Behavior of organelle nuclei (nucleoids) in generative and vegetative cells during maturation of pollen inLilium longiflorum andPelargonium zonale

Summary The behavior of organelle nuclei during maturation of the male gametes ofLilium longiflorum andPelargonium zonale was examined by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and Southern hybridization. The organelle nuclei in both generative and vegetative cells inL. longiflorum were preferentially degraded during the maturation of the male gametes. In the mature pollen grains ofL. longiflorum, there were absolutely no organelle nuclei visible in the cytoplasm of the generative cells. In the vegetative cells, almost all the organelle nuclei were degraded. However, in contrast to the situation in generative cells, the last vestiges of organelle nuclei in vegetative cells did not disappear completely. They remained in evidence in the vegetative cells during germination of the pollen tubes. InP. zonale, however, no evidence of degradation of organelle nuclei was ever observed. As a result, a very large number of organelle nuclei remained in the sperm cells during maturation of the pollen grains. When the total DNA isolated from the pollen or pollen tubes was analyzed by Southern hybridization with a probe that contained therbc L gene, for detection of the plastid DNA and a probe that contained thecox I gene, for detection of the mitochondrial DNA, the same results were obtained. Therefore, the maternal inheritance of the organelle genes inL. longiflorum is caused by the degradation of the organelle DNA in the generative cells while the biparental inheritance of the organelle genes inP. zonale is the result of the preservation of the organelle DNA in the generative and sperm cells. To characterize the degradation of the organelle nuclei, nucleolytic activities in mature pollen were analyzed by an in situ assay on an SDS-DNA-gel after electrophoresis. The results revealed that a 40kDa Ca²⁺-dependent nuclease and a 23 kDa Zn²⁺ -dependent nuclease were present specifically among the pollen proteins ofL. longiflorum. By contrast, no nucleolytic activity was detected in a similar analysis of pollen proteins ofP. zonale.     

The FtsH protease heterocomplex in Arabidopsis: dispensability of type-B protease activity for proper chloroplast development

FtsH is an ATP-dependent metalloprotease present as a hexameric heterocomplex in thylakoid membranes. Encoded in the Arabidopsis thaliana YELLOW VARIEGATED2 (VAR2) locus, FtsH2 is one isoform among major Type A (FtsH1/5) and Type B (FtsH2/8) isomers. Mutants lacking FtsH2 (var2) and FtsH5 (var1) are characterized by a typical leaf-variegated phenotype. The functional importance of the catalytic center (comprised by the zinc binding domain) in FtsH2 was assessed in this study by generating transgenic plants that ectopically expressed FtsH2(488), a proteolytically inactive version of FtsH2. The resulting amino acid substitution inhibited FtsH protease activity in vivo when introduced into Escherichia coli FtsH. By contrast, expression of FtsH2(488) rescued not only leaf variegation in var2 but also seedling lethality in var2 ftsh8, suggesting that the protease activity of Type B isomers is completely dispensable, which implies that the chloroplastic FtsH complex has protease sites in excess and that they act redundantly rather than coordinately. However, expression of FtsH2(488) did not fully rescue leaf variegation in var1 var2 because the overall FtsH levels were reduced under this background. Applying an inducible promoter to our complementation analysis revealed that rescue of leaf variegation indeed depends on the overall amount of FtsH. Our results elucidate protein activity and its amount as important factors for the function of FtsH heterocomplexes that are composed of multiple isoforms in the thylakoid membrane.     

柏科4个属(Fokienia、Cupressus、Chamaecyparis和Juniperus)植物雌球果的发育(英文)

通过扫描电镜和常规石蜡切片技术,观察了柏科4个属(Fokienia、Cupressus、Chamaecyparis和Juniperus)植物雌球果中胚珠的发育及苞片的结构变化。在所有研究的种类中,可育苞片腋部最先观察到的结构是一扁平的突起,并在其上分化出发育为胚珠的胚珠原基。在雌球果的发育过程中,未观察到独立的珠鳞发育。不同的种中,胚珠的数量和发育顺序有所不同,但苞片的发育是相似的。传粉前,苞片的结构与叶相似。传粉后,由于剧烈的居间生长,苞片发育为盾形,形成球形的球果。另外,在发育后期,苞片内维管系统变得复杂,并且在近轴面有反向的维管束发育。我们还对柏科植物雌球果的形态学特性及其可能的演化趋势进行了讨论。     

Mitochondrial dynamics in plant male gametophyte visualized by fluorescent live imaging

