| 1. |
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| 2. |
- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Research review (peer-reviewed)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 3. |
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| 4. |
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| 5. |
- Bozhkov, Peter
(author)
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A Bipartite Molecular Module Controls Cell Death Activation in the Basal Cell Lineage of Plant Embryos
- 2013
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In: PLoS Biology. - : Public Library of Science (PLoS). - 1544-9173 .- 1545-7885. ; 11
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Journal article (peer-reviewed)abstract
- 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.
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| 6. |
- Bozhkov, Peter, et al.
(author)
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Aspasing Out Metacaspases and Caspases: Proteases of Many Trades
- 2010
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In: Science Signaling. - : American Association for the Advancement of Science (AAAS). - 1945-0877 .- 1937-9145. ; 3
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Journal article (peer-reviewed)abstract
- 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.
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| 7. |
- Bozhkov, Peter, et al.
(author)
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Autophagy-related approaches for improving nutrient use efficiency and crop yield protection
- 2018
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In: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 69, s. 1335-1353
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Research review (peer-reviewed)abstract
- Autophagy is a eukaryotic catabolic pathway essential for growth and development. In plants, it is activated in response to environmental cues or developmental stimuli. However, in contrast to other eukaryotic systems, we know relatively little regarding the molecular players involved in autophagy and the regulation of this complex pathway. In the framework of the COST (European Cooperation in Science and Technology) action TRANSAUTOPHAGY (2016-2020), we decided to review our current knowledge of autophagy responses in higher plants, with emphasis on knowledge gaps. We also assess here the potential of translating the acquired knowledge to improve crop plant growth and development in a context of growing social and environmental challenges for agriculture in the near future.
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| 8. |
- Bozhkov, Peter, et al.
(author)
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Concentrating and sequestering biomolecules in condensates: impact on plant biology
- 2023
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In: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 74, s. 1303-1308
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Journal article (peer-reviewed)abstract
- Biomolecules can exist in a variety of forms, ranging from single entities to mesoscale assemblies akin to small organelles, also known as ‘biomolecular condensates’. The formation of biomolecular condensates is expedited by phase separation, in which molecules de-mix to form dilute and condensed phases. Phase separation results in concentrating or sequestering certain molecules, thus altering their abundance or other features in the phases and in this way inhibiting or promoting biochemical reactions. Here, we discuss recent research implicating biomolecular condensates in the regulation of biochemical reactions in plants.
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| 9. |
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| 10. |
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| 11. |
- Bozhkov, Peter
(author)
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Impact of salt stress, cell death, and autophagy on peroxisomes: quantitative and morphological analyses using small fluorescent probe N-BODIPY
- 2017
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Journal article (peer-reviewed)abstract
- Plant peroxisomes maintain a plethora of key life processes including fatty acid beta-oxidation, photorespiration, synthesis of hormones, and homeostasis of reactive oxygen species (ROS). Abundance of peroxisomes in cells is dynamic; however mechanisms controlling peroxisome proliferation remain poorly understood because measuring peroxisome abundance is technically challenging. Counting peroxisomes in individual cells of complex organs by electron or fluorescence microscopy is expensive and time consuming. Here we present a simple technique for quantifying peroxisome abundance using the small probe Nitro-BODIPY, which in vivo fluoresces selectively inside peroxisomes. The physiological relevance of our technique was demonstrated using salinity as a known inducer of peroxisome proliferation. While significant peroxisome proliferation was observed in wildtype Arabidopsis leaves following 5-hour exposure to NaCl, no proliferation was detected in the salt-susceptible mutants fry1-6, sos1-14, and sos1-15. We also found that N-BODIPY detects aggregation of peroxisomes during final stages of programmed cell death and can be used as a marker of this stage. Furthermore, accumulation of peroxisomes in an autophagy-deficient Arabidopsis mutant atg5 correlated with N-BODIPY labeling. In conclusion, the technique reported here enables quantification of peroxisomes in plant material at various physiological settings. Its potential applications encompass identification of genes controlling peroxisome homeostasis and capturing stress-tolerant genotypes.
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| 12. |
- Bozhkov, Peter
(author)
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Plant autophagy: mechanisms and functions
- 2018
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In: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 69, s. 1281-1285
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Other publication (peer-reviewed)abstract
- Autophagy (from Greek: 'self-eating') is a major catabolic process in eukaryotic cells in which cytoplasmic components are collected and delivered to the lysosomes or vacuoles for recycling. It plays a paramount role in plant fitness and immunity. At present, the frontiers of our understanding of the process are extending exponentially, with new, plant-specific mechanisms and functions being uncovered. In this special issue, original research articles and reviews enlighten this knowledge from lipid metabolism and dynamics, membrane trafficking and proteolysis to pathogen-mediated modulation of the process and the emerging role of autophagy-related approaches in crop improvement.
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| 13. |
- Bozhkov, Peter
(author)
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Somatic embryogenesis: life and death processes during apical - basal patterning
- 2014
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In: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 65, s. 1343-1360
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Research review (peer-reviewed)abstract
- Early embryos consist of the apical domain which gives rise to the mature embryo and the basal domain which is gradually eliminated by PCD. Molecular pathways regulating development of each domain are reviewed.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 apicalbasal 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.
