Label-free functional analysis of root-associated microbes with dynamic quantitative oblique back-illumination microscopy.

The increasing global demand for food, coupled with concerns about the environmental impact of synthetic fertilizers, underscores the urgency of developing sustainable agricultural practices. Nitrogen-fixing bacteria, known as diazotrophs, offer a potential solution by converting atmospheric nitrogen into bioavailable forms, reducing the reliance on synthetic fertilizers. However, a deeper understanding of their interactions with plants and other microbes is needed. In this study, we introduce a recently developed label-free 3D quantitative phase imaging technology called dynamic quantitative oblique back-illumination microscopy (DqOBM) to assess the functional dynamic activity of diazotrophs in vitro and in situ. Our experiments involved three different diazotrophs (Sinorhizobium meliloti, Azotobacter vinelandii, and Rahnella aquatilis) cultured on media with amendments of carbon and nitrogen sources. Over 5 days, we observed increased dynamics in nutrient-amended media. These results suggest that the observed bacterial dynamics correlate with their metabolic activity. Furthermore, we applied qOBM to visualize microbial dynamics within the root cap and elongation zone of Arabidopsis thaliana primary roots. This allowed us to identify distinct areas of microbial infiltration in plant roots without the need for fluorescent markers. Our findings demonstrate that DqOBM can effectively characterize microbial dynamics and provide insights into plant-microbe interactions in situ, offering a valuable tool for advancing our understanding of sustainable agriculture.

Label-Free Functional Analysis of Root-Associated Microbes with Dynamic Quantitative Oblique Back-illumination Microscopy.

The increasing global demand for food, coupled with concerns about the environmental impact of synthetic fertilizers, underscores the urgency of developing sustainable agricultural practices. Nitrogen-fixing bacteria, known as diazotrophs, offer a potential solution by converting atmospheric nitrogen into bioavailable forms, reducing the reliance on synthetic fertilizers. However, a deeper understanding of their interactions with plants and other microbes is needed. In this study, we introduce a recently developed label-free 3D quantitative phase imaging technology called dynamic quantitative oblique back-illumination microscopy (DqOBM) to assess the dynamic activity of diazotrophs in vitro and in situ. Our experiments involved three different diazotrophs (Sinorhizobium meliloti, Azotobacter vinelandii, and Rahnella aquatilis) cultured on media with amendments of carbon and nitrogen sources. Over five days, we observed increased dynamic activity in nutrient-amended media. These results suggest that the observed bacterial dynamics correlate with their metabolic activity. Furthermore, we applied qOBM to visualize bacterial activity within the root cap and elongation zone of Arabidopsis thaliana primary roots. This allowed us to identify distinct areas of microbial infiltration in plant roots without the need for fluorescent markers. Our findings demonstrate that DqOBM can effectively characterize microbial activity and provide insights into plant-microbe interactions in situ, offering a valuable tool for advancing our understanding of sustainable agriculture.

The Arabidopsis SWEET1 and SWEET2 uniporters recognize similar substrates while differing in subcellular localization.

Sugars Will Eventually be Exported Transporters (SWEETs) are central for sugar allocation in plants. The SWEET family has approximately 20 homologs in most plant genomes, and despite extensive research on their structures and molecular functions, it is still unclear how diverse SWEETs recognize different substrates. Previous work using SweetTrac1, a biosensor constructed by the intramolecular fusion of a conformation-sensitive fluorescent protein in the plasma membrane transporter SWEET1 from Arabidopsis thaliana, identified common features in the transporter's substrates. Here, we report SweetTrac2, a new biosensor based on the Arabidopsis vacuole membrane transporter SWEET2, and use it to explore the substrate specificity of this second protein. Our results show that SWEET1 and SWEET2 recognize similar substrates but some with different affinities. Sequence comparison and mutagenesis analysis support the conclusion that the differences in affinity depend on nonspecific interactions involving previously uncharacterized residues in the substrate-binding pocket. Furthermore, SweetTrac2 can be an effective tool for monitoring sugar transport at vacuolar membranes that would be otherwise challenging to study.

Exploring the Substrate Specificity of a Sugar Transporter with Biosensors and Cheminformatics.

