The circadian clock organizes physiological processes in plants to occur at specific times of the day, optimizing efficient use of resources. Nitrate is a crucial inorganic nitrogen source for agricultural systems to sustain crop productivity. However, because nitrate fertilization has a negative impact on the environment, it is important to carefully manage nitrate levels. Understanding crop biological rhythms can lead to more ecologically friendly agricultural practices. Gating responses through the circadian clock could be a strategy to enhance root nitrate uptake and to limit nitrate runoff. In Arabidopsis, the NITRATE TRANSPORTER 2.1 (NRT2.1) gene encodes a key component of the high-affinity nitrate transporter system. Our study reveals that NRT2.1 exhibits a rhythmic expression pattern, with daytime increases and nighttime decreases. The NRT2.1 promoter activity remains rhythmic under constant light, indicating a circadian regulation. The clock-associated transcription factor LUX ARRHYTHMO (LUX) binds to the NRT2.1 promoter in vivo. Loss-of-function of LUX leads to increased NRT2.1 transcript levels and root nitrate uptake at dusk. This supports LUX acting as a transcriptional repressor and modulating NRT2.1 expression in a time-dependent manner. Furthermore, applying nitrate at different times of the day results in varying magnitudes of the transcriptional response in nitrate-regulated genes. We also demonstrate that a defect in the high-affinity nitrate transport system feeds back to the central oscillator by modifying the LUX promoter activity. In conclusion, this study uncovers a molecular pathway connecting the root nitrate uptake and circadian clock, with potential agro-chronobiological applications.

Optimizing root system architecture is a strategy for coping with soil fertility, such as low nitrogen input. An ample number of Arabidopsis thaliana natural accessions have set the foundation for studies on mechanisms that regulate root morphology. This report compares the Columbia-0 (Col-0) reference and Pyla-1 (Pyl-1) from a coastal zone in France, known for having the tallest sand dune in Europe. Seedlings were grown on vertical agar plates with different nitrate concentrations. The lateral root outgrowth of Col-0 was stimulated under mild depletion and repressed under nitrate enrichment. The Pyl-1 produced a long primary root and any or very few visible lateral roots across the nitrate supplies. This could reflect an adaptation to sandy soil conditions, where the primary root grows downwards to the lower strata to take up water and mobile soil resources without elongating the lateral roots. Microscopic observations revealed similar densities of lateral root primordia in both accessions. The Pyl-1 maintained the ability to initiate lateral root primordia. However, the post-initiation events seemed to be critical in modulating the lateral-root-less phenotype. In Pyl-1, the emergence of primordia through the primary root tissues was slowed, and newly formed lateral roots stayed stunted. In brief, Pyl-1 is a fascinating genotype for studying the nutritional influences on lateral root development.

Developmental thymic waves of innate-like and adaptive-like γδ T cells have been described, but the current understanding of γδ T cell development is mainly limited to mouse models. Here, we combine single cell (sc) RNA gene expression and sc γδ T cell receptor (TCR) sequencing on fetal and pediatric γδ thymocytes in order to understand the ontogeny of human γδ T cells. Mature fetal γδ thymocytes (both the Vγ9Vδ2 and nonVγ9Vδ2 subsets) are committed to either a type 1, a type 3 or a type 2-like effector fate displaying a wave-like pattern depending on gestation age, and are enriched for public CDR3 features upon maturation. Strikingly, these effector modules express different CDR3 sequences and follow distinct developmental trajectories. In contrast, the pediatric thymus generates only a small effector subset that is highly biased towards Vγ9Vδ2 TCR usage and shows a mixed type 1/type 3 effector profile. Thus, our combined dataset of gene expression and detailed TCR information at the single-cell level identifies distinct functional thymic programming of γδ T cell immunity in human.

The regulation of signalling capacity, combined with the spatiotemporal distribution of developmental signals themselves, is pivotal in setting developmental responses in both plants and animals1. The hormone auxin is a key signal for plant growth and development that acts through the AUXIN RESPONSE FACTOR (ARF) transcription factors2-4. A subset of these, the conserved class A ARFs5, are transcriptional activators of auxin-responsive target genes that are essential for regulating auxin signalling throughout the plant lifecycle2,3. Although class A ARFs have tissue-specific expression patterns, how their expression is regulated is unknown. Here we show, by investigating chromatin modifications and accessibility, that loci encoding these proteins are constitutively open for transcription. Through yeast one-hybrid screening, we identify the transcriptional regulators of the genes encoding class A ARFs from Arabidopsis thaliana and demonstrate that each gene is controlled by specific sets of transcriptional regulators. Transient transformation assays and expression analyses in mutants reveal that, in planta, the majority of these regulators repress the transcription of genes encoding class A ARFs. These observations support a scenario in which the default configuration of open chromatin enables a network of transcriptional repressors to regulate expression levels of class A ARF proteins and modulate auxin signalling output throughout development.

