Systemic Upregulation of MTP2- and HMA2-Mediated Zn Partitioning to the Shoot Supplements Local Zn Deficiency Responses.

Low bioavailable concentrations of the micronutrient zinc (Zn) limit agricultural production on 40% of cultivated land. Here, we demonstrate that plant acclimation to Zn deficiency involves systemic regulation. Physiological Zn deficiency of Arabidopsis thaliana shoots results in increased root transcript levels of the membrane transport protein-encoding genes METAL TRANSPORT PROTEIN2 (MTP2) and HEAVY METAL ATPASE2 (HMA2), which are unresponsive to the local Zn status of roots. MTP2 and HMA2 act additively in the partitioning of Zn from roots to shoots. Chimeric GFP fusion proteins of MTP2 complement an mtp2 mutant and localize in the endoplasmic reticulum (ER) membrane of the outer cell layers from elongation to root hair zone of lateral roots. MTP2 restores Zn tolerance in a hypersensitive yeast mutant. These results are consistent with cell-to-cell movement of Zn toward the root vasculature inside the ER-luminal continuum through the desmotubules of plasmodesmata, under Zn deficiency. The previously described Zn deficiency response comprises transcriptional activation of target genes, including ZINC-REGULATED TRANSPORTER IRON-REGULATED TRANSPORTER PROTEIN genes ZIP4 and ZIP9, by the F-group bZIP transcription factors bZIP19 and bZIP23. We show that ZIP4 and ZIP9 respond to the local Zn status in both roots and shoots, in contrast to the systemic regulation identified here. Our findings are relevant for crop management and improvement toward combating human nutritional Zn deficiency that affects 30 to 50% of the world's population.

Determination of Substrate Preferences for Desaturases and Elongases for Production of Docosahexaenoic Acid from Oleic Acid in Engineered Canola.

Production of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in plant seed oils has been pursued to improve availability of these omega-3 fatty acids that provide important human health benefits. Canola (Brassica napus), through the introduction of 10 enzymes, can convert oleic acid (OLA) into EPA and ultimately DHA through a pathway consisting of two elongation and five desaturation steps. Herein we present an assessment of the substrate specificity of the seven desaturases and three elongases that were introduced into canola by expressing individual proteins in yeast. In vivo feeding experiments were conducted with 14 potential fatty acid intermediates in an OLA to DHA pathway to determine the fatty acid substrate profiles for each enzyme. Membrane fractions were prepared from yeast expression strains and shown to contain active enzymes. The elongases, as expected, extended acyl-CoA substrates in the presence of malonyl-CoA. To distinguish between enzymes that desaturate CoA- and phosphatidylcholine-linked fatty acid substrates, we developed a novel in vitro method. We show that a delta-12 desaturase from Phytophthora sojae, an omega-3 desaturase from Phytophthora infestans and a delta-4 desaturase from Thraustochytrium sp., all prefer phosphatidylcholine-linked acyl substrates with comparatively low use of acyl-CoA substrates. To further validate our method, a delta-9 desaturase from Saccharomyces cerevisiae was confirmed to use acyl-CoA as substrate, but could not use phosphatidylcholine-linked substrates. The results and the assay methods presented herein will be useful in efforts to improve modeling of fatty acid metabolism and production of EPA and DHA in plants.

Four amino acid residues influence the substrate chain-length and regioselectivity of Siganus canaliculatus Δ4 and Δ5/6 desaturases.

Although ω3- and ω6- desaturases have been well studied in terms of substrate preference and regiospecificity, relatively little is known about the membrane-bound, "front-end" long chain fatty acid desaturases, such as ∆4, Δ5 or Δ6 desaturases. The first vertebrate ∆4 desaturase was recently identified in the marine teleost fish Siganus canaliculatus (S. canaliculatus), which also possesses a bifunctional Δ5/6 desaturase. These two long chain polyunsaturated fatty acid desaturases are very different in terms of regiospecificity and substrate chain-length, but share an unusually high degree of amino acid identity (83 %). We took advantage of this similarity by constructing a series of chimeric enzymes, replacing regions of one enzyme with the corresponding sequence of the other. Heterologous expression of the chimeric series of enzymes in yeast indicated that the substitution of a four amino acid region was sufficient to convert a ∆4 desaturase to an enzyme with ∆6 desaturase activity, and convert a ∆5/6 desaturase to an enzyme with a low level of ∆4 desaturase activity. In addition, enzymes having both ∆4 and ∆6 desaturase activities were produced by single or double amino acid substitutions within this four-amino acid region.

