A peptide-mediated, multilateral molecular dialogue for the coordination of pollen wall formation.

The surface of pollen grains is reinforced by pollen wall components produced noncell autonomously by tapetum cells that surround developing pollen within the male floral organ, the anther. Here, we show that tapetum activity is regulated by the GASSHO (GSO) receptor-like kinase pathway, controlled by two sulfated peptides, CASPARIAN STRIP INTEGRITY FACTOR 3 (CIF3) and CIF4, the precursors of which are expressed in the tapetum itself. Coordination of tapetum activity with pollen grain development depends on the action of subtilases, including AtSBT5.4, which are produced stage specifically by developing pollen grains. Tapetum-derived CIF precursors are processed by subtilases, triggering GSO-dependent tapetum activation. We show that the GSO receptors act from the middle layer, a tissue surrounding the tapetum and developing pollen. Three concentrically organized cell types, therefore, cooperate to coordinate pollen wall deposition through a multilateral molecular dialogue.

Embryo-Endosperm Interactions.

In angiosperms, double fertilization triggers the concomitant development of two closely juxtaposed tissues, the embryo and the endosperm. Successful seed development and germination require constant interactions between these tissues, which occur across their common interface. The embryo-endosperm interface is a complex and poorly understood compound apoplast comprising components derived from both tissues, across which nutrients transit to fuel embryo development. Interface properties, which affect molecular diffusion and thus communication, are themselves dynamically regulated by molecular and physical dialogues between the embryo and endosperm. We review the current understanding of embryo-endosperm interactions, with a focus on the structure, properties, and function of their shared interface. Concentrating on Arabidopsis, but with reference to other species, we aim to situate recent findings within the broader context of seed physiology, developmental biology, and genetic factors such as parental conflicts over resource allocation.

Family plot: the impact of the endosperm and other extra-embryonic seed tissues on angiosperm zygotic embryogenesis.

The zygotic embryos of angiosperms develop buried deep within seeds and surrounded by two main extra-embryonic tissues: the maternally derived seed coat tissues and the zygotic endosperm. Generally, these tissues are considered to play an important role in nurturing the developing embryo by acting as conduits for maternally derived nutrients. They are also critical for key seed traits (dormancy establishment and control, longevity, and physical resistance) and thus for seed and seedling survival. However, recent studies have highlighted the fact that extra-embryonic tissues in the seed also physically and metabolically limit embryonic development and that unique mechanisms may have evolved to overcome specific developmental and genetic constraints associated with the seed habit in angiosperms. The aim of this review is to illustrate how these studies have begun to reveal the highly complex physical and physiological relationship between extra-embryonic tissues and the developing embryo. Where possible I focus on Arabidopsis because of space constraints, but other systems will be cited where relevant.

Transcriptomics at Maize Embryo/Endosperm Interfaces Identifies a Transcriptionally Distinct Endosperm Subdomain Adjacent to the Embryo Scutellum.

Seeds are complex biological systems comprising three genetically distinct tissues nested one inside another (embryo, endosperm, and maternal tissues). However, the complexity of the kernel makes it difficult to understand intercompartment interactions without access to spatially accurate information. Here, we took advantage of the large size of the maize (Zea mays) kernel to characterize genome-wide expression profiles of tissues at different embryo/endosperm interfaces. Our analysis identifies specific transcriptomic signatures in two interface tissues compared with whole seed compartments: the scutellar aleurone layer and the newly named endosperm adjacent to scutellum (EAS). The EAS, which appears around 9 d after pollination and persists for around 11 d, is confined to one to three endosperm cell layers adjacent to the embryonic scutellum. Its transcriptome is enriched in genes encoding transporters. The absence of the embryo in an embryo specific mutant can alter the expression pattern of EAS marker genes. The detection of cell death in some EAS cells together with an accumulation of crushed cell walls suggests that the EAS is a dynamic zone from which cell layers in contact with the embryo are regularly eliminated and to which additional endosperm cells are recruited as the embryo grows.

