Spatial organization and dynamics of chromosomes and microtubules during barley mitosis.

Mitosis and cytokinesis are fundamental processes through which somatic cells increase their numbers and allow plant growth and development. Here, we analyzed the organization and dynamics of mitotic chromosomes, nucleoli, and microtubules in living cells of barley root primary meristems using a series of newly developed stable fluorescent protein translational fusion lines and time-lapse confocal microscopy. The median duration of mitosis from prophase until the end of telophase was 65.2 and 78.2 min until the end of cytokinesis. We showed that barley chromosomes frequently start condensation before mitotic pre-prophase as defined by the organization of microtubules and maintain it even after entering into the new interphase. Furthermore, we found that the process of chromosome condensation does not finish at metaphase, but gradually continues until the end of mitosis. In summary, our study features resources for in vivo analysis of barley nuclei and chromosomes and their dynamics during mitotic cell cycle.

Spatiotemporal distribution of reactive oxygen species production, delivery, and use in Arabidopsis root hairs.

Fluorescent selective probes for reactive oxygen species (ROS) detection in living cells are versatile tools for the documentation of ROS production in plant developmental or stress reactions. We employed high-resolution live-cell imaging and semiquantitative analysis of Arabidopsis (Arabidopsis thaliana) stained with CM-H2DCFDA, CellROX Deep Red, and Amplex Red for functional characterization of the spatiotemporal mode of ROS production, delivery, and utilization during root hair formation. Cell viability marker fluorescein diacetate served as a positive control for dye loading and undisturbed root hair tip growth after staining. Using a colocalization analysis with subcellular molecular markers and two root hair mutants with similar phenotypes of nonelongating root hairs, but with contrasting reasons for this impairment, we found that: (i) CM-H2DCFDA is a sensitive probe for ROS generation in the cytoplasm, (ii) CellROX Deep Red labels ROS in mitochondria, (iii) Amplex Red labels apoplastic ROS and mitochondria and shows high selectivity to root hairs, (iv) the root hair defective 2-1 (rhd2-1) mutant with nonfunctional NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN C/ROOT HAIR-DEFECTIVE 2 (AtRBOHC/RHD2) has a low level of CM-H2DCFDA-reactive ROS in cytoplasm and lacks Amplex Red-reactive ROS in apoplast, and (v) the ACTIN2-deficient deformed root hairs1-3 (der1-3) mutant is not altered in these aspects. The sensitivity of CellROX Deep Red was documented by discrimination between larger ROS-containing mitochondria and small, yet ROS-free premature mitochondria in the growing tip of root hairs. We characterized spatial changes in ROS production and compartmentalization induced by external ROS modulators, ethylene precursor 1-aminocyclopropane-1-carboxylic acid, and ionophore valinomycin. This dynamic and high-resolution study of ROS production and utilization opens opportunities for precise speciation of particular ROS involved in root hair formation.

Methyl viologen-induced changes in the Arabidopsis proteome implicate PATELLIN 4 in oxidative stress responses.

The photosynthesis-induced accumulation of reactive oxygen species in chloroplasts can lead to oxidative stress, triggering changes in protein synthesis, degradation, and the assembly/disassembly of protein complexes. Using shot-gun proteomics, we identified methyl viologen-induced changes in protein abundance in wild-type Arabidopsis and oxidative stress-hypersensitive fsd1-1 and fsd1-2 knockout mutants, which are deficient in IRON SUPEROXIDE DISMUTASE 1 (FSD1). The levels of proteins that are localized in chloroplasts and the cytoplasm were modified in all lines treated with methyl viologen. Compared with the wild-type, fsd1 mutants showed significant changes in metabolic protein and chloroplast chaperone levels, together with increased ratio of cytoplasmic, peroxisomal, and mitochondrial proteins. Different responses in proteins involved in the disassembly of photosystem II-light harvesting chlorophyll a/b binding proteins were observed. Moreover, the abundance of PATELLIN 4, a phospholipid-binding protein enriched in stomatal lineage, was decreased in response to methyl viologen. Reverse genetic studies using patl4 knockout mutants and a PATELLIN 4 complemented line indicate that PATELLIN 4 affects plant responses to oxidative stress by effects on stomatal closure.

Zebularine induces enzymatic DNA-protein crosslinks in 45S rDNA heterochromatin of Arabidopsis nuclei.

