| 1. |
- Arnqvist, Lisa, 1970-, et al.
(author)
-
Overexpression of CYP710A1 and CYP710A4 in transgenic Arabidopsis plants increases the level of stigmasterol at the expense of sitosterol
- 2008
-
In: Planta. - : Springer Science and Business Media LLC. - 0032-0935 .- 1432-2048. ; 227:2, s. 309-317
-
Journal article (peer-reviewed)abstract
- Sitosterol and stigmasterol are major sterols in vascular plants. An altered stigmasterol:sitosterol ratio has been proposed to influence the properties of cell membranes, particularly in relation to various stresses, but biosynthesis of stigmasterol is poorly understood. Recently, however, Morikawa et al. (Plant Cell 18:1008–1022, 2006) showed in Arabidopsis thaliana that synthesis of stigmasterol and brassicasterol is catalyzed by two separate sterol C-22 desaturases, encoded by the genes CYP710A1 and CYP710A2, respectively. The proteins belong to a small cytochrome P450 subfamily having four members, denoted by CYP710A1-A4, and are related to the yeast sterol C-22 desaturase Erg5p acting in ergosterol synthesis. Here, we report on our parallel investigation of the Arabidopsis CYP710A family. To elucidate the function of CYP710A proteins, transgenic Arabidopsis plants were generated overexpressing CYP710A1 and CYP710A4. Compared to wild-type plants, both types of transformant displayed a normal phenotype, but contained increased levels of free stigmasterol and a concomitant decrease in the level of free sitosterol. CYP710A1 transformants also displayed higher levels of esterified forms of stigmasterol, cholesterol, 24-methylcholesterol and isofucosterol. The results confirm the findings of Morikawa et al. (Plant Cell 18:1008–1022, 2006) regarding the function of CYP710A1 in stigmasterol synthesis, and show that CYP710A4 also has this capacity. Furthermore, our results suggest that an increased stigmasterol level alone is sufficient to stimulate esterification of other major sterols.
|
|
| 2. |
- Aslan, Selcuk, et al.
(author)
-
Increased production of wax esters in transgenic tobacco plants by expression of a fatty acid reductase:wax synthase gene fusion
- 2015
-
In: Transgenic Research. - : Springer Science and Business Media LLC. - 0962-8819 .- 1573-9368. ; 24, s. 945-953
-
Journal article (peer-reviewed)abstract
- Wax esters are hydrophobic lipids consisting of a fatty acid moiety linked to a fatty alcohol with an ester bond. Plant-derived wax esters are today of particular concern for their potential as cost-effective and sustainable sources of lubricants. However, this aspect is hampered by the fact that the level of wax esters in plants generally is too low to allow commercial exploitation. To investigate whether wax ester biosynthesis can be increased in plants using transgenic approaches, we have here exploited a fusion between two bacterial genes together encoding a single wax ester-forming enzyme, and targeted the resulting protein to chloroplasts in stably transformed tobacco (Nicotiana benthamiana) plants. Compared to wild-type controls, transgenic plants showed both in leaves and stems a significant increase in the total level of wax esters, being eight-fold at the whole plant level. The profiles of fatty acid methyl ester and fatty alcohol in wax esters were related, and C16 and C18 molecules constituted predominant forms. Strong transformants displayed certain developmental aberrations, such as stunted growth and chlorotic leaves and stems. These negative effects were associated with an accumulation of fatty alcohols, suggesting that an adequate balance between formation and esterification of fatty alcohols is crucial for a high wax ester production. The results show that wax ester engineering in transgenic plants is feasible, and suggest that higher yields may become achieved in the near future.
|
|
| 3. |
- Aslan, Selcuk, et al.
