Video-intensified Microscope Photon-counting System Research Articles

DNA Content of Individual Mitochondrial Nucleoids Varies Depending on the Culture Conditions of the Yeast Saccharomyces cerevisiae

The DNA content of individual mitochondrial nucleoids (mt-nucleoids) in the stationary-phase cells of the yeast Saccharomyces cerevisiae was quantitatively measured by a combination of DAPI-staining and use of a video-intensified microscope photon counting system (VIMPCS). The mt-nucleoids in the cells which were grown aerobically in AN medium contained, on average, 1.5 mitochondrial DNA (mtDNA) molecules per mt-nucleoid, and the mt-nucleoids that were isolated from these cells contained 1.1 mtDNA molecules per mt-nucleoid. In contrast, the giant mt-nucleoids that appeared in the cells grown anaerobically in the same medium contained, on average, 20.3 mtDNA molecules per mt-nucleoid, and the giant mt-nucleoids that were isolated from these cells contained, on average, 13.1 mtDNA molecules per mt-nucleoid. These results, together with the previous data, demonstrated that the DNA content of individual mt-nucleoids and the number of mt-nucleoids in a cell were distinctly changed by the culture conditions of the yeast cells, whereas the total mtDNA content per cell did not vary significantly when the culture conditions were changed.

Pelargonium embryogenesis: cytological investigations of organelles in early embryogenesis from the egg to the two-celled embryo

Changes in the distribution of organelles and organelle-DNA in Pelargonium zonale from the mature egg cell stage to the first zygotic division during the early stages of embryogenesis were investigated using electron microscopy and fluorescence microscopy. The mature egg is a large, polarized bulbous-shaped cell, tapering toward its micropylar end. The wide chalazal region has a large nucleus that is surrounded by cytoplasm containing many giant mitochondria and large amyloplasts. The mitochondria contain a large amount of mitochondrial DNA and appear as long stretched rods or complex rings, sometimes consisting of several concentric or half-concentric circles in sections. The time from pollination to cell fusion is approximately 6–9 h and it is 20–24 h until the first zygotic division. The changes in the zygote and its organelles preparatory to division occur in 3 stages. At stage 1 (6–9 h after pollination), cell fusion occurs and the zygote begins to elongate. Many vacuoles of varying size appear surrounding the nucleus. At stage 2 (9–15 h), the zygote nucleus migrates to a central position in the cell and the mitochondria form a single ring that becomes either irregularly crushed or appears as long thin strings. Amyloplasts exhibit a gradual decrease in the number of starch grains. At stage 3 (15–20 h), the vacuoles disappear, except for a few that remain in the micropylar region, and cell size decreases. Mitochondria become short, fine strings or small rings. Amyloplasts with starch grains are no longer observed, but are transformed into large proplastids. Following the first division of the zygote, approximately equal-sized apical and basal cells are formed. Short rod-shaped or small ring-shaped mitochondria are randomly distributed near the nucleus of the apical cell, whereas mitochondria in the basal cell are long and rod-shaped. In the electron microscope, two types of plastids can be distinguished: dark oval plastids originating from the sperm cell, which are observed in both the apical and basal cell, and others with a less dense, amorphous matrix, believed to originate from egg amyloplasts, which are unevenly distributed in the micropylar region of the basal cell. Fluorometry using a video-intensified microscope photon counting system reveals that, correlated with changes in mitochondrial morphology, DNA amount within the mitochondrion decreases linearly during these stages.

Large amounts of apicoplast nucleoid DNA and its segregation in Toxoplasma gondii.