Biosynthesis of long chain polyunsaturated fatty acids in the marine ichthyosporean Sphaeroforma arctica.

Sphaeroforma arctica is a unique, recently discovered marine protist belonging to a group falling close to the yeast/animal border. S. arctica is found in cold environments, and accordingly has a fatty acid composition containing a high proportion of very long chain polyunsaturated fatty acids, including the ω3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA). Two elongases and five desaturases, representing the complete set of enzymes necessary for the synthesis of DHA from oleic acid, were isolated from this species and characterized in yeast. One elongase showed high conversion rates on a wide range of 18 and 20 carbon substrates, and was capable of sequential elongation reactions. The second elongase had a strong preference for the 20-carbon fatty acids EPA and arachidonic acid, with over 80 % of EPA converted to docosapentaenoic acid (DPA) in the heterologous yeast host. The isolation of a Δ8-desaturase, along with the detection of eicosadienoic acid in S. arctica cultures indicated that this species uses the alternate Δ8-pathway for the synthesis of long-chain polyunsaturated fatty acids. S. arctica also carried a Δ4-desaturase that proved to be very active in the production of DHA from DPA. Finally, a long chain acyl-CoA synthetase from S. arctica improved DHA uptake in the heterologous yeast host and led to an improvement in desaturation and elongation efficiencies.

The Arabidopsis metal tolerance protein AtMTP3 maintains metal homeostasis by mediating Zn exclusion from the shoot under Fe deficiency and Zn oversupply.

Zinc ions are required to maintain the biological activity of numerous proteins. However, when mislocalized or accumulated in excess, Zn(2+) ions are toxic because of adventitious binding to proteins and displacement of other metal ions, among them Fe(2+), from their binding sites. Heterologous expression of a previously uncharacterized Arabidopsis thaliana metal tolerance protein, MTP3, in the zrc1 cot1 mutant of budding yeast restores tolerance to, and cellular accumulation of, zinc and cobalt. An MTP3-GFP fusion protein localizes to the vacuolar membrane when expressed in Arabidopsis. Ectopic over-expression of MTP3 increases Zn accumulation in both roots and rosette leaves of A. thaliana, and enhances Zn tolerance. Exposure of wild-type plants to high but non-toxic concentrations of Zn or Co, or Fe deficiency, strongly induce MTP3 expression specifically in epidermal and cortex cells of the root hair zone. Silencing of MTP3 by RNA interference causes Zn hypersensitivity and enhances Zn accumulation in above-ground organs of soil-grown plants and of seedlings exposed to excess Zn or to Fe deficiency. Our data indicate that, in wild-type A. thaliana, the AtMTP3 protein contributes to basic cellular Zn tolerance and controls Zn partitioning, particularly under conditions of high rates of Zn influx into the root symplasm.

A multifunctional lipoxygenase with fatty acid hydroperoxide cleaving activity from the moss Physcomitrella patens.

A complex mixture of fatty acid-derived aldehydes, ketones, and alcohols is released upon wounding of the moss Physcomitrella patens. To investigate the formation of these oxylipins at the molecular level we isolated a lipoxygenase from P. patens, which was identified in an EST library by sequence homology to lipoxygenases from plants. Sequence analysis of the cDNA showed that it exhibits a domain structure similar to that of type2 lipoxygenases from plants, harboring an N-terminal import signal for chloroplasts. The recombinant protein was identified as arachidonate 12-lipoxygenase and linoleate 13-lipoxygenase with a preference for arachidonic acid and eicosapentaenoic acid. In contrast to any other lipoxygenase cloned so far, this enzyme exhibited in addition an unusual high hydroperoxidase and also a fatty acid chain-cleaving lyase activity. Because of these unique features the pronounced formation of (2Z)-octen-1-ol, 1-octen-3-ol, the dienal (5Z,8Z,10E)-12-oxo-dodecatrienoic acid and 12-keto eicosatetraenoic acid was observed when arachidonic acid was administered as substrate. 12-Hydroperoxy eicosatetraenoic acid was found to be only a minor product. Moreover, the P. patens LOX has a relaxed substrate tolerance accepting C(18)-C(22) fatty acids giving rise to even more LOX-derived products. In contrast to other lipoxygenases a highly diverse product spectrum is formed by a single enzyme accounting for most of the observed oxylipins produced by the moss. This single enzyme might, in a fast and effective way, be involved in the formation of signal and/or defense molecules thus contributing to the broad resistance of mosses against pathogens.