| 1. |
- Alvarez, José M, et al.
(författare)
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The WUSCHEL-RELATED HOMEOBOX 3 gene PaWOX3 regulates lateral organ formation in Norway spruce
- 2015
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Ingår i: New Phytologist. - : Wiley. - 0028-646X .- 1469-8137. ; 208:4, s. 1078-1088
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Tidskriftsartikel (refereegranskat)abstract
- In angiosperms, WUSCHEL-RELATED HOMEOBOX 3 (WOX3) genes are required for the recruitment of founder cells from the lateral domains of shoot meristems that form lateral regions of leaves. However, the regulation of the formation of lateral organs in gymnosperms remains unknown. By using somatic embryos of Norway spruce ( Picea abies) we have studied the expression and function of PaWOX3 during embryo development. The mRNA abundance of PaWOX3 was determined by quantitative real-time PCR, and the spatial expression of PaWOX3 was analysed by histochemical beta-glucuronidase (GUS) assays and in situ mRNA hybridization. To investigate the function of PaWOX3, we analysed how downregulation of PaWOX3 in RNA interference lines affected embryo development and morphology. PaWOX3 was highly expressed in mature embryos at the base of each cotyledon close to the junction between the cotyledons, and in the lateral margins of cotyledons and needles, separating them into an adaxial and an abaxial side. Downregulation of the expression of PaWOX3 caused defects in lateral margin outgrowth in cotyledons and needles, and reduced root elongation. Our data suggest that the WOX3 function in margin outgrowth in lateral organs is conserved among the seed plants, whereas its function in root elongation may be unique to gymnosperms.
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| 2. |
- Bozhkov, Peter, et al.
(författare)
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Concentrating and sequestering biomolecules in condensates: impact on plant biology
- 2023
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Ingår i: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 74, s. 1303-1308
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Tidskriftsartikel (refereegranskat)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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| 3. |
- Bozhkov, Peter, et al.
(författare)
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Transcriptional stimulation of autophagy improves plant fitness
- 2017
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Patent (övrigt vetenskapligt/konstnärligt)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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| 4. |
- Dölfors, Fredrik, et al.
(författare)
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The RsRlpA Effector Is a Protease Inhibitor Promoting Rhizoctonia solani Virulence through Suppression of the Hypersensitive Response
- 2020
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 21
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Tidskriftsartikel (refereegranskat)abstract
- Rhizoctonia solani (Rs) is a soil-borne pathogen with a broad host range. This pathogen incites a wide range of disease symptoms. Knowledge regarding its infection process is fragmented, a typical feature for basidiomycetes. In this study, we aimed at identifying potential fungal effectors and their function. From a group of 11 predicted single gene effectors, a rare lipoprotein A (RsRlpA), from a strain attacking sugar beet was analyzed. The RsRlpA gene was highly induced upon early-stage infection of sugar beet seedlings, and heterologous expression in Cercospora beticola demonstrated involvement in virulence. It was also able to suppress the hypersensitive response (HR) induced by the Avr4/Cf4 complex in transgenic Nicotiana benthamiana plants and functioned as an active protease inhibitor able to suppress Reactive Oxygen Species (ROS) burst. This effector contains a double-psi beta-barrel (DPBB) fold domain, and a conserved serine at position 120 in the DPBB fold domain was found to be crucial for HR suppression. Overall, R. solani seems to be capable of inducing an initial biotrophic stage upon infection, suppressing basal immune responses, followed by a switch to necrotrophic growth. However, regulatory mechanisms between the different lifestyles are still unknown.
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| 5. |
- Elander, Pernilla, et al.
(författare)
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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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Ingår i: The Embo Journal. - : EMBO. - 0261-4189 .- 1460-2075. ; 40
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Tidskriftsartikel (refereegranskat)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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| 6. |
- Gutierrez, Emilio, et al.
(författare)
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Tudor Staphylococcal Nuclease Links Formation of Stress Granules and Processing Bodies with mRNA Catabolism in Arabidopsis
- 2015
-
Ingår i: Plant Cell. - : Oxford University Press (OUP). - 1040-4651 .- 1532-298X. ; 27, s. 926-943
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Tidskriftsartikel (refereegranskat)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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| 7. |
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| 8. |
- Liu, Chen, et al.
