The 2003 article by Li et al. identified subunits of the plant Arp2/3 complex and functionally characterized three of these subunits. The foundation laid by this article aided in the identification of Arp2/3 regulatory proteins, which suggests that plant Arp2/3 may be regulated in a similar manner to animal systems. In addition, this article furthered the understanding of polarity establishment in fucoid zygotes.
The influence of indoleacetic acid, 0.03% CO(2), and malate on protein metabolism of etiolated Avena sativa coleoptile sections has been investigated. All three were found to elevate both the rate of incorporation of labeled leucine into protein, and the level of soluble protein. The combination of indoleacetic acid and CO(2) stimulated these values in an additive or weakly synergistic manner, in contrast to the nonadditive influence of malate and CO(2). Evidence is presented that cyclo-heximide inhibited the stimulation of protein synthesis by CO(2), and that indoleacetic acid increased the incorporation of (14)C-bicarbonate into protein. These data are discussed in the context of CO(2)-stimulated growth of etiolated tissue, and proposals that CO(2)-stimulated growth involves dark CO(2) fixation.
A procedure for isolating spinach (Spinacia oleracea L.) leaf peroxisomes in 0.25 molar sucrose solution by Percoll density gradient centrifugation followed by removal of the Percoll by washing and centrifugation was established. The preparation contains more than 90% peroxisomes as intact organelles with no detectable chlorophyll or cytochrome oxidase contamination. The peroxisomes are stable at 0 to 4 degrees C or 25 degrees C for at least 2 hours.
The number of nodules produced per clover seedling inoculated with Rhizobium trifolii 0403 can be increased almost 2-fold by the addition of penicillin or mecillinam. Two-day-old dutch white clover seedlings grown in 250 milliliter boston round jars containing agar-solidified plant growth medium were inoculated with exponentially growing Rhizobium trifolii 0403 cells. Penicillin or mecillinam (100 micrograms per milliliter) were added immediately or after 24 hours. Following 42 days growth, 10 replicate sets of 5 plants for each treatment were assayed for nodule number, plant dry weight, and Kjeldahl nitrogen. Both antibiotics increased nodule number, plant dry weight, and Kjeldahl nitrogen. Increases in nodule number and dry weight were statistically significant. The range of values in Kjeldahl nitrogen was so extensive as to make the data insignificant at the P < 0.05 level, however nodule number, plant dry weight, and Kjeldahl nitrogen displayed a significant correlation with each other. There were no significant differences in treatment with either antibiotic or with time of treatment. Nodule number increased by about 85%, and plant dry weight and nitrogen increased by about 30%.
The genomic response to low levels of nitrate was studied in Arabidopsis using the Affymetrix ATH1 chip containing more than 22,500 probe sets. Arabidopsis plants were grown hydroponically in sterile liquid culture on ammonium as the sole source of nitrogen for 10 d, then treated with 250 microm nitrate for 20 min. The response to nitrate was much stronger in roots (1,176 genes showing increased or decreased mRNA levels) than in shoots (183 responding genes). In addition to known nitrate-responsive genes (e.g. those encoding nitrate transporters, nitrate reductase, nitrite reductase, ferredoxin reductase, and enzymes in the pentose phosphate pathway), genes encoding novel metabolic and potential regulatory proteins were found. These genes encode enzymes in glycolysis (glucose-6-phosphate isomerase and phosphoglycerate mutase), in trehalose-6-P metabolism (trehalose-6-P synthase and trehalose-6-P phosphatase), in iron transport/metabolism (nicotianamine synthase), and in sulfate uptake/reduction. In many cases, only a few select genes out of several in small gene families were induced by nitrate. These results show that the effect of nitrate on gene expression is substantial (affecting almost 10% of the genes with detectable mRNA levels) yet selective and affects many genes involved in carbon and nutrient metabolism.
In the red alga Porphyra perforata, the level of chlorophyll fluorescence in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) decreased during illumination of the thallus. The results showed that: (a) this decay was related to the photooxidative activity of photosystem I; (b) Q, the primary electron acceptor of photosystem II, became oxidized during the decay of the fluorescence; (c) reagents which inhibit the back reaction of photosystem II inhibited the decay.From these results, it is suggested that, when conditions in the chloroplasts of this red alga become too oxidative, excess light energy can be converted to heat as a result of an accelerated back reaction of photosystem II. This may be one of the mechanisms by which this alga can cope with the high salt and high light conditions that can occur in its natural habitat.
Illuminated pea (Pisum sativum) chloroplasts can convert glutamate to glutamine using ATP generated by photophosphorylation to drive the glutamine-synthetase reaction. Light-dependent glutamine synthesis is sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), but only at concentrations higher than are necessary to suppress photoreduction of ferricyanide or phosphoglycerate. Conversely, glutamine synthesis is far more sensitive to antimycin A than is photoconversion of phosphoglycerate to triosephosphate. When 3.8 mm phosphoglycerate is supplied, glutamine synthesis is stimulated in both the presence and absence of antimycin A.These data seem to be consistent with the operation of an endogenous, DCMU-sensitive, phosphorylation process-possibly cyclic-which can support glutamine synthesis in white light under aerobic conditions. The stimulatory effect of phosphoglycerate suggests that noncyclic phosphorylation is initiated or accelerated when this substrate is supplied. This noncyclic process evidently provides ATP over and above the amount required for phosphoglycerate photoreduction, i.e. the ATP/e(2) ratio exceeds 1.0. The additional ATP produced under these conditions is available for glutamine synthesis and lessens its dependence on cyclically (or pseudocyclically) generated ATP.
The light-dependent quenching of the chlorophyll a fluorescence at room temperature by N-methylphenazinium methyl sulfate (PMS) was investigated with isolated chloroplasts inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Other investigators have considered this quenching to be a consequence of the formation of a high energy membrane state related to photophosphorylation.It was found that the fluorescence quenching was accompanied by a reversible bleaching of PMS which could not be attributed to its photo-reduction. Both fluorescence quenching and PMS bleaching, and their dark reversal, were similarly affected by the experimental conditions. In particular, they were only slightly sensitive to the presence of uncouplers of photophosphorylation. However, bleaching and fluorescence quenching were strongly inhibited by uncouplers when the creation of a proton gradient across the thylakoid membrane was made possible by a presence of some PMS in its reduced form.It is suggested that the bleaching of PMS resulted from its binding to the thylakoid membranes when charges became available during conformational changes as a consequence of the light reaction in photo-system I. The same conformational changes were apparently responsible for the fluorescence quenching, but a large pH gradient across the membranes was not essential.
