Enhancing microRNA167A expression in seed decreases the α-linolenic acid content and increases seed size in Camelina sativa.

Despite well established roles of microRNAs in plant development, few aspects have been addressed to understand their effects in seeds especially on lipid metabolism. In this study, we showed that overexpressing microRNA167A (miR167OE) in camelina (Camelina sativa) under a seed-specific promoter changed fatty acid composition and increased seed size. Specifically, the miR167OE seeds had a lower α-linolenic acid with a concomitantly higher linoleic acid content than the wild-type. This decreased level of fatty acid desaturation corresponded to a decreased transcriptional expression of the camelina fatty acid desaturase3 (CsFAD3) in developing seeds. MiR167 targeted the transcription factor auxin response factor (CsARF8) in camelina, as had been reported previously in Arabidopsis. Chromatin immunoprecipitation experiments combined with transcriptome analysis indicated that CsARF8 bound to promoters of camelina bZIP67 and ABI3 genes. These transcription factors directly or through the ABI3-bZIP12 pathway regulate CsFAD3 expression and affect α-linolenic acid accumulation. In addition, to decipher the miR167A-CsARF8 mediated transcriptional cascade for CsFAD3 suppression, transcriptome analysis was conducted to implicate mechanisms that regulate seed size in camelina. Expression levels of many genes were altered in miR167OE, including orthologs that have previously been identified to affect seed size in other plants. Most notably, genes for seed coat development such as suberin and lignin biosynthesis were down-regulated. This study provides valuable insights into the regulatory mechanism of fatty acid metabolism and seed size determination, and suggests possible approaches to improve these important traits in camelina

An analysis of expressed sequence tags of developing castor endosperm using a full-length cDNA library

<p>Abstract</p> <p>Background</p> <p>Castor seeds are a major source for ricinoleate, an important industrial raw material. Genomics studies of castor plant will provide critical information for understanding seed metabolism, for effectively engineering ricinoleate production in transgenic oilseeds, or for genetically improving castor plants by eliminating toxic and allergic proteins in seeds.</p> <p>Results</p> <p>Full-length cDNAs are useful resources in annotating genes and in providing functional analysis of genes and their products. We constructed a full-length cDNA library from developing castor endosperm, and obtained 4,720 ESTs from 5'-ends of the cDNA clones representing 1,908 unique sequences. The most abundant transcripts are genes encoding storage proteins, ricin, agglutinin and oleosins. Several other sequences are also very numerous, including two acidic triacylglycerol lipases, and the oleate hydroxylase (<it>FAH12</it>) gene that is responsible for ricinoleate biosynthesis. The role(s) of the lipases in developing castor seeds are not clear, and co-expressing of a lipase and the FAH12 did not result in significant changes in hydroxy fatty acid accumulation in transgenic Arabidopsis seeds. Only one oleate desaturase (<it>FAD2</it>) gene was identified in our cDNA sequences. Sequence and functional analyses of the castor <it>FAD2 </it>were carried out since it had not been characterized previously. Overexpression of castor FAD2 in a FAH12-expressing <it>Arabidopsis </it>line resulted in decreased accumulation of hydroxy fatty acids in transgenic seeds.</p> <p>Conclusion</p> <p>Our results suggest that transcriptional regulation of <it>FAD2 </it>and <it>FAH12 </it>genes maybe one of the mechanisms that contribute to a high level of ricinoleate accumulation in castor endosperm. The full-length cDNA library will be used to search for additional genes that affect ricinoleate accumulation in seed oils. Our EST sequences will also be useful to annotate the castor genome, which whole sequence is being generated by shotgun sequencing at the Institute for Genome Research (TIGR).</p

Cloning of a cDNA encoding diacylglycerol acyltransferase from Arabidopsis thaliana and its functional expression

