Monocotyledonous plants graft at the embryonic root-shoot interface.

Grafting is possible in both animals and plants. Although in animals the process requires surgery and is often associated with rejection of non-self, in plants grafting is widespread, and has been used since antiquity for crop improvement1. However, in the monocotyledons, which represent the second largest group of terrestrial plants and include many staple crops, the absence of vascular cambium is thought to preclude grafting2. Here we show that the embryonic hypocotyl allows intra- and inter-specific grafting in all three monocotyledon groups: the commelinids, lilioids and alismatids. We show functional graft unions through histology, application of exogenous fluorescent dyes, complementation assays for movement of endogenous hormones, and growth of plants to maturity. Expression profiling identifies genes that unify the molecular response associated with grafting in monocotyledons and dicotyledons, but also gene families that have not previously been associated with tissue union. Fusion of susceptible wheat scions to oat rootstocks confers resistance to the soil-borne pathogen Gaeumannomyces graminis. Collectively, these data overturn the consensus that monocotyledons cannot form graft unions, and identify the hypocotyl (mesocotyl in grasses) as a meristematic tissue that allows this process. We conclude that graft compatibility is a shared ability among seed-bearing plants.

ISRES+: an improved evolutionary strategy for function minimization to estimate the free parameters of systems biology models.

MOTIVATION: Mathematical models in systems biology help generate hypotheses, guide experimental design, and infer the dynamics of gene regulatory networks. These models are characterized by phenomenological or mechanistic parameters, which are typically hard to measure. Therefore, efficient parameter estimation is central to model development. Global optimization techniques, such as evolutionary algorithms (EAs), are applied to estimate model parameters by inverse modeling, i.e. calibrating models by minimizing a function that evaluates a measure of the error between model predictions and experimental data. EAs estimate model parameters "fittest individuals" by generating a large population of individuals using strategies like recombination and mutation over multiple "generations." Typically, only a few individuals from each generation are used to create new individuals in the next generation. Improved Evolutionary Strategy by Stochastic Ranking (ISRES), proposed by Runnarson and Yao, is one such EA that is widely used in systems biology to estimate parameters. ISRES uses information at most from a pair of individuals in any generation to create a new population to minimize the error. In this article, we propose an efficient evolutionary strategy, ISRES+, which builds on ISRES by combining information from all individuals across the population and across all generations to develop a better understanding of the fitness landscape. RESULTS: ISRES+ uses the additional information generated by the algorithm during evolution to approximate the local neighborhood around the best-fit individual using linear least squares fits in one and two dimensions, enabling efficient parameter estimation. ISRES+ outperforms ISRES and results in fitter individuals with a tighter distribution over multiple runs, such that a typical run of ISRES+ estimates parameters with a higher goodness-of-fit compared with ISRES. AVAILABILITY AND IMPLEMENTATION: Algorithm and implementation: Github-https://github.com/gtreeves/isres-plus-bandodkar-2022.

Using breeding and quantitative genetics to understand the C4 pathway.

Reducing photorespiration in C3 crops could significantly increase rates of photosynthesis and yield. One method to achieve this would be to integrate C4 photosynthesis into C3 species. This objective is challenging as it involves engineering incompletely understood traits into C3 leaves, including complex changes to their biochemistry, cell biology, and anatomy. Quantitative genetics and selective breeding offer underexplored routes to identify regulators of these processes. We first review examples of natural intraspecific variation in C4 photosynthesis as well as the potential for hybridization between C3 and C4 species. We then discuss how quantitative genetic approaches including artificial selection and genome-wide association could be used to better understand the C4 syndrome and in so doing guide the engineering of the C4 pathway into C3 crops.

A Rubisco-binding protein is required for normal pyrenoid number and starch sheath morphology in Chlamydomonas reinhardtii.

