inhibitor of ferroptosis. We further reveal that the lack of protoplast shrinkage at 85 °C, the only striking morphological difference between cell deaths induced by 40–55 °C or 85 °C heat shock, is a consequence of the fixative effect of the high temperature on intracellular contents. Conclusions We conclude that heat shock-induced cell death is an energy-independent process best matching definition of necrosis. Although the initial steps of this necrotic cell death could be genetically regulated, classifying it as apoptosis or AL-PCD is a terminological misnomer. Our work supports the viewpoint that apoptosis is not conserved across animal and plant kingdoms and demonstrates the importance of focusing on plant-specific aspects of cell death pathways.

Metacaspases are part of an evolutionarily broad family of multifunctional cysteine proteases, involved in disease and normal development. As the structure-function relationship of metacaspases remains poorly understood, we solved the X-ray crystal structure of an Arabidopsis thaliana type II metacaspase (AtMCA-IIf) belonging to a particular subgroup not requiring calcium ions for activation. To study metacaspase activity in plants, we developed an in vitro chemical screen to identify small molecule metacaspase inhibitors and found several hits with a minimal thioxodihydropyrimidine-dione structure, of which some are specific AtMCA-IIf inhibitors. We provide mechanistic insight into the basis of inhibition by the TDP-containing compounds through molecular docking onto the AtMCA-IIf crystal structure. Finally, a TDP-containing compound (TDP6) effectively hampered lateral root emergence in vivo, probably through inhibition of metacaspases specifically expressed in the endodermal cells overlying developing lateral root primordia. In the future, the small compound inhibitors and crystal structure of AtMCA-IIf can be used to study metacaspases in other species, such as important human pathogens, including those causing neglected diseases.

Hybridization of labelled cDNA from various cell types with high-density arrays of expressed sequence tags is a powerful technique for investigating gene expression. Few conifer cDNA libraries have been sequenced. Because of the high level of sequence conservation between Pinus and Picea we have investigated the use of arrays from one genus for studies of gene expression in the other. The partial cDNAs from 384 identifiable genes expressed in differentiating xylem of Pinus taeda were printed on nylon membranes in randomized replicates. These were hybridized with labelled cDNA from needles or embryogenic cultures of Pinus taeda, P. sylvestris and Picea abies, and with labelled cDNA from leaves of Nicotiana tabacum. The Spearman correlation of gene expression for pairs of conifer species was high for needles (r(2) = 0.78 - 0.86), and somewhat lower for embryogenic cultures (r(2) = 0.68 - 0.83). The correlation of gene expression for tobacco leaves and needles of each of the three conifer species was lower but sufficiently high (r(2) = 0.52 - 0.63) to suggest that many partial gene sequences are conserved in angiosperms and gymnosperms. Heterologous probing was further used to identify tissue-specific gene expression over species boundaries. To evaluate the significance of differences in gene expression, conventional parametric tests were compared with permutation tests after four methods of normalization. Permutation tests after Z-normalization provide the highest degree of discrimination but may enhance the probability of type I errors. It is concluded that arrays of cDNA from loblolly pine are useful for studies of gene expression in other pines or spruces.

Somatic embryogenesis of a gymnosperm, Picea abies, represents a sequence of specifically regulated developmental stages including proembryogenic mass (PEM), PEM-to-embryo transition, and early and late embryogeny. Here, we report cDNA array analysis of expression patterns of 373 genes in the beginning of P. abies embryo development. The analysis revealed a group of 107 genes (29% of arrayed cDNAs) which were upregulated upon PEM-to-embryo transition, then downregulated during early embryogeny and finally upregulated again at the beginning of late embryogeny. This major gene expression pattern was abrogated in a developmentally arrested cell line that is unable to pass through the PEM-to-embryo transition. Thirty-five genes (9.4% of arrayed cDNAs) were found to be differentially expressed during normal embryonic pattern formation. Among them, 22 genes (5.9% of arrayed cDNAs) were directly associated with embryo pattern formation and can be considered as marker genes for early stages of P. abies embryogenesis. The majority of the marker genes encode for proteins involved in translation and posttranslational modification. Among them, 18 genes displayed the major expression pattern.

• Here, embryo-specific patterns of glutamine synthetase (GS) genes were studied for the first time using pine somatic and zygotic embryogenesis as model systems. • GS1a expression was absent in zygotic embryos whereas it was detected in the cotyledons of somatic embryos at late developmental stages along with transcripts for photosynthesis genes and arginase. These findings suggest that germination was initiated in maturing somatic embryos. • GS1b transcripts were found mainly in procambial cells in both zygotic and somatic embryos. Expression of the GS1b in procambial cells before the differentiation of mature vascular elements indicated that this gene could be useful as a molecular marker for early stages of vascular differentiation in pine. Accordingly, a correlation was found between the quality of somatic embryos generated from three different cell lines and the pattern and level of GS1b expression. • Our data suggest that GS1a and GS1b genes play distinct functional roles in the biosynthesis and mobilization of seed nitrogen reserves. Furthermore, the results presented may have potential application for improving conifer somatic embryogenesis.