AbstractTriacylglycerols are the most important storage lipids in most plants and animals. Acyl-CoA:diacylglycerol acyltransferase (EC 2.3.1.20) catalyzes the final step of the pathway of triacylglycerol synthesis and is the only step which is unique to this process. Diacylglycerol acyltransferase is required for the synthesis of storage oil in a wide range of oil-bearing seeds and fruits and in floral structures such as petals, anthers and pollen. We describe the first cloning and functional expression of a cDNA encoding diacylglycerol acyltransferase from a plant. The cDNA, cloned from Arabidopsis thaliana, encodes a 520 amino acid protein with a predicted molecular mass of 59.0 kDa which shares 38% amino acid sequence identity with diacylglycerol acyltransferase from mouse. When expressed in insect cell cultures, the protein catalyzes the synthesis of [14C]triacylglycerol from [14C]diacylglycerol and acyl-CoA. Primer extension analysis revealed that the transcription begins 225 bases before the translation start site, yielding an unusually long 5′ untranslated region. The gene is expressed in a wide range of tissues but most strongly in developing embryos and petals of flowers

A Phospholipase C-Like Protein From Ricinus communis Increases Hydroxy Fatty Acids Accumulation in Transgenic Seeds of Camelina sativa

There have been strong interests in producing unusual fatty acids in oilseed crops to provide renewable industrial feedstock. Results are so far largely disappointing since much lower amounts of such fatty acids accumulate in genetically engineered seeds than in their original natural sources. It has been suggested that the flux of unusual fatty acids through phosphatidylcholine (PC) represents a major bottleneck for high accumulation of such fatty acids in triacylglycerol (TAG). We show here that a phospholipase C-like protein (RcPLCL1) from castor bean, which accumulates nearly 90% of the hydroxylated ricinoleic acid in its seed TAG, increases the amount of hydroxy fatty acids (HFAs) when co-expresses with the fatty acid hydroxylase (RcFAH12) in transgenic seed of Camelina sativa. RcPLCL1 shows hydrolyzing activities on both PC and phosphatidylinositol substrates in our in vitro assay conditions. The PC-PLC activity of the RcPLCL1 may have increased the efficiency of HFA-PC to diacylglycerol conversion, which explains our observation of increased HFA contents in TAG concomitant with decreased HFA in the membrane lipid PC during seed development. Consequently, this may also alleviate the potential detrimental effect of HFA on germination of the engineered camelina seeds. Our results provide new knowledge that will help design effective strategies to engineer high levels of HFAs in transgenic oilseeds

Towards the synthetic design of camelina oil enriched in tailored acetyl-triacylglycerols with medium-chain fatty acids

The ability to manipulate expression of key biosynthetic enzymes has allowed the development of genetically modified plants that synthesise unusual lipids that are useful for biofuel and industrial applications. By taking advantage of the unique activities of enzymes from different species, tailored lipids with a targeted structure can be conceived. In this study we demonstrate the successful implementation of such an approach by metabolically engineering the oilseed crop Camelina sativa to produce 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) with medium-chain fatty acids (MCFAs). Different transgenic camelina lines that had been genetically modified to produce MCFAs through the expression of MCFA-specific thioesterases and acyltransferases were retransformed with the Euonymus alatus gene for diacylglycerol acetyltransferase (EaDAcT) that synthesises acetyl-TAGs. Concomitant RNAi suppression of acyl-CoA:diacylglycerol acyltransferase increased the levels of acetyl-TAG, with up to 77 mole percent in the best lines. However, the total oil content was reduced. Analysis of the composition of the acetyl-TAG molecular species using electrospray ionisation mass spectrometry demonstrated the successful synthesis of acetyl-TAG containing MCFAs. Field growth of high-yielding plants generated enough oil for quantification of viscosity. As part of an ongoing design–test–learn cycle, these results, which include not only the synthesis of ‘designer’ lipids but also their functional analysis, will lead to the future production of such molecules tailored for specific applications

Heat stress during reproductive stages reduces camelina seed productivity and changes seed composition

