EFFECT OF SHORT-TERM SHADING ON THE CYTOLOGY OF PALISADE TISSUE IN MATURE LEAVES OF THE SUNFLOWER HELIANTHUS ANNUUS

Mature sunflower leaves were exposed to partial shading (35 or 14% of normal sun) or darkness (0% of normal sun) for approximately 8 hr. During this period one-half of each test leaf was shaded; the other half was used as a normal sun control. Palisade cell structure from both halves of each leaf was compared. Shading of leaves had little effect on organelle percent volume values (V_v) with exception of the starch compartment which decreased as shading increased. The surface to volume ratio (S_v) of the chloroplast thylakoids increased while the S_v of the mitochondrial membranes decreased as shading increased. Palisade cell volume did not change in shaded portions of the leaf, except in the fully shaded (dark) tissues where cell volume decreased. Changes in the actual volume of organelle compartments were strongly correlated with changes in cell volume. Thus a general osmotic response may account for some of the volume changes associated with differences in light intensity. Shading increased thylakoid surface areas 10–30% over the full sun controls. The ratio of stromal to granal thylakoid surface area remained constant in both the control and partially shaded samples. However, in darkened samples this ratio decreased as stromal membranes increased more than granal membranes. Changes observed in thylakoid surface areas associated with shading did not support thylakoid models which propose the interconversion of granal membranes to stromal membranes and vice versa.     

A Quantitative Study of Daily Variation in the Cellular Ultrastructure of Palisade Chlorenchyma from Sunflower Leaves

Stereology was used to describe cytological changes which occurin palisade cells of fully expanded leaves as part of theirnormal daily activity. These changes were evaluated by describingthe relationship between organelle volume and cell volume asratio values (i.e. percentage volumes, V_v; surface-to-volume,S_v). These ratios describe an average cell in terms of its volumecommitment to each organelle compartment. Cells were also describedin terms of actual volume (µm³) or surface area (µm²)of membrane present in an average cell. ANOVA-LSD and Mann-Whitneystatistics indicate significant changes occur in the ratio valuesof the vacuole, chloroplast, oil, starch and microbody compartmentsover the 24 h period. This indicates a re-allocation of cellspace to these compartments during this period. The S_v ratioof internal membranes of the chloroplast and mitochondria showedno significant change over 24 h indicating that there is a constantrelationship between volume and membrane surface area in theseorganelles. Significant changes occurred in average cell volumeover 24 h with maximum volume during the dark period. Sincechanges in cell volume affected the actual volume expressionof all of the organelle compartments there were diurnal variationin the actual size of these compartments, including the internalmembranes of chloroplasts and mitochondria which more than doubledin surface area. Helianthus annuus L, sunflower, cytology, stereology, quantitative microscopy, diurnal, morphometrics, ultrastructure, chlorenchyma, chloroplast, mitochondria, microbodies     

SUBCELLULAR MORPHOMETRIC AND BIOCHEMICAL ANALYSES OF DEVELOPING RAT HEPATOCYTES

Livers of rats between the 16th gestational and 100th postnatal day of age were subjected to quantitative biochemical and electron microscope, morphometric analyses. The amount of total mitochondrial protein per gram of liver remained at 34% of the adult level throughout the last 4 days of gestation but this was the period of rapid rise in the levels of cytochrome c oxidase, aspartate aminotransferase, and glutamate dehydrogenase in mitochondria; the nuclear fraction also acquired some glutamate dehydrogenase but lost most of it during postnatal development. During early postnatal life the amount of mitochondrial protein rose in parallel with the levels of cytochrome c oxidase and glutamate dehydrogenase but the upsurges of glutaminase and, later, of ornithine aminotransferase were accompanied by relatively little change in total mitochondrial protein. The surface area of rough endoplasmic reticulum per unit volume of hepatocyte cytoplasm (S_v^RER) did not change significantly throughout the period of development studied. From the 16th day of gestation to term the surface area of smooth ER (S_v^SER), the volume occupied by mitochondria (V_v^MT) and their number (N_v^MT) remained at 30, 66, and 45% of their adult values, respectively. V_v^MT and N_v^MT attained their maximal levels by the 2nd postnatal day and S_v^SER between days 2 and 12. Mitochondria of adult liver are thus smaller and contain more protein per unit volume than do those of fetal liver. After the 12th postnatal day, hepatocytes treble their size; they acquire more cytoplasm with additional enzymes but without further change in organelle concentration. The data reveal several distinct phases in the differentiation of hepatocytes. Each phase can be characterized by the extent to which the quantity and composition of various subcellular compartments evolve.     

