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All land plants-the embryophytes-produce multicellular embryos, as do other multicellular organisms, such as brown algae and animals. A unique characteristic of plant embryos is their immobile and confined nature. Their embedding in maternal tissues may offer protection from the environment, but also physically constrains development. Across the different land plants, a huge discrepancy is present between their reproductive structures whilst leading to similarly complex embryos. Therefore, we review the roles that maternal tissues play in the control of embryogenesis across land plants. These nurturing, constraining, and protective roles include both direct and indirect effects. In this review, we explore how the maternal surroundings affect embryogenesis and which chemical and mechanical barriers are in place. We regard these questions through the lens of evolution, and identify key questions for future research., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

In many flowering plants, asymmetric division of the zygote generates apical and basal cells with different fates. In Arabidopsis thaliana , the apical cell generates the embryo while the basal cell divides anticlinally, leading to a suspensor of six to nine cells that remain extra-embryonic and eventually senesce. In some genetic backgrounds, or upon ablation of the embryo, suspensor cells can undergo periclinal cell divisions and eventually form a second twin embryo. Likewise, embryogenesis can be induced from somatic cells by various genes, but the relationship with suspensor-derived embryos is unclear. Here, we addressed the nature of the suspensor to embryo fate transformation and its genetic triggers. We expressed most known embryogenesis-inducing genes specifically in suspensor cells. We next analyzed morphology and fate-marker expression in embryos in which suspensor division was activated by different triggers to address the developmental paths towards reprogramming. Our results show that reprogramming of Arabidopsis suspensor cells towards embryonic identity is a specific cellular response that is triggered by defined regulators, follows a conserved developmental trajectory and shares similarity to the process of somatic embryogenesis from post-embryonic tissues., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Virtually all growth, developmental, physiological, and defense responses in plants are accompanied by reorganization of subcellular structures to enable altered cellular growth, differentiation or function. Visualizing cellular reorganization is therefore critical to understand plant biology at the cellular scale. Fluorescently labeled markers for organelles, or for cellular components are widely used in combination with confocal microscopy to visualize cellular reorganization. Early during plant embryogenesis, the precursors for all major tissues of the seedling are established, and in Arabidopsis, this entails a set of nearly invariant switches in cell division orientation and directional cell expansion. Given that these cellular reorganization events are genetically regulated and coupled to formative events in plant development, they offer a good model to understand the genetic control of cellular reorganization in plant development. Until recently, it has been challenging to visualize subcellular structures in the early Arabidopsis embryo for two reasons: embryos are deeply embedded in seed coat and fruit, and in addition, no dedicated fluorescent markers, expressed in the embryo, were available. We recently established both an imaging approach and a set of markers for the early Arabidopsis embryo. Here, we describe a detailed protocol to use these new tools in imaging cellular reorganization.

Cells differentiate from undifferentiated precursors in order to establish the tissues of vascular plants. The different cell types and stem cells are first specified in the early embryo. How cell type specification is instructed by transcriptional control on a genome-wide level is poorly understood. A major hurdle has been the technical challenge associated with obtaining cellular transcriptomes in this inaccessible tissue. Recently, we adapted a two-component genetic labeling system called INTACT to isolate nuclei and generate a microarray-based expression atlas of the cell types in the early Arabidopsis thaliana embryo. Here we present a step-by-step description of the adapted INTACT protocol, as well as the approach to generate transcriptomic profiles. This protocol has been adapted to account for using seeds with embryos of various developmental stages as a starting material, and the relatively few cell type-specific nuclei that can be isolated from embryos.

In the early embryo of vascular plants, the different cell types and stem cells of the seedling are specified as the embryo develops from a zygote towards maturity. How the key steps in cell and tissue specification are instructed by genome-wide transcriptional activity is poorly understood. Progress in defining transcriptional regulation at the genome-wide level in plant embryos has been hampered by difficulties associated with capturing cell-type-specific transcriptomes in this small and inaccessible structure. We recently adapted a two-component genetic nucleus labelling system called INTACT to isolate nuclei from distinct cell types at different stages of Arabidopsis thaliana embryogenesis. We have used these to generate a transcriptomic atlas of embryo development following microarray-based expression profiling. Here, we present a general description of the adapted INTACT procedure, including the two-component labelling system, seed isolation, nuclei preparation and purification, as well as transcriptomic profiling. We also compare nuclear and cellular transcriptomes from the early Arabidopsis embryo to assess nucleocytoplasmic differences and discuss how these differences can be used to infer regulation of gene activity.

