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Alternative transcription start sites (TSS) are widespread in eukaryotes and can alter the 5' UTR length and coding potential of transcripts. Here we show that inorganic phosphate (Pi) availability regulates the usage of several alternative TSS in Arabidopsis (Arabidopsis thaliana). In comparison to phytohormone treatment, Pi had a pronounced and specific effect on the usage of many alternative TSS. By combining short-read RNA sequencing with long-read sequencing of full-length mRNAs, we identified a set of 45 genes showing alternative TSS under Pi deficiency. Alternative TSS affected several processes, such as translation via the exclusion of upstream open reading frames present in the 5' UTR of RETICULAN LIKE PROTEIN B1 mRNA, and subcellular localization via removal of the plastid transit peptide coding region from the mRNAs of HEME OXYGENASE 1 and SULFOQUINOVOSYLDIACYLGLYCEROL 2. Several alternative TSS also generated shorter transcripts lacking the coding potential for important domains. For example, the EVOLUTIONARILY CONSERVED C-TERMINAL REGION 4 (ECT4) locus, which encodes an N 6 -methyladenosine (m 6 A) reader, strongly expressed under Pi deficiency a short noncoding transcript (named ALT ECT4 ) ~550 nt long with a TSS in the penultimate intron. The specific and robust induction of ALT ECT4 production by Pi deficiency led to the identification of a role for m 6 A readers in primary root growth in response to low phosphate that is dependent on iron and is involved in modulating cell division in the root meristem. Our results identify alternative TSS usage as an important process in the plant response to Pi deficiency., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Aims/hypothesis: tRNAs play a central role in protein synthesis. Besides this canonical function, they were recently found to generate non-coding RNA fragments (tRFs) regulating different cellular activities. The aim of this study was to assess the involvement of tRFs in the crosstalk between immune cells and beta cells and to investigate their contribution to the development of type 1 diabetes., Methods: Global profiling of the tRFs present in pancreatic islets of 4- and 8-week-old NOD mice and in extracellular vesicles released by activated CD4 + T lymphocytes was performed by small RNA-seq. Changes in the level of specific fragments were confirmed by quantitative PCR. The transfer of tRFs from immune cells to beta cells occurring during insulitis was assessed using an RNA-tagging approach. The functional role of tRFs increasing in beta cells during the initial phases of type 1 diabetes was determined by overexpressing them in dissociated islet cells and by determining the impact on gene expression and beta cell apoptosis., Results: We found that the tRF pool was altered in the islets of NOD mice during the initial phases of type 1 diabetes. Part of these changes were triggered by prolonged exposure of beta cells to proinflammatory cytokines (IL-1β, TNF-α and IFN-γ) while others resulted from the delivery of tRFs produced by CD4 + T lymphocytes infiltrating the islets. Indeed, we identified several tRFs that were enriched in extracellular vesicles from CD4 + /CD25 - T cells and were transferred to beta cells upon adoptive transfer of these immune cells in NOD.SCID mice. The tRFs delivered to beta cells during the autoimmune reaction triggered gene expression changes that affected the immune regulatory capacity of insulin-secreting cells and rendered the cells more prone to apoptosis., Conclusions/interpretation: Our data point to tRFs as novel players in the crosstalk between the immune system and insulin-secreting cells and suggest a potential involvement of this novel class of non-coding RNAs in type 1 diabetes pathogenesis., Data Availability: Sequences are available from the Gene Expression Omnibus (GEO) with accession numbers GSE242568 and GSE256343., (© 2024. The Author(s).)

