白车轴草和紫花苜蓿根瘤的显微及超微结构

通过对白车轴草(Trifolium repens Linn.)和紫花苜蓿(Medicago sativa Linn.)根瘤的显微及超微结构进行观察,发现其根瘤显微结构都由4部分组成,由外向内依次为：保护层、皮层、鞘细胞层和中心组织(侵染组织)。在中心组织的侵染细胞中,分布有大量的线粒体、高尔基体、核糖体及内质网,白车轴草根瘤侵染细胞中的细菌圆形或椭圆形,有明显的周膜、细菌细胞壁和质膜,在细菌发育过程中,周膜活动旺盛,有时相邻细菌的周膜发生融合,在周膜附近常分布有大量的内质网、高尔基体以及高尔基体小泡,似与周膜融合有关。紫花苜蓿细菌椭圆形、长棒形,甚至有的细菌呈分枝状。二者细菌细胞质中分布着大量的核糖体和纤维状的核物质。在白车轴草中还有染色很深的多聚磷酸盐颗粒。     

尼泊尔马桑放线菌共生固氮根瘤的感染和发育

弗兰克氏放线菌通过感染尼泊尔马桑的根毛侵入根的皮层细胞。由于内生菌侵入的刺激,部分皮层细胞分裂和增大,产生前根瘤原基。根瘤原基分裂、分化,形成初生根瘤瘤片。瘤片顶端分生组织不断双叉分枝,发育,并伴随着内生菌感染的寄主细胞,产生多次双叉分枝的珊瑚状根瘤。观察瘤片的横切面,含菌组织是一个马蹄形的致密整体,不完全地包围着稍偏的中柱。观察瘤片的纵切面,可将其划分为6个区域,即顶端分生组织,未感染皮层细胞组织、新感染含菌组织、成熟含菌组织、衰老含菌组织和中柱及其外围数层富含淀粉粒的皮层细胞。     

几种罗汉松属植物根瘤的形态与结构

对分布在广西的几种罗汉松属植物自然结瘤的根瘤形态和显微结构进行了观察。结果表明:几种罗汉松属植物所结的根瘤形态是一致的,均为球形或近球形,直径约1.0～1.5mm。根瘤的组织结构从内向外包括:维管组织、薄壁细胞、侵染细胞和表皮(周皮);侵染细胞与薄壁细胞呈不均匀的相间分布。侵染细胞中所含菌体为细菌。此外还发现细叶罗汉松根瘤的侵染细胞中有变形膨大的细胞核。总体上看,罗汉松属植物根瘤在形态与结构上与豆科植物的根瘤有许多相似之处。     

田菁营养器官的解剖结构特征

运用石蜡切片法与组织离析法研究田菁营养器官解剖结构特征。结果表明,田菁根部显示典型的次生结构特征,内皮层具凯氏带;根表面有大量根瘤,根瘤菌分布在根部皮层,维管组织相连形成输导组织网。茎部表皮细胞壁出现角质层,皮层含有特化的厚角组织,由大型薄壁细胞构成,维管组织具有含晶韧皮纤维。茎瘤3～4个,大小不等且不连续分布,茎瘤外层为皮层,内层为含菌组织。叶片为异面叶,角质层与表皮层较厚,栅栏组织发达,近叶厚的1/2,海绵组织细胞间隙大,气孔小且多。田菁根、茎的木质部发达,有较多大孔径的导管及丰富的木纤维,叶片栅海比、组织结构紧密度(CTR)较大。田菁的解剖特点显示出其具有的固氮特性以及耐盐、抗旱的生态适应性。     