(author)
-
Transient silencing of the KASII genes is feasible in Nicotiana benthamiana for metabolic engineering of wax ester composition
- 2015
-
In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 5
-
Journal article (peer-reviewed)abstract
- The beta-ketoacyl-ACP synthase II (KASII) is an enzyme in fatty acid biosynthesis, catalyzing the elongation of 16:0-acyl carrier protein (ACP) to 18:0-ACP in plastids. Mutations in KASII genes in higher plants can lead to lethality, which makes it difficult to utilize the gene for lipid metabolic engineering. We demonstrated previously that transient expression of plastid-directed fatty acyl reductases and wax ester synthases could result in different compositions of wax esters. We hypothesized that changing the ratio between C16 (palmitoyl-compounds) and C18 (stearoyl-compounds) in the plastidic acyl-ACP pool by inhibition of KASII expression would change the yield and composition of wax esters via substrate preference of the introduced enzymes. Here, we report that transient inhibition of KASII expression by three different RNAi constructs in leaves of N. benthamiana results in almost complete inhibition of KASII expression. The transient RNAi approach led to a shift of carbon flux from a pool of C18 fatty acids to C16, which significantly increased wax ester production in AtFAR6-containing combinations. The results demonstrate that transient inhibition of KASII in vegetative tissues of higher plants enables metabolic studies towards industrial production of lipids such as wax esters with specific quality and composition.
|
|
| 4. |
- Beste, Lisa, et al.
(author)
-
Synthesis of Hydroxylated Sterols in Transgenic Arabidopsis Plants Alters Growth and Steroid Metabolism
- 2011
-
In: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 157:1, s. 426-440
-
Journal article (peer-reviewed)abstract
- To explore mechanisms in plant sterol homeostasis, we have here increased the turnover of sterols in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum) plants by overexpressing four mouse cDNA encoding cholesterol hydroxylases (CHs), hydroxylating cholesterol at the C-7, C-24, C-25, or C-27 positions. Compared to the wild type, the four types of Arabidopsis transformant showed varying degrees of phenotypic alteration, the strongest one being in CH25 lines, which were dark-green dwarfs resembling brassinosteroid-related mutants. Gas chromatography-mass spectrometry analysis of extracts from wild-type Arabidopsis plants revealed trace levels of alpha and beta forms of 7-hydroxycholesterol, 7-hydroxycampesterol, and 7-hydroxysitosterol. The expected hydroxycholesterol metabolites in CH7-, CH24-, and CH25 transformants were identified and quantified using gas chromatography-mass spectrometry. Additional hydroxysterol forms were also observed, particularly in CH25 plants. In CH24 and CH25 lines, but not in CH7 ones, the presence of hydroxysterols was correlated with a considerable alteration of the sterol profile and an increased sterol methyltransferase activity in microsomes. Moreover, CH25 lines contained clearly reduced levels of brassinosteroids, and displayed an enhanced drought tolerance. Equivalent transformations of potato plants with the CH25 construct increased hydroxysterol levels, but without the concomitant alteration of growth and sterol profiles observed in Arabidopsis. The results suggest that an increased hydroxylation of cholesterol and/or other sterols in Arabidopsis triggers compensatory processes, acting to maintain sterols at adequate levels.
|
|
| 5. |
- Dahlin, Paul, 1983-
(author)
-
Analysis of sterol metabolism in the pathogenic oomycetes Saprolegnia parasitica and Phytophthora infestans
- 2016
-
Doctoral thesis (other academic/artistic)abstract
- The primary objective of this thesis was to investigate the sterol metabolism of two pathogenic oomycetes, specifically the processes of sterol synthesis and sterol acquisition in the fish pathogen Saprolegnia parasitica (Saprolegniales) and the plant pathogen Phytophthora infestans (Peronosporales). Furthermore, the effects of steroidal glycoalkaloids from Solanaceous plants, on P. infestans, were examined. The improved understanding of these processes should help to identify approaches for the identification of new oomycete inhibitors targeting sterol metabolism in agriculture and aquaculture farming systems, and to guide plant-breeding strategies to defend solanaceous plants against oomycetes.For these reasons, the molecular basis of the metabolic pathways of sterol synthesis and/or sterol acquisition was investigated. Sterols are derived from isoprenoids and indispensable in various biological processes. Our biochemical investigation of an oxidosqualene cyclase revealed that sterol synthesis in S. parasitica begins with the formation of lanosterol (Paper I), and a reconstruction of the complete sterol synthesis pathway to the final compound, fucosterol, in S. parasitica was performed using bioinformatics (Paper II). Complementary to this work, the extent to which P. infestans, which is incapable of de novo sterol synthesis, is able to modify exogenously provided sterols was investigated by determining the growth impact of various sterol supplements in the growth media (Paper II). Building on the sterol investigations, the solanaceous sterol derivatives from the glycoalkaloid family were analysed. These compounds contain both a steroidal and a carbohydrate (glycan) moiety. Data obtained by feeding various deuterium-labeled sterols to potato shoots, supported the theory that steroidal glycoalkaloids in Solanum tuberosum are produced from cholesterol (Paper III). Since these steroidal glycoalkaloids are thought to play a role in plant defense, their physiological effects on P. infestans were investigated (Paper IV). Unexpectedly we found that non-glycosylated steroidal alkaloids had a greater inhibitory effect than steroidal glycoalkaloids. Steroidal glycoalkaloids derived from other Solanaceous species exhibited different physiological effects on the growth of P. infestans. This research was conducted on two oomycete species belonging to the Saprolegniales and Peronosporales orders, hence the results presented are likely to be representative of each of these two oomycete orders.
