Mesorhizobium loti and Rhizobium etli are microsymbionts of the Lotus and Phaseolus spp., respectively, and secrete essentially the same Nod factors. Lotus japonicus efficiently formed root nodules with R. etli CE3, irrespective of the presence or absence of a flavonoid-independent transcription activator nodD gene. On a nitrogen-free medium, however, the host plant inoculated with R. etli showed a severe nitrogen deficiency symptom. Initially, the nodules formed with R. etli were pale pink and leghemoglobin mRNA was detectable at significant levels. Nevertheless, the nodules became greenish with time. Acetylene-reduction activity of nodules formed with R. etli was comparable with that formed by M. loti 3 weeks postinoculation, but thereafter it decreased rapidly. The nodules formed with R. etli contained much more starch granules than those formed with M. loti. R. etli developed into bacteroids in the L. japonicus nodules, although the density of bacteroids in the infected cells was lower than that in the nodules formed with M. loti. The nodules formed with R. etli were of the early senescence type, in that membrane structures were drastically disintegrated in the infected cells of the greenish nodules. Thus, L. japonicus started and then ceased a symbiotic relationship with R. etli at the final stage.

Attempts were made to clarify the precise location and possible site of production of the K-glucan pullulan in different morphological forms of the fungus Aureobasidium pullulans. Gold-conjugated pullulanase was used as the specific probe for this purpose. No cell wall pullulan-like material was detected by transmission electron microscopy (TEM) in any morphological form of this fungus, although intracellular electron transparent material bound this probe. When silver enhancement of this gold-conjugated pullulanase probe was used, the data strongly suggested that only swollen cells and chlamydospores, and neither hyphae nor unicellular blastospores, often held responsible for pullulan formation, appeared to produce pullulan-like material. ß

The mitochondrial phosphate transporter of Saccharomyces cerevisiae, encoded by MIR1 (YJR077C) gene, shows divergence among the transporters in various eukaryotes. We have characterized another gene, YER053C, that appeared to encode an orthologous mitochondrial phosphate transporter of yeast. The predicted amino acid sequence of the YER053C protein is much more similar to that of mitochondrial phosphate transporters of other species than that of MIR1. RNA gel blot analysis indicated that, like the MIR1 promoter, the YER053C promoter is functional and that its activity varies according to aeration. An MIR1 gene null mutant did not grow on glycerol medium, whereas a YER053C null mutant grew well on the medium, suggesting that the YER053C gene is not essential for the mitochondrial function. YER053C also did not support the growth of the MIR1 null mutant on glycerol. The MIR1 and YER053C proteins were expressed in Escherichia coli and then reconstituted into liposomes. Unlike the proteoliposomes of MIR1, those of YER053C did not exhibit significant phosphate transport activity. Unexpectedly, it was shown that YER053C is localized in vacuoles, not mitochondria, by immunological electron microscopy. These results suggest that, during evolution, yeast lost the function and/or mitochondrial targeting of YER053C and then recruited an atypical MIR1 as the only transporter.

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The peanut plant (Arachis hypogaea L.), unlike other known legumes, can sustain nitrogen fixation when prolonged periods of darkness or detopping curtail the supply of photosynthate to the nodule. This ability to withstand photosynthate stress is attributed to the presence of lipid bodies in infected nodule cells. In both dark-treated and detopped plants, the lipid bodies show a gradual decrease in numbers, suggesting their utilization as a source of energy and carbon for nitrogen fixation. Lipolytic activity can be localized in the lipid bodies, and the existence of ,Boxidation pathway and glyoxylate cycle is shown by the release of 14C02 from 14C lineoleoyl coenzyme A by the nodule homogenate.Symbiotic association of leguminous plants and the soil microbe Rhizobium results in the formation of root nodules where rhizobia fix atmospheric nitrogen. Carbon compounds derived from the host plant are essential for the reduction of N2 and for the assimilation of the fixed nitrogenous products (7). In symbiotic N2 fixing systems, blockage in the import of photosynthate availability drastically affects N2 fixation (5, 6, 12. 14, 23, 29) as measured by ARA2 (10). Diurnal variation in N2 fixation activity due to the dark period of night has been reported in various legumes (16,19 Such blockage in photosynthate supply could be induced experimentally by keeping the plants in dark or by detopping the aerial part. This paper deals with the results of such experiments supplemented by cytological and radioisotope tracer studies. MATERIALS AND METHODS Plants and BacteriaSeeds of peanut (Arachis hypogaea L. var Jumbo virginia) and cowpea (Vigna unguiculata L. var California blackeye) were obtained from W. Atlee Burpee Co. Warminster, PA. The seeds were planted in pots (6 inch standard) containing vermiculite, and the seedlings were inoculated with a broth culture of Bradyrhizobium sp. 32H 1 provided by the Nitragin Co. Ltd, Milwaukee, WI. The potted plants were kept in an environment chamber with approximately 700 ,tmol m-2 s-1 PPFD under day/night conditions of 16 h/8 h, 27°C/22°C, and 70%/50% RH and were irrigated with nitrogen-free nutrient solution according to Elfolk (8). Well-nodulated healthy plants were used for dark treatment and detopping at 45 d after inoculation. The plants were kept in the dark for 96 h, during which samples were taken at 0,3,6,12,24, 48, 72, and 96 h. Detopping was achieved by cutting the stem 10 cm above ground, and the root system was sampled at 0, 24, 48, 72, and 96 h.