The cell cycle-regulated DNA methyltransferase CcrM is conserved in most Alphaproteobacteria, but its role in bacteria with complex or multicentric genomes remains unexplored. Here, we compare the methylome, the transcriptome and the phenotypes of wild-type and CcrM-depleted Agrobacterium tumefaciens cells with a dicentric chromosome with two essential replication origins. We find that DNA methylation has a pleiotropic impact on motility, biofilm formation and viability. Remarkably, CcrM promotes the expression of the repABCCh2 operon, encoding proteins required for replication initiation/partitioning at ori2, and represses gcrA, encoding a conserved global cell cycle regulator. Imaging ori1 and ori2 in live cells, we show that replication from ori2 is often delayed in cells with a hypo-methylated genome, while ori2 over-initiates in cells with a hyper-methylated genome. Further analyses show that GcrA promotes the expression of the RepCCh2 initiator, most likely through the repression of a RepECh2 anti-sense RNA. Altogether, we propose that replication at ori1 leads to a transient hemi-methylation and activation of the gcrA promoter, allowing repCCh2 activation by GcrA and contributing to initiation at ori2. This study then uncovers a novel and original connection between CcrM-dependent DNA methylation, a conserved epigenetic regulator and genome maintenance in an Alphaproteobacterial pathogen., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: We previously described the KINSSHIP syndrome, an autosomal dominant disorder associated with intellectual disability (ID), mesomelic dysplasia and horseshoe kidney, caused by de novo variants in the degron of AFF3. Mouse knock-ins and overexpression in zebrafish provided evidence for a dominant-negative mode of action, wherein an increased level of AFF3 resulted in pathological effects., Methods: Evolutionary constraints suggest that other modes-of-inheritance could be at play. We challenged this hypothesis by screening ID cohorts for individuals with predicted-to-be damaging variants in AFF3. We used both animal and cellular models to assess the deleteriousness of the identified variants., Results: We identified an individual with a KINSSHIP-like phenotype carrying a de novo partial duplication of AFF3 further strengthening the hypothesis that an increased level of AFF3 is pathological. We also detected seventeen individuals displaying a milder syndrome with either heterozygous Loss-of-Function (LoF) or biallelic missense variants in AFF3. Consistent with semi-dominance, we discovered three patients with homozygous LoF and one compound heterozygote for a LoF and a missense variant, who presented more severe phenotypes than their heterozygous parents. Matching zebrafish knockdowns exhibit neurological defects that could be rescued by expressing human AFF3 mRNA, confirming their association with the ablation of aff3. Conversely, some of the human AFF3 mRNAs carrying missense variants identified in affected individuals did not rescue these phenotypes. Overexpression of mutated AFF3 mRNAs in zebrafish embryos produced a significant increase of abnormal larvae compared to wild-type overexpression further demonstrating deleteriousness. To further assess the effect of AFF3 variation, we profiled the transcriptome of fibroblasts from affected individuals and engineered isogenic cells harboring + / + , KINSSHIP/KINSSHIP, LoF/ + , LoF/LoF or KINSSHIP/LoF AFF3 genotypes. The expression of more than a third of the AFF3 bound loci is modified in either the KINSSHIP/KINSSHIP or the LoF/LoF lines. While the same pathways are affected, only about one third of the differentially expressed genes are common to the homozygote datasets, indicating that AFF3 LoF and KINSSHIP variants largely modulate transcriptomes differently, e.g. the DNA repair pathway displayed opposite modulation., Conclusions: Our results and the high pleiotropy shown by variation at this locus suggest that minute changes in AFF3 function are deleterious., (© 2024. The Author(s).)

Encephalitis is a rare and potentially fatal manifestation of herpes simplex type 1 infection. Following genome-wide genetic analyses, we identified a previously uncharacterized and very rare heterozygous variant in the E3 ubiquitin ligase WWP2, in a 14-month-old girl with herpes simplex encephalitis. The p.R841H variant (NM_007014.4:c.2522G > A) impaired TLR3 mediated signaling in inducible pluripotent stem cells-derived neural precursor cells and neurons; cells bearing this mutation were also more susceptible to HSV-1 infection compared to control cells. The p.R841H variant increased TRIF ubiquitination in vitro. Antiviral immunity was rescued following the correction of p.R841H by CRISPR-Cas9 technology. Moreover, the introduction of p.R841H in wild type cells reduced such immunity, suggesting that this mutation is linked to the observed phenotypes., (© 2024. The Author(s).)