|
|
| 6. |
|
|
| 7. |
- Jin, Yunkai, et al.
(author)
-
Improved bioenergy value of residual rice straw by increased lipid levels from upregulation of fatty acid biosynthesis
- 2023
-
In: Biotechnology for Biofuels and Bioproducts. - 2731-3654. ; 16
-
Journal article (peer-reviewed)abstract
- BackgroundRice (Oryza sativa) straw is a common waste product that represents a considerable amount of bound energy. This energy can be used for biogas production, but the rate and level of methane produced from rice straw is still low. To investigate the potential for an increased biogas production from rice straw, we have here utilized WRINKLED1 (WRI1), a plant AP2/ERF transcription factor, to increase triacylglycerol (TAG) biosynthesis in rice plants. Two forms of Arabidopsis thaliana WRI1 were evaluated by transient expression and stable transformation of rice plants, and transgenic plants were analyzed both for TAG levels and biogas production from straw.ResultsBoth full-length AtWRI1, and a truncated form lacking the initial 141 amino acids (including the N-terminal AP2 domain), increased fatty acid and TAG levels in vegetative and reproductive tissues of Indica rice. The stimulatory effect of the truncated AtWRI1 was significantly lower than that of the full-length protein, suggesting a role for the deleted AP2 domain in WRI1 activity. Full-length AtWRI1 increased TAG levels also in Japonica rice, indicating a conserved effect of WRI1 in rice lipid biosynthesis. The bio-methane production from rice straw was 20% higher in transformants than in the wild type. Moreover, a higher producing rate and final yield of methane was obtained for rice straw compared with rice husks, suggesting positive links between methane production and a high amount of fatty acids.ConclusionsOur results suggest that heterologous WRI1 expression in transgenic plants can be used to improve the metabolic potential for bioenergy purposes, in particular methane production.
|
|
| 8. |
- Larsson, Emma, et al.
(author)
-
Differential regulation of Knotted1-like genes during establishment of the shoot apical meristem in Norway spruce (Picea abies)
- 2012
-
In: Plant Cell Reports. - : Springer Science and Business Media LLC. - 0721-7714 .- 1432-203X. ; 31, s. 1053-1060
-
Journal article (peer-reviewed)abstract
- Establishment of the shoot apical meristem (SAM) in Arabidopsis embryos requires the KNOXI transcription factor SHOOT MERISTEMLESS. In Norway spruce (Picea abies), four KNOXI family members (HBK1, HBK2, HBK3 and HBK4) have been identified, but a corresponding role in SAM development has not been demonstrated. As a first step to differentiate between the functions of the four Norway spruce HBK genes, we have here analyzed their expression profiles during the process of somatic embryo development. This was made both under normal embryo development and under conditions of reduced SAM formation by treatment with the polar auxin transport inhibitor NPA. Concomitantly with the formation of an embryonic SAM, the HBK2 and HBK4 genes displayed a significant up-regulation that was delayed by NPA treatment. In contrast, HBK1 and HBK3 were up-regulated prior to SAM formation, and their temporal expression was not affected by NPA. Ectopic expression of the four HBK genes in transgenic Arabidopsis plants further supported similar functions of HBK2 and HBK4, distinct from those of HBK1 and HBK3. Together, the results suggest that HBK2 and HBK4 exert similar functions related to the SAM differentiation and somatic embryo development in Norway spruce, while HBK1 and HBK3 have more general functions during embryo development.
|
|
| 9. |
- Larsson, Emma, et al.
