A fossil ovule with wind dispersal mechanisms and a probable micropyle.

Anemochory (wind dispersal) represents a key dispersal strategy that has allowed seed plants (spermatophytes) to expand habitats. To facilitate anemochory, the spermatophytes use diverse wind dispersal mechanisms, including wings or plumes of the ovule or seed1,2. Seed wings are integument outgrowths of an ovule, while seed plumes refer to a bundle of filaments. These two wind dispersal mechanisms are independently employed by many seed plants1,2, but are very rarely combined in a single taxon. Seed wings originated in the Late Devonian (Famennian)3,4,5 while seed plumes first appeared in the Early Carboniferous (Mississippian)6,7. An opening, or micropyle, in the integument characterizes the ovules of seed plants7. This essential structure had not appeared until the Carboniferous, and it evolved through the gradual fusion of integumentary lobes7,8. Famennian ovules demonstrate great diversity in integumentary lobes. Nevertheless, notably few of these earliest ovules exhibit wings or wing-like integumentary lobes and none possess plumes or micropyles. Here, we document a Famennian ovule Gnetopsis quadria sp. nov. from Anhui Province, China. It combines wing-like lobes and plumes as two dispersal mechanisms, and probably has a micropyle.

Pollination efficiency and the pollen-ovule ratio.

Pollination presents a risky journey for pollen grains. Pollen loss is sometimes thought to favour greater pollen investment to compensate for the inefficiency of transport. Sex allocation theory, to the contrary, has consistently concluded that postdispersal loss should have no selective effect on investment in either sex function. But the intuitively appealing compensation idea continues to be raised despite the lack of theoretical endorsement. We address the theoretical issue with a model that directly represents pollen loss (and ovule loss through floral demise or loss of receptivity) as rate-dependent dynamical processes. These loss rates can be varied to examine the effect of pollination efficiency on optimal sex allocation. Pollen-ovule ratios follow from the sex allocation based on the resource costs of pollen and ovule production. This model confirms conventional findings that pollen loss should have essentially no effect on sexual resource allocation in large, panmictic populations. Pollen limitation of seed set does not alter this conclusion. These results force us to rethink the empirical association of pollination efficiency with low pollen-ovule ratios. This pattern could arise if efficient pollen transport commonly results in stigmatic deposition of cohorts of related pollen. Empirical evidence of correlated paternity supports this explanation.

Deep imaging reveals dynamics and signaling in one-to-one pollen tube guidance.

In the pistil of flowering plants, each ovule usually associates with a single pollen tube for fertilization. This one-to-one pollen tube guidance, which contributes to polyspermy blocking and efficient seed production, is largely different from animal chemotaxis of many sperms to one egg. However, the functional mechanisms underlying the directional cues and polytubey blocks in the depths of the pistil remain unknown. Here, we develop a two-photon live imaging method to directly observe pollen tube guidance in the pistil of Arabidopsis thaliana, clarifying signaling and cellular behaviors in the one-to-one guidance. Ovules are suggested to emit multiple signals for pollen tubes, including an integument-dependent directional signal that reaches the inner surface of the septum and adhesion signals for emerged pollen tubes on the septum. Not only FERONIA in the septum but ovular gametophytic FERONIA and LORELEI, as well as FERONIA- and LORELEI-independent repulsion signal, are involved in polytubey blocks on the ovular funiculus. However, these funicular blocks are not strictly maintained in the first 45 min, explaining previous reports of polyspermy in flowering plants.

Life table parameters of Amblyseius largoensis, Amblyseius swirskii and Proprioseiopsis lenis (Acari: Phytoseiidae) fed on eggs and larvae of Frankliniella occidentalis.

