Preferential digestion of chloroplast DNA in male gametangia during the late stage of gametogenesis in the anisogamous algaBryopsis maxima

Summary The fate of chloroplast nuclei (cp-nuclei) and mitochondrial nuclei (mt-nuclei) was followed during gametogenesis in male and female coenocytic thalli in the anisogamous algaBryopsis maxima by epifluorescence microscopy, after staining with 4,6-diamidino-2-phenylindole (DAPI), by quantification of chloroplast DNA (cp-DNA) by fluorimetry using a video-intensified, photon-counting system (VIMPICS), and by CsCl density gradient centrifugation. The male and female coenocytic thalli, 48 h before the release of gametes, contain a large number of chloroplasts, each of which is larger in size than the cell nucleus and the mitochondria and contains about 150 cp-nuclei. The size of each chloroplast in the female and male gametangia decreases markedly during gametogenesis as a result of continuous divisions till about 10 h before the release of gametes and, eventually, the numbers of cp-nuclei per chloroplast in the male and female gametangia fall to about 20 and 5, respectively. Two hours later, as the preferential digestion of cp-DNA in the male gametangium occurs, the number of cp-nuclei in the chloroplast of each male gamete falls to zero while the number of cp-nuclei in female gamete does not change, even after release of female gametes. Several mt-nuclei are observed in all of the female gametes. By contrast, the mt-nuclei in the bulk of the male gametes disappear but those in a few gametes remain. The profiles after CsCl density gradient centrifugation of DNAs extracted from male and female plants and gametes support the cytological data. The results suggest that the preferential digestion of cp-DNA in male plants occurs about 8 h before the release of gametes and that there is differential digestion of cp-DNA and mitochondrial DNA (mt-DNA).     

Behavior of chloroplasts and chloroplast nuclei during spermatogenesis in the fern,Pteris vittata L.

Summary The fate of the chloroplasts and chloroplast nuclei (cp-nuclei) was followed during spermatogenesis in the fernPteris vittata L. by epifluorescence microscopy after staining with 4-6-diamidino-2-phenylindole (DAPI) and by quantitation of chloroplast DNA (cp-DNA) by fluorimetry using a video intensified microscope photon counting system (VIMPICS). The spores were grown on solid medium that contained antheridiogen (An_ptd), and formed an antheridium initial on the protonema cell. The antheridium initial divided and produced 16 spermatocytes and 3 surrounding cells. The chloroplasts in the spermatocytes decreased in volume as cell division was repeated, until finally the volume of each chloroplast was 1/15 of that of the primary chloroplasts. The DNA content of the chloroplasts was also reduced to 1/5 of the original value and when the sperm matured, the fluorescence of cp-DNA disappeared. In the 16-cell spermatocyte, the recognition of the fluorescence of chlorophyll in the chloroplasts with a green excitation filter became difficult. But, the plastids could be observed until the final stage of the sperm. From these observations, it appears that there are two steps in the metamorphosis of chloroplasts during spermatogenesis in the fern. The first step involves the decrease in the volume of chloroplasts, accompanied by reduction of the DNA content, and the second step involves the change of the physical state of chloroplasts to amyloplasts and the disappearance of the cp-DNA from the amyloplasts.     

Destruction of Chloroplast Nuclei of the Male Gamete by Calcium and Nuclease C In a Cell Model of Chlamydomonas reinhardtii

A cell model was prepared for each mating type cell of Chlamydomonasreinhardtii in order to investigate the molecular mechanismfor the maternal inheritance of chloroplast genes. Chloroplastnuclei (cp-nuclei) in the chloroplast were well preserved. Therefore,their disappearance after a change in the external medium couldbe monitored by high resolution fluorescence microscopy afterstaining the cells with 4'-6-diamidino-2-phenyl indole. Thenumber of cp-nuclei in the cell model diminished when Ca²⁺ wasadded to a cell suspension. Presumably, this ion activated theendogenous nuclease C that remained in the cytoplasm, thus settingup the destruction of chloroplast DNA. Almost all disappearedwhen Ca²⁺ was given together with nuclease C. From the kineticsof the disappearance of cp-nuclei after combining the cell modelwith nuclease C, we found that male gametes begin to lose theircp-nuclei after 20 min, whereas female gametes lose them after40 min. Furthermore, as a culture aged, the female cp-nucleibecame resistant to the destructive attack by nuclease C, butthe male cp-nuclei remained sensitive. (Received October 8, 1984; Accepted January 21, 1985)     