Sugars will eventually be exported transporters (SWEETs) are conserved sugar transporters that play crucial roles in plant physiology and biotechnology. The genomes of flowering plants typically encode about 20 SWEET paralogs that can be classified into four clades. Clades I, II, and IV have been reported to favor hexoses, while clade III SWEETs prefer sucrose. However, the molecular features of substrates required for recognition by members of this family have not been investigated in detail. Here, we show that SweetTrac1, a previously reported biosensor constructed from the Clade I Arabidopsis thaliana SWEET1, can provide insight into the structural requirements for substrate recognition. The biosensor translates substrate binding to the transporter into a change in fluorescence, and its application in a small-molecule screen combined with cheminformatics uncovered 12 new sugars and their derivatives capable of eliciting a response. Furthermore, we confirmed that the wild-type transporter mediates cellular uptake of three of these species, including the diabetes drugs 1-deoxynojirimycin and voglibose. Our results show that SWEETs can recognize different furanoses, pyranoses, and acyclic sugars, illustrating the potential of combining biosensors and computational techniques to uncover the basis of substrate specificity.

Toolboxes for plant systems biology research.

The terms 'systems' and 'synthetic biology' are often used together, with most scientists striding between the two fields rather than adhering to a single side. Often too, scientists want to understand a system to inform the design of gene circuits that could endow it with new functions. However, this does not need to be the progression of research, as synthetic constructs can help improve our understanding of a system. Here, we review synthetic biology tool kits with the potential to overcome pleiotropic effects, compensatory mechanisms, and redundancy in plants. Combined with -omics techniques, these tools could reveal novel insights on plant growth and development, an aim that has gained renewed urgency given the impact of climate change on crop productivity.

Development and quantitative analysis of a biosensor based on the Arabidopsis SWEET1 sugar transporter.

SWEETs are transporters with homologs in Archeae, plants, some fungi, and animals. As the only transporters known to facilitate the cellular release of sugars in plants, SWEETs play critical roles in the allocation of sugars from photosynthetic leaves to storage tissues in seeds, fruits, and tubers. Here, we report the design and use of genetically encoded biosensors to measure the activity of SWEETs. We created a SweetTrac1 sensor by inserting a circularly permutated green fluorescent protein into the Arabidopsis SWEET1, resulting in a chimera that translates substrate binding during the transport cycle into detectable changes in fluorescence intensity. We demonstrate that a combination of cell sorting and bioinformatics can accelerate the design of biosensors and formulate a mass action kinetics model to correlate the fluorescence response of SweetTrac1 with the transport of glucose. Our analysis suggests that SWEETs are low-affinity, symmetric transporters that can rapidly equilibrate intra- and extracellular concentrations of sugars. This approach can be extended to SWEET homologs and other transporters.

When SWEETs Turn Tweens: Updates and Perspectives.

Sugar translocation between cells and between subcellular compartments in plants requires either plasmodesmata or a diverse array of sugar transporters. Interactions between plants and associated microorganisms also depend on sugar transporters. The sugars will eventually be exported transporter (SWEET) family is made up of conserved and essential transporters involved in many critical biological processes. The functional significance and small size of these proteins have motivated crystallographers to successfully capture several structures of SWEETs and their bacterial homologs in different conformations. These studies together with molecular dynamics simulations have provided unprecedented insights into sugar transport mechanisms in general and into substrate recognition of glucose and sucrose in particular. This review summarizes our current understanding of the SWEET family, from the atomic to the whole-plant level. We cover methods used for their characterization, theories about their evolutionary origins, biochemical properties, physiological functions, and regulation. We also include perspectives on the future work needed to translate basic research into higher crop yields.

Tuning self-renewal in the Arabidopsis stomatal lineage by hormone and nutrient regulation of asymmetric cell division.

Asymmetric and self-renewing divisions build and pattern tissues. In the Arabidopsis stomatal lineage, asymmetric cell divisions, guided by polarly localized cortical proteins, generate most cells on the leaf surface. Systemic and environmental signals modify tissue development, but the mechanisms by which plants incorporate such cues to regulate asymmetric divisions are elusive. In a screen for modulators of cell polarity, we identified CONSTITUTIVE TRIPLE RESPONSE1, a negative regulator of ethylene signaling. We subsequently revealed antagonistic impacts of ethylene and glucose signaling on the self-renewing capacity of stomatal lineage stem cells. Quantitative analysis of cell polarity and fate dynamics showed that developmental information may be encoded in both the spatial and temporal asymmetries of polarity proteins. These results provide a framework for a mechanistic understanding of how nutritional status and environmental factors tune stem-cell behavior in the stomatal lineage, ultimately enabling flexibility in leaf size and cell-type composition.

Quantitative and dynamic cell polarity tracking in plant cells.

