The Role of Anaphase-Promoting Complex/Cyclosome (APC/C) in Plant Reproduction

Most eukaryotic species propagate through sexual reproduction that requires male and female gametes. In flowering plants, it starts through a single round of DNA replication (S phase) and two consecutive chromosome segregation (meiosis I and II). Subsequently, haploid mitotic divisions occur, which results in a male gametophyte (pollen grain) and a female gametophyte (embryo sac) formation. In order to obtain viable gametophytes, accurate chromosome segregation is crucial to ensure ploidy stability. A precise gametogenesis progression is tightly regulated in plants and is controlled by multiple mechanisms to guarantee a correct evolution through meiotic cell division and sexual differentiation. In the past years, research in the field has shown an important role of the conserved E3-ubiquitin ligase complex, Anaphase-Promoting Complex/Cyclosome (APC/C), in this process. The APC/C is a multi-subunit complex that targets proteins for degradation via proteasome 26S. The functional characterization of APC/C subunits in Arabidopsis, which is one of the main E3 ubiquitin ligase that controls cell cycle, has revealed that all subunits investigated so far are essential for gametophytic development and/or embryogenesis.

Gametophyte and embryonic ontogeny: understanding the reproductive calendar of Cypripedium japonicum Thunb. (Cypripedoideae, Orchidaceae), a lady’s slipper orchid endemic to East Asia

Background The genus Cypripedium L. is one of the five genera of the subfamily Cypripedioideae, members of which are commonly known as lady’s slipper orchids. Cypripedium japonicum is a perennial herb native to East Asia, specifically China, Japan, and Korea. Due to its limited distribution, the species is included in the Endangered category of the IUCN Red List. Results We investigated gametophyte development, including complete embryogenesis, in C. japonicum. The complete reproductive cycle is presented based on our observations. Anther development begins under the soil, and meiosis of pollen mother cells begins 3 weeks before anthesis, possibly during early April. The megaspore mother cells develop just after pollination in early May and mature in mid–late June. The pattern of embryo sac formation is bisporic, and there are six nuclei: three forming the egg apparatus, two polar nuclei, and an antipodal cell in the mature embryo sac. Triple fertilization results in the endosperm nucleus, which degenerates when the proembryo reaches the eight-to-sixteen-cell stage. Conclusion Our overall comparisons of the features of gametophyte and embryo development in C. japonicum suggest that previous reports on the embryology of Cypripedium are not sufficient for characterization of the entire genus. Based on the available information, a reproductive calendar showing the key reproductive events leading to embryo formation has been prepared.

Gibberellin-mediated RGA-LIKE1 degradation regulates embryo sac development in Arabidopsis

Ovule development is essential for plant survival, as it allows correct embryo and seed development upon fertilization. The female gametophyte is formed in the central area of the nucellus during ovule development, in a complex developmental programme that involves key regulatory genes and the plant hormones auxins and brassinosteroids. Here we provide novel evidence of the role of gibberellins (GAs) in the control of megagametogenesis and embryo sac development, via the GA-dependent degradation of RGA-LIKE1 (RGL1) in the ovule primordia. YPet-rgl1Δ17 plants, which express a dominant version of RGL1, showed reduced fertility, mainly due to altered embryo sac formation that varied from partial to total ablation. YPet-rgl1Δ17 ovules followed normal development of the megaspore mother cell, meiosis, and formation of the functional megaspore, but YPet-rgl1Δ17 plants had impaired mitotic divisions of the functional megaspore. This phenotype is RGL1-specific, as it is not observed in any other dominant mutants of the DELLA proteins. Expression analysis of YPet-rgl1Δ17 coupled to in situ localization of bioactive GAs in ovule primordia led us to propose a mechanism of GA-mediated RGL1 degradation that allows proper embryo sac development. Taken together, our data unravel a novel specific role of GAs in the control of female gametophyte development.

