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.

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.

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.

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.

Workflow to Characterize Mutants with Reproductive Defects.

Reverse genetics approaches for characterizing phenotypes of mutants in a gene of interest (GOI) require thorough genotyping and phenotypic analysis. However, special challenges are encountered when a GOI is expressed in reproductive tissues: a variety of assays are required to characterize the phenotype and a mutant may show sporophytic and/or gametophytic defects in male and/or female reproductive tissues, which are structurally and functionally intertwined. Here, we present a streamlined workflow to characterize mutants with reproductive defects, primarily using Arabidopsis as a model, which can also be adapted to characterize mutants in other flowering plants. Procedures described here can be used to distinguish different kinds of reproductive defects and pinpoint the defective reproductive step(s) in a mutant. Although our procedures emphasize the characterization of mutants with male reproductive defects, they can nevertheless be used to identify female reproductive defects, as those defects could manifest alongside, and sometimes require, male reproductive tissues.

Fertilization in flowering plants: an odyssey of sperm cell delivery.

KEY MESSAGE: In light of the available discoveries in the field, this review manuscript discusses on plant reproduction mechanism and molecular players involved in the process. Sperm cells in angiosperms are immotile and are physically distant to the female gametophytes (FG). To secure the production of the next generation, plants have devised a clever approach by which the two sperm cells in each pollen are safely delivered to the female gametophyte where two fertilization events occur (by each sperm cell fertilizing an egg cell and central cell) to give rise to embryo and endosperm. Each of the successfully fertilized ovules later develops into a seed. Sets of macromolecules play roles in pollen tube (PT) guidance, from the stigma, through the transmitting tract and funiculus to the micropylar end of the ovule. Other sets of genetic players are involved in PT reception and in its rupture after it enters the ovule, and yet other sets of genes function in gametic fusion. Angiosperms have come long way from primitive reproductive structure development to today's sophisticated, diverse, and in most cases flamboyant organ. In this review, we will be discussing on the intricate yet complex molecular mechanism of double fertilization and how it might have been shaped by the evolutionary forces focusing particularly on the model plant Arabidopsis.

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization.

Flowering plants have a unique sexual reproduction system called 'double fertilization', in which each of the sperm cells precisely fuses with an egg cell or a central cell. Thus, two independent fertilization events take place almost simultaneously. The fertilized egg cell and central cell develop into zygote and endosperm, respectively. Therefore, precise control of double fertilization is essential for the ensuing seed development. Double fertilization occurs in the female gametophyte (embryo sac), which is deeply hidden and covered with thick ovule and ovary tissues. This pistil tissue construction makes observation and analysis of double fertilization quite difficult and has created the present situation in which many questions regarding the mechanism of double fertilization remain unanswered. For the functional evaluation of a potential candidate for fertilization regulator, phenotypic analysis of fertilization is important. To judge the completion of fertilization in Arabidopsis thaliana, the shapes of fluorescence signals labeling sperm nuclei are used as indicators. A sperm cell that fails to fertilize is indicated by a condensed fluorescence signal outside of the female gametes, whereas a sperm cell that successfully fertilizes is indicated by a decondensed signal due to karyogamy with the female gametes' nucleus. The method described here provides a tool to determine successful or failed fertilization under in vivo conditions.

Both male and female gametogenesis require a fully functional protein S-acyl transferase 21 in Arabidopsis thaliana.

S-Acylation is a reversible post-translational lipid modification in which a long chain fatty acid covalently attaches to specific cysteine(s) of proteins via a thioester bond. It enhances the hydrophobicity of proteins, contributes to their membrane association and plays roles in protein trafficking, stability and signalling. A family of Protein S-Acyl Transferases (PATs) is responsible for this reaction. PATs are multi-pass transmembrane proteins that possess a catalytic Asp-His-His-Cys cysteine-rich domain (DHHC-CRD). In Arabidopsis, there are currently 24 such PATs, five having been characterized, revealing their important roles in growth, development, senescence and stress responses. Here, we report the functional characterization of another PAT, AtPAT21, demonstrating the roles it plays in Arabidopsis sexual reproduction. Loss-of-function mutation by T-DNA insertion in AtPAT21 results in the complete failure of seed production. Detailed studies revealed that the sterility of the mutant is caused by defects in both male and female sporogenesis and gametogenesis. To determine if the sterility observed in atpat21-1 was caused by upstream defects in meiosis, we assessed meiotic progression in pollen mother cells and found massive chromosome fragmentation and the absence of synapsis in the initial stages of meiosis. Interestingly, the fragmentation phenotype was substantially reduced in atpat21-1 spo11-1 double mutants, indicating that AtPAT21 is required for repair, but not for the formation, of SPO11-induced meiotic DNA double-stranded breaks (DSBs) in Arabidopsis. Our data highlight the importance of protein S-acylation in the early meiotic stages that lead to the development of male and female sporophytic reproductive structures and associated gametophytes in Arabidopsis.

