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Swarup R Kargul J Marchant A Zadik D Rahman A Mills R Yemm A May S Williams L Millner P Tsurumi S Moore I Napier R Kerr ID Bennett MJ 《The Plant cell》2004,16(11):3069-3083
We have investigated the subcellular localization, the domain topology, and the amino acid residues that are critical for the function of the presumptive Arabidopsis thaliana auxin influx carrier AUX1. Biochemical fractionation experiments and confocal studies using an N-terminal yellow fluorescent protein (YFP) fusion observed that AUX1 colocalized with plasma membrane (PM) markers. Because of its PM localization, we were able to take advantage of the steep pH gradient that exists across the plant cell PM to investigate AUX1 topology using YFP as a pH-sensitive probe. The YFP-coding sequence was inserted in selected AUX1 hydrophilic loops to orient surface domains on either apoplastic or cytoplasmic faces of the PM based on the absence or presence of YFP fluorescence, respectively. We were able to demonstrate in conjunction with helix prediction programs that AUX1 represents a polytopic membrane protein composed of 11 transmembrane spanning domains. In parallel, a large aux1 allelic series containing null, partial-loss-of-function, and conditional mutations was characterized to identify the functionally important domains and amino acid residues within the AUX1 polypeptide. Whereas almost all partial-loss-of-function and null alleles cluster in the core permease region, the sole conditional allele aux1-7 modifies the function of the external C-terminal domain. 相似文献
23.
Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation 总被引:21,自引:0,他引:21
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Abbadi A Domergue F Bauer J Napier JA Welti R Zähringer U Cirpus P Heinz E 《The Plant cell》2004,16(10):2734-2748
Omega6- and omega3-polyunsaturated C20 fatty acids represent important components of the human diet. A more regular consumption and an accordingly sustainable source of these compounds are highly desirable. In contrast with the very high levels to which industrial fatty acids have to be enriched in plant oils for competitive use as chemical feedstocks, much lower percentages of very-long-chain polyunsaturated fatty acids (VLCPUFA) in edible plant oils would satisfy nutritional requirements. Seed-specific expression in transgenic tobacco (Nicotiana tabacum) and linseed (Linum usitatissimum) of cDNAs encoding fatty acyl-desaturases and elongases, absent from all agronomically important plants, resulted in the very high accumulation of Delta6-desaturated C18 fatty acids and up to 5% of C20 polyunsaturated fatty acids, including arachidonic and eicosapentaenoic acid. Detailed lipid analyses of developing seeds from transgenic plants were interpretated as indicating that, after desaturation on phosphatidylcholine, Delta6-desaturated products are immediately channeled to the triacylglycerols and effectively bypass the acyl-CoA pool. Thus, the lack of available Delta6-desaturated acyl-CoA substrates in the acyl-CoA pool limits the synthesis of elongated C20 fatty acids and disrupts the alternating sequence of lipid-linked desaturations and acyl-CoA dependent elongations. As well as the successful production of VLCPUFA in transgenic oilseeds and the identification of constraints on their accumulation, our results indicate alternative strategies to circumvent this bottleneck. 相似文献
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Beaudoin F Michaelson LV Lewis MJ Shewry PR Sayanova O Napier JA 《Biochemical Society transactions》2000,28(6):661-663
Using a combination of database-mining and functional characterization, we have identified a component of the polyunsaturated fatty acid (PUFA) elongase. Co-expression of this elongating activity with fatty acid desaturases has allowed us to heterologously reconstitute the PUFA biosynthetic pathway. Both these enzymes (desaturases and elongase components) have undergone gene-duplication events which provide a paradigm for the diverged nature of PUFA biosynthetic activities. 相似文献
26.
Longman AJ Michaelson LV Sayanova O Napier JA Stobart AK 《Biochemical Society transactions》2000,28(6):641-643
Aquilegia vulgaris seed oil contains high levels of the rare fatty acid columbinic acid (18:3 Delta(5,9,12)), which is unusual in having the double bond at the Delta(5) carbon in the trans configuration. Columbinic acid was found to be a seed-specific fatty acid not only present in the storage oil but also in membrane lipids. Several putative gene fragments have been isolated from plant RNA with sequences similar to previously characterized 'front-end' desaturases. Functional characterization of the Aquilegia cDNA is underway. 相似文献
27.
Fran?ois Roudier Lionel Gissot Frédéric Beaudoin Richard Haslam Louise Michaelson Jessica Marion Diana Molino Amparo Lima Liên Bach Halima Morin Frédérique Tellier Jean-Christophe Palauqui Yannick Bellec Charlotte Renne Martine Miquel Marco DaCosta Julien Vignard Christine Rochat Jonathan E. Markham Patrick Moreau Johnathan Napier Jean-Denis Faure 《The Plant cell》2010,22(2):364-375
Very-long-chain fatty acids (VLCFAs) are essential for many aspects of plant development and necessary for the synthesis of seed storage triacylglycerols, epicuticular waxes, and sphingolipids. Identification of the acetyl-CoA carboxylase PASTICCINO3 and the 3-hydroxy acyl-CoA dehydratase PASTICCINO2 revealed that VLCFAs are important for cell proliferation and tissue patterning. Here, we show that the immunophilin PASTICCINO1 (PAS1) is also required for VLCFA synthesis. Impairment of PAS1 function results in reduction of VLCFA levels that particularly affects the composition of sphingolipids, known to be important for cell polarity in animals. Moreover, PAS1 associates with several enzymes of the VLCFA elongase complex in the endoplasmic reticulum. The pas1 mutants are deficient in lateral root formation and are characterized by an abnormal patterning of the embryo apex, which leads to defective cotyledon organogenesis. Our data indicate that in both tissues, defective organogenesis is associated with the mistargeting of the auxin efflux carrier PIN FORMED1 in specific cells, resulting in local alteration of polar auxin distribution. Furthermore, we show that exogenous VLCFAs rescue lateral root organogenesis and polar auxin distribution, indicating their direct involvement in these processes. Based on these data, we propose that PAS1 acts as a molecular scaffold for the fatty acid elongase complex in the endoplasmic reticulum and that the resulting VLCFAs are required for polar auxin transport and tissue patterning during plant development. 相似文献
28.
Mónica Venegas-Calerón Olga Sayanova Johnathan A. Napier 《Progress in lipid research》2010,49(2):108-119
It is now accepted that omega-3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA; 20:5Δ5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) play important roles in a number of aspects of human health, with marine fish rich in these beneficial fatty acids our primary dietary source. However, over-fishing and concerns about pollution of the marine environment indicate a need to develop alternative, sustainable sources of very long chain polyunsaturated fatty acids (VLC-PUFAs) such as EPA and DHA. A number of different strategies have been considered, with one of the most promising being transgenic plants “reverse-engineered” to produce these so-called fish oils. Considerable progress has been made towards this goal and in this review we will outline the recent achievements in demonstrating the production of omega-3 VLC-PUFAs in transgenic plants. We will also consider how these enriched oils will allow the development of nutritionally-enhanced food products, suitable either for direct human ingestion or for use as an animal feedstuff. In particular, the requirements of aquaculture for omega-3 VLC-PUFAs will act as a strong driver for the development of such products. In addition, biotechnological research on the synthesis of VLC-PUFAs has provided new insights into the complexities of acyl-channelling and triacylglycerol biosynthesis in higher plants. 相似文献
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García-Maroto F Garrido-Cárdenas JA Michaelson LV Napier JA Alonso DL 《Plant molecular biology》2007,64(3):241-250