ABSTRACT
The CDC48 protein, highly conserved in the living kingdom, is a player of the ubiquitin proteasome system and contributes to various cellular processes. In plants, CDC48 is involved in cell division, plant growth and, as recently highlighted in several reports, in plant immunity. In the present study, to further extend our knowledge about CDC48 functions in plants, we analysed the incidence of its overexpression on tobacco development and immune responses. CDC48 overexpression disrupted plant development and morphology, induced changes in plastoglobule appearance and exacerbated ROS production. In addition, levels of salicylic acid (SA) and glycosylated SA were higher in transgenic plants, both in the basal state and in response to cryptogein, a protein produced by the oomycete Phytophthora cryptogea triggering defence responses. The expression of defence genes, notably those coding for some pathogenesis-related (PR) proteins, was also exacerbated in the basal state in transgenic plant lines. Finally, tobacco plants overexpressing CDC48 did not develop necrosis in response to tobacco mosaic virus (TMV) infection, suggesting a role for CDC48 in virus resistance.
Subject(s)
Nicotiana , Plant Immunity , Plant Proteins , Plants, Genetically Modified , Nicotiana/genetics , Nicotiana/virology , Nicotiana/immunology , Nicotiana/metabolism , Plant Proteins/metabolism , Plant Proteins/genetics , Valosin Containing Protein/metabolism , Valosin Containing Protein/genetics , Plant Diseases/virology , Plant Diseases/immunology , Salicylic Acid/metabolism , Gene Expression Regulation, Plant , Reactive Oxygen Species/metabolism , Fungal Proteins/metabolism , Fungal Proteins/genetics , Tobacco Mosaic Virus/physiology , Phytophthora/physiology , Phytophthora/pathogenicityABSTRACT
Protein homeostasis, namely the balance between protein synthesis and degradation, must be finely controlled to ensure cell survival, notably through the ubiquitin-proteasome system (UPS). In all species, including plants, homeostasis is disrupted by biotic and abiotic stresses. A key player in the maintenance of protein balance, the protein CDC48, shows emerging functions in plants, particularly in response to biotic stress. In this review on CDC48 in plants, we detail its highly conserved structure, describe a gene expansion that is only present in Viridiplantae, discuss its various functions and regulations, and finally highlight its recruitment, still not clear, during the plant immune response.
Subject(s)
Plant Immunity , Plant Proteins , Plant Proteins/metabolism , Plant Proteins/genetics , Valosin Containing Protein/metabolism , Valosin Containing Protein/genetics , Plants/immunology , Plants/metabolism , Plants/geneticsABSTRACT
Ectopic expression of defensins in plants correlates with their increased capacity to withstand abiotic and biotic stresses. This applies to Arabidopsis thaliana, where some of the seven members of the PLANT DEFENSIN 1 family (AtPDF1) are recognised to improve plant responses to necrotrophic pathogens and increase seedling tolerance to excess zinc (Zn). However, few studies have explored the effects of decreased endogenous defensin expression on these stress responses. Here, we carried out an extensive physiological and biochemical comparative characterization of (i) novel artificial microRNA (amiRNA) lines silenced for the five most similar AtPDF1s, and (ii) a double null mutant for the two most distant AtPDF1s. Silencing of five AtPDF1 genes was specifically associated with increased aboveground dry mass production in mature plants under excess Zn conditions, and with increased plant tolerance to different pathogens - a fungus, an oomycete and a bacterium, while the double mutant behaved similarly to the wild type. These unexpected results challenge the current paradigm describing the role of PDFs in plant stress responses. Additional roles of endogenous plant defensins are discussed, opening new perspectives for their functions.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Stress, Physiological/genetics , Zinc/metabolism , Defensins/genetics , Defensins/metabolism , Defensins/pharmacology , Gene Expression Regulation, Plant , Plant Diseases/geneticsABSTRACT
Microalgae have recently emerged as a key research topic, especially as biological models. Among them, the green alga Klebsormidium nitens, thanks to its particular adaptation to environmental stresses, represents an interesting photosynthetic eukaryote for studying the transition stages leading to the colonization of terrestrial life. The tolerance to different stresses is manifested by changes in gene expression, which can be monitored by quantifying the amounts of transcripts by RT-qPCR. The identification of optimal reference genes for experiment normalization was therefore necessary. In this study, using four statistical algorithms followed by the RankAggreg package, we determined the best reference gene pairs suitable for normalizing RT-qPCR data in K. nitens in response to three abiotic stresses: high salinity, PEG-induced dehydration and heat shock. Based on these reference genes, we were able to identify marker genes in response to the three abiotic stresses in K. nitens.