Arabinogalactan proteins mediate intercellular crosstalk in the ovule of apple flowers.

KEY MESSAGE: AGP-rich glycoproteins mediate pollen-ovule interactions and cell patterning in the embryo sac of apple before and after fertilization. Glycoproteins are significant players in the dialog that takes place between growing pollen tubes and the stigma and style in the angiosperms. Yet, information is scarce on their possible involvement in the ovule, a sporophytic organ that hosts the female gametophyte. Apple flowers have a prolonged lapse of time between pollination and fertilization, offering a great system to study the developmental basis of glycoprotein secretion and their putative role during the last stages of the progamic phase and early seed initiation. For this purpose, the sequential pollen tube elongation within the ovary was examined in relation to changes in arabinogalactan proteins (AGPs) in the tissues of the ovule before and after fertilization. To evaluate what of these changes are developmentally regulated, unpollinated and pollinated flowers were compared. AGPs paved the pollen tube pathway in the ovules along the micropylar canal, and the nucellus entrance toward the synergids, which also developmentally accumulated AGPs at the filiform apparatus. Glycoproteins vanished from all these tissues following pollen tube passage, strongly suggesting a role in pollen-ovule interaction. In addition, AGPs marked the primary cell walls of the haploid cells of the female gametophyte, and they further built up in the cell walls of the embryo sac and developing embryo, layering the interactive walls of the three generations hosted in the ovule, the maternal sporophytic tissues, the female gametophyte, and the developing embryo.

Gamete fusion is facilitated by two sperm cell-expressed DUF679 membrane proteins.

Successful double fertilization in flowering plants relies on two coordinated gamete fusion events, but the underlying molecular processes are not well understood. We show that two sperm-specific DOMAIN OF UNKNOWN FUNCTION 679 membrane proteins (DMP8 and DMP9) facilitate gamete fusion, with a greater effect on sperm-egg fusion than on sperm-central cell fusion. We also show that sperm adhesion and sperm cell separation depend on egg cell-secreted EGG CELL 1 proteins.

Comparative development of staminate and pistillate flowers in the dioecious cactus Opuntia robusta.

KEY MESSAGE: PCD role in unisexual flowers. The developmental processes underlying the transition from hermaphroditism to unisexuality are key to understanding variation and evolution of floral structure and function. A detailed examination of the cytological and histological patterns involved in pollen and ovule development of staminate and pistillate flowers in the dioecious Opuntia robusta was undertaken, and the potential involvement of programmed cell death in the abortion of the sex whorls was explored. Flowers initiated development as hermaphrodites and became functionally unisexual by anthesis. Female individuals have pistillate flowers with a conspicuous stigma, functional ovary, collapsed stamens and no pollen grains. Male individuals have staminate flowers, with large yellow anthers, abundant pollen grains, underdeveloped stigma, style and an ovary that rarely produced ovules. In pistillate flowers, anther abortion resulted from the premature degradation of the tapetum by PCD, followed by irregular deposition of callose wall around the microsporocytes, and finally by microspore degradation. In staminate flowers, the stigma could support pollen germination; however, the ovaries were reduced, with evidence of placental arrest and ovule abortion through PCD, when ovules were present. We demonstrate that PCD is recruited in both pistillate and staminate flower development; however, it occurs at different times of floral development. This study contributes to the understanding of the nature of the O. robusta breeding system and identifies developmental landmarks that contribute to sexual determination in Cactaceae.

Breeding systems in Balsaminaceae in relation to pollen/ovule ratio, pollination syndromes, life history and climate zone.

Pollen/ovule (P/O) ratios are often used as proxy for breeding systems. Here, we investigate the relations between breeding systems and P/O ratios, pollination syndromes, life history and climate zone in Balsaminaceae. We conducted controlled breeding system experiments (autonomous and active self-pollination and outcrossing tests) for 65 Balsaminaceae species, analysed pollen grain and ovule numbers and evaluated the results in combination with data on pollination syndrome, life history and climate zone on a phylogenetic basis. Based on fruit set, we assigned three breeding systems: autogamy, self-compatibility and self-incompatibility. Self-pollination led to lower fruit set than outcrossing. We neither found significant P/O differences between breeding systems nor between pollination syndromes. However, the numbers of pollen grains and ovules per flower were significantly lower in autogamous species, but pollen grain and ovule numbers did not differ between most pollination syndromes. Finally, we found no relation between breeding system and climate zone, but a relation between climate zone and life history. In Balsaminaceae reproductive traits can change under resource or pollinator limitation, leading to the evolution of autogamy, but are evolutionary rather constant and not under strong selection pressure by pollinator guild and geographic range changes. Colonisation of temperate regions, however, is correlated with transitions towards annual life history. Pollen/ovule-ratios, commonly accepted as good indicators of breeding system, have a low predictive value in Balsaminaceae. In the absence of experimental data on breeding system, additional floral traits (overall pollen grain and ovule number, traits of floral morphology) may be used as proxies.