Loss of genome stability leads to reduced fitness, fertility and a high mutation rate. Therefore, the genome is guarded by the pathways monitoring its integrity and neutralizing DNA lesions. To analyze the mechanism of DNA damage induction by cytidine analog zebularine, we performed a forward-directed suppressor genetic screen in the background of Arabidopsis thaliana zebularine-hypersensitive structural maintenance of chromosomes 6b (smc6b) mutant. We show that smc6b hypersensitivity was suppressed by the mutations in EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 3 (ENT3), DNA METHYLTRANSFERASE 1 (MET1) and DECREASE IN DNA METHYLATION 1 (DDM1). Superior resistance of ent3 plants to zebularine indicated that ENT3 is likely necessary for the import of the drug to the cells. Identification of MET1 and DDM1 suggested that zebularine induces DNA damage by interference with the maintenance of CG DNA methylation. The same holds for structurally similar compounds 5-azacytidine and 2-deoxy-5-azacytidine. Based on our genetic and biochemical data, we propose that zebularine induces enzymatic DNA-protein crosslinks (DPCs) of MET1 and zebularine-containing DNA in Arabidopsis, which was confirmed by native chromatin immunoprecipitation experiments. Moreover, zebularine-induced DPCs accumulate preferentially in 45S rDNA chromocenters in a DDM1-dependent manner. These findings open a new avenue for studying genome stability and DPC repair in plants.

Spatial proteomics of vesicular trafficking: coupling mass spectrometry and imaging approaches in membrane biology.

In plants, membrane compartmentalization requires vesicle trafficking for communication among distinct organelles. Membrane proteins involved in vesicle trafficking are highly dynamic and can respond rapidly to changes in the environment and to cellular signals. Capturing their localization and dynamics is thus essential for understanding the mechanisms underlying vesicular trafficking pathways. Quantitative mass spectrometry and imaging approaches allow a system-wide dissection of the vesicular proteome, the characterization of ligand-receptor pairs and the determination of secretory, endocytic, recycling and vacuolar trafficking pathways. In this review, we highlight major proteomics and imaging methods employed to determine the location, distribution and abundance of proteins within given trafficking routes. We focus in particular on methodologies for the elucidation of vesicle protein dynamics and interactions and their connections to downstream signalling outputs. Finally, we assess their biological applications in exploring different cellular and subcellular processes.

Cell surface receptor kinase FERONIA linked to nutrient sensor TORC signaling controls root hair growth at low temperature linked to low nitrate in Arabidopsis thaliana.

Root hairs (RH) are excellent model systems for studying cell size and polarity since they elongate several hundred-fold their original size. Their tip growth is determined both by intrinsic and environmental signals. Although nutrient availability and temperature are key factors for a sustained plant growth, the molecular mechanisms underlying their sensing and downstream signaling pathways remain unclear. We use genetics to address the roles of the cell surface receptor kinase FERONIA (FER) and the nutrient sensing TOR Complex 1 (TORC) in RH growth. We identified that low temperature (10°C) triggers a strong RH elongation response in Arabidopsis thaliana involving FER and TORC. We found that FER is required to perceive limited nutrient availability caused by low temperature. FERONIA interacts with and activates TORC-downstream components to trigger RH growth. In addition, the small GTPase Rho of plants 2 (ROP2) is also involved in this RH growth response linking FER and TOR. We also found that limited nitrogen nutrient availability can mimic the RH growth response at 10°C in a NRT1.1-dependent manner. These results uncover a molecular mechanism by which a central hub composed by FER-ROP2-TORC is involved in the control of RH elongation under low temperature and nitrogen deficiency.

ROOT HAIR DEFECTIVE 2 vesicular delivery to the apical plasma membrane domain during Arabidopsis root hair development.

