128 research outputs found
Lazarus1, a DUF300 Protein, Contributes to Programmed Cell Death Associated with Arabidopsis acd11 and the Hypersensitive Response
Programmed cell death (PCD) is a necessary part of the life of multi-cellular organisms. A type of plant PCD is the defensive hypersensitive response (HR) elicited via recognition of a pathogen by host resistance (R) proteins. The lethal, recessive accelerated cell death 11 (acd11) mutant exhibits HR-like accelerated cell death, and cell death execution in acd11 shares genetic requirements for HR execution triggered by one subclass of R proteins
Current European Labyrinthula zosterae Are Not Virulent and Modulate Seagrass (Zostera marina) Defense Gene Expression
Pro- and eukaryotic microbes associated with multi-cellular organisms are receiving increasing attention as a driving factor in ecosystems. Endophytes in plants can change host performance by altering nutrient uptake, secondary metabolite production or defense mechanisms. Recent studies detected widespread prevalence of Labyrinthula zosterae in European Zostera marina meadows, a protist that allegedly caused a massive amphi-Atlantic seagrass die-off event in the 1930's, while showing only limited virulence today. As a limiting factor for pathogenicity, we investigated genotype×genotype interactions of host and pathogen from different regions (10–100 km-scale) through reciprocal infection. Although the endophyte rapidly infected Z. marina, we found little evidence that Z. marina was negatively impacted by L. zosterae. Instead Z. marina showed enhanced leaf growth and kept endophyte abundance low. Moreover, we found almost no interaction of protist×eelgrass-origin on different parameters of L. zosterae virulence/Z. marina performance, and also no increase in mortality after experimental infection. In a target gene approach, we identified a significant down-regulation in the expression of 6/11 genes from the defense cascade of Z. marina after real-time quantitative PCR, revealing strong immune modulation of the host's defense by a potential parasite for the first time in a marine plant. Nevertheless, one gene involved in phenol synthesis was strongly up-regulated, indicating that Z. marina plants were probably able to control the level of infection. There was no change in expression in a general stress indicator gene (HSP70). Mean L. zosterae abundances decreased below 10% after 16 days of experimental runtime. We conclude that under non-stress conditions L. zosterae infection in the study region is not associated with substantial virulence
The Interactions Between Rapeseed Lipoxygenase and Native Polyphenolic Compounds in a Model System
The focus of the present research was to study inhibition of lipoxygenase activity by rapeseed native polyphenols and the interactions between those compounds and the enzyme. The enzyme and polyphenolic compounds (polyphenols, phenolic acids) were extracted from rapeseed (Brassica napus) varieties Aviso and PR45DO3. The total phenolic compounds concentration in tested rapeseed was 1,485–1,691 mg/100 g d.m. (dry matter) and the free phenolic acids content in both rapeseed varieties was about 76 μg/100 g d.m. The isolated proteins showed lipoxygenase activity. Prooxidant properties of phenolic compounds in the presence of lipoxygenase and linoleic acid were observed rather in the case of extracts containing a relatively high concentration of miscellaneous polyphenols. Antioxidant properties were recorded in the case of phenolic acid extracts which contain only 1.4–1.9% of phenolics present in raw phenolic extracts. We propose that the prooxidant effect of phenolic compounds comes from quinone and oxidized polyphenols formation. The observed antioxidant activity of phenolic acid extracts is probably due to their ability to scavenge free radicals formed from linoleic acid. However, reduction of lipoxygenase ferric to ferrous ions, which prevent the activation of the enzyme and inhibited its activity, was also observed
Catalase and NO CATALASE ACTIVITY1 Promote Autophagy-Dependent Cell Death in Arabidopsis
The LSD1-Interacting Protein GILP Is a LITAF Domain Protein That Negatively Regulates Hypersensitive Cell Death in Arabidopsis
Hypersensitive cell death, a form of avirulent pathogen-induced programmed cell death (PCD), is one of the most efficient plant innate immunity. However, its regulatory mechanism is poorly understood. AtLSD1 is an important negative regulator of PCD and only two proteins, AtbZIP10 and AtMC1, have been reported to interact with AtLSD1.To identify a novel regulator of hypersensitive cell death, we investigate the possible role of plant LITAF domain protein GILP in hypersensitive cell death. Subcellular localization analysis showed that AtGILP is localized in the plasma membrane and its plasma membrane localization is dependent on its LITAF domain. Yeast two-hybrid and pull-down assays demonstrated that AtGILP interacts with AtLSD1. Pull-down assays showed that both the N-terminal and the C-terminal domains of AtGILP are sufficient for interactions with AtLSD1 and that the N-terminal domain of AtLSD1 is involved in the interaction with AtGILP. Real-time PCR analysis showed that AtGILP expression is up-regulated by the avirulent pathogen Pseudomonas syringae pv. tomato DC3000 avrRpt2 (Pst avrRpt2) and fumonisin B1 (FB1) that trigger PCD. Compared with wild-type plants, transgenic plants overexpressing AtGILP exhibited significantly less cell death when inoculated with Pst avrRpt2, indicating that AtGILP negatively regulates hypersensitive cell death.These results suggest that the LITAF domain protein AtGILP localizes in the plasma membrane, interacts with AtLSD1, and is involved in negatively regulating PCD. We propose that AtGILP functions as a membrane anchor, bringing other regulators of PCD, such as AtLSD1, to the plasma membrane. Human LITAF domain protein may be involved in the regulation of PCD, suggesting the evolutionarily conserved function of LITAF domain proteins in the regulation of PCD
Autoimmunity in Arabidopsis acd11 Is Mediated by Epigenetic Regulation of an Immune Receptor
Certain pathogens deliver effectors into plant cells to modify host protein targets and thereby suppress immunity. These target modifications can be detected by intracellular immune receptors, or Resistance (R) proteins, that trigger strong immune responses including localized host cell death. The accelerated cell death 11 (acd11) “lesion mimic” mutant of Arabidopsis thaliana exhibits autoimmune phenotypes such as constitutive defense responses and cell death without pathogen perception. ACD11 encodes a putative sphingosine transfer protein, but its precise role during these processes is unknown. In a screen for lazarus (laz) mutants that suppress acd11 death we identified two genes, LAZ2 and LAZ5. LAZ2 encodes the histone lysine methyltransferase SDG8, previously shown to epigenetically regulate flowering time via modification of histone 3 (H3). LAZ5 encodes an RPS4-like R-protein, defined by several dominant negative alleles. Microarray and chromatin immunoprecipitation analyses showed that LAZ2/SDG8 is required for LAZ5 expression and H3 lysine 36 trimethylation at LAZ5 chromatin to maintain a transcriptionally active state. We hypothesize that LAZ5 triggers cell death in the absence of ACD11, and that cell death in other lesion mimic mutants may also be caused by inappropriate activation of R genes. Moreover, SDG8 is required for basal and R protein-mediated pathogen resistance in Arabidopsis, revealing the importance of chromatin remodeling as a key process in plant innate immunity
Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)
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. For example, 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 versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost 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 fi eld 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. 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. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. 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
Distribution pattern of antioxidants in white cabbage heads (Brassica oleracea L. var. capitata f. alba)
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