(författare)
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A proxitome-RNA-capture approach reveals that processing bodies repress coregulated hub genes
- 2024
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Ingår i: Plant Cell. - 1040-4651 .- 1532-298X. ; 36, s. 59–584-
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Tidskriftsartikel (refereegranskat)abstract
- Cellular condensates are usually ribonucleoprotein assemblies with liquid- or solid-like properties. Because these subcellular structures lack a delineating membrane, determining their compositions is difficult. Here we describe a proximity-biotinylation approach for capturing the RNAs of the condensates known as processing bodies (PBs) in Arabidopsis (Arabidopsis thaliana). By combining this approach with RNA detection, in silico, and high-resolution imaging approaches, we studied PBs under normal conditions and heat stress. PBs showed a much more dynamic RNA composition than the total transcriptome. RNAs involved in cell wall development and regeneration, plant hormonal signaling, secondary metabolism/defense, and RNA metabolism were enriched in PBs. RNA-binding proteins and the liquidity of PBs modulated RNA recruitment, while RNAs were frequently recruited together with their encoded proteins. In PBs, RNAs follow distinct fates: in small liquid-like PBs, RNAs get degraded while in more solid-like larger ones, they are stored. PB properties can be regulated by the actin-polymerizing SCAR (suppressor of the cyclic AMP)-WAVE (WASP family verprolin homologous) complex. SCAR/WAVE modulates the shuttling of RNAs between PBs and the translational machinery, thereby adjusting ethylene signaling. In summary, we provide an approach to identify RNAs in condensates that allowed us to reveal a mechanism for regulating RNA fate.A proxitome-RNA-capture approach captures the transcriptome of the archetypal condensates known as processing bodies and reveals a translational hub based on liquid-liquid phase separation.
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| 9. |
- Liu, Chen, et al.
(författare)
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An actin remodeling role for Arabidopsis processing bodies revealed by their proximity interactome
- 2023
-
Ingår i: EMBO Journal. - : EMBO. - 0261-4189 .- 1460-2075. ; 42
-
Tidskriftsartikel (refereegranskat)abstract
- Cellular condensates can comprise membrane-less ribonucleoprotein assemblies with liquid-like properties. These cellular condensates influence various biological outcomes, but their liquidity hampers their isolation and characterization. Here, we investigated the composition of the condensates known as processing bodies (PBs) in the model plant Arabidopsis thaliana through a proximity-biotinylation proteomics approach. Using in situ protein-protein interaction approaches, genetics and high-resolution dynamic imaging, we show that processing bodies comprise networks that interface with membranes. Surprisingly, the conserved component of PBs, DECAPPING PROTEIN 1 (DCP1), can localize to unique plasma membrane subdomains including cell edges and vertices. We characterized these plasma membrane interfaces and discovered a developmental module that can control cell shape. This module is regulated by DCP1, independently from its role in decapping, and the actin-nucleating SCAR-WAVE complex, whereby the DCP1-SCAR-WAVE interaction confines and enhances actin nucleation. This study reveals an unexpected function for a conserved condensate at unique membrane interfaces.
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| 10. |
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| 11. |
- Liu, Chen, et al.
(författare)
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Establishment of Proximity-Dependent Biotinylation Approaches in Different Plant Model Systems([OPEN])
- 2020
-
Ingår i: Plant Cell. - : Oxford University Press (OUP). - 1040-4651 .- 1532-298X. ; 32, s. 3388-3407
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Tidskriftsartikel (refereegranskat)abstract
- Proximity labeling is a powerful approach for detecting protein-protein interactions. Most proximity labeling techniques use a promiscuous biotin ligase or a peroxidase fused to a protein of interest, enabling the covalent biotin labeling of proteins and subsequent capture and identification of interacting and neighboring proteins without the need for the protein complex to remain intact. To date, only a few studies have reported on the use of proximity labeling in plants. Here, we present the results of a systematic study applying a variety of biotin-based proximity labeling approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols that combine mass spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using Lotus japonicus symbiotically active receptor kinases as a test case. We further benchmark the efficiency of various promiscuous biotin ligases in comparison with one-step affinity purification approaches. We identified both known and novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both nonbiotinylated and biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to suggest structural information of protein complexes.A systematic study applying a variety of biotin-based proximity labeling approaches in several plant systems using various conditions and bait proteins.
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| 12. |
- Liu, Chen, et al.