When grown in medium containing dl-lactate at 27 C in the light, Euglena gracilis Z populations underwent modifications of the pigment system in response to 0.05 to 250 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU).Chlorophyll content dropped dramatically, the only remaining form being Chl a(673). Light-driven O(2) evolution was no longer detectable for the two highest DCMU concentrations tested. The energy-capture cross-section of detectable photosystem II units remained unchanged, although intersystem energy transfer no longer occurred. Euglena at this stage had chloroplast membranes destacked and swollen. A recovery phase then occurred, marked by enhanced photosynthetic properties. The initial forms of chlorophyll which were accumulated were highly efficient for O(2) evolution. The newly formed photosystem II antennae were connected and of small size. Finally, the third phase involved the recovery of photosynthetic capacity similar to that of the controls as the thylakoids regained their normal structures.Since these modifications occurred in the entire population and DCMU resistance persisted through successive cell generations, these adapted Euglena were considered to be a variant of the Z strain, designated ZR.
The cell cycle of the photosynthetic unicellular alga Euglena gracilis growing in phototrophic medium is regulated by light. To investigate the relationship of this cell cycle response to light stimulated photosynthesis, we have tested the effect of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on Euglena cell cycle transit. While DCMU does not block light stimulated cells from entering the S phase of the cell cycle, it does inhibit the transit through G(2)/M. The specificity of this response and its relationship to photosynthesis was studied by looking at the effect of DCMU on dark grown wild-type cells, and on two bleached variants of Euglena (W(3)BUL and W(10)BSmL) that lack chloroplasts. The drug does block G(2)/M in these cells, but not entrance into the cell cycle. Our studies show that entrance of cells into the cell cycle from a quiescent state does not require active photosynthesis, and that DCMU has effects on G(2)/M transit that are independent of the photosynthetic capacity of the cells.
The effects of light, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and ammonium ion on pool sizes of ATP were studied in Lemna paucicostata 6746 (wild type) and a photosynthetic mutant (strain 1073) with abnormal flowering responses. Wild type fronds were capable of endogenous and phenazine methosulfate-catalyzed cyclic photophosphorylation. The endogenous cyclic photophosphorylation was inhibited by DCMU. The mutant fronds showed little endogenous but appreciable rates of phenazine methosulfate-catalyzed cyclic photophosphorylation. Treatment with DCMU during prolonged exposure to light did not result in elevated levels of ATP. Ammonium ion in the medium did not inhibit light-induced increases in pool sizes of ATP. It is concluded that the previously reported effects on flowering of DCMU, the photosynthetic mutation or ammonium ion, were not due to altered pool sizes of ATP.
The light saturated rate of photosystem I-dependent electron transport (ascorbate/dichlorophenol-indophenol --> methyl vilogen in presence of 1 micromolar 3-[3,4-dichlorophenyl]-1,1-dimethyl urea [DCMU]) was increased by a high concentration of DCMU added to broken and uncoupled chloroplasts isolated from pea (Pisum sativum). At 50 micromolar DCMU, the increase was around 50%. No stimulation was observed under limiting intensity of illumination, indicating that the relative quantum yield of electron transport was not affected by high DCMU. The light-saturated rate in coupled (to proton gradient formation) chloroplasts was unchanged by 50 micromolar DCMU, suggesting that the rate-limitation imposed by energy coupling was not affected. Using N,N,N',N'-tetramethyl-p-phenylene diamine as electron donor, essentially no DCMU stimulation of the rate was observed, indicating further that the electron donation at a site close to P700 was not affected by high DCMU. It is concluded that DCMU, in the range of 10 to 50 micromolar, affected the thylakoid membranes in such a way that the rate constant of electron donation by dichlorophenol-indophenol at the site prior to the site of energy coupling increased. Further observations that DCMU at 100 micromolar stimulated the rate in coupled chloroplasts indicated an additional DCMU action, presumably by uncoupling the chloroplasts from phosphorylation, as suggested by Izawa (Shibata et al., eds, Comprehensive Biochemistry and Biophysics of Photosynthesis, University Press, State College, Pennsylvania, pp 140-147, 1968). A scheme has been proposed for multiple sites of DCMU action on the electron transport system in chloroplasts.
The possibility that photosynthetic competence is gratuitous for light-induced chloroplast development in Euglena gracilis var. bacillaris was examined by incubating dark-grown resting cells in the light with DCMU, an inhibitor of photosynthesis. Under these conditions photosynthetic carbon dioxide fixation was inhibited essentially completely at all times during chloroplast development, but about 70% of the chlorophyll was formed with essentially the same pattern of accumulation found for cells incubated in the absence of the inhibitor. Electron microscopy of cells incubated with DCMU in the light revealed the formation of morphologically recognizable chloroplasts having comparable overall dimensions and structural elements to those found in normally developed chloroplasts, but frequently lacking a readily detectable pyrenoid with paramylum sheaths, and often containing increased numbers of discs per lamella. Such abnormalities are considered minor since upon removal of DCMU by centrifugation, the cells usually regained almost full photosynthetic competence on a chlorophyll basis.It is concluded that photosynthetic competence is not necessary for chloroplast development in Euglena and supports the hypothesis, already suggested from other evidence, that light induction results in activation of synthetic machinery external to the developing chloroplast.
