Dinitrogen fixation in male-sterile soybeans

Partial male-sterile (ms₄/ms₄) soybeans (Glycine max L. Merr.) and their fertile isoline (Ms₄/Ms₄) were grown in adjoining field plots. From 62 until 92 days after emergence, the nitrogenase activity, assayed by acetylene reduction, of the average male-sterile plant was approximately twice that of the average fertile plant. At approximately 100 days after emergence, the assayable nitrogenase activity of the fertile plants fell to zero, whereas the nitrogenase of the partial male-sterile plants continued to be active for two additional weeks. Thus, this male-sterile plant seems to fix dinitrogen both at a higher rate and over a longer duration than does its fertile isoline.     

Hydroponic growth and the nondestructive assay for dinitrogen fixation

Hydroponic growth medium must be well buffered if it is to support sustained plant growth. Although 1.0 millimolar phosphate is commonly used as a buffer for hydroponic growth media, at that concentration it is generally toxic to a soybean plant that derives its nitrogen solely from dinitrogen fixation. On the other hand, we show that 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid, pK_a 6.1, has excellent buffering capacity, and it neither interferes with nor contributes nutritionally to soybean plant growth. Furthermore, it neither impedes nodulation nor the assay of dinitrogen fixation. Hence, soybean plants grown hydroponically on a medium supplemented with 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid and 0.1 millimolar phosphate achieve an excellent rate of growth and, in the absence of added fixed nitrogen, attain a very high rate of dinitrogen fixation. Combining the concept of hydroponic growth and the sensitive acetylene reduction technique, we have devised a simple, rapid, reproducible assay procedure whereby the rate of dinitrogen fixation by individual plants can be measured throughout the lifetime of those plants. The rate of dinitrogen fixation as measured by the nondestructive acetylene reduction procedure is shown to be approximately equal to the rate of total plant nitrogen accumulation as measured by Kjeldahl analysis. Because of the simplicity of the procedure, one investigator can readily assay 50 plants individually per day.     

Independent spontaneous mitochondrial malate dehydrogenase null mutants in soybean are the result of deletions

The mitochondrial malate dehydrogenase-1 (Mdh1) gene of soybean [Glycine max (L.) Merr.] spontaneously mutates to a null phenotype at a relatively high rate. To determine the molecular basis for the instability of the Mdh1 gene, the gene was cloned and sequenced. The null phenotype correlated with the deletion of specific genomic restriction fragments that encode the Mdh1 gene. The composition of the Mdh1 gene and its environs were compared with those of the more stable MDH2 gene. Several possible causes of the observed instability were found, including duplications, repeats, and two regions with similarity to a soybean catalase. The most likely cause of instability, however, appeared to be a 1233 bp region with 58.9% identity to the Cyclops retrotransposons. Translation of a 714 bp segment of this region produced a peptide composed of 238 amino acid residues that showed 35-40% identity and 55-60% similarity to several putative Cyclops gag-pol proteins (group-specific antigen polyprotein). This short peptide also contained a segment that corresponded to the protease active site of the gag-pol protein. Thus in an appropriate genetic background, a retrotransposon, whether whole or fractured, could promote genetic rearrangements.     

Effect of N source during soybean pod filling on nitrogen and sulfur assimilation and remobilization

During pod filling, a grain legume remobilizes vegetative nitrogen and sulfur to its developing fruit. This study was conducted to determine whether different nitrogen sources affected N and S assimilation and remobilization during pod filling. Well-nodulated plants fed 1.0 mM KNO₃, 0.5 mM urea, or 2.5 mM urea assimilated 0%, 37%, or 114% more N, respectively, and 25%, 46%, or 56% more S, respectively, than did the average non-nodulated control plant fed 5.0 mM KNO₃. Thus, N source during pod filling greatly affected both N and S assimilation. Depending upon N source, plant N concentration during pod filling decreased from 2.96% to between 1.36% and 1.82%. Non-nodulated control plants fed 5.0 mM KNO₃ had the highest residual N at harvest. During the same treatments, plant S concentration decreased from 0.246% to a relatively uniform 0.215%. Thus, during pod filling, vegetative N was seemingly remobilized more efficiently (38–54%) than was S (13%). N source also affected seed yield and seed quality. Non-nodulated control plants fed 5.0 mM KNO₃ produced the lowest yield (21.1 g seeds plant^-1), whereas well nodulated plants fed 1.0 mM KNO₃, 0.5 mM urea, or 2.5 mM urea produced yields of 26.2 g, 31.8 g, and 36.7 g seeds plant^-1, respectively. Non-nodulated plants fed 2.5 mM urea yielded 28.6 g of seeds plant^-1. Seed N concentrations of non-nodulated plants and nodulated plants fed 2.5 mM urea were high, 6.30% and 6.11% N, respectively, whereas their seed S concentrations were low, 0.348% and 0.330% S, respectively. N sources that produced both a relatively high seed yield and seed N concentration (i.e., a relatively high total seed N plant^-1) produced a proportionately smaller increase in total seed sulfur. Consequently, seed quality, as judged solely by seed S concentration, was lowered.     