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| 14. |
- Bozhkov, Peter
(author)
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The Life and Death Signalling Underlying Cell Fate Determination During Somatic Embryogenesis
- 2014
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In: Applied Plant Cell Biology : Cellular Tools and Approaches for Plant Biotechnology. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 9783642417870 ; 22:22, s. 131-178
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Book chapter (other academic/artistic)abstract
- 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.
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| 15. |
- Bozhkov, Peter, et al.
(author)
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Transcriptional stimulation of autophagy improves plant fitness
- 2017
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Patent (other academic/artistic)abstract
- The present invention provides to a method for enhancing the productivity of a plant by genetically modifying the genome of the plant to over-express at least one autophagy-related (ATG) protein selected from the group consisting of ATG5 and ATG7. The invention further provides a genetically modified plant characterized by over-expression of least one autophagy related (ATG) protein selected from the group consisting of ATG5 and ATG7.Additionally the use of a transgene encoding atleast one autophagy related (ATG) protein selected from the group consisting of ATG5 and ATG7for enhancing the productivity of a plantis disclosed.
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| 16. |
- Dauphinee, Adrian N., et al.
(author)
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Chemical Screening Pipeline for Identification of Specific Plant Autophagy Modulators
- 2019
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In: Plant Physiology. - : AMER SOC PLANT BIOLOGISTS. - 0032-0889 .- 1532-2548. ; 181:3, s. 855-866
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Journal article (peer-reviewed)abstract
- Autophagy is a major catabolic process in eukaryotes with a key role in homeostasis, programmed cell death, and aging. In plants, autophagy is also known to regulate agronomically important traits such as stress resistance, longevity, vegetative biomass, and seed yield. Despite its significance, there is still a shortage of reliable tools modulating plant autophagy. Here, we describe the first robust pipeline for identification of specific plant autophagy-modulating compounds. Our screening protocol comprises four phases: (1) high-throughput screening of chemical compounds in cell cultures of tobacco (Nicotiana tabacum); (2) confirmation of the identified hits in planta using Arabidopsis (Arabidopsis thaliana); (3) further characterization of the effect using conventional molecular biology methods; and (4) verification of chemical specificity on autophagy in planta. The methods detailed here streamline the identification of specific plant autophagy modulators and aid in unraveling the molecular mechanisms of plant autophagy.
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| 17. |
- Eisele-Bürger, Anna Maria, et al.
(author)
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Calmodulin regulates protease versus co-chaperone activity of a metacaspase
- 2023
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In: Cell Reports. - 2211-1247. ; 42:11
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Journal article (peer-reviewed)abstract
- Metacaspases are ancestral homologs of caspases that can either promote cell death or confer cytoprotection. Furthermore, yeast (Saccharomyces cerevisiae) metacaspase Mca1 possesses dual biochemical activity: proteolytic activity causing cell death and cytoprotective, co-chaperone-like activity retarding replicative aging. The molecular mechanism favoring one activity of Mca1 over another remains elusive. Here, we show that this mechanism involves calmodulin binding to the N-terminal pro-domain of Mca1, which prevents its proteolytic activation and promotes co-chaperone-like activity, thus switching from pro-cell death to anti-aging function. The longevity-promoting effect of Mca1 requires the Hsp40 co-chaperone Sis1, which is necessary for Mca1 recruitment to protein aggregates and their clearance. In contrast, proteolytically active Mca1 cleaves Sis1 both in vitro and in vivo, further clarifying molecular mechanism behind a dual role of Mca1 as a cell-death protease versus gerontogene.
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| 18. |
- Elander, Pernilla, et al.
(author)
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Autophagy in turnover of lipid stores: trans-kingdom comparison
- 2018
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In: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 69, s. 1301-1311
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Research review (peer-reviewed)abstract
- Lipids and their cellular utilization are essential for life. Not only are lipids energy storage molecules, but their diverse structural and physical properties underlie various aspects of eukaryotic biology, such as membrane structure, signalling, and trafficking. In the ever-changing environment of cells, lipids, like other cellular components, are regularly recycled to uphold the housekeeping processes required for cell survival and organism longevity. The ways in which lipids are recycled, however, vary between different phyla. For example, animals and plants have evolved distinct lipid degradation pathways. The major cell recycling system, autophagy, has been shown to be instrumental for both differentiation of specialized fat storing-cells, adipocytes, and fat degradation in animals. Does plant autophagy play a similar role in storage and degradation of lipids? In this review, we discuss and compare implications of bulk autophagy and its selective route, lipophagy, in the turnover of lipid stores in animals, fungi, and plants.
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| 19. |
- Elander, Pernilla, et al.