Caspases are essential in animal programmed cell death both as initiator and executioner proteases. Plants do not have close caspase homologues, but several instances of caspase-like proteolytic activity have been demonstrated in connection with programmed cell death in plants. It was asked if caspase-like proteases are involved during development of the barley caryopsis. The presence of a caspase-6-like proteolytic activity that preferentially cleaved the sequence VEID was demonstrated. A range of protease inhibitors was tested and only caspase-specific inhibitors showed major inhibitory effects. The profile of VEIDase activity in developing starchy endosperm, embryo, and whole caryopsis was measured and showed a general trend of higher activity in young, rapidly developing tissues. The VEIDase activity was localized in vivo to vesicles, shown to be autophagosomes, in randomly distributed cells of the starchy endosperm. The VEIDase activity detected in barley caryopsis is similar to activities described previously in mammals, spruce, yeast, and thale cress. In mammals, spruce, and yeast, VEIDase activity has been shown to be positively correlated with the occurrence of programmed cell death. Several manifestations of programmed cell death exist in developing barley caryopsis, indicating a connection between VEIDase activity and developmental programmed cell death in barley.

Lipids and their cellular utilization are essential for life. Not only are lipids energy storage molecules, but their diverse structural and physical properties underlie various aspects of eukaryotic biology, such as membrane structure, signalling, and trafficking. In the ever-changing environment of cells, lipids, like other cellular components, are regularly recycled to uphold the housekeeping processes required for cell survival and organism longevity. The ways in which lipids are recycled, however, vary between different phyla. For example, animals and plants have evolved distinct lipid degradation pathways. The major cell recycling system, autophagy, has been shown to be instrumental for both differentiation of specialized fat storing-cells, adipocytes, and fat degradation in animals. Does plant autophagy play a similar role in storage and degradation of lipids? In this review, we discuss and compare implications of bulk autophagy and its selective route, lipophagy, in the turnover of lipid stores in animals, fungi, and plants.

Autophagy is a catabolic pathway capable of degrading cellular components ranging from individual molecules to organelles. Autophagy helps cells cope with stress by removing superfluous or hazardous material. In a previous work, we demonstrated that transcriptional upregulation of two autophagy-related genes, ATG5 and ATG7, in Arabidopsis thaliana positively affected agronomically important traits: biomass, seed yield, tolerance to pathogens and oxidative stress. Although the occurrence of these traits correlated with enhanced autophagic activity, it is possible that autophagy-independent roles of ATG5 and ATG7 also contributed to the phenotypes. In this study, we employed affinity purification and LC-MS/MS to identify the interactome of wild-type ATG5 and its autophagy-inactive substitution mutant, ATG5K128R Here we present the first interactome of plant ATG5, encompassing not only known autophagy regulators but also stress-response factors, components of the ubiquitin-proteasome system, proteins involved in endomembrane trafficking, and potential partners of the nuclear fraction of ATG5. Furthermore, we discovered post-translational modifications, such as phosphorylation and acetylation present on ATG5 complex components that are likely to play regulatory functions. These results strongly indicate that plant ATG5 complex proteins have roles beyond autophagy itself, opening avenues for further investigations on the complex roles of autophagy in plant growth and stress responses.

Microspore embryogenesis is a biotechnological process that allows us to rapidly obtain doubled-haploid plants for breeding programs. The process is initiated by the application of stress treatment, which reprograms microspores to embark on embryonic development. Typically, a part of the microspores undergoes cell death that reduces the efficiency of the process. Metacaspases (MCAs), a phylogenetically broad group of cysteine proteases, and autophagy, the major catabolic process in eukaryotes, are critical regulators of the balance between cell death and survival in various organisms. In this study, we analyzed the role of MCAs and autophagy in cell death during stress-induced microspore embryogenesis in Brassica napus. We demonstrate that this cell death is accompanied by the transcriptional upregulation of three BnMCA genes (BnMCA-Ia, BnMCA-IIa and BnMCA-IIi), an increase in MCA proteolytic activity and the activation of autophagy. Accordingly, inhibition of autophagy and MCA activity, either individually or in combination, suppressed cell death and increased the number of proembryos, indicating that both components play a pro-cell death role and account for decreased efficiency of early embryonic development. Therefore, MCAs and/or autophagy can be used as new biotechnological targets to improve in vitro embryogenesis in Brassica species and doubled-haploid plant production in crop breeding and propagation programs.

Proteases can regulate myriad biochemical pathways by digesting or processing target proteins. While up to 3% of eukaryotic genes encode proteases, only a tiny fraction of proteases are mechanistically understood. Furthermore, most of the current knowledge about proteases is derived from studies of a few model organisms, including Arabidopsis thaliana in the case of plants. Proteases in other plant model systems are largely unexplored territory, limiting our mechanistic comprehension of post-translational regulation in plants and hampering integrated understanding of how proteolysis evolved. We argue that the unicellular green alga Chlamydomonas reinhardtii has a number of technical and biological advantages for systematic studies of proteases, including reduced complexity of many protease families and ease of cell phenotyping. With this end in view, we share a genome-wide inventory of proteolytic enzymes in Chlamydomonas, compare the protease degradomes of Chlamydomonas and Arabidopsis, and consider the phylogenetic relatedness of Chlamydomonas proteases to major taxonomic groups. Finally, we summarize the current knowledge of the biochemical regulation and physiological roles of proteases in this algal model. We anticipate that our survey will promote and streamline future research on Chlamydomonas proteases, generating new insights into proteolytic mechanisms and the evolution of digestive and limited proteolysis.

