Interactive CLV3, CLE16 and CLE17 signaling mediates stem cell homeostasis in the Arabidopsis shoot apical meristem.

The ability of plants to grow and form organs throughout their lifetime is dependent on their sustained stem cell activity. These stem cell populations are maintained by intricate networks of intercellular signaling pathways. In Arabidopsis thaliana, the small secreted peptide CLAVATA3 (CLV3) controls shoot apical meristem (SAM) maintenance by activating a signal transduction pathway that modulates the expression of the homeodomain transcription factor WUSCHEL (WUS). Here, we demonstrate that two CLV3-related peptides, CLE16 and CLE17, restrict stem cell accumulation in the absence of CLV3. CLE16 and CLE17 contribute independently to SAM maintenance and organ production in clv3 plants at all stages of development. We show that CLE16 and CLE17 signal through a subset of CLV3 receptors, the BARELY ANY MERISTEM (BAM) receptor kinases, and act upstream of WUS. Our study reveals that CLE16 and CLE17 function in a mechanism that partially compensates for CLV3 to maintain stem cell homeostasis and plant resiliency, and expands the potential targets for enhancing yield traits in crop species.

First Plant Cell Atlas symposium report.

The Plant Cell Atlas (PCA) community hosted a virtual symposium on December 9 and 10, 2021 on single cell and spatial omics technologies. The conference gathered almost 500 academic, industry, and government leaders to identify the needs and directions of the PCA community and to explore how establishing a data synthesis center would address these needs and accelerate progress. This report details the presentations and discussions focused on the possibility of a data synthesis center for a PCA and the expected impacts of such a center on advancing science and technology globally. Community discussions focused on topics such as data analysis tools and annotation standards; computational expertise and cyber-infrastructure; modes of community organization and engagement; methods for ensuring a broad reach in the PCA community; recruitment, training, and nurturing of new talent; and the overall impact of the PCA initiative. These targeted discussions facilitated dialogue among the participants to gauge whether PCA might be a vehicle for formulating a data synthesis center. The conversations also explored how online tools can be leveraged to help broaden the reach of the PCA (i.e., online contests, virtual networking, and social media stakeholder engagement) and decrease costs of conducting research (e.g., virtual REU opportunities). Major recommendations for the future of the PCA included establishing standards, creating dashboards for easy and intuitive access to data, and engaging with a broad community of stakeholders. The discussions also identified the following as being essential to the PCA's success: identifying homologous cell-type markers and their biocuration, publishing datasets and computational pipelines, utilizing online tools for communication (such as Slack), and user-friendly data visualization and data sharing. In conclusion, the development of a data synthesis center will help the PCA community achieve these goals by providing a centralized repository for existing and new data, a platform for sharing tools, and new analytical approaches through collaborative, multidisciplinary efforts. A data synthesis center will help the PCA reach milestones, such as community-supported data evaluation metrics, accelerating plant research necessary for human and environmental health.

Plant single-cell solutions for energy and the environment.

Progress in sequencing, microfluidics, and analysis strategies has revolutionized the granularity at which multicellular organisms can be studied. In particular, single-cell transcriptomics has led to fundamental new insights into animal biology, such as the discovery of new cell types and cell type-specific disease processes. However, the application of single-cell approaches to plants, fungi, algae, or bacteria (environmental organisms) has been far more limited, largely due to the challenges posed by polysaccharide walls surrounding these species' cells. In this perspective, we discuss opportunities afforded by single-cell technologies for energy and environmental science and grand challenges that must be tackled to apply these approaches to plants, fungi and algae. We highlight the need to develop better and more comprehensive single-cell technologies, analysis and visualization tools, and tissue preparation methods. We advocate for the creation of a centralized, open-access database to house plant single-cell data. Finally, we consider how such efforts should balance the need for deep characterization of select model species while still capturing the diversity in the plant kingdom. Investments into the development of methods, their application to relevant species, and the creation of resources to support data dissemination will enable groundbreaking insights to propel energy and environmental science forward.

Network analyses identify a transcriptomic proximodistal prepattern in the maize leaf primordium.

The formation of developmental boundaries is a common feature of multicellular plants and animals, and impacts the initiation, structure and function of all organs. Maize leaves comprise a proximal sheath that encloses the stem, and a distal photosynthetic blade that projects away from the plant axis. An epidermally derived ligule and a joint-like auricle develop at the blade/sheath boundary of maize leaves. Mutations disturbing the ligule/auricle region disrupt leaf patterning and impact plant architecture, yet it is unclear how this developmental boundary is established. Targeted microdissection followed by transcriptomic analyses of young leaf primordia were utilized to construct a co-expression network associated with development of the blade/sheath boundary. Evidence is presented for proximodistal gradients of gene expression that establish a prepatterned transcriptomic boundary in young leaf primordia, before the morphological initiation of the blade/sheath boundary in older leaves. This work presents a conceptual model for spatiotemporal patterning of proximodistal leaf domains, and provides a rich resource of candidate gene interactions for future investigations of the mechanisms of blade/sheath boundary formation in maize.