Mass cytometry and full spectrum flow cytometry have recently emerged as new promising single cell proteomic analysis tools that can be exploited to decipher the extensive diversity of immune cell repertoires and their implication in human diseases. In this study, we evaluated the performance of mass cytometry against full spectrum flow cytometry using an identical 33-color antibody panel on four healthy individuals. Our data revealed an overall high concordance in the quantification of major immune cell populations between the two platforms using a semi-automated clustering approach. We further showed a strong correlation of cluster assignment when comparing manual and automated clustering. Both comparisons revealed minor disagreements in the quantification and assignment of rare cell subpopulations. Our study showed that both single cell proteomic technologies generate highly overlapping results and substantiate that the choice of technology is not a primary factor for successful biological assessment of cell profiles but must be considered in a broader design framework of clinical studies., Competing Interests: Declaration of competing interest All authors declare that there are no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The transforming growth factor-β (TGF-β) family member activin A (hereafter Activin-A) is overexpressed in many cancer types, often correlating with cancer-associated cachexia and poor prognosis. Activin-A secretion by melanoma cells indirectly impedes CD8 + T cell-mediated anti-tumor immunity and promotes resistance to immunotherapies, even though Activin-A can be proinflammatory in other contexts. To identify underlying mechanisms, we here analyzed the effect of Activin-A on syngeneic grafts of Braf mutant YUMM3.3 mouse melanoma cells and on their microenvironment using single-cell RNA sequencing. We found that the Activin-A-induced immune evasion was accompanied by a proinflammatory interferon signature across multiple cell types, and that the associated increase in tumor growth depended at least in part on pernicious STING activity within the melanoma cells. Besides corroborating a role for proinflammatory signals in facilitating immune evasion, our results suggest that STING holds considerable potential as a therapeutic target to mitigate tumor-promoting Activin-A signaling at least in melanoma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Pinjusic, Ambrosini, Lourenco, Fournier, Iseli, Guex, Egorova, Nassiri and Constam.)

Mycobacterium tuberculosis (Mtb) cultured axenically without detergent forms biofilm-like cords, a clinical identifier of virulence. In lung-on-chip (LoC) and mouse models, cords in alveolar cells contribute to suppression of innate immune signaling via nuclear compression. Thereafter, extracellular cords cause contact-dependent phagocyte death but grow intercellularly between epithelial cells. The absence of these mechanopathological mechanisms explains the greater proportion of alveolar lesions with increased immune infiltration and dissemination defects in cording-deficient Mtb infections. Compression of Mtb lipid monolayers induces a phase transition that enables mechanical energy storage. Agent-based simulations demonstrate that the increased energy storage capacity is sufficient for the formation of cords that maintain structural integrity despite mechanical perturbation. Bacteria in cords remain translationally active despite antibiotic exposure and regrow rapidly upon cessation of treatment. This study provides a conceptual framework for the biophysics and function in tuberculosis infection and therapy of cord architectures independent of mechanisms ascribed to single bacteria., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: 5-methylcytosine RNA modifications are driven by NSUN methyltransferases. Although variants in NSUN2 and NSUN3 were associated with neurodevelopmental diseases, the physiological role of NSUN6 modifications on transfer RNAs and messenger RNAs remained elusive., Methods: We combined exome sequencing of consanguineous families with functional characterization to identify a new neurodevelopmental disorder gene., Results: We identified 3 unrelated consanguineous families with deleterious homozygous variants in NSUN6. Two of these variants are predicted to be loss-of-function. One maps to the first exon and is predicted to lead to the absence of NSUN6 via nonsense-mediated decay, whereas we showed that the other maps to the last exon and encodes a protein that does not fold correctly. Likewise, we demonstrated that the missense variant identified in the third family has lost its enzymatic activity and is unable to bind the methyl donor S-adenosyl-L-methionine. The affected individuals present