(author)
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Interactome of Arabidopsis ATG5 Suggests Functions beyond Autophagy
- 2023
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In: International Journal of Molecular Sciences. - 1661-6596 .- 1422-0067. ; 24
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Journal article (peer-reviewed)abstract
- Autophagy is a catabolic pathway capable of degrading cellular components ranging from individual molecules to organelles. Autophagy helps cells cope with stress by removing superfluous or hazardous material. In a previous work, we demonstrated that transcriptional upregulation of two autophagy-related genes, ATG5 and ATG7, in Arabidopsis thaliana positively affected agronomically important traits: biomass, seed yield, tolerance to pathogens and oxidative stress. Although the occurrence of these traits correlated with enhanced autophagic activity, it is possible that autophagy-independent roles of ATG5 and ATG7 also contributed to the phenotypes. In this study, we employed affinity purification and LC-MS/MS to identify the interactome of wild-type ATG5 and its autophagy-inactive substitution mutant, ATG5(K128R) Here we present the first interactome of plant ATG5, encompassing not only known autophagy regulators but also stress-response factors, components of the ubiquitin-proteasome system, proteins involved in endomembrane trafficking, and potential partners of the nuclear fraction of ATG5. Furthermore, we discovered post-translational modifications, such as phosphorylation and acetylation present on ATG5 complex components that are likely to play regulatory functions. These results strongly indicate that plant ATG5 complex proteins have roles beyond autophagy itself, opening avenues for further investigations on the complex roles of autophagy in plant growth and stress responses.
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| 20. |
- Elander, Pernilla, et al.
(author)
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Tudor staphylococcal nuclease is a docking platform for stress granule components and is essential for SnRK1 activation in Arabidopsis
- 2021
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In: The Embo Journal. - : EMBO. - 0261-4189 .- 1460-2075. ; 40
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Journal article (peer-reviewed)abstract
- Tudor staphylococcal nuclease (TSN; also known as Tudor-SN, p100, or SND1) is a multifunctional, evolutionarily conserved regulator of gene expression, exhibiting cytoprotective activity in animals and plants and oncogenic activity in mammals. During stress, TSN stably associates with stress granules (SGs), in a poorly understood process. Here, we show that in the model plant Arabidopsis thaliana, TSN is an intrinsically disordered protein (IDP) acting as a scaffold for a large pool of other IDPs, enriched for conserved stress granule components as well as novel or plant-specific SG-localized proteins. While approximately 30% of TSN interactors are recruited to stress granules de novo upon stress perception, 70% form a protein-protein interaction network present before the onset of stress. Finally, we demonstrate that TSN and stress granule formation promote heat-induced activation of the evolutionarily conserved energy-sensing SNF1-related protein kinase 1 (SnRK1), the plant orthologue of mammalian AMP-activated protein kinase (AMPK). Our results establish TSN as a docking platform for stress granule proteins, with an important role in stress signalling.
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| 21. |
- Gutierrez, Emilio, et al.
(author)
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Phenolic acid-induced phase separation and translation inhibition mediate plant interspecific competition
- 2023
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In: Nature Plants. - 2055-026X .- 2055-0278. ; 9, s. 1481–1499-
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Journal article (peer-reviewed)abstract
- Phenolic acids (PAs) secreted by donor plants suppress the growth of their susceptible plant neighbours. However, how structurally diverse ensembles of PAs are perceived by plants to mediate interspecific competition remains a mystery. Here we show that a plant stress granule (SG) marker, RNA-BINDING PROTEIN 47B (RBP47B), is a sensor of PAs in Arabidopsis. PAs, including salicylic acid, 4-hydroxybenzoic acid, protocatechuic acid and so on, directly bind RBP47B, promote its phase separation and trigger SG formation accompanied by global translation inhibition. Salicylic acid-induced global translation inhibition depends on RBP47 family members. RBP47s regulate the proteome rather than the absolute quantity of SG. The rbp47 quadruple mutant shows a reduced sensitivity to the inhibitory effect of the PA mixture as well as to that of PA-rich rice when tested in a co-culturing ecosystem. In this Article, we identified the long sought-after PA sensor as RBP47B and illustrated that PA-induced SG-mediated translational inhibition was one of the PA perception mechanisms.Allelopathy-the chemical competition between neighbouring plants-has been observed for centuries. This study identifies a sensor of phenolic allelochemicals and reveals translational control as a key mechanism.
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| 22. |
- Gutierrez, Emilio, et al.
(author)
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Tudor staphylococcal nuclease: biochemistry and functions
- 2016
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In: Cell Death and Differentiation. - : Springer Science and Business Media LLC. - 1350-9047 .- 1476-5403. ; 23, s. 1739-1748
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Research review (peer-reviewed)abstract
- Tudor staphylococcal nuclease (TSN, also known as Tudor-SN, SND1 or p100) is an evolutionarily conserved protein with invariant domain composition, represented by tandem repeat of staphylococcal nuclease domains and a tudor domain. Conservation along significant evolutionary distance, from protozoa to plants and animals, suggests important physiological functions for TSN. It is known that TSN is critically involved in virtually all pathways of gene expression, ranging from transcription to RNA silencing. Owing to its high protein-protein binding affinity coexistent with enzymatic activity, TSN can exert its biochemical function by acting as both a scaffolding molecule of large multiprotein complexes and/or as a nuclease. TSN is indispensible for normal development and stress resistance, whereas its increased expression is closely associated with various types of cancer. Thus, TSN is an attractive target for anti-cancer therapy and a potent tumor marker. Considering ever increasing interest to further understand a multitude of TSN-mediated processes and a mechanistic role of TSN in these processes, here we took an attempt to summarize and update the available information about this intriguing multifunctional protein.
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| 23. |
- Gutierrez, Emilio, et al.