A phase-separated, liquid-like organelle called the pyrenoid mediates CO2 fixation in the chloroplasts of nearly all eukaryotic algae. While most algae have 1 pyrenoid per chloroplast, here we describe a mutant in the model alga Chlamydomonas that has on average 10 pyrenoids per chloroplast. Characterization of the mutant leads us to propose a model where multiple pyrenoids are favored by an increase in the surface area of the starch sheath that surrounds and binds to the liquid-like pyrenoid matrix. We find that the mutant's phenotypes are due to disruption of a gene, which we call StArch Granules Abnormal 1 (SAGA1) because starch sheath granules, or plates, in mutants lacking SAGA1 are more elongated and thinner than those of wild type. SAGA1 contains a starch binding motif, suggesting that it may directly regulate starch sheath morphology. SAGA1 localizes to multiple puncta and streaks in the pyrenoid and physically interacts with the small and large subunits of the carbon-fixing enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), a major component of the liquid-like pyrenoid matrix. Our findings suggest a biophysical mechanism by which starch sheath morphology affects pyrenoid number and CO2-concentrating mechanism function, advancing our understanding of the structure and function of this biogeochemically important organelle. More broadly, we propose that the number of phase-separated organelles can be regulated by imposing constraints on their surface area.

Mechanism and implications of morphogen shuttling: Lessons learned from dorsal and Cactus in Drosophila.

In a developing animal, morphogen gradients act to pattern tissues into distinct domains of cell types. However, despite their prevalence in development, morphogen gradient formation is a matter of debate. In our recent publication, we showed that the Dorsal/NF-κB morphogen gradient, which patterns the DV axis of the early Drosophila embryo, is partially established by a mechanism of facilitated diffusion. This mechanism, also known as "shuttling," occurs when a binding partner of the morphogen facilitates the diffusion of the morphogen, allowing it to accumulate at a given site. In this case, the inhibitor Cactus/IκB facilitates the diffusion of Dorsal/NF-κB. In the fly embryo, we used computation and experiment to not only show that shuttling occurs in the embryo, but also that it enables the viability of embryos that inherit only one copy of dorsal maternally. In this commentary, we further discuss our evidence behind the shuttling mechanism, the previous literature data explained by the mechanism, and how it may also be critical for robustness of development. Finally, we briefly provide additional experimental data pointing toward an interaction between Dorsal and BMP signaling that is likely affected by shuttling.

Robustness of the Dorsal morphogen gradient with respect to morphogen dosage.

In multicellular organisms, the timing and placement of gene expression in a developing tissue assigns the fate of each cell in the embryo in order for a uniform field of cells to differentiate into a reproducible pattern of organs and tissues. This positional information is often achieved through the action of spatial gradients of morphogens. Spatial patterns of gene expression are paradoxically robust to variations in morphogen dosage, given that, by definition, gene expression must be sensitive to morphogen concentration. In this work we investigate the robustness of the Dorsal/NF-κB signaling module with respect to perturbations to the dosage of maternally-expressed dorsal mRNA. The Dorsal morphogen gradient patterns the dorsal-ventral axis of the early Drosophila embryo, and we found that an empirical description of the Dorsal gradient is highly sensitive to maternal dorsal dosage. In contrast, we found experimentally that gene expression patterns are highly robust. Although the components of this signaling module have been characterized in detail, how their function is integrated to produce robust gene expression patterns to variations in the dorsal maternal dosage is still unclear. Therefore, we analyzed a mechanistic model of the Dorsal signaling module and found that Cactus, a cytoplasmic inhibitor for Dorsal, must be present in the nucleus for the system to be robust. Furthermore, active Toll, the receptor that dissociates Cactus from Dorsal, must be saturated. Finally, the vast majority of robust descriptions of the system require facilitated diffusion of Dorsal by Cactus. Each of these three recently-discovered mechanisms of the Dorsal module are critical for robustness. These mechanisms synergistically contribute to changing the amplitude and shape of the active Dorsal gradient, which is required for robust gene expression. Our work highlights the need for quantitative understanding of biophysical mechanisms of morphogen gradients in order to understand emergent phenotypes, such as robustness.

Ancient duons may underpin spatial patterning of gene expression in C4 leaves.

If the highly efficient C4 photosynthesis pathway could be transferred to crops with the C3 pathway there could be yield gains of up to 50%. It has been proposed that the multiple metabolic and developmental modifications associated with C4 photosynthesis are underpinned by relatively few master regulators that have allowed the evolution of C4 photosynthesis more than 60 times in flowering plants. Here we identify a component of one such regulator that consists of a pair of cis-elements located in coding sequence of multiple genes that are preferentially expressed in bundle sheath cells of C4 leaves. These motifs represent duons as they play a dual role in coding for amino acids as well as controlling the spatial patterning of gene expression associated with the C4 leaf. They act to repress transcription of C4 photosynthesis genes in mesophyll cells. These duons are also present in the C3 model Arabidopsis thaliana, and, in fact, are conserved in all land plants and even some algae that use C3 photosynthesis. C4 photosynthesis therefore appears to have coopted an ancient regulatory code to generate the spatial patterning of gene expression that is a hallmark of C4 photosynthesis. This intragenic transcriptional regulatory sequence could be exploited in the engineering of efficient photosynthesis of crops.

