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。     

濒危药用植物降香檀根瘤的解剖学研究

采用显微观察法对国家二级保护植物降香檀根瘤的结构进行细胞学研究。结果表明,降香檀的根瘤生长在侧根上,外表金黄色,呈球形或扁球形;根瘤结构由周皮、皮层、维管系统和侵染组织等4部分组成;根瘤菌在皮层中形成多个侵染区域,增大了根瘤的体积,增强了根瘤菌的固氮能力,更利于降香檀植物的生长发育。     

几种非豆科植物根瘤内生菌侵染特征的研究

自不同科、属、种的非豆科植物根瘤分离内生菌,对其寄主植物进行了交叉侵染,结果表明,这些Frankia菌对不同寄主的侵染没有明显的专一性,供试菌可以进行跨越科、属、种的侵染,但有的菌株对于某些植物的侵染,可能存在一些特殊情况,相同菌株对不同植物的侵染能力,以及不同菌株对同一寄主的侵染能力是有差异的。从同一种植物根瘤中分离的不同菌株,侵染能力也有高低之分,供试菌随寄主植物的改变,侵染能力及所建立的共生系统固氮活性有所降低,侵染原寄主植物所形成的根瘤固氮活性较高的菌株,在改变寄主后所形成的根瘤固氮活性也比较高,在一定条件下,寄主植物的结瘤量与根瘤固氮活性呈正相关,而侵染不同寄主后,根瘤中菌体孢子的表面结构也发生了一定变化。     

几种非豆科植物根瘤内生菌侵染特征的研究

自不同科、属、种的非豆科植物根瘤分离内生菌,对其寄主植物进行了交叉侵染,结果表明,这些Frankia菌对不同寄主的侵染没有明显的专一性,供试菌可以进行跨越科、属、种的侵染,但有的菌株对于某些植物的侵染,可能存在一些特殊情况,相同菌株对不同植物的侵染能力,以及不同菌株对同一寄主的侵染能力是有差异的。从同一种植物根瘤中分离的不同菌株,侵染能力也有高低之分,供试菌随寄主植物的改变,侵染能力及所建立的共生系统固氮活性有所降低,侵染原寄主植物所形成的根瘤固氮活性较高的菌株,在改变寄主后所形成的根瘤固氮活性也比较高,在一定条件下,寄主植物的结瘤量与根瘤固氮活性呈正相关,而侵染不同寄主后,根瘤中菌体孢子的表面结构也发生了一定变化。     

骆驼刺根瘤菌的超微结构研究

用透射电子显微镜研究骆驼刺根瘤中的根瘤菌。结果表明。在成熟的骆驼刺根瘤中,根瘤菌的大小、数量、形态、分布位置及精细结构随寄主细胞的发育程度不同而异。早期侵染细胞中,根瘤菌小,数量少,一般呈球形或椭球形,位于细胞壁附近及靠近核区的地方,没有聚磷酸盐颗粒和聚羟基丁酸。成熟侵染细胞中,根瘤菌个体较大,数量较多．多呈棒状,少数为球形或椭球形。有很多根瘤菌还呈现明显的“T”形、“Y”形或“V”形,菌体占满了整个细胞,这时的根瘤菌大多数含有聚羟基丁酸和聚磷酸盐颗粒。而在衰老的侵染细胞中,根瘤菌细胞质收缩,电子密度增高．形状变得很不规则,有的根瘤菌解体,呈现膜泡状结构,菌体中含有数量不等的聚羟基丁酸和聚磷酸盐颗粒。球状根瘤菌从侵染初期到侵染细胞裂解的整个阶段中都仔在。并且观察到的处于分裂状态的根瘤菌都是球状菌,因此可以推测骆驼刺根瘤中是以球状根瘤菌来进行增殖的。     

