小麦珠心细胞衰退过程酸性磷酸酶的超微细胞化学定位_英文_

小麦珠心细胞衰退过程酸性磷酸酶的超微细胞化学定位_英文_ () 植 物 学 报 1999 ,41 8:791,794 Acta B ota nica Si nica Ξ 小麦珠心细胞衰退过程酸性磷酸酶的超微细胞化学定位 1 2 1 田国伟申家恒尤瑞麟1 ( )北京大学生命科学学院北京 100871 2 ( ) 哈尔滨师范大学生物系 哈尔滨 150080 ( ) 摘要 采用磷酸铅盐沉淀技术对小麦 Triticum aestivum L . 珠心细胞衰退过程进行了酸性磷酸酶的超微细胞化学 定位研究 。结果显示 ,在未有明显衰退迹象的一些珠心细胞中 ,酸性磷酸酶只出现在细胞核轻微凝聚的染色质上 。 随珠心细胞衰退程度的逐渐增大 ,其衰退特征越来越明显 ,酸性磷酸酶依次在细胞质中较小液泡 、细胞壁 、线粒体 、 质体以及内质网等结构上出现活性反应 。紧连胚囊的珠心细胞衰退程度最大 ,细胞严重变形 ,酸性磷酸酶定位于细 胞绝大部分结构中 ,但此时变形的细胞核则无酸性磷酸酶活性反应 。研究结果明 ,小麦珠心细胞的衰退过程中 , 酸性磷酸酶存在一个有规律的变化 ,支持珠心细胞的衰退是属于细胞程序性死亡类型的观点 。 关键词 小麦 ,珠心细胞 ,衰退 ,酸性磷酸酶 ,超微细胞化学定位 Ultracytochemical Localization of Acid Phosphata se in Nucellar Cells Ξ of Wheat During Degeneration 1 2 1 TIAN Guo2Wei SHEN J ia2Heng YOU Rui2Lin 1 ( )College of Life Sciences , Peking University , Beijing 100871 2 ( )Department of Biology , Harbin Normal University , Harbin 150080 ( )The distribution of acid phosphatase activity in nucellar cells of wheat Triticum aestivum L . Abstract during degeneration has been studied using the lead precipitation method at the electron microscopic level . Acid phosphatase was localized in the slightly condensed nuclear chromatin in nucellar cells without any sign of ultrastructural degeneration. As the nucellar cells started degenerating , the enzyme activity in the cell was ob2 served , in the order from small vacuoles to cell walls , mitochondria , plastids and endoplasmic reticulum. En2 zyme activity was the highest in most components of the nucellar cells adjacent to the embryo sac where the de2 generation of nucellar cells was the strongest , but it was not observed in the nuclei of the degenerated nucellar cells. The results indicated that the degeneration of nucellar cells was a progressive and orderly process and supported that the degeneration of nucellar cells was a programmed cell death. Key words Triticum aestivum , Nucellar cell , Degeneration , Acid phosphatase , Ultracytochemical localiza2 tion The nucellus , being an ephemeral tissue , degener2 2 the mechanism of pcd in plant cells because the tissue deates with the development of the female gametophyte and generates during the process of embryo sac development growth of the embryo and endosperm in angiosperms. The and different degenerating stages can be observed in an degeneration of the nucellar cells is a normal physiological ovule . There are a few reports about nucellus degenera2 1 ,4 ] process of ovule development , and , therefore , it is con2 tion. Furthermore , little is known about the struc2 1 ,4 ] () sidered as a programmed cell death pcd. Consid2 tural localization of enzymes in the degenerative process. erable literatures have reported the existence of pcd in an2 Based on previous studies of ATPase on the nucellus de2 5 ,8 ] 4 ] imal cells, but in comparison , much less research generation of wheat , the present study is designed to 9 ,12 ] has been done in plant cells. However , the mecha2 reveal the ultrastructural localization of acid phosphatase () nism of pcd is still not completely clear in either animal or AcPase in the process , and the relationship between plant cells. Nucellar tissue is an ideal material to explore pcd and AcPase activity in plant cells. Ξ Supported by a grant from the National Natural Science Foundation of China and partly by Biological Science Development Foundation of Cai Huo- Shi . Received :1998211226 Revised :1999203208 eration were easily observed at the chalazal end of an 1 Materials and Methods ovule . With slight OsOstaining , most components of the 4 nucellar cells could be visualized. The stage difference in Plants of spring wheat , Triticum aestivum L . , were the ultrastructural localization of AcPase in the degenera2 cultivated in the field of Harbin Normal University , tion of nucellar cells are described as followings. Harbin , Heilongjiang Province . The pistils were dissected No structural signs of degeneration were found in nu2 during anthesis , and immediately fixed in a mixture of cellar cells 4,6 cell layers away from the antipodal cells 4 % paraformaldehyde and 2 . 