Burden tests can be used to map causal genes for a simple metabolic trait in an exome-sequenced polyploid mutant population

Forward genetic screens are an excellent tool to assign gene function, but it is often necessary to employ map-based cloning to identify the causal genes. This can be laborious and represents a bottleneck in plant fundamental and applied research. With advances in DNA technology, it is becoming increasingly affordable to sequence large populations. Krasileva et al. (2017) exome sequenced tetraploid and hexaploid wheat ethyl methanesulfonate (EMS) mutagenized populations, primarily to facilitate reverse genetic screens. Gene redundancy allows a very high mutant load of 35–40 mutations per kilobase, and the populations of ~1500 and ~1200 lines each harbour ~22–23 missense or truncation mutations per gene. Here, we show that burden tests, a simple form of rare-variant association analysis developed for human disease genetics (Lee et al., 2014), can be used to identify causal genes in the hexaploid wheat (Triticum aestivum) cv. Cadenza mutant population, without the need for map-based cloning

A native promoter–gene fusion created by CRISPR/Cas9-mediated genomic deletion offers a transgene-free method to drive oil accumulation in leaves

Achieving gain-of-function phenotypes without inserting foreign DNA is an important challenge for plant biotechnologists. Here we show that a gene can be brought under the control of a promoter from an upstream gene by deleting the intervening genomic sequence using dual-guide CRISPR/Cas9. We fuse the promoter of a non-essential photosynthesis-related gene to DIACYLGLYCEROL ACYLTRANSFERASE 2 (DGAT2) in the lipase-deficient sugar-dependent 1 mutant of Arabidopsis thaliana to drive ectopic oil accumulation in leaves. DGAT2 expression is enhanced more than twenty-fold and the triacylglycerol content increases by around thirty-fold. This deletion strategy offers a transgene-free route to engineering traits that rely on transcriptional gain-of-function, such as producing high lipid forage to increase the productivity and sustainability of ruminant farming

Laboratory phenomics predicts field performance and identifies superior indica haplotypes for early seedling vigour in dry direct-seeded rice

Seedling vigour is an important agronomic trait and is gaining attention in Asian rice (Oryza sativa) as cultivation practices shift from transplanting to forms of direct seeding. To understand the genetic control of rice seedling vigour in dry direct seeded (aerobic) conditions we measured multiple seedling traits in 684 accessions from the 3000 Rice Genomes (3K-RG) population in both the laboratory and field at three planting depths. Our data show that phenotyping of mesocotyl length in laboratory conditions is a good predictor of field performance. By performing a genome wide association study, we found that the main QTL for mesocotyl length, percentage seedling emergence and shoot biomass are co-located on the short arm of chromosome 7. We show that haplotypes in the indica subgroup from this region can be used to predict the seedling vigour of 3K-RG accessions. The selected accessions may serve as potential donors in genomics-assisted breeding programs

Down‐regulation of key genes involved in carbon metabolism in Medicago truncatula results in increased lipid accumulation in vegetative tissue

Alfalfa (Medicago sativa L.), is the most widely grown perennial forage crop, which is a close relative of the model diploid legume Medicago truncatula. However, use of alfalfa lead to substantial greenhouse gas emissions and economic losses related to inefficiencies in rumen fermentation. The provision of supplemental lipids has been used as a strategy to mitigate these issues, but it is a costly approach. The ability to enhance lipid content within the vegetative tissues of alfalfa would therefore be very advantageous. As such, our aim was to assess and select gene candidates to increase total shoot lipid content in M. truncatula using a virus‐induced gene silencing (VIGS) approach. We targeted gene homologs of the SUGAR‐DEPENDANT 1 (SDP1), ADP‐GLUCOSE‐PYROPHOSPHORYLASE SMALL SUBUNIT 1 (APS1), TRIGALACTOSYLDIACYLGLYCEROL 5 (TGD5) and PEROXISOMAL ABC TRANSPORTER 1 (PXA1) in M. truncatula for silencing. Reduced target transcript levels were confirmed and changes of shoot lipid content and fatty acid composition were measured. Silencing of SDP1, APS1 and PXA1 each resulted in significant increases in shoot total lipid content. Significantly increased proportions of α‐linolenic acid (18:3Δ9cis,12cis,15cis) were observed and stearic acid (18:0) levels significantly decreased in the total acyl lipids extracted from vegetative tissues of each of the M. truncatula silenced plants. In contrast, palmitic acid (16:0) levels were significantly decreased in only SDP1 and PXA1‐silenced plants. Genes of PXA1 and SDP1 would be ideal targets for mutation as a means of improving the quality of alfalfa for increasing feed efficiency and minimizing greenhouse gas emissions from livestock production in the future

