Genome Expression during Normal Leaf Development : 2. Direct Correlation between Ribulose Bisphosphate Carboxylase Content and Nuclear Ploidy in a Polyploid Series of Wheat

The quantitative relationships between ribulose bisphosphate carboxylase, nuclear ploidy, and plastid DNA content were examined in the nonisogenic polyploid series Triticum monococcum (2×), Triticum dicoccum (4×), and Triticum aestivum (6×). Ribulose bisphosphate carboxylase per mesophyll cell increased in step with each increase in nuclear ploidy so the ratios of ribulose bisphosphate carboxylase per mesophyll cell (picograms) to nuclear DNA per mesophyll cell (picograms) were almost identical in the three species. Ribulose bisphosphate carboxylase per plastid was 14.1, 14.7, and 16.8 picograms in the 2×, 4×, and 6× ploidy levels, respectively. Plastid area in these three species decreased with increasing nuclear ploidy so the concentration of ribulose bisphosphate carboxylase in the plastoids was 60% higher in the hexaploid compared to the diploid species. DNA levels per plastid were 64 and 67 femtograms for the diploid and tetraploid species, respectively, but were 40% less in the plastids of the hexaploid species. These relationships are discussed in terms of cellular and plastid control of ribulose bisphosphate carboxylase content.     

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     

Some factors controlling step-wise variation of organelle number in Euglena gracilis

Both nuclear and plastid ploidy double during the first 2–3 cell generations during the phosphate-induced shift from half ploidy to full ploidy in mixotrophic Euglena. Growth conditions can affect nuclear and plastid ploidy differently; plastid ploidy is more labile than nuclear ploidy during heterotrophic growth of both wild type and mutant cells, while nuclear ploidy is more labile during photoautotrophic growth.     

Plastid DNA during grain filling in wheat

During grain filling in wheat (Triticum aestivum L.) there is a progressive increase in the number of amyloplasts in the endosperm, as well as in cell number, DNA content and nuclear ploidy as the grain increases in size. The plastid DNA content also rises initially, and then there is a levelling off in the amount, with the percentage plastid DNA finally making up approximately 0.9% of the total endosperm DNA.     

Expression of the Plastid ndhF Gene Product in Photosynthetic and Non-Photosynthetic Tissues of Developing Barley Seedlings

A fragment of the NDH-F subunit of the plastid NAD(P)H dehydrogenasecomplex (NAD(P)H-plastoquinone-oxidoreductase) from barley wasexpressed as a fusion protein in Escherichia coli and an antibodyto the fusion protein was prepared. Western blot analysis usingthe anti-NDH-F antibody showed specificity towards a plastidpolypeptide of approximately 70 kDa present in both photosyntheticand non-photosynthetic barley tissue. The polypeptide was foundin thylakoid membranes of green leaves whereas in etiolatedleaves it was shown to be associated with the membrane fractionof etioplasts. NDH-F levels were higher in roots and etiolatedtissue than in greening or young leaves. During leaf ontogeny,NDH-F levels decreased from young to mature tissue but increasedduring senescence. The accumulation of NDH-F in thylakoids ofyoung leaves was stimulated by photooxidative treatment. Theresults indicate a high degree of expression of plastid ndhgenes (which encode NAD(P)H dehydrogenase sub-units) in non-photosyntheticplastids and under conditions which impair the photosyntheticactivity of chloroplasts. In addition to its putative implicationin photosynthetic electron transport, a non-photosynthetic role,such as chloro-respiration, is proposed for the plastid NAD(P)Hdehydrogenase complex. (Received May 20, 1997; Accepted October 8, 1997)     