Camelina (Camelina sativa L. Crantz) is a low-input oilseed crop with great potential in bioenergy and industrial oils. Improving tolerance to high temperatures is essential for camelina agronomic sustainability. Two genotypes, Suneson and Pryzeth, were exposed to a transient 14-day heat stress at 37 °C during the reproductive stages. Four cohorts of pods along the main stem, which were at different stages from fully developed pods (C1), young pods (C2), open flowers (C3) and flowering buds (C4) at the time of heat treatment, were examined for morphological and seed quality traits at maturity. The main stem length was shortened in both genotypes. Pods and seeds in all cohorts were negatively affected by heat, resulting in lower seed yield and reduced oil content. Seed size and seed weight had the greatest reduction in C1, pod size reduction was found the most in C3, and the number of fertile pods that contain at least one seed was reduced in C3 and C4. These results suggest that heat stress effects are developmental stage specific. Heat stress significantly reduced fertility during flowering and inhibited storage product biosynthesis and accumulation during seed filling which resulted in smaller and lighter seeds. Analyzing seed composition indicated that oil content decreased while protein content increased in seeds from heat treated plants. In addition, fatty acid composition was altered with the reduction of omega-3 α-linolenic acid and concomitantly increased omega-6 linoleic acid being the most significantly affected. Our results also revealed the different responses in the two genotypes examined, suggesting genetic variation in camelina germplasm which can be explored to improve heat tolerance. This study provides resources and guidance for future studies to understand genetic and physiological mechanisms of heat stress and to assist in improving the sustainability of camelina production facing climate change

The Phosphatidylcholine Diacylglycerol Cholinephosphotransferase Is Required for Efficient Hydroxy Fatty Acid Accumulation in Transgenic Arabidopsis1[W][OA]

We previously identified an enzyme, phosphatidylcholine diacylglycerol cholinephosphotransferase (PDCT), that plays an important role in directing fatty acyl fluxes during triacylglycerol (TAG) biosynthesis. The PDCT mediates a symmetrical interconversion between phosphatidylcholine (PC) and diacylglycerol (DAG), thus enriching PC-modified fatty acids in the DAG pool prior to forming TAG. We show here that PDCT is required for the efficient metabolism of engineered hydroxy fatty acids in Arabidopsis (Arabidopsis thaliana) seeds. When a fatty acid hydroxylase (FAH12) from castor (Ricinus communis) was expressed in Arabidopsis seeds, the PDCT-deficient mutant accumulated only about half the amount of hydroxy fatty acids compared with that in the wild-type seeds. We also isolated a PDCT from castor encoded by the RcROD1 (Reduced Oleate Desaturation1) gene. Seed-specific coexpression of this enzyme significantly increased hydroxy fatty acid accumulation in wild type-FAH12 and in a previously produced transgenic Arabidopsis line coexpressing a castor diacylglycerol acyltransferase 2. Analyzing the TAG molecular species and regiochemistry, along with analysis of fatty acid composition in TAG and PC during seed development, indicate that PDCT acts in planta to enhance the fluxes of fatty acids through PC and enrich the hydroxy fatty acids in DAG, and thus in TAG. In addition, PDCT partially restores the oil content that is decreased in FAH12-expressing seeds. Our results add a new gene in the genetic toolbox for efficiently engineering unusual fatty acids in transgenic oilseeds

Acyl Editing and Headgroup Exchange Are the Major Mechanisms That Direct Polyunsaturated Fatty Acid Flux into Triacylglycerols