Spatiotemporal control of gene expression boundaries using a feedforward loop.

BACKGROUND: A feedforward loop (FFL) is commonly observed in several biological networks. The FFL network motif has been mostly studied with respect to variation of the input signal in time, with only a few studies of FFL activity in a spatially distributed system such as morphogen-mediated tissue patterning. However, most morphogen gradients also evolve in time. RESULTS: We studied the spatiotemporal behavior of a coherent FFL in two contexts: (a) a generic, oscillating morphogen gradient and (b) the dorsal-ventral patterning of the early Drosophila embryo by a gradient of the NF-κB homolog dorsal with its early target Twist. In both models, we found features in the dynamics of the intermediate node-phase difference and noise filtering-that were largely independent of the parameterization of the models, and thus were functions of the structure of the FFL itself. In the dorsal gradient model, we also found that proper target gene expression was not possible without including the effect of maternal pioneer factor Zelda. CONCLUSIONS: An FFL buffers fluctuation to changes in the morphogen signal ensuring stable gene expression boundaries.

A facilitated diffusion mechanism establishes the Drosophila Dorsal gradient.

The transcription factor NF-κB plays an important role in the immune system, apoptosis and inflammation. Dorsal, a Drosophila homolog of NF-κB, patterns the dorsal-ventral axis in the blastoderm embryo. During this stage, Dorsal is sequestered outside the nucleus by the IκB homolog Cactus. Toll signaling on the ventral side breaks the Dorsal/Cactus complex, allowing Dorsal to enter the nucleus to regulate target genes. Fluorescent data show that Dorsal accumulates on the ventral side of the syncytial blastoderm. Here, we use modeling and experimental studies to show that this accumulation is caused by facilitated diffusion, or shuttling, of the Dorsal/Cactus complex. We also show that active Toll receptors are limiting in wild-type embryos, which is a key factor in explaining global Dorsal gradient formation. Our results suggest that shuttling is necessary for viability of embryos from mothers with compromised dorsal levels. Therefore, Cactus not only has the primary role of regulating Dorsal nuclear import, but also has a secondary role in shuttling. Given that this mechanism has been found in other, independent, systems, we suggest that it might be more prevalent than previously thought.

Discovery of Membrane-Permeating Cyclic Peptides via mRNA Display.

Small synthetic peptides capable of crossing biological membranes represent valuable tools in cell biology and drug delivery. While several cell-penetrating peptides (CPPs) of natural or synthetic origin have been reported, no peptide is currently known to cross both cytoplasmic and outer embryonic membranes. Here, we describe a method to engineer membrane-permeating cyclic peptides (MPPs) with broad permeation activity by screening mRNA display libraries of cyclic peptides against embryos at different developmental stages. The proposed method was demonstrated by identifying peptides capable of permeating Drosophila melanogaster (fruit fly) embryos and mammalian cells. The selected peptide cyclo[Glut-MRKRHASRRE-K*] showed a strong permeation activity of embryos exposed to minimal permeabilization pretreatment, as well as human embryonic stem cells and a murine fibroblast cell line. Notably, in both embryos and mammalian cells, the cyclic peptide outperformed its linear counterpart and the control MPPs. Confocal microscopy and single cell flow cytometry analysis were utilized to assess the degree of permeation both qualitatively and quantitatively. These MPPs have potential application in studying and nondisruptively controlling intracellular or intraembryonic processes.

Natural Variation within a Species for Traits Underpinning C4 Photosynthesis.

