(1.10.2)--油松细胞质遗传的细胞学机理雄配子的超微结构和细胞器类核.pdf

油松细胞质遗传的细胞学机理：雄配子的超微结构和细胞器类核摘要：证明了油松（Pinus tabulaeformis Carr）雄配子存在质体和线粒体及细胞器 DNA 提供了油松具父系质体和线粒体遗传基础的确切的细胞学证据结果与松科植物在遗传学上已确定的父系质体遗传的一般规律是一致的。但精细胞中的线粒体是否能传递至胚 还需要追踪其后的发育过程。另一重要的结果是揭示了油松的雄配子是细胞这与以前将松科植物雄配子归入雄核（精核）的类型不同。精细胞无壁 仅被质膜包围。在切面上 第一精细胞体积大 长形 具大量的细胞质；第二精细胞体积较小 球形 细胞质量少。根据油松雄配子是细胞的类型 对松科植物雄配子是细胞还是雄核进行了讨论 提出了雄配子的类型是否能代表科的特征 还需扩大研究种类才能下结论。关键词：裸子植物；细胞质遗传；父系质体遗传；母系线粒体遗传；裸子植物精细胞；油松中图分类号：Q 9 4 3 文献标识码：A文章编号：0 5 7 7-7 4 9 6（2 0 0 0）0 1-0 0 2 3-0 9Cytological Mechanism of Cytoplasmic Inheritance in Pinus tabulaeformis：Ultrastructural Aspects and Nucleoids of the Male GametesHU Sh-i Yi1 GUO Feng-Li2 YUAN Zong-Fei1 XU Sh-i Xiong（S Y Zee）3（1 College of Life SciencesPeking UniversityBeijing1 0 0 8 7 1 China；2 College of Biological SciencesChina Agricultural UniversityBeijing1 0 0 0 9 4 China；3 Department of BotanyThe University of Hong KongPokfulam RoadHong KongChina）Abstract：The cytological mechanism of plastid and mitochondrion inheritance in Pinus is an interesting re-search topic with only a limited number of published articlesThe results indicate that the sperms of Pinustabulaeformis Carrcontain abundant plastidsmitochondria and organelle DNA These data provide reliablecytological evidence of paternal plastid and mitochondrion inheritance in Pinus The results are in line with theconfirmed general rule of paternal plastid inheritance in Pinaceae But whether mitochondria in sperm cells canbe transmitted into the embryos is an issue needs further developmental studiesAnother important finding isthat contrary to earlier classification of the male gamete of Pinaceae into the male nuclei type the results revealthat male gametes in Pinus tabulaeformis are actually cellsHowever the sperm cells are only surrounded byplasma membranes without cell walls The larger leading sperm cell in a pollen tube section is long in shapewith a large amount of cytoplasm；while the second sperm cell is smaller round in shape and contains less cy-toplasm Whether this feature of the male gamete type could be considered as a representative characteristic ofthe family is discussed and further conclusions await more experimental evidences from studies on plants fromdifferent speciesKey words：gymnosperm；cytoplasmic inheritance；paternal plastid inheritance；maternal mitochondrion in-heritance；gymnosperm sperm cell；Pinus tabulaeformis By using restrict fragment length polymorphism（RFLP）techniqueit has been demonstrated that pater-nal plastid inheritance1 8and maternal mitochondrioninheritance are prevalent in PseudotsugaPinusPiceaand Larix3 9 1 3 Paternal plastid inheritance appears tobe dominant in Pinaceae and has been considered as ageneral trait in gymnosperms1 4 1 5 This feature is differ-ent in angiosperm alga and moss where maternal plastidinheritance is prevalent1 6The mechanism of organelleinheritance modes in angiosperms has been extensivelystudied1 4 1 7These studies indicate that the exclusionand or degeneration of paternal plastid or decompositionReceived：1 9 9 9-0 9-2 7 Accepted：1 9 9 9-1 1-2 2Foundation item：The National Natural Science Foundation of China（3 9 6 7 0 0 9 4）Author for correspondence植物学报 2 0 0 0 4 2（1）：2 3 3 1Acta Botanica Sinicaof plastid DNA during male gamete development and fer-tilization are the major reasons to the failure of paternalplastid inheritance1 4 1 7However little is known aboutthe mechanisms of paternal plastid inheritance and mater-nal mitochondrion inheritance in Pinaceae This situationis most probably due to the large size of its reproductiveorgan and cells which makes them comparatively difficultfor ultrastructural studiesUp to nowdetailed ultrastructural investigations onthe development of the male gametophyte and the archego-niumfertilization