Search

A continuous cuticle is required for normal development of Arabidopsis embryos. Despite this barrier, embryos remain sensitive to outside signals. In this issue of Developmental Cell, De Giorgi et al. show that the embryonic cuticle is relatively permeable and becomes sealed during germination in response to peptide signaling.

Genome editing in plants typically relies on T-DNA plasmids that are mobilized by Agrobacterium-mediated transformation to deliver the CRISPR/Cas9 machinery. Here, we introduce a series of CRISPR/Cas9 T-DNA vectors for minimal lab settings, such as in the classroom or citizen science projects. Spacer sequences targeting genes of interest can be inserted as annealed short oligonucleotides in a single straightforward cloning step. Fluorescent markers expressed in mature seeds enable reliable selection of transgenic as well as transgene-free individuals using a combination of inexpensive LED lamps and colored-glass alternative filters. Testing these tools on the Arabidopsis GROWTH-REGULATING FACTOR (GRF) gene family, we found that Cas9 expression from an EGG CELL1 (EC1) promoter resulted in tenfold lower mutation rates than expression from a UBIQUITIN10 (UBQ10) promoter. A collection of bona fide null mutations in all nine GRF genes could be established with little effort. Finally, we explored the effects of simultaneously targeting two, four and eight GRF genes on the rate of induced mutations at each target locus. Multiplexing caused strong interference effects: while mutation rates at some loci remained consistently high, mutation rates at other loci dropped dramatically with increasing number of single guide RNA species. Our results suggest potential detrimental genetic interaction between induced mutations as well as competition of CRISPR RNAs for a limiting amount of Cas9 apoprotein.

Activation of mitogen-activated protein kinases (MAPKs) is one of the earliest responses after plants sense an invading pathogen. Here, we show that MPK3 and MPK6, two Arabidopsis thaliana pathogen-responsive MAPKs, and their upstream MAPK kinases, MKK4 and MKK5, are essential to both stomatal and apoplastic immunity. Loss of function of MPK3 and MPK6, or their upstream MKK4 and MKK5, abolishes pathogen/microbe-associated molecular pattern- and pathogen-induced stomatal closure. Gain-of-function activation of MPK3/MPK6 induces stomatal closure independently of abscisic acid (ABA) biosynthesis and signaling. In contrast, exogenously applied organic acids such as malate or citrate are able to reverse the stomatal closure induced by MPK3/MPK6 activation. Gene expression analysis and in situ enzyme activity staining revealed that malate metabolism increases in guard cells after activation of MPK3/MPK6 or inoculation of pathogen. In addition, pathogen-induced malate metabolism requires functional MKK4/MKK5 and MPK3/MPK6. We propose that the pathogen-responsive MPK3/MPK6 cascade and ABA are two essential signaling pathways that control, respectively, the organic acid metabolism and ion channels, two main branches of osmotic regulation in guard cells that function interdependently to control stomatal opening/closure.

The major tissue types and stem-cell niches of plants are established during embryogenesis, and thus knowledge of embryo development is essential for a full understanding of plant development. Studies of seed development are also important for human health, because the nutrients stored in both the embryo and endosperm of plant seeds provide an essential part of our diet. Arabidopsis and maize have evolved different types of seeds, opening a range of experimental opportunities. Development of the Arabidopsis embryo follows an almost invariant pattern, while cell division patterns of maize embryos are variable. Embryo-endosperm interactions are also different between the two species: in Arabidopsis, the endosperm is consumed during seed development, while mature maize seeds contain an enormous endosperm. Genetic screens have provided important insights into seed development in both species. In the genomic era, genetic analysis will continue to provide important tools for understanding embryo and endosperm biology in plants, because single gene functional studies can now be integrated with genome-wide information. Here, we lay out important factors to consider when designing genetic screens to identify new genes or to probe known pathways in seed development. We then highlight the technical details of two previous genetic screens that may serve as useful examples for future experiments.

The ethylating agent ethyl methanesulfonate (EMS) is widely used for inducing random point mutations. In Arabidopsis, treatment with EMS causes GC-to-AT transitions with great efficiency: it has been estimated that a population of 50,000 well-mutagenized plants harbors one or more transitions in almost every GC pair of the genome. These properties, combined with ease of use, make EMS a mutagen of choice for genetic screens. Here, we describe a protocol for mutagenizing Arabidopsis seed with EMS. In addition, we briefly consider the germ line sectors typically induced by this treatment, and approaches for estimating the rate of induced mutations.

The ethylating agent ethyl methanesulfonate (EMS) is widely used for inducing random point mutations. In Arabidopsis, treatment with EMS causes GC-to-AT transitions with great efficiency: it has been estimated that a population of 50,000 well-mutagenized plants harbors one or more transitions in almost every GC pair of the genome. These properties, combined with ease of use, make EMS a mutagen of choice for genetic screens. Here, we describe a protocol for mutagenizing Arabidopsis seed with EMS. In addition, we briefly consider the germ line sectors typically induced by this treatment, and approaches for estimating the rate of induced mutations.

