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.

A time-dependent mechanism mediated by SPL9 highlights how age-related growth reprogramming influences leaf shape modifications in simple- and complex-leaved, related plants. Extending cell proliferation and delaying differentiation drives the mechanism, offering insights into the shared developmental processes underlying leaf form diversification.

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 grass Zea mays subsp. 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 in WUSCHEL‐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.

SUMMARYStudying the genetic basis of leaf wax composition and its correlation with leaf cuticular conductance (gc) is crucial for improving crop water-use efficiency. 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 leaf cuticular waxes and their connection togc, we conducted statistical genetic analyses using leaf transcriptomic, metabolomic, and physiological data sets collected from a maize (Zea maysL.) 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 predictinggc. 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 bothgcand at least one wax trait, with four considered plausible candidates. Our study uncovered the genetic control of maize leaf waxes, establishing a link between wax composition andgc, with implications for potentially breeding more water-use efficient maize.SIGNIFICANCE STATEMENTWe exploited natural variation in the abundance of maize leaf cuticular waxes to identify genetic determinants of wax composition and its relationship to cuticle function as a barrier against water loss. We identified a set of strongly supported candidate genes with plausible functions in cuticular wax biosynthesis or deposition and added to the evidence for wax esters as the most important wax for water barrier function, offering new tools for modification of cuticle-dependent traits.

Crosstalk between auxin and cytokinin contributes to widespread developmental processes, including root and shoot meristem maintenance, phyllotaxy, and vascular patterning. Although auxin and cytokinin are potent regulators of plant development, our understanding of crosstalk between these hormones is limited to few model systems. The moss Physcomitrium patens (formerly Physcomitrella patens ) is a powerful system for studying plant hormone function. Auxin and cytokinin play similar roles in regulating moss gametophore (upright shoot) architecture, as they do in flowering plant shoots. However, auxin-cytokinin crosstalk is poorly understood in moss. Here we find that the ratio of auxin to cytokinin is an important determinant of development in P. patens , especially during leaf development and branch stem cell initiation. Addition of high levels of auxin to P. patens gametophores blocks leaf outgrowth. However, simultaneous addition of high levels of both auxin and cytokinin partially restores leaf outgrowth, suggesting that the ratio of these hormones is the overriding factor. Likewise, during branch initiation and outgrowth, chemical inhibition of auxin synthesis phenocopies cytokinin application. Finally, cytokinin insensitive mutants resemble plants with altered auxin signaling and are hypersensitive to auxin. In summary, our results suggest that the ratio between auxin and cytokinin signaling is the basis for developmental decisions in the moss gametophore.

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 maize Wox3 genes 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.

Grass inflorescence development is diverse and complex and involves sophisticated but poorly understood interactions of genes regulating branch determinacy and leaf growth. Here, we use a combination of transcript profiling and genetic and phylogenetic analyses to investigate tasselsheath1 (tsh1) and tsh4, two maize genes that simultaneously suppress inflorescence leaf growth and promote branching. We identify a regulatory network of inflorescence leaf suppression that involves the phase change gene tsh4 upstream of tsh1 and the ligule identity gene liguleless2 (lg2). We also find that a series of duplications in the tsh1 gene lineage facilitated its shift from boundary domain in nongrasses to suppressed inflorescence leaves of grasses. Collectively, these results suggest that the boundary domain genes tsh1 and lg2 were recruited to inflorescence leaves where they suppress growth and regulate a nonautonomous signaling center that promotes inflorescence branching, an important component of yield in cereal grasses.

