Studies on the biochemistry and fine structure of silica shell formation in diatoms

Summary Cell division in Navicula pelliculosa (Bréb.) Hilse, strain 668 was synchronized with an alternating regime of 5 h light and 7 h dark. Cell volume and dry weight increased only during the light period. DNA synthesis, which began during the third h of light, was followed sequentially by mitosis, cytokinesis, silicic acid uptake, cell wall formation, and cell separation. Silicification and a small amount of net synthesis of DNA, RNA and protein occurred during the dark at the expense of carbohydrate reserves accumulated during the light period. Cells kept in continuous light, after synchronization with the light-dark regime, remained synchronized through a second division cycle; the sequence of morphological events was the same as that in the light-dark division cycle, but the biosynthesis of macromolecular components changed from a stepwise to a linear pattern. The silicon-starvation synchrony was improved by depriving light-dark synchronized cells of silicic acid at the beginning of their division cycle, then resupplying silicic acid to cells blocked at wall formation.Abbreviation L light - D dark Portions based on a thesis submitted by W.M.D. to the University of California, San Diego in partial fulfillment of the requirements for the PH.D degree     

Inhibition of mitosis in pisum root meristems by continuous gamma radiation: the influence of temperature on the synthesis of DNA, RNA and protein during inhibition

Tritiated precursors of DNA, RNA and protein were used to measure synthesis at 10 and 20C in root meristem cells of Pisum after they were mitotically arrested by continuous irradiation with gamma rays. The experiments were designed to determine if the arrested cells accumulated in a certain part of interphase, to determine the effect on DNA, RNA and protein synthesis, to find out if the effects were temperature dependent, and finally to reveal possible relationships between growth inhibition and altered synthesis. The results showed that the incorporation of DNA and RNA precursors was impaired by irradiation and that decreased temperature further increased radiation impairment of DNA synthesis. Protein synthesis on the other hand was not impaired by irradiation at either temperature. Irradiation at 20C reduced the number of DNA-synthesizing cells; at 10C this number was reduced to near zero. Although irradiated cells synthesizing RNA showed a reduction in grain counts when compared to the controls, they still retained the ability to incorporate tritiated uridine at 10C. It was hypothesized that the combination of reduced DNA and RNA synthesis and unaffected protein synthesis resulted in precocious maturation of the arrested meristem cells. Growth which occurred in the absence of cell division was attributed to meristematic cells which precociously matured and cells which were in the region of elongation.     

Effect of irradiance on the course of RNA synthesis in the cell cycle ofScenedesmus quadricauda

In synchronous populations ofScenedesmus quadricauda the RNA amount in the cells increases in waves: periods of a high rate of RNA synthesis alternate with periods of a low rate in the course of the cell cycle. Each wave usually leads to the doubling of the RNA amount per cell. In cells growing under normal conditions the waves of RNA synthesis seem to be linked with consecutive rounds of DNA replication. The pattern of RNA synthesis in the course of the cell cycle, however, does not change, if DNA replication is prevented by application of 5-fluoro-deoxyuridine. In darkness the rate of RNA synthesis drops to zero and thereafter the RNA amount per cell decreases. In cells which have been induced to cellular division RNA synthesis may become restored in the dark in newly formed daughter cells. The lowering of RNA amount and its new increase during the dark period become more pronounced with increasing irradiance in the previous light period as well as with its increasing length. In the period of protoplast fissions RNA synthesis is arrested even if the cells divide in the light; whether a similar inhibition occurs during mitoses is not clear.     

