Chloroplast nucleoids are highly dynamic in ploidy,number, and structure during angiosperm leaf development

Chloroplast nucleoids are large, compact nucleoprotein structures containing multiple copies of the plastid genome. Studies on structural and quantitative changes of plastid DNA (ptDNA) during leaf development are scarce and have produced controversial data. We have systematically investigated nucleoid dynamics and ptDNA quantities in the mesophyll of Arabidopsis, tobacco, sugar beet, and maize from the early post‐meristematic stage until necrosis. DNA of individual nucleoids was quantified by DAPI‐based supersensitive epifluorescence microscopy. Nucleoids occurred in scattered, stacked, or ring‐shaped arrangements and in recurring patterns during leaf development that was remarkably similar between the species studied. Nucleoids per organelle varied from a few in meristematic plastids to >30 in mature chloroplasts (corresponding to about 20–750 nucleoids per cell). Nucleoid ploidies ranged from haploid to >20‐fold even within individual organelles, with average values between 2.6‐fold and 6.7‐fold and little changes during leaf development. DNA quantities per organelle increased gradually from about a dozen plastome copies in tiny plastids of apex cells to 70–130 copies in chloroplasts of about 7 μm diameter in mature mesophyll tissue, and from about 80 plastome copies in meristematic cells to 2600–3300 copies in mature diploid mesophyll cells without conspicuous decline during leaf development. Pulsed‐field electrophoresis, restriction of high‐molecular‐weight DNA from chloroplasts and gerontoplasts, and CsCl equilibrium centrifugation of single‐stranded and double‐stranded ptDNA revealed no noticeable fragmentation of the organelle DNA during leaf development, implying that plastid genomes in mesophyll tissues are remarkably stable until senescence.     

DNA Levels in Dividing and Developing Plastids in Expanding Primary Leaves of Avena sativa

The amounts of plastid DNA in the primary leaves of 4-d-oldlight- and dark-grown seedlings of Avena sativa were measuredby microspectrofluorometry using the DNA-fluorochrome DAPI (4',6-diamidino-2-phenylindole). In the light-grown primary leaves (40–45 mm long) therewas a marked increase in DNA level per plastid from 10.2 to18.5 ? 10^–15 g between 2.0 mm and 10 mm from the leafbase, resulting from the rate of plastid DNA synthesis beinghigher than the rate of plastid division. Beyond 30 mm the plastidDNA level was reduced to 14 ? 10^–15g due to chloroplastdivision rates being higher than the rate of plastid DNA synthesis,while from 20 mm plastid DNA levels were constant at 2.2 ? 10^–12g per cell, which corresponds to 16000 plastome copies per cell. Observations of dark-grown leaves establish that, in Avena,light is not necessary for plastid division and the dark-grownleaf cells accumulate higher amounts of plastid DNA than light-grownleaf cells. Plastid nucleoids showed a change of distribution after completionof plastid DNA synthesis in light-grown leaves. A change inthe distribution of plastid nucleoids was also observed duringthe greening of etioplasts of dark-grown leaves while plastidDNA level remained constant. Such changes in plastid nucleoiddistribution appear to be independent of plastid DNA synthesisand correlate with the formation of grana stacks. Key words: Avena sativa, microspectrofluorometry, plastid DNA     

Heterogeneity of chloroplasts in Acetabularia mediterranea. Heterogeneous distribution and morphology of chloroplast DNA

A high percentage of chloroplasts in the siphonaceous green alga Acetabularia mediterranea lacks DNA: staining with the sensitive DNA-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) revealed that DNA was present only in 47–51% of the total plastid population. The distribution of DNA-containing chloroplasts appeared heterogeneous, exhibiting an apicobasal gradient. DNA could be detected in 64, 46, 35, and 36% of the plastids from the apical, subapical, middle, and basal part of the cell, respectively. DNA morphology also exhibited heterogeneity. Three types of nucleoid were distinguished: (1) round compact nucleoids; (2) long thin nucleoids characteristic of chloroplasts of the cell apex and the whorls; and (3) elaborate nucleoids appearing to consist of several subunits, which were more typical of the middle and basal part of the cell. On the basis of the nucleoid morphology and the decrease in DNA-containing plastids from the apex towards the basis of the cell, we propose a model for the development of plastids lacking DNA in relation to chloroplast replication.     