Studies on Plastid-Nuclei (Nucleoids) in Nicotiana tabacum L. IV. Association of Chloroplast-DNA with Proteins at Several Specific Sites in Isolated Chloroplast-Nuclei

When chloroplast-nuclei (cp-nuclei), isolated from tobacco mesophyllprotoplasts, were directly digested by restriction enzymes andanalyzed by agarose gel electrophoresis, the migration of severalrestriction fragments was greatly inhibited during the electrophoresis.When the digested cp-nuclei were treated with SDS and proteinaseK prior to electrophoresis, all the restriction fragments migratednormally and the electrophoretic pattern was the same as thatgenerated by similarly restricted, purified chloroplast-DNA(cp-DNA). Use of a newly developed method, "reciprocal electrophoresis",proved that these fragments were bound so tightly to certainDNA-binding protein(s) that the fragments were trapped in thegel slot. These fragments corresponded to at least four sitesof the cp-DNA. By contrast, when proplastid-nuclei (pp-nuclei)isolated from tobacco cultured cells were analyzed in the sameway, no restriction fragments were trapped in the gel slot.These results, taken together, suggest that a DNA-binding protein(s)is present in cp-nuclei, but not in pp-nuclei, and binds tightlyto the four sites of the cp-DNA. Therefore, the molecular architectureof cp-nuclei is clearly different from that of pp-nuclei. TheDNA-binding protein(s) may have some function(s) specific tocp-nuclei. ⁴ Present address: Department of Biotechnology, Faculty of Technology,Tokyo University of Agriculture and Technology, Nakamachi, Koganei,Tokyo, 184 Japan (Received July 30, 1990; Accepted November 29, 1990)     

Reduction of Chloroplast DNA Content in Solanum nigrum Suspension Cells by Treatment with Chloroplast DNA Synthesis Inhibitors

Suspension cell cultures of Solanum nigrum were grown in the presence of six different chloroplast DNA synthesis inhibitors in order to determine whether the pool size of chloroplast DNA (cpDNA) could be selectively reduced relative to the nuclear DNA content. One of the effects of the inhibitors was a reduction in cell growth and viability. Cell growth (fresh weight) was reduced 50% (in 8 day cultures) by: 100 micromolar bisbenzimide, 8 micromolar ethidium bromide, 0.3 micromolar 5-fluordeoxyuridine (Fudr), 200 micromolar nalidixic acid, 30 micromolar novobiocin, or 10 micrograms per milliliter rifampicin. At these concentrations, three of the inhibitors, ethidium bromide, Fudr, and rifampicin, also substantially reduced the viability of the cultures. Analyses of the chloroplast and nuclear DNA content per gram fresh weight by dot blot hybridizations indicated that the reduction of cpDNA content was greatest at inhibitor concentrations which reduced cell growth by more than 50% but this depended on the culture conditions. For example, the two DNA gyrase inhibitors, nalidixic acid and novobiocin, were more effective in lowering cpDNA content in cultures which were transferred (2 × 4 days) once during the eight day incubation. Because several inhibitors were toxic to cell growth, the DNA content of treated cells was also determined on the basis of cell (protoplasts) number. Analyses of nuclear and cpDNA content per cell for each treatment indicated that only the DNA gyrase inhibitors, nalidixic acid, and novobiocin reduced cpDNA content. Neither inhibitor reduced nuclear DNA content. These results suggest that DNA gyrases participate in cpDNA replication. The selective reduction of cpDNA content in regeneratable cultures may facilitate the generation and selection of cpDNA mutants or transformants from higher plants.     

