Lysis of bacterioids in the vicinity of the host cell nucleus in an ineffective (fix-) root nodule of soybean (Glycine max)

In nodules of Glycine max cv. Mandarin infected with a nod ⁺fix^- mutant of Rhizobium japonicum (RH 31-Marburg), lysis of bacteroids was observed 20 d after infection, but occurred in the region around the host cell nucleus, where lytic compartments were formed. Bacteroids, and peribacteroid membranes in other parts of the host cell remained stable until senescence (40d after infection). With two other nod⁺ fix^- mutants of R. japonicum either stable bacteroids and peribacteroid membranes were observed throughout the cell (strain 61-A-165) or a rapid degeneration of bacteroids without an apparent lysis (strain USDA 24) occurred. The size distribution of RH 31-Marburg-infected nodules exhibited only two maxima compared with four in wild-type nodules and nodule leghaemoglobin content was found to be reduced to about one half that of the wild type. The RH 31-Marburg-nodule type is discussed in relation to the stability of the bacteroids and the peribacteroid membrane system in soybean.     

A dominant function of CCaMK in intracellular accommodation of bacterial and fungal endosymbionts

In legumes, Ca²⁺/calmodulin‐dependent protein kinase (CCaMK) is a component of the common symbiosis genes that are required for both root nodule (RN) and arbuscular mycorrhiza (AM) symbioses and is thought to be a decoder of Ca²⁺ spiking, one of the earliest cellular responses to microbial signals. A gain‐of‐function mutation of CCaMK has been shown to induce spontaneous nodulation without rhizobia, but the significance of CCaMK activation in bacterial and/or fungal infection processes is not fully understood. Here we show that a gain‐of‐function CCaMK^T265D suppresses loss‐of‐function mutations of common symbiosis genes required for the generation of Ca²⁺ spiking, not only for nodule organogenesis but also for successful infection of rhizobia and AM fungi, demonstrating that the common symbiosis genes upstream of Ca²⁺ spiking are required solely to activate CCaMK. In RN symbiosis, however, CCaMK^T265D induced nodule organogenesis, but not rhizobial infection, on Nod factor receptor (NFRs) mutants. We propose a model of symbiotic signaling in host legume plants, in which CCaMK plays a key role in the coordinated induction of infection thread formation and nodule organogenesis.     

Spontaneous oscillations of extracellular electrical potentials measured on Lepidium sativum L. roots

Abstract. Electrical potential differences (PDs) were measured continuously at six points in line on the surface of primary roots of cress ( Lepidium sativum L.). The root-to-hypocotyl transition was used as the reference. A gradient of extracellular potentials existed along the root axis. The most negative potentials were observed at the root tip. Whenever the growth rate declined to 0·10 mm h⁻¹ or less, the PDs were nearly constant in space and time. Otherwise, the PDs showed different types of changes with time. Roots with growth rates between 0·65 and 1·10 mm h⁻¹ preferably showed PDs characterized by approximately sinoidal oscillations. Maximal amplitudes appeared in the middle of the elongation zone. Roots which were exposed to a N₂-atmosphere exhibited a reversible disappearance of the electrical oscillations and a decrease in the PD-gradient. Similar effects were obtained by the application of the metabolic inhibitor azide (N₃⁻). These results proved that the electrical oscillations are dependent on the aerobic cell metabolism.     

The Impact of Scale,Recycling Boundary,and Type of Waste on Symbiosis and Recycling

Innovative waste recycling through industrial processes such as industrial and urban symbiosis has long been practiced and recently received much attention in the field of industrial ecology, with researchers making efforts to identify key contributing factors to successful industrial symbiosis. By analyzing 88 sample recycling projects in 23 eco‐towns in Japan, this article focuses on the factors of project scale, recycling boundary, and types of waste in relationship to environmental benefits and operational performance. The results showed that larger eco‐towns achieved more savings of virgin materials and higher stability in operation. Large‐scale projects tended to locate closer to the users of recycled products than did small‐scale projects. For treating similar types of waste, projects producing recycled products for special users (e.g., feedstock to a blast furnace for iron production) tended to locate closer to the users than those not producing for special users. The type of waste had a strong effect on the savings of virgin materials and recycling boundaries, while local factors had significant impacts on operational performance. The results also showed that agglomeration did not significantly contribute to the environmental benefits or operational performance of eco‐town projects. Another finding was that national agencies were helpful for facilitating cross‐prefecture transportation and long‐distance transaction of wastes. Implications of the findings are also discussed.     