Land plants reproduce sexually by developing an embryo from a fertilized egg cell. However, embryos can also be formed from other cell types in many plant species. Thus, a key question is how embryo identity in plants is controlled, and how this process is modified during nonzygotic embryogenesis. The Arabidopsis ( Arabidopsis thaliana ) zygote divides to produce an embryonic lineage and an extra-embryonic suspensor. Yet, normally quiescent suspensor cells can develop a second embryo when the initial embryo is damaged, or when response to the signaling molecule auxin is locally blocked. Here we used auxin-dependent suspensor embryogenesis as a model to determine transcriptome changes during embryonic reprogramming. We found that reprogramming is complex and accompanied by large transcriptomic changes before anatomical changes. This analysis revealed a strong enrichment for genes encoding components of auxin homeostasis and response among misregulated genes. Strikingly, deregulation among multiple auxin-related gene families converged upon the re-establishment of cellular auxin levels or response. This finding points to a remarkable degree of feedback regulation to create resilience in the auxin response during embryo development. Starting from the transcriptome of auxin-deregulated embryos, we identified an auxin-dependent basic Helix Loop Helix transcription factor network that mediates the activity of this hormone in suppressing embryo development from the suspensor., (© 2018 American Society of Plant Biologists. All rights reserved.)

Oriented cell divisions are significant in plant morphogenesis because plant cells are embedded in cell walls and cannot relocate. Cell divisions follow various regular orientations, but the underlying mechanisms have not been clarified. We propose that cell-shape-dependent self-organization of cortical microtubule arrays is able to provide a mechanism for determining planes of early tissue-generating divisions and may form the basis for robust control of cell division orientation in the embryo. To show this, we simulate microtubules on actual cell surface shapes, from which we derive a minimal set of three rules for proper array orientation. The first rule captures the effects of cell shape alone on microtubule organization, the second rule describes the regulation of microtubule stability at cell edges, and the third rule includes the differential effect of auxin on local microtubule stability. These rules generate early embryonic division plane orientations and potentially offer a framework for understanding patterned cell divisions in plant morphogenesis., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Considerable progress has been made in understanding the influence of physical and genetic factors on the patterns of cell division in various model systems. However, how each of these factors directs changes in subcellular structures has remained unclear. Generic machineries for the execution of cell expansion and division have been characterized, but how these are influenced by genetic regulators and physical cell properties remains an open question. To a large degree, the complexity of growing post-embryonic tissues and a lack of precise predictability have prevented the extraction of rigid correlations between subcellular structures and future orientation of cell division. The Arabidopsis embryo offers an exquisitely predictable and simple model for studying such correlations, but so far the tools and methodology for studying subcellular structures in the early embryo have been lacking. Here, we describe a set of markers to visualize a range of subcellular structures in the early Arabidopsis embryo. We have designed a series of fluorescent cellular reporters optimized for embryos, and demonstrate the effectiveness of using these 'ACE' reporters with simple three-dimensional imaging procedures that preserve delicate cellular structures. We describe the ontogeny of subcellular structures in the early embryo and find that central/peripheral cell polarity is established much earlier than suspected. In addition, we show that the actin and microtubule cytoskeleton has distinct topologies in the embryo. These tools and methods will allow detailed analysis of the events of cellular reorganization that underlie morphogenesis in the Arabidopsis embryo., (© 2018 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.)

During early plant embryogenesis, precursors for all major tissues and stem cells are formed. While several components of the regulatory framework are known, how cell fates are instructed by genome-wide transcriptional activity remains unanswered-in part because of difficulties in capturing transcriptome changes at cellular resolution. Here, we have adapted a two-component transgenic labelling system to purify cell-type-specific nuclear RNA and generate a transcriptome atlas of early Arabidopsis embryo development, with a focus on root stem cell niche formation. We validated the dataset through gene expression analysis, and show that gene activity shifts in a spatio-temporal manner, probably signifying transcriptional reprogramming, to induce developmental processes reflecting cell states and state transitions. This atlas provides the most comprehensive tissue- and cell-specific description of genome-wide gene activity in the early plant embryo, and serves as a valuable resource for understanding the genetic control of early plant development.