Social Impact StatementAlthough tremendously successful at feeding humanity, row crop agriculture based on annuals contributes to numerous ecosystem dis‐services, ranging from soil degradation and aquatic eutrophication to greenhouse gas production. In contrast, perennial grain crops (which produce harvests for multiple seasons from single plantings) have the potential to provide valuable regulating and supporting ecosystem services in addition to food production. In particular, losses of ecological capital that threaten permanent food insecurity such as ~1% of global soil per year are expected to be mitigated or even reversed by crops that combine the high yield realized by scientific breeding via multiple cropping cycles from single plantings.Summary Perennial herbaceous may provide food and biomass while preserving ecological capital and reducing energy inputs. Sorghum has two perennial relatives and rich morphological diversity being used to breed for perenniality. We elucidate genetic determinants of rhizomatousness and survival, in two BC1F2 populations totaling 246 genotypes derived from backcrossing different annual Sorghum bicolor X perennial S. halepense F1 plants to a tetraploidized S. bicolor. RNA‐seq assisted in identifying candidate genes for rhizomatousness. Correspondence of rhizomatousness quantitative trait loci (QTLs) with those from two populations derived from crosses between S. halepense progenitors S. bicolor X S. propinquum suggests either the preservation of interspecific polymorphism or the formation of novel alleles following polyploid S. halepense formation. Correspondence of tillering and branching QTLs further supports their developmental. Identification of genes from RNA‐seq study within QTL intervals provides insight toward discovery of causal rhizomatous genes. An unexpected finding from both S. halepense‐ and S. propinquum‐derived populations is that alleles contributing to late flowering are related to reduced rhizomatousness. Twelve of 16 QTL regions conferring rhizomatousness fall in paleo‐duplicated regions tracing to single ancestral regions 96 million years ago, indicating that corresponding genes in these regions have retained similar functions since the duplication event.

The cuticle, a hydrophobic layer of cutin and waxes synthesized by plant epidermal cells, is the major barrier to water loss when stomata are closed. Dissecting the genetic architecture of natural variation for maize (Zea mays L.) leaf cuticular conductance (gc) is important for identifying genes relevant to improving crop productivity in drought-prone environments. To this end, we performed an integrated genome- and transcriptome-wide association studies (GWAS and TWAS) to identify candidate genes putatively regulating variation in leaf gc. Of the 22 plausible candidate genes identified, 4 were predicted to be involved in cuticle precursor biosynthesis and export, 2 in cell wall modification, 9 in intracellular membrane trafficking, and 7 in the regulation of cuticle development. A gene encoding an INCREASED SALT TOLERANCE1-LIKE1 (ISTL1) protein putatively involved in intracellular protein and membrane trafficking was identified in GWAS and TWAS as the strongest candidate causal gene. A set of maize nested near-isogenic lines that harbor the ISTL1 genomic region from eight donor parents were evaluated for gc, confirming the association between gc and ISTL1 in a haplotype-based association analysis. The findings of this study provide insights into the role of regulatory variation in the development of the maize leaf cuticle and will ultimately assist breeders to develop drought-tolerant maize for target environments.

SignificancePlants grow from their tips. The gametophore (shoot-like organ) tip of the moss Physcomitrium patens is a single cell that performs the same functions as those of multicellular flowering plants, producing the cells that make leaves and regenerating new stem cells to maintain the shoot tip. Several pathways, including CLAVATA and cytokinin hormonal signaling, regulate stem cell abundance in flowering plants and in mosses, although the mechanisms whereby these pathways regulate stem cell abundance and their conservation between these plant lineages is poorly understood. Using moss, we investigated how PpCLAVATA and cytokinin signaling interact. Overall, we found evidence that PpCLAVATA and cytokinin signaling interact similarly in moss and flowering plants, despite their distinct anatomies, life cycles, and evolutionary distance.

The sheathing leaf found in grasses and other monocots is an evolutionary innovation, yet its origin has been a subject of long-standing debate. Here, we revisit the problem in the light of developmental genetics and computational modeling. We show that the sheathing leaf likely arose through WOX-gene-dependent extension of a primordial zone straddling concentric domains around the shoot apex. Patterned growth within this zone, oriented by two polarity fields, accounts for wild-type, mutant and mosaic grass leaf development, whereas zone contraction and growth remodeling accounts for eudicot leaf development. In contrast to the prevailing view, our results suggest that the sheath derives from petiole, whereas the blade derives from the lamina of the eudicot leaf, consistent with homologies proposed in the 19th century.