Arabidopsis (Arabidopsis thaliana) root hairs develop as long tubular extensions from the rootward pole of trichoblasts and exert polarized tip growth. The establishment and maintenance of root hair polarity is a complex process involving the local apical production of reactive oxygen species generated by A. thaliana nicotinamide adenine dinucleotide phosphate (NADPH) oxidase respiratory burst oxidase homolog protein C/ROOT HAIR-DEFECTIVE 2 (AtRBOHC/RHD2). Loss-of-function root hair defective 2 (rhd2) mutants have short root hairs that are unable to elongate by tip growth, and this phenotype is fully complemented by GREEN FLUORESCENT PROTEIN (GFP)-RHD2 expressed under the RHD2 promoter. However, the spatiotemporal mechanism of AtRBOHC/RHD2 subcellular redistribution and delivery to the plasma membrane (PM) during root hair initiation and tip growth are still unclear. Here, we used advanced microscopy for detailed qualitative and quantitative analysis of vesicular compartments containing GFP-RHD2 and characterization of their movements in developing bulges and growing root hairs. These compartments, identified by an independent molecular marker mCherry-VTI12 as the trans-Golgi network (TGN), deliver GFP-RHD2 to the apical PM domain, the extent of which corresponds with the stage of root hair formation. Movements of TGN/early endosomes, but not late endosomes, were affected in the bulging domains of the rhd2-1 mutant. Finally, we revealed that structural sterols might be involved in the accumulation, docking, and incorporation of TGN compartments containing GFP-RHD2 to the apical PM of root hairs. These results help in clarifying the mechanism of polarized AtRBOHC/RHD2 targeting, maintenance, and recycling at the apical PM domain, coordinated with different developmental stages of root hair initiation and growth.

Imaging plant cells and organs with light-sheet and super-resolution microscopy.

The documentation of plant growth and development requires integrative and scalable approaches to investigate and spatiotemporally resolve various dynamic processes at different levels of plant body organization. The present update deals with vigorous developments in mesoscopy, microscopy and nanoscopy methods that have been translated to imaging of plant subcellular compartments, cells, tissues and organs over the past 3 years with the aim to report recent applications and reasonable expectations from current light-sheet fluorescence microscopy (LSFM) and super-resolution microscopy (SRM) modalities. Moreover, the shortcomings and limitations of existing LSFM and SRM are discussed, particularly for their ability to accommodate plant samples and regarding their documentation potential considering spherical aberrations or temporal restrictions prohibiting the dynamic recording of fast cellular processes at the three dimensions. For a more comprehensive description, advances in living or fixed sample preparation methods are also included, supported by an overview of developments in labeling strategies successfully applied in plants. These strategies are practically documented by current applications employing model plant Arabidopsis thaliana (L.) Heynh., but also robust crop species such as Medicago sativa L. and Hordeum vulgare L. Over the past few years, the trend towards designing of integrative microscopic modalities has become apparent and it is expected that in the near future LSFM and SRM will be bridged to achieve broader multiscale plant imaging with a single platform.

Knockout of MITOGEN-ACTIVATED PROTEIN KINASE 3 causes barley root resistance against Fusarium graminearum.

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.

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.

HEAT SHOCK PROTEIN 90 proteins and YODA regulate main body axis formation during early embryogenesis.

The YODA (YDA) kinase pathway is intimately associated with the control of Arabidopsis (Arabidopsis thaliana) embryo development, but little is known regarding its regulators. Using genetic analysis, HEAT SHOCK PROTEIN 90 (HSP90) proteins emerge as potent regulators of YDA in the process of embryo development and patterning. This study is focused on the characterization and quantification of early embryonal traits of single and double hsp90 and yda mutants. HSP90s genetic interactions with YDA affected the downstream signaling pathway to control the development of both basal and apical cell lineage of embryo. Our results demonstrate that the spatiotemporal expression of WUSCHEL-RELATED HOMEOBOX 8 (WOX8) and WOX2 is changed when function of HSP90s or YDA is impaired, suggesting their essential role in the cell fate determination and possible link to auxin signaling during early embryo development. Hence, HSP90s together with YDA signaling cascade affect transcriptional networks shaping the early embryo development.

Analysis of formin functions during cytokinesis using specific inhibitor SMIFH2.

The phragmoplast separates daughter cells during cytokinesis by constructing the cell plate, which depends on interaction between cytoskeleton and membrane compartments. Proteins responsible for these interactions remain unknown, but formins can link cytoskeleton with membranes and several members of formin protein family localize to the cell plate. Progress in functional characterization of formins in cytokinesis is hindered by functional redundancies within the large formin gene family. We addressed this limitation by employing Small Molecular Inhibitor of Formin Homology 2 (SMIFH2), a small-molecule inhibitor of formins. Treatment of tobacco (Nicotiana tabacum) tissue culture cells with SMIFH2 perturbed localization of actin at the cell plate; slowed down both microtubule polymerization and phragmoplast expansion; diminished association of dynamin-related proteins with the cell plate independently of actin and microtubules; and caused cell plate swelling. Another impact of SMIFH2 was shortening of the END BINDING1b (EB1b) and EB1c comets on the growing microtubule plus ends in N. tabacum tissue culture cells and Arabidopsis thaliana cotyledon epidermis cells. The shape of the EB1 comets in the SMIFH2-treated cells resembled that of the knockdown mutant of plant Xenopus Microtubule-Associated protein of 215 kDa (XMAP215) homolog MICROTUBULE ORGANIZATION 1/GEMINI 1 (MOR1/GEM1). This outcome suggests that formins promote elongation of tubulin flares on the growing plus ends. Formins AtFH1 (A. thaliana Formin Homology 1) and AtFH8 can also interact with EB1. Besides cytokinesis, formins function in the mitotic spindle assembly and metaphase to anaphase transition. Our data suggest that during cytokinesis formins function in: (1) promoting microtubule polymerization; (2) nucleating F-actin at the cell plate; (3) retaining dynamin-related proteins at the cell plate; and (4) remodeling of the cell plate membrane.