Light‐induced stabilization of ACS contributes to hypocotyl elongation during the dark‐to‐light transition in Arabidopsis seedlings

Hypocotyl growth during seedling emergence is a crucial developmental transition influenced by light and phytohormones such as ethylene. Ethylene and light antagonistically control hypocotyl growth in either continuous light or darkness. However, how ethylene and light regulate hypocotyl growth, including seedling emergence, during the dark‐to‐light transition remains elusive. Here, we show that ethylene and light cooperatively stimulate a transient increase in hypocotyl growth during the dark‐to‐light transition via the light‐mediated stabilization of 1‐aminocyclopropane‐1‐carboxylic acid (ACC) synthases (ACSs), the rate‐limiting enzymes in ethylene biosynthesis. We found that, in contrast to the known inhibitory role of light in hypocotyl growth, light treatment transiently increases hypocotyl growth in wild‐type etiolated seedlings. Moreover, ACC, the direct precursor of ethylene, accentuates the effects of light on hypocotyl elongation during the dark‐to‐light transition. We determined that light leads to the transient elongation of hypocotyls by stabilizing the ACS5 protein during the dark‐to‐light transition. Furthermore, biochemical analysis of an ACS5 mutant protein bearing an alteration in the C‐terminus indicated that light stabilizes ACS5 by inhibiting the degradation mechanism that acts through the C‐terminus of ACS5. Our study reveals that plants regulate hypocotyl elongation during seedling establishment by coordinating light‐induced ethylene biosynthesis at the post‐translational level. Moreover, the stimulatory role of light on hypocotyl growth during the dark‐to‐light transition provides additional insights into the known inhibitory role of light in hypocotyl development.     

Macromolecular syntheses and the course of cell cycle events in the chlorococcal algascenedesmus quadricauda under nutrient starvation: Effect of sulphur starvation

Daughter cells of the chlorococcal algaScenedesmus quadricauda were incubated under photosynthesizing conditions in a sulphur-free medium. The course of the cell cycle under these conditions was changed in daughter cells which differed in their stage of development. In absence of sulphur, advanced daughter cells with two nuclei and 2 or 4 genomes passed a cycle identical with that of control in sulphur containing medium. Each cell yielded eight binuclear daughter cells. With less advanced daughter cells (one nucleus and 1 or 2 genomes) restriction of RNA synthesis occurred near to the end of the cell cycle and protein synthesis ceased two hours later (practically at the time of the protoplast fission). The last round of DNA replication found in the control culture was not initiated in sulphur-starved culture and uninuclear daughter cells with one genome were released. If the daughter cells coming from the starved populations were kept further in the sulphur-free medium, macromolecular syntheses were dramatically restricted. Only photosynthesis continued to produce starch at a similar rate as in normally grown cells. Thus, a very large amount of starch accumulated. Supported by these reserves, starved cells refed with sulphur passed an entire cell cycle in the dark and divided into eight daughter cells. In sulphur-supplied cells, both in the dark and in light, RNA, protein and DNA synthesis started without any delay in a similar way as in the control culture. Competition for sulphur reserves occurred between the growth and division processes; the former were preferred in the light and the latter in the dark.     

Dependence of gibberellic acid-induced dark germination of lettuce seed on RNA synthesis

Summary Gibberellicacid-induced dark germination of Grand Rapids lettude seed was completely inhibited by 6-azauracil and partly by 2-thiouracil. Other inhibitors of nucleic acid and protein synthesis used were without effect. Inhibition of gibberellic acid-induced dark germination was reversed by uracil but not by thymine, deoxycytidine and orotic acid. The results suggest that gibberellic acid-induced dark germination is dependent on RNA synthesis and not on DNA synthesis.Gibberellic acid-induced lettuce hypocotyl growth was inhibited by all the inhibiters of nucleic acid and protein synthesis used, including actinomycin D, puromycin, chloramphenicol and p-fluorophenylalanine.Approved by the Director of the New York State Agricultural Experiment Station for publication as Journal Paper No. 1507.     

