Evidence of Ultrastructure and Physiology of F-actin as Component of Plasmodesmata

The characters and ultrastructure of the intercellular connection were revealed in the outer epidermis of the garlic clove sheath by means of fluorescent probe TRITC_Phalloidin (TRITC_Ph) labeling combined with confocal laser scanning microscopy (CLSM), immuno_gold labeling and transmission electron microscopy. These results show that transcellular channel is a complex of rod_like cytoplasm channel and grouped plasmodesmata (PDs) in pit. The former remains a portion of the cell protoplast. The diameter of PD is normally 60-70 nm. The PDs are the real intercellular symplasmic connections of the cells. The transcellular fibers labeled with the TRITC_Ph obviously become narrow in the primary pit fields, which is the same as the characters observed under the electron microscope. The bright fluorescent spot in the primary wall reflects the grouped PDs in pit, and hence the presence of F_actin in the PDs can be confirmed. In immuno_gold labeling experiment, a lot of gold particles were massively distributed in the rod_like cytoplasm channel and grouped PDs. The result provides effective support that these fluorescent filaments possibly are the existing form of F_actin.

OSMOTIC STRESS DECREASES THE ACTIVITY OF ATPASE ASSOCIATED WITH THE ROOT CAP PLASMODESMATAIN ZEA MAYS

With light and electron microscopy the substructural change and the ATPase activity of corn (Zea mays L.) root cap cells after short-term osmotic stress were studied. Some spoke-like fine strands originating from the departed periplasm and stretching towards cell wall could be observed even after plasmolysis. By observing the precipitation of ATPase activity product (lead phosphate) at plasma membrane and plasmodesmata, it was found that the fine strands were plasma membrane-lined channels surrounding the cytoplasm and that they still firmly connected to the plasmodesmata during plasmolysis. Compared with the control (unstressed), a sharp decrease of ATPase activity in the plasmodesmata of the stressed cells was observed. Inhibition of energy metabolism in these limited locales would affect the physiological activity, maybe including the regulation of permeability and the change of size exclusion limit (SEL) of plasmodesmata.

Plasmodesmata: Dynamic Channels for Symplastic Transport

Plasmodesmata (PDs) are cytoplasmic structures that link adjacent cells to form the symplast of a plant. PDs are involved extensively in a plant's life by mediating symplastic transport of a wide range of ions and molecules. Major components of a plasmodesma (PD) include a plasma membrane, a desmotubule, and a cytoplasmic annulus, all of which are readily detectable by electron microscopy. Both the plasma membrane and the desmotubule contain proteinaceous particles, thought to be involved in altering the size of the cytoplasmic annulus. Cytoskeleton elements (actin and myosin) are essential for maintaining the integrity of PDs. Together with these elements, calcium_binding proteins probably play a significant role in regulating PD function. Symplastic transport occurs through the cytoplasmic annulus for the great majority of solutes, while other substances may traverse through the desmotubule internal compartment, the desmotubule shell, or the plasma membrane. The symplast is subdivided into several domains with varying molecular size exclusion limits (ranging from <1 kD to >10 kD). Plasmodesmata can be either primary or secondary; the former are developed during new wall formation and the latter are made in existing walls. The dynamic nature of plasmodesmata is also reflected by their changing frequencies, which, in turn, depend on the developmental and physiological status of the tissue or the entire plant. While diffusion is the major mechanism of symplastic transport, plasmodesmata are selective for certain ions and molecules. Upon viral infection, viral movement proteins interact with PD receptor proteins and, as a result of yet unknown mechanisms, the plasmodesmata are remarkably dilated to allow viral movement proteins and the bound viral genome to enter healthy cells. Some proteins of plant origin are also able to traverse plasmodesmata, presumably in ways similar to viral movement proteins. Some of these plant proteins are probably signal molecules contributing to cell differentiation and other activities. Other proteins move cell_to_cell in a non_specific manner.

