Nuclear DNA content and the control of chloroplast replication in wheat leaves

During development of the first leaf of breadwheat (Triticum aestivum L.) the number of chloroplasts per mesophyll cell increases between three- and four-fold. To establish if chloroplast replication is accompanied by endoreduplication, the nuclear DNA content of the cells was determined by chemical assay of isolated nuclei from mesophyll protoplasts and by microdensitometry of nuclei in mesophyll tissue. The DNA content of the nuclei was constant (27 to 32 pg) at each phase of chloroplast replication. Approximately 93% of the cells had a nuclear DNA content close to the 2C value of 32 pg. It is concluded that chloroplast replication is not dependent on nuclear endoreduplication in seedling leaves of wheat.     

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.     

Changes in chloroplast number during pea leaf development

Protoplasts were prepared from pea (Pisum sativum L.) leaves throughout development and their contents spread in a monolayer to determine the number of chloroplasts per cell. This approach permitted the rapid analysis of more than 100 cells at each stage of development. The average number of chloroplasts per cell increased from 24±10 to 64±20 during greening and expansion of the first true foliage leaves; all cells containing chloroplasts apparently increase their chloroplast number. A parallel increase in the amount of DNA per nucleus was not observed. As the leaves senesced the chloroplast number gradually decreased to 44±12. We have correlated these changes with our previous results on the percentage of chloroplast DNA per cell. Chloroplast multiplication resulted in a 2.7-fold dilution (from 272 to 102) of the number of copies of the chloroplast DNA molecule per plastid.     

DNA Content of Beta vulgaris Chloroplasts during Leaf Cell Expansion

During the growth of beet leaves from 2 to 3 to 25 to 30 centimeters, the leaf cells increase in size, the average number of chloroplasts per cell increases from 11 to 65 and the amount of chloroplast DNA per cell increases from 1100 to 1900 plastome copies. The average number of copies of the plastome per chloroplast decreases from 104 in 2 to 3-centimeter leaves to 29 in 25 to 30-centimeter leaves during a period when the chloroplasts undergo two to three rounds of division and increase diameter from 1.5 to 4.9 micrometers. This result is at variance with previously published studies of beet chloroplasts but agrees with the conclusions reached in more recent studies of pea and spinach and wheat leaf cell expansion.     

Changes in the structure of DNA molecules and the amount of DNA per plastid during chloroplast development in maize

We examined the DNA from chloroplasts obtained from different tissues of juvenile maize seedlings (from eight to 16 days old) and adult plants (50-58 days old). During plastid development, we found a striking progression from complex multigenomic DNA molecules to simple subgenomic molecules. The decrease in molecular size and complexity of the DNA paralleled a progressive decrease in DNA content per plastid. Most surprising, we were unable to detect DNA of any size in most chloroplasts from mature leaves, long before the onset of leaf senescence. Thus, the DNA content per plastid is not constant but varies during development from hundreds of genome copies in the proplastid to undetectable levels in the mature chloroplast. This loss of DNA from isolated, mature chloroplasts was monitored by three independent methods: staining intact chloroplasts with 4',6-diamidino-2-phenylindole (DAPI); staining at the single-molecule level with ethidium bromide after exhaustive deproteinization of lysed chloroplasts; and blot-hybridization after standard DNA isolation procedures. We propose a mechanism for the production of multigenomic chloroplast chromosomes that begins at paired DNA replication origins on linear molecules to generate a head-to-tail linear concatemer, followed by recombination-dependent replication.     

Changes in mitochondrial DNA levels during development of pea (Pisum sativum L.)

The percentage of mitochondrial DNA (mtDNA) present in total DNA isolated from pea tissues was determined using labeled mtDNA in reassociation kinetics reactions. Embryos contained the highest level of mtDNA, equal to 1.5% of total DNA. This value decreased in light- and dark-grown shoots and leaves, and roots. The lowest value found was in dark-grown shoots; their total DNA contained only 0.3% mtDNA. This may be a reflection of increased nuclear ploidy levels without concomitant mtDNA synthesis. It was possible to compare the mtDNA values directly with previous estimates of the amount of chloroplast DNA (ctDNA) per cell because the same preparations of total DNA were used for both analyses. The embryo contained 1.5% of both mtDNA and ctDNA; this equals 410 copies of mtDNA and 1200 copies of ctDNA per diploid cell. Whereas mtDNA levels decreased to 260 copies in leaf cells of pea, the number of copies of ctDNA increased to 10300. In addition, the levels of ctDNA in first leaves of dark-grown and light-transferred pea were determined, and it was found that leaves of plants maintained in the dark had the same percentage of ctDNA as those transferred to the light.Abbreviations ctDNA chloroplast DNA - mtDNA mitochondrial DNA     