Comparative stereology of the lizard and frog myocardium

The atria and ventricles of the frog and lizard were quantitated using stereologic techniques. The volume fraction (V_v) and surface density (S_v) of the free, junctional and total sarcoplasmic reticulum and mitochondria of the lizard atrium and ventricle were greater than in the corresponding chambers in the frog. Myofibrillar volume fraction and plasmalemmal surface density did not differ between the two species. The volume fraction and surface density of the free and total SR, and myocardial granules were greater in the lizard atrium than ventricle but the myofibrillar V_v and mitochondrial V_v and S_v were less. The S_v of the free SR, total SR, and the V_v and S_v of myocardial granules of the frog atrium were greater than in the frog ventricle. There were no differences between myofibrils and mitochondria in the frog atrium and ventricle.     

Comparative stereology of mouse atria

The left and right atria of the mouse were compared to each other and to the mouse left ventricle using stereologic techniques. The volume fraction (V_v) and surface area per unit cell volume (S_v) of the interior junctional sarcoplasmic reticulum (IJSR), total JSR and extended JSR were greater in the left atrium than in right. The V_v and S_v of the free SR, transverse tubules, and mitochondria were similar in the two atria. It is suggested that the differences in junctional sarcoplasmic reticulum between the atria can be accounted for by a difference in distribution of two types of cells whose anatomy is analogous to working and conducting fibers in the ventricle. The S_v and V_v of the transverse tubules, mitochondria, and all the components of the sarcoplasmic reticulum except for the free SR were greater in the left ventricle than in either atrium. The greater calcium content and sensitivity to extracellular calcium of the atria may explain the greater volume of free SR in the atria as compared to the left ventricle. The S_v of the plasmalemma of the atria and of the S_v of the plasmalemma of the transverse tubules of the left ventricles supports the suggestion of others that there is a constant ratio of surface area to cell volume in cardiac cells.     

Ultrastructure of Mesophyll Cells and Pigment Content in Senescing Leaves of Maize and Barley

Leaf senescence is a genetically regulated stage in the plant life cycle leading to death. Ultrastructural analysis of a particular region of the leaf and even of a particular mesophyll cell can give a clear picture of the time development of the process. In this study we found relations between changes in mesophyll cell ultrastructure and pigment concentration in every region of the leaf during leaf senescence in maize and barley. Our observations demonstrated that each mesophyll cell undergoes a similar senescence sequence of events: a) chromatin condensation, b) degradation of thylakoid membranes and an increase in the number of plastoglobules, c) damage to internal mitochondrial membrane and chloroplast destruction. Degradation of chloroplast structure is not fully correlated with changes in photosynthetic pigment content; chlorophyll and carotenoid content remained at a rather high level in the final stage of chloroplast destruction. We also compared the dynamics of leaf senescence between maize and barley. We showed that changes to the mesophyll cells do not occur at the same time in different parts of the leaf. The senescence damage begins at the base and moves to the top of the leaf. The dynamics of mesophyll cell senescence is different in leaves of both analyzed plant species; in the initial stages, the process was faster in barley whereas in the later stages the process occurred more quickly in maize. At the final stage, the oldest barley mesophyll cells were more damaged than maize cells of the same age.     

The course of chloroplast DNA replication and its relationship to other reproductive processes in the chloroplast and nucleocytoplasmic compartment during the cell cycle of the algaScenedesmus quadricauda

Summary DNA containing structures (cellular, chloroplast and mitochondrial nuclei) were stained with the fluorochrome DAPI. Fluorescence intensity, as a measure of DNA content, was estimated during the mitotic cycle in synchronized populations of the chlorococcal alga,Scenedesmus quadricauda. In cells yielding eight daughter cells, three consecutive steps in chloroplast DNA increase occurred over one mitotic cycle. The first step was performed shortly after releasing the daughter cells, the second and third steps occurred consecutively during the first half of the mitotic cycle. Commitment to chloroplast DNA replication was chronologically separated from commitment to division of chloroplast nuclei, revealing that these two chloroplast reproductive steps were under different control mechanisms. The replication of chloroplast DNA occurred at a different time to that of cell-nuclear DNA. The coordination of chloroplast reproductive processes and those in the nucleocytoplasmic compartment were governed by the mutual trophic and metabolic dependency of these compartments rather than by any direct or feedback control controlled by either of them.Abbreviations DAPI 46-diamidino-2-phenylindole - ptDNA DNA in chloroplast nuclei - nucDNA DNA in cell nuclei     

