Recent progress and potential future directions to enhance biological nitrogen fixation in faba bean (Vicia faba L.).

The necessity for sustainable agricultural practices has propelled a renewed interest in legumes such as faba bean (Vicia faba L.) as agents to help deliver increased diversity to cropped systems and provide an organic source of nitrogen (N). However, the increased cultivation of faba beans has proven recalcitrant worldwide as a result of low yields. So, it is hoped that increased and more stable yields would improve the commercial success of the crop and so the likelihood of cultivation. Enhancing biological N fixation (BNF) in faba beans holds promise not only to enhance and stabilize yields but also to increase residual N available to subsequent cereal crops grown on the same field. In this review, we cover recent progress in enhancing BNF in faba beans. Specifically, rhizobial inoculation and the optimization of fertilizer input and cropping systems have received the greatest attention in the literature. We also suggest directions for future research on the subject. In the short term, modification of crop management practices such as fertilizer and biochar input may offer the benefits of enhanced BNF. In the long term, natural variation in rhizobial strains and faba bean genotypes can be harnessed. Strategies must be optimized on a local scale to realize the greatest benefits. Future research must measure the most useful parameters and consider the economic cost of strategies alongside the advantages of enhanced BNF.

Material properties in regenerating axolotl limbs using inverse finite element analysis.

BACKGROUND: The extracellular mechanical environment plays an important role in the skeletal development process. Characterization of the material properties of regenerating tissues that recapitulate development, provides insights into the mechanical environment experienced by the cells and the maturation of the matrix. In this study, we estimated the viscoelastic material properties of regenerating forelimbs in the axolotl (Ambystoma mexicanum) at three different regeneration stages: 27 days post-amputation (mid-late bud) and 41 days post-amputation (palette stage), and fully-grown time points. A stress-relaxation indentation test followed by two-term Prony series viscoelastic inverse finite element analysis was used to obtain material parameters. Glycosaminoglycan (GAG) content was estimated using a 1,9- dimethyl methylene blue assay. RESULTS: The instantaneous and equilibrium shear moduli significantly increased with regeneration while the short-term stress relaxation time significantly decreased with limb regeneration. The long-term stress relaxation time in the fully-grown time point was significantly lower than 27 and 41 DPA groups. The GAG content was not significantly different between 27 and 41 DPA but the GAG content of cartilage in the fully-grown group was significantly greater than in 27 and 41 DPA. CONCLUSIONS: The mechanical environment of the proliferating cells changes drastically during limb regeneration. Understanding how the tissue's mechanical properties change during limb regeneration is critical for linking molecular-level matrix production of the cells to tissue-level behavior and mechanical signals.

Vacuolar Ca2+/H+ exchanger and Ca2+-ATPase homologues are differentially regulated in tipburn-resistant and susceptible lettuce (Lactuca sativa) cultivars.

Tipburn is a physiological disorder of lettuce (Lactuca sativa) and other leafy crops that causes external and internal leaf discolouration and results in serious quality issues for the fresh produce industry. Tipburn occurrence is difficult to predict and no completely effective control methods exist. This is compounded by poor knowledge of the underlying physiological and molecular basis of the condition, which appears to be associated with deficiency of calcium and other nutrients. Vacuolar calcium transporters, which are involved in calcium homeostasis in Arabidopsis, show differential expression in tipburn-resistant and susceptible Brassica oleracea lines. We therefore investigated expression of a subset of L. sativa vacuolar calcium transporter homologues, belonging to the Ca2+/H+ exchanger and Ca2+-ATPase classes, in tipburn-resistant and susceptible cultivars. This indicated that some L. sativa vacuolar calcium transporter homologues belonging to these gene classes exhibited higher expression levels in resistant cultivars, whilst others had higher expression in susceptible cultivars or were independent of tipburn phenotype. In addition, some homologues were more highly expressed in symptomatic versus asymptomatic leaves in susceptible cultivars, suggesting that tipburn-induced increases in expression are unsuccessful in conferring resistance and that differential baseline expression of such genes is important for tipburn resistance. Knowledge of individual genes associated with tipburn resistance will improve breeding for such traits and the development of resistant lettuce varieties.

Tipburn resilience in lettuce (Lactuca spp.) - the importance of germplasm resources and production system-specific assays.

