兵豆萌发过程中初生根皮层细胞的细胞周期活动的动态分析

观察了兵豆(Lens culinaris Medic.)初生根原皮层组织的细胞周期在其种子萌发过程中时间和空间上的动态变化.免疫组织化学和细胞学证据表明,原皮层细胞分别在种子吸胀大约13 h和17 h开始DNA复制和细胞分裂.最早进行DNA复制和细胞分裂的细胞位于远基端1 mm附近,但这些分裂细胞的DNA复制是在种子成熟过程中完成的,而不是在萌发后.第一个细胞周期的激活样式表明,这些细胞并不同步激活,而是依次进入细胞周期,且进入的次序与自身在根尖中的相对位置有关.在兵豆初生根原皮层组织中,邻近位置上的细胞的细胞周期同步化程度较高.     

值得注意的细胞周期调节蛋白

细胞周期调节蛋白在细胞周期的G_1期和S期交界处开始合成,S期结束后消失。细胞内定位在核内,又称分裂细胞核抗原。细胞周期调节蛋白含量与细胞分裂状况直接有关。细胞周期调节蛋白为细胞DNA复制所必须,是DNA聚合酶δ的活化蛋白。因此细胞周期调节蛋白对于真核生物DNA复制的调控及细胞分裂具有重要意义,值得重视。     

用ribozyme抑制增殖细胞核抗原的表达对HaLa细胞增殖的影响

增殖细胞核抗原(PCNA)是DNA聚合酶δ的辅助蛋白,它是细胞染色体DNA复制所必需的。人工设计的ribozyme具有可特异地切割PCNA mRNA的性质,将此ribozyme的自修剪体内表达质粒导入HeLa细胞,从细胞总RNA中分离相应部分能在体外切割靶RNA片段,证明此表达质粒在细胞内能表达出有活性的ribozyme分子。与对照相比,导入ribo-zyme表达质粒的HeLa细胞进入S期的时间从12 h推迟到20 h,而突变ribozyme的对照表明反义抑制对细胞进入S期的影响较小(推迟到15 h)。证明该ribozyme能有效抑制He-La细胞DNA复制,同时亦证明PCNA对于细胞DNA复制及细胞周期进程的重要性。     

预浸和发芽过程中番茄种子细胞核的倍性变化

用细胞流检仪（ｆｌｏｗｃｙｔｏｍｅｔｅｒ）检测番茄种子细胞核倍性水平时发现：当年成熟的番茄种子胚细胞核ＤＮＡ绝大多数为２Ｃ水平,胚乳细胞核则为３Ｃ水平,说明成熟番茄种子细胞一般休止停留在Ｇ１期。同时我们也发现极少量的胚和胚乳细胞核分别为４Ｃ和６Ｃ水平,说明这些细胞已经进行了ＤＮＡ内复制。供试番茄种子浸种后１２ｈ左右完成吸水过程,２ｄ后胚根可突破种皮发芽。随着种子吸水过程的完成,胚根尖部分细胞开始进入ＤＮＡ复制期（Ｓ期）,而且此类细胞的数量增加迅速,一直到种子发芽。番茄胚根尖细胞进入４Ｃ的数量的多少与种子萌发时期有明显相关,４Ｃ／２Ｃ比率越大说明越接近发芽。渗控处理可以增加番茄种子胚根细胞４Ｃ／２Ｃ比率,因而明显提高种子的发芽速率。结果还表明;渗控处理的番茄种子再度干燥后４Ｃ／２Ｃ比率不变,这说明干燥可以固定细胞周期。     

天麻种子与兰小菇共生萌发过程中超微结构变化研究*

天麻Ｇａｓｔｒｏｄｉａｅｌａｔａ种子与兰小菇Ｍｙｃｅｎａｏｒｃｈｉｄｉｃｏｌａ的共生萌发试验表明兰小菇可与天麻种子共生促进天麻种子萌发并形成原球茎。菌丝自胚柄端的柄状细胞侵入天麻种子原胚,其分布被限制在天麻原球茎基部的柄状细胞、外皮层细胞和内皮层细胞内,均被电子透明物质和原球茎细胞质膜包围而与原球茎细胞质相隔离。菌丝在外皮层细胞中形成菌丝结,在内皮层细胞中则被消化,形成扁化、衰败的菌丝或菌丝四块。含有衰败菌丝的原球茎细胞可被菌丝重新定殖,新近定殖的菌丝又被原球茎细胞消化。     