Subject(s)
Gene Expression Regulation, Plant , Streptophyta , Stress, Physiological/genetics , Streptophyta/genetics , Genes, Plant , Salinity , Real-Time Polymerase Chain Reaction , Reference Standards , Gene Expression ProfilingABSTRACT
Nitric oxide (NO) is a multifunctional gaseous signal that modulates the growth, development and stress tolerance of higher plants. NO donors have been used to boost plant endogenous NO levels and to activate NO-related responses, but this strategy is often hindered by the relative instability of donors. Alternatively, nanoscience offers a new, promising way to enhance NO delivery to plants, as NO-releasing nanomaterials (e.g. S-nitrosothiol-containing chitosan nanoparticles) have many beneficial physicochemical and biochemical properties compared to non-encapsulated NO donors. Nano NO donors are effective in increasing tissue NO levels and enhancing NO effects both in animal and human systems. The authors believe, and would like to emphasize, that new trends and technologies are essential for advancing plant NO research and nanotechnology may represent a breakthrough in traditional agriculture and environmental science. Herein, we aim to draw the attention of the scientific community to the potential of NO-releasing nanomaterials in both basic and applied plant research as alternatives to conventional NO donors, providing a brief overview of the current knowledge and identifying future research directions. We also express our opinion about the challenges for the application of nano NO donors, such as the environmental footprint and stakeholder's acceptance of these materials.
Subject(s)
Chitosan , Nitric Oxide , Agriculture , Animals , Biotechnology , Nanotechnology , PlantsABSTRACT
Tyrosine-specific protein tyrosine phosphatases (Tyr-specific PTPases) are key signaling enzymes catalyzing the removal of the phosphate group from phosphorylated tyrosine residues on target proteins. This post-translational modification notably allows the regulation of mitogen-activated protein kinase (MAPK) cascades during defense reactions. Arabidopsis thaliana protein tyrosine phosphatase 1 (AtPTP1), the only Tyr-specific PTPase present in this plant, acts as a repressor of H2O2 production and regulates the activity of MPK3/MPK6 MAPKs by direct dephosphorylation. Here, we report that recombinant histidine (His)-AtPTP1 protein activity is directly inhibited by H2O2 and nitric oxide (NO) exogenous treatments. The effects of NO are exerted by S-nitrosation, i.e., the formation of a covalent bond between NO and a reduced cysteine residue. This post-translational modification targets the catalytic cysteine C265 and could protect the AtPTP1 protein from its irreversible oxidation by H2O2. This mechanism of protection could be a conserved mechanism in plant PTPases.
ABSTRACT
Calcium is a ubiquitous second messenger regulating numbers of cellular processes in living organisms. It encodes and transmits information perceived by cells to downstream sensors, including calmodulin (CaM), that initiate cellular responses. In plants, CaM has been involved in the regulation of plant responses to biotic and abiotic environmental cues. Plant CaMs possess a cysteine residue in their first calcium-binding motif EF-hand, which is not conserved in other eucaryotic organisms. In this work, we report the near-complete backbone chemical shift assignment of tobacco CaM2 with calcium. These results will be useful to study the impact of this particular EF-hand domain regarding CaM interaction with partners involved in stress responses.