The potential to use somatic embryos for large-scale propagation of elite genotypes, for integration into breeding programmes and for connecting breeding and mass propagation, is receiving much attention. However, before the methods are applied it is important that the plants regenerated via somatic embryogenesis grow as expected, i.e. as seedlings or cuttings. Growth of somatic embryo plants is under a cumulative influence of a number of treatments given during the in vitro phase and during the ex vitro establishment phase. The aim of this study was to identify treatments with a negative influence on the subsequent growth of somatic embryo plants of Norway spruce (Picea abies L. Karst.). Based on the results, the time of contact with abscisic acid during somatic embryo maturation and the length of continuous light treatment (CLT) during the first growth period strongly affect the height growth during two successive growth periods. In both cases longer treatments exerted negative effects. Based on these results a new method was set up, which includes: (1) prematuration treatment of the suspension culture in a growth regulator-free medium, by which the maturation step is synchronized and contracted; and (2) a two-phase germination treatment, first on a solidified medium and then in a liquid medium. This treatment avoids extended CLT during the first growth period. Another advantage of the two-phase germination treatment is a better root-system development. Somatic embryo plants produced according to this method can be transferred directly from in vitro conditions to the greenhouse.

Two Norway spruce (Picea abies (L.) Karst.) genes belonging to class I of the KNOTTED1-like homeobox (KNOX) genes, HBK2 and HBK3, were cloned with PCR-based methods. The expression of these and a previously characterised related gene, HBK1, in different organs and during somatic embryogenesis was studied with RT-PCR. Transcripts of all three genes were detected in stems, roots and in cone buds, but not in needles. HBK1 and HBK3 are expressed throughout development in a normal cell line with embryogenic potential and in a cell line unable to form somatic embryos. HBK2 is expressed in the normal cell line, but not in the developmentally arrested cell line. This suggests that the HBK2 gene is involved in the somatic embryo development.

Apoptosis is an evolutionarily young cell-death strategy evolved to disassemble animal cells through the action of the caspase family of proteases and phagocytic clearance. This strategy does not work in plants, which instead feature a phylogenetically older autophagic programmed cell death (PCD), as a bona fide type of cellular suicide. Recent work has begun to address the mechanistic roles for autophagic and proteolytic components, as well as their possible cooperation in plant PCD. A recent study has shown autophagosomal localization of a key cell-death proteolytic activity at the early stage of plant PCD. Here we focus on the relationship between autophagic and proteoloytic components in plant PCD at the cellular and organismal levels.

Individual mature stored seeds of Pinus koraiensis sometimes contain several viable zygotic embryos originated through the processes of simple and cleavage polyembryony. To induce the embryonic process, isolated zygotic embryos were cultured on five different media all supplemented with 10 μM 2,4-dichlorophenoxyacetic acid and 5 μM 6-benzyladenine. Two alternative pathways of somatic embryo origin were revealed. The first pathway was associated with the production of a friable, translucent callus in the hypocotyls–cotyledon region of the dominant zygotic embryo. The second pathway was related to the proliferation of a translucent, moist, and mucilaginous tissue (termed embryonal–suspensor mass) in the suspensor region of the dominant zygotic embryo. Both types of tissues contained early somatic embryos. Regression analysis has shown a strong negative correlation between the frequencies of formation of embryogenic callus and embryonal–suspensor mass both at 3 and 8 weeks of culture (r = − 0.85; p = 0.07 and r = −0.71; p = 0.17, respectively). Key words: Pinus koraiensis; polyembryonal seeds; somatic embryogenesis; embryogénie callus; embryonal–suspensor mass.

Somatic embryos of Norway spruce (Picea abies (L.) Karst.) differentiate from proembryogenic masses (PEMs), which are subject to autodestruction through programmed cell death. In PEMs, somatic embryo formation and activation of programmed cell death are interrelated processes. We sought to determine if activation of programmed cell death in PEMs is caused by genetic aberrations during somatic embryogenesis. Based on the finding that withdrawal of auxin and cytokinin induces programmed cell death in PEMs, 1-week-old cell suspensions were cultured in medium either with or without auxin and cytokinin and then transferred to maturation medium containing abscisic acid. We analyzed the stability of three nuclear simple sequence repeat (SSR) microsatellite markers at successive stages of somatic embryogenesis in two cell lines. There were no mutations at the SSR loci at any of the successive developmental stages from PEMs to cotyledonary embryos, irrespective of whether or not the proliferation medium in which cell suspensions had been cultured contained auxin or cytokinin. The morphologies of plants regenerated from the cultures were similar, although withdrawal of auxin and cytokinin significantly stimulated the yield of both embryos and plants. We conclude, therefore, that the high genetic stability of somatic embryos in Norway spruce is unaffected by the induction of programmed cell death caused by withdrawal of auxin and cytokinin.

