Differential regulation of serine acetyltransferase is involved in nickel hyperaccumulation in Thlaspi goesingense

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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

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

Dissecting the components controlling root-to-shoot arsenic translocation in Arabidopsis thaliana

Arsenic (As) is an important environmental and food-chain toxin. We investigated the key components controlling As accumulation and tolerance in Arabidopsis thaliana. We tested the effects of different combinations of gene knockout, including arsenate reductase (HAC1), γ-glutamyl-cysteine synthetase (γ-ECS), phytochelatin synthase (PCS1) and phosphate effluxer (PHO1), and the heterologous expression of the As-hyperaccumulator Pteris vittata arsenite efflux (PvACR3), on As tolerance, accumulation, translocation and speciation in A. thaliana. Heterologous expression of PvACR3 markedly increased As tolerance and root-to-shoot As translocation in A. thaliana, with PvACR3 being localized to the plasma membrane. Combining PvACR3 expression with HAC1 mutation led to As hyperaccumulation in the shoots, whereas combining HAC1 and PHO1 mutation decreased As accumulation. Mutants of γ-ECS and PCS1 were hypersensitive to As and had higher root-to-shoot As translocation. Combining γ-ECS or PCS1 with HAC1 mutation did not alter As tolerance or accumulation beyond the levels observed in the single mutants. PvACR3 and HAC1 have large effects on root-to-shoot As translocation. Arsenic hyperaccumulation can be engineered in A. thaliana by knocking out the HAC1 gene and expressing PvACR3. PvACR3 and HAC1 also affect As tolerance, but not to the extent of γ-ECS and PCS1

The role of serine acetyltransferase in nickel and selenium assimilation and tolerance in metal hyperaccumulators

When growing in its native habitat, hyperaccumulators can accumulate two to three orders of magnitude higher concentration of metals in their shoots when compared to other plants grown in the same habitat, without showing symptoms of metal toxicity. We previously reported that the constitutively elevated concentration of the antioxidant glutathione (GSH) is involved in the ability of Ni hyperaccumulating Thlaspi species to protect against nickel (Ni)-induced oxidative stress. Given the central role of SAT in regulating sulfur (S) assimilation and GSH biosynthesis we propose that SAT is differentially regulated in Thlaspi goesingense to allow for enhanced GSH biosynthesis. Specifically, we hypothesize that SAT in T. goesingense is less sensitive to inhibition by cysteine. Such reduced sensitivity to cysteine would provide a mechanism to allow enhanced steady-state levels of O-acetylserine (OAS), leading to increased cysteine and ultimately GSH biosynthesis. We have previously cloned three SAT cDNAs from T. goesingense. Based on the predicted amino acid sequences of these TgSATs, and their alignment to the SATs from the nonaccumulator A. thaliana, we predicted which SAT isoform was homologous to the cysteine sensitive cytosolic SAT from A. thaliana. We found that recombinant cytosolic SAT from T. goesingense (TgSAT-c) is not inhibited by cysteine unlike the homologous cytosolic SAT from A. thaliana (AtSAT-c) which is sensitive to cysteine. Using domain swapping and site-directed mutagenesis we identified two amino acid residues in the C-terminus of TgSAT-c that are critical for the cysteine insensitivity of this enzyme compared to AtSAT-c. A change of cysteine to proline is most critical for this loss of cysteine sensitivity, with alanine to glycine playing a secondary role. Moreover, we also observed that this proline residue in TgSAT-c was also related to the competitive binding site for cysteine and the SAT substrate serine. Heterologous expression of the engineered and native cysteine insensitive SAT was observed to lead to increased Ni tolerance in both E. coli and A. thaliana when compared to expression of the cysteine sensitive SAT. Interestingly, the proline and alanine residues in SAT-c are well conserved in Thlaspi species, occurring in hyperaccumulator and nonaccumulator species, and this may partially explain the elevated OAS and GSH contents we observe across the Thlaspi genus. Furthermore, this may suggest why the Thlaspi genus appears to be evolutionally preadapt for Ni hyperaccumulation. Selenium (Se) hyperaccumulators also have an elevated OAS content compared to nonaccumulators. Given the central role of OAS in S assimilation, we hypothesized that this elevated OAS plays an important role in up-regulating selenate uptake and reduction in Se hyperaccumulating species of Astragalus. Furthermore, we hypothesized that differential regulation of SAT in the Se hyperaccumulator leads to this elevation of OAS content compared to nonaccumulator. To directly test this, we cloned SAT cDNAs from the Se hyperaccumulator A. bisulcatus and the nonaccumulator A. drummondii. We found that recombinant cytosolic SAT from A. bisulcatus is less sensitive to cysteine and has a higher maximum enzyme activity compared to recombinant cytosolic SAT from A. drummondii. Furthermore, we found that the subcellular localization of SAT in A. bisulcatus differs from A. drummondii and this may also affect the production of OAS. Expression of Astragalus SAT in A. thaliana was not sufficient to produce Se hyperaccumulation. However, our preliminary results suggest that the expression of SAT and selenocysteine methyltransferase (SMT) together may be sufficient to induce Se hyperaccumulation. We demonstrate that the expression of SAT in combination with SMT leads to increased OAS, possibly up-regulating the expression of sulfate transporters, and increasing Se uptake in plants, although more work is needed to confirm these results

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

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

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