The Phenotype of a Virescent Chloroplast Mutation in Tobacco Is Associated with the Absence of a 37.5 kD Thylakoid Polypeptide

The Vir-c mutation is a virescent chloroplast mutation found in a line of plants derived from protoplast fusions between a Nicotina tabacum line and a line containing N. tabacum nuclei with Nicotiana suaveolens cytoplasm. Vir-c displays a lag period in chlorophyll accumulation and granal stack formation in young leaves. We examined total chloroplast protein in young leaves and showed the mutant contains 1.3 to 2.1 times less stromal protein, and 2.9 to 4.3 times less thylakoid protein when compared to the N. tabacum var “Turkish Samsun” control. Electrophoretic patterns of total thylakoid proteins indicated three polypeptides were specifically decreased in amount within the context of the overall reduction in thylakoid protein. Electrophoresis of thylakoid proteins synthesized by chloroplasts isolated from half-expanded leaves demonstrated that mutant chloroplasts did not synthesize a 37.5 kilodalton polypeptide which was synthesized by “Samsun” chloroplasts. A polypeptide of this molecular weight was synthesized by Vir-c chloroplasts isolated from mature leaves which had recovered the normal phenotype. Restriction digestion and electrophoresis of the mutant's chloroplast DNA produced a pattern of restriction fragments different from either N. tabacum or N. suaveolens chloroplast DNA.     

AUTORADIOGRAPHIC EVIDENCE FOR MANY SEGREGATING DNA MOLECULES IN THE CHLOROPLAST OF OCHROMONAS DANICA

Light-grown cells of Ochromonas danica, which contain a single chloroplast per cell, were labeled with [methyl-³H]thymidine for 3 h (0.36 generations) and the distribution of labeled DNA among the progeny chloroplasts was followed during exponential growth in unlabeled medium for a further 3.3 generations using light microscope autoradiography of serial sections of entire chloroplasts. Thymidine was specifically incorporated into DNA in both nuclei and chloroplasts. Essentially all the chloroplasts incorporated label in the 3-h labeling period, indicating that chloroplast DNA is synthesized throughout the cell cycle. Nuclear DNA has a more limited S period. Both chloroplast DNA and nuclear DNA are conserved during 3.3 generations. After 3.3 generations in unlabeled medium, grains per chloroplast followed a Poisson distribution indicating essentially equal labeling of all progeny chloroplasts. It is concluded that the average chloroplast in cells of Ochromonas growing exponentially in the light contains at least 10 segregating DNA molecules.     

A Vertical Gradient of the Chloroplast Abundance among Leaves of Chenopodium album

The abundances of chloroplasts in leaves on the main stems ofChenopodium album at different height levels were investigatedin relation to the photosynthetic capacity and light environmentof the leaves. (1) The number of chloroplasts per mesophyllcell decreased with descending position of leaves, except foryoung developing leaves at the top of plants that had smallerchloroplast numbers per cell than matured leaves beneath them.Contents of chlorophyll and ribulose-1,5-bisphosphate carboxylase/oxygenaseper leaf area that were highest in the topmost young leavesand decreased with decreasing height level indicate that thereis a vertical gradient of chloroplast abundance per leaf areadecreasing from the top of the leaf canopy with depth. (2) Light-saturatingrate of photosynthetic oxygen evolution per leaf area of maturedleaves decreased more steeply with decreasing leaf positionthan the chloroplast number per cell. Gradients of chlorophylland the enzyme protein contents were also steeper than thatof the chloroplast number. Loss of photosynthesis in lower leavesis, therefore, ascribed partly to loss of whole chloroplastsand partly to reduced photosynthetic capacities of the remainingchloroplasts. (3) The chloroplast number per cell in newly expandedsecond leaves was comparable to those in leaves that have developedat later stages of the plant growth but decreased graduallyduring leaf senescence both in the dark and light. The formationof the vertical gradient of chloroplast abundance is, therefore,ascribed to loss of whole chloroplasts during senescence ofleaves. (4) Irradiance a leaf receives decreased sharply fromthe top of the canopy with depth. The physiological or ecophysiologicalsignificance of the vertical distribution of chloroplasts amongleaves was discussed taking light environments of leaves intoconsideration. (Received July 31, 1995; Accepted October 20, 1995)     

Dismantling of Arabidopsis thaliana mesophyll cell chloroplasts during natural leaf senescence

