Killing me softly - Programmed cell death in plant reproduction from sporogenesis to fertilization.

Regulated or programmed cell death (RCD or PCD) is a fundamental biological principle integral to a considerable variety of functions in multicellular organisms. In plants, different PCD processes are part of biotic and abiotic stress responses, but also occur as an essential aspect of unperturbed plant development. PCD is particularly abundant during plant reproduction, eliminating unwanted or no longer needed cells, tissues, or organs in a precisely controlled manner. Failure in reproductive PCD can have detrimental consequences for plant reproduction. Here we shed a light on the latest research into PCD mechanisms in plant reproduction from sex determination over sporogenesis to pollination and fertilization.

Morphological and Ultrastructural Features of Formation of the Skin of Wheat (Triticum aestivum L.) Kernel.

The integumentary tissues of plant seeds protect the embryo (new sporophyte) forming in them from unfavorable external conditions; therefore, comprehensive knowledge about the structural and functional specificity of seed covers in various plants may be of both theoretical and practical interest. As a result of our study, additional data were obtained on the morphological and ultrastructural features of the formation of a multilayer skin of wheat (Triticum aestivum L.) kernel (caryopsis). The ultrastructure research analysis showed that differentiation of the pericarp and inner integument of the ovule leads to the formation of functionally different layers of the skin of mature wheat grain. Thus, the differentiation of exocarp and endocarp cells is accompanied by a significant thickening of the cell walls, which reliably protect the ovule from adverse external conditions. The cells of the two-layer inner integument of the ovule differentiate into cuticular and phenolic layers, which are critical for protecting daughter tissues from various pathogens. The epidermis of the nucellus turns into a layer of mucilage, which apparently helps to maintain the water balance of the seed. Morphological and ultrastructural data showed that the formation of the kernel's skin occurs in coordination with the development of the embryo and endosperm up to the full maturity of the kernel. This is evidenced by the structure of the cytoplasm and nucleus, characteristic of metabolically active protoplasts of cells, which is observed in most integumentary layers at the late stages of maturation. This activity can also be confirmed by a significant increase in the thickness of the cell walls in the cells of two layers of the exocarp and in cross cells in comparison with the earlier stages. Based on these results, we came to the conclusion that the cells of a majority in the covering tissues of the wheat kernel during its ontogenesis are transformed into specialized layers of the skin by terminal differentiation.

APETALA2 functions as a temporal factor together with BLADE-ON-PETIOLE2 and MADS29 to control flower and grain development in barley.

Cereal grain develops from fertilised florets. Alterations in floret and grain development greatly influence grain yield and quality. Despite this, little is known about the underlying genetic control of these processes, especially in key temperate cereals such as barley and wheat. Using a combination of near-isogenic mutant comparisons, gene editing and genetic analyses, we reveal that HvAPETALA2 (HvAP2) controls floret organ identity, floret boundaries, and maternal tissue differentiation and elimination during grain development. These new roles of HvAP2 correlate with changes in grain size and HvAP2-dependent expression of specific HvMADS-box genes, including the B-sister gene, HvMADS29 Consistent with this, gene editing demonstrates that HvMADS29 shares roles with HvAP2 in maternal tissue differentiation. We also discovered that a gain-of-function HvAP2 allele masks changes in floret organ identity and grain size due to loss of barley LAXATUM.A/BLADE-ON-PETIOLE2 (HvBOP2) gene function. Taken together, we reveal novel pleiotropic roles and regulatory interactions for an AP2-like gene controlling floret and grain development in a temperate cereal.

Evolution and patterning of the ovule in seed plants.

The ovule and its developmental successor, the seed, together represent a highly characteristic feature of seed plants that has strongly enhanced the reproductive and dispersal potential of this diverse group of taxa. Ovules encompass multiple tissues that perform various roles within a highly constrained space, requiring a complex cascade of genes that generate localized cell proliferation and programmed cell death during different developmental stages. Many heritable morphological differences among lineages reflect relative displacement of these tissues, but others, such as the second (outer) integuments of angiosperms and Gnetales, represent novel and apparently profound and independent innovations. Recent studies, mostly on model taxa, have considerably enhanced our understanding of gene expression in the ovule. However, understanding its evolutionary history requires a comparative and phylogenetic approach that is problematic when comparing extant angiosperms not only with phylogenetically distant extant gymnosperms but also with taxa known only from fossils. This paper reviews ovule characters across a phylogenetically broad range of seed plants in a dynamic developmental context. It discusses both well-established and recent theories of ovule and seed evolution and highlights potential gaps in comparative data that will usefully enhance our understanding of evolutionary transitions and developmental mechanisms.

