Leguminous plants have established mutualistic endosymbiotic interactions with nitrogen-fixing rhizobia to secure nitrogen sources in root nodules. Before nodule formation, the development of early symbiotic structures is essential for rhizobia docking, internalization, targeted delivery, and intracellular accommodation. We recently reported that overexpression of stress-induced mitogen-activated protein kinase (SIMK) in alfalfa affects root hair, nodule, and shoot formation, raising the question of how SIMK modulates these processes. In particular, detailed subcellular spatial distribution, activation, and developmental relocation of SIMK during early stages of alfalfa nodulation remain unclear. Here, we characterized SIMK distribution in Ensifer meliloti-infected root hairs using live-cell imaging and immunolocalization, employing alfalfa stable transgenic lines with genetically manipulated SIMK abundance and kinase activity. In the SIMKK-RNAi line, showing down-regulation of SIMKK and SIMK, we found considerably decreased accumulation of phosphorylated SIMK around infection pockets and infection threads. However, this was strongly increased in the GFP-SIMK line, constitutively overexpressing green fluorescent protein (GFP)-tagged SIMK. Thus, genetically manipulated SIMK modulates root hair capacity to form infection pockets and infection threads. Advanced light-sheet fluorescence microscopy on intact plants allowed non-invasive imaging of spatiotemporal interactions between root hairs and symbiotic E. meliloti, while immunofluorescence detection confirmed that SIMK was activated in these locations. Our results shed new light on SIMK spatiotemporal participation in early interactions between alfalfa and E. meliloti, and its internalization into root hairs, showing that local accumulation of active SIMK modulates early nodulation in alfalfa.

• Klíčová slova
• Ensifer meliloti, Alfalfa, MAPKs, SIMK, immunolocalization, infection pocket, infection thread, light-sheet fluorescence microscopy, root hairs, subcellular localization,
• MeSH
• Medicago sativa genetika   metabolismus   MeSH
• mikroskopie MeSH
• mitogenem aktivované proteinkinasy * metabolismus   MeSH
• rostliny metabolismus   MeSH
• Sinorhizobium meliloti * metabolismus   MeSH
• symbióza fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitogenem aktivované proteinkinasy * MeSH

Nitrogen-fixing rhizobia and legumes have developed complex mutualistic mechanism that allows to convert atmospheric nitrogen into ammonia. Signalling by mitogen-activated protein kinases (MAPKs) seems to be involved in this symbiotic interaction. Previously, we reported that stress-induced MAPK (SIMK) shows predominantly nuclear localization in alfalfa root epidermal cells. Nevertheless, SIMK is activated and relocalized to the tips of growing root hairs during their development. SIMK kinase (SIMKK) is a well-known upstream activator of SIMK. Here, we characterized production parameters of transgenic alfalfa plants with genetically manipulated SIMK after infection with Sinorhizobium meliloti. SIMKK RNAi lines, causing strong downregulation of both SIMKK and SIMK, showed reduced root hair growth and lower capacity to form infection threads and nodules. In contrast, constitutive overexpression of GFP-tagged SIMK promoted root hair growth as well as infection thread and nodule clustering. Moreover, SIMKK and SIMK downregulation led to decrease, while overexpression of GFP-tagged SIMK led to increase of biomass in above-ground part of plants. These data suggest that genetic manipulations causing downregulation or overexpression of SIMK affect root hair, nodule and shoot formation patterns in alfalfa, and point to the new biotechnological potential of this MAPK.

