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International audience; Production of in planta haploid embryos that inherit chromosomes from only one parent can greatly increase breeding efficiency via quickly generating homozygous plants, called doubled haploid. One of the main players of in planta haploid induction is a pollen-specific phospholipase A, which is able, when mutated, to induce in vivo haploid induction in numerous monocots. However, no functional orthologous gene has been identified in dicots plants. Here, we show that loss-of-function of gynoecium-expressed phospholipase AII (pPLAIIγ) triggers maternal haploid plants in Arabidopsis, at an average rate of 1.07%. Reciprocal crosses demonstrate that haploid plants are triggered from the female side and not from the pollen, and the haploid plants carry the maternal genome. Promoter activity of pPLAIIγ shows enriched expression in the funiculus of flower development stages 13 and 18, and pPLAIIγ fused to yellow fluorescent protein reveals a plasma-membrane localization Interestingly, the polar localized PIN1 at the basal plasma membrane of the funiculus was all internalized in pplaIIγ mutants, suggesting that altered PIN1 localization in female organ could play a role in maternal haploid induction.

Pollen-specific phospholipase A-mediated in vivo haploid induction system was functionally well conserved in numerous monocots. However, no functional orthologous gene has been identified in dicots plants. Here, we show that loss-of-function of gynoecium-expressed phospholipase AII (pPLAIIγ) triggers haploid plants in Arabidopsis, at an average rate of 1.46%. Reciprocal crosses demonstrate that haploid plants are triggered from the female side and not from the pollen, and the haploid plants carry the maternal genome.

Mixing maternal and paternal genomes in embryos is not only responsible for the evolutionary success of sexual reproduction, but is also a cornerstone of plant breeding. However, once an interesting gene combination is obtained, further genetic mixing is problematic. To rapidly fix genetic information, doubled haploid plants can be produced: haploid embryos having solely the genetic information from one parent are allowed to develop, and chromosome doubling generates fully homozygous plants. A powerful path to the production of doubled haploids is based on haploid inducer lines. A simple cross between a haploid inducer line and the line with gene combinations to be fixed will trigger haploid embryo development. However, the exact mechanism behind in planta haploid induction remains an enduring mystery. The recent discoveries of molecular actors triggering haploid induction in the maize crop and the model Arabidopsis thaliana pinpoint an essential role of processes related to gamete development, gamete interactions and genome stability. These findings enabled translation of haploid induction capacity to other crops as well as the use of haploid inducer lines to deliver genome editing machinery into various crop varieties. These recent advances not only hold promise for the next generations of plant breeding strategies, but they also provide a deeper insight into the fundamental bases of sexual reproduction in plants. This Perspective summarizes recent advances made on in planta haploid induction systems and how these advances contribute to our understanding of plant reproduction and innovations of plant breeding.

Doubled haploid technology has been widely applied to multiple plant species and is recognized as one of the most important technologies for improving crop breeding efficiency. Although mutations in MATRILINEAL/Zea mays PHOSPHOLIPASE A1/NOT LIKE DAD (MTL/ZmPLA1/NLD) and Zea mays DOMAIN OF UNKNOWN FUNCTION 679 MEMBRANE PROTEIN (ZmDMP) have been shown to generate haploids in maize, knowledge of the genetic basis of haploid induction (HI) remains incomplete. Therefore, cloning of new genes underlying HI is important for further elucidating its genetic architecture. Here, we found that loss-of-function mutations of Zea mays PHOSPHOLIPASE D3 (ZmPLD3), one of the members from the phospholipase D subfamily, could trigger maternal HI in maize. ZmPLD3 was identified through a reverse genetic strategy based on analysis of pollen-specifically expressed phospholipases, followed by validation through the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR–Cas9) system. Mutations of ZmPLD3 resulted in a haploid induction rate (HIR) similar to that of mtl/zmpla1/nld and showed synergistic effects rather than functional redundancy on tripling the HIR (from 1.19% to 4.13%) in the presence of mtl/zmpla1/nld. RNA-seq profiling of mature pollen indicated that a large number of pollen-specific differentially expressed genes were enriched in processes related to gametogenesis development, such as pollen tube development and cell communication, during the double-fertilization process. In addition, ZmPLD3 is highly conserved among cereals, highlighting the potential application of these in vivo haploid-inducer lines for other important crop plant species. Collectively, our discovery identifies a novel gene underlying in vivo maternal HI and provides possibility of breeding haploid inducers with further improved HIR., This study revealed a novel regulator, ZmPLD3, for maternal haploid induction in maize. Loss-of-function mutations of ZmPLD3 showed an induction rate similar to that of mtl/zmpla1/nld and induced haploids in a synergistic manner with the mtl mutations.