Chloroplast DNA in Expanding Spinach Leaves

The proportion of chloroplast DNA in total DNA from spinachleaves has been measured using the second order reassociationkinetics of a ³H-labelled chloroplast DNA probe in total DNAextracts. There was no significant difference between the proportionof chloroplast DNA in the basal and distal halves of 2 cm leavesand in the distal halves of 5, 8, and 10 cm leaves. The meanof all the observations was 21.1 ± 0.7%. There was littlechange in the average total DNA content of cells from any ofthe leaves but cells from larger leaves contained 130–170chloroplasts while cells from the basal half of 2 cm leavescontained about 20 chloroplasts which were smaller than thosefrom the larger leaves. Consequently the average number of copiesof the plastome per chloroplast in large leaves was about 30(5 x 10^–15 g DNA) and in the smaller chloroplasts in thebase of 2 cm leaves was 200 (32 x 10^–15 g DNA). Stainingwith the DNA fluorochrome 4, 6-diamidino-2 phenyl indole (DAPI)showed 10–15 plastid nucleoid areas in chloroplasts oflarger leaves, suggesting there are 2–3 copies of theplastome per plastid nucleoid.     

Photoperiodic Regulation of Cell Division and Chloroplast Replication in Heterosigma akashiwo

Cell division and chloroplast replication in Heterosigma akashiwo(Hada) Hada occurred as separate synchronous events during thecell cycle when cells were subjected to light-dark regimes.Under three different photoperiodic cycles of 10L/14D (10 hlight/14 h dark), 12L/12D or 16L/8D, cell division began athour 19–20 and finished at hour 23–26 after theonset of the light period, while chloroplast replication beganat hour 20–22 after the onset of the dark period. Almostall the cells divided only once in the 12L/12D cycle. The rateof increase in chloroplast number during one light-anddark cyclewas always equal to that in cell number in every photoperiodexamined. Light was essential for both cell division and chloroplast replication,but the minimum light period necessary for each event differed.When the light period was shorter than 6 h, no cell divisionoccurred; when it was shorter than 3 h, no chloroplast replicationoccurred. (Received February 26, 1987; Accepted June 17, 1987)     

Effect of Benzyladenine on Diurnal Changes in DNA Content per Chloroplast and Chloroplast Replication in Intact Bean Leaves

The effect of benzyladenine (BA) on the diurnal changes in DNAand Chl contents per chloroplast and chloroplast replicationin primary leaves of bean plants (Phaseolus vulgaris L.) grownunder a 16 h light/8 h dark cycle was studied. Experiments weremade on primary leaves in the early expansion phase, where celldivision had been completed but chloroplasts were replicating.In untreated controls, chloroplast number, Chl content and freshweight per leaf showed daily periodic changes. Chl content perchloroplast increased in the light period every day, and freshweight per leaf increased most rapidly in the early dark period.Chloroplast number per leaf increased rapidly in the early darkperiod on day 9, though the increase began a little earlierand was less sharp on days 8 and 10. During these periods, DNAcontent per chloroplast was decreasing due to chloroplast divisionas chloroplast DNA (ctDNA) per leaf remained unchanged throughoutthe experimental period. BA induced increases in Chi contentper chloroplast, ctDNA content and fresh weight per leaf within6 h of its application, regardless of whether it was appliedat or 10 h after the beginning of the light period. Applicationof BA at 10 h in the light period shifted the start of chloroplastreplication by 6 h compared to that in untreated controls. However,when BA was applied at the beginning of illumination, the startof chloroplast replication showed the same relative change intime as above. 5-Fluorodeoxyuridine (5-FdU) promptly preventedBA-induced increase in Chl content and chloroplast number perleaf as well as ctDNA content per leaf.     

