Cytokinin control of growth of Spirodela oligorrhiza in darkness

Summary Continuous heterotrophic growth of Spirodela oligorrhiza cultures following transfer to darkness requires cytokinins, or periodic brief treatment with red light. In the absence of cytokinins or red light growth ceases after 2–3 days. However, growth resumes spontaneously after 3–4 weeks in darkness to produce etiolated plants. The growth rate of these etiolated plants is not stimulated by kinetin.Although the kinetin concentration in treated plants reaches a plateau 30–60 min after adding kinetin to dormant plants in darkness new fronds do not appear for 24 h. Dormant colonies treated with kinetin in darkness for only 6–12 h subsequently grow in darkness at the same rate as plants treated with kinetin for 1, 2 or 3 days. Treatments which inhibit growth in the light, for example cold, chloramphenicol or actidione, eliminate the requirement for cytokinin and allow subsequent growth in darkness. The results suggest that a growth inhibitor may be present but ineffective in Spirodela growing in the light. The inhibitor is active in darkness but slowly decays. Kinetin appears to inactivate the inhibitor in darkness.     

Phytochrome-mediated growth responses in green and etiolated Lemna minor

Summary The growth of Lemna minor in darkness is log-linear, at a much reduced rate compared to growth in white or red light. This rate of frond production in darkness is stimulated by kinetin, yeast extract, and thiamine either in green plants transferred directly from the light or in plants which had been grown in the dark for 54 days. (Fig. 1).The magnitude of the stimulation of frond production by interruption of darkgrowth with red light (Fig. 2) is smaller in green than in etiolated plants, and is shown to depend upon the length of time that initially green plants were held in darkness (Fig. 4, Table 2). The stimulation of frond production in either green or etiolated plants does, however, obey the reciprocity law (Fig. 3).The stimulation by red light can be fully and repeatedly nullified by far red light only in etiolated plants, but the efficiency of nullification of the red effect by far red seems to increase in green plants with increasing sets of red + far red exposures (Fig. 5).As the dark-interval between red and far red exposures is lengthened, the efficiency of nullification is lessened significantly for etiolated plants only after 30 min (Fig. 6).     

The effect of glyphosate on frond regeneration, bud development and survival, and storage rhizome starch content in bracken

In addition to very effectively suppressing bracken frond regeneration for two years at levels of 2.24 kg a. i./ha and above, glyphosate had considerable effects on the rhizome system. Dry weight of frond-bearing rhizomes, number of living apices and developed buds, number and viability of dormant buds and starch content of storage rhizomes were all markedly reduced. The effects on dormant buds and starch content are particularly important when considering frond regeneration and as a consequence of these, glyphosate is likely to give long lasting control of bracken. Low levels of glyphosate (0.02 and 0.07 kg a. i./ha) did not stimulate bracken growth, while the addition of NH₄SCN apparently inhibited the action of the sub-lethal rate of 0.56 kg a. i./ha.     

纳米银对紫萍休眠芽萌发、存活和生长的影响

纳米银(AgNPs)是一种潜在的新型环境污染物,本研究以紫萍(Spirodela polyrhiza (L.) Schleid)休眠芽为实验材料,研究AgNPs对休眠芽萌发率、存活率及叶状体数目、面积和叶绿素含量等指标的影响,并对各项指标的半数效应浓度(EC₅₀)进行比较。结果显示,AgNPs可以抑制紫萍休眠芽的萌发,高浓度(10 mg/L)时可造成休眠芽死亡率显著增加。紫萍休眠芽萌发后叶状体数目和叶状体面积、色素含量均随浓度增加逐渐降低,表现出剂量效应,且叶绿素a对AgNPs最敏感。研究结果表明AgNPs对水生植物紫萍无性繁殖体的萌发和生长都具有抑制作用,具有一定的生态风险。     