Visualization of organelles in living cells is a powerful method for studying their dynamic behavior. Here we attempted to visualize mitochondria in angiosperm male gametophyte (pollen grain from Arabidopsis thaliana) that are composed of one vegetative cell (VC) and two sperm cells (SCs). Combination of mitochondria-targeted fluorescent proteins with VC- or SC-specific expression allowed us to observe the precise number and dynamic behavior of mitochondria in the respective cell types. Furthermore, live imaging of SC mitochondria during double fertilization confirmed previous observations, demonstrated by electron microscopy in other species, that sperm mitochondria enter into the egg and central cells. We also attempted to visualize mutant mitochondria that were elongated due to a defect in mitochondrial division. This mutant phenotype was indeed detectable in VC mitochondria of a heterozygous F(1) plant, suggesting active mitochondrial division in male gametophyte. Finally, we performed mutant screening and isolated a putative mitochondrial protein transport mutant whose phenotype was detectable only in haploid cells. The transgenic materials presented in this work are useful not only for live imaging but also for studying mitochondrial functions by mutant analysis.     

The model plant Medicago truncatula exhibits biparental plastid inheritance

The plastid, which originated from the endosymbiosis of a cyanobacterium, contains its own plastid DNA (ptDNA) that exhibits a unique mode of inheritance. Approximately 80% of angiosperms show maternal inheritance, whereas the remainder exhibit biparental inheritance of ptDNA. Here we studied ptDNA inheritance in the model legume, Medicago truncatula. Cytological analysis of mature pollen with DNA-specific fluorescent dyes suggested that M. truncatula is one of the few model plants potentially showing biparental inheritance of ptDNA. We further examined pollen by electron microscopy and revealed that the generative cell (a mother of sperm cells) indeed has many DNA-containing plastids. To confirm biparental inheritance genetically, we crossed two ecotypes (Jemalong A17 and A20), and the transmission mode of ptDNA was investigated by a PCR-assisted polymorphism. Consistent with the cytological observations, the majority of F(1) plants possessed ptDNAs from both parents. Interestingly, cotyledons of F(1) plants tended to retain a biparental ptDNA population, while later emergent leaves tended to be uniparental with either one of the parental plastid genotypes. Biparental transmission was obvious in the F(2) population, in which all plants showed homoplasmy with either a paternal or a maternal plastid genotype. Collectively, these data demonstrated that M. truncatula is biparental for ptDNA transmission and thus can be an excellent model to study plastid genetics in angiosperms.     

柏科4个属(Fokienia、Cupressus、Chamaeyparis 和Juniperus)植物雌球果的发育

通过扫描电镜和常规石蜡切片技术,观察了柏科4个属(Fokienia、Cupressus、Chamaeyparis 和Juniperus)植物雌球果中胚珠的发育及苞片的结构变化.在所有研究的种类中,可育苞片腋部最先观察到的结构是一扁平的突起,并在其上分化出发育为胚珠的胚珠原基.在雌球果的发育过程中,未观察到独立的珠鳞发育.不同的种中,胚珠怕数量和发育顺序有所不同,但苞片的发育是相似的.传粉前,苞片的结构与叶相似.传粉后,由于剧烈的居间维管束发育.我们还对柏科植物雌球果的形态学特性及其可能的演化趋势进行了讨论.     

银杏小孢子中的微核形成及其在进化过程中的意义

通过ＤＡＰＩ（４’,６－ｄｉａｍｉｄｉｎｏ－２－ｐｈｅｎｙｌｉｎｄｏｌｅ）染色的压片,对银杏小孢子的形成过程进行了观察,发现小孢子母细胞减数分裂过程中,有的细胞在中期Ｉ、中期Ⅱ时出现染色体排列异常和后期染色体桥等畸变。所形成的四分体中,具有一定数量的微核;个别植株的四分体中可有高达６．５％的微核;具微核的小孢子很可能败育。结合古植物方面的研究结果,银杏小孢子形成过程的这些异常规象为认识银杏的演化趋     

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.     

Application of YO-PRO-1 as an Epifluorescent Dye for in situ Detection of Small Amount DNA in Plant Cells

i.e. plastid and mitochondrial DNA in the plant cells such as the sperm cell of Jasminum nudiflorum, the generative cell of Pharbitis lim-bata, the cultured cell of Nicotiana tabacum and the root cell of Vicia faba with epifluorescence microscopy and laser confocal microscopy using YO-PRO-1 as a fluorescent dye. The excitation for YO-PRO-1 was blue light in epifluorescence microscopy and 488 nm Kr/Ar ion laser in confocal microscopy. Dimorphic epifluorescent spots that corresponded plastid DNA and mitochondrial DNA were distinctly detected in the cells of each species examined. In this report, we introduce YO-PRO-1 as a new epifluorescent dye for successful in situ detection of small amount DNA in plant live cells and cell sections with perticular emphasis on the importance of sample preparation. Received 10 November 1998/ Accepted in revised form 13 January 1999