Turning sugar into oil - making photosynthesis blind to feedback inhibition

Since the advent of metabolic engineering 30 years ago it has been a goal to modify crops to accumulate alternative higher value products and/or to increase yield. Direct targeting of metabolic enzymes has been difficult because of strong endogenous regulatory mechanisms that can confound such changes, either impeding the metabolic alteration or producing a growth and yield penalty. Beechey-Gradwell et al. (2020) show that in Lolium perenne, an important temperate pasture and forage grass, introduction of two genes involved in lipid synthesis and storage results in significant accumulation of oil and increases photosynthesis and growth, elevating calorific value and overall yield

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

Big data from small tissue extraction of high-quality RNA for RNA-Sequencing from different oilseed Brassica seed tissues during seed development

Background Obtaining high-quality RNA for gene expression analyses from different seed tissues is challenging due to the presence of various contaminants, such as polyphenols, polysaccharides and lipids which interfere with RNA extraction methods. At present, the available protocols for extracting RNA from seeds require high amounts of tissue and are mainly focused on extracting RNA from whole seeds. However, extracting RNA at the tissue level enables more detailed studies regarding tissue specific transcriptomes during seed development. Results Seeds from heart stage embryo to mature developmental stages of Brassica napus and B. oleracea were sampled for isolation of the embryo, endosperm and seed coat tissues. Ovules and ovary wall tissue were also collected from pre-fertilized buds. Subsequent to testing several RNA extraction methods, we determined that modifications applied to E.Z.N.A. Plant RNA and Picopure RNA Isolation kit extraction methods resulted in RNA with high yield and quality. Furthermore, the use of polyvinylpolypyrrolidone for the extraction of RNA from seed coats and endosperm at green seed stage resulted in high-quality RNA. As a result of the introduced modifications to established RNA extraction methods, the RNA from all the above-mentioned tissues presented clear 28S and 18S bands and high RIN values, ranging from 7.0 to 10.0. The protocols reported in this study are not only suitable for different and challenging seed tissue types, but also enable the extraction of high-quality RNA using only 2 to 3 mg of starting tissue. Conclusions Here, we present efficient, reproducible and reliable high-quality RNA extraction methods for diverse oilseed Brassica spp reproductive tissue types including pre-fertilization and developing seed tissues for diploid and polyploid species. The high-quality RNA obtained is suitable for RNA-Sequencing and subsequent gene expression analysis

DES2 is a fatty acid Delta 11 desaturase capable of synthesizing palmitvaccenic acid in the arbuscular mycorrhizal fungus Rhizophagus irregularis

Arbuscular mycorrhizal (AM) fungi are oleaginous organisms, and the most abundant fatty acyl moiety usually found in their lipids is palmitvaccenic acid (16:1Δ11cis). However, it is not known how this uncommon fatty acid species is made. Here, we have cloned two homologs of Lepidopteran fatty acyl-CoenzymeA Δ11 desaturases from the AM fungus Rhizophagus irregularis. Both enzymes, DES1 and DES2, are expressed in intraradicle mycelium and can complement the unsaturated fatty acid-requiring auxotrophic growth phenotype of the Saccharomyces cerevisiae ole1Δ mutant. DES1 expression leads almost exclusively to oleic acid (18:1Δ9cis) production, whereas DES2 expression results in the production of 16:1Δ11cis and vaccenic acid (18:1Δ11cis). DES2 therefore encodes a Δ11 desaturase that is likely to be responsible for the synthesis of 16:1Δ11cis in R. irregularis

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

A causal inference and Bayesian optimisation framework for modelling multi-trait relationships—Proof-of-concept using Brassica napus seed yield under controlled conditions

The improvement of crop yield is a major breeding target and there is a long history of research that has focussed on unravelling the mechanisms and processes that contribute to yield. Quantitative prediction of the interplay between morphological traits, and the effects of these trait-trait relationships on seed production remains, however, a challenge. Consequently, the extent to which crop varieties optimise their morphology for a given environment is largely unknown. This work presents a new combination of existing methodologies by framing crop breeding as an optimisation problem and evaluates the extent to which existing varieties exhibit optimal morphologies under the test conditions. In this proof-of-concept study using spring and winter oilseed rape plants grown under greenhouse conditions, we employ causal inference to model the hierarchically structured effects of 27 morphological yield traits on each other. We perform Bayesian optimisation of seed yield, to identify and quantify the morphologies of ideotype plants, which are expected to be higher yielding than the varieties in the studied panels. Under the tested growth conditions, we find that existing spring varieties occupy the optimal regions of trait-space, but that potentially high yielding strategies are unexplored in extant winter varieties. The same approach can be used to evaluate trait (morphology) space for any environment

Uncovering Trait Associations Resulting in Maximal Seed Yield in Winter and Spring Oilseed Rape