天麻种子与兰小菇共生萌发过程中超微结构变化研究

天麻Ｇａｓｔｒｏｄｉａｅｌａｔａ种子与兰小菇Ｍｙｃｅｎａｏｒｃｈｉｄｉｃｏｌａ的共生萌发试验表明兰小菇可与天麻种子共生促进天麻种子萌发并形成原球茎。菌丝自胚柄端的柄状细胞侵入天麻种子原胚,其分布被限制在天麻原球茎基部的柄状细胞、外皮层细胞和内皮层细胞内,均被电子透明物质和原球茎细胞质膜包围而与原球茎细胞质相隔离。菌丝在外皮层细胞中形成菌丝结,在内皮层细胞中则被消化,形成扁化、衰败的菌丝或菌丝四块。含有衰败菌丝的原球茎细胞可被菌丝重新定殖,新近定殖的菌丝又被原球茎细胞消化。     

脱落酸对绿豆下胚轴不定根发生的影响

以10^-4 mol/L脱落酸(ABA)处理绿豆种子24 h,在幼苗下胚轴长6 cm时,切除根部作为插条,研究ABA对插条不定根发生及插条基部细胞周期时相的影响。结果表明,ABA可促进下胚轴插条不定根发生,增加生根数和生根范围;ABA提高插条基部细胞色氨酸转氨酶、吲哚丙酮酸脱羧酶和吲哚乙醛脱氢酶的比活性,增加吲哚乙酸含量,同时进入细胞周期S期的基部细胞数目增加,促进DNA合成,有利于不定根的发生。     

天麻种子萌发过程与开唇兰小菇的相互作用*

实验表明开唇兰小菇Mycena anoectochila可与天麻Gastrodia elata种子共生促进其萌发形成原球茎。 菌丝自胚柄端的柄状细胞侵入天麻种子原胚,进一步在皮层细胞中扩展,在外皮层细胞中形成发育良好的菌丝结,菌丝完整而有活力; 在内皮层细胞中则被消化,菌丝衰败、扁化。菌丝在原球茎细胞内的分布被限制在原球茎基部的柄状细胞、外皮层细胞和内皮层细胞,菌丝均被电子透明物质包围, 外围环绕有原球茎细胞质膜, 该界面使侵入的菌丝与原球茎细胞质相隔离,也是两共生生物间进行物质交换的所在。上述菌丝侵入至被消化的过程在整个原球茎发育过程中可反复进行。     

细胞周期检控点

细胞周期是高度有组织的时序调控过程,受到DNA损伤检控点、DNA复制检控点和纺锤体检控点等细胞周期检控点的精确调控。细胞周期检控点的作用主要是调节细胞周期的时序转换,以确保DNA复制、染色体分离等细胞重要生命活动的高度精确性,并对DNA损伤、DNA复制受阻、纺锤体组装和染色体分离异常等细胞损伤及时做出反应,以防止突变和遗传不稳定的发生。细胞周期检控点的功能缺陷,将导致细胞基因组的不稳定,与细胞癌变密切相关。因此细胞周期检控点对于维持细胞遗传信息的稳定性和完整性以及防止细胞癌变和遗传疾病的发生起着至关重要的作用。     

天麻种子萌发过程与开唇兰小菇的相互作用

实验表明开唇兰小菇Mycena anoectochila可与天麻Gastrodia elata种子共生促进其萌发形成原球茎,菌丝自胚柄端的柄状细胞侵入天麻种子原胚,进一步在皮层细胞中扩展,在外皮层细胞中形成发育良好的菌丝结,菌丝完整而有活力;在内皮层细胞中则被消化,菌丝衰败,扁化,菌丝在原球茎细胞内的分布被限制在原球茎基部的柄状细胞,外皮层细胞和内皮层细胞,菌丝均被电子透明物质包围,外围环绕有原球茎细胞质膜,该界面使侵入的菌丝与原球茎细胞质相隔离,也是两共生生物间进行物质交换的所在,上述菌丝仞入至被消化的过程在整个原球茎发育过程中可反应进行。     