Iron, sulfur, and chlorophyll deficiencies: A need for an integrative approach in plant physiology

Green plants deficient in nitrogen, sulfur, or iron develop a similar yellow coloration. In each case, the yellow coloration is accompanied by a lowered chlorophyll concentration. This review attempts to collate some of the biochemical information concerning these three seemingly diverse nutritive deficiencies and bares a need for a more integrative approach to plant physiology. The biochemical and biological roles of nitrogen, sulfur and iron in living systems are examined, with emphasis on sulfur and iron. Mechanistically, iron and/or sulfur are highly reactive components of many enzymes. Indeed, iron and sulfur sometimes form Fe₂S₂, Fe₃S₄, or Fe₄S₄ clusters which are very active electron transfer agents. Recently, iron‐sulfur clusters have been reported to serve as sensors of oxidative stress, to couple photosynthesis with several metabolic pathways, to participate in the reduction of sulfite and nitrite, and to participate in regulation of gene expression. Thus, there are several mechanisms by which a deficiency of nitrogen, sulfur, or iron could produce the same low‐chlorophyll, yellow phenotype in plants. Unless the interactions and coordination of the various pathways connected to chlorophyll synthesis are elucidated, it is unlikely that we will select the quickest and most direct path to plant improvement.     

A soybean plastid-targeted NADH-malate dehydrogenase: cloning and expression analyses

A typical soybean (Glycine max) plant assimilates nitrogen rapidly both in active root nodules and in developing seeds and pods. Oxaloacetate and 2-ketoglutarate are major acceptors of ammonia during rapid nitrogen assimilation. Oxaloacetate can be derived from the tricarboxylic acid (TCA) cycle, and it also can be synthesized from phosphoenolpyruvate and carbon dioxide by phosphoenolpyruvate carboxylase. An active malate dehydrogenase is required to facilitate carbon flow from phosphoenolpyruvate to oxaloacetate. We report the cloning and sequence analyses of a complete and novel malate dehydrogenase gene in soybean. The derived amino acid sequence was highly similar to the nodule-enhanced malate dehydrogenases from Medicago sativa and Pisum sativum in terms of the transit peptide and the mature subunit (i.e., the functional enzyme). Furthermore, the mature subunit exhibited a very high homology to the plastid-localized NAD-dependent malate dehydrogenase from Arabidopsis thaliana, which has a completely different transit peptide. In addition, the soybean nodule-enhanced malate dehydrogenase was abundant in both immature soybean seeds and pods. Only trace amounts of the enzyme were found in leaves and nonnodulated roots. In vitro synthesized labeled precursor protein was imported into the stroma of spinach chloroplasts and processed to the mature subunit, which has a molecular mass of ～34 kDa. We propose that this new malate dehydrogenase facilitates rapid nitrogen assimilation both in soybean root nodules and in developing soybean seeds, which are rich in protein. In addition, the complete coding region of a geranylgeranyl hydrogenase gene, which is essential for chlorophyll synthesis, was found immediately upstream from the new malate dehydrogenase gene.     

Regulation of Staphylococcal Penicillinase Synthesis

5-Methyl tryptophan was found to be an efficient inducer of penicillinase synthesis in Staphylococcus aureus. Addition of actinomycin D or tryptophan to the culture medium shuts off the 5-methyl tryptophan-induced synthesis of penicillinase with an apparent half-life of approximately 1 to 2 min, respectively. Hence, in the induction of penicillinase synthesis, 5-methyl tryptophan seems to function as a structural analogue of penicillin rather than by becoming incorporated in proteins and thereby creating faulty penicillinase repressor or antirepressor. This conclusion is supported by similarities in the structures of the two compounds as revealed by solid atomic models. The fact that S. aureus exposed to (14)C-penicillin in the absence of protein synthesis failed to synthesize penicillinase at an increased level when cell growth was resumed strongly suggests that a protein involved in the regulation of penicillinase synthesis must be synthesized in the presence of the penicillinase inducer. In turn, this observation suggests that the penicillinase inducer promotes penicillinase synthesis by directing the penicillinase regulatory protein (i.e., the penicillinase antirepressor) to acquire a different conformation when it is synthesized in the presence of the penicillinase inducer. A working model for the regulation of penicillinase synthesis based on these and other data has been constructed and is presented.     

Characterization of mutations in the penicillinase operon of Staphylococcus aureus

Summary Mutant penicillinase plasmids, in which penicillinase synthesis is not inducible by penicillin or a penicillin analogue, were examined by biochemical and genetic analyses. In five of the six mutants tested, penicillinase synthesis could be induced by growth in the presence of 5-methyltryptophan. It is known that the tryptophan analogue 5-methyltryptophan is readily incorporated into protein by S. aureus and that staphylococcal penicillinase lacks tryptophan. 5-methyltryptophan seems to induce penicillinase synthesis in wild-type plasmids by becoming incorporated into the repressor and thereby inactivating the operator binding function of the penicillinase repressor. Therefore, induction of penicillinase synthesis in the mutant plasmids by 5-methyltryptophan strongly suggests that the noninducible phenotype of these five plasmids is due to a mutation that inactivates the effector binding site of the penicillinase repressor (i.e., the five mutant plasmids carry an i^s genotype for the penicillinase repressor). This conclusion was supported by heterodiploid analysis. The mutant plasmid that did not respond to 5-methyltryptophan either produces an exceedingly low basal level of penicillinase or does not produce active enzyme. This plasmid seems to carry a mutation in the penicillinase structural gene or in the promoter for the structural gene. Thus, a genetic characterization of many mutations in the penicillinase operon can be accomplished easily and rapidly by biochemical analysis.Journal Paper No. J-7994 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2029