The circadian clock is synchronized by environmental cues, mostly by light and temperature. Explaining how the plant circadian clock responds to temperature oscillations is crucial to understanding plant responsiveness to the environment. Here, we found a prevalent temperature-dependent function of the Arabidopsis clock component EARLY FLOWERING 4 (ELF4) in the root clock. Although the clocks in roots are able to run in the absence of shoots, micrografting assays and mathematical analyses show that ELF4 moves from shoots to regulate rhythms in roots. ELF4 movement does not convey photoperiodic information, but trafficking is essential for controlling the period of the root clock in a temperature-dependent manner. Low temperatures favour ELF4 mobility, resulting in a slow-paced root clock, whereas high temperatures decrease movement, leading to a faster clock. Hence, the mobile ELF4 delivers temperature information and establishes a shoot-to-root dialogue that sets the pace of the clock in roots.

A Correction to this paper has been published: https://doi.org/10.1038/s41586-020-03066-x

The regulation of signalling capacity plays a pivotal role in setting developmental patterns in both plants and animals ([1][1]). The hormone auxin is a key signal for plant growth and development that acts through the AUXIN RESPONSE FACTOR (ARF) transcription factors ([2][2]). A subset of these ARFs comprises transcriptional activators of target genes in response to auxin, and are essential for regulating auxin signalling throughout the plant lifecycle ([3][3]). While ARF activators show tissue-specific expression patterns, it is unknown how their expression patterns are established. Chromatin modifications and accessibility studies revealed the chromatin of loci encoding ARF activators is constitutively open for transcription. Using a high-throughput yeast one-hybrid (Y1H) approach, we discovered a network of transcriptional regulators of ARF activator genes from Arabidopsis thaliana . Expression analyses demonstrated that the majority of these regulators act as repressors of ARF transcription in planta . Our observations support a scenario where the default configuration of open chromatin enables a network of transcriptional repressors to shape the expression pattern of ARF activators and provide specificity in auxin signalling output throughout development. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3

Centre for Plant Integrative Biology, School of Biosciences, University of Nottingham, Nottingham LE12 5RD, UK; Laboratoire de Reproduction et Développement des Plantes, CNRS, INRA, ENS Lyon, UCBL, Université de Lyon, Lyon, France; Molecular Genetics, Department of Biology, Faculty of Science, Utrecht University, 3584 CH Utrecht, The Netherlands; Department of Plant Biology and Genome Center, University of California Davis, One Shields Avenue, Davis, California 95616, USA; Department of Biology, Graduate School of Science, Kobe University, Kobe, 657-8501 Japan Institute of Plant Sciences, 21 Altenbergrain, 3006 Bern, Switzerland Departamento Anatomia, Biologia Celular Y Zoologia, Facultad de Ciencias, Universidad de Extremadura, Badajoz 06006, Spain; Institute of Science and Technology Austria, Am Campus 1, 3400 Klosterneuburg, Austria; Centre National de la Recherche Scientifique, Biochimie et Physiologie Moléculaire des Plantes, Montpellier SupAgro, 2 Place Pierre Viala, 34060 Montpellier, France

Lateral root primordia (LRP) originate from pericycle stem cells located deep within parental root tissues. LRP emerge through overlying root tissues by inducing auxin-dependent cell separation and hydraulic changes in adjacent cells. The auxin-inducible auxin influx carrier LAX3 plays a key role concentrating this signal in cells overlying LRP. Delimiting LAX3 expression to two adjacent cell files overlying new LRP is crucial to ensure that auxin-regulated cell separation occurs solely along their shared walls. Multiscale modeling has predicted that this highly focused pattern of expression requires auxin to sequentially induce auxin efflux and influx carriers PIN3 and LAX3, respectively. Consistent with model predictions, we report that auxin-inducible LAX3 expression is regulated indirectly by AUXIN RESPONSE FACTOR 7 (ARF7). Yeast one-hybrid screens revealed that the LAX3 promoter is bound by the transcription factor LBD29, which is a direct target for regulation by ARF7. Disrupting auxin-inducible LBD29 expression or expressing an LBD29-SRDX transcriptional repressor phenocopied the lax3 mutant, resulting in delayed lateral root emergence. We conclude that sequential LBD29 and LAX3 induction by auxin is required to coordinate cell separation and organ emergence.