Single and multiple gene knockouts by CRISPR-Cas9 in maize.

KEY MESSAGE: The analysis of 93 mutant alleles in 18 genes demonstrated that CRISPR-Cas9 is a robust tool for targeted mutagenesis in maize, permitting efficient generation of single and multiple knockouts. CRISPR-Cas9 technology is a simple and efficient tool for targeted mutagenesis of the genome. It has been implemented in many plant species, including crops such as maize. Here we report single- and multiple-gene mutagenesis via stably transformed maize plants. Two different CRISPR-Cas9 vectors were used allowing the expression of multiple guide RNAs and different strategies to knockout either independent or paralogous genes. A total of 12 plasmids, representing 28 different single guide RNAs (sgRNAs), were generated to target 20 genes. For 18 of these genes, at least one mutant allele was obtained, while two genes were recalcitrant to sequence editing. 19% (16/83) of mutant plants showed biallelic mutations. Small insertions or deletions of less than ten nucleotides were most frequently observed, regardless of whether the gene was targeted by one or more sgRNAs. Deletions of defined regions located between the target sites of two guide RNAs were also reported although the exact deletion size was variable. Double and triple mutants were created in a single step, which is especially valuable for functional analysis of genes with strong genetic linkage. Off-target effects were theoretically limited due to rigorous sgRNA design and random experimental checks at three potential off-target sites did not reveal any editing. Sanger chromatograms allowed to unambiguously class the primary transformants; the majority (85%) were fully edited plants transmitting systematically all detected mutations to the next generation, generally following Mendelian segregation.

A mechanosensitive Ca2+ channel activity is dependent on the developmental regulator DEK1.

Responses of cells to mechanical stress are thought to be critical in coordinating growth and development. Consistent with this idea, mechanically activated channels play important roles in animal development. For example, the PIEZO1 channel controls cell division and epithelial-layer integrity and is necessary for vascular development in mammals. In plants, the actual contribution of mechanoperception to development remains questionable because very few putative mechanosensors have been identified and the phenotypes of the corresponding mutants are rather mild. Here, we show that the Arabidopsis Defective Kernel 1 (DEK1) protein, which is essential for development beyond early embryogenesis, is associated with a mechanically activated Ca2+ current in planta, suggesting that perception of mechanical stress plays a critical role in plant development.

Regulation of cell wall genes in response to DEFECTIVE KERNEL1 (DEK1)-induced cell wall changes.

Defective Kernel1 (DEK1) is a plant-specific calpain involved in epidermis specification and maintenance. DEK1 regulation of the epidermal cell wall is proposed to be key to ensure tissue integrity and coordinated growth. Changes in the expression of DEK1 are correlated with changes in the expression of cell wall-related genes. For example, we have found that Lipid transfer protein 3 (LTP3), EXPANSIN 11 (EXP11), and an AP2 transcription factor (AP2TF) are misexpressed in plants with constitutively altered levels of DEK1 activity. RT-qPCR studies show that LTP3 and AP2TF may respond to a DEK1-generated signal whereas EXP11 is not altered immediately after dexamethasone induction of CALPAIN suggesting it is not in the direct signaling pathway downstream of DEK1. Our data suggest these genes are regulated by a feedback mechanism in response to DEK1-induced changes in the cell wall, and contribute to the phenotypes seen in plants with altered DEK1 expression.

Arabidopsis DEFECTIVE KERNEL1 regulates cell wall composition and axial growth in the inflorescence stem.

