Dusty Post-Beittenmiller

Isolation and Characterization of Homogentisate Phytyltransferase Genes from Synechocystis sp. PCC 6803 and Arabidopsis

Tocopherols, synthesized by photosynthetic organisms, are micronutrients with antioxidant properties that play important roles in animal and human nutrition. Because of these health benefits, there is considerable interest in identifying the genes involved in tocopherol biosynthesis to allow transgenic alteration of both tocopherol levels and composition in agricultural crops. Tocopherols are generated from the condensation of phytyldiphosphate and homogentisic acid (HGA), followed by cyclization and methylation reactions. Homogentisate phytyltransferase (HPT) performs the first committed step in this pathway, the phytylation of HGA. In this study, bioinformatics techniques were used to identify candidate genes,slr1736 and HPT1, that encode HPT fromSynechocystis sp. PCC 6803 and Arabidopsis, respectively. These two genes encode putative membrane-bound proteins, and contain amino acid residues highly conserved with other prenyltransferases of the aromatic type. A Synechocystissp. PCC 6803 slr1736 null mutant obtained by insertional inactivation did not accumulate tocopherols, and was rescued by the Arabidopsis HPT1 ortholog. The membrane fraction of wild-type Synechocystis sp. PCC 6803 was capable of catalyzing the phytylation of HGA, whereas the membrane fraction from the slr1736 null mutant was not. The microsomal membrane fraction of baculovirus-infected insect cells expressing the Synechocystis sp. PCC 6803slr1736 were also able to perform the phytylation reaction, verifying HPT activity of the protein encoded by this gene. In addition, evidence that antisense expression of HPT1in Arabidopsis resulted in reduced seed tocopherol levels, whereas seed-specific sense expression resulted in increased seed tocopherol levels, is presented.

Cloning of a cDNA Encoding 3-Ketoacyl-Acyl Carrier Protein Synthase III from Arabidopsis

The initial step of fatty acid biosynthesis in both plants and Escherichia coli is the condensation of malonyl-acyl carrier protein with acetyl-COA, a reaction that is catalyzed by KAS 111 (Jackowski and Rock, 1987; Jackowski et al., 1989; Jaworski et al., 1989; Clough et al., 1992). In E. coli, KAS 111 has been implicated as a site of regulation for de novo fatty acid synthesis (Jackowski and Rock, 1987; Jackowski et al., 1989). At present, the regulation of fatty acid biosynthesis has not been clearly delineated in plants. Alteration of KAS 111 expression in Arabidopsis by sense and antisense constructs will help elucidate its regulatory role in vivo. The first plant cDNA clone of KAS 111 was recently isolated from spinach (Tai and Jaworski, 1993). The spinach clone showed a high degree of amino acid identity to the E. coli KAS 111 (Tsay et al., 1992) and the putative KAS I11 from Porphyra umbilicalis (red alga) (Reith, 1993), but no similarity to clones of other KAS isozymes from either plants or E. coli. We have isolated an Arabidopsis KAS 111 cDNA clone using the spinach clone as a heterologous probe (Table I). An Arabidopsis cDNA library was screened using moderatestringency conditions with a radiolabeled 612-bp probe generated from the spinach cDNA clone using PCR. Positive X clones were excised into plasmids and rescreened. One of the putative KAS I11 clones showed a strong positive signal in the second screening, and the initial sequencing of the clone revealed a high sequence identity to the 3‘ end of the spinach KAS I11 clone. The full-length clone was sequenced on both strands with customized primers. The Arabidopsis cDNA clone of KAS I11 contains a single open reading frame of 1215 bp encoding a polypeptide that has 71.8% identity to the deduced amino acid sequence of spinach KAS 111. Its active site Cys was identified at position 179 by comparing conserved sequences near the active site of other KAS 111 proteins. The highest degree of homology to spinach KAS 111 was observed near the C tenninus of the peptide (84.9% identity at the residues 300-405), whereas the N tenninus shows more divergence (50% identity at the residues 1-lOO), mainly due to the transit peptide sequence.