三种白及属植物不同生长发育时期的菌根显微结构

采用石蜡切片技术对白及Bletilla striata、黄花白及B. ochracea和小白及B. formosana的栽培种在生长期、花期、果期和休眠期的菌根解剖结构特征、菌根真菌入侵方式和菌丝特征等进行观察研究,以进一步了解菌根真菌与白及属植物的共生关系。结果表明,3种白及属植物的菌根真菌均是通过通道细胞侵入根皮层薄壁细胞,侵入后菌丝靠近皮层细胞的细胞核分布,最终在皮层细胞形成菌丝团;真菌侵染率和菌丝形态随着植物生长发育变化而变化,3种白及属植物均表现为花期和生长期的真菌侵染率较高,以丝状菌丝团为主,而果期和休眠期较低,以团块状菌丝团居多;同一时期不同植物类型的菌根特征无显著差异。     

刺槐核糖体蛋白同源基因RpRPL22在共生结瘤过程中功能研究

目的: 核糖体蛋白(RPs)属于多功能蛋白,能够参与调控细胞生长和响应胁迫条件。RpRPL22是一个从豆科植物刺槐中分离得到的结瘤相关基因,通过序列比对发现其与核糖体大亚基蛋白RPL22高度同源。对其如何通过调控根瘤菌侵染而在共生结瘤过程中发挥重要作用进行了较为深入的探索。方法: 利用实时荧光定量PCR技术(qRT-PCR)分析RpRPL22在接菌后不同时间及不同植物组织的表达变化。利用cDNA末端快速扩增技术(RACE)获得目的基因cDNA全长。通过GFP报告基因进行RpRPL22亚细胞定位分析。通过Gateway BP重组技术构建RNA干扰(RNAi)重组载体,借助电转化法将重组载体转至农杆菌K599,利用农杆菌介导植物根部,接菌后观察和测量植株表型。首先从宏观水平统计观察目的基因是否对结瘤过程有影响,其次从分子水平揭示目的基因在共生结瘤过程的重要功能。结果: 不同接菌时期、不同植物组织目的基因qRT-PCR相对表达量结果显示,几乎在所有取样的接菌时间,目的基因RpRPL22在接菌根中的相对表达量都低于未接菌对照根,只有接菌后第25天除外。在成熟的根瘤中,接菌后第25天该基因的表达量也最高。洋葱表皮和毛状根亚细胞定位结果均显示在椰菜花叶病毒(CaMV)的35S启动子控制下,RpRPL22融合绿色荧光蛋白GFP的荧光信号在细胞核和细胞质有明显的表达。RNAi转化植株的表型统计观察结果,比如植株鲜重、植株的有效结瘤数目较对照组均有明显的降低;同时RNAi转化植株在根瘤菌侵染过程形成的侵染线数目和根瘤原基数目较对照均显著降低。根瘤切片实验用于观察根瘤显微超微结构,结果显示RNAi植株根瘤中固氮区的受菌侵染细胞数目与对照相比明显减少。电镜观察根瘤单个受菌侵染细胞中类菌体形态显示,RNAi根瘤中类菌体侵染细胞胞体多呈不规则形状,皱缩变形严重,环类菌体周间隙空间增大,多共生体融合,表现出细胞凋亡的迹象。对照根瘤中的受菌侵染细胞胞体多呈圆形椭圆形,胞质饱满丰富且分布均匀,细胞发育正常,表明RNAi植株根瘤发育过程明显受阻。结论: 核糖体蛋白(RP)能够参与调控豆科植物共生结瘤过程,相关同源基因RpRPL22可能在起始根瘤菌侵染植物和阻止类菌体降解过程中起重要作用。     

羊奶果根瘤侵入线和被侵染细胞的亚显微结构

羊奶果根瘤的侵入线,总是在几个相邻细胞的细胞间隙中出现。在根瘤发生部位未出现根毛的变形。侵入线似一囊状物,其内含物有丝状物和基质,侵入线的壁部分或全部加厚,并与周围的寄主细胞壁相连。侵入线呈多种形态,在被侵染细胞中,细胞核膨大,并分叉变成指状核。随着细胞的衰老,被侵染细胞中的内生菌、细胞器也逐渐消失。     

