1. Cells of Chlamydomonas reinhardi grown in the light or dark on standard medium require an additional exposure to light in the absence of a nitrogen source, in order to become sexually active. As the culture ages, the light requirement decreases. 2. This light requirement is a function of nitrogen depletion, as shown by the observation that cells from cultures grown to maturity on a low nitrogen medium in the light or in the dark, have no additional light requirement for zygote formation. The withholding of no other component of the medium has this effect. 3. In cells requiring light for zygote formation, the light can be supplied before the mating types are mixed, indicating that light is required, not for mating per se, but for the conversion of vegetative cells to gametes. 4. Gametes can be dedifferentiated to the vegetative state by any nitrogen compound which the cells can use for growth; and by further exposure to light in the absence of a nitrogen source, these vegetative cells can again become gametic. 5. Cells grown at different nitrogen levels become gametic at widely different cell concentrations of nitrogen and carbon and C/N ratios. 6. It is postulated that the role of light in gametic differentiation is indirect, providing by photosynthesis, energy for the mating process and carbohydrates to tie up excess nitrogenous reserves; and that the concentration of some particular nitrogen fraction or compound determines whether or not gametic differentiation is initiated.

At present, we are investigating the genetic mechanisms involved in chlorophyll biosynthesis and chloroplast development. For this project, it has been necessary to use a unicellular organism which is photosynthetic but can also be grown in the dark, and which has a sexual phase in its life-cycle. The ability of the organism to grow in the dark has been of particular importance because of our interest in mutants unable to synthesize chlorophyll and which are therefore non-photosynthetic. The sexual requirement is necessary for carrying out the genetic analysis. The organism chosen, Chlamydomonas reinhardi, a unicellular green alga, was isolated by Professor G. M. Smith, to whom we are indebted for the mating type strains used in this work.The nutritional studies to be reported here are concerned, first, with vegetative growth of the organism in light and dark and, second, with the nutritional conditions which we have found to be of critical importance in controlling the sexual phases of the life-cycle.

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In 7-to 10-day-old leaves of etiolate(I barley (Hordeum vulgare), all of tile eiizymes that convert 1-aminolevulinic acid to chlorophyll are nonlimiting duri-ing the first 6 to 12 hours of illumination, even in the presence of iinhibitors of protein synthesis. The limiting activity for chlorophyll synthesis appears to be a protein (or proteins) related to the synthesis of 6-aminolevulinic acid, prestumably i-aininolevtulinic acid synthetase. Protein Schiff and Epstein (26), and others have suggested that the rate of ALA synthesis is controlled by a negative feedback in which protochlorophyllide or the protochlorophyllide holochrome inhibits the activity of ALA-synthetase. Gassman and Bogorad (5, 6) have proposed the hypothesis that light regulates chlorophyll synthesis in young bean leaves but that synthesis of RNA was required as a precursor step in this process.In this paper we shall present further evidence to support the hypotheses that light stimulates the synthesis of an enzyme which forms 6-aminolevulinic acid, that this is the rate-limiting enzyme in chlorophyll synthesis, and that, at least in barley, RNA synthesis may not be required for early chlorophyll synthesis. METHODS AND MATERIALSBarley (Hordeum vulgare var. Wong; W. A. Burpee Co., Philadelphia, Pa.) seedlings were grown in vermiculite beds in the dark in closed cabinets. The seedlings were watered with tap water every second day. The temperature in the growth room was regulated at 25 i 2 C. The growth rate of the seedlings is shown in Figure 1. The period of most rapid growth was over after 7 days. The first leaf was almost fully formed at this time. A second, smaller leaf appeared after 10 days. Unless noted otherwise, seedlings, 7 to 10 days old, were used.All handling of the seedlings was performed under dim, green safelight (less than 1 ft-c with a filter combination that passed only 540-560 nm of light). The dark-grown seedling "tops" were harvested by cutting them off just above the bed; the lowest inch (approximately the coleoptile region) was discarded in seedlings older than 7 days. Approximately 10 g of seedling tops, prepared as above, were placed in 15-mi beakers containing 6 ml of solution, with the bottom 3 cm of the tops immersed in the solution usually for 2 hr in the dark before illumination. The uptake of solutions was facilitated by gently circulating air around the tops with a small fan (40 cubic feet per min). The solutions in the beakers were replenished to ensure maintenance of turgor of the seedlings throughout the experiment. That a 2-hr dark preincubation permitted adequate uptake of solution by the leaves was shown by the following observations. (a) ALA-fed leaves synthesized enough PCHLD in the dark preincubation period to form more CHL than controls in a subsequent 2-hr period of low intensity illumination; (b) CHX decreased the rate of CHL formation within 1 1 hr after it was fed to the leaves.AU illuminations were performed with 7 ft-c of tungsten light unless otherwise noted. The effect of the slight v...

The customary route in animals and bacteria for 6-aminolevulinic acid biosynthesis is from glycine and succinyl CoA, tamine were more effective in labeling the ALA (9, 10). Therefore, an alternative route of synthesis of ALA in plants appeared to be indicated. In this paper we report the labeling of specific carbon atoms of ALA by specifically labeled glutamate, clearly indicating that the five-carbon skeleton of glutamate is incorporated intact into ALA. We discuss possible biochemical routes of the labeling pattern observed. days of growth in complete darkness at 250, the terminal 80 mm of the leaves were excised with a razor blade and placed in 10 ml (25 mm diameter) beakers containing 5 ml of distilled water. Samples comprising 5 g of leaves (approximately 60 leaves) per beaker were then subjected to experimental treatment. MATERIALS AND METHODSIncubation Conditions. The leaf samples were exposed to 400 ,uW/cm2 of cool white fluorescent light as measured with a YSI model 65 radiometer (350 lux as measured with a General Electric DW58 exposure meter) at 250 for 3 hr. Then the water from the beakers was removed and replaced with incubation solutions consisting of the appropriate [14C]glutamic acid (5 Ci/mol) and, in some cases, 25 mM levulinic acid, pH adjusted to 6.5 with KOH. In the presence of levulinic acid, ALA-dehydratase [porphobilinogen synthase; b-aminolevulinate hydro-lyase (adding b-aminolevulinate and cyclizing); EC 4.2.1.24] is inhibited, thus permitting the accumulation of ALA. Total volume of the incubation solutions was 2.0 ml in all cases. The leaf samples were placed back in the same light and in the air current of a fume hood to facilitate uptake of the solution into the leaves via transpiration. After 2 hr, the incubation solutions were almost completely taken up by the leaves, and at this time the incubation was terminated by placing the leaves in stoppered plastic vials and then freezing them in powdered dry ice.