Novel alphaproteobacterial root nodule symbiont associated with Lupinus texensis

Phylogenetic analysis of rRNA gene, recA, nodA, nifD, and nifH sequences suggested that nitrogen-fixing symbionts from two populations of Lupinus texensis acquired the capacity for nodule symbiosis separately from other rhizobia in the alphaproteobacteria. Their closest 16S rRNA relatives were the nonsymbiotic taxa Chelatococcus, Bosea, and Balneomonas.     

Flavonoids of Lupinus texensis and L. subcarnosus

Lupinus texensis and L.- subcarnosus exhibit very similar flavonoid profiles based on O-glycosylflavones, Cglycosylflavones, an isoflavone and a flavanone. Differences between the two species amount to the presence of a diglucoside of diosmetin in L. texensis and comparatively higher concentrations of C-glycosylflavones in L. subcarnosus. The two species have a higher degree of flavonoid pigment similarity than any other pair of lupines examined in our studies. The similar profiles support the view that these two endemic Texan species are closely related.     

Root Nodule Symbiosis II

Nodule-roots of Myrica cerifera (Southern Wax Myrtle) and Casuarina cunning hamiana (Australian Pine) have a negative geotropic curvature. Studies of their endogenotts auxin content revealed a pattern of correlation: the absence of detectable auxin when the geotropisni was negative. Non-nodulated roots of Myrica exhibited a normal positive geotropic curvature and possessed an auxin content within an anticipated range (10 mg/kg). Root nodules of Alnus species, whose roots exhibit a positive geotropic curvature, contained measurable endogenous auxin (20 mg IAA/kg). The presence of an indoleaectic acid oxidase system in Myrica and Casuarina root nodules has heen described and correlations are drawn between non-detectable endogenous auxin concentrations and high enzymatic activities. It is suggested that the negative geotropic curvature of the nodule-roots of Myrica and Casuarina is due to the presence of a sub-optimal concentration of auxin which in turn results from the activity of an indoleacetic aeid destroying system.     

Genetic load,nutrient limitation,and seed production in Lupinus texensis (Fabaceae)

The interaction of mating system and nutrient limitation in determining seed production was investigated in the annual, self-compatible plant Lupinus texensis (Fabaceae). Abortion of developing seeds is a major factor limiting seed production in natural populations (17-28%). Selfing rates are generally low (0.02-0.21), suggesting that deleterious recessive genes may be maintained at significant levels in natural populations. The average inbreeding depression associated with seed development is δ = 0.24. Nutrient limitation reduced seed output across experimental treatments by a factor of 0.22 through decreased production of inflorescences, flowers, and ovules, and by a factor of 0.29 through increased abortion of fruits and of seeds within fruits. Competition for resources among fruits increased the frequency of seed abortion. Moreover, a greater proportion of selfed seeds were aborted as the overall abortion rate increased. Estimates of genetic load may therefore only be appropriate if undertaken in the field, and inbreeding depression may vary from year to year simply due to changes in environmental conditions rather than to underlying genetic changes in populations. The existence of inbreeding depression and the high frequency of abortions suggest that selective abortion favoring outcrossed progeny occurs in natural populations of L. texensis.     

Characterization of a Culturable Alphaproteobacterial Symbiont Common to Many Marine Sponges and Evidence for Vertical Transmission via Sponge Larvae

A closely related group of alphaproteobacteria were found to be present in seven genera of marine sponges from several locations and were shown to be transferred between sponge generations through the larvae in one of these sponges. Isolates of the alphaproteobacterium were cultured from the sponges Axinella corrugata, Mycale laxissima, Monanchora unguifera, and Niphates digitalis from Key Largo, Florida; Didiscus oxeata and Monanchora unguifera from Discovery Bay, Jamaica; an Acanthostronglyophora sp. from Manado, Indonesia; and Microciona prolifera from the Cheasapeake Bay in Maryland. Isolates were very similar to each other on the basis of 16S rRNA gene sequence (>99% identity) and are closely related to Pseudovibrio denitrificans. The bacterium was never isolated from surrounding water samples and was cultured from larvae of M. laxissima, indicating that it is a vertically transmitted symbiont in this sponge. Denaturing gradient gel electrophoresis, 16S rRNA gene clone library analysis, and fluorescent in situ hybridization with probes specific to the alphaproteobacterium confirmed the presence of this bacterium in the M. laxissima larvae. The alphaproteobacterium was densely associated with the larvae rather than being evenly distributed throughout the mesohyl. This is the first report of the successful culture of a bacterial symbiont of a sponge that is transferred through the gametes.     

