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Excessive gibberellin (GA) signalling, mediated through the DELLA proteins, has a negative impact on plant fertility. Loss of DELLA activity in the monocot rice (Oryza sativa) causes complete male sterility, but not in the dicot model Arabidopsis (Arabidopsis thaliana) ecotype Landsberg erecta (Ler), in which DELLA function has been studied most extensively, leading to the assumption that DELLA activity is not essential for Arabidopsis pollen development. A novel DELLA fertility phenotype was identified in the Columbia (Col-0) ecotype that necessitates re-evaluation of the general conclusions drawn from Ler. Fertility phenotypes were compared between the Col-0 and Ler ecotypes under conditions of chemical and genetic GA overdose, including mutants in both ecotypes lacking the DELLA paralogues REPRESSOR OF ga1-3 (RGA) and GA INSENSITIVE (GAI). Ler displays a less severe fertility phenotype than Col-0 under GA treatment. Col-0 rga gai mutants, in contrast with the equivalent Ler phenotype, were entirely male sterile, caused by post-meiotic defects in pollen development, which were rescued by the reintroduction of DELLA into either the tapetum or developing pollen. We conclude that DELLA activity is essential for Arabidopsis pollen development. Differences between the fertility responses of Col-0 and Ler might be caused by differences in downstream signalling pathways or altered DELLA expression.

During land plant evolution, determinate spore-bearing axes (retained in extant bryophytes such as mosses) were progressively transformed into indeterminate branching shoots with specialized reproductive axes that form flowers. The LEAFY transcription factor, which is required for the first zygotic cell division in mosses and primarily for floral meristem identity in flowering plants, may have facilitated developmental innovations during these transitions. Mapping the LEAFY evolutionary trajectory has been challenging, however, because there is no functional overlap between mosses and flowering plants, and no functional data from intervening lineages. Here, we report a transgenic analysis in the fern Ceratopteris richardii that reveals a role for LEAFY in maintaining cell divisions in the apical stem cells of both haploid and diploid phases of the lifecycle. These results support an evolutionary trajectory in which an ancestral LEAFY module that promotes cell proliferation was progressively co-opted, adapted and specialized as novel shoot developmental contexts emerged. The first plants colonized land around 500 million years ago. These plants had simple shoots with no branches, similar to the mosses that live today. Later on, some plants evolved more complex structures including branched shoots and flowers (collectively known as the “flowering plants”). Ferns are a group of plants that evolved midway between the mosses and flowering plants and have branched shoots but no flowers. The gradual transition from simple to more complex plant structures required changes to the way in which cells divide and grow within plant shoots. Whereas animals produce new cells throughout their body, most plant cells divide in areas known as meristems. All plants grow from embryos, which contain meristems that will form the roots and shoots of the mature plant. A gene called LEAFY is required for cells in moss embryos to divide. However, in flowering plants LEAFY does not carry out this role, instead it is only required to make the meristems that produce flowers. How did LEAFY transition from a general role in embryos to a more specialized role in making flowers? To address this question, Plackett, Conway et al. studied the two LEAFY genes in a fern called Ceratopteris richardii. The experiments showed that at least one of these LEAFY genes was active in the meristems of fern shoots throughout the lifespan of the plant. The shoots of ferns with less active LEAFY genes could not form the leaves seen in normal C. richardii plants. This suggests that as land plants evolved, the role of LEAFY changed from forming embryos to forming complex shoot structures. Most of our major crops are flowering plants. By understanding how the role of LEAFY has changed over the evolution of land plants, it might be possible to manipulate LEAFY genes in crop plants to alter shoot structures to better suit specific environments.

Background Learning tools using virtual patients can be used to teach clinical reasoning (CR) skills and overcome limitations of using face-to-face methods. However, the adoption of new tools is often challenging. The aim of this study was to explore UK medical educators’ perspectives of what influences the adoption of virtual patient learning tools to teach CR. Methods A qualitative research study using semi-structured telephone interviews with medical educators in the UK with control over teaching materials of CR was conducted. The Consolidated Framework for Implementation Research (CFIR), commonly used in healthcare services implementation research was adapted to inform the analysis. Thematic analysis was used to analyse the data. Results Thirteen medical educators participated in the study. Three themes were identified from the data that influenced adoption: the wider context (outer setting); perceptions about the innovation; and the medical school (inner context). Participants’ recognition of situations as opportunities or barriers related to their prior experiences of implementing online learning tools. For example, participants with experience of teaching using online tools viewed limited face-to-face placements as opportunities to introduce innovations using virtual patients. Beliefs that virtual patients may not mirror real-life consultations and perceptions of a lack of evidence for them could be barriers to adoption. Adoption was also influenced by the implementation climate of the setting, including positioning of CR in curricula; relationships between faculty, particularly where faculty were dispersed. Conclusions By adapting an implementation framework for health services, we were able to identify features of educators, teaching processes and medical schools that may determine the adoption of teaching innovations using virtual patients. These include access to face-to-face teaching opportunities, positioning of clinical reasoning in the curriculum, relationship between educators and institutions and decision-making processes. Framing virtual patient learning tools as additional rather than as a replacement for face-to-face teaching could reduce resistance. Our adapted framework from healthcare implementation science may be useful in future studies of implementation in medical education.