The substrates of the metacaspase type of proteases are beginning to be identified.

Tight regulation of cell cycle is of critical importance for eukaryotic biology and is achieved through a combined action of a large number of highly specialized proteins. Separases are evolutionarily conserved caspase‐like proteases playing a crucial role in cell cycle regulation, as they execute sister chromatid separation at metaphase to anaphase transition. In contrast to extensively studied yeast and metazoan separases, very little is known about the role of separases in plant biology. Here we describe the molecular mechanisms of separase‐mediated chromatid segregation in yeast and metazoan models, discuss new emerging but less‐understood functions of separases and highlight major gaps in our knowledge about plant separases.

The terminal differentiation and elimination of the embryo-suspensor is the earliest manifestation of programmed cell death (PCD) during plant ontogenesis. Molecular regulation of suspensor PCD remains poorly understood. Norway spruce (Picea abies) embryos provide a powerful model for studying embryo development because of their large size, sequenced genome, and the possibility to obtain a large number of embryos at a specific developmental stage through somatic embryogenesis. Here, we have carried out global gene expression analysis of the Norway spruce embryo-suspensor versus embryonal mass (a gymnosperm analogue of embryo proper) using RNA sequencing. We have identified that suspensors have enhanced expression of the NAC domain-containing transcription factors, XND1 and ANAC075, previously shown to be involved in the initiation of developmental PCD in Arabidiopsis. The analysis has also revealed enhanced expression of Norway spruce homologues of the known executioners of both developmental and stress-induced cell deaths, such as metacaspase 9 (MC9), cysteine endopeptidase-1 (CEP1) and ribonuclease 3 (RNS3). Interestingly, a spruce homologue of bax inhibitor-1 (PaBI-1, for Picea abies BI-1), an evolutionarily conserved cell death suppressor, was likewise up-regulated in the embryo-suspensor. Since Arabidopsis BI-1 so far has been implicated only in the endoplasmic reticulum (ER)-stress induced cell death, we investigated its role in embryogenesis and suspensor PCD using RNA interference (RNAi). We have found that PaBI-1-deficient lines formed a large number of abnormal embryos with suppressed suspensor elongation and disturbed polarity. Cytochemical staining of suspensor cells has revealed that PaBI-1 deficiency suppresses vacuolar cell death and induces necrotic type of cell death previously shown to compromise embryo development. This study demonstrates that a large number of cell-death components are conserved between angiosperms and gymnosperms and establishes a new role for BI-1 in the progression of vacuolar cell death.

Height growth during the first and second growth periods (i.e., the June–September period in consecutive years) and intraclonal variation were assessed in 13 Norway spruce (Picea abies (L.) Karst.) clones propagated by somatic embryogenesis. The plants were acclimatized and grown in a greenhouse until mid-July and then transferred outdoors. The clonal mean heights after the first and second growth periods were lower for somatic embryo plants than for seedlings from corresponding families sown at the time of somatic embryo plant ex vitro transfer, because a large proportion of somatic embryo plants were small. We determined whether certain selection criteria at ex vitro transfer can be used to identify somatic embryo plants with height growth characteristics comparable with those of seedlings. Epicotyl length and presence of lateral roots proved to be important parameters for selection, whereas main root length was less useful. A combined selection for somatic embryo plants with lateral roots and with an epicotyl length exceeding 8 mm resulted in taller plants and reduced intraclonal variation after the first and second growth periods. The growth of somatic embryo plants selected in this way was similar to that of seedlings from the corresponding families. We conclude that selection according to these criteria at ex vitro transfer can result in improved performance of clonal stock propagated by somatic embryogenesis.

Abstract Clonal propagation by somatic embryogenesis is a promising approach to preserve and multiply élite genotypes in tree breeding and reforestation programmes of conifers. The principal requirement for the large-scale application of this approach is near uniformity of the plants regenerated from somatic embryos. Picea abies families varied significantly in genetic stability, as shown by allele frequencies at four variable nuclear microsatellite loci in 314 plants regenerated from somatic embryos from six families. Furthermore, in some clones only one or two plants carried the mutated allele, while in other clones all the plants carried the same mutation. Relative growth increment was not affected in somatic embryo plants carrying mutated microsatellite alleles. For comparison, seedlings from half-sib families were analysed for microsatellite variation. Mutated alleles were detected in three of the 208 seedlings analysed. The allelic distribution of microsatellites in clones derived from somatic embryos was not significantly different from that of the parents. This suggests that the procedure for somatic embryogenesis does not give any large changes in allele frequency.