From noble beginnings as a prospective forage, polyploid Sorghum halepense (‘Johnsongrass’) is both an invasive species and one of the world’s worst agricultural weeds. Formed by S. bicolor x S. propinquum hybridization, we show S. halepense to have S. bicolor-enriched allele composition and striking mutations in 5,957 genes that differentiate it from representatives of its progenitor species and an outgroup. The spread of S. halepense may have been facilitated by introgression from closely-related cultivated sorghum near genetic loci affecting rhizome development, seed size, and levels of lutein, a photochemical protectant and abscisic acid precursor. Rhizomes, subterranean stems that store carbohydrates and spawn clonal propagules, have growth correlated with reproductive rather than other vegetative tissues, and increase survival of both temperate cold seasons and tropical dry seasons. Rhizomes of S. halepense are more extensive than those of its rhizomatous progenitor S. propinquum, with gene expression including many alleles from its non-rhizomatous S. bicolor progenitor. The first surviving polyploid in its lineage in ∼96 million years, its post-Columbian spread across six continents carried rich genetic diversity that in the United States has facilitated transition from agricultural to non-agricultural niches. Projected to spread another 200–600 km northward in the coming century, despite its drawbacks S. halepense may offer novel alleles and traits of value to improvement of sorghum.

Histology incorporates microtechnique, the preparation of living material for observation and microscopy to study the anatomy of cells, tissues and organs. Interpreting histological patterns in a specimen may help to correlate form and function at the cellular, tissue and organ levels. Many stain options and staining methods (“schedules”) for plant tissues have been described previously (Johansen, 1940; Jensen, 1962; O’Brien and McCully, 1981; Sylvester and Ruzin, 1994; Ruzin, 1999). Here, we present a straightforward, inexpensive protocol that uses Toluidine Blue O (TBO) to stain maize tissue. TBO is a general metachromatic stain that can be used on paraffin-embedded histological sections, as well as fixed, hand-sectioned material. The value of TBO as a stain is the inherent metachromasia, whereby the dye is altered by pH resulting in violet blue color variants. This property is particularly useful even in sectioned material for identifying acidic components of plant cells. We demonstrate the utility of TBO staining for cellular-, tissue- and organ-level studies using maize seedling root, shoot apex and mature leaf (Figure 2)., [摘要] 组织学结合了微技术，用于观察和显微镜检查的活材料的制备，以研究细胞，组织和器官的解剖学，解释标本的组织学模式可能有助于在细胞，组织和器官水平上关联形式和功能。先前已经描述了植物组织的染色选择和染色方法（“时间表”）（Johansen ，1940； Jensen，1962； O'Brien和McCully，1981； Sylvester和Ruzin ，1994；Ruzin ，1999）。 在此，我们提出了一种简单，便宜的方案，该方案使用甲苯胺蓝O（TBO）对玉米组织进行染色.TBO是一种普通的变色染色剂，可用于石蜡包埋的组织学切片以及固定的手工切片材料TBO作为染色剂的价值是固有的变色，通过pH改变染料会导致紫蓝色变色。即使在用于鉴定植物细胞酸性成分的切片材料中，该特性也特别有用。使用玉米幼苗根，茎尖和成熟叶对细胞，组织和器官水平的染色进行染色（图2 ）。

Plants maintain populations of stem cells to generate new organs throughout the course of their lives. The pathways that regulate plant stem cell maintenance have garnered great interest over the past decades, as variation in these pathways contributes plant morphological diversity and can be harnessed for crop improvement. In order to facilitate cross-species comparisons of gene function and better understand how these stem cell regulatory pathways evolved, we undertook a functionally informed phylogenetic analysis of leucine-rich receptor-like kinases (LRR-RLK) and related proteins across diverse land plant model systems. Based on our phylogenetic analysis and on functional data, we propose a naming scheme for these stem cell signaling genes. We discovered evidence for frequent loss of protein domains in angiosperms but not in bryophytes. In addition, several clades of stem cell signaling genes are closely related to genes that function in immunity, although these distinct developmental and immune functions likely separated or after the divergence of lycophytes and angiosperms. Overall, the phylogenetic framework and evolutionary hypotheses we provide here will empower future research on cross-species comparisons of stem cell signaling pathways.