BACKGROUND: Tipburn is a physiological disorder of lettuce (Lactuca spp.). It causes discoloration and collapse of leaf margins, leading to unsaleable crops in both protected (glasshouse, hydroponic) and outdoor production systems. The occurrence of tipburn is hard to predict and is sensitive to environmental conditions. Phenotyping for tipburn resilience requires diverse germplasm resources and, to date, limited material has been investigated for this condition. RESULTS: Using a Lactuca diversity fixed foundation set (DFFS) under glasshouse conditions, we identified a significant (P < 0.001) genotypic effect on tipburn resilience across both the entire population and across lines belonging to the cultivated species L. sativa alone. Latuca sativa lines exhibited significantly (P < 0.05) higher average tipburn severity than those belonging to the wild species L. saligna, L. serriola, and L. virosa but we were able to identify both cultivated and wild tipburn-resilient lines. Leaf morphology factors, which included pigmentation, width, and serration, also significantly (P < 0.05) influenced tipburn resilience. Using a recombinant inbred line (RIL) mapping population derived from two DFFS lines, different small-effect quantitative trait loci (QTLs) accounting for 12.3% and 25.2% of total tipburn variation were identified in glasshouse and field conditions, respectively. CONCLUSIONS: These results reflect the advantages of phenotyping under production-system-specific conditions for the examination of environmentally sensitive traits and highlight genetic markers and germplasm resources for the development of tipburn resilient lines for use in both protected and outdoor lettuce production. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Establishing a New Research Axolotl Colony.

The field of regenerative biology has taken a keen interest in the Mexican axolotl (Ambystoma mexicanum) over the past few decades, as this salamander successfully regenerates amputated limbs and injured body parts. Recent progress in research tool development has also made possible axolotl genetic manipulation and single-cell analysis, which will help understand the molecular mechanisms of complex tissue regeneration. To support the growing popularity of this model, we describe how to set up a new axolotl housing facility at a research laboratory. We also review husbandry practices for raising axolotls and using them in biological research, with a focus on diet, water quality, breeding, and anesthesia.

Hybridization Chain Reaction Fluorescence In Situ Hybridization (HCR-FISH) in Ambystoma mexicanum Tissue.

In situ hybridization is a standard procedure for visualizing mRNA transcripts in tissues. The recent adoption of fluorescent probes and new signal amplification methods have facilitated multiplexed RNA imaging in tissue sections and whole tissues. Here we present protocols for multiplexed hybridization chain reaction fluorescence in situ hybridization (HCR-FISH) staining, imaging, cell segmentation, and mRNA quantification in regenerating axolotl tissue sections. We also present a protocol for whole-mount staining and imaging of developing axolotl limbs.

Click Chemistry-Enabled Conjugation Strategy for Producing Dibenzodiazepinone-Type Fluorescent Probes To Target M2 Acetylcholine Receptors.

The development of fluorescently labeled receptor-targeting compounds represents a powerful pharmacological tool to study and characterize ligand-receptor interactions. Despite significant advances in developing sub-type-specific antagonists for muscarinic acetylcholine receptors (mAChRs), reports on antagonists feasible for click chemistry are less common. Here, we designed and synthesized an antagonist suitable for probe attachment through click chemistry, namely, dibenzodiazepinone (DIBA)-alkyne, based on a previously reported DIBA scaffold with a high binding affinity to type-2 mAChR (M2R). To demonstrate the versatility of DIBA-alkyne as a building block for bioconjugates, we assembled DIBA-alkyne with Cyanine5 fluorophores (Cy5) and polyethylene glycol (PEG) biomolecules to obtain fluorescent DIBA antagonist (DIBA-Cy5) and fluorescent DIBA PEG derivatives. Flow cytometric analysis showed that DIBA-Cy5 possessed a high binding affinity to M2R (Kd = 1.80 nM), a two-order magnitude higher binding affinity than M1R. Fluorescent DIBA PEG derivatives maintained a potent binding to the M2R (Kd ≤ 4 nM), confirmed by confocal microscopic imaging. Additionally, DIBA-Cy5 can serve as a fluorescent ligand in the receptor-ligand competitive binding assay for other mAChR ligands, an attractive alternative to the traditional radioligand-based assay. The competitive binding mode between DIBA-Cy5 and orthosteric antagonist atropine/allosteric modulator LY2119620 indicated a dualsteric binding mode of the DIBA-type antagonist to M2R. Lastly, we demonstrated the direct staining of DIBA-Cy5 to M2R receptors in the sinoatrial node of a mouse heart. The adaptability of the clickable DIBA antagonist to a wide range of fluorophores and biomolecules can facilitate its use in various biomedical applications such as binding assays that screen compounds for M2R as the receptor target.

