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Nucleic Acid Metabolism during Greening and Unrolling of Barley Leaf Segments
Abstract
Barley (Hordeum vulgare) leaf segments unroll and green when illuminated. Illuminated segments also have an increased capacity for RNA synthesis. Part of this increased RNA synthesis may be attributed to an increased RNA polymerase activity. In addition, following illumination there is an increased formation of polysomes.Analyses of RNA synthesized during illumination showed that the radioactive RNA which accumulated was predominantly associated with cytoplasmic ribosomal RNAs. It appears that the early phases of greening are achieved without chloroplast RNA synthesis. Following extended illumination there was evidence of chloroplast ribosomal RNA synthesis; however, this occurred after appreciable chlorophyll synthesis. Actinomycin D, which effectively inhibits RNA synthesis and leaf unrolling, restricts chlorophyll synthesis only during the later stages of illumination. 5-Fluorouracil inhibits the bulk of RNA synthesis but not greening, unrolling, or polysome formation. Studies with inhibitors of protein synthesis have demonstrated a requirement for protein synthesis concomitant with chlorophyll production and leaf unrolling.
... This observation is not in agreement with that of Ingle (12) who reported a lag of 24 hr between the initial 'P labeling of cytoplasmic rRNA and chloroplast rRNA in the cotyledons of germinating radishes. Several recent papers (12,13,32) have presented data which suggest that chloroplast rRNA is synthesized in the chloroplast, and that light enhances the synthesis of chloroplast rRNA over that of cytoplasmic rRNA. That light is not an absolute requirement for proplastic rRNA synthesis was suggested by Vedel (38) in work with cucumber seedlings and by Ingle (13) using radish cotyledons. ...
Nucleic acid synthesis in the G(1) cell population of the 1-millimeter apex of the Allium cepa embryo was studied during the initial 73 hours of germination. Quantitative data indicate that the total amount of RNA per cell began to increase after 18 hours of germination while the initial DNA per cell increase did not occur until some 20 hours later. Polyacrylamide gel electrophoresis patterns of (3)H-uridine-labeled total nucleic acid samples indicated that synthesis of all detectable RNA fractions present in the pre-emergent 1-millimeter apex (i.e., cytoplasmic and "chloroplast-like" RNA) began at approximately the same time (18 hours). Synthesis of the various cytoplasmic RNA fractions continued throughout the germination period. Data indicating synthesis of the "chloroplast-like" RNA were obtained only for the initial 36 hours of germination. Specific radioactivity of (3)H-uridine-labeled total nucleic acid increased during the first 41.5 hours of germination but then decreased while the accumulation of RNA per cell continued to increase throughout the 73-hour period. In addition, a method is described which reduced the bacterial contamination of Allium seed to a level not detectable by incorporation of radioactive precursors into bacterial ribosomal RNA.
Polyacrylamide gel electrophoresis revealed a specific reduction or elimination of plastid 1.1 and 0.56 - 10⁶ mol.wt. rRNA in chlorotic leaf segments of amitrole-treated, light-grown oat plants, while cytoplasmic rRNAs remained unaffected. Dark-grown amitrole-treated oat seedlings and untreated controls gave similar electrophoretic patterns of rRNA with about 30% plastid rRNA. The chloroplast rRNA content decreased after the appearance of chlorotic symptoms.
Nitrate reductase is one of the most important enzymes in the assimilation of exogenous nitrate—the predominant form of nitrogen available to green plants growing in soil. Activity of this enzyme in plants gives a good estimate of the nitrogen status of the plant and is very often correlated with growth and yield. Although it is difficult to explain the physiological significance and the mechanism of effects of several factors on the enzyme activity, in some cases suitable postulates have been advanced. In general, the enzyme activity in a plant tissue is a balance between its relative rates of synthesis/degradation and activation/inactivation. Factors may affect the overall activity by interfering with either of these processes.
