Search

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.

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 betweenPinusandPiceawe 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 ofPinus taedawere printed on nylon membranes in randomized replicates. These were hybridized with labelled cDNA from needles or embryogenic cultures ofPinus taeda,P. sylvestrisandPicea abies, and with labelled cDNA from leaves ofNicotiana tabacum. The Spearman correlation of gene expression for pairs of conifer species was high for needles (r2= 0.78 − 0.86), and somewhat lower for embryogenic cultures (r2= 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 (r2= 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 is defined as a process in which a bipolar structure, resembling a zygotic embryo, develops from a non-zygotic cell without vascular connection with the original tissue. Somatic embryos are used for studying regulation of embryo development, but also as a tool for large scale vegetative propagation. Somatic embryogenesis is a multi-step regeneration process starting with formation of proembryogenic masses, followed by somatic embryo formation, maturation, desiccation and plant regeneration. Although great progress has been made in improving the protocols used, it has been revealed that some treatments, coinciding with increased yield of somatic embryos, can cause adverse effects on the embryo quality, thereby impairing germination and ex vitro growth of somatic embryo plants. Accordingly, ex vitro growth of somatic embryo plants is under a cumulative influence of the treatments provided during the in vitro phase. In order to efficiently regulate the formation of plants via somatic embryogenesis it is important to understand how somatic embryos develop and how the development is influenced by different physical and chemical treatments. Such knowledge can be gained through the construction of fate maps representing an adequate number of morphological and molecular markers, specifying critical developmental stages. Based on this fate map, it is possible to make a model of the process. The mechanisms that control cell differentiation during somatic embryogenesis are far from clear. However, secreted, soluble signal molecules play an important role. It has long been observed that conditioned medium from embryogenic cultures can promote embryogenesis. Active components in the conditioned medium include endochitinases, arabinogalactan proteins and lipochitooligosaccharides.

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.

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.

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.

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.

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.

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.

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 (ψs) 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 (ψs=−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 ψs 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 ψs of the maturation medium, nor on the genotype.

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 embryogenesis (SE) is a sequence of stereotypical morphological transformations, which results in differentiation of cells into a plant body bypassing the fusion of gametes. As such, it represents a very powerful tool in biotechnology to propagate species with long reproductive cycles or low seed set and the production of genetically modified plants with improved traits. The initiation of SE can be divided into five major stages: (i) perception of extracellular signals or stress stimuli, (ii) transduction of the extracellular signal through the cytoplasm into the nucleus, (iii) induction of gene transcription required for embryogenesis, (iv) reorganisation of cytoplasm and (v) onset of embryonic development. The further embryonic development during SE resembles its zygotic counterpart and begins with the 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, becomes a subject of terminal differentiation and gradually degrades via vacuolar programmed cell death (PCD). This PCD is essential for normal development of the apical domain. Some signalling events in the apical and basal domains share homologous components. Here, we describe our current knowledge on the control of life and death processes during SE.

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.

Stable growing callus cultures were recovered from stem segments of Taxus baccata. Callus developed from the peridermal cells. The content of taxol in callus tissues varied in the range of [0.0003–0.001% on dry weight basis] and was higher in fast growing calli compared to slow growing calli.

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.

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.

Execution of programmed cell death (PCD) in nonmetazoan organisms is morphologically different from apoptotic PCD in animals and lacks a number of key molecular components of apoptotic machinery, including caspases. Yet protozoan, fungal, and plant cells exhibit caspase-like proteolytic activities, which increase in a PCD-dependent manner. This poses a question whether nonmetazoan organisms contain structurally dissimilar proteases that functionally substitute for caspases. Putative ancestors of caspases, metacaspases, are candidates for this role; however, their distinct substrate specificity raises doubts. The identification of a common biological target of caspases and metacaspases and previously unknown functions unrelated to cell death of metacaspases provide new food for thought.

Programmed cell death (PCD) is executed by proteases, which cleave diverse proteins thus modulating their biochemical and cellular functions. Proteases of the caspase family and hundreds of caspase substrates constitute a major part of the PCD degradome in animals. Plants lack close homologues of caspases, but instead possess an ancestral family of cysteine proteases, metacaspases. Although metacaspases are essential for PCD, their natural substrates remain unknown. Here we show that metacaspase mcII-Pa cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), during both developmental and stress-induced PCD. TSN knockdown leads to activation of ectopic cell death during reproduction, impairing plant fertility. Surprisingly, human TSN (also known as p100 or SND1), a multifunctional regulator of gene expression, is cleaved by caspase-3 during apoptosis. This cleavage impairs the ability of TSN to activate mRNA splicing, inhibits its ribonuclease activity and is important for the execution of apoptosis. Our results establish TSN as the first biological substrate of metacaspase and demonstrate that despite the divergence of plants and animals from a common ancestor about one billion years ago and their use of distinct PCD pathways, both have retained a common mechanism to compromise cell viability through the cleavage of the same substrate, TSN.