Apicoplasts (apicomplexan plastids) are nonphotosynthetic, secondary endosymbiotic plastids that are found in most apicomplexans. Although these organelles are essential for parasite survival, their functions, activities, and structures are not well understood. We examined the apicoplast nucleoid of Toxoplasma gondii from a morphological aspect by high-resolution epifluorescence microscopy and electron microscopy. We found unexpectedly large amounts of DNA in the nucleoid and the presence of several division-related structures. Initially, we identified the organellar nucleoids by staining with the DNA-specific dye 4',6-diamidino-2-phenylindole. A single nucleoid was observed per apicoplast, and the fluorescent spot representing the nucleoid was bright and spherical in contrast to the weak and filamentous spot representing the mitochondrial nucleoid. We also measured the DNA content of each apicoplast nucleoid by a video-intensified microscope photon-counting system and determined that the genomic copy number was at least 25, a figure over four times greater than that reported previously. Moreover, several groups of apicoplasts had significantly higher genomic copy numbers. The DNA molecules were accurately divided into two daughter apicoplasts just before nuclear division. In addition, we examined nucleoid segregation and the division apparatus using electron microscopy. However, we failed to observe nucleoid structures, suggesting that the apicoplasts are predominantly composed of nucleoid material. In addition, we observed "cap" structures at the termini of dividing apicoplasts, a possible plastid-dividing ring, and a microbody-like granule around the constriction. These structures may be involved in apicoplast division.

Characterization of Novel Cytokinesis-defective Mutants of Chlamydomonas reinhardtii.

We isolated 2 independent cytokinesis-defective mutants, inc-1 and inc-2, of the unicellular green alga Chlamydomonas reinhardtii through DNA insertional mutagenesis. Characterization of the mutants, using fluorescence microscopy and microfluorometry, revealed that progression of furrowing begins in inc cells after nuclear division. Unlike wild-type cells, however, the cleavage furrow of the dividing cells disappears before cytokinesis. Successive nuclear divisions with incomplete cytokinesis produce multinucleate cells. Additionally, chloroplast autofluorescence revealed that the chloroplast fails to divide throughout the process of cytokinesis in the mutant cells. We also examined the number of chloroplast nucleoids (cp-nucleoids) and chloroplast DNA (cpDNA) content per chloroplast as indicators of cell cycle. We observed cp-nucleoids through DAPI staining and measured the intensity of DAPI fluorescence with a video-intensified microscope photon-counting system (VIMPCS). Both the number of cp-nucleoids and cpDNA content per chloroplast increased under incomplete cytokinesis in inc cells. The inc mutants represent novel cytokinesis-defective mutants of C. reinhardtii.

Decrease in mitochondrial DNA and concurrent increase in plastid DNA in generative cells of Pharbitis nil during pollen development

The amount of organellar DNA in a generative cell of Pharbitis nil was observed when squashed pollen grains collected on the day of flowering were stained with the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI). Using both DAPI-fluorescence microscopy and electron microscopy, observation of the same thin section of Technovit 7100 resin-embedded material revealed that all of the organellar DNA in mature generative cells is plastid DNA, and there is no mitochondrial DNA. During pollen development, we observed organellar DNA in fluorescence microscopic images using double-staining with DAPI and 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and quantified the DNA using a video-intensified microscope photon counting system (VIMPCS). In the vegetative cells, the amounts of both mitochondrial and plastid DNA progressively decreased and had disappeared by 2 days before flowering. In the generative cells, mitochondrial DNA disappeared sooner than in the vegetative cells, indicating a more active mechanism for the decrease in mitochondrial DNA in the generative cells. In contrast, plastid DNA in the generative cells increased markedly. The DNA content per plastid was at a minimum value (corresponding to one copy of the plastid genome) 7 days before flowering, but it increased to a maximum value (corresponding to over 10 copies of the plastid genome) 2 days before flowering. Similar results were also obtained with immunogold electron microscopy using an anti-DNA antibody. These results suggest that the DNA content of mitochondria and plastids in P. nil is controlled independently during pollen development.

