Light-induced Polysome Formation in Etiolated Leaves: Kinetics of Inhibition by Antibiotics.

Red light-induced, far red light-reversible increase in etiolated bean (Phaseolus vulgaris, var. Asgrow Valentine) leaf polyribosomes was shown to be sensitive to actinomycin D, cycloheximide, and rifampicin inhibition. Actinomycin prevented response to red light if administered simultaneously with a 10-minute illumination, had no immediate effect if given 2 hours after illumination, but was again rapidly inhibitory at 4 and 6 hours. The effects of actinomycin and far red light were more than additive.Cycloheximide consistently inhibited polysome formation within 1 hour at all times tested.Formation of both cytoplasmic and organelle polysomes was depressed by rifampicin, an antibiotic whose action is primarily on RNA synthesis in organelles.The results are interpreted to show that light-triggered polysome formation exhibits a changing need for RNA synthesis and an obligate dependence on protein synthesis during the 1st hours following brief illumination.

Isolation and translation of plant messenger RNA.

A fraction of the RNA species isolated from Lemna gibba G-3 consists of molecules with attached sequences of polyadenylic acid. This polyadenylic acid-containing fraction, separated from total RNA by adsorption onto oligothymidylic acid-cellulose, was shown to be mRNA by its ability to serve as template in a cell-free translation system derived from wheat germ. The products of translation were characterized by electrophoresis. This method permitted the comparison of mRNA from plants grown under different light conditions. Such plants were shown to possess qualitative and quantitative differences in their mRNA complements.

Relationship between Photoconvertible and Nonphotoconvertible Protochlorophyllides.

Two forms of protochlorophyllide are found in dark-grown bean (Phaseolus vulgaris, var. Black Velentine) leaves, one (protochlorophyllide(650)) which is directly photoconvertible to chlorophyllide and another (protochlorophyllide(632)) which is not. Dark-grown leaves placed in solutions of delta-aminolevulinic acid accumulate protochlorophyllide(632). Protochlorophyllide(650) and protochlorophyllide(632) can be partially separated on sucrose density gradients. A nitrogen atmosphere blocks chlorophyll synthesis in light or the regeneration of protochlorophyllide(650) in the dark, even in the presence of excess delta-aminolevulinic acid, except when a stockpile of protochlorophyllide(632) is present in the leaf. Under the latter conditions chlorophyll synthesis or protochlorophyllide(650) regeneration is accompanied by a decrease in protochlorophyllide(632). These experiments suggest that protochlorophyllide(632) may be converted to protochlorophyllide(650).Cycloheximide inhibited greening only after an "action-dependent" delay, requiring a predictable minimal period of illumination. This inhibition could be relieved for a time by feeding delta-aminolevulinic acid.

Persistent photoreversibility of leaf development.

Far red light reversal of red light induced leaf expansion and enzyme changes were investigated in seedlings of Phaseolus vulgaris var. Black Valentine. In etiolated plants growth, anthocyanin accumulation and increases in glyceraldehyde-3-phosphate dehydrogenase and glycolic acid oxidase activities induced by a 10 min red irradiation were stopped by a 7 min far red irradiation given 17, 24, or 48 hr after activation. Etiolated seedlings illuminated for 24 hr with white light and seedlings grown in continuous light remained sensitive to far red reversal. This suggests that the far red sensitive receptor does not decay with time but remains associated with the site of its regulatory functions.

Changes in enzymatic activities in etiolated bean seedling leaves after a brief illumination.

The phytochrome controlled increase in total protein in the primary leaf pair of etiolated bean (Phaseolus vulgaris var. Black Valentine) seedlings, which occurs during growth in the dark subsequent to a brief illumination, was investigated. Enzymes from the chloroplasts, the mitochondria, and the soluble cytoplasm all increase in total activity after the illumination.The total protein and the ribulose carboxylase increases are not inhibited by FUdR, an inhibitor of DNA synthesis. Cycloheximide, an inhibitor of protein synthesis, applied at a time when the ribulose carboxylase activity increase has already commenced, blocks further increase. It was concluded that the total protein and the enzyme increases in the leaf are the result of increases in the per cell levels.The initial brief illumination is saturating, but 40 minutes later the seedlings have acquired the ability to respond to a second brief illumination. The rate of increase in ribulose carboxylase activity in seedlings that have been illuminated twice is greater than the rate in seedlings that have been illuminated only once.Far-red light prevents further increase in enzyme activity 48 hours after the initial illumination. There is a lag period interposed between the time of illumination with far-red light and the time at which the seedlings show the greatest effect of far-red light. It was concluded that the phytochrome influence on protein synthesis is not at the terminal steps.