Phytochrome-mediated assembly of polyribosomes in etiolated bean leaves. Evidence for post-transciptional regulation of development.

Light operating through phytochrome controls the proportion of total ribosomes present as polyribosomes in etiolated leaves of Phaseolus vulgaris. Similar responses but with slightly different time courses are elicited by brief red light treatment and by continuous far-red or white light. The increase in polyribosome proportions after red light treatment reaches a maximum within 2 h, after which the proportion steadily declines. Light treatment appears to lead to increased proportions of polyribosomes in higher size classes. This is most evident with continuous white light, but probably also occurs with red and far-red light. The increase in polyribosomes is due principally to cytoplasmic ribosomes although proportionately greater effects are observed in chloroplast ribosomes. Although cordycepin inhibits RNA synthesis by 85-90% within 3 h there is no depression of the light-mediated increase in polyribosome proportions when leaves are preincubated in the inhibitor for 3 h. The data therefore indicate that phytochrome is capable of controlling protein synthesis, and thus development, at a post-transciptional level.     

Non-synchronous increases in activities of peroxisomal enzymes in etiolated mung bean seedling leaves after illumination

When etiolated 4-day-old seedlings of mung bean were illuminated,catalase, glycolate oxidase and hydroxypyruvate reductase activitiesin the primary leaves increased non-synchronously. Under intenselight, glycolate oxidase activity increased while catalase activitydid not. Phytochrome seems to be involved in increases in allthree enzyme activities. (Received August 10, 1977; )     

Greening of etiolated bean leaves in far red light

Eight-day-old dark-grown bean leaves were greened by prolonged irradiation with far red light. Growth, chlorophyll content, oxygen-evolving capacity, photophosphorylation capacity, chloroplast structure (by electron microscopy), and in vivo forms of chlorophyll (by low temperature absorption and derivative spectroscopy on intact leaves) were followed during the greening process. Chlorophyll a accumulated slowly but continuously during the 7 days of the experiment (each day consisted of 12 hours of far red light and 12 hours of darkness). Chlorophyll b was not detected until the 5th day. The capacity for oxygen evolution and photophosphorylation began at about the 2nd day. Electron microscopy showed little formation of grana during the 7 days but rather unfused stacks of primary thylakoids. The thylakoids would fuse to give grana if the leaves were placed subsequently in white light. The low temperature spectroscopy of intact leaves showed that the chlorophyll a was differentiated into three forms with absorption maxima near 670, 677, and 683 nanometers at −196 C during the first few hours and that these forms accumulated throughout the greening process. Small amounts of two longer wavelength forms with maxima near 690 and 698 nanometers appeared at about the same time as photosynthetic activity.     

Absorbance and fluorescence properties of protochlorophyllide in etiolated bean leaves

Primary leaves of 7-to-9 day-old etiolated bean seedlings contain a species of protochlorophyllide which is not transformed to chlorophyllide by light; this pigment species exhibits an absorption peak at 631nm $\text{in}\text{vivo}$ at ?196° and a fluorescence emission peak at 639nm $\text{in}\text{vivo}$ at room temperature. Heat-treatment of etiolated leaves converts the phototransformable protochlorophyllide holochrome to a pigment species with $\text{in}\text{vivo}$ absorption and fluorescence peaks identical to those of endogenous nontransformable protochlorophyllide. Administration of δ-amino-levulinic acid to etiolated leaves causes the synthesis of non-transformable protochlorophyllide with an absorption peak also at 631nm $\text{in}\text{vivo}$ at ?196° but with a fluorescence emission peak at 643nm $\text{in}\text{vivo}$ at room temperature. Heat-treatment of such leaves does not affect the position of these bands. The results indicate that protochlorophyllide which is derived from exogenous δ-amino-levulinic acid is in a physically different state from other forms of protochlorophyllide in the leaf.     

Relationships between glycollate and formate metabolism in greening barley leaves

The activities of enzymes catalysing glycollate oxidation, formate production and folate-dependent formate utilization were examined in the primary leaves of Hordeum vulgare cv Galt. Seedlings were grown for 6 days in darkness and then transferred to continuous light (500 μinsteins/m² per sec) for up to 5 days. Cell-free extracts of the primary leaves contained glycollate oxidase (EC 1.1.3.1), 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5, 10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) and ability to enzymically decarboxylate glyoxylate. These activities increased during greening and at the end of the light treatment were 70–450% higher than etiolated controls. Greened primary leaves also incorporated [¹⁴C]formate at rates that were three- to four-fold higher than shown by etiolated leaves. The specific activity of 10-formyltetrahydrofolate synthetase was decreased by 20–35% when the leaves were greened in the presence of 10 mM hydroxysulphonate. This inhibitor also reduced the incorporation of [¹⁴C]formate by up to 45%. A potential flow of carbon from glycollate to 10-formyltetrahydrofolate via glyoxylate and formate was suggested by the data.     

The greening of etiolated bean leaves II. Secondary and further photoconversion processes

1. Photoconversions in light-dark stages in etiolated bean leaves show that protochlorophyllide is resynthesized to the same level at each stage.     