In the primary leaf sections of etiolated wheat (Triticum aestivum L.) seedlings, red light-induced unrolling is accompanied by an increase in incorporation of (14)C-leucine into protein. By differential centrifugation, the unrolling response was found to be closely related to incorporation of the amino acid into the supernatant fraction (105,000g). Cycloheximide and chloramphenicol inhibit both leaf unrolling and synthesis of the supernatant protein, although chloramphenicol exerts its effect more strongly on the fraction which presumably contains the plastids. In a barley (Hordeum vulgare L.) albino mutant completely devoid of ribulose diphosphate carboxylase activity, only incorporation of (14)C-leucine into the supernatant fraction is substantially promoted by red light. This mutant exhibits the photoresponse of leaf unrolling.Both unrolling and increased incorporation of (14)C-leucine induced by red light are prevented by indoleacetic and abscisic acids. Kinetin promotes leucine incorporation into protein and can induce unrolling in complete darkness. Protein synthesis is still promoted by red light when unrolling is almost completely inhibited by an osmoticum. It is suggested that the action of red light on leaf unrolling is dependent on synthesis of a soluble protein in the tissue.
Nitrate reductase can be induced in excised shoots of 3-day-old dark-grown Zea mays (var. WF9 x M14) seedlings in the absence of light. In contrast, leaves of 10-day-old dark-grown seedlings require a light treatment in order to induce enzymatic activity. Leaves of 10-day-old dark-grown seedlings contain a very low level of polyribosomes while 3-day-old shoots contain a very high level of polyribosomes. There is a gradual loss of polyribosomes from 3 to 10 days and a gradual loss of in vitro protein synthetic activity of the ribosome preparations. The loss of polyribosomes and decrease in their amino acid-incorporating activity correlate positively with the loss of ability to induce nitrate reducase activity as leaves of dark-grown corn seedlings age. These results corroborate and extend our previous results, in that light is not required for nitrate reductase induction per se in leaves of dark-grown seedlings but is required to reactivate the protein synthetic apparatus of older leaves.
The effect of illumination on the incorporation of labeled precursors into RNA of dark-grown maize (Zea mays) leaves was studied using either (32)P-phosphate or double labeling with (14)C- and (3)H-uridine. In the dark, label was preferentially incorporated into etioplast ribosomal RNAs. Incorporation into this fraction and into lower molecular weight fractions was strongly and preferentially stimulated by light during the first 2 hours of illumination. The effect persisted after illumination was terminated. The possibility that light-induced alterations in plastid ribosomal RNA metabolism may not be required for chlorophyll accumulation in maize is discussed.Sucrose density gradient analyses of ribosomes and of extracted RNA did not reveal light-induced incorporation of label into messenger-like RNA associated with polyribosomes during brief illumination. However, newly produced RNA which seems to be neither ribosomal RNA nor transfer RNA is detectable after illumination for 2.5 hours or longer.
The effect of light on protein synthesis during the early stages of greening of etiolated maize (Zea mays) leaves was studied using double labeling with leucine and fractionation of proteins by gel filtration and acrylamide gel electrophoresis. The incorporation of labeled leucine into a relatively small number of plastid proteins is effected within the first 30 to 60 minutes of illumination. These proteins do not accumulate with time. When illumination is prolonged, additional proteins are effected.Experiments using inhibitors of protein synthesis suggest that at least some of the proteins effected by 1 hour of illumination might be synthesized in the cytoplasm and not in plastids. Actinomycin D inhibits the incorporation of labeled leucine into some of the protein fractions, but enhances the incorporation into other fractions far above the effect exerted by light.
Twelve years ago, when we wrote a similar survey on nucleic acids and protein synthesis in plastids (Parthier and Wollgiehn 1966), the experimental evidence available at that time was suited to prove the flow of genetic information from DNA into protein inside the organelles. These data favored suggestions on the genetic autonomy of plastids which were also supported by earlier investigations demonstrating the non-Mendelian mode of chloroplast inheritance. During the last decade, an extensive literature has been accumulated dealing with occurrence, properties, coding, and synthesis of those macromolecular plastid constituents necessary to replicate, transcribe, and translate the genetic information for the construction of functional photosynthetic organelles.