Acorn production in oak ( Quercus spp.) shows considerable inter-annual variation, known as masting, which provides a natural defence against seed predators but a highly-variable supply of acorns for uses such as in commercial tree planting each year. Anthropogenic emissions of greenhouse gases have been very widely reported to influence plant growth and seed or fruit size and quantity via the ‘fertilisation effect’ that leads to enhanced photosynthesis. To examine if acorn production in mature woodland communities will be affected by further increase in CO 2 , the contents of litter traps from a Free Air Carbon Enrichment (FACE) experiment in deciduous woodland in central England were analysed for numbers of flowers and acorns of pedunculate oak ( Quercus robur L.) at different stages of development and their predation levels under ambient and elevated CO 2 concentrations. Inter-annual variation in acorn numbers was considerable and cyclical between 2015 and 2021, with the greatest numbers of mature acorns in 2015, 2017 and 2020 but almost none in 2018. The numbers of flowers, enlarged cups, immature acorns, empty acorn cups, and galls in the litter traps also varied amongst years; comparatively high numbers of enlarged cups were recorded in 2018, suggesting Q. robur at this site is a fruit maturation masting species (i.e., the extent of abortion of pollinated flowers during acorn development affects mature acorn numbers greatly). Raising the atmospheric CO 2 concentration by 150 μL L −1 , from early 2017, increased the numbers of immature acorns, and all acorn evidence (empty cups + immature acorns + mature acorns) detected in the litter traps compared to ambient controls by 2021, but did not consistently affect the numbers of flowers, enlarged cups, empty cups, or mature acorns. The number of flowers in the elevated CO 2 plots’ litter traps was greater in 2018 than 2017, one year after CO 2 enrichment began, whereas numbers declined in ambient plots. Enrichment with CO 2 also increased the number of oak knopper galls ( Andricus quercuscalicis Burgsdorf). We conclude that elevated CO 2 increased the occurrence of acorns developing from flowers, but the putative benefit to mature acorn numbers may have been hidden by excessive pre- and/or post-dispersal predation. There was no evidence that elevated CO 2 altered masting behaviour.

Advanced therapy medicinal products (ATMPs) are ground-breaking genetic and cell/tissue-based therapies with the potential to treat and even cure myriad health conditions. However, they have faced multiple challenges in decision making with relatively low success in achieving reimbursement. The aim of this study was to review the published preference-based research on ATMPs to identify value attributes as a starting point in a research workstream aimed at understanding the true value of these innovative therapies. A literature review was used to identify studies across MedLine, Embase, Allied and Complementary Medicine Database, Global Health, ProQuest Central, Social Sciences Premium Collection, Applied Social Sciences Index and Abstracts, Econ Lit, Web of Science and SCOPUS from inception to 3rd March 2024. Search terms for genetic and cell/tissue-based therapies were conducted separately. Studies were included if they were a peer-reviewed primary research study or systematic literature review using stated preference measures to elicit preferences for ATMPs. Thirteen studies were included with 8 pertaining to genetic therapies and 5 to cell/tissue therapies. Attributes of value were similar across the studies and ATMP categories. Attributes were grouped into the following for genetic therapies: clinical benefits, uncertainty, risk, treatment burden, and quality of life; for cell/tissue therapies clinical benefit, uncertainty, risk and treatment burden. There is limited research on the preferences for ATMPs; but these findings suggest that the value attributes of ATMPs are consistent across gene and cell/tissue therapies with clinical benefit, uncertainty, risk, treatment burden, and quality of life being important.