Auxin represents a key signal in plants, regulating almost every aspect of their growth and development. Major breakthroughs have been made dissecting the molecular basis of auxin transport, perception, and response. In contrast, how plants control the metabolism and homeostasis of the major form of auxin in plants, indole-3-acetic acid (IAA), remains unclear. In this paper, we initially describe the function of the Arabidopsis thaliana gene DIOXYGENASE FOR AUXIN OXIDATION 1 (AtDAO1). Transcriptional and translational reporter lines revealed that AtDAO1 encodes a highly root-expressed, cytoplasmically localized IAA oxidase. Stable isotope-labeled IAA feeding studies of loss and gain of function AtDAO1 lines showed that this oxidase represents the major regulator of auxin degradation to 2-oxoindole-3-acetic acid (oxIAA) in Arabidopsis. Surprisingly, AtDAO1 loss and gain of function lines exhibited relatively subtle auxin-related phenotypes, such as altered root hair length. Metabolite profiling of mutant lines revealed that disrupting AtDAO1 regulation resulted in major changes in steady-state levels of oxIAA and IAA conjugates but not IAA. Hence, IAA conjugation and catabolism seem to regulate auxin levels in Arabidopsis in a highly redundant manner. We observed that transcripts of AtDOA1 IAA oxidase and GH3 IAA-conjugating enzymes are auxin-inducible, providing a molecular basis for their observed functional redundancy. We conclude that the AtDAO1 gene plays a key role regulating auxin homeostasis in Arabidopsis, acting in concert with GH3 genes, to maintain auxin concentration at optimal levels for plant growth and development.

Emergence of new lateral roots from within the primary root in Arabidopsis has been shown to be regulated by the phytohormone auxin, via the expression of the auxin influx carrier LAX3, mediated by the ARF7/19 IAA14 signalling module (Swarup et al., 2008). A single cell model of the LAX3 and IAA14 auxin response was formulated and used to demonstrate that hysteresis and bistability may explain the experimentally observed 'all-or-nothing' LAX3 spatial expression pattern in cortical cells containing a gradient of auxin concentrations. The model was tested further by using a parameter fitting algorithm to match model output with qRT-PCR mRNA expression data following exogenous auxin treatment. It was found that the model is able to show good agreement with the data, but only when the exogenous auxin signal is degraded over time, at a rate higher than that measured in the experimental medium, suggesting the triggering of an endogenous auxin homeostasis mechanism. Testing the model over a more physiologically relevant range of extracellular auxin shows bistability and hysteresis still occur when using the optimised parameters, providing the rate of LAX3 active auxin transport is sufficiently high relative to passive diffusion.

Genetic and genomic approaches in model organisms have advanced our understanding of root biology over the last decade. Recently, however, systems biology and modeling have emerged as important approaches, as our understanding of root regulatory pathways has become more complex and interpreting pathway outputs has become less intuitive. To relate root genotype to phenotype, we must move beyond the examination of interactions at the genetic network scale and employ multiscale modeling approaches to predict emergent properties at the tissue, organ, organism, and rhizosphere scales. Understanding the underlying biological mechanisms and the complex interplay between systems at these different scales requires an integrative approach. Here, we describe examples of such approaches and discuss the merits of developing models to span multiple scales, from network to population levels, and to address dynamic interactions between plants and their environment.

In Arabidopsis , lateral roots originate from pericycle cells deep within the primary root. New lateral root primordia (LRP) have to emerge through several overlaying tissues. Here, we report that auxin produced in new LRP is transported towards the outer tissues where it triggers cell separation by inducing both the auxin influx carrier LAX3 and cell‐wall enzymes. LAX3 is expressed in just two cell files overlaying new LRP. To understand how this striking pattern of LAX3 expression is regulated, we developed a mathematical model that captures the network regulating its expression and auxin transport within realistic three‐dimensional cell and tissue geometries. Our model revealed that, for the LAX3 spatial expression to be robust to natural variations in root tissue geometry, an efflux carrier is required—later identified to be PIN3. To prevent LAX3 from being transiently expressed in multiple cell files, PIN3 and LAX3 must be induced consecutively, which we later demonstrated to be the case. Our study exemplifies how mathematical models can be used to direct experiments to elucidate complex developmental processes.

Laboratory of Plant Physiology and Molecular Genetics, Universite´ Libre de Bruxelles, B–1050 Brussels,Belgium (C.H., S.P., J.D.P., N.V.); Department of Biology, Colorado State University, Fort Collins, Colorado80523 (S.P., D.R.B.); Laboratory of Functional Plant Biology, Department of Physiology, Ghent University,B–9000 Ghent, Belgium (F.V., D.V.D.S.); and Energy Biosciences Institute, University of California, Berkeley,California 94720 (S.G.)