Axial growth in plant stems requires a fine balance between elongation and stem mechanical reinforcement to ensure mechanical stability. Strength is provided by the plant cell wall, the deposition of which must be coordinated with cell expansion and elongation to ensure that integrity is maintained during growth. Coordination of these processes is critical and yet poorly understood. The plant-specific calpain, DEFECTIVE KERNEL1 (DEK1), plays a key role in growth coordination in leaves, yet its role in regulating stem growth has not been addressed. Using plants overexpressing the active CALPAIN domain of DEK1 (CALPAIN OE) and a DEK1 knockdown line (amiRNA-DEK1), we undertook morphological, biochemical, biophysical, and microscopic analyses of mature inflorescence stems. We identify a novel role for DEK1 in the maintenance of cell wall integrity and coordination of growth during inflorescence stem development. CALPAIN OE plants are significantly reduced in stature and have short, thickened stems, while amiRNA-DEK1 lines have weakened stems that are unable to stand upright. Microscopic analyses of the stems identify changes in cell size, shape and number, and differences in both primary and secondary cell wall thickness and composition. Taken together, our results suggest that DEK1 influences primary wall growth by indirectly regulating cellulose and pectin deposition. In addition, we observe changes in secondary cell walls that may compensate for altered primary cell wall composition. We propose that DEK1 activity is required for the coordination of stem strengthening with elongation during axial growth.

Dying to live: cell elimination as a developmental strategy in angiosperm seeds.

The complete elimination of unwanted cells during development is a repeated theme in both multicellular animals and in plants. In plants, such events have been extensively studied and reviewed in terms of their molecular regulation, of marker genes and proteins expressed, and in terms of cellular changes associated with their progression. This review will take a slightly different view of developmental cell elimination and will concentrate specifically on the numerous elimination events that occur during ovule and seed development (here grouped together as seed development). It asks why this cell elimination occurs in specific seed tissues, in order to understand something about the commonalities underlying how seemingly disparate events are triggered and regulated. Finally, by placing the seed in its broader evolutionary context, the question of why cell elimination may have emerged as such a key component of the seed developmental toolbox will be considered.

DEFECTIVE KERNEL1 (DEK1) Regulates Cell Walls in the Leaf Epidermis.

The plant epidermis is crucial to survival, regulating interactions with the environment and controlling plant growth. The phytocalpain DEFECTIVE KERNEL1 (DEK1) is a master regulator of epidermal differentiation and maintenance, acting upstream of epidermis-specific transcription factors, and is required for correct cell adhesion. It is currently unclear how changes in DEK1 lead to cellular defects in the epidermis and the pathways through which DEK1 acts. We have combined growth kinematic studies, cell wall analysis, and transcriptional analysis of genes downstream of DEK1 to determine the cause of phenotypic changes observed in DEK1-modulated lines of Arabidopsis (Arabidopsis thaliana). We reveal a novel role for DEK1 in the regulation of leaf epidermal cell wall structure. Lines with altered DEK1 activity have epidermis-specific changes in the thickness and polysaccharide composition of cell walls that likely underlie the loss of adhesion between epidermal cells in plants with reduced levels of DEK1 and changes in leaf shape and size in plants constitutively overexpressing the active CALPAIN domain of DEK1. Calpain-overexpressing plants also have increased levels of cellulose and pectins in epidermal cell walls, and this is correlated with the expression of several cell wall-related genes, linking transcriptional regulation downstream of DEK1 with cellular effects. These findings significantly advance our understanding of the role of the epidermal cell walls in growth regulation and establish a new role for DEK1 in pathways regulating epidermal cell wall deposition and remodeling.

Developing a 'thick skin': a paradoxical role for mechanical tension in maintaining epidermal integrity?

Plant aerial epidermal tissues, like animal epithelia, act as load-bearing layers and hence play pivotal roles in development. The presence of tension in the epidermis has morphogenetic implications for organ shapes but it also constantly threatens the integrity of this tissue. Here, we explore the multi-scale relationship between tension and cell adhesion in the plant epidermis, and we examine how tensile stress perception may act as a regulatory input to preserve epidermal tissue integrity and thus normal morphogenesis. From this, we identify parallels between plant epidermal and animal epithelial tissues and highlight a list of unexplored questions for future research.

