The roles of mitogen-activated protein kinases (MAPKs) in plant-fungal pathogenic interactions are poorly understood in crops. Here, microscopic, phenotypic, proteomic, and biochemical analyses revealed that roots of independent transcription activator-like effector nuclease (TALEN)-based knockout lines of barley (Hordeum vulgare L.) MAPK 3 (HvMPK3 KO) were resistant against Fusarium graminearum infection. When co-cultured with roots of the HvMPK3 KO lines, F. graminearum hyphae were excluded to the extracellular space, the growth pattern of extracellular hyphae was considerably deregulated, mycelia development was less efficient, and number of appressoria-like structures and their penetration potential were substantially reduced. Intracellular penetration of hyphae was preceded by the massive production of reactive oxygen species (ROS) in attacked cells of the wild-type (WT), but ROS production was mitigated in the HvMPK3 KO lines. Suppression of ROS production in these lines coincided with elevated abundance of catalase (CAT) and ascorbate peroxidase (APX). Moreover, differential proteomic analysis revealed downregulation of several defense-related proteins in WT, and the upregulation of pathogenesis-related protein 1 (PR-1) and cysteine proteases in HvMPK3 KO lines. Proteins involved in suberin formation, such as peroxidases, lipid transfer proteins (LTPs), and the GDSL esterase/lipase (containing "GDSL" aminosequence motif) were differentially regulated in HvMPK3 KO lines after F. graminearum inoculation. Consistent with proteomic analysis, microscopic observations showed enhanced suberin accumulation in roots of HvMPK3 KO lines, most likely contributing to the arrested infection by F. graminearum. These results suggest that TALEN-based knockout of HvMPK3 leads to barley root resistance against Fusarium root rot.

• MeSH
• Fusarium * fyziologie   MeSH
• ječmen (rod) * genetika   mikrobiologie   MeSH
• mitogenem aktivovaná proteinkinasa 3 metabolismus   MeSH
• nemoci rostlin genetika   mikrobiologie   MeSH
• proteomika MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• TALENs metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitogenem aktivovaná proteinkinasa 3 MeSH
• reaktivní formy kyslíku MeSH
• TALENs MeSH

Mitogen activated protein kinases (MAPKs) integrate elicitor perception with both early and late responses associated with plant defense and innate immunity. Much of the existing knowledge on the role of plant MAPKs in defense mechanisms against microbes stems from extensive research in the model plant Arabidopsis thaliana. In the present study, we investigated the involvement of barley (Hordeum vulgare) MPK3 in response to flagellin peptide flg22, a well-known bacterial elicitor. Using differential proteomic analysis we show that TALEN-induced MPK3 knock-out lines of barley (HvMPK3 KO) exhibit constitutive downregulation of defense related proteins such as PR proteins belonging to thaumatin family and chitinases. Further analyses showed that the same protein families were less prone to flg22 elicitation in HvMPK3 KO plants compared to wild types. These results were supported and validated by chitinase activity analyses and immunoblotting for HSP70. In addition, differential proteomes correlated with root hair phenotypes and suggested tolerance of HvMPK3 KO lines to flg22. In conclusion, our study points to the specific role of HvMPK3 in molecular and root hair phenotypic responses of barley to flg22.

• Klíčová slova
• HvMPK3, PR proteins, TALEN, barley, chitinases, flagellin, proteomics, root hairs,
• Publikační typ
• časopisecké články MeSH

Reactive oxygen species (ROS) are signaling molecules essential for plant responses to abiotic and biotic stimuli as well as for multiple developmental processes. They are produced as byproducts of aerobic metabolism and are affected by adverse environmental conditions. The ROS content is controlled on the side of their production but also by scavenging machinery. Antioxidant enzymes represent a major ROS-scavenging force and are crucial for stress tolerance in plants. Enzymatic antioxidant defense occurs as a series of redox reactions for ROS elimination. Therefore, the deregulation of the antioxidant machinery may lead to the overaccumulation of ROS in plants, with negative consequences both in terms of plant development and resistance to environmental challenges. The transcriptional activation of antioxidant enzymes accompanies the long-term exposure of plants to unfavorable environmental conditions. Fast ROS production requires the immediate mobilization of the antioxidant defense system, which may occur via retrograde signaling, redox-based modifications, and the phosphorylation of ROS detoxifying enzymes. This review aimed to summarize the current knowledge on signaling processes regulating the enzymatic antioxidant capacity of plants.