Necrosis plays a fundamental role in plant physiology and pathology. When plants or plant cell cultures are subjected to abiotic stress they initiate rapid cell death with necrotic morphology. Likewise, when plants are attacked by pathogens, they develop necrotic lesions, the reaction known as hypersensitive response. Great advances in the understanding of signaling pathways that lead to necrosis during plant–pathogen interaction have been made in the last two decades using Arabidopsis thaliana as a model plant. Further understanding of these signaling pathways, as well as those regulating the execution phase of necrotic cell death per se would require a robust set of readout assays to detect and measure necrosis in various plant model systems. Here we provide description of such assays, beginning from electron microscopy, as the “gold standard” to diagnose necrosis. This is followed by two groups of biochemical and cytochemical assays used by our group to detect and quantify mitochondrial dysfunction and the loss of protoplast integrity during necrosis in Arabidopsis plants and cell suspension cultures of both Arabidopsis and Norway spruce.

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 Arabidopsis homolog of Scc4, which we denote Arabidopsis thaliana (At)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 the suspensor overproliferation 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 the Scc2-Scc4 complex as an evolutionary conserved machinery controlling cohesin loading and chromatin structure maintenance, and provide new insight into the plant-specific role of this complex in controlling cell fate during embryogenesis.

Summary 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.

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.

Abstract 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.

Abstract Caspases are restricted to animals, while other organisms, including plants possess metacaspases (MCAs), a more ancient and broader class of structurally-related yet biochemically distinct proteases. Our current understanding of plant MCAs is derived from studies in streptophytes, and mostly in Arabidopsis expressing nine MCAs with partly redundant activities. In contrast to streptophytes, most chlorophytes contain only one or two hitherto uncharacterized MCAs, providing an excellent platform for MCA research. Here we investigate CrMCA-II, a single type II MCA from a model chlorophyte Chlamydomonas reinhardtii . Surprisingly, unlike other studied MCAs and similar to caspases, CrMCA-II dimerizes both in vitro and in vivo . Furthermore, activation of CrMCA-II in vivo correlates with the dimerization. Most of CrMCA-II in the cell is present as a zymogen attached to the plasma membrane (PM). Deletion of CrMCA-II by CRISPR/Cas9 compromises thermotolerance leading to increased cell death under heat stress. Adding back either wild-type or catalytically dead CrMCA-II restores thermoprotection, suggesting that its proteolytic activity is dispensable for this effect. Finally, we link the non-proteolytic role of CrMCA-II in thermotolerance to the ability to modulate PM fluidity. Our study reveals an ancient, MCA-dependent thermotolerance mechanism retained by Chlamydomonas and probably lost during the evolution of multicellularity.

Abstract Intracellular recycling via autophagy is governed by post-translational modifications of the autophagy-related (ATG) proteins. One notable example is ATG4-dependent delipidation of ATG8, a process that plays critical but distinct roles in autophagosome formation in yeast and mammals. Here, we aimed to elucidate the specific contribution of this process to autophagosome formation in species representative of evolutionary distant green plant lineages: unicellular green alga Chlamydomonas reinhardtii , with a relatively simple set of ATG genes, and a vascular plant Arabidopsis thaliana , harboring expanded ATG gene families. Remarkably, the more complex autophagy machinery of Arabidopsis rendered ATG8 delipidation entirely dispensable for the maturation of autophagosomes, autophagic flux and related stress tolerance; whereas autophagy in Chlamydomonas strictly depended on the ATG4-mediated delipidation of ATG8. Importantly, we uncovered the distinct impact of different Arabidopsis ATG8 orthologs on autophagosome formation, especially prevalent under nitrogen depletion, providing a new insight into potential drivers behind the expansion of the ATG8 family in higher plants. Our findings underscore the evolutionary diversification of the molecular mechanism governing the maturation of autophagosomes in eukaryotic lineages and highlight how this conserved pathway is tailored to diverse organisms.

Abstract Caspases are restricted to animals, while other organisms, including plants, possess metacaspases (MCAs), a more ancient and broader class of structurally related yet biochemically distinct proteases. Our current understanding of plant MCAs is derived from studies in streptophytes, and mostly in Arabidopsis (Arabidopsis thaliana) with 9 MCAs with partially redundant activities. In contrast to streptophytes, most chlorophytes contain only 1 or 2 uncharacterized MCAs, providing an excellent platform for MCA research. Here we investigated CrMCA-II, the single type-II MCA from the model chlorophyte Chlamydomonas (Chlamydomonas reinhardtii). Surprisingly, unlike other studied MCAs and similar to caspases, CrMCA-II dimerizes both in vitro and in vivo. Furthermore, activation of CrMCA-II in vivo correlated with its dimerization. Most of CrMCA-II in the cell was present as a proenzyme (zymogen) attached to the plasma membrane (PM). Deletion of CrMCA-II by genome editing compromised thermotolerance, leading to increased cell death under heat stress. Adding back either wild-type or catalytically dead CrMCA-II restored thermoprotection, suggesting that its proteolytic activity is dispensable for this effect. Finally, we connected the non-proteolytic role of CrMCA-II in thermotolerance to the ability to modulate PM fluidity. Our study reveals an ancient, MCA-dependent thermotolerance mechanism retained by Chlamydomonas and probably lost during the evolution of multicellularity.