5 % glutaraldehyde in 0 . 05 at the chalazal end of the embryo sac . Each nucellar cell contained a prominent nucleus with a large nucleolus and () mmol/ L cacodylate buffer p H 7 . 2for 2 h at room tem2 dense cytoplasm in which some smaller vacuoles appearing perature under a partial vacuum. After fixation , the sam2 as fused with each other to form median sized vacuoles. ples were washed in the same buffer at 4 ?for 3 h and The nuclear chromatin was slightly condensed showing were cut with a razor blade into slices of about 0 . 5 mm in ( ) AcPase Pl . ?, 3. Occasionally , a little enzyme activi2 the buffer , then rinsed with 0 . 05 mmol/ L acetate buffer ( ) ty could be found in some small vacuoles Pl . ?, 4. There were also no distinct structural signs of degen2 () p H 5 . 0for 1 h prior to the enzyme reaction step . eration in most nucellar cells 3,4 cell layers away from The enzyme reaction procedure followed that reported 13 ] the antipodal cells of the embryo sac . But AcPase activity by Sexton and Hall . The specimens were incubated in was observed in the cell walls and some unidentified ( a complete reaction medium 50 mmol/ L acetate buffer , membranous organelles. The activity was stronger in some () p H 5 . 0 , 3 . 6 mmol/ L Pb NOand 10 mmol/ L sodium 32 of the smaller vacuoles of the nucellar cells than that men2 β) 2glycerophosphateat 36,37 ? for 3 h. Controls in2 ( ) tioned above Pl . ?, 5 . At the same site , however , ( ) cluded samples 1 incubated in medium without sub2 some structural signs of degeneration such as thin cyto2 () strate ; 2pretreated in 0 . 1 mmol/ L NaF for 30 min , plasm could be observed in other nucellar cells. In addi2 then incubated with complete reaction medium plus 0 . 1 tion , the nuclear chromatin was more condensed in cells mmol/ L NaF. ( ) with strong AcPase activity Pl . ?, 6. After incubation , all samples were rinsed with 0 . 05 The nucellar cells that were 1 , 2 layers of cells mmol/ L cacodylate buffer at p H 7 . 2 for 4 h at 4 ?, then fixed at 4 ? with 2 % osmium tetroxide in the same away from the antipodal cells at the chalazal end showed buffer overnight . Following this , samples were dehydrated distinct structural signs of degeneration , including the ir2 stepwise in an acetone series at 4 ? until the step in regularity of cell shape , much scanty of cytoplasm , 70 % acetone solution and the subsequent step s were at shrunkage of mitochondria and plastids , and dilation or room temperature . Samples were embedded in Epon 812 . ring- shape formation of ER cisterna embracing some cyto2 Thin sections were cut on a L KB- NOVA ultramicrotome ( ) plasm and organelles Pl . ?, 7 , 8. AcPase activity and the unstained sections were viewed and photographed existed strongly in the cell walls , ER cisterna and the under a Hitachi 300 electron microscope . condensed chromatin , but varied in plastids and mito2 chondria . Little enzyme activity was found in the vacuoles 2 Results ( ) and the thin cytoplasm matrix Pl . ?, 7 , 8. The nucellar cells laid closely to the embryo sac As the nucellar cells degenerated along with the de2 showed the most prominent degeneration. These cells were velopment of the female gametophyte , the formation of strongly shrunken exhibiting distinguishable cell shapes. embryo and endosperm , therefore , there were a few layers Strong AcPase