Yair M. Heimer

Regulation of the nitrate assimilation pathway in cultured tobacco cells: III. The nitrate uptake system

Abstract 1. 1. In the presence of tungstate, nitrate reductase develops in a non-functional form. The development of the nitrate uptake system is not inhibited by tungstate. This makes it possible to measure nitrate uptake rates as nitrate accumulation rates. 2. 2. The nitrate uptake system of tobacco XD cells has a v max of 2–5 μmoles nitrate/h per fresh wt. and an apparent K m for nitrate of 0.4 mM. It can concentrate nitrate 80-fold and the process is energy dependent. 3. 3. Development of an active nitrate uptake system is not initiated by nitrogen starvation nor by slow growth on a poor nitrogen source, urea. Nitrate specifically induces development of an active nitrate uptakes system. 4. 4. The rate of development of the nitrate uptake system is maximal immediately upon exposure of the cells to nitrate, before nitrate has accumulated appreciably. Upon removal of the exogenous supply of nitrate from fully induced cells, the accumulated nitrate is slowly assimilated and the nitrate reductase activity decays. The initially high concentrations of accumulated nitrate do not maintain nitrate reductase induction. It is assumed that distinct substrate and inducing pools of nitrate exist in the cells. 5. 5. Casein hydrolysate inhibits nitrate accumulation. Thus the nitrate uptake system in tobacco XD cells is subject to end product regulation by amino acids.

Mild salinity stimulates a stress-induced morphogenic response in Arabidopsis thaliana roots

Plant roots exhibit remarkable developmental plasticity in response to local soil conditions. It is shown here that mild salt stress stimulates a stress-induced morphogenic response (SIMR) in Arabidopsis thaliana roots characteristic of several other abiotic stresses: the proliferation of lateral roots (LRs) with a concomitant reduction in LR and primary root length. The LR proliferation component of the salt SIMR is dramatically enhanced by the transfer of seedlings from a low to a high NO3− medium, thereby compensating for the decreased LR length and maintaining overall LR surface area. Increased LR proliferation is specific to salt stress (osmotic stress alone has no stimulatory effect) and is due to the progression of more LR primordia from the pre-emergence to the emergence stage, in salt-stressed plants. In salt-stressed seedlings, greater numbers of LR primordia exhibit expression of a reporter gene driven by the auxin-sensitive DR5 promoter than in unstressed seedlings. Moreover, in the auxin transporter mutant aux1-7, the LR proliferation component of the salt SIMR is completely abrogated. The results suggest that salt stress promotes auxin accumulation in developing primordia thereby preventing their developmental arrest at the pre-emergence stage. Examination of ABA and ethylene mutants revealed that ABA synthesis and a factor involved in the ethylene signalling network also regulate the LR proliferation component of the salt SIMR.

Elucidation of the Biosynthesis of Eicosapentaenoic Acid in the Microalga Porphyridium cruentum (II. Studies with Radiolabeled Precursors).

In the course of the study of the biosynthesis of the fatty acid eicosapentaenoic acid (EPA) in the microalga Porphyridium cruentum, cells were pulse-labeled with various radiolabeled fatty acid precursors. Our data show that the major end products of the biosynthesis are EPA-containing galactolipids of a eukaryotic and prokaryotic nature. The prokaryotic molecular species contain EPA and arachidonic acid at the sn-1 position and C16 fatty acids, mainly 16:0, at the sn-2 positions, whereas in the eukaryotic species both positions are occupied by EPA or arachidonic acid. However, we suggest that both the eukaryotic and prokaryotic molecular species are formed in two pathways, [omega]6 and [omega]3, which involve cytoplasmic and chloroplastic lipids. In the [omega]6 pathway, cytoplasmic 18:2-phosphatidylcholine (PC) is converted to 20:4[omega]6-PC by a sequence that includes a [delta]6 desaturase, an elongation step, and a [delta]5 desaturase. In the minor [omega]3 pathway, 18:2-PC is presumably desaturated to 18:3[omega]3, which is sequentially converted by the enzymatic sequence of the [omega]6 pathway to 20:5[omega]3-PC. The products of both pathways are exported, as their diacylglycerol moieties, to the chloroplast to be galactosylated into their respective monogalactosyldiacylglycerol molecular species. The 20:4[omega]6 in both eukaryotic and prokaryotic monogalactosyldiacylglycerol can be further desaturated to EPA by a chloroplastic [delta]17 ([omega]3) desaturase.