Alfalfa Root Nodule Carbon Dioxide Fixation : II. Partial Purification and Characterization of Root Nodule Phosphoenolpyruvate Carboxylase

A nonphotosynthetic phosphoenolpyruvate carboxylase (EC 4.1.1.31) was partially purified from the cytosol of root nodules of alfalfa. The enzyme was purified 86-fold by ammonium sulfate fractionation, DEAE-cellulose, hydroxylapatite chromatography, and reactive agarose with a final yield of 32%. The enzyme exhibited a pH optimum of 7.5 with apparent K_m values for phosphoenolpyruvate and magnesium of 210 and 100 micromolar, respectively. Two isozymes were resolved by nondenaturing polyacrylamide disc gel electrophoresis. Subsequent electrophoresis of these isozymes in a second dimension by sodium dodecyl sulfate slab gel electrophoresis yielded identical protein patterns for the isozymes with one major protein band at molecular weight 97,000. Malate and AMP were slightly inhibitory (about 20%) to the partially purified enzyme. Phosphoenolpyruvate carboxylase comprised approximately 1 to 2% of the total soluble protein in actively N₂-fixing alfalfa nodules.     

Diurnal Functioning of the Legume Root Nodule

Diurnal changes in plant and nodule performance were studiedin 28–9 d plants of Pisum sativum L. in two environments,both with a 12 h (27 000 lx):12 h::light:dark cycle, but one(A) with a fluctuating temperature-humidity regime (photoperiod18 ?C, 60 per cent relative humidity:night 12 ?C, 85 per cent),the other (B) with constant temperature (18 ?C) and humidity(75 per cent). Fixation rate (C₂H₂ reduction), respiratory output of the nodulatedroot, and nodule sugar level increased throughout the photoperiod,whereas nodule soluble nitrogen level declined steadily. Reversalof these trends in the night period led, at its end, to minimain fixation rate, sugar level and respiration, but a maximumin soluble nitrogen. The A environment produced the greaterday:night fluctuations in transpiration and nodule soluble nitrogen,but B, with its higher night temperature, induced the more pronounceddecrease in fixation at night. Slightly less nitrogen was fixed during the photoperiod thanduring the night in the A environment, yet since some fixationproducts were retained in the nodules at night and not releaseduntil the next photoperiod, the day: night difference in nitrogenexport from nodules was 1.8:1. The photoperiod of A was alsoa time of higher nodule respiration and replenishment of nodulesugar and starch, so that the nodules' requirement for translocatedcarbohydrate was more than twice that at night. Humidity decrease in the photoperiod (of A) elicited higherrates of transpiration and a more rapid than normal emptyingof soluble nitrogen from the nodules: elevation of humidityhad the opposite effects. Shoot removal (A-grown plants) causednodule sugar levels to fall rapidly below those normally encounteredin intact plants.     

Glucose and Glycogen Metabolism in Brugia malayi Is Associated with Wolbachia Symbiont Fitness