Usability of deep learning pipelines for 3D nuclei identification with Stardist and Cellpose.

Segmentation of 3D images to identify cells and their molecular outputs can be difficult and tedious. Machine learning algorithms provide a promising alternative to manual analysis as emerging 3D image processing technology can save considerable time. For those unfamiliar with machine learning or 3D image analysis, the rapid advancement of the field can make navigating the newest software options confusing. In this paper, two open-source machine learning algorithms, Cellpose and Stardist, are compared in their application on a 3D light sheet dataset counting fluorescently stained proliferative cell nuclei. The effects of image tiling and background subtraction are shown through image analysis pipelines for both algorithms. Based on our analysis, the relative ease of use of Cellpose and the absence of need to train a model leaves it a strong option for 3D cell segmentation despite relatively longer processing times. When Cellpose's pretrained model yields results that are not of sufficient quality, or the analysis of a large dataset is required, Stardist may be more appropriate. Despite the time it takes to train the model, Stardist can create a model specialized to the users' dataset that can be iteratively improved until predictions are satisfactory with far lower processing time relative to other methods.

Manipulation of the Phytochemical Profile of Tenderstem® Broccoli Florets by Short Duration, Pre-Harvest LED Lighting.

Light quality has been reported to influence the phytochemical profile of broccoli sprouts/microgreens; however, few studies have researched the influence on mature broccoli. This is the first study to investigate how exposing a mature glasshouse grown vegetable brassica, Tenderstem® broccoli, to different light wavelengths before harvest influences the phytochemical content. Sixty broccoli plants were grown in a controlled environment glasshouse under ambient light until axial meristems reached >1 cm diameter, whereupon a third were placed under either green/red/far-red LED, blue LED, or remained in the original compartment. Primary and secondary spears were harvested after one and three weeks, respectively. Plant morphology, glucosinolate, carotenoid, tocopherol, and total polyphenol content were determined for each sample. Exposure to green/red/far-red light increased the total polyphenol content by up to 13% and maintained a comparable total glucosinolate content to the control. Blue light increased three of the four indole glucosinolates studied. The effect of light treatments on carotenoid and tocopherol content was inconclusive due to inconsistencies between trials, indicating that they are more sensitive to other environmental factors. These results have shown that by carefully selecting the wavelength, the nutritional content of mature broccoli prior to harvest could be manipulated according to demand.

Local mechanical stimuli correlate with tissue growth in axolotl salamander joint morphogenesis.

Movement-induced forces are critical to correct joint formation, but it is unclear how cells sense and respond to these mechanical cues. To study the role of mechanical stimuli in the shaping of the joint, we combined experiments on regenerating axolotl (Ambystoma mexicanum) forelimbs with a poroelastic model of bone rudiment growth. Animals either regrew forelimbs normally (control) or were injected with a transient receptor potential vanilloid 4 (TRPV4) agonist during joint morphogenesis. We quantified growth and shape in regrown humeri from whole-mount light sheet fluorescence images of the regenerated limbs. Results revealed significant differences in morphology and cell proliferation between groups, indicating that TRPV4 desensitization has an effect on joint shape. To link TRPV4 desensitization with impaired mechanosensitivity, we developed a finite element model of a regenerating humerus. Local tissue growth was the sum of a biological contribution proportional to chondrocyte density, which was constant, and a mechanical contribution proportional to fluid pressure. Computational predictions of growth agreed with experimental outcomes of joint shape, suggesting that interstitial pressure driven from cyclic mechanical stimuli promotes local tissue growth. Predictive computational models informed by experimental findings allow us to explore potential physical mechanisms involved in tissue growth to advance our understanding of the mechanobiology of joint morphogenesis.

Wnt Signaling Coordinates the Expression of Limb Patterning Genes During Axolotl Forelimb Development and Regeneration.