Actin and Myosin Co-Localize in Plasmodesmata and Ectodesmata-Like Structure

Actin and myosin were found to be associated with the cytoplasmic sleeve of plasmodesmata. As cytoskeletal proteins, actin and myosin are believed to regulate the conductivity of plasmodesmata （PDs） in higher plants. Using immunocytochemical methods, we found the two proteins to be co-localized - and closely linked to each other - in plasmodesmata and ectodesmata-like structure in ageing parenchymatous cells of Allium sativum L. We suggest that intercellular communication is affected by the interaction between actin and myosin.

α1-COP modulates plasmodesmata function through sphingolipid enzyme regulation

Callose,aβ-1,3-glucan plant cell wall polymer,regulates symplasmic channel size at plasmodesmata(PD)and plays a crucial role in a variety of plant processes.However,elucidating the molecular mechanism of PD callose homeostasis is limited.We screened and identified an Arabidopsis mutant plant with excessive callose deposition at PD and found that the mutated gene wasα1-COP,a member of the coat protein I(COPI)coatomer complex.We report that loss of function ofα1-COP elevates the callose accumulation at PD by affecting subcellular protein localization of callose degradation enzyme Pd BG2.This process is linked to the functions of ERH1,an inositol phosphoryl ceramide synthase,and glucosylceramide synthase through physical interactions with theα1-COP protein.Additionally,the loss of function ofα1-COP alters the subcellular localization of ERH1 and GCS proteins,resulting in a reduction of Glc Cers and Glc HCers molecules,which are key sphingolipid(SL)species for lipid raft formation.Our findings suggest thatα1-COP protein,together with SL modifiers controlling lipid raft compositions,regulates the subcellular localization of GPI-anchored PDBG2 proteins,and hence the callose turnover at PD and symplasmic movement of biomolecules.Our findings provide the first key clue to link the COPI-mediated intracellular trafficking pathway to the callose-mediated intercellular signaling pathway through PD.

Arabidopsis PDLP7 modulated plasmodesmata function is related to BG10-dependent glucosidase activity required for callose degradation

The microdomains of plasmodesmata,specialized cell-wall channels responsible for communications between neighboring cells,are composed of various plasmodesmata-located proteins(PDLPs)and lipids.Here,we found that,among all PDLP or homologous proteins in Arabidopsis thaliana genome,PDLP5 and PDLP7 possessed a C-terminal sphingolipid-binding motif,with the latter being the only member that was significantly upregulated upon turnip mosaic virus and cucumber mosaic virus infections.pdlp7mutant plants exhibited significantly reduced callose deposition,larger plasmodesmata diameters,and faster viral transmission.These plants exhibited increased glucosidase activity but no change in callose synthase activity.PDLP7 interacted specifically with glucan endo-1,3-β-glucosidase 10(BG10).Consistently,higher levels of callose deposition and slower virus transmission in bg10 mutants were observed.The interaction between PDLP7 and BG10 was found to depend on the presence of the Gnk2-homologous 1(Gn K2-1)domain at the N terminus of PDLP7 with Asp-35,Cys-42,Gln-44,and Leu-116 being essential.In vitro supplementation of callose was able to change the conformation of the Gn K2-1 domain.Our data suggest that the Gn K2-1 domain of PDLP7,in conjunction with callose and BG10,plays a key role in plasmodesmata opening and closure,which is necessary for intercellular movement of various molecules.

The Fusarium oxysporum Avr2-Six5 Effector Pair Alters Plasmodesmatal Exclusion Selectivity to Facilitate Cell-to-Cell Movement of Avr2

Pathogens use effector proteins to manipulate their hosts. During infection of tomato, the fungus Fusarium oxysporum secretes the effectors Avr2 and Six5. Whereas Avr2 suffices to trigger I-2-mediated cell death in heterologous systems, both effectors are required for I-2-mediated disease resistance in tomato. How Six5 participates in triggering resistance is unknown. Using bimolecular fluorescence complementation assays we found that Avr2 and Six5 interact at plasmodesmata. Single-cell transformation revealed that a 2xRFP marker protein and Avr2-GFP only move to neighboring cells in the presence of Six5. Six5 alone does not alter plasmodesmatal transduction as 2xRFP was only translocated in the presence of both effectors. In SIX5-expressing transgenic plants, the distribution of virally expressed Avr2-GFP, and subsequent onset of I-2-mediated cell death, differed from that in wild-type tomato. Taken together, our data show that in the presence of Six5, Avr2 moves from cell to cell, which in susceptible plants contributes to virulence, but in I-2 containing plants induces resistance.