Quantitative information on the spatiotemporal distribution of polarised proteins is central for understanding cell-fate determination, yet collecting sufficient data for statistical analysis is difficult to accomplish with manual measurements. Here we present Polarity Measurement (Pome), a semi-automated pipeline for the quantification of cell polarity and demonstrate its application to a variety of developmental contexts. Pome analysis reveals that, during asymmetric cell divisions in the Arabidopsis thaliana stomatal lineage, polarity proteins BASL and BRXL2 are more asynchronous and less mutually dependent than previously thought. A similar analysis of the linearly arrayed stomatal lineage of Brachypodium distachyon revealed that the MAPKKK BdYDA1 is segregated and polarised following asymmetrical divisions. Our results demonstrate that Pome is a versatile tool, which by itself or combined with tissue-level studies and advanced microscopy techniques can help to uncover new mechanisms of cell polarity.

GRIBCG: a software for selection of sgRNAs in the design of balancer chromosomes.

BACKGROUND: Balancer chromosomes are tools used by fruit fly geneticists to prevent meiotic recombination. Recently, CRISPR/Cas9 genome editing has been shown capable of generating inversions similar to the chromosomal rearrangements present in balancer chromosomes. Extending the benefits of balancer chromosomes to other multicellular organisms could significantly accelerate biomedical and plant genetics research. RESULTS: Here, we present GRIBCG (Guide RNA Identifier for Balancer Chromosome Generation), a tool for the rational design of balancer chromosomes. GRIBCG identifies single guide RNAs (sgRNAs) for use with Streptococcus pyogenes Cas9 (SpCas9). These sgRNAs would efficiently cut a chromosome multiple times while minimizing off-target cutting in the rest of the genome. We describe the performance of this tool on six model organisms and compare our results to two routinely used fruit fly balancer chromosomes. CONCLUSION: GRIBCG is the first of its kind tool for the design of balancer chromosomes using CRISPR/Cas9. GRIBCG can accelerate genetics research by providing a fast, systematic and simple to use framework to induce chromosomal rearrangements.

Structure of a eukaryotic SWEET transporter in a homotrimeric complex.

Eukaryotes rely on efficient distribution of energy and carbon skeletons between organs in the form of sugars. Glucose in animals and sucrose in plants serve as the dominant distribution forms. Cellular sugar uptake and release require vesicular and/or plasma membrane transport proteins. Humans and plants use proteins from three superfamilies for sugar translocation: the major facilitator superfamily (MFS), the sodium solute symporter family (SSF; only in the animal kingdom), and SWEETs. SWEETs carry mono- and disaccharides across vacuolar or plasma membranes. Plant SWEETs play key roles in sugar translocation between compartments, cells, and organs, notably in nectar secretion, phloem loading for long distance translocation, pollen nutrition, and seed filling. Plant SWEETs cause pathogen susceptibility possibly by sugar leakage from infected cells. The vacuolar Arabidopsis thaliana AtSWEET2 sequesters sugars in root vacuoles; loss-of-function mutants show increased susceptibility to Pythium infection. Here we show that its orthologue, the vacuolar glucose transporter OsSWEET2b from rice (Oryza sativa), consists of an asymmetrical pair of triple-helix bundles, connected by an inversion linker transmembrane helix (TM4) to create the translocation pathway. Structural and biochemical analyses show OsSWEET2b in an apparent inward (cytosolic) open state forming homomeric trimers. TM4 tightly interacts with the first triple-helix bundle within a protomer and mediates key contacts among protomers. Structure-guided mutagenesis of the close paralogue SWEET1 from Arabidopsis identified key residues in substrate translocation and protomer crosstalk. Insights into the structure-function relationship of SWEETs are valuable for understanding the transport mechanism of eukaryotic SWEETs and may be useful for engineering sugar flux.

Transport of sugars.

Soluble sugars serve five main purposes in multicellular organisms: as sources of carbon skeletons, osmolytes, signals, and transient energy storage and as transport molecules. Most sugars are derived from photosynthetic organisms, particularly plants. In multicellular organisms, some cells specialize in providing sugars to other cells (e.g., intestinal and liver cells in animals, photosynthetic cells in plants), whereas others depend completely on an external supply (e.g., brain cells, roots and seeds). This cellular exchange of sugars requires transport proteins to mediate uptake or release from cells or subcellular compartments. Thus, not surprisingly, sugar transport is critical for plants, animals, and humans. At present, three classes of eukaryotic sugar transporters have been characterized, namely the glucose transporters (GLUTs), sodium-glucose symporters (SGLTs), and SWEETs. This review presents the history and state of the art of sugar transporter research, covering genetics, biochemistry, and physiology-from their identification and characterization to their structure, function, and physiology. In humans, understanding sugar transport has therapeutic importance (e.g., addressing diabetes or limiting access of cancer cells to sugars), and in plants, these transporters are critical for crop yield and pathogen susceptibility.