Visualization of plastid nucleoids in situ using the PEND-GFP fusion protein

Plastid DNA is a circular molecule of 120-150 kbp, which is organized into a protein-DNA complex called a nucleoid. Although various plastids other than chloroplasts exist, such as etioplasts, amyloplasts and chromoplasts, it is not easy to observe plastid nucleoids within the cells of many non-green tissues. The PEND (plastid envelope DNA-binding) protein is a DNA-binding protein in the inner envelope membrane of developing chloroplasts, and a DNA-binding domain called cbZIP is present at its N-terminus. We made various PEND-green fluorescent protein (GFP) fusion proteins using the cbZIP domains from various plants, and found that they were localized in the chloroplast nucleoids in transient expression in leaf protoplasts. In stable transformants of Arabidopsis thaliana, PEND-GFP fusion proteins were also localized in the nucleoids of various plastids. We have succeeded in visualizing plastid nucleoids in various intact tissues using this stable transformant. This technique is useful in root, flower and pollen, in which it had been difficult to observe plastid nucleoids. The relative arrangement of nucleoids within a chloroplast was kept unchanged when the chloroplast moved within a cell. During the division of plastid, nucleoids formed a network structure, which made possible equal partition of nucleoids.     

Changes in distribution of nucleoids in developing and dividing chloroplasts and etioplasts ofArena sativa

Summary Nucleoid distribution in chloroplasts and etioplasts at the different developmental stages was examined with the first leaves ofAvena sativa by using a DNA-specific fluorescent probe, 46-diamidino-2-phenylindole (DAPI). In light-grown first leaves, three types of plastid nucleoid distribution were recognized. 1. Peripheral distribution in undeveloped chloroplasts which contain only a few thylakoids in the middle region of the leaf sheath. 2. Ring-like arrangement along the rim of developing and dividing young chloroplasts, of which grana were composed of four to eight layers of thylakoids, at the base of the leaf blade. The plane of the nucleoids' ring is in parallel with the face of the thylakoids. 3. Scattered distribution of 10 to 20 discrete spherular nucleoids in the stroma of fully developed chloroplasts, of which grana were composed of up to 20 thylakoids, in the regions of the middle and the tip of the leaf blade. In dark-grown first leaves two types were recognized. 1. Peripheral distribution in developing and dividing young etioplasts in the leaf sheath and the base of the leaf blade. 2. Scattered distribution of 10 or more discrete spherular nucleoids in fully developed etioplasts, containing extended prothylakoids, in the regions of the middle and the tip of the leaf blade. Ring-like arrangement of nucleoids was not observed in any etioplasts. The results indicates that spatial arrangement of plastid nucleoids dynamically changes in close relationship with the development of the inner membrane systems of plastids.     

Dynamics of localization and protein composition of plastid nucleoids in light-grown pea seedlings

Summary Localization and protein composition of plastid nucleoids was analyzed in light-grown pea seedlings at various stages of leaf development. In young plastids of unopened leaf buds, nucleoids were abundant and localized in the periphery of plastids, whereas, in mature leaves, chloroplasts contained nucleoids within narrow spaces restricted by thylakoids or grana. The migration of nucleoids into the interior of plastids preceded the formation of grana, and hence, the maturation of the photosynthetic apparatus. The protein composition of nucleoids was considerably different in young plastids and mature chloroplasts. Polypeptides with a molecular mass of 70–100 kDa predominated in the nucleoids of young plastids, whereas polypeptides with molecular mass of 20–30 kDa were abundant in the nucleoids of mature chloroplasts. Immuno-blot analysis with antibodies against the nucleoids of young plastids identified various polypeptides that were significantly more abundant in the nucleoids of young plastids than in the nucleoids of mature chloroplasts. These results demonstrate that plastid nucleoids are subject to dynamic changes in both localization and composition during the normal development of chloroplasts in the light.Abbreviations DAPI 4,6-diamidino-2-phenylindol - DiOC₆ 3,3-dihexyloxacarbocyanine iodide     

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.     

