Premise Cleomaceae is emerging as a promising family to investigate a wide range of phenomena, such as C 4 photosynthesis and floral diversity. However, functional techniques are lacking for elucidating this diversity. Herein, we establish virus‐induced gene silencing (VIGS) as a method of generating functional data for Cleome violacea , bolstering Cleomaceae as a model system. Methods We leveraged the sister relationship of Cleomaceae and Brassicaceae by using constructs readily available for Arabidopsis thaliana to provide initial information about the feasibility of VIGS in C . violacea . We then developed endogenous constructs to optimize VIGS efficiency and viability for fruit development. Results PHYTOENE DESATURASE was successfully downregulated in C . violacea using both heterologous and endogenous constructs. The endogenous construct had the highest degree of downregulation, with many plants displaying strong photobleaching. FRUITFULL ‐treated plants were also successfully downregulated, with a high rate of survival but less effective silencing; only a small percentage of survivors showed a strong phenotype. Discussion Our optimized VIGS protocol in C . violacea enables functional gene analyses at different developmental stages. Additionally, C . violacea is amenable to heterologous knockdown, which suggests that a first pass using non‐endogenous constructs is a possible route to test additional species of Cleomaceae.

Comparative gene expression studies are invaluable for predicting how existing genetic pathways may be modified or redeployed to produce novel and variable phenotypes. Fruits are ecologically important organs because of their impact on plant fitness and seed dispersal, modifications in which results in morphological variation across species. A novel fruit type in the Brassicaceae known as heteroarthrocarpy enables distinct dispersal methods in a single fruit through segmentation via a lateral joint and variable dehiscence at maturity. Given the close relationship to Arabidopsis, species that exhibit heteroarthrocarpy are powerful models to elucidate how differences in gene expression of a fruit patterning pathway may result in novel fruit types. Transcriptomes of distal, joint, and proximal regions from Erucaria erucarioides and Cakile lanceolata were analyzed to elucidate within fruit and between species differences in whole transcriptome, gene ontology, and fruit patterning expression profiles. Whole transcriptome expression profiles vary between fruit regions in patterns that are consistent with fruit anatomy. These transcriptomic variances do not correlate with changes in gene ontology, as they remain generally stable within and between both species. Upstream regulators in the fruit patterning pathway, FILAMENTOUS FLOWER and YABBY3 , are expressed in the distal and proximal regions of E . erucarioides , but not in the joint, implicating alterations in the pathway in heteroarthrocarpic fruits. Downstream gene, INDEHISCENT , is significantly upregulated in the abscissing joint region of C . lanceolata , which suggests repurposing of valve margin genes for novel joint disarticulation in an otherwise indehiscent fruit. In summary, these data are consistent with modifications in fruit patterning genes producing heteroarthrocarpic fruits through different components of the pathway relative to other indehiscent, non-heteroarthrocarpic, species within the family. Our understanding of fruit development in Arabidopsis is now extended to atypical siliques within the Brassicaceae, facilitating future studies on seed shattering in important Brassicaceous crops and pernicious weeds.

Nectaries are a promising frontier for plant evo-devo research, and are particularly fascinating given their diversity in form, position, and secretion methods across angiosperms. Emerging model systems permit investigations of the molecular basis for nectary development and nectar secretion across a range of taxa, which addresses fundamental questions about underlying parallelisms and convergence. Herein, we explore nectary development and nectar secretion in the emerging model taxa, Cleome violacea (Cleomaceae), which exhibits a prominent adaxial nectary. First, we characterized nectary anatomy and quantified nectar secretion to establish a foundation for quantitative and functional gene experiments. Next, we leveraged RNA-seq to establish gene expression profiles of nectaries across three key stages of development: pre-anthesis, anthesis, and post-fertilization. We then performed functional studies on five genes that were putatively involved in nectary and nectar formation: CvCRABSCLAW (CvCRC), CvAGAMOUS (CvAG), CvSHATTERPROOF (CvSHP), CvSWEET9, and a highly expressed but uncharacterized transcript. These experiments revealed a high degree of functional convergence to homologues from other core Eudicots, especially Arabidopsis. CvCRC, redundantly with CvAG and CvSHP, are required for nectary initiation. Concordantly, CvSWEET9 is essential for nectar formation and secretion, which indicates that the process is eccrine based in C. violacea. While demonstration of conservation is informative to our understanding of nectary evolution, questions remain. For example, it is unknown which genes are downstream of the developmental initiators CvCRC, CvAG, and CvSHP, or what role the TCP gene family plays in nectary initiation in this family. Further to this, we have initiated a characterization of associations between nectaries, yeast, and bacteria, but more research is required beyond establishing their presence. Cleome violacea is an excellent model for continued research into nectary development because of its conspicuous nectaries, short generation time, and close taxonomic distance to Arabidopsis.