activity was found in most components of of continuously degenerating nucellar cells surrounding the these cells but little enzyme activity existed in the shrunk2 embryo sac . The degree of degeneration of nucellar cells ( ) en and still recognizable nuclei Pl . ?, 1 , 2. was associated with their position rather than the develop2 Similar enzyme localization existed in the degenerat2 mental stages of the ovules. Generally , the closer to the ing nucellar cells close to both antipodal cells and other embryo sac , especially at the chalazal end , the greater portions of the embryo sac . But no enzyme activity was degree and the larger number of degenerating nucellar detected in the vacuoles of the nucellar cells near the mi2 ( ) cells Pl . ?, 1. However , the degeneration of the nu2 ( cropyle of the embryo sac at all stages Pl . ?, 2 ; Pl . cellar cells at the micropylar end was comparatively much ) ?, 9 , 10.( less prominent both in degree and in cell number Pl . Little enzyme activity was observed in all sites of the ) ?, 2. So the different stages of the nucellar cell degen2 793 ()8 期 田国伟等 : 小麦珠心细胞衰退过程酸性磷酸酶的超微细胞化学定位 英 nucellar cells in both controls indicating that our results Thus we concluded that the distribution and change ( ) are reliable Pl . ?, 11 , 12. 2 of AcPase activity is a consistent process during the de generation of nucellar cells in wheat . AcPase activity is 3 Discussion initially found in the nuclear chromatin , from where it A body of literatures reported on programmed cell proceeds from the small vacuoles to cell walls and other 5 ,8 ] ( ) death pcdor apoptosis in animal cellsindicated ( ) organelles mitochondria , plastids , ER , etc, and , fi2 that reasonable judgement of cell death , regardless of be2 nally , disappears in the shrunken nuclei . The results also ing pcd or not , should be based on different angles of evi2 indicate that the degeneration process of nucellar cells is dences including morphological features , DNA fragmenta2 tion and biochemical changes. In animals the cells under2 gene controlled. going pcd exhibited a series of morphological hallmarks , such as cell shrinkage , nuclear coagulation , chromatin References 5 , 14 ,16 ] condensation and formation of apoptotic bodies. 1 Norstog K. Nucellus during early embryogeny in barley : fine Recent studies on the senescence in plant cells have structure . B ot Gaz , 1974 , 135 :97,103 demonstrated similar senescent features as those in animal Phillipson M N. Nucellar degeneration on Cortaderia 2 9 ,12 ] ( ) cells. Previous description of the morphological fea2 Gramineae. Protoplasma , 1978 , 95 :361,3691 ,3 ] () You R-L 尤瑞麟. Ultrastructural studies on the degeneration tures of the degenerating nucellar in angiospermshas 3 ( ) process in wheat nucellar cells. Acta B ot Sin 植 物 学 报 , suggested a characteristic pcd of the nucellar cells during () 1985 , 27 :345,353 in Chinesedegeneration which was supported by the work on the ul2 () () Tian G- W田国伟, Shen J- H 申家恒. Ultracytochemical 4 trastructural localization of ATPase of the degenerating nu2 localization of ATPase activity on the degenerating nucellar 4 ] cellar cells in wheat . The present work also supports () cells of wheat during degeneration. Acta B ot Sin 植物学报,this viewpoint . () 1996 , 38 :100,104 in Chinese Kerr J F R , Wyllie A H , Currie A R. Apoptosis : a basic bio2 AcPase was found to be localized cytochemically in 5 4 ,17 ,23 ] many organelles, and occur as goup s of logical phenomenon with wide- ranging implications in tissue ki2 13 ] netics. B ritish J Cancer , 1972 , 26 :239,357isozymeswhich distributed unevenly in various or2 Williams G T , Smith C A , McCarthy J J , Grimes E A. Apop2 6 ganelles