Cultures of Euglena gracilis Klebs strain Z Pringsheim were grown photoorganotrophically in the presence of different concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) in the range of 0.05 to 250 micromolar. Cultures were serially transferred and various metabolic parameters were followed for 10 weeks. A process of adaptation occurred which was divided operationally into three phases. A phase of ultrastructural disorganization occurred, succeeded by a recovery phase; their intensity and duration were functions of the dose of DCMU. A stable adaptation phase then ensued. This phase was observed in all cultures except that exposed to the highest DCMU concentration. Adapted cells from all of the DCMU cultures contained twice the protein and half the paramylon of the control cells and thus utilized the carbon source to accumulate cellular reserves with only half the efficiency of controls. DCMU affected cellular metabolism as well as photosynthesis.The energy charge remained at high levels throughout adaptation, although the size of the adenylate pool was half that of controls at the disorganized phase. At this stage the ultrastructure of chloroplasts and mitochondria was considerably modified. The progressive changes of the parameters studied appeared to affect all of the cells in a given culture.
A uniparentally inherited 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-resistant mutant of Chlamydomonas reinhardii, Dr2, which has a resistance mechanism of the type defined as ;primary,' has been isolated. In vitro Hill reactions catalyzed by isolated thylakoid membranes reveal a reduced apparent affinity of the thylakoids for DCMU. These changes in membrane properties quantitatively account for the resistance of mutant Dr2 to herbicide inhibition of growth. The properties of this mutant show that all of the Hill reaction-inhibiting DCMU binding sites are under identical genetic control. Mutant Dr2 is a useful new uniparental genetic marker, since it has a novel phenotype and it may be possible to identify its altered gene product. The low cross-resistance of Dr2 to atrazine suggests that there may be considerable flexibility in exploiting induced herbicide resistance of crop plants for improving herbicide specificity.Four mendelian mutants in at least three loci all have resistance mechanisms in the class we define as ;secondary.' They are as sensitive as wild type to in vitro inhibition of the Hill reaction, and must acquire resistance in vivo by preventing the active form of the herbicide from reaching the sensitive site.
The effects of cyanide and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on photosynthesis and respiration of intact chlorophyllic moss (Funaria hygrometrica) spore was investigated. Thirty micromolar cyanide strongly inhibited dark respiration, was without effect on photosynthesis at high light intensities (above the saturation plateau values), and stimulated photosynthesis at low light intensities (below the saturation plateau values). Three hundred nanomolar DCMU inhibited the photosynthesis and was without effect, even under light conditions, on the dark respiration. It seems likely, therefore, that in the chlorophyllic moss spore the cytochrome oxidase pathway is not functioning under high light intensities unless the photosynthesis is inhibited by DCMU.
The ultrastructure of light-grown Euglena gracilis var. bacillaris was examined by the techniques of thin sectioning and freeze-etching. Thin sectioning revealed the typical organelles previously observed in chemically fixed Euglena. In addition to confirming the observations on thin sections, the freeze-etch technique has revealed the presence in E. gracilis of a complex multilaminar pellicle, and an ordered arrangement to the paramylon granule. The chloroplast thylakoids are particulate and similar to those observed in higher plants.
3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) inhibition of (14)CO(2) fixation in isolated intact spinach (Spinacia oleracea L.) chloroplasts was reversed (by about 34%) by l-malate but not by oxaloacetate (OAA). However, OAA reversed the DCMU inhibition in spinach protoplasts indicating an extrachloroplastic enzyme requirement. Extrachloroplastic OAA reduction was coupled with external dihydroxyacetone phosphate (DHAP) oxidation, and the malate formed from such coupling might then enter the chloroplasts. Evidence was presented using ruptured protoplasts that the export of recently formed 3-phosphoglyceric acid (PGA) out of chloroplasts in exchange for external DHAP was reversed by excess OAA. The PGA/DHAP shuttle across the chloroplast envelope was found to be regulated by the external concentrations of DHAP and OAA.
The effects of DDT (2,2-bis-(p-chlorophenyl)-1, 1, 1-trichloroethane) on the growth of seven marine phytoplankters, representative of five algal divisions, were studied. At a concentration of 80 parts per billion (0.23 mum) DDT, growth of Dunaliella tertiolecta was unaffected, and there was slight, if any, influence on the development of Cyclotella nana, Thalassiosira fluviatilis, Amphidinium carteri, Coccolithus huxleyi, and Porphyridium sp. Skeletonema costatum exhibited a 9 day lag before cell division commenced, the rate of growth subsequently being the same as in the control (no DDT). A further inoculation of this culture of S. costatum into 80 parts per billion DDT gave another 9-day lag before initiation of normal growth.The ability of marine phytoplankton to metabolize DDT varied. DDE (2,2-bis-(p-chlorophenyl)-1, 1-dichloroethylene) was the only significant hexane-soluble metabolite detected. It occurred in cells of S. costatum, C. nana, T. fluviatilis and D. tertiolecta. Maximum degree of conversion was 7.5% and was based on the total DDT found in the cell-water system of 9-day D. tertiolecta cultures. The total amount of DDT recovered from cultures in 2- to 3-week experiments ranged from 63.5% for T. fluviatilis to 90.7% for S. costatum. The amount of DDT found associated with the cells, collected by centrifugation, in the cell-water system ranged from 70.8 to 99.5%.Chloroplast particles were isolated from a "resistant" species, D. tertiolecta. Noncyclic electron flow, as measured by ferricyanide reduction, was inhibited by DDT and DDE, and could explain growth inhibition in other phytoplankters. Fifty percent inhibition occurred at 20 mum DDT. Sensitivity of phytoplankton to toxic hydrophobic chlorinated hydrocarbons may be dependent upon penetration of the molecules to active sites within membranes.
Enrichment of the cell wall in hydroxyproline-rich glycoprotein is involved in the defense of muskmelon (Cucumis melo) seedlings to Colletotrichum lagenarium, the causative agent of anthracnose. The extent to which this accumulation proceeds may be experimentally modified by treating plants with ethylene or growing them in the presence of free l-trans-hydroxyproline. It appears that the increase in the wall hydroxyproline-rich glycoprotein mediated through ethylene is paralleled by an increasing resistance of the host to the pathogen. Inversely, inhibiting the synthesis of this glycoprotein in diseased plants is strictly correlated to an accelerated and more intense colonization of the host by the pathogen.In both cases, the inverse relationship between the accumulation of hydroxyproline-rich glycoproteins and the ability of the pathogen to develop in the host has been checked by the quantification, in infected tissues, of glucosamine, a characteristic component of chitin-containing fungi.
