Michael J. Scanlon

Founder cells comprise the earliest state of commitment to form one organ or branch, and no other [1]. Comparative lineage analyses concur that cell fate is established later in flowering plants than in animals, and is determined by position rather than lineage, but both systems harbor large numbers of organ progenitors in lieu of a few stem cells [2]. An exciting new serial-editing strategy, applied here for the first time in plants, holds great promise for determining the cell lineage history of every structure in the plant body [3]. Unfortunately, the authors' conclusion that every shoot branch and lateral organ of the Arabidopsis plant derives from exactly three founder cells is unsupported by both the classical plant literature and more recent studies of plant cell lineage, calling for more cautious interpretation of the data and for better integration with complementary experimental strategies.

We present our morphological and transcriptomic evidence for a model of the bipartite grass cotyledon, wherein the scutellum comprises the distal, upper zone and the coleoptile is the sheathing, lower zone of the single grass cotyledon. We assert that homology is best evaluated by examining ontogeny, and not mature adult stages of development. The scutellum is the first lateral organ to arise in the grass embryo, accompanied by leaf homologous gene expression; thereafter the scutellum forms a highly-modified, non-foliar, digestive organ. Lateral organs in grasses exhibit distichous phyllotaxy, where successive organs arise on opposite sides of the shoot axis and in two ranks. Accordingly, scutellum and coleoptile arise on the same side of the shoot, as expected in a bipartite, single lateral organ that is fused medially. Likewise, transcriptomic data identifies expression of the sheath identity marker BOP1a in the coleoptile but not the scutellum, supporting the homology of the coleoptile and the grass sheathing leaf base. Taken together, these data support the bipartite model on the evolution of the grass cotyledon.

Plant architecture is a crucial component of maize productivity. Tailoring architectural component traits like leaf area and angle can increase productivity by promoting deeper light penetration into the canopy and better resource utilization. Novel genetic variants can increase the rate of gain for optimized plant architecture. Here, we map a moderate-effect mutation denoted reduced leaf area1 ( rdla1 ) to the RAGGED5 ( RGD5 ) locus and characterize it as a transposon insertion allele. Mutant leaf area reductions were most extreme in mid-upper canopy positions. Photosynthetic gas exchange rates were not significantly impacted in rdla1 relative to wild-type, indicating that mutant leaf structure, but not function, is altered. Functional annotations of RDLA1 were supported by metabolite profiles suggesting a role in cuticular wax biosynthesis. Introgression of the rdla1 allele into 27 commercially relevant genetic backgrounds identified differences in effect size across genotypes, revealing modifier effects that could serve as targets for modulating plant architecture.

The formation of boundaries separating developmental fields with distinct gene expression and cell fate trajectories is a universal feature of noncolonial 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, 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.

Plant embryogenesis encompasses the biological processes wherein the zygote (fertilized egg) undergoes cell division, cell expansion, and cell differentiation to develop histological tissue layers, meristems, and various organs comprising the primordial body plan of the organism. Studies of embryogenesis in the agronomically important maize crop advance our understanding of the fundamental mechanism of plant development, which, upon translation, may advance agronomic improvement, optimization of conditions for somatic embryogenesis, and plant synthetic biology. Maize embryo development is coordinated temporally and spatially and is regulated by interactive genetic networks. Single-cell RNA sequencing (RNA-seq) and spatial transcriptomics are powerful tools to examine gene expression patterns and regulatory networks at single-cell resolution and in a spatial context, respectively. Single-cell technology enables profiling of three-dimensional samples with high cellular resolution, but it can be difficult to identify specific cell clusters due to a lack of known markers in most plant species. In contrast, spatial transcriptomics provide transcriptomic profiling of discrete regions within a sectioned, two-dimensional sample, although single-cell resolution is typically not obtained and fewer transcripts per cell are detected than in single-cell RNA-seq. In this review, we describe the combined use of these two transcriptomic strategies to study maize embryogenesis with synergistic results.

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.

Zoologists have adduced morphological convergence among embryonic stages of closely related taxa, which has been called the phylotypic stage of embryogenesis. Transcriptomic analyzes reveal an hourglass pattern of gene expression during plant and animal embryogenesis, characterized by the accumulation of evolutionarily older and conserved transcripts during mid-embryogenesis, whereas younger less-conserved transcripts predominate at earlier and later embryonic stages. In contrast, comparisons of embryonic gene expression among different animal phyla describe an inverse hourglass pattern, where expression is correlated during early and late stages but not during mid-embryo development. Here, multiplexed spatial-transcriptomic analyzes is used to investigate embryogenesis and homology in maize, which has grass-specific morphology. A set of shared, co-expressed genes is identified during initiation of maize embryonic organs, replete for ancient/conserved genes manifesting an hourglass pattern during mid-embryogenesis. Transcriptomic comparisons of maize and Arabidopsis embryogenesis with that of the moss Physcomitrium patens identify an inverse hourglass pattern across plant phyla, as in animals. The data suggest that the phylotypic stages in plants and animals are characterized by expression of ancient and conserved genes during histogenesis, organization of embryonic axes, and initial morphogenesis. We propose a mechanism for gene evolution during the innovation of morphological novelty.

Studying the genetic basis of leaf wax composition and its correlation with leaf cuticular conductance (g_c) is crucial for improving crop productivity. The leaf cuticle, which comprises a cutin matrix and various waxes, functions as an extracellular hydrophobic layer, protecting against water loss upon stomatal closure. To address the limited understanding of genes associated with the natural variation of adult leaf cuticular waxes and their connection to g_c, we conducted statistical genetic analyses using leaf transcriptomic, metabolomic, and physiological data sets collected from a maize (Zea mays L.) panel of ∼300 inbred lines. Through a random forest analysis with 60 cuticular wax traits, it was shown that high molecular weight wax esters play an important role in predicting g_c. Integrating results from genome-wide and transcriptome-wide studies (GWAS and TWAS) via a Fisher’s combined test revealed 231 candidate genes detected by all three association tests. Among these, 11 genes exhibit known or predicted roles in cuticle-related processes. Throughout the genome, multiple hotspots consisting of GWAS signals for several traits from one or more wax classes were discovered, identifying four additional plausible candidate genes and providing insights into the genetic basis of correlated wax traits. Establishing a partially shared genetic architecture, we identified 35 genes for both g_c and at least one wax trait, with four considered plausible candidates. Our study enhances the understanding of how adult leaf cuticle wax composition relates to g_c and implicates both known and novel candidate genes as potential targets for optimizing productivity in maize.