Plastid-DNA levels in the different tissues of potato

The plastid-(pt) DNA levels in the different tissues of potato (Solanum tuberosum L.), including tubers of differing ages, have been studied. The DNA could be detected as a single nucleoid in amyloplasts of cells from young potato tubers by fluorescence microscopy, following staining of glutaraldehyde-fixed tissue with 4,6-diamidino-2-phenyl indole (DAPI). The renaturation kinetics of spinach ptDNA in the presence of total DNA from potato tissues and the fragments generated by restriction-enzyme digestion of potato-tuber DNA and chloroplast DNA indicated that the ptDNA of potato-tuber amyloplasts and of potato-leaf chloroplasts is essentially the same. Expressed as a percentage of the total DNA the level of ptDNA (5.2%) found in tubers, while less than that found in leaves (7.6%) was more than that found in petioles (3.4%), stems (3.0%) and roots (1.0%). There was a high level of both nuclear and plastid ploidy in mature potato-tuber cells and, on average, nuclei contained 32 pg of DNA (equivalent to 14C) and the 40 amyloplasts per cell contained DNA equivalent to 7800 copies of ptDNA, or 195 copies per amyloplast.Abbreviations DAPI 4,6-diamidino-2-phenyl-indole - LSU large sub-unit of ribulose-1,5-bisphosphate carboxylase - mtDNA mitochondrial DNA - ptDNA chloroplast or plastid DNA     

Endoreduplication Occurs during Pod Wall Development in Temperate Grain Legumes

Differentiation to a specialized function in plant tissue isassociated with an increase in the DNA content of the cellsover the diploid state. Using flow cytometry, ploidy levelswere assessed during pod development in three white lupin genotypesunder three environmental conditions and in other lupin speciesand temperate grain legumes in one environmental condition.Endoreduplication was found in the pod walls of all genotypesstudied. Higher ploidy levels coincided with maximal growthof the pod. DNA replication was not related to the proportionof the pod that comprised walls. Endoreduplication also occurredin other temperate grain legumes with at least two DNA duplicationsduring the development of the pod walls. The biological significanceof endoreduplication in the pod walls of grain legumes is unknown,but could be related to the storage function of these organs.Copyright2000 Annals of Botany Company Lupinus species, Pisum sativum, Phaseolus vulgaris, Vicia faba, pod wall development, ploidy level, flux cytometry, Bis-benzimide Hoechst 33258     

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.     

Independent effects of leaf growth and light on the development of the plastid and its DNA content in Zea species

In maize (Zea mays L.), chloroplast development progresses from the basal meristem to the mature leaf tip, and light is required for maturation to photosynthetic competence. During chloroplast greening, it was found that chloroplast DNA (cpDNA) is extensively degraded, falling to undetectable levels in many individual chloroplasts for three maize cultivars, as well as Zea mexicana (the ancestor of cultivated maize) and the perennial species Zea diploperennis. In dark-grown maize seedlings, the proplastid-to-etioplast transition is characterized by plastid enlargement, cpDNA replication, and the retention of high levels of cpDNA. When dark-grown seedlings are transferred to white light, the DNA content per plastid increases slightly during the first 4 h of illumination and then declines rapidly to a minimum at 24 h during the etioplast-to-chloroplast transition. Plastid autofluorescence (from chlorophyll) continues to increase as cpDNA declines, whereas plastid size remains constant. It is concluded that the increase in cpDNA that accompanies plastid enlargement is a consequence of cell and leaf growth, rather than illumination, whereas light stimulates photosynthetic capacity and cpDNA instability. When cpDNA from total tissue was monitored by blot hybridization and real-time quantitative PCR, no decline following transfer from dark to light was observed. The lack of agreement between DNA per plastid and cpDNA per cell may be attributed to nupts (nuclear sequences of plastid origin).     

Protein Synthesis in Isolated Plastids during Chloroplast Development in Wheat

Light-driven protein synthesis in isolated plastids was studiedduring the greening of etiolated wheat (Triticum aestivum L.)seedlings. The process was divided into five phases (I to V)according to the recovery of plastids from the leaf tissue.The activity was not detected in the etioplasts, but rapidlyincreased to the maximum level in phase I and remained at thislevel through phase II. During the transition from phase IIto III, the activity rapidly decreased to one-third and thencontinued to decrease slowly. The plastid polypeptides synthesizedduring the greening were analyzed by SDS-polyacrylamide gelelectrophoresis. In phase I, membrane polypeptides having molecularweights of about 21k were synthesized, while 23 k membrane polypeptidewas synthesized in phases III, IV and V. Synthesis of solublepolypeptides of 50–60 k and membrane polypeptides of 15k and 30–35 k was active in phases I and II, but decreasedbetween phases II and III. (Received October 31, 1983; Accepted May 14, 1984)     