Subject(s)
Calmodulin , Nicotiana , Calcium/metabolism , Calmodulin/metabolism , Magnetic Resonance Spectroscopy , Nuclear Magnetic Resonance, Biomolecular , Nicotiana/metabolismABSTRACT
The degradation of misfolded proteins is mainly mediated by the ubiquitin-proteasome system (UPS). UPS can be assisted by the protein Cdc48 but the relationship between UPS and Cdc48 in plants has been poorly investigated. Here, we analysed the regulation of UPS by Cdc48 in tobacco thanks to two independent cell lines overexpressing Cdc48 constitutively and plant leaves overexpressing Cdc48 transiently. In the cell lines, the accumulation of ubiquitinated proteins was affected both quantitatively and qualitatively and the number of proteasomal subunits was modified, while proteolytic activities were unchanged. Similarly, the over-expression of Cdc48 in planta impacted the accumulation of ubiquitinated proteins. A similar process occurred in leaves overexpressing transiently Rpn3, a proteasome subunit. Cdc48 being involved in plant immunity, its regulation of UPS was also investigated in response to cryptogein, an elicitor of immune responses. In the cell lines stably overexpressing Cdc48 and in leaves transiently overexpressing Cdc48 and/or Rpn3, cryptogein triggered a premature cell death while no increase of the proteasomal activity occurred. Overall, this study highlights a role for Cdc48 in ubiquitin homeostasis and confirms its involvement, as well as that of Rpn3, in the processes underlying the hypersensitive response.
Subject(s)
Nicotiana/metabolism , Plant Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Ubiquitin/metabolism , Valosin Containing Protein/metabolism , Fungal Proteins/pharmacology , Plant Immunity , Plant Proteins/genetics , Plants, Genetically Modified , Nicotiana/cytology , Nicotiana/drug effects , Ubiquitinated Proteins/metabolism , Valosin Containing Protein/geneticsABSTRACT
In animals, NO is synthesized from L-arginine by three isoforms of nitric oxide synthase (NOS) enzyme. NO production and effects have also been reported in plants but the identification of its sources, especially the enzymatic ones, remains one of the critical issues in the field. NOS-like activities have been reported, although there are no homologs of mammalian NOS in the land plant genomes sequenced so far. However, several NOS homologs have been found in algal genomes and transcriptomes. A first study has characterized a functional NOS in the chlorophyte Ostreococcus tauri and the presence of NOS homologs was later confirmed in a dozen algae. These results raise the questions of the significance of the presence of NOS and their molecular diversity in algae. We hypothesize that comparisons among protein structures of the two KnNOS, together with the identification of their interacting partner proteins, might allow a better understanding of the molecular diversification and functioning of NOS in different physiological contexts and, more generally, new insights into NO signaling in photosynthetic organisms. We recently identified two NOS homologs sequences in the genome of the streptophyte Klebsormidium nitens, a model alga in the study of plant adaptation to terrestrial life. The first sequence, named KnNOS1, contains canonical NOS signatures while the second, named KnNOS2, presents a large C-ter extension including a globin domain. In order to identify putative candidates for KnNOSs partner proteins, we draw the protein-protein interaction networks of the three human NOS using the BioGRID database and hypothesized on the biological role of K. nitens orthologs. Some of these conserved partners are known to be involved in mammalian NOSs regulation and functioning. In parallel, our methodological strategy for the identification of partner proteins of KnNOS1 and KnNOS2 by in vitro pull-down assay is presented.
ABSTRACT
Nitric oxide (NO) was the first identified gaseous messenger and is now well established as a major ubiquitous signalling molecule. The rapid development of our understanding of NO biology in embryophytes came with the partial characterization of the pathways underlying its production and with the decrypting of signalling networks mediating its effects. Notably, the identification of proteins regulated by NO through nitrosation greatly enhanced our perception of NO functions. In comparison, the role of NO in algae has been less investigated. Yet, studies in Chlamydomonas reinhardtii have produced key insights into NO production through the identification of NO-forming nitrite reductase and of S-nitrosated proteins. More intriguingly, in contrast to embryophytes, a few algal species possess a conserved nitric oxide synthase, the main enzyme catalysing NO synthesis in metazoans. This latter finding paves the way for a deeper characterization of novel members of the NO synthase family. Nevertheless, the typical NO-cyclic GMP signalling module transducing NO effects in metazoans is not conserved in algae, nor in embryophytes, highlighting a divergent acquisition of NO signalling between the green and the animal lineages.