In flowering plants, the asymmetrical division of the zygote is the first hallmark of apical-basal polarity of the embryo and is controlled by a MAP kinase pathway that includes the MAPKKK YODA (YDA). In Arabidopsis, YDA is activated by the membrane-associated pseudokinase SHORT SUSPENSOR (SSP) through an unusual parent-of-origin effect: SSP transcripts accumulate specifically in sperm cells but are translationally silent. Only after fertilization is SSP protein transiently produced in the zygote, presumably from paternally inherited transcripts. SSP is a recently diverged, Brassicaceae-specific member of the BRASSINOSTEROID SIGNALING KINASE (BSK) family. BSK proteins typically play broadly overlapping roles as receptor-associated signaling partners in various receptor kinase pathways involved in growth and innate immunity. This raises two questions: How did a protein with generic function involved in signal relay acquire the property of a signal-like patterning cue, and how is the early patterning process activated in plants outside the Brassicaceae family, where SSP orthologs are absent? Here, we show that Arabidopsis BSK1 and BSK2, two close paralogs of SSP that are conserved in flowering plants, are involved in several YDA-dependent signaling events, including embryogenesis. However, the contribution of SSP to YDA activation in the early embryo does not overlap with the contributions of BSK1 and BSK2. The loss of an intramolecular regulatory interaction enables SSP to constitutively activate the YDA signaling pathway, and thus initiates apical-basal patterning as soon as SSP protein is translated after fertilization and without the necessity of invoking canonical receptor activation.

Vascular plants have an open body plan and continuously generate new axes of growth, such as shoot or root branches. Apical-to-basal transport of the hormone auxin is a hallmark of every axis, and the resulting pattern of auxin distribution affects plant development across scales, from overall architecture to cellular differentiation. How the first axis is initiated in the early embryo is a long-standing question. While our knowledge is still sparse, some of the key players of axialization have emerged, and recent work points to specific models for connecting cellular polarity to the asymmetric division of the zygote and domain specific gene expression to the organization of basipetal auxin flux.

Summary In flowering plants, developing embryos reside in maternal sporophytes. It is known that maternal generation influences the development of next-generation embryos; however, little is known about the signaling components in the process. Previously, we demonstrated that Arabidopsis mitogen-activated protein kinase 6 (MPK6) and MPK3 play critical roles in plant reproduction. In addition, we noticed that a large fraction of seeds from mpk6 single-mutant plants showed a wrinkled seed coat or a burst-out embryo phenotype. Here, we report that these seed phenotypes can be traced back to defective embryogenesis. The defective embryos have shorter suspensors and reduced growth along the longitudinal axis. Furthermore, the cotyledons fail to bend over to progress to the bent-cotyledon stage. As a result of the uneven circumference along the axis, the seed coat wrinkles to develop raisin-like morphology after dehydration. In more severe cases, the embryo can be pushed out from the micropylar end, resulting in the burst-out embryo seed phenotype. Genetic analyses demonstrated that the defective embryogenesis of the mpk6 mutant is a maternal effect. Heterozygous or homozygous mpk6 embryos have defects only in mpk6 homozygous maternal plants, but not in wild-type or heterozygous maternal plants. The loss of function of MKK4/MKK5 also results in the same phenotypes, suggesting that MKK4/MKK5 might act upstream of MPK6 in this pathway. The maternal-mediated embryo defects are associated with changes in auxin activity maxima and PIN localization. In summary, this research demonstrates that the Arabidopsis MKK4/MKK5–MPK6 cascade is an important player in the maternal control of embryogenesis.

SummaryThe 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.

Summary Background The division of plant zygotes is typically asymmetric, generating daughter cells with different developmental fates. In Arabidopsis , the apical daughter cell produces the proembryo, whereas the basal daughter cell forms the mostly extraembryonic suspensor. Establishment of apical and basal fates is known to depend on the YODA ( YDA ) mitogen-associated protein (MAP) kinase cascade and WUSCHEL-LIKE HOMEOBOX ( WOX ) homeodomain transcription factors. Results Mutations in GROUNDED ( GRD ) cause anatomical defects implying a partial loss of developmental asymmetry in the first division. Subsequently, suspensor-specific WOX8 expression disappears while proembryo-specific ZLL expression expands in the mutants, revealing that basal fates are confounded. GRD encodes a small nuclear protein of the RWP-RK family and is broadly transcribed in the early embryo. Loss of GRD eliminates the dominant effects of hyperactive YDA variants, indicating that GRD is required for YDA-dependent signaling in the embryo. However, GRD function is not regulated via direct phosphorylation by MAP kinases, and forced expression of GRD does not suppress the effect of yda mutations. In a strong synthetic interaction, grd;wox8;wox9 triple mutants arrest as zygotes or one-cell embryos lacking apparent polarity. Conclusions The predicted transcription factor GRD acts cooperatively with WOX homeodomain proteins to establish embryonic polarity in the first division. Like YDA , GRD promotes zygote elongation and basal cell fates. GRD function is required for YDA-dependent signaling but apparently not regulated by the YDA MAP kinase cascade. Similarity of GRD to Chlamydomonas MID suggests a conserved role for small RWP-RK proteins in regulating the transcriptional programs of generative cells and the zygote.