Wolbachia are endosymbiotic bacteria found in the majority of arthropods and filarial nematodes of medical and veterinary importance. They have evolved a wide range of symbiotic associations. In filarial nematodes that cause human lymphatic filariasis (Wuchereria bancrofti, Brugia malayi) or onchocerciasis (Onchocerca volvulus), Wolbachia are important for parasite development, reproduction and survival. The symbiotic bacteria rely in part on nutrients and energy sources provided by the host. Genomic analyses suggest that the strain of Wolbachia found in B. malayi (wBm) lacks the genes for two glycolytic enzymes—6-phosphofructokinase and pyruvate kinase—and is thus potentially unable to convert glucose into pyruvate, an important substrate for energy generation. The Wolbachia surface protein, wBm00432, is complexed to six B. malayi glycolytic enzymes, including aldolase. In this study we characterized two B. malayi aldolase isozymes and found that their expression is dependent on Wolbachia fitness and number. We confirmed by immuno-transmission electron microscopy that aldolase is associated with the Wolbachia surface. RNAi experiments suggested that aldolase-2 plays a significant role in both Wolbachia survival and embryogenesis in B. malayi. Treatment with doxycycline reduced Wolbachia fitness and increased the amount of both glucose and glycogen detected in the filarial parasite, indicating that glucose metabolism and glycogen storage in B. malayi are associated with Wolbachia fitness. This metabolic co-dependency between Wolbachia and its filarial nematode indicates that glycolysis could be a shared metabolic pathway between the bacteria and B. malayi, and thus a potential new target for anti-filarial therapy.     

Some Aspects of Translocation in Root Nodule Plants

By the use of ¹⁶N it is shown that the removal, over a shortzone at the base of the shoot of a typical root nodule plant(alder), of the tissues external to the xylem does not interferewith the upward movement of fixed nitrogen from the nodulesinto the shoot. It is concluded that the fixed nitrogen, whichis probably in organic form, can be translocated in the xylemin the transpiration stream, and that this is most likely itsnormal route. It is also shown that in unringed plants substantialenrichment in fixed ¹⁶N is detectable in the shoot within 6hours from the commencement of the exposure of the nodules toexcess free ¹⁶N. Structural and experimental evidence showsthat the alder nodule is not a water-absorbing organ, and themechanism of transfer of fixed nitrogen from the nodule intothe transpiration stream is not obvious.     

The Cobalt Requirement of Non-legume Root Nodule Plants

As already shown for Alnus glutinosa, cobalt is found to beessential for the proper growth of nodulated plants of Casuarinacunninghamiana and Myrica gale in a nitrogen-free rooting medium.If cobalt is not supplied, the plants develop symptoms of nitrogendeficiency; under the conditions of the experiments such symptomsbecame pronounced during the second season of growth of theseperennial plants. No cobalt requirement could be detected innon-nodulated plants of Alnus and Myrica supplied with nitrateor ammonium-nitrogen, and this suggests that in nodulated plantsthe need for cobalt is confined to the nodules. Vitamin B₁₂analogues are shown to be present in the nodules in relativelylarge amounts when cobalt is supplied, their formation beingattributed to the endophytes, which may therefore require cobaltfor their growth. The great reduction in fixation of atmosphericnitrogen in cobalt-deficient nodules may be due to a retardedgrowth of the endophyte, though this is not the only possibility.The cobalt relation of these non-legumes appears to be basicallysimilar to that of legumes.     

Callose-Regulated Symplastic Communication Coordinates Symbiotic Root Nodule Development

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Alfalfa Root Nodule Carbon Dioxide Fixation : III. Immunological Studies of Nodule Phosphoenolpyruvate Carboxylase

Antiserum was prepared in rabbits against purified alfalfa (Medicago sativa L.) nodule phosphoenolpyruvate carboxylase (PEPC). Immunotitration assays revealed that the antiserum recognized the enzyme from alfalfa nodules, uninoculated alfalfa roots, and from soybean nodules. Tandem-crossed immunoelectrophoresis showed that the PEPC protein from alfalfa roots and nodules was immunologically indistinguishable. The 101 kilodalton polypeptide subunit of alfalfa nodule PEPC was identified on Western blots. The PEPC polypeptide was detected in low quantities in young alfalfa roots and nodules but was present at increased levels in mature nodules. Senescent nodules appeared to contain a reduced amount of the PEPC polypeptide. PEPC was also detected by western blot in some plant- and bacterially-conditioned ineffective alfalfa nodules but was not detected in bacteroids isolated from effective nodules. Alfalfa nodule PEPC is constitutively expressed in low levels in roots. In nodules, expression of PEPC polypeptide increases several-fold, resulting in increased PEPC activity. Antiserum prepared against the C₄ PEPC from maize leaves recognized the PEPC enzyme in all legume nodules and roots tested, while the antiserum prepared against alfalfa nodule PEPC also recognized the leaf PEPC of several C₄ plant species. Neither antiserum reacted strongly with any C₃ leaf proteins. The molecular weight of the PEPC polypeptide from C₄ leaves and legume nodules appears to be similar.     