(author)
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Tudor Staphylococcal Nuclease Links Formation of Stress Granules and Processing Bodies with mRNA Catabolism in Arabidopsis
- 2015
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In: Plant Cell. - : Oxford University Press (OUP). - 1040-4651 .- 1532-298X. ; 27, s. 926-943
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Journal article (peer-reviewed)abstract
- Tudor Staphylococcal Nuclease (TSN or Tudor-SN; also known as SND1) is an evolutionarily conserved protein involved in the transcriptional and posttranscriptional regulation of gene expression in animals. Although TSN was found to be indispensable for normal plant development and stress tolerance, the molecular mechanisms underlying these functions remain elusive. Here, we show that Arabidopsis thaliana TSN is essential for the integrity and function of cytoplasmic messenger ribonucleoprotein (mRNP) complexes called stress granules (SGs) and processing bodies (PBs), sites of posttranscriptional gene regulation during stress. TSN associates with SGs following their microtubule-dependent assembly and plays a scaffolding role in both SGs and PBs. The enzymatically active tandem repeat of four SN domains is crucial for targeting TSN to the cytoplasmic mRNA complexes and is sufficient for the cytoprotective function of TSN during stress. Furthermore, our work connects the cytoprotective function of TSN with its positive role in stress-induced mRNA decapping. While stress led to a pronounced increase in the accumulation of uncapped mRNAs in wild-type plants, this increase was abrogated in TSN knockout plants. Taken together, our results establish TSN as a key enzymatic component of the catabolic machinery responsible for the processing of mRNAs in the cytoplasmic mRNP complexes during stress.
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| 24. |
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| 25. |
- Lager, Ida, et al.
(author)
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Subcellular Localization of Acyl-CoA: Lysophosphatidylethanolamine Acyltransferases (LPEATs) and the Effects of Knocking-Out and Overexpression of Their Genes on Autophagy Markers Level and Life Span of A. thaliana
- 2021
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In: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 22
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Journal article (peer-reviewed)abstract
- Arabidopsis thaliana possesses two acyl-CoA:lysophosphatidylethanolamine acyltransferases, LPEAT1 and LPEAT2, which are encoded by At1g80950 and At2g45670 genes, respectively. Both single lpeat2 mutant and double lpeat1 lpeat2 mutant plants exhibit a variety of conspicuous phenotypes, including dwarfed growth. Confocal microscopic analysis of tobacco suspension-cultured cells transiently transformed with green fluorescent protein-tagged versions of LPEAT1 or LPEAT2 revealed that LPEAT1 is localized to the endoplasmic reticulum (ER), whereas LPEAT2 is localized to both Golgi and late endosomes. Considering that the primary product of the reaction catalyzed by LPEATs is phosphatidylethanolamine, which is known to be covalently conjugated with autophagy-related protein ATG8 during a key step of the formation of autophagosomes, we investigated the requirements for LPEATs to engage in autophagic activity in Arabidopsis. Knocking out of either or both LPEAT genes led to enhanced accumulation of the autophagic adaptor protein NBR1 and decreased levels of both ATG8a mRNA and total ATG8 protein. Moreover, we detected significantly fewer membrane objects in the vacuoles of lpeat1 lpeat2 double mutant mesophyll cells than in vacuoles of control plants. However, contrary to what has been reported on autophagy deficient plants, the lpeat mutants displayed a prolonged life span compared to wild type, including delayed senescence.
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| 26. |
- Liu, Chen, et al.
(author)
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The Proteolytic Landscape Of An Arabidopsis Separase-Deficient Mutant Reveals Novel Substrates Associated With Plant Development
- 2017
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Other publication (other academic/artistic)abstract
- Digestive proteolysis executed by the proteasome plays an important role in plant development. Yet, the role of limited proteolysis in this process is still obscured due to the absence of studies. Previously, we showed that limited proteolysis by the caspase-related protease separase (EXTRA SPINDLE POLES [ESP]) modulates development in plants through the cleavage of unknown substrates. Here we used a modified version of the positional proteomics method COmbined FRActional DIagonal Chromatography (COFRADIC) to survey the proteolytic landscape of wild-type and separase mutant RADIALLY SWOLLEN 4 (rsw4) root tip cells, as an attempt to identify targets of separase. We have discovered that proteins involved in the establishment of pH homeostasis and sensing, and lipid signalling in wild-type cells, suggesting novel potential roles for separase. We also observed significant accumulation of the protease PRX34 in rsw4 which negatively impacts growth. Furthermore, we observed an increased acetylation of N-termini of rsw4 proteins which usually comprise degrons identified by the ubiquitin-proteasome system, suggesting that separase intersects with additional proteolytic networks. Our results hint to potential pathways by which separase could regulate development suggesting also novel proteolytic functions.
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| 27. |
- Minina, Alyona, et al.
