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During greening of detached leaves from dark-grown barley seedlings, the linolenic acid content of the lipids increases in the early stages of the formation of the chloroplast lamellar system. Primarily the fraction containing monogalactosyl diglyceride is enriched with linolenic acid. Incorporation of (14)C-labeled acetate into the leaf lipids of detached whole leaves is low, but increases 10- to 20-fold during greening. Increasing percentages of label appear in linolenic acid during the first 15 hr of greening, whereafter they remain constant. A constant, relatively high amount of acetate is incorporated into lipids when slices of leaves at various stages of greening are incubated by submersion in acetate solution, a treatment that blocks further chlorophyll synthesis during incubation. At the initial greening stages 75% of the label is channeled into steroids and other unsaponifiable lipids, but in advanced stages of chloroplast development 75% of the incorporated acetate is built into phospho-, sulfo- and galacto-lipids, and only 25% is channeled into unsaponifiable lipids. Experimental variation of the physiological conditions of the tissue during incubation resulted in differences in the amount of label found in the various phospho- and galacto-lipids. The amounts of labeling of the individual fatty acids in the lipid classes studied differ markedly and could be changed by varying the conditions of incubation. Labeling of linolenic acid was found to be highest in the monogalactosyl diglyceride fraction at all stages of greening.

The cell organelle contents of chloroplast preparations made from barley leaves with salt and sucrose isolation media at pH 6 and 8 were determined and compared with the acetate incorporating activity of these preparations. A chloroplast preparation obtained with 0.5 m sucrose at pH 8 gave the highest number of intact chloroplasts (with envelope and stroma), the lowest number of contaminating mitochondria, and the highest activity in light dependent acetate incorporation into lipids. In the preparations observed, the light induced lipid synthesizing capacity correlates well with the percentage of intact chloroplasts. It is suggested that the intact chloroplasts are responsible for the light induced lipid synthesis of the preparations and that the synthesizing enzymes are localized in the chloroplast stroma. Acetate is mainly incorporated into palmitic and oleic acids. The low yield of intact chloroplasts and of light induced lipid synthesis in preparations isolated at pH 6 seem to result from the action of galactolipid lipase(s).

The capacity of leaf slices from light-grown seedlings of wild type barley and 10 xantha mutants at six different gene loci to incorporate acetate-(14)C into various lipids has been investigated. The fatty acid composition of the leaf lipids in these lethal mutants was similar to that of the wild type, but the fatty acid labeling pattern in the individual lipid classes can be drastically altered by these mutations, which affect chloroplast differentiation. A genetic block in chlorophyll synthesis, caused by mutations in the xan-f locus, leads to a repression of the formation of chloroplast membranes and of acetate incorporation into phospho-, sulfo-, and galacto-lipids (the acetate being preferentially channeled into a lipid fraction containing steroids and free fatty acids). Two leucine "auxotrophs" at different loci, which in the absence of leucine in the growth medium produce giant grana and accumulate some chlorophyll, differed considerably in the amount of labeling of their polar lipids during incubation. Leaves of xan-a(11), containing plastids with little chlorophyll, highly disorganized membrane systems, and large bodies with osmiophilic deposits, were nonetheless equal to wild type in their capacity to incorporate acetate-(14)C into phospho-, sulfo-, and galacto-lipids. The mutants at the xan-m locus have plastids with undispersed prolamellar bodies and osmiophilic packages of grana-like membranes associations. Leaf slices of these mutants synthesized considerably more linolenic acid-(14)C, which was incorporated into monogalactosyl diglycerides, than did slices of the wild type. This led to a labeling pattern of the fatty acids in the monogalactolipids which was remarkably similar to their endogenous fatty acid composition.