Subject(s)
Chlorophyta/metabolism , Nitric Oxide Synthase , Nitric Oxide , Cyclic GMP , Nitric Oxide Synthase/metabolism , Nitrites , Signal TransductionABSTRACT
Nitric oxide (NO) is essential for plant growth and development, as well as interactions with abiotic and biotic environments. Its importance for multiple functions in plants means that tight regulation of NO concentrations is required. This is of particular significance in roots, where NO signalling is involved in processes, such as root growth, lateral root formation, nutrient acquisition, heavy metal homeostasis, symbiotic nitrogen fixation and root-mycorrhizal fungi interactions. The NO signal can also be produced in high levels by microbial processes in the rhizosphere, further impacting root processes. To explore these interesting interactions, in the present review, we firstly summarize current knowledge of physiological processes of NO production and consumption in roots and, thereafter, of processes involved in NO homeostasis in root cells with particular emphasis on root growth, development, nutrient acquisition, environmental stresses and organismic interactions.
Subject(s)
Nitric Oxide/physiology , Plant Roots/growth & development , Atmosphere , Nitric Oxide/metabolism , Plant Development , Plant Physiological Phenomena , Plant Roots/metabolism , Plant Roots/physiology , Plants/metabolismABSTRACT
Nitric oxide (NO) is perfectly suited for the role of a redox signalling molecule. A key route for NO bioactivity occurs via protein S-nitrosation, and involves the addition of a NO moiety to a protein cysteine (Cys) thiol (-SH) to form an S-nitrosothiol (SNO). This process is thought to underpin a myriad of cellular processes in plants that are linked to development, environmental responses and immune function. Here we collate emerging evidence showing that NO bioactivity regulates a growing number of diverse post-translational modifications including SUMOylation, phosphorylation, persulfidation and acetylation. We provide examples of how NO orchestrates these processes to mediate plant adaptation to a variety of cellular cues.
Subject(s)
Nitric Oxide , S-Nitrosothiols , Nitric Oxide/metabolism , Nitrosation , Oxidation-Reduction , Plants/metabolism , Protein Processing, Post-TranslationalABSTRACT
Among Plant Protection Products (PPP), a new emerging category of pesticides act by stimulating plant defense in order to improve plant resistance against microbial pathogens. Given that these compounds, the so-called Plant Defense Stimulators (PDS) act on innate immunity, we tested, using an in vitro approach on human mononuclear leucocytes (PBMC), the potential toxicity (XTT assay) and inflammatory effects (production of IL-1ß) of 4 PPP belonging to different chemical families. We found that two products (LBG-01F34® and Regalis®) did not induce any cytotoxicity or IL-1 ß production. The product BION-50 WG®, that contains Acibenzolar-S-methyl (ASM) and silica particles did not present any cytotoxicity but induced a significant increase in the production of the inflammatory cytokine IL-1 ß. Finally, Vacciplant® that contains laminarin, was highly cytotoxic and pro-inflammatory. It induced a strong production of IL-1 ß when used at a concentration in the culture medium, as low as 0.02 mg/mL. We also tested the potential toxic effect of these 4 PPP on 4 days old zebra fish larvae. After 24 h of exposure, our results indicate that Vacciplant® induced zebra fish larvae mortality at concentration of 20 µg/mL. LBG did not induced significant mortality at concentrations up to 1 mg/mL whereas Regalis was lethal for 0,3 mg/mL concentrations and BION-50 WG began to induce mortality at 2,5 mg/mL. Our results indicate possible effects of PDS on IL-1ß production in human cells and fish survival, calling for more studies on the potential noxious side effects of these compounds.