International audience; Mixing maternal and paternal genomes is the base of plant sexual reproduction, but some so-called 'haploid inducer lines' lead to the formation of seeds bearing well-developed embryos with solely the maternal genome. A recent study adds a new piece to the puzzle of this enigmatic in planta haploid embryo induction process.

International audience; Phospholipases cleave phospholipids, major membrane constituents. They are thus essential for many developmental processes, including male gamete development. In flowering plants, mutation of phospholipase NOT-LIKE-DAD (NLD, also known as MTL or ZmPLA1) leads to peculiar defects in sexual reproduction, notably the induction of maternal haploid embryos. Contrary to previous reports, NLD does not localize to cytosol and plasma membrane of sperm cells but to the pollen endo-plasma membrane (endo-PM), a specific membrane derived from the PM of the pollen vegetative cell that encircles the two sperm cells. After pollen tube burst, NLD localizes at the apical region of the egg apparatus. Pharmacological approaches coupled with targeted mutagenesis revealed that lipid anchoring together with electrostatic interactions are involved in the attachment of NLD to this atypical endo-PM. Membrane surface-charge and lipid biosensors indicated that phosphatidylinositol-4,5-bisphosphate is enriched in the endo-PM, uncovering a unique example of how membrane electrostatic properties can define a specific polar domain (i.e., endo-PM), which is critical for plant reproduction and gamete formation.

International audience; Doubled haploids (DH) have become a powerful tool to assist in different basic research studies, and also in applied research. The principal (but not the only) and routine use of DH by breeding companies is to produce pure lines for hybrid seed production in different crop species. Several decades after the discovery of haploid inducer lines in maize and of anther culture as a method to produce haploid plants from pollen precursors, the biotechnological revolution of the last decades allowed to the development of a variety of approaches to pursue the goal of doubled haploid production. Now, it is possible to produce haploids and DHs in many different species, because when a method does not work properly, there are several others to test. In this chapter, we overview the currently available approaches used to produce haploids and DHs by using methods based on in vitro culture, or involving the in vivo induction of haploid embryo development, or a combination of both.

Doubled haploids (DH) have become a powerful tool to assist in different basic research studies, and also in applied research. The principal (but not the only) and routine use of DH by breeding companies is to produce pure lines for hybrid seed production in different crop species. Several decades after the discovery of haploid inducer lines in maize and of anther culture as a method to produce haploid plants from pollen precursors, the biotechnological revolution of the last decades allowed to the development of a variety of approaches to pursue the goal of doubled haploid production. Now, it is possible to produce haploids and DHs in many different species, because when a method does not work properly, there are several others to test. In this chapter, we overview the currently available approaches used to produce haploids and DHs by using methods based on in vitro culture, or involving the in vivo induction of haploid embryo development, or a combination of both.

Doubled haploid (DH) technology produces strictly homozygous fertile plant thanks to doubling the chromosomes of a haploid embryo/seedling. Haploid embryos are derived from either male or female germ line cells and hold only half the number of chromosomes found in somatic plant tissues, albeit in a recombinant form due to meiotic genetic shuffling. DH production allows to rapidly fix these recombinant haploid genomes in the form of perfectly homozygous plants (inbred lines), which are produced in two rather than six or more generations. Thus, DH breeding enables fast evaluation of phenotypic traits on homogenous progeny. While for most crops haploid embryos are produced by costly and often genotype-dependent in vitro methods, for maize, two unique in planta systems are available to induce haploid embryos directly in the seed. Two "haploid inducer lines", identified from spontaneous maize mutants, are able to induce embryos of paternal or maternal origin. Although effortless crosses with lines of interest are sufficient to trigger haploid embryos, substantial improvements were necessary to bring DH technology to large scale production. They include the development of modern haploid inducer lines with high induction rates (8-12%), and methods to sort kernels with haploid embryos from the normal ones. Chromosome doubling represents also a crucial step in the DH process. Recent identification of genomic loci involved in spontaneous doubling opens up perspectives for a fully in planta DH pipeline in maize. Although discovered more than 60 years ago, maize haploid inducer lines still make headlines thanks to novel applications and findings. Indeed, maternal haploid induction was elegantly diverted to deliver genome editing machinery in germplasm recalcitrant to transformation techniques. The recent discovery of two molecular players controlling haploid induction allowed to revisit the mechanistic basis of maize maternal haploid induction and to successfully translate haploid induction ability to other crops.

