Iceland is an episodic source of atmospheric ice-nucleating particles relevant for mixed-phase clouds.

Ice-nucleating particles (INPs) have the potential to remove much of the liquid water in climatically important mid- to high-latitude shallow supercooled clouds, markedly reducing their albedo. The INP sources at these latitudes are very poorly defined, but it is known that there are substantial dust sources across the high latitudes, such as Iceland. Here, we show that Icelandic dust emissions are sporadically an important source of INPs at mid to high latitudes by combining ice-nucleating active site density measurements of aircraft-collected Icelandic dust samples with a global aerosol model. Because Iceland is only one of many high-latitude dust sources, we anticipate that the combined effect of all these sources may strongly contribute to the INP population in the mid- and high-latitude northern hemisphere. This is important because these emissions are directly relevant for the cloud-phase climate feedback and because high-latitude dust emissions are expected to increase in a warmer climate.

Plant coenzyme A biosynthesis: characterization of two pantothenate kinases from Arabidopsis.

In bacterial and animal coenzyme A (CoA) biosynthesis, pantothenate kinase (PANK) activity is critical in regulating intracellular CoA levels. Less is known about the role of PANK in plants, although a single plant isozyme from Arabidopsis, AtPANK1, was previously cloned and analyzed in vitro. We report here the characterization of a second pantothenate kinase of Arabidopsis, AtPANK2, as well as characterization of the physiological roles of both plant enzymes. The activity of the second pantothenate kinase, AtPANK2, was confirmed by its ability to complement the temperature-sensitive mutation of the bacterial pantothenate kinase in E. coli strain ts9. Knock-out mutation of either AtPANK1 or AtPANK2 did not inhibit plant growth, whereas pank1-1/pank2-1 double knockout mutations were embryo lethal. The phenotypes of the mutant plants demonstrated that only one of the AtPANK enzymes is necessary and sufficient for producing adequate CoA levels, and that no other enzyme can compensate for the loss of both isoforms. Real-time PCR measurements of AtPANK1 and AtPANK2 transcripts indicated that both enzymes are expressed with similar patterns in all tissues examined, further suggesting that AtPANK1 and AtPANK2 have complementary roles. The two enzymes have homologous pantothenate kinase domains, but AtPANK2 also carries a large C-terminal protein domain. Sequence comparisons indicate that this type of "bifunctional" pantothenate kinase is conserved in other higher eukaryotes as well. Although the function of the C-terminal domain is not known, homology structure modeling suggests it contains a highly conserved cluster of charged residues that likely constitute a metal-binding site.

Plant defense in the absence of jasmonic acid: the role of cyclopentenones.

The Arabidopsis opr3 mutant is defective in the isoform of 12-oxo-phytodienoate (OPDA) reductase required for jasmonic acid (JA) biosynthesis. Oxylipin signatures of wounded opr3 leaves revealed the absence of detectable 3R,7S-JA as well as altered levels of its cyclopentenone precursors OPDA and dinor OPDA. In contrast to JA-insensitive coi1 plants and to the fad3 fad7 fad8 mutant lacking the fatty acid precursors of JA synthesis, opr3 plants exhibited strong resistance to the dipteran Bradysia impatiens and the fungus Alternaria brassicicola. Analysis of transcript profiles in opr3 showed the wound induction of genes previously known to be JA-dependent, suggesting that cyclopentenones could fulfill some JA roles in vivo. Treating opr3 plants with exogenous OPDA powerfully up-regulated several genes and disclosed two distinct downstream signal pathways, one through COI1, the other via an electrophile effect of the cyclopentenones. We conclude that the jasmonate family cyclopentenone OPDA (most likely together with dinor OPDA) regulates gene expression in concert with JA to fine-tune the expression of defense genes. More generally, resistance to insect and fungal attack can be observed in the absence of JA.

Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes.

Polyunsaturated fatty acids (PUFAs) are essential membrane components in higher eukaryotes and are the precursors of many lipid-derived signaling molecules. Here, pathways for PUFA synthesis are described that do not require desaturation and elongation of saturated fatty acids. These pathways are catalyzed by polyketide synthases (PKSs) that are distinct from previously recognized PKSs in both structure and mechanism. Generation of cis double bonds probably involves position-specific isomerases; such enzymes might be useful in the production of new families of antibiotics. It is likely that PUFA synthesis in cold marine ecosystems is accomplished in part by these PKS enzymes.

Temperature sensing and cold acclimation.