Determination of ploidy levels in Ipheion uniflorum (R. C. Graham) Rafin (Liliaceae)

In this study, chromosome number and ploidy levels of Ipheion uniflorum cv. "Wisley Blue" (spring starflower) were determined. In meristematic root tip cells, chromosome number was found as 2n = 12 and 4n = 24. The ratios of diploid and tetraploid cells were found as 80.74% and 19.26%, respectively. In differentiated root tissues and mature leaf tissues ploidy levels were analysed by flow cytometry and polysomaty were found in both organs. In differentiated root tissues, ploidy levels were found as 2C, 4C, 8C and 16C DNA. In root tissues percentages of 2C, 4C, 8C and 16C nuclear DNA content were observed as 57.2%, 33.1%, 2.47% and 7.23%, respectively. In mature leaf tissues, ploidy levels were determined 2C, 4C, 8C and 16C DNA. In this tissue the frequency of 4C DNA was found very higher (74.3%) and 2C DNA content was determined as 19.2%. In mature leaf tissue, 8C and 16C nuclear DNA contents were observed as 2.72% and 3.78%, respectively. When nuclear DNA contents in leaves and roots were compared, an apparent difference in 2C and 4C DNA contents was found.     

Stereological Analysis of Cotyledon Cell Development in Phaseolus: II. THE DEVELOPING COTYLEDON

Quantitative changes in organelle volume, number, and area duringcotyledon cell development in Phaseolus vulgaris are describedduring the early period of reserve protein synthesis. RoughER undergoes a 400-fold increase in area per cell, and alsochanges in shape; changes in mitochondrial volume do not parallelthe changes which occur in respiration rate; nuclear volumeincreases, and relationships between cell and nuclear volume,DNA content, and cell weight are discussed; plastid divisionappears to occur in steps.     

Comparative analysis of 343 plastid genomes of Solanum section Petota: Insights into potato diversity,phylogeny, and species discrimination

Common potato (Solanum tuberosum L.) and its wild relatives belong to Solanum section Petota. This section's phylogeny and species delimitation are complicated due to various ploidy levels, high heterozygosity, and frequent interspecific hybridization. Compared to the nuclear genome, the plastid genome is more conserved, has a haploid nature, and has a lower nucleotide substitution rate, providing informative alternative insights into the phylogenetic study of section Petota. Here, we analyzed 343 potato plastid genomes from 53 wild and four cultivated species. The diversity of sequences and genomes was comprehensively analyzed. A total of 24 species were placed in a phylogenetic tree based on genomic data for the first time. Overall, our results not only confirmed most existing clades and species boundaries inferred by nuclear evidence but also provided some distinctive species clade belonging and the maternally inherited evidence supporting the hybrid origin of some species. Furthermore, the divergence times between the major potato clades were estimated. In addition, the species discriminatory power of universal barcodes, nuclear ribosomal DNA, and whole and partial plastid genomes and their combinations were thoroughly evaluated; the plastid genome performed best but had limited discriminatory power for all survey species (40%). Overall, our study provided not only new insights into phylogeny and DNA barcoding of potato but also provided valuable genetic data resources for further systematical research of Petota.     

Ribulose Bisphosphate Carboxylase Activity in Anther-Derived Plants of Saintpaulia ionantha Wendl. Shag

Plants obtained from anther culture of the African violet, Saintpaulia ionantha Wendl. `Shag' and vegetatively cloned copies of the parent anther donor plant were examined for their ploidy and ribulose-1,5-biphosphate carboxylase (RuBPcase) activity. The cloned parent plants were all diploid and did not vary much in their nuclear DNA, chlorophyll, and RuBPcase activity. Some of the anther-derived plants were similar to the parent plants while others were not. Different levels of ploidy were observed among the androgenetic plants. RuBPcase activities higher than that of the parent plants were found in some anther-derived plants. However, there was no direct correlation between ploidy and RuBPcase activity. Expression of nuclear genes from a single parent in the anther-derived plants and it's diploidization or plastid changes during early stages of microsporogenesis or androgenesis are suggested as possible reasons for the variations observed among them. This could be a useful technique to obtain physiological variants which could be agronomically desirable.     