Triacylglycerols (TAG) in seeds of Arabidopsis (Arabidopsis thaliana) and many plant species contain large amounts of polyunsaturated fatty acids (PUFA). These PUFA are synthesized on the membrane lipid phosphatidylcholine (PC). However, the exact mechanisms of how fatty acids enter PC and how they are removed from PC after being modified to participate in the TAG assembly are unclear, nor are the identities of the key enzymes/genes that control these fluxes known. By reverse genetics and metabolic labeling experiments, we demonstrate that two genes encoding the lysophosphatidylcholine acyltransferases LPCAT1 and LPCAT2 in Arabidopsis control the previously identified “acyl-editing” process, the main entry of fatty acids into PC. The lpcat1/lpcat2 mutant showed increased contents of very-long-chain fatty acids and decreased PUFA in TAG and the accumulation of small amounts of lysophosphatidylcholine in developing seeds revealed by [(14)C]acetate-labeling experiments. We also showed that mutations in LPCATs and the PC diacylglycerol cholinephosphotransferase in the reduced oleate desaturation1 (rod1)/lpcat1/lpcat2 mutant resulted in a drastic reduction of PUFA content in seed TAG, accumulating only one-third of the wild-type level. These results indicate that PC acyl editing and phosphocholine headgroup exchange between PC and diacylglycerols control the majority of acyl fluxes through PC to provide PUFA for TAG synthesis

Towards the synthetic design of camelina oil enriched in tailored acetyl-triacylglycerols with medium-chain fatty acids

The ability to manipulate expression of key biosynthetic enzymes has allowed the development of genetically modified plants that synthesise unusual lipids that are useful for biofuel and industrial applications. By taking advantage of the unique activities of enzymes from different species, tailored lipids with a targeted structure can be conceived. In this study we demonstrate the successful implementation of such an approach by metabolically engineering the oilseed crop Camelina sativa to produce 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) with medium-chain fatty acids (MCFAs). Different transgenic camelina lines that had been genetically modified to produce MCFAs through the expression of MCFA-specific thioesterases and acyltransferases were retransformed with the Euonymus alatus gene for diacylglycerol acetyltransferase (EaDAcT) that synthesises acetyl-TAGs. Concomitant RNAi suppression of acyl-CoA:diacylglycerol acyltransferase increased the levels of acetyl-TAG, with up to 77 mole percent in the best lines. However, the total oil content was reduced. Analysis of the composition of the acetyl-TAG molecular species using electrospray ionisation mass spectrometry demonstrated the successful synthesis of acetyl-TAG containing MCFAs. Field growth of high-yielding plants generated enough oil for quantification of viscosity. As part of an ongoing design–test–learn cycle, these results, which include not only the synthesis of ‘designer’ lipids but also their functional analysis, will lead to the future production of such molecules tailored for specific applications

Seed-specific suppression of ADP-glucose pyrophosphorylase in Camelina sativa increases seed size and weight

Abstract Background Camelina (Camelina sativa L.) is a promising oilseed crop that may provide sustainable feedstock for biofuel production. One of the major drawbacks of Camelina is its smaller seeds compared to other major oil crops such as canola, which limit oil yield and may also pose challenges in successful seedling establishment, especially in dryland cultivation. Previous studies indicate that seed development may be under metabolic control. In oilseeds, starch only accumulates temporarily during seed development but is almost absent in mature seeds. In this study, we explored the effect of altering seed carbohydrate metabolism on Camelina seed size through down-regulating ADP-glucose pyrophosphorylase (AGPase), a major enzyme in starch biosynthesis. Results An RNAi construct comprising sequences of the Camelina small subunit of an AGPase (CsAPS) was expressed in Camelina cultivar Suneson under a seed-specific promoter. The RNAi suppression reduced AGPase activities which concurred with moderately decreased starch accumulation during seed development. Transcripts of genes examined that are involved in storage products were not affected, but contents of sugars and water were increased in developing seeds. The transgenic seeds were larger than wild-type plants due to increased cell sizes in seed coat and embryos, and mature seeds contained similar oil but more protein contents. The larger seeds showed advantages on seedling emergence from deep soils. Conclusions Changing starch and sugar metabolism during seed development may increase the size and mass of seeds without affecting their final oil content in Camelina. Increased seed size may improve seedling establishment in the field and increase seed yield