Hao Wu

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.

WUSCHEL-related homeobox plays important roles in diverse biological processes, such as plant growth and development, hormonal homeostasis, and abiotic stress adaptation. Lycium barbarum (goji berry) is a model species for studying regeneration in woody plants; however, the LbWOX gene family has yet to be characterized. This work reports a genomic and transcriptomic characterization of the LbWOX gene family in Lycium barbarum. Eighteen LbWOX genes were identified with uneven distribution across eight chromosomes. These genes were grouped into three subfamilies via phylogenetic classification. Additionally, cis-regulatory element characterization suggests that the expression of LbWOX genes is mainly influenced by plant differentiation, phytohormones, and various abiotic stresses. Expression profiles derived from RNA-Seq of root, stem, leaf, and fruit revealed that all eighteen genes were expressed. Notably, LbWOX1 and LbWOX4 were highly expressed in leaves, suggesting a role in leaf growth and a potential to enhance differentiation capacity. Furthermore, LbWOX4 showed elevated expression in roots and stems, an association with vascular development that implicates them as prime candidates for enhancing adventitious root formation during cutting propagation. This work represents the first genome-wide analysis of the LbWOX genes, integrating high-throughput RNA-Seq to characterize the function of all eighteen identified members. Our research provides further insights for future studies of LbWOX gene functions in wolfberry.

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.

Zoologists have adduced morphological convergence among embryonic stages of closely related taxa, which has been called the phylotypic stage of embryogenesis. Transcriptomic analyses reveal a phylotypic hourglass pattern of gene expression during plant as well as 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. However, widespread comparisons of embryonic gene expression across animal phyla describe an inverse hourglass pattern, where gene expression is correlated during early and late stages but not during mid-development. Here, multiplexed spatial-transcriptomic analyses is used to investigate embryo ontogeny and homology in maize, which has novel, grass-specific morphology. An embryonic-organ genetic network is identified, replete for ancient/conserved genes manifesting a phylotypic hourglass during mid-embryogenesis. Transcriptomic comparisons of grass embryo ontogeny with that of a moss Physcomitrium patens identify a conserved, inverse hourglass pattern across plant phyla, as in animals. The data indicate that the plant phylotypic stage and that of animals is characterized by ancient gene network expression during early histo– and morphogenesis and polarized embryonic development. The data reveal an ancient, convergent mechanism for the evolution of morphological novelty.

NAKED ENDOSPERM1 (NKD1), NKD2, and OPAQUE2 (O2) are transcription factors important for cell patterning and nutrient storage in maize (Zea mays) endosperm. To study the complex regulatory interrelationships among these 3 factors in coregulating gene networks, we developed a set of nkd1, nkd2, and o2 homozygous lines, including all combinations of mutant and wild-type genes. Among the 8 genotypes tested, we observed diverse phenotypes and gene interactions affecting cell patterning, starch content, and storage proteins. From ∼8 to ∼16 d after pollination, maize endosperm undergoes a transition from cellular development to nutrient accumulation for grain filling. Gene network analysis showed that NKD1, NKD2, and O2 dynamically regulate a hierarchical gene network during this period, directing cellular development early and then transitioning to constrain cellular development while promoting the biosynthesis and storage of starch, proteins, and lipids. Genetic interactions regulating this network are also dynamic. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) showed that O2 influences the global regulatory landscape, decreasing NKD1 and NKD2 target site accessibility, while NKD1 and NKD2 increase O2 target site accessibility. In summary, interactions of NKD1, NKD2, and O2 dynamically affect the hierarchical gene network and regulatory landscape during the transition from cellular development to grain filling in maize endosperm.

The naked endosperm1 ( nkd1 ), naked endosperm2 ( nkd2 ), and thick aleurone1 ( thk1 ) genes are important regulators of maize ( Zea mays L.) endosperm development. Double mutants of nkd1 and nkd2 ( nkd1,2 ) show multiple aleurone (AL) cell layers with disrupted AL cell differentiation, whereas mutants of thk1 cause multiple cell layers of fully differentiated AL cells. Here, we conducted a comparative analysis of nkd1,2 and thk1 mutant endosperm transcriptomes to study how these factors regulate gene networks to control AL layer specification and cell differentiation. Weighted gene coexpression network analysis was incorporated with published laser capture microdissected transcriptome datasets to identify a coexpression module associated with AL development. In this module, both Nkd1,2+ and Thk1+ appear to regulate cell cycle and division, whereas Nkd1,2+ , but not Thk1+ , regulate auxin signaling. Further investigation of nkd1,2 differentially expressed genes combined with published putative targets of auxin response factors (ARFs) identified 61 AL‐preferential genes that may be directly activated by NKD‐modulated ARFs. All 61 genes were upregulated in nkd1,2 mutant and the enriched Gene Ontology terms suggested that they are associated with hormone crosstalk, lipid metabolism, and developmental growth. Expression of a transgenic DR5–red fluorescent protein auxin reporter was significantly higher in nkd1,2 mutant endosperm than in wild type, supporting the prediction that Nkd1,2+ negatively regulate auxin signaling in developing AL. Overall, these results suggest that Nkd1,2+ and Thk1+ may normally restrict AL development to a single cell layer by limiting cell division, and that Nkd1,2+ restrict auxin signaling in the AL to maintain normal cell patterning and differentiation processes.

The sophistication of gene prediction algorithms and the abundance of RNA-based evidence for the maize genome may suggest that manual curation of gene models is no longer necessary. However, quality metrics generated by the MAKER-P gene annotation pipeline identified 17,225 of 130,330 (13%) protein-coding transcripts in the B73 Reference Genome V4 gene set with models of low concordance to available biological evidence. Working with eight graduate students, we used the Apollo annotation editor to curate 86 transcript models flagged by quality metrics and a complimentary method using the Gramene gene tree visualizer. All of the triaged models had significant errors–including missing or extra exons, non-canonical splice sites, and incorrect UTRs. A correct transcript model existed for about 60% of genes (or transcripts) flagged by quality metrics; we attribute this to the convention of elevating the transcript with the longest coding sequence (CDS) to the canonical, or first, position. The remaining 40% of flagged genes resulted in novel annotations and represent a manual curation space of about 10% of the maize genome (~4,000 protein-coding genes). MAKER-P metrics have a specificity of 100%, and a sensitivity of 85%; the gene tree visualizer has a specificity of 100%. Together with the Apollo graphical editor, our double triage provides an infrastructure to support the community curation of eukaryotic genomes by scientists, students, and potentially even citizen scientists.