Loss-of-function of gynoecium-expressed phospholipase pPLAIIγ triggers maternal haploid induction in Arabidopsis.

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.

Evaluation of genome and base editing tools in maize protoplasts.

Introduction: Despite its rapid worldwide adoption as an efficient mutagenesis tool, plant genome editing remains a labor-intensive process requiring often several months of in vitro culture to obtain mutant plantlets. To avoid a waste in time and money and to test, in only a few days, the efficiency of molecular constructs or novel Cas9 variants (clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9) prior to stable transformation, rapid analysis tools are helpful. Methods: To this end, a streamlined maize protoplast system for transient expression of CRISPR/Cas9 tools coupled to NGS (next generation sequencing) analysis and a novel bioinformatics pipeline was established. Results and discussion: Mutation types found with high frequency in maize leaf protoplasts had a trend to be the ones observed after stable transformation of immature maize embryos. The protoplast system also allowed to conclude that modifications of the sgRNA (single guide RNA) scaffold leave little room for improvement, that relaxed PAM (protospacer adjacent motif) sites increase the choice of target sites for genome editing, albeit with decreased frequency, and that efficient base editing in maize could be achieved for certain but not all target sites. Phenotypic analysis of base edited mutant maize plants demonstrated that the introduction of a stop codon but not the mutation of a serine predicted to be phosphorylated in the bHLH (basic helix loop helix) transcription factor ZmICEa (INDUCER OF CBF EXPRESSIONa) caused abnormal stomata, pale leaves and eventual plant death two months after sowing.

Maize In Planta Haploid Inducer Lines: A Cornerstone for Doubled Haploid Technology.

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.

Overview of In Vitro and In Vivo Doubled Haploid Technologies.

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.

Lipid anchoring and electrostatic interactions target NOT-LIKE-DAD to pollen endo-plasma membrane.

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.

Haploid Embryos: Being Like Mommy or Like Daddy?

In planta haploid embryo induction is a powerful plant breeding tool, but is limited to very few crops. Two recent studies by Wang et al. and Lv et al. report seed-based haploid systems that produce paternal haploid embryos by engineering CENTROMERIC HISTONE H3 (CENH3). Together with recent translation of maize maternal haploid induction ability into wheat and rice, this extends our collection of haploid inducer lines.

Puzzling out plant reproduction by haploid induction for innovations in plant breeding.

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.

The Endosperm-Derived Embryo Sheath Is an Anti-adhesive Structure that Facilitates Cotyledon Emergence during Germination in Arabidopsis.

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 [3-5]. 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.

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

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

A stress-response-related inter-compartmental signalling pathway regulates embryonic cuticle integrity in Arabidopsis.

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, cuticle staining and permeability assays to show that cuticle deposition initiates de novo in 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.

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

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

The Physcomitrella patens chromosome-scale assembly reveals moss genome structure and evolution.

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.

Haploid induction in plants.

Gilles et al. introduce the technique of haploid induction in plant breeding.

ZHOUPI and KERBEROS Mediate Embryo/Endosperm Separation by Promoting the Formation of an Extracuticular Sheath at the Embryo Surface.

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.

Signaling in Early Maize Kernel Development.

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.

Loss of pollen-specific phospholipase NOT LIKE DAD triggers gynogenesis in maize.

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.

Fast virtual histology using X-ray in-line phase tomography: application to the 3D anatomy of maize developing seeds.

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.

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

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