菜豆根瘤,类根瘤以及根颈瘤的细胞学观察

菜豆根瘤菌(Rhizobium leguminosarum bv.phaseoli)的共生质粒 pSym 3622转移到具有 C58染色体背景的农杆菌(Agrobacterium tumefaciens)中以后,杂交子可以通过伤口感染菜豆(Phaseolus vulgaris)根系,并在接种位点周围诱导类根瘤形成。类根瘤的结构与根瘤和菜豆根颈瘤的结构完全不同,其维管组织位于中央,周围为含有丰富淀粉粒的、高度液泡化的薄壁细胞,而且它的任何细胞以及胞间隙内均不含细菌。但是菜豆有效根瘤则含有大片含菌细胞区,不仅其细胞含有类菌体,而且在胞间隙中也存在细菌。由农杆菌 A208(pTiT37)所诱导的菜豆根颈瘤的任何细胞和胞间隙内也不含细菌。肿瘤的内部结构可以明显地分成许多不同的细胞层次,它们分别起源于不同的分生区;并且其内还可以分化出根伏突起,通过继续发育而形成新根系。此外肿瘤内许多区域的细胞都有明显解体的现象。     

豆科根瘤非侵染细胞的形态结构研究

非侵染细胞是豆科根瘤中一种非常重要的细胞,它不仅参与了根瘤共生固氮,而且还在其中起了极为重要的作用,这些作用的发挥与其具有与此相适应的形态结构和分布有关。非侵染细胞体积小、数量少,分散于侵染细胞之间。在大豆根瘤中,它们彼此相连接,形成一条条的线和一个个的面,由根瘤中心向四周辐射,直至与皮层细胞相接,其作用可能与根瘤中心组织和皮层细胞之间的物质运输,特别是固氮产物的输出密切有关。非侵染细胞与侵染细胞之间的主要差异在于它不含细菌,中央有一个大液泡,细胞质中含有大量的过氧化物酶体和许多膨大的管状内质网,而且细胞壁上还有丰富的胞间连丝和纹孔,但主要存在于非侵染细胞与非侵染细胞之间的连接壁上。     

根瘤菌在大麦和水稻根上形成拟瘤的细胞结构

用3种方法使紫云英根瘤菌(Rhizobium astragali Huikui)、田菁根瘤菌(R.sesbania sp.)分别入侵大麦(hordeum vulgare L.)和水稻(Oryza sativa L.),形成拟瘤状组织。一是用一定磁场强度处理根瘤菌和植物,并接种培养。二是用含有水稻幼苗根提取物的培养基培养根瘤菌,接种水稻。三是重复别人用2,4-D外源激素处理植物,接种根瘤菌。镜检观察,用紫云英根瘤菌接种形成的大麦根拟瘤细胞结构非常精细,保持各种细胞器。有侵入线结构和根瘤菌从侵入线释放。根瘤菌被宿主细胞来源的膜包围,成为拟菌体。这些形态结构与豆科根瘤细胞相似,有共生状态特征,但拟菌体有泡状化现象。田菁根瘤菌入侵水稻根形成的拟瘤,在细胞间隙和细胞内都有细菌分布。受侵染的细胞结构粗糙,根瘤菌裸露,无胞膜包围。用2,4-D处理接种根瘤菌的拟瘤细胞结构也如此,但在维管系统内有大量密集的细菌存在。这种结构完全不同于豆科根瘤细胞结构,细菌与植物细胞的形态学相互关系是一种非共生联合作用。     

七叶一枝花根的显微结构及其内生真菌分布研究

本文采用石蜡永久制片和光学显微摄像的方法对七叶一枝花Parispolyphylla根的显微结构及其内生真菌的分布进行了研究。结果表明,七叶一枝花的根茎由栓皮层、薄壁组织及维管组织组成,其中栓皮层由4层细胞组成;薄壁组织的细胞含有丰富的营养物质,其内有时分布有针状结晶束。不定根由表皮层、皮层、内皮层及维管束构成,表皮上有根毛,皮层所占根径的比例达80%以上;木质部为三原型。在七叶一枝花的根茎和不定根的皮层细胞中均有内生真菌的分布。真菌由表皮、外皮层侵入到皮层薄壁组织,在皮层薄壁细胞中形成菌丝结,并扩展成一定的侵染区域,部分皮层细胞中菌丝结已被消化吸收。内生真菌只侵染皮层薄壁细胞,不侵染维管柱。七叶一枝花可以通过消化细胞内的菌丝作为营养的来源之一。     