Light-Regulated Hypocotyl Elongation Involves Proteasome-Dependent Degradation of the Microtubule Regulatory Protein WDL3 in Arabidopsis

Light significantly inhibits hypocotyl cell elongation, and dark-grown seedlings exhibit elongated, etiolated hypocotyls. Microtubule regulatory proteins function as positive or negative regulators that mediate hypocotyl cell elongation by altering microtubule organization. However, it remains unclear how plants coordinate these regulators to promote hypocotyl growth in darkness and inhibit growth in the light. Here, we demonstrate that WAVE-DAMPENED 2–LIKE3 (WDL3), a microtubule regulatory protein of the WVD2/WDL family from Arabidopsis thaliana, functions in hypocotyl cell elongation and is regulated by a ubiquitin-26S proteasome–dependent pathway in response to light. WDL3 RNA interference Arabidopsis seedlings grown in the light had much longer hypocotyls than controls. Moreover, WDL3 overexpression resulted in overall shortening of hypocotyl cells and stabilization of cortical microtubules in the light. Cortical microtubule reorganization occurred slowly in cells from WDL3 RNA interference transgenic lines but was accelerated in cells from WDL3-overexpressing seedlings subjected to light treatment. More importantly, WDL3 protein was abundant in the light but was degraded through the 26S proteasome pathway in the dark. Overexpression of WDL3 inhibited etiolated hypocotyl growth in regulatory particle non-ATPase subunit-1a mutant (rpn1a-4) plants but not in wild-type seedlings. Therefore, a ubiquitin-26S proteasome–dependent mechanism regulates the levels of WDL3 in response to light to modulate hypocotyl cell elongation.     

THE EFFECT OF HYDROXYUREA AND FLUORODEOXYURIDINE ON CELL CYCLE EVENTS IN THE CHLOROCOCCAL ALGA SCENEDESMUS QUADRICAUDA (CHLOROPHYTA)1

The effect of hydroxyurea and 5-fluorodeoxyuridine (FdUrd) on the course of growth (RNA and protein synthesis) and reproductive (DNA replication and nuclear and cellular division) processes was studied in synchronous cultures of the chlorococcal alga Scenedesmus quadricauda (Turp.) Bréb. The presence of hydroxyurea (5 mg·L^?1)from the beginning of the cell cycle prevented growth and further development of the cells because of complete inhibition of RNA synthesis. In cells treated later in the cell cycle at the time when the cells were committed to division, hydroxyurea present in light affected the cells in the same way as a dark treatment without hydroxyurea; i. e. RNA synthesis was immediately inhibited followed after a short time period by cessation of protein synthesis. Reproductive processes including DNA replication to which the commitment was attained, however, were initiated and completed. DNA synthesis continued until the constant minimal ratio of RNA to DNA was reached. FdUrd (25 mg·L^?1) added before initiation of DNA replication in control cultures prevented DNA synthesis in treated cells. Addition of FdUrd at any time during the cell cycle prevented or immediately stopped DNA replication. However, by adding excess thymidine (100 mg·L^?1), FdUrd inhibition of DNA replication could be prevented. FdUrd did not affect synthesis of RNA, protein, or starch for at least one cell cycle. After removal of FdUrd, DNA synthesis was reinitiated with about a 2-h delay. The later in the cell cycle FdUrd was removed, the longer it took for DNA synthesis to resume. At exposures to FdUrd longer than two or three control cell cycles, cells in the population were gradually damaged and did not recover at all.     

Effect of white light on meristematic activity in developing sunflower hypocotyls

Summary To determine whether hypocotyl elongation in sunflower seedlings (Helianthus annuus L.) is dependent on cell divisions (meristematic activity), we used a specific inhibitor of DNA synthesis (fluorodeoxyuridine). The seedlings were either grown for 6 days in darkness or continuous white light (WL). Under both conditions hypocotyl growth was retarded by 30–70% in the presence of the inhibitor. Because the nuclei do not become endopolyploid we conclude that hypocotyl growth is dependent on cell reproduction. In the next step an immunocytochemical method was used to detect the percentage of nuclei in S-phase (meristematic activity) in different regions and tissues of the hypocotyls. In the peripheral cell layers (epidermis, cortex) meristematic activity was much greater than in the pith of the organ. In rapidly growing (etiolated) hypocotyls meristematic activity is largely restricted to the closed apical hook of the stem. After transfer to WL the hook opens and hypocotyl elongation is inhibited. In the epidermis and cortex of the apical hook a large WL-induced enhancement in the percentage of nuclei in S-phase occurred, which was followed by a light-mediated retardation of meristematic activity. Our data show that WL exerts a transient stimulatory effect on meristematic activity during photomorphogenesis of the sunflower seedling.Abbreviations BrdUrd 5-bromo-2-deoxyuridine - D darkness - FdUrd 5-fluoro-2-deoxyuridine - TRITC tetramethyl-rhodamine-isothiocyanate - WL white light     