One of the earliest events in the process of leaf senescence is dismantling of chloroplasts. Mesophyll cell chloroplasts from rosette leaves were studied in Arabidopsis thaliana undergoing natural senescence. The number of chloroplasts decreased by only 17% in fully yellow leaves, and chloroplasts were found to undergo progressive photosynthetic and ultrastructural changes as senescence proceeded. In ultrastructural studies, an intact tonoplast could not be visualized, thus, a 35S-GFP::δ-TIP line with a GFP-labeled tonoplast was used to demonstrate that chloroplasts remain outside of the tonoplast even at late stages of senescence. Chloroplast DNA was measured by real-time PCR at four different chloroplast loci, and a fourfold decrease in chloroplast DNA per chloroplast was noted in yellow senescent leaves when compared to green leaves from plants of the same age. Although chloroplast DNA did decrease, the chloroplast/nuclear gene copy ratio was still 31:1 in yellow leaves. Interestingly, mRNA levels for the four loci differed: psbA and ndhB mRNAs remained abundant late into senescence, while rpoC1 and rbcL mRNAs decreased in parallel to chloroplast DNA. Together, these data demonstrate that, during senescence, chloroplasts remain outside of the vacuole as distinct organelles while the thylakoid membranes are dismantled internally. As thylakoids were dismantled, Rubisco large subunit, Lhcb1, and chloroplast DNA levels declined, but variable levels of mRNA persisted.     

Increase in DNA Content of Primary Leaves of Phaseolus vulgaris upon Decapitation

Changes in DNA content of bean (Phaseolus vulgaris) primaryleaves after decapitation were investigated. When apical budswere removed at 11 d, DNA content per leaf increased by about20% at 15 d and then decreased in parallel with the controls.The RNA and chlorophyll contents, fresh weight, and leaf areaexpressed on a single leaf basis changed in the same manneras the DNA content in response to decapitation. But when bothapical and lateral buds were removed, all these values continuedincreasing during the test period. Thus, growing lateral budsand apical buds have the same effect on the DNA change in primaryleaves as that due to ageing of the leaves. Cell number perleaf was not increased by any treatment, indicating that theobserved increase in the DNA content of primary leaves is ascribableto an increase in DNA per cell. Next, the whole shoots above the nodes of primary leaves wereremoved at various ages. The response of primary leaves to decapitationvaried according to their age. With age, they lost the abilityto increase their fresh weight, area, and chlorophyll contentbut not their DNA and RNA contents in response to decapitation.Decapitation stimulated chloroplast replication only withinthe period in which chloroplasts were replicating in controlleaves, but it induced chloroplast enlargement at any age. Therefore,the increase in DNA content after decapitation may be partiallydue to an increase in the amount of chloroplast DNA. When stems were heat-girdled above the nodes of the primaryleaves, these leaves showed responses similar to but smallerthan those to decapitation. The senescence of primary leavesseems to be controlled by the distribution of substances whichare transported from the roots.     

Distinction between Nuclear Satellite DNAs and Chloroplast DNA in Higher Plants

Triton X-100 solubilized chloroplast DNA but not nuclear DNA from a mixture of chloroplasts and nuclei. The buoyant density of chloroplast DNA was different from that of the satellite DNA in all of the species examined (Phaseolus coccineus, Cucumis sativus, Cucumis melo, Antirrhinum majus, Vicia faba, Oenothera fruiticosa youngii). Chloroplast DNA constituted between 4.3% and 0.25% of the total leaf DNA in these species, and was present as 5 to 20 copies in each chloroplast.     

Site of Fluoride Accumulation in Navel Orange Leaves

Fluoride-polluted navel orange leaves, Citrus sinensis (Linn.) Osbeck, were fractionated into the subcellular components in hexane/carbon tetrachloride mixtures having various densities. Fluoride was determined at each fraction. Analyses were also made for the subcellular distribution of chlorophyll, nitrogen, and DNA to assess the extent of cross-contamination of each component.The fraction containing cell wall, nuclei, and partly broken cells apparently contained a major amount of fluoride. However, if allowance was made for the cross-contamination of chloroplasts and chloroplast fragments, the fraction of chloroplasts was found to be the site of the highest fluoride accumulation. When each particulate component was washed with water after drying, the combined washings contained more than 50% of the total fluoride of the isolated fractions.The usual method of subcellular fractionation with aqueous solvent shifted the major site of fluoride accumulation from the fraction of chloroplasts to that of the supernatant.     