with developmental delay, intellectual disability, motor delay, and behavioral anomalies. Homozygous ablation of the NSUN6 ortholog in Drosophila led to locomotion and learning impairment., Conclusion: Our data provide evidence that biallelic pathogenic variants in NSUN6 cause one form of autosomal recessive intellectual disability, establishing another link between RNA modification and cognition., Competing Interests: Conflict of Interest Radoslava Saraeva-Lamri and Laure Raymond are employees of Eurofins Biomnis. Stylianos E. Antonarakis is a co-founder and CEO of Medigenome, Swiss Institute of Genomic Medicine. All other authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Previous studies have identified topologically associating domains (TADs) as basic units of genome organization. We present evidence of a previously unreported level of genome folding, where distant TAD pairs, megabases apart, interact to form meta-domains. Within meta-domains, gene promoters and structural intergenic elements present in distant TADs are specifically paired. The associated genes encode neuronal determinants, including those engaged in axonal guidance and adhesion. These long-range associations occur in a large fraction of neurons but support transcription in only a subset of neurons. Meta-domains are formed by diverse transcription factors that are able to pair over long and flexible distances. We present evidence that two such factors, GAF and CTCF, play direct roles in this process. The relative simplicity of higher-order meta-domain interactions in Drosophila, compared with those previously described in mammals, allowed the demonstration that genomes can fold into highly specialized cell-type-specific scaffolds that enable megabase-scale regulatory associations., Competing Interests: Declaration of interests S.R. developed MUSE technology marketed by arcoris bio AG, has shares, and sits on the board., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Acinetobacter baumannii is a dangerous nosocomial pathogen, especially due to its ability to rapidly acquire new genetic traits, including antibiotic resistance genes (ARG). In A. baumannii, natural competence for transformation, one of the primary modes of horizontal gene transfer (HGT), is thought to contribute to ARG acquisition and has therefore been intensively studied. However, knowledge regarding the potential role of epigenetic DNA modification(s) on this process remains lacking. Here, we demonstrate that the methylome pattern of diverse A. baumannii strains differs substantially and that these epigenetic marks influence the fate of transforming DNA. Specifically, we describe a methylome-dependent phenomenon that impacts intra- and inter-species DNA exchange by the competent A. baumannii strain A118. We go on to identify and characterize an A118-specific restriction-modification (RM) system that impairs transformation when the incoming DNA lacks a specific methylation signature. Collectively, our work contributes towards a more holistic understanding of HGT in this organism and may also aid future endeavors towards tackling the spread of novel ARGs. In particular, our results suggest that DNA exchanges between bacteria that share similar epigenomes are favored and could therefore guide future research into identifying the reservoir(s) of dangerous genetic traits for this multi-drug resistant pathogen., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

GEMIN5 is a multifunctional RNA-binding protein required for the assembly of survival motor neurons. Several bi-allelic truncating and missense variants in this gene are reported to cause a neurodevelopmental disorder characterized by cerebellar atrophy, intellectual disability (ID), and motor dysfunction. Whole exome sequencing of a Pakistani consanguineous family with three brothers affected by ID, cerebral atrophy, mobility, and speech impairment revealed a novel homozygous 3bp-deletion NM_015465.5:c.3162_3164del that leads to the loss of NM_015465.5 (NP_056280.2):p. (Asp1054_Ala1055delinsGlu) amino acid in one of the α-helixes of the tetratricopeptide repeats of GEMIN5. In silico 3D representations of the GEMIN5 dimerization domain show that this variant likely affects the orientation of the downstream sidechains out of the helix axis, which would affect the packing with neighboring helices. The phenotype of all affected siblings overlaps well with previously reported patients, suggesting that NM_015465.5: c.3162_3164del (NP_056280.2):p. (Asp1054_Ala1055delinsGlu) is a novel GEMIN5 pathogenic variant. Overall, our data expands the molecular and clinical phenotype of the recently described neurodevelopmental disorder with cerebellar atrophy and motor dysfunction (NEDCAM) syndrome.