and proembryo development have onlybeen conducted in Douglas-fir（Pseudotsuga menziesii）and interior spruce（Picea）providing cytological evidencefor paternal plastid inheritance and maternal mitochondri-on inheritance in these plants1 5 1 8 2 1In Abiesultra-structural observation of the egg cell during fertilization il-lustrates the mechanism of maternal plastid elimination inthe zygote2 2 In order to evaluate the general rule of or-ganelle transmission in gymnospermsit is necessary toinvestigate the dynamic changes of plastid mitochondrionand their DNA during gametogenesisfertilizationandearly embryo development in more speciesIn this pro-ject we choose Pinus tabulaeformisa special species ofPinaceae in China to trace the fate of organelles in maleand female gametes and during the fertilization process byusing electron microscopy（EM）and DNA epifluorescencemicroscopy Our aim is to clarify the cytological mecha-nism of paternal and maternal plastid and mitochondriontransmission or exclusion during sexual reproduction ofthis plant The current paper covers the ultrastructure andDNA fluorescence observations in the male gametes Oth-er results concerning the female gametes and parentalplastids and mitochondria during fertilization will be re-ported separately1 Material and Methods1 1 MaterialPlants of Pinus tabulaeformis Carrused are grownnaturally on the campus of Peking University The femalecones were hand pollinated with fresh pollens in March of1 9 9 7 For transmission electron microscope（EM）andDNA epifluorescence microscope observationsthe ovuleswere collected at intervals of3days in May 1 9 9 8 1 2 Sample processing for EMBased on previous experienceswe knew that someof the ovules in the female cones may be sterile To en-sure the sample was normally pollinated and developedwe selected ovules only at the middle part of the cones forprocessing The nucellus with pollen tube（PT）was dis-sected out and fixed in4 glutaraldehyde in0 1mol Lcacodylate buffer（pH7 2）for6h at room temperaturePost fixation was conducted with 2 osmium acid（OsO4）in the same buffer for1 2h at4 After wash-ing with ddH2O the samples were dehydrated through anethanol seriesinfiltrated and embedded in Epon 8 1 2resin Ultrathin sections were made on a Reichert UltracutS7 0 2 5 1 0ultratome with a diamond knife stained with u-ranyl acetate and modified Santo s lead citrate methodObservation and photography were conducted under aPhilips EM2 0 8 S electron microscopeIn additionsomesemithin sections were made and stained in toluidine blueO（TBO）for routine light microscopic observation1 3 DNA epifluorescence observationMaterial collected was the same as above and fixationwas carried out as shown in1 2 After thorough washingin ddH2O the sample was dehydrated through an ethanolseriesinfiltrated and embedded in Technovitt 7 1 0 0resin Sections of0 5 1 0 m in thickness were cut ona Reichert-Jung microtome with glass knife and stained inDAPI（0 1 g ml）water solution for1 2h in darknessThe sections were observed and photographed with a LeicaDMRXA microscope under UV light2 Results2 1 Light microscopic observations of male gametesin the pollen tube（PT）One year after pollination the PT has penetrated in-to the nucellar tissue of the ovuleThe tip of the PT isapproaching the archegonium of the female gametophyteThe spermatogenous cell（body cell）has divided to formtwo male gametes which are located in the cytoplasm-filled-tip of the PT together with the tube nucleus（Fig1）There is no obvious difference observed between thenuclei of the two male gametes The nuclei are sphericalor oval in shape and about4 0 m in diameter with cyto-plasm around each of themIn the sem-i thin sectionthere are plenty of round and thread-like structures（strongly stained