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Membrane contact sites (MCS) are regions where two membranes of different organelles are close but not fused; they coordinate non-vesicular communication between organelles and are involved in a wide variety of physiological functions, including membrane lipid homeostasis. Amongst proteins localized at MCS are those containing a lipid transport domain known as synaptotagmin-like mitochondrial-lipid binding protein (SMP), being the mammalian Extended Synaptotagmins, the yeast Tricalbins and the plant Synaptotagmin 1 (SYT1) the best SMP proteins characterized so far. They are all localized at endoplasmic reticulum-plasma membrane contact sites (ER-PM CS). We have carried outin-silicogenome-wide identification of genes encoding SMP proteins in Arabidopsis and tomato. We have identified the plant exclusive NTMC2T5 proteins as ER-chloroplast CS components which make them extremely interesting as the route for lipid trafficking into and out of chloroplasts remains unknown. Additionally,NTMC2T5over-expressions caused a significant clustering of chloroplast around nucleus. Moreover, SYT6, NTMC2T6 and TEX2 have been identified as ER-Trans-Golgi Network CS proteins. These proteins associated between them and with the exocytosis related proteins VAMP721 and VAMP727. Since the functional roles of many of these genes are unknown, this gene collection provides a useful resource for future studies.HIGHLIGHTPlant exclusive lipid transport proteins were identified at membrane contact sites. SYT6, TEX2 and NTMC2T6 proteins are localized at ER-TGN. NTMC2T5 proteins are localized at ER-Chloroplast and induced chloroplast-nucleus clustering.

SummaryThe Arabidopsis Plant HomeoDomain (PHD) proteins AtMS1 and AtMMD1 provide chromatin-mediated transcriptional regulation essential for tapetum-dependent pollen formation. Such pollen-based male gametogenesis is a derived trait of seed plants. Male gametogenesis in the common ancestors of land plants is instead likely to have been reminiscent of that in extant bryophytes where flagellated sperms are produced by an elaborate gametophyte generation. Still, also bryophytes possess MS1/MMD1-related PHD proteins.We addressed the function of two MS1/MMD1-homologs in the bryophyte model moss Physcomitrium patens by the generation and analysis of reporter and loss-of-function lines.The two genes are together essential for both male and female fertility by providing cell autonomous functions in the gamete-producing inner cells of antheridia and archegonia. They are furthermore expressed in the diploid sporophyte generation suggesting a function during sporogenesis, a process proposed related by descent to pollen formation in angiosperms.We propose that the moss MS1/MMD1-related regulatory network required for completion of male and female gametogenesis and possibly for sporogenesis, represent a heritage from ancestral land plants.

Although land plant germ cells have received much attention, knowledge about their specification is still limited. We thus identified transcripts enriched in egg cells of the bryophyte model species Physcomitrium patens, compared the results with angiosperm egg cells, and selected important candidate genes for functional analysis. We used laser-assisted microdissection to perform a cell-type-specific transcriptome analysis on egg cells for comparison with available expression profiles of vegetative tissues and male reproductive organs. We made reporter lines and knockout mutants of the two BONOBO (PbBNB) genes and studied their role in reproduction. We observed an overlap in gene activity between bryophyte and angiosperm egg cells, but also clear differences. Strikingly, several processes that are male-germline specific in Arabidopsis are active in the P. patens egg cell. Among those were the moss PbBNB genes, which control proliferation and identity of both female and male germlines. Pathways shared between male and female germlines were most likely present in the common ancestors of land plants, besides sex-specifying factors. A set of genes may also be involved in the switches between the diploid and haploid moss generations. Nonangiosperm gene networks also contribute to the specification of the P. patens egg cell.