Macromolecular syntheses and the course of cell cycle events in the chlorococcal algaScenedesmus quadricauda under nutrient starvation: Effect of nitrogen starvation

Daughter cells of the chlorococcal algaScenedesmus quadricauda incubated under photosynthesizing conditions in a nitrogen-free medium did not make any progress in the cell cycle. Photosynthetic starch formation continued for a period corresponding to a half of the cell cycle and then levelled off. Protein synthesis was very slow and it did not surpass double the initial amount. RNA content decayed from the start of treatment and approached about 2 pg/cell. When a synchronous population was deprived of nitrogen or of light in the middle of the cell cycle RNA synthesis stopped immediately or very soon afterwards and, in spite ofabundant intracellular nitrogen reserves, RNA content slowly declined. This degradation was much extensive in nitrogen starved cells where, eventually, the RNA content attained about half the starting value. In both experimental variants, DNA replications started at the same time as in control culture, but the final amount of DNA attained only half the control value. Protein synthesis stopped immediately in the dark. In the nitrogen-starved cells, it continued for several hours and protein content increased about 70 % of the amount present at the start of starvation. The number of daughter cells formed was proportional to the final protein content in the nitrogen-and light-deprived cells (corresponding division numbers were 6 and 4, respectively). Upon refeeding of daughter cells formed under nitrogen starvation, RNA synthesis started immediately, while protein synthesis displayed a lag of about 5 h. DNA replications were triggered at the time when the ratio of RNA to DNA content attained the same value as in the control culture.     

Synchronous growth and synthesis of macromolecules in a naturally wall-less volvocale,Dunaliella bioculata

Summary Dunaliella bioculata, a naturally wall-less unicellular green alga, can be induced to divide synchronously when subjected to a 12 hours light-12 hours dark cycle. This rhythmic cell division will last for at least 15 days under a subsequent constant illumination. Synchronization can be improved when cells are submitted to 8 hours light-16 hours dark cycles under bright white light (10,000 lux). In these conditions the cell division gives rise to two daughter cells: The chronology of DNA, RNA and proteins synthesis has been studied during such a synchronized cell cycle. DNA synthesis begins 4 hours before the outset of cell division and is completed after two hours in the dark; in difference, illumination seems necessary to the synthesis of RNA and proteins.     

Phytochrome mediated induction of nitrate reductase activity in etiolated maize leaves

The effects of red and far-red light on the enhancement of in vitro nitrate reductase activity and on nitrate accumulation in etiolated excised maize leaves were examined. Illumination for 5 min with red light followed by a 4-h dark period caused a marked increase in nitrate reductase activity, whereas a 5-min illumination with far-red light had no effect on the enzyme activity. The effect of red light was completely reversed by a subsequent illumination with the same period of far-red light. Continuous far-red light also enhanced nitrate reductase activity. Both photoreversibility by red and far-red light and the operation of high intensity reaction under continuous far-red light indicated that the induction of nitrate reductase was mediated by phytochrome. Though nitrate accumulation was slightly enhanced by red and continuous far-red light treatments by 17% and 26% respectively, this is unlikely to account for the entire increase of nitrate reductase activity. The far-red light treatments given in water, to leaves preincubated in nitrate, enhanced nitrate reductase activity considerably over the dark control. The presence of a lag phase and inhibition of increase in enzyme activity under continuous far-red light-by tungstate and inhibitors of RNA synthesis and protein synthesis-rules out the possibility of activation of nitrate reductase and suggests de novo synthesis of the enzyme affected by phytochrome.     

