Nitrogenase-dependent hydrogen evolution from detached legume nodules and from reaction mixtures containing cell-free nitrogenase has been well established, but the overall effect of hydrogen evolution on the efficiency of nitrogen fixation in vivo has not been critically assessed. This paper describes a survey which revealed that hydrogen evolution is a general phenomenon associated with nitrogen fixation by many nodulated nitrogen-fixing symbionts. An evaluation of the magnitude of energy loss in terms of the efficiency of electron transfer to nitrogen, via nitirogenase, in excised nodules suggested that hydrogen production may severely reduce nitrogen fixation in many legumes where photosynthate supply is a factor limiting fixation. With most symbionts. including soybeans, only 40-60% of the electron fow to nitrogenase was transferred to nitrogen. The remainder was lost through hydrogen evolution. In situ measurements of hydrogen evolution and acetylene reduction by nodulated soybeans confirmed the results obtained with excised nodules. In an atmosphere of air, a major portion of the total electron flux available for the reduction of atmospheric nitrogen by either excised nodules or intact nodulated plants was utilized in the production of hydrogen gas. Some nonleguminous symbionts, such as Alnus rubra, and a few legumes (i.e., Vigns sinensis) apparently have evolved mechanisms of minimizing net hydrogen production, thus increasin their efficiency of electron. transfer to nitrogen. Our res ts indicate that the extent of hydrogen evolution during nitrogen reduction is a major factor affecting the efficiency of nitrogen fixation by many agronomically important legumes. The increasing world population and depletion of fossil fuel supplies have stimulated renewed interest in methods of increasing agricultural productivity while minimizing the consumption of fossil fuels. A major factor limiting agricultural production is nitrogen fertilizer, the synthesis of which consumes major quantities of energy. Approximately 3% (600 X 109 cubic feet; 16.8 X 109 meter3) of the natural gas consumed in the United States in 1973 was used for the synthesis of 17 X 106 U.S. tons (153 X 108 kg) of anhydrous ammonia (1, 2). About 10 X 106 U.S. tons of synthetic ammonia were used for nitrogen fertilizer to supply a portion of an annual agricultural nitrogen demand of about 18 X 106 U.S. tons (1, 2). A large part of the remaining need for nitrogen in agriculture was supplied by nitrogen-fixing organisms, such as legumes, which utilize photosynthetically stored solar energy to reduce atmospheric nitrogen to ammonia. Because the biological nitrogen-fixing process is not dependent upon nonrenewable energy resources, its use in agriculture should be maximized. Factors limiting biological nitrogen fixation therefore deserve thorough investigation (3,4).One characteristic of all cell-free nitrogenase preparations that might limit nitrogen fixation is the release of hydrogen gas concomitant with nitrogen reduction (5-11). During thi...

We previously demonstrated, in transgenic tobacco plants, that the role of the movement protein (MP) of tobacco mosaic virus is to facilitate the cell-to-cell spread of viral progeny during infection. An analysis of different tissues of these transgenic plants indicated that the MP accumulated in leaf, stem, and root tissue. The highest levels were detected in older leaves. The relative levels of MP in leaf tissue from transgenic plants were equivalent to, or higher than, the levels of MP in tobacco mosaic virus-infected leaf tissue. Results of subcellular fractionation of homogenates of transgenic leaf tissue showed that the MP was most abundant in the cell wall fraction of older leaves and that the protein remained at high levels in the cell wall fraction as the leaves continued to age. Significant levels of the MP were detected in a crude membrane/organelle fraction and a soluble fraction in younger leaves but decreased to low levels in older leaves. These results suggest that the MP accumulates and is stable in cell walls. We have previously shown that the MP modifies the molecular exclusion limit of plasmodesmata, which is consistent with the hypothesis that plant viruses move from cell to cell through altered plasmodesmata. We show here that the ability of the tobacco mosaic virus MP to modify the molecular exclusion limit of plasmodesmata in tobacco depends on the developmental stage of the leaf. The implications of these findings on understanding virus movement and how plasmodesmata function are discussed.The movement of a plant virus from the initial site of infection into adjacent healthy cells is essential for establishing a productive virus infection. In some virus-host interactions, pathogenesis includes systemic movement of virus by means of the vascular tissue. Although little is known about the virus-host interactions necessary for virus spread, recent studies have provided direct evidence that a protein encoded by tobacco mosaic virus (TMV) is required for cell-to-cell movement of the virus. When expressed in transgenic plants, this protein, the movement protein (MP; 32 kDa), complemented the temperature-sensitive defect in movement of the Lsl mutant of the L strain of TMV (1). Using a different approach, Meshi et al. (2) showed that when the single amino acid change that distinguishes the MP gene of the Lsl virus from that of the parental L strain was introduced into the MP gene of the L virus, the mutated L strain showed the same phenotype as the Lsl virus.Plant viruses (or nucleic acids) move from cell to cell through plasmodesmata, channels that extend through cell walls and provide cytoplasmic continuity between adjacent cells. However, on the basis of present knowledge of plasmodesmata architecture (3, 4) and the molecular size exclusion limits of plasmodesmata (5-7), it is generally assumed that the structure must be modified during virus infection for virus progeny to move from cell to cell. In the case of TMV in tobacco, the MP may be responsible for altering plasmodesmata. At p...