Effects of phylloxera infestation on the root ultrastructure of grape cultivars with different resistance

[目的]探讨不同抗性葡萄品种对葡萄根瘤蚜Daktulosphaira vitifolia Fitch侵染后的组织结构响应.[方法]以砧木140Ru(Vitis rupestris×V.berlandieri)和栽培品种赤霞珠Vitis vinifera cv.Cabernet Sauvignon离体三级根及140Ru盆栽苗为试材接种根瘤蚜卵,接种两周后取刺吸位点组织作超薄切片并观察超微结构变化,取盆栽140Ru不同时期根结测定多酚含量.[结果]超微结构显示,砧木140Ru根系周皮层最外层细胞壁的厚度(1 031.25 nm)及周皮层细胞层数(6～7层)显著高于赤霞珠品种的周皮层细胞厚度(543.75 nm)及层数(3～4层),140Ru根系韧皮部酚类物质含量比赤霞珠高出35％;侵染后,砧木140Ru周皮层细胞第3层以内的细胞壁加厚并积聚大量的多酚类物质,140Ru葡萄新根被根瘤蚜侵染后多酚含量呈升高趋势,在接种20d时是对照的2.4倍;赤霞珠粗根被根瘤蚜侵染后薄壁细胞中细胞质变浓,出现大量淀粉粒,线粒体及内质网数量增多.[结论]砧木140Ru的周皮层组织结构较赤霞珠不利于根瘤蚜口针穿刺,被根瘤蚜侵染后发生了不利于根瘤蚜侵染取食的变化.     

密花石斛等六种兰科植物菌根的显微结构研究

本文对密花石斛等六种兰科植物根的显微结构进行了比较观察。结果表明,它们具有典型的兰科植物根,具根被和发达的皮层组织,皮层细胞内分布有针状结晶和菌根真菌形成的菌丝结,发现菌根真菌通过外皮层薄壁通道细胞或破坏根被组织和外皮层细胞侵入皮层细胞,形成内生菌根。     

Structural Analysis of Nodule-Like Tissue on Rice Roots Collected from Wenzhou District of Zhejiang Province

Structural analysis of nodule-like tissues on rice roots collected from Wenzhou District of Zhejiang Province were conducted by using LM, SEM and TEM, and compared with that of nodules on leguminous plants. The observation showed the followings: During the stage of young rice shoots the bacteria were distributed in either dispersed or aggregated form in a few of the host cells of the nodule-like tissue, but the host cells showed their defence reaction to the bacteria, resulting in cytoplasmic agglutination and fibrillation, from which the fine structure of cytoplasm became undistinguishable, indicating the death of infected cells; during the booting or earing stage of rice, the surfaces of the nodule-like tissues were dark-brown with phellem matrial. In the exo-cortex there were no vascular tissue. In the endo-cortex the parenchyma cells were expanded and no bacteria were observed. In some of the nodule-like tissues the exo-and endo-cortical cells were basically lignified. It was clearly shown that there were gear wheel-like structures especially existing in the endo-cortical cells. Under the observations by LM, SEM, these structures were identified as coral-like balls of fibre-like material. In fact, they are spores of a kind of fungi with hyphae. Therefore, the nodule-like tissues on rice roots collected from Wenzhou District of Zhejiang Province are completely different from the genuine nodules on leguminous roots. In fact, they are calluses infected by bacteria and fungi.     

Rhizobium nod factors reactivate the cell cycle during infection and nodule primordium formation, but the cycle is only completed in primordium formation.