Illumination or gibberellic acid treatment of etiolated barley leaf segments stimulates unrolling and results in an increased level of RNA. In contrast, segments treated with abscisic acid do not unroll and have a lower content of RNA. Gibberellic acid treatment enhanced the capacity of segments to incorporate radioactivity from (32)P-orthophosphate into all the RNA components detected by gel electrophoresis; abscisic acid greatly restricted the incorporation of precursors into all the RNA fractions. In conjunction with a changed capacity for RNA synthesis it was observed that abscisic acid-treated segments had a lowered soluble DNA-dependent RNA polymerase level in comparison to gibberellic acid-treated or illuminated segments. However, the influence of growth regulators on RNA polymerase content of the segments was associated with general effects on protein level rather than a specific effect on the synthesis of polymerase enzyme. Ribosomal preparations from gibberellic acid-treated segments had a greater percentage of polysomes and a greater capacity for amino acid incorporation in vitro into proteins than similar preparations from abscisic acid-treated segments.
Changes in RNA synthesis in cotyledons of Pisum sativum L. during seed development and maturation were studied. The bulk of the total RNA accumluated by 21 days after flowering preceding by nearly 1 week the maximum accumulation of protein which occurred at 27 days. Gel electrophoresis of total RNA indicated that exogenously supplied [32P]orthophosphate was incorporated into heavy and light rRNAs and the 4–5-S RNA component but incorporation into these fractions became restricted as the seed matured. At maturity [32P]orthophosphate was incorporated increasingly into a non-nucleic acid contaminant in addition to the accumulation of radioactive material into an apparently novel RNA component. Ribosomes occurred in polysomic configurations in young cotyledons but at seed maturity there was a preponderance of monosomes. These changes presumably reflected changes in the mRNA population. Analysis of low molecular weight messenger-like RNA species associated with ribosomes indicated that both quantitative and qualitative changes occurred in these components during seed development.
1. Buffers of high ionic strength, e.g. containing 0.33 M KCl, have been shown to be effective for the preservation of the in vivo distribution of polyribosomes extracted from Neurospora crassa.2. Polyribosomes collected by centrifugation prior to analysis on sucrose gradients have an altered size distribution in comparison to those found in crude extracts.3. Storage of mycelial samples in liquid N2 for up to 10 days was shown to have little or no effect on the activity and size distribution of extracted polyribosomes.
Photomorphogenesis in the barley leaf is characterised by an increased synthesis of chlorophyll and an associated increase in leaf width. These changes are accompanied by an increased capacity for RNA and protein synthesis. We have previously demonstrated that photoinduced unrolling can be enhanced by GA and, furthermore, this growth regulator can induce unrolling of segments maintained in the dark. ABA prevents photoinduced unrolling (Poulson and Beevers 1970). In view of the light-induced changes in RNA metabolism of barley leaf segments (Poulson and Beevers 1970) it was of interest to determine whether the hormonal treatments which influenced unrolling affected the pattern of RNA synthesis of the segments.
A method was developed to measure the solubilized DNA-dependent RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) activity in pea buds (Pisum sativum, var. Alaska). The method, involving solubilization of enzymes and their partial purification by a step-wise elution via a DEAE-Sephadex column, was used to study the effect of illumination time on enzyme activity. The amounts and proportions of the RNA polymerases I and II were almost constant until 48 h of illumination. Properties of isolated RNA polymerases I and II were not observed to alter during illumination. These results suggest that several factors are involved in photomorphogenesis.
Nucleic acid metabolism in the coleoptile and primary leaf tissues of the germinating oat (Avena sativa L.) seedling was studied. The concentrations of the different species of nucleic acid present at various stages of development were determined and the amounts of each compared. All species of nucleic acid in the coleaptile increased as the tissue elongated; but, with the onset of senescence all species decreased, especially rRNA. Exposing dark grown coleoptiles to light did not modify their capacity to synthesize nucleic acids. In the rapidly developing leaf, all species of nucleic. acid increased throughout early germination. A general enhancement in the synthesis of all species of nucleic acids resulted when dark-grown-leaves were exposed to light. Furthermore, the tRNA/DNA ratio remained constant in both tissues during development, whereas the rRNA/DNA ratio changed.