Background: Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by altered cellular metabolism in the brain. Several of these alterations have been found to be exacerbated in females, known to be disproportionately affected by AD. We aimed to unravel metabolic alterations in AD at the metabolic pathway level and evaluate whether they are sex-specific through integrative metabolomic, lipidomic, and proteomic analysis of mouse brain tissue., Methods: We analyzed male and female triple-transgenic mouse whole brain tissue by untargeted mass spectrometry-based methods to obtain a molecular signature consisting of polar metabolite, complex lipid, and protein data. These data were analyzed using multi-omics factor analysis. Pathway-level alterations were identified through joint pathway enrichment analysis or by separately evaluating lipid ontology and known proteins related to lipid metabolism., Results: Our analysis revealed significant AD-associated and in part sex-specific alterations across the molecular signature. Sex-dependent alterations were identified in GABA synthesis, arginine biosynthesis, and in alanine, aspartate, and glutamate metabolism. AD-associated alterations involving lipids were also found in the fatty acid elongation pathway and lysophospholipid metabolism, with a significant sex-specific effect for the latter., Conclusions: Through multi-omics analysis, we report AD-associated and sex-specific metabolic alterations in the AD brain involving lysophospholipid and amino acid metabolism. These findings contribute to the characterization of the AD phenotype at the molecular level while considering the effect of sex, an overlooked yet determinant metabolic variable., (© 2023. The Author(s).)

Antibodies against phosphorylated alpha-synuclein (aSyn) at S129 have emerged as the primary tools to investigate, monitor, and quantify aSyn pathology in the brain and peripheral tissues of patients with Parkinson's disease and other neurodegenerative diseases. Herein, we demonstrate that the co-occurrence of multiple pathology-associated C-terminal post-translational modifications (PTMs) (e.g., phosphorylation at Tyrosine 125 or truncation at residue 133 or 135) differentially influences the detection of pS129-aSyn species by pS129-aSyn antibodies. These observations prompted us to systematically reassess the specificity of the most commonly used pS129 antibodies against monomeric and aggregated forms of pS129-aSyn in mouse brain slices, primary neurons, mammalian cells and seeding models of aSyn pathology formation. We identified two antibodies that are insensitive to pS129 neighboring PTMs. Although most pS129 antibodies showed good performance in detecting aSyn aggregates in cells, neurons and mouse brain tissue containing abundant aSyn pathology, they also showed cross-reactivity towards other proteins and often detected non-specific low and high molecular weight bands in aSyn knock-out samples that could be easily mistaken for monomeric or high molecular weight aSyn species. Our observations suggest that not all pS129 antibodies capture the biochemical and morphological diversity of aSyn pathology, and all should be used with the appropriate protein standards and controls when investigating aSyn under physiological conditions. Finally, our work underscores the need for more pS129 antibodies that are not sensitive to neighboring PTMs and more thorough characterization and validation of existing and new antibodies., (© 2022. The Author(s).)

Boundaries in animal genomes delimit contact domains with enhanced internal contact frequencies and have debated functions in limiting regulatory cross-talk between domains and guiding enhancers to target promoters. Most mammalian boundaries form by stalling of chromosomal loop-extruding cohesin by CTCF, but most Drosophila boundaries form CTCF independently. However, how CTCF-independent boundaries form and function remains largely unexplored. Here, we assess genome folding and developmental gene expression in fly embryos lacking the ubiquitous boundary-associated factor Cp190. We find that sequence-specific DNA binding proteins such as CTCF and Su(Hw) directly interact with and recruit Cp190 to form most promoter-distal boundaries. Cp190 is essential for early development and prevents regulatory cross-talk between specific gene loci that pattern the embryo. Cp190 was, in contrast, dispensable for long-range enhancer-promoter communication at tested loci. Cp190 is thus currently the major player in fly boundary formation and function, revealing that diverse mechanisms evolved to partition genomes into independent regulatory domains.