Rhizobia induce the formation of root nodules on the roots of leguminous plants. In temperate legumes, nodule organogenesis starts with the induction of cell divisions in regions of the root inner cortex opposite protoxylem poles, resulting in the formation of nodule primordia. It has been postulated that the susceptibility of these inner cortical cells to Rhizobium nodulation (Nod) factors is conferred by an arrest at a specific stage of the cell cycle. Concomitantly with the formation of nodule primordia, cytoplasmic rearrangement occurs in the outer cortex. Radially aligned cytoplasmic strands form bridges, and these have been called preinfection threads. It has been proposed that the cytoplasmic bridges are related to phragmosomes. By studying the in situ expression of the cell cycle genes cyc2, H4, and cdc2 in pea and alfalfa root cortical cells after inoculation with Rhizobium or purified Nod factors, we show that the susceptibility of inner cortical cells to Rhizobium is not conferred by an arrest at the G2 phase and that the majority of the dividing cells are arrested at the G0/G1 phase. Furthermore, the outer cortical cells forming a preinfection thread enter the cell cycle although they do not divide.     

Nodules are induced on alfalfa roots by Agrobacterium tumefaciens and Rhizobium trifolii containing small segments of the Rhizobium meliloti nodulation region.

Regions of the Rhizobium meliloti nodulation genes from the symbiotic plasmid were transferred to Agrobacterium tumefaciens and Rhizobium trifolii by conjugation. The A. tumefaciens and R. trifolii transconjugants were unable to elicit curling of alfalfa root hairs, but were able to induce nodule development at a low frequency. These were judged to be genuine nodules on the basis of cytological and developmental criteria. Like genuine alfalfa nodules, the nodules were initiated from divisions of the inner root cortical cells. They developed a distally positioned meristem and several peripheral vascular bundles. An endodermis separated the inner tissues of the nodule from the surrounding cortex. No infection threads were found to penetrate either root hairs or the nodule cells. Bacteria were found only in intercellular spaces. Thus, alfalfa nodules induced by A. tumefaciens and R. trifolii transconjugants carrying small nodulation clones of R. meliloti were completely devoid of intracellular bacteria. When these strains were inoculated onto white clover roots, small nodule-like protrusions developed that, when examined cytologically, were found to more closely resemble roots than nodules. Although the meristem was broadened and lacked a root cap, the protrusions had a central vascular bundle and other rootlike features. Our results suggest that morphogenesis of alfalfa root nodules can be uncoupled from infection thread formation. The genes encoded in the 8.7-kilobase nodulation fragment are sufficient in A. tumefaciens or R. trifolii backgrounds for nodule morphogenesis.     

Nodule formation in soybeans by exopolysaccharide mutants of Rhizobium fredii USDA 191

Production of exopolysaccharides by Rhizobium has been linked with efficient invasion and nodulation of leguminous plant roots by the bacteria. Exopolysaccharide-deficient (exo) mutants of Rhizobium fredii USDA 191 were isolated following Tn5-insertion mutagenesis. Five phenotypically unique exo mutants were investigated for exopolysaccharide synthesis and their ability to nodulate soybeans. The exopolysaccharides produced by these mutants were analysed for polysaccharide composition by column chromatography and thin-layer chromatography. Two mutants designed exo-3 and exo-5 were deficient in both neutral glucan and exopolysaccharide synthesis, but each induced some functional nodules on Glycine max (Peking). The remaining three mutants (exo-1, exo-2 and exo-4) synthesized neutral glucans at levels higher or lower than those in wild-type and exhibited partial exopolysaccharide deficiencies. The data imply that neither exopolysaccharides nor neutral glucans are essential for the induction of determinate nodules by R. fredii.     