After amputation, axolotl salamanders can regenerate their limbs, but the degree to which limb regeneration recapitulates limb development remains unclear. One limitation in answering this question is our lack of knowledge about salamander limb development. Here, we address this question by studying expression patterns of genes important for limb patterning during axolotl salamander limb development and regeneration. We focus on the Wnt signaling pathway because it regulates multiple functions during tetrapod limb development, including limb bud initiation, outgrowth, patterning, and skeletal differentiation. We use fluorescence in situ hybridization to show the expression of Wnt ligands, Wnt receptors, and limb patterning genes in developing and regenerating limbs. Inhibition of Wnt ligand secretion permanently blocks limb bud outgrowth when treated early in limb development. Inhibiting Wnt signaling during limb outgrowth decreases the expression of critical signaling genes, including Fgf10, Fgf8, and Shh, leading to the reduced outgrowth of the limb. Patterns of gene expression are similar between developing and regenerating limbs. Inhibition of Wnt signaling during regeneration impacted patterning gene expression similarly. Overall, our findings suggest that limb development and regeneration utilize Wnt signaling similarly. It also provides new insights into the interaction of Wnt signaling with other signaling pathways during salamander limb development and regeneration.

A constitutively expressed fluorescent ubiquitination-based cell-cycle indicator (FUCCI) in axolotls for studying tissue regeneration.

Regulation of cell cycle progression is essential for cell proliferation during regeneration following injury. After appendage amputation, the axolotl (Ambystoma mexicanum) regenerates missing structures through an accumulation of proliferating cells known as the blastema. To study cell division during blastema growth, we generated a transgenic line of axolotls that ubiquitously expresses a bicistronic version of the fluorescent ubiquitination-based cell-cycle indicator (FUCCI). We demonstrate near-ubiquitous FUCCI expression in developing and adult tissues, and validate these expression patterns with DNA synthesis and mitosis phase markers. We demonstrate the utility of FUCCI for live and whole-mount imaging, showing the predominantly local contribution of cells during limb and tail regeneration. We also show that spinal cord amputation results in increased proliferation at least 5 mm from the site of injury. Finally, we use multimodal staining to provide cell type information for cycling cells by combining fluorescence in situ hybridization, EdU click-chemistry and immunohistochemistry on a single FUCCI tissue section. This new line of animals will be useful for studying cell cycle dynamics using in situ endpoint assays and in vivo imaging in developing and regenerating animals.

A matter of nerves.

Regrowing new body parts requires neural input to restore appropriately sized limbs in salamanders.

Imaging in vivo acetylcholine release in the peripheral nervous system with a fluorescent nanosensor.

The ability to monitor the release of neurotransmitters during synaptic transmission would significantly impact the diagnosis and treatment of neurological diseases. Here, we present a DNA-based enzymatic nanosensor for quantitative detection of acetylcholine (ACh) in the peripheral nervous system of living mice. ACh nanosensors consist of DNA as a scaffold, acetylcholinesterase as a recognition component, pH-sensitive fluorophores as signal generators, and α-bungarotoxin as a targeting moiety. We demonstrate the utility of the nanosensors in the submandibular ganglia of living mice to sensitively detect ACh ranging from 0.228 to 358 µM. In addition, the sensor response upon electrical stimulation of the efferent nerve is dose dependent, reversible, and we observe a reduction of ∼76% in sensor signal upon pharmacological inhibition of ACh release. Equipped with an advanced imaging processing tool, we further spatially resolve ACh signal propagation on the tissue level. Our platform enables sensitive measurement and mapping of ACh transmission in the peripheral nervous system.

Lung injury in axolotl salamanders induces an organ-wide proliferation response.

BACKGROUND: Ambystoma mexicanum, the axolotl salamander, is a classic model organism used to study vertebrate regeneration. It is assumed that axolotls regenerate most tissues, but the exploration of lung regeneration has not been performed until now. RESULTS: Unlike the blastema-based response used during appendage regeneration, lung amputation led to organ-wide proliferation. Pneumocytes and mesenchymal cells responded to injury by increased proliferation throughout the injured lung, which led to a recovery in lung mass and morphology by 56 days post-amputation. Receptors associated with the Neuregulin signaling pathway were upregulated at one and 3 weeks post lung amputation. We show expression of the ligand, neuregulin, in the I/X cranial nerve that innervates the lung and cells within the lung. Supplemental administration of Neuregulin peptide induced widespread proliferation in the lung similar to an injury response, suggesting that neuregulin signaling may play a significant role during lung regeneration. CONCLUSION: Our study characterizes axolotl lung regeneration. We show that the lung responds to injury by an organ-wide proliferative response of multiple cell types, including pneumocytes, to recover lung mass.

HDAC Inhibitor Titration of Transcription and Axolotl Tail Regeneration.

