Carbon cost of nitrogenase activity in Frankia–Alnus incana root nodules

The carbon cost of nitrogenase activity was investigated to determine symbiotic efficiency of the actinorhizal root nodule symbiosis between the woody perennial Alnus incana and the soil bacterium Frankia. Respiration (CO₂ production) and nitrogenase activity (H₂ production) by intact nodulated root systems were continuously recorded in short-term assays in an open-flow gas exchange system. The assays were conducted in N₂:O₂, thus under N₂-fixing conditions, in all experiments except for one. This avoided the declines in nitrogenase activity and respiration due to N₂ deprivation that occur in acetylene reduction assays and during extended Ar:O₂ exposures in H₂ assays. Two approaches were used: (i) direct estimation of root and nodule respiration by removing nodules, and (ii) decreasing the partial pressure of O₂ from 21 to 15% to use the strong relationship between respiration and nitrogenase activity to calculate CO₂/H₂. The electron allocation of nitrogenase was determined to be 0.6 and used to convert the results into moles of CO₂ produced per 2e^– transferred by nitrogenase to reduction of N₂. The results ranged from 2.6 to 3.4mol CO₂ produced per 2e^–. Carbon cost expressed as gC produced per gN reduced ranged from 4.5 to 5.8. The result for this actinorhizal tree symbiosis is in the low range of estimates for N₂-fixing actinorhizal symbioses and crop legumes. Methodology and comparisons of root nodule physiology among actinorhizal and legume plants are discussed.     

Survival ofFrankia strains introduced into soil

Factors affecting the establishment of Alnus/Frankia symbioses were studied partly by following the survival ofFrankia strains exposed to different soil conditions, and partly by investigating the effect of pH on nodulation. TwoFrankia strains were used, both of the Sp⁻ type (sporangia not formed in nodules). One of the strains sporulated heavily, while the other formed mainly hyphae. The strains originated fromAlnus incana root nodules growing in soils of pH 3.5 and 5.0. The optimum pH for their growth in pure culture was found to be 6.7 and 6.2, respectively. The strains were introduced into twoFrankia-free soils, peat and fine sand. Their survival, measured as the persistance of nodulation capacity using the plant infection technique, was followed for 14 months. The survival curves of the strains were similar despite the morphological differences between the strains in pure culture. The nodulation capacities declined over time both at 14 and 22°C. Survival was better in soils limed to a pH above 6 than in soils at their original pH (peat 2.9, fine sand 4.2). The effect of pH on nodule formation in Alnus seedlings by theFrankia strains was studied in liquid culture. The number of nodules increased linearly within the pH range studied (3.5–5.8). No nodules were formed at pH 3.5.     

An assessment of soil enrichment by actinorhizal N2 fixation using δ15N values in a chronosequence of deglaciation at Glacier Bay,Alaska

The extent of transfer of fixed N between N₂-fixing and non-N₂-fixing plant species is largely unknown in successional studies. In order to redress this deficiency at a locale intensively studied ecologically, leaf tissue samples were collected from actinorhizal N₂-fixing (Alnus, Shepherdia, and Dryas) and two non-N₂-fixing (Salix) woody species within research plots located along a chronosequence of deglaciated fjord in Glacier Bay National Park, Alaska. The tissue samples were analyzed for ¹⁵N content, and the resulting data analyzed for trends in plant tissue N. Among the non-N₂-fixing Salix species, ¹⁵N values increased from the most recently deglaciated sites to converge with the temporally more-stable values for the symbiotic N₂-fixing species on sites at about 40 years after deglaciation. The lower ¹⁵N values of sequestered N in plant tissues suggested that N derived from N₂-fixing plants accounts for the major portion of N in associated plants up to 40 years after deglaciation. The ¹⁵N isotopic data also suggested that Shepherdia canadensis depends least on soil N, D. drummondii the most, and A. viridis ssp. sinuata somewhere between those two species. The presence of a sere dominated by dense thickets of A. viridis ssp. sinuata at the convergence of ¹⁵N values for the N₂-fixing and non-N₂-fixing species indicated that this species is most responsible for accumulation of fixed N in soil at Glacier Bay. This paper is dedicated to the memory of Steven J. Kohls who died prior to publication of this research.     

The evolution of nodulation

In this review we will first describe the different steps leading to nodule formation, and these will be compared with processes of non-symbiotic plant development and growth. In general, aspects of both actinorhizal as well as rhizobial symbiosis are described, but in several cases, the emphasis will be on the Rhizobium-legume symbiosis because more knowledge of this system is available. Subsequently, the phylogeny of nodulating plants is described and a comparison is made between several aspects of legume and actinorhizal nodulation. At the end of this paper the relationship between nodule symbiosis and endomycorrhizal symbiosis is described, and it is discussed to what extent the development of root nodules involves unique properties, or whether processes and genes have been recruited from common plant development and the endomycorrhizal symbiosis.     