基于高通量测序分析西藏沙棘根瘤内生菌的多样性

根瘤是微生物侵染植物根部并与之形成的共生结构,这些微生物都可被称为植物内生菌。豆科植物根瘤中的内生菌常常又被称为根瘤菌,而侵染非豆科植物形成根瘤的主要是放线菌弗兰克氏菌,这些非豆科植物又被称为放线菌结瘤植物。西藏沙棘是一种典型的放线菌结瘤植物,由于其分布生境的特殊性,对其根瘤内生菌的研究具有重要的生态意义。对于西藏沙棘根瘤内生菌的研究,培养方法因难以模拟自然条件而不易获得纯培养,高通量测序技术对其多样性的研究提供了便利。因此,本研究以生长在甘肃省天祝县金强河河滩地的西藏沙棘根瘤为材料,采用16S rRNA基因扩增子高通量测序方法,结合OTU分析,对西藏沙棘根瘤内生菌的多样性进行探讨。实验结果表明,西藏沙棘根瘤内生菌具有丰富的多样性,根瘤内的优势属为共生固氮的弗兰克氏菌属（Frankia）,其相对丰度为47.63%,共检测到7个弗兰克氏菌属的OTUs;根瘤内除弗兰克氏菌外,还存在大量的非弗兰克氏菌,共检测到1523个OTUs,隶属于22个门、33个纲、69个目、113个科和202个属,相对丰度排名前9的属中有25个非弗兰克氏菌属的OTUs。该研究也表明,西藏沙棘根瘤内生菌具有丰富的多样性,西藏沙棘根瘤中不仅存在着可共生固氮的弗兰克氏菌,并且还分布着非弗兰克氏菌;在同一根瘤样品中,弗兰克氏菌属还具有不同的物种。本研究不仅拓展了西藏沙棘根瘤内生菌多样性的研究方法,还为同一寄主植物中弗兰克氏菌多样性的研究提供了分析思路。     

中国芸香科植物叶分泌囊比较解剖学研究

利用整体透明、石腊和薄切片方法对芸香科22属,40种和2变种植物叶分泌囊的形态结构和分 布进行了比较研究。成熟分泌囊都由鞘细胞和一层上皮细胞围绕圆形腔隙构成,上皮细胞扁平,细胞壁 薄、完整,故分泌囊属裂生方式发生。鞘细胞1～5层,不同种类的层数有变化,个别种缺乏。内层鞘细 胞为扁平的薄壁细胞,外层的细胞壁较厚。分泌囊的形态结构、着生位置和分布密度等在不同属或不同 种间存在一定差异。根据分泌囊在叶中的分布位置和形态结构特点,可将其划分为:叶缘齿缝分泌囊, 叶肉分泌囊和两者混合型。叶肉分泌囊又可分海绵组织分泌囊和栅栏组织分泌囊。在此基础上对该科各类型分泌囊的形态演化关系以及各亚科或各属间的亲缘关系进行了探讨。     

Drought Stress,Permeability to O2 Diffusion,and the Respiratory Kinetics of Soybean Root Nodules

In legume nodules, treatments such as detopping or nitrate fertilization inhibit nodule metabolism and N2 fixation by decreasing the nodule's permeability to O2 diffusion, thereby decreasing the infected cell O2 concentration (Oi) and increasing the degree to which nodule metabolism is limited by O2 availability. In the present study we used nodule oximetry to assess and compare the role of O2 limitation in soybean (Glycine max L. Merr) nodules inhibited by either drought or detopping. Compared to detopping, drought caused only minor decreases in Oi, and when the external O2 concentration was increased to raise Oi, the infected cell respiration rate in the drought-stressed plants was not stimulated as much as it was in the nodules of the detopped plants. Unlike those in detopped plants, nodules exposed to moderate drought stress displayed an O2-sufficient respiration rate that was significantly lower than that in control nodules. Despite possible side effects of oximetry in altering nodule metabolism, these results provided direct evidence that, compared to detopping, O2 limitation plays a minor role in the inhibition of nodule metabolism during drought stress and changes in nodule permeability are the effect, not the cause, of a drought-induced inhibition of nodule metabolism and the O2-suffiecient rate of respiration.     

Is There a Relationship between Infection by Rhizobia and Occurrence of Disomatic Nuclei in Nodules of Alfalfa (Medicago sativa L.)?