ABSTRACT
Both plants and animals are endowed with sophisticated innate immune systems to combat microbial attack. In these multicellular eukaryotes, innate immunity implies the presence of cell surface receptors and intracellular receptors able to detect danger signal referred as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). Membrane-associated pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), C-type lectin receptors (CLRs), receptor-like kinases (RLKs), and receptor-like proteins (RLPs) are employed by these organisms for sensing different invasion patterns before triggering antimicrobial defenses that can be associated with a form of regulated cell death. Intracellularly, animals nucleotide-binding and oligomerization domain (NOD)-like receptors or plants nucleotide-binding domain (NBD)-containing leucine rich repeats (NLRs) immune receptors likely detect effectors injected into the host cell by the pathogen to hijack the immune signaling cascade. Interestingly, during the co-evolution between the hosts and their invaders, key cross-kingdom cell death-signaling macromolecular NLR-complexes have been selected, such as the inflammasome in mammals and the recently discovered resistosome in plants. In both cases, a regulated cell death located at the site of infection constitutes a very effective mean for blocking the pathogen spread and protecting the whole organism from invasion. This review aims to describe the immune mechanisms in animals and plants, mainly focusing on cell death signaling pathways, in order to highlight recent advances that could be used on one side or the other to identify the missing signaling elements between the perception of the invasion pattern by immune receptors, the induction of defenses or the transmission of danger signals to other cells. Although knowledge of plant immunity is less advanced, these organisms have certain advantages allowing easier identification of signaling events, regulators and executors of cell death, which could then be exploited directly for crop protection purposes or by analogy for medical research.
Subject(s)
Cell Death/immunology , Plant Cells/immunology , Plant Immunity/immunology , Plants/immunology , Receptors, Immunologic/immunology , Signal Transduction/immunology , Animals , Humans , Immunity, Innate/immunology , Inflammasomes/immunology , Receptors, Pattern Recognition/immunologyABSTRACT
Nitric oxide (NO) emerged as a key signal molecule in plants. During the last two decades impressive progress has been made in plant NO research. This small, redox-active molecule is now known to play an important role in plant immunity, stress responses, environmental interactions, plant growth and development. To more accurately and robustly establish the full spectrum of NO bioactivity in plants, it will be essential to apply methodological best practice. In addition, there are some instances of conflicting nomenclature within the field, which would benefit from standardization. In this context, we attempt to provide some helpful guidance for best practice associated with NO research and also suggestions for the cognate terminology.
Subject(s)
Nitric Oxide , Plants , Plant DevelopmentABSTRACT
There is increasing evidence that the chaperone-like protein CDC48 (cell division cycle 48) plays a role in plant immunity. Cytosolic ascorbate peroxidase (cAPX), which is a major regulator of the redox status of plant cells, has previously been shown to interact with CDC48. In this study, we examined the regulation of cAPX by the ATPase NtCDC48 during the cryptogein-induced immune response in tobacco cells. Our results not only confirmed the interaction between the proteins but also showed that it occurs in the cytosol. cAPX accumulation was modified in cells overexpressing NtCDC48, a process that was shown to involve post-translational modification of cAPX. In addition, cryptogein-induced increases in cAPX activity were suppressed in cells overexpressing NtCDC48 and the abundance of the cAPX dimer was below the level of detection. Furthermore, the levels of both reduced (GSH) and oxidized glutathione (GSSG) and the GSH/GSSG ratio decreased more rapidly in response to the elicitor in these cells than in controls. A decrease in cAPX activity was also observed in response to heat shock in the cells overexpressing NtCDC48, indicating that the regulation of cAPX by NtCDC48 is not specific to the immune response.