New patterns of gene expression are enacted and regulated during tissue regeneration. Histone deacetylases (HDACs) regulate gene expression by removing acetylated lysine residues from histones and proteins that function directly or indirectly in transcriptional regulation. Previously we showed that romidepsin, an FDA-approved HDAC inhibitor, potently blocks axolotl embryo tail regeneration by altering initial transcriptional responses to injury. Here, we report on the concentration-dependent effect of romidepsin on transcription and regeneration outcome, introducing an experimental and conceptual framework for investigating small molecule mechanisms of action. A range of romidepsin concentrations (0-10 µM) were administered from 0 to 6 or 0 to 12 h post amputation (HPA) and distal tail tip tissue was collected for gene expression analysis. Above a threshold concentration, romidepsin potently inhibited regeneration. Sigmoidal and biphasic transcription response curve modeling identified genes with inflection points aligning to the threshold concentration defining regenerative failure verses success. Regeneration inhibitory concentrations of romidepsin increased and decreased the expression of key genes. Genes that associate with oxidative stress, negative regulation of cell signaling, negative regulation of cell cycle progression, and cellular differentiation were increased, while genes that are typically up-regulated during appendage regeneration were decreased, including genes expressed by fibroblast-like progenitor cells. Using single-nuclei RNA-Seq at 6 HPA, we found that key genes were altered by romidepin in the same direction across multiple cell types. Our results implicate HDAC activity as a transcriptional mechanism that operates across cell types to regulate the alternative expression of genes that associate with regenerative success versus failure outcomes.

Combinatorial Transcriptional Profiling of Mouse and Human Enteric Neurons Identifies Shared and Disparate Subtypes In Situ.

BACKGROUND & AIMS: The enteric nervous system (ENS) coordinates essential intestinal functions through the concerted action of diverse enteric neurons (ENs). However, integrated molecular knowledge of EN subtypes is lacking. To compare human and mouse ENs, we transcriptionally profiled healthy ENS from adult humans and mice. We aimed to identify transcripts marking discrete neuron subtypes and visualize conserved EN subtypes for humans and mice in multiple bowel regions. METHODS: Human myenteric ganglia and adjacent smooth muscle were isolated by laser-capture microdissection for RNA sequencing. Ganglia-specific transcriptional profiles were identified by computationally subtracting muscle gene signatures. Nuclei from mouse myenteric neurons were isolated and subjected to single-nucleus RNA sequencing, totaling more than 4 billion reads and 25,208 neurons. Neuronal subtypes were defined using mouse single-nucleus RNA sequencing data. Comparative informatics between human and mouse data sets identified shared EN subtype markers, which were visualized in situ using hybridization chain reaction. RESULTS: Several EN subtypes in the duodenum, ileum, and colon are conserved between humans and mice based on orthologous gene expression. However, some EN subtype-specific genes from mice are expressed in completely distinct morphologically defined subtypes in humans. In mice, we identified several neuronal subtypes that stably express gene modules across all intestinal segments, with graded, regional expression of 1 or more marker genes. CONCLUSIONS: Our combined transcriptional profiling of human myenteric ganglia and mouse EN provides a rich foundation for developing novel intestinal therapeutics. There is congruency among some EN subtypes, but we note multiple species differences that should be carefully considered when relating findings from mouse ENS research to human gastrointestinal studies.

Postharvest temperature and water status influence postharvest splitting susceptibility in summer radish (Raphanus sativus L.).

BACKGROUND: Splitting is a problem that seriously affects appearance and marketability in a number of fruit and vegetables. In summer radish (Raphanus sativus L.), splitting can occur during growth, harvesting and postharvest. We investigated the factors affecting splitting susceptibility in summer radish cv. Celesta during postharvest handling. RESULTS: Splitting susceptibility was negatively related to temperature, with higher temperature reducing splitting due to dropping impact. Radish diameter was positively associated with compression failure force, suggesting that larger radishes are more resistant to compressive splitting. An increase in radish hypocotyl water content (WC) was associated with an increase in splitting susceptibility due to impact and decrease in failure force for both compression and puncture forces. Increased hypocotyl WC may increase splitting susceptibility by increasing the water potential of the radish tissue. In agreement, we found that increased hypocotyl WC was associated with higher internal water potential in radish tissue. CONCLUSIONS: We therefore recommend that the hypocotyl WC of summer radish crops be managed during the harvest and postharvest phases, and that crops are processed at higher, ambient, temperature in order to reduce splitting, before storing at low temperature and high humidity to maintain quality and shelf life. © 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.