closely associated with the physiological state of tosis : final control point in cell biology. Trends Cell Biol , 19 , 22 , 23 ] the organelles. Thus it seems difficult and inap2 1992 , 2 :263,267propriate to define a general rule for AcPase function in Vaux D L , Haecker G , Strasser A. An evolutionary perspec2 7 all organelles. Generally , it has been suggested that the tive on apoptosis. Cell , 1994 , 76 :777,779function of AcPase in cell walls is involved in intercellular Steller H. Mechanisms and genes of cellular suicide . Science , 8 1995 , 267 :1445,1499transport , phosphate transfer reaction and cell wall 18 , 22 , 23 ] Greenberg J T , Guo A , Klessig D F , Ausubel F M. Pro2 metabolism. AcPase in the nucleus is considered 9 19 , 22 , 23 ] grammed cell death in plants : a pathogen- triggered response as a non- lysosome enzymethat may be associated activated coordinately with multiple defense functions. Cell , with dephosphorylation of nuclear protein influencing ei2 1994 , 77 :551,563 ther the quality or quantity of gene transcription , or Mittler R , Lam E. In situ detection of cDNA fragmentation 10 22 , 23 ] 19 , 20 ] both. Lytic function of AcPase in ER, mito2 during the differentiation of treachery elements in higher 20 , 23 ] 17 , 19 , 21 ] 18 , 19 ] plants. Plant Physiol , 1995 , 108 :489,493 chondria , plastids, vacuoles, etc , Ryerson D E , Heath M C. Cleavage of nuclear DNA into in association with the respective physiological state of the 11 oligonucleosomal fragments during cell deaths induced by fun2 organelles was postulated. The present study shows that gal infection or by abiotic treatments. Plant Cell , 1996 , 8 : the nuclear chromatin of the nucellar cells of wheat exhib2 393,402 ited AcPase activity , although the nucellar cells showed Wang H , Li J , Bostock R M , Gichrist D G. Apoptosis : a 12 no structural signs of degeneration at the early stage of de2 functional paradigm for programmed cell death induced by a velopment. Furthermore , the fact that no AcPase but host selective phytotoxin and invoked during development . 4 ] Plant Cell , 1996 , 8 :375,391 strong ATPase activity in the nucleiwas observed at the Sexton R , Hall J L . Enzyme cytochemistry. In : Hall J L ed. late stage of degeneration suggests that the AcPase in the 13 Electron Microscopy and Cytochemistry of Plant Cells. Elsevi2 nuclei was not a lysosome enzyme . These results suggest er : North- Holland Biomedical Press , 1978. 63,147 that the degeneration of nucellar cells is programmed un2 Wyllie A H , Morris R G , Smith A L , Dunlop D. Chromatin 14 der genetic control . cleavage in apoptosis : association with condensed chromatin morphology and dependence on macromolecular synthesis. J Pathol , 1984 , 142 :67,77 Plate ? Fig. 1. Degenerated nucellar cells adjacent to the em2 15 Gorczya W , Gong J , Darzynkiewies A. Detection of DNA strand breaks in individual apoptotic cells by the in situ termi2 bryo sac at chalazal end of an ovule , showing 3,4 layers of degen2 nal deoxynucleotidyl transferase and nick translation assays. erated nucellar cells with dense lead phosphate deposits in cell walls Cancer Roes , 1993 , 53 :1445,1451 and ER but not in the strongly degenerated nuclei . ×3 400 Fig. Cohen G M , Sun X , Fearnhead H , MacFarlane M , Brown D 2. Nucellar cells adjacent to embryo sac in submicropyle , showing 16 G , Snowden R T , Dinsdale D. Formation of large molecular only 1,2 layers of the degenerated nucellar cells with dense lead weight fragments of DNA is a key committed step of apoptosis phosphate deposits in cell walls and nuclear chromatin but not in the on thymocytes. J Immunology , 1994 , 153 :507,516 strongly degenerated nuclei . ×1 600 