Disappearance of plastid and mitochondrial nucleoids during the formation of generative cells of higher plants revealed by fluorescence microscopy

Summary The fate of plastid and mitochondrial nucleoids (pt and mt nucleoids) ofTriticum aestivum was followed during the reproductive organ formation using fluorescence microscopy after staining with 4'6-diamidino-2-phenylindole (DAPI). This investigation showed a drastic morphological change of pt nucleoids during the differentiation of reproductive organs from the shoot apex. Dot-shaped pt nucleoids grew into ring-shaped ones, which divided into small pieces in the monocellular pollen grain, as observed in this plant's earlier stage of leaf development. During the development of mature pollen grain from monocellular pollen grain, pt and/or mt nucleoids disappeared through the division of the male generative cell ofT. aestivum. Cytologically, this observation is direct evidence of the maternal inheritance of higher plants. Thus far, cytological evidence of this phenomenon has been found mostly by morphological criteria using electron microscopy, which admits some ambiguity. In the plants exemplified byLilium longiflorum, pt and/or mt nucleoids disappeared after the first pollen grain mitosis, which precededT. aestivum. In the plants exemplified byTrifolium repens, pt and/or mt nucleoids existed in the generative cells of the mature pollen grain.The significance of these observations was discussed in relation to the interaction between nuclear and organelle genomes during plant development.Abbreviations DAPI 4'6 diamidino-2-phenylindole - Mt DNA Mitochondrial DNA - Mt nucleoid Mitochondrial nucleoid - Pt DNA Plastid DNA - Pt nucleoid Plastid nucleoid On leave from Department of Biology, Nagoya University, Furocho, Chikusaku, Nagoya 464, Japan.     

Variable amounts of DNA related to the size of chloroplasts III. Biochemical determinations of DNA amounts per organelle

Plastid genomes (plastomes) are part of the integrated compartmentalised genetic system of photoautotrophic eukaryotes. They are highly redundant and generally dispersed in several regions (nucleoids) within organelles. DNA quantities and number of DNA-containing regions per plastid vary and are developmentally regulated in a way not yet understood. Reliable quantitative data describing these patterns are scarce. We present a protocol to isolate fractions of pure plastids with varying average sizes from leaflets (≤1 mm) and leaves of different developmental stages continuously up to maturity (25 cm) from Beta vulgaris L. (sugar beet) to determine DNA amounts per organelle. The approach is based on plastid purification from homogenates of moderately fixed tissue by differential and isopycnic gradient centrifugations and on application of two different DNA specific colorimetric reactions after removing potentially interfering compounds. The sensitive fluorochrome DAPI (4′,6-diamidino-2-phenylindole) was used to estimate numbers and emission intensity of nucleoids per plastid. The amounts determined ranged from 0.15 to 4.9 × 10⁻² pg DNA for plastids of 1→8 μm average diameter, corresponding from approximately a dozen to 330 genome equivalents per organelle and on average four to seven copies per nucleoid. The ratio of plastid/nuclear DNA changed continuously during leaf development from as little as 0.4% to about 20% in fully developed leaves. On the other hand, mesophyll cells of mature leaves differing in ploidy (di-, tri- and tetraploid) appeared to maintain a relatively constant nuclear genome/plastome ratio, equivalent to about 1,700 copies per C-value.     

Changes of Anatomical Features, Photosynthesis and Ribulose Bisphosphate Carboxylase-Oxygenase Content of Mango Leaves

Changes in anatomical and physiological features, includingchanges in amount per unit area of anthocyanin and chlorophyll,in leaves of seedling mango (Mangifera indica L. cv. Irwin)trees were determined to understand what controls the rate ofphotosynthesis (Pn) at various stages of development. The youngleaves of seedling trees contained high concentrations of anthocyanin.During enlargement of leaves, the disappearance of anthocyaninand the accumulation of chlorophyll occurred concomitantly;the anthocyanin content began to decrease markedly once theleaf area had reached a maximum. During the early period ofleaf development, the thickness of mesophyll tissue decreasedtemporarily, but when the length of the leaf reached half thatof a mature leaf, the mesophyll began to thicken again. Smallstarch grains appeared in the chloroplasts of the young leavesand chloroplast nucleoids (ct-nuclei) were distributed throughoutthe chloroplasts. When leaves matured, ct-nuclei were displacedto the periphery of chloroplasts because of the accumulationof large starch grains. Compared with young leaves, green andmature leaves contained greater concentrations of ribulose bisphosphatecarboxylase-oxygenase (RuBisCO) protein. The results of immunocytochemicalexamination of RuBisCO under the light microscope reflectedthe results of electrophoresis measurements of RuBisCO. Pn waslow during the chocolate-coloured stage of early leaf development.In green and mature leaves Pn was higher; the average Pn was7·6 mg CO₂ dm^-2 h^-1 under light at intensities above500 µmol m^-2 s^-1.Copyright 1995, 1999 Academic Press Mangifera indica L., mango leaf, chloroplast nucleoids, chloroplast ultrastructure, starch accumulation, anthocyanin, chlorophyll, DAPI staining, SDS-PAGE, immunocytochemical technique     