Protein profile of Lupinus texensis phloem sap exudates: Searching for Fe‐ and Zn‐containing proteins

The aim of this study was to obtain a comprehensive overview of the phloem sap protein profile of Lupinus texensis, with a special focus on proteins binding Fe and Zn. L. texensis was chosen as model plant given the simplicity to obtain exudates from sieve elements. Protein profiling by 2DE revealed 249 spots, and 54 of them were unambiguously identified by MALDI‐MS and ESI‐MS/MS. The largest number of identified protein species belongs to protein modification/turnover and general metabolism (19–21%), followed by redox homeostasis (9%) and defense and cell structural components (7%). This protein profile is similar to that reported in other plant species, suggesting that the phloem sap proteome is quite conserved. Staining of 2DE gels for Fe‐containing proteins and affinity chromatography experiments revealed the presence of two low molecular weight Fe‐binding proteins in phloem sap: a metallothionein‐like protein type 2B identified in the Fe‐affinity chromatography, and a second protein identified with both Fe staining methods. This protein species had a molecular weight of 13.5 kDa, a pI of 5.6 and 51% homology to a phloem‐specific protein from Medicago truncatula. Zinc affinity chromatography revealed four Zn‐binding proteins in phloem sap, one belonging to the dehydrin family and three Zn finger proteins.     

Detection of S-Nitrosothiol and Nitrosylated Proteins in Arachis hypogaea Functional Nodule: Response of the Nitrogen Fixing Symbiont

To detect the presence of NO, ROS and RNS in nodules of crack entry legumes, we used Arachis hypogaea functional nodule. The response of two cognate partner rhizobia was compared towards NO and GSNO using S. meliloti and Bradyrhizobium sp NC921001. ROS, NO, nitrosothiol and bacteroids were detected by fluorescence microscopy. Redox enzymes and thiol pools were detected biochemically. Nitrosothiols were found to be present but ROS and NO were absent in A. hypogaea nodule. A number of S-nitrosylated proteins were also detected. The total thiol pool and most of the redox enzymes were low in nodule cytosolic extract but these were found to be high in the partner microorganisms indicating partner rhizobia could protect the nodule environment against the nitrosothiols. Both S. meliloti and Bradyrhizobium sp NC921001 were found to contain GSNO reductase. Interestingly, there was a marked difference in growth pattern between S. meliloti and Bradyrhizobium sp in presence of sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO). Bradyrhizobium sp was found to be much more tolerant to NO donor compounds than the S. meliloti. In contrast, S. meliloti showed resistance to GSNO but was sensitive to SNP. Together our data indicate that nodule environment of crack entry legumes is different than the nodules of infection mode entry in terms of NO, ROS and RNS. Based on our biochemical characterization, we propose that exchange of redox molecules and reactive chemical species is possible between the bacteroid and nodule compartment.     

Physiological Role of Leghaemoglobin Heterogeneity in Pea Root Nodule Development

Disk-gel-electrophoresis of the leghaemoglobin isolated frompea root nodules revealed two major (Lb I and Lb IV) and threeminor components. The ratio of the two major components (LbI/Lb IV) decreased with increasing age. This ratio was higherin the distal than in proximal region when nodules were cutinto distal and proximal sections. In young nodules, the incorporationof radioactive leucine into Lb I was much higher than into LbIV. In older nodules, the radioactivities were much higher inLb IV than in Lb I. These data suggest that the change in thisratio is due mainly to differences in the rates of biosynthesis. Two major components (Lb I and Lb IV) which were isolated separatelyhad different O₂-binding affinities. The O₂-binding affinityof the component synthesized mainly in older nodules (Lb IV)was higher than that of the component synthesized mainly inyoung nodules (Lb I). These results indicate that changes inthe relative contents of leghaemoglobin during nodule developmentcontribute to more effective nitrogen fixation which is mediatedby changes in the capacities of oxygen transport. (Received July 7, 1981; Accepted November 2, 1981)