International audience; Doubled haploid (DH) technology produces strictly homozygous fertile plant thanks to doubling the chromosomes of a haploid embryo/seedling. Haploid embryos are derived from either male or female germ line cells and hold only half the number of chromosomes found in somatic plant tissues, albeit in a recombinant form due to meiotic genetic shuffling. DH production allows to rapidly fix these recombinant haploid genomes in the form of perfectly homozygous plants (inbred lines), which are produced in two rather than six or more generations. Thus, DH breeding enables fast evaluation of phenotypic traits on homogenous progeny. While for most crops haploid embryos are produced by costly and often genotype-dependent in vitro methods, for maize, two unique in planta systems are available to induce haploid embryos directly in the seed. Two "haploid inducer lines", identified from spontaneous maize mutants, are able to induce embryos of paternal or maternal origin. Although effortless crosses with lines of interest are sufficient to trigger haploid embryos, substantial improvements were necessary to bring DH technology to large scale production. They include the development of modern haploid inducer lines with high induction rates (8-12%), and methods to sort kernels with haploid embryos from the normal ones. Chromosome doubling represents also a crucial step in the DH process. Recent identification of genomic loci involved in spontaneous doubling opens up perspectives for a fully in planta DH pipeline in maize. Although discovered more than 60 years ago, maize haploid inducer lines still make headlines thanks to novel applications and findings. Indeed, maternal haploid induction was elegantly diverted to deliver genome editing machinery in germplasm recalcitrant to transformation techniques. The recent discovery of two molecular players controlling haploid induction allowed to revisit the mechanistic basis of maize maternal haploid induction and to successfully translate haploid induction ability to other crops.

Phospholipases are ubiquitous enzymes that cleave phospholipids, one major constituent of membranes. They are thus essential for many developmental processes, including male gamete development. In flowering plants, mutation of phospholipase NOT-LIKE-DAD (NLD) leads to peculiar defects in sexual reproduction. Indeed, pollination of a wild-type female with mutant pollen generates haploid embryos containing solely maternal genetic information. Contrary to previous reports NLD does not localize to cytosol and plasma membrane (PM) of sperm cells but to the pollen endo-plasma membrane (endo-PM), a specific membrane derived from the PM of the pollen vegetative cell that encircles the two sperm cells. Pharmacological approaches coupled with targeted mutagenesis revealed that lipid anchoring together with electrostatic interactions between membrane and NLD are involved in the attachment of NLD to this atypical endo-PM. Membrane surface-charge and anionic lipid bio-sensors indicated that phosphatidylinositol-4,5-bisphosphate (PIP(4,5)P2) is enriched in the endo-PM as compared to the PM. Our results uncover a unique example of how membrane electrostatic properties can specify a unique polar domain (i.e. endo-PM), which is critical for plant reproduction and gamete formation.

International audience; A population of endosperm cells adjacent to the embryo scutellum shows transcriptomic enrichment in transport functions and is influenced by embryo development.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.

Filling in the gaps In a plant seed, the embryo lies dormant surrounded by nutritive endosperm while awaiting suitable conditions to germinate. A hydrophobic cuticle around the embryo protects it from catastrophic water loss during the early days of growth. Doll et al. identified a back-and-forth signaling pathway that ensures an intact cuticle. The precursor of a signaling peptide is made in the embryo and transferred to the endosperm, where it is processed into an active form. The activated peptide diffuses back into the embryo to activate receptor-like kinases that drive cuticle development. Serve and return continues until all leaks in the cuticle are filled in and the peptide can no longer cross the barrier. Science , this issue p. 431

International audience; 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.

International audience; Introduce the technique of haploid induction in plant breeding.

The plant embryonic cuticle is a hydrophobic barrier deposited de novo by the embryo during seed development. At germination it protects the seedling from water loss and is thus critical for survival. Embryonic cuticle formation is controlled by a signaling pathway involving the protease ALE1 and the receptor-like kinases GSO1 and GSO2. We show that a sulfated peptide, TWISTED SEED1 (TWS1) acts as a GSO1/GSO2 ligand. Cuticle surveillance depends on the action of ALE1 which, unlike TWS1 and GSO1/2, is not produced in the embryo but in the neighboring endosperm. Cleavage of an embryo-derived TWS1 precursor by ALE1 releases the active peptide, triggering GSO1/2-dependent cuticle reinforcement in the embryo. A bidirectional molecular dialogue between embryo and endosperm thus safeguards cuticle integrity prior to germination.One Sentence SummarySubtilase-mediated activation of the TWISTED SEED1 peptide provides spatial cues during embryo cuticle integrity monitoring.