Patterns of protein synthesis in dormant and growing vegetative buds of pea

Lateral buds on intact pea plants (Pisum sativum L. cv. Alaska) remain dormant until they are stimulated to develop by decapitating the terminal bud. Using two-dimensional gel electrophoresis, we have examined the protein content of terminal and lateral buds from intact plants and from plants at various times after decapitation. Silver-staining and in-vivo-labeling demonstrated very different sets of proteins. The level of expression of 18 stained and 25 labeled proteins was altered when growth was stimulated; this represents 3.4% and 9.1% of the total proteins detected by each method, respectively. Within 24 h of being stimulated, lateral buds doubled in length and their protein content was qualitatively nearly the same as that of terminal buds. Six hours after decapitation, before the onset of detectable growth, the overall pattern of protein synthesis in lateral buds was more like that of growing lateral buds or of terminal buds than that of dormant lateral buds. Direct application of N⁶-furfurylaminopurine (kinetin) to buds on intact plants stimulated their growth and resulted in the same pattern of protein synthesis as did decapitation. Inhibition of bud growth by addition of indole-3-acetic acid to the stumps of decapitated plants resulted in the synthesis of dormancy-related proteins. Lateral buds at all stages of development incorporated labeled amino acids at similar rates, indicating that metabolic activity is not a component of dormancy in these buds.Abbreviations IAA indole-3-acetic acid - IEF isoelectric focusing - KIN kinetin (N⁶-furfurylaminopurine) - SDS sodium dodecylsulfate - TCA trichloroacetic acid - 2D-PAGE two-dimensional polyacrylamide gel electrophoresis     

Das Verhalten von CO2-Assimilation,Nadpund PGS-Reduktion und ATP-Synthese Intakter Blattzellen in Abhängigkeit vom Wassergehalt

Summary The carrot-root tissue culture assay for cytokinin activity has been improved by changing the site of explant excision and eliminating certain vitamins from the basal medium. These modifications increased its sensitivity and enabled zeatin [6-(4-hydroxy-3-methylbut-trans-2-enyl)aminopurine] to be detected at concentrations less than 5×10^-5M. In the improved assay, zeatin was markedly more active than kinetin, 6-benzylaminopurine, 6-(o-methylbenzyl)aminopurine and 6-(3-methylbut-2-enyl)aminopurine.The activity of zeatin also exceeded that of kinetin in the etiolated bean-leaf disk expansion assay. Zeatin was markedly more effective than kinetin and 6-(3-methylbut-2-enyl)aminopurine in promoting frond expansion and increasing frond number of Spirodela oligorrhiza cultures grown under continuous illumination. Zeatin was also more active than kinetin and 6-(3-methylbut-2-enyl)aminopurine in increasing frond number of Spirodela cultures grown in darkness. In retarding the senescence of disks of leaves of several species, kinetin was considerably more effective than zeatin which was more active than 6-(3-methylbut-2-enyl)aminopurine. The allylic hydroxyl group in zeatin is therefore a structural feature associated with high cytokinin activity.The relative activities of cytokinins can be very different and even in reverse order in different bioassays. It is suggested that this is due to the mechanism of cytokinin action varying in the different biological systems used.Part IV: Shannon and Letham (1966).     

Decreased sucrose-6-phosphate phosphatase level in transgenic tobacco inhibits photosynthesis,alters carbohydrate partitioning,and reduces growth

The aim of this work was to examine the role of sucrose-6-phosphate phosphatase (SPP; EC 3.1.3.24) in photosynthetic carbon partitioning. SPP catalyzes the final step in the pathway of sucrose synthesis; however, until now the importance of this enzyme in plants has not been studied by reversed-genetics approaches. With the intention of conducting such a study, transgenic tobacco plants with reduced SPP levels were produced using an RNA interference (RNAi) strategy. Transformants with less than 10% of wild-type SPP activity displayed a range of phenotypes, including those that showed inhibition of photosynthesis, chlorosis, and reduced growth rates. These plants had strongly reduced levels of sucrose and hexoses but contained 3–5 times more starch than the control specimens. The leaves were unable to export transient starch during extended periods of darkness and as consequence showed a starch- and maltose-excess phenotype. This indicates that no alternative mechanism for carbon export was activated. Inhibition of SPP resulted in an approximately 1,000-fold higher accumulation of sucrose-6-phosphate (Suc6P) compared to wild-type leaves, whereas the content of hexose-phosphates was reduced. Although the massive accumulation of Suc6P in the cytosol of transgenic leaves was assumed to impair phosphate-recycling into the chloroplast, no obvious signs of phosphate-limitation of photosynthesis became apparent. 3-Phosphoglycerate (3-PGA) levels dropped slightly and the ATP/ADP ratio was not reduced in the transgenic lines under investigation. It is proposed that in SPP-deficient plants, long-term compensatory responses give rise to the observed acceleration of starch synthesis, increase in total cellular Pi content, decrease in protein content, and related reduction in photosynthetic activity.     