Male and female mating types of Chlamydomonas eugametos Moewus show an absolute light requirement for gametogenesis. Increasing light intensity from 0.3 to 1.2 mw cm(-2) during nitrogen starvation (a precondition for gametogenesis) caused an increase in gametogenesis throughout a 28-hour period. Gametogenesis was measured by determining the percentage of paired cells after a 1-hour mixing period. Light requirements for the male and female differed. There was a 9-hour lag period in gametogenesis in the male, but no lag in the female. Gametogenesis was reduced 50% in the female and 90% in the male when 6.0 mum 3-(3,4-dichlorophenyl)-1, 1-dimethyl-urea was in the N-starvation medium. Sodium acetate, 1.8 mm, in the N-starvation medium increased gametogenesis in both mating types and eliminated the 9-hour lag in the male for cells irradiated for 3, 6, 9, 12, 15, 18, or 23 hours during the last part of a 23-hour N-starvation period. Sodium acetate concentrations higher than 1.8 mm inhibited the mating process. 3-(3,4-Dichlorophenyl)-1, 1-dimethylurea inhibition of gametogenesis was decreased in the male but increased in the female, when sodium acetate was added to the N-starvation medium. These results indicate a nonphotosynthetic as well as a photosynthetic role for light in the gametogenesis of both mating types. Also, the male will not undergo gametogenesis unless a required amount of energy is provided either in the medium or through photosynthesis.
The synergistic inhibition of the growth of Marchantia polymorpha gemmalings by lysine and threonine and its prevention by methionine has been investigated utilizing (14)C-labeled amino acids. Experiments involving the uptake of (14)C-lysine or (14)C-threonine in the presence or absence of methionine indicated that the synergistic growth effects were not a result of altered amino acid uptake. These data, as well as direct chemical analysis, indicated that growth inhibition was correlated with an inhibition of protein synthesis. Experiments utilizing (14)C-aspartic acid revealed that the presence of lysine and threonine resulted in increased (14)CO(2) production and an accumulation of soluble (14)C-aspartic acid and labeled ninhydrin-positive compounds. These metabolic alterations were prevented when methionine was also included in the growth media. A model depicting a sequence of events which involve the interaction of regulatory mechanisms is suggested to account for the effects of specific amino acids on plant growth.
The utilization of 2,4-dichlorophenoxyacetic acid (2,4-D) molecules by Acer pseudoplatanus cells is governed mainly by a glucosylation process. Evidence that 2,4-D glucoside molecules are biologically inactive is presented. 2,3,5-Triiodobenzoic acid (TIBA), by inhibiting 2,4-D glucosylation, has a sparing effect on 2,4-D molecules; thus TIBA treatments increase growth yield (expressed as the ratio of the maximum number of cells produced to the initial concentration of 2,4-D in the culture medium).Significant amounts of intact 2,4-D molecules remain outside and inside the cells when cell division stops at the onset of the stationary phase. This result and the previous demonstration that, at the onset of the stationary phase, 2,4-D is the specific limiting factor of cell division (Leguay JJ, J Guern 1975 Plant Physiol 56: 356-359) suggest that a threshold concentration of auxin is needed for cell division to proceed.The distribution of 2,4-D molecules between the cells and the culture medium is dependent on the population density at the stationary phase. The extracellular 2,4-D concentration at that time is a linear function of the population density whereas intracellular amounts of 2,4-D and 2,4-D metabolites are constant. By using a modified 2-(14)C,-5,5-dimethyloxazolidine-2,4-dione technique, it has been shown that the intracellular pH is markedly lowered as the population density at the plateau is increased. This intracellular pH modification is likely to be responsible for a large modification of the ratio between intracellular and extracellular auxin concentrations.The intracellular auxin concentration reaches a constant value (about 3 x 10(-7)m), independent of population density when cell division stops at the onset of the stationary phase suggesting that it represents the threshold value of the control for cell division.
Four ecotypes of the species Lycopersicon cheesmanii ssp. minor (Hook.) C.H. Mull. from the Galapagos Islands were compared with L. esculentum Mill cv. VF 36 with respect to salt tolerance. The L. cheesmanii ecotype that proved most salt-tolerant was selected for detailed comparison with the L. esculentum cultivar. Plants were grown in modified Hoagland solution salinized with synthetic seawater salt mix. Growth rates under saline conditions were examined and amino acid, sugar, total amino nitrogen, free acidity, and Na and K levels in the tissues of the most and least tolerant plants were measured under salt stress and nonstress conditions. Results indicate that all Galapagos ecotypes were far more salt-tolerant than was the esculentum cultivar. They could survive in full strength seawater nutrient solution while the esculentum cultivar could not in most cases withstand levels higher than 50% seawater. Growth rates were reduced in both species under saline conditions but the esculentum cultivar was more severely affected. High levels of total amino nitrogen, specific amino acids, and free acidity along with low sodium content were found in the salt stressed VF 36 cultivar. The opposite responses were noted in the salt stressed treatments of the Galapagos ecotype. Tissue sugar levels did not appear to be similarly correlated with salt stress in either species. Potassium content fell sharply during salinization in the Galapagos ecotype while in the esculentum cultivar it declined relatively little even at high levels of salinity.
Several peaks of aldolase activity are found in the isoelectric focusing pattern of pea (Pisum sativum) leaf chloroplast extracts. One peak, separated by 0.5 pH unit from the major chloroplast aldolase peak, is found when cytoplasmic extracts are focused. The chloroplast and cytoplasmic enzymes have a pH 7.4 optimum with fructose 1,6-diphosphate. The Michaelis constant for fructose-1,6-diphosphate is 19 muM for the chloroplast, 21 muM for the cytoplasmic enzyme, and for sedoheptulose 1,7-diphosphate, 8 muM for the chloroplast enzyme, 18 muM for the cytoplasmic enzyme. Both enzymes are inhibited by d-glyceraldehyde 3-phosphate and by ribulose 1,5-diphosphate. The similarity in the catalytic properties of the isoenzymes suggests that both enzymes have an amphibolic role in carbon metabolism in the green leaf.