N2-fixing root nodules of soybean (Glycine max L. Merr.) convert atmospheric N2 to ammonia(um) in an energy-intensive enzymatic reaction. These nodules synthesize large quantities of purines because nitrogen fixed by bacteria contained within this tissue is transferred to the shoots in the form of ureides, which are degradation products of purines. In animal systems, it has been proposed that proline biosynthesis by pyrroline-5-carboxylate reductase (P5CR) is used to generate the NADP+ required for the synthesis of the purine precursor ribose 5-phosphate. We have examined the levels, properties, and location of P5CR and proline dehydrogenase (ProDH) in soybean nodules. Nodule P5CR was found in the plant cytosol. Its activity was substantially higher than that reported for other animal and plant tissues and is 4-fold higher than in pea (Pisum sativum) nodules (which export amides).The Km for NADPH was lower by a factor of 25 than the Km for NADH, while the Vm. with NADPH was one-third of that with NADH. P5CR activity was diminished by NADP+ but not by proline. These characteristics are consistent with a role for P5CR in supporting nodule purine biosynthesis rather than in producing proline for incorporation into protein. ProDH activity was divided between the bacteroids and plant cytosol, but <2% was in the mitochondria-rich fractions. The specific activity of ProDH in soybean nodule bacteroids was comparable to that in rat liver mitochondria. In addition, we propose that some of the proline synthesized in the plant cytosol by P5CR is catabolized within the bacteroids by ProDH and that this represents a novel mechanism for transferring energy from the plant to its endosymbiont. N2 fixation in legumes is a symbiotic process in which bacteria of the genus Bradyrhizobium or Rhizobium infect root cells and form specialized organs (nodules) within which N2 is reduced to NH'. Fixation of N2 is an energyintensive process, requiring a total of 25-30 ATP per N atom fixed. Of this total, 12-14 ATP per N are required within the bacteroid to reduce N2. As much as 10-30% of the total photosynthetic capacity of the plant is used to support this process (1). The energy-yielding metabolite(s) supplied by the host to the bacteroid, the symbiotic form of the bacterium, is not known. However, one attractive suggestion is that bacteroids import and oxidize a nitrogenous compound, such as glutamate (2). The nitrogen fixed in the bacteroid is exported as ammonia(um) to the infected host cell, where it is packaged for export to the rest of the plant. Legumes of temperate origin (e.g., peas) export nitrogen as amides (principally asparagine), whereas legumes of tropical origin (e.g., soybeans) export the ureides, allantoin, and allantoic acid. Ureide biogenesis proceeds by way of synthesis of purine ribonucleotides (3). This pathway is the same as that found in microorganisms, fungi, and animals. The purines so formed are then oxidatively degraded to ureides (3). The estimated peak rate of de novo purine biosynthesis necessary t...

Humans depend on plants as a major source of dietary folates. Inadequate dietary levels of the vitamin folate can lead to megaloblastic anemia, birth defects, impaired cognitive development, and increased risk of cardiovascular disease and cancer. The biofortification of folate levels in food crops is a target for metabolic engineering. Folates are synthesized de novo from pterins and para-amino benzoic acid, which are subsequently combined to form dihydropteroate, the direct precursor to dihydrofolate. We postulated that GTP cyclohydrolase-1, which catalyzes the first committed step in pterin biosynthesis, was a rate-limiting step in pterin synthesis in plants and, therefore, in folate synthesis. On this basis, we proposed that the expression of an unregulated bacterial GTP cyclohydrolase-1 in plants would increase pterin biosynthesis with a concomitant enhancement of folate levels. The folE gene encoding GTP cyclohydrolase-1 was cloned from Escherichia coli and introduced into Arabidopsis thaliana through plant transformation. The expression of bacterial GTP cyclohydrolase-1 in transgenic Arabidopsis resulted in a 1,250-fold and 2-to 4-fold enhancement of pterins and folates, respectively. These results helped to identify other potential factors regulating folate synthesis, suggesting ways to further enhance folate levels in food crops.

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The intraceliular location of enzymes involved in the synthesis of the ureides, allantoin and allantoic acid, was investigated in nodules of Glycuie max L. Merr. Ceilular organelles were separated on isopycnic sucrose density gradients. Xanthine dehydrogenase activity (270 nanomoles per min per gram fresh weight) was totally soluble, whereas approximately 15% of the total uricase and catalase activities (1 and 2000 micromoles per minute per gram fresh weight, respectively) was in the fraction containing intact peroxisomes. Aliantoinase activity (680 nanomoles per minute per gram fresh weight) was associated with the microsomal fraction, which apparently originates from the endoplasmic reticulum.

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