Protists are abundant and play key trophic functions in soil. Documenting how their trophic contributions vary across large environmental gradients is essential to understand and predict how biogeochemical cycles will be impacted by global changes. Here, using amplicon sequencing of environmental DNA in open habitat soil from 161 locations spanning 2600 m of elevation in the Swiss Alps (from 400 to 3000 m), we found that, over the whole study area, soils are dominated by consumers, followed by parasites and phototrophs. In contrast, the proportion of these groups in local communities shows large variations in relation to elevation. While there is, on average, three times more consumers than parasites at low elevation (400-1000 m), this ratio increases to 12 at high elevation (2000-3000 m). This suggests that the decrease in protist host biomass and diversity toward mountains tops impact protist functional composition. Furthermore, the taxonomic composition of protists that infect animals was related to elevation while that of protists that infect plants or of protist consumers was related to soil pH. This study provides a first step to document and understand how soil protist functions vary along the elevational gradient., (© 2021 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The study of insular populations was key in the development of evolutionary theory. The successful colonisation of an island depends on the geographic context, and specific characteristics of the organism and the island, but also on stochastic processes. As a result, apparently identical islands may harbour populations with contrasting histories. Here, we use whole genome sequences of 65 barn owls to investigate the patterns of inbreeding and genetic diversity of insular populations in the eastern Mediterranean Sea. We focus on Crete and Cyprus, islands with similar size, climate and distance to mainland, that provide natural replicates for a comparative analysis of the impacts of microevolutionary processes on isolated populations. We show that barn owl populations from each island have a separate origin, Crete being genetically more similar to other Greek islands and mainland Greece, and Cyprus more similar to the Levant. Further, our data show that their respective demographic histories following colonisation were also distinct. On the one hand, Crete harbours a small population and maintains very low levels of gene flow with neighbouring populations. This has resulted in low genetic diversity, strong genetic drift, increased relatedness in the population and remote inbreeding. Cyprus, on the other hand, appears to maintain enough gene flow with the mainland to avoid such an outcome. Our study provides a comparative population genomic analysis of the effects of neutral processes on a classical island-mainland model system. It provides empirical evidence for the role of stochastic processes in determining the fate of diverging isolated populations., (© 2021 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Single-cell transcriptomics allows the study of immune cell heterogeneity at an unprecedented level of resolution. The Swiss portal for immune cell analysis (SPICA) is a web resource dedicated to the exploration and analysis of single-cell RNA-seq data of immune cells. In contrast to other single-cell databases, SPICA hosts curated, cell type-specific reference atlases that describe immune cell states at high resolution, and published single-cell datasets analysed in the context of these atlases. Additionally, users can privately analyse their own data in the context of existing atlases and contribute to the SPICA database. SPICA is available at https://spica.unil.ch., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The climate fluctuations of the Quaternary shaped the movement of species in and out of glacial refugia. In Europe, the majority of species followed one of the described traditional postglacial recolonization routes from the southern peninsulas towards the north. Like most organisms, barn owls are assumed to have colonized the British Isles by crossing over Doggerland, a land bridge that connected Britain to northern Europe. However, while they are dark rufous in northern Europe, barn owls in the British Isles are conspicuously white, a contrast that could suggest selective forces are at play on the islands. Yet, our analysis of known candidate genes involved in coloration found no signature of selection. Instead, using whole genome sequences and species distribution modelling, we found that owls colonised the British Isles soon after the last glaciation, directly from a white coloured refugium in the Iberian Peninsula, before colonising northern Europe. They would have followed a hitherto unknown post-glacial colonization route to the Isles over a westwards path of suitable habitat in now submerged land in the Bay of Biscay, thus not crossing Doggerland. As such, they inherited the white colour of their Iberian founders and maintained it through low gene flow with the mainland that prevents the import of rufous alleles. Thus, we contend that neutral processes probably explain this contrasting white colour compared to continental owls. With the barn owl being a top predator, we expect future research will show this unanticipated route was used by other species from its paleo community., (© 2021 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

The effects of brassinosteroid signaling on shoot and root development have been characterized in great detail but a simple consistent positive or negative impact on a basic cellular parameter was not identified. In this study, we combined digital 3D single-cell shape analysis and single-cell mRNA sequencing to characterize root meristems and mature root segments of brassinosteroid-blind mutants and wild type. The resultant datasets demonstrate that brassinosteroid signaling affects neither cell volume nor cell proliferation capacity. Instead, brassinosteroid signaling is essential for the precise orientation of cell division planes and the extent and timing of anisotropic cell expansion. Moreover, we found that the cell-aligning effects of brassinosteroid signaling can propagate to normalize the anatomy of both adjacent and distant brassinosteroid-blind cells through non-cell-autonomous functions, which are sufficient to restore growth vigor. Finally, single-cell transcriptome data discern directly brassinosteroid-responsive genes from genes that can react non-cell-autonomously and highlight arabinogalactans as sentinels of brassinosteroid-dependent anisotropic cell expansion., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)