Effect of microgravity on the cell cycle in the lentil root

Characteristics of the cell cycle in cortical regions (0–0.6 mm from the root-cap junction) of the primary root of lentil (Lens culinaris L.) during germination in the vertical position on earth were determined by iododeoxyuridine labelling and image analysis. All cells were in the G1 phase at the beginning of germination and the duration of the first cell cycle was about 25 h. At 29 h, around 14% of the cortical nuclei were still in the G2 or M phases of the first cell cycle, whereas 53 and 33% of the nuclei were respectively in the G1 or S phase of the second cell cycle. In parallel, the cell cycle was analysed in root tips of lentil seedlings grown in space during the IML 2 mission (1994), (1) on the 1-g centrifuge for 29 h, (2) on the 1-g centrifuge for 25 h and placed in microgravity for 4 h, (3) in microgravity for 29 h, (4) in microgravity for 25 h and placed on the 1-g centrifuge for 4 h. The densitometric analysis of nuclear DNA content showed that in microgravity there were less cells in DNA synthesis and more cells in G1 than in the controls on the 1-g centrifuge (flight and ground). The comparison of the sample grown continuously on the 1-g centrifuge in space and of the sample grown first in 1-g and then in microgravity indicated that 4 h of microgravity modified cell cycle, increasing the percentage of cells in the G1 phase. On the contrary, the transfer from microgravity to the 1-g centrifuge (for 4 h) did not provoke any significant change in the distribution of the nuclear DNA content. Thus the effect of microgravity could not be reversed by a 4 h centrifugation. As the duration of the first cell cycle in the lentil root meristem is about 25 h, the results obtained are in agreement with the hypothesis that the first cell cycle and/or the second G1 phase was lengthened in absence of gravity. The difference observed in the distribution of the nuclear DNA content in the two controls could be due to the fact that the 1g control on board was subjected to a period of 15 min of microgravity for photography 25 h after the hydration of the seeds, which indicated an effect of short exposure to weightlessness. The mitotic index of cortical cells was greater on the 1-g centrifuge in space than in any other sample (flight and ground) which could show an effect of the centrifugation on the mitosis.     

The effects of Fusilade (Fluazifop-<Emphasis Type="Italic">p</Emphasis>-butyl) on germination,mitotic frequency and α-amylase activity of lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> Medik.) seeds

In this study, seed germination percentages, effects on phases of mitosis and α-amylase enzyme activity of lentil seeds treated with four different concentrations (0.25, 0.5, 1 and 1.5%) of Fusilade (Fluazifop-p-butyl) were determined. Median EC (effective concentration) values were calculated according to seed germination percentages after treatment for 72 h. Germination percentages of primary lentil roots decreased with increasing Fusilade concentrations. Cytological observations showed that the mitotic frequency in root meristematic cells were decreased parallel to the increase in concentrations and all Fusilade concentrations applied decreased the activity of α-amylase enzyme in lentil seeds. The obtained results indicate that the herbicide Fusilade had the ability to cause reduction in seed germination, mitotic frequency and also α-amylase activity of lentil seeds.     

Densitometric analysis of nuclear DNA content in lentil roots grown in space

Lentil seedlings were grown for 28 h in space, on board Spacelab (IML 1 Mission) and growth of the primary root was analysed. The length of cortical cells was less in near weightlessness than on the 1 g centrifuge (flight control) and mitotic index was lower but there was no apparent perturbation in the mitosis. To further investigate which phase of cell cycle was modified, densitometric analysis of nuclear DNA content in cortical cells was carried out by the mean of an image processing system (SAMBA). In microgravity there was a decrease in DNA synthesis and a promotion of the arrest in the G2 phase of cell cycle. These results, and other ones obtained elsewhere on a slowly rotating clinostat, led us to think that in microgravity the perturbation of the gravisensing cells and/or the absence of convection could account for the modification of cell growth registered in the primary root.     

Cell proliferation and cell cycle dependent changes in the methylthioadenosine phosphorylase activity in mammalian cells