The immature development and reproduction of the predatory mites Amblyseius largoensis (Muma), Proprioseiopsis lenis (Corpuz and Rimando), and Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae) were investigated using both thrips eggs and first instars of the western flower thrips, Frankliniella occidentalis Pergande, as prey in a controlled laboratory environment at 25 °C and 60% relative humidity. When provided with thrips eggs as food, A. largoensis exhibited a notably shorter immature development period for both males (7.05 days) and females (6.51 days) as compared with A. swirskii (8.05 and 7.19 days, respectively) and P. lenis (8.10 days and 7.05 days, respectively). Amblyseius largoensis also displayed a higher oviposition rate (2.19 eggs/female/day) than A. swirskii and P. lenis (1.79 and 1.78 eggs/female/day, respectively). Moreover, it exhibited the highest fecundity (25.34 eggs/female), followed by P. lenis (24.23 eggs/female) and A. swirskii (22.86 eggs/female). These variations led to A. largoensis having the highest intrinsic rate of increase (rm) at 0.209, followed by A. swirskii at 0.188, and P. lenis at 0.165. However, when the predatory mites were provided with first instars of F. occidentalis, A. swirskii demonstrated a faster immature development period for both males (7.67 days) and females (7.59 days) as compared with P. lenis (9.00 days and 7.86 days, respectively) and A. largoensis (8.47 days and 8.61 days, respectively). While the oviposition rates of P. lenis (1.92 eggs/female/day) and A. swirskii (1.90 eggs/female/day) were similar when feeding on this prey, A. largoensis produced fewer eggs (1.83 eggs/female/day). Further, A. swirskii exhibited the highest fecundity (31.93 eggs/female), followed by A. largoensis (25.71 eggs/female) and P. lenis (23 eggs/female). Consequently, the intrinsic rate of increase (rm) on thrips first instars was highest in A. swirskii (0.190), followed by A. largoensis (0.186), and P. lenis (0.176). In summary, our findings indicate that in terms of life history parameters A. largoensis performs optimally when feeding on thrips eggs, whereas A. swirskii performs best when preying on the mobile first instars of the thrips. These insights into the dietary preferences and reproductive capabilities of the studied predatory mite species have important implications for their potential use as biological control agents against F. occidentalis in agricultural settings.

Development of pollinated and unpollinated ovules in Ginkgo biloba: unravelling the role of pollen in ovule tissue maturation.

In gymnosperms such as Ginkgo biloba, the arrival of pollen plays a key role in ovule development, before fertilization occurs. Accordingly, G. biloba female plants geographically isolated from male plants abort all their ovules after the pollination drop emission, which is the event that allows the ovule to capture pollen grains. To decipher the mechanism induced by pollination required to avoid ovule senescence and then abortion, we compared the transcriptomes of pollinated and unpollinated ovules at three time points after the end of the emission of pollination drop. Transcriptomic and in situ expression analyses revealed that several key genes involved in programmed cell death such as senescence and apoptosis, DNA replication, and cell cycle regulation were differentially expressed in unpollinated ovules compared to pollinated ovules. We provide evidence that the pollen captured by the pollination drop affects auxin local accumulation and might cause deregulation of key genes required for the ovule's programmed cell death, activating both the cell cycle regulation and DNA replication genes.

JAGGER localization and function are dependent on GPI anchor addition.

KEY MESSAGE: GPI anchor addition is important for JAGGER localization and in vivo function. Loss of correct GPI anchor addition in JAGGER, negatively affects its localization and function. In flowering plants, successful double fertilization requires the correct delivery of two sperm cells to the female gametophyte inside the ovule. The delivery of a single pair of sperm cells is achieved by the entrance of a single pollen tube into one female gametophyte. To prevent polyspermy, Arabidopsis ovules avoid the attraction of multiple pollen tubes to one ovule-polytubey block. In Arabidopsis jagger mutants, a significant number of ovules attract more than one pollen tube to an ovule due to an impairment in synergid degeneration. JAGGER encodes a putative arabinogalactan protein which is predicted to be anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Here, we show that JAGGER fused to citrine yellow fluorescent protein (JAGGER-cYFP) is functional and localizes mostly to the periphery of ovule integuments and transmitting tract cells. We further investigated the importance of GPI-anchor addition domains for JAGGER localization and function. Different JAGGER proteins with deletions in predicted ω-site regions and GPI attachment signal domain, expected to compromise the addition of the GPI anchor, led to disruption of JAGGER localization in the cell periphery. All JAGGER proteins with disrupted localization were also not able to rescue the polytubey phenotype, pointing to the importance of GPI-anchor addition to in vivo function of the JAGGER protein.

Architectural effects regulate resource allocation within the inflorescences with nonlinear blooming patterns.