Plastid and nuclear DNA synthesis are not coupled in suspension cells ofNicotiana tabacum

The relationship between nuclear and plastid DNA synthesis in cultured tobacco cells was measured by following³H-thymidine incorporation into total cellular DNA in the absence or presence of specific inhibitors. Plastid DNA synthesis was determined by hybridization of total radiolabeled cellular DNA to cloned chloroplast DNA. Cycloheximide, an inhibitor of nuclear encoded cytoplasmic protein synthesis, caused a rapid and severe inhibition of nuclear DNA synthesis and a delayed inhibition of plastid DNA synthesis. By contrast, chloramphenicol which only inhibits plastid and mitochondrial protein production, shows little inhibition of either nuclear or plastid DNA synthesis even after 24 h of exposure to the cells. The inhibition of nuclear DNA synthesis by aphidicolin, which specifically blocks the nuclear DNA polymeraseα, has no significant effect on plastid DNA formation. Conversely, the restraint of plastid DNA synthesis exerted by low levels of ethidium bromide has no effect on nuclear DNA synthesis. These results show that the synthesis of plastid and nuclear DNA are not coupled to one another. However, both genomes require the formation of cytoplasmic proteins for their replication, though our data suggest that different proteins regulate the biosynthesis of nuclear and plastid DNA.     

A microspectrofluorometric analysis of nuclear and chloroplast DNA in Volvox

Nuclear division immediately follows nuclear DNA doubling in all stages of the life cycle examined in the green alga Volvox; fluorescence microfluorometry of individual cells revealed no evidence of prolonged accumulation of nuclear DNA prior to mitosis in reproductive cells. Somatic cell nuclear DNA quantity is unaffected by developmental events in gonidia of the same spheroid; it remains constant from the end of cleavage until the death of the cell. In reproductive cells, chloroplast DNA replication precedes nuclear replication. The sites of plastid DNA accumulation, made visible by use of the fluorochrome 4′,6-diamidino-2-phenylindole, increase in number during the prolonged growth phase of the V. carteri gonidium. Microspectrofluorometry of fluorochrome-stained DNA in situ shows that plastid DNA increases exponentially throughout this phase. The continuous plastid DNA accumulation during gonidial growth appears to represent a prokaryote-like instead of a eukaryote-like control of DNA synthesis. Most somatic cells contain plastid DNA, and this does not increase in amount during colony growth and reproduction. Most sperm cells also contain plastid DNA, although approximately 5% of somatic cells and up to 20% of sperm cells have no discernable plastid DNA. This is the second group of organisms in which DNA-free plastids have been observed.     

A method for determining ploidy distributions in liver tissue by stereological analysis of nuclear size calibrated by flow cytometric DNA analysis

A method is presented for determining ploidy distributions in mouse liver from image analysis with stereological estimations of nuclear size in tissue sections. Nuclear profile distributions obtained from profile measurements were subjected to a mathematical unfolding procedure in order to obtain the nuclear size distributions. Based on the assumption that nuclear size increases monotonically with nuclear DNA content, flow cytometric DNA analysis of suspensions of liver cell nuclei was used to calibrate the method, thus yielding the mean nuclear size of each ploidy class, i.e., diploid, tetraploid, and octaploid nuclei. After the size interval for each of the ploidy classes was determined, the method allowed determination of ploidy distributions in mouse liver by stereological image analysis alone. The method was established from combined stereological and flow cytometric measurements on liver tissue representing two different stages of liver regeneration after two-thirds partial hepatectomy, and it was tested against an independent set of data representing a marked increase in the portion of S-phase cells.