Phytosphinganine Affects Plasmodesmata Permeability via Facilitating PDLP5-Stimulated Callose Accumulation in Arabidopsis

Plant plasmodesmata (PDs) are specialized channels that enable communication between neighboring cells. The intercellular permeability of PDs, which affects plant development, defense, and responses to stimuli, must be tightly regulated. However, the lipid compositions of PD membrane and their impact on PD permeability remain elusive. Here, we report that the Arabidopsis sld1 sld2 double mutant, lacking sphingolipid long-chain base 8 desaturases 1 and 2, displayed decreased PD permeability due to a significant increase in callose accumulation. PD-located protein 5 (PDLP5) was significantly enriched in the leaf epidermal cells of sld1 sld2 and showed specific binding affinity to phytosphinganine (t18:0), suggesting that the enrichment of t18:0-based sphingolipids in sld1 sld2 PDs might facilitate the recruitment of PDLP5 proteins to PDs. The sld1 sld2 double mutant seedlings showed enhanced resistance to the fungal-wilt pathogen Verticillium dahlia and the bacterium Pseudomonas syringae pv. tomato DC3000, which could be fully rescued in sld1 sld2 pdlp5 triple mutant . Taken together, these results indicate that phytosphinganine might regulate PD functions and cell-to-cell communication by modifying the level of PDLP5 in PD membranes.

浙贝母鳞茎衰退过程的解剖学研究初报

采用徒手制片、石蜡切片和电子显微镜技术，系统而深入地研究了浙贝母（ＦｒｉｔｉｌｌａｒｉａｔｈｕｎｂｅｒｇｉｉＢａｋｅｒ）鳞茎衰退过程中的形态、组织结构和细胞内部的变化情况．看到了大分子物质进行运输时采用集装囊泡形式的可能性。进一步论证了胞间连丝作为细胞间原生质通道的观点．

玉米胚乳传递细胞的结构观察研究

以玉米品种‘登海11号’为材料,分别于授粉后8、10、15和20 d采集颖果,取所需部位并采用树脂包埋的方法及半薄和超薄切片技术,对玉米胚乳传递细胞进行了显微和超微结构观察。结果显示:(1)胚乳传递细胞的壁内突从外层向内层依次递减,溶质浓度逐步降低,形成了明显的溶质浓度梯度,有利于溶质的运输;(2)中层胚乳传递细胞和内层胚乳传递细胞的邻壁上存在胞间连丝或一些孔径较大的胞壁孔道,从而使溶质更快的进入内层胚乳传递细胞;(3)在壁内突周围存在许多线粒体。研究表明,玉米胚乳传递细胞的结构适合溶质运输。

水稻胚囊壁的形成与发育观察

通过透射电镜对水稻(Oryza sativa L.)功能大孢子形成开始至胚囊成熟期间胚囊壁的形成与发育进行观察,结果表明:水稻胚囊壁是在原有功能大孢子壁的基础上,通过与其周围退化珠心细胞留下的壁相叠合,使壁加厚。功能大孢子近合点端壁存在胞间连丝,其中个别胞间连丝可保留到八核胚囊。胚囊壁上内突最早于四核胚囊近珠孔端发生。八核胚囊形成后,内突的发育在胚囊不同的细胞中表现不同,其中以中央细胞最具特点,表现为先在中央细胞与珠心相接的近珠孔端和近合点端两个区域的胚囊壁上形成,以后近珠孔端胚囊壁上的内突大量增加,而近合点端的却增加不明显,中部胚囊壁上的内突出现的时间相对较晚。到胚囊成熟时,近珠孔端胚囊壁上内突的分布密度最大,中部次之,近合点端的最小,三个区域上内突的形态各异。反足细胞与珠心相接的胚囊壁上内突的形成时间较早,但以后的发育却相对缓慢,数量增加不明显。2个助细胞交界处胚囊壁上的丝状器在胚囊未明显膨大时已形成。卵细胞除在与助细胞交界处的壁外,其它部位不形成明显的内突结构。