Metacaspases are essential for cell death regulation in plants. Further understanding of biochemistry of metacaspases and their molecular function in plant biology requires a set of robust methods for detection of metacaspase activation and quantitative analysis of corresponding proteolytic activity. Here we describe methods for purification of recombinant metacaspases, measurement of enzymatic activity of recombinant and endogenous metacaspases in vitro and in cell lysates, respectively, and finally detection of metacaspase activation in vivo. Additionally, an in vitro metacaspase protein substrate cleavage assay based on the cell-free production of substrate protein followed by proteolysis with recombinant metacaspase is presented. These methods have been originally developed for type II metacaspases from Arabidopsis and Norway spruce (Picea abies), but they can be used as templates for type I metacaspases, as well as for type II metacaspases from other species.

Arabidopsis thaliana possesses two acyl-CoA:lysophosphatidylethanolamine acyltransferases, LPEAT1 and LPEAT2, which are encoded by At1g80950 and At2g45670 genes, respectively. Both single lpeat2 mutant and double lpeat1 lpeat2 mutant plants exhibit a variety of conspicuous phenotypes, including dwarfed growth. Confocal microscopic analysis of tobacco suspension-cultured cells transiently transformed with green fluorescent protein-tagged versions of LPEAT1 or LPEAT2 revealed that LPEAT1 is localized to the endoplasmic reticulum (ER), whereas LPEAT2 is localized to both Golgi and late endosomes. Considering that the primary product of the reaction catalyzed by LPEATs is phosphatidylethanolamine, which is known to be covalently conjugated with autophagy-related protein ATG8 during a key step of the formation of autophagosomes, we investigated the requirements for LPEATs to engage in autophagic activity in Arabidopsis. Knocking out of either or both LPEAT genes led to enhanced accumulation of the autophagic adaptor protein NBR1 and decreased levels of both ATG8a mRNA and total ATG8 protein. Moreover, we detected significantly fewer membrane objects in the vacuoles of lpeat1 lpeat2 double mutant mesophyll cells than in vacuoles of control plants. However, contrary to what has been reported on autophagy deficient plants, the lpeat mutants displayed a prolonged life span compared to wild type, including delayed senescence.

Abstract Phenotyping of model organisms grown on Petri plates is often carried out manually, despite the procedures being time-consuming and laborious. The main reason for this is the limited availability of automated phenotyping facilities, whereas constructing a custom automated solution can be a daunting task for biologists. Here, we describe SPIRO, the Smart Plate Imaging Robot, an automated platform that acquires time-lapse photos of up to four vertically oriented Petri plates in a single experiment, corresponding to 192 seedlings for a typical root growth assay and up to 2500 seeds for a germination assay. SPIRO is catered specifically to biologists’ needs, requiring no engineering or programming expertise for assembly and operation. Its small footprint is optimized for standard incubators, the inbuilt green LED enables imaging under dark conditions, and remote control provides access to the data without interfering with sample growth. SPIRO’s excellent image quality is suitable for automated image processing, which we demonstrate on the example of seed germination and root growth assays. Furthermore, the robot can be easily customized for specific uses, as all information about SPIRO is released under open-source licenses. Importantly, uninterrupted imaging allows considerably more precise assessment of seed germination parameters and root growth rates compared to manual assays. Moreover, SPIRO enables previously technically challenging assays such as phenotyping in the dark. We illustrate the benefits of SPIRO in proof-of-concept experiments which yielded a novel insight on the interplay between autophagy, nitrogen sensing and photoblastic response.

Proliferating embryonal-suspensor masses (ESMs) were initiated from mature open-pollinated seeds of a northern Norway spruce (Picea abies (L.) Karst) family possessed a relatively low level of embryogenic activity. Different nitrogen content modifications of the half-strength von Arnold and Eriksson(1/2 LP) basal medium were tested for both the induction of ESM formation and the establishment of ESM lines. Both organic and inorganic nitrogen strongly affected the ESM line establishment. Amide nitrogen (asparagine or glutamine) was an obligatory prerequisite for the ESM line establishment and prolonged maintenance. The optimal ammonium/nitrate molar ratio in the presence of 3.0 mM glutamine was about 0.2. The highest frequency ESM line establishment (33 %) was obtained with 7.5 mM ammonium, 33.8 mM nitrate and 3.0 mM glutamine. Somatic embryo maturation was determined by the combined effects of the nitrogen balances of maintenance and maturation media. After a two-step maturation procedure, mature somatic embryos were successfully germinated and transferred ex vitrum.

Type II metacaspases (MCAs) are proteases, belonging to the C14B MEROPS family. Like the MCAs of type I and type III, they preferentially cleave their substrates after the positively charged amino acid residues (Arg or Lys) at the P1 position. Type II MCAs from various higher plants have already been successfully overexpressed in E. coli mostly as His-tagged proteins and were shown to be proteolytically active after the purification. Here we present a protocol for expression and purification of the only type II MCA from the model green alga Chlamydomonas reinhardtii. The two-step purification, which consists of immobilized metal affinity chromatography using cobalt as ion followed by size-exclusion chromatography, can be performed in 1 day and yields 4 mg CrMCA-II protein per liter of overexpression culture.