Cytological examination of nodules from diploid, tetraploid, and octoploid alfalfa (Medicago sativa L.) plants revealed that the proportion of nodule cells infected by rhizobia was not significantly affected by nuclear ploidy of the host plant. Flow cytometry was used to determine the influence of host plant nuclear ploidy on the nuclear ploidy of infected cells. In nodules from diploid plants, most of the nuclei were tetraploid, whereas in nodules from tetraploid plants, about half of the nodule nuclei were tetraploid and half were octoploid; in octoploid plants, most of the nodule nuclei were octoploid. The occurrence of disomatic nuclei was independent of infection of nodule cells by rhizobia, because diploid plants had mostly disomatic nodule nuclei, and octoploid plants had mostly monosomatic nodule nuclei, whereas all nodules maintained a constant proportion of infected to uninfected cells. These results do not support the earlier hypothesis that infected nodule cells contain disomatic nuclei.     

Nodulation of Crotalaria podocarpa DC. by Methylobacterium nodulans displays very unusual features

Crotalaria are plants of the Fabaceae family whose nodulation characteristics have been little explored despite the recent discovery of their unexpected ability to be efficiently nodulated in symbiosis with bacteria of the genus Methylobacterium. It has been shown that methylotrophy plays a key role in this unusual symbiotic system, as it is expressed within the nodule and as non-methylotroph mutants had a depleting effect on plant growth response. Within the nodule, Methylobacterium is thus able to obtain carbon both from host plant photosynthesis and from methylotrophy. In this context, the aim of the present study was to show the histological and cytological impacts of both symbiotic and methylotrophic metabolism within Crotalaria podocarpa nodules. It was established that if Crotalaria nodules are multilobed, each lobe has the morphology of indeterminate nodules but with a different anatomy; that is, without root hair infection or infection threads. In the fixation zone, bacteroids display a spherical shape and there is no uninfected cell. Crotalaria nodulation by Methylobacterium displayed some very unusual characteristics such as starch storage within bacteroid-filled cells of the fixation zone and also the complete lysis of apical nodular tissues (where bacteria have a free-living shape and express methylotrophy). This lysis could possibly reflect the bacterial degradation of plant wall pectins through bacterial pectin methyl esterases, thus producing methanol as a substrate, allowing bacterial multiplication before release from the nodule.     

Cadmium-stress in white lupin: effects on nodule structure and functioning

Cadmium effects on nodule structure and changes in organic and amino acids, proteins, nutrients and some stress indicators were studied in nodules of white lupin (Lupinus albus L., cv. Multolupa). Plants were grown hydroponically on perlite for 49 d with (18 μM) or without Cd in the nutrient solution. Cadmium-treated plants showed decreases in leaf chlorophyll and shoot sucrose concentrations, but sucrose did not change in nodules. Cadmium application produced alterations in nodule cortex and infected zone structure. Furthermore, Cd supply caused a marked decrease in P, K, leghemoglobin, N–amino compounds, malate, succinate and soluble protein in the nodules. Conversely, the levels of lipid peroxidation and total thiols increased strongly. Results obtained suggest that white lupin nodules are Cd sensitive, in spite of Cd sequestering by cell walls and thiols. The main phytotoxic effects of Cd on nodule structure and function were the occlusion with glycoprotein of intracellular spaces of nodule cortex, alterations in symbiosomes, enrichment in Cd of cell walls and oxidative stress. Glycoprotein accumulation and leghemoglobin depletion may be considered useful indicators of Cd stress in white lupin nodules.     

Biosynthesis of ascorbic acid in legume root nodules

Ascorbic acid (vitamin C) is a major antioxidant and redox buffer, but is also involved in other critical processes of plants. Recently, the hypothesis has been proposed that legume nodules are unable to synthesize ascorbate and have to import it from the shoot or root, thus providing a means by which the plant regulates nodule senescence. The last step of ascorbate biosynthesis in plants is catalyzed by L-galactono-1,4-lactone dehydrogenase (GalLDH). The mRNAs encoding GalLDH and three other enzymes involved in ascorbate biosynthesis are clearly detectable in nodules. Furthermore, an active membrane-bound GalLDH enzyme is present in nodule mitochondria. Biochemical assays on dissected nodules reveal that GalLDH activity and ascorbate are correlated in nodule tissues and predominantly localized in the infected zone, with lower levels of both parameters (relative to the infected tissues) in the apex (87%) and senescent region (43%) of indeterminate nodules and in the peripheral tissues (65%) of determinate nodules. In situ RNA hybridization showed that the GalLDH mRNA is particularly abundant in the infected zone of indeterminate and determinate nodules. Thus, our results refute the hypothesis that ascorbate is not synthesized in nodules and lend support to a previous conclusion that ascorbate in the infected zone is primarily involved in the protection of host cells against peroxide damage. Likewise, the high ascorbate and GalLDH activity levels found in the apex of indeterminate nodules strongly suggest a participation of ascorbate in additional functions during symbiosis, possibly related to cell growth and division and to molecular signaling.     