蒜鳞片休眠进程中胞间联络的变化及类外连丝结构与功能的研究

利用电子显微镜术,对蒜休眠进程中鳞片薄壁细胞间胞间联络的特征进行了实验观察,发现不同时期胞间联络具有随细胞间生理关系密切程度而呈现相应结构变化的特点。并观察到萌芽期鳞片中衰败细胞与存活细胞之间有类外连丝型胞间连丝的存在;以激光共聚焦荧光显微镜结合荧光标记物示踪检测,发现不透膜荧光物质分子量为457Da 的萤黄(Lucifer yellow,LYCH),可以共质体运输方式进入存活细胞内,论证了类外连丝这一胞间连丝特定修饰态的存在,并可在一段时间内继续保持生理活性,起到进行物质共质运输的功能。

番茄/番茄和苋菜/番茄嫁接组合形成过程的细胞学观察

用光镜和电镜对亲和性自体嫁接组合(番茄/番茄)和非亲和性异体嫁接组合(苋菜/番茄)进行了组织学细胞学比较观察。嫁接组合形成的早期阶段,两种组合隔离层两侧细胞中高尔基体、线粒体数量增多,细胞分裂形成愈份组织,表现出与愈伤反应一致的特征。后期,亲和性嫁接组合隔离层大部分消失,两侧细胞恢复正常状态,接穗砧木间有新分化的维管束桥相连,且有胞间连丝的次生形成,环状片层和壁旁体活跃地参与嫁接组合的形成过程;非亲和性嫁接组合中,隔离层不消失,附近出现死细胞层,接穗砧木之间无维管束桥相连。对观察的结果和有关问题作了讨论。

Cr^(6+)污染对水鳖的超微结构及菱、莼菜、黑藻细胞膜的影响

Cr6+污染对水鳖超微结构的影响主要表现在 :胞间连丝呈现不同程度的断裂 ;细胞核出现各种各样的变形 ;叶绿体膨胀 ,其基粒解体。细胞损伤程度与 Cr6+ 培养浓度呈正相关。Cr6+ 对水鳖、菱、莼菜和黑藻这几种水生高等植物的细胞膜影响主要表现在 :质壁发生分离 ,质壁之间有黑色颗粒存在。在做生理验证时发现处理组的膜脂过氧化产物丙二醛 (MDA)和超氧阴离子 (O 2 )均高于对照。

鹤顶兰胚囊发育的超微结构观察

运用电子显微镜技术对鹤顶兰(Phaius tankervilliae(Aiton)Bl.)胚囊发育过程中功能大孢子、二核胚囊、四核胚囊、成熟胚囊的超微结构进行观察,捕捉到了功能大孢子的三个阶段、成熟胚囊的两个阶段,进一步积累了鹤顶兰生殖生物学研究的基础资料。在功能大孢子、四核胚囊时期的合点端壁上可观察到胞间连丝,与体细胞间有物质及信息的交换,胚囊发育并非处于完全"隔离"状态。功能大孢子早期可见明显大液泡,随后进入第一次有丝分裂时大液泡消失,移向两极的染色体之间可见大量体积较小的液泡,成熟胚囊前期助细胞及卵细胞内也可见明显液泡,但当助细胞解体时,卵细胞内的大液泡也消失,液泡形态的变化可能是细胞生理状态发生改变的结果。