Ultrastructural analysis of Rhynchospora ovules: The first record of Cyperaceae megagametophyte on transmission electron microscope.

Microsporogenesis and microgametogenesis are unusual in sedges (Cyperaceae), the third largest monocotyledonous family, as three microspores are aborted in favor of a single functional microspore. However, studies using light microscopy show that megasporogenesis and megagametogenesis occur normally. Nevertheless, the lack of ultrastructural details limits our knowledge of female gametophyte development in this family. Given the importance of morphological studies of reproductive structures, ovules and megagametophytes of Rhynchospora pubera were analyzed under transmission electron microscopy for the first time. Overall, ovules presented features similar to those described for the family, but ultrastructural details revealed an absence of a clear boundary between the egg cell and the central cell cytoplasm. Most interestingly, antipodal and nucellar cells showed several signs of vacuolar cell death, which suggest that programmed autolysis in sporogenous and gametophytic tissue is common in gametophyte development in the Cyperaceae. This may be related to the reproductive success of this family.

Megasporogenesis, megagametogenesis, and embryogenesis in Dendrobium nobile (Orchidaceae).

The orchid reproductive strategy, including the formation of numerous tiny seeds, is achieved by the elimination of some stages in the early plant embryogenesis. In this study, we documented in detail the formation of the maternal tissues (the nucellus and integuments), the structures of female gametophyte (megaspores, chalazal nuclei, synergids, polar nuclei), and embryonic structures in Dendrobium nobile. The ovary is unilocular, and the ovule primordia are formed in the placenta before the pollination. The ovule is medionucellate: the two-cell postament and two rows of nucellar cells persist until the death of the inner integument. A monosporic eight-nucleated embryo sac is developed. After the fertilization, the most common central cell nucleus consisted of two joined but not fused polar nuclei. The embryogenesis of D. nobile is similar to the Caryophyllad-type, and it is characterized by the formation of all embryo cells from the apical cell (ca) of a two-celled proembryo. The only exception is that there is no formation of the radicle and/or cotyledons. The basal cell (cb) does not divide during the embryogenesis, gradually transforming into the uninuclear suspensor. Then the suspensor goes through three main stages: it starts with an unbranched cell within the embryo sac, followed by a branched stage growing into the integuments, and it ends with the cell death. The stage-specific development of the female gametophyte and embryo of D. nobile is discussed.

Natural Variation in Ovule Morphology Is Influenced by Multiple Tissues and Impacts Downstream Grain Development in Barley (Hordeum vulgare L.).

The ovule plays a critical role in cereal yield as it is the site of fertilization and the progenitor of the grain. The ovule primordium is generally comprised of three domains, the funiculus, chalaza, and nucellus, which give rise to distinct tissues including the integuments, nucellar projection, and embryo sac. The size and arrangement of these domains varies significantly between model eudicots, such as Arabidopsis thaliana, and agriculturally important monocotyledonous cereal species, such as Hordeum vulgare (barley). However, the amount of variation in ovule development among genotypes of a single species, and its functional significance, remains unclear. To address this, wholemount clearing was used to examine the details of ovule development in barley. Nine sporophytic and gametophytic features were examined at ovule maturity in a panel of 150 European two-row spring barley genotypes, and compared with grain traits from the preceding and same generation. Correlations were identified between ovule traits and features of grain they produced, which in general highlighted a negative correlation between nucellus area, ovule area, and grain weight. We speculate that the amount of ovule tissue, particularly the size of the nucellus, may affect the timing of maternal resource allocation to the fertilized embryo sac, thereby influencing subsequent grain development.

Differential activity of F-box genes and E3 ligases distinguishes sexual versus apomictic germline specification in Boechera.

Germline specification is the first step during sexual and apomictic plant reproduction, and takes place in the nucellus of the ovule, a specialized domain of the reproductive flower tissues. In each case, a sporophytic cell is determined to form the sexual megaspore mother cell (MMC) or an apomictic initial cell (AIC). These differ in their developmental fates: while the MMC undergoes meiosis, the AIC modifies or omits meiosis to form the female gametophyte. Despite great interest in these distinct developmental processes, little is known about their gene regulatory basis. To elucidate the gene regulatory networks underlying germline specification, we conducted tissue-specific transcriptional profiling using laser-assisted microdissection and RNA sequencing to compare the transcriptomes of nucellar tissues between different sexual and apomictic Boechera accessions representing four species and two ploidy levels. This allowed us to distinguish between expression differences caused by genetic background or reproductive mode. Statistical data analysis revealed 45 genes that were significantly differentially expressed, and which potentially play a role for determination of the reproductive mode. Based on annotations, these included F-box genes and E3 ligases that most likely relate to genes previously described as regulators important for germline development. Our findings provide novel insights into the transcriptional basis of sexual and apomictic reproduction.