Secretion of Phospholipase Dδ Functions as a Regulatory Mechanism in Plant Innate Immunity.

Plant phospholipase Ds (PLDs), essential regulators of phospholipid signaling, function in multiple signal transduction cascades; however, the mechanisms regulating PLDs in response to pathogens remain unclear. Here, we found that Arabidopsis (Arabidopsis thaliana) PLDδ accumulated in cells at the entry sites of the barley powdery mildew fungus, Blumeria graminis f. sp hordei Using fluorescence recovery after photobleaching and single-molecule analysis, we observed higher PLDδ density in the plasma membrane after chitin treatment; PLDδ also underwent rapid exocytosis. Fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy showed that the interaction between PLDδ and the microdomain marker AtREMORIN1.3 (AtREM1.3) increased in response to chitin, indicating that exocytosis facilitates rapid, efficient sorting of PLDδ into microdomains upon pathogen stimulus. We further unveiled a trade-off between brefeldin A (BFA)-resistant and -sensitive pathways in secretion of PLDδ under diverse conditions. Upon pathogen attack, PLDδ secretion involved syntaxin-associated VAMP721/722-mediated exocytosis sensitive to BFA. Analysis of phosphatidic acid (PA), hydrogen peroxide, and jasmonic acid (JA) levels and expression of related genes indicated that the relocalization of PLDδ is crucial for its activation to produce PA and initiate reactive oxygen species and JA signaling pathways. Together, our findings revealed that the translocation of PLDδ to papillae is modulated by exocytosis, thus triggering PA-mediated signaling in plant innate immunity.plantcell;31/12/3015/FX1F1fx1.

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.

In vivo light-sheet microscopy resolves localisation patterns of FSD1, a superoxide dismutase with function in root development and osmoprotection.

Superoxide dismutases (SODs) are enzymes detoxifying superoxide to hydrogen peroxide while temporal developmental expression and subcellular localisation are linked to their functions. Therefore, we aimed here to reveal in vivo developmental expression, subcellular, tissue- and organ-specific localisation of iron superoxide dismutase 1 (FSD1) in Arabidopsis using light-sheet and Airyscan confocal microscopy. FSD1-GFP temporarily accumulated at the site of endosperm rupture during seed germination. In emerged roots, it showed the highest abundance in cells of the lateral root cap, columella, and endodermis/cortex initials. The largest subcellular pool of FSD1-GFP was localised in the plastid stroma, while it was also located in the nuclei and cytosol. The majority of the nuclear FSD1-GFP is immobile as revealed by fluorescence recovery after photobleaching. We found that fsd1 knockout mutants exhibit reduced lateral root number and this phenotype was reverted by genetic complementation. Mutant analysis also revealed a requirement for FSD1 in seed germination during salt stress. Salt stress tolerance was coupled with the accumulation of FSD1-GFP in Hechtian strands and superoxide removal. It is likely that the plastidic pool is required for acquiring oxidative stress tolerance in Arabidopsis. This study suggests new developmental and osmoprotective functions of SODs in plants.

Single Amino Acid Exchange in ACTIN2 Confers Increased Tolerance to Oxidative Stress in Arabidopsis der1-3 Mutant.