Preferential degradation of plastid DNA with preservation of mitochondrial DNA in the sperm cells ofPelargonium zonale during pollen development

In the present study, we studied changes in organellar DNA in the sperm cells of maturing pollen ofPelargonium zonale, a plant typical to exhibit biparental inheritance, by fluorescence microscopy after staining with 4′,6-diamidino-2-phenylindole (DAPI) and by immunogold electron microscopy using anti-DNA antibody. Fluorescence intensities of DAPI-stained plastid nuclei in generative and sperm cells at various developmental stages were quantified with a video-intensified microscope photon counting system (VIMPCS). Results indicated that the amount of DNA per plastid in generative cells increased gradually during pollen development and reached a maximum value (about 70 T per plastid; 1 T represents the amount of DNA in a particle of T4 phage) in young sperm cells at 5 days before flowering. However, the DNA content of plastids was subsequently reduced to about 20% of the maximum value on the day of flowering. Moreover, the DNA content of the plastid further decreased to 4% of the maximum value when pollen grains were cultured for 6 h in germination medium. In contrast, the amount of DNA per mitochondrion did not decrease significantly around the flowering day. Similar results were also obtained by immunogold electron microscopy using anti-DNA antibody. The density of gold particles on plastids decreased during pollen maturation whereas labelling density on mitochondria remained relatively constant. The number of plastids and mitochondria per generative cell or per pair of sperm cells did not change significantly, indicating that the segregation of DNA by plastid division was not responsible for the decrease in the amount of DNA per plastid. These results indicate that the plastid DNA is preferentially degraded, but the mitochondrial DNA is preserved, in the sperm cells ofP. zonale. While the plastid DNA of the sperm cells decreased before fertilization, it was also suggested that the low DNA contents that remain in the plastids of the sperm cells are enough to account for the biparental inheritance of plastids inP. zonale.

Studies on the development and three-dimensional reconstruction of giant mitochondria and their nuclei in egg cells ofPelargonium zonale Ait.

The preferential development of giant mitochondria and their nuclei (nucleoids) in the egg cells ofPelargonium zonale Ait. during megasporogenesis and megagametogenesis was examined by fluorescence microscopy, after Technovit embedding and 4′,6-diamidino-2-phenylindole (DAPI) staining, fluorimetry for DNA content, using a video-intensified microscope photon-counting system (VIMPICS), and by three-dimensional reconstruction of mitochondrial nuclei (mt-nuclei). Reproductive cells during the megaspore mother cell, meiosis, tetrad, and functioning megaspore stages contained many small mitochondria with characteristic, uniformly DAPI-stained mt-nuclei about 0.3 μm in diameter, containing a small amount of DNA (0.3 Mbp). During formation of the 2-, 4-, and 8-nucleate embryo sac, mt-nuclei did not markedly change in shape or DNA content. When the embryo sac formed and differentiation of each cell began, mitochondria and their nuclei in the egg cell took on a small ring or string-like shape. Accompanying the maturation of the embryo sac, they underwent progressive enlargement and gradually altered to long thick strings, or stacks of concentric or half concentric rings. By flower opening, they have developed to an extremely large size. One of these stacks of mt-nuclei was reconstructed in three dimensions; each ring in the stack was cup- or plate-shaped; 5 to 10 rings made up the stack, though each remained discontinuous from the others. From serial sections, we counted 44 mitochondria in one egg cell. Fluorometry using VIMPICS revealed that DNA amount within the stacked mitochondrion increased to 40 times that of the megaspore mother cell stage; a single stack of mitochondria contained 340–1700 Mbp DNA; which means that one egg cell contains at least 15000 Mbp mt-DNA, a value greater than the cell-nuclear DNA content.

Behavior of organelles and their nucleoids in the shoot apical meristem during leaf development in Arabidopsis thaliana L.

