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

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 metacaspase­autophagy 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.

Detection and measurement of necrosis in plants.

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.

Affinity maturation generates greatly improved xyloglucan-specific carbohydrate binding modules.

BACKGROUND: Molecular evolution of carbohydrate binding modules (CBM) is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology. RESULTS: Two modified proteins with greatly improved affinity for xyloglucan, a key polysaccharide abundant in the plant kingdom crucial for providing plant support, were generated. Both improved modules differ from other existing xyloglucan probes by binding to galactose-decorated subunits of xyloglucan. The usefulness of the evolved binders was verified by staining of plant sections, where they performed better than the xyloglucan-binding module from which they had been derived. They discriminated non-fucosylated from fucosylated xyloglucan as shown by their ability to stain only the endosperm, rich in non-fucosylated xyloglucan, but not the integument rich in fucosylated xyloglucan, on tamarind seed sections. CONCLUSION: We conclude that affinity maturation of CBM selected from molecular libraries based on the CBM4-2 scaffold is possible and has the potential to generate new analytical tools for detection of plant carbohydrates.

Detection of programmed cell death in plant embryos.

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.

Synthetic xylan-binding modules for mapping of pulp fibres and wood sections.

BACKGROUND: The complex carbohydrate composition of natural and refined plant material is not known in detail but a matter that is of both basic and applied importance. Qualitative assessment of complex samples like plant and wood tissues requires the availability of a range of specific probes. Monoclonal antibodies and naturally existing carbohydrate binding modules (CBMs) have been used in the past to assess the presence of certain carbohydrates in plant tissues. However, the number of natural CBMs is limited and development of carbohydrate-specific antibodies is not always straightforward. We envisage the use of sets of very similar proteins specific for defined targets, like those developed by molecular evolution of a single CBM scaffold, as a suitable strategy to assess carbohydrate composition. An advantage of using synthetic CBMs lies in the possibility to study fine details of carbohydrate composition within non-uniform substrates like plant cell walls as made possible through minor differences in CBM specificity of the variety of binders that can be developed by genetic engineering. RESULTS: A panel of synthetic xylan-binding CBMs, previously selected from a molecular library based on the scaffold of CBM4-2 from xylanase Xyn10A of Rhodothermus marinus, was used in this study. The wild type CBM4-2 and evolved modules both showed binding to wood sections. However, differences were observed in the staining patterns suggesting that these modules have different xylan-binding properties. Also the staining stability varied between the CBMs, the most stable staining being obtained with one (X-2) of the synthetic modules. Treatment of wood materials resulted in altered signal intensities, thereby also demonstrating the potential application of engineered CBMs as analytical tools for quality assessment of diverse plant material processes. CONCLUSION: In this study we have demonstrated the usefulness of synthetic xylan-binding modules as specific probes in analysis of hemicelluloses (xylan) in wood and fibre materials.

Characteristics of Gloeophyllum trabeum alcohol oxidase, an extracellular source of H2O2 in brown rot decay of wood.

A novel alcohol oxidase (AOX) has been purified from mycelial pellets of the wood-degrading basidiomycete Gloeophyllum trabeum and characterized as a homooctameric nonglycosylated protein with native and subunit molecular masses of 628 and 72.4 kDa, containing noncovalently bonded flavin adenine dinucleotide. The isolated AOX cDNA contained an open reading frame of 1,953 bp translating into a polypeptide of 651 amino acids displaying 51 to 53% identity with other published fungal AOX amino acid sequences. The enzyme catalyzed the oxidation of short-chain primary aliphatic alcohols with a preference for methanol (K(m) = 2.3 mM, k(cat) = 15.6 s(-1)). Using polyclonal antibodies and immunofluorescence staining, AOX was localized on liquid culture hyphae and extracellular slime in sections from degraded wood and on cotton fibers. Transmission electron microscopy immunogold labeling localized the enzyme in the hyphal periplasmic space and wall and on extracellular tripartite membranes and slime, while there was no labeling of hyphal peroxisomes. AOX was further shown to be associated with membranous or slime structures secreted by hyphae in wood fiber lumina and within the secondary cell walls of degraded wood fibers. The differences in AOX targeting compared to the known yeast peroxisomal localization were traced to a unique C-terminal sequence of the G. trabeum oxidase, which is apparently responsible for the protein's different translocation. The extracellular distribution and the enzyme's abundance and preference for methanol, potentially available from the demethylation of lignin, all point to a possible role for AOX as a major source of H(2)O(2), a component of Fenton's reagent implicated in the generally accepted mechanisms for brown rot through the production of highly destructive hydroxyl radicals.

Analysis of the surfaces of wood tissues and pulp fibers using carbohydrate-binding modules specific for crystalline cellulose and mannan.

Carbohydrate binding modules (CBMs) are noncatalytic substrate binding domains of many enzymes involved in carbohydrate metabolism. Here we used fluorescent labeled recombinant CBMs specific for crystalline cellulose (CBM1(HjCel7A)) and mannans (CBM27(TmMan5) and CBM35(CjMan5C)) to analyze the complex surfaces of wood tissues and pulp fibers. The crystalline cellulose CBM1(HjCel7A) was found as a reliable marker of both bacterially produced and plant G-layer cellulose, and labeling of spruce pulp fibers with CBM1(HjCel7A) revealed a signal that increased with degree of fiber damage. The mannan-specific CBM27(TmMan5) and CBM35(CjMan5C) CBMs were found to be more specific reagents than a monoclonal antibody specific for (1-->4)-beta-mannan/galacto-(1-->4)-beta-mannan for mapping carbohydrates on native substrates. We have developed a quantitative fluorometric method for analysis of crystalline cellulose accumulation on fiber surfaces and shown a quantitative difference in crystalline cellulose binding sites in differently processed pulp fibers. Our results indicated that CBMs provide useful, novel tools for monitoring changes in carbohydrate content of nonuniform substrate surfaces, for example, during wood or pulping processes and possibly fiber biosynthesis.

Expression patterns of two glutamine synthetase genes in zygotic and somatic pine embryos support specific roles in nitrogen metabolism during embryogenesis.

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.

Cysteine protease mcII-Pa executes programmed cell death during plant embryogenesis.

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.

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.

Re-organisation of the cytoskeleton during developmental programmed cell death in Picea abies embryos.

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.

A key developmental switch during Norway spruce somatic embryogenesis is induced by withdrawal of growth regulators and is associated with cell death and extracellular acidification.

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.