Diverting phenylpropanoid pathway flux from sinapine to produce industrially useful 4-vinyl derivatives of hydroxycinnamic acids in Brassicaceous oilseeds

Sinapine (sinapoylcholine) is an antinutritive phenolic compound that can account for up to 2% of seed weight in brassicaceous oilseed crops and reduces the suitability of their protein-rich seed meal for use as animal feed. Sinapine biosynthesis draws on hydroxycinnamic acid precursors produced by the phenylpropanoid pathway. The 4-vinyl derivatives of several hydroxycinnamic acids have industrial applications. For example, 4-vinyl phenol (4-hydroxystyrene) is a building block for a range of synthetic polymers applied in resins, inks, elastomers, and coatings. Here we have expressed a modified bacterial phenolic acid decarboxylase (PAD) in developing seed of Camelina sativa to redirect phenylpropanoid pathway flux from sinapine biosynthesis to the production of 4-vinyl phenols. PAD expression led to a ∼95% reduction in sinapine content in seeds of both glasshouse and field grown C. sativa and to an accumulation of 4-vinyl derivatives of hydroxycinnamic acids, primarily as glycosides. The most prevalent aglycone was 4-vinyl phenol, but 4-vinyl guaiacol, 6-hydroxy-4-vinyl guaiacol and 4-vinylsyringol (Canolol) were also detected. The molar quantity of 4-vinyl phenol glycosides was more than twice that of sinapine in wild type seeds. PAD expression was not associated with an adverse effect on seed yield, harvest index, seed morphology, storage oil content or germination in either glasshouse or field experiments. Our data show that expression of PAD in brassicaceous oilseeds can supress sinapine accumulation, diverting phenylpropanoid pathway flux into 4-vinyl phenol derivatives, thereby also providing a non-petrochemical source of this class of industrial chemicals

Ketocarotenoid production in tomato triggers metabolic reprogramming and cellular adaptation: The quest for homeostasis

Plants are sessile and therefore have developed an extraordinary capacity to adapt to external signals. Here, the focus is on the plasticity of the plant cell to respond to new intracellular cues. Ketocarotenoids are high-value natural red pigments with potent antioxidant activity. In the present study, system-level analyses have revealed that the heterologous biosynthesis of ketocarotenoids in tomato initiated a series of cellular and metabolic mechanisms to cope with the formation of metabolites that are non-endogenous to the plant. The broad multilevel changes were linked to, among others, (i) the remodelling of the plastidial membrane, where the synthesis and storage of ketocarotenoids occurs; (ii) the recruiting of core metabolic pathways for the generation of metabolite precursors and energy; and (iii) redox control. The involvement of the metabolites as regulators of cellular processes shown here reinforces their pivotal role suggested in the remodelled ‘central dogma’ concept. Furthermore, the role of metabolic reprogramming to ensure cellular homeostasis is propose