(författare)
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Phenotypic novelty by CRISPR in plants
- 2018
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Ingår i: Developmental Biology. - : Elsevier BV. - 0012-1606 .- 1095-564X. ; 435, s. 170-175
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Tidskriftsartikel (refereegranskat)abstract
- Genome editing by CRISPR is now routinely used in plant biology for unravelling gene functions and improving agronomical traits. CRISPR opens up the possibility of genome manipulations which would have been unthinkable a few years ago. In this perspective, we discuss and suggest CRISPR-mediated approaches for steering plant development, also highlighting potential challenges.
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| 13. |
- Liu, Chen, et al.
(författare)
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Potassium transporter TRH1/KUP4 contributes to distinct auxin-mediated root system architecture responses
- 2022
-
Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 188, s. 1043-1060
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Tidskriftsartikel (refereegranskat)abstract
- A proton-coupled potassium transporter regulates root hair development and root gravitropism in a cell-file-specific manner by facilitating polar auxin transport in Arabidopsis root tips.In plants, auxin transport and development are tightly coupled, just as hormone and growth responses are intimately linked in multicellular systems. Here we provide insights into uncoupling this tight control by specifically targeting the expression of TINY ROOT HAIR 1 (TRH1), a member of plant high-affinity potassium (K+)/K+ uptake/K+ transporter (HAK/KUP/KT) transporters that facilitate K+ uptake by co-transporting protons, in Arabidopsis root cell files. Use of this system pinpointed specific root developmental responses to acropetal versus basipetal auxin transport. Loss of TRH1 function shows TRHs and defective root gravitropism, associated with auxin imbalance in the root apex. Cell file-specific expression of TRH1 in the central cylinder rescued trh1 root agravitropism, whereas positional TRH1 expression in peripheral cell layers, including epidermis and cortex, restored trh1 defects. Applying a system-level approach, the role of RAP2.11 and ROOT HAIR DEFECTIVE-LIKE 5 transcription factors (TFs) in root hair development was verified. Furthermore, ERF53 and WRKY51 TFs were overrepresented upon restoration of root gravitropism supporting involvement in gravitropic control. Auxin has a central role in shaping root system architecture by regulating multiple developmental processes. We reveal that TRH1 jointly modulates intracellular ionic gradients and cell-to-cell polar auxin transport to drive root epidermal cell differentiation and gravitropic response. Our results indicate the developmental importance of HAK/KUP/KT proton-coupled K+ transporters.
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| 14. |
- Liu, Chen, et al.
(författare)
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Proteolytic Proteoforms: Elusive Components of Hormonal Pathways?
- 2020
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Ingår i: Trends in Plant Science. - : Elsevier BV. - 1360-1385 .- 1878-4372. ; 25, s. 325-328
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Tidskriftsartikel (refereegranskat)abstract
- Hormonal pathways often converge on transcriptional repressors that can be degraded by the proteasome to initiate a response. We wish to draw attention to developments in a less-explored proteolytic branch called 'limited proteolysis' that, in addition to the classical proteolytic pathways, seems to regulate auxin and ethylene signaling.
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| 15. |
- Liu, Chen, et al.
(författare)
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SEC14-like condensate phase transitions at plasma membranes regulate root growth in Arabidopsis
- 2023
-
Ingår i: PLoS biology. - : Public Library of Science (PLoS). - 1544-9173 .- 1545-7885. ; 21:9
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Tidskriftsartikel (refereegranskat)abstract
- Protein function can be modulated by phase transitions in their material properties, which can range from liquid- to solid-like; yet, the mechanisms that drive these transitions and whether they are important for physiology are still unknown. In the model plant Arabidopsis, we show that developmental robustness is reinforced by phase transitions of the plasma membrane-bound lipid-binding protein SEC14-like. Using imaging, genetics, and in vitro reconstitution experiments, we show that SEC14-like undergoes liquid-like phase separation in the root stem cells. Outside the stem cell niche, SEC14-like associates with the caspase-like protease separase and conserved microtubule motors at unique polar plasma membrane interfaces. In these interfaces, SEC14-like undergoes processing by separase, which promotes its liquid-to-solid transition. This transition is important for root development, as lines expressing an uncleavable SEC14-like variant or mutants of separase and associated microtubule motors show similar developmental phenotypes. Furthermore, the processed and solidified but not the liquid form of SEC14-like interacts with and regulates the polarity of the auxin efflux carrier PINFORMED2. This work demonstrates that robust development can involve liquid-to-solid transitions mediated by proteolysis at unique plasma membrane interfaces.