20Comparative gene expression studies are invaluable for predicting how existing genetic 21 pathways may be modified or redeployed to produce novel and variable phenotypes. Fruits are 22 ecologically important organs because of their impact on plant fitness and seed dispersal, 42 siliques within the Brassicaceae, facilitating future studies on seed shattering in important 43 Brassicaceous crops and pernicious weeds.44 45 3 46 62Arabidopsis, especially amongst close relatives e.g., the loss of dehiscence in many species 63 across the Brassicaceae (1). 65Brassicaceae fruits vary markedly in shape, structure, and size (1,8). Their variation in 66 dehiscence is a focal point for research because it fundamentally changes fruit structure, 67 subsequently affecting dispersal and diversification (9). A prerequisite for exploring how 68 differences in fruit morphology are achieved across the Brassicaceae is familiarity with both the 69 fruit structure and underlying genetic pathways in Arabidopsis (10,11). Arabidopsis fruits, 102 results in indehiscent fruits in Arabidopsis: SHP1/2, SPT,. Overexpression 103 of FUL or NO TRANSMITTING TRACT (NTT) also results in indehiscent fruits (28,29); FUL 104 overexpression completely suppresses SHP1/2, resulting in reduced lignification in the enb layer 105 and reduced valve margin formation; overexpression of NTT phenocopies the ful mutation 106 resulting in valve margin specific genes being expressed throughout valve. In summary, a 107 modification of many components in this pathway results in a loss of dehiscence. Because 108 indehiscence is observed in at least 20 different lineages across the family, it is likely that this 109 phenotype evolved via multiple modifications to this pathway (30). As such, there is no singular 110 alteration to the fruit patterning pathway implicated in this shift for all tribes. 111 112 To date, little is known about the genetic basis of indehiscence in the Brassicaceae, although it is 113 currently being bridged by studies in taxa with varying indehiscent morphologies. Recently, a114 study demonstrated a deviation in expression of eight key genes between pod shatter sensitive 115 species and shatter resistant species of Brassica and Sinapis (2). In Lepidium, there has been an 116 evolutionary shift from dehiscence to indehiscence, e.g., valve margin genes that are conserved 6 117 between the dehiscent L. campestre and Arabidopsis have been lost in the indehiscent L.118 apellianum (31,32). Upregulation in upstream regulator AP2 has been suggested as a factor in 119 this indehiscence (32).120 121 A notable morphological adaptation is the evolution of a complex fruit type known as 122 heteroarthrocarpy, which is only found in some members of the tribe Brassiceae (30,33,34). This 123 modified silique is defined by the presence of a variably abscising central joint, an indehiscent 124 distal region, and a variably dehiscent proximal region (Fig 2). As such, this novel morphology 125 offers an opportunity to investigate fruit variation beyond shifts from ...

Aims: This study investigated the acceptability and feasibility of delivering the On The Level (OTL) mental health intervention to a sample of young people (aged 11-18 years) in secondary schools in London and Essex, UK.Methods: Participants were offered a OTL show intervention session as part of their class. Participants were followed up 4-6 weeks post-intervention session. Measures were completed for intervention acceptability and engagement, mental health outcomes and intervention evaluation at baseline/intervention (T1) and at follow-up (T2; 4-6 weeks post-intervention). Results: In total, 10,315 participants participated at T1. The results indicated good feasibility and acceptability of the OTL intervention among participants. It was feasible to recruit a large sample of young people and the OTL could be delivered during class at UK-based secondary schools as planned. Some adaptations to the study methods were identified in this study. Some preliminary differences were found for the mental health outcome measures between T1 and T2, notably with a significant reduction in levels of stress.Conclusion: This feasibility study provides preliminary evidence for the feasibility and acceptability of the OTL mental health intervention for young people in secondary schools in the UK.