The biochemical events utilized by transport proteins to convert the chemical energy from the hydrolysis of ATP into an electro-chemical gradient are poorly understood. The inhibition of the plasma membrane ATPase from corn (Zea mays L.) roots by N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) was compared to that of ATPase solubilized with N-tetradecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate (3-14) to provide insight into the minimal functional unit. The chromatographic behavior of the 3-14-solubilized ATPase activity during size exclusion chromatography and glycerol gradient centrifugation indicated that the solubilized enzyme was in a monomeric form. Both plasma membrane-bound and solubilized ATPase were inhibited by EEDQ in a time- and concentration-dependent manner consistent with a first-order reaction. When the log of the reciprocal of the half-time for inhibition was plotted as a function of the log of the EEDQ concentration, straight lines were obtained with slopes of approximately 0.5 and 1.0 for membrane-bound and 3-14-solubilized ATPase, respectively, indicating a change in the number of polypeptides per functional ATPase complex induced by solubilization with 3-14.
Thirty-nine plant species representing 20 families from the four major divisions of plants were surveyed for the presence of proteinase inhibitor-inducing factor activity in leaves or other tissues. Tissue juices were assayed for their capacity to induce accumulation of proteinase inhibitor I in excised tomato (Lycopersico esculentum) leaves. In tissues of only 2 of the 39 species was proteinase inhibitor-inducing factor-like activity not found. The activity was absent in cabbage leaves and celery stalks. Fruiting bodies from one of three fungi genera assayed contained exceptionally large quantities of proteinase inhibitor-inducing factor-like activity. Extracts from Agraricus campestris fruiting bodies contained over 20 times more activity than tomato leaf juice. The survey confirms that substances with proteinase inhibitor-inducing factor-like activity are widespread in the plant kingdom.
In order to investigate the relationship between malate oxidation and subsequent cycle reactions, the effects of oxaloacetate, pyruvate, and thiamine pyrophosphate on malate oxidation in mung bean (Phaseolus aureus var. Jumbo) hypocotyl mitochondria were quantitatively examined. Malate oxidation was optimally stimulated by addition of pyruvate and thiamine pyrophosphate, whose addition lowered the apparent Km for malate from 5 mm to 0.1 mm. Intermediate analysis showed that the stimulatory effect was correlated with removal of oxaloacetate to citrate. Oxaloacetate added alone was shown not to be metabolized until addition of pyruvate and thiamine pyrophosphate; then oxaloacetate was converted in part to pyruvate and also to citrate. These results establish that malate oxidation in mung bean mitochondria is subject to control by oxaloacetate levels, which are primarily determined by the resultant of the activities of malate dehydrogenase, citrate synthase, and pyruvate dehydrogenase.
When the detached first leaves of green or etiolated oat (Avena sativa cv. Victory) seedlings senesce in the dark, their oxygen consumption shows a large increase, beginning after 24 hours and reaching a peak of up to 2.5 times the initial rate by the 3rd day. This effect takes place while the chlorophyll of green leaves, or the carotenoid of etiolated leaves, is steadily decreasing. Kinetin, at a concentration which inhibits the decrease in pigment, completely prevents the respiratory rise; instead, the oxygen consumption drifts downwards. Lower kinetin concentrations have a proportional effect, 50% reduction of respiration being given by about 0.1 mg/l. About one-fifth of the respiratory rise may be attributed to the free amino acids which are liberated during senescence; several amino acids are shown to cause increases of almost 50% in the oxygen consumption when supplied at the concentrations of total amino acid present during senescence. A smaller part of the rise may also be due to soluble sugars liberated during senescence, largely coming from the hydrolysis of a presumptive fructosan. The remainder, and the largest part, of the increase is ascribed to a natural uncoupling of respiration from phosphorylation. This is deduced from the fact that dinitrophenol causes a similar large rise in the oxygen consumption of the fresh leaves or of leaf segments kept green with kinetin, but causes only a very small rise when the oxygen consumption is near its peak in senescent controls. The respiration of these leaves is resistant to cyanide, and 10 mm KCN even increases it by some 30%; in contrast, etiolated leaves of the same age, which undergo a similar rise in oxygen consumption over the same time period, show normal sensitivity to cyanide. The respiratory quotient during senescence goes down as low as 0.7, both with and without kinetin, though it is somewhat increased by supplying sugars or amino acids; glucose or alanine at 0.3 m bring it up to 1.0 and 0.87, respectively.N(6)-Benzylaminopurine and Delta-2-isopentenylaminopurine act similarly to kinetin in repressing the respiratory rise, the former being five times as active as kinetin, while the latter has only 1% of the activity of kinetin. Zeatin also powerfully prevents senescence. Because the repression of the respiratory rise is shown by each cytokinin at the concentration at which it inhibits senescence, the action is ascribed in both cases to the maintenance of a tight coupling between respiration and phosphorylation. It is pointed out that such an effect would explain many features of cytokinin action.A change in the methodology of the senescence experiments is described and compared with the method previously used, and the influence of temperature and age of the plants on the course of leaf senescence are presented in detail.
In a prior study (E. Chinn and J. Silverthorne  Plant Physiol 103: 727-732) we showed that the gymnosperm Ginkgo biloba was completely dependent on light for chlorophyll synthesis and chloroplast development and that expression of light-harvesting complex b (Lhcb) mRNAs was substantially increased by light. However, dark-grown seedlings that were transferred to constant white light took significantly longer than angiosperm seedlings to initiate a program of photomorphogenesis and the stems failed to green completely. We have prepared type-specific probes for mRNAs encoding major polypeptides of light-harvesting complex II (Lhcb1, Lhcb2, and Lhcb3) and have used these to analyze the expression of individual Lhcb mRNAs during greening. All three sequences accumulated in the top portions of dark-grown seedlings transferred to light, but, as was seen previously for total Lhcb mRNAs, there was a transient, reproducible decline in the levels of all three mRNAs after 4 d in the light. This transient decrease in Lhcb mRNA levels was not paralleled by a decrease in Chl accumulation. By contrast, there were significantly lower levels of all three Lhcb mRNAs in the lower portions of greening dark-grown stems as well as lower Chl levels. We conclude that although the tops of the plants have the capacity to etiolate and green, Gingko seedling stems continue a program of development into woody tissue in darkness that precludes greening when the seedlings are transferred to the light.