Transcriptomic profiling identifies differentially expressed genes associated with programmed cell death of nucellar cells in Ginkgo biloba L.

BACKGROUND: Previously, we demonstrated that pollen chamber formation (PCF) in G. biloba ovules was a process of programmed cell death (PCD) within the nucellar cells at the micropylar end. However, the signal triggering the cascades of the programmed events in these nucellar cells remains unexplored. RESULTS: A transcriptomic strategy was employed to unravel the mechanism underlying the nucellar PCD via the comparative profiles of RNA-seq between pre-PCF and post-PCF ovules. A total of 5599 differentially expressed genes (DEGs) with significance was identified from G. biloba ovules and classified into three main categories of GO annotation, including 17 biological processes, 15 cellular components and 17 molecular functions. KEGG analysis showed that 72 DEGs were enriched in "Plant hormone signal transduction". Furthermore, 99 DEGs were found to be associated with the PCD process, including the genes involved in ethylene signaling pathway, PCD initiation, and PCD execution. Moreover, calcium-cytochemical localization indicated that calcium could play a role in regulating PCD events within the nucellar cells during pollen chamber formation in G. biloba ovules. CONCLUSIONS: A putative working model, consisting of three overlapping processes, is proposed for the nucellar PCD: at the stage of PCD preparation, ethylene signaling pathway is activated for transcriptional regulation of the downstream targets; subsequently, at the stage of PCD initiation, the upregulated expression of several transcription factors, i.e., NAC, bHLH, MADS-box, and MYB, further promotes the corresponding transcript levels of CYTOCHROME C and CALMODULINs, thereby, leads to the PCD initiation via the calcium-dependent signaling cascade; finally, at the stage of PCD execution, some proteases like metacaspases and vacuolar processing enzyme for hydrolysis, together with the process of autophagy, play roles in the clearance of cellular components. Afterwards, a pollen chamber is generated from the removal of specific nucellar cells in the developing ovule.

Revisiting the Female Germline and Its Expanding Toolbox.

The Arabidopsis thaliana ovule arises as a female reproductive organ composed solely of somatic diploid cells. Among them, one cell will acquire a unique identity and initiate female germline development. In this review we explore the complex network that facilitates differentiation of this single cell, and consider how it becomes committed to a distinct developmental program. We highlight recent progress towards understanding the role of intercellular communication, cell competency, and cell-cycle regulation in the ovule primordium, and we discuss the possibility that distinct pathways restrict germline development at different stages. Importantly, these recent findings suggest a renaissance in plant ovule research, restoring the female germline as an attractive model to study cell communication and cell fate establishment in multicellular organs.

Development and evolution of the unique ovules of flowering plants.

Ovules are the precursors to seeds and as such are critical to plant propagation and food production. Mutant studies have led to the identification of numerous genes regulating ovule development. Genes encoding transcription factors have been shown to direct ovule spacing, ovule identity and integument formation. Particular co-regulators have now been associated with activities of some of these transcription factors, and other protein families including cell surface receptors have been shown to regulate ovule development. Hormone levels and transport, especially of auxin, have also been shown to play critical roles in ovule emergence and morphogenesis and to interact with the transcriptional regulators. Ovule diversification has been studied using orthologs of regulatory genes in divergent angiosperm groups. Combining modern genetic evidence with expanding knowledge of the fossil record illuminates the possible origin of the unique bitegmic ovules of angiosperms.

Seed Evolution, A 'Simpler' Story.

Seed evolution is often presented as the evolution of morphological complexity. Following the steps of Wilhelm Hofmeister, I argue that changes in the development of one tissue, the megasporangium/nucellus, can explain the origin of the seed habit. Here, I lay down a 'simpler' story that correlates seed evolution to nucellus cell fate.

Seed tissue and nutrient partitioning, a case for the nucellus.

Flowering plants display a large spectrum of seed architectures. The volume ratio of maternal versus zygotic seed tissues changes considerably among species and underlies different nutrient-storing strategies. Such diversity arose through the evolution of cell elimination programs that regulate the relative growth of one tissue over another to become the major storage compartment. The elimination of the nucellus maternal tissue is regulated by developmental programs that marked the origin of angiosperms and outlined the most ancient seed architectures. This review focuses on such a defining mechanism for seed evolution and discusses the role of nucellus development in seed tissues and nutrient partitioning at the light of novel discoveries on its molecular regulation.