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| 16. |
- Liu, Chen, et al.
(författare)
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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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Annan publikation (övrigt vetenskapligt/konstnärligt)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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| 17. |
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| 18. |
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| 19. |
- Minina, Alyona, et al.
(författare)
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Limited and digestive proteolysis: crosstalk between evolutionary conserved pathways
- 2017
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Ingår i: New Phytologist. - : Wiley. - 0028-646X .- 1469-8137. ; 215, s. 958-964
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Forskningsöversikt (refereegranskat)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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| 20. |
- Minina, Alyona, et al.
(författare)
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The Arabidopsis homolog of Scc4/MAU2 is essential for embryogenesis
- 2017
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 130, s. 1051-1063
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Tidskriftsartikel (refereegranskat)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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| 21. |
- Moschou, Panagiotis Nikolaou
(författare)
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An Epigenetic Alphabet of Crop Adaptation to Climate Change
- 2022
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Ingår i: Frontiers in Genetics. - : Frontiers Media SA. - 1664-8021. ; 13
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Forskningsöversikt (refereegranskat)abstract
- Crop adaptation to climate change is in a part attributed to epigenetic mechanisms which are related to response to abiotic and biotic stresses. Although recent studies increased our knowledge on the nature of these mechanisms, epigenetics remains under-investigated and still poorly understood in many, especially non-model, plants, Epigenetic modifications are traditionally divided into two main groups, DNA methylation and histone modifications that lead to chromatin remodeling and the regulation of genome functioning. In this review, we outline the most recent and interesting findings on crop epigenetic responses to the environmental cues that are most relevant to climate change. In addition, we discuss a speculative point of view, in which we try to decipher the "epigenetic alphabet" that underlies crop adaptation mechanisms to climate change. The understanding of these mechanisms will pave the way to new strategies to design and implement the next generation of cultivars with a broad range of tolerance/resistance to stresses as well as balanced agronomic traits, with a limited loss of (epi)genetic variability.
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| 22. |
- Moschou, Panagiotis Nikolaou
(författare)
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An NADPH-Oxidase/Polyamine Oxidase Feedback Loop Controls Oxidative Burst Under Salinity
- 2016
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 172, s. 1418-1431
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Tidskriftsartikel (refereegranskat)abstract
- The apoplastic polyamine oxidase (PAO) catalyzes the oxidation of the higher polyamines spermidine and spermine, contributing to hydrogen peroxide (H2O2) accumulation. However, it is yet unclear whether apoplastic PAO is part of a network that coordinates the accumulation of reactive oxygen species (ROS) under salinity or if it acts independently. Here, we unravel that NADPH oxidase and apoplastic PAO cooperate to control the accumulation of H2O2 and superoxides (O-2(center dot-)) in tobacco (Nicotiana tabacum). To examine to what extent apoplastic PAO constitutes part of a ROS-generating network, we examined ROS accumulation in guard cells of plants overexpressing or down-regulating apoplastic PAO (lines S2.2 and A2, respectively) or down-regulating NADPH oxidase (line AS-NtRbohD/F). The H2O2-specific probe benzene sulfonyl-H2O2 showed that, under salinity, H2O2 increased in S2.2 and decreased in A2 compared with the wild type. Surprisingly, the O-2(center dot-)-specific probe benzene sulfonyl-So showed that O-2(center dot-) levels correlated positively with that of apoplastic PAO (i.e. showed high and low levels in S2.2 and A2, respectively). By using AS-NtRbohD/F lines and a pharmacological approach, we could show that H2O2 and O-2(center dot-) accumulation at the onset of salinity stress was dependent on NADPH oxidase, indicating that NADPH oxidase is upstream of apoplastic PAO. Our results suggest that NADPH oxidase and the apoplastic PAO form a feed-forward ROS amplification loop, which impinges on oxidative state and culminates in the execution of programmed cell death. We propose that the PAO/NADPH oxidase loop is a central hub in the plethora of responses controlling salt stress tolerance, with potential functions extending beyond stress tolerance.
