Advanced microscopy resolves dynamic localization patterns of stress-induced mitogen-activated protein kinase (SIMK) during alfalfa root hair interactions with Ensifer meliloti.

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.

Overexpression of alfalfa SIMK promotes root hair growth, nodule clustering and shoot biomass production.

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.

Tissue culture, genetic transformation, interaction with beneficial microbes, and modern bio-imaging techniques in alfalfa research.

Current research needs to be more focused on agronomical plants to effectively utilize the knowledge obtained from model plant species. Efforts to improve legumes have long employed common breeding tools. Recently, biotechnological approaches facilitated the development of improved legumes with new traits, allowing them to withstand climatic changes and biotic stress. Owing to its multiple uses and profits, alfalfa (Medicago sativa L.) has become a prominent forage crop worldwide. This review provides a comprehensive research summary of tissue culture-based genetic transformation methods, which could be exploited for the development of transgenic alfalfa with agronomically desirable traits. Moreover, advanced bio-imaging approaches, including cutting-edge microscopy and phenotyping, are outlined here. Finally, characterization and the employment of beneficial microbes should help to produce biotechnologically improved and sustainable alfalfa cultivars.

Super-resolution imaging of microtubules in Medicago sativa.

Study of microtubules on cellular and subcellular levels is compromised by limited resolution of conventional fluorescence microscopy. However, it is possible to improve Abbe's diffraction-limited resolution by employment of super-resolution microscopy methods. Two of them, described herein, are structured-illumination microscopy (SIM) and Airyscan laser scanning microscopy (AM). Both methods allow high-resolution imaging of cortical microtubules in plant cells, thus contributing to the current knowledge on plant morphogenesis, growth and development. Both SIM and AM provide certain advantages and characteristic features, which are described here. We present immunofluorescence localization methods for microtubules in fixed plant cells achieving high signal efficiency, superb sample stability and sub-diffraction resolution. These protocols were developed for whole-mount immunolabeling of root samples of legume crop species Medicago sativa. They also contain tips for optimal sample preparation of plants germinated from seeds as well as plantlets regenerated from somatic embryos in vitro. We describe in detail all steps of optimized protocols for sample preparation, microtubule immunolabeling and super-resolution imaging.

Advanced Microscopy Reveals Complex Developmental and Subcellular Localization Patterns of ANNEXIN 1 in Arabidopsis.

Annexin 1 (ANN1) is the most abundant member of the evolutionary conserved multigene protein superfamily of annexins in plants. Generally, annexins participate in diverse cellular processes, such as cell growth, differentiation, vesicle trafficking, and stress responses. The expression of annexins is developmentally regulated, and it is sensitive to the external environment. ANN1 is expressed in almost all Arabidopsis tissues, while the most abundant is in the root, root hairs, and in the hypocotyl epidermal cells. Annexins were also occasionally proposed to associate with cytoskeleton and vesicles, but they were never developmentally localized at the subcellular level in diverse plant tissues and organs. Using advanced light-sheet fluorescence microscopy (LSFM), we followed the developmental and subcellular localization of GFP-tagged ANN1 in post-embryonic Arabidopsis organs. By contrast to conventional microscopy, LSFM allowed long-term imaging of ANN1-GFP in Arabidopsis plants at near-environmental conditions without affecting plant viability. We studied developmental regulation of ANN1-GFP expression and localization in growing Arabidopsis roots: strong accumulation was found in the root cap and epidermal cells (preferentially in elongating trichoblasts), but it was depleted in dividing cells localized in deeper layers of the root meristem. During root hair development, ANN1-GFP accumulated at the tips of emerging and growing root hairs, which was accompanied by decreased abundance in the trichoblasts. In aerial plant parts, ANN1-GFP was localized mainly in the cortical cytoplasm of trichomes and epidermal cells of hypocotyls, cotyledons, true leaves, and their petioles. At the subcellular level, ANN1-GFP was enriched at the plasma membrane (PM) and vesicles of non-dividing cells and in mitotic and cytokinetic microtubular arrays of dividing cells. Additionally, an independent immunolocalization method confirmed ANN1-GFP association with mitotic and cytokinetic microtubules (PPBs and phragmoplasts) in dividing cells of the lateral root cap. Lattice LSFM revealed subcellular accumulation of ANN1-GFP around the nuclear envelope of elongating trichoblasts. Massive relocation and accumulation of ANN1-GFP at the PM and in Hechtian strands and reticulum in plasmolyzed cells suggest a possible osmoprotective role of ANN1-GFP during plasmolysis/deplasmolysis cycle. This study shows complex developmental and subcellular localization patterns of ANN1 in living Arabidopsis plants.

Biotechnological Perspectives of Omics and Genetic Engineering Methods in Alfalfa.

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.