• Klíčová slova
• Medicago sativa, SIMK, SIMKK, infection thread, nodule, root hair,
• MeSH
• biomasa MeSH
• Medicago sativa * genetika   MeSH
• mitogenem aktivované proteinkinasy kinas MeSH
• rostlinné proteiny * genetika   MeSH
• shluková analýza MeSH
• symbióza genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitogenem aktivované proteinkinasy kinas MeSH
• rostlinné proteiny * MeSH

Water scarcity is the primary constraint on crop productivity in arid and semiarid tropical areas suffering from climate alterations; in accordance, agricultural systems have to be optimized. Several concepts and strategies should be considered to improve crop yield and quality, particularly in vulnerable regions where such environmental changes cause a risk of food insecurity. In this work, we review two strategies aiming to increase drought stress tolerance: (i) the use of natural genes that have evolved over time and are preserved in crop wild relatives and landraces for drought tolerance breeding using conventional and molecular methods and (ii) exploiting the reservoir of neglected and underutilized species to identify those that are known to be more drought-tolerant than conventional staple crops while possessing other desired agronomic and nutritive characteristics, as well as introducing them into existing cropping systems to make them more resilient to water deficiency conditions. In the past, the existence of drought tolerance genes in crop wild relatives and landraces was either unknown or difficult to exploit using traditional breeding techniques to secure potential long-term solutions. Today, with the advances in genomics and phenomics, there are a number of new tools available that facilitate the discovery of drought resistance genes in crop wild relatives and landraces and their relatively easy transfer into advanced breeding lines, thus accelerating breeding progress and creating resilient varieties that can withstand prolonged drought periods. Among those tools are marker-assisted selection (MAS), genomic selection (GS), and targeted gene editing (clustered regularly interspaced short palindromic repeat (CRISPR) technology). The integration of these two major strategies, the advances in conventional and molecular breeding for the drought tolerance of conventional staple crops, and the introduction of drought-tolerant neglected and underutilized species into existing production systems has the potential to enhance the resilience of agricultural production under conditions of water scarcity.

• Klíčová slova
• crop diversity, drought tolerance, genetic approaches, neglected and underutilized species,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

For several decades, researchers are working to develop improved major crops with better adaptability and tolerance to environmental stresses. Forage legumes have been widely spread in the world due to their great ecological and economic values. Abiotic and biotic stresses are main factors limiting legume production, however, alfalfa (Medicago sativa L.) shows relatively high level of tolerance to drought and salt stress. Efforts focused on alfalfa improvements have led to the release of cultivars with new traits of agronomic importance such as high yield, better stress tolerance or forage quality. Alfalfa has very high nutritional value due to its efficient symbiotic association with nitrogen-fixing bacteria, while deep root system can help to prevent soil water loss in dry lands. The use of modern biotechnology tools is challenging in alfalfa since full genome, unlike to its close relative barrel medic (Medicago truncatula Gaertn.), was not released yet. Identification, isolation, and improvement of genes involved in abiotic or biotic stress response significantly contributed to the progress of our understanding how crop plants cope with these environmental challenges. In this review, we provide an overview of the progress that has been made in high-throughput sequencing, characterization of genes for abiotic or biotic stress tolerance, gene editing, as well as proteomic and metabolomics techniques bearing biotechnological potential for alfalfa improvement.

• Klíčová slova
• Medicago sativa, alfalfa, genomics, metabolomics, proteomics, stress resistance genes,
• Publikační typ
• časopisecké články MeSH
• přehledy 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

Disentanglement of functional complexity associated with plant mitogen-activated protein kinase (MAPK) signaling has benefited from transcriptomic, proteomic, phosphoproteomic, and genetic studies. Published transcriptomic analysis of a double homozygous recessive anp2anp3 mutant of two MAPK kinase kinase (MAPKKK) genes called Arabidopsis thaliana Homologues of Nucleus- and Phragmoplast-localized Kinase 2 (ANP2) and 3 (ANP3) showed the upregulation of stress-related genes. In this study, a comparative proteomic analysis of anp2anp3 mutant against its respective Wassilevskaja ecotype (Ws) wild type background is provided. Such differential proteomic analysis revealed overabundance of core enzymes such as FeSOD1, MnSOD, DHAR1, and FeSOD1-associated regulatory protein CPN20, which are involved in the detoxification of reactive oxygen species in the anp2anp3 mutant. The proteomic results were validated at the level of single protein abundance by Western blot analyses and by quantitative biochemical determination of antioxidant enzymatic activities. Finally, the functional network of proteins involved in antioxidant defense in the anp2anp3 mutant was physiologically linked with the increased resistance of mutant seedlings against paraquat treatment.

