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.

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.

Crop plants with improved culture and quality traits for food, feed and other uses.

The large French research project GENIUS (2012-2019, https://www6.inra.genius-project_eng/ ) provides a good showcase of current genome editing techniques applied to crop plants. It addresses a large variety of agricultural species (rice, wheat, maize, tomato, potato, oilseed rape, poplar, apple and rose) together with some models (Arabidopsis, Brachypodium, Physcomitrella). Using targeted mutagenesis as its work horse, the project is limited to proof of concept under confined conditions. It mainly covers traits linked to crop culture, such as disease resistance to viruses and fungi, flowering time, plant architecture, tolerance to salinity and plant reproduction but also addresses traits improving the quality of agricultural products for industrial purposes. Examples include virus resistant tomato, early flowering apple and low-amylose starch potato. The wide range of traits illustrates the potential of genome editing towards a more sustainable agriculture through the reduction of pesticides and to the emergence of innovative bio-economy sectors based on custom tailored quality traits.

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.

Characterization of GMO or glyphosate effects on the composition of maize grain and maize-based diet for rat feeding.

INTRODUCTION: In addition to classical targeted biochemical analyses, metabolomic analyses seem pertinent to reveal expected as well as unexpected compositional differences between plant genetically modified organisms (GMO) and non-GMO samples. Data previously published in the existing literature led to divergent conclusions on the effect of maize transgenes on grain compositional changes and feeding effects. Therefore, a new study examining field-grown harvested products and feeds derived from them remains useful. OBJECTIVES: Our aim was to use a metabolomics approach to characterize grain and grain-based diet compositional changes for two GMO events, one involving Bacillus thuringiensis toxin to provide insect resistance and the other one conferring herbicide tolerance by detoxification of glyphosate. We also investigated the potential compositional modifications induced by the use of a glyphosate-based herbicide on the transgenic line conferring glyphosate tolerance. RESULTS: The majority of statistically significant differences in grain composition, evidenced by the use of 1H-NMR profiling of polar extracts and LC-ESI-QTOF-MS profiling of semi-polar extracts, could be attributed to the combined effect of genotype and environment. In comparison, transgene and glyphosate effects remained limited in grain for the compound families studied. Some but not all compositional changes observed in grain were also detected in grain-based diets formulated for rats. CONCLUSION: Only part of the data previously published in the existing literature on maize grains of plants with the same GMO events could be reproduced in our experiment. All spectra have been deposited in a repository freely accessible to the public. Our grain and diet characterization opened the way for an in depth study of the effects of these diets on rat health.

Regulation of a maize HD-ZIP IV transcription factor by a non-conventional RDR2-dependent small RNA.

Small non-coding RNAs are versatile riboregulators that control gene expression at the transcriptional or post-transcriptional level, governing many facets of plant development. Here we present evidence for the existence of a 24 nt small RNA (named small1) that is complementary to the 3' UTR of OCL1 (Outer Cell Layer1), the founding member of the maize HD-ZIP IV gene family encoding plant-specific transcription factors that are mainly involved in epidermis differentiation and specialization. The biogenesis of small1 depends on DICER-like 3 (DCL3), RNA-dependent RNA polymerase 2 (RDR2) and RNA polymerase IV, components that are usually required for RNA-dependent DNA-methylation. Unexpectedly, GFP sensor experiments in transient and stable transformation systems revealed that small1 may regulate its target at the post-transcriptional level, mainly through translational repression. This translational repression is attenuated in an rdr2 mutant background in which small1 does not accumulate. Our experiments further showed the possible involvement of a secondary stem-loop structure present in the 3' UTR of OCL1 for efficient target repression, suggesting the existence of several regulatory mechanisms affecting OCL1 mRNA stability and translation.

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.

PPR2263, a DYW-Subgroup Pentatricopeptide repeat protein, is required for mitochondrial nad5 and cob transcript editing, mitochondrion biogenesis, and maize growth.

RNA editing plays an important role in organelle gene expression in various organisms, including flowering plants, changing the nucleotide information at precise sites. Here, we present evidence that the maize (Zea mays) nuclear gene Pentatricopeptide repeat 2263 (PPR2263) encoding a DYW domain-containing PPR protein is required for RNA editing in the mitochondrial NADH dehydrogenase5 (nad5) and cytochrome b (cob) transcripts at the nad5-1550 and cob-908 sites, respectively. Its putative ortholog, MITOCHONDRIAL EDITING FACTOR29, fulfills the same role in Arabidopsis thaliana. Both the maize and the Arabidopsis proteins show preferential localization to mitochondria but are also detected in chloroplasts. In maize, the corresponding ppr2263 mutation causes growth defects in kernels and seedlings. Embryo and endosperm growth are reduced, leading to the production of small but viable kernels. Mutant plants have narrower and shorter leaves, exhibit a strong delay in flowering time, and generally do not reach sexual maturity. Whereas mutant chloroplasts do not have major defects, mutant mitochondria lack complex III and are characterized by a compromised ultrastructure, increased transcript levels, and the induction of alternative oxidase. The results suggest that mitochondrial RNA editing at the cob-908 site is necessary for mitochondrion biogenesis, cell division, and plant growth in maize.