离体细胞共培养中科间细胞共质体的形成

离体培养下选出的绿色胡萝卜（Ｄａｕｃｕｓ ｃａｒｏｔａ）细胞系和白色普通烟草（Ｎｉｃｏｔｉａｎａｔａｂａｃｕｍ ）细胞系，各自具有独特的细胞结构标志，在愈伤组织、光镜和电镜３ 个水平上均可区分。对两个细胞系进行分散、混合、Ｋ＋ 液低渗处理后在固体培养基上共培养，１０—１５ ｄ 后可观察到两种细胞的镶嵌生长。光镜和电镜下均观察到烟草细胞和胡萝卜细胞之间隔离层的存在与消失。在隔离层消失的区域可见到异种细胞间次生胞间连丝的形成，从而将独立的两个共质体连成一个统一的共质体。对科间细胞共质联系的建立过程进行了讨论，认为细胞接触后首先非特异粘连——以隔离层形成并适度加厚为标志，然后特异的细胞识别在隔离层中启动，从而导致隔离层或消失而重新建立共质联系或加厚、木质化。

紫竹梅雄蕊毛细胞发育过程中胞间连丝超微结构的变化

紫竹梅（Ｓｅｔｃｒｅａｓｅａ ｐｕｒｐｕｒｅａ）雄蕊毛细胞间的胞间连丝随着细胞的生长、发育、衰老而呈现动态变化的过程．花蕾和开放花的雄蕊毛细胞间的胞间连丝，具备胞间连丝的一般结构，直径约５０ ｎｍ ．衰老花雄蕊毛细胞间的胞间连丝拓宽，内部结构逐步降解、撤离，呈开放式通道，直径约１００ ｎｍ ． 在胞间连丝的动态开放过程中，细胞内的细胞器也发生相应变化．

百合小孢子母细胞形成和发育过程中细胞间连接形态和结构的变化

超显微结构水平观察发现造孢时期造胞细胞的壁较厚，由中间层、初生和次生壁组成，细胞间的连接为典型的胞间连丝。减数分裂前间期的细胞壁主要由中间和初生壁组成，典型的胞间连丝消失，但局部位置上的中间层和初生壁组分缺失，构成约２００～８００ｎｍ宽的“豁口”。进入分裂期后，次生壁组分急剧增加。当整个细胞壁加厚到２４０—５００ｎｍ时，细胞壁出现穿孔。穿孔过程似乎有下列两种方式：１）在细胞壁的一侧或两侧出现电子致密物，这种电子致密物因不断积累，在与细胞壁垂直的方向上向壁内延伸。它们穿过次生壁、初生壁和中间层，最后整个细胞壁被贯通；２）细胞壁的两侧同时出现类似分泌泡的结构，可能由于这些分泌泡的作用使它们所处部位的细胞壁变薄，最终被贯通。当细胞壁进一步加厚至４７０—１７００ｎｍ时（偶线－粗线期），细胞壁出现很宽阔（２００～１５００ｎｍ）的细胞融合道（ｃｙｔｏｍｉｃｔｉｃｃｈａｎｎｅｌ），其中并伴随有核物质或细胞器通过。文章讨论了细胞发育过程中次生胞间连丝与细胞融合道形成的机理及其相互联系。

紫竹梅雄蕊毛发育过程中胞间连丝通透能力的动态变化

运用显微注射法将不同分子量的荧光分子探针注入紫竹梅雄蕊毛细胞中，在荧光显微镜下观察其通过胞间连丝转移情况。表明，开放花和花蕾Ⅳ期胞间连丝允许通过物质的分子量不超过１０００Ｄ，衰老花可允许ＦＩＴＣ－ｉｎｓｕｌｉｎＡ链（２９２１Ｄ）通过，而花蕾Ⅰ期可允许ＦＩＴＣ－ｄｅｘｔｒａｎ（４４００Ｄ）通过。说明胞间连丝允许通过物质的分子量极限不是固定不变的，它随组织、细胞的发育进程而改变。

胞间连丝的次生形成和次生变化

本文介绍了胞间连丝次生形成和次生变化的研究进展,用统计特定细胞壁区段上胞间连丝数量与密度的变化,电镜观察嫁接组合中接穗与砧水间细胞壁上胞间连丝的形成等方法,证明了在植物生长发育过程中,存在着胞间连丝的次生形成。在某些特定部位,某一发育阶段,已形成的胞间连丝常会发生可逆的次生变化,这种变化和植物发育过程中的共质体隔离以及物质运输的调节有关。