Arabidopsis embryos follow a predictable sequence of cell divisions, facilitating a genetic analysis of their early development. Both asymmetric divisions and cell-to-cell communication are probably involved in generating specific gene expression domains along the main axis within the first few division cycles. The function of these domains is not always understood, but recent work suggests that they may serve as a basis for organizing polar auxin flux. Auxin acts as systemic signal throughout the life cycle and, in the embryo, has been demonstrated to direct formation of the main axis and root initiation at the globular stage. At about the same time, root versus shoot fates are imposed on the incipient meristems by the expression of antagonistic regulators at opposite poles of the embryo. Some of the key features of the embryonic patterning process have emerged over the past few years and may provide the elements of a coherent conceptual framework.

SummaryDivision of the Arabidopsis zygote defines two fundamentally different developmental domains, the proembryo and suspensor. The resulting boundary separates domain-specific gene expression, and a signal originating from the proembryo instructs the suspensor to generate the root stem cell niche. While root induction is known to require the phytohormone auxin and the Auxin Response Factor MONOPTEROS, it has remained largely elusive how the two domains involved in this process are initially specified. Here, we show that the GATA factor HANABA TARANU (HAN) is required to position the inductive proembryo boundary. Mutations in HAN cause a coordinated apical shift of gene expression patterns, revealing that HAN regulates transcription in the basal proembryo. Key auxin transporters are affected as early as the 8 cell stage, resulting in apical redistribution of auxin. Remarkably, han embryos eventually organize a root independent of MONOPTEROS and the suspensor around a new boundary marked by the auxin maximum.

The YODA ( YDA ) mitogen-activated protein kinase pathway promotes elongation of the Arabidopsis zygote and development of its basal daughter cell into the extra-embryonic suspensor. Here, we show that the interleukin-1 receptor–associated kinase (IRAK)/Pelle-like kinase gene SHORT SUSPENSOR ( SSP ) regulates this pathway through a previously unknown parent-of-origin effect. SSP transcripts are produced in mature pollen but do not appear to be translated. Instead, they are delivered via the sperm cells to the zygote and the endosperm, where SSP protein transiently accumulates. Ectopic expression of SSP protein in the leaf epidermis is sufficient to activate YDA -dependent signaling. We propose that SSP protein produced from paternal transcripts upon fertilization triggers zygotic YDA activity, providing an essential temporal cue for the regulation of the asymmetric first division.

The primary axis and main tissue types of Arabidopsis are laid down in the early embryo. Apical-to-basal auxin flux functions as a global organizer of the axis, and recent reports are clarifying our mechanistic understanding of how a graded auxin distribution is generated and interpreted. Polar targeting of PIN transporters in the cells of the embryo is dynamic and linked to their phosphorylation status, suggesting a flexible mechanism for regulating auxin flux in space and time. PLETHORA transcription factors then interpret the graded auxin distribution to provide positional values along the axis in a dose-dependent manner. A comparable framework for tissue patterning in the radial dimension is still lacking, although cell surface signaling probably plays a key role.

Organ formation in animals and plants relies on precise control of cell state transitions to turn stem cell daughters into fully differentiated cells. In plants, cells cannot rearrange due to shared cell walls. Thus, differentiation progression and the accompanying cell expansion must be tightly coordinated across tissues. PLETHORA (PLT) transcription factor gradients are unique in their ability to guide the progression of cell differentiation at different positions in the growing Arabidopsis thaliana root, which contrasts with well-described transcription factor gradients in animals specifying distinct cell fates within an essentially static context. To understand the output of the PLT gradient, we studied the gene set transcriptionally controlled by PLTs. Our work reveals how the PLT gradient can regulate cell state by region-specific induction of cell proliferation genes and repression of differentiation. Moreover, PLT targets include major patterning genes and autoregulatory feedback components, enforcing their role as master regulators of organ development.

Cytokinesis in plants involves the formation of unique cellular structures such as the phragmoplast and the cell plate, both of which are required to divide the cell after nuclear division. In order to isolate genes that are involved in de novo cell wall formation, we performed a large-scale, microscope-based screen for Arabidopsis mutants that severely impair cytokinesis in the embryo. We recovered 35 mutations that form abnormally enlarged cells with multiple, often polyploid nuclei and incomplete cell walls. These mutants represent seven genes, four of which have previously been implicated in phragmoplast or cell plate function. Mutations in two loci show strongly reduced transmission through the haploid gametophytic generation. Molecular cloning of both corresponding genes reveals that one is represented by hypomorphic alleles of the kinesin-5 gene RADIALLY SWOLLEN 7 (homologous to tobacco kinesin-related protein TKRP125), and that the other gene corresponds to the Arabidopsis FUSED ortholog TWO-IN-ONE (originally identified based on its function in pollen development). No mutations that completely abolish the formation of cross walls in diploid cells were found. Our results support the idea that cytokinesis in the diploid and haploid generations involve similar mechanisms.

