Etiolated Wheat Seedlings Research Articles

N6-Methyladenine in mitochondrial DNA of higher plants

After incubation of etiolated wheat seedlings in the presence of [8- 14C]adenine the radioactive N 6-methyladenine (m 6A) has been detected in the newly synthesized mitochondrial DNA (mtDNA) (ML = 100·m 6A/(m 6A+A)=0.4–0.6). This DNA is a low molecular mass (7.7 S) fraction of the mtDNA population. The detection of N 6-methyladenine in the mtDNA of wheat seedlings indicates the presence of adenine DNA methylase in mitochondria of higher plants. The presence of m 6A in plant mtDNAs makes them distinct from animal mtDNAs in which, as we know, this additional base has not been found.

The Effect of Light and Phytochrome on 1-Aminocyclopropane-1-Carboxylic Acid Metabolism in Etiolated Wheat Seedling Leaves

While light-grown wheat leaves produced ethylene at a low rate of <0.1 nanomoles per gram per hour and contained 1-aminocyclopropane-1-carboxylic acid (ACC) at low levels of <2.5 nanomoles per gram, etiolated wheat leaves produced ethylene at a rate of 2 nanomoles per gram per hour and accumulated concentrations of ACC at levels of 40 nanomoles per gram. Upon illumination of 8-day-old etiolated wheat seedlings with white light, the ethylene production rate increased initially, due to the activation of ethylene-forming activity, but subsequently declined to a low level (0.1 nanomoles per gram per hour) at the end of the 6-hour illumination. This light-induced decline in ethylene production rate resulted from a decline (more than 35 nanomoles per gram) in ACC level, which was accompanied by a corresponding increase in 1-(malonylamino)cyclopropane-1-carboxylic acid content. These data indicate that illumination promoted ACC malonylation, resulting in reduced ACC level and consequently reduced ethylene production. However, light did not cause any significant increase in the extractable ACC-malonyltransferase activity. The effect of continuous white light on promotion of ACC malonylation was also observed in intermittent white light or red light. A far-red light treatment following red light partially reversed the red light effect, indicating that phytochrome participates in the promotion of ACC malonylation.

Characterization of the mode of action of the experimental herbicide LS 82–556: [( S)3- N-(Methylbenzyl)carbamoyl-5-propionyl-2,6-lutidine

The herbicidal activity of the pyridine derivative LS 82–556 [( S)3- N-(methylbenzyl)carbamoyl-5-propionyl-2,6-lutidine] has been studied on green or etiolated seedlings of cucumber ( Cucumis sativus L. cv Vert Long Maraîcher), wheat ( Triticum aestivum L. cv Fidel), and maize ( Zea mays L. cv Monclair). The symptoms were strictly light dependent, and consisted of bleaching, wilting, and desiccation of leaves and stems. In green seedlings pretreated for 24 hr in the dark with 10 μ M LS 82–556, increases in ethane evolution and thiobarbituric acid-reacting material content could be detected after a 3-hr exposure to high light intensity (400 μE m −2 sec −1 photosynthetically active radiation). Similar increases were also detected in etiolated seedlings. Electron microscope observations of treated tissues exposed to light for 6 hr or more revealed structural damage at the level of cellular membranes (chloroplast envelope, tonoplast, and plasma membrane). In cotyledons or leaves grown in the light, activity of LS 82–556 required the presence of chloroplastic pigments. All the above results suggest that as for diphenyl ether herbicides, the mechanism of action of LS 82–556 involves photooxidative reactions leading to the degradation of fatty acids. A further analogy between the two types of herbicide action comes from their similar requirement for an operating photosynthetic electron flow when they are administered to green tissues.

Compartmentation of chlorophyll, phytol, and carotenoid syntheses in greening leaves of etiolated wheat seedlings (Triticum aestivum L.)

Compartmentation of chlorophyll, phytol, and carotenoid syntheses in greening leaves of etiolated wheat seedlings (Triticum aestivum L.)

Functional aspects of carotenoid accumulation in greening leaves of etiolated wheat seedlings (Triticum aestivum L.)

Functional aspects of carotenoid accumulation in greening leaves of etiolated wheat seedlings (Triticum aestivum L.)