Summary Germination sensu stricto in Arabidopsis involves seed-coat and endosperm rupture by the emerging seedling root. Subsequently, the cotyledons emerge rapidly from the extra-embryonic tissues of the seed, allowing autotrophic seedling establishment [ 1 , 2 ]. Seedling survival depends upon the presence of an intact seedling cuticle that prevents dehydration, which has hitherto been assumed to form the interface between the newly germinated seedling and its environment [ 3 , 4 , 5 ]. Here, we show that in Arabidopsis, this is not the case. The primary interface between the emerging seedling and its environment is formed by an extra-cuticular endosperm-derived glycoprotein-rich structure called the sheath, which is maintained as a continuous layer at seedling surfaces during germination and becomes fragmented as cotyledons expand. Mutants lacking an endosperm-specific cysteine-rich peptide (KERBEROS [KRS]) show a complete loss of sheath production [ 6 ]. Although krs mutants have no defects in germination sensu stricto, they show delayed cotyledon emergence, a defect not observed in seedlings with defects in cuticle biosynthesis. Biophysical analyses reveal that the surfaces of wild-type cotyledons show minimal adhesion to silica beads in an aqueous environment at cotyledon emergence but that adhesion increases as cotyledons expand. In contrast, krs mutant cotyledons show enhanced adhesion at germination. Mutants with defects in cuticle biosynthesis, but no sheath defects, show a similar adhesion profile to wild-type seedlings at germination. We propose that the sheath reduces the adhesiveness of the cotyledon surface under the humid conditions necessary for seed germination and thus promotes seed-coat shedding and rapid seedling establishment.

The embryonic cuticle is necessary for normal seed development and seedling establishment in Arabidopsis. Although mutants with defective embryonic cuticles have been identified, neither the deposition of cuticle material, nor its regulation, has been described during embryogenesis. Here we use electron microscopy, lipid staining and permeability assays to show that cuticle deposition initiatesde novoin patches on globular embryos. By combining these techniques with genetics and gene expression analysis, we show that successful patch coalescence to form a continuous cuticle requires a signalling involving the endosperm-specific subtilisin protease ALE1 and the receptor kinases GSO1 and GSO2, which are expressed in the developing embryonic epidermis. Transcriptome analysis shows that this pathway regulates stress-related gene expression in seeds. Consistent with these findings we show genetically, and through activity analysis, that the stress-associated MPK6 protein acts downstream of GSO1 and GSO2 in the developing embryo. We propose that a stress-related signalling pathway has been hijacked in some angiosperm seeds through the recruitment of endosperm-specific components. Our work reveals the presence of an inter-compartmental dialogue between the endosperm and embryo that ensures the formation of an intact and functional cuticle around the developing embryo through an “auto-immune” type interaction.