Early embryonic development in the flowering plant Arabidopsis thaliana follows a predictable sequence of cell divisions. Anatomical hallmarks and the expression of marker genes in dynamic patterns indicate that new cell fates are established with virtually every round of mitosis. Although some of the factors regulating these early patterning events have been identified, the overall process remains relatively poorly understood. Starting at the globular stage, when the embryo has approximately 100 cells, the organization of development appears to be taken over by programs that regulate postembryonic patterning throughout the life cycle.

The mitotic spindle of vascular plants is assembled and maintained by processes that remain poorly explored at a molecular level. Here, we report that AtKRP125c, one of four kinesin-5 motor proteins in arabidopsis, decorates microtubules throughout the cell cycle and appears to function in both interphase and mitosis. In a temperature-sensitive mutant, interphase cortical microtubules are disorganized at the restrictive temperature and mitotic spindles are massively disrupted, consistent with a defect in the stabilization of anti-parallel microtubules in the spindle midzone, as previously described in kinesin-5 mutants from animals and yeast. AtKRP125c introduced into mammalian epithelial cells by transfection decorates microtubules throughout the cell cycle but is unable to complement the loss of the endogenous kinesin-5 motor (Eg5). These results are among the first reports of any motor with a major role in anastral spindle structure in plants and demonstrate that the conservation of kinesin-5 motor function throughout eukaryotes extends to vascular plants.

Somatic embryos of Arabidopsis thaliana can be produced from explants of developing zygotic embryos. Cultivation of explants on maturation medium results in development of three main classes of regenerative structures: adventitious shoots, fused shoots, and complete somatic embryos. The ontogeny and anatomy of these structures was examined using serial plastic sections. Furthermore, two molecular markers were assayed to monitor transcriptional auxin responses and formation of a root meristem in this process: the LENNY allele of PIN4, a transposon insertion creating a fusion to the reporter gene GUS; and DR5::GUS, a synthetic reporter of auxin-induced transcription. In zygotic embryogenesis, PIN4 expression is confined to the center of the root meristem and begins to be detectable by the globular stage of embryogenesis, while DR5::GUS expression marks an "auxin perception maximum" in the more distal regions of the root. Adventitious and fused shoots develop no anatomically recognizable root meristem and do not express either of the two markers at their basal pole. Instead, the vasculature of their axis is directly connected to the vasculature of the explant. By contrast, complete somatic embryos were only loosely attached to the explant, had an anatomically defined root meristem and showed expression of both markers at their root pole. Our results suggest that the establishment of a root meristem in somatic embryos required appropriate auxin levels during the course of their development.

Mutations at five loci named PEANUT1-5 (PNT) were identified in a genetic screen for radially swollen embryo mutants. pnt1 cell walls showed decreased crystalline cellulose, increased pectins, and irregular and ectopic deposition of pectins, xyloglucans, and callose. Furthermore, pnt1 pollen is less viable than the wild type, and pnt1 embryos were delayed in morphogenesis and showed defects in shoot and root meristems. The PNT1 gene encodes the Arabidopsis thaliana homolog of mammalian PIG-M, an endoplasmic reticulum-localized mannosyltransferase that is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor. All five pnt mutants showed strongly reduced accumulation of GPI-anchored proteins, suggesting that they all have defects in GPI anchor synthesis. Although the mutants are seedling lethal, pnt1 cells are able to proliferate for a limited time as undifferentiated callus and do not show the massive deposition of ectopic cell wall material seen in pnt1 embryos. The different phenotype of pnt1 cells in embryos and callus suggest a differential requirement for GPI-anchored proteins in cell wall synthesis in these two tissues and points to the importance of GPI anchoring in coordinated multicellular growth.

The gravitropism defective 2 (grv2) mutants of Arabidopsis show reduced shoot phototropism and gravitropism. Amyloplasts in the shoot endodermal cells of grv2 do not sediment to the same degree as in wild type. The GRV2 gene encodes a 277-kD polypeptide that is 42% similar to the Caenorhabditis elegans RME-8 protein, which is required for endocytosis. We hypothesize that a defect in endocytosis may affect both the initial gravity sensing via amyloplasts sedimentation and the subsequent more general tropic growth response.