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)     

Patterns of Assimilate Distribution and Source--sink Relationships in the Young Reproductive Tomato Plant (Lycopersicon esculentum Mill.)

Patterns of distribution of ¹⁴C were determined in 47-day-oldtomato plants (Lycopersicon esculentum Mill.) 24 h after theapplication of [¹⁴C]sucrose to individual source leaves fromleaves 1–10 (leaf 1 being the first leaf produced abovethe cotyledons). The first inflorescence of these plants wasbetween the ‘buds visible’ and the ‘firstanthesis’ stages of development. The predominant sink organs in these plants were the root system,the stem, the developing first inflorescence and the shoot ‘apex’(all tissues above node 10). The contribution made by individualsource leaves to the assimilate reaching these organs dependedupon the vertical position of the leaf on the main-stem axisand upon its position with respect to the phyllotactic arrangementof the leaves about this axis. The root system received assimilateprincipally from leaf 5 and higher leaves, and the stem apexfrom the four lowest leaves. The developing first inflorescencereceived assimilates mainly from leaves in the two orthostichiesadjacent to the radial position of the inflorescence on thevertical axis of the plant; these included leaves which weremajor contributors of ¹⁴C to the root system (leaves 6 and 8)and to the shoot apex (leaves 1 and 3). This pattern of distributionof assimilate may explain why root-restriction treatments andremoval of young leaves at the shoot apex can reduce the extentof flower bud abortion in the first inflorescence under conditionsof reduced photoassimilate availability. Lycopersicon esculentum Mill, tomato, assimilate distribution, source-sink relationships     

Photosynthesis by developing embryos of oilseed rape (Brassica napus L.)

The aim of this study was to assess the photosynthetic potentialof developing seeds of oilseed rape (Brassica napus L.) andto compare photosynthetic properties of embryo plastids withthose of leaf chloroplasts from the same species. Measurementsof CO₂-dependent O₂ evolution show that developing seeds ofB. napus are photosynthetically active in vitro. Essentially,all of the photosynthetic activity of the developing seed isaccounted for by the embryo. The rate of photosynthesis by developingembryos increased until the onset of desiccation, after whichit declined, so that by maturity embryos were no longer photosyntheticallyactive. Photosynthetic activity was positively correlated withchlorophyll content throughout development. Comparison of thephotosynthetic characteristics of leaf and embryo chloroplastsrevealed that rates of uncoupled electron transport were 2.5-foldgreater in those from the embryo. Light-saturated rates of CO₂-dependentO₂ evolution, per unit chlorophyll, and CO₂ saturation pointswere similar for chloroplasts from both tissues. However, light-saturationpoints and chlorophyll a/b ratios were lower for embryo thanfor leaf choroplasts. Embryos and embryo chloroplasts also containedconsiderably less ribulose 1,5-bisphosphate carboxylase/oxygenaseprotein per unit total protein, than leaves. Although excisedembryos were capable of high rates of CO₂-dependent O₂ evolution(90–100 mol mg^–1 chlorophyll h^–1) under asaturating photosynthetic photon flux density (PPFD), low transmittanceof light through the silique wall (30%), together with the highPPFD required to achieve light compensation points in developingseeds (500 mol m^–2 s^–1), suggests that photosynthesisin vivo is unlikely to make a net contribution to carbon economyunder normal environmental conditions. Key words: Embryo, development, photosynthesis, chloroplast, Brassica napus L.     