Nucleic Acid, Protein, and Starch Synthesis in Developing Cotyledons of Pisum arvense L.

During the cell-division period of cotyledon development inPisum arvense L. cell volume increases slightly but nuclearvolume shows little variation and the DNA content remains atthe 2C to 4C level. During the main period of cell expansionthere is a close correlation between cell volume, nuclear volume,and nuclear DNA content, the nuclei of the largest storage cellsfinally attaining the 64C level. The rate of RNA synthesis increasesseveral days after the increase in DNA has begun and at thesame time accumulation of reserve protein and starch begins.RNA and starch synthesis apparently cease some time before maturationbut protein synthesis continues until the seeds are ripe. Cotyledondevelopment was found to comprise two distinct phases: an initialphase of cell division and differentiation during which DNA,RNA, and protein per unit volume of cell decline; and a phaseof reserve accumulation in which DNA per unit volume of cellremains constant but RNA and protein per unit volume increase,starch synthesis is initiated, and all the cotyledon cells assumethe properties of storage cells.     

DNA, RNA, protein and heterochromatin changes during embryo development and germination of soybean (Glycine max L.)

DNA, RNA, protein and heterochromatin were measured cytophotometrically in developing soybean (Glycine max) seeds. The average 2C DNA content for the soybean genome was 2.64 pg. The amounts of nuclear DNA in embryo axes showed no significant change during embryo development, whereas the DNA content in cotyledon nuclei increased significantly from 3.58 pg to 5.49 pg. The number of endopolyploid nuclei increased from 26% to 48% and the DNA content from 4.45 to 5.49 pg after cessation of cell division. The changes in RNA and protein content during embryo development were in general similar to those in DNA content. This can be interpreted that increased DNA levels in soybean cotyledons generated during embryogeny increase the protein synthesizing capacity. During the first 15 days of germination, the number of endopolyploid nuclei in cotyledons declined from 46% to 4%, and this decline is interpreted as DNA degradation providing a ready source of nucleosides and phosphates during early embryo growth. A later decline, however, between 15 and 20 days after germination, was age related similar to leaf senescence, because the percentage of endopolyploid nuclei remained unchanged while the number of non-viable cells increased. In senescing cotyledons, 73% and 80% of RNA and protein but only 20% of DNA were lost, as compared to dormant cotyledons. The heterochromatin (condensed chromatin) measurements indicated that nuclei of metabolically inactive dormant and senescent cotyledon nuclei contained an average of 33% more heterochromatin than nuclei from the green cotyledons of seedlings.     

Effects of hydroxyurea on Crithidia fasciculata.

Hydroxyurea (HU) inhibits increase in cell number in cultures of Crithidia fasciculata. Complete inhibition is produced by 8 mM and higher concentrations. If HU is not removed, population growth resumes in 45-50 h; if HU is removed, partially synchronous growth occurs through 2 cycles. During HU inhibition, the rate of DNA synthesis is reduced to 1% of that in exponentially growing cultures; protein and RNA syntheses continue at slightly reduced rates. Mean cell size and protein and RNA contents per cell increase; rate of oxygen consumption per mg cell protein remains constant. The behavior of a culture upon addition of HU and upon its removal agrees with predictions based on the hypothesis that the only direct effect of HU is to block DNA synthesis. The synchrony produced by HU is judged satisfactory for investigations of kinetoplast and nuclear replication but not for biochemical characterization of other aspects of the cell cycle.     