Cellular Levels of Ribulose 1,5 Bisphosphate Carboxylase and Chloroplast Compartment Size in Wheat Mesophyll Cells

Pyke, K. A. and Leech, R. M. 1987. Cellular levels of ribulose1,5 bisphosphate carboxylase and chloroplast compartment sizein wheat mesophyll cells.—J. exp. Bot. 38: 1949–1956. The amount of the photosynthetic enzyme ribulose 1,5 bisphosphatecarboxylase (RUBISCO),as determined in mesophyll cells in primarywheat leaves was related to the size of the chloroplast compartmentwithin the cell for wheat species of three ploidy levels. Asimilar comparison was made for several genotypes of the hexaploidbreadwheat Triticum aestivum. Estimation of total chloroplastvolume per mesophyll cell was made assuming chloroplasts tobe oblate spheroid in shape. A significant correlation was found between the amount of RUBISCOper cell and the total chloroplast volume per cell for diploid,tetraploid and hexaploid wheat species. A significant correlationbetween cellular RUBISCO level and total chloroplast volumeper cell was also observed for a range of genotypes of the hexaploidT. aestivum but these genotypes of T. aestivutn accumulate agreater amount of RUBISCO per unit chloroplast volume than doany other wheat species. For these genotypes of T. aestivumthe stromal concentration of RUBISCO was estimated at 0·5mol m^–3 with a ribulose Msphosphate binding site concentrationof 4·0 mol m^–3. These results are discussed with respect to a gene dosage hypothesisto explain the accumulation of RUBISCO in leaf mesophyll cells. Key words: Ribulose, bisphosphate carboxylase, wheat chloroplasts, mesophyll cells     

Factors Affecting Chloroplast Replication in Spinach

Chloroplast replication has been studied in discs cut from thebase of young spinach leaves and cultured on sterile nutrientagar. In discs grown in a growth cabinet chloroplast numbersper cell increased logarithmically with time over a 7-day cultureperiod. Chloroplast replication proceeds in a similar way incultured discs and in intact leaves. Cytokinins do not affect chloroplast replication in this systembut they stimulate the fresh-weight growth of discs. Chloroplastreplication is temperature dependent, having an optimum at 25°C. By contrast chloroplast size is at a maximum in discscultured at 12 °C. Light stimulates chloroplast replication, a linear relationshipoccurring between chloroplast number per cell and the dailyquantity of light given to discs up to a saturating value of250 J d^–1. Daylength does not affect chloroplast formationin spinach. In a number of experiments a general relationship was establishedbetween chloroplast number per cell and cell size but no evidenceis available to suggest that this correlation is causal. Theresults of experiments in which discs were transferred fromdark to light suggest that some of the events which precedechloroplast replication may occur at similar rates in both lightand dark.     

Studies on Plastid-Nuclei (Nucleoids) in Nicotiana tabacum L. III. Isolation of Chloroplast-Nuclei from Mesophyll Protoplasts and Identification of Chloroplast DNA-Binding Proteins

We have developed a method of isolating morphologically intactchloroplast-nuclei (nucleoids) in large quantities from mesophyllprotoplasts of Nicotiana tabacum L. cv. Bright Yellow-2. The isolated chloroplast-nuclei (cp-nuclei) were dispersed bythe treatments with proteinase K, 2 M NaCl and 2 M KCL, whichsuggested that the cp-nuclei are compactly organized by an electrostaticinteraction between the chloroplast-DNA and some protein(s).However, the four proplastid DNA-binding proteins identifiedpreviously (Nemoto et al. 1988) were not found in the cp-nuclei,and a different set of DNA-binding proteins (mol wt: 35 kDa,28 kDa and 26 kDa) was detected in the cp-nuclei by a DNA-bindingassay. On the other hand, the chloroplast-DNA was not differentfrom the proplastid-DNA. These findings indicate that the cp-nuclei are constituted fromthe plastid-DNA and the chloroplast-specific DNA-binding proteins.This suggests that the DNA-binding proteins in proplastids aredynamically replaced with the chloroplast DNA-binding proteinsduring the differentiation of plastids from proplastids to chloroplasts.The change of DNA-binding proteins may be involved in the morphologicalchange of plastid-nuclei and/or the regulation of plastid-DNAreplication and gene expression during the differentiation processof plastids. ⁶Present address: Department of Biotechnology, Faculty of Technology,Tokyo University of Agriculture and Technology, Nakamachi, Koganei,Tokyo, 184 Japan ⁷Present address: Department of Biology, Faculty of Science,University of Tokyo, Hongo, Tokyo, 113 Japan (Received April 4, 1990; Accepted May 18, 1990)     