DYNAMICS OF ARBUSCULE DEVELOPMENT AND DEGENERATION IN A ZEA MAYS MYCORRHIZA

A quantitative light and electron microscope study of developing and degenerating mycorrhizal arbuscules of Glomus fasciculatum in Zea mays was carried out in order to estimate three parameters during the colonization cycle. These were: 1) V_v(f,c), the fraction of the host cell volume occupied by a volume of fungus; 2) V_v(cy,c), the fraction of the host cell volume occupied by host cytoplasm; 3) S_v(pr,c), the surface-area-to-volume ratio of the host protoplast to the whole host cell. Uninfected cortical cells had an S_v(pr,c) of 0.13 μm²/μm³. As the fungus penetrates the cell wall, the protoplast invaginates, causing a decrease in protoplast volume and an increase in protoplast S_v. The S_v(pr,c) of a cell containing a mature arbuscule is 1.275 μm²/μm³. Because of the shrinkage of the protoplast, the S_v of the protoplast to its own volume rather than the original cell volume is 2.55 μm²/μm³, or almost a 20-fold increase. Total cell size is unaffected. When the arbuscule is mature, the fungus occupies 42% of the cell, with 24% as 1-μm-diam branches, and 18% as trunk. Arbuscular branch formation progresses at a linear rate and is the most important factor in causing the increased host S_v. The correlation coefficient for V_v(br,c) the volume fraction for arbuscular branches, vs. Sv(pr,c) is r = 0.932 (P < 0.001). Degeneration of the arbuscule is marked by a rapid decrease in branches, host S_v, and host cytoplasm. The trunk develops and degenerates at a slower rate than the branches.     

Organelle redox autonomy during environmental stress

Oxidative stress is generated in plants because of inequalities in the rate of reactive oxygen species (ROS) generation and scavenging. The subcellular redox state under various stress conditions was assessed using the redox reporter roGFP2 targeted to chloroplastic, mitochondrial, peroxisomal and cytosolic compartments. In parallel, the vitality of the plant was measured by ion leakage. Our results revealed that during certain physiological stress conditions the changes in roGFP2 oxidation are comparable to application of high concentrations of exogenous H₂O₂. Under each stress, particular organelles were affected. Conditions of extended dark stress, or application of elicitor, impacted chiefly on the status of peroxisomal redox state. In contrast, conditions of drought or high light altered the status of mitochondrial or chloroplast redox state, respectively. Amalgamation of the results from diverse environmental stresses shows cases of organelle autonomy as well as multi‐organelle oxidative change. Importantly, organelle‐specific oxidation under several stresses proceeded cell death as measured by ion leakage, suggesting early roGFP oxidation as predictive of cell death. The measurement of redox state in multiple compartments enables one to look at redox state connectivity between organelles in relation to oxidative stress as well as assign a redox fingerprint to various types of stress conditions.     

Oxygen consumption and the composition of skeletal muscle tissue after training and inactivation in the European woodmouse (Apodemus sylvaticus)

Summary In European woodmice the amount and intensity of daily activity was compared to oxygen uptake and to the potential for oxidative metabolism of heart and skeletal muscle. One group of animals was inactivated by exposition to light during night time; another group of animals was trained by enforced running on a treadmill. The oxidative potential of the muscle tissue was assessed by morphometry of capillaries and mitochondria. A novel sampling technique was used which allowed us to obtain morphological data related to single muscles, to muscle groups, and finally to whole body muscle mass.Reducing the spontaneous activity by ten fold had no effect on oxygen uptake nor on capillaries or mitochondria in locomotory muscles. Mitochondrial volume was reduced, however, in heart and diaphragm. Enforced running increased the weight specific maximal oxygen uptake significantly. It also increased the mitochondrial volume in heart and diaphragm as well as in M. tibialis anterior. Capillary densities were neither affected by training nor by inactivation. A significant correlation was found between the capillary density and the volume density of mitochondria in all muscles analysed morphometrically. For the whole skeletal muscle mass of a European woodmouse the inner mitochondrial membranes were estimated to cover 30 m². The oxygen consumption per unit time and per unit volume of muscle mitochondrion was found to be identical in all groups of animals (4.9 ml O₂ min^–1 cm^–3).Symbols S _v (im,m) surface area of inner mitochondrial membranes per unit mitochondrial volume - V _v (mt, f) volume density of mitochondria (mitochondrial volume per fiber volume) - V (mt) total mitochondrial volume - V (f) muscle volume - N _A (c, f) capillary density - (f) mean fiber cross-sectional area     