The objective of this study is to investigate the activity of methylthioadenosine phosphorylase (MTA-Pase) in mammalian cells stimulated by serum to proliferate and during their cell cycle. A direct correlation between growth rate and MTA-Pase activity in chinese hamster ovary (CHO) cells was observed. High MTA-Pase activity was observed during the exponential growth phase followed by a low enzyme activity during plateau phase of growth. To understand whether the fluctuations in the enzyme activity was cell cycle dependent, initially the activity of MTA-Pase was studied in plateau phase (G0) CHO cells as they synchronously go into S phase upon plating in fresh medium. The MTA-Pase activity in G0 cells before initiation of growth was 10.3 n.mol/mg protein/30'. A peak activity of 16.0 n.mol/mg/30 min was found at 12 hr after stimulation of proliferation by serum. These results indicate a peak MTA-Pase activity between 10-12 hr after stimulation of proliferation coinciding with the initiation of DNA synthesis. The activity of the enzyme slowly decreased as the cells completed their DNA synthesis. To understand whether these fluctuations are cell cycle specific, HeLa cells were synchronized in different phases and MTA-Pase activity was studied. The specific activities of the enzyme were 2.76, 2.99, 3.97, 3.28 and 3.65 n.moles/mg/30 min. in mitosis, early G1, late G1, S and G2 phases of the cell cycle respectively. These results indicate that MTA-Pase activity peaks in late G1 phase before the initiation of DNA synthesis, similar to the polyamine biosynthetic enzymes and might play a role in the initiation of DNA synthesis by salvage of adenine into nucleotide pools.     

Life Cycle of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

Plasmodiphora brassicae is a soil-borne obligate parasite. The pathogen has three stages in its life cycle: survival in soil, root hair infection, and cortical infection. Resting spores of P. brassicae have a great ability to survive in soil. These resting spores release primary zoospores. When a zoospore reaches the surface of a root hair, it penetrates through the cell wall. This stage is termed the root hair infection stage. Inside root hairs the pathogen forms primary plasmodia. A number of nuclear divisions occur synchronously in the plasmodia, followed by cleavage into zoosporangia. Later, 4–16 secondary zoospores are formed in each zoosporangium and released into the soil. Secondary zoospores penetrate the cortical tissues of the main roots, a process called cortical infection. Inside invaded roots cells, the pathogen develops into secondary plasmodia which are associated with cellular hypertrophy, followed by gall formation in the tissues. The plasmodia finally develop into a new generation of resting spores, followed by their release back into soil as survival structures. In vitro dual cultures of P. brassicae with hairy root culture and suspension cultures have been developed to provide a way to nondestructively observe the growth of this pathogen within host cells. The development of P. brassicae in the hairy roots was similar to that found in intact plants. The observations of the cortical infection stage suggest that swelling of P. brassicae-infected cells and abnormal cell division of P. brassicae-infected and adjacent cells will induce hypertrophy and that movement of plasmodia by cytoplasmic streaming increases the number of P. brassicae-infected cells during cell division.     

Directional cell-to-cell communication in theArabidopsis root apical meristem III. Plasmodesmata turnover and apoptosis in meristem and root cap cells during four weeks after germination

Summary Plasmodesmata frequency and distribution in root cap cells ofArabidopsis thaliana root tips were characterized during four weeks after germination to understand the symplasmic control of apoptosis. Apoptotic cells in some of the root apical-meristem cells and in root cap cells were identified by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling reaction and characterized by electron microscopy. Starting at the second week after germination, cells in the outermost layers of the root cap showed typical apoptotic features, including nuclear DNA fragmentation, chromatin condensation, cytoplasmic vacuolation, and organelle destruction. Intercellular connections, indicated by the frequency and number of plasmodesmata per cell length, were significantly reduced in the walls of outer root cap cells. This shows that cells become symplasmically isolated during the apoptosis process. In apoptotic root cap cells, the majority of nonfunctional plasmodesmata were observed to be associated with degenerated endoplasmic reticulum; this state was prior to the detection of any nuclear DNA fragmentation. Other nonfunctional plasmodesmata were sealed by heterogeneous cell wall materials. However, in immature epidermal and cortical cells in 4-week-old arrested roots the endoplasmic reticulum associated with plasmodesmata became disconnected as a result of protoplast condensation and shrinkage. No degenerated endoplasmic reticulum was observed in these cells. These observations suggest that the apoptotic processes in the root body and the root cap are different.     