(author)
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Apoptosis is not conserved in plants as revealed by critical examination of a model for plant apoptosis-like cell death
- 2021
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In: BMC biology. - : Springer Science and Business Media LLC. - 1741-7007. ; 19:1, s. 100-
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Journal article (peer-reviewed)abstract
- BackgroundAnimals and plants diverged over one billion years ago and evolved unique mechanisms for many cellular processes, including cell death. One of the most well-studied cell death programmes in animals, apoptosis, involves gradual cell dismantling and engulfment of cellular fragments, apoptotic bodies, through phagocytosis. However, rigid cell walls prevent plant cell fragmentation and thus apoptosis is not applicable for executing cell death in plants. Furthermore, plants are devoid of the key components of apoptotic machinery, including phagocytosis as well as caspases and Bcl-2 family proteins. Nevertheless, the concept of plant “apoptosis-like programmed cell death” (AL-PCD) is widespread. This is largely due to superficial morphological resemblances between plant cell death and apoptosis, and in particular between protoplast shrinkage in plant cells killed by various stimuli and animal cell volume decrease preceding fragmentation into apoptotic bodies.ResultsHere, we provide a comprehensive spatio-temporal analysis of cytological and biochemical events occurring in plant cells subjected to heat shock at 40–55 °C and 85 °C, the experimental conditions typically used to trigger AL-PCD and necrotic cell death, respectively. We show that cell death under both conditions was not accompanied by membrane blebbing or formation of apoptotic bodies, as would be expected during apoptosis. Instead, we observed instant and irreversible permeabilization of the plasma membrane and ATP depletion. These processes did not depend on mitochondrial functionality or the presence of Ca2+and could not be prevented by an inhibitor of ferroptosis. We further reveal that the lack of protoplast shrinkage at 85 °C, the only striking morphological difference between cell deaths induced by 40–55 °C or 85 °C heat shock, is a consequence of the fixative effect of the high temperature on intracellular contents.ConclusionsWe conclude that heat shock-induced cell death is an energy-independent process best matching definition of necrosis. Although the initial steps of this necrotic cell death could be genetically regulated, classifying it as apoptosis or AL-PCD is a terminological misnomer. Our work supports the viewpoint that apoptosis is not conserved across animal and plant kingdoms and demonstrates the importance of focusing on plant-specific aspects of cell death pathways.
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| 28. |
- Minina, Alyona, et al.
(author)
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Autophagy and metacaspase determine the mode of cell death in plants
- 2013
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In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 203, s. 917-927
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Journal article (peer-reviewed)abstract
- 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.
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| 29. |
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| 30. |
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| 31. |
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| 32. |
- Minina, Alyona, et al.
(author)
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Detection and measurement of necrosis in plants
- 2013
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In: Necrosis : methods and protocols. - Totowa, NJ : Humana Press. - 9781627033824 ; :1004, s. 229-248
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Book chapter (other academic/artistic)abstract
- 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 micros- copy, 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.
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| 33. |
- Minina, Alyona, et al.
(author)
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Limited and digestive proteolysis: crosstalk between evolutionary conserved pathways
- 2017
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In: New Phytologist. - : Wiley. - 0028-646X .- 1469-8137. ; 215, s. 958-964
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Research review (peer-reviewed)abstract
- Proteases can either digest target proteins or perform the so-called 'limited proteolysis' by cleaving polypeptide chains at specific site(s). Autophagy and the ubiquitin-proteasome system (UPS) are two main mechanisms carrying out digestive proteolysis. While the net outcome of digestive proteolysis is the loss of function of protein substrates, limited proteolysis can additionally lead to gain or switch of function. Recent evidence of crosstalk between autophagy, UPS and limited proteolysis indicates that these pathways are parts of the same proteolytic nexus. Here, we focus on three emerging themes within this area: limited proteolysis as a mechanism modulating autophagy; interplay between autophagy and UPS, including autophagic degradation of proteasomes (proteophagy); and specificity of protein degradation during bulk autophagy.
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| 34. |
- Minina, Alyona, et al.
(author)
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Metacaspases versus caspases in development and cell fate regulation
- 2017
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In: Cell Death and Differentiation. - : Nature Publishing Group. - 1350-9047 .- 1476-5403. ; 24:8, s. 1314-1325
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Research review (peer-reviewed)abstract
- Initially found to be critically involved in inflammation and apoptosis, caspases have since then been implicated in the regulation of various signaling pathways in animals. How caspases and caspase-mediated processes evolved is a topic of great interest and hot debate. In fact, caspases are just the tip of the iceberg, representing a relatively small group of mostly animal-specific enzymes within a broad family of structurally related cysteine proteases (family C14 of CD clan) found in all kingdoms of life. Apart from caspases, this family encompasses para-and metacaspases, and all three groups of proteases exhibit significant variation in biochemistry and function in vivo. Notably, metacaspases are present in all eukaryotic lineages with a remarkable absence in animals. Thus, metacaspases and caspases must have adapted to operate under distinct cellular and physiological settings. Here we discuss biochemical properties and biological functions of metacaspases in comparison to caspases, with a major focus on the regulation of developmental aspects in plants versus animals.
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| 35. |
- Minina, Alyona, et al.
(author)
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Plant metacaspase activation and activity : Caspases,Paracaspases, and Metacaspases
- 2014
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In: Methods in Molecular Biology. - New York, NY : Springer New York. - 9781493903566 ; 1133:1133, s. 237-253
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Book chapter (peer-reviewed)abstract
- 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.
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| 36. |
- Minina, Alyona, et al.