The behavior of organelle nucleoids and cell nuclei was studied in the shoot apical meristem and developing first foliage leaves of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4′-6-diamidino-2-phenylindole to observe DNA. Fluorimetry was performed using a video-intensified microscope photon-counting system. The DNA content of individual mitochondria was more than 1 Mbp in the shoot apical meristem and the young leaf primordium, and decreased to approximately 170 kbp in the mature foliage leaf. In contrast, the DNA content of individual plastids was low in the shoot apical meristem and increased until day 7 after sowing. Application of 5-bromo-2′-deoxyuridine, an analogue of thymidine, was usesd to investigate DNA synthesis in situ. The activities of DNA synthesis in the mitochondria and plastids changed according to the stage of development. Mitochondrial DNA was actively synthesized in the shoot apical meristem and young leaf primordia. This strongly suggests that the amount of mitochondrial DNA per mitochondrion, which has been synthesized in the shoot apical meristem and young leaf primordium, is gradually reduced due to continual divisions of the mitochondria during low levels of mitochondrial DNA synthesis. Synthesis of DNA in the plastid became active in the leaf primordia following DNA synthesis in the mitochondria, and the small plastids were filled with large plastid nucleotids. This enlargement of the plastid nucleoids occurred before the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase and the development of thylakoids.

Cytological Analyses in Melandrium album: Genome Size, C.hromosome Size and Fluorescence in situ Hybridization.

Melandrium album, a dioecious plant, has two sex chromosomes (X and Y). The haploid genome size of M. album was determined to be 4700 Mbp based on the amount of DNA in sperm nuclei in pollen by fluorimetry using a video-intensified microscope photon counting system (VIMPCS) after propidium iodide and 4', 6-diamidino-2-phenylindole staining. This value is about 33 times as much as that of Arabidopsis thaliana. The DNA content of each mitotic chromosome was also determined by VIMPCS. The size of the Y chromosome was 860 Mbp, which was 1.4 times as much as that of the X chromosome. Moreover, we visualized biotin-labeled 18S-5.8S-26S rRNA gene (large rRNA gene) probe and 5S rRNA gene probe in mitotic chromosomes by fluorescence in situ hybridization. Both of the rRNA genes were located on four pairs of autosomes, respectively. The sex chromosomes exhibited neither large rRNA genes nor 5S rRNA genes.

Studies on the behavior of organelles and their nucleoids in the root apical meristem of Arabidopsis thaliana (L.) Col.

The behavior of cell nuclei, mitochondrial nucleoids (mt-nucleoids) and plastid nucleoids (ptnucleoids) was studied in the root apical meristem of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4'-6-diamidino-2-phenylindole for light-microscopic autoradiography and microphotometry. Synthesis of cell nuclear DNA and cell division were both active in the root apical meristem between 0 μm and 300 μm from the central cells. It is estimated that the cells generated in the lower part of the root apical meristem enter the elongation zone after at least four divisions. Throughout the entire meristematic zone, individual cells had mitochondria which contained 1-5 mt-nucleoids. The number of mitochondria increased gradually from 65 to 200 in the meristem of the central cylinder. Therefore, throughout the meristem, individual mitochondria divided either once or twice per mitotic cycle. By contrast, based on the incorporation of [(3)H]thymidine into organelle nucleoids, syntheses of mitochondrial DNA (mtDNA) and plastid DNA (ptDNA) occurred independently of the mitotic cycle and mainly in a restricted region (i.e., the lower part of the root apical meristem). Fluorimetry, using a videointensified microscope photon-counting system, revealed that the amount of mtDNA per mt-nucleoid in the cells in the lower part of the meristem, where mtDNA synthesis was active, corresponded to more than 1 Mbp. By contrast, in the meristematic cells just below the elongation zone of the root tip, the amount of mtDNA per mt-nucleoid fell to approximately 170 kbp. These findings strongly indicate that the amount of mtDNA per mitochondrion, which has been synthesized in the lower part of the meristem, is gradually reduced as a result of continual mitochondrial divisions during low levels of mtDNA synthesis. This phenomenon would explain why differentiated cells in the elongation zone have mitochondria that contain only extremely small amounts of mtDNA.