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| 23. |
- Moschou, Panagiotis Nikolaou
(författare)
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Analyses of Protein Turnover at the Cell Plate by Fluorescence Recovery After Photobleaching During Cytokinesis
- 2022
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Ingår i: Plant Cell Division : Methods and Protocols. - New York, NY : Springer US. - 9781071617434 ; 2382:2382, s. 233-243
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Bokkapitel (refereegranskat)abstract
- Membrane trafficking is central to cell plate construction during plant cytokinesis. Studies on cell plate formation can provide answers to basic biology questions including molecular mechanisms of membrane trafficking, tissue patterning, and cytoskeletal dynamics. Consequently, a detailed understanding of cytokinesis depends on the characterization of molecules that function in the formation, transport, targeting, and fusion of membrane vesicles and delivery of proteins to the developing and maturing plate. This chapter describes a pipeline based on fluorescence recovery after photobleaching (FRAP) to measure and analyze turnover of peripheral or transmembrane proteins on the cell plate. The approach described here can also be applied in other biological contexts.
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| 24. |
- Moschou, Panagiotis Nikolaou, et al.
(författare)
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Characterization of Cytokinetic Mutants Using Small Fluorescent Probes
- 2016
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 9781493931415 ; 1370:1370, s. 199-208
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Bokkapitel (övrigt vetenskapligt/konstnärligt)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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| 25. |
- Moschou, Panagiotis Nikolaou
(författare)
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Deep inside the epigenetic memories of stressed plants
- 2023
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Ingår i: Trends in Plant Science. - : Elsevier BV. - 1360-1385 .- 1878-4372. ; 28, s. 142-153
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Forskningsöversikt (refereegranskat)abstract
- Recent evidence sheds light on the peculiar type of plant intelligence. Plants have developed complex molecular networks that allow them to remember, choose, and make decisions depending on the stress stimulus, although they lack a nervous system. Being sessile, plants can exploit these networks to optimize their resources cost-effectively and maximize their fitness in response to multiple environmental stresses. Even more interesting is the capability to transmit this experience to the next generation(s) through epigenetic modifications that add to the classical genetic inheritance. In this opinion article, we present concepts and perspectives regarding the capabilities of plants to sense, perceive, remember, re-elaborate, respond, and to some extent transmit to their progeny information to adapt more efficiently to climate change.
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| 26. |
- Moschou, Panagiotis Nikolaou
(författare)
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Determination of di-/Polyamine Oxidase Activity in Plants by an In-Gel Spermidine Oxidation Assay
- 2018
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1064-3745 .- 1940-6029. ; 1694, s. 141-147
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Tidskriftsartikel (refereegranskat)abstract
- Diamine and polyamine catabolism controls plant development, resistance to pathogens and stress responses. Diamine and polyamine oxidases control the catabolism of diamines and polyamines, respectively. Two major routes of di-/polyamine catabolism exist: the terminal and the interconverting. The in vitro activity of each route is assayed by the colorimetric or chemiluminescent determination of hydrogen peroxide produced by oxidation of di-/polyamine substrates. However, these assays fail to estimate activity of individual di-/polyamine oxidase isoenzymes. Herein, I describe an assay for the simultaneous in-gel determination of terminal and interconverting di-/polyamine oxidase isoenzyme activities.
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| 27. |
- Moschou, Panagiotis Nikolaou
(författare)
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Do Microplastics Enter Our Food Chain Via Root Vegetables? A Raman Based Spectroscopic Study on Raphanus sativus
- 2021
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Ingår i: Materials. - : MDPI AG. - 1996-1944. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- The outburst of plastic pollution in terrestrial ecosystems poses a potential threat to agriculture and food safety. Studies have already provided evidence for the uptake of plastic microparticles by several plant species, accompanied by numerous developmental effects, using fluorescence labelling techniques. Here, we introduce the implementation of confocal Raman spectroscopy, a label-free method, for the effective detection of microplastics (MPs) accumulation in the roots of a common edible root vegetable plant, Raphanus sativus, after treatment with acrylonitrile butadiene styrene (ABS) powder. We also demonstrate the concomitant occurrence of phenotypic defects in the polymer-treated plants. We anticipate that this work can provide new insights not only into the extent of the impact this widespread phenomenon has on crop plants but also on the methodological requirements to address it.
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| 28. |
- Moschou, Panagiotis Nikolaou, et al.