(author)
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Suppression of Metacaspase- and Autophagy-Dependent Cell Death Improves Stress-Induced Microspore Embryogenesis in Brassica napus
- 2020
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In: Plant and Cell Physiology. - : Oxford University Press (OUP). - 0032-0781 .- 1471-9053. ; 61, s. 2097-2110
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Journal article (peer-reviewed)abstract
- Microspore embryogenesis is a biotechnological process that allows us to rapidly obtain doubled-haploid plants for breeding programs. The process is initiated by the application of stress treatment, which reprograms microspores to embark on embryonic development. Typically, a part of the microspores undergoes cell death that reduces the efficiency of the process. Metacaspases (MCAs), a phylogenetically broad group of cysteine proteases, and autophagy, the major catabolic process in eukaryotes, are critical regulators of the balance between cell death and survival in various organisms. In this study, we analyzed the role of MCAs and autophagy in cell death during stress-induced microspore embryogenesis in Brassica napus. We demonstrate that this cell death is accompanied by the transcriptional upregulation of three BnMCA genes (BnMCA-Ia, BnMCA-IIa and BnMCA-IIi), an increase in MCA proteolytic activity and the activation of autophagy. Accordingly, inhibition of autophagy and MCA activity, either individually or in combination, suppressed cell death and increased the number of proembryos, indicating that both components play a pro-cell death role and account for decreased efficiency of early embryonic development. Therefore, MCAs and/or autophagy can be used as new biotechnological targets to improve in vitro embryogenesis in Brassica species and doubled-haploid plant production in crop breeding and propagation programs.
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| 37. |
- Minina, Alyona, et al.
(author)
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The Arabidopsis homolog of Scc4/MAU2 is essential for embryogenesis
- 2017
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In: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 130, s. 1051-1063
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Journal article (peer-reviewed)abstract
- Factors regulating dynamics of chromatin structure have direct impact on expression of genetic information. Cohesin is a multi-subunit protein complex that is crucial for pairing sister chromatids during cell division, DNA repair and regulation of gene transcription and silencing. In non-plant species, cohesin is loaded on chromatin by the Scc2-Scc4 complex (also known as the NIBPL-MAU2 complex). Here, we identify the Arabidopsis homolog of Scc4, which we denote Arabidopsis thaliana (At) SCC4, and show that it forms a functional complex with AtSCC2, the homolog of Scc2. We demonstrate that AtSCC2 and AtSCC4 act in the same pathway, and that both proteins are indispensable for cell fate determination during early stages of embryo development. Mutant embryos lacking either of these proteins develop only up to the globular stage, and show the suspensor overproliferation phenotype preceded by ectopic auxin maxima distribution. We further establish a new assay to reveal the AtSCC4-dependent dynamics of cohesin loading on chromatin in vivo. Our findings define the Scc2-Scc4 complex as an evolutionary conserved machinery controlling cohesin loading and chromatin structure maintenance, and provide new insight into the plant-specific role of this complex in controlling cell fate during embryogenesis.
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| 38. |
- Minina, Alyona, et al.
(author)
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Transcriptional stimulation of rate-limiting components of the autophagic pathway improves plant fitness
- 2018
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In: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 69, s. 1415-1432
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Journal article (peer-reviewed)abstract
- 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-over-expressing 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.
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| 39. |
- Minina, Alyona, et al.
(author)
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Vacuolar cell death in plants Metacaspase releases the brakes on autophagy
- 2014
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In: Autophagy. - : Informa UK Limited. - 1554-8627 .- 1554-8635. ; 10, s. 926-927
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Research review (peer-reviewed)abstract
- 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.
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| 40. |
- Moschou, Panagiotis Nikolaou, et al.
(author)
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Characterization of Cytokinetic Mutants Using Small Fluorescent Probes
- 2016
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In: Methods in Molecular Biology. - New York, NY : Springer New York. - 9781493931415 ; 1370:1370, s. 199-208
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Book chapter (other academic/artistic)abstract
- Cytokinesis is a powerful paradigm for addressing fundamental questions of plant biology including molecular mechanisms of development, cell division, cell signaling, membrane trafficking, cell wall synthesis, and cytoskeletal dynamics. Genetics was instrumental in identification of proteins regulating cytokinesis. Characterization of mutant lines generated using forward or reverse genetics includes microscopic analysis for defects in cell division. Typically, failure of cytokinesis results in appearance of multinucleate cells, formation of cell wall stubs, and isotropic cell expansion in the root elongation zone. Small fluorescent probes served as a very effective tool for the detection of cytokinetic defects. Such probes stain living or formaldehyde-fixed specimens avoiding complex preparatory steps. Although resolution of the fluorescence probes is inferior to electron microscopy, the procedure is fast, easy, and does not require expensive materials or equipment. This chapter describes techniques for staining DNA with the probes DAPI and SYTO82, for staining membranes with FM4-64, and for staining cell wall with propidium iodide.
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| 41. |
- Moschou, Panagiotis Nikolaou, et al.
(author)
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EXTRA SPINDLE POLES (Separase) controls anisotropic cell expansion in Norway spruce (Picea abies) embryos independently of its role in anaphase progression
- 2016
-
In: New Phytologist. - : Wiley. - 0028-646X .- 1469-8137. ; 212, s. 232-243
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Journal article (peer-reviewed)abstract
- 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 cellexpansion are conserved between gymnosperms and angiosperms. However, the mechanisms of ESP-mediated regulation of cell expansion seem to be lineage-specific.
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| 42. |
- Moschou, Panagiotis Nikolaou, et al.
