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Summary The homology of the single cotyledon of grasses and the ontogeny of the scutellum and coleoptile as the initial, highly modified structures of the grass embryo are investigated using leaf developmental genetics and targeted transcript analyses in the model grassZea mayssubsp.mays.Transcripts of leaf developmental genes are identified in both the initiating scutellum and the coleoptile, while mutations disrupting mediolateral leaf development also disrupt scutellum and coleoptile morphology, suggesting that these grass‐specific organs are modified leaves.Higher‐order mutations inWUSCHEL‐LIKE HOMEOBOX3(WOX3) genes, involved in mediolateral patterning of plant lateral organs, inform a model for the fusion of coleoptilar margins during maize embryo development.Genetic, RNA‐targeting, and morphological evidence supports models for cotyledon evolution where the scutellum and coleoptile, respectively, comprise the distal and proximal domains of the highly modified, single grass cotyledon. 

Abstract Grass leaves develop from a ring of primordial initial cells within the periphery of the shoot apical meristem, a pool of organogenic stem cells that generates all of the organs of the plant shoot. At maturity, the grass leaf is a flattened, strap-like organ comprising a proximal supportive sheath surrounding the stem and a distal photosynthetic blade. The sheath and blade are partitioned by a hinge-like auricle and the ligule, a fringe of epidermally derived tissue that grows from the adaxial (top) leaf surface. Together, the ligule and auricle comprise morphological novelties that are specific to grass leaves. Understanding how the planar outgrowth of grass leaves and their adjoining ligules is genetically controlled can yield insight into their evolutionary origins. Here we use single-cell RNA-sequencing analyses to identify a ‘rim’ cell type present at the margins of maize leaf primordia. Cells in the leaf rim have a distinctive identity and share transcriptional signatures with proliferating ligule cells, suggesting that a shared developmental genetic programme patterns both leaves and ligules. Moreover, we show that rim function is regulated by genetically redundant Wuschel-like homeobox3 (WOX3) transcription factors. Higher-order mutations in maizeWox3genes greatly reduce leaf width and disrupt ligule outgrowth and patterning. Together, these findings illustrate the generalizable use of a rim domain during planar growth of maize leaves and ligules, and suggest a parsimonious model for the homology of the grass ligule as a distal extension of the leaf sheath margin. 

The formation of boundaries separating developmental fields with distinct gene expression and cell-fate trajectories is a universal feature of non-colonial multicellular organisms. Developmental boundaries arise reiteratively during ontogeny and are characterized by stiff, slowly dividing cells that demarcate adjacent and divergent morphogenetic domains; the genetic mechanisms of cell-fate acquisition within these boundaries are incompletely understood. Grass leaves are initiated at a developmental boundary in the periphery of the shoot apical meristem (SAM), an organogenic pool of plant stem cells that generates all lateral organs in the plant shoot. During later primordial growth, maize leaves form a de novo developmental boundary that ultimately separates the distal, photosynthetic leaf blade from the proximal, clasping leaf sheath. Morphogenesis at this blade/sheath boundary in maize leaves generates an epidermal outgrowth called the ligule and two tissue-wedges forming the auricle, a hinge-like structure with major effects on leaf angle, light capture, and yield. Here, we use cell-lineage mapping, morphometric measures of cell division and expansion, cell-specific multidimensional transcriptomic analyses, and topological landscape modeling to investigate the mechanisms of cell-fate acquisition at the ligule/auricle morphogenetic boundary in the maize leaf. The data suggest a model where auricle initial cells are recruited from blade founder cells at this boundary, via repression of blade identity during early stages in auricle ontogeny. Thereafter, auricle primordial cells assume a developmental genetic trajectory that is distinct from the blade, sheath, and ligule, thereby acquiring a unique auricle cell-fate in the maize leaf. 

Maize is an important crop that contributes to the modern economy in various ways, including use for human consumption, as animal feed, and in industrial products. Research on maize is crucial for understanding plant development, which in turn provides valuable insight into improvement of maize crops to meet the food demands of a growing population. Maize embryogenesis, which is the primordial stage of the corn life cycle, determines the fundamental body plan and developmental programs that organize the tissue patterning and subsequent growth and reproduction of the corn plant. Investigating maize embryogenesis at high cellular resolution can enhance our understanding of the homology, ontogeny, and developmental genetic mechanisms of embryonic organ morphogenesis. However, until recently, no published studies have used methods for analyzing maize embryo development at single-cell resolution. This protocol describes single-cell RNA sequencing (scRNA-seq) and spatial transcriptomic analyses, which are powerful, combinatorial tools that can be used to study maize embryogenesis at the single-cell level within a spatial context. These tools have the power to reveal transcriptomic relationships between tissues/organs, and to provide insight into the gene regulatory networks operating during embryogenesis. In this protocol, we describe a detailed procedure to prepare maize embryo samples for construction of scRNA-seq and Visium spatial transcriptomic libraries that are suitable for massively parallel sequencing. Our protocol borrows from prior published studies and manufacturer's instructions and is optimized for studies of the maize embryo. 

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Plants maintain populations of pluripotent stem cells in shoot apical meristems (SAMs), which continuously produce new aboveground organs. We used single-cell RNA sequencing (scRNA-seq) to achieve an unbiased characterization of the transcriptional landscape of the maize shoot stem-cell niche and its differentiating cellular descendants. Stem cells housed in the SAM tip are engaged in genome integrity maintenance and exhibit a low rate of cell division, consistent with their contributions to germline and somatic cell fates. Surprisingly, we find no evidence for a canonical stem-cell organizing center subtending these cells. In addition, trajectory inference was used to trace the gene expression changes that accompany cell differentiation, revealing that ectopic expression of KNOTTED1 (KN1) accelerates cell differentiation and promotes development of the sheathing maize leaf base. These single-cell transcriptomic analyses of the shoot apex yield insight into the processes of stem-cell function and cell-fate acquisition in the maize seedling and provide a valuable scaffold on which to better dissect the genetic control of plant shoot morphogenesis at the cellular level.