The fundamental question in cold acclimation is how do plants perceive the low but nonfreezing temperatures that activate cold acclimation responses. New findings in the past year suggest that changes in membrane fluidity, cytoskeleton rearrangement, and calcium influxes are among the earliest events taking place in plants upon exposure to low nonfreezing temperatures. In the cyanobacterium Synechocystis PCC6803, temperature change is detected by at least two separate sensors. One of these measures membrane fluidity using a classical two-component system involving histidine kinases and a response regulator in a His-to-Asp phosphorelay. Although these Synechocystis results may not be directly relevant to cold acclimation, they can guide our thinking as we search for biological thermometers in higher plants.

Trienoic fatty acids are required to maintain chloroplast function at low temperatures.

The chloroplast membranes of all higher plants contain very high proportions of trienoic fatty acids. To investigate how these lipid structures are important in photosynthesis, we have generated a triple mutant line of Arabidopsis that contains negligible levels of trienoic fatty acids. For mutant plants grown at 22 degrees C, photosynthetic fluorescence parameters were indistinguishable from wild type at 25 degrees C. Lowering the measurement temperature led to a small decrease in photosynthetic quantum yield, Phi(II), in the mutant relative to wild-type controls. These and other results indicate that low temperature has only a small effect on photosynthesis in the short term. However, long-term growth of plants at 4 degrees C resulted in decreases in fluorescence parameters, chlorophyll content, and thylakoid membrane content in triple-mutant plants relative to wild type. Comparisons among different mutant lines indicated that these detrimental effects of growth at 4 degrees C are strongly correlated with trienoic fatty acid content with levels of 16:3 + 18:3, approximately one-third of wild type being sufficient to sustain normal photosynthetic function. In total, our results indicate that trienoic fatty acids are important to ensure the correct biogenesis and maintenance of chloroplasts during growth of plants at low temperatures.

The Arabidopsis male-sterile mutant, opr3, lacks the 12-oxophytodienoic acid reductase required for jasmonate synthesis.

Jasmonic acid (JA) and its precursor 12-oxophytodienoic acid (OPDA) act as plant growth regulators and mediate responses to environmental cues. To investigate the role of these oxylipins in anther and pollen development, we characterized a T-DNA-tagged, male-sterile mutant of Arabidopsis, opr3. The opr3 mutant plants are sterile but can be rendered fertile by exogenous JA but not by OPDA. Cloning of the mutant locus indicates that it encodes an isozyme of 12-oxophytodienoate reductase, designated OPR3. All of the defects in opr3 are alleviated by transformation of the mutant with an OPR3 cDNA. Our results indicate that JA and not OPDA is the signaling molecule that induces and coordinates the elongation of the anther filament, the opening of the stomium at anthesis, and the production of viable pollen. Just as importantly, our data demonstrate that OPR3 is the only isoform of OPR capable of reducing the correct stereoisomer of OPDA to produce JA required for male gametophyte development.

Antifungal compounds from idioblast cells isolated from avocado fruits.

(E,Z,Z)-1-Acetoxy-2-hydroxy-4-oxo-heneicosa-5,12,15-triene was isolated from avocado, Persea americana Mill., idioblast cells. It inhibited spore germination of the fungal pathogen Colletotrichum gloeosporioides. Full characterization is also reported for two additional compounds that have been described and partially characterized previously.

A palmitoyl-CoA-specific delta9 fatty acid desaturase from Caenorhabditis elegans.

Biosynthesis of polyunsaturated fatty acids in C. elegans is initiated by the introduction of a double bond at the delta9 position of a saturated fatty acid. We identified three C. elegans fatty acid desaturase genes related to the yeast delta9 desaturase OLE1 and the rat stearoyl-CoA desaturase SCD1. Heterologous expression of all three genes rescues the fatty acid auxotrophy of the yeast delta9 desaturase mutant ole1. Examination of the fatty acid composition of the transgenic yeast reveals striking differences in the substrate specificities of these desaturases. Two desaturases, FAT-6 and FAT-7, readily desaturate stearic acid (18:0) and show less activity on palmitic acid (16:0). In contrast, the other desaturase, FAT-5, readily desaturates palmitic acid (16:0), but shows nearly undetectable activity on the common delta9 substrate stearic acid. This is the first report of a palmitoyl-CoA-specific membrane fatty acid desaturase.

Identification and characterization of an animal delta(12) fatty acid desaturase gene by heterologous expression in Saccharomyces cerevisiae.

