Plant reproduction meetings often deal either with pre-fertilization processes such as flowering and pollen biology or post-fertilization processes such as embryogenesis and seed development. The Biochemical Society Focused Meeting entitled ‘Regulation of Fertilization and Early Seed Development’ was organized to close this gap and to discuss mechanistic similarities and future research directions in the reproductive processes shortly before, during and after fertilization. As an outcome of the workshop, invited speakers and a few selected oral communication presenters contributed focused reviews and technical articles for this issue of Biochemical Society Transactions. We provide here a short overview of the contents and highlights of the various articles.
The life cycle of flowering plants (angiosperms) begins with a single cell, the fertilized egg cell or zygote. During the successive stages of embryo development, which involve the establishment of cell polarity, cell identity, asymmetric cell divisions and pattern formation, an embryo is formed inside the tissues of the seed coat containing the basic body plan and cell types of an adult plant (see the reviews by Weijers  and Musielak and Bayer ). However, in contrast with most animal species, flowering plants continuously generate new organs after embryogenesis is completed and develop a series of additional specialized cell types that do not occur in the embryo. A major characteristic of angiosperms is a second fertilization event generating the endosperm. Both processes, i.e. embryogenesis and endosperm formation, occur inside the maternal tissues of the seed coat and mutually depend on cross-talk between both structures. Although the endosperm is consumed during seed development in eudicots, it serves as a reservoir of nutrients and energy in the monocots that will be used up by the germinating seedling. After germination, most angiosperms grow vegetatively for almost their entire life, eventually forming reproductive structures towards the end of their life cycle in annual species or periodically in perennial species. Plant reproduction is thus initiated with the formation of flowering meristems from vegetative meristems, the generation of flower organs that contain the male and female germlines producing the gametes. Similar to early seed development, these reproductive processes require the establishment and maintenance of cell polarity, cell identity, asymmetric cell divisions and patterning. Moreover, recent studies indicate that the same plant hormones as well as small non-coding RNAs play key roles during reproduction both before and after fertilization (see, for example, the reviews by Chen et al. , Lituiev and Grossniklaus  and Vashisht and Nodine ).
Despite these similarities and mechanistic overlaps, the focus of most plant reproduction meetings in the past was on flowering and flower organ development, development and function of the germlines, or on seed development. The process of fertilization itself was either considered as a final event of reproduction or the initial process of seed development. A major aim of the Regulation of Fertilization and Early Seed Development Biochemical Society Focused Meeting that took place on 11–13 September 2013 at the University of Bath was to bridge these processes and to bring together leading researchers from both fields to report the most recent developments and to discuss mechanistic similarities in the various processes and future directions. This exciting meeting was attended by approximately 60 scientists. Most invited speakers and a few selected oral communication presenters contributed articles to this issue of Biochemical Society Transactions. Although a number of reviews summarize the current state-of-the-art knowledge of fertilization mechanisms and early seed development in angiosperms, there are also reviews with a focus on methods and recent technical progress whose application will further our understanding on the underlying molecular mechanisms.
Self-incompatibility mechanisms and pollen tube growth towards the egg apparatus
Recent findings have shown that plants have evolved species-specific communication systems to achieve successful fertilization and to maximize reproductive success during pollen tube germination, growth through the maternal tissues of the stigma and style as well as for pollen tube reception and sperm release. Moreover, a large number of genetic controls exist to prevent fertilization of egg and central cell by alien sperm cells. Two articles in this issue cover the molecular mechanisms of SI (self-incompatibility), a widespread phenomenon among angiosperms that permits the recognition and rejection of ‘self’ pollen thus preventing inbreeding. SI systems have evolved independently multiple times in angiosperm lineages and those that have been characterized to date feature specificity proteins in pollen and stigma that on interaction trigger pollen rejection. The article by Nasrallah and Nasrallah  focuses on the SI system found in the Brassicaceae and gives particular attention to the role of the stigmatic determinant SRK (S-locus receptor kinase), which, when activated, leads to pollen rejection. Importantly, the authors point out some perplexing differences between the downstream targets of SRK in Arabidopsis and Brassica revealed by mutational studies in a transgenic Arabidopsis thaliana line engineered to have a functional SI system. They consider whether this may indicate that multiple cellular pathways are involved in pollen rejection with different species favouring a particular mechanism. This article also discusses the exciting observation that SRK signalling also modulates developmental process in the stigma with a role for auxin in enhancing SI. The second article on SI by Eaves et al.  moves to Papaver rhoeas (poppy), which is arguably the best-characterized SI system to date and differs from the system found in the Brassicaceae. Here, pollen rejection is centred in the pollen and ultimately results in PCD (programmed cell death) via signalling pathways involving a Ca2+-dependent signalling network. Eaves et al.  review the incredible finding that transfer of the male determinant, PrpS (Papaver rhoeas pollen S), into A. thaliana resulted in functional SI in Arabidopsis pollen and that the inhibition of pollen featured all the key hallmarks of pollen rejection in poppy. This observation is remarkable given that these two species diverged some 144 million years ago. These data highlight the fact that SI determinants (SRK in the Brassicaceae and PrpS in poppy) seem to plug into common cellular systems that are conserved across great evolutionary distances. This opens up the real possibility of moving functional SI into plants of agronomic importance to facilitate processes such as the production of hybrid F1 seed.