Rhizobial infection does not require cortical expression of upstream common symbiosis genes responsible for the induction of Ca2+ spiking

For the establishment of an effective root nodule symbiosis, a coordinated regulation of the infection processes between the epidermis and cortex is required. However, it remains unclear whether the symbiotic genes identified so far are involved in epidermal and/or cortical infection, e.g. epidermal and cortical infection thread formation or cortical cell division. To analyze the symbiotic gene requirements of the infection process, we have developed an epidermis‐specific expression system (pEpi expression system) and examined the symbiotic genes NFR1, NFR5, NUP85, NUP133, CASTOR, POLLUX, CCaMK, CYCLOPS, NSP1 and NSP2 for involvement in the infection process in the epidermis and cortex. Our study shows that expression of the upstream common symbiosis genes CASTOR, POLLUX, NUP85 and NUP133 in the epidermis is sufficient to induce formation of infection threads and cortical cell division, leading to the development of fully effective nodules. Our system also shows a requirement of CCaMK, CYCLOPS, NSP1 and NSP2 for the entire nodulation process, and the different contributions of NFR1 and NFR5 to cortical infection thread formation. Based on these analyses using the pEpi expression system, we propose a functional model of symbiotic genes for epidermal and cortical infection.     

丛枝菌根真菌与植物共生对植物水分关系的影响及机理

自1885年Frank首次提到菌根(mykorhiza)概念以来,大量的试验证实了丛枝菌根真菌(AMF)与植物根系之间形成具有一定结构和功能的共生体,促进植物生长并提高干旱耐受能力,在干旱生态系统中发挥重要的作用。该研究多集中在对宿主植物生理生态的影响及其机制方面,然而菌根共生对宿主植物水分吸收和信号产生、传递的影响研究少而分散,缺少系统总结。综述了最近四十多年丛枝菌根真菌与植物共生体对宿主植物干旱适应性影响研究进展,讨论了菌根共生对植物根冠通讯的影响及机理。干旱胁迫下AMF与植物共生,通过影响宿主植物一系列生理生态过程,提高宿主植物横向根压和纵向蒸腾拉力。经典的Ohm吸水模型是该方向最有代表性的研究成果,该模型揭示了菌根共生的根外菌丝具有不同于根细胞的细胞结构和水分运输性能,这为宿主植物提供一种特殊的快速吸水方式,可提高植物对土壤水分的吸收和运输能力。研究表明,AMF会影响宿主植物根冠通讯过程,如诱发信号级联反应,诱导根系尽早感知水分胁迫并产生非水力根源信号,提高宿主对干旱的耐受性。讨论了AMF在根冠通讯分子机制研究方面存在的问题及可能的解决途径,展望了AMF在干旱农业生产中的应用潜力。     

Evolution of root nodule symbiosis: Focusing on the transcriptional regulation from the genomic point of view

Since molecular phylogenetics recognized root nodule symbiosis (RNS) of all lineages as potentially homologous, scientists have tried to understand the “when” and the “how” of RNS evolution. Initial progress was made on understanding the timing of RNS evolution, facilitating our progress on understanding the underlying genomic changes leading to RNS. Here, we will first cover the different hypotheses on the timings of gains/losses of RNS and show how this has helped us understand how RNS has evolved. Finally, we will discuss how our improved understanding of the genetic changes that led to RNS is now helping us refine our understanding on when RNS has evolved.     

The influence of ectomycorrhiza on nitrogen nutrition and growth of Pinus sylvestris seedlings