Engineering C4 photosynthesis into C3 crops could substantially increase their yield by alleviating photorespiratory losses. This objective is challenging because the C4 pathway involves complex modifications to the biochemistry, cell biology, and anatomy of leaves. Forward genetics has provided limited insight into the mechanistic basis of these properties, and there have been no reports of significant quantitative intraspecific variation of C4 attributes that would allow trait mapping. Here, we show that accessions of the C4 species Gynandropsis gynandra collected from locations across Africa and Asia exhibit natural variation in key characteristics of C4 photosynthesis. Variable traits include bundle sheath size and vein density, gas-exchange parameters, and carbon isotope discrimination associated with the C4 state. The abundance of transcripts encoding core enzymes of the C4 cycle also showed significant variation. Traits relating to water use showed more quantitative variation than those associated with carbon assimilation. We propose that variation in these traits likely adapted the hydraulic system for increased water use efficiency rather than improving carbon fixation, indicating that selection pressure may drive C4 diversity in G. gynandra by modifying water use rather than photosynthesis. The accessions analyzed can be easily crossed and produce fertile offspring. Our findings, therefore, indicate that natural variation within this C4 species is sufficiently large to allow genetic mapping of key C4 traits and regulators.

A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle.

Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere's composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO2 Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO2 We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1's four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency.

Regulatory gateways for cell-specific gene expression in C4 leaves with Kranz anatomy.

C4 photosynthesis is a carbon-concentrating mechanism that increases delivery of carbon dioxide to RuBisCO and as a consequence reduces photorespiration. The C4 pathway is therefore beneficial in environments that promote high photorespiration. This pathway has evolved many times, and involves restricting gene expression to either mesophyll or bundle sheath cells. Here we review the regulatory mechanisms that control cell-preferential expression of genes in the C4 cycle. From this analysis, it is clear that the C4 pathway has a complex regulatory framework, with control operating at epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels. Some genes of the C4 pathway are regulated at multiple levels, and we propose that this ensures robust expression in each cell type. Accumulating evidence suggests that multiple genes of the C4 pathway may share the same regulatory mechanism. The control systems for C4 photosynthesis gene expression appear to operate in C3 plants, and so it appears that pre-existing mechanisms form the basis of C4 photosynthesis gene expression.

Broadening the spectrum of photosynthesis in the grass, Alloteropsis semialata.

This article comments on: Evolutionary implications of C3 -C4 intermediates in the grass Alloteropsis semialata.

The presence of nuclear cactus in the early Drosophila embryo may extend the dynamic range of the dorsal gradient.

In a developing embryo, the spatial distribution of a signaling molecule, or a morphogen gradient, has been hypothesized to carry positional information to pattern tissues. Recent measurements of morphogen distribution have allowed us to subject this hypothesis to rigorous physical testing. In the early Drosophila embryo, measurements of the morphogen Dorsal, which is a transcription factor responsible for initiating the earliest zygotic patterns along the dorsal-ventral axis, have revealed a gradient that is too narrow to pattern the entire axis. In this study, we use a mathematical model of Dorsal dynamics, fit to experimental data, to determine the ability of the Dorsal gradient to regulate gene expression across the entire dorsal-ventral axis. We found that two assumptions are required for the model to match experimental data in both Dorsal distribution and gene expression patterns. First, we assume that Cactus, an inhibitor that binds to Dorsal and prevents it from entering the nuclei, must itself be present in the nuclei. And second, we assume that fluorescence measurements of Dorsal reflect both free Dorsal and Cactus-bound Dorsal. Our model explains the dynamic behavior of the Dorsal gradient at lateral and dorsal positions of the embryo, the ability of Dorsal to regulate gene expression across the entire dorsal-ventral axis, and the robustness of gene expression to stochastic effects. Our results have a general implication for interpreting fluorescence-based measurements of signaling molecules.

Size-dependent regulation of dorsal-ventral patterning in the early Drosophila embryo.