ZmZHOUPI, an endosperm-specific basic helix-loop-helix transcription factor involved in maize seed development.

In angiosperm seeds the embryo is embedded within the endosperm, which is in turn enveloped by the seed coat, making inter-compartmental communication essential for coordinated seed growth. In this context the basic helix-loop-helix domain transcription factor AtZHOUPI (AtZOU) fulfils a key role in both the lysis of the transient endosperm and in embryo cuticle formation in Arabidopsis thaliana. In maize (Zea mays), a cereal with a persistent endosperm, a single gene, ZmZOU, falls into the same phylogenetic clade as AtZOU. Its expression is limited to the endosperm where it peaks during the filling stage. In ZmZOU-RNA interference knock-down lines embryo size is slightly reduced and the embryonic suspensor and the adjacent embryo surrounding region show retarded breakdown. Ectopic expression of ZmZOU reduces stomatal number, possibly due to inappropriate protein interactions. ZmZOU forms functional heterodimers with AtICE/AtSCREAM and the closely related maize proteins ZmICEb and ZmICEc, but its interaction is more efficient with the ZmICEa protein, which shows sequence divergence and only has close homologues in other monocotyledonous species. Consistent with the observation that these complexes can trans-activate target gene promoters from Arabidopsis, ZmZOU partially complements the Atzou-4 mutant. However, structural, trans-activation and gene expression data support the hypothesis that ZmZOU and ZmICEa may have coevolved to form a functional complex unique to monocot seeds. This divergence may explain the reduced functionality of ZmZOU in Arabidopsis, and reflect functional specificities which are unique to the monocotyledon lineage.

DEFECTIVE KERNEL 1 promotes and maintains plant epidermal differentiation.

During plant epidermal development, many cell types are generated from protodermal cells, a process requiring complex co-ordination of cell division, growth, endoreduplication and the acquisition of differentiated cellular morphologies. Here we show that the Arabidopsis phytocalpain DEFECTIVE KERNEL 1 (DEK1) promotes the differentiated epidermal state. Plants with reduced DEK1 activity produce cotyledon epidermis with protodermal characteristics, despite showing normal growth and endoreduplication. Furthermore, in non-embryonic tissues (true leaves, sepals), DEK1 is required for epidermis differentiation maintenance. We show that the HD-ZIP IV family of epidermis-specific differentiation-promoting transcription factors are key, albeit indirect, targets of DEK1 activity. We propose a model in which DEK1 influences HD-ZIP IV gene expression, and thus epidermis differentiation, by promoting cell adhesion and communication in the epidermis.

Communication is key: Reducing DEK1 activity reveals a link between cell-cell contacts and epidermal cell differentiation status.

Plant epidermis development requires not only the initial acquisition of tissue identity, but also the ability to differentiate specific cell types over time and to maintain these differentiated states throughout the plant life. To set-up and maintain differentiation, plants activate specific transcriptional programs. Interfering with these programs can prevent differentiation and/or force differentiated cells to lose their identity and re-enter a proliferative state. We have recently shown that the Arabidopsis Defective Kernel 1 (DEK1) protein is required both for the differentiation of epidermal cells and for the maintenance of their fully differentiated state. Defects in DEK1 activity lead to a deregulation of the expression of epidermis-specific differentiation-promoting HD-ZIP IV transcription factors. Here we propose a working model in which DEK1, by maintaining cell-cell contacts, and thus communication between neighboring cells, influences HD-ZIP IV gene expression and epidermis differentiation.

ZHOUPI controls embryonic cuticle formation via a signalling pathway involving the subtilisin protease ABNORMAL LEAF-SHAPE1 and the receptor kinases GASSHO1 and GASSHO2.