Ectomycorrhizal seedlings of Scots pine ( Pinus sylvestris L. cv.), inoculated with the fungus Suillus bovinus (L. ex Fr.) O. Kuntze, and non-mycorrhizal controls were grown in growth units with a circulating culture solution. Steady-state nutrition and constant relative growth rates were achieved by means of varied relative nutrient addition rates and free access of nutrients. Typical mycorrhizas always formed within a short period of time after inoculation. The nutrition/growth relationships were in principle similar to previous studies under steady-state conditions: there were close linear relationships between relative addition rate, relative growth rate and internal nitrogen concentration, i.e. an equilibrium established between nutrients added and taken up. This occurred when infected and uninfected seedlings were grown separately. When grown together in the same growth unit, there are indications that the fungus influenced the exudation pattern of the uninfected seedlings. More carbon was thus provided to the unspecified microflora in the cultivation system, and it was able to grow and withhold nitrogen from the seedlings. The mycorrhizal infection did not increase the specific uptake capacity of the roots, and the fungus constituted a sink for carbon. However, the nitrogen productivity (growth rate per unit of nitrogen per unit of time) was similar for mycorrhizal and non-mycorrhizal seedlings, so that there might be mechanisms which compensate for the carbon cost.     

Apoplastic plant subtilases support arbuscular mycorrhiza development in Lotus japonicus

In the arbuscular mycorrhiza (AM) symbiosis, plant roots accommodate Glomeromycota fungi within an intracellular compartment, the arbuscule. At this symbiotic interface, fungal hyphae are surrounded by a plant membrane, which creates an apoplastic compartment, the periarbuscular space (PAS) between fungal and plant cell. Despite the importance of the PAS for symbiotic signal and metabolite exchange, only few of its components have been identified. Here we show that two apoplastic plant proteases of the subtilase family are required for AM development. SbtM1 is the founder member of a family of arbuscular mycorrhiza-induced subtilase genes that occur in at least two clusters in the genome of the legume Lotus japonicus . A detailed expression analysis by RT-PCR revealed that SbtM1 , SbtM3 , SbtM4 and the more distantly related SbtS are all rapidly induced during development of arbuscular mycorrhiza, but only SbtS and SbtM4 are also up-regulated during root nodule symbiosis. Promoter–reporter fusions indicated specific activation in cells that are adjacent to intra-radical fungal hyphae or in cells that harbour them. Venus fluorescent protein was observed in the apoplast and the PAS when expressed from a fusion construct with the SbtM1 signal peptide or the full-length subtilase. Suppression of SbtM1 or SbtM3 by RNAi caused a decrease in intra-radical hyphae and arbuscule colonization, but had no effect on nodule formation. Our data indicate a role for these subtilases during the fungal infection process in particular arbuscule development.     

Mycobiont-cyanobiont interactions during dark nitrogen fixation by the lichen Peltigera aphthosa

Acetylene reduction (nitrogenase activity) by excised cephalodia of Peltigera aphthosa Willd. slowly declined on transfer of the cephalodia from light to darkness. The decline was more rapid in the absence of CO₂ or when phosphoenolpyruvate carboxylase activity was inhibited by adding maleic acid or malonic acid. When glutamine synthetase (GS) activity was totally inhibited by adding l -methionine- dl -sulphoximine (MSX) the decline in nitrogenase activity in the absence of CO₂ still occurred. However, this loss of activity did not occur when the mycobiont was disrupted using digitonin (0.01 % w/v) and the fixed NH₄⁺ was released into the medium. The data suggest that dark CO₂ fixation by the fungus supplies carbon skeletons which remove newly fixed NH₄⁺ produced by the cyanobacterium. When such carbon skeletons are not available MH₄⁺ accumulates and inhibits nitrogenase activity even in the absence of GS activity. It is probable that NH₄⁺ and a product of GS exert independent inhibitory effects on nitrogenase activity.     

Solar UV-B radiation affects below-ground parameters in a fen ecosystem in Tierra del Fuego, Argentina: implications of stratospheric ozone depletion

Stratospheric ozone depletion caused by the release of chlorofluorocarbons is most pronounced at high latitudes, especially in the Southern Hemisphere (including the so‐called ‘ozone hole’). The consequent increase in solar ultraviolet‐B radiation (UV‐B, 280–315 nm) reaching the earth's surface may cause a variety of alterations in terrestrial ecosystems. Most effects might be expected to occur above‐ground since sunlight does not penetrate effectively below‐ground. Here, we demonstrate that solar UV‐B radiation in a fen in Tierra del Fuego (Argentina), where the ozone hole passes overhead several times during the Austral spring, is causing large changes of below‐ground processes of this ecosystem. During the third and fourth year of a manipulative field experiment, we investigated root systems in these plots and found that when the ambient solar UV‐B radiation was substantially reduced, there was a 30% increase in summer root length production and as much as a threefold decrease in already low symbiotic mycorrhizal colonization frequency of the roots compared with plots receiving near‐ambient solar UV‐B. There was also an apparent shift toward older age classes of roots under reduced solar UV‐B. Such large changes in root system behaviour may have decided effects on competition and other ecological interactions in this ecosystem.     