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.

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.

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.

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.Addendum to:Developmental Regulation of a VEIDase Caspase-Like Proteolytic Activity in Barley CaryopsisM. Boren, A.S. Hoglund, P. Bozhkov and C. JanssonJ Exp Bot; In press

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 p roteases a re i nvolved d uring 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 caspasespecific 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 caryopses 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.

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.

Summary • 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.

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.

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].

Somatic embryogenesis combined with cryopreservation is an attractive method to propagate Norway spruce (Picea abies) vegetatively both as a tool in the breeding programme and for large-scale clonal propagation of elite material. Somatic embryos are also a valuable tool for studying regulation of embryo development. Embryogenic cell lines of Norway spruce are established from zygotic embryos. The cell lines proliferate as proembryogenic masses (PEMs). Somatic embryos develop from PEMs. PEM-to-somatic embryo transition is a key developmental switch that determines the yield and quality of mature somatic embryos. Withdrawal of plant growth regulators stimulates PEM-to-somatic embryo transition accompanied by programmed cell death (PCD) in PEMs. This PCD is mediated by a marked decrease in extracellular pH. If the acidification is abolished by buffering the culture medium, PEM-to-somatic embryo transition together with PCD is inhibited. Cell death, induced by withdrawal of PGRs, can be suppressed by extra supply of lipo-chitooligosaccharides (LCOs). Extracellular chitinases are probably involved in production and degradation of LCOs. During early embryogeny, the embryos form an embryonal mass surrounded by a surface layer. The formation of a surface layer is accompanied by a switch in the expression pattern of an Ltp-like gene (Pa18) and a homeobox gene (PaHB1), from ubiquitous expression in PEMs to surface layer-specific in somatic embryos. Ectopic expression of Pa18 and PaHB1 leads to an early developmental block. Transgenic embryos and plants of Norway spruce are routinely produced by using a biolistic approach. The transgenic material is used for studying the importance of specific genes for regulating plant development, but transgenic plants can also be used for identification of candidate genes for use in the breeding programme.

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.

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.

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.

The biotechnology of somatic embryogenesis holds considerable promise for clonal propagation and breeding programes 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.

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.

Understanding the consequence of characteristic morphological stages during embryo-genesis is required for any project aimed at isolation of genes which control embryonic pattern formation and post-embryonic development. Besides, selection and classification of embryo mutants, as another prerequisite for the most molecular studies, is impossible without the reference fate map showing correct progression of embryogenesis. Construction of such map in somatic embryogenesis can be achieved by two alternative approaches: (1) using synchronous cell-division systems (e.g., Fukuda et al. 1994), or (2) by time-lapse tracking of development of individual embryogenic protoplasts, cells or multicellular structures (Toonen et al. 1994).

Plant zygote divides asymmetrically into an apical cell that develops into the embryo proper and a basal cell that generates the suspensor, a vital organ functioning as a conduit of nutrients and growth factors to the embryo proper. After the suspensor has fulfilled its function, it is removed by programmed cell death (PCD) at the late stages of embryogenesis. The molecular trigger of this PCD is unknown. Here we use tobacco (Nicotiana tabacum) embryogenesis as a model system to demonstrate that the mechanism triggering suspensor PCD is based on the antagonistic action of two proteins: a protease inhibitor, cystatin NtCYS, and its target, cathepsin H-like protease NtCP14. NtCYS is expressed in the basal cell of the proembryo, where encoded cystatin binds to and inhibits NtCP14, thereby preventing precocious onset of PCD. The anti-cell death effect of NtCYS is transcriptionally regulated and is repressed at the 32-celled embryo stage, leading to increased NtCP14 activity and initiation of PCD. Silencing of NtCYS or overexpression of NtCP14 induces precocious cell death in the basal cell lineage causing embryonic arrest and seed abortion. Conversely, overexpression of NtCYS or silencing of NtCP14 leads to profound delay of suspensor PCD. Our results demonstrate that NtCYS-mediated inhibition of NtCP14 protease acts as a bipartite molecular module to control initiation of PCD in the basal cell lineage of plant embryos.

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 agriculturally 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: (i) high-throughput screening of chemical compounds in cell cultures of Nicotiana tabacum; (ii) confirmation of the identified hits in planta using Arabidopsis thaliana; (iii) further characterization of the effect using conventional molecular biology methods; (iv) verification of chemical specificity on autophagy in planta. The methods detailed here streamline identification of specific plant autophagy modulators and aid in unravelling the molecular mechanisms of plant autophagy.