Complex reproductive secretions occur in all extant gymnosperm lineages: a proteomic survey of gymnosperm pollination drops.

KEY MESSAGE: Complex protein-containing reproductive secretions are a conserved trait amongst all extant gymnosperms; the pollination drops of most groups include carbohydrate-modifying enzymes and defence proteins. Pollination drops are aqueous secretions that receive pollen and transport it to the ovule interior in gymnosperms (Coniferales, Cycadales, Ginkgoales, Gnetales). Proteins are well established as components of pollination drops in conifers (Coniferales) and Ephedra spp. (Gnetales), but it is unknown whether proteins are also present in the pollination drops of cycads (Cycadales), Ginkgo (Ginkgoales), Gnetum (Gnetales), or in the pollination drops produced by sterile ovules occurring on pollen plants in the Gnetales. We used liquid chromatography-tandem mass spectrometry followed by database-derived protein identification to conduct proteomic surveys of pollination drops collected from: Ceratozamia hildae, Zamia furfuracea and Cycas rumphii (Cycadales); Ginkgo biloba (Ginkgoales); Gnetum gnemon and Welwitschia mirabilis, including pollination drops from both microsporangiate and ovulate plants (Gnetales). We identified proteins in all samples: C. hildae (61), Z. furfuracea (40), C. rumphii (9), G. biloba (57), G. gnemon ovulate (17) and sterile ovules from microsporangiate plants (25) and W. mirabilis fertile ovules (1) and sterile ovules from microsporangiate plants (138). Proteins involved in defence and carbohydrate modification occurred in the drops of most groups, indicating conserved functions for proteins in pollination drops. Our study demonstrates that all extant gymnosperm groups produce complex reproductive secretions containing proteins, an ancient trait that likely contributed to the evolutionary success of seed plants.

Arabidopsis PIRL6 Is Essential for Male and Female Gametogenesis and Is Regulated by Alternative Splicing.

Plant intracellular Ras-group leucine-rich repeat (LRR) proteins (PIRLs) are related to Ras-interacting animal LRR proteins that participate in developmental cell signaling. Systematic knockout analysis has implicated some members of the Arabidopsis (Arabidopsis thaliana) PIRL family in pollen development. However, for PIRL6, no bona fide knockout alleles have been recovered, suggesting that it may have an essential function in both male and female gametophytes. To test this hypothesis, we investigated PIRL6 expression and induced knockdown by RNA interference. Knockdown triggered defects in gametogenesis, resulting in abnormal pollen and early developmental arrest in the embryo sac. Consistent with this, PIRL6 was expressed in gametophytes: functional transcripts were detected in wild-type flowers but not in sporocyteless (spl) mutant flowers, which do not produce gametophytes. A genomic PIRL6-GFP fusion construct confirmed expression in both pollen and the embryo sac. Interestingly, PIRL6 is part of a convergent overlapping gene pair, a scenario associated with an increased likelihood of alternative splicing. We detected multiple alternative PIRL6 mRNAs in vegetative organs and spl mutant flowers, tissues that lacked the functionally spliced transcript. cDNA sequencing revealed that all contained intron sequences and premature termination codons. These alternative mRNAs accumulated in the nonsense-mediated decay mutant upf3, indicating that they are normally subjected to degradation. Together, these results demonstrate that PIRL6 is required in both male and female gametogenesis and suggest that sporophytic expression is negatively regulated by unproductive alternative splicing. This posttranscriptional mechanism may function to minimize PIRL6 protein expression in sporophyte tissues while allowing the overlapping adjacent gene to remain widely transcribed.

Establishing the cell biology of apomictic reproduction in diploid Boechera stricta (Brassicaceae).