Figs. 3, 6. Degenerated 17 Pritchard H N , Bergstrasser K A. The cytochemistry of some nucellar cells at different stages at the chalazal end of ovules. Figs. enzyme activities in Stellaria media embryos. Experientia , 3 , 4. The cells 4,6 layers of degenerated nucellar cells away from1969 , 25 :1116,1117 the embryo sac . Fig. 3. No distinct structural signs of degeneration Hall J L , Davie C A. Localization of acid hydrolase activity in are seen , but enzyme activity exists in the slightly condensed nucle2 18 Zea mays L . root tips. Ann B ot , 1971 , 35 :849,855ar chromatin. ×3 370 Fig. 4. Weak AcPase activity in some 19 smaller vacuoles. ×6 000 Figs. 5 , 6. Nucellar cells being 3,4 Cartner P G , Nagl W. Acid phosphatase activity in plastids () plastolysomesof senescing embryo- suspensor cells. Planta , layers of cells away from embryo sac . Fig. 5. AcPase activity in cell 1980 , 149 :341,349walls , small vacuoles and some unidentified membranous organelles () 20 Schulz P , J ensen W A. Pre-fertilization ovule development in arrow. ×12 000 Fig. 6. Thin cytoplasm and strong AcPase ac2 Capsella : ultrastructure and ultracytochemical localization of tivity in cell walls , condensed nuclear chromatin and smaller vac2 acid phosphatase in meiocytes. Protoplasma , 1981 , 107 : 27 uoles. ×4 800 ,45 Plate ? Figs. 7 , 8. Degenerated nucellar cells at the chalazal 21 Schulz P , J ensen W A. Pre-fertilization ovule development in end of ovules. Nucellar cells 1,2 cell layers away from embryo sac Capsella : the dyad , tetrad , developing megaspore , and two- with distinct structural signs of degeneration showing thin cytoplasm , nucleate gametophyte . Can J B ot , 1986 , 64 :875,884 shrunken mitochondria and plastids , and ringed and dilated ER. β22 Joshi P A , Stewart J M , Graham E T. Localization of- glyc2 Fig. 7. Strong AcPase activity in cell walls and ER and weak Ac2 (Pase activity in some other unidentified membranous organelles per2 erophosphatase activity in cotton fiber during differentiation. ) () Protoplasma , 1985 , 125 :75,85 haps M or Parrow. ×8 400 Fig. 8. Conspicious AcPase ac2 ( ) ( ) 23 tivity in cell walls , ringed and dilated ER , and some mitochondria Tian G- W 田 国 伟 , Shen J- H 申 家 恒 . The ultracyto2 chemical localization of acid phosphatase activity in wheat dur2 and plastids. ×12 000 Figs. 9 , 10. Degenerated nucellar cells in () ing fertilization. the submicropylar portion of an ovule . Fig. 9. AcPase in the cell Acta B ot Sin 植物学报, 1994 , 36 : 251, ()256 in Chinese walls , condensed nuclear chromatin and some unidentified organelles () arrowbut non in the vacuoles. ×8 750 Fig. 10. Fewer layers of Explanation of Plates the degenerated nucellar cells are seen than that in the chalazal ER. A. Antipodal cell CC. Central cell CW. Cell wall portion of an ovule . No AcPase activity occurs in the vacuoles at all Endoplasmic reticulum M. Mitochondrion N. Nucleus NC. stages , and strong AcPase activity occurs in most components of the Nucellar cell P. Plastid V. Vacuole intensely degenerated nucellar cells. ×3 750 Figs. 11 , 12. Con2 nucellar Figs. 1 , 12. Transmission electron micrographs of trols. In both cases , no AcPase is observed. Fig. 11. Sample incu2 ( ) cells of wheat ovules with acid phosphatase AcPaselocalized as bated in a medium containing NaF. ×3 000 Fig. 12. Sample in2 electron dense lead phosphate deposits. βcubated in a medium without2glycerophosphate . ×3 000 图版 ? 田国伟等 :小麦珠心细胞衰退过程酸性磷酸酶的超微细胞化学定位 TIAN Guo2Wei et al . : Ultracytochemical Localization of Acid Phosphatase in Nucellar ? Plate Cells of Wheat During Degeneration See explanation at the end of text TIAN Guo2Wei ? 田国伟等 :图版 ? et al . : Plate See explanation at the end of text