油茶叶肿病变态叶片的形态特征和超微结构观察

利用扫描电镜(SEM)对油茶叶肿病变态叶叶片表面和横切面进行观察,利用透射电镜(TEM)对其细胞超微结构进行观察,以期探明油茶叶肿病变态叶的形态特征和细胞学特征。结果表明:(1)变态叶是受感染油茶幼叶组织增生形成的,肿大的叶片厚度比正常叶片厚度增加3~5倍,细胞体积增大3~8倍,细胞数增加1~2倍,叶片细胞形态和结构发生了变化。(2)叶片受细丽外担菌侵染后,菌丝存在于下表皮向内的4~7层细胞间隙中,感染后期叶片下表面脱落露出子实层。(3)变态叶细胞出现叶绿体膜破裂、类囊体片层膜数目减少及细胞器成分被破坏等异常现象。     

EVIDENCE THAT THE NUCLEOMORPHS OF CHLORARACHNION REPTANS (CHLORARACHNIOPHYCEAE) ARE VESTIGIAL NUCLEI: MORPHOLOGY,DIVISION AND DNA-DAPI FLUORESCENCE1

Chlorarachnion reptans Geitler shows affinities to both the Chlorophyceae and the chloroplast endoplasmic reticulum-containing chromophyte algae in possessing chlorophyll b and chloroplasts which are limited by four membranes, respectively. In the periplastidal compartment surrounding each of the four to eight chloroplasts of a C. reptans cell are putative eukaryotic-sized ribosomes, scattered tubules and vesicles, and a small double-membrane-limited nucleus-like organelle named the nucleomorph. The nucleomorphs display 4′-6-diamidino-2-phenylindole (DAPI)fluorescence which is sensitive to DNase digestion, but not to treatment with RNase. The nucleomorphs also contain a fibrillogranular body which resembles a nucleolus. Nucleomorph division occurs by the sequential infolding of the inner and outer envelope membranes and subsequent constriction in two, with no involvement of microtubules. In all these characteristics, the nucleomorphs of C. reptans are similar to the cryptomonad nucleomorph which has been hypothesized to be the vestigial nucleus of an ancestral red alga which gave rise to the chloroplasts of the Cryptophyceae. The presence of chlorophyll b and the contents and morphology of C. reptans chloroplast compartments suggest a green algal origin for the chloroplasts of these cells. The discovery of a second organism with a DNA-containing, nucleus-like organelle in its chloroplast compartment lends strong support to the hypothesis that the chloroplasts of many algae have evolved from eukaryotic endosymbionts.     

Structural changes in the vacuole and cytoskeleton are key to development of the two cytoplasmic domains supporting single-cell C<Subscript>4</Subscript> photosynthesis in <Emphasis Type="Italic">Bienertia sinuspersici</Emphasis>

Bienertia sinuspersici Akhani has an unusual mechanism of C₄ photosynthesis which occurs within individual chlorenchyma cells. To perform C₄, the mature cells have two cytoplasmic compartments consisting of a central (CCC) and a peripheral (PCC) domain containing dimorphic chloroplasts which are interconnected by cytoplasmic channels. Based on leaf development studies, young chlorenchyma cells have not developed the two cytoplasmic compartments and dimorphic chloroplasts. Fluorescent dyes which are targeted to membranes or to specific organelles were used to follow changes in cell structure and organelle distribution during formation of C₄-type chlorenchyma. Chlorenchyma cell development was divided into four stages: 1—the nucleus and chloroplasts occupy much of the cytoplasmic space and only small vacuoles are formed; 2—development of larger vacuoles, formation of a pre-CCC with some scattered chloroplasts; 3—the vacuole expands, cells have directional growth; 4—mature stage, cells have become elongated, with a distinctive CCC and PCC joined by interconnecting cytoplasmic channels. By staining vacuoles with a fluorescent dye and constructing 3D images of chloroplasts, and by microinjecting a fluorescence dye into the vacuole of living cells, it was demonstrated that the mature cell has only one vacuole, which is traversed by cytoplasmic channels connecting the CCC with the PCC. Immunofluorescent studies on isolated chlorenchyma cells treated with cytoskeleton disrupting drugs suspended in different levels of osmoticum showed that both microtubules and actin filaments are important in maintaining the cytoplasmic domains. With prolonged exposure of plants to dim light, the cytoskeleton undergoes changes and there is a dramatic shift of the CCC from the center toward the distal end of the cell.     