The Arabidopsis zygote divides asymmetrically into an embryonic apical cell and a basal cell with mostly extra-embryonic fate. This fundamental asymmetry sets the stage for further embryonic development, but the events mediating it are poorly understood. We have identified a MAPKK kinase gene, named YODA, that promotes extra-embryonic cell fates in the basal lineage. In loss-of-function mutants, the zygote does not elongate properly, and the cells of the basal lineage are eventually incorporated into the embryo instead of differentiating the extra-embryonic suspensor. Gain-of-function alleles cause exaggerated growth of the suspensor and can suppress embryonic development to a degree where no recognizable proembryo is formed. Our results imply that a MAP kinase cascade acts as a molecular switch promoting extra-embryonic fate.

Background The inherent recalcitrance of woody bioenergy feedstocks is a major challenge for their use as a source of second-generation biofuel. Secondary cell walls that constitute the majority of hardwood biomass are rich in cellulose, xylan, and lignin. The interactions among these polymers prevent facile accessibility and deconstruction by enzymes and chemicals. Plant biomass that can with minimal pretreatment be degraded into sugars is required to produce renewable biofuels in a cost-effective manner. Results GAUT12/IRX8 is a putative glycosyltransferase proposed to be involved in secondary cell wall glucuronoxylan and/or pectin biosynthesis based on concomitant reductions of both xylan and the pectin homogalacturonan (HG) in Arabidopsis irx8 mutants. Two GAUT12 homologs exist in Populus trichocarpa, PtGAUT12.1 and PtGAUT12.2. Knockdown expression of both genes simultaneously has been shown to reduce xylan content in Populus wood. We tested the proposition that RNA interference (RNAi) downregulation of GAUT12.1 alone would lead to increased sugar release in Populus wood, that is, reduced recalcitrance, based on the hypothesis that GAUT12 synthesizes a wall structure required for deposition of xylan and that cell walls with less xylan and/or modified cell wall architecture would have reduced recalcitrance. Using an RNAi approach, we generated 11 Populus deltoides transgenic lines with 50 to 67% reduced PdGAUT12.1 transcript expression compared to wild type (WT) and vector controls. Ten of the eleven RNAi lines yielded 4 to 8% greater glucose release upon enzymatic saccharification than the controls. The PdGAUT12.1 knockdown (PdGAUT12.1-KD) lines also displayed 12 to 52% and 12 to 44% increased plant height and radial stem diameter, respectively, compared to the controls. Knockdown of PdGAUT12.1 resulted in a 25 to 47% reduction in galacturonic acid and 17 to 30% reduction in xylose without affecting total lignin content, revealing that in Populus wood as in Arabidopsis, GAUT12 affects both pectin and xylan formation. Analyses of the sugars present in sequential cell wall extracts revealed a reduction of glucuronoxylan and pectic HG and rhamnogalacturonan in extracts from PdGAUT12.1-KD lines. Conclusions The results show that downregulation of GAUT12.1 leads to a reduction in a population of xylan and pectin during wood formation and to reduced recalcitrance, more easily extractable cell walls, and increased growth in Populus. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0218-y) contains supplementary material, which is available to authorized users.

Positional (or map-based) cloning techniques are widely used to identify the protein products of genes defined by mutation. In Arabidopsis the information generated by the Genome Initiative is giving this approach a decisive boost. A wealth of sequence polymorphisms and molecular markers is now available and can be exploited for fine mapping with technically simple and robust polymerase chain reaction-based methods. As a result it has become possible to complete positional cloning projects in a short time and with relatively little effort.

Mitogen-activated protein (MAP) kinase signaling cascades play important roles in the regulation of plant defense. The Raf-like MAP kinase kinase kinase (MAPKKK) EDR1 negatively regulates plant defense responses and cell death. However, how EDR1 functions, and whether it affects the regulation of MAPK cascades, are not well understood. Here, we showed that EDR1 negatively regulates the MKK4/MKK5-MPK3/MPK6 kinase cascade in Arabidopsis. We found that edr1 mutants have highly activated MPK3/MPK6 kinase activity and higher levels of MPK3/MPK6 proteins than wild type. EDR1 physically interacts with MKK4 and MKK5, and this interaction requires the N-terminal domain of EDR1. EDR1 also negatively affects MKK4/MKK5 protein levels. In addition, the mpk3, mkk4 and mkk5 mutations suppress edr1-mediated resistance, and over-expression of MKK4 or MKK5 causes edr1-like resistance and mildew-induced cell death. Taken together, our data indicate that EDR1 physically associates with MKK4/MKK5 and negatively regulates the MAPK cascade to fine-tune plant innate immunity., Author Summary Plant immunity must be tightly regulated, as over- or constitutive activation of plant defenses can cause detrimental effects, such as dwarf stature and enhanced cell death. EDR1, a Raf-like mitogen-activated protein kinase (MAPK) kinase kinase, negatively regulates defenses in Arabidopsis. The highly conserved MAPK cascades modulate diverse biological processes, including plant immunity. However, whether EDR1 affects the regulation of one of the MAPK pathways was not previously known. Here, we show that EDR1 physically associates with MKK4 and MKK5, two MAP kinase kinases, and negatively regulates the protein levels of MKK4, MKK5, MPK3 and MPK6. We further show that edr1-mediated disease resistance requires MKK4, MKK5 and MPK3 function. Over-expression of MKK4 or MKK5 in wild-type increased resistance to powdery mildew and caused mildew-induced cell death. Our study suggests that EDR1 negatively regulates defenses and directly modulates the MKK4/MKK5-MPK3/MPK6 cascade to fine-tune plant immunity.