PREMISE: Spatial and temporal resource allocations within inflorescences have been well-studied in many plants based on flowering sequence or floral position. However, there had been few attempts to investigate architectural effects and resource competition in species where the blooming pattern does not follow a linear positional pattern within the inflorescence. Moreover, most flowering plants show female-biased sex allocation in early or basal flowers, but it is unclear in species with inherent and changeless ovule production. METHODS: We investigated intra-inflorescence variation in reproductive traits of Salvia przewalskii, a perennial herb with 4-ouvle ovary flowers and flowering sequence-floral position decoupled inflorescences. To detect the effects of resource competition and architectural effects on reproductive success, we manipulated inflorescence (removed floral buds by position and flowering sequence) and pollination (opened and supplemented pollination). RESULTS: Pollen production and dry mass deceased from bottom to top flowers but did not significantly differ following flowering sequence, resulting in male-biased sex allocation in basal flowers. The seed production, fruit set, and bud development exhibited significant declining trends from proximal to distal positions regardless of the thinning and pollen treatments. Meanwhile, the seed production, fruit set, and bud development success did not significant differ when thinning was conducted according to flowering sequence. CONCLUSIONS: Architectural effects plays a crucial role in resource allocation within decoupled flowering inflorescences. Moreover, our results highlighted that inherent floral traits such as changeless ovule production, may modify architectural effects on sex allocation.

Screening of Differentially Expressed Genes and Localization Analysis of Female Gametophyte at the Free Nuclear Mitosis Stage in Pinus tabuliformis Carr.

Female sterility is a common phenomenon in the plant world, and systematic research has not been carried out in gymnosperms. In this study, the ovules of No. 28 sterile line and No. 15 fertile line Pinus tabuliformis were used as materials, and a total of 18 cDNA libraries were sequenced by the HiSeqTM 4000 platform to analyze the differentially expressed genes (DEGs) and simple sequence repeats (SSRs) between the two lines. In addition, this study further analyzed the DEGs involved in the signal transduction of plant hormones, revealing that the signal pathways related to auxin, cytokinin, and gibberellin were blocked in the sterile ovule. Additionally, real-time fluorescent quantitative PCR verified that the expression trend of DEGs related to plant hormones was consistent with the results of high-throughput sequencing. Frozen sections and fluorescence in situ hybridization (FISH) were used to study the temporal and spatial expression patterns of PtRab in the ovules of P. tabuliformis. It was found that PtRab was significantly expressed in female gametophytes and rarely expressed in the surrounding diploid tissues. This study further explained the molecular regulation mechanism of female sterility in P. tabuliformis, preliminarily mining the key factors of ovule abortion in gymnosperms at the transcriptional level.

RALF peptide signaling controls the polytubey block in Arabidopsis.

Fertilization of an egg by multiple sperm (polyspermy) leads to lethal genome imbalance and chromosome segregation defects. In Arabidopsis thaliana, the block to polyspermy is facilitated by a mechanism that prevents polytubey (the arrival of multiple pollen tubes to one ovule). We show here that FERONIA, ANJEA, and HERCULES RECEPTOR KINASE 1 receptor-like kinases located at the septum interact with pollen tube-specific RALF6, 7, 16, 36, and 37 peptide ligands to establish this polytubey block. The same combination of RALF (rapid alkalinization factor) peptides and receptor complexes controls pollen tube reception and rupture inside the targeted ovule. Pollen tube rupture releases the polytubey block at the septum, which allows the emergence of secondary pollen tubes upon fertilization failure. Thus, orchestrated steps in the fertilization process in Arabidopsis are coordinated by the same signaling components to guarantee and optimize reproductive success.

RH17 restricts reproductive fate and represses autonomous seed coat development in sexual Arabidopsis.

Plant sexual and asexual reproduction through seeds (apomixis) is tightly controlled by complex gene regulatory programs, which are not yet fully understood. Recent findings suggest that RNA helicases are required for plant germline development. This resembles their crucial roles in animals, where they are involved in controlling gene activity and the maintenance of genome integrity. Here, we identified previously unknown roles of Arabidopsis RH17 during reproductive development. Interestingly, RH17 is involved in repression of reproductive fate and of elements of seed development in the absence of fertilization. In lines carrying a mutant rh17 allele, development of supernumerary reproductive cell lineages in the female flower tissues (ovules) was observed, occasionally leading to formation of two embryos per seed. Furthermore, seed coat, and putatively also endosperm development, frequently initiated autonomously. Such induction of several features phenocopying distinct elements of apomixis by a single mutation is unusual and suggests that RH17 acts in regulatory control of plant reproductive development. Furthermore, an in-depth understanding of its action might be of use for agricultural applications.