Plant cuticles are composed of wax and cutin and evolved in the land plants as a hydrophobic boundary that reduces water loss from the plant epidermis. The expanding maize adult leaf displays a dynamic, proximodistal gradient of cuticle development, from the leaf base to the tip. Laser microdissection RNA Sequencing (LM-RNAseq) was performed along this proximodistal gradient, and complementary network analyses identified potential regulators of cuticle biosynthesis and deposition. A weighted gene coexpression network (WGCN) analysis suggested a previously undescribed function for PHYTOCHROME-mediated light signaling during the regulation of cuticular wax deposition. Genetic analyses reveal that phyB1 phyB2 double mutants of maize exhibit abnormal cuticle composition, supporting the predictions of our coexpression analysis. Reverse genetic analyses also show that phy mutants of the moss Physcomitrella patens exhibit abnormal cuticle composition, suggesting an ancestral role for PHYTOCHROME-mediated, light-stimulated regulation of cuticle development during plant evolution.

Plants maintain populations of pluripotent stem cells in shoot apical meristems (SAMs), which continuously produce new aboveground organs. We used single-cell RNA sequencing 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, we use trajectory inference to trace the gene expression changes that accompany cell differentiation. These data provide a valuable scaffold on which to better dissect the genetic control of plant shoot morphogenesis.

The mechanisms whereby leaf anlagen undergo proliferative growth and expansion to form wide, flat leaves are unclear. The maize gene NARROWSHEATH1 (NS1) is a WUSCHEL-related homeobox3 (WOX3) homolog expressed at the margins of leaf primordia, and is required for mediolateral outgrowth. To investigate the mechanisms of NS1 function, we used chromatin immunoprecipitation and laser-microdissection RNAseq of leaf primordial margins to identify gene targets bound and modulated by NS1. Microscopic analyses of cell division and gene expression in expanding leaves, and reverse genetic analyses of homologous NS1 target genes in Arabidopsis, reveal that NS1 controls mediolateral outgrowth by repression of a growth inhibitor and promotion of cell division at primordial leaf margins. Intriguingly, homologous WOX gene products are expressed in stem-cell organizing centers and traffic to adjoining cells to activate stem-cell identity non-autonomously. In contrast, WOX3/NS1 does not traffic, and stimulates cell divisions in the same cells where it is transcribed.

The cuticle, a hydrophobic layer of cutin and waxes synthesized by plant epidermal cells, is the major barrier to water loss when stomata are closed at night and under water-limited conditions. Elucidating the genetic architecture of natural variation for leaf cuticular conductance (gc) is important for identifying genes relevant to improving crop productivity in drought-prone environments. To this end, we conducted a genome-wide association study of gc of adult leaves in a maize inbred association panel that was evaluated in four environments (Maricopa, AZ, and San Diego, CA, in 2016 and 2017). Five genomic regions significantly associated with gc were resolved to seven plausible candidate genes (ISTL1, two SEC14 homologs, cyclase-associated protein, a CER7 homolog, GDSL lipase, and β-D-XYLOSIDASE 4). These candidates are potentially involved in cuticle biosynthesis, trafficking and deposition of cuticle lipids, cutin polymerization, and cell wall modification. Laser microdissection RNA sequencing revealed that all these candidate genes, with the exception of the CER7 homolog, were expressed in the zone of the expanding adult maize leaf where cuticle maturation occurs. With direct application to genetic improvement, moderately high average predictive abilities were observed for whole-genome prediction of gc in locations (0.46 and 0.45) and across all environments (0.52). The findings of this study provide novel insights into the genetic control of gc and have the potential to help breeders more effectively develop drought-tolerant maize for target environments.