In higher plant cytokinesis, plasma membrane and cell wall originate by vesicle fusion in the plane of cell division. The Arabidopsis KNOLLE gene, which is required for cytokinesis, encodes a protein related to vesicle-docking syntaxins. We have raised specific rabbit antiserum against purified recombinant KNOLLE protein to show biochemically and by immunoelectron microscopy that KNOLLE protein is membrane associated. Using immunofluorescence microscopy, KNOLLE protein was found to be specifically expressed during mitosis and, unlike the plasma membrane H+-ATPase, to localize to the plane of division during cytokinesis. Arabidopsis dynamin-like protein ADL1 accumulates at the plane of cell plate formation in knolle mutant cells as in wild-type cells, suggesting that cytokinetic vesicle traffic is not affected. Furthermore, electron microscopic analysis indicates that vesicle fusion is impaired. KNOLLE protein was detected in mitotically dividing cells of various parts of the developing plant, including seedling root, inflorescence meristem, floral meristems and ovules, and the cellularizing endosperm, but not during cytokinesis after the male second meiotic division. Thus, KNOLLE is the first syntaxin-like protein that appears to be involved specifically in cytokinetic vesicle fusion.

The segmentation gene hunchback (hb) is involved in setting up the anterior-posterior axis of the Drosophila embryo. It is expressed maternally and zygotically and it plays a key role in integrating the effects of the anterior and posterior maternal systems. The hb gene from D. virilis has previously been cloned and was shown to be well conserved in its coding region, but less so in its upstream region which shows a more patchy pattern of conserved and diverged sequences. This work deals with the functional conservation of hb between the two species. We have mapped two additional regulatory elements for the expression of hb in the early embryo, namely the enhancer for the maternal expression and the enhancer region for the late blastoderm expression. Fragments containing these two elements, the previously identified bicoid dependent element for the early blastoderm expression of hb and the coding region were taken from D. virilis and tested in the D. melanogaster background. We find that all enhancer elements as well as the coding region are functionally conserved between the two species. Comparison of the upstream sequences that include the enhancer region for the late blastoderm expression reveal seven highly conserved blocks. Some of these contain consensus binding sites for transregulatory factors that are likely to control the respective expression domains.

Apical-to-basal auxin flux is a defining feature of land plants and determines their main body axis. How is the axis first set up in the embryo? Recent studies reveal that the establishment of embryonic polarity with the asymmetric first division as well as the separation of shoot and root fates within the proembryo depend on transcriptional regulation in the zygote and early embryo. Although the functional connections need to be better defined, this transcriptional network likely provides the positional information required for initiating the machinery capable of processing the systemic signal auxin in a context-dependent manner.

Mitogen-activated protein kinase (MAPK) signaling networks regulate numerous eukaryotic biological processes. In Arabidopsis thaliana, signaling networks that contain MAPK kinases MKK4/5 and MAPKs MPK3/6 function in abiotic and biotic stress responses and regulate embryonic and stomatal development. However, how single MAPK modules direct specific output signals without cross-activating additional downstream processes is largely unknown. Studying relationships between MAPK components and downstream signaling outcomes is difficult because broad experimental manipulation of these networks is often lethal or associated with multiple phenotypes. Stomatal development in Arabidopsis follows a series of discrete, stereotyped divisions and cell state transitions. By expressing a panel of constitutively active MAPK kinase (MAPKK) variants in discrete stomatal lineage cell types, we identified a new inhibitory function of MKK4 and MKK5 in meristemoid self-renewal divisions. Furthermore, we established roles for MKK7 and MKK9 as both negative and (unexpectedly) positive regulators during the major stages of stomatal development. This has expanded the number of known MAPKKs that regulate stomatal development and allowed us to build plausible and testable subnetworks of signals. This in vivo cell type–specific assay can be adapted to study other protein families and thus may reveal insights into other complex signal transduction pathways in plants.