Inoculation of cranberry (Vaccinium macrocarpon) with the ericoid mycorrhizal fungus Rhizoscyphus ericae increases nitrate influx

Despite the ubiquitous presence of ericoid mycorrhizal (ERM) fungi in cranberry (Vaccinium macrocarpon), no prior studies have examined the effect of ERM colonization on NO(3)(-) influx kinetics. Here, (15)NO(3)(-) influx was measured in nonmycorrhizal and mycorrhizal cranberry in hydroponics. Mycorrhizal cranberry were inoculated with the ERM fungus Rhizoscyphus (syn. Hymenoscyphus) ericae. (15)NO(3)(-) influx by R. ericae in solution culture was also measured. Rhizoscyphus ericae NO(3)(-) influx kinetics were linear when mycelium was exposed for 24 h to 3.8 mm NH(4)(+), and saturable when pretreated with 3.8 mm NO(3)(-), 50 microm NO(3)(-), or 50 microm NH(4)(+). Both low-N pretreatments induced greater NO(3)(-) influx than either of the high-N pretreatments. Nonmycorrhizal cranberry exhibited linear NO(3)(-) influx kinetics. By contrast, mycorrhizal cranberry had saturable NO(3)(-) influx kinetics, with c. eightfold greater NO(3)(-) influx than nonmycorrhizal cranberry at NO(3)(-) concentrations from 20 microm to 2 mm. There was no influence of pretreatments on cranberry NO(3)(-) influx kinetics, regardless of mycorrhizal status. Inoculation with R. ericae increased the capacity of cranberry to utilize NO(3)(-)-N. This finding is significant both for understanding the potential nutrient niche breadth of cranberry and for management of cultivated cranberry when irrigation water sources contain nitrate.     

环境因子对豆科共生固氮影响的研究进展

环境因子的限制一直是豆科植物一根瘤菌共生固氮体系没有在农业生产中充分发挥作用的重要原因之一。目前,研究涉及的环境因子主要行水分、矿质营养元素、温度、重金属、钠盐、CO2、土壤类型以及pH等。水分胁迫会导致豆科植物根瘤减少和固氮效率低下;矿质元素方面,除氮磷钾外,微量死素对固氮影响也很明显;不适的温度会对豆科植物的结瘤固氮产生一定的限制;重金属能从不同方面直接和间接地影响共生同氮,寻找适合作尾矿先锋植物的豆科植物是当前的一个研究热点。本文除详细阐述了这方面开展的研究以外,还浅析了这方而研究目前国内外存在的一些主要问题和发展趋势。     

Lotus japonicus symRK-14 uncouples the cortical and epidermal symbiotic program

SYMRK is a leucine-rich-repeat (LRR)-receptor kinase that mediates intracellular symbioses of legumes with rhizobia and arbuscular mycorrhizal fungi. It participates in signalling events that lead to epidermal calcium spiking, an early cellular response that is typically considered as central for intracellular accommodation and nodule organogenesis. Here, we describe the Lotus japonicus symRK-14 mutation that alters a conserved GDPC amino-acid sequence in the SYMRK extracellular domain. Normal infection of the epidermis by fungal or bacterial symbionts was aborted in symRK-14. Likewise, epidermal responses of symRK-14 to bacterial signalling, including calcium spiking, NIN gene expression and infection thread formation, were significantly reduced. In contrast, no major negative effects on the formation of nodule primordia and cortical infection were detected. Cumulatively, our data show that the symRK-14 mutation uncouples the epidermal and cortical symbiotic program, while indicating that the SYMRK extracellular domain participates in transduction of non-equivalent signalling events. The GDPC sequence was found to be highly conserved in LRR-receptor kinases in legumes and non-legumes, including the evolutionarily distant bryophytes. Conservation of the GDPC sequence in nearly one-fourth of LRR-receptor-like kinases in the genome of Arabidopsis thaliana suggests, however, that this sequence might also play an important non-symbiotic function in this plant.