BMP-dependent gene repression cascade in Drosophila eggshell patterning.

Bone Morphogenetic Proteins (BMPs) signal by activating Smad transcription factors to control a number of decisions during animal development. In Drosophila, signaling by the BMP ligand Decapentaplegic (Dpp) involves the activity of brinker (brk) which, in most contexts, is repressed by Dpp. Brk encodes a transcription factor which represses BMP signaling output by antagonizing Smad-dependent target gene activation. Here, we study BMP-dependent gene regulation during Drosophila oogenesis by following the signal transmission from Dpp to its target broad (br), a gene with a crucial function in eggshell patterning. We identify regulatory sequences that account for expression of both brk and br, and connect these to the transcription factors of the pathway. We show that Dpp directly regulates brk transcription through Smad- and Schnurri (Shn)-dependent repression. Brk is epistatic to Dpp in br expression and activates br indirectly, through removal of a repressor, which is yet to be identified. Our work provides first cis-regulatory insights into transcriptional interpretation of BMP signaling in eggshell morphogenesis and defines a transcriptional cascade that connects Dpp to target gene regulation.

A multiplex fluorescent in situ hybridization protocol for clonal analysis of Drosophila oogenesis.

Fluorescent in situ hybridization (FISH) is a common inmunohistochemical method used to examine the distribution of RNAs in tissue samples. In mosaic tissues composed of a mixed population of wild-type and loss-of- or gain-of-function mutant cells, FISH allows comparison of the effect of the perturbation on gene expression patterns in a mutant cell and its wild-type neighbors. Here, we provide a protocol for the detection of RNA in Drosophila mosaic follicular epithelia, where the mosaic analysis with a repressible cell marker (MARCM) technique is used for expression of transgenes.

Structures of bacterial homologues of SWEET transporters in two distinct conformations.

SWEETs and their prokaryotic homologues are monosaccharide and disaccharide transporters that are present from Archaea to plants and humans. SWEETs play crucial roles in cellular sugar efflux processes: that is, in phloem loading, pollen nutrition and nectar secretion. Their bacterial homologues, which are called SemiSWEETs, are among the smallest known transporters. Here we show that SemiSWEET molecules, which consist of a triple-helix bundle, form symmetrical, parallel dimers, thereby generating the translocation pathway. Two SemiSWEET isoforms were crystallized, one in an apparently open state and one in an occluded state, indicating that SemiSWEETs and SWEETs are transporters that undergo rocking-type movements during the transport cycle. The topology of the triple-helix bundle is similar yet distinct to that of the basic building block of animal and plant major facilitator superfamily (MFS) transporters (for example, GLUTs and SUTs). This finding indicates two possibilities: that SWEETs and MFS transporters evolved from an ancestral triple-helix bundle or that the triple-helix bundle represents convergent evolution. In SemiSWEETs and SWEETs, two triple-helix bundles are arranged in a parallel configuration to produce the 6- and 6 + 1-transmembrane-helix pores, respectively. In the 12-transmembrane-helix MFS transporters, four triple-helix bundles are arranged into an alternating antiparallel configuration, resulting in a much larger 2 × 2 triple-helix bundle forming the pore. Given the similarity of SemiSWEETs and SWEETs to PQ-loop amino acid transporters and to mitochondrial pyruvate carriers (MPCs), the structures characterized here may also be relevant to other transporters in the MtN3 clan. The insight gained from the structures of these transporters and from the analysis of mutations of conserved residues will improve the understanding of the transport mechanism, as well as allow comparative studies of the different superfamilies involved in sugar transport and the evolution of transporters in general.

Dynamic model for the coordination of two enhancers of broad by EGFR signaling.

Although it is widely appreciated that a typical developmental control gene is regulated by multiple enhancers, coordination of enhancer activities remains poorly understood. We propose a mechanism for such coordination in Drosophila oogenesis, when the expression of the transcription factor Broad (BR) evolves from a uniform to a two-domain pattern that prefigures the formation of two respiratory eggshell appendages. This change reflects sequential activities of two enhancers of the br gene, early and late, both of which are controlled by the epidermal growth factor receptor (EGFR) pathway. The late enhancer controls br in the appendage-producing cells, but the function of the early enhancer remained unclear. We found that the early enhancer is essential for the activity of the late enhancer and induction of eggshell appendages. This requirement can be explained by a mechanism whereby the BR protein produced by the early enhancer protects the late enhancer from EGFR-dependent repression. We illustrate this complex mechanism using a computational model that correctly predicts the wild-type dynamics of BR expression and its response to genetic perturbations.