We have cloned a Caenorhabditis elegans cDNA encoding a Delta12 fatty acid desaturase and demonstrated its activity by heterologous expression in Saccharomyces cerevisiae. The predicted protein is highly homologous both to the cloned plant genes with similar function and to the published sequence of the C. elegans omega-3 fatty acid desaturase. In addition, it conforms to the structural constraints expected of a membrane-bound fatty acid desaturase including the canonical histidine-rich regions. This is the first report of a cloned animal Delta(12) desaturase gene. Expression of this cDNA in yeast resulted in the accumulation of 16:2 and 18:2 (linoleic) acids. The increase of membrane fluidity brought about by this change in unsaturation was measured. The production of polyunsaturated fatty acids in yeast cells and the concomitant increase in membrane fluidity was correlated with a modest increase in growth rate at low temperature and with increased resistance to ethanol and oxidative stress.

Two families of acyl-CoA thioesterases in Arabidopsis.

We have identified two families of acyl-CoA thioesterase (ACHs) in Arabidopsis thaliana. One family, consisting of AtACH1 and AtACH2, appears to be peroxisomal, as they have type-1 peroxisomal targeting sequences. The other family, consisting of AtACH4 and AtACH5, resides in the endoplasmic reticulum, as shown by green fluorescent protein studies. AtACH2 has been overexpressed in Escherichia coli and shows high levels of acyl-CoA thioesterase activity against both 16:0-CoA and 18:1-CoA. AtACH5 has also been overexpressed in E. coli, and shows thioesterase activity as well. ACHs have been characterized in other many other organisms and in various subcellular locations, but their true physiological role is not yet understood. Indeed, atach5 gene knockout mutants have no observable phenotype.

Characterization of the AMP-binding protein gene family in Arabidopsis thaliana: will the real acyl-CoA synthetases please stand up?

One of the most prominent and important topics in modern agricultural biotechnology is the manipulation of oilseed triacylglycerol composition. Towards this goal, we have sought to identify and characterize acyl-CoA synthetases (ACSs), which play an important role in both de novo synthesis and modification of existing lipids. We have identified and cloned 20 different genes that bear strong sequence homology to known ACSs from other organisms. Through sequence comparisons and functional characterization, we have identified several members of this group that encode ACSs, while the other genes fall into the broader category of genes for AMP-binding proteins (AMPBPs). Distinguishing ACSs from AMPBPs will simplify our efforts to understand the role of ACS in triacylglycerol metabolism.

Characterization of an acyl-CoA synthetase from Arabidopsis thaliana.

One of the major goals of modern plant biotechnology is to manipulate lipid metabolism in oilseed crops to produce new and improved edible and industrial vegetable oils. Lipids constitute the structural components of cellular membranes and act as sources of energy for the germinating seed and are therefore essential to plant cell function. Both de novo synthesis and modification of existing lipids are dependent on the activity of acyl-CoA synthetases (ACSs). To date, ACSs have been recalcitrant to traditional methods of purification due to their association with membranes. In our laboratory, several isoforms of ACSs have been identified in Arabidopsis thaliana. Reverse genetics allowed us to identify a mutant containing a transfer DNA-interrupted ACS gene. Results will be presented that describe the isolation and characterization of this mutant. The elucidation of the specific roles of ACSs will lead to a greater understanding of plant lipid metabolism.

The Delta8-desaturase of Euglena gracilis: an alternate pathway for synthesis of 20-carbon polyunsaturated fatty acids.

Desaturation of fatty acids is an important metabolic process. In mammals, 20-carbon and longer polyunsaturated fatty acids are not only incorporated into cellular membranes in a tissue-specific manner, but also serve as the precursors to synthesis of eicosanoid metabolic regulators. The processes of desaturation and elongation in human liver are well characterized, but an alternate Delta8 desaturation pathway that may be important in certain tissues or in cancer cells is less well examined. The Delta8-desaturase enzyme introduces a double bond at the 8-position in 20-carbon fatty acids that have an existing Delta11 unsaturation. We have isolated the first fatty acid Delta8-desaturase, from the protist Euglena gracilis, in order to explore this alternate pathway. A full-length cDNA was obtained after reverse transcription of mRNA purified from heterotrophically grown Euglena, followed by PCR amplification with primers degenerate to conserved histidine-rich regions of microsomal desaturases. The protein predicted from the cDNA sequence is highly homologous to Delta5 and Delta6 desaturases of Caenhorabditis elegans. When the cDNA was expressed in Saccharomyces cerevisiae, the yeast cultures readily desaturated appropriate 20-carbon fatty acids by inserting an additional double bond at the Delta8-position. The enzyme demonstrated a preference for substrates of metabolic significance, 20:3 Delta11,14,17 and 20:2 Delta11,14. Cloning of a Delta8 fatty acid desaturase offers the opportunity to examine an alternate pathway of long chain fatty acid biosynthesis.