The Evolution of Sexual Fluids in Gymnosperms From Pollination Drops to Nectar.

A current synthesis of data from modern and fossil plants paints a new picture of sexual fluids, including nectar, as a foundational component of gymnosperm reproductive evolution. We review the morpho-anatomical adaptations, their accompanying secretions, and the functional compounds involved. We discuss two types of secretions: (1) those involved in fertilization fluids produced by gametophytes and archegonia of zooidogamous gymnosperms, i.e., Ginkgo and cycads, and (2) those involved in pollen capture mechanisms (PCMs), i.e., pollination drops. Fertilization fluids provide both liquid in which sperm swim, as well as chemotactic signals that direct sperm to the egg. Such fertilization fluids were probably found among many extinct plants such as ancient cycads and others with swimming sperm, but were subsequently lost upon the evolution of siphonogamy (direct delivery of sperm to the egg by pollen tubes), as found in modern gnetophytes, conifers, and Pinaceae. Pollination drops are discussed in terms of three major types of PCMs and the unique combinations of morphological and biochemical adaptations that define each. These include their amino acids, sugars, calcium, phosphate and proteins. The evolution of PCMs is also discussed with reference to fossil taxa. The plesiomorphic state of extant gymnosperms is a sugar-containing pollination drop functioning as a pollen capture surface, and an in ovulo pollen germination medium. Additionally, these drops are involved in ovule defense, and provide nectar for pollinators. Pollination drops in anemophilous groups have low sugar concentrations that are too low to provide insects with a reward. Instead, they appear to be optimized for defense and microgametophyte development. In insect-pollinated modern Gnetales a variety of tissues produce sexual fluids that bear the biochemical signature of nectar. Complete absence of fluid secretions is restricted to a few, poorly studied modern conifers, and is presumably derived. Aspects of pollination drop dynamics, e.g., regulation of secretion and retraction, are reviewed. Lastly, we discuss pollination drops' control of pollen germination. Large gaps in our current knowledge include the composition of fertilization fluids, the pollination drops of Podocarpaceae, and the overall hydrodynamics of sexual fluids in general.

Programmed cell death in seeds of angiosperms.

During the diversification of angiosperms, seeds have evolved structural, chemical, molecular and physiologically developing changes that specially affect the nucellus and endosperm. All through seed evolution, programmed cell death (PCD) has played a fundamental role. However, examples of PCD during seed development are limited. The present review examines PCD in integuments, nucellus, suspensor and endosperm in those representative examples of seeds studied to date.

Use of diffusion magnetic resonance imaging to correlate the developmental changes in grape berry tissue structure with water diffusion patterns.

BACKGROUND: Over the course of grape berry development, the tissues of the berry undergo numerous morphological transformations in response to processes such as water and solute accumulation and cell division, growth and senescence. These transformations are expected to produce changes to the diffusion of water through these tissues detectable using diffusion magnetic resonance imaging (MRI). To assess this non-invasive technique diffusion was examined over the course of grape berry development, and in plant tissues with contrasting oil content. RESULTS: In this study, the fruit of Vitis vinfera L. cv. Semillon at seven different stages of berry development, from four weeks post-anthesis to over-ripe, were imaged using diffusion tensor and transverse relaxation MRI acquisition protocols. Variations in diffusive motion between these stages of development were then linked to known events in the morphological development of the grape berry. Within the inner mesocarp of the berry, preferential directions of diffusion became increasingly apparent as immature berries increased in size and then declined as berries progressed through the ripening and senescence phases. Transverse relaxation images showed radial striation patterns throughout the sub-tissue, initiating at the septum and vascular systems located at the centre of the berry, and terminating at the boundary between the inner and outer mesocarp. This study confirms that these radial patterns are due to bands of cells of alternating width that extend across the inner mesocarp. Preferential directions of diffusion were also noted in young grape seed nucelli prior to their dehydration. These observations point towards a strong association between patterns of diffusion within grape berries and the underlying tissue structures across berry development. A diffusion tensor image of a post-harvest olive demonstrated that the technique is applicable to tissues with high oil content. CONCLUSION: This study demonstrates that diffusion MRI is a powerful and information rich technique for probing the internal microstructure of plant tissues. It was shown that macroscopic diffusion anisotropy patterns correlate with the microstructure of the major pericarp tissues of cv. Semillon grape berries, and that changes in grape berry tissue structure during berry development can be observed.