Unrolling of 1 cm sections, taken between 3 and 4 cm from the apex, of 6-day-old, etiolated barley leaves, was promoted by blue (426 nm) and red (658 nm) light. Accompanying such unrolling was a reduction in the level of the free proline of the tissue. When leaf unrolling was prevented by irradiation with far-red (728 nm) light, or treatment with abscisic acid (ABA) following red light irradiation, the level of proline remained more or less unchanged, at the level of the untreated, dark controls. The proline analogue, azetidine carboxylic acid (AZC) powerfully inhibited the light induced leaf opening, emphasizing the significance of proline-containing, structural and functional proteins in barley leaf unrolling. The inhibition imposed by AZC is partially reversible by added proline.
Anthocyanin pigments in hypocotyls and cotyledons of Raphanus sativus L. seedlings. - Raphanus sativus L. seedlings growing in darkness do not form anthocyanins. Etiolated seedlings grown in CAP 10M, exposed 48 h after sowing to far red light accumulate large quantity of anthocyanins in hypocotyls and cotyledons. This amount of pigments is bigger than that formed by seedlings grown in water and irradiated with white light. However, in both cases, the only anthocyanidin present was pelargonidin and pelargonin the only detectable anthocyanin.
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 isolation of RNA requires an effective means of cell disruption, a procedure for separating the nucleic acid from the protein or lipoprotein with which it is intimately associated, and a method for purification. To avoid degradation or denaturation, the RNA must be protected throughout from liberated nucleases, strong mechanical forces, high temperatures and extremes of pH and ionic strength. A number of methods which have been reported appear to meet these requirements when applied to one or a number of plants, animals or micro-organisms. However, none of them are universally applicable, due to a number of tissue and species specific differences. For example, variations in cell or organelle structure dictate different methods of disruption. Differences between tissues in the pH optima and inhibitor sensitivity of their nucleases necessitate procedures adapted to each material which minimize enzymic breakdown. Other variables of which a satisfactory method must take account include the resistance to denaturation of the RNA to be recovered and the nature of the principal contaminants. As a guide to the isolation of RNA from tissues for which no satisfactory procedure has been reported, a number of methods of cell disruption, nucleoprotein dissociation and RNA purification are described and evaluated.
The RNA molecules are stable under physiological conditions, and can be broken down in vivo only by enzymatic hydrolysis. All RNA molecules are subject to hydrolytic breakdown; this makes their turnover possible. The hydrolytic breakdown of RNA, in addition to the regulation of RNA synthesis at the transcriptional level, plays a major role in the selection of those molecular
Table 1
Classification of RNA-splitting enzymes
species of RNA which are needed by the cell at a given moment of its development. Therefore, a thorough knowledge of the RNA-splitting enzymes in pivotal for the understanding of a number of regulatory and developmental processes in which RNA molecules are involved.
Development involves the spatial and temporal, selective regulation of gene expression and thus information about the photocontrol of gene expression is of central importance to our understanding of the cellular transduction of environmental stimuli leading to the initiation of developmental change during photomorphogenesis. The aim of this chapter is to review from a biochemical point of view our current understanding of the photocontrol of gene expression, paying particular attention to those systems where the underlying molecular mechanisms have been elucidated. Hence the treatment is not comprehensive but rather represents a critical consideration of selected experimental results and theoretical concepts currently under intense investigation and debate. In the last few years there have been a number of reviews related to the present topic and the reader is referred to these for historical details (Mohr 1974, Schopfer 1977, Zucker 1972).