Induction of porphyrin synthesis in etiolated bean leaves by chelators of iron

Primary leaves of 7- to 9-day-old etiolated seedlings of Phaseolus vulgaris L. var. Red Kidney infiltrated in darkness with aqueous solutions of alpha, alpha'-dipyridyl, o-phenanthroline, pyridine-2-aldoxime, pyridine-2-aldehyde, 8-hydroxyquinoline, or picolinic acid synthesize large amounts of magnesium protoporphyrin monomethyl ester and lesser amounts of magnesium protoporphyrin, protoporphyrin, and protochlorophyllide. Pigment formation proceeds in a linear manner for up to 21 hours after vacuum infiltration with 10 mm alpha, alpha'-dipyridyl. Etiolated tissues of Zea mays L., Cucumis sativus L., and Pisum sativum L. respond in the same way to dipyridyl treatment. Compounds active in eliciting this response are aromatic heterocyclic nitrogenous bases which also act as bidentate chelators and form extremely stable complexes with iron; other metal ion chelators, such as ethylenediaminetetraacetic acid, salicylaldoxime, and sodium diethyldithiocarbamate, do not elicit any pigment synthesis. The ferrous, ferric, cobaltous, and zinc chelates of alpha, alpha'-dipyridyl are similarly ineffective. If levulinic acid is supplied to etiolated bean leaves together with alpha, alpha'-dipyridyl, porphyrin production is inhibited and delta-aminolevulinic acid accumulates in the tissue. Synthesis of porphyrins proceeds in the presence of 450 micrograms per milliliter chloramphenicol or 50 micrograms per milliliter cycloheximide with only partial diminution. We propose that heme or an iron-protein complex blocks the action of the enzyme(s) governing the synthesis of delta-aminolevulinic acid in etiolated leaves in the dark and that iron chelators antagonize this inhibition, leading to the biosynthesis of delta-aminolevulinic acid and porphyrins.     

Regulatory aspects of chloroplast growth in leaves ofXanthium pensylvanicum and etiolated red kidney bean seedling leaves

Summary The average chloroplast size was studied as a function of leaf growth in leaves of cocklebur (Xanthium, pensylvanicum) and the primary leaves of 9-day old seedlings of red kidney bean (Phaseolus vulgaris).Diameters of chloroplasts were measured in crude tissue homogenates with the aid of a fluorescence microscope. Chlorophyll content of the leaves was determined spectrophotometrically in acetone extracts.For cocklebur, data are presented to show the relationship of average chloroplast diameter to morphological age of leaves (Leaf Plastochron Index) and are discussed in relation to the available leaf growth analyses. In bean, the increase in chloroplast diameter in response to illumination of etiolated leaves of various size was studied as a function of the duration of continuous illumination. The size of the etiolated bean leaves was varied experimentally by exposing the seedlings in darkness to low energy red light. Average diameter of the chloroplasts was found to be related to the size of leaf lamina.In both cocklebur and bean, a definite relationship of chloroplast size to leaf area and morphological age was established. The observed patterns of chloroplast size increase are interpreted to be a reflection of the integration of growth at three levels of organization: the leaf, its cells and the chloroplasts.This study was performed during the tenure of a U. S. Public Health Service postdoctoral fellowship by the senior author and was supported in part by research grant number GM-08145, from the National Institutes of Health, Public Health Service.Predoctoral fellow of the National Science Foundation.     

The effects of actinomycin D on the greening of etiolated bean leaves

The effects of actinomycin D on chlorophyll formation in beanleaves were studied. Chlorophyll formation was inhibited inilluminated bean leaves. In etiolated leaves and actinomycinD-treated illuminated leaves, we observed the accumulating ofRNA components, probable precursors of ribosomal RNA. (Received August 14, 1971; )     

Photoconvertible and non-photoconvertible forms of protochlorophyll(ide) in etiolated bean leaves

Precursors of chlorophylls in etiolated bean leaves were studiedby a sensitive technique of dual wavelength scanning of thinlayer chromatograms of pigments. The photoconvertible pigmentswith absorption maxima at 650 and 638 nm, respectively, wereidentified as protochlorophyllide. A minor non-photoconvertiblepigment with a maximum at 628 nm was found to be protochlorophyll. (Received July 3, 1974; )     

Glycine metabolism in etiolated barley leaves on exposure to light

Of a large number of amino acids examined, changes in glycine were the only ones which were correlated with the ability of dark-grown barley leaves to synthesise protochlorophyllide, δ-aminolaevulinic acid and chlorophyll on exposure to light. A rapid depletion was found in endogenous glycine in barley leaves after day 7. Illumination of the leaves increased the rate of glycine depletion. Glycine concentrations were high throughout the young leaf. The top and middle leaf sections however, which had maximal chlorophyll synthesising potential exhibited the most pronounced decrease in glycine as the leaf aged. Using glycine-[¹⁴C] pulse techniques the half life of glycine in 7 and 14-day-old dark-grown leaves was 3.5 and 4.4 min respectively. Light treatment lengthened the half life to 6.9 and 12.1 min in 7 day and 14-day-old-leaves. Sustained illumination continued to decrease glycine turnover.     

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.