Some Properties and Surface Localization of a Mg2+-ATPase in Microsomal Vesicles of Wheat Seedlings

Summary Microsomal membranes isolated from etiolated wheat seedlings were centrifuged on a discontinuous sucrose density gradient. A band of ionophore-stimulated and nitrate-sensitive ATPase activity was found in low density peak (1.09-1.11 g · ccm-1). There is an evident correlation between the degree of fluorescence quench and ATPase activity, proving that this enzyme acts as a proton pump. The enzyme (opt. ρH 6.2-6.8) preferentialy hydrolysed ATP and was strongly stimulated by gramicidin and nigericin. It was inhibited by DES, DCCD, Dio-9 and KN0 3 and to a slight extent by vanadate, oligomycin and sodium azide. With the use of affinity chromatography on a column with lectin-Sepharose 6 MB it was demonstrated that membrane vesicles are oriented right-side-out, i.e. that their cytoplasmic side is turned outside. Activity measurement of ATPase of microsomal vesicles digested with trypsin in the absence or presence of DOC, as well as upon use of Ca 2+ -ions and ATP (protecting the catalytic centre) permit the assumption that either the whole ATPase molecule or only its catalytic centre occurs on the extracytoplasmic side of microsomal membranes.

Auxin Binding and Proton Translocating ATPase in Microsomal Membranes from Wheat Seedlings

The microsomal vesicles of etiolated wheat seedlings exhibit an IAA-stimulated ability of quinacrine fluorescence quench, as an effect of proton transfer through membranes. There is an evident correlation between the degree of fluorescence quench and ATPase activity, proving that this enzyme acts as a proton pump. Microsomal membranes contain auxin-binding sites. Agarose chromatography of 14C-IAA-labelled extracts of microsomal membranes shows that this hormone does not bind with ATPase, whereas relatively high radioactivity is found in case of acid phosphatase. These results strongly suggest, that in wheat microsomes, the acid phosphatase is an auxin receptor.

Cyanide-insensitive and Cyanide-sensitive O2 Uptake in Wheat

Enriched mitochondrial fractions isolated from durum wheat seedlings via differential centrifugation exhibited classical cyanide- or antimycin A-insensitive O(2) uptake which was inhibited by either salicylhydroxamic acid or propyl gallate. Further purification of this fraction using Percoll density gradients resulted in two discrete bands which were essentially homogeneous mitochondrial populations, as verified by electron microscopy. Respiratory O(2) uptake in these two fractions was completely inhibited by cyanide or antimycin A. Addition of linoleic acid to a third-step gradient band, which was shown to contain virtually no mitochondria, resulted in demonstrable cyanide-insensitive O(2) uptake. This O(2) consumption was completely inhibited by propyl gallate or salicylhydroxamic acid, two known lipoxygenase inhibitors. In contrast, addition of linoleic acid to the two purified mitochondrial fractions did not stimulate O(2) uptake. These data indicate that lipoxygenase oxygenation, the enzyme physically separable from the mitochondria, is responsible for the cyanide-insensitive component of O(2) uptake that was observed in subcellular fractions isolated from etiolated wheat seedlings.

Influence of Cell Age on Chlorophyll Formation in Light-grown and Etiolated Wheat Seedlings

A method is described for relating the age of a cereal leaf cell to its distance from the leaf base. The rates of chlorophyll synthesis per plastid in the first leaf of light-grown and of greening etiolated seedlings of wheat (Triticum aestivum, var. Maris Dove) increase with cell age. Normally developing plastids of light-grown wheat take over 24 hours to reach the chlorophyll a/b ratio characteristic of mature wheat chloroplasts (4.5), but mature etioplasts need only 8 hours light to achieve this a/b ratio. Plastid greening potential depends only on cell age, whereas the chlorophyll a/b ratio is influenced both by cell age and by light.

Effect of light on the nucleotide composition of rRNA of wheat seedlings

Both qualitative and quantitative differences in the minor nucleotide constituents of rRNA from normally grown and from etiolated wheat plants (Triticum aestivum L.) were established. Using different degradation methods and separation techniques the 18S+26S RNA of 8-day-old wheat seedlings grown in the light was found to contain 5-methylcytidine, 3-methylcytidine, 5-methyluridine, 3-methyluridine, 5-carboxymethyluridine, 1-methyladenine, N-methyladenine, 5-hydroxymethylcytidine, O(2')-methyluridine, O(2')-methylcytidine, pseudouridine, O(2')-methylpseudouridine, N(2),N(2)-dimethylguanine, 1-methylguanine, ribothymidine and some unknown minor constituents. On the other hand, there were only a few minor nucleotides in the rRNA of etiolated wheat seedlings. Cycloheximide, a cytoplasmic protein synthesis inhibitor, simulated etiolation in that it reduced the number of minor nucleotides in rRNA, whereas chloramphenicol, a chloroplast protein synthesis inhibitor, had no significant effect on the minor nucleotide content of rRNA. This finding suggests that illumination may cause de novo synthesis of cytoplasmic modifying enzymes leading to the formation of highly modified rRNAs.