Effects of Hydroxyurea on Crithidia fasciculata

SYNOPSIS Hydroxyurea (HU) inhibits increase in cell number in cultures of Crithidia fasciculata. Complete inhibition is produced by 8 mM and higher concentrations. If HU is not removed, population growth resumes in 45–50 h: if HU is removed, partially synchronous growth occurs through 2 cycles. During HU inhibition, the rate of DNA synthesis is reduced to 1% of that in exponentially growing cultures; protein and RNA syntheses continue at slightly reduced rates. Mean cell size and protein and RNA contents per cell increase; rate of oxygen consumption per mg cell protein remains constant. The behavior of a culture upon addition of HU and upon its removal agrees with predictions based on the hypothesis that the only direct effect of HU is to block DNA synthesis. The synchrony produced by HU is judged satisfactory for investigations of kinetoplast and nuclear replication but not for biochemical characterization of other aspects of the cell cycle.     

Über die Wirkung kurzfristiger Kinetingaben auf Protein,Aminosäuren und RNS bei Lemna minor L.

Summary Lemna minor L. was cultivated on nutrient medium with kinetin (10^-5 m) for 3 hours at 28°C and ca. 5,000 lux. Samples were taken at different times and their amino acid, protein and RNA content was determined.The amino acid content in the cultures with kinetin corresponds to that in the cultures without kinetin. The amino acid concentration seems to be regulated by mechanisms independent of kinetin. On the other hand, kinetin increases the quantity of protein immediately i.e., within the first two minutes. This increase stops after ca. 30 minutes, but the excess of protein remains for a long time. In the first 30 minutes the RNA concentration also rises. This increase is not as strong as that of the protein nor does it remain. After 30 minutes, when the rise of protein content stops, the RNA concentration begins to drops within 1 hour to that of the control cultures without kinetin.The extent of the protein and RNA increase induced by kinetin depends strongly upon the physiological state of the plants. This may be because the plants produce different quantities of endogenous cytokinins under different conditions.The protein induced by kinetin was then examined after centrifugation at 100,000 x g. In samples taken during the first 5 minutes, the quantity of protein in the supernatant and in the sediment is increased. In samples taken after this period, the increase of protein in the supernatant disappears and only that in the sediment remains. After some time, therefore, all the excess protein consists of structural protein, which is found in the sediment. The findings are compared with those of other authors and an unspecific stimulation of RNA is suggested.     

Protein synthesis by intact Coxiella burnetii cells.

Coxiella burnetii was isolated from persistently infected fibroblast host cells by a rapid mechanical lysis technique. Macromolecular synthesis was initiated in these otherwise dormant cells by incubation at pH 4.5. The synthesis of protein proceeded for as long as 24 h. Initiation of protein synthesis in C. burnetii was dependent upon RNA synthesis. Approximately 24 species of polypeptides were synthesized, and some of these appeared to be major synthetic products. Increases in protein biomass of 15 to 30% were calculated to occur during incubation. Inhibition of DNA synthesis affected protein synthesis after 12 h of incubation. The results suggest that although these parasitic bacteria did not grow in the axenic media devised, significant biosynthetic processes occurred.     

Hormone-like effect of vascular tissue on starch accumulation in stem explants of kale, Brassica oleracea

Explants of stem pith of kale ( Brassica oleracea L. var. medullosa cv. Krasa), cultured for several days on agar medium containing sucrose, accumulate starch. Application of streptomycin, 5-fluorouracil and other inhibitors indicates that starch accumulation depends on protein synthesis on 80 S ribosomes. If explants derived from plants grown under natural long-day conditions contained vascular tissue, including cambium, in addition to pith parenchyma, the amount of starch formed in the pith tissue increased up to seven fold when compared with explants without vascular tissue. Similar increase of starch content as caused by vascular tissue was achieved by the addition of kinetin or trans -zeatin (10 μ) in the presence of 5 μ indole-3-acetic acid or 1-naphthaleneacetic acid. A further three-fold increase in starch accumulation could be achieved by application of cytokinin and auxin to explants containing vascular tissue. When explants were derived from plants grown under natural short-day conditions cytokinins and auxins had little or no effect, but vascular tissue enhanced starch formation significantly. The spreading of starch inducing stimulus from vascular tissue (probably from its meristematic region) to the pith parenchyma up to a distance of at least 20 mm was demonstrated. It was concluded that a hormone-like factor other than cytokinin and auxin was involved in the stimulatory action of vascular tissue. The effects of this factor on protein accumulation and growth in the explants and its possible production by meristematic tissues in vivo are discussed.     

