The ch4 mutant of sweetclover (Melilotus alba) has previously been demonstrated to be temperature-and photoperiod-sensitive for the accumulation of chlorophyll. Pigment content of ch4 mutant leaves was examined as a function of trifoliolate mass, an index of leaf development. Inhibition of chlorophyll accumulation caused by increased growth temperature or decreased photoperiod can be attenuated during the earliest leaf development stages but is almost complete after the trifoliolates have reached 10 milligrams in size. Once this size is reached, the total amount of chlorophyll per trifoliolate remains constant, and the amount of chlorophyll per gram of leaf decreases as the leaf expands. We have evidence for several other sweetclover mutants with this general phenotype, all with alterations previously determined to be located in different genes as evidenced by complementation following genetic crosses. This mutant phenotype may represent lesions in the chlorophyll biosynthetic pathway or the assembly of the photosynthetic apparatus.During a recent study on nuclear mutants of sweetclover (Melilotus alba) with less than normal amounts of Chl b, it was discovered that the ch4 mutant, which is normally characterized by somewhat reduced amounts of Chl and an elevated ratio of Chl alb, was temperature-and photoperiod-sensitive (8). Growth of the plant at 23°C and 16 h photoperiod (termed a permissive condition) resulted in its usual phenotype, leaves containing about one-third the normal amount of Chl and a somewhat elevated ratio of Chl alb. However, growth at 26°C and 16 h photoperiod or at 23°C and 8 h photoperiod (termed nonpermissive conditions) resulted in production of stunted plants with yellow leaves. The effect of the mutation on plants grown under these latter conditions appeared to be relatively specific for Chl content, which was reduced over 20-fold relative to normal plants; the amount of ribulose 1,5-bisphosphate carboxylase activity, an indicator of cytoplasmic and chloroplastic protein synthesis, was only half of normal. It was suggested that the molecular basis of this mutation was some developmentally regulated process involved in pigment production.The data in this manuscript will demonstrate that the ch4 mutation appears to set limitations on RESULTSWhile studies on the temperature-sensitive nature of the ch4 sweetclover mutant were in progress, it was noticed that, whereas the large and medium-sized trifoliolates on the ch4 mutant plants grown at 26°C with 16 h photoperiod were clearly Chl deficient, the very small trifoliolates appeared to be more green in color. Consequently, the pigment content of the ch4 mutant was examined as a function of trifoliolate mass, which should be an indirect indicator of developmental state; smaller trifoliolates should generally be at an earlier developmental state than larger trifoliolates on the same plant. For comparison, the pigment content was also measured in trifoliolates of normal and ch5 mutant plants grown under identical conditions. All mat...