Isolation and symbiotic characterization of transposon Tn5-induced arginine auxotrophs of Sinorhizobium meliloti

Seventeen arginine auxotrophic mutants of Sinorhizobium meliloti Rmd201 were isolated by random transposon Tn5 mutagenesis using Tn5 delivery vector pGS9. Based on intermediate feeding studies, these mutants were designated as argA/argB/argC/argD/argE (ornithine auxotrophs), argF/argI, argG and argH mutants. The ornithine auxotrophs induced ineffective nodules whereas all other arginine auxotrophs induced fully effective nodules on alfalfa plants. In comparison to the parental strain induced nodule, only a few nodule cells infected with rhizobia were seen in the nitrogen fixation zone of the nodule induced by the ornithine auxotroph. TEM studies showed that the bacteroids in the nitrogen fixation zone of ornithine auxotroph induced nodule were mostly spherical or oval unlike the elongated bacteroids in the nitrogen fixation zone of the parental strain induced nodule. These results indicate that ornithine or an intermediate of ornithine biosynthesis, or a chemical factor derived from one of these compounds is required for the normal development of nitrogen fixation zone and transformation of rhizobial bacteria into bacteroids during symbiosis of S. meliloti with alfalfa plants.     

A novel fix- symbiotic mutant of Lotus japonicus, Ljsym105, shows impaired development and premature deterioration of nodule infected cells and symbiosomes

Nitrogen-fixing symbiosis between legume plants and rhizobia is established through complex interactions between two symbiotic partners. To identify the host legume genes that play crucial roles in such interactions, we isolated a novel Fix- mutant, Ljsym105, from a model legume Lotus japonicus MG-20. The Ljsym105 plants displayed nitrogen-deficiency symptoms after inoculation with Mesorhizobium loti under nitrogen-free conditions, but their growth recovered when supplied with nitrogen-rich nutrients. Ljsym105 was recessive and monogenic and mapped on the upper portion of chromosome 4. The mutant Ljsym105 formed an increased number of small and pale-pink nodules. Nitrogenase (acetylene reduction) activity per nodule fresh weight was low but retained more than 50% of that of the wild-type nodules. Light and electron microscopic observations revealed that the Ljsym105 nodule infected cells were significantly smaller than those of wild-type plants, contained enlarged symbiosomes with multiple bacteroids, and underwent deterioration of the symbiosomes prematurely as well as disintegration of the whole infected cell cytoplasm. These results indicate that the ineffectiveness of the Ljsym105 nodules is primarily due to impaired growth of infected cells accompanied with the premature senescence induced at relatively early stages of nodule development. These symbiotic phenotypes are discussed in respect to possible functions of the LjSym105 locus in the symbiotic interactions required for establishment of the nitrogen-fixing symbiosis.     

Soybean root nodule ultrastructure during dark-induced stress and recovery

Summary Root-nodules of soybean plants dark-stressed for 8 days and then allowed to recover for up to 17 days were examined by transmission electron microscopy. Control nodules possessed all the ultrastructural features characteristic of infected and uninfected nodule cells. Minimal changes in the appearance of host cells and bacteroids occurred during the first four days of dark stress. After 8 days of dark stress, damage was observed in the cellular and organelle membranes; however, very few changes were observed in the bacteroids. Nodule structure continued to degrade during the first two days of recovery after which time nodules either recovered or completely degraded. In the former case, structural integrity returned to all nodule cells. In the latter case all structural integrity of the host cell disappeared; however, bacteroids appeared intact suggesting that they remained viable.Published as Paper no. 7974, Journal Series, Nebraska Agriculture Research Division.