EFFECT OF LIGHT HISTORY ON THE ULTRASTRUCTURE AND PHYSIOLOGY OF PROROCENTRUM MARIAE-LEBOURIAE (DINOPHYCEAE)1

Ultrastructural and physiological responses of Prorocentrum mariae-lebouriae (Parke & Ballantine) Faust are reported for cultures maintained at growth irradiances (I_g) ranging from 20.6 to 0.3 E m^?2.d^?1 and following downward shifts in light intensity. We tested the hypothesis that Prorocentrum grown under light regimes that elicit different responses in photosynthesis and pigmentation exhibit distinctive cell ultrastructures. Prorocentrum from high-light conditions had high saturation intensities for photosynthesis (I_k) and low levels of Chl a, Chl c and peridinin-cell^?1 These cultures were morphologically distinguished by a large starch volume fraction (V_v), small chloroplast V_v and fewer thylakoids lamella^?1. I_k values were lower and pigment concentrations higher in low-light treatments, and cells showed reduced starch V_v, large chloroplast V_v, and higher numbers of thylakoids · lamella ^?1. Cells grown under extremely low-light conditions appeared stressed as indicated by the absence of starch reserves and the presence of large vacuoles within the cytoplasm. Results for presence of large vacuoles within the cytoplasm. Results for quantiative electron microscopy, photosynthesis-irradiance (P-I) relations and cell pigmentation indicate that photoadaptation in P. mariae-lebouriae involves a strategy that encompasses changes in both the “size” and “number” of photosynthetic units.     

The conquest of fresh water by the palaemonid shrimps: an evolutionary history scripted in the osmoregulatory epithelia of the gills and antennal glands

Extant Palaemonidae occupy aquatic environments that have generated physiological diversity during their evolutionary history. We analyze ultrastructural traits in gills and antennal glands of palaemonid species from distinct osmotic niches, and employ phylogenetic comparative methods to ascertain whether transformations in their osmoregulatory epithelia have evolved in tandem, driven by salinity. Gill pillar cells exhibit apical evaginations whose surface density (S_v, μm² plasma membrane area/μm³ cytoplasmic volume) ranges from 6.3–7.1 in Palaemon, and 0.7–38.4 in Macrobrachium. In the septal cells, S_v varies from 8.9–10.0 in Palaemon, and 3.3–21.6 in Macrobrachium; mitochondrial volumes (V_mit) range from 43.3–46.8% in Palaemon and 34.9–53.4% in Macrobrachium. In the renal proximal tubule cells, apical microvilli S_v varies from 27.0–34.3 in Palaemon, and 38.3–47.8 in Macrobrachium; basal invagination S_v ranges from 18.7–20.0 in Palaemon and 30.8–40.8 in Macrobrachium. Septal cell S_v shows phylogenetic signal; evagination height/density, apical S_v, and V_mit vary independently of species relatedness. Salt transport capability by the gill and renal epithelia has increased during palaemonid evolution, reflecting amplified membrane availability for ion transporter insertion. These traits underpin the increased osmotic gradients maintained against the external media. Gill ultrastructure and osmotic gradient have evolved in tandem, driven by salinity at the genus level. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 673–688.     

Stereological analyses of the structure of mitochondria in pigeon skeletal muscle

Summary The mitochondria in type-I and -II muscle fibres in the pectoralis major muscle of the pigeon (Columba livia) have been analysed using stereological techniques not previously applied in muscle biology.Mitochondrial volume fractions (V_V) were estimated in different regions of each type of muscle fibre using randomly orientated sampling sectors within fibre profiles. These sectors were sub-divided into smaller sampling regions to provide accurate data on the intracellular distribution of mitochondria. Estimates of the external surface densities of mitochondria per unit volume of fibre, S_{V total} ^surface , and also the densities of mitochondrial cristae, S_{V total} ^cristae , were obtained using a specific technique derived for analysing anisotropic structures (Saltykov, 1958). The relative amounts of the random and orientated mitochondrial membranes were also estimated.Significiant differences were found to exist between the different types of muscle fibres and considerable though constant variations in the intracellular arrangement of mitochondria were also found.     