(författare)
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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
-
Ingår i: New Phytologist. - : Wiley. - 0028-646X .- 1469-8137. ; 212, s. 232-243
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Tidskriftsartikel (refereegranskat)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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| 29. |
- Moschou, Panagiotis Nikolaou
(författare)
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Induced proximity of a TIR signaling domain on a plant-mammalian NLR chimera activates defense in plants
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 117, s. 18832-18839
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Tidskriftsartikel (refereegranskat)abstract
- Plant and animal intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors detect pathogen-derived molecules and activate defense. Plant NLRs can be divided into several classes based upon their N-terminal signaling domains, including TIR (Toll-like, Interleukin-1 receptor, Resistance protein)- and CC (coiled-coil)-NLRs. Upon ligand detection, mammalian NAIP and NLRC4 NLRs oligomerize, forming an inflammasome that induces proximity of its N-terminal signaling domains. Recently, a plant CC-NLR was revealed to form an inflammasome-like hetero-oligomer. To further investigate plant NLR signaling mechanisms, we fused the N-terminal TIR domain of several plant NLRs to the N terminus of NLRC4. Inflammasome-dependent induced proximity of the TIR domain in planta initiated defense signaling. Thus, induced proximity of a plant TIR domain imposed by oligomerization of a mammalian inflammasome is sufficient to activate authentic plant defense. Ligand detection and inflammasome formation is maintained when the known components of the NLRC4 inflammasome is transferred across kingdoms, indicating that NLRC4 complex can robustly function without any additional mammalian proteins. Additionally, we found NADase activity of a plant TIR domain is necessary for plant defense activation, but NADase activity of a mammalian or a bacterial TIR is not sufficient to activate defense in plants.
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| 30. |
- Moschou, Panagiotis Nikolaou
(författare)
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Peroxisomal polyamine oxidase and NADPH-oxidase cross-talk for ROS homeostasis which affects respiration rate in Arabidopsis thaliana
- 2014
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Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- Homeostasis of reactive oxygen species (ROS) in the intracellular compartments is of critical importance as ROS have been linked with nearly all cellular processes and more importantly with diseases and aging. PAs are nitrogenous molecules with an evolutionary conserved role in the regulation of metabolic and energetic status of cells. Recent evidence also suggests that polyamines (PA) are major regulators of ROS homeostasis. In Arabidopsis the backconversion of the PAs spermidine (Spd) and spermine to putrescine and Spd, respectively, is catalyzed by two peroxisomal PA oxidases (AtPAO). However, the physiological role of this pathway remains largely elusive. Here we explore the role of peroxisomal PA backconversion and in particular that catalyzed by the highly expressed AtPAO3 in the regulation of ROS homeostasis and mitochondrial respiratory burst. Exogenous PAs exert an NADPH-oxidase dependent stimulation of oxygen consumption, with Spd exerting the strongest effect. This increase is attenuated by treatment with the NADPH-oxidase blocker diphenyleneiodonium iodide (DPI). Loss-of-function of AtPAO3 gene results to increased NADPH-oxidase-dependent production of superoxide anions (O-2(center dot-)), but not H2O2, which activate the mitochondrial alternative oxidase pathway (AOX). On the contrary, overexpression of AtPAO3 results to an increased but balanced production of both H2O2 and O-2(center dot-) . These results suggest that the ratio of O-2(center dot-)/H2O2 regulates respiratory chain in mitochondria, with PA-dependent production of O-2(center dot-) by NADPH-oxidase tilting the balance of electron transfer chain in favor of the AOX pathway. In addition, AtPAO3 seems to be an important component in the regulating module of ROS homeostasis, while a conserved role for PA backconversion and ROS across kingdoms is discussed.
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| 31. |
- Moschou, Panagiotis Nikolaou
(författare)
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Polyamines and programmed cell death
- 2014
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Ingår i: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 65, s. 1285-1296
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Forskningsöversikt (refereegranskat)abstract
- Polyamines (PAs) have been considered as important molecules for survival. However, evidence reinforces that PAs are also implicated, directly or indirectly, in pathways regulating programmed cell death (PCD). Direct correlation of PAs with cell death refers to their association with particular biological processes, and their physical contact with molecules or structures involved in cell death. Indirectly, PAs regulate PCD through their metabolic derivatives, such as catabolic and interconversion products. Cytotoxic products of PA metabolism are involved in PCD cascades, whereas it remains largely elusive how PAs directly control pathways leading to PCD. In this review, we present and compare advances in PA-dependent PCD in animals and plants.