Seed yield is a complex trait for many crop species including oilseed rape (OSR) (Brassica napus), the second most important oilseed crop worldwide. Studies have focused on the contribution of distinct factors in seed yield such as environmental cues, agronomical practices, growth conditions, or specific phenotypic traits at the whole plant level, such as number of pods in a plant. However, how female reproductive traits contribute to whole plant level traits, and hence to seed yield, has been largely ignored. Here, we describe the combined contribution of 33 phenotypic traits within a B. napus diversity set population and their trade-offs at the whole plant and organ level, along with their interaction with plant level traits. Our results revealed that both Winter OSR (WOSR) and Spring OSR (SOSR); the two more economically important OSR groups in terms of oil production; share a common dominant reproductive strategy for seed yield. In this strategy, the main inflorescence is the principal source of seed yield, producing a good number of ovules, a large number of long pods with a concomitantly high number of seeds per pod. Moreover, we observed that WOSR opted for additional reproductive strategies than SOSR, presenting more plasticity to maximise seed yield. Overall, we conclude that OSR adopts a key strategy to ensure maximal seed yield and propose an ideal ideotype highlighting crucial phenotypic traits that could be potential targets for breeding

Highlights of recent progress in plant lipid research

Raw fossil material reserves are not inexhaustible and as prices continue to raise it is necessary to find new sources of alternative and renewable energy. Oils from oleaginous field crops (sunflower and rape) with properties close to those of fossil fuel could constitute an alternative source of energy for the production of raw materials. This is the context in which the 18th International Symposium on Plant lipids (ISPL) was held in Bordeaux from 20th to 25th July 2008 at “La Cité Mondiale”. The 18th ISPL gathered 270 researchers from 33 countries. Sixty nine oral communications and 136 posters were presented during the 12 sessions of the Symposium. The sessions have covered all the different aspects of the Plant Lipid field including: Surface lipids: suberin, cutin and waxes, Fatty acids, Glycerolipids, Plant lipids as renewable sources of energy, Seed oils and bioengineering of metabolic pathways, Lipid catabolism, Models for lipid studies: lower plants, micro-organisms and others, Modifications of proteins by lipids, Sphingolipids, sterols and isoprenoids, Lipid signaling and plant stress responses, Lipid trafficking and membrane dynamics, New methods and technologies: functional lipidomics, fluxome, modelling

ACYL-ACYL CARRIER PROTEIN DESATURASE2 and 3 are responsible for making omega-7 fatty acids in the Arabidopsis aleurone

Omega-7 monounsaturated fatty acids (ω-7s) are specifically enriched in the aleurone of Arabidopsis (Arabidopsis thaliana) seeds. We found significant natural variation in seed ω-7 content and used a Multiparent Advanced Generation Inter-Cross population to fine-map a major quantitative trait loci to a region containing ACYL-ACYL CARRIER PROTEIN DESATURASE1 (AAD1) and AAD3. We found that AAD3 expression is localized to the aleurone where mutants show an approximately 50% reduction in ω-7 content. By contrast, AAD1 is localized to the embryo where mutants show a small reduction in ω-9 content. Enzymatic analysis has previously shown that AAD family members possess both stearoyl- and palmitoyl-ACP Δ9 desaturase activity, including the predominant isoform SUPPRESSOR OF SALICYLIC ACID INSENSITIVE2. However, aad3 ssi2 aleurone contained the same amount of ω-7s as aad3. Within the AAD family, AAD3 shares the highest degree of sequence similarity with AAD2 and AAD4. Mutant analysis showed that AAD2 also contributes to ω-7 production in the aleurone, and aad3 aad2 exhibits an approximately 85% reduction in ω-7s. Mutant analysis also showed that FATTY ACID ELONGASE1 is required for the production of very long chain ω-7s in the aleurone. Together, these data provide genetic evidence that the ω-7 pathway proceeds via Δ9 desaturation of palmitoyl-ACP followed by elongation of the product. Interestingly, significant variation was also identified in the ω-7 content of Brassica napus aleurone, with the highest level detected being approximately 47% of total fatty acids

Cyclin-dependent kinase activity enhances phosphatidylcholine biosynthesis in Arabidopsis by repressing phosphatidic acid phosphohydrolase activity

Coordination of endomembrane biogenesis with cell cycle progression is considered to be important in maintaining cell function during growth and development. We previously showed that disruption of PHOSPHATIDIC ACID PHOSPHOHYDROLASE (PAH) activity in Arabidopsis thaliana stimulates biosynthesis of the major phospholipid phosphatidylcholine (PC) and causes expansion of the endoplasmic reticulum. Here we show that PC biosynthesis is repressed by disruption of the core cell cycle regulator CYCLIN-DEPENDENT KINASE A;1 (CDKA;1) and that this repression is reliant on PAH. Furthermore, we show that CDKs phosphorylate PAH1 at serine 162, which reduces both its activity and membrane association. Expression of a CDK-insensitive version of PAH1 with a serine 162 to alanine substitution represses PC biosynthesis and also reduces the rate of cell division in early leaf development. Together our findings reveal a physiologically important mechanism that couples the rate of phospholipid biosynthesis and endomembrane biogenesis to cell cycle progression in Arabidopsis