Genome-wide characterization of the HD-ZIP IV transcription factor family in maize: preferential expression in the epidermis.

Transcription factors of the plant-specific homeodomain leucine zipper IV (HD-ZIP IV) family have been found from moss to higher plants, and several family members have been associated with epidermis-related expression and/or function. In maize (Zea mays), four of the five characterized HD-ZIP IV family members are expressed specifically in the epidermis, one contributes to trichome development, and target genes of another one are involved in cuticle biosynthesis. Assessing the phylogeny, synteny, gene structure, expression, and regulation of the entire family in maize, 12 novel ZmHDZIV genes were identified in the recently sequenced maize genome. Among the 17 genes, eight form homeologous pairs duplicated after the split of maize and sorghum (Sorghum bicolor), whereas a fifth duplication is shared with sorghum. All 17 ZmHDZIV genes appear to be derived from a basic module containing seven introns in the coding region. With one possible exception, all 17 ZmHDZIV genes are expressed and show preferential expression in immature reproductive organs. Fourteen of 15 ZmHDZIV genes with detectable expression in laser-dissected tissues exhibit a moderate to very strong expression preference for the epidermis, suggesting that at least in maize, the majority of HD-ZIP IV family members may have epidermis-related functions. Thirteen ZmHDZIV genes carry conserved motifs of 19 and 21 nucleotides in their 3' untranslated region. The strong evolutionary conservation and the size of the conserved motifs in the 3' untranslated region suggest that the expression of HD-ZIP IV genes may be regulated by small RNAs.

Duplicate maize Wrinkled1 transcription factors activate target genes involved in seed oil biosynthesis.

WRINKLED1 (WRI1), a key regulator of seed oil biosynthesis in Arabidopsis (Arabidopsis thaliana), was duplicated during the genome amplification of the cereal ancestor genome 90 million years ago. Both maize (Zea mays) coorthologs ZmWri1a and ZmWri1b show a strong transcriptional induction during the early filling stage of the embryo and complement the reduced fatty acid content of Arabidopsis wri1-4 seeds, suggesting conservation of molecular function. Overexpression of ZmWri1a not only increases the fatty acid content of the mature maize grain but also the content of certain amino acids, of several compounds involved in amino acid biosynthesis, and of two intermediates of the tricarboxylic acid cycle. Transcriptomic experiments identified 18 putative target genes of this transcription factor, 12 of which contain in their upstream regions an AW box, the cis-element bound by AtWRI1. In addition to functions related to late glycolysis and fatty acid biosynthesis in plastids, the target genes also have functions related to coenzyme A biosynthesis in mitochondria and the production of glycerol backbones for triacylglycerol biosynthesis in the cytoplasm. Interestingly, the higher seed oil content in ZmWri1a overexpression lines is not accompanied by a reduction in starch, thus opening possibilities for the use of the transgenic maize lines in breeding programs.

PPR8522 encodes a chloroplast-targeted pentatricopeptide repeat protein necessary for maize embryogenesis and vegetative development.

The pentatricopeptide repeat (PPR) domain is an RNA binding domain allowing members of the PPR superfamily to participate in post-transcriptional processing of organellar RNA. Loss of PPR8522 from maize (Zea mays) confers an embryo-specific (emb) phenotype. The emb8522 mutation was isolated in an active Mutator (Mu) population and co-segregation analysis revealed that it was tightly linked to a MuDR insertion in the first exon of PPR8522. Independent evidence that disruption of PPR8522 caused the emb phenotype was provided by fine mapping to a region of 116kb containing no other gene than PPR8522 and complementation of the emb8522 mutant by a PPR8522 cDNA. The deduced PPR8522 amino acid sequence of 832 amino acids contains 10 PPR repeats and a chloroplast target peptide, the function of which was experimentally demonstrated by transient expression in Nicotiana benthamiana. Whereas mutant endosperm is apparently normal, mutant embryos deviate from normal development as early as 3 days after pollination, are reduced in size, exhibit more or less severe morphological aberrations depending on the genetic background, and generally do not germinate. The emb8522 mutation is the first to associate the loss of a PPR gene with an embryo-lethal phenotype in maize. Analyses of mutant plantlets generated by embryo-rescue experiments indicate that emb8522 also affects vegetative plant growth and chloroplast development. The loss of chloroplast transcription dependent on plastid-encoded RNA polymerase is the likely cause for the lack of an organized thylakoid network and an albino, seedling-lethal phenotype.