Background and aims: In the Brassicaceae family, apomictic development is characteristic of the genus Boechera. Hybridization, polyploidy and environmental adaptation that arose during the evolution of Boechera may serve as (epi)genetic regulators of apomictic initiation in this genus. Here we focus on Boechera stricta, a predominantly diploid species that reproduces sexually. However, apomictic development in this species has been reported in several studies, indicating non-obligate sexuality. Methods: A progressive investigation of flower development was conducted using three accessions to assess the reproductive system of B. stricta. We employed molecular and cyto-embryological identification using histochemistry, transmission electron microscopy and Nomarski and epifluorescence microscopy. Key Results: Data from internal transcribed spacer (ITS) and chloroplast haplotype sequencing, in addition to microsatellite variation, confirmed the B. stricta genotype for all lines. Embryological data indicated irregularities in sexual reproduction manifested by heterochronic ovule development, longevity of meiocyte and dyad stages, diverse callose accumulation during meiocyte-to-gametophyte development, and the formation of triads and tetrads in several patterns. The arabinogalactan-related sugar epitope recognized by JIM13 immunolocalized to one or more megaspores. Furthermore, pollen sterility and a high frequency of seed abortion appeared to accompany reproduction of the accession ES512, along with the initiation of parthenogenesis. Data from flow cytometric screening revealed both sexual and apomictic seed formation. Conclusion: These results imply that B. stricta is a species with an underlying ability to initiate apomixis, at least with respect to the lines examined here. The existence of apomixis in an otherwise diploid sexual B. stricta may provide the genomic building blocks for establishing highly penetrant apomictic diploids and hybrid relatives. Our findings demonstrate that apomixis per se is a variable trait upon which natural selection could act.

High temperature exposure did not affect induced 2n pollen viability in Populus.

High temperature exposure is widely used as a physical mutagenic agent to induce 2n gametes in Populus. However, whether high temperature exposure affects induced 2n pollen viability remains unknown. To clarify whether high temperature exposure affected the induced 2n pollen viability, 2n pollen induced by 38 and 41 °C temperatures, pollen morphology, 2n pollen germination in vitro, and crossing induced 2n pollen with normal gametes to produce a triploid was, based on observations of meiosis, conducted in Populus canescens. We found that the dominant meiotic stages (F = 56.6, p < .001) and the treatment duration (F = 21.4, p < .001) significantly affected the occurrence rate of induced 2n pollen. A significant decrease in pollen production and an increase in aborted pollen were observed (p < .001). High temperature sometimes affected in ectexine deposition and some narrow furrows were also analysed via details of ectexine structure. However, no significant difference in 2n pollen germination rate was observed between natural 2n pollen (26.7%) and high-temperature-induced 2n pollen (26.2%), and 42 triploids were created by crossing high-temperature-induced 2n pollen, suggesting that 38 and 41 °C temperatures exposure will not result in dysfunctional induced 2n pollen.

Effect of flower orientation on the male and female traits of Myrtillocactus geometrizans (Cactaceae).

Intra-individual variation in the production and size of reproductive traits has been documented in columnar cacti, being higher in equator-facing flowers. Such variation is attributed to the high amount of PAR intercepted by stems oriented towards the equator. Most studies focused on this phenomenon have documented the existence of intra-individual variation on traits associated with the female function; however, its impact on traits associated with the male function has been neglected. We tested the hypothesis that equator-facing flowers of Myrtillocactus geometrizans exhibit higher values on traits associated with both male and female functions than flowers facing against it. Number and size of anthers and ovaries, pollen:ovule ratio and number and quality of pollen grains (diameter, germinability, viability and pollen tube length) were estimated from reproductive structures facing north and south, and compared with t-tests between orientations. Number of anthers per flower, number of pollen grains per anther and per floral bud; pollen size, viability and germinability; pollen tube length; ovary length and pollen:ovule ratio were significantly higher in reproductive structures oriented towards the south (i.e. equator). These findings suggest that intra-individual variation in floral traits of M. geometrizans might be associated with different availability of resources in branches with contrasting orientation. Our results provide new evidence of the existence of a response to an orientation-dependent extrinsic gradient. To our knowledge, this is the first study documenting the existence of intra-individual variation on pollen quality and P:O ratio in Cactaceae species.

Novel computed tomography-based tools reliably quantify plant reproductive investment.

The flower is a bisexual reproductive unit where both genders compete for resources. Counting pollen and ovules in flowers is essential to understand how much is invested in each gender. Classical methods to count very numerous pollen grains and ovules are inefficient when pollen grains are tightly aggregated, and when fertilization rates of ovules are unknown. In this study we have therefore developed novel counting techniques based on computed tomography. In order to demonstrate the potential of our methods in very difficult cases, we counted pollen and ovules across inflorescences of deceptive and rewarding species of European orchids, which possess both very large numbers of pollen grains (tightly aggregated) and ovules. Pollen counts did not significantly vary across inflorescences and pollination strategies, whereas deceptive flowers had significantly more ovules than rewarding flowers. The within-inflorescence variance of pollen-to-ovule ratios in rewarding flowers was four times higher than in deceptive flowers, possibly demonstrating differences in the constraints acting on both pollination strategies. We demonstrate the inaccuracies and limitations of previously established methods, and the broad applicability of our new techniques: they allow measurement of reproductive investment without restriction on object number or aggregation, and without specimen destruction.