The signaling pathways that allow plants to mount defenses against chewing insects are known to be complex. To investigate the role of jasmonate in wound signaling in Arabidopsis and to test whether parallel or redundant pathways exist for insect defense, we have studied a mutant (fad3-2 fad7-2 fad8) that is deficient in the jasmonate precursor linolenic acid. Mutant plants contained negligible levels of jasmonate and showed extremely high mortality (Ϸ80%) from attack by larvae of a common saprophagous fungal gnat, Bradysia impatiens (Diptera: Sciaridae), even though neighboring wild-type plants were largely unaffected. Application of exogenous methyl jasmonate substantially protected the mutant plants and reduced mortality to Ϸ12%. These experiments precisely define the role of jasmonate as being essential for the induction of biologically effective defense in this plant-insect interaction. The transcripts of three wound-responsive genes were shown not to be induced by wounding of mutant plants but the same transcripts could be induced by application of methyl jasmonate. By contrast, measurements of transcript levels for a gene encoding glutathione S-transferase demonstrated that wound induction of this gene is independent of jasmonate synthesis. These results indicate that the mutant will be a good genetic model for testing the practical effectiveness of candidate defense genes.

The very high proportlons of trlenolc fatty acids found in chloroplast membranes of all higher plants suggest that these llpld otructurtis mlght be essentlal for photosynthasis. We report here on the productisn of Arabidopsis triple mutants that contaln negllglble levels of trlenolc fatty acids. Photosynthesls at 22OC was barely affected, and vegetative growth of the mutants was ldentlcal wlth that of tha wlld type, dm"StratIng that any requlrement for trienoic acyl groups in membrane structure and functlon 1s relatively subtle. Although vegetative growth and development were unaffected, the triple mutants are male sterlle and produce no seed under normal conditlons. Comparisons of psllen development In wlld-type and triple mutant flowersestablished that pallan gralns in the mutant develeped to the tslcellular stege. Exogenous applicatlens of a-llnolenata or jasmonate restered fertlllty. T a k n tsgether, the results demonstrate that the critical role of trlenolc acids ln tha llfe cycle of plants 1s as the precursor of sxylipln, a signallng compound that regulates final maturatlon processes and the release of pollen. INTRODUCTIONThe biophysical reactions of light harvesting and electron transport during photosynthesis take place in a uniquely constructed bilayer membrane, the thylakoid. In all photosynthetic eukaryotes, the complement of atypical glyceralipid molecules that form the foundation of this membrane are characterized by sugar headgroups and avery high leve1 of unsaturation in the fatty acid chains, which compose the central portion of the thylakoid lamella bilayer. For example, menogalactosyldiacylglycerol, the major thylakoid lipid, typically contains >90% af a-linolenic acid (183) er a combination of 183 and hexadecatrienoic (163) acids, depending on the plant species (Jamieson and Reid, 1971). These very high levels of trienoic fatty asids are noteworthy because free radicals that are byproducts of the photosynthetic llght reactions stimulate oxidation of polyunsaturated fatty acids. Because this oxidation might be expected ta medlate against a high degree of unsaturation, it has been inferred that there is a strong selective advantage to having such high levels of trienoic fatty acids in the thylakoid. Therefore, it could be reasoned that these lipid structures must have some critical role in maintaining photosynthetic functlon.To test this line of reasoning directly, we set out to develop Arabldopsis llnes with reduced levels of 18:3 and 163 fatty acids. There are two dlstinct pathways in plant 6811s for the biosynthesis of glycerolipids Rnd the associated praduction To whom correspondence should be addressed. of polyunsaturated fatty acids (Roughan et al., 1980;Browse and Somerville, 1991). 60th pathways are initiated by the synthesis of 16:O-acyl carrier protein (ACP) and 18:l-ACP by the combined action of a type II fatty acid synthase (Shimakata and Stumpf, 1982) and a soluble stearoyl-ACP desaturase (McKeon and Stumpf, 1982;Shanklin and Somerville, 1991) located in the chloroplasts or other p...

The very high proportions of trienoic fatty acids found in chloroplast membranes of all higher plants suggest that these lipid structures might be essential for photosynthesis. We report here on the production of Arabidopsis triple mutants that contain negligible levels of trienoic fatty acids. Photosynthesis at 22[deg]C was barely affected, and vegetative growth of the mutants was identical with that of the wild type, demonstrating that any requirement for trienoic acyl groups in membrane structure and function is relatively subtle. Although vegetative growth and development were unaffected, the triple mutants are male sterlle and produce no seed under normal conditions. Comparisons of pollen development in wild-type and triple mutant flowers established that pollen grains in the mutant developed to the tricellular stage. Exogenous applications of [alpha]-llnolenate or jasmonate restored fertility. Taken together, the results demonstrate that the critical role of trienoic acids in the life cycle of plants is as the precursor of oxylipin, a signaling compound that regulates final maturation processes and the release of pollen.