et al., The draft genome of the moss model, Physcomitrella patens, comprised approximately 2000 unordered scaffolds. In order to enable analyses of genome structure and evolution we generated a chromosome-scale genome assembly using genetic linkage as well as (end) sequencing of long DNA fragments. We find that 57% of the genome comprises transposable elements (TEs), some of which may be actively transposing during the life cycle. Unlike in flowering plant genomes, gene- and TE-rich regions show an overall even distribution along the chromosomes. However, the chromosomes are mono-centric with peaks of a class of Copia elements potentially coinciding with centromeres. Gene body methylation is evident in 5.7% of the protein-coding genes, typically coinciding with low GC and low expression. Some giant virus insertions are transcriptionally active and might protect gametes from viral infection via siRNA mediated silencing. Structure-based detection methods show that the genome evolved via two rounds of whole genome duplications (WGDs), apparently common in mosses but not in liverworts and hornworts. Several hundred genes are present in colinear regions conserved since the last common ancestor of plants. These syntenic regions are enriched for functions related to plant-specific cell growth and tissue organization. The P. patens genome lacks the TE-rich pericentromeric and gene-rich distal regions typical for most flowering plant genomes. More non-seed plant genomes are needed to unravel how plant genomes evolve, and to understand whether the P. patens genome structure is typical for mosses or bryophytes., The work conducted by the US Department of Energy Joint Genome Institute is supported by the Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231. Support to RR and SAR by the German Research Foundation (DFG RE 837/10-2), the Excellence Initiative of the German Federal and State Governments (EXC 294), and by the German Federal Ministry of Education and Research (BMBF FRISYS), is highly appreciated. CoGe is supported by the US National Science Foundation under Award Numbers IOS-339156 and IOS-1444490, CyVerse is supported by the U.S. National Science Foundation under Award Numbers DBI-0735191 and DBI-1265383. YK and ACC are grateful for support from the UK Biological Sciences and Biotechnology Research Council (Grant BB/F001797/1). KV acknowledges the Multidisciplinary Research Partnership ‘Bioinformatics: from nucleotides to networks’ Project (no 01MR0410W) of Ghent University. JC is grateful for support from the Spanish Ministerio de Economía y Competitividad (Grant AGL2013-43244-R). RSQ is grateful to Monsanto (St. Louis, MO, USA) for sequencing genomic DNA of P. patens accession Kaskaskia. The 1000 Plants (1 KP) initiative, led by GKSW, is funded by the Alberta Ministry of Innovation and Advanced Education, Alberta Innovates Technology Futures (AITF), Innovates Centres of Research Excellence (iCORE), Musea Ventures, BGI-Shenzhen and China National Genebank (CNGB). TW was supported by EMBO Long-Term Fellowships (ALTF 1166-2011) and by Marie Curie Actions (European Commission EMBOCOFUND2010, GA-2010-267146). The work conducted at PGSB was supported by the German Research Foundation (SFB924) and German Ministry of Education and Research (BMBF, 031A536/de.NBI).

Gynogenesis is an asexual mode of reproduction common to animals and plants, in which stimuli from the sperm cell trigger the development of the unfertilized egg cell into a haploid embryo. Fine mapping restricted a major maize QTL (quantitative trait locus) responsible for the aptitude of inducer lines to trigger gynogenesis to a zone containing a single gene NOT LIKE DAD ( NLD ) coding for a patatin‐like phospholipase A. In all surveyed inducer lines, NLD carries a 4‐bp insertion leading to a predicted truncated protein. This frameshift mutation is responsible for haploid induction because complementation with wild‐type NLD abolishes the haploid induction capacity. Activity of the NLD promoter is restricted to mature pollen and pollen tube. The translational NLD::citrine fusion protein likely localizes to the sperm cell plasma membrane. In Arabidopsis roots, the truncated protein is no longer localized to the plasma membrane, contrary to the wild‐type NLD protein. In conclusion, an intact pollen‐specific phospholipase is required for successful sexual reproduction and its targeted disruption may allow establishing powerful haploid breeding tools in numerous crops. ![][1] The function of the patatin‐like phospholipase A NOT LIKE DAD (NLD) in the sperm cells of maize pollen is necessary for successful fertilization, whereas its disruption promotes the development of haploid embryos, which represent an important plant breeding tool. [1]: /embed/graphic-1.gif

Developing the next plant generation within the seed requires the coordination of complex programs driving pattern formation, growth, and differentiation of the three main seed compartments: the embryo (future plant), the endosperm (storage compartment), representing the two filial tissues, and the surrounding maternal tissues. This review focuses on the signaling pathways and molecular players involved in early maize kernel development. In the 2 weeks following pollination, functional tissues are shaped from single cells, readying the kernel for filling with storage compounds. Although the overall picture of the signaling pathways regulating embryo and endosperm development remains fragmentary, several types of molecular actors, such as hormones, sugars, or peptides, have been shown to be involved in particular aspects of these developmental processes. These molecular actors are likely to be components of signaling pathways that lead to transcriptional programming mediated by transcriptional factors. Through the integrated action of these components, multiple types of information received by cells or tissues lead to the correct differentiation and patterning of kernel compartments. In this review, recent advances regarding the four types of molecular actors (hormones, sugars, peptides/receptors, and transcription factors) involved in early maize development are presented.