Variations in biochemical parameters of Heterocapsa sp. and Olisthodiscus luteus grown in 12:12 light:dark cycles I. Cell cycle and nucleic acid composition

The division cycle of two phytoplankton species, Olisthodiscus luteus and Heterocapsa sp. was studied in relation to a 12:12 light:dark cycle. Batch cultures in exponential phase were sampled every three hours during 48 hours. Cell number, cellular volume and DNA and RNA concentrations were measured. Microscopic observations of the nuclei of Heterocapsa sp. were also performed. In both species, cell division took place in the dark. In Heterocapsa sp., DNA and RNA showed a similar diel variability pattern, with synthesis starting at the end of the light period, previously to mitosis and cytokinesis. In O. luteus. Major RNA synthesis occurred during darkness, and DNA was produced almost continuously. Both species presented different values and diel rhythmicity on the RNA/DNA ratios.     

Changes in Average Cell Concentrations of Various Constituents During Synchronous Division of Platymonas striata Butcher (Prasinophyceae)

The changes in the average cell composition of the green algaPlatymonas striata Butcher have been determined during a singlecell cycle in synchronous culture induced by an alternatinglight/dark regime. The cells divided into two at the onset ofdarkness, but remained attached until exposed to light 10 hlater. There appeared to be virtually no net synthesis of constituentsduring the dark period. On exposure to light most components(apart from DNA) showed some continuing net synthesis, but inthe majority of cases there was a short part of this light syntheticperiod in which there was very active net synthesis. The activesynthetic period was frequently immediately prior to the onsetof division. DNA synthesis occurred only in the 6 h precedingdivision. The major period of net protein synthesis occurredwhilst the divided cells were separating, at the commencementof the light period. The other factors studied were RNA, carbohydrate,chlorophyll a, chlorophyll b, carotenoids, phospholipids, acid-solublecompounds, and phosphorus uptake. The results are discussed.     

Macromolecular syntheses and the course of cell cycle events in the chlorococcal algaScenedesmus quadricauda under nutrient starvation: Effect of phosphorus starvation

Daughter cells of the chlorococcal algaScenedesmus quadricauda incubated under photosynthesizing conditions in a phosphate-free medium accomplished one cell cycle but divided into a lower number of daughter cells compared to the control. RNA synthesis was restricted early in the cell cycle while protein synthesis was retarded as compared to the control only at the end of the cycle. The number of DNA replication rounds (and consequently the number of divisions) was reduced in proportion to the lower content of RNA per cell. Daughter cells produced by phosphorus-starved mother cells and grown further in a phosphorus free medium performed no net RNA, DNA and protein synthesis within the period corresponding to the duration of control cell cycle an o were unable to develop. They accumulated, however, about half the amount of starch found in normally developed mother cells. In a complete medium, the phosphorus-starved daughter cells resumed macromolecular syntheses with a lag of about 5 h. Thereafter, their development and reproductive processes were comparable to those in a healthy population. A similar course of recovery was obtained with starved daughter cells exposed to light in phosphorus-free medium for the period corresponding to one cell cycle. Thanks to the large amount of starch accumulated in these cells, they were able to run through an entire cell cycle in the dark after being supplied with phosphorus. The first response to phosphorus withdrawal from the nutrient medium was the restriction of RNA synthesis. This occurred in spite of the fact that phosphorus reserves in the cell were still abundant, which suggests an intimate link between the supply of exogenous phosphorus to the cell and RNA synthesis.     