(author)
-
Expression of PaNAC01, a Picea abies CUP-SHAPED COTYLEDON orthologue, is regulated by polar auxin transport and associated with differentiation of the shoot apical meristem and formation of separated cotyledons
- 2012
-
In: Annals of Botany. - : Oxford University Press (OUP). - 0305-7364 .- 1095-8290. ; 110, s. 923-934
-
Journal article (peer-reviewed)abstract
- During embryo development in most gymnosperms, the establishment of the shoot apical meristem (SAM) occurs concomitantly with the formation of a crown of cotyledons surrounding the SAM. It has previously been shown that the differentiation of cotyledons in somatic embryos of Picea abies is dependent on polar auxin transport (PAT). In the angiosperm model plant, Arabidopsis thaliana, the establishment of cotyledonary boundaries and the embryonal SAM is dependent on PAT and the expression of the CUP-SHAPED COTYLEDON (CUC) genes, which belong to the large NAC gene family. The aim of this study was to characterize CUC-like genes in a gymnosperm, and to elucidate their expression during SAM and cotyledon differentiation, and in response to PAT.Sixteen Picea glauca NAC sequences were identified in GenBank and deployed to different clades within the NAC gene family using maximum parsimony analysis and Bayesian inference. Motifs conserved between angiosperms and gymnosperms were analysed using the motif discovery tool MEME. Expression profiles during embryo development were produced using quantitative real-time PCR. Protein conservation was analysed by introducing a P. abies CUC orthologue into the A. thaliana cuc1cuc2 double mutant.Two full-length CUC-like cDNAs denoted PaNAC01 and PaNAC02 were cloned from P. abies. PaNAC01, but not PaNAC02, harbours previously characterized functional motifs in CUC1 and CUC2. The expression profile of PaNAC01 showed that the gene is PAT regulated and associated with SAM differentiation and cotyledon formation. Furthermore, PaNAC01 could functionally substitute for CUC2 in the A. thaliana cuc1cuc2 double mutant.The results show that CUC-like genes with distinct signature motifs existed before the separation of angiosperms and gymnosperms approx. 300 million years ago, and suggest a conserved function between PaNAC01 and CUC1/CUC2.
|
|
| 10. |
- Liu, Qinsong, et al.
(author)
-
Vacuole Integrity Maintained by DUF300 Proteins Is Required for Brassinosteroid Signaling Regulation
- 2018
-
In: Molecular Plant. - : Cell Press. - 1674-2052 .- 1752-9867. ; 11:4, s. 553-567
-
Journal article (peer-reviewed)abstract
- Brassinosteroid (BR) hormone signaling controls multiple processes during plant growth and development and is initiated at the plasma membrane through the receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1) together with co-receptors such as BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1). BRI1 abundance is regulated by endosomal recycling and vacuolar targeting, but the role of vacuole-related proteins in BR receptor dynamics and BR responses remains elusive. Here, we show that the absence of two DUF300 domain-containing tonoplast proteins, LAZARUS1 (LAZ1) and LAZ1 HOMOLOG1 (LAZ1H1), causes vacuole morphology defects, growth inhibition, and constitutive activation of BR signaling. Intriguingly, tonoplast accumulation of BAK1 was substantially increased and appeared causally linked to enhanced BRI1 trafficking and degradation in laz1 laz1h1 plants. Since unrelated vacuole mutants exhibited normal BR responses, our findings indicate that DUF300 proteins play distinct roles in the regulation of BR signaling by maintaining vacuole integrity required to balance subcellular BAK1 pools and BR receptor distribution.
|
|