Time course of nodule development in the Discaria trinervis (Rhamnaceae) –Frankia symbiosis

The time course of initiation and development of root nodules was investigated in the South American actinorhizal shrub Discaria trinervis (Rhamnaceae). A local strain of Frankia (BCU110501) which was isolated from D. trinervis nodules, was used as inoculum. Inoculated seedlings were periodically studied under the light microscope after clearing with aqueous NaClO. In parallel, semithin and ultrathin sections were analysed by light and electron microscopy. Infection by Frankia BCU110501 involved intercellular penetration among epidermal and cortical root cells. Nodule primordia were detected from 6 d after inoculation, while bacteria were progressing through intercellular spaces of the outer layers of cortical cells. Invasion of host cells by the symbiont occurred 7–9 d after inoculation, and hypertrophy of the primordium cells was associated with Frankia penetration. Root hairs were not deformed during the early events of nodule formation. From 13 to 16 d after inoculation, the proximal cellular zone of the primordia behaved differently from the other tissues after NaClO treatment and remained darkly pigmented. At the same time, differentiation of Frankia vesicles started to occur inside already infected cells. By 16 d after inoculation, spherical vesicles of BCU110501 were homogeneously distributed in the host cells. These vesicles were septate and surrounded by void space. Frankia spores or sporangia were not observed in the nodule tissue. This study has clarified the mode of Frankia penetration in D. trinervis , one of the Rhamnaceae which also includes Ceanothus . The events involved in infection, nodule induction, host-cell infection and vesicle differentiation have been characterized and identified as time-segregated developmental processes in the ontogeny of D. trinervis root nodules.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration of <Emphasis Type="Italic">Alnus acuminata</Emphasis> H.B.K. ssp. <Emphasis Type="Italic">acuminata</Emphasis> and its root nodulation by <Emphasis Type="Italic">Frankia</Emphasis>

A procedure for in vitro plant regeneration of Alnus acuminata from epicotyls with cotyledonary buds was developed using different media formulations with different growth regulators and carbon sources. The development of multiple buds on explants at the initiation step was obtained with MS at 1/2 strength with either 1 or 2M of BAP but not without it. Multiplication gave up to 15 elongating shoots by explant, the best medium being MS supplemented with vitamins from B5 medium, 1M of BAP and 87mM sucrose. Rooting of about 88% occurred in the medium MS with 83 mM sucrose and 1M IBA. Alnus acuminata did not developed well on WPM. Roots of in vitro propagated plants were nodulated by Alnus-infective Frankia. The root nodules show a typical alder root nodule anatomy and differentiation pattern and effectively fixed nitrogen. Rhamnaceae-infective Frankia did not nodulate in vitro cultivated Alnus acuminata suggesting that symbiotic recognition was not altered by in vitro regeneration of the plant.     

Feedback regulation of nodule formation in Hippophaë rhamnoides

Seedlings of Hippophaë rhamnoides possessing two equally infectible root systems (split roots) were used in conjunction with specific Frankia strains to investigate plant control over nodulation. When a wild-type Frankia strain was inoculated onto both root systems simultaneously or 1, 2, 4, or 8 weeks apart, an inhibitory response occurred which retarded nodulation on the root exposed to the delayed inoculum. Similar suppressive responses were also observed when two different wild-type Frankia inocula were applied onto opposite sides of a split-root system at different times. The depressed response shown by the delayed inoculum was more pronounced as the delay period was increased. The roots exposed to the delayed inoculum displayed a complete lack of nodulation when the delay was 4 or 8 weeks. The nodulation response on the root inoculated first depended on subsequent inoculation of the second root system of the plant, so that maximum nodulation of the first root was observed when the second root was unnodulated. These results provide evidence that sea buckthorn has an active, systemic mechanism for feedback control of nodulation that suppresses further nodule formation and prevents excessive nodulation. The significance of these results to the understanding of nodule ontogeny is discussed.     

The physiology of spore-negative and spore-positive nodules ofMyrica gale

The physiology of spore-negative and spore-positive root nodules was investigated inMyrica gale L. grown in water culture in a growth chamber. Spore(–) nodules were induced withFrankia cultures and spore(+) nodules with crushed nodules. Gas exchange was measured in a flow-through system.The time course of acetylene reduction following addition of acetylene was essentially the same in both spore(–) and spore(+) nodules with a stable maximum between 2 and 4 minutes followed by a steep decline to a minimum (37% of the maximum) between 9 and 30 minutes depending on the plant. The minimum was followed by a partial recovery. Nodule CO₂ evolution showed a similar pattern but the minimum rate (83% of the maximum) was not nearly as low.Plants nodulated with one spore(–) and one spore(+) strain were compared at 6, 8 and 10 weeks after inoculation. At 6 weeks the spore(–) plants had 52% greater specific nitrogenase activity and 46% more biomass than the spore(+) plants. At 8 and 10 weeks, however, the differences between plants with spore(–) and spore(+) nodules became smaller.Plants nodulated with 4 spore(–) and 5 spore(+) strains were compared at 8 weeks after inoculation. Collectively the spore(–) plants exhibited a 32% greater specific nitrogenase activity, a 15% lower energy cost of nitrogenase activity (CO₂/C₂H₄), and invested 31% less biomass in nodules than the spore(+) plants. The spore(–) plants also produced 16% more biomass indicating that spore(–) strains are generally more desirable than spore(+) strains. However, two spore(+) strains were as effective as the spore(–) strains.