Arabidopsis thaliana seed development requires the concomitant development of two zygotic compartments, the embryo and the endosperm. Following fertilization, the endosperm expands and the embryo grows invasively through the endosperm, which breaks down. Here, we describe a structure we refer to as the embryo sheath that forms on the surface of the embryo as it starts to elongate. The sheath is deposited outside the embryonic cuticle and incorporates endosperm-derived material rich in extensin-like molecules. Sheath production is dependent upon the activity of ZHOUPI, an endosperm-specific transcription factor necessary for endosperm degradation, embryo growth, embryo-endosperm separation, and normal embryo cuticle formation. We show that the peptide KERBEROS, whose expression is ZHOUPI dependent, is necessary both for the formation of a normal embryo sheath and for embryo-endosperm separation. Finally, we show that the receptor-like kinases GSO1 and GSO2 are required for sheath deposition at the embryo surface but not for production of sheath material in the endosperm. We present a model in which sheath formation depends on the coordinated production of material in the endosperm and signaling within the embryo, highlighting the complex molecular interaction between these two tissues during early seed development.

Maize seeds, like those of all other angiosperms, are highly complex biological systems. This complexity is a consequence of the fact that the angiosperm seed is composed of tissues that evolved from three genetically distinct organisms: the mother plant (maternal sporophyte-specifically the nucellus, integuments, and in the case of maize and other cereals, other floral organs that fuse with the integuments to form the pericarp); the developing embryo (zygotic sporophyte); and the endosperm (arising through fertilization-dependent proliferation of a second fertilization competent cell of the female gametophyte). In evolutionary terms, the two main interactions that will be addressed in this chapter initially arose well before emergence of the angiosperms. The first and probably most ancient association is "retention and nourishment" of the sporophytic embryo by the female gametophyte. The second association is derived from the "retention of the megaspore" by the maternal sporophyte, and the subsequent extended interaction of maternal tissues (the nucellus and integuments) with the developing female gametophyte (and subsequently, in angiosperms, the endosperm). This chapter addresses interactions that occur at compartment interfaces. After a short description of their structures, it focuses mainly on three aspects: (i) the potential role of signalling peptides (and other mobile substances) and their receptors as signalling components in cell fate decisions and intercompartmental communication; (ii) the role of sugars both as nutrients and as signalling molecules; and (iii) the role and control of cell death processes as a consequence of physical interactions and as a potential source of signalling molecules for intercompartmental communication.

International audience; Caffeoyl CoA O-methyltransferases (OMTs) have been characterized from numerous plant species and have been demonstrated to be involved in lignin biosynthesis. Higher plant species are known to have additional caffeoyl CoA OMT-like genes, which have not been well characterized. Here, we identified two new caffeoyl CoA OMT-like genes by screening a cDNA library from specialized hair cells of pods of the orchid Vanilla planifolia. Characterization of the corresponding two enzymes, designated Vp-OMT4 and Vp-OMT5, revealed that in vitro both enzymes preferred as a substrate the flavone tricetin, yet their sequences and phylogenetic relationships to other enzymes are distinct from each other. Quantitative analysis of gene expression indicated a dramatic tissue-specific expression pattern for Vp-OMT4, which was highly expressed in the hair cells of the developing pod, the likely location of vanillin biosynthesis. Although Vp-OMT4 had a lower activity with the proposed vanillin precursor, 3,4-dihydroxybenzaldehyde, than with tricetin, the tissue specificity of expression suggests it may be a candidate for an enzyme involved in vanillin biosynthesis. In contrast, the Vp-OMT5 gene was mainly expressed in leaf tissue and only marginally expressed in pod hair cells. Phylogenetic analysis suggests Vp-OMT5 evolved from a cyanobacterial enzyme and it clustered within a clade in which the sequences from eukaryotic species had predicted chloroplast transit peptides. Transient expression of a GFP-fusion in tobacco demonstrated that Vp-OMT5 was localized in the plastids. This is the first flavonoid OMT demonstrated to be targeted to the plastids.

The Arabidopsis thaliana AtNRT2.1 gene, which encodes a NO(3)(-) transporter involved in high-affinity uptake by the roots, is a molecular target of several mechanisms responsible for the regulation of root NO(3)(-) acquisition by the N status of the plant. All levels of AtNRT2.1 expression (promoter activity, transcript level, protein accumulation, transport activity) are coordinately up-regulated in the presence of NO(3)(-), and repressed by downstream N metabolites. Transgenic plants expressing the GUS reporter gene under the control of upstream sequences of AtNRT2.1 have been studied to identify elements targeted by these two regulatory mechanisms. A 150 bp sequence located upstream of the TATA box that is required for both stimulation by NO(3)(-) and repression by N metabolites of the promoter has been identified. This sequence is able to confer these two regulations to a minimal promoter. Split-root experiments indicate that the stimulation of the chimaeric promoter by NO(3)(-) occurs only at the local level, whereas its repression by N metabolites is mediated by a systemic signal spread to the whole plant. The activity of the cis-acting 150 bp element is also regulated by sucrose supply to the roots, suggesting a possible interaction between N and C signalling within this short region. Accordingly, multiple motifs potentially involved in regulations by N and/or C status are identified within this sequence by bioinformatic approaches. This is the first report of such a cis-acting element in higher plants.