Understanding how pollen tubes grow and function at the cellular level is not only crucial to understanding SI (see Eaves et al. ), but also compatible growth and its regulation. On pollen hydration, there are massive metabolic changes that are likely to be accompanied by protein phosphorylation and dephosphorylation events that shape subsequent processes relating to pollen germination and various stages of growth through maternal tissues. In relation to this, the article by Fíla et al.  considers techniques available to characterize the pollen phosphoproteome and highlights the crucial importance of standardizing the methodology utilized to enrich for phosphorylated proteins and their subsequent identification. They point out that standardization would permit more useful comparisons to be made not only between different developmental time points, but also between species as is highlighted by their comparison of Arabidopsis and tobacco datasets.
The pollen grains in angiosperms carry two sperm cells, the male gametes, and the article by Pereira et al.  considers evolutionarily conserved mechanisms of male germline development in angiosperms and animals. This process involves two gene expression programmes: (i) a pre-meiotic phase, which commits cells to meiosis, and (ii) a post-meiotic phase, which permits acquisition of factors that provide fertilizing ability. In addition to this, the authors discuss the essential role of small RNAs, which epigenetically silence mobile genetic elements and contribute to regulation of the differentiation program (see also the review by Vashisht and Nodine ). Importantly, they also point out the growing evidence that small RNAs are also likely to have a significant role post-fertilization as complex populations of these molecules have been identified in mature gametes of both plants and animals. Recent studies have also shown that the pollen tube changes its gene expression programme during its path towards the egg apparatus where the two sperm cells are released for double fertilization. The review by Leydon et al.  highlights especially a subgroup of MYB transcriptional regulators expressed during pollen tube growth, which seem to represent major components regulating the transcriptional programme during pollen tube growth through the maternal tissues. Using Arabidopsis as a model, they discuss further intra- and inter-specific hybridization barriers (see also the reviews by Nasrallah and Nasrallah  and Eaves et al. ) and the requirement to express genes required to distinguish self- from alien pollen (tubes) and to enable their perception in the egg apparatus. The egg apparatus is a component of the FG (female gametophyte). Patterning of the FG, a syncytium forming both female gametes (egg and central cell) and a number of accessory cells at both poles is the topic of the review by Lituiev and Grossniklaus . On the basis of the analysis of a large number of FG mutants and corresponding phenotypes, they discuss the establishment of FG polarity, the role of extrinsic and intrinsic positional information, and specification of nuclei before and after final localization at certain positions in the maturating FG. After cellularization and cell specification has occurred, maintenance of cellular differentiation and cell fate determination involves, for example, non-cell-autonomous signalling. Positional information is discussed further in line with three paradigms for pattern formation derived from the study of animal model systems.
Technical advances to understand cell–cell communication during reproduction
However, despite a lot of progress in the last few years, the handling of the above-described reproductive structures and quantitative measurements during the various processes remains difficult. To circumvent some of these problems, Arata and Higashiyama  suggest the usage of microfabrication technologies to generate, for example, poly(dimethylsiloxane)-based microdevices such as a T-shaped microchannel that allows the study of pollen tube growth and attraction at two different conditions. Moreover, the fabrication of a microcage is reported for positioning reproductive structures such as ovules and developing embryos for long-term live-cell imaging. Such devices are likely to become very powerful tools for future studies in plant reproductive biology. Two additional technical articles deal with methodological approaches to identify and investigate ligands and receptors with roles during cell–cell communication in plant reproduction. Hafidh et al.  summarize the small number of ligands and receptors identified to date with roles during pollen tube growth, guidance and perception. Although significant progress has been made in recent years to understand the communication of FG cells with growing pollen tubes, most pollen-tube-derived ligands and plasma-membrane-localized receptors, as well as receptors and ligands of interacting cells such as transmitting tract and synergid cells, remain to be identified and their role(s) to be elucidated. Hafidh et al.  describe an improved semi-in vivo technique combined with gel-free LC–MS/MS to identify the pollen tube secretome. Once secreted proteinaceous ligands (usually peptides and small proteins) are identified, methods are required to identify their cell-surface receptors. A review by Uebler and Dresselhaus  follows this line and report most promising recent methods for identifying cell-surface receptor interactions. A focus is on biochemical methods such as ligand-based cross-linking and -capture and alternative ‘classical’ methods such as the generation of mRNA-display libraries combined with peptide-ligand exposure. New reagents such as TRICEPS and their usage for affinity purification are also discussed. The identification of cell-surface receptors or other interacting proteins is also essential to find out the role(s) of the many secreted small CRPs (cysteine-rich proteins) which are produced by the individual cells of the FG, such as the ECA1 (early culture abundant 1) gametogenesis-related family of CRPs. Sprunck et al.  report on CRPs expressed in the FG, and about similarities and differences between egg-cell-secreted EC1 (egg cell 1) proteins, which are known to promote sperm activation and gamete fusion, and other members of the ECA1 gametogenesis-related family with as yet unknown functions. Functional diversification within this family of CRPs is discussed, on the basis of their different expression patterns and low primary sequence conservation.