Gametophytes and embryo ontogeny: understanding the reproductive calendar of Cypripedium japonicum Thunb. (Cypripedoideae, Orchidaceae)

Among the flowering plants, the gametophyte development and reproductive biology of orchids is particularly poorly understood. Cypripedium japonicum is a perennial herb, native to East Asia. Due to its limited distribution, the species is included in the Endangered category of the IUCN Red List. Light microscopy and SEM methods were used to study the development of the gametes and embryo. The complete reproductive cycle was developed based on our observations. Anther development begins under the soil and meiosis of pollen cells begins 3 weeks before anthesis, possibly during early April. The megaspore mother cells develop just after pollination in early May and mature in mid–late June. The pattern of embryo sac formation is bisporic and there are six nuclei. Triple fertilization results in the endosperm nucleus. A globular embryo is formed after multiple cell division and 9 weeks after pollination the entire embryo sac is occupied by embryo. Overall comparisons of the features of gametophyte and embryo development in C. japonicum suggest that previous reports on the embryology of Cypripedium are not sufficient to characterize the entire genus. Based on the available information a reproductive calendar showing the key reproductive events leading to embryo formation has been prepared.HighlightManual pollination, reproductive biology and seed development process in Cypripedium japonicum Thunb., a lady’s slipper orchid endemic to East Asia

Embryology in Helosis cayennensis (Balanophoraceae): Structure of Female Flowers, Fruit, Endosperm and Embryo

Helosis cayennensis (Balanophoraceae s.str.) is a holoparasite characterised by aberrant vegetative bodies and tiny, reduced unisexual flowers. Here, we analysed the development of female flowers to elucidate their morpho-anatomy and the historical controversy on embryo sac formation. We also studied the developmental origin of inflorescences and the ontogeny of fruits, embryo and endosperm and discussed in a phylogenetic framework. Inflorescences were analysed by optical, fluorescence and scanning electron microscopy. Inflorescences of H. cayennensis arise endogenously. Female flowers lack perianth organs, thus only consist of the ovary, two styles and stigmata. Ovules are undifferentiated; two megaspore mother cells develop inside a nucellar complex. The female gametophyte, named Helosis-type, is a bisporic four-celled embryo sac, provided with a typical egg apparatus and a uni-nucleated central cell. Fertilization was not observed, yet a few-celled embryo and cellular endosperm developed. In sum, results confirm that, among Santalales holoparasites, Helosis is intermediate in the reduction series of its floral organs. Although perianth absence best supports the Balanophoraceae s.str. clade, our literature survey on female flower developmental data across Balanophoraceae s.l. highlights the many gaps that need to be filled to really understand these features in the light of new phylogenetic relationships.

Megaspore competition in F1 and F2 hybrids between Oenothera hookeri and Oe. suaveolens

Megasporogenesis and development of the embryo sac were investigated in F₁ and F₂ hybrids from crosses of <em>Oe. hookeri</em> and <em>Oe. suaveolens</em>. All hybrids form heteropolar and homopolar magaspore tetrads; the embryo sac, however, usually develops from the micropylar megaspore. Its development may occur immediately after degeneration of three other megaspores or after a period of competition between both apical megaspores. They develop simultaneously for a relatively short time, after which the growth of the chalazal megaspore is inhibited, although the latter does not degenerate. The micropylar megaspore as a rule develops without disturbances into the embryo sac, but in some ovules it is formed from the chalazal megaspore or double ones arise from both apical megaspores of the tetrad. The frequency of the micropylar embryo sac formation seems to be dependent above all on the hybrid plant genome and not on the haploid genome of the megaspore.

Factors affecting the production of seeds in fully fertile tomatoes (Lycopersicon esculentum L. Mill.) and those showing a tendency to parthenocarpy

Comparative studies on the development of the female gametophyte, pollination and fertilization in two lines of <em>Lycopersicon esculentum</em>, Kholodostoykye (Kh, fertile) and A33 (with a tendency to parthenocarpy) have revealed that seed production is affected by disturbances in embryo sac formation but mainly by its degeneration after anthesis, which is especially visible in line A33. Moreover, delayed development of some embryo sacs and incomplete pollination due to various stigma levels seem to be responsible for the diminution of seed number in line A33. Deep fluorescence of numerous pollen grains as well as whole pollen tubes in 83.3 per cent of A33 stigmas and only 24.1 per cent in the Kh line points to the heterogeneity of pollen. This could be one more reason for reduced fertility. The results of application of plant growth regulators (auxin, PCIB) which affect seed production in tomato of line A33 remain inconclusive.

Megasporogenesis and early megagametogenesis

Qualitative and quantitative data on organelle distribution during megasporogenesis and megagametogenesis were discussed. Megaspocyte and megaspore ultrastructural and cytochemical characters were related to developmental stages and embryo sac formation. The role of nucellus was considered