Production of (10E,12Z)-conjugated linoleic acid in yeast and tobacco seeds

Hornung, Ellen; Krueger, Claudia; Pernstich, Christian; Gipmans, Martijn; Porzel, Andrea; Feußner, Ivo

doi:10.1016/j.bbalip.2005.11.004

Cited by 72 publications

(107 citation statements)

References 32 publications

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“…The insert was introduced by Gateway cloning (Invitrogen) into the binary plasmid pCAMBIA modified for resistance to ammonium glufosinate and seed-specific expression using the USP promoter (22). The construct was transformed into chemically competent Agrobacteria (strain EH105), and introduced into Arabidopsis thaliana ecotype Columbia by floral dipping (23).…”

Section: Methodsmentioning

confidence: 99%

“…Cloning and Vector Construction for Production of Transgenic Arabidopsis Plants-The cDNA ORF of An⌬12/3 was moved as an EcoRI/NotI-fragment into the Gateway Entry vector pUC18-Entry (22). The insert was introduced by Gateway cloning (Invitrogen) into the binary plasmid pCAMBIA modified for resistance to ammonium glufosinate and seed-specific expression using the USP promoter (22).…”

Section: Methodsmentioning

confidence: 99%

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A Small Membrane-peripheral Region Close to the Active Center Determines Regioselectivity of Membrane-bound Fatty Acid Desaturases from Aspergillus nidulans

Hoffmann¹,

Hornung²,

Busch

et al. 2007

Journal of Biological Chemistry

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Fatty acid desaturases catalyze the introduction of double bonds at specific positions of an acyl chain and are categorized according to their substrate specificity and regioselectivity. The current understanding of membrane-bound desaturases is based on mutant studies, biochemical topology analysis, and the comparison of related enzymes with divergent functionality. Because structural information is lacking, the principles of membrane-bound desaturase specificity are still not understood despite of substantial research efforts. Here we compare two membrane-bound fatty acid desaturases from Aspergillus nidulans: a strictly monofunctional oleoyl-⌬12 desaturase and a processive bifunctional oleoyl-⌬12/linoleoyl-3 desaturase. The high similarities in the primary sequences of the enzymes provide an ideal starting point for the systematic analysis of factors determining substrate specificity and bifunctionality. Based on the most current topology models, both desaturases were divided into nine domains, and the domains of the monofunctional ⌬12 desaturase were systematically exchanged for their respective corresponding matches of the bifunctional sister enzyme. Catalytic capacities of hybrid enzymes were tested by heterologous expression in yeast, followed by biochemical characterization of the resulting fatty acid patterns. The individual exchange of two domains of a length of 18 or 49 amino acids each resulted in bifunctional ⌬12/3 activity of the previously monofunctional parental enzyme. Sufficient determinants of fatty acid desaturase substrate specificity and bifunctionality could, thus, be narrowed down to a membrane-peripheral region close to the catalytic site defined by conserved histidine-rich motifs in the topology model.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A Small Membrane-peripheral Region Close to the Active Center Determines Regioselectivity of Membrane-bound Fatty Acid Desaturases from Aspergillus nidulans

Hoffmann¹,

Hornung²,

Busch

et al. 2007

Journal of Biological Chemistry

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“…For constructs for particle bombardment, +2EYFP amplicons were moved into the plasmid pUC18Entry (Hornung et al, 2005) as NotI/ EcoRV fragments, creating the plasmid pUC18Entry-EYFP. Prior to ligation, the ccdb gene was eliminated from pUC18Entry by EcoRI digestion and religation.…”

Section: Fusion Constructs For Stable Expression In Arabidopsismentioning

confidence: 99%

Type B Phosphatidylinositol-4-Phosphate 5-Kinases MediateArabidopsisandNicotiana tabacumPollen Tube Growth by Regulating Apical Pectin Secretion