Background The inability to genetically transform any fern species has been a major technical barrier to unlocking fern biology. Initial attempts to overcome this limitation were based on transient transformation approaches or achieved very low efficiencies. A highly efficient method of stable transformation was recently reported using the fern Ceratopteris richardii , in which particle bombardment of callus tissue achieved transformation efficiencies of up to 72%. As such, this transformation method represents a highly desirable research tool for groups wishing to undertake fern genetic analysis. Results We detail an updated and optimized protocol for transformation of C. richardii by particle bombardment, including all necessary ancillary protocols for successful growth and propagation of this species in a laboratory environment. The C. richardii lifecycle comprises separate, free-living gametophyte and sporophyte stages. Callus is induced from the sporophyte apex through growth on cytokinin-containing tissue culture medium and can be maintained indefinitely by sub-culturing. Transgene DNA is introduced into callus cells through particle bombardment, and stable genomic integration events are selected by regeneration and growth of T 0 sporophytes for a period of 8 weeks on medium containing antibiotics. Selection of T 1 transgenic progeny is accomplished through screening T 1 gametophytes for antibiotic resistance. In many cases sexual reproduction and development of transgenic embryos requires growth and fertilization of gametophytes in the absence of antibiotics, followed by a separate screen for antibiotic resistance in the resultant sporophyte generation. Conclusions Genetic transformation of C. richardii using this protocol was found to be robust under a broad range of bombardment and recovery conditions. The successful expansion of the selection toolkit to include a second antibiotic for resistance screening (G-418) and different resistance marker promoters increases the scope of transformations possible using this technique and offers the prospect of more complex analysis, for example the creation of lines carrying more than one transgene. The introduction of a robust and practicable transformation technique is a very important milestone in the field of fern biology, and its successful implementation in C. richardii paves the way for adoption of this species as the first fern genetic model.

• How plant seeds originated remains unresolved, in part due to disconnects between fossil intermediates and developmental genetics in extant species. The Carboniferous fossil Genomosperma is considered among the most primitive known seeds, with highly lobed integument and exposed nucellus. We have used this key fossil taxon to investigate the evolutionary origins of seed development. • We examined sectioned Genomosperma specimens using modern digital 3D reconstruction techniques and established population-level measurements of Genomosperma ovules for quantitative analysis. • Genomosperma ovules show significant variation in integumentary lobe fusion and curvature. Our analysis suggests that this variation represents a single species with significant variations in lobe number and fusion, reminiscent of floral development in extant species. We conclude that changes in lobe flexure occurred late in development, consistent with a previously hypothesized function in pollen guidance/retention. We also identify seeds of Genomosperma within cupules for the first time. • The presence of a cupule adds evidence towards the plesiomorphy of cupules within seed plants. Together with the similarities identified between the Genomosperma lobed integument and floral organs, we propose that the cupule, integument and nucellus together developed in a shoot-like fashion, potentially ancestral to extant seed plant reproductive shoots.

Gibberellin (GA) biosynthesis is necessary for normal plant development, with later GA biosynthetic stages being governed by multigene families. Arabidopsis thaliana contains five GA 20-oxidase (GA20ox) genes, and past work has demonstrated the importance of GA20ox1 and -2 for growth and fertility. Here, we show through systematic mutant analysis that GA20ox1, -2, and -3 are the dominant paralogs; their absence results in severe dwarfism and almost complete loss of fertility. In vitro analysis revealed that GA20ox4 has full GA20ox activity, but GA20ox5 catalyzes only the first two reactions of the sequence by which GA12 is converted to GA9. GA20ox3 functions almost entirely redundantly with GA20ox1 and -2 at most developmental stages, including the floral transition, while GA20ox4 and -5 have very minor roles. These results are supported by analysis of the gene expression patterns in promoter:β-glucuronidase reporter lines. We demonstrate that fertility is highly sensitive to GA concentration, that GA20ox1, -2, and -3 have significant effects on floral organ growth and anther development, and that both GA deficiency and overdose impact on fertility. Loss of GA20ox activity causes anther developmental arrest, with the tapetum failing to degrade. Some phenotypic recovery of late flowers in GA-deficient mutants, including ga1-3, indicated the involvement of non-GA pathways in floral development.

Ferns represent the most closely related extant lineage to seed plants. The aquatic fern Ceratopteris richardii has been subject research for a considerable period of time, but analyses of the genetic programs underpinning developmental processes have been hampered by a large genome size, a lack of available mutants, and an inability to create stable transgenic lines. In this paper, we report a protocol for efficient stable genetic transformation of C. richardii and a closely related species Ceratopteris thalictroides microparticle bombardment. Indeterminate callus was generated and maintained from the sporophytes of both species using cytokinin treatment. In proof-of-principle experiments, a 35S::β-glucuronidase (GUS) expression cassette was introduced into callus cells via tungsten microparticles, and stable transformants were selected via a linked hygromycin resistance marker. The presence of the transgene in regenerated plants and in subsequent generations was validated using DNA-blot analysis, reverse transcription-polymerase chain reaction, and GUS staining. GUS staining patterns in most vegetative tissues corresponded with constitutive gene expression. The protocol described in this paper yields transformation efficiencies far greater than those previously published and represents a significant step toward the establishment of a tractable fern genetic model.