No abstract

Abstract. The phytochrome controlled increase in total protein in the primary leaf pair of etiolated bean (Phaseolus vidlgaris 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 inhib'itor 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 celli levels.The initial brief illumination is Eaturating, but 40 minutes later the sezdlings 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.

Abstract. 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.In considering models for the physiological actioii of phvtochrome the stability of the "active" forni of the pigment plays a crucial role. Designated as P730 this form hais been considered alternately as ral)idly reverting to P660 (1), decaying (9) or both (2). On the other hand Downs reported in 1955 that far-red reversibility of red light activated leaf elongation was lost very slowlv. In 8 hr only about 20 % of the reversibility was lost when adjustnients were made for the intervening growth of the seedling (5I). This points to an extremely stable "active" pliotoreceptor.Recently e l)resenited evideiice (7) that lightactivated increases in total protein and ribulose diphosphate carboxylase activity of 'etiolated bean leaves remain partially reversible for over 24 hr. The remarkably long-lived susceptibility of developmental changes to reversal has interesting iniplications for models of growth regulation by phytochrome, particularly when contrasted with the lability of spectrophotonietrically detectable P730 (4,12). WVe, therefore. undertook an exploration of the limits within which red light activated developmental responses of bean leaves remain far red reversible.

Two forms of protochlorophyllide are found in dark-grown bean (Phaseolus vulgaris, var (13,14,17).ALA treatment of the leaves of dark-grown seedlings causes accumulation of a form of PChld with an absorbance peak

A fraction of the RN-A 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 comnplements.Many attempts have been made to discover the level at which specific developmental changes are controlled in plants. Some of these attempts have focused on the possibility of transcriptional control (5, 8, 9, 14-16, 18, 19, 21, 24, 32, 36). Until recently, attempts to isolate and study mRNA from plants depended on the rapid labeling of certain fractions which were not fully characterized (12,16,20). With the discovery that most eucaryotic mRNA contains poly(A)3 sequences, a simple procedure for mRNA isolation has become available. Poly(A) RNA has been found now in a number of plant species (10,13,23,30,(33)(34)(35) for 2 min at top speed. After adding isoamyl alcohol (1-2%,) to control foaming, the homogenate was extracted with 2 volumes of hot phenol (60 C) containing 1 % 8-hydroxyquinoline and saturated with the grinding buffer. The phenol phase was extracted with 0.5 volume of the grinding buffer, and the combined aqueous phases were extracted two more times with phenol. Nucleic acids were precipitated overnight with 2 volumes of cold ethanol, dissolved in 0.01 M tris, pH 7.4, and reprecipitated in the presence of 0.1 M sodium acetate with ethanol.When RNA uncontaminated by DNA was required, the nucleic acid precipitate was dissolved in 0.01 M MgCl2, 0.01 M tris, pH 7.4, and was treated with ribonuclease-free DNase (25 ,ug/rnl [Worthington]) for 1 hr at 37 C. The solution was then brought to 0.01 M EDTA, 0.2 M LiCl, and 0.5% SDS, extracted twice with phenol, and the RNA was precipitated with ethanol. The precipitate was dissolved and reprecipitated in the presence of 4 M potassium acetate.The RNA was further purified in all cases by extraction with 2-methoxyethanol and precipitation with cetyltrimethylammonium bromide, according to the procedure of Bellamy and Ralph (3).Poly(A) containing RNA was isolated on oligo (dT) cellulose columns (2). Total RNA was dissolved in 0.5 M salt (KCI or NaCI) in 0.01 M tris, pH 7.5, and applied to a column containing 0.2 to 0.25 g of oligo (dT) cellulose Collaborative Research, Waltham, Mass.). Poly(A) RNA binds to the oligo (dT) cellulose under this high salt condition, but the bulk of the RNA does not bind. The sample was washed onto the column with additional high salt buffer until the eluate did not contain any RNA. The column was then washed with 0.1 M salt, 0.01 M tris, pH 7.5, and the poly(A) RNA was eluted with 0.01 M tris, pH 7.5. The co...