Meta-analysis of RNA-Seq studies reveals genes with dominant functions during flower bud endo- to eco-dormancy transition in Prunus species.

In deciduous fruit trees, entrance into dormancy occurs in later summer/fall, concomitantly with the shortening of day length and decrease in temperature. Dormancy can be divided into endodormancy, ecodormancy and paradormancy. In Prunus species flower buds, entrance into the dormant stage occurs when the apical meristem is partially differentiated; during dormancy, flower verticils continue their growth and differentiation. Each species and/or cultivar requires exposure to low winter temperature followed by warm temperatures, quantified as chilling and heat requirements, to remove the physiological blocks that inhibit budburst. A comprehensive meta-analysis of transcriptomic studies on flower buds of sweet cherry, apricot and peach was conducted, by investigating the gene expression profiles during bud endo- to ecodormancy transition in genotypes differing in chilling requirements. Conserved and distinctive expression patterns were observed, allowing the identification of gene specifically associated with endodormancy or ecodormancy. In addition to the MADS-box transcription factor family, hormone-related genes, chromatin modifiers, macro- and micro-gametogenesis related genes and environmental integrators, were identified as novel biomarker candidates for flower bud development during winter in stone fruits. In parallel, flower bud differentiation processes were associated to dormancy progression and termination and to environmental factors triggering dormancy phase-specific gene expression.

Organ geometry channels reproductive cell fate in the Arabidopsis ovule primordium.

In multicellular organisms, sexual reproduction requires the separation of the germline from the soma. In flowering plants, the female germline precursor differentiates as a single spore mother cell (SMC) as the ovule primordium forms. Here, we explored how organ growth contributes to SMC differentiation. We generated 92 annotated 3D images at cellular resolution in Arabidopsis. We identified the spatio-temporal pattern of cell division that acts in a domain-specific manner as the primordium forms. Tissue growth models uncovered plausible morphogenetic principles involving a spatially confined growth signal, differential mechanical properties, and cell growth anisotropy. Our analysis revealed that SMC characteristics first arise in more than one cell but SMC fate becomes progressively restricted to a single cell during organ growth. Altered primordium geometry coincided with a delay in the fate restriction process in katanin mutants. Altogether, our study suggests that tissue geometry channels reproductive cell fate in the Arabidopsis ovule primordium.

Formins control dynamics of F-actin in the central cell of Arabidopsis thaliana.

In the female gamete of flowering plants, sperm nuclear migration is controlled by a constant inward movement of actin filaments (F-actin) for successful fertilization. This dynamic F-actin movement is ARP2/3-independent, raising the question of how actin nucleation and polymerization is controlled in the female gamete. Using confocal microscopy live-cell imaging in combination with a pharmacological approach, we assessed the involvement of another group of actin nucleators, formins, in F-actin inward movement in the central cell of Arabidopsis thaliana. We identify that the inhibition of the formin function, by formin inhibitor SMIFH2, significantly reduced the dynamic inward movement of F-actin in the central cell, indicating that formins play a major role in actin nucleation required for F-actin inward movement in the central cell.

Paving the Way for Fertilization: The Role of the Transmitting Tract.

Angiosperm reproduction relies on the precise growth of the pollen tube through different pistil tissues carrying two sperm cells into the ovules' embryo sac, where they fuse with the egg and the central cell to accomplish double fertilization and ultimately initiate seed development. A network of intrinsic and tightly regulated communication and signaling cascades, which mediate continuous interactions between the pollen tube and the sporophytic and gametophytic female tissues, ensures the fast and meticulous growth of pollen tubes along the pistil, until it reaches the ovule embryo sac. Most of the pollen tube growth occurs in a specialized tissue-the transmitting tract-connecting the stigma, the style, and the ovary. This tissue is composed of highly secretory cells responsible for producing an extensive extracellular matrix. This multifaceted matrix is proposed to support and provide nutrition and adhesion for pollen tube growth and guidance. Insights pertaining to the mechanisms that underlie these processes remain sparse due to the difficulty of accessing and manipulating the female sporophytic tissues enclosed in the pistil. Here, we summarize the current knowledge on this key step of reproduction in flowering plants with special emphasis on the female transmitting tract tissue.