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.

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

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

Overexpression of the epidermis-specific homeodomain-leucine zipper IV transcription factor Outer Cell Layer1 in maize identifies target genes involved in lipid metabolism and cuticle biosynthesis.

Transcription factors of the homeodomain-leucine zipper IV (HD-ZIP IV) family play crucial roles in epidermis-related processes. To gain further insight into the molecular function of OUTER CELL LAYER1 (OCL1), 14 target genes up- or down-regulated in transgenic maize (Zea mays) plants overexpressing OCL1 were identified. The 14 genes all showed partial coexpression with OCL1 in maize organs, and several of them shared preferential expression in the epidermis with OCL1. They encoded proteins involved in lipid metabolism, defense, envelope-related functions, or cuticle biosynthesis and include ZmWBC11a (for white brown complex 11a), an ortholog of AtWBC11 involved in the transport of wax and cutin molecules. In support of the annotations, OCL1-overexpressing plants showed quantitative and qualitative changes of cuticular wax compounds in comparison with wild-type plants. An increase in C24 to C28 alcohols was correlated with the transcriptional up-regulation of ZmFAR1, coding for a fatty acyl-coenzyme A reductase. Transcriptional activation of ZmWBC11a by OCL1 was likely direct, since transactivation in transiently transformed maize kernels was abolished by a deletion of the activation domain in OCL1 or mutations in the L1 box, a cis-element bound by HD-ZIP IV transcription factors. Our data demonstrate that, in addition to AP2/EREBP and MYB-type transcription factors, members of the HD-ZIP IV family contribute to the transcriptional regulation of genes involved in cuticle biosynthesis.

Functional characterization of the HD-ZIP IV transcription factor OCL1 from maize.

OCL1 (OUTER CELL LAYER1) encodes a maize HD-ZIP class IV transcription factor (TF) characterized by the presence of a homeo DNA-binding domain (HD), a dimerization leucine zipper domain (ZIP), and a steroidogenic acute regulatory protein (StAR)-related lipid transfer domain (START) involved in lipid transport in animals but the function of which is still unknown in plants. By combining yeast and plant trans-activation assays, the transcriptional activation domain of OCL1 was localized to 85 amino acids in the N-terminal part of the START domain. Full-length OCL1 devoid of this activation domain is unable to trans-activate a reporter gene under the control of a minimal promoter fused to six repeats of the L1 box, a cis-element present in target genes of HD-ZIP IV TFs in Arabidopsis. In addition, ectopic expression of OCL1 leads to pleiotropic phenotypic aberrations in transgenic maize plants, the most conspicuous one being a strong delay in flowering time which is correlated with the misexpression of molecular markers for floral transition such as ZMM4 (Zea Mays MADS-box4) or DLF1 (DELAYED FLOWERING1). As suggested by the interaction in planta between OCL1 and SWI3C1, a bona fide subunit of the SWI/SNF complex, OCL1 may modulate transcriptional activity of its target genes by interaction with a chromatin remodelling complex.

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.

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.

The HD-ZIP IV transcription factor OCL4 is necessary for trichome patterning and anther development in maize.

Among the genes controlling the differentiation and maintenance of epidermal cell fate are members of the HD-ZIP IV class family of plant-specific transcription factors, most of which are specifically expressed in the epidermis of tissues. Here, we report the functional analysis of the maize HD-ZIP IV gene OCL4 (outer cell layer 4) via the phenotypic analysis of two insertional mutants, and of OCL4-RNAi transgenic plants. In all three materials, the macrohairs, one of the three types of trichomes present on adult maize leaf blades, developed ectopically at the margin of juvenile and adult leaves. Consistent with this phenotype, OCL4 is expressed in the epidermis of the leaf blade, with a maximum at the margin of young leaf primordia. Expression of OCL4 in the model plant Arabidopsis under the control of the GLABRA2 (GL2) promoter, a member of the Arabidopsis HD-ZIP IV family involved in trichome differentiation, did not complement the gl2-1 mutant, but instead aggravated its phenotype. The construct also caused a glabrous appearance of rosette leaves in transformed control plants of the Ler ecotype, suggesting that OCL4 inhibits trichome development both in maize and Arabidopsis. Furthermore, insertional mutants showed a partial male sterility that is likely to result from the presence of an extra subepidermal cell layer with endothecium characteristics in the anther wall. Interestingly, the epidermis-specific OCL4 expression in immature anthers was restricted to the region of the anther locule where the extra cell layer differentiated. Taken together these results suggest that OCL4 inhibits trichome development and influences division and/or differentiation of the anther cell wall.

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.