Seed production in angiosperms requires tight coordination of the development of the embryo and the endosperm. The endosperm-specific transcription factor ZHOUPI has previously been shown to play a key role in this process, by regulating both endosperm breakdown and the formation of the embryonic cuticle. To what extent these processes are functionally linked is, however, unclear. In order to address this issue we have concentrated on the subtilisin-like serine protease encoding gene ABNORMAL LEAF-SHAPE1. Expression of ABNORMAL LEAF-SHAPE1 is endosperm specific, and dramatically decreased in zhoupi mutants. We show that, although ABNORMAL LEAF-SHAPE1 is required for normal embryonic cuticle formation, it plays no role in regulating endosperm breakdown. Furthermore, we show that re-introducing ABNORMAL LEAF-SHAPE1 expression in the endosperm of zhoupi mutants partially rescues embryonic cuticle formation without rescuing their persistent endosperm phenotype. Thus, we conclude that ALE1 can normalize cuticle formation in the absence of endosperm breakdown, and that ZHOUPI thus controls two genetically separable developmental processes. Finally, our genetic study shows that ZHOUPI and ABNORMAL LEAF-SHAPE1 promotes formation of embryonic cuticle via a pathway involving embryonically expressed receptor kinases GASSHO1 and GASSHO2. We therefore provide a molecular framework of inter-tissue communication for embryo-specific cuticle formation during embryogenesis.

Epidermis: the formation and functions of a fundamental plant tissue.

Epidermis differentiation and maintenance are essential for plant survival. Constant cross-talk between epidermal cells and their immediate environment is at the heart of epidermal cell fate, and regulates epidermis-specific transcription factors. These factors in turn direct epidermal differentiation involving a whole array of epidermis-specific pathways including specialized lipid metabolism necessary to build the protective cuticle layer. An intact epidermis is crucial for certain key processes in plant development, shoot growth and plant defence. Here, we discuss the control of epidermal cell fate and the function of the epidermal cell layer in the light of recent advances in the field.

A signaling module controlling the stem cell niche in Arabidopsis root meristems.

The niches of the Arabidopsis shoot and root meristems, the organizing center (OC) and the quiescent center (QC), orchestrate the fine balance of stem cell maintenance and the provision of differentiating descendants. They express the functionally related homeobox genes WUSCHEL (WUS) and WOX5, respectively, that promote stem cell fate in adjacent cells. Shoot stem cells signal back to the OC by secreting the CLAVATA3 (CLV3) dodecapeptide, which represses WUS expression. However, the signals controlling homeostasis of the root stem cell system are not identified to date. Here we show that the differentiating descendants of distal root stem cells express CLE40, a peptide closely related to CLV3. Reducing CLE40 levels delays differentiation and allows stem cell proliferation. Conversely, increased CLE40 levels drastically alter the expression domain of WOX5 and promote stem cell differentiation. We report that the receptor kinase ACR4, previously shown to control cell proliferation, is an essential component, and also a target, of CLE40 signaling. Our results reveal how, in contrast to the shoot system, signals originating from differentiated cells, but not the stem cells, determine the size and position of the root niche.

Receptor-like kinase ACR4 restricts formative cell divisions in the Arabidopsis root.

During the development of multicellular organisms, organogenesis and pattern formation depend on formative divisions to specify and maintain pools of stem cells. In higher plants, these activities are essential to shape the final root architecture because the functioning of root apical meristems and the de novo formation of lateral roots entirely rely on it. We used transcript profiling on sorted pericycle cells undergoing lateral root initiation to identify the receptor-like kinase ACR4 of Arabidopsis as a key factor both in promoting formative cell divisions in the pericycle and in constraining the number of these divisions once organogenesis has been started. In the root tip meristem, ACR4 shows a similar action by controlling cell proliferation activity in the columella cell lineage. Thus, ACR4 function reveals a common mechanism of formative cell division control in the main root tip meristem and during lateral root initiation.

Cell signalling: the merry lives of BAK1.

Plant receptor-like kinases characterised by leucine-rich repeats have been shown to play dual roles in seemingly unrelated biological processes, inviting comparison with TOLL-like receptors of animals.