Hemmung der Phytol-Akkumulation mit Herbiziden. Geranylgeraniol-und Dihydrogeranylgeraniol-haltiges Chlorophyll aus Weizenkeimlingen

The accumulation of carotenoids, phytol and chlorophyll in etiolated wheat seedlings during exposure to white light was determined after treatment with the herbicides amitrol'l), Sandoz 6706 and Sandoz 9789. The Sandoz herbicides inhibit the accumulation of carotenoid, phytol, and chlorophyll to the same extent. The accumulation of chlorophyll is, however, more strongly inhibited by the action of amitrol than that of carotenoids and less strongly than that of phytol. The main carotene to be detected after amitrol inhibition was lycopene. Amitrol inhibition leads to the accumulation of new chlorophyll species (isolated as pheophytins) which contain all-trans-geranylgeraniol and dihydrogeranylgeraniol instead of phytol. The alcohols were isolated after saponification of the corresponding pheophytin fractions and identified by thin layer and gas chromatography and mass spectrometry. The new chlorophylls are considered to be intermediates in chlorophyll biosynthesis.

Identification of the small subunit of ribulose 1,5-bisphosphate carboxylase as a product of wheat leaf cytoplasmic ribosomes

Polyribosomes from greening wheat seedlings ( Triticum vulgaris) were allowed to incorporate [ 3H]leucine into proteins in the presence of a wheat germ supernatant fraction under conditions permitting the completion and release of polypeptides by cytoplasmic polyribosomes. The released proteins were analyzed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Discrete proteins, as well as a variety of poorly resolved proteins, were observed to have been labeled. The molecular weight distribution of the labeled proteins correlated well with the distribution of polyribosome size classes present in the samples. Neither the large nor the small subunit of ribulosebisphosphate carboxylase were detected as labeled peaks by this procedure. Immune precipitates formed by the addition of carrier small subunit, detergent, and anti-small subunit serum to the released proteins contained a substantial proportion of nonspecifically precipitated material resembling the population of released proteins, but they also contained two discrete peaks not resolved previously, one having mobility slightly faster than the light chain of immunoglobulin (20,000 daltons) and the other having mobility identical to that of small subunit carrier (ca. 12,000 daltons). Samples containing the latter material were shown to contain labeled tryptic peptides corresponding to those of the small subunit carrier. The results establish that the small subunit of ribulose bisphosphate carboxylase is the product of a small proportion of cytoplasmic polyribosomes during greening of etiolated wheat seedlings.

APPEARANCE AND DEVELOPMENT OF PHOTOSYNTHETIC ACTIVITIES IN WHEAT ETIOPLASTS GREENED UNDER CONTINUOUS OR INTERMITTENT LIGHT—EVIDENCE FOR WATER‐SIDE PHOTOSYSTEM II DEFICIENCY AFTER GREENING UNDER INTERMITTENT LIGHT*

Abstract— Etiolated wheat seedlings are greened under continuous or intermittent light. Under continuous light the onset of photosystem II (PS II) activity appears after 4 h of illumination. Under intermittent light (1 ms flashes alternating with 15 min dark periods), PS II activity cannot be detected after 300–400 flashes, although the pigment composition and structural development of these plastids are similar to those observed after 4 h of continuous light. However, the appearance of PS II activity in isolated plastids can be observed in two different ways: (1) in vivo by exposing the seedlings to a short period of continuous light after the intermittent light; or (2) in vitro by addition to the isolated plastids of an electron donor for PS II, such as diphenylcarbazide. It is concluded that the intermittent light induces development of the electron transport chain from PS II to PS I, but that a deficiency occurs on the water‐side of PS II.

Effect of various gas atmospheres upon the greening of etiolated seedlings

Summary The greening response of etiolated wheat seedlings upon exposure to light has been studied in atmospheres of various gases and gas mixtures. In the presence of high levels (> 95 %) of oxygen, nitrogen, hydrogen or helium, greening occurs normally, and there is a requirement for small amounts of oxygen. Greening is completely inhibited, however, by high levels of carbon dioxide, methane, or ethylene, and is partially inhibited by carbon monoxide. The inhibition of greening by carbon dioxide is due in part to pH, and in part to interference with one or more of the decarboxylation reactions of chlorophyll biosynthesis. Experiments with cyanide, azide, and dinitrophenol demonstrate the requirement for metabolic energy derived from respiration and oxidative phosphorylation.