Parallel global profiling of plant TOR dynamics reveals a conserved role for LARP1 in translation.

Target of rapamycin (TOR) is a protein kinase that coordinates eukaryotic metabolism. In mammals, TOR specifically promotes translation of ribosomal protein (RP) mRNAs when amino acids are available to support protein synthesis. The mechanisms controlling translation downstream from TOR remain contested, however, and are largely unexplored in plants. To define these mechanisms in plants, we globally profiled the plant TOR-regulated transcriptome, translatome, proteome, and phosphoproteome. We found that TOR regulates ribosome biogenesis in plants at multiple levels, but through mechanisms that do not directly depend on 5' oligopyrimidine tract motifs (5'TOPs) found in mammalian RP mRNAs. We then show that the TOR-LARP1-5'TOP signaling axis is conserved in plants and regulates expression of a core set of eukaryotic 5'TOP mRNAs, as well as new, plant-specific 5'TOP mRNAs. Our study illuminates ancestral roles of the TOR-LARP1-5'TOP metabolic regulatory network and provides evolutionary context for ongoing debates about the molecular function of LARP1.

Genomic prediction of maize microphenotypes provides insights for optimizing selection and mining diversity.

Effective evaluation of millions of crop genetic stocks is an essential component of exploiting genetic diversity to achieve global food security. By leveraging genomics and data analytics, genomic prediction is a promising strategy to efficiently explore the potential of these gene banks by starting with phenotyping a small designed subset. Reliable genomic predictions have enhanced selection of many macroscopic phenotypes in plants and animals. However, the use of genomicprediction strategies for analysis of microscopic phenotypes is limited. Here, we exploited the power of genomic prediction for eight maize traits related to the shoot apical meristem (SAM), the microscopic stem cell niche that generates all the above-ground organs of the plant. With 435 713 genomewide single-nucleotide polymorphisms (SNPs), we predicted SAM morphology traits for 2687 diverse maize inbreds based on a model trained from 369 inbreds. An empirical validation experiment with 488 inbreds obtained a prediction accuracy of 0.37-0.57 across eight traits. In addition, we show that a significantly higher prediction accuracy was achieved by leveraging the U value (upper bound for reliability) that quantifies the genomic relationships of the validation set with the training set. Our findings suggest that double selection considering both prediction and reliability can be implemented in choosing selection candidates for phenotyping when exploring new diversity is desired. In this case, individuals with less extreme predicted values and moderate reliability values can be considered. Our study expands the turbocharging gene banks via genomic prediction from the macrophenotypes into the microphenotypic space.

A high-resolution gene expression atlas links dedicated meristem genes to key architectural traits.

The shoot apical meristem (SAM) orchestrates the balance between stem cell proliferation and organ initiation essential for postembryonic shoot growth. Meristems show a striking diversity in shape and size. How this morphological diversity relates to variation in plant architecture and the molecular circuitries driving it are unclear. By generating a high-resolution gene expression atlas of the vegetative maize shoot apex, we show here that distinct sets of genes govern the regulation and identity of stem cells in maize versus Arabidopsis. Cell identities in the maize SAM reflect the combinatorial activity of transcription factors (TFs) that drive the preferential, differential expression of individual members within gene families functioning in a plethora of cellular processes. Subfunctionalization thus emerges as a fundamental feature underlying cell identity. Moreover, we show that adult plant characters are, to a significant degree, regulated by gene circuitries acting in the SAM, with natural variation modulating agronomically important architectural traits enriched specifically near dynamically expressed SAM genes and the TFs that regulate them. Besides unique mechanisms of maize stem cell regulation, our atlas thus identifies key new targets for crop improvement.

Reconstructing the Transcriptional Ontogeny of Maize and Sorghum Supports an Inverse Hourglass Model of Inflorescence Development.

Assembling meaningful comparisons between species is a major limitation in studying the evolution of organismal form. To understand development in maize and sorghum, closely related species with architecturally distinct inflorescences, we collected RNA-seq profiles encompassing inflorescence body-plan specification in both species. We reconstructed molecular ontogenies from 40 B73 maize tassels and 47 BTx623 sorghum panicles and separated them into transcriptional stages. To discover new markers of inflorescence development, we used random forest machine learning to determine stage by RNA-seq. We used two descriptions of transcriptional conservation to identify hourglass-like stages during inflorescence development. Despite a relatively short 12 million years since their last common ancestor, we found maize and sorghum inflorescences are most different during their hourglass-like stages of development, following an inverse-hourglass model of development. We discuss whether agricultural selection may account for the rapid divergence signatures in these species and the observed separation of evolutionary pressure and developmental reprogramming.