Programmed cell death (PCD) is an integral part of embryogenesis. In plant embryos, PCD functions during terminal differentiation and elimination of the temporary organ, suspensor, as well as during establishment of provascular system. Embryo abortion is another example of embryonic PCD activated at pathological situations and in polyembryonic seeds. Recent studies identified the sequence of cytological events leading to cellular self-destruction in plant embryos. As in most if not all the developmental cell deaths in plants, embryonic PCD is hallmarked by autophagic degradation of the cytoplasm and nuclear disassembly that includes breakdown of the nuclear envelope and DNA fragmentation. The optimized setup of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) allows the routine in situ analysis of nuclear DNA fragmentation in plant embryos. This chapter provides step-by-step procedure of how to process embryos for TUNEL and how to combine TUNEL with immunolocalization of the protein of interest.

Crassinucellate ovules are initiated in <em>Taxus</em>, directly from the shoot apex. The rudimentary pollen chamber is formed in the nucellus. A linear tetrad of megaspores with a functional chalazal megaspore is formed. A free-nuclear stage is charac-teristic at the beginning of megagametophyte development. Archegonia without ventral canal cell are solitary or in complexes. The embryo has a very long suspensor even after maturation. Two types of polyembryony have been revealed: i) embryogenic redifferentiation of suspensor cells and ii) cleavage of embryonic region in the early embryo. In the northern temperate climate of St. Petersburg one month delay in development of reproductive structures has been noted.

Cytokinesis is a powerful paradigm for addressing fundamental questions of plant biology including molecular mechanisms of development, cell division, cell signaling, membrane trafficking, cell wall synthesis, and cytoskeletal dynamics. Genetics was instrumental in identification of proteins regulating cytokinesis. Characterization of mutant lines generated using forward or reverse genetics includes microscopic analysis for defects in cell division. Typically, failure of cytokinesis results in appearance of multinucleate cells, formation of cell wall stubs, and isotropic cell expansion in the root elongation zone. Small fluorescent probes served as a very effective tool for the detection of cytokinetic defects. Such probes stain living or formaldehyde-fixed specimens avoiding complex preparatory steps. Although resolution of the fluorescence probes is inferior to electron microscopy, the procedure is fast, easy, and does not require expensive materials or equipment. This chapter describes techniques for staining DNA with the probes DAPI and SYTO82, for staining membranes with FM4-64, and for staining cell wall with propidium iodide.

Scientific Reports 7: Article number: 39069; published online: 01 February 2017; updated: 03 May 2017. This Article contains errors. In Table 1, the polymorphism for sos1-15 “SALK_114744” should read “SALK_149947”. Additionally, the legend for Figure 7 is incorrect: “(A) Model of the SOS1 gene and positions of T-DNA insertions.

Abstract Metacaspases are part of an evolutionarily broad family of multifunctional cysteine proteases, involved in disease and normal development. Despite the extensive study of metacaspases in the two decades since their discovery, the structure-function relationship of metacaspases remains poorly understood. Furthermore, previous studies on their function have been thwarted by the redundancy in gene copy number and potential phenotypic suppression of genetic mutations, especially in plants. Here, we have solved the X-ray crystal structure of an Arabidopsis thaliana type II metacaspase (AtMCA-IIf) that belongs to a particular sub-group that does not require calcium ions for activation. Compared to crystal structures of other metacaspases and caspases, the AtMCA-IIf active site is structurally similar and poses a conundrum for the catalytic mechanism of the cysteine-histidine dyad. To study metacaspase activity in plants, we developed an in vitro chemical screen to identify small molecule metacaspase inhibitors. Several hits with a minimal thioxodihydropyrimidine-dione (TDP) structure were identified, some being specific inhibitors of AtMCA-IIf. We provide a mechanistic basis for inhibition by the TDP-containing compounds through molecular docking onto the AtMCA-IIf crystal structure. Finally, a TDP-containing compound (TDP6) was effective at inhibiting lateral root emergence in vivo , likely through the inhibition of metacaspases that are specifically expressed in the endodermal cells overlaying developing lateral root primordia. In the future, the small compound inhibitors and crystal structure of AtMCA-IIf can be used to study metacaspases in various other species, such as important human pathogens including those causing neglected diseases.

Abstract To survive extreme desiccation, seeds enter dormancy that can last millennia. This dormancy involves the accumulation of protective but structurally disordered storage proteins through unknown adjustments of proteolytic surveillance mechanisms. Mutation of all six types II metacaspases (MCAs)-II in the model plant Arabidopsis revealed their essential role in modulating these proteolytic mechanisms. MCA-II mutant seeds fail to properly target at the endoplasmic reticulum (ER) the AAA ATPase Cell Division Cycle 48 (CDC48) to dispose of misfolded proteins. MCA-IIs cleave a CDC48 adaptor, the ubiquitination regulatory X domain-containing (PUX) responsible for localizing CDC48 to the lipid droplets. When cleaved, CDC48-PUX is inactivated and allows a lipid droplet-to-ER shuttling of CDC48, an important step in the regulation of seeds’ lifespan. In sum, we uncover antagonism between proteolytic pathways bestowing longevity. One-Sentence Summary Metacaspase proteases confer seed longevity by antagonizing CDC48 activity.