DNA Biosynthesis in Chloroplasts: I. CHARACTERIZATION OF AND DEVELOPMENTAL VARIATION IN BOTH LIGHT- AND ATP-DRIVEN THYMIDINE INCORPORATION INTO DNA IN ISOLATED INTACT CHLOROPLASTS

Intact chloroplasts from young pea leaves were able to incorporate[³H]thymidine into DNA at relatively high rates (50 pmol mg^–1chlorophyll h^–1 or more), using light as the sole energysource. The intact plastids were also able to synthesize DNAin darkness, but only if ATP and MgCl₂ (MgATP) were both present.The rates of MgATP-driven assimilation in darkness were equalto or greater than light-driven activity. Neither light nordithiothreitol pretreatments enhanced thymidine incorporationin darkness, suggesting that enzymes of chloroplast DNA (ctDNA)biosynthesis are not regulated via a thioredoxin-type system.Although exogenous nucleosides (other than [³H]thymidine) werenot an absolute requirement, dramatically elevated rates ofincorporation (over 300 pmol mg^–1 chlorophyll h^–1)were seen when adenosine, cytidine, guanosine and thymidinewere supplied in combination (500 mmol m^–3 each). RadiolabelledDNA synthesized by the isolated chloroplasts was prepared usinga new heat extraction method. After digestion by restrictionendonucleases, ctDNA synthesized in organello was found to givetypical autoradiography patterns for chloroplast DNA. ExonucleaseIII studies suggested that 5% to 15% of the newly synthesizedDNA might be in a closed circular form. MgATP-driven synthesisin darkness was highly age-dependent. Chloroplasts from young(6 to 8-d-old) plants, or alternatively the youngest leavesof more mature plants, were 4–10 times more active thanthose from older tissues. Although these data do not establishconclusively that replication-type synthesis was occurring inthe isolated chloroplasts, they are consistent with this suggestion. Key words: Chloroplast DNA replication, isolated chloroplasts, chloroplast DNA synthesis     

Interactive effects of salinity, nitrogen and sulphur on the organic solutes in Spartina alterniflora leaf blades

Glycinebetaine, proline, asparagine, sucrose, glucose, and dimethylsulphoniopropionate(DMSP) were the major organic solutes in Spartina alternifloraleaf blades. To investigate the physiological role(s) of thesesolutes, the effects of salinity, nitrogen, and sulphur treatmentson leaf blade solute levels were examined. Glycinebetaine wasthe major organic solute accumulated in leaf blades grown at500 mol m^–3 NaCl, although asparagine and proline alsoaccumulated when the supply of nitrogen was sufficient. Thesesolutes may play a role in osmotic adjustment. In contrast,DMSP levels either did not change or were reduced in responseto the 500 mol m^–3 NaCl treatment. Furthermore, elevatednitrogen supply decreased leaf blade DMSP levels, which wasopposite to the response of glycinebetaine, proline, and asparagine.A 1000-fold increase in external sulphate concentration hadno effect on the leaf blade levels of DMSP, glycinebetaine,proline, or asparagine. These findings suggest that the majorphysiological role of DMSP in S. alterniflora leaf blades isnot for osmotic adjustment, even under conditions of nitrogendeficit and excess sulphur. Instead, DMSP which was presentat 45—130 µmol g^–1 dry weight, may play arole as a constitutive organic osmoticum. Key words: Spartina alterniflora, dimethylsulphoniopropionate, glycinebetaine, nitrogen, salinity     

The division of pleomorphic plastids with multiple FtsZ rings in tobacco BY-2 cells

Plastids, an essential group of plant cellular organelles, proliferate by division to maintain continuity through cell lineages in plants. In recent years, it was revealed that the bacterial cell division protein FtsZ is encoded in the nuclear genome of plant cells, and plays a major role in the plastid division process forming a ring along the center of plastids. Although the best-characterized type of plastid division so far is the division with a single FtsZ ring at the plastid midpoint, it was recently reported that in some plant organs and tissues, plastids are pleomorphic and form multiple FtsZ rings. However, the pleomorphic plastid division mechanism, such as the formation of multiple FtsZ rings, the constriction of plastids and the behavior of plastid (pt) nucleoids, remains totally unclear. To elucidate these points, we used the cultured cell line, tobacco (Nicotiana tabacum L.) Bright Yellow-2, in which plastids are pleomorphic and show dynamic morphological changes during culture. As a result, it was revealed that as the plastid elongates from an ellipsoid shape to a string shape after medium renewal, FtsZ rings are multiplied almost orderly and perpendicularly to the long axis of plastids. Active DNA synthesis of pt nucleoids is induced by medium transfer, and the division and the distribution of pt nucleoids occur along with plastid elongation. Although it was thought that the plastid divides with simultaneous multiple constrictions at all the FtsZ ring sites, giving rise to many small plastids, we found that the plastids generally divide constricting at only one FtsZ ring site. Moreover, using electron microscopy, we revealed that plastid-dividing (PD) rings are observed only at the constriction site, and not at swollen regions. These results indicate that in the pleomorphic plastid division with multiple FtsZ rings, the formation of PD rings occurs at a limited FtsZ ring site for one division. Multiplied FtsZ rings seem to localize in advance at the expected sites of division, and the formation of a PD ring at each FtsZ ring site occurs in a certain order, not simultaneously. Based on these results, a novel model for the pleomorphic plastid division with multiple FtsZ rings is proposed.     