The root phenotype of an Arabidopsis (Arabidopsis thaliana) mutant of CHITINASE-LIKE1 (CTL1), called arm (for anion-related root morphology), was previously shown to be conditional on growth on high nitrate, chloride, or sucrose. Mutants grown under restrictive conditions displayed inhibition of primary root growth, radial swelling, proliferation of lateral roots, and increased root hair density. We found here that the spatial pattern of CTL1 expression was mainly in the root and root tips during seedling development and that the protein localized to the cell wall. Fourier-transform infrared microspectroscopy of mutant root tissues indicated differences in spectra assigned to linkages in cellulose and pectin. Indeed, root cell wall polymer composition analysis revealed that the arm mutant contained less crystalline cellulose and reduced methylesterification of pectins. We also explored the implication of growth regulators on the phenotype of the mutant response to the nitrate supply. Exogenous abscisic acid application inhibited more drastically primary root growth in the arm mutant but failed to repress lateral branching compared with the wild type. Cytokinin levels were higher in the arm root, but there were no changes in mitotic activity, suggesting that cytokinin is not directly involved in the mutant phenotype. Ethylene production was higher in arm but inversely proportional to the nitrate concentration in the medium. Interestingly, eto2 and eto3 ethylene overproduction mutants mimicked some of the conditional root characteristics of the arm mutant on high nitrate. Our data suggest that ethylene may be involved in the arm mutant phenotype, albeit indirectly, rather than functioning as a primary signal.

The root phenotype of an Arabidopsis (Arabidopsis thaliana) mutant of CHITINASE-LIKE1 (CTL1), called arm (for anion-related root morphology), was previously shown to be conditional on growth on high nitrate, chloride, or sucrose. Mutants grown under restrictive conditions displayed inhibition of primary root growth, radial swelling, proliferation of lateral roots, and increased root hair density. We found here that the spatial pattern of CTL1 expression was mainly in the root and root tips during seedling development and that the protein localized to the cell wall. Fourier-transform infrared microspectroscopy of mutant root tissues indicated differences in spectra assigned to linkages in cellulose and pectin. Indeed, root cell wall polymer composition analysis revealed that the arm mutant contained less crystalline cellulose and reduced methylesterification of pectins. We also explored the implication of growth regulators on the phenotype of the mutant response to the nitrate supply. Exogenous abscisic acid application inhibited more drastically primary root growth in the arm mutant but failed to repress lateral branching compared with the wild type. Cytokinin levels were higher in the arm root, but there were no changes in mitotic activity, suggesting that cytokinin is not directly involved in the mutant phenotype. Ethylene production was higher in arm but inversely proportional to the nitrate concentration in the medium. Interestingly, eto2 and eto3 ethylene overproduction mutants mimicked some of the conditional root characteristics of the arm mutant on high nitrate. Our data suggest that ethylene may be involved in the arm mutant phenotype, albeit indirectly, rather than functioning as a primary signal.

Plant root architecture is highly responsive to changes in nutrient availability. However, the molecular mechanisms governing the adaptability of root systems to changing environmental conditions is poorly understood. A screen for abnormal root architecture responses to high nitrate in the growth medium was carried out for a population of ethyl methanesulfonate-mutagenized Arabidopsis (Arabidopsis thaliana). The growth and root architecture of the arm (for anion altered root morphology) mutant described here was similar to wild-type plants when grown on low to moderate nitrate concentrations, but on high nitrate, arm exhibited reduced primary root elongation, radial swelling, increased numbers of lateral roots, and increased root hair density when compared to the wild-type control. High concentrations of chloride and sucrose induced the same phenotype. In contrast, hypocotyl elongation in the dark was decreased independently of nitrate availability. Positional cloning identified a point mutation in the AtCTL1 gene that encodes a chitinase-related protein, although molecular and biochemical analysis showed that this protein does not possess chitinase enzymatic activity. CTL1 appears to play two roles in plant growth and development based on the constitutive effect of the arm mutation on primary root growth and its conditional impact on root architecture. We hypothesize that CTL1 plays a role in determining cell wall rigidity and that the activity is differentially regulated by pathways that are triggered by environmental conditions. Moreover, we show that mutants of some subunits of the cellulose synthase complex phenocopy the conditional effect on root architecture under nonpermissive conditions, suggesting they are also differentially regulated in response to a changing environment.