John Imsande

N Demand and the Regulation of Nitrate Uptake

Uptake of nitrate by root cells followed by reduction and assimilation in plant tissues is the main route by which mineral N is converted into organic N by living organisms. Like photosynthesis, these are life-dependent processes that members of the animal kingdom are unable to perform for themselves. Nitrate and other mineral nutrients required for optimal plant growth and development frequently exist at relatively low concentrations in soil. To thrive on these dilute nutrients, plants have developed high-performance uptake systems in their root cells. To cope with wide variations in mineral concentrations in soil, plants have evolved mechanisms to regulate the activity of uptake systems so that net intake of a nutrient depends on the plants need for this element rather than its concentration in the rooting medium. Indeed, uptake rates of most ions are seemingly controlled by specific demand-driven regulatory mechanisms. Such processes set the uptake rate of a given element to match the plants current growth rate and developmental stage. Nitrate uptake is of special interest because nitrate is absorbed at a relatively high rate and because compounds that function as uptake sensors may have been identified. This paper focuses on whole-plant signaling processes involved in the regulation of nitrate uptake by N demand.

Iron-sulfur clusters: Formation, perturbation, and physiological functions

Abstract Iron-sulfur proteins occur in all life forms. Ferredoxins and Rieske proteins each contain a (2Fe2S) cluster whereas photosystem I (PSI) contains three (4Fe4S) clusters. Essential enzymes such as sulfite reductase, nitrite reductase, nitrogenase, glutamate synthase, aconitase, succinate dehydrogenase, ferredoxin/thioredoxin reductase, as well as many other vital proteins, each contain a (4Fe4S) cluster. Iron-sulfur clusters are formed enzymatically from cysteinyl-sulfur and ferritin-sequestered iron. Many iron-sulfur clusters are inactivated by O 2 and/or reactive oxygen species (ROS) such as O 2 •− . Perhaps 0.1 % of the electrons passing through either the mitochondrial electron transport chain or PSI result in the formation of O 2 •− . Many plant stresses increase ROS formation, which subsequently may perturb iron-sulfur clusters. Plants have evolved three different superoxide dismutases (SODs) to control the internal concentrations of harmful ROS. Possible roles of functional and non-functional iron-sulfur clusters in the coordination of metabolic activities of stressed and non-stressed plants are discussed.

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.

Nitrogen deficit during soybean pod fill and increased plant biomass by vigorous N2 fixation

Abstract The N concentration of a soybean Glycine max. [(L.) Merr.] seed frequently is greater than 60 mg g −1 . Because of high seed-N demand, a soybean plant may become N-deficient during pod fill. To test the effect of N source during pod fill on seed N, plants were grown hydroponically so that N source could be controlled. From seedling stage to mid-R4, all plants were provided an excess of nitrate-N. From mid-R4 to harvest (R7), randomized sets of 20 plants were fed excess N in the form of any one of 4.0 mM KNO 3 , 2.0 mM NH 4 NO 3 , 2.0 mM KNO 3 + 1.0 mM urea, 1.0 mM KNO 3 + 1.5 mM urea, or 2.0 mM urea. The mean rate of N assimilation during pod fill for plants fed only nitrate was approximately 65% of the mean rates of plants fed the same amount, but different forms, of N during pod fill. Likewise, seed analysis showed that plants fed only nitrate during pod fill frequently produced smaller seeds that contained a lower seed-N concentration (experiment 1, 63.3 mg N g −1 vs. 67.7–73.4 mg N g −1 ; experiment 2, 61.6 mg N g −1 vs. 63.0–67.2 mg N g −1 ) than those fed other forms of N. Thus, the total N content of seeds produced by plants fed only nitrate during pod fill was approximately 77% of the mean values of plants fed the same amount, but different forms, of N during pod fill. On the other hand, nodulated plants provided a low level of nitrate or urea and fixing approximately 600 mg N 2 plant −1 frequently had a higher gain in harvested biomass (169%) than did non-inoculated plants fed excess nitrate (139%). It is concluded that rapid N 2 fixation during pod filling enhances net photosynthetic output of soybean.

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.

Characterization of mutations in the penicillinase operon of Staphylococcus aureus

SummaryMutant 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 is 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.

Plant Genotype and the Control of Nitrogen Fixation

Recently a nondestructive assay for nitrogenase activity was described that permits the monitoring of nitrogen fixation periodically throughout the lifetime of the nodulated soybean plant (Imsande, Ralston, 1981). This procedure, which relies upon hydroponic growth and the repeated measurement of acetylene reduction by the nitrogenase complex, is as follows: 1) seeds are sprouted aseptically in rolls of moistened germination paper; 2) approximately 3 days after emergence, the seedlings are transferred, aseptically, to hydroponic growth in a plant growth chamber; 3) after 21 days of aseptic hydroponic growth, the entire root system of each plant is immersed for 10 minutes in a pure culture Rhizobium japonicum, rinsed, and returned to hydroponic growth; 4) twice each week for approximately 8 weeks individual plants are weighed, assayed for acetylene reduction activity, and returned to hydroponic growth. Growth medium supplemented with 1.0 to 3.0 mM nitrate is changed twice each week (Ralston, Imsande, 1983). Using this procedure I attempted to identify individual soybean plants that fix nitrogen at a relatively high rate and to determine whether or not the capacity for enhanced nitrogen fixation is controlled by the plant genotype.

Nature of the plasmid-linked penicillinase regulatory region in Staphylococcus aureus

SummaryFour noninducible staphylococcal penicillinase plasmids reported to produce a very low basal level of penicillinase were investigated. Incorporation of 5-methyltryptophan, which is known to inactivate the operator binding site of wild-type penicillinase repressor and thereby elicit penicillinase synthesis, did not induce penicillinase synthesis in any of these “micro” mutants. Therefore, these plasmids are not simply peniS mutants. Heterodiploid strains composed of a plasmid fully constitutive for penicillinase synthesis and one of the various “micro” penicillinase plasmids were constructed. Three of these heterodiploids produce a normal basal level of penicillinase and are inducible by 5-methyltryptophan but not by the standard gratuitous penicillinase inducer. Therefore, each of these three noninducible “micro” plasmids produces a peniS repressor, but in addition, each must bear a mutation in the penZ region. The fourth heterodiploid produces a fully constitutive level of penicillinase. The noninducible “micro” plasmid present in this heterodiploid must contain a penI− mutation and a mutation in the penZ region. Consequently, each of these four noninducible “micro” plasmids contains at least two mutants genes. Hence, the phenotype of noninducibility plus low basal penicillinase is not due to a point mutation in a second penicillinase regulatory region as has been proposed. Instead, these results strongly suggest that there is only one penicillinase regulatory gene located on the penicillinase plasmid and that this gene (penI) specifies the penicillinase repressor.