Embryo patterning and seed development
After fertilization, the single-celled zygote develops into a simple organism, the embryo, already containing the basic cell types and tissues of adult plants such as epidermal, ground and vascular tissues, and various types of stem cells. Patterning during early embryogenesis depends on asymmetric cell divisions, establishment of polarity and cell fates, which are highlighted by two review articles. Musielak and Bayer  summarize the signalling and transcriptional network required to specify apical–basal polarity in the very early Arabidopsis embryo. Asymmetric division of the zygote depends on the WRKY2, WOX2, WOX8 and WOX9 transcriptional regulators as well as the YDA (YODA)/MAPK (mitogen-activated protein kinase) signalling pathway. The correlation and activation of these pathways as well as the cross-talk with the activity of plant hormones auxin and brassinosteroid is discussed. Weijers  further summarizes patterning of the early embryo with a focus on the molecules and mechanisms that specify the embryonic root. Activity of auxin via ARFs (auxin-response factors) is required for many of the above-described processes in the eudicot model plant Arabidopsis. In contrast with patterning in Arabidopsis, Chen et al.  report that neither an auxin nor a cytokinin response is detectable in the early embryo of the grass model plant maize. Using the auxin and cytokinin response markers DR5 and TCSv2 (two-component system, cytokinin-responsive promoter version #2), as well as the major auxin efflux carrier PIN1a (PINFORMED1a), they report strong auxin responses within adaxial endosperm cells and cytokinin responses in the basal endosperm transfer layer shortly after fertilization, but signals do not appear in the embryo until the transition stage. Interestingly, auxin responses in the endosperm cells surrounding the developing embryo correlate with adaxial embryo differentiation and outgrowth, suggesting that exogenous auxin may also play a role in patterning the early embryo in grasses. Most of our knowledge about embryonic patterning and developmental timing was obtained from genetic mutant studies of transcriptional regulators and the corresponding upstream activity of plant hormones. Temporal and spatial degradation of transcripts encoding many of these transcription factor families during early embryogenesis is regulated at the post-transcriptional level by the activity of miRNAs. Vashisht and Nodine  summarize our current knowledge about the role of miRNAs in Arabidopsis embryos. A focus is on the role of miR165/166 and miR164 that define the localization of HD-ZIPIII (class III homeodomain-leucine zipper gene) and CUC (CUP-SHAPED COTYLEDON) transcription factors. miR394 restricts the F-box protein transcript LCR (LEAF CURLING RESPONSIVENESS). Transcripts encoding auxin receptors and response factors are targets of miR393, miR160 and miR167, whereas transcription factor genes involved in temporal control of differentiation are miR156, miR166 and miR394 targets. miRNAs are highly conserved in plants and the authors therefore suggest that the mechanisms observed may function analogously in other plant species.
There has been much focus on the regulation of seed development over the last 20 years, with great progress being made in identifying key regulators acting in the zygotic tissues of the embryo and endosperm, and the maternal tissues of the seed coat. Figueiredo and Köhler  provide a review of how co-ordination of signals from different tissues of the seed orchestrates seed development, with a particular focus on the role of the seed coat in seed development. They introduce how epigenetic mechanisms operate in the endosperm to regulate both endosperm and seed coat development. These authors also highlight the importance of bidirectional signalling between this maternal seed tissue and the zygotic endosperm. Doughty et al.  develop this theme further and examine the function of the endothelium, the innermost layer of the seed coat, which is adjacent to the endosperm, in regulating seed size. This layer is distinctive in that it is the site of flavonoid biosynthesis and oxidized PAs (proanthocyanidins) accumulate in the vacuoles of this layer, giving seeds their brown colour on maturity. They discuss recent data that suggest that flavonoids may have an important regulatory role in modulating signalling between the seed coat and the endosperm. Mutants of the pathway have altered seed development featuring early cellularization of the endosperm. The authors suggest that these developmental changes may result from modifications to normal auxin movement in the developing seed as flavonoids are known to be potent auxin transport regulators. Moving from the identification of key transcriptional regulators to a greater understanding of the cellular processes that regulate seed development is now clearly an important goal for researchers in the field and is likely to form a large part of work in the future.
This summary of contributions to the Regulation of Fertilization and Early Seed Development Biochemical Society Focused Meeting wonderfully highlights the breadth and depth of studies in this field. Although much is understood, there is still much to understand and, gratifyingly, this meeting revealed that new discoveries illuminating this fascinating area of biology continue apace. We thank all who took part in this exciting meeting and who contributed to this issue of Biochemical Society Transactions, and offer special thanks to The Biochemical Society whose sponsorship and organizational support made for a really excellent Focused Meeting.
Regulation of Fertilization and Early Seed Development: A Biochemical Society Focused Meeting held at the University of Bath, U.K., 11–13 September 2013. Organized and Edited by James Doughty (University of Bath, U.K.) and Thomas Dresselhaus (University of Regensburg, Germany).