Growth and synthesis of nucleic Acid and protein by excised radish cotyledons

Nutritional and light requirements for growth and synthesis of RNA, DNA, and protein by cotyledons excised from 5-day-old seedlings of Raphanus sativus L. were investigated, and the course of synthesis was followed through the cell cycle. The minimum requirements for a net increase in nucleic acid and protein were sugar, nitrate, and light. The cotyledons used nitrite at low concentration, but not ammonium ion. Light was required for preliminary steps in synthesis of RNA, DNA, and protein, but the actual polymerization reactions occurred in the dark. The cotyledons contained sufficient endogenous growth factors for about half of the cells to complete 1 cycle on a medium of 1% sucrose, 80 mm KNO₃. The increase in DNA was limited to about 50% and was accompanied by a comparable increase in cell number. Fresh weight, RNA, and protein tended to increase in proportion to DNA. Growth of the isolated cotyledons commenced with cell enlargement. RNA began to increase after about 4 hours, DNA after about 12. The major increase in protein also began at about 12 hours. The maximum rate of increase for all 3 occurred between 12 and 16 hours. Cell counts indicated that by 28 hours most of the cells which had replicated DNA had also completed cell division.     

CHANGES IN ENDOGENOUS CYTOKININ CONCENTRATIONS IN CHLORELLA (CHLOROPHYCEAE) IN RELATION TO LIGHT AND THE CELL CYCLE1

Endogenous cytokinins were quantified in synchronized Chlorella minutissima Fott et Novákova (MACC 361) and Chlorella sp. (MACC 458) grown in a 14:10 light:dark (L:D) photoperiod. In 24 h experiments, cell division occurred during the dark period, and cells increased in size during the light period. Cytokinin profiles were similar in both strains, consisting of five cis‐zeatin (cZ) and three N⁶‐(2‐isopentenyl)adenine (iP) derivatives. Cytokinin concentrations were low during the dark period and increased during the light period. In 48 h experiments using synchronized C. minutissima (MACC 361), half the cultures were maintained in continuous dark conditions for the second photoperiod. Cell division occurred during both dark periods, and cells increased in size during the light periods. Cultures kept in continuous dark did not increase in size following cell division. DNA analysis confirmed these results, with cultures grown in light having increased DNA concentrations prior to cell division, while cultures maintained in continuous dark had less DNA. Cytokinins (cZ and iP derivatives) were detected in all samples with concentrations increasing over the first 24 h. This increase was followed by a large increase, especially during the second light period where cytokinin concentrations increased 4‐fold. Cytokinin concentrations did not increase in cultures maintained in continuous dark conditions. In vivo deuterium‐labeling technology was used to measure cytokinin biosynthetic rates during the dark and light periods in C. minutissima with highest biosynthetic rates measured during the light period. These results show that there is a relationship between light, cell division, and cytokinins.     

Evidence against the involvement of DNA synthesis in phytochrome-mediated photomorphogenesis

In order to clarify the relationship between photomorphogenesis and DNA replication we investigated the effect of continuous far-red or white light on the synthesis of DNA in the cotyledons and the hypocotyl of mustard seedlings between 36 and 108 h after sowing. The total DNA content of the cotyledons (about 2.2 pg cell^-1) did not significantly change during this period although long-term labeling experiments revealed newly synthesized DNA of nuclear, plastid, and mitochondrial origin. Light had no detectable effect on total DNA content and on the labeling of either DNA fraction. Histoautoradiography indicated that nuclear DNA synthesis was exclusively localized in dividing stomatal cells and in sieve tube companion cells undergoing endopolyploidization. The DNA content of the hypocotyl increased continuously but likewise showed no detectable effect of light. It is concluded that cell growth and differentiation during photomorphogenesis is independent of DNA synthesis.Abbreviation DABA 3,5-diaminobenzoic acid     

The Effect of Benzimidazole and Red Light on the Senescence of Detached Leaves of Oryza sativa cv. Ratna

Excised rice leaves (Oryza sativa L. cv. Ratna) werefloated on a 10^–3M solution of benzirnidazole under dark or continuous red light. Compared to the water control a degradation of chlorophyll, protein, RNA, DNA and a decrease in the activity of alkaline inorganic pyrophosphatase was delayed at the same time as an increase of α-amino nitrogen and the activity of acid inorganic pyrophosphatase occurred, Benzimidazole was more effective under red light than in the dark in retarding senescence. The possible role of inorganic pyrophosphatases is discussed with respect to biosynthesis during leaf senescence.