The Second Site Modifier, Sympathy for the ligule, Encodes a Homolog of Arabidopsis ENHANCED DISEASE RESISTANCE4 and Rescues the Liguleless narrow Maize Mutant.

Liguleless narrow1 encodes a plasma membrane-localized receptor-like kinase required for normal development of maize (Zea mays) leaves, internodes, and inflorescences. The semidominant Lgn-R mutation lacks kinase activity, and phenotypic severity is dependent on inbred background. We created near isogenic lines and assayed the phenotype in multiple environments. Lgn-R plants that carry the B73 version of Sympathy for the ligule (Sol-B) fail to grow under hot conditions, but those that carry the Mo17 version (Sol-M) survive at hot temperatures and are significantly taller at cool temperatures. To identify Sol, we used recombinant mapping and analyzed the Lgn-R phenotype in additional inbred backgrounds. We identified amino acid sequence variations in GRMZM2G075262 that segregate with severity of the Lgn-R phenotypes. This gene is expressed at high levels in Lgn-R B73, but expression drops to nonmutant levels with one copy of Sol-M An EMS mutation solidified the identity of SOL as a maize homolog of Arabidopsis (Arabidopsis thaliana) ENHANCED DISEASE RESISTANCE4 (EDR4). SOL, like EDR4, is induced in response to pathogen-associated molecular patterns such as flg22. Integrated transcriptomic and phosphoproteomic analyses suggest that Lgn-R plants constitutively activate an immune signaling cascade that induces temperature-sensitive responses in addition to defects in leaf development. We propose that aspects of the severe Lgn-R developmental phenotype result from constitutive defense induction and that SOL potentially functions in repressing this response in Mo17 but not B73. Identification of LGN and its interaction with SOL provides insight into the integration of developmental control and immune responses.

Tasselseed5 overexpresses a wound-inducible enzyme, ZmCYP94B1, that affects jasmonate catabolism, sex determination, and plant architecture in maize.

Maize is monecious, with separate male and female inflorescences. Maize flowers are initially bisexual but achieve separate sexual identities through organ arrest. Loss-of-function mutants in the jasmonic acid (JA) pathway have only female flowers due to failure to abort silks in the tassel. Tasselseed5 (Ts5) shares this phenotype but is dominant. Positional cloning and transcriptomics of tassels identified an ectopically expressed gene in the CYP94B subfamily, Ts5 (ZmCYP94B1). CYP94B enzymes are wound inducible and inactivate bioactive jasmonoyl-L-isoleucine (JA-Ile). Consistent with this result, tassels and wounded leaves of Ts5 mutants displayed lower JA and JA-lle precursors and higher 12OH-JA-lle product than the wild type. Furthermore, many wounding and jasmonate pathway genes were differentially expressed in Ts5 tassels. We propose that the Ts5 phenotype results from the interruption of JA signaling during sexual differentiation via the upregulation of ZmCYP94B1 and that its proper expression maintains maize monoecy.

Morphological Plant Modeling: Unleashing Geometric and Topological Potential within the Plant Sciences.

The geometries and topologies of leaves, flowers, roots, shoots, and their arrangements have fascinated plant biologists and mathematicians alike. As such, plant morphology is inherently mathematical in that it describes plant form and architecture with geometrical and topological techniques. Gaining an understanding of how to modify plant morphology, through molecular biology and breeding, aided by a mathematical perspective, is critical to improving agriculture, and the monitoring of ecosystems is vital to modeling a future with fewer natural resources. In this white paper, we begin with an overview in quantifying the form of plants and mathematical models of patterning in plants. We then explore the fundamental challenges that remain unanswered concerning plant morphology, from the barriers preventing the prediction of phenotype from genotype to modeling the movement of leaves in air streams. We end with a discussion concerning the education of plant morphology synthesizing biological and mathematical approaches and ways to facilitate research advances through outreach, cross-disciplinary training, and open science. Unleashing the potential of geometric and topological approaches in the plant sciences promises to transform our understanding of both plants and mathematics.

Modeling the Morphometric Evolution of the Maize Shoot Apical Meristem.

