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 '

Two different mutants of Arabidopsis thaliana deficient in chloroplast membrane lipid polyunsaturation were indistinguishable in appearance from the wild-type when grown at 22 degrees C. By contrast, leaf tissues of the mutants that developed during growth at 5 degrees C were chlorotic, whereas the wild type was not. This is the first direct evidence that chloroplast lipid polyunsaturation contributes to low-temperature fitness. Chloroplasts from mutant lines grown at 5 degrees C were much smaller than those of the wild-type, and the thylakoid membrane content was reduced by up to 70%. However, there was no discernible effect of low temperature on chloroplasts that developed prior to exposure to low temperatures. These and related observations suggest that the high degree of chloroplast membrane lipid polyunsaturation is required for some aspect of chloroplast biogenesis.

A mutant of Arabidopsis thaliana, deficient in activity of the chloroplast n-6 desaturase, accumulated high levels of C16:1 and C18:1 lipids and had correspondingly reduced levels of polyunsaturated lipids. The altered lipid composition of the mutant had pronounced effects on chloroplast ultrastructure, thylakoid membrane protein and chlorophyll content, electron transport rates, and the thermal stability of the photosynthetic membranes. The change in chloroplast ultrastructure was due to a 48% decrease in the amount of appressed membranes that was not compensated for by an increased amount of nonappressed membrane. This resulted in a net loss of 36% of the thylakoid membrane per chloroplast and a corresponding reduction in chlorophyll and protein content. Electrophoretic analysis of the chlorophyll-protein complexes further revealed a small decrease in the amount of light-harvesting complex. Relative levels of whole chain and protosystem 11 electron transport rates were also reduced in the mutant. In addition, the mutation resulted in enhanced thermal stability of photosynthetic electron transport. These observations suggest a central role of polyunsaturated lipids in determining chloroplast structure and maintaining normal photosynthetic function and demonstrate that lipid unsaturation directly affects the thermal stability of photosynthetic membranes.The photosynthetic membranes of higher plants contain an unusually high proportion of polyunsaturated fatty acids.Depending on the plant species, trienoic acids (C16:3 and C18:3) and dienoic acids (C162 and C182) may account for 85% of the total fatty acids found within the chloroplast (1 1

The transfer of specific Ti (tumor-inducing) plasmid sequences, the T-DNA, from Agrobacterium tumefaciens to a wide range of plants results in the formation of crown gall tumors. These tissues differ from most plant cells in that they can be grown in vitro in the absence of added phytohormones. Here, data are presented that offer an explanation for the auxin-independent phenotype of crown gall tissues. It is shown that crude cell-free extracts prepared from three bacterial species harboring pTiA6 gene 1 could convert L-tryptophan to indole-3-acetamide; control extracts lacking gene 1 could not carry out the reaction. Other reports indicate that the pTiA6 gene 2 product can convert indole-3-acetamide to indole-3-acetic acid, a naturally occurring auxin of plants. It is concluded that the auxin-independent phenotype of crown gall tissue involves the introduction of Ti plasmid sequences encoding a two-step pathway for auxin synthesis.

Leaf tissue of a mutant of Arabidopsis thaliana contains reduced levels of both 18-carbon and 16-carbon polyunsaturated fatty acids and increased levels of the 18:1 and cis-16:1 precursors due to a single nuclear mutation at a locus designated WadC. Analysis of the fatty acid compositions of individual lipids and the kinetics of lipid labeling with [14Cjacetate in vivo indicate that the mutant lacks activity of the chloroplast glycerolipid w0-6 desaturase. As a result, lipids synthesized by the prokaryotic pathway are not desaturated further than 18:1 and 16:1. Lipids derived from the eukaryotic pathway are desaturated-presumably by the endoplasmic reticulum 18:1 phosphatidylcholine desaturase. However, an increase in the level of 18:1 on all the phospholipids derived from the eukaryotic pathway in leaves of the mutant suggests that the mutation does exert an effect on the composition of extrachloroplast membranes. Synthesis of monogalactosyldiacylglycerol (MGD) by the prokaryotic pathway is reduced 30 to 35% in the mutant and there is a corresponding increase in MGD synthesis by the eukaryotic pathway. This shift in metabolism which results in a more unsaturated MGD pool, may reflect the existence of a regulatory mechanism which apportions lipid synthesis between the two pathways in response to alterations in the physical properties of the chloroplast membranes.It is now generally accepted that there are two distinct pathways in plant cells for the biosynthesis of glycerolipids and the associated production of polyunsaturated fatty acids (10,23,29). Both pathways are initiated by the synthesis of 16:0-ACP2 in the plastid. 16:0-ACP may be elongated to 18:0-ACP and then desaturated to 18: 1-ACP by a soluble desaturase so that 16:0-ACP and 18: 1-ACP are the primary products of plastid fatty acid synthesis. In 16:3 species such as Arabidopsis thaliana, these thioesters may be used within the chloroplast for the acylation of glycerol-3-P and the subsequent synthesis, via the prokaryotic pathway, of chloroplast