Subject(s)
Ascorbate Peroxidases/genetics , Gene Expression Regulation, Plant , Nicotiana/genetics , Valosin Containing Protein/genetics , Ascorbate Peroxidases/metabolism , Cytosol/metabolism , Molecular Chaperones/metabolism , Nicotiana/enzymology , Valosin Containing Protein/metabolismABSTRACT
Nitric oxide (NO) is a ubiquitous signalling molecule with widespread distribution in prokaryotes and eukaryotes where it is involved in countless physiological processes. While the mechanisms governing nitric oxide (NO) synthesis and signalling are well established in animals, the situation is less clear in the green lineage. Recent investigations have shown that NO synthase, the major enzymatic source for NO in animals, is absent in land plants but present in a limited number of algae. The first detailed analysis highlighted that these new NO synthases are functional but display specific structural features and probably original catalytic activities. Completing this picture, analyses were undertaken in order to investigate whether major components of the prototypic NO/cyclic GMP signalling cascades mediating many physiological effects of NO in animals were also present in plants. Only a few homologues of soluble guanylate cyclases, cGMP-dependent protein kinases, cyclic nucleotide-gated channels, and cGMP-regulated phosphodiesterases were identified in some algal species and their presence did not correlate with that of NO synthases. In contrast, S-nitrosoglutathione reductase, a critical regulator of S-nitrosothiols, was recurrently found. Overall, these findings highlight that plants do not mediate NO signalling through the classical NO/cGMP signalling module and support the concept that S-nitrosation is a ubiquitous NO-dependent signalling mechanism.
Subject(s)
Evolution, Molecular , Nitric Oxide/metabolism , Plants/metabolism , Signal Transduction , Animals , Cyclic GMP , Nitric Oxide Synthase/metabolismABSTRACT
The evolutionally conserved chaperone-like protein CDC48 (cell division cycle 48) is a major component of ubiquitin-dependent protein degradation pathways in animal and yeast and, more generally, of the protein quality control machinery. In plants, CDC48 plays essential regulatory functions in development and the possibly that it contributes to protein degradation through the ubiquitin-proteasome system (UPS) and the endoplasmic reticulum-associated protein degradation (ERAD) system has been reported. In this review we described recent findings highlighting a role for CDC48 in plant immunity. First data indicated that CDC48 is S-nitrosylated in plant cells undergoing an immune response, regulates the turnover of immune receptors and mediates the degradation of viral proteins. Furthermore its overexpression was associated to an exacerbated hypersensitive-like cell death. We also designed and reported here the first CDC48 interactome. The corresponding data confirm the closed interaction of CDC48 with components of the UPS and shed light on its putative regulatory function of S-adenosyl-methionine synthesis and metabolism. More generally, these investigations further support the concept that plant cells facing pathogen attack finely regulate the protein quality control machinery.
Subject(s)
Plant Immunity , Plant Proteins/physiology , Valosin Containing Protein/physiology , Plant Proteins/metabolism , Plants/immunology , Plants/metabolism , Proteasome Endopeptidase Complex/metabolism , Valosin Containing Protein/metabolismABSTRACT
Type-2 HDACs (HD2s) are plant-specific histone deacetylases that play diverse roles during development and in responses to biotic and abiotic stresses. In this study we characterized the six tobacco genes encoding HD2s that mainly differ by the presence or the absence of a typical zinc finger in their C-terminal part. Of particular interest, these HD2 genes exhibit a highly conserved intron/exon structure. We then further investigated the phylogenetic relationships among the HD2 gene family, and proposed a model of the genetic events that led to the organization of the HD2 family in Solanaceae. Absolute quantification of HD2 mRNAs in N. tabacum and in its precursors, N. tomentosiformis and N. sylvestris, did not reveal any pseudogenization of any of the HD2 genes, but rather specific regulation of HD2 expression in these three species. Functional complementation approaches in Arabidopsis thaliana demonstrated that the four zinc finger-containing HD2 proteins exhibit the same biological function in response to salt stress, whereas the two HD2 proteins without zinc finger have different biological function.