The maize (Zea mays subsp. mays L.) shoot apical meristem (SAM) is a self-replenishing pool of stem cells that produces all above-ground plant tissues. Improvements in image acquisition and processing techniques have allowed high-throughput, quantitative genetic analyses of SAM morphology. As with other large-scale phenotyping efforts, meaningful descriptions of genetic architecture depend on the collection of relevant measures. In this study, we tested two quantitative image processing methods to describe SAM morphology within the genus Zea, represented by 33 wild relatives of maize and 841 lines from a domesticated maize by wild teosinte progenitor (MxT) backcross population, along with previously reported data from several hundred diverse maize inbred lines. Approximating the MxT SAM as a paraboloid derived eight parabolic estimators of SAM morphology that identified highly overlapping quantitative trait loci (QTL) on eight chromosomes, which implicated previously identified SAM morphology candidate genes along with new QTL for SAM morphological variation. Using a Fourier-transform related method of comprehensive shape analysis, we detected cryptic SAM shape variation that identified QTL on six chromosomes. We found that Fourier transform shape descriptors and parabolic estimation measures are highly correlated and identified similar QTL. Analysis of shoot apex contours from 73 anciently diverged plant taxa further suggested that parabolic shape may be a universal feature of plant SAMs, regardless of evolutionary clade. Future high-throughput examinations of SAM morphology may benefit from the ease of acquisition and phenotypic fidelity of modeling the SAM as a paraboloid.

Genetic control of morphometric diversity in the maize shoot apical meristem.

The maize shoot apical meristem (SAM) comprises a small pool of stem cells that generate all above-ground organs. Although mutational studies have identified genetic networks regulating SAM function, little is known about SAM morphological variation in natural populations. Here we report the use of high-throughput image processing to capture rich SAM size variation within a diverse maize inbred panel. We demonstrate correlations between seedling SAM size and agronomically important adult traits such as flowering time, stem size and leaf node number. Combining SAM phenotypes with 1.2 million single nucleotide polymorphisms (SNPs) via genome-wide association study reveals unexpected SAM morphology candidate genes. Analyses of candidate genes implicated in hormone transport, cell division and cell size confirm correlations between SAM morphology and trait-associated SNP alleles. Our data illustrate that the microscopic seedling SAM is predictive of adult phenotypes and that SAM morphometric variation is associated with genes not previously predicted to regulate SAM size.

Ontogeny of the sheathing leaf base in maize (Zea mays).

Leaves develop from the shoot apical meristem (SAM) via recruitment of leaf founder cells. Unlike eudicots, most monocot leaves display parallel venation and sheathing bases wherein the margins overlap the stem. Here we utilized computed tomography (CT) imaging, localization of PIN-FORMED1 (PIN1) auxin transport proteins, and in situ hybridization of leaf developmental transcripts to analyze the ontogeny of monocot leaf morphology in maize (Zea mays). CT imaging of whole-mounted shoot apices illustrates the plastochron-specific stages during initiation of the basal sheath margins from the tubular disc of insertion (DOI). PIN1 localizations identify basipetal auxin transport in the SAM L1 layer at the site of leaf initiation, a process that continues reiteratively during later recruitment of lateral leaf domains. Refinement of these auxin transport domains results in multiple, parallel provascular strands within the initiating primordium. By contrast, auxin is transported from the L2 toward the L1 at the developing margins of the leaf sheath. Transcripts involved in organ boundary formation and dorsiventral patterning accumulate within the DOI, preceding the outgrowth of the overlapping margins of the sheathing leaf base. We suggest a model wherein sheathing bases and parallel veins are both patterned via the extended recruitment of lateral maize leaf domains from the SAM.

Arabidopsis thaliana GEX1 has dual functions in gametophyte development and early embryogenesis.

GEX1 is a plasma membrane protein that is conserved among plant species, and has previously been shown to be expressed in sperm cells and some sporophytic tissues. Here we show that GEX1 is also expressed in the embryo sac before cellularization, in the egg cell after cellularization, in the zygote/embryo immediately after fertilization and in the pollen vegetative cell. We functionally characterize GEX1 in Arabidopsis thaliana, and show that it is a versatile protein that performs functions during male and female gametophyte development, and during early embryogenesis. gex1-1/+ plants, which synthesize a truncated GEX1 mRNA encoding a protein lacking the predicted cytoplasmic domain, but still targeted to the plasma membrane, had embryos that arrested before the pre-globular stage. gex1-3/+ plants, carrying a null GEX1 allele, had defects during male and female gametophyte development, and during early embryogenesis. Using an antisense GEX1 transgenic line we demonstrate that the predicted GEX1 extracellular domain is sufficient and necessary for GEX1 function during the development of both gametophytes. The predicted cytoplasmic domain is necessary for correct early embryogenesis and mediates homodimer formation at the plasma membrane. We propose that dimerization of GEX1 in the zygote might be an upstream step in a signaling cascade regulating early embryogenesis.