Abstract To survive extreme desiccation, seeds enter a period of dormancy that can last millennia. Seed dormancy involves the accumulation of protective storage proteins through unknown adjustments in proteasomal degradation. Mutating all six type II metacaspase (MCA-II) proteases in Arabidopsis thaliana revealed their essential roles in modulating proteasomal degradation. MCA-II mutant seeds fail to target the AAA ATPase CELL DIVISION CYCLE 48 (CDC48) at the endoplasmic reticulum to discard misfolded proteins, compromising their storability. Moreover, we show that MCA-IIs cleave a PUX (ubiquitination regulatory X domain-containing) adaptor, which is responsible for the localization of CDC48 to lipid droplets. This cleavage enables the shuttling of CDC48 between lipid droplets and the endoplasmic reticulum, constituting an important step in the regulation of spatiotemporal proteolysis. In summary, we uncovered a proteolytic pathway conferring seed longevity.

Metacaspases are ancestral homologs of caspases that can either promote cell death or confer cytoprotection. Furthermore, yeast (Saccharomyces cerevisiae) metacaspase Mca1 possesses dual biochemical activity: proteolytic activity causing cell death and cytoprotective, co-chaperone-like activity retarding replicative aging. The molecular mechanism favoring one activity of Mca1 over another remains elusive. Here, we show that this mechanism involves calmodulin binding to the N-terminal pro-domain of Mca1, which prevents its proteolytic activation and promotes co-chaperone-like activity, thus switching from pro-cell death to anti-aging function. The longevity-promoting effect of Mca1 requires the Hsp40 co-chaperone Sis1, which is necessary for Mca1 recruitment to protein aggregates and their clearance. In contrast, proteolytically active Mca1 cleaves Sis1 both in vitro and in vivo, further clarifying molecular mechanism behind a dual role of Mca1 as a cell-death protease versus gerontogene.

Phenotyping of model organisms grown on Petri plates is often carried out manually, despite the procedures being time-consuming and laborious. The main reason for this is the limited availability of automated phenotyping facilities, whereas constructing a custom automated solution can be a daunting task for biologists. Here, we describe SPIRO, the Smart Plate Imaging Robot, an automated platform that acquires time-lapse photographs of up to four vertically oriented Petri plates in a single experiment, corresponding to 192 seedlings for a typical root growth assay and up to 2500 seeds for a germination assay. SPIRO is catered specifically to biologists' needs, requiring no engineering or programming expertise for assembly and operation. Its small footprint is optimized for standard incubators, the inbuilt green LED enables imaging under dark conditions, and remote control provides access to the data without interfering with sample growth. SPIRO's excellent image quality is suitable for automated image processing, which we demonstrate on the example of seed germination and root growth assays. Furthermore, the robot can be easily customized for specific uses, as all information about SPIRO is released under open-source licenses. Importantly, uninterrupted imaging allows considerably more precise assessment of seed germination parameters and root growth rates compared with manual assays. Moreover, SPIRO enables previously technically challenging assays such as phenotyping in the dark. We illustrate the benefits of SPIRO in proof-of-concept experiments which yielded a novel insight on the interplay between autophagy, nitrogen sensing, and photoblastic response.

As plants grow they not only form new tissues and structures using highly coordinated cell-division and cell-differentiation programs but also continuously kill many of their own cells through activation of programmed cell death (PCD). The earliest functions of PCD in plant life are fulfilled during embryogenesis. Here PCD governs two major developmental processes. First is the elimination of a transient embryonic structure – suspensor, which functions at early stages of embryo development as a conduit of nutrients and growth factors, but is not required at later stages [1]. Another function of embryonic PCD applies to embryo abortion, which is not only a response to stress or mutagens, but also a normal feature of those plant species, which produce polyembryonic seeds. In the latter case competition among multiple embryos for survival often induces PCD resulting in the elimination of all but one embryo in a seed [2]. At the demise, both suspensor and entire embryos display a gradient of successive stages of PCD along apical-to-basal axis [2,3]. This PCD implicates active role of autophagy in complete removal of cell protoplast. Autophagosomes are formed via Golgi and proplastids [1]. Autophagocytosis depends on the dynamic reorganization of the cytoskeleton. Microtubule network is disrupted early in PCD pathway. F-actin is gradually reorganised into thick longitudinal cables and is present till the vacuole collapse and fragmentation of nuclear DNA [1,3]. Type II metacapase is critically involved in the regulation of the cell death pathway, which is essential for normal embryo development. Metacaspase gene silencing results in the suppression of cell death and failure of embryonic pattern formation [4].

SUMMARY Adaptation 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.

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.