The DNA dependent RNA Polymerase(EC 2.7.7.6) responsible for the synthesis of RNA catalyzes the formation of phosphodiester bonds using the four ribonucleoside triphosphates as substrate and DNA as template in the presence of a divalent metal ion. The RNA Polymeraseis a key enzyme implicated in the first step of gene expression and the control of transcription is of central importance during differentiation processes.
Light stimulates the accumulation of cytoplasmic and plastid rRNA in the cotyledons of mustard (Sinapis alba L.) seedlings. This effect is mediated by phytochrome. Continuous far red irradiation and brief red light pulses (the effect of which is reversible by brief far red light pulses) increase the amount of 1.3 x 10(6) + 0.7 x 10(6) and 1.1 x 10(6) + 0.56 x 10(6) molecular weight rRNA species. Large and small ribosomal subunit RNAs are maintained in a 1:1 molar ratio in both cytoplasm and plastids, irrespective of seedling age and light treatment. Continuous white fluorescent light (which saturates chlorophyll synthesis) is less effective than continuous far red light (essentially no chlorophyll synthesis) in producing the response, indicating that the accumulation of plastid rRNA is independent of the build-up of a functional photosynthetic apparatus. We conclude that the transcription of cytoplasmic and plastid rRNA cistrons is under phytochrome control.
Unrolling of etiolated wheat leaf segments is stimulated by short periods of exposure to red light. Both gibberellic acid and kinetin will stimulate unrolling in the dark, whereas abscisic acid (ABA) inhibits the unrolling response to these two hormones and to red light. Exposure to 5 minutes of red light leads to a rapid increase in endogenous gibberellin levels in etiolated wheat leaves, and this increase is followed by a rapid decline. Pre-treatment with ABA inhibits the increase in gibberellin levels in response to red light, but the ihibitory effect of ABA on unrolling cannot be ascribed only to its effect on gibberellin levels. Pre-treatment with red light reduces the lag-phase in chlorophyll development when wheat leaf segments are subsequently exposed to white light; the effect of red light may be replaced by pre-treatment with kinetin, but gibberellic acid is relatively ineffective in this respect.
A rapid and sensitive method has been described for the measurement of amino acid incorporation in cell-free systems. Samples of a reaction mixture were acid-precipitated on filter-paper disks, washed and extracted to remove unincorporated radioactivity, and then counted directly in a liquid scintillation spectrometer. The method has been applied to both C14- and H3-labeled proteins.
1. A DNA-dependent RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) has been purified 90-fold from young etiolated maize seedlings. The enzyme has been partially purified from the supernatant fraction by the standard enzymological techniques of ultracentrifugation, salt fractionation and adsorption chromatography.2. The RNA polymerase from maize requires the simultaneous presence of 4 ribonucleoside triphosphates for the incorporation of any ribonucleotide into acid-insoluble product. Both native and heat-denatured DNA satisfy the template requirement, whereas RNA does not.3. The product formed is an oligonucleotide having a base composition complementary with that of the template DNA.
A number of morphological and biochemical changes occur in the leaves of most higher plants after dark-grown shoots are exposed to light. One characteristic effect is a relatively rapid and extensive increase in total leaf protein (De Deken-Grenson, 1954; Mego and Jagendorf, 1961). This change is accompanied by the development of a number of enzymatic activities not found in the etiolated leaf (Marcus, 1960; Tolbert and Gailey, 1955; and Keister, Jagendorf, and San Pietro, 1962). Because this effect of light represents a unique and presumably specific regulation of protein synthesis, we have attempted to study the phenomenon further . This preliminary report is concerned with the effect of light on dark-grown seedlings as reflected by the amino acid incorporating activity of subsequently prepared ribosomes.
The amino acid incorporating system isolated from maize seedlings has been stabilized and its ability to incorporate labeled leucine enhanced 10-fold. By treatment of the microsomal pellet with sodium deoxycholate, a reproducible and stable ribosomal component is obtained routinely. A moderately stable supernatant component is prepared from a concentrated homogenate of the maize plumules. The increased activity and stability of these components correlate with a decrease in endogenous levels of ribonuclease (EC 2.7.7.16) present in both components. No net increase in protein or formation of labeled soluble protein has been demonstrated.