Some factors affecting the chloroplast replication in the mossPlagiomnium trichomanes

Summary Some factors affecting the chloroplast replication were studied using the leaf cells of the mossPlagiomnium trichomanes. There was a significant positive correlation between chloroplast number per cell and cell volume in leaves of any developmental stage. However, when the detached leaves were cultured on nutrient agar, it was observed that the chloroplast replication occurred without cell enlargement regardless of the developmental stage of leaves. This implies that cell enlargement is not an essential factor for the chloroplast replication, but one of the environmental factors affecting it. Light is essential for the chloroplast replication which response to the light intensity. In the dark, there was little increase in chloroplast number per cell. With a light intensity of 50 lux, the increase rate of chloroplast number per cell was about half of that with 3,000 lux. Day length also affected significantly the chloroplast replication.     

The density of apical cells of dark-grown protonemata of the mossCeratodon purpureus

Summary Determinations of plant or algal cell density (cell mass divided by volume) have rarely accounted for the extracellular matrix or shrinkage during isolation. Three techniques were used to indirectly estimate the density of intact apical cells from protonemata of the mossCeratodon purpureus. First, the volume fraction of each cell component was determined by stereology, and published values for component density were used to extrapolate to the entire cell. Second, protonemal tips were immersed in bovine serum albumin solutions of different densities, and then the equilibrium density was corrected for the mass of the cell wall. Third, apical cell protoplasts were centrifuged in low-osmolarity gradients, and values were corrected for shrinkage during protoplast isolation. Values from centrifugation (1.004 to 1.015 g/cm³) were considerably lower than from other methods (1.046 to 1.085 g/cm³). This work appears to provide the first corrected estimates of the density of any plant cell. It also documents a method for the isolation of protoplasts specifically from apical cells of protonemal filaments.Abbreviations BSA bovine serum albumin - ER endoplasmic reticulum - V_v volume fraction     

Changes of dictyosome ultrastructure during the cell cycle in onion root meristematic cells. A morphometric and stereological study

Summary Dictyosome ultrastructure changes during the cell cycle in onion root meristematic cells. Changes were mainly related to cisternae and intercisternal spaces morphology. Taking each dictyosome to be composed of three different regions (CIS, medial, and TRANS), several quantitative changes were detected in some of the compartments. Many of the planimetric parameters evaluated showed higher values in medical cisternae, while CIS and TRANS remained nearly constant. We have also found an increased activity of dictyosomes, as indicated by increase in the volume fraction of vesicular attached structures. This reaches maximum values at ana-telophase in coincidence with the onset and progression of cytokinesis.Abbreviations A anaphase - Ac mean area occupied by cisternae per stack section - C CIS - CCS cell cycle stage - D_A mean total dictyosome area - I_SA mean area occupied by intercisternal spaces per stack section - M metaphase - N mean number of cisternae per stack - P prophase - S.E. standard error - T telophase - T TRANS - V_v volume density     

Constancy of organellar genome copy numbers during leaf development and senescence in higher plants

In higher plants, plastid and mitochondrial genomes occur at high copy numbers per cell. Several recent publications have suggested that, in higher plants like Arabidopsis and maize, chloroplast DNA is virtually absent in mature and old leaves. This conclusion was mainly based on DAPI staining of isolated chloroplasts. If correct, the finding that chloroplasts in mature leaves lack DNA would change dramatically our understanding of gene expression, mRNA stability and protein stability in chloroplasts. In view of the wide implications that the disposal of chloroplast DNA during leaf development would have, we have reinvestigated the age dependency of genome copy numbers in chloroplasts and, in addition, tested for possible changes in mitochondrial genome copy number during plant development. Analyzing chloroplast and mitochondrial DNA amounts in Arabidopsis and tobacco plants, we find that organellar genome copy numbers remain remarkably constant during leaf development and are present in essentially unchanged numbers even in the senescing leaves. We conclude that, during leaf development, organellar gene expression in higher plants is not significantly regulated at the level of genome copy number and we discuss possible explanations for the failure to detect DNA in isolated chloroplasts stained with DAPI.