Imputation integrates single-cell and spatial gene expression data to resolve transcriptional networks in barley shoot meristem development

nature plants Article https://doi.org/10.1038/s41477-025-02176-6 Imputation integrates single-cell and spatial gene expression data to resolve transcriptional networks in barley shoot meristem development Received: 2 June 2025 A list of authors and their affiliations appears at the end of the paper Accepted: 17 November 2025 Published online: 7 January 2026 Check for updates Grass inflorescences are composite structures, featuring complex sets of meristems as stem cell niches that are initiated in a repetitive manner. Meristems differ in identity and longevity, generate branches or split to form flower meristems that finally produce seeds. Within meristems, distinct cell types are determined by positional information and the regional activity of gene regulatory networks. Understanding these local microenvironments requires precise spatio-temporal information on gene expression profiles, which current technology cannot achieve. Here we investigate transcriptional changes during barley development, from the specification of meristem and organ founder cells to the initiation of distinct floral organs, on the basis of an imputation approach integrating deep single-cell RNA sequencing with spatial gene expression data. The expression profiles of more than 40,000 genes can now be analysed at cellular resolution in multiple barley tissues using the new web-based graphical interface BARVISTA, which enables precise virtual microdissection to analyse any sub-ensemble of cells. Our study pinpoints previously inaccessible key transcriptional events in founder cells during primordia initiation and specification, characterizes complex branching mutant phenotypes by barcoding gene expression profiles, and defines spatio-temporal trajectories during flower development. We thus uncover the genetic basis of complex developmental processes, providing novel opportunities for precisely targeted manipulation of barley traits. Plant development depends on the activities of stem cell systems, the meristems, that give rise to organ primordia or new meristems in distinct patterns. In the grass family, flower development involves the sequential specification of meristematic identities at the inflorescence meristem, leading to diverse architectures1,2. Rice and oat plants develop multiple branched panicles, whereas teosinte, rye, wheat and barley form simpler spikes that generate rows of floret meristems, probably reflecting an ancestral state3. There is a strong genetic basis for these inflorescence architectures which can be explored by studying mutants or genetic variation affecting meristem behaviour during domestication. For example, the evolution of maize from teosinte involved the repression of axillary meristems (AMs), enlargement of the inflorescence meristem (IM), and a switch from distichous to spiral inflorescence phyllotaxis, forming multiple kernels at each node4. In barley, a spike-type inflorescence axis (rachis) generates an AM at each node in a distichous pattern subtended by a developmentally repressed leaf meristem. AMs differentiate into triple spikelet meristems (TSMs) that separate to form three distinct spikelet meristems e-mail: Nature Plants | Volume 12 | January 2026 | 107–124 107 Article (SMs), two lateral (LSM) and one central (CSM)5. Each spikelet initiates a determinate meristem that forms a short axis (rachilla), subtending bracts and a single floret meristem (FM) that generates the floral organs. When the indeterminate IM at the tip of the rachis has generated a finite number of TSMs, the IM and several SMs undergo gradual degeneration in a basipetal sequence, indicating that position along the rachis is a key fate determinant. Each CSM forms a fertile flower and a single grain, whereas LSMs develop to fertility only in six-rowed barley varieties, but remain small and sterile in two-rowed barley. Analysis of barley mutants uncovered that LSM fertility and the determinacy of TSMs is regulated by the LOB-domain transcription factor (TF) VRS4 (Hordeum vulgare RAMOSA2, or HvRA2) and the TCP-family TF INT-C6,7. Loss-of-function mutations in the INT-M gene, encoding an AP2-like TF, or in COMPOSITUM1 (COM1), encoding a TB1/CYC/PCF (TCP)-like TF whose expression at the IM–SM boundary depends on HvRA2, cause the rachilla to give rise to a new branch or generate more florets per spikelet8,9, indicating that COM1 promotes meristem determinacy. Mutations in HvCLV1, encoding a CLAVATA1 family receptor kinase, or in HvFCP1, encoding a secreted signalling peptide that acts through HvCLV1, enhance rachilla indeterminacy and fail to maintain COM2 expression and AM initiation at the IM10. At higher temperatures, rachilla determinacy is promoted by the MADS-box transcription factor (MADS TF) HvMADS33/HvMADS1, a member of the LOFSEP clade associated with inflorescence architecture in rice and several dicot species11. The homeodomain TF KNOTTED1 (KN1), first described in maize, promotes meristem indeterminacy during vegetative and reproductive development and is expressed in vegetative shoot apical meristems (vSAM) and IMs, but not in organ founder cells12. Lateral meristems later regain KN1 expression. KN1 RNA is mostly absent from the outermost cell layer (the L1) of meristems, but KN1 protein together with KN1-mRNA can enter the L1 via plasmodesmata13–15. Local auxin accumulation and absence of KN1 expression mark organ initiation in meristems of many species16. However, gene regulatory networks (GRNs) responsible for primordia specification, their identities, determinacy and fates are still unknown1,2,8,17. To explore GRNs underlying meristem specification and organ initiation in an unbiased manner, we generated single-cell RNA-sequencing (scRNA-seq) data for the barley vSAM and inflorescence (spike). Several scRNA-seq datasets are available in grass species18–31, and deduced developmental trajectories can illustrate shifts in cell fate and gene expression patterns. However, the origin and fate of cells within complex tissues can only be inferred indirectly from the known expression profiles of prominent marker genes32. We could precisely localize and quantify transcripts of 86 genes on tissue sections at cellular resolution using the Molecular Cartography (MC) platform for multiplexed single-molecule RNA fluorescence in situ hybridization (smRNA-FISH). We integrated the scRNA-seq and smRNA-FISH data to generate imputed transcriptome-wide single-cell expression matrices. The combined datasets are presented in the user-friendly, searchable online database BARVISTA (barley virtual in situ transcriptome atlas), developed in-house as part of this study, which visualizes the expression of 48,904 barley genes at cellular resolution on tissue sections representing different developmental stages. BARVISTA enables the virtual microdissection of cell populations from these sections, followed by reclustering and mining for regionally specific gene expression profiles. We used B (...truncated)