By phase microscopic observation of living palisade parenchyma cells in sections of Nicotiana excelsior leaves from plants previously placed in the dark for 72 hours, 30 to 45 minutes of light is found to induce mitochondria to remain stationary within the concavity of the chloroplasts and become round. Extending the illumination period to 60 to 90 minutes causes the stationary mitochondria in the concavity to change from a translucent to an opaque appearance, the change coinciding with the first appearance of starch as detected by blue staining of the grains with I(2)-KI. It is speculated that an interaction bearing some resemblance to the previously described interaction between mitochondria and the mobile phase of the chloroplasts may also operate in the starch grain phenomenon.
In Clark and Shelby soybean (Glycine max [L.] Merr.) seedlings, hypocotyl elongation was inhibited and hypocotyl swelling and root dry weight were increased by a temperature of 25 C. At 20 and 30 C, development was normal, as was development of Hawkeye and Mandarin soybean seedlings at all three temperatures. Dry matter distribution at 9 days indicates that inhibition of hypocotyl elongation is not due to a lack of translocation from cotyledons, but to a diversion of dry matter from hypocotyl to root. Ethylene evolution by Clark seedlings at 25 C exceeds that at 20 and 30 C. At all three temperatures, Mandarin seedlings' ethylene evolution is at the same low rates as those of Clark at 20 and 30 C. Clark's enhanced rate at 25 C precedes the deceleration of hypocotyl elongation occurring at 5 days. The abnormal effects of a temperature of 25 C on Clark seedlings' development is partially reversed by CO(2) and GA(3) and can be duplicated in Mandarin by applying ethylene. In Clark, effects of the temperature can be further accentuated by indoleacetic acid, which stimulates ethylene evolution, and by applying ethylene to the seedlings. It is concluded that the temperature-induced symptoms, similar in most respects to the well known "triple response" of legume seedlings, are caused by abnormally high levels of ethylene in tissues of the anomalous cultivars.
Sugar uptake by sugarcane cells in suspension culture was measured over short incubation time spans (5 seconds to 4 minutes), and membrane transport rates were calculated. A relatively high proportion of labeled products in cell extracts after incubation of cells with (14)C-glucose for 5 seconds was sugar phosphates (56%); fructose and sucrose began to appear after 15 and 30 seconds, respectively. Galactose and 3-O-methylglucose competed appreciably with glucose uptake, but ketohexoses and pentoses did not; there was no detectable uptake of sucrose. It is postulated that besides endogenous phosphorylation and further metabolism of glucose the configuration of the hydroxyl on the carbon-2 may be important for efficient membrane transport. The cells had a particularly high affinity for glucose and 3-O-methylglucose (Km = 15 and 16 mum, respectively).
A technique for obtaining large numbers of root hair cells in cell cultures from soybeans is described. The cells were grown on agar containing the Prairie Regional Laboratory B5 (PRL-B5) medium for periods longer than 60 days. Mixed populations of cultured root hair cells and cortical cells were used to study the in vitro association between soybean cells and Rhizobium japonicum. The advantages of these types of root cell cultures in studies of symbiosis are discussed.
Grana and stroma lamellae fractions prepared from illuminated chloroplasts (Lactuca sativa L. var. Manoa) by French press treatment contained less violaxanthin and more zeaxanthin than the corresponding fractions from dark controls. In both fractions, only part of the total violaxanthin was de-epoxidized under illumination, and the ratio of de-epoxidized and unchanged violaxanthin was similar. This not only shows that the de-epoxidation system is present in both grana and stroma thylakoids but also that violaxanthin is heterogeneous in both membranes. The presence and similarity of the de-epoxidation system in grana and stroma lamellae suggest that the function of the violaxanthin cycle is linked to photosynthetic activities which are common to both types of membranes.
A chlorosis-inducing toxin of Pseudomonas phaseolicola was examined for inhibition of ornithine carbamoyltransferease prepared from acetone powder of bean (Phaseolus vulgaris L.) plants. The enzyme has a pH optimum at 8.5, involves a ternary complex reaction mechanism, and shows Michaelis constants of 5.0 mm and 1.7 mm for ornithine and carbamoylphosphate, respectively. Assuming reversible catalysis, Michaelas constants of 11 mm and 3.3 mm are calculated for citrulline and arsenate. Toxin induces allosteric competitive inhibition in relation to carbamoylphosphate and a noncompetitive mode of inhibition in relation to ornithine, except at high toxin concentrations where uncompetitive inhibition is observed. In the backward assay, competitive inhibition is observed for both arsenate and citrulline. Inhibition is increased with preincubation time and shows saturation kinetics with regard to toxin concentration.
The phosphate translocator protein of C(3) and C(4) mesophyll chloroplast envelopes was specifically labeled using the anion exchange inhibitor, 1,2-ditritio-1,2-(2,2' -disulfo-4,4' -diisothiocyano) diphenylethane ([(3)H](2)-DIDS). Intact mesophyll chloroplasts were isolated from the C(3) plants, Spinacia oleracea L. (spinach) and Pisum sativum L. (pea), and the C(4) plant, Zea mays L. (corn). Chloroplasts were incubated with 5 to 50 mum [(3)H](2)-DIDS and, in addition, pea chloroplasts were also incubated with pyridoxal phosphate/tritiated sodium borohydride. The chloroplasts were washed, the envelopes isolated and solubilized. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis, label from bound [(3)H](2)-DIDS was detected only in the 28- to 30-kilodalton protein (proposed C(3) phosphate translocator) for both C(3) and C(4) chloroplasts, as demonstrated by fluorography. In contrast, when pyridoxal phosphate/tritiated sodium borohydride was used to label pea chloroplasts, radioactivity was detected in several other bands in addition to the 29-kilodalton polypeptide. These findings suggest that DIDS is a much more specific inhibitor than reagents previously employed to study the phosphate translocator and could be used to isolate and characterize the differences in the C(3) and C(4) phosphate translocator protein(s).