by TBO）in the cytoplasm of the malegametes（Figs 4 6）They are probably plastids and mi-tochondriaThe male gametes can be identified as cellstructuresThere are remarkable differences in size andshape between the two sperm cellsThe leading spermcell（SC1）in the PT is long and larger The following one（SC2）is round and smaller with nucleus occupying mostpart of the cell（Figs 2 6）2 2 Cytoplasmic nucleoids in sperm cellsIn the DAPI stained sections a great number of dot-ted and thread-like DNA fluorescence can be observed2 4植物学报Acta Botanica Sinica4 2卷Figs 1 4 Fig 1 Light micrograph（LM）longitudinal section of upper portion of the ovuleshowing a pollen tube（PT）penetrating thenucellar tissue and a pair of sperms in the end of the tube（arrow heads）7 0 Figs 2 3 LM of DAPI stained semithin sections Fig 2 A pair of sperms in PT of the earlier stage their nuclear DNA are mostly distributed along the inner nuclear envelop 7 0 0 Fig 3 SC1atthe tip of PTNote its organelle DNA is visible in the cytoplasm 7 0 0 Fig 4 The same section as Fig 3stained with TBO the sperm nu-cleus and organelles（arrowhead）are also visible 7 0 0 Figure abbreviations：CyCytoplasm；MMitochondrion；NNucleus；NuNucellus；PPlastid；PPPeriplasm；PTPollen tube；SC1The leading sperm cell；SC2 The following sperm cell；SP Starch-containing plastid；V Vacuole；Vc Vesicle1期胡适宜等：油松细胞质遗传的细胞学机理：雄配子的超微结构和细胞器类核2 5in the cytoplasm of the spermsThe brightness of theseDNA appears weaker and quenched more easily than thatin the nucleus Distribution of the DNA fluoresent dots inthe cytoplasm was at random The dots often gathered inclusters（Figs 3 5）With reference to observations un-der the light microscope（LM）and EMit may be con-cluded that there are abundant DNA-containing plastidsand mitochondria in the sperm cellsNuclear DNA of the sperms distribute mainly alongthe nuclear envelope（Figs 2 3 5）The sperm nucleusis evenly stained as observed by LM（Fig 3）This differsfrom the surrounding nucellar tissue nuclei which possessFigs 5 7 Figs 5 6 Another section of the same PT as Fig 3stained with DAPI and TBO separatelyShowing SC2and portion of SC1cytoplasm The boundary line between SC2and SC1is difficult to recognize（arrowhead）7 0 0 Fig 7 Electron micrograph of a PTshow-ing most portions of SC1and SC2Note the plasmalemma between them（arrowhead）There are some organelles in the thin layer cytoplasm ofSC2Note groups of organelles and a large inclusion in the cytoplam of SC 1 2 0 0 0 Figure abbreviations：see Figs 1 4 2 6植物学报Acta Botanica Sinica4 2卷Fig 8 Higher magnification of a portion in Fig 7 showing details of the plasmalemma between the two sperm cells Note the curved or fin-ger-like protrusions of the sperm plasmalemma abutted to the tube cell（arrowhead）5 7 0 0 Abbreviations see Figs 1 4 condensed chromatin and show strong DNA fluorescenceThe volume of the sperm nucleus is about5 0 0times of thenucellar nucleusBecause the volume is large and theDNA content is only one half of that in the somatic cellnucleus the dispersed DNA in the sperm nucleus gener-ally display weaker fluorescence as compared to the sur-rounding nuclei of the nucellar tissue It has been report-ed that the nucleus of male gamete in Proxburghii ap-pears negative by Feulgen reaction2 3 which is a generalcytochemical method for DNA detection By usingthe1期胡适宜等：油松细胞质遗传的细胞学机理：雄配子的超微结构和细胞器类核2 7Figs 9 1 2 9 Portion of SC2showing the distribution of mitochondriaA double membrane bounded inclusion of unclear origin is seen（）1 5 0 0 0 Fig 1 0 Portion of SC1showing many plastids and few mitochondria near the nucleus 8 6 0 0 Fig 1 1 Portion of SC1interfacing the tube cell showing in much more detail the sperm plasmalemma evagination（arrowhead）There are abundent organelles includ-ing plastids mitochondria vesicles