Isolation and phenotypic characterization ofChlamydomonas reinhardtii mutants defective in chloroplast DNA segregation

Summary Each wild-typeChlamydomonas reinhardtii cell has one large chloroplast containing several nuclei (nucleoids). We used DNA insertional mutagenesis to isolate Chlamydomonas mutants which contain a single, large chloroplast (cp) nucleus and which we namedmoc (monokaryotic chloroplast). DAPI-fluorescence microscopy and microphotometry observations revealed thatmoc mutant cells only contain one cp-nucleus throughout the cell division cycle, and that unequal segregation of cpDNA occurred during cell division in themoc mutant. One cell with a large amount of cpDNA and another with a small amount of cpDNA were produced after the first cell division. Unequal segregation also occurred in the second cell division, producing one cell with a large amount (about 70 copies) of cpDNA and three other cells with a small amount (only 2–8 copies) of cpDNA. However, most individualmoc cells contained several dozen cpDNA copies 12 h after the completion of cell division, suggesting that cpDNA synthesis was activated immediately after chloroplast division. In contrast to the cpDNA, the mitochondrial (mt) DNA of themoc mutants was observed as tiny granules scattered throughout the entire cell. These segregated to each daughter cell equally during cell division. Electron-microscopic observation of the ultrastructure ofmoc mutants showed that a low-electron-density area, which was identified as the cp-nucleus by immunoelectron microscopy with anti-DNA antibody, existed near the pyrenoid. However, there were no other structural differences between the chloroplasts of wild-type cells andmoc mutants. The thylakoid membranes and pyrenoid were identical. Therefore, we propose that the novelmoc mutants are only defective in the dispersion and segregation of cpDNA. This strain should be useful to elucidate the mechanism for the segregation of cpDNA.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon-counting system     

Influence des pretraitements par KCl et des rapports Ca/Na du milieu sur la croissance de vitroplants de tomate en milieu NaCl

The 18-day-old tomato vitroplants were obtained in axenic conditions by culture of expiants (including the terminal bud and the last internode of the stem) on agar-agar nutritive medium with 0 or 75 mM NaCl. The growth and the mineral content of the vitroplants were compared when the expiants were grown on media either with low or high K/Na and Ca/Na ratios, or with low K/Na and Ca/Na ratios after pretreatments of expiants by KC1, NaCl or CaCl₂ (from 0 to – 4.5 bar). The KCl pretreatment (-1.1 bar) during one day brings about an increase in vitroplant growth greater than that produced by a high Ca/Na ratio medium. The Cl accumulation was similar in expiants pretreated by KCl or NaCl. Ion content per gram of fresh matter was similar in 18-day-old vitroplants pretreated by KCl, NaCl or CaCl₂; the Na accumulation by KC1 pretreated vitroplants was not lower than that of 18-day-old vitroplants grown on a high Ca/Na ratio medium. These results show the relation between Na content of expiants and the growth of vitroplants in a NaCl medium.     