Epicuticular wax on leek in vitro developmental stages and seedlings under varied growth conditions

Abstract Epicuticular wax was analyzed on regenerated shoots and plants as well as seedlings and market plants of Allium porrum L. (leek). Wax compositions were similar for tissue culture stages 1–4 and for seedlings and market plants as revealed by gas chromatography–mass spectrometry analysis. The amount of hentriacontan-16-one, the primary wax component, increased with seedling and regenerated shoot developmental stages. It was also substantially higher on shoots regenerated in moderate relative humidity conditions than on those from a relatively high humidity environment. No hentriacontan-16-one was found in the chloroform-extractable lipids from callus or 15-day etiolated seedlings. The total amount of epicuticular wax on etiolated seedlings was 20% lower than on seedlings grown in the light, and the level of hentriacontane was reduced 73%, while the levels of nonacosane and heptacosane were reduced 40 and 36%, respectively. Shoots and seedlings grown in high relative humidity conditions had reduced levels of total wax but elevated levels of primary alcohols. A correlation was found between the level of hentriacontan-16-one and the abundance of crystalline wax structures on leaf epidermis observed by scanning electron microscopic examination. The branched rods and waffle-shaped crystals characteristic of leek waxes were present at levels of hentriacontan-16-one >5 and >100 ng/mm2, respectively. The optimal stage for epicuticular wax studies following transformation to alter cuticular waxes was considered to be stage three shoots cultured under moderate relative humidity conditions due to substantial levels of wax and the reduced variability of total wax composition.

Is Acetylcarnitine a Substrate for Fatty Acid Synthesis in Plants

Long-chain fatty acid synthesis from [1–14C]acetylcarnitine by chloroplasts isolated from spinach (Spinacia oleracea), pea (Pisum sativum), amaranthus (Amaranthus lividus), or maize (Zea mays) occurred at less than 2% of the rate of fatty acid synthesis from [1–14C]acetate irrespective of the maturity of the leaves or whether the plastids were purified using sucrose or Percoll medium. [1–14C]-Acetylcarnitine was not significantly utilized by highly active chloroplasts rapidly prepared from pea and spinach using methods not involving density gradient centrifugation. [1–14C]Acetylcarnitine was recovered quantitatively from chloroplast incubations following 10 min in the light. Unlabeled acetyl-L-carnitine (0.4 mM) did not compete with [1–14C]acetate (0.2 mM) as a substrate for fatty acid synthesis by any of the more than 70 chloroplast preparations tested in this study. Carnitine acetyltransferase activity was not detected in any chloroplast preparation and was present in whole leaf homogenates at about 0.1% of the level of acetyl-coenzyme A synthetase activity. When supplied to detached pea shoots and detached spinach, amaranthus, and maize leaves via the transpiration stream, 1 to 4% of the [1–14C]acetylcarnitine and 47 to 57% of the [1–14C]acetate taken up was incorporated into lipids. Most (78–82%) of the [1–14C]acetylcarnitine taken up was recovered intact. It is concluded that acetylcarnitine is not a major precursor for fatty acid synthesis in plants.

Epicuticular wax on the included air channel of Gloriosa rothschildiana L.

Abstract Histological examination of the basal internode of Gloriosa rothschildiana L. revealed an enclosed, highly specialized crescent-shaped air channel from the underground rhizome through the axil of the first aerial leaf. The air channel was enveloped by a single layer of epidermal cells and a cuticle. The aim of these studies was to determine if the included air channel was also covered by an epicuticular wax layer. Chemical analysis revealed that the channel was covered with chloroform-extractable lipids comprised of similar levels of fatty acids, aldehydes and alkanes. In comparison, stem samples had primarily aldehydes and alkanes, and leaf wax had substantial levels of 1-octacosanol and alkanes. Scanning electron microscopic analyses of the channel and leaf surfaces showed the presence of an amorphous film which was removed with chloroform. Stem and leaf tip tendrils showed irregularly shaped plates and ribbons, respectively. The stomata of the stem, channel, and abaxial leaf surfaces were covered with wax domes which had an elliptical slit running parallel to the stomatal pore. These chemical and electron microscopic studies are the first demonstration that an included system, such as the Gloriosa air channel, is covered with a wax layer.

Plant Fatty Acid Biosynthesis and Its Potential For Manipulation

In the American diet, plant oils have gradually replaced animal fats and now account for 15–20 percent of total calories consumed. In addition, plant oils are employed in the manufacture of a wide range of specialty products including lubricants, plastics and detergents. Success in altering seed oils through mutation breeding has indicated that relatively large alterations can be made in triacylglycerol fatty acid composition without exerting any obvious deleterious effect on plant growth and development. It, therefore, may be possible to use molecular genetic methods to provide more directed and extensive modifications of the plant fatty acid biosynthetic pathway. Alterations in the amount and type of unsaturated fatty acids may have particular importance in obtaining optimum nutritional value from plant oils. Our current understanding of the biochemistry and molecular biology of plant fatty acid production will be reviewed, and the potential for its modification will be discussed.