Cadmium toxicity and resistance in Chlorella sp.

Abstract The Cd 2+ tolerance of Chlorella sp. isolated from an urban waste water treatment plant was studied. The growth of this alga was severely inhibited at 10 μM Cd 2+ with an LD 50 of 3 μM. Addition of 100 μM glutathione or 100 μM cysteine reduced the inhibitory effect of Cd 2+ . The amelioration of Cd 2+ inhibition by GSH was not due to direct complexing of the two compounds in the growth medium, indicating that an intracellular mechanism involving GSH or its metabolites is responsible for Cd 2+ detoxification. A Cd 2+ resistant line (CdR-DK), with an LD 50 of 35 μM, was selected by growing the alga on solid medium in the presence of increasing concentrations of the ion. Phytochelatins (PCs), a family of peptides synthesized by plants in response to cadmium, were detected in extracts of the CdR-DK cells. The possible role that these peptides, which are derived from glutathione, may play in cadmium detoxification in Chlorella is discussed.

Triacylglycerols of the red microalga Porphyridium cruentum can contribute to the biosynthesis of eukaryotic galactolipids.

A mutant of the red microalga Porphyridium cruentum was selected on the basis of impaired growth at suboptimal temperatures (15 vs. 25°C). Fatty acid and lipid analyses revealed diminished proportions of eicosapentaenoic acid (from 41 to 30%) and of the eukaryotic molecular species (from 38 to 28% of monogalactosyldiacylglycerol (MGDG) and elevated proportion (10 vs. 2%) of triacylglycerols (TAG) in the mutant, as compared with the wild type. Pulse labeling of the wild type cells with radioactive fatty acid precursors indicated an initial incorporation of the fatty acids into phosphatidylcholine (PC) and TAG. Following the pulse, the label of PC and TAG decreased with time (from 25 to 5% of the total dpm in TAG) while that of chloroplastic polar lipids, mainly MGDG, continued to increase. In the mutant, however, the labeling of TAG after the pulse was higher (30% of the total dpm) than that of the wild type and decreased only slightly to 20%. This may indicate that in P. cruentum, TAG can contribute to the biosynthesis of eukaryotic species of MGDG.

Biosynthesis of eicosapentaenoic acid in the microalgaPorphyridium cruentum. I: The use of externally supplied fatty acids

The biosynthetic pathways of eicosapentaenoic acid (20:5n-3) in microalgae, in general, and inPorphyridium cruentum, in particular, are not known. Some of the putative intermediates along the suggested pathways could not be detected probably due to their low endogenous level. In order to increase the endogenous levels of the intermediates, we provided various fatty acids in the growth medium. Exogenously supplied fatty acids were indeed incorporated into algal lipids and were further metabolized along the n-6 and n-3 pathways. In the n-6 pathway, 18:2 was desaturated to 18:3n-6, elongated to 20:3n-6, and subsequently desaturated to 20:4n-6 and then to 20:5n-3. In the n-3 pathway, 18:2 was first desaturated to 18:3n-3 which was then sequentially converted, apparently by the same enzymatic sequence of the n-6 pathway to 18:4n-3, 20:4n-3, and 20:5n-3.