Land plants are called 'embryophytes' and thus, their collective name is defined by their ability to form embryos. Indeed, embryogenesis is a widespread phenomenon in plants, and much of our diet is composed of embryos (just think of grains, beans or nuts; Figure 1). However, in addition to embryos as a source of nutrition, they are also a fascinating study object. Some of the most fundamental decisions on fate and identity, as well as patterning and morphogenesis, are taken during the first days of plant life. Yet, embryos are diverse in structure and function, and embryogenesis in plants is by no means restricted to the zygote - the product of fertilization. In this Primer, we discuss the adventures of the young plant. We will consider what it means to be a plant embryo and how to become one. We will next highlight how the study of early embryogenesis can reveal principles underlying oriented cell division and developmental pattern formation in plants., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Land plants can grow to tremendous body sizes, yet even the most complex architectures are the result of iterations of the same developmental processes: organ initiation, growth, and pattern formation. A central question in plant biology is how these processes are regulated and coordinated to allow for the formation of ordered, 3D structures. All these elementary processes first occur in early embryogenesis, during which, from a fertilized egg cell, precursors for all major tissues and stem cells are initiated, followed by tissue growth and patterning. Here we discuss recent progress in our understanding of this phase of plant life. We consider the cellular basis for multicellular development in 3D and focus on the genetic regulatory mechanisms that direct specific steps during early embryogenesis.

The flowering plant female gametophyte carries two gametes, an egg cell and a central cell, whose double fertilization gives rise to embryo and endosperm, respectively. In this issue of Developmental Cell, Yuan et al. (2016) identify the protein CKI1 as a key switch that controls the differential female gamete identities., (Copyright © 2016 Elsevier Inc. All rights reserved.)

As the start of a new life cycle, activation of the first division of the zygote is a critical event in both plants and animals. Because the zygote in plants is difficult to access, our understanding of how this process is achieved remains poor. Here we report genetic and cell biological analyses of the zygote-arrest 1 (zyg1) mutant in Arabidopsis, which showed zygote-lethal and over-accumulation of cyclin B1 D-box-GUS in ovules. Map-based cloning showed that ZYG1 encodes the anaphase-promoting complex/cyclosome (APC/C) subunit 11 (APC11). Live-cell imaging studies showed that APC11 is expressed in both egg and sperm cells, in zygotes and during early embryogenesis. Using a GFP-APC11 fusion construct that fully complements zyg1, we showed that GFP-APC11 expression persisted throughout the mitotic cell cycle, and localized to cell plates during cytokinesis. Expression of non-degradable cyclin B1 in the zygote, or mutations of either APC1 or APC4, also led to a zyg1-like phenotype. Biochemical studies showed that APC11 has self-ubiquitination activity and is able to ubiquitinate cyclin B1 and promote degradation of cyclin B1. These results together suggest that APC/C-mediated degradation of cyclin B1 in Arabidopsis is critical for initiating the first division of the zygote., (© 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The plant hormone auxin and its directional transport are known to play a crucial role in defining the embryonic axis and subsequent development of the body plan. Although the role of PIN auxin efflux transporters has been clearly assigned during embryonic shoot and root specification, the role of the auxin influx carriers AUX1 and LIKE-AUX1 (LAX) proteins is not well established. Here, we used chemical and genetic tools on Brassica napus microspore-derived embryos and Arabidopsis thaliana zygotic embryos, and demonstrate that AUX1, LAX1 and LAX2 are required for both shoot and root pole formation, in concert with PIN efflux carriers. Furthermore, we uncovered a positive-feedback loop between MONOPTEROS (ARF5)-dependent auxin signalling and auxin transport. This MONOPTEROS-dependent transcriptional regulation of auxin influx (AUX1, LAX1 and LAX2) and auxin efflux (PIN1 and PIN4) carriers by MONOPTEROS helps to maintain proper auxin transport to the root tip. These results indicate that auxin-dependent cell specification during embryo development requires balanced auxin transport involving both influx and efflux mechanisms, and that this transport is maintained by a positive transcriptional feedback on auxin signalling., (© 2015. Published by The Company of Biologists Ltd.)

Multicellular plant development requires strict control of cell division orientation. A key unanswered question is how developmental regulators interact with the generic cell division machinery to trigger oriented divisions. We discuss the Arabidopsis embryo as a model for addressing this question. Recent progress in 3D imaging and computation now allows sketching of a framework for the developmental control of division orientation in which the signaling molecule auxin controls oriented division by preventing a geometrically defined default plane. We expect that the identification of auxin effectors, together with the identification of novel regulators of cell division will help to link developmental regulators to the division machinery., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Although plant embryogenesis is usually studied in the context of seed development, there are many alternative roads to embryo initiation. These include somatic embryogenesis in tissue culture and microspore embryogenesis, both widely used in breeding and crop propagation, but also include other modes of ectopic embryo initiation. In the past decades several genes, mostly transcription factors, were identified that can induce embryogenesis in somatic cells. Because the genetic networks in which such regulators operate to promote embryogenesis are largely unknown, a key question is how their activity relates to zygotic and alternative embryo initiation. We describe here the many roads to plant embryo initiation and discuss a framework for defining the developmental roles and mechanisms of plant embryogenesis regulators., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The first vascular tissue precursors are specified early during embryogenesis. These precursors give rise to the multi-layered cylinder of hypocotyl and root through controlled, oriented divisions. Concomitant with its growth, the bundle is patterned into xylem and phloem tissues, and intervening procambial cells. These patterns are later maintained during post-embryonic growth and vascular cells will eventually differentiate, displaying characteristic secondary cell wall modifications. Given that the vascular system forms de novo in a simple yet predictable fashion, the embryo provides an excellent model system to study early developmental aspects of vascular tissue formation. However, the benefits of this model are only beginning to be exploited, and most knowledge about the vascular development is derived from growing post-embryonic tissues. Importantly, it is unclear how much of these established post-embryonic mechanisms can be extrapolated to tissue formation during embryogenesis. Here we review concepts established in the model plant Arabidopsis thaliana and focus on recent advances made in understanding embryonic vascular development., (© 2013 Scandinavian Plant Physiology Society.)