Cytoskeletal arrays in the cells of soybean root nodules: The role of actin microfilaments in the organisation of symbiosomes

Summary Within the infected cells of root nodules there is evidence of stratification and organisation of symbiosomes and other organelles. This organisation is likely to be important for the efficient exchange of nutrients and metabolites during functioning of the nodules. Using immunocytochemical labelling and confocal microscopy we have determined the organisation of cytoskeletal elements, micro tubules and actin microfilaments in soybean nodule cells, with a view to assessing their possible role in organelle distribution. Most microtubule arrays occurred in the cell cortex where they formed disorganised arrays in both uninfected and infected cells from mature nodules. In infected cells from developing nodules, parallel arrays of microtubules, transverse to the long axis of the cell, were observed. In incipient nodules, before release of rhizobia into the plant cells, the cells also had an array of microtubules which radiated from the nucleus into the cytoplasm. Three actin arrays were identified in the infected cells of mature nodules: an aster-like array which emanated from the surface of the nucleus, a cortical array which had an arrangement similar to that of the cortical microtubules, and, throughout the cytoplasm, an array of fine filaments which had a honeycomb arrangement consistent with a distribution between adjacent symbiosomes. Uninfected cells from mature nodules had only a random cortical array of actin filaments. In incipient nodules, the density of actin microfilaments associated with the nucleus and radiating through the cytoplasm was much less than that seen in mature infected cells. The cortical array of actin also differed, being composed of swirling configurations of filaments. After invasion of nodule cells by the rhizobia, the number of actin filaments emanating from the nucleus increased markedly and formed a network through the cytoplasm. Conversely, the cytoplasmic array in uninfected cells of developing nodules was identical to that in the cells of incipient nodules. The cytoplasmic network in infected cells of developing nodules is likely to be the precursor of the honeycomb array seen in mature nodule cells. We propose that this actin array plays a role in the spatial organisation of symbiosomes and that the microtubules are involved in the localisation of mitochondria and plastids at the cell periphery in the infected cells of root nodules.     

Initial proliferation of cortical cells in the formation of root nodules in Pisum sativum L.

Summary Root nodule initiation in Pisum sativum begins with cell divisions in the inner cortex at some distance from the advancing infection thread. After penetrating almost the entire cortex, the branches of the thread infiltrate the meristematic area previously initiated in the inner cortical cells. These cells are soon invaded by bacteria released from the infection thread and subsequently differentiate into non-dividing, bacteriod-containing cells. As the initial meristematic centre in the inner cortex is thus lost to bacteroid formation, new meristematic activity is initiated in neighbouring cortical cells. As development proceeds, more cortical layers contribute to the nodule, with the peripheral layer and apical meristem of the nodule not invaded by bacteria.Lateral root primordia are initiated in a region separate from that in which nodules are formed, with the lateral primordia being closer to the root apex. This is interpreted to indicate that the physiological basis for nodule initiation is distinct from that for initiation of lateral roots. The role of a single tetraploid cell in nodule initiation is refuted, as is the existence of incipient meristematic foci in the root. It is suggested that the tetraploid cells in nodule meristems arise from pre-existing endoreduplicated cells, or by the induction of endoreduplication in diploid cortical cells by Rhizobium.     

Ontogeny and ultrastructure of spontaneous nodules in alfalfa (Medicago sativa)

Summary The development of spontaneous nodules, formed in the absence ofRhizobium and combined nitrogen, on alfalfa (Medicago sativa L. cv. Vernal) was investigated at the light and electron microscopic level and compared to that ofRhizobium-induced normal nodules. Spontaneous nodules were initiated from cortical cell divisions in the inner cortex next to the endodermis, i.e., the site of normal nodule development. These nodules, on uninoculated roots, were white multilobed structures, histologically composed of nodule meristems, cortex, endodermis, central zone and vascular strands. Nodules were devoid of intercellular or intracellular bacteria confirming microbiological tests. Early development of spontaneous nodules was initiated by series of anticlinal followed by periclinal divisions of dedifferentiated cells in the inner cortex of the root. These cells formed the nodular meristem from which the nodule developed. The cells in the nodule meristems divided unequally and differentiated into two distinct cell types, one larger type being filled with numerous membrane-bound starch grains, and the other smaller type with very few starch grains. There were no infection threads or bacteria in the spontaneous nodules at any stage of development. This size differentiation is suggestive of the different cell sizes seen inRhizobium-induced nodules, where the larger cell type harbours the invading bacteria and the smaller type is essential in supportive metabolic roles. The ontogenic studies further support the claim that these structures are nodules rather than aberrant lateral roots, and that plant possess all the genetic information needed to develop a nodule with distinct cell types. Our results suggest that bacteria and therefore theirnod genes are not necessarily involved in the ontogeny and morphogenesis of spontaneous and normal nodules in alfalfa.Abbreviations EH smallest emergent root hair - EM electron microscope - enod2 early nodulin2 gene - RT root tip - RER rough endoplasmic reticulum - YEMG yeast extract-mannitol-gluconate     