EGFR-dependent network interactions that pattern Drosophila eggshell appendages.

Similar to other organisms, Drosophila uses its Epidermal Growth Factor Receptor (EGFR) multiple times throughout development. One crucial EGFR-dependent event is patterning of the follicular epithelium during oogenesis. In addition to providing inductive cues necessary for body axes specification, patterning of the follicle cells initiates the formation of two respiratory eggshell appendages. Each appendage is derived from a primordium comprising a patch of cells expressing broad (br) and an adjacent stripe of cells expressing rhomboid (rho). Several mechanisms of eggshell patterning have been proposed in the past, but none of them can explain the highly coordinated expression of br and rho. To address some of the outstanding issues in this system, we synthesized the existing information into a revised mathematical model of follicle cell patterning. Based on the computational model analysis, we propose that dorsal appendage primordia are established by sequential action of feed-forward loops and juxtacrine signals activated by the gradient of EGFR signaling. The model describes pattern formation in a large number of mutants and points to several unanswered questions related to the dynamic interaction of the EGFR and Notch pathways.

Transcriptional interpretation of the EGF receptor signaling gradient.

Epidermal growth factor receptor (EGFR) controls a wide range of developmental events, from body axes specification in insects to cardiac development in humans. During Drosophila oogenesis, a gradient of EGFR activation patterns the follicular epithelium. Multiple transcriptional targets of EGFR in this tissue have been identified, but their regulatory elements are essentially unknown. We report the regulatory elements of broad (br) and pipe (pip), two important targets of EGFR signaling in Drosophila oogenesis. br is expressed in a complex pattern that prefigures the formation of respiratory eggshell appendages. We found that this pattern is generated by dynamic activities of two regulatory elements, which display different responses to Pointed, Capicua, and Mirror, transcription factors involved in the EGFR-mediated gene expression. One of these elements is active in a pattern similar to pip, a gene repressed by EGFR and essential for establishing the dorsoventral polarity of the embryo. We demonstrate that this similarity of expression depends on a common sequence motif that binds Mirror in vitro and is essential for transcriptional repression in vivo.

Pattern formation by receptor tyrosine kinases: analysis of the Gurken gradient in Drosophila oogenesis.

Spatial patterns of cell differentiation in developing tissues can be controlled by receptor tyrosine kinase (RTK) signaling gradients, which may form when locally secreted ligands activate uniformly expressed receptors. Graded activation of RTKs can span multiple cell diameters, giving rise to spatiotemporal patterns of signaling through the Extracellular Signal Regulated/Mitogen Activated Protein Kinase (ERK/MAPK), which connects receptor activation to multiple aspects of tissue morphogenesis. This general mechanism has been identified in numerous developmental contexts, from body axis specification in insects to patterning of the mammalian neocortex. We review recent quantitative studies of this mechanism in Drosophila oogenesis, an established genetic model of signaling through the Epidermal Growth Factor Receptor (EGFR), a highly conserved RTK.

Pattern formation by a moving morphogen source.

During Drosophila melanogaster oogenesis, the follicular epithelium that envelops the germline cyst gives rise to an elaborate eggshell, which houses the future embryo and mediates its interaction with the environment. A prominent feature of the eggshell is a pair of dorsal appendages, which are needed for embryo respiration. Morphogenesis of this structure depends on broad, a zinc-finger transcription factor, regulated by the EGFR pathway. While much has been learned about the mechanisms of broad regulation by EGFR, current understanding of processes that shape the spatial pattern of broad expression is incomplete. We propose that this pattern is defined by two different phases of EGFR activation: an early, posterior-to-anterior gradient of EGFR signaling sets the posterior boundary of broad expression, while the anterior boundary is set by a later phase of EGFR signaling, distributed in a dorsoventral gradient. This model can explain the wild-type pattern of broad in D. melanogaster, predicts how this pattern responds to genetic perturbations, and provides insight into the mechanisms driving diversification of eggshell patterning. The proposed model of the broad expression pattern can be used as a starting point for the quantitative analysis of a large number of gene expression patterns in Drosophila oogenesis.