Characteristics of Photosynthate Partitioning during Chloroplast Development in Avena Leaves

The first leaves (40 millimeters long) of 4-day-old light-grown Avena sativa L. cv Victory I seedlings contained a complete age sequence of cells from the base to the tip, and within these tissues all stages of chloroplast development could be observed. Although chloroplasts underwent progressive development, a marked increase in number of thylakoids per granum, in chloroplast volume, and in chlorophyll content occurred in the region between 20 and 30 millimeters from the base. Photosynthetic CO₂ fixation (per unit chlorophyll) increased markedly during chloroplast development and closely followed structural changes in chloroplasts. It was also found that the partitioning of photosynthates differed greatly in the segment from 30 to 40 millimeters (at the tip of the leaf) compared with the segment nearer to the leaf base, although both total ¹⁴CO₂ fixation and chlorophyll content per segment did not change significantly along the length of the leaves. As the thylakoid system reached full maturation, partitioning of photosynthates into sucrose increased but partitioning decreased into starch, lipids, and phosphorylated intermediates.     

Chloroplast autonomy in pigment synthesis

Summary An experimental design developed usingAcetabularia permits a novel approach to studies of cytoplasmic genetic functions. The site of the genes for the enzymes of the plastid pigment pathways were examined by 1. determining pigment content per cell and per chloroplast in intact cells and during long periods of enucleate cell growth, 2. comparing pigment synthetic activities of plastids isolated from intact and enucleate cells at various times postenucleation and 3. comparing the ability of intact and enucleate cells to modulate pigment content in response to various light regimens.Intact cells grow and increase their pigment content exponentially. Enucleate cells grow at the control rate for several weeks and increase their chloroplast number and pigment content proportionally. Pigment content per plastid remains constant in both intact and enucleate cells. Pigment synthesis in isolated chloroplasts from enucleate cells is normal up to 65 days post-enucleation when compared with isolated chloroplasts from intact control cells. Enucleate cells differentially modulate their pigment content in response to various light regimens in a manner indistinguishable from normal cells.The problems in interpreting these and other results are discussed and it is concluded that plastid autonomy in pigment synthesis is the simplest explanation.     

Light-Triggered Changes of Membrane Potential in Cells of Anthoceros punctatus and their Relation to Activation of Chloroplast ATPase

Post-illumination transients of the membrane potential wererevealed in cells of Anthoceros punctatus upon short (1–2s) irradiation with photosynthetically active light. An initialfast depolarization of the cell by 20–30 mV after a lagperiod of 2 s and a subsequent slow repolarization were recordedwith micro-electrodes positioned both in the chloroplast andin transparent parts of the cell. The potential changes of similarkinetics were also observed upon continuous illumination. Post-illuminationpotential changes were abolished by 3-(3, 4-dichlorophenyl)-l,l-dimethylurea and dicyclohexylcarbodiimide and were stimulatedby the addition of NH₄C1. It is assumed that the light-triggeredpotential change across the plasmalemma of A. punctatus aredetermined by the light activation of chloroplast ATPase. Thisassumption is further supported by observations of post-illuminationtransient increase of the chlorophyll fluorescence in preparationsof A. punctatus. Key words: Cell membrane potential, Chloroplast, Photosynthesis     

Chloroplast Nucleoids during Greening of Cyanidium caldarium M-8 Type

Cyanidium caldarium M-8 type grown in the dark was illuminatedfor 3 days, and changes of its cell and cell organelle structuresand of photosynthetic activity were observed quantitatively.Dark grown (DG) cells showed no photosynthetic activity andno phycocyanin. During 3 day illumination they fully recoveredtheir photosynthetic activities as measured by Hill reaction,and also synthesized chlorophyll a and phycocyanins. Sizes ofcell, cell nucleus, chloroplast and its nucleoid observed byfluorescence microscopy after staining with DAPI increased simultaneouslyupon illumination. The chloroplast and its ring shaped nucleoidsizes increased especially rapidly, concomitant with the recoveryof Hill activity. In fully recovered cells after 3 days, a goodcorrelation was found among the sizes of cells, chloroplastsand chloroplast nucleoids. ( Revision received June 28, 1986. Accepted December 23, 1986)     

Chloroplast assemblage by mechanical stimulation and its intercellular transmission in diatom cells