and vacuoles in the tube cell 6 8 6 0 0 Fig 1 2 A group of organelles in the cytoplasm of SC1 Note thepresence of many abnormal mitochondria with dark-stained substance（arrowhead）Some abnormal plastids can be seen 8 6 0 0 DAPI staining method we have proved undoubtedly thepresence of DNA in sperm nuclei of Ptabulaeformis2 3 Ultrastructure of the sperm cellThe pair of male gametes observed by using LM and2 8植物学报Acta Botanica Sinica4 2卷EM is at the stage of about one year after pollination Un-der the EMit clearly shows that the male gametes arecellular in structureeach one being bounded by theirown plasma membrane without any structured cell wallThe membranes are discontinuous with some gaps betweenthe two male gametes as seen in some sections（Fig 8）Although the division process of the spermatogenous cellhas not been observedbased on the fact that the borderbetween the two sperms is always near the rear one（SC2）we infer that the nucleus of the spermatogenouscell before mitosis is not located at centerthus resultingin the unequal division The SC1is larger and longac-quires more cytoplasm of the former cell（spermatogenouscell）Its nucleus occupies near the central position of thecelland the cytoplasm extends to both ends with abun-dent organelles The SC2is smaller and round in shapesurrounded by a thin layer of cytoplasmThe two spermcells are different in size shape and amount of cytoplasm（Figs 7 1 3）A pair of sperm cells of Ptabulaeformisis enclosed together by an inner membrane of tube cellwhich maybeconsideredsimilarasthatinan-giosperms2 4The sperm plasma membrane next to thetube cell cytoplasm often evaginates or becomes curved orforms finger-shaped protrusions（Figs 8 1 1）Fiber-likematerials and vesicles may be found in the periplasmicspace The tube cytoplasm around the sperms is extremelycondensed with abundent organelles including starch-con-taining plastidsmitochondriaendoplasmic reticulumdictyosomeslipid droplets vesicles covered with a darkcoating and small va（ER）cuoles（Figs 8 1 1）Both sperm cells contain plastidsmitochondriasmall vesicles and dark stainning granules that are proba-bly protein bodiesRibosomes in sperm cytoplasm areabundant and often gathered in thread-like structuresInsections the nucleus of the SC2has been seen to occupymost part of the cell The total amount of organelles in thesheath of cytoplasm in the SC2is less than that in the SC1（Fig 7）The plastids are typical proplastids in appear-ancebeing long and round or ovalSome of them areconstricted in the middleforming the shape of a dumb-bellindicating that they are in the proliferation state（Fig 1 0）The plastids contain obvious lamellaedensematrix and some osmium-stained particlesMost of themitochondria with developed cristae are round and smallerthan plastids（Fig 1 0）The SC1contains more plastidsand less mitochondria which often gathered into groups（Figs 8 1 2）Among those organelle groupsthe mito-chondria often deposit electron-opaque substancesome-times filling the entire matrix They appear to be degener-ating（Figs 9 1 2）Similar abnormal structures are rarelyseen in plastidsOther than organelles described abovethere aresome short tube-like ER cisternae in the sperm cyto-plasm The SC1often contains few spherical structureseach surrounded by a single membranefilled with someground cytoplasm This type of inclusion varies greatly insizesome of which contain distinguishable organelles（Figs 79）They are presumed to function as lyso-somesFig 1 3 Diagram of a pair of sperm cells in the pollen tube of Pi-nus tabulaeformis showing difference in size between the leadingsperm cell（SC1）and the following sperm cell（SC2）with plastids（hollowed）and mitochondria（solid）in their cytoplasmNspermnucleus1期胡适宜等：油松细胞质遗传的细胞学机理：雄配子的超微结构和细胞器类核2