The reduction of vinyl side-chains of Mg-protoporphyrin IX monomethyl ester in vitro

Portions of crude homogenates of etiolated wheat seedlings incubated with Mg-protoporphyrin IX and S- adenosyl- L- methionine and then added to other portions of the same crude homogenates that were pretreated with [1- 3H]ethanol and yeast alcohol dehydrogenase provided, after a short reaction period, 3H-labeled Mg-protoporphyrin IX monomethyl ester. The 3H-labeled Mg-protoporphyrin IX monomethyl ester thus obtained was shown to contain the 3H in one reduced (to ethyl) vinyl side-chain. Subsequently, 3H-labeled Mg-monoethyl-(monodivinyl)-protoporphyrin IX monomethyl ester was obtained when Mg-protoporphyrin IX monomethyl ester and [ 3H]NADH were added to dialyzed crude homogenates of etiolated wheat seedlings. Insignificant amounts of 3H were incorporated into poprhyrin substrates when Mg-2,4-divinylpheoporphyrin a 5 or [ 3H]NADPH were substituted in reaction mixtures for Mg-protoporphyrin IX monomethyl ester or [ 3H]NADPH, respectively. The results of these and further experiments suggest that an NADPH-dependent enzyme in the crude homogenates of etiolated wheat seedlings was capable of catalyzing the reduction to ethyl of one vinyl side-chain of Mg-protoporphyrin IX monomethyl ester. These findings suggest that the 4-vinyl side-chain reductive reaction likely occurs after the biosynthesis IX monomethyl ester, but before isocyclic ring formation in the pathway to chlorophyll a.

Activity and properties of (−)- S-adenosyl- l-methionine:magnesium-protoporphyrin IX methyltransferase in crude homogenates from wheat seedlings

A direct, rapid assay for the enzyme S-adenosylmethionine:magnesium-protoporphyrin IX methyltransferase (EC 2.1.1.11) is presented. Using the assay developed, the effect of temperature and pH on the activity of the enzyme in a crude homogenate of etiolated wheat seedlings was studied. The pH optimum was found to be 7.7 using phosphate buffer; the enzyme exhibited maximum activity at 37 °C although activity was found to be nearly optimal at 25 °C. The K m (pH 7.7, 25 °C) values determined for Mg-protoporphyrin IX and S-adenosylmethionine were 36 and 48 μM, respectively. Treatment of the crude preparation from etiolated wheat with n-butanol to remove endogenous pigments did not result in a decrease in specific activity of the preparation. A crude preparation from green wheat seedlings when treated with butanol exhibited an activity comparable with that of the etiolated wheat preparation so treated thereby demonstrating that this assay could be used in green plant systems.

Uptake of labeled glucose by root tips from etiolated wheat seedlings

Wheat root tips from germinatyed seedlings were incubated in labeled D-glucose-U- L- 14C for periods up to 30 min. Uptake of activity into glucose-1-phosphate, glucose-6-phosphate, sucrose and UDP-glucose was monitored. Extremely low, but significant, activities were found in ADP-glucose and GDP-glucose. A Golgi fraction was isolated. Activity was found in a particulate form but it could be solubilized in mild base or acid. Sephadex chromatography of the base solubilized material demonstrated that it had high mol. wt. Acid hydrolysis yielded mainly radioactive xylose, arabinose, glucose, galactose, with lesser quantities of mannose and uronic acids.

Evidence for the Presence of Protochlorophyllase in Etiolated Wheat Seedlings

The biosynthetic pathway to chlorophyll a (chl a) is now quite complete in terms of porphyrin intermediates (3, 8). The biosynthetic pathway to protochlorophyll a (Pchl a) is undoubtedly identical with that of chl a to the protochlorophyllide a (Pchlide a) step, however, the sequence of steps from Pchlide a to Pchl a still remains unknown (1, 9). The production of Pchl a by etiolated plants (devoid of chl a and b) can be explained most simply as an oxidation of chl a (9) or a phytylation of Pchlide a (1,9) by an esterase enzyme (e.g. protochlorophyllase). As the formation of chlorophyllide a (chlide a) requires light, the former pathway seems unlikely in etiolated plants, and the latter becomes the simplest hypothesis, namely, that an esterase catalyzes the formation of Pchl a from Pchlide a.

Carotenoid Synthesis in Leaves of Etiolated Wheat Seedlings after Varying Light and Dark Treatments

AbstractA short impulse of red light has a varying effect on carotenoid synthesis in dark‐grown wheat seedlings. Except for β‐carotene, which remains unchanged in the dark (it increases in continuous light), the carotenoid synthesis shows the same tendency as during constant irradiation. Thus, lutein and neoxanthin slowly increases, while violaxanthin decreases. During a period of constant light following various periods of darkness after the short impulse of light, the pigment changes correspond to those occurring in the dark, but are much more pronounced. The changes are discussed on the basis of phytochrome action.

A colorimetric test for gibberellic acid and evidence from a dwarf pea assay for the occurrence of a gibberellin-like substance in wheat seedlings.

A RECENT communication by McComb and Carr1, reporting the use of a dwarf pea bioassay for naturally occurring gibberellins, prompts this report of some results of an experiment designed to investigate whether gibberellic acid occurs naturally in etiolated wheat seedlings.