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.

Intracellular Levels of Free Linolenic and Linoleic Acids Increase in Tomato Leaves in Response to Wounding.

An intracellular signaling pathway for activating plant defense genes against attacking herbivores and pathogens is mediated by a lipid-based signal transduction cascade. In this pathway, linolenic acid (18:3) is proposed to be liberated from cell membranes and is converted to cyclopentanones that are involved in transcriptional regulation of defense genes, analogously to prostaglandin synthesis and function in animals. Levels of 18:3 and linoleic acid in tomato (Lycopersicon esculentum) leaves increased within 1 h when the leaves were wounded with a hemostat across the main vein to simulate herbivore attacks. The increase correlated with the time course of accumulation of jasmonic acid, a cyclopentanone product of 18:3, that had previously been shown to increase in leaves in response both to wounding and to elicitors of plant defense genes. One hour after wounding, at least a 15-fold excess of 18:3 was found over that required to account for the levels of newly synthesized jasmonic acid. The free fatty acids in both control and wounded leaves accounted for less than 0.25% of the total fatty acids. However, the total lipid contents of the leaves remained relatively unchanged up to 8 h after wounding, indicating that extensive loss of lipids did not occur, although a gradual decrease in polar lipids was observed, mainly in monogalactosyl diacylglycerol of chloroplast lipids. The data support a role for lipid release as a key step in the signaling events that activate defense genes in tomato leaves in response to wounding by attacking herbivores.

High-Oleate Oilseeds Fail to Develop at Low Temperature.

The fad2 mutants of Arabidopsis thaliana are deficient in activity of the endoplasmic reticulum oleate desaturase that is the main enzyme responsible for polyunsaturated lipid synthesis in developing seeds of oil crops. A comparison of wild-type and fad2 seeds developing on heterozygous (FAD2/-) plants was used as a model for genetically engineered high-oleate oilseeds of species such as soybean and canola. When fad2 seeds developed at normal temperatures (22[deg]C), they showed high viability compared to wild-type seeds. When a portion of seed development took place at 6[deg]C, germination of the wild-type siblings remained high but germination of fad2 segregants declined considerably. This was true even when exposure to low temperature was limited to the final stages of seed filling and maturation. Compared to wild-type seeds, fully viable fad2 seeds produced at 22[deg]C had reduced lipid contents and were slower to germinate at 10 and 6[deg]C. Taken together, these results indicate that for some oilseed species at least, molecular genetic manipulation of oleate levels in the oil may result in plant lines with unacceptable performance in the field.

Escherichia coli beta-hydroxydecanoyl thioester dehydrase reacts with native C10 acyl-acyl-carrier proteins of plant and bacterial origin.

beta-Hydroxydecanoyl-[acyl-carrier-protein] dehydrase catalyzes the essential step in the formation of unsaturated fatty acids in Escherichia coli. This reaction was characterized with native C10 acyl-acyl-carrier protein (ACP) structures in both an aqueous phase system and a substrate immobilization assay system. The dehydrase is equally active with E. coli ACP, recombinant ACP-I derived from spinach, or protein A:ACP-I fusion (acyl-thioesters). There were differences among the substrates in terms of the equilibrium product distribution. Both E. coli acyl-ACP and recombinant acyl-ACP-I as cosubstrates with beta-OH 10:0, trans-2 10:1, or cis-3 10:1 yielded about equal amounts (37 mol%) of the two monoenes regardless of the initial substrate. In contrast, the fusion acyl-ACP-I yielded only 17 mol% cis-3 10:1 with 49 mol% trans-2 10:1 present at equilibrium. These equilibrium values for native cis-3 10:1 are higher than those reported previously for the dehydrase using N-acetylcysteamine thioesters as substrates. The Km values for each beta-OH 10:0 ACP substrate were similar to each other and within the range of in vivo concentrations (5-10 microM). Dehydrase reactivity depends more on acyl chain length than ACP structure or origin and is therefore different from other branch point ACP-utilizing enzymes (plant and bacterial) which discriminate according to ACP structure (D. J. Guerra, J. B. Ohlrogge, and M. Frentzen, 1986, Plant Physiol. 82, 448-453).

Spinach carbonic anhydrase primary structure deduced from the sequence of a cDNA clone.

A cDNA clone 1,156 base pairs in length was selected by screening a lambda gt11 library with antibodies directed against spinach chloroplast carbonic anhydrase (carbonate dehydratase, EC 4.2.1.1). Sequence analysis revealed an open reading frame of 957 base pairs encoding a polypeptide containing 319 amino acids with a molecular weight of 34,569. This polypeptide is of sufficient size to represent the precursor of spinach chloroplast carbonic anhydrase. The polypeptide contains a sequence of 19 amino acids identical to the sequence of a cyanogen bromide fragment from spinach carbonic anhydrase. In addition, Escherichia coli was transformed with a plasmid that expresses spinach carbonic anhydrase. Lysates prepared from transformed E. coli contain acetazolamide-inhibitable carbonic anhydrase activity. The amino acid sequence of spinach carbonic anhydrase is distinct from those reported for the mammalian isozymes.

Spinach Leaves Desaturate Exogenous [C]Palmitate to Hexadecatrienoate : Evidence that de Novo Glycerolipid Synthesis in Chloroplasts Can Utilize Free Fatty Acids Imported from Other Cellular Compartments.

Long-chain (14)C-fatty acids applied to the surface of expanding spinach leaves were incorporated into all major lipid classes. When applied in diethyleneglycol monomethyl ether solution, as done by previous workers, [(14)C]palmitic acid uptake was much lower than that of [(14)C] oleic acid. However, when applied in a thin film of liquid paraffin the rate of [(14)C] palmitic acid metabolism was rapid and virtually complete. Considerable radioactivity from [(14)C]palmitate incorporated into lipids following either application method gradually appeared in polyunsaturated C(16) fatty acids esterified to those molecular species of galactolipids previously thought to be made using only fatty acids synthesized and retained within the chloroplast. Evidence for the incorporation of radioactivity from exogenous [(14)C]oleate into those same molecular species of galactolipids was less compelling. The unexpected availability of fatty acids bound to extrachloroplastidal lipids for incorporation into galactolipids characteristically assembled entirely within the chloroplast emphasizes the need to reassess interrelations between the "prokaryotic" and "eukaryotic" pathways of galactolipid formation.