Jonathan Lightner

Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis.

The polyunsaturated fatty acids linoleate and alpha-linolenate are important membrane components and are the essential fatty acids of human nutrition. The major enzyme responsible for the synthesis of these compounds is the plant oleate desaturase of the endoplasmic reticulum, and its activity is controlled in Arabidopsis by the fatty acid desaturation 2 (fad2) locus. A fad2 allele was identified in a population of Arabidopsis in which mutations had been created by T-DNA insertions. Genomic DNA flanking the T-DNA was cloned by plasmid rescue and used to isolate cDNA and genomic clones of FAD2. A cDNA containing the entire FAD2 coding sequence was expressed in fad2 mutant plants and shown to complement the mutant fatty acid phenotype. The deduced amino acid sequence from the cDNA showed homology to other plant desaturases, and this confirmed that FAD2 is the structural gene for the desaturase. Gel blot analyses of FAD2 mRNA levels showed that the gene is expressed throughout the plant and suggest that transcript levels are in excess of the amount needed to account for oleate desaturation. Sequence analysis identified histidine-rich motifs that could contribute to an iron binding site in the cytoplasmic domain of the protein. Such a position would facilitate interaction between the desaturase and cytochrome b5, which is the direct source of electrons for the desaturation reaction, but would limit interaction of the active site with the fatty acyl substrate.

An octadecanoid pathway mutant (JL5) of tomato is compromised in signaling for defense against insect attack.

The activation of defense genes in tomato plants has been shown to be mediated by an octadecanoic acid-based signaling pathway in response to herbivore attack or other mechanical wounding. We report here that a tomato mutant (JL5) deficient in the activation of would-inducible defense genes is also compromised in resistance toward the lepidopteran predator Manduca sexta (tobacco hornworm). Thus we propose the name defenseless1 (def1) for the mutation in the JL5 line that mediates this altered defense response. In experiments designed to define the normal function of DEF1, we found that def1 plants are defective in defense gene signaling initiated by prosystemin overexpression in transgenic plants as well as by oligosaccharide (chitosan and polygalacturonide) and polypeptide (systemin) elicitors. Supplementation of plants through their cut stems with intermediates of the octadecanoid pathway indicates that def1 plants are affected in octadecanoid metabolism between the synthesis of hydroperoxylinolenic acid and 12-oxo-phytodienoic acid. Consistent with this defect, def1 plants are also compromised in their ability to accumulate jasmonic acid, the end product of the pathway, in response to wounding and the aforementioned elicitors. Taken together, these results show that octadecanoid metabolism plays an essential role in the transduction of upstream would signals to the activation of antiherbivore plant defenses.

A Mutant of Arabidopsis with Increased Levels of Stearic Acid

A mutation at the fab2 locus of Arabidopsis caused increased levels of stearate in leaves. The increase in leaf stearate in fab2 varied developmentally, and the largest increase occurred in young leaves, where stearate accounted for almost 20% of total leaf fatty acids. The fatty acid composition of leaf lipids isolated from the fab2 mutant showed increased stearate in all the major glycerolipids of both the chloroplast and extrachloroplast membranes. Although the stearate content was increased, the fab2 mutant still contained abundant amounts of 18:1, 18:2, and 18:3 fatty acids. These results are consistent with the expectations for a mutation partially affecting the action of the stromal stearoyl-acyl carrier protein desaturase. Positional analysis indicated that the extra 18:0 is excluded with high specificity from the sn-2 position of both chloroplast and extrachloroplast glycerolipids. Although stearate content was increased in all the major leaf membrane lipids, the amount of increase varied considerably among the different lipids, from a high of 25% of fatty acids in phosphatidylcholine to a low of 2.9% of fatty acids in monogalactosyldiacylglycerol.