Identification of a mutation in chloroplast DNA correlated with formation of defective chloroplasts in a variegated mutant of Nicotiana tabacum

Summary As in wild-type Nicotiana tabacum L., two satellite DNAs having densities of 1.700 and 1.705 g cm^–3 in CsCl were identified in the organelle fraction of homogenates made from variegated leaves of a cytoplasmic mutant of N. tabacum. As the proportion of white to green tissue increased a great reduction in the 1.700 chloroplast DNA occurred correlated with a concomitant reduction in the total number of defective and normal chloroplasts per cell. At the same time, there was an absolute increase in the 1.705 satellite DNA. Separation of the two satellite DNAs was achieved by one cycle of purification on NaI gradients. When the 1.700 chloroplast DNAs from white and from green tissue of variegated leaves were compared, identical properties were found by the conventional buoyant density, T _m and renaturation kinetics measurements. However, using a specially constructed difference melting system, the 1.700 DNA from defective chloroplasts was shown to have an approximately 1% higher GC composition than the DNA from normal chloroplasts. Also, by renaturation of a mixture of alkali denatured normal and defective chloroplast DNAs and subsequent spreading in formamide for electron microscopy, internal regions of mismatching were observed. The nonhomologous region corresponded to about 500–1000 base pairs. No differences in composition of the 1.705 satellite DNA derived from white or green tissues were detected either by difference melting or formation of heteroduplexes.     

Chloroplasts Isolated from Kidney Bean Leaves Are Capable of Phenolic Compound Biosynthesis

The homogenate and chloroplast fractions isolated from the leaves of 10–14-day-old kidney-bean (Phaseolus vulgaris L.) seedlings were incubated with ¹⁴C-L-phenylalanine for 30 min in the light, and the incorporation of radioactivity into phenolic compounds was determined. Label incorporation into phenolic compounds of the homogenate and chloroplast fractions amounted to 15–17 and 4–5% of the introduced radioactivity, respectively. The chloroplasts were about an order of magnitude higher than the homogenate in the specific radioactivity of phenolic compounds. Chloroplasts contained four flavonol glycosides (kaempferol and quercetin aglycones), which were the major components of soluble phenolic compounds of leaves. It was concluded that kidney-bean leaf chloroplasts were capable of performing phenolic compound biosynthesis.     

Use of the fluorochrome 4''6-diamidino-2-phenylindole in genetic and developmental studies of chloroplast DNA

Use of the DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) makes it possible to examine in situ the structure of chloroplast DNA (chDNA) with the fluorescence microscope. This simplifies the study of genetic and developmental changes in chloroplast DNA. Three examples are presented. (a) Wild-type Euglena gracilis B contains several chloroplast DNA nucleoids per chloroplast. A yellow mutant lacking functional chloroplasts is similar, but such nucleoids are absent in an aplastidic mutant strain known from biochemical studies to have lost its chDNA. (b) In vegetative cells of the giant-celled marine algae Acetabularia and Batophora, only about a quarter of the chloroplasts have even one discernible chloroplast DNA particle, and such particles vary in size, showing a 30-fold variation in the amount of DNA-bound DAPI fluorescence detected per chloroplast. By contrast, 98% of chloroplasts in developing Acetabularia cysts contain chDNA, with as many as nine nucleoids per chloroplast. (c) DAPI-stained chloroplasts of chromophyte algae display the peripheral ring of DNA expected from electron microscope studies. However, these rings are not uniform in thickness, but are necklace-like, with the appearance of beads on a string. Since the multiple nucleoids in plastids of chlorophyte algae also appear to be interconnected throughout the chloroplast, a common structural plan may underlie chDNA morphology in both groups of algae.     

The Isolation of DNA from Plant Materials

Total DNA was isolated from seedling and tissues of many plants. The nuclei and chloroplasts were prepared from plant tissues, and then the nuclear DNA and chloroplast DNA were isolated from them. According to the chemical analysis and the physical properties determined by ultraviolet absorbance, hyperchromicities, ultracentrifugation, gel electrophoresis and the electro-microscopical observations it is suggested that DNA obtained possessed a considerable purity and to a certain extent retained the natural status of the large molecule.     

