Peter V. Bozhkov

Developmental pathways of somatic embryogenesis

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.

Morphological classification of plant cell deaths

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.

Metacaspase-dependent programmed cell death is essential for plant embryogenesis

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. We explored the role of plant metacaspases in PCD using a model system of somatic embryogenesis of Norway spruce (Picea abies), where the pathway of embryo development (Figure 1A) resembles zygotic embryogeny, even though the embryo origin is different in each case (i.e., somatic cells in proembryogenic mass (PEM) versus zygote) [6]. In this developmental pathway autophagic PCD ablates PEMs at the time of their differentiation to embryos and then eliminates terminally differentiated embryo suspensor as the embryos enter late embryogeny [6,7] (Figure 1A). We have isolated a 1687 bp cDNA sequence from the embryogenic cell cultures (EMBL database accession number AJ534970). The encoded protein shows a significant degree of conservation with metacaspases and falls into the type II plant metacaspase subfamily (Figure S1A). The protein was named mcII-Pa. The predicted secondary structure of mcII-Pa contains conserved domains and motifs present in all members of the caspase/metacaspase/paracaspase superfamily [5] (Figure S1B). The putative mcII-Pa catalytic diad of cysteine and histidine is placed in the α α/β β fold characteristic for the caspase-hemoglobinase fold (CHF)-containing proteins [5]. The predicted mcII-Pa protein lacks both the death-effector domain and the caspase-activating recruitment domain found in classical initiator caspases, but has a p20-like domain including the active-site pentapeptide DXCHS (where X is A or S) shared by all metacaspases [5] (Figure S1B). This domain is fused to the 268 amino acid carboxy-terminal region consisting of a large insert of approximately 180 amino acids and a p10-like domain. In situ hybridization analysis has revealed restricted accumulation …

Tudor staphylococcal nuclease is an evolutionarily conserved component of the programmed cell death degradome

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.

VEIDase is a principal caspase-like activity involved in plant programmed cell death and essential for embryonic pattern formation

AbstractPlant 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 Zn2+ 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.

4 – Programmed Cell Death in Plant 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.

Programmed cell death eliminates all but one embryo in a polyembryonic plant seed

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.

Autophagy and metacaspase determine the mode of cell death in plants.

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

Somatic embryogenesis: life and death processes during apical–basal patterning

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.

Up, down and up again is a signature global gene expression pattern at the beginning of gymnosperm embryogenesis

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.