• Klíčová slova
• antioxidant enzymes, calcium, mitogen-activated protein kinases, oxidative stress, plants, reactive oxygen species, signaling, stress,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Phospholipase D alpha 1 (PLDα1, AT3G15730) and mitogen-activated protein kinases (MAPKs) participate on signaling-dependent events in plants. MAPKs are able to phosphorylate a wide range of substrates putatively including PLDs. Here we have focused on functional regulations of PLDα1 by interactions with MAPKs, their co-localization and impact on salt stress and abscisic acid (ABA) tolerance in Arabidopsis. Yeast two-hybrid and bimolecular fluorescent assays showed that PLDα1 interacts with MPK3. Immunoblotting analyses likewise confirmed connection between both these enzymes. Subcellularly we co-localized PLDα1 with MPK3 in the cortical cytoplasm close to the plasma membrane and in cytoplasmic strands. Moreover, genetic interaction studies revealed that pldα1mpk3 double mutant was resistant to a higher salinity and showed a higher tolerance to ABA during germination in comparison to single mutants and wild type. Thus, this study revealed importance of new biochemical and genetic interactions between PLDα1 and MPK3 for Arabidopsis stress (salt and ABA) response.

• Klíčová slova
• Arabidopsis thaliana, abscisic acid, genetic interaction, localization, mitogen-activated protein kinase 3, phospholipase D alpha 1, protein interaction, salt stress,
• Publikační typ
• časopisecké články MeSH

Phospholipase D alpha 1 (PLDα1) is a phospholipid hydrolyzing enzyme playing multiple regulatory roles in stress responses of plants. Its signaling activity is mediated by phosphatidic acid (PA) production, capacity to bind, and modulate G-protein complexes or by interaction with other proteins. This work presents a quantitative proteomic analysis of two T-DNA insertion pldα1 mutants of Arabidopsis thaliana. Remarkably, PLDα1 knockouts caused differential regulation of many proteins forming protein complexes, while PLDα1 might be required for their stability. Almost one third of differentially abundant proteins (DAPs) in pldα1 mutants are implicated in metabolism and RNA binding. Latter functional class comprises proteins involved in translation, RNA editing, processing, stability, and decay. Many of these proteins, including those regulating chloroplast protein import and protein folding, share common functions in chloroplast biogenesis and leaf variegation. Consistently, pldα1 mutants showed altered level of TIC40 (a major regulator of protein import into chloroplast), differential accumulation of photosynthetic protein complexes and changed chloroplast sizes as revealed by immunoblotting, blue-native electrophoresis, and microscopic analyses, respectively. Our proteomic analysis also revealed that genetic depletion of PLDα1 also affected proteins involved in cell wall architecture, redox homeostasis, and abscisic acid signaling. Taking together, PLDα1 appears as a protein integrating cytosolic and plastidic protein translations, plastid protein degradation, and protein import into chloroplast in order to regulate chloroplast biogenesis in Arabidopsis.

• Klíčová slova
• Arabidopsis, chloroplast biogenesis, chloroplast protein import, phospholipase D alpha 1, proteomics, translation,
• Publikační typ
• časopisecké články MeSH

Phospholipase Dα1 (PLDα1) belongs to phospholipases, a large phospholipid hydrolyzing protein family. PLDα1 has a substrate preference for phosphatidylcholine leading to enzymatic production of phosphatidic acid, a lipid second messenger with multiple cellular functions. PLDα1 itself is implicated in biotic and abiotic stress responses. Here, we present a shot-gun differential proteomic analysis on roots of two Arabidopsis pldα1 mutants compared to the wild type. Interestingly, PLDα1 deficiency leads to altered abundances of proteins involved in diverse processes related to membrane transport including endocytosis and endoplasmic reticulum-Golgi transport. PLDα1 may be involved in the stability of attachment sites of endoplasmic reticulum to the plasma membrane as suggested by increased abundance of synaptotagmin 1, which was validated by immunoblotting and whole-mount immunolabelling analyses. Moreover, we noticed a robust abundance alterations of proteins involved in mitochondrial import and electron transport chain. Notably, the abundances of numerous proteins implicated in glucosinolate biosynthesis were also affected in pldα1 mutants. Our results suggest a broader biological involvement of PLDα1 than anticipated thus far, especially in the processes such as endomembrane transport, mitochondrial protein import and protein quality control, as well as glucosinolate biosynthesis.

• Klíčová slova
• Arabidopsis, cytoskeleton, mitochondrial protein import, phospholipase D alpha1, proteomics, quality control, vesicular transport,
• MeSH
• Arabidopsis metabolismus   MeSH
• endocytóza MeSH
• fosfolipasa D genetika   metabolismus   MeSH
• genová ontologie MeSH
• glukosinoláty biosyntéza   MeSH
• kořeny rostlin metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteom metabolismus   MeSH
• proteomika * MeSH
• synaptotagmin I metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• transport proteinů MeSH
• uncoupling protein 1 metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfolipasa D MeSH
• glukosinoláty MeSH
• mitochondriální proteiny MeSH
• PLDA1 protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• proteom MeSH
• synaptotagmin I MeSH
• SYT1 protein, Arabidopsis MeSH Prohlížeč
• uncoupling protein 1 MeSH