Synthesis of proteins,RNA and DNA in dormant and after-ripenedCaryophyllaceae seeds

Macromolecule syntheses, especially incorporation of radioactive labelled precursors into proteins, RNA and DNA were investigated. Some results on the action of phytohormones applied to dormant seeds and on the influence on water stress conditions by interruption of imbibition even before the radicle protrudes, on germination as well as on RNA and DNA synthesis were analysed. Benzylaminopurine and ethylene, applied in combination, could break dormancy of dormant seeds; a process which is correlated with the onset of DNA synthesis. Interruption of the imbibition during the time of onset of DNA synthesis (after 16 h of imbibition) did not impair the germination, and the protein, RNA and DNA syntheses started after reimbibition at that level which was reached at the interruption point. Only after a break in later phases (after 22 h of imbibition) a weak impairment of germination could be observed.     

Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: evidence for differential gene expression.

The kinetics of retroviral DNA and RNA synthesis are parameters vital to understanding viral growth, especially for human immunodeficiency virus (HIV), which encodes several of its own regulatory genes. We have established a single-cycle growth condition for HIV in H9 cells, a human CD4+ lymphocyte line. The full-length viral linear DNA is first detectable by 4 h postinfection. During a one-step growth of HIV, amounts of viral DNA gradually increase until 8 to 12 h postinfection and then decrease. The copy number of unintegrated viral DNA is not extraordinarily high even at its peak. Most strikingly, there is a temporal program of RNA accumulation: the earliest RNA is greatly enriched in the 2-kilobase subgenomic mRNA species, while the level of 9.2-kilobase RNA which is both genomic RNA and mRNA remains low until after 24 h of infection. Virus production begins at about 24 h postinfection. Thus, viral DNA synthesis is as rapid as for other retroviruses, but viral RNA synthesis involves temporal alteration in the species that accumulate, presumably as a consequence of viral regulatory genes.     

Abscisic-acid-induced turion formation in Spirodela polyrrhiza L III. Specific changes in protein synthesis and translatable RNA during turion development

Abstract The developmental process leading to the formation of the abscisic acid (ABA) induced turion of Spirodela polyrrhiza was accompanied by a repression of nucleic acid and protein synthesis. DNA synthesis in the developing lurion (induced by 10⁻⁴mol m⁻³ ABA) was inhibited within 3h of ABA addition, followed by a repression of protein synthesis after 24 h, while RNA synthesis was not inhibited until 3 d. The inhibitory effect of ABA on protein synthesis was found to be selective and the synthesis of several novel proteins appeared to be induced. These effects were specific to ABA-sensitive tissue. The relationship between the changes in the protein and mRNA profiles during the development of the turion was investigated. The rapid general inhibition of protein synthesis at early stages of lurion formation could not be accounted for by the level of translatable mRNA, indicating an effect of ABA at the translational level. The specific alteration to the pattern of in vivo labelled proteins could have resulted, however, from control of the level of specific mRNAs for those particular proteins. Only after 3 d in ABA, when the developing primordium is committed to the turion developmental pathway, is there a total inhibition in the production of mRNA leading to the shutdown of all primary processes and the onset of the irreversible events leading to the dormant state.     

Effect of low temperature on eutrophicated waterbody restoration by Spirodela polyrhiza

Spirodela polyrhiza exposed to low temperature was investigated to study the possible application for recovering eutrophicated waterbody at 10-12 degrees C. The cell growth and enzymatic resistance to oxidative stress were compared with that grown at optimum temperature (26-28 degrees C). The frond number, fresh weight, soluble protein, chlorophyll, carrot pigment, root length, peroxidase (POD), catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA) were measured. The results demonstrated that the cell growth, the synthesis and absorption ability decreased and the protective enzymes increased. S. polyrhiza encountered seriously oxidative damage at such low temperature condition, and it was not suitable for recovering the eutrophicated natural waterbody at low temperature of 10-12 degrees C.