Effects of light and gibberellic acid (GA(3)) application on the germination of Dioscorea tokoro Makino and Dioscorea tenuipes Franch. et Savat. were observed. For complete germination, seeds of both species required prechilling in moist condition before incubation at a higher temperature. Red light irradiation during the incubation after the prechilling promoted germination; blue, green, or far red light markedly inhibited the germination of both species.Application of GA(3) induced complicated changes in the germination of both species in relation to light quality. In the germination of D. tokoro, GA(3) inhibited in the dark and red; however, it promoted germination in blue and far red light. GA(3) promoted germination of D. tenuipes in the dark and in blue, green, or far red light. These phenomena are explainable by assuming two counteractive reactions (germination-promoting and germination-inhibiting) which are both activated by applied GA(3).
A galactolipid lipase has been isolated and partially purified from the chloroplast fraction of the primary leaves of Phaseolus vulgaris var. Kentucky Wonder. The lipase hydrolyzed monogalactosyl diglyceride rapidly and phosphatidyl choline relatively slowly. Triolein and p-nitrophenyl stearate were not hydrolyzed.Spinach subchloroplast particles were excellent substrates for the lipase. Initial rates of fatty acid release from subchloroplast particles at 30 C by the lipase as high as 60 microequivalents per minute per milligram protein were observed. At completion of the reaction, about 2.7 microequivalents of fatty acid were liberated per milligram of chlorophyll in the subchloroplast particles, indicating that major amounts of lipid in the particles were rapidly attacked by the lipase.The treatment of subchloroplast particles with the lipase resulted in a rapid inhibition of light-dependent electron flow. This inhibition was largely prevented when the incubation was carried out in the presence of high concentrations of defatted bovine serum albumin. These results suggest that when precautions are taken to prevent the binding of fatty acids to the subchloroplast particles, large amounts of lipid may be removed without a marked effect on electron flow.
Plants are increasingly being employed to clean up environmental pollutants such as heavy metals; however, a major limitation of phytoremediation is the inability of plants to mineralize most organic pollutants. A key component of organic pollutants is halogenated aliphatic compounds that include 1,2-dichloroethane (1,2-DCA). Although plants lack the enzymatic activity required to metabolize this compound, two bacterial enzymes, haloalkane dehalogenase (DhlA) and haloacid dehalogenase (DhlB) from the bacterium Xanthobacter autotrophicus GJ10, have the ability to dehalogenate a range of halogenated aliphatics, including 1,2-DCA. We have engineered the dhlA and dhlB genes into tobacco (Nicotiana tabacum 'Xanthi') plants and used 1,2-DCA as a model substrate to demonstrate the ability of the transgenic tobacco to remediate a range of halogenated, aliphatic hydrocarbons. DhlA converts 1,2-DCA to 2-chloroethanol, which is then metabolized to the phytotoxic 2-chloroacetaldehyde, then chloroacetic acid, by endogenous plant alcohol dehydrogenase and aldehyde dehydrogenase activities, respectively. Chloroacetic acid is dehalogenated by DhlB to produce the glyoxylate cycle intermediate glycolate. Plants expressing only DhlA produced phytotoxic levels of chlorinated intermediates and died, while plants expressing DhlA together with DhlB thrived at levels of 1,2-DCA that were toxic to DhlA-expressing plants. This represents a significant advance in the development of a low-cost phytoremediation approach toward the clean-up of halogenated organic pollutants from contaminated soil and groundwater.
When the temperature of incorporation of sodium acetate-1, 2-(14)C into lipids of alfalfa (Medicago media Pers. var. Rambler and Medicago sativa L. var. Caliverde) roots was lowered from 22 C to 1 C, elongation and desaturation of fatty acids and the labeling of phosphatidylcholine were strongly stimulated.Controlled hardening of alfalfa stimulated strongly the incorporation of sodium acetate-1, 2-(14)C into root lipids of the hardy variety Rambler, but only slightly in the case of the frost-sensitive variety Caliverde. When incorporation was done at 1 C at various times of hardening, labeling decreased significantly in linoleic acid with a corresponding increase in oleic acid. Hardening, therefore, repressed specifically the initial low temperature stimulation of oleic acid desaturation, without affecting the stimulation of elongation of palmitic acid and the desaturation of stearic acid at low temperature. The radioactivity in linoleic acid was slightly greater in hardy Rambler than in Caliverde throughout hardening.When feedings were done at 1 C at various times of hardening, labeling of phosphatidylcholine increased in Rambler while it decreased in Caliverde. Throughout the hardening period, when incorporation was done at 1 C, linoleic acid represented a higher percentage of the label in phosphatidylcholine than in phosphatidylethanolamine or triglycerides and its specific radioactivity was much greater in phosphatidylcholine than in phosphatidylethanolamine and triglycerides and in Rambler than in Caliverde. Phosphatidylcholine seems, therefore, to play a special part in linoleic acid synthesis and in its control during the acquisition of frost hardiness.
Arabidopsis seeds were germinated on sterile mineral agar supplemented with 1% glucose and cultured under continuous light regimes. With 4-hour incandescent plus 20-hour monochromatic illumination in the region from 400 to 485 nanometers there was effective floral induction at an intensity of 100 microwatts per square centimeter. Exclusion of far red wave lengths from the 4-hour incandescent period sharply reduced the effectiveness of subsequent monochromatic blue light in promoting floral induction. Delayed floral induction occurred under continuous incandescent light lacking far red and was attributable to the blue wave lengths. Continuous 485 nanometer (100 microwatts per square centimeter) exposure without any white light treatment during the postgermination growth period was ineffective in floral induction and meristem development. Light at 730 nanometers under the same conditions was partially effective, whereas energy between 500 and 700 nanometers was completely ineffective. When continuous monochromatic light at a 3-fold higher energy level was administered, all photomorphogenic responses were accomplished with 485 nanometer light, including germination and 100% floral induction without any white light treatment at any time during the experiment. Almost equal quantum effectiveness was calculated when equivalent quantum flux densities in the region from 710 to 740 nanometers or at 485 nanometers were used. It is postulated that floral induction in Arabidopsis may be the result of a continuous excitation of a stable form of far red-absorbing phytochrome localized in or on a membrane, and that excitation can be either by direct absorption of energy by far red-absorbing phytochrome or by transfer from an accessory pigment.