Sucrose synthase and enolase expression in actinorhizal nodules ofAlnus glutinosa: comparison with legume nodules

Two different types of nitrogen-fixing root nodules are known — actinorhizal nodules induced byFrankia and legume nodules induced by rhizobia. While legume nodules show a stem-like structure with peripheral vascular bundles, actinorhizal nodule lobes resemble modified lateral roots with a central vascular bundle. To compare carbon metabolism in legume and actinorhizal nodules, sucrose synthase and enolase cDNA clones were isolated from a cDNA library, obtained from actinorhizal nodules ofAlnus glutinosa. The expression of the corresponding genes was markedly enhanced in nodules compared to roots. In situ hybridization showed that, in nodules, both sucrose synthase and enolase were expressed at high levels in the infected cortical cells as well as in the pericycle of the central vascular bundle of a nodule lobe. Legume sucrose synthase expression was studied in indeterminate nodules from pea and determinate nodules fromPhaseolus vulgaris by usingin situ hybridization.     

INITIATION AND DEVELOPMENT OF ROOT NODULES OF CASUARINA (CASUARINACEAE)

Roots of seedlings of the “beefwood” tree, Casuarina cunninghamiana Miq. grown in nitrogen-free nutrient solution were inoculated with a suspension prepared from crashed root nodules taken from mature plants. Marked deformation of root hairs was evident but no infection threads were observed in root hairs. The mode of infection remains undetermined. Root nodules were initiated within three weeks and thereafter numerous upward-growing nodule roots developed from each nodule. Nodules in this symbiotic nitrogen-fixing plant resulted from an infection caused by an unidentified actinomycete-like soil microorganism. Anatomical analysis of nodule formation showed that nodules are the result of repeated endogenous lateral root initiations, one placed upon another in a complexly branched and truncated root system. The endophyte-infected cortical tissues derived from successive root primordia form the swollen nodular mass. Nodule roots develop from nodule lobes after escaping from the initial inhibitory effects of the endophyte. Included is a discussion of the anatomical similarities between nodules of Casuarina which produce nodule roots and those of Alnus which form coralloid nodules usually lacking nodule roots.     

A γ-TIP cross-reacting protein is abundant in the cortex of soybean N2-fixing nodules

The distribution and abundance of tonoplast intrinsic protein (γ-TIP), a putative aquaporin which is abundant in the tonoplast of osmoregulated pulvinus motor cells, were determined in nodules of Glycine max (L.) Merr. using chemical fixation and immunolocalization. This protein was highly expressed in the tonoplast of the inner cortical cells of the nodules but poorly expressed in the vascular transfer cells and in infected cells. It is concluded that the differentiation of the inner cortical cells of the nodules like that of pulvinus motor cells, is accompanied by an increased expression of γ-TIP. This result is consistent with our previous hypothesis that a reversible exchange of intercellular water by the inner cortical cells plays a role in the regulation of nodule conductance to O₂ diffusion, and hence in subsequent N₂-fixing activity. Received: 7 February 1998 / Accepted: 22 May 1998