Establishment of the embryonic axis foreshadows the main body axis of adults both in plants and in animals, but underlying mechanisms are considered distinct. Plants utilize directional, cell-to-cell transport of the growth hormone auxin to generate an asymmetric auxin response that specifies the embryonic apical-basal axis. The auxin flow directionality depends on the polarized subcellular localization of PIN-FORMED (PIN) auxin transporters. It remains unknown which mechanisms and spatial cues guide cell polarization and axis orientation in early embryos. Herein, we provide conceptually novel insights into the formation of embryonic axis in Arabidopsis by identifying a crucial role of localized tryptophan-dependent auxin biosynthesis. Local auxin production at the base of young embryos and the accompanying PIN7-mediated auxin flow toward the proembryo are required for the apical auxin response maximum and the specification of apical embryonic structures. Later in embryogenesis, the precisely timed onset of localized apical auxin biosynthesis mediates PIN1 polarization, basal auxin response maximum, and specification of the root pole. Thus, the tight spatiotemporal control of distinct local auxin sources provides a necessary, non-cell-autonomous trigger for the coordinated cell polarization and subsequent apical-basal axis orientation during embryogenesis and, presumably, also for other polarization events during postembryonic plant life., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Four basic ingredients of morphogenesis, oriented cell division and expansion, cell-cell communication and cell fate specification allow plant cells to develop into a wide variety of organismal architectures. A central question in plant biology is how these cellular processes are regulated and orchestrated. Here, we present the advantages of the early Arabidopsis embryo as a model for studying the control of morphogenesis. All ingredients of morphogenesis converge during embryogenesis, and the highly predictable nature of embryo development offers unprecedented opportunities for understanding their regulation in time and space. In this review we describe the morphogenetic principles underlying embryo patterning and discuss recent advances in their regulation. Morphogenesis is under tight transcriptional control and most genes that were identified as important regulators of embryo patterning encode transcription factors or components of signaling pathways. There exists, therefore, a large gap between the transcriptional control of embryo morphogenesis and the cellular execution. We describe the first such connections, and propose future directions that should help bridge this gap and generate comprehensive understanding of the control of morphogenesis., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

The cell types of the plant root are first specified early during embryogenesis and are maintained throughout plant life. Auxin plays an essential role in embryonic root initiation, in part through the action of the ARF5/MP transcription factor and its auxin-labile inhibitor IAA12/BDL. MP and BDL function in embryonic cells but promote auxin transport to adjacent extraembryonic suspensor cells, including the quiescent center precursor (hypophysis). Here we show that a cell-autonomous auxin response within this cell is required for root meristem initiation. ARF9 and redundant ARFs, and their inhibitor IAA10, act in suspensor cells to mediate hypophysis specification and, surprisingly, also to prevent transformation to embryo identity. ARF misexpression, and analysis of the short suspensor mutant, demonstrates that lineage-specific expression of these ARFs is required for normal embryo development. These results imply the existence of a prepattern for a cell-type-specific auxin response that underlies the auxin-dependent specification of embryonic cell types., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The shoot and root apical meristems (SAM and RAM) formed during embryogenesis are crucial for postembryonic plant development. We report the identification of POPCORN (PCN), a gene required for embryo development and meristem organization in Arabidopsis thaliana. Map-based cloning revealed that PCN encodes a WD-40 protein expressed both during embryo development and postembryonically in the SAM and RAM. The two pcn alleles identified in this study are temperature sensitive, showing defective embryo development when grown at 22°C that is rescued when grown at 29°C. In pcn mutants, meristem-specific expression of WUSCHEL (WUS), CLAVATA3, and WUSCHEL-RELATED HOMEOBOX5 is not maintained; SHOOTMERISTEMLESS, BODENLOS (BDL) and MONOPTEROS (MP) are misexpressed. Several findings link PCN to auxin signaling and meristem function: ectopic expression of DR5(rev):green fluorescent protein (GFP), pBDL:BDL-GFP, and pMP:MP-β-glucuronidase in the meristem; altered polarity and expression of pPIN1:PIN1-GFP in the apical domain of the developing embryo; and resistance to auxin in the pcn mutants. The bdl mutation rescued embryo lethality of pcn, suggesting that improper auxin response is involved in pcn defects. Furthermore, WUS, PINFORMED1, PINOID, and TOPLESS are dosage sensitive in pcn, suggesting functional interaction. Together, our results suggest that PCN functions in the auxin pathway, integrating auxin signaling in the organization and maintenance of the SAM and RAM.