Transgenic exploitation of bacterial degradative genes in plants has been considered a favorable strategy for degrading organic pollutants in the environment. The aromatic ring characteristic of these pollutants is mainly responsible for their recalcitrance to degradation. In this study, a Plesiomonas-derived chlorocatechol 1,2-dioxygenase (TfdC) gene (tfdC), capable of cleaving the aromatic ring, was introduced into Arabidopsis (Arabidopsis thaliana). Morphology and growth of transgenic plants are indistinguishable from those of wild-type plants. In contrast, they show significantly enhanced tolerances to catechol. Transgenic plants also exhibit strikingly higher capabilities of removing catechol from their media and high efficiencies of converting catechol to cis,cis-muconic acid. As far-less-than-calculated amounts of cis,cis-muconic acid were accumulated within the transgenic plants, existence of endogenous TfdD- and TfdE-like activities was postulated and, subsequently, putative orthologs of bacterial tfdD and tfdE were detected in Arabidopsis. However, no TfdC activity and no putative orthologs of either tfdC or tfdF were identified. This work indicates that the TfdC activity, conferred by tfdC in transgenic Arabidopsis, is a key requirement for phytoremoval and degradation of catechol, and also suggests that microbial degradative genes may be transgenically exploited in plants for bioremediation of aromatic pollutants in the environment.
Glyoxysomes, a form of microbody, are present in castor bean endosperm during the first 8 days of seed germination. They have a "typical" microbody form and are shown histochemically to contain catalase. The catalase label is distributed throughout the microbody and is not an exclusive feature of the crystalline or amorphous core.Castor bean endosperm contain a second cytosome, only slightly larger than the glyoxysomes, which is bound by a rough-surfaced membrane and which does not label for catalase. We have not observed these cytosomes in other tissues, suggesting that they may have a specific cellular function characteristic of castor bean endosperm.
Two zein proteins (Z1 and Z2) represent the majority of the protein synthesized during maize endosperm development. Undegraded membrane-bound polysomes isolated from normal maize synthesized these proteins when incubated in a cell-free protein-synthesizing system from wheat germ. The proteins synthesized in vitro were similar to authentic zein in ethanol solubility and electrophoretic mobility. Zein synthesis was associated with large size classes of membrane bound polysomes in normal maize.Membrane-bound polysomes isolated from developing kernels of opaque-2 mutant synthesized less total zein in vitro, and dramatically reduced incorporation into the Z1 component. The reduction in total zein corresponded to a 50% reduction in the level of membrane-bound polysomes in opaque-2, and the near absence of the large polysome size classes, which synthesized zein in normal maize. We concluded that the opaque-2 mutation results in a decreased "availability" of the zein mRNAs, reflected in a reduced level of membrane-bound polysomes.
Superoxide dismutase was purified from pea (Pisum sativum L., cv. Wando) seeds and corn (Zea mays L., cv. Michigan 500) seedlings. The purified pea enzyme eluting as a single peak from gel exclusion chromatography columns contained the three electrophoretically distinct bands of superoxide dismutase characterizing the crude extract. The purified corn enzyme eluted as the same peak as the pea enzyme, and contained five of the seven active bands found in the crude extract. The similar molecular weights and the cyanide sensitivities of these bands indicated that they are probably isozymes of a cupro-zinc superoxide dismutase. One of the remaining corn bands was shown to be a peroxidase.Superoxide dismutase accounted for 1.6 to 2.4% of the water-soluble protein in seedlings of corn, peas, and oats (Avena sativa L., cv. Au Sable). The superoxide dismutase activity per plant and per milligram water-soluble protein considerably increased during germination of oats and during greening and hook opening of peas.
Ribosomal subunits prepared by NH(4)Cl dissociation (0.5 m) of the monomeric ribosomes were much less active in in vitro protein synthesis than those prepared by KCl dissociation. The decrease in activity correlated with a detachment of some proteins (L(2) and L(9) as shown by gel electrophoresis) within the 60S ribosomal subunits. Subunits prepared with 0.3 m NH(4)Cl retained L(2) and L(9), but the activity remained low. Incubation of these 60S subunits in TKM buffer (50 mm tris [pH 7.5], 20 mm KCl, and 5 mm MgCl(2)) for 20 min at 37 C restored the activity almost to the level of those obtained by KCl dissociation. Treatment of the 0.3 m NH(4)Cl-derived 60S subunits with a protein reagent, Procion brilliant blue, prior to extraction of the ribosomal proteins resulted in the loss of L(2) and L(9), showing that these proteins were made accessible for dye binding. These observations suggest that a considerable degree of unfolding of the 60S subunit occurs at 0.3 m NH(4)Cl (this apparently leads to a preferential detachment of L(2) and L(9) at 0.5 m NH(4)Cl) and that the activity of the purified subunits depends not only on the presence of L(2) and L(9) but also on the organization of these proteins within the 60S subunits.
Gibberellins A(1) and A(34) (possibly A(2)) were found as products of metabolism of 1,2-[(3)H]GA(4) during germination of Pinus attenuata pollen. The conversion from GA(4) to GA(1) and GA(34) occurred as hydroxylations at atoms C-13 and C-2 of the ent-gibberellane skeleton, respectively. Percentage interconversion of the GA(4) absorbed was in the range of 0.15 to 0.43% for GA(1) and 1.54 to 3.22% for GA(34). Identifications were made on a gas-liquid chromatograph with radioactive monitoring by comparison with standards.