Effect of water stress on root meristems in woody and herbaceous plants during the first stage of development

We investigated the effect of water stress on the root system architecture of pine saplings and pea seedlings during the first stage of development. Attention was focused on meristematic tissue situated at the root tip because of the leading role played by the tissue in the planning of root system architecture. The data showed that both species are extremely sensitive and that plants arrest their growth immediately during water stress treatment. When stress treatment was not intense, both species recovered growth but presented modifications in the root system architecture. In pine saplings, the modification in root system architecture was the consequence of fine root meristems not recovering from water stress. The saplings survived by producing new lateral meristems from the cortical tannin zone above the fine root tip. In the case of pea seedlings, the meristematic tissues in the primary root arrested proliferation during water stress although they recovered when the event occurred during the first hours of germination. The response was different when water stress was enforced on older seedlings. In this case, root meristems never completely recovered their proliferation despite the increase in proline content observed in the cells. The modification of root system architecture in pea seedlings depended on the arrest of primary root elongation and the formation of new root laterals. As regards the primary roots, water stress treatment induced along the axis the formation of irregular ‘swellings’ in the cortical zone above the meristematic zone. Anatomical investigations suggested that such swellings may have derived from the changes in elongation direction of derivatives. The formation of new laterals was observed in hydroponic cultures when water stress treatment was enforced slowly and prolonged for a long time. The production of new lateral meristems may have been a similar response of woody and herbaceous plants to water stress conditions. It is not known whether these new meristems present characteristics of resistance to water stress. This revised version was published online in June 2006 with corrections to the Cover Date.     

Programmed cell death in the root cortex of soybean root necrosis mutants

The soybean root necrosis (rn) mutation causes a progressive browning of the root soon after germination that is associated with accumulation of phytoalexins and pathogenesis-related proteins and an increased tolerance to root-borne infection by the fungal pathogen, Phytophthora sojae. Grafting and decapitation experiments indicate that the rn phenotype is root-autonomous at the macroscopic level. However, the onset and severity of browning was modulated in intact plants by exposure to light, as was the extent of lateral root formation, suggesting that both lateral roots and the rn phenotype could be directly or indirectly controlled by similar shoot-derived factors. Browning first occurs in differentiated inner cortical cells adjacent to the stele and is preceded by a wave of autofluorescence that emanates from cortical cells opposite the xylem poles and spreads across the cortex. Before any visible changes in autofluorescence or browning, fragmented DNA was detected by TUNEL (T erminal deoxynucleotidyl transferase-mediated dU TP-digoxigenin n ick e nd l abeling) in small clusters of inner cortical cells that subsequently could be distinguished cytologically from neighboring cells throughout rn root development. Inner cortical cells overlying lateral root primordia in either Rn or rn plants also were stained by TUNEL. Features commonly observed in animal cell apoptosis were confirmed by electron microscopy but, surprisingly, cells with a necrotic morphology were detected alongside apoptotic cells in the cortex of rn roots when TUNEL-positive cells were first observed. The two morphologies may represent different stages of a common pathway for programmed cell death (pcd) in plant roots, or two separate pathways of pcd could be involved. The phenotype of rn plants suggests that the Rn gene could either negatively regulate cortical cell death or be required for cortical cell survival. The possibility of a mechanistic link between cortical cell death in rn plants and during lateral root emergence is discussed.     

Resumption of DNA Synthesis and Cell Division in Wheat Roots as Related to Lateral Root Initiation

The arrest of DNA synthesis and termination of cell division in basal meristematic cells as well as the resumption of these processes as related to the initiation of lateral root primordia (LRP) were studied in tissues of Triticum aestivumroots incubated with ³H-thymidine. All cells of the stelar parenchyma and cortex as well as most endodermal and pericycle cells left the mitotic cycle and ceased proliferative activity at the basal end of the meristem and at the beginning of the elongation zone. Some endodermal and pericycle cells started DNA synthesis in the basal part of the meristem and completed it later on during their elongation, but they did not divide. In the cells of these tissues, DNA synthesis resumed above the elongation zone, the cells being located much closer to the root tip than the first newly dividing cells. Thus, the initiation of LRP started much closer to the root tip than it was previously believed judging from the distance of the first dividing pericycle cells from the root tip. DNA synthesizing and dividing cells first appeared in the stelar parenchyma, then, in the pericycle, and later, in the endodermis and cortex. It seems likely that a release from the inhibition of DNA synthesis allows the cells that completed mitotic cycle in the basal part of meristem in the G₁phase to cease the proliferative arrest above the elongation zone and to continue their cycling. The location of the first DNA synthesizing and dividing cells in the stelar parenchyma and pericycle did not strictly correspond to the LRP initiation sites and proximity to the xylem or phloem poles. This indicates that LRP initiation results from the resumption of DNA synthesis in all pericycle and stelar parenchyma cells that retained the ability to synthesize DNA and occurs only in the pericycle sector situated between the two tracheal protoxylem strands, all cells of which terminated their mitotic cycles in the G₁phase.