Treatment of dark-grown Arabidopsis thaliana with a brassinosteroid-biosynthesis inhibitor, brassinazole, induces some characteristics of light-grown plants

When a brassinosteroid biosynthesis inhibitor, brassinazole (Brz), was applied at concentrations ranging from 0.1 to 2 μM, Arabidopsis thaliana (L.) Heynh seedlings grown in the dark exhibited morphological features of light-grown plants, i.e. short hypocotyls, expanded cotyledons, and true leaves, in a dose-dependent manner. Control (non Brz-treated) seedlings grown in the dark for 40 d did not develop leaf primordia. However, treatment with the lowest concentration of Brz induced the development of leaf buds, although it hardly induced any short hypocotyls, and treatment with the highest concentration of Brz induced both short hypocotyls and leaves. Labeling experiments with the thymidine analogue 5-bromo-2′-deoxyuridine revealed that amplification of cell nuclei and organellar nucleoids is activated in the shoot apical meristems of dark-grown Brz-treated seedlings. These results suggest that Brz-treatment induces development of true leaves. Furthermore, condensation and scattering of plastid nucleoids, which is known to occur during the differentiation of etioplasts into chloroplasts, was observed in the plastids of dark-grown Brz-treated cotyledons. In addition, high levels of ribulose-1,5-bisphosphate carboxylase-oxygenase proteins accumulated in the plastids of the cotyledons. Electron microscopy showed that the plastids were etioplasts with a prolamellar body and few thylakoid membranes. These results suggest that Brz treatment in the dark induces the initial steps of plastid differentiation, which occur prior to the development of thylakoid membranes. This is a novel presumed function of brassinosteroids. These cytological changes seen in Brz-treated Arabidopsis were exactly the same as those seen in a brassinosteroid-biosynthesis-deficient mutant, det2, supporting the hypothesis that Brz has no side-effects except inhibiting brassinosteroid biosynthesis, and should prove a useful tool in clarifying the role of brassinosteroids. Received: 10 February 2000 / Accepted: 11 April 2000     

Studies on nucleic acids in plastids of the pigment mutant C-2A{acute} of Scenedesmus obliquus

Pigment mutant C-2A{acute} of Scenedesmus obliquus whose chlorophyllformation and chloroplast development are light dependent, wasstudied for the nucleic acid content of its plastids. The ribosomalRNA of plastids of the achlorophyllous or greened mutant C-2A{acute},did not show any difference from that of the wild type. Incorporationof [5-³H] uridine into mutant cells was partially inhibitedby rifampicin, indicating this part as being plastidial incorporation.Since there were no significant differences in the ribosomalRNA of plastids between the mutant and the wild type of Scenedesmus,the ribosomal system in the plastids of mutant C-2A' seemednot to be affected by the mutation. C_sCl gradient patterns ofScenedesmus mutant and wild-type DNA were almost identical withthose of Chlorella DNA. A peak at a buoyant density of 1.69g/cm³, the same as that of Chlorella chloroplast DNA, couldbe identified in Scenedesmus also as plastid DNA because itdisappeared after prolonged treatment with myxin and hybridizedwith rifampicin-sensitive pulse-labelled RNA. This peak waspresent to nearly the same degree in the mutant and the wildtype, indicating that a larger deficit of plastid DNA did notoccur in the mutant. Whether or not the mutation might be localizedin the plastid genome is discussed. (Received March 19, 1976; )