Background Despite increasing demand, imaging the internal structure of plant organs or tissues without the use of transgenic lines expressing fluorescent proteins remains a challenge. Techniques such as magnetic resonance imaging, optical projection tomography or X-ray absorption tomography have been used with various success, depending on the size and physical properties of the biological material. Results X-ray in-line phase tomography was applied for the imaging of internal structures of maize seeds at early stages of development, when the cells are metabolically fully active and water is the main cell content. This 3D imaging technique with histology-like spatial resolution is demonstrated to reveal the anatomy of seed compartments with unequalled contrast by comparison with X-ray absorption tomography. An associated image processing pipeline allowed to quantitatively segment in 3D the four compartments of the seed (embryo, endosperm, nucellus and pericarp) from 7 to 21 days after pollination. Conclusion This work constitutes an innovative quantitative use of X-ray in-line phase tomography as a non-destructive fast method to perform virtual histology and extends the developmental stages accessible by this technique which had previously been applied in seed biology to more mature samples. Electronic supplementary material The online version of this article (doi:10.1186/s13007-015-0098-y) contains supplementary material, which is available to authorized users.

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 Zm-ZOURNA 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.

International audience; Fusarium head blight (FHB) or scab is one of the most important plant diseases worldwide, affecting wheat, barley and other small grains. Trichothecene mycotoxins such as deoxynivalenol (DON) accumulate in the grain, presenting a food safety risk and health hazard to humans and animals. Despite considerable breeding efforts, highly resistant wheat or barley cultivars are not available. We screened an activation tagged Arabidopsis thaliana population for resistance to trichothecin (Tcin), a type B trichothecene in the same class as DON. Here we show that one of the resistant lines identified, trichothecene resistant 1 (trr1) contains a T-DNA insertion upstream of two nonspecific lipid transfer protein (nsLTP) genes, AtLTP4.4 and AtLTP4.5. Expression of both nsLTP genes was induced in trr1 over 10-fold relative to wild type. Overexpression of AtLTP4.4 provided greater resistance to Tcin than AtLTP4.5 in Arabidopsis thaliana and in Saccharomyces cerevisiae relative to wild type or vector transformed lines, suggesting a conserved protection mechanism. Tcin treatment increased reactive oxygen species (ROS) production in Arabidopsis and ROS stain was associated with the chloroplast, the cell wall and the apoplast. ROS levels were attenuated in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls. Exogenous addition of glutathione and other antioxidants enhanced resistance of Arabidopsis to Tcin while the addition of buthionine sulfoximine, an inhibitor of glutathione synthesis, increased sensitivity, suggesting that resistance was mediated by glutathione. Total glutathione content was significantly higher in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls, highlighting the importance of AtLTP4.4 in maintaining the redox state. These results demonstrate that trichothecenes cause ROS accumulation and overexpression of AtLTP4.4 protects against trichothecene-induced oxidative stress by increasing the glutathione-based antioxidant defense.

International audience; The moss Physcomitrella patens is an important model organism for evo-devo studies. Here, we determined the genome-wide chromatin landscape of five important histone three (H3) modifications (H3K4me3, H3K27me3, H3K27Ac, H3K9Ac and H3K9me2) and describe the changes to these histone marks in two contrasted situations, developmental transition and abiotic (drought) stress. Integrative analysis of these histone H3 modifications revealed their preferential association into 15 chromatin states (CS) in genic regions of the P. patens genome. Synergistic relationships that influence expression levels were revealed for the three activating marks H3K4me3, H3K27Ac and H3K9Ac, while an antagonistic relationship was found between CS containing the H3K27me3 and H3K27Ac marks, suggesting that H3K27 is a key indexing residue regarding transcriptional output. Concerning the alteration of histone marks in response to developmental transition (juvenile to adult) and drought stress, the three activating marks H3K4me3, H3K27Ac and H3K9Ac show significant changes in both situations. However, changes to H3K27me3 are central only for genes differentially expressed during development. Interestingly, genes induced during drought stress show significant histone mark toggling during developmental transition. This situation suggests that drought induced adult (gametophore expressed) genes are primed to respond to this stress during the juvenile to adult transition.