(author)
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Separase Promotes Microtubule Polymerization by Activating CENP-E-Related Kinesin Kin7
- 2016
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In: Developmental Cell. - : Elsevier BV. - 1534-5807 .- 1878-1551. ; 37, s. 350-361
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Journal article (peer-reviewed)abstract
- Microtubules play an essential role in breaking cellular symmetry. We have previously shown that separase associates with microtubules and regulates microtubule-dependent establishment of cell polarity in Arabidopsis. However, separase lacks microtubule-binding activity, raising questions about mechanisms underlying this phenomenon. Here we report that the N-terminal non-catalytic domain of separase binds to the C-terminal tail domain of three homologs of the centromeric protein CENP-E Kinesin 7 (Kin7). Conformational changes of Kin7 induced upon binding to separase facilitate recruitment of Kin7/separase complex (KISC) onto microtubules. KISC operates independently of proteolytic activity of separase in promoting microtubule rescue and pauses, as well as in suppressing catastrophes. Genetic complementation experiments in conditional separase mutant rsw4 background demonstrate the importance of KISC for the establishment of cell polarity and for plant development. Our study establishes a mechanism governing microtubule dynamics via the separase-dependent activation of CENP-E-related kinesins.
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| 43. |
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| 44. |
- Moschou, Panagiotis Nikolaou, et al.
(author)
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Separases: biochemistry and function
- 2012
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In: Physiologia Plantarum. - : Wiley. - 0031-9317 .- 1399-3054. ; 145, s. 67-76
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Journal article (peer-reviewed)abstract
- 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.
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| 45. |
- Moschou, Panagiotis Nikolaou, et al.
(author)
-
Stress-related biomolecular condensates in plants
- 2023
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In: Plant Cell. - 1040-4651 .- 1532-298X. ; 35, s. 3187-3204
-
Research review (peer-reviewed)abstract
- This review describes the mechanism, regulation, composition, and properties of stress-related biomolecular condensates in plants.Biomolecular condensates are membraneless organelle-like structures that can concentrate molecules and often form through liquid-liquid phase separation. Biomolecular condensate assembly is tightly regulated by developmental and environmental cues. Although research on biomolecular condensates has intensified in the past 10 years, our current understanding of the molecular mechanisms and components underlying their formation remains in its infancy, especially in plants. However, recent studies have shown that the formation of biomolecular condensates may be central to plant acclimation to stress conditions. Here, we describe the mechanism, regulation, and properties of stress-related condensates in plants, focusing on stress granules and processing bodies, 2 of the most well-characterized biomolecular condensates. In this regard, we showcase the proteomes of stress granules and processing bodies in an attempt to suggest methods for elucidating the composition and function of biomolecular condensates. Finally, we discuss how biomolecular condensates modulate stress responses and how they might be used as targets for biotechnological efforts to improve stress tolerance.
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| 46. |
- Moschou, Panagiotis Nikolaou, et al.
(author)
-
The Caspase-Related Protease Separase (EXTRA SPINDLE POLES) Regulates Cell Polarity and Cytokinesis in Arabidopsis
- 2013
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In: Plant Cell. - : Oxford University Press (OUP). - 1040-4651 .- 1532-298X. ; 25, s. 2171-2186
-
Journal article (peer-reviewed)abstract
- Vesicle trafficking plays an important role in cell division, establishment of cell polarity, and translation of environmental cues to developmental responses. However, the molecular mechanisms regulating vesicle trafficking remain poorly understood. Here, we report that the evolutionarily conserved caspase-related protease separase (EXTRA SPINDLE POLES [ ESP]) is required for the establishment of cell polarity and cytokinesis in Arabidopsis thaliana. At the cellular level, separase colocalizes with microtubules and RabA2a (for RAS GENES FROM RAT BRAINA2a) GTPase-positive structures. Separase facilitates polar targeting of the auxin efflux carrier PIN-FORMED2 (PIN2) to the rootward side of the root cortex cells. Plants with the radially swollen4 (rsw4) allele with compromised separase activity, in addition to mitotic failure, display isotropic cell growth, perturbation of auxin gradient formation, slower gravitropic response in roots, and cytokinetic failure. Measurements of the dynamics of vesicle markers on the cell plate revealed an overall reduction of the delivery rates of KNOLLE and RabA2a GTPase in separase-deficient roots. Furthermore, dissociation of the clathrin light chain, a protein that plays major role in the formation of coated vesicles, was slower in rsw4 than in the control. Our results demonstrate that separase is a key regulator of vesicle trafficking, which is indispensable for cytokinesis and the establishment of cell polarity.
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| 47. |
- Ohlsson, Jonas A., et al.