Studies on the behavior of mitochondrial DNA: Synthesis of mitochondrial dna occurs actively in a specific region just above the quiescent center in the root meristem of Pelargonium zonale

ABSTRACT The fate of mitochondrial nuclei (known as nucleoids or mt-nuclei), which contain extremely small amounts of DNA, was followed in thin sections of the root meristem of Pelargonium zonale by embedding of samples in Technovit 7100 resin and double staining with 4′-6-diamidino-2-phenylindole (DAPI) and acridine orange, in combination with light-microscopic autoradiography and microphotometry. The synthesis of cell-nuclear DNA and cell division occurs actively in the root meristem, between 150 μm and 700 μm from the tip of the root. For simplicity, cells in S phase in the cortex were selected for main analysis as the model system for examination of cell proliferation. It is estimated, on the basis of the length of the cells in longitudinal median sections, that the cells in the cortex, which are generated in the area just above the quiescent center (QC) about 150 μm from the tip, enter the elongation zone after at least five divisions. In the entire cortex, individual cells in S phase have approximately 230 mitochondria that each contain one mt-nucleus. The observation suggests that individual mitochondria divide once per mitotic cycle in the entire region of the meristem. By contrast, on the basis of incorporation of [3H]thymidine into mt-nuclei, the synthesis of mitochondrial DNA (mtDNA) occurs independently of the mitotic cycle in a restricted region just above the QC. Fluorimetry, using a videointensified microscope photon-counting system (VIM-PICS), revealed that the mtDNA content per mt-nucleus in the cells just above QC, where the synthesis of mtDNA is active, corresponds to approximately 3000 kilobase pairs (kbp) but, in the meristematic cells just below the elongation zone of the root it falls to less than 170 kbp. These findings strongly suggest that the amount of mtDNA per mitochondrion which has been synthesized in the region just above the QC is reduced stepwise as a result of continuous divisions of mitochondria in the absence of the synthesis of mtDNA. This phenomenon would explain why differentiated cells with a large vacuole in the elongation zone have mitochondria that contain only extremely small amounts of mtDNA.

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

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.

Absence of DNA in the basal body ofChlamydomonas reinhardtii by fluorimetry using a video-intensified microscope photon-counting system

A search was made for DNA in both the basal bodies (BBs) in situ and BBs isolated from cells ofChlamydomonas reinhardtii by high-resolution epifluorescence microscopy after staining with 4′-6-diamidino-2-phenylindole (DAPI), by fluorimetry using a video-intensified microscope photon-counting system (VIMPICS) and by immunofluorescence microscopy after staining with a monoclonal tubulin-specific antibody. The flagella and intracellular microtubules radiate from the BBs. The BBs in young vegetative cells, gametes and young zygotes do not emit fluorescence after staining with DAPI but the spherical cell nucleus, the ovoid chloroplast nuclei and the tiny mitochondrial nuclei emit bright, blue-white fluorescence. Thus, it appears that BBs do not contain larger amounts of DNA than do the other organelles. To avoid the halation effects of fluorescence from the cell debris and cytoplasm and to measure carefully any extremely low levels of DNA that might be present in the organelles, a complex, composed of two flagella, a pair of BBs and the cell nucleus, was isolated from the gametes by treatment with autolysin and 0.1% Triton X-100. After staining with DAPI, the BBs of such complexes exhibit faint fluorescence while the cell nucleus emits strong fluorescence. The point and total intensities of the fluorescence emitted from each portion of the complex were measured with the VIMPICS. When the fluorescence intensity “T” of T 4 phage is taken as a standard, the fluorescence intensities of the flagella, the pair of BBs, the cell nucleus and the nucleus ofEscherichia coli are respectively 0.2 T, 0.40 T, 1452.2 T and 20.4 T. The slight fluorescence emitted from the BB seems to be due to the halation of the fluorescence emitted from the cell nucleus. The intensity of the fluorescence from the BBs is reduced to the intensity of the fluorescence of the flagella when the cell nucleus is removed from the complex. From these results, we conclude that the BBs do not contain DNA. Discrepancies related to the reported presence of DNA in the BBs are discussed.