Interference with the citrulline-based nitric oxide synthase assay by argininosuccinate lyase activity in Arabidopsis extracts

There are many reports of an arginine‐dependent nitric oxide synthase activity in plants; however, the gene(s) or protein(s) responsible for this activity have yet to be convincingly identified. To measure nitric oxide synthase activity, many studies have relied on a citrulline‐based assay that measures the formation of l‐citrulline from l‐arginine using ion exchange chromatography. In this article, we report that when such assays are used with protein extracts from Arabidopsis, an arginine‐dependent activity was observed, but it produced a product other than citrulline. TLC analysis identified the product as argininosuccinate. The reaction was stimulated by fumarate (> 500 µm), implicating the urea cycle enzyme argininosuccinate lyase (EC 4.3.2.1), which reversibly converts arginine and fumarate to argininosuccinate. These results indicate that caution is needed when using standard citrulline‐based assays to measure nitric oxide synthase activity in plant extracts, and highlight the importance of verifying the identity of the product as citrulline.

Fatty acid unsaturation in the red alga Porphyridium cruentum. Is the methylene interrupted nature of polyunsaturated fatty acids an intrinsic property of the desaturases

Polyunsaturated fatty acids (PUFAs) exogenously supplied to the red microalga Porphyridium cruentum were incorporated into cellular lipids. All the C18 PUFAs studied were desaturated by a delta 6-desaturase and all the C20 PUFAs by a delta 5-desaturase. The latter enzyme desaturated even 20:2(11, 14) to 20:3(5, 11, 14) and 20:3(11, 14, 17) to 20:4(5, 11, 14, 17). We infer the existence of several fatty acid desaturases, with different chain length specificities. Furthermore, the introduction of double bonds in a methylene interrupted pattern, at least for alpha-type desaturase such as the delta 5- and delta 6-desaturases, is not an intrinsic property of the enzyme but a consequence of the arrangement of the pre-existing double bonds in the fatty acid substrate.

The relation between low K+/Na+ ratio and salt-tolerance in the wild tomato species Lycopersicon pennellii

Summary The cultivated tomato Lycopersicon esculentum cultivar M82 and its wild salt-tolerant relative L. pennellii accession Atico were analyzed for: (a) dry weight, content of Na + and K + ions (in young and old leaves), and dry weight/potassium ratio (in young leaves) in plants grown in standard Hoagland and K-free solutions, without or with 100 mmol L −1 NaCl; and (b) dry weight in plants grown in K-free medium, in which K + was substituted by the same concentration (5 mmol L −1 ) of Na + . Plants of L. pennellii , as expected: (a) responded better than the cultivated species to the high salinity in the standard solution with respect to biomass production; and (b) accumulated less K + and more Na + onder this condition. Furthermore, L. pennellii plants were characterized by: (a) a more efficient substitution of K + function by Na + , as expressed by the fact that Na + added to the K-free medium caused a greater increase of dry weight in the wild species; (b) a higher retranslocation of K + from old to young leaves, and consequently (c) a higher K-efficiency (dry weight/K + ) ratio. It is suggested that. (a) the use of less energy in L. pennellii , as compared with the cultivated species, to exclude Na + and to accumulate K + under salt stress contributes to the mechanism of salt tolerance in that species, and (b) the higher Na + in the wild species is used as a cheap osmoticum in the vacuole and, possibly, as a partial substitute for K + in some of its functions, and the lower K + is compensated, at least in part, by the higher K-efficiency ratio.

Herbicide-resistant lines of microalgae: Growth and fatty acid composition

Cell lines of Spirulina platensis and Porphyridium cruentum resistant to growth inhibition by the herbicide SAN 9785 had a significantly higher growth rate than their respective wild-type strains. These lines were also shown to overproduce y-linolenic acid (GLA) and eicosapentaenoic acid (EPA), respectively, in the presence and absence of the inhibitor, as compared with wild-type cultures under similar conditions. The effect was most conspicuous in polar lipids. Thus, the proportion of GLA in the galactolipid (GL) fraction of the SAN 9785-resistant strain of S. platensis, SRS-1, increased in the absence of the inhibitor from 33.3% in the wild-type to 39.0%. Similarly, the proportion of EPA in the GL fraction of the resistant strain of P. cruentum, SRP, increased in the presence of the inhibitor from 29.1 to 45.4%.