In plants, root nitrate uptake systems are under systemic feedback repression by the N satiety of the whole organism, thus adjusting the N acquisition capacity to the N demand for growth; however, the underlying molecular mechanisms are largely unknown. We previously isolated the Arabidopsis high nitrogen-insensitive 9-1 ( hni9-1 ) mutant, impaired in the systemic feedback repression of the root nitrate transporter NRT2.1 by high N supply. Here, we show that HNI9 encodes Arabidopsis INTERACT WITH SPT6 (AtIWS1), an evolutionary conserved component of the RNA polymerase II complex. HNI9/AtIWS1 acts in roots to repress NRT2.1 transcription in response to high N supply. At a genomic level, HNI9/AtIWS1 is shown to play a broader role in N signaling by regulating several hundred N-responsive genes in roots. Repression of NRT2.1 transcription by high N supply is associated with an HNI9/AtIWS1-dependent increase in histone H3 lysine 27 trimethylation at the NRT2.1 locus. Our findings highlight the hypothesis that posttranslational chromatin modifications control nutrient acquisition in plants.

L'article original est publié par The American Society of Plant Biologists; International audience; Nitrate uptake by the roots is under systemic feedback repression by high nitrogen (N) status of the whole plant. The NRT2.1 gene, which encodes a NO(3)(-) transporter involved in high-affinity root uptake, is a major target of this N signaling mechanism. Using transgenic Arabidopsis (Arabidopsis thaliana) plants expressing the pNRT2.1::LUC reporter gene (NL line), we performed a genetic screen to isolate mutants altered in the NRT2.1 response to high N provision. Three hni (for high nitrogen insensitive) mutants belonging to three genetic loci and related to single and recessive mutations were selected. Compared to NL plants, these mutants display reduced down-regulation of both NRT2.1 expression and high-affinity NO(3)(-) influx under repressive conditions. Split-root experiments demonstrated that this is associated with an almost complete suppression of systemic repression of pNRT2.1 activity by high N status of the whole plant. Other mechanisms related to N and carbon nutrition regulating NRT2.1 or involved in the control of root SO(4)(-) uptake by the plant sulfur status are not or are slightly affected. The hni mutations did not lead to significant changes in total N and NO(3)(-) contents of the tissues, indicating that hni mutants are more likely regulatory mutants rather than assimilatory mutants. Nevertheless, hni mutations induce changes in amino acid, organic acid, and sugars pools, suggesting a possible role of these metabolites in the control of NO(3)(-) uptake by the plant N status. Altogether, our data indicate that the three hni mutants define a new class of N signaling mutants specifically impaired in the systemic feedback repression of root NO(3)(-) uptake.

Background Pods of the vanilla orchid (Vanilla planifolia) accumulate large amounts of the flavor compound vanillin (3-methoxy, 4-hydroxy-benzaldehyde) as a glucoside during the later stages of their development. At earlier stages, the developing seeds within the pod synthesize a novel lignin polymer, catechyl (C) lignin, in their coats. Genomic resources for determining the biosynthetic routes to these compounds and other flavor components in V. planifolia are currently limited. Results Using next-generation sequencing technologies, we have generated very large gene sequence datasets from vanilla pods at different times of development, and representing different tissue types, including the seeds, hairs, placental and mesocarp tissues. This developmental series was chosen as being the most informative for interrogation of pathways of vanillin and C-lignin biosynthesis in the pod and seed, respectively. The combined 454/Illumina RNA-seq platforms provide both deep sequence coverage and high quality de novo transcriptome assembly for this non-model crop species. Conclusions The annotated sequence data provide a foundation for understanding multiple aspects of the biochemistry and development of the vanilla bean, as exemplified by the identification of candidate genes involved in lignin biosynthesis. Our transcriptome data indicate that C-lignin formation in the seed coat involves coordinate expression of monolignol biosynthetic genes with the exception of those encoding the caffeoyl coenzyme A 3-O-methyltransferase for conversion of caffeoyl to feruloyl moieties. This database provides a general resource for further studies on this important flavor species. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-964) contains supplementary material, which is available to authorized users.