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| 32. |
- Moschou, Panagiotis Nikolaou, et al.
(författare)
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Separase Promotes Microtubule Polymerization by Activating CENP-E-Related Kinesin Kin7
- 2016
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Ingår i: Developmental Cell. - : Elsevier BV. - 1534-5807 .- 1878-1551. ; 37, s. 350-361
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Tidskriftsartikel (refereegranskat)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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| 33. |
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| 34. |
- Moschou, Panagiotis Nikolaou, et al.
(författare)
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Separases: biochemistry and function
- 2012
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Ingår i: Physiologia Plantarum. - : Wiley. - 0031-9317 .- 1399-3054. ; 145, s. 67-76
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Tidskriftsartikel (refereegranskat)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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| 35. |
- Moschou, Panagiotis Nikolaou
(författare)
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Silencing S-Adenosyl-L-Methionine Decarboxylase (SAMDC) in Nicotiana tabacum Points at a Polyamine-Dependent Trade-Off between Growth and Tolerance Responses
- 2016
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Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Polyamines (PAs) are nitrogenous molecules that are indispensable for cell viability and with an agreed-on role in the modulation of stress responses. Tobacco plants with downregulated SAMDC (AS-SAMDC) exhibit reduced PAs synthesis but normal levels of PA catabolism. We used AS-SAMDC to increase our understanding on the role of PAs in stress responses. Surprisingly, at control conditions AS-SAMDC plants showed increased biomass and altered developmental characteristics, such as increased height and leaf number. On the contrary, during salt stress AS-SAMDC plants showed reduced vigor when compared to the WT. During salt stress, the AS-SAMDC plants although showing compensatory readjustments of the antioxidant machinery and of photosynthetic apparatus, they failed to sustain their vigor. AS-SAMDC sensitivity was accompanied by inability to effectively control H2O2 levels and concentrations of monovalent and divalent cations. In accordance with these findings, we suggest that PAs may regulate the trade-off between growth and tolerance responses.
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| 36. |
- Moschou, Panagiotis Nikolaou, et al.
(författare)
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Stress-related biomolecular condensates in plants
- 2023
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Ingår i: Plant Cell. - 1040-4651 .- 1532-298X. ; 35, s. 3187-3204
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Forskningsöversikt (refereegranskat)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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| 37. |
- Moschou, Panagiotis Nikolaou, et al.
(författare)
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The Caspase-Related Protease Separase (EXTRA SPINDLE POLES) Regulates Cell Polarity and Cytokinesis in Arabidopsis
- 2013
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Ingår i: Plant Cell. - : Oxford University Press (OUP). - 1040-4651 .- 1532-298X. ; 25, s. 2171-2186
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Tidskriftsartikel (refereegranskat)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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| 38. |
- Moschou, Panagiotis Nikolaou
(författare)
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The host exocyst complex is targeted by a conserved bacterial type-III effector that promotes virulence
- 2022
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Ingår i: Plant Cell. - : Oxford University Press (OUP). - 1040-4651 .- 1532-298X.
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Tidskriftsartikel (refereegranskat)abstract
- XopP, a core bacterial effector of Xanthomonas campestris, manipulates the plant exocyst complex bypassing several host defense responses.For most Gram-negative bacteria, pathogenicity largely depends on the type-III secretion system that delivers virulence effectors into eukaryotic host cells. The subcellular targets for the majority of these effectors remain unknown. Xanthomonas campestris, the causal agent of black rot disease of crucifers such as Brassica spp., radish, and turnip, delivers XopP, a highly conserved core-effector protein produced by X. campestris, which is essential for virulence. Here, we show that XopP inhibits the function of the host-plant exocyst complex by direct targeting of Exo70B, a subunit of the exocyst complex, which plays a significant role in plant immunity. XopP interferes with exocyst-dependent exocytosis and can do this without activating a plant NOD-like receptor that guards Exo70B in Arabidopsis. In this way, Xanthomonas efficiently inhibits the host's pathogen-associated molecular pattern (PAMP)-triggered immunity by blocking exocytosis of pathogenesis-related protein-1A, callose deposition, and localization of the FLAGELLIN SENSITIVE2 (FLS2) immune receptor to the plasma membrane, thus promoting successful infection. Inhibition of exocyst function without activating the related defenses represents an effective virulence strategy, indicating the ability of pathogens to adapt to host defenses by avoiding host immunity responses.