• Klíčová slova
• ANP2, ANP3, Arabidopsis, antioxidant defense, mitogen-activated protein kinase kinase kinase, oxidative stress, proteomics, signaling,
• MeSH
• antioxidancia metabolismus   MeSH
• Arabidopsis účinky léků   genetika   metabolismus   MeSH
• biologické modely MeSH
• chromatografie kapalinová MeSH
• herbicidy farmakologie   MeSH
• imunoblotting MeSH
• MAP kinasy kinas (kinas) genetika   metabolismus   MeSH
• mutace MeSH
• paraquat farmakologie   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteom genetika   metabolismus   MeSH
• proteomika metody   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• semenáček účinky léků   genetika   metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• superoxiddismutasa metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• ANP2 protein, Arabidopsis MeSH Prohlížeč
• ANP3 protein, Arabidopsis MeSH Prohlížeč
• antioxidancia MeSH
• herbicidy MeSH
• MAP kinasy kinas (kinas) MeSH
• paraquat MeSH
• proteiny huseníčku MeSH
• proteom MeSH
• reaktivní formy kyslíku MeSH
• superoxiddismutasa MeSH

Dual-specificity mitogen-activated protein kinases kinases (MAPKKs) are the immediate upstream activators of MAPKs. They simultaneously phosphorylate the TXY motif within the activation loop of MAPKs, allowing them to interact with and regulate multiple substrates. Often, the activation of MAPKs triggers their nuclear translocation. However, the spatiotemporal dynamics and the physiological consequences of the activation of MAPKs, particularly in plants, are still poorly understood. Here, we studied the activation and localization of the Medicago sativa stress-induced MAPKK (SIMKK)-SIMK module after salt stress. In the inactive state, SIMKK and SIMK co-localized in the cytoplasm and in the nucleus. Upon salt stress, however, a substantial part of the nuclear pool of both SIMKK and SIMK relocated to cytoplasmic compartments. The course of nucleocytoplasmic shuttling of SIMK correlated temporally with the dual phosphorylation of the pTEpY motif. SIMKK function was further studied in Arabidopsis plants overexpressing SIMKK-yellow fluorescent protein (YFP) fusions. SIMKK-YFP plants showed enhanced activation of Arabidopsis MPK3 and MPK6 kinases upon salt treatment and exhibited high sensitivity against salt stress at the seedling stage, although they were salt insensitive during seed germination. Proteomic analysis of SIMKK-YFP overexpressors indicated the differential regulation of proteins directly or indirectly involved in salt stress responses. These proteins included catalase, peroxiredoxin, glutathione S-transferase, nucleoside diphosphate kinase 1, endoplasmic reticulum luminal-binding protein 2, and finally plasma membrane aquaporins. In conclusion, Arabidopsis seedlings overexpressing SIMKK-YFP exhibited higher salt sensitivity consistent with their proteome composition and with the presumptive MPK3/MPK6 hijacking of the salt response pathway.

• Klíčová slova
• Arabidopsis, MAPK, Medicago, SIMK, SIMKK, proteomics, salt stress, subcellular relocation.,
• MeSH
• aktivace enzymů MeSH
• Arabidopsis genetika   růst a vývoj   metabolismus   MeSH
• exprese genu MeSH
• geneticky modifikované rostliny genetika   růst a vývoj   metabolismus   MeSH
• Medicago sativa enzymologie   genetika   MeSH
• mitogenem aktivované proteinkinasy kinas genetika   metabolismus   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• semenáček genetika   růst a vývoj   metabolismus   MeSH
• soli metabolismus   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitogenem aktivované proteinkinasy kinas MeSH
• rostlinné proteiny MeSH
• soli MeSH