The Arabidopsis Plant HomeoDomain (PHD) proteins AtMS1 and AtMMD1 provide chromatin-mediated transcriptional regulation essential for tapetum-dependent pollen formation. This pollen-based male gametogenesis is a derived trait of seed plants. Male gametogenesis in the common ancestors of land plants is instead likely to have been reminiscent of that in extant bryophytes where flagellated sperms are produced by an elaborate gametophyte generation. Still, also bryophytes possess MS1/MMD1-related PHD proteins. We addressed the function of two MS1/MMD1-homologues in the bryophyte model moss Physcomitrium patens by the generation and analysis of reporter and loss-of-function lines. The two genes are together essential for both male and female fertility by providing functions in the gamete-producing inner cells of antheridia and archegonia. They are furthermore expressed in the diploid sporophyte generation suggesting a function during sporogenesis, a process proposed related by descent to pollen formation in angiosperms. We propose that the moss MS1/MMD1-related regulatory network required for completion of male and female gametogenesis, and possibly for sporogenesis, represent a heritage from ancestral land plants.

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

Autophagy, a major catabolic process in eukaryotes, was initially related to cell tolerance to nutrient depletion. In plants autophagy has also been widely related to tolerance to biotic and abiotic stresses (through the induction or repression of programmed cell death, PCD) as well as to promotion of developmentally regulated PCD, starch degradation or caloric restriction important for life span. Much less is known regarding its role in plant cell differentiation. Here we show that macroautophagy, the autophagy pathway driven by engulfment of cytoplasmic components by autophagosomes and its subsequent degradation in vacuoles, is highly active during germ cell differentiation in the early diverging land plant Physcomitrella patens. Our data provide evidence that suppression of ATG5-mediated autophagy results in reduced density of the egg cell-mediated mucilage that surrounds the mature egg, pointing toward a potential role of autophagy in extracellular mucilage formation. In addition, we found that ATG5- and ATG7-mediated autophagy is essential for the differentiation and cytoplasmic reduction of the flagellated motile sperm and hence for sperm fertility. The similarities between the need of macroautophagy for sperm differentiation in moss and mouse are striking, strongly pointing toward an ancestral function of autophagy not only as a protector against nutrient stress, but also in gamete differentiation.

Metacaspase-dependent autophagy in plants promotes cell disassembly during vacuolar cell death and inhibits necrosis., 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.

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

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

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.

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.

Autophagy is a catabolic and recycling pathway that maintains cellular homeostasis under normal growth and stress conditions. Two major types of autophagy, microautophagy and macroautophagy, have been described in plants. During macroautophagy, cellular content is engulfed by a double-membrane vesicle called autophagosome. This vesicle fuses its outer membrane with the tonoplast and releases the content into the vacuole for degradation. During certain developmental processes, autophagy is enhanced by induction of several autophagy-related genes (ATG genes). Autophagy in crop development has been studied in relation to leaf senescence, seed and reproductive development, and vascular formation. However, its role in fruit ripening has only been partially addressed. Strawberry is an important berry crop, representative of non-climacteric fruit. We have analyzed the occurrence of autophagy in developing and ripening fruits of the cultivated strawberry. Our data show that most ATG genes are conserved in the genome of the cultivated strawberry Fragaria x ananassa and they are differentially expressed along the ripening of the fruit receptacle. ATG8-lipidation analysis proves the presence of two autophagic waves during ripening. In addition, we have confirmed the presence of autophagy at the cellular level by the identification of autophagy-related structures at different stages of the strawberry ripening. Finally, we show that blocking autophagy either biochemically or genetically dramatically affects strawberry growth and ripening. Our data support that autophagy is an active and essential process with different implications during strawberry fruit ripening. This work has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie to VS-V grant agreement 844365 project FrATGaria: Identification of the role of autophagy during fruit ripening and stress resistance in Strawberry: Autophagy manipulation as a way to improve Strawberry fitness, by the Ministerio de Economía y Competitividad, co-financed by the European Regional Development Fund (grant BIO2017-82609-R) to MB and by the Plan Propio of the University of Malaga.