Single-point mutation in the ACTIN2 gene of the der1-3 mutant revealed that ACTIN2 is an essential actin isovariant required for root hair tip growth, and leads to shorter, thinner and more randomly oriented actin filaments in comparison to the wild-type C24 genotype. The actin cytoskeleton has been linked to plant defense against oxidative stress, but it is not clear how altered structural organization and dynamics of actin filaments may help plants to cope with oxidative stress. In this study, we characterized root growth, plant biomass, actin organization and antioxidant activity of the der1-3 mutant under oxidative stress induced by paraquat and H2O2. Under these conditions, plant growth was better in the der1-3 mutant, while the actin cytoskeleton in the der1-3 carrying pro35S::GFP:FABD2 construct showed a lower bundling rate and higher dynamicity. Biochemical analyses documented a lower degree of lipid peroxidation, and an elevated capacity to decompose superoxide and hydrogen peroxide. These results support the view that the der1-3 mutant is more resistant to oxidative stress. We propose that alterations in the actin cytoskeleton, increased sensitivity of ACTIN to reducing agent dithiothreitol (DTT), along with the increased capacity to decompose reactive oxygen species encourage the enhanced tolerance of this mutant against oxidative stress.

Phosphorylation-Mediated Dynamics of Nitrate Transceptor NRT1.1 Regulate Auxin Flux and Nitrate Signaling in Lateral Root Growth.

The dual-affinity nitrate transceptor NITRATE TRANSPORTER1.1 (NRT1.1) has two modes of transport and signaling, governed by Thr-101 (T101) phosphorylation. NRT1.1 regulates lateral root (LR) development by modulating nitrate-dependent basipetal auxin export and nitrate-mediated signal transduction. Here, using the Arabidopsis (Arabidopsis thaliana) NRT1.1T101D phosphomimetic and NRT1.1T101A nonphosphorylatable mutants, we found that the phosphorylation state of NRT1.1 plays a key role in NRT1.1 function during LR development. Single-particle tracking revealed that phosphorylation affected NRT1.1 spatiotemporal dynamics. The phosphomimetic NRT1.1T101D form showed fast lateral mobility and membrane partitioning that facilitated auxin flux under low-nitrate conditions. By contrast, nonphosphorylatable NRT1.1T101A showed low lateral mobility and oligomerized at the plasma membrane (PM), where it induced endocytosis via the clathrin-mediated endocytosis and microdomain-mediated endocytosis pathways under high-nitrate conditions. These behaviors promoted LR development by suppressing NRT1.1-controlled auxin transport on the PM and stimulating Ca2+-ARABIDOPSIS NITRATE REGULATED1 signaling from the endosome.

Multifaceted roles of HEAT SHOCK PROTEIN 90 molecular chaperones in plant development.

HEAT SHOCK PROTEINS 90 (HSP90s) are molecular chaperones that mediate correct folding and stability of many client proteins. These chaperones act as master molecular hubs involved in multiple aspects of cellular and developmental signalling in diverse organisms. Moreover, environmental and genetic perturbations affect both HSP90s and their clients, leading to alterations of molecular networks determining respectively plant phenotypes and genotypes and contributing to a broad phenotypic plasticity. Although HSP90 interaction networks affecting the genetic basis of phenotypic variation and diversity have been thoroughly studied in animals, such studies are just starting to emerge in plants. Here, we summarize current knowledge and discuss HSP90 network functions in plant development and cellular homeostasis.

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.

Acceleration of Carbon Fixation in Chilling-Sensitive Banana under Mild and Moderate Chilling Stresses.

Banana is one of the most important food and fruit crops in the world and its growth is ceasing at 10-17 °C. However, the mechanisms determining the tolerance of banana to mild (>15 °C) and moderate chilling (10-15 °C) are elusive. Furthermore, the biochemical controls over the photosynthesis in tropical plant species at low temperatures above 10 °C is not well understood. The purpose of this research was to reveal the response of chilling-sensitive banana to mild (16 °C) and moderate chilling stress (10 °C) at the molecular (transcripts, proteins) and physiological levels. The results showed different transcriptome responses between mild and moderate chilling stresses, especially in pathways of plant hormone signal transduction, ABC transporters, ubiquinone, and other terpenoid-quinone biosynthesis. Interestingly, functions related to carbon fixation were assigned preferentially to upregulated genes/proteins, while photosynthesis and photosynthesis-antenna proteins were downregulated at 10 °C, as revealed by both digital gene expression and proteomic analysis. These results were confirmed by qPCR and immunofluorescence labeling methods. Conclusion: Banana responded to the mild chilling stress dramatically at the molecular level. To compensate for the decreased photosynthesis efficiency caused by mild and moderate chilling stresses, banana accelerated its carbon fixation, mainly through upregulation of phosphoenolpyruvate carboxylases.