Chloroplast division without DNA synthesis during the life cycle of the unicellular algaCyanidium caldarium M-8 as revealed by quantitative fluorescence microscopy

The behavior and DNA content of the cell and chloroplast nuclei (synonymous with nucleoids; ct-nuclei) during the life cycle have been studied in a synchronized population of cells of the unicellular algaCyanidium caldarium M-8. Cells were examined by epifluorescence microscopy after staining with 4′,6-diamidino-2-phenylindole (DAPI), and by fluorimetry using a video-intensified microscope photon-counting system (VIMPICS). The young cell contains a single petal-like chloroplast, a spherical cell nucleus and several mitochondria, and the cell nucleus and the chloroplast divide in that order just prior to cytokinesis. The chloroplast contains a ring-shaped ct-nucleus which is located at the periphery of the chloroplast during the life cycle. During the first 40 h after the initiation of sychronous cultures, the young cell and its chloroplast increase markedly in size, and the DNA contents per cell nucleus and per ct-nucleus increase approximately two times and 16 times the value in 16-endospore cells, respectively. Four endospore divisions then occur, at intervals of approximately 12 h between 40 h and 90 h, after the initiation of synchronous cultures. The volume of each cell, the volume of each chloroplast, the amount of chloroplast DNA (ct-DNA), and the level of pigmented material in the chloroplast are reduced stepwise after each endospore division until finally, at the 16-endospore stage, they reach approximately 1/16 of the original values for the mother cell. The size of the mitotic spindle also is reduced stepwise as the cell divisions proceed. By contrast, the cell nuclei duplicate their DNA during each endospore division cycle. These results and analysis of other components indicate that the chloroplasts divide into two daughter chloroplasts without any DNA synthesis during four successive cycles of endospore division and also that the DNA content of the chloroplast is intimately related to the volume of the chloroplast and the cell and to the level of pigmented material in the cloroplast, but is not related to the DNA content of the cell nuclei.

No effect of preferential digestion of chloroplast genome of male origin on chloroplast genome of female origin in young zygotes of Chlamydomon as reinhardtii as revealed by a video-intensified microscope photon counting system.

It has been examined using a video-intensified microscope photon counting system (VIMPCS) whether preferential digestion of chloroplast gemone of male origin in young zygotes of Chlamydomonas reinhardtii affects the chloroplast genomes of female origins in number. To distinguish between the chloroplasts of male origin and the chloroplasts of female origin in young zygotes, a cross, that is, mt+ (gametes with normal chlorophyll, 137c wild type)×mt- (gametes with diminished chlorophyll, ac29a mutant) was prepared. The timing of initiation of the digestion of the male chloroplast genome differs among zygotes but usually occurs during 50-120min. The degrees of the preferential destruction of male chloroplast genomes in young zygotes were quantitatively examined using VIMPCS after 4′-6-diamidino-2-phenyindole (DAPI) staining. The results show that each gamete (mt+, mt-) has a cu-pshaped chloroplast including 7-8 chloroplast nuclei (ct-nuclei). Each ct-nucleus (mt-, mt+) is composed of 1-43 copies of the chloroplast genome (mean number, 10 copies) and female and male chloroplasts contain about 83 and 86 copies, respectively. In most zygotes, all copies of the chloroplast genome from the male gamete are completely digested during the first 40-180min after mating, while all copies from the female gamete persit till at least 4 days later when 7ct-nuclei have fused together to form one large ct-nucleus. This suggests that the preferential digestion of the chloroplast genome of male origin does not affect the chloroplast genome of female origin in zygotes.