Preparations from cotyledons of unimbibed peanut seed show a low level of amino acid incorporation into protein. Similar preparations from imbibed seed exhibit increased activity. RNA extracted from germinating peanut cotyledons and then eluted after ribosomal RNA on the methylated albumin-kieselguhr (MAK) column had the highest template activity. This fraction stimulates amino acid incorporation into protein in an in vitro system. Isolated messenger RNA binds to monoribosomes obtained from dry seed and the polyribosomes thus produced are active in protein synthesis. It is suggested from these results that native mRNA from peanut cotyledons forms a stable complex with monoribosomes in the presence of Mg2+. The reaction does not require an ATP energy source or 105 000 ×g supernatant enzymes.
1.1. An adaptation of the phenol procedure is described for the isolation of relatively undegraded ribonucleic acid from plant tissue, which is also useful for the isolation of ribonucleic acid from bacterial and animal cells. The novel aspects that make the isolation procedure feasible is the use of a basic buffer (pH 9.5) in the presence of versene. Ribonucleic acid was isolated from white potato tubers, the leaves, stems and roots of etiolated peas, Escherichia coli K-12, rat fat pads, ribosomes from human reticulocytes, rabbit reticulocytes and rabbit liver.2.2. The preparations, analyzed by sucrose-gradient centrifugation, revealed that the two ribosomal RNA fractions of pea seedlings were present in equimolar concentrations and had molecular weights of 1.25 · 106 and 5.7 · 105 estimated from sedimentation coefficients of 24.5 S and 16 S.3.3. The molar ratios (cytidine:adenine:guanine:uridine) of the 25 S, 16 S, and 4 S RNA of pea seedlings were 22.7:23.6:32.1:21.5, 20.1:23.7:31.1:25.2, and 25.5:20.8:32.4:21.2, respectively.4.4. Pea seedling leaves, which have a higher metabolic activity than stems and roots of the same tissue, also have a higher content of soluble ribonucleic acid (% of the total ribonucleic acid).
The amino acid incorporating activity in vitro of ribosomes prepared from dark-grown plants is stimulated up to 200 % following exposure of the seedlings to low levels of light, and the percentage of polyribosomes is increased following this treatment. Red light is most effective in promoting this response. Polyuridylic acid (poly (U)) strongly enhances the incorporation of phenylalanine by ribosomes from both treated and control plants, and a portion of the observed stimulation by light is retained in the presence of an excess of this synthetic messenger.
Chloroplast RNA is synthesized in dark-grown radish cotyledons at about one-third the rate of that in the light. The synthesis, however, continues for longer in the dark and the percentage of chloroplast RNA can approach that in light-grown tissue. Light stimulates the synthesis and accumulation of both cytoplasmic and chloroplast RNA, but shows a 4-fold greater stimulation of the chloroplast RNA. Chloramphenicol, streptomycin and cycloheximide inhibit the synthesis of chloroplast RNA with little effect on cytoplasmic RNA. 5-Fluorouracil inhibits the synthesis of cytoplasmic more than chloroplast RNA. Synthesis of the 0.56 x 10(6) mol wt chloroplast RNA is inhibited much less than the other ribosomal RNA components by actinomycin D.
The effects of several base analogues and cycloheximide on RNA synthesis, protein synthesis, and cell elongation were studied in excised soybean hypocotyl. None of the pyrimidine analogues tested affected growth or protein synthesis; only 5-fluorouracil appreciably inhibited RNA synthesis. 8-Azaguanine and 6-methylpurine markedly inhibited RNA and protein synthesis and cell elongation. Cycloheximide effectively inhibited both cell elongation and protein synthesis.
The results show that 5-fluorouracil selectively inhibited ribosomal and soluble RNA synthesis without affecting the synthesis of D-RNA. These results indicate that the requirement for RNA synthesis to support continued protein synthesis and cell elongation is restricted to the synthesis of D-RNA.