Low-Temperature Damage and Subsequent Recovery of fab1 Mutant Arabidopsis Exposed to 2[deg]C

The fab1 mutant of Arabidopsis thaliana, which contains increased levels of saturated fatty acids, was indistinguishable from the wild type when it was grown at 22 or 12[deg]C. During the first 7 to 10 d after transfer to 2[deg]C, the growth and photosynthetic characteristics of the fab1 plants remained indistinguishable from the wild type, with values for the potential quantum efficiency of photosystem II decreasing from 0.8 to 0.7 in plants of both lines. Whereas wild-type plants maintained quantum efficiency of photosystem II at approximately 0.7 for at least 35 d at 2[deg]C, this parameter declined rapidly in the mutant after 7 d and reached a value of less than 0.1 after 28 d at 2[deg]C. This decline in photosynthetic capacity was accompanied by reductions in chlorophyll content and the amount of chloroplast glycerolipids per gram of leaf. Electron microscopic examination of leaf samples revealed a rapid and extensive disruption of the thylakoid and chloroplast structure in the mutant, which is interpreted here as a form of selective autophagy. Despite the almost complete loss of photosynthetic function and the destruction of photosynthetic machinery, fab1 plants retained a substantial capacity for recovery following transfer to 22[deg]C. These results provide a further demonstration of the importance of chloroplast membrane unsaturation to the proper growth and development of plants at low temperature.

Isolation of signaling mutants of tomato (Lycopersicon esculentum)

As a first step towards developing a genetic system for investigating signaling processes in plants, we have developed a screen for signaling mutants deficient in a wound response. We have isolated two mutants of tomato that lack detectable production of proteinase inhibitors induced systemically in leaves by wounding. The mutants are deficient in the induction of both proteinase Inhibitor I and proteinase Inhibitor II but can be induced to respond at near wild-type levels by methyl jasmonate, a known elicitor of inhibitor production in tomato. While completely deficient in systemic production of proteinase inhibitors, both mutants produce some proteinase inhibitor in wounded leaves. This evidence suggests the existence of two signaling pathways, one local and one systemic, that regulate the induction of proteinase inhibitor snythesis in response to wounding.

Novel mutations affecting leaf stearate content and plant size in Arabidopsis

Abstractu2002The fab2-1 mutant of Arabidopsis is an extreme dwarf as a direct result of an increase in the levels of stearate (18u2009:u20090) in membrane lipids. We isolated a series of lines in which second-site suppressor mutations partly alleviate the dwarf phenotype. In all four of the suppressor lines examined, restoration of more normal morphology is accompanied by decreases in leaf 18u2009:u20090 content. Three of the isolated suppressors suppress the high stearate phenotype in both leaves and seeds. The effects of one of the suppressors, TW2-1, is limited to the leaves. A second allele at the fab2 locus, fab2-2, was also identified and plants homozygous for this allele where intermediate in both plant size and 18u2009:u20090 content between wild-type Arabidopsis and fab2-1 mutants. The alleles at fab2 and the suppressor mutations provided a total of nine genotypes which were analyzed to demonstrate a clear-cut relationship between leaf 18u2009:u20090 content (0.7–19.6% of total leaf fatty acids) and reductions in plant size (24–4u2005mm). These results illustrate the utility of suppressor analysis for addressing problems in biochemistry and plant biology. They also indicate that the genetic control of plant lipid composition is more complex than previously appreciated.

Membrane Lipid Structure and Plant Function: What are The Relationships?

Membranes are of central importance to all biology. Membranes not only define the limits of cells but also allow for the subcellular compartmentation of various biochemical functions. The ability to establish chemical potential gradients across membranes is essential to photosynthesis, the primary means of energy capture in the biosphere. Explaining membrane lipid diversity is a central problem of membrane biology. In plants, as well as in animals, membrane lipids are extremely heterogeneous in the molecular species present. However, our capacity to explain how the structure lipid molecules affects membrane function is very limited. In this paper we shall summarize recent information about polyunsaturated lipid synthesis and discuss two mutant lines of Arabidopsis that have expanded our understanding of the roles of lipids in plant biology.