Summary By touching a cell ofPleurosira laevis, a centric diatom, the chloroplasts in the cortical cytoplasm immediately migrate to the nuclear cytoplasm which is located at the center of the cell. The time required for migration, is about 10 s and that for restoration is about 15 min. The touch could be replaced by an electric stimulation. A 7 V pulse for 1 ms was required for the induction of chloroplast assemblage. To obtain chloroplast assemblage in 50% of the cells, the amount of time and voltage required were inversely proportional. Higher concentrations of KCl also induced chloroplast assemblage. Neither contact nor electric stimulation in Ca²⁺ free medium induced chloroplast assemblage. Verapamil, a Ca²⁺ channel blocker, in medium suppressed the chloroplast assemblage. Ca²⁺ ionophore caused the chloroplast assemblage. These results suggested that the depolarization of the plasma membrane was a trigger of the induction of chloroplast assemblage and the influx of Ca²⁺ to the cytoplasm through the channel was essential for this. The chloroplast assemblage was transmitted successively to the other cells of the filamentous colony, though these cells were linked only by a mucilaginous substance. Furthermore, the transmission occurred in cells which were placed separately. The maximum distance possible for this jumping transmission was 200 m in the culture medium. To search for the release of any chemical transmitter, contact stimulation was applied to one cell at the middle of a filamentous colony which was placed in water streaming along the axis of the filament. Chloroplast assemblage was induced more in downstream cells than in upstream ones. This result suggested the existence of some substance which mediated the transmission of the stimulation.     

Isolation of the cell-nuclear,mitochondrial, and chloroplast DNA from the ultra-small eukaryoteCyanidioschyzon merolae

Summary The amounts of cell-nuclear DNA (cl-DNA), mitochondrial DNA (mt-DNA) and chloroplast DNA (cp-DNA) inCyanidioschyzon merolae were estimated by using a video-intensified microscope (VIM) system.C. merolae had the smallest amount of cell-nuclear DNA among eukaryotes. The results show that a cell-nucleus, a mitochondrion and a chloroplast contain an average 8.0×10³kbp, 1.6×10³kbp, and 5.0×10³kbp, respectively. To confirm these results, cl-DNA, mt-DNA, and cp-DNA were isolated from cells by density centrifugation on Hoechst 33258/CsCl after density centrifugation on ethidium bromide/CsCl. The amounts of cl-DNA, mt-DNA, and cp-DNA obtained from the bands supported the data shown by the VIM-system. The cytochemical and biochemical characteristics were compared with those ofCyanidium caldarium RK-1 andC. caldarium Forma A. The values of cl-DNA and cp-DNA ofC. merolae were about 1.716 and 1.709, respectively. The order in density was different from that ofC. caldarium Forma A but very similar to that ofC. caldarium RK-1. However, the restriction patterns of cp-DNA inC. merolae differed from those ofC. caldarium RK-1.     

Chloroplast Dedifferentiation in Mechanically Isolated Asparagus Cells during Culture Initiation

Mechanically isolated asparagus (Asparagus officinalis) mesophyll cells dedifferentiate and divide when cultured in the dark in a medium containing sucrose. A strong correlation was observed between the onset of cell division and a loss of photosynthetic capacity. For the first 8 to 9 d of culture, there was no change in chloroplast size or morphology. However, following this period, the chloroplasts divided to form smaller proplastid-like structures. The gross chlorophyll content of the cell population did not change, suggesting that the loss of photosynthetic potential was not by senescence. Northern analysis showed that mRNA of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase was undetectable within 1 d postisolation, which was quicker than in dark-treated plants. The mRNA of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase decreased to low levels within 2 d of cell isolation. Both the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase protein showed a gradual reduction in abundance, falling to basal levels by days 6 to 7, which coincided with the onset of rapid cell division. A similar trend was observed with chloroplast rRNA molecules, which decreased to basal levels by day 6 in culture.