Changes in Chloroplast DNA Levels during Growth of Spinach Leaves

In young spinach leaves, 1–4 mm long, 7–10% of thetotal DNA of the leaf was chloroplast (pt) DNA. Growth in theseleaves was mainly by cell division with plastid division keepingpace with cell division and maintaining about 10 plastids percell. About 1% of the leaf cells were formed in 4.0 mm leaves.Both cell division and cell expansion contribute to the nextstage of leaf growth, which was quantitatively the major periodof new cell formation, nuclear DNA synthesis and ptDNA synthesis.Relative to the nuclear DNA level ptDNA levels rose to 21% ofthe total DNA and chloroplast.plastome copy numbers from 1500to 5000 per cell while chloroplast numbers rose from 10 to 30per cell. In the final period of leaf growth, cell expansionwas the main determinant of growth and chloroplast number percell rose to 180. In contrast to young leaves, newly emergedcotyledons contained 20% of their DNA as ptDNA and, during cellexpansion, cell number per cotyledon doubled. On average, thecells became octoploid, and chloroplast numbers and plastomecopy numbers rose to 500 and 22 000 per cell respectively. Similarlevels of nuclear ploidy, chloroplast number and plastome copynumber were induced in the first leaf pair of spinach followingdecapitation. When senescence was induced in mature leaves byshading, no loss of nuclear or ptDNA occurred. Following theonset of leaf yellowing and a form of senescence induced bynitrogen deficiency in leaves which had not fully expanded,there was preferential loss of ptDNA which fell from 8200 to3700 plastome copies per cell over an 11 d period. Key words: Spinach, Chloroplast, DNA, Ploidy     

Rice chloroplast DNA: a physical map and the location of the genes for the large subunit of ribulose 1,5-bisphosphate carboxylase and the 32 KD photosystem II reaction center protein

Summary By homogenizing rice leaves in liquid nitrogen, it was possible to isolate intact chloroplasts and, subsequently, pure rice chloroplast DNA from the purified chloroplasts. The DNA was digested by several restriction enzymes and fragments were fractionated by agarose gel electrophoresis. The sum of the fragment sizes generated by the restriction enzymes showed that the total length of the DNA is 130 kb. A circular physical map of fragments, generated by digestion with SalI, PstI, and PvuII, has been constructed. The circular DNA contains two inverted repeats of about 20 kb separated by a large, single copy region of about 75 kb and a short, single copy region of about 15 kb. The location of the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase (Fraction I protein) and the 32 KD photosystem II reaction center gene were determined by using as probes tobacco chloroplast DNAs containing these genes. Rice chloroplast DNA differs from chloroplast DNAs of wheat and corn as well as from dicot chloroplast DNAs by having the 32 KD gene located 20 kb removed from the end of an inverted repeat instead of close to the end, as in other plants.     

Degradation of chloroplast DNA in second leaves of rice (Oryza sativa) before leaf yellowing

Summary The second leaf ofOryza sativa develops, grows and ages within the 10 days that follow imbibition under our controlled continuous-light conditions. Proplastids in the leaf cells develop, mature to become chloroplasts and then age and disintegrate. In an examination of this life process, we studied first the behavior and the number of copies of plastid DNA and levels of chlorophyll by epifluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI), and by fluorimetry with a video-intensified microscope photon-counting system (VIMPCS). The results indicated that the number of copies of the plastid DNA per plastid increased and reached to plateau value of approximately 100 at the time when the elongation of the mesophyll cells and the enlargement of chloroplasts ceased 96 h after imbibition. However, 24 h later, the number of copies of plastid DNA per chloroplast began to decrease and fell rapidly to approximately 30 copies within 168 h after imbibition. Our examination of the number of chloroplasts per mesophyll cell indicated that no division of chloroplasts occurred more than 72 h after imbibition. The results suggest that the decrease in number of copies of plastid DNA per chloroplast was not due to an increase in the number of chloroplasts, but that this decrease was caused by degradation by unidentified enzymes. Since visible senescence of leaves, which was characterized by development of a yellowish color, began 168 h after imbibition, the degradation of plastid DNA seemed to occur 48 h before the visible leaf senescence. When we tested the nucleolytic activities in the second leaves after imbibition by digestion of plasmids in vitro and DNA-SDS polyacrylamide gel electrophoresis, five Ca²⁺–, four Zn²⁺–, and four Mn²⁺–dependent nucleases were detected in the leaf blades, and one of the Ca²⁺–, two of the Zn²⁺–, and two of the Mn²⁺–dependent nucleases were also identified in a purified preparation of intact chloroplasts. When the activity of the Zn²⁺–dependent nucleases (51 kDa and 13 kDa) increased markedly, degradation of the plastid DNA occurred. These results suggest that the destruction of chloroplast DNA, which occurs approximately 48 h before leaf yellowing, could be due to the activation of some metallo-nucleases and, furthermore, this enzymatic degradation propels the leaf towards senescence.