Fine mapping and candidate gene analysis of a major locus controlling ovule abortion and seed number per silique in Brassica napus L.

KEY MESSAGE: A major QTL controlling ovule abortion and SN was fine-mapped to a 80.1-kb region on A8 in rapeseed, and BnaA08g07940D and BnaA08g07950D are the most likely candidate genes. The seed number per silique (SN), an important yield determining trait of rapeseed, is the final consequence of a complex developmental process including ovule initiation and the subsequent ovule/seed development. To explore the genetic mechanism regulating the natural variation of SN and its related components, quantitative trait locus (QTL) mapping was conducted using a doubled haploid (DH) population derived from the cross between C4-146 and C4-58B, which showed significant differences in SN and aborted ovule number (AON), but no obvious differences in ovule number (ON). QTL analysis identified 19 consensus QTLs for six SN-related traits across three environments. A novel QTL on chromosome A8, un.A8, which associates with multiple traits, except for ON, was stably detected across the three environments. This QTL explained more than 50% of the SN, AON and percentage of aborted ovules (PAO) variations as well as a moderate contribution on silique length (SL) and thousand seed weight (TSW). The C4-146 allele at the locus increases SN and SL but decreases AON, PAO and TSW. Further fine mapping narrowed down this locus into an 80.1-kb interval flanked by markers BM1668 and BM1672, and six predicted genes were annotated in the delimited region. Expression analyses and DNA sequencing showed that two homologs of Arabidopsis photosystem I subunit F (BnaA08g07940D) and zinc transporter 10 precursor (BnaA08g07950D) were the most promising candidate genes underlying this locus. These results provide a solid basis for cloning un.A8 to reduce the ovule abortion and increase SN in the yield improvement of rapeseed.

AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube guidance.

Reproductive isolation is a prerequisite to form and maintain a new species. Multiple prezygotic and postzygotic reproductive isolation barriers have been reported in plants. In the model plant, Arabidopsis thaliana conspecific pollen tube precedence controlled by AtLURE1/PRK6-mediated signaling has been recently reported as a major prezygotic reproductive isolation barrier. By accelerating emergence of own pollen tubes from the transmitting tract, A. thaliana ovules promote self-fertilization and thus prevent fertilization by a different species. Taking advantage of a septuple atlure1null mutant, we now report on the role of AtLURE1/PRK6-mediated signaling for micropylar pollen tube guidance. Compared with wild-type (WT) ovules, atlure1null ovules displayed remarkably reduced micropylar pollen tube attraction efficiencies in modified semi-in vivo A. thaliana ovule targeting assays. However, when prk6 mutant pollen tubes were applied, atlure1null ovules showed micropylar attraction efficiencies comparable to that of WT ovules. These findings indicate that AtLURE1/PRK6-mediated signaling regulates micropylar pollen tube attraction in addition to promoting emergence of own pollen tubes from the transmitting tract. Moreover, semi-in vivo ovule targeting competition assays with the same amount of pollen grains from both A. thaliana and Arabidopsis lyrata showed that A. thaliana WT and xiuqiu mutant ovules are mainly targeted by own pollen tubes and that atlure1null mutant ovules are also entered to a large extent by A. lyrata pollen tubes. Taken together, we report that AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube attraction representing an additional prezygotic isolation barrier.

Role of ethylene in the regulatory mechanism underlying the abortion of ovules after fertilization in Xanthoceras sorbifolium.