5-Fluorouracil was incorporated into all classes of RNA in a form believed to be 5-fluorouridylic acid.
Cycloheximide markedly inhibited the accumulation of ribosomal RNA, but the results indicate that CH did not inhibit, per se, the synthesis of ribosomal RNA. The accumulation of newly synthesized D-RNA was only slightly affected by cycloheximide. These results show that the inhibition of cell elongation by cycloheximide correlates with the inhibition of protein synthesis, but not with the effect on RNA metabolism.
Gel electrophoresis of ribosomal RNA from green plant tissues provides evidence consistent with the suggestion that chloroplasts have evolved from symbiotic blue-green algae, while the plant cytoplasmic RNA is distinct from both the bacterial and mammalian.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
The kinetics of development of protein-synthesizing capacity in the imbibing wheat embryo, were studied both in vivo and in vitro. During the first 30 minutes of imbibition protein-synthesizing capacity rises rapidly, lagging about 10 minutes behind water uptake. This rise in synthesizing capacity is accompanied by an increase in polysome content. As imbibition continues, both protein-synthesizing capacity and polysome content increase. With embryos from aged seed, the rate of protein synthesis is initially limited by another, presumably nonribosomal, reaction.
The chloroplast ribosomal-RNAs (1.1 x 10(6) and 0.56 x 10(6) mol wt) are synthesized in the normal ratio of 2:1. The non-ribosomal distribution observed after extraction and fractionation results from the lability of the 1.1 x 10(6) component, and a correction for this breakdown can be applied in certain cases. Newly synthesized 1.1 x 10(6) RNA is more stable than the older accumulated 1.1 x 10(6) RNA. Accumulation of the chloroplast RNA during growth of radish cotyledons occurs at a later time than the accumulation of cytoplasmic RNA, and its turnover is much less than that of the cytoplasmic ribosomal-RNA.
The influence of light, gibberellic acid, and abscisic acid on unrolling of etiolated barley leaf segments was investigated. Gibberellic acid stimulated unrolling of both illuminated and nonilluminated leaf segments. In contrast, abscisic acid prevented light-stimulated unrolling and abolished the slight unrolling of segments maintained in the dark.Analysis of the time course of photoinduced unrolling showed a lag period of 6 hours before the onset of rapid leaf unrolling. The application of gibberellic acid to illuminated segments shortened this lag phase. Also, incubating the segments in buffer in darkness prior to illumination eliminated the lag period. Inhibitors of RNA and protein synthesis prevented light- and gibberellic acid induced unrolling, indicating a requirement for macromolecular synthesis. The possibilities that the lag period of unrolling following illumination represents the time required for the removal of endogeneous inhibitor or the synthesis of essential macromolecules are discussed.
Diversity of RNA components in green plant tissues Protein measurements with the Folin phenol reagent
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An increase in RNA polymerase activity after illumination of dark-grown maize seedlings Stimulation of an in vitro amino acid incorporating system by illumination of dark-grown plants Ribosome changes following illumina-tion of dark-grown plants
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STOUT, E. R., R. PARENTI, AND R. J. MANS. 1967. An increase in RNA polymerase activity after illumination of dark-grown maize seedlings. Biochem. Biophys. Res. Commun. 29: 322-326. 24. WILUAMS, G. AND G. D. NOVELLI. 1964. Stimulation of an in vitro amino acid incorporating system by illumination of dark-grown plants. Biochem. Biophys. Res. Commun. 17: 23-27. 25. WrLLLus, G. R. AND G. D. NOVELLI. 1968. Ribosome changes following illumina-tion of dark-grown plants. Biochim. Biophys. Acta 155: 183-195. 319
RNA metabolism in chloroplasts
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Uber Entfeltung und Einrollen eines mesophilen Grasblattes
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Chloroplast structure and development
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York. pp. 191-210.
Stimulation of an in vitro amino acid incorporating system by illumination of dark-grown plants
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