How natural variation in embryo size affects patterning of the Drosophila embryo dorsal-ventral (DV) axis is not known. Here we examined quantitatively the relationship between nuclear distribution of the Dorsal transcription factor, boundary positions for several target genes, and DV axis length. Data were obtained from embryos of a wild-type background as well as from mutant lines inbred to size select embryos of smaller or larger sizes. Our data show that the width of the nuclear Dorsal gradient correlates with DV axis length. In turn, for some genes expressed along the DV axis, the boundary positions correlate closely with nuclear Dorsal levels and with DV axis length; while the expression pattern of others is relatively constant and independent of the width of the Dorsal gradient. In particular, the patterns of snail (sna) and ventral nervous system defective (vnd) correlate with nuclear Dorsal levels and exhibit scaling to DV length; while the pattern of intermediate neuroblasts defective (ind) remains relatively constant with respect to changes in Dorsal and DV length. However, in mutants that exhibit an abnormal expansion of the Dorsal gradient which fails to scale to DV length, only sna follows the Dorsal distribution and exhibits overexpansion; in contrast, vnd and ind do not overexpand suggesting some additional mechanism acts to refine the dorsal boundaries of these two genes. Thus, our results argue against the idea that the Dorsal gradient works as a global system of relative coordinates along the DV axis and suggest that individual targets respond to changes in embryo size in a gene-specific manner.

Image analysis and empirical modeling of gene and protein expression.

Protein gradients and gene expression patterns are major determinants in the differentiation and fate map of the developing embryo. Here we discuss computational methods to quantitatively measure the positions of gene expression domains and the gradients of protein expression along the dorsal-ventral axis in the Drosophila embryo. Our methodology involves three layers of data. The first layer, or the primary data, consists of z-stack confocal images of embryos processed by in situ hybridization and/or antibody stainings. The secondary data are relationships between location, usually an x-axis coordinate, and fluorescent intensity of gene or protein detection. Tertiary data comprise the optimal parameters that arise from fits of the secondary data to empirical models. The tertiary data are useful to distill large datasets of imaged embryos down to a tractable number of conceptually useful parameters. This analysis allows us to detect subtle phenotypes and is adaptable to any set of genes or proteins with a canonical pattern. For example, we show how insights into the Dorsal transcription factor protein gradient and its target gene ventral-neuroblasts defective (vnd) were obtained using such quantitative approaches.

Dynamics of BMP signaling and stable gene expression in the early Drosophila embryo.

In developing tissues, morphogen gradients are thought to initialize gene expression patterns. However, the relationship between the dynamics of morphogen-encoded signals and gene expression decisions is largely unknown. Here we examine the dynamics of the Bone Morphogenetic Protein (BMP) pathway in Drosophila blastoderm-stage embryos. In this tissue, the BMP pathway is highly dynamic: it begins as a broad and weak signal on the dorsal half of the embryo, then 20-30 min later refines into a narrow, intense peak centered on the dorsal midline. This dynamical progression of the BMP signal raises questions of how it stably activates target genes. Therefore, we performed live imaging of the BMP signal and found that dorsal-lateral cells experience only a short transient in BMP signaling, after which the signal is lost completely. Moreover, we measured the transcriptional response of the BMP target gene pannier in live embryos and found it to remain activated in dorsal-lateral cells, even after the BMP signal is lost. Our findings may suggest that the BMP pathway activates a memory, or 'ratchet' mechanism that may sustain gene expression.

Optimal performance objectives in the highly conserved bone morphogenetic protein signaling pathway.

Throughout development, complex networks of cell signaling pathways drive cellular decision-making across different tissues and contexts. The transforming growth factor ß (TGF-ß) pathways, including the BMP/Smad pathway, play crucial roles in determining cellular responses. However, as the Smad pathway is used reiteratively throughout the life cycle of all animals, its systems-level behavior varies from one context to another, despite the pathway connectivity remaining nearly constant. For instance, some cellular systems require a rapid response, while others require high noise filtering. In this paper, we examine how the BMP-Smad pathway balances trade-offs among three such systems-level behaviors, or "Performance Objectives (POs)": response speed, noise amplification, and the sensitivity of pathway output to receptor input. Using a Smad pathway model fit to human cell data, we show that varying non-conserved parameters (NCPs) such as protein concentrations, the Smad pathway can be tuned to emphasize any of the three POs and that the concentration of nuclear phosphatase has the greatest effect on tuning the POs. However, due to competition among the POs, the pathway cannot simultaneously optimize all three, but at best must balance trade-offs among the POs. We applied the multi-objective optimization concept of the Pareto Front, a widely used concept in economics to identify optimal trade-offs among various requirements. We show that the BMP pathway efficiently balances competing POs across species and is largely Pareto optimal. Our findings reveal that varying the concentration of NCPs allows the Smad signaling pathway to generate a diverse range of POs. This insight identifies how signaling pathways can be optimally tuned for each context.