KEY MESSAGE: Genes related to the MAPK cascade, ethylene signaling pathway, Pi starvation response, and NAC TFs were differentially expressed between normal and abortive ovules. Receptor-mediated ethylene signal perception and transmission play an important role in regulating fruit and ovule development. Xanthoceras sorbifolium, a small to medium-sized tree endemic to northern China, is an emerging dedicated oilseed crop designed for applications in advanced biofuel, engine oil, and functional food, as well as for pharmaceutical and cosmetic applications. Despite the importance of Xanthoceras seed oil, low seed productivity has constricted commercial exploitation of the species. The abortion of developing seeds (ovules after fertilization) is a major factor limiting fruit and seed production in the plant. To increase fruit and seed yields, a better understanding of the mechanisms underlying the abortion of fertilized ovules is critical. This study revealed differences in nucellus degeneration, endosperm development, and starch grain content between normally and abnormally developing ovules after fertilization. We constructed 6 RNA-sequencing (RNA-seq) libraries from normally and abnormally developing ovules at the onset of their abortion process. Comparative transcriptome analysis between the normal and abnormal ovules identified 818 differentially expressed genes (DEGs). Among DEGs, many genes involved in mitogen-activated protein kinase (MAPK) cascades, ethylene signaling pathway, and NAC transcription factor genes showed up-regulated expression in abnormal ovules. The RNA-seq data were validated using quantitative reverse-transcription PCR. Using virus-induced gene silencing (VIGS) methods, evaluation of an ethylene receptor gene (XsERS) function indicated that the gene was closely related to early development of fruits and seeds. Based on the data presented here, we propose a model for a MAPK-ethylene signaling-NAC2 gene regulatory cascade that plays an important role in the regulation of the ovule abortion process in X. sorbifolium. The present study is imperative for understanding the mechanisms of ovule abortion after fertilization and identifying the critical genes and gene networks involved in determining the fate of ovule development.

Regulation of Female Germline Specification via Small RNA Mobility in Arabidopsis.

In the ovules of most sexually reproducing plants, one hypodermal cell differentiates into a megaspore mother cell (MMC), which gives rise to the female germline. Trans-acting small interfering RNAs known as tasiR-ARFs have been suggested to act non-cell-autonomously to prevent the formation of multiple MMCs by repressing AUXIN RESPONSE FACTOR3 (ARF3) expression in Arabidopsis (Arabidopsis thaliana), but the underlying mechanisms are unknown. Here, we examined tasiR-ARF-related intercellular regulatory mechanisms. Expression analysis revealed that components of the tasiR-ARF biogenesis pathway are restricted to distinct ovule cell types, thus limiting tasiR-ARF production to the nucellar epidermis. We also provide data suggesting tasiR-ARF movement along the mediolateral axis into the hypodermal cells and basipetally into the chalaza. Furthermore, we used cell type-specific promoters to express ARF3m, which is resistant to tasiR-ARF regulation, in different ovule cell layers. ARF3m expression in hypodermal cells surrounding the MMC, but not in epidermal cells, led to a multiple-MMC phenotype, suggesting that tasiR-ARFs repress ARF3 in these hypodermal cells to suppress ectopic MMC fate. RNA sequencing analyses in plants with hypodermally expressed ARF3m showed that ARF3 potentially regulates MMC specification through phytohormone pathways. Our findings uncover intricate spatial restriction of tasiR-ARF biogenesis, which together with tasiR-ARF mobility enables cell-cell communication in MMC differentiation.

A Toolkit for Teasing Apart the Early Stages of Pollen-Stigma Interactions in Arabidopsis thaliana.

In hermaphroditic flowering plants, the female pistil serves as the main gatekeeper of mate acceptance as several mechanisms are present to prevent fertilization by unsuitable pollen. The characteristic Brassicaceae dry stigma at the top of pistil represents the first layer that requires pollen recognition to elicit appropriate physiological responses from the pistil. Successful pollen-stigma interactions then lead to pollen hydration, pollen germination, and pollen tube entry into the stigmatic surface. To assess these early stages in detail, our lab has used three experimental procedures to quantitatively and qualitatively characterize the outcome of compatible pollen-stigma interactions that would ultimately lead to the successful fertilization. These assays are also useful for assessing self-incompatible pollinations and mutations that affect these pathways. The model organism, Arabidopsis thaliana, offers an excellent platform for these investigations as loss-of-function or gain-of-function mutants can be easily generated using CRISPR/Cas9 technology, existing T-DNA insertion mutant collections, and heterologous expression constructs, respectively. Here, we provide a detailed description of the methods for these inexpensive assays that can be reliably used to assess pollen-stigma interactions and used to identify new players regulating these processes.