Summary Cytokinesis partitions the cytoplasm of dividing eukaryotic cells. In higher plants, a dynamic microtubule array—phragmoplast—mediates the formation of the partitioning membrane—cell plate—in a centrifugal fashion [1, 2]. This phragmoplast dynamic involves microtubule-associated proteins [3–7]. Mutations in a novel Arabidopsis gene RUNKEL ( RUK ) result in cytokinesis defects caused by abnormal phragmoplast organization and arrested cell plate expansion. RUK encodes an essential cell-cycle-regulated 152 kDa protein with a putative serine/threonine kinase domain and a large microtubule-binding domain, both of which are largely conserved in uncharacterized proteins from protozoa, plants, and animals. RUK directly bound to microtubules in vitro and colocalized with mitotic preprophase band, spindle, and phragmoplast in vivo. An engineered RUK fusion protein that was degraded before telophase did not rescue the ruk mutant phenotype, demonstrating RUK action during cytokinesis. Both microtubule-binding domain and putative kinase domain were essential for RUK function. Surprisingly, RUK did not show kinase activity in vitro, and transgenically expressed "kinase-dead" RUK rescued the seedling lethality of ruk mutants. Our results suggest that RUK plays a regulatory, rather than catalytic, role in phragmoplast microtubule organization during cell plate expansion in cytokinesis.

The anatomical terminology that is used to describe the orientation and polarity in the plant body (for distinction indicated here in italics) is based on the consensus agreement that the transition zone between the aerial and the soil-borne part (the rootshoot junction) is defined as the base of the plant, and the ends of a body axis root and shoot tips are defined as apices. For embryos, another terminology is equally well established (indicated here by underlining), which defines the root embryo pole as the base and its opposite end as the apex. Over the past few years, studies dealing with issues of cellular polarity in plants have used either anatomical or embryological terminology to describe the upper and lower side of individual cells. Recently, this confusion in terminology was highlighted, and it was proposed that the anatomical terminology should be used in all cases. However, we believe that the anatomical terminology, when used for the description of cellular polarity, has important deficiencies. Here, based on a number of arguments, we propose what we consider to be a suitable compromise.

Stomata are epidermal structures that modulate gas exchange between a plant and its environment. During development, stomata are specified and positioned nonrandomly by the integration of asymmetric cell divisions and intercellular signaling. The Arabidopsis mitogen-activated protein kinase kinase kinase gene, YODA , acts as part of a molecular switch controlling cell identities in the epidermis. Null mutations in YODA lead to excess stomata, whereas constitutive activation of YODA eliminated stomata. Transcriptome analysis of seedlings with altered YODA activity was used to identify potential stomatal regulatory genes. A putative transcription factor from this set was shown to regulate the developmental behavior of stomatal precursors.

Distinct forms of cytokinesis characterise specific phases of development in plants. In Arabidopsis, as in many other species, the endosperm that nurtures the embryo in the seed initially develops as a syncytium. This syncytial phase ends with simultaneous partitioning of the multinucleate cytoplasm into individual cells, a process referred to as cellularisation. Our in vivo observations show that, as in cytokinesis, cellularisation of theArabidopsis endosperm is coupled to nuclear division. A genetic analysis reveals that most Arabidopsis mutations affecting cytokinesis in the embryo also impair endosperm cellularisation. These results imply that cellularisation and cytokinesis share multiple components of the same basic machinery. We further report the identification of mutations in a novel gene, SPÄTZLE, that specifically interfere with cellularisation of the endosperm, but not with cytokinesis in the embryo. The analysis of this mutant might identify a specific checkpoint for the onset of cellularisation.

Arabidopsis cyt1 mutants have a complex phenotype indicative of a severe defect in cell wall biogenesis. Mutant embryos arrest as wide, heart-shaped structures characterized by ectopic accumulation of callose and the occurrence of incomplete cell walls. Texture and thickness of the cell walls are irregular, and unesterified pectins show an abnormally diffuse distribution. To determine the molecular basis of these defects, we have cloned the CYT1 gene by a map-based approach and found that it encodes mannose-1-phosphate guanylyltransferase. A weak mutation in the same gene, called vtc1 , has previously been identified on the basis of ozone sensitivity due to reduced levels of ascorbic acid. Mutant cyt1 embryos are deficient in N-glycosylation and have an altered composition of cell wall polysaccharides. Most notably, they show a 5-fold decrease in cellulose content. Characteristic aspects of the cyt1 phenotype, including radial swelling and accumulation of callose, can be mimicked with the inhibitor of N-glycosylation, tunicamycin. Our results suggest that N-glycosylation is required for cellulose biosynthesis and that a deficiency in this process can account for most phenotypic features of cyt1 embryos.

Partitioning of the cytoplasm during cytokinesis or cellularisation requires syntaxin-mediated membrane fusion [1–3]. Whereas in animals, membrane fusion promotes ingression of a cleavage furrow from the plasma membrane [4,5], somatic cells of higher plants form de novo a transient membrane compartment, the cell plate, which is initiated in the centre of the division plane and matures into a new cell wall and its flanking plasma membranes [6,7]. Cell plate formation results from the fusion of Golgi-derived vesicles delivered by a dynamic cytoskeletal array, the phragmoplast. Mutations in two Arabidopsis genes, KNOLLE ( KN ) and KEULE ( KEU ), cause abnormal seedlings with multinucleate cells and incomplete cell walls [1,8]. The KN gene encodes a cytokinesis-specific syntaxin which localises to the cell plate [9]. Here, we show that KN protein localisation is unaffected in keu mutant cells, which, like kn , display phragmoplast microtubules and accumulate ADL1 protein in the plane of cell division but vesicles fail to fuse with one another. Genetic interactions between KN and KEU were analysed in double mutant embryos. Whereas the haploid gametophytes gave rise to functional gametes, the embryos behaved like single cells displaying multiple, synchronously cycling nuclei, cell cycle-dependent microtubule arrays and ADL1 accumulation between pairs of daughter nuclei. This complete inhibition of cytokinesis from fertilisation indicates that KN and KEU , have partially redundant functions and interact specifically in vesicle fusion during cytokinesis of somatic cells.