Isolation and characterization of a Delta 5-fatty acid desaturase from Caenorhabditis elegans.

Arachidonic acid and eicosapentaenoic acid are important precursors for the production of prostaglandins and other hormone-like eicosanoid molecules. These fatty acids are synthesized by animals by elongating and desaturating precursor fatty acids such as linoleic acid (18:2Delta9,12) and alpha-linolenic acid (18:3Delta9, 12,15). We have identified a Delta5 fatty acid desaturase gene (fat-4) from the nematode Caenorhabditis elegans. We have expressed this gene product in Saccharomyces cerevisiae and demonstrate that it readily converts di-homo-gamma-linolenic acid (20:3Delta8,11,14) to arachidonic acid (20:4Delta5,8,11,14). The FAT-4 Delta5-desaturase also acts on a number of other substrates, including fatty acids that do not contain a double bond at the Delta8 position.

Polyunsaturated membranes are required for photosynthetic competence in a mutant of Arabidopsis.

High levels of polyunsaturation are characteristic of all the membranes of plant and animal cells. For example, the chloroplasts of leaf cells contain about 75-80% polyunsaturated fatty acids. For the extra-chloroplast membranes in leaf cells and the membranes of non-photosynthetic tissues, values of 60-65% are typical. We report here the production of Arabidopsis double mutants that contain negligible levels of polyunsaturated fatty acids. The mutants were not capable of autotrophic growth and produced extremely chlorotic cotyledons and leaves. However, on sucrose media, the double mutants were robust plants showing strong leaf and root development. These observations indicate that the vast majority of receptor-mediated and transport-related membrane functions required to sustain the organism and induce proper development are adequately supported in the absence of polyunsaturated lipids. By contrast, photosynthesis is one process that does require high levels of membrane polyunsaturation.

A new class of Arabidopsis mutants with reduced hexadecatrienoic acid fatty acid levels.

Chloroplast glycerolipids in a number of higher-plant species, including Arabidopsis thaliana, are synthesized by two distinct pathways termed the prokaryotic and eukaryotic pathways. The molecules of galactolipids produced by the prokaryotic pathway contain substantial amounts of hexadecatrienoic acid fatty acid. Here we describe a new class of mutants, designated gly1, with reduced levels of hexadecatrienoic acid. Lipid fatty acid profiles indicated that gly1 mutants exhibited a reduced carbon flux through the prokaryotic pathway that was compensated for by an increased carbon flux through the eukaryotic pathway. Genetic and biochemical approaches revealed that the gly1 phenotype could not be explained by a deficiency in the enzymes of the prokaryotic pathway. The flux of fatty acids into the prokaryotic pathway is sensitive to changes in glycerol-3-phosphate (G3P) availability, and the chloroplast G3P pool can be increased by exogenous application of glycerol to leaves. Exogenous glycerol treatment of gly1 plants allowed chemical complementation of the mutant phenotype. These results are consistent with a mutant lesion affecting the G3P supply within the chloroplast. The gly1 mutants may therefore help in determining the pathway for synthesis of chloroplast G3P.

Chloroplast development at low temperatures requires a homolog of DIM1, a yeast gene encoding the 18S rRNA dimethylase.

Poikilothermic organisms require mechanisms that allow survival at chilling temperatures (2 to 15 degreesC). We have isolated chilling-sensitive mutants of Arabidopsis, a plant that is very chilling resistant, and are characterizing them to understand the genes involved in chilling resistance. The T-DNA-tagged mutant paleface1 (pfc1) grows normally at 22 degrees C but at 5 degrees C exhibits a pattern of chilling-induced chlorosis consistent with a disruption of chloroplast development. Genomic DNA flanking the T-DNA was cloned and used to isolate wild-type genomic and cDNA clones. The PFC1 transcript is present at a low level in wild-type plants and was not detected in pfc1 plants. Wild-type Arabidopsis expressing antisense constructs of PFC1 grew normally at 22 degrees C but showed chilling-induced chlorosis, confirming that the gene is essential for low-temperature development of chloroplasts. The deduced amino acid sequence of PFC1 has identity with rRNA methylases found in bacteria and yeast that modify specific adenosines of pre-rRNA transcripts. The pfc1 mutant does not have these modifications in the small subunit rRNA of the plastid.