DORNROSCHEN (DRN), which encodes a member of the AP2-type transcription factor family, contributes to auxin transport and perception in the Arabidopsis embryo. Live imaging performed with transcriptional or translational GFP fusions shows DRN to be activated in the apical cell after the first zygotic division, to act cell-autonomously and to be expressed in single cells extending laterally from the apical shoot stem-cell zone at the position of incipient leaf primordia. Here, we show that the Auxin response factor (ARF) MONOPTEROS (MP) directly controls DRN transcription in the tips of the embryonic cotyledons, which depends on the presence of canonical Auxin response elements (AuxREs), potential ARF-binding sites flanking the DRN transcription unit. Chromatin immunoprecipitation experiments show that MP binds in vivo to two AuxRE-spanning fragments in the DRN promoter, and that MP is required for expression of DRN in cotyledon tips. Hence, DRN represents a direct target of MP and functions downstream of MP in cotyledon development.

Fertilization of the female gametophyte in angiosperm plants initiates a process of coordinated development of embryo, endosperm, and seed coat that ensures the production of a viable seed. Mutant analysis has suggested that communication between the endosperm and the seed coat is an important determinant in this process. In addition, cell groups within the embryo, derived from the apical and from the basal cell, respectively, after zygote division, concertedly establish a functional root meristem, and cells in the apical region of the embryo are hypothesized to repress cell divisions in the basal cell-derived suspensor. The available evidence for these interregional communication events mostly relies on the analysis of mutant phenotypes in Arabidopsis. To provide independent and direct evidence for communication events, we used conditional domain-specific expression of the diphtheria toxin A chain (DTA) in developing Arabidopsis seeds. By using a collection of cell- or tissue-type-specific promoters, we show that the mGAL4:VP16/UAS two-component gene expression allows reliable spatiotemporal and conditional expression of the GFP:GUS reporter and the DTA gene in the developing embryo and endosperm. Expression of DTA in the protoderm of the embryo proper led to excessive proliferation of suspensor cells, sometimes resulting in the formation of secondary embryos. Endosperm-specific expression of DTA caused complete cessation of seed growth, followed by pattern defects in the embryo and embryo arrest. Taken together, the results presented here substantiate the evidence for and underline the importance of interregional communication in embryo and seed development and demonstrate the usefulness of conditional toxin expression as a method complementary to phenotypic analysis of developmental mutants.

Mutations in ribosomal protein (RP) genes in Drosophila lead to strong developmental phenotypes, expressed in the semi-dominant Minute syndrome. In plants, however, mutations in RP genes have so far only been reported to result in recessive developmental phenotypes. We present the analysis of an Arabidopsis promoter-trap line, in which a T-DNA insertion in an RPS5 gene (AtRPS5A) causes semi-dominant developmental phenotypes. Most cell-division processes are delayed or disturbed in the heterozygous mutant, and development is completely arrested at an early embryonic stage in the homozygous mutant. By analogy with Drosophila rp mutants, we have named this mutant Arabidopsis Minute-like 1 (aml1). As with other Arabidopsis RPs, RPS5 is represented by a small gene family, but in contrast to other described plant RPs, this family comprises only two members. The AtRPS5A gene (mutated in aml1) is strongly expressed in dividing cells, whereas expression of the second RPS5 gene, AtRPS5B, is lower than that of AtRPS5A, and is correlated with cell differentiation rather than cell division. From expression analyses we conclude that AtRPS5A is the most abundantly expressed RPS5 gene in Arabidopsis. The Minute-like defects in the aml1 mutant provide the first evidence that ribosome insufficiency leads to similar consequences in both plants and insects, and emphasize the general importance of efficient protein translation for cell proliferation in higher eukaryotes.