CRISPR/Cas9 mutagenesis of the Arabidopsis GROWTH-REGULATING FACTOR (GRF) gene family.

Genome editing in plants typically relies on T-DNA plasmids that are mobilized by Agrobacterium-mediated transformation to deliver the CRISPR/Cas machinery. Here, we introduce a series of CRISPR/Cas9 T-DNA vectors for minimal settings, such as teaching labs. Gene-specific targeting sequences can be inserted as annealed short oligonucleotides in a single straightforward cloning step. Fluorescent markers expressed in mature seeds enable reliable selection of transgenic or 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) genes, we were able to create a collection of predicted null mutations in all nine family members with little effort. We then explored the effects of simultaneously targeting two, four and eight GRF genes on the rate of induced mutations at each target locus. In our hands, multiplexing was associated with pronounced disparities: while mutation rates at some loci remained consistently high, mutation rates at other loci dropped dramatically with increasing number of single guide RNA species, thereby preventing a systematic mutagenesis of the family.

A game of hide and peek.

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.

EMS Mutagenesis of Arabidopsis Seeds.

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.

Genetic Screens to Target Embryo and Endosperm Pathways in Arabidopsis and Maize.

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.

Constitutive signaling activity of a receptor-associated protein links fertilization with embryonic patterning in Arabidopsis thaliana.

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.

Maternal control of embryogenesis by MPK6 and its upstream MKK4/MKK5 in Arabidopsis.

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.

Regulation of Stomatal Immunity by Interdependent Functions of a Pathogen-Responsive MPK3/MPK6 Cascade and Abscisic Acid.

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 PLETHORA Gene Regulatory Network Guides Growth and Cell Differentiation in Arabidopsis Roots.

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.

Going mainstream: How is the body axis of plants first initiated in the embryo?

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.

A Genetic Screen for Mutations Affecting Cell Division in the Arabidopsis thaliana Embryo Identifies Seven Loci Required for Cytokinesis.

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.

Embryos, camera, laser, action!

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.

Downregulation of GAUT12 in Populus deltoides by RNA silencing results in reduced recalcitrance, increased growth and reduced xylan and pectin in a woody biofuel feedstock.

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.

Loss of Arabidopsis GAUT12/IRX8 causes anther indehiscence and leads to reduced G lignin associated with altered matrix polysaccharide deposition.

GAlactUronosylTransferase12 (GAUT12)/IRregular Xylem8 (IRX8) is a putative glycosyltransferase involved in Arabidopsis secondary cell wall biosynthesis. Previous work showed that Arabidopsis irregular xylem8 (irx8) mutants have collapsed xylem due to a reduction in xylan and a lesser reduction in a subfraction of homogalacturonan (HG). We now show that male sterility in the irx8 mutant is due to indehiscent anthers caused by reduced deposition of xylan and lignin in the endothecium cell layer. The reduced lignin content was demonstrated by histochemical lignin staining and pyrolysis Molecular Beam Mass Spectrometry (pyMBMS) and is associated with reduced lignin biosynthesis in irx8 stems. Examination of sequential chemical extracts of stem walls using 2D (13)C-(1)H Heteronuclear Single-Quantum Correlation (HSQC) NMR spectroscopy and antibody-based glycome profiling revealed a reduction in G lignin in the 1 M KOH extract and a concomitant loss of xylan, arabinogalactan and pectin epitopes in the ammonium oxalate, sodium carbonate, and 1 M KOH extracts from the irx8 walls compared with wild-type walls. Immunolabeling of stem sections using the monoclonal antibody CCRC-M138 reactive against an unsubstituted xylopentaose epitope revealed a bi-lamellate pattern in wild-type fiber cells and a collapsed bi-layer in irx8 cells, suggesting that at least in fiber cells, GAUT12 participates in the synthesis of a specific layer or type of xylan or helps to provide an architecture framework required for the native xylan deposition pattern. The results support the hypothesis that GAUT12 functions in the synthesis of a structure required for xylan and lignin deposition during secondary cell wall formation.

EDR1 physically interacts with MKK4/MKK5 and negatively regulates a MAP kinase cascade to modulate plant innate immunity.

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.

Different auxin response machineries control distinct cell fates in the early plant embryo.

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.

Axis formation in Arabidopsis - transcription factors tell their side of the story.

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.

The RWP-RK factor GROUNDED promotes embryonic polarity by facilitating YODA MAP kinase signaling.

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.

Taking the very first steps: from polarity to axial domains in the early Arabidopsis embryo.

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.

The GATA factor HANABA TARANU is required to position the proembryo boundary in the early Arabidopsis embryo.

Division 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.

Novel and expanded roles for MAPK signaling in Arabidopsis stomatal cell fate revealed by cell type-specific manipulations.

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.