Ischebeck¹,

Stenzel²,

Heilmann³

2008

The Plant Cell

175

262

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Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P 2 ] occurs in the apical plasma membrane of growing pollen tubes. Because enzymes responsible for PtdIns(4,5)P 2 production at that location are uncharacterized, functions of PtdIns(4,5)P 2 in pollen tube tip growth are unresolved. Two candidate genes encoding pollen-expressed Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinases (PI4P 5-kinases) of Arabidopsis subfamily B were identified (PIP5K4 and PIP5K5), and their recombinant proteins were characterized as being PI4P 5-kinases. Pollen of T-DNA insertion lines deficient in both PIP5K4 and PIP5K5 exhibited reduced pollen germination and defects in pollen tube elongation. Fluorescence-tagged PIP5K4 and PIP5K5 localized to an apical plasma membrane microdomain in Arabidopsis and tobacco (Nicotiana tabacum) pollen tubes, and overexpression of either PIP5K4 or PIP5K5 triggered multiple tip branching events. Further studies using the tobacco system revealed that overexpression caused massive apical pectin deposition accompanied by plasma membrane invaginations. By contrast, callose deposition and cytoskeletal structures were unaltered in the overexpressors. Morphological effects depended on PtdIns(4,5)P 2 production, as an inactive enzyme variant did not produce any effects. The data indicate that excessive PtdIns(4,5)P 2 production by type B PI4P 5-kinases disturbs the balance of membrane trafficking and apical pectin deposition. Polar tip growth of pollen tubes may thus be modulated by PtdIns(4,5)P 2 via regulatory effects on membrane trafficking and/or apical pectin deposition.

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“…To create constructs for mutant complementation tests, the 1500-bp promoter fragment promPIP5K3 was moved as a SalI-NotI fragment into the vector pUC18-Entry (Hornung et al, 2005), yielding the plasmid promPIP5K3puc18entry. The full-length PIP5K3 cDNA sequence was moved from PIP5K3-pGEM-Teasy2 as a NotI-NotI fragment into the vector promPIP5K3puc18entry, yielding the plasmid promPIP5K3-PIP5K3puc18entry.…”

Section: Mutant Complementation Constructsmentioning

confidence: 99%

“…The PCR product was moved as a SalI-NotI fragment into the vector pUC18-Entry (Hornung et al, 2005), yielding the plasmid prom-EXP7-puc18entry. The complete cDNA sequence of PIP5K3 was amplified with the primer combination PIP5K3Nco_for/PIP5K3_rev2 using the plasmid PIP5K3-pGEM-Teasy as a template, and the PCR product was cloned into pGEM-Teasy.…”

Section: Pip5k3 Overexpression Constructsmentioning

confidence: 99%

The Type B Phosphatidylinositol-4-Phosphate 5-Kinase 3 Is Essential for Root Hair Formation inArabidopsis thaliana

et al. 2008

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Root hairs are extensions of root epidermal cells and a model system for directional tip growth of plant cells. A previously uncharacterized Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinase gene (PIP5K3) was identified and found to be expressed in the root cortex, epidermal cells, and root hairs. Recombinant PIP5K3 protein was catalytically active and converted phosphatidylinositol-4-phosphate to phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P 2 ]. Arabidopsis mutant plants homozygous for T-DNA-disrupted PIP5K3 alleles were compromised in root hair formation, a phenotype complemented by expression of wild-type PIP5K3 cDNA under the control of a 1500-bp PIP5K3 promoter fragment. Root hair-specific PIP5K3 overexpression resulted in root hair deformation and loss of cell polarity with increasing accumulation of PIP5K3 transcript. Using reestablishment of root hair formation in T-DNA mutants as a bioassay for physiological functionality of engineered PIP5K3 variants, catalytic activity was found to be essential for physiological function, indicating that PtdIns(4,5)P 2 formation is required for root hair development. An N-terminal domain containing membrane occupation and recognition nexus repeats, which is not required for catalytic activity, was found to be essential for the establishment of root hair growth. Fluorescencetagged PIP5K3 localized to the periphery of the apical region of root hair cells, possibly associating with the plasma membrane and/or exocytotic vesicles. Transient heterologous expression of full-length PIP5K3 in tobacco (Nicotiana tabacum) pollen tubes increased plasma membrane association of a PtdIns(4,5)P 2 -specific reporter in these tip-growing cells. The data demonstrate that root hair development requires PIP5K3-dependent PtdIns(4,5)P 2 production in the apical region of root hair cells.

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Production of (10E,12Z)-conjugated linoleic acid in yeast and tobacco seeds

Cited by 72 publications

References 32 publications

A Small Membrane-peripheral Region Close to the Active Center Determines Regioselectivity of Membrane-bound Fatty Acid Desaturases from Aspergillus nidulans

A Small Membrane-peripheral Region Close to the Active Center Determines Regioselectivity of Membrane-bound Fatty Acid Desaturases from Aspergillus nidulans

Type B Phosphatidylinositol-4-Phosphate 5-Kinases MediateArabidopsisandNicotiana tabacumPollen Tube Growth by Regulating Apical Pectin Secretion

The Type B Phosphatidylinositol-4-Phosphate 5-Kinase 3 Is Essential for Root Hair Formation inArabidopsis thaliana

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