(author)
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SPIRO : the automated Petri plate imaging platform designed by biologists, for biologists
- 2024
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In: The Plant Journal. - : John Wiley & Sons. - 0960-7412 .- 1365-313X. ; 118:2, s. 584-600
-
Journal article (peer-reviewed)abstract
- Phenotyping of model organisms grown on Petri plates is often carried out manually, despite the procedures being time-consuming and laborious. The main reason for this is the limited availability of automated phenotyping facilities, whereas constructing a custom automated solution can be a daunting task for biologists. Here, we describe SPIRO, the Smart Plate Imaging Robot, an automated platform that acquires time-lapse photographs of up to four vertically oriented Petri plates in a single experiment, corresponding to 192 seedlings for a typical root growth assay and up to 2500 seeds for a germination assay. SPIRO is catered specifically to biologists' needs, requiring no engineering or programming expertise for assembly and operation. Its small footprint is optimized for standard incubators, the inbuilt green LED enables imaging under dark conditions, and remote control provides access to the data without interfering with sample growth. SPIRO's excellent image quality is suitable for automated image processing, which we demonstrate on the example of seed germination and root growth assays. Furthermore, the robot can be easily customized for specific uses, as all information about SPIRO is released under open-source licenses. Importantly, uninterrupted imaging allows considerably more precise assessment of seed germination parameters and root growth rates compared with manual assays. Moreover, SPIRO enables previously technically challenging assays such as phenotyping in the dark. We illustrate the benefits of SPIRO in proof-of-concept experiments which yielded a novel insight on the interplay between autophagy, nitrogen sensing, and photoblastic response.
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| 48. |
- Ortiz Rios, Rodomiro Octavio, et al.
(author)
-
Oil crops for the future
- 2020
-
In: Current Opinion in Plant Biology. - : Elsevier BV. - 1369-5266 .- 1879-0356. ; 56, s. 181-189
-
Research review (peer-reviewed)abstract
- Agriculture faces enormous challenges including the need to substantially increase productivity, reduce environmental footprint, and deliver renewable alternatives that are being addressed by developing new oil crops for the future. The efforts include domestication of Lepidium spp. using genomics-aided breeding as a cold hardy perennial high-yielding oil crop that provides substantial environmental benefits, expands the geography for oil crops, and improves farmers’ economy. In addition, genetic engineering in Crambe abyssinica may lead to a dedicated industrial oil crop to replace fossil oil. Redirection of photosynthates from starch to oil in plant tubers and cereal endosperm also provides a path for enhancing oil production to meet the growing demands for food, fuel, and biomaterials. Insect pheromone components are produced in seed oil plants in a cost-effective and environmentally friendly pest management replacing synthetically produced pheromones. Autophagy is explored for increasing crop fitness and oil accumulation using genetic engineering in Arabidopsis.
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| 49. |
- Reza, Salim Hossain, et al.
(author)
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Transcriptome analysis of embryonic domains in Norway spruce reveals potential regulators of suspensor cell death
- 2018
-
In: PLOS ONE. - San Francisco : Public Library of Science. - 1932-6203. ; 13:3
-
Journal article (peer-reviewed)abstract
- The terminal differentiation and elimination of the embryo-suspensor is the earliest manifestation of programmed cell death (PCD) during plant ontogenesis. Molecular regulation of suspensor PCD remains poorly understood. Norway spruce (Picea abies) embryos provide a powerful model for studying embryo development because of their large size, sequenced genome, and the possibility to obtain a large number of embryos at a specific developmental stage through somatic embryogenesis. Here, we have carried out global gene expression analysis of the Norway spruce embryo-suspensor versus embryonal mass (a gymnosperm analogue of embryo proper) using RNA sequencing. We have identified that suspensors have enhanced expression of the NAC domain-containing transcription factors, XND1 and ANAC075, previously shown to be involved in the initiation of developmental PCD in Arabidiopsis. The analysis has also revealed enhanced expression of Norway spruce homologues of the known executioners of both developmental and stress-induced cell deaths, such as metacaspase 9 (MC9), cysteine endopeptidase-1 (CEP1) and ribonuclease 3 (RNS3). Interestingly, a spruce homologue of bax inhibitor-1 (PaBI-1, for Picea abies BI-1), an evolutionarily conserved cell death suppressor, was likewise up-regulated in the embryosuspensor. Since Arabidopsis BI-1 so far has been implicated only in the endoplasmic reticulum (ER)-stress induced cell death, we investigated its role in embryogenesis and suspensor PCD using RNA interference (RNAi). We have found that PaBI-1-deficient lines formed a large number of abnormal embryos with suppressed suspensor elongation and disturbed polarity. Cytochemical staining of suspensor cells has revealed that PaBI-1 deficiency suppresses vacuolar cell death and induces necrotic type of cell death previously shown to compromise embryo development. This study demonstrates that a large number of cell-death components are conserved between angiosperms and gymnosperms and establishes a new role for BI-1 in the progression of vacuolar cell death.
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| 50. |
- Sabljić, Igor, et al.
(author)
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Expression and purification of the type II metacaspase from a unicellular green alga Chlamydomonas reinhardtii
- 2022
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In: Plant proteases and plant cell death. - New York, NY : Humana Press. - 9781071620786 - 9781071620793 ; , s. 13-20
-
Book chapter (peer-reviewed)abstract
- Type II metacaspases (MCAs) are proteases, belonging to the C14B MEROPS family. Like the MCAs of type I and type III, they preferentially cleave their substrates after the positively charged amino acid residues (Arg or Lys) at the P1 position. Type II MCAs from various higher plants have already been successfully overexpressed in E. coli mostly as His-tagged proteins and were shown to be proteolytically active after the purification. Here we present a protocol for expression and purification of the only type II MCA from the model green alga Chlamydomonas reinhardtii. The two-step purification, which consists of immobilized metal affinity chromatography using cobalt as ion followed by size-exclusion chromatography, can be performed in 1 day and yields 4 mg CrMCA-II protein per liter of overexpression culture.
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