Two independently isolated mutations at the fad7 locus in Arabidopsis produced plants with a temperature-conditional phenotype. Leaves of fad7 mutants grown at 28'C contained less than 30% of wild-type levels of trienoic fatty acids (163 plus 183) compared with more than 70% of wild-type levels for plants grown at 15°C. Screening of an Mz population derived from the fad7-l line led to the identification of a line, SH1, in which the proportion of trienoic acids was much less than in fad7 plants. l h e segregation pattern of Fz progeny from a cross between SH1 and wild type indicated that the additional fatty acid mutation in SH1 is at a new locus, designated fad8. In a genetic background that was wild type at the FAD7 locus, the fad8 mutation had no detedable effed on overall leaf fatty acid composition irrespedive of the temperature at which plants were grown. However, fatty acid analyses of individual leaf lipids revealed small decreases in the levels of 18:3 in two chloroplast lipids. In fad8 plants grown at 22'C, phosphatidylglycerol contained 22.5% 183 compared with 33.5% in wildtype Arabidopsis. For sulfoquinovosyldiacylglycerol, the values were 31.4 and 44.5%, respedively. logether with information from studies of the cloned FADB gene (S. Cibson, V. Arondel, K. Iba, C. Somerville [1994] Plant Physiol 106 1615-1621), these results indicate that the FADB locus encodes a chloroplast-localized 162/ 18:2 desaturase that has a substrate specificity similar to the FAD7 gene produd but that is induced by low temperature.Fatty acids containing three double bonds (trienoic fatty acids) are the dominant acyl components of chloroplast membranes in a11 higher plants (Harwood, 1982). The major chloroplast glycerolipid, MGD, typically contains more than 90% of a-linolenic acid (18:3) or a combination of a-linolenic and hexadecatrienoic (16:3) acids depending on the plant species (Jamieson and Reid, 1971). These observations have been taken as inferential evidence that trienoic fatty acids have an important, possibly essential, role in assuring photosynthetic competence of the light-harvesting thylakoid membranes. One attractive approach to investigating the role of trienoic fatty acids in photosynthesis and other processes is to isolate mutants that are deficient in 16:3 and 18:3 synthesis. In Arabidopsis, two classes of mutants have been isolated that have decreased capacities for conversion of 1 8 2 '

Plants accumulate crystals of calcium oxalate in a variety of shapes, sizes, amounts, and spatial locations. How and why many plants form crystals of calcium oxalate remain largely unknown. To gain insight into the regulatory mechanisms of crystal formation and function, we have initiated a mutant screen to identify the genetic determinants. Leaves from a chemically mutagenized Medicago truncatula population were visually screened for alterations in calcium oxalate crystal formation. Seven different classes of calcium oxalate defective mutants were identified that exhibited alterations in crystal nucleation, morphology, distribution and/or amount. Genetic analysis suggested that crystal formation is a complex process involving more than seven loci. Phenotypic analysis of a mutant that lacks crystals, cod 5, did not reveal any difference in plant growth and development compared with controls. This finding brings into question the hypothesized roles of calcium oxalate formation in supporting tissue structure and in regulating excess tissue calcium.

SummaryHigh 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 receptormediated 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.

The pads (nopales) of the prickly pear cactus are considered to be a good source of minerals and other nutrients on the basis of compositional analysis. In this study, this analysis is taken a step further by assessing the availability of selected minerals in nopales using an in vitro digestion and dialysis method. The results obtained suggest that although nopales are enriched in a number of minerals, their tissue calcium is not freely available. Microscopic analysis, energy-dispersive X-ray microanalysis, and oxalate measurements suggest that this reduction in available calcium is a result of its sequestration in the form of calcium oxalate crystals. The issue of mineral availability in plant foods is important when the dependence of many populations around the world on plant foods as their main source of minerals and other nutrients is considered.

Bioavailable calcium affects bone formation and calcification. Here we investigate how a single gene mutation altering calcium partitioning in the model forage crop Medicago truncatula affects calcium bioavailability. Previously, the cod5 M. truncatula mutant was identified which contains identical calcium concentrations to wild-type, but contains no oxalate crystals. In this study, equal number of male and female mice were randomly grouped and then fed one of four 45Ca-containing diets: M. truncatula extrinsically or intrinsically labeled, and cod5 extrinsically or intrinsically labeled. Absorption of the tracer was determined in the legs one day after consumption. The absorption was similar in the M. truncatula and cod5 extrinsically labeled diets; however, in the intrinsically labeled diets, calcium absorption was 22.87% (P < 0.001) higher in mice fed cod5. Our study presents the first genetic evidence demonstrating the nutritional impact of removing oxalate crystals from foods.