Genome wide analysis of fatty acid desaturation and its response to temperature

Plants modify the polyunsaturated fatty acid content of their membrane and storage lipids in order to adapt to changes in temperature. In developing seeds, this response is largely controlled by the activities of the microsomal ω-6 and ω-3 fatty acid desaturases, FAD2 and FAD3. Although temperature regulation of desaturation has been studied at the molecular and biochemical levels, the genetic control of this trait is poorly understood. Here, we have characterized the response of Arabidopsis (Arabidopsis thaliana) seed lipids to variation in ambient temperature and found that heat inhibits both ω-6 and ω-3 desaturation in phosphatidylcholine, leading to a proportional change in triacylglycerol composition. Analysis of the 19 parental accessions of the multiparent advanced generation intercross (MAGIC) population showed that significant natural variation exists in the temperature responsiveness of ω-6 desaturation. A combination of quantitative trait locus (QTL) analysis and genome-wide association studies (GWAS) using the MAGIC population suggests that ω-6 desaturation is largely controlled by cis-acting sequence variants in the FAD2 5′ untranslated region intron that determine the expression level of the gene. However, the temperature responsiveness of ω-6 desaturation is controlled by a separate QTL on chromosome 2. The identity of this locus is unknown, but genome-wide association studies identified potentially causal sequence variants within ∼40 genes in an ∼450-kb region of the QTL

Arabidopsis uses two gluconeogenic gateways for organic acids to fuel seedling establishment.

Gluconeogenesis is a fundamental metabolic process that allows organisms to make sugars from non-carbohydrate stores such as lipids and protein. In eukaryotes only one gluconeogenic route has been described from organic acid intermediates and this relies on the enzyme phosphoenolpyruvate carboxykinase (PCK). Here we show that two routes exist in Arabidopsis, and that the second uses pyruvate, orthophosphate dikinase (PPDK). Gluconeogenesis is critical to fuel the transition from seed to seedling. Arabidopsis pck1 and ppdk mutants are compromised in seed-storage reserve mobilization and seedling establishment. Radiolabelling studies show that PCK predominantly allows sugars to be made from dicarboxylic acids, which are products of lipid breakdown. However, PPDK also allows sugars to be made from pyruvate, which is a major product of protein breakdown. We propose that both routes have been evolutionarily conserved in plants because, while PCK expends less energy, PPDK is twice as efficient at recovering carbon from pyruvate.We thank the Biotechnology and Biology Sciences Research Council for funding J.M.H. (P18931 and a studentship to B.P.W.) and P.J.E. (BB/G009724/1 and BB/K002147/1), the Isaac Newton Trust and the Max-Planck Gesellschaft for funding and ATC for a CASE studentship to H.M.A.This is the final published version. It first appeared at http://www.nature.com/ncomms/2015/150410/ncomms7659/full/ncomms7659.html

Transporting an evidence-based program to a new country: a narrative description and analysis of pre-implementation adaptation

There is a pressing need to prevent and address youth crime and violence owing to its prevalence, harms and cost to society. Interventions with proven effectiveness in doing this exist. Adopting and adapting them in new contexts is potentially cost-effective. However, more research is needed into how to make adaptations that enhance intervention implementation, effectiveness and maintenance in new settings. This article reports the pre-implementation adaptation work involved in transporting Becoming a Man (BAM) from the US to the UK. BAM is a selective school-based youth development program for 12–18 year-old boys that aims to improve school engagement and reduce interactions with the criminal justice system. We describe the nature of and rationale for adaptations and identify learning for future adaptation efforts. An adaptation team comprising the intervention developers, new providers and the evaluators met weekly for 10 weeks, applying a structured, pragmatic and evidence-informed approach to adapt the BAM curriculum and implementation process. Changes were informed by documentary analysis, group-based discussions and site visits. The group agreed 27 changes to the content of 17/30 lessons, at both surface (e.g., cultural references) and deep (key mechanisms or concepts) levels. Of 28 contextual factors considered, 15 discrepancies between the US and UK were identified and resolved (e.g., differences in staffing arrangements). Strengths of the process were the blend of expertise on the adaptation team in the program and local context, and constant reference to and ongoing refinement of the program theory of change. Limitations included the lack of involvement of school staff or students. Further research is needed into potential conflicts between stakeholder perspectives during adaptation and whose views to prioritise and when