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| 39. |
- Törnkvist, Anna, et al.
(författare)
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Proteolysis and nitrogen: emerging insights
- 2019
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Ingår i: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 70, s. 2009-2019
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Tidskriftsartikel (refereegranskat)abstract
- Nitrogen (N) is a core component of fertilizers used in modern agriculture to increase yields and thus to help feed a growing global population. However, this comes at a cost to the environment, through run-off of excess N as a result of poor N-use efficiency (NUE) by crops. An obvious remedy to this problem would therefore be the improvement of NUE, which requires advancing our understanding on N homeostasis, sensing, and uptake. Proteolytic pathways are linked to N homeostasis as they recycle proteins that contain N and carbon; however, emerging data suggest that their functions extend beyond this simple recycling. Here, we highlight roles of proteolytic pathways in non-symbiotic and symbiotic N uptake and in systemic N sensing. We also offer a novel view in which we suggest that proteolytic pathways have roles in N homeostasis that differ from their accepted function in recycling.
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| 40. |
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| 41. |
- Zhu, Tianqing, et al.
(författare)
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WUSCHEL-RELATED HOMEOBOX 2 is important for protoderm and suspensor development in the gymnosperm Norway spruce
- 2016
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Ingår i: BMC Plant Biology. - : Springer Science and Business Media LLC. - 1471-2229. ; 16
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Tidskriftsartikel (refereegranskat)abstract
- Background: Distinct expression domains of WUSCHEL-RELATED HOMEOBOX (WOX) gene family members are involved in patterning and morphogenesis of the early embryo in Arabidopsis. However, the role of WOX genes in other taxa, including gymnosperms, remains elusive. Here, we use somatic embryos and reverse genetics for studying expression and function of PaWOX2, the corresponding homolog of AtWOX2 in the gymnosperm Picea abies (Pa; Norway spruce). Results: The mRNA level of PaWOX2 was transiently up-regulated during early and late embryogeny. PaWOX2 mRNA in early and early late embryos was detected both in the embryonal mass and in the upper part of the suspensor. Down-regulation of PaWOX2 during development of early embryos resulted in aberrant early embryos, which failed to form a proper protoderm. Cells on the surface layer of the embryonal mass became vacuolated, and new embryogenic tissue differentiated from the embryonal mass. In addition, the aberrant early embryos lacked a distinct border between the embryonal mass, and the suspensor and the length of the suspensor cells was reduced. Down-regulation of PaWOX2 in the beginning of embryo development, before late embryos were formed, caused a significant decrease in the yield of mature embryos. On the contrary, down-regulation of PaWOX2 after late embryos were formed had no effect on further embryo development and maturation. Conclusions: Our data suggest an evolutionarily conserved function of WOX2 in protoderm formation early during embryo development among seed plants. In addition, PaWOX2 might exert a unique function in suspensor expansion in gymnosperms.
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| 42. |
- Zhu, Tianqing, et al.
(författare)
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WUSCHEL-RELATED HOMEOBOX 8/9 is important for proper embryo patterning in the gymnosperm Norway spruce
- 2014
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Ingår i: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 65, s. 6543-6552
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Tidskriftsartikel (refereegranskat)abstract
- Proper embryo development is crucial as that is when the primary body axes are established. In Arabidopsis, AtWOX8 and AtWOX9, members of the WUSCHEL-RELATED HOMEOBOX (WOX) gene family, are critical for embryo development. In Norway spruce, PaWOX8/9, which is expressed in embryos, is the homologue of AtWOX8 and AtWOX9. In this work, it is shown that the transcript abundance of PaWOX8/9 is high during early and late embryogeny and that it decreases when the maturation phase starts. To address the function of PaWOX8/9 during embryo development, RNAi lines were established to down-regulate the transcript level of PaWOX8/9, using both constitutive and inducible promoters. Embryos in the PaWOX8/9 RNAi lines show an aberrant morphology caused by disturbed orientation of the cell division plane at the basal part of the embryonal mass during early and late embryogeny. In addition, the transcript level of several key cell-cycle-regulating genes, for example, PaE2FAB-LIKE and PaCYCLIN B-LIKE, are affected in the PaWOX8/9 RNAi lines. Taken together, our results suggest that PaWOX8/9 may perform an evolutionarily conserved function as a regulator of the establishment of the apical-basal embryo pattern.
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