The embryo of the flowering plant Arabidopsis develops by a regular pattern of cell divisions and cell shape changes. Mutations in the KNOLLE (KN) gene affect the rate and plane of cell divisions as well as cell morphology, resulting in mutant seedlings with a disturbed radial organization of tissue layers. At the cellular level, mutant embryos are characterized by incomplete cross walls and enlarged cells with polyploid nuclei. The KN gene was isolated by positional cloning. The predicted KN protein has similarity to syntaxins, a protein family involved in vesicular trafficking. During embryogenesis, KN transcripts are detected in patches of single cells or small cell groups. Our results suggest a function for KN in cytokinesis.

During embryogenesis, a single cell gives rise to different cell types, tissues and organs which are arranged in a biologically meaningful context, or pattern. The resulting basic body organization of higher plants, which is expressed in the seedling, provides a reference system for postembryonic development during which the meristems of the shoot and the root produce the adult body. The seedling may be viewed as the superimposition of two patterns: one along the apical-basal axis of polarity and the other perpendicular to the axis. To analyse mechanisms underlying pattern formation in the embryo, a genetic approach has been taken inArabidopsis. Mutations in a small number of genes alter one or the other of the two patterns. The mutant phenotypes suggest that early partitioning of the axis is followed by region-specific development, including the formation of the primary shoot and root meristems. The cloning of two genes involved in pattern formation provides a basis for mechanistic studies of how cells adopt specific fates in the developing embryo.

Plant cytokinesis starts in the center of the division plane, with vesicle fusion generating a new membrane compartment, the cell plate, that subsequently expands laterally by continuous fusion of newly arriving vesicles to its margin [1–4]. Targeted delivery of vesicles is assisted by the dynamic reorganization of a plant-specific cytoskeletal array, the phragmoplast, from a solid cylinder into an expanding ring-shaped structure [5]. This lateral translocation is brought about by depolymerization of microtubules in the center, giving way to the expanding cell plate, and polymerization of microtubules along the edge [6, 7]. Whereas several components are known to mediate cytokinetic vesicle fusion [8–10], no gene function involved in phragmoplast dynamics has been identified by mutation. Mutations in the Arabidopsis HINKEL gene cause cytokinesis defects, such as enlarged cells with incomplete cell walls and multiple nuclei. Proper targeting of the cytokinesis-specific syntaxin KNOLLE [8] and lateral expansion of the phragmoplast are not affected. However, the phragmoplast microtubules appear to persist in the center, where vesicle fusion should result in cell plate formation. Molecular analysis reveals that the HINKEL gene encodes a plant-specific kinesin-related protein with a putative N-terminal motor domain and is expressed in a cell cycle-dependent manner similar to the KNOLLE gene. Our results suggest that HINKEL plays a role in the reorganization of phragmoplast microtubules during cell plate formation.

hunchback (hb) is a key regulatory gene in the early segmentation gene hierarchy of Drosophila. It codes for a transcription factor of the Cys2-His2 zinc finger type and shows two separate zinc finger domains in its coding region. hb forms a morphogenetic gradient in the middle of the embryo that is required for setting the spatial boundaries of several target genes. We have analyzed the molecular lesions found in the different hb alleles and have studied the differential effects of these alleles on a number of such target genes. We find that in mutants in which the HB protein lacks a functional second finger domain, the regulation of the target genes Krüppel (Kr) and knirps (kni) is differentially affected. While this domain is required for the correct regulation of Kr, it is not necessary for the repression of kni. Furthermore, mutations affecting this domain lead to a decreased protein stability. The integration of the expression pattern of target genes was found to be distorted in a second class of mutants between the two finger domains which lead to gain of function or neomorphic phenotypes. The effects of these mutations were studied in detail and it was found that they fall into two classes, the first one interfering with the function of the maternal hb product, the second leading to a delayed segmentation. The function of the latter class appears to be linked to the secondary expression of hb in the parasegment 4 (PS4) stripe at blastoderm stage.

The Arabidopsis embryo establishes polarity and main tissue types with the first five rounds of cell division. In this issue of Developmental Cell, Gooh et al. (2015) provide tools toward elucidating this poorly understood process through the first movies and targeted manipulations of early embryos developing inside cultured seeds.