José Ronaldo Magalhães

Nitrite as the major source of nitric oxide production by Arabidopsis thaliana in response to Pseudomonas syringae

The origin of nitric oxide (.NO) in plants is unclear and an .NO synthase (NOS)‐like enzyme and nitrate reductase (NR) are claimed as potential sources. Here we used wild‐type and NR‐defective double mutant plants to investigate .NO production in Arabidopsis thaliana in response to Pseudomonas syringae pv maculicola. NOS activity increased substantially in leaves inoculated with P. syringae. However, electron paramagnetic resonance experiments showed a much higher .NO formation that was dependent on nitrite and mitochondrial electron transport rather than on arginine or nitrate. Overall, these results indicate that NOS, NR and a mitochondrial‐dependent nitrite‐reducing activity cooperate to produce .NO during A. thaliana–P. syringae interaction.

Response of ammonium assimilation enzymes to nitrogen form treatments in different plant species 1

Abstract This series of experiments studied N metabolism in tomato, rice and corn. Ammonium (NH4 +), as a sole source of N, reduced tomato and corn growth, but not rice growth. Tomato showed the most severe NH4 + toxicity. Ammonium assimilation enzyme activity differed greatly among the species. Rice had much higher glutamine synthetase (GS) activity than corn and tomato with NH4 + nutrition. GS activity was especially high in shoot tissue. Ammonium induced high activity of glutamate dehydrogenase (GDH) in roots of tomato but not in rice. GS activity in rice increased as the level of NH4 + increased; and it was higher in shoots than roots, indicating GS activity as a key factor in the detoxification and metabolism of NH4 + in green tissues of efficient plant species.

Ammonium toxicity development in tomato plants relative to nitrogen form and light intensity 1

Abstract Tomato plants were grown in sand culture with NH4 + and NO3 – forms of N and three levels of light established by using shade cloth. Plants supplied with NH4 + nutrition under high light intensity showed symptoms of stunting, leaf‐roll, wilting, interveinal chlorosis of the older leaves, and produced one third the dry weight of NO3 – fed plants. In contrast, plants receiving NH4 + nutrition with light reduced over 50% appeared normal. NH4‐N increased NH4 and free amino acid contents of the tissue as compared to NO3‐N nutrition. With shading there was a greatly decreased level of free NH4 and increased levels of free amino acids, mainly asparagine and glutamine, in shoots of NH4‐treated plants. The NO3 content in shoots of NO3‐treated plants increased sharply with shade. The results suggest that the increased NH4 concentration in leaves from photorespiration can he a major factor in the ammonium toxicity expression under high light intensity.

Evidence of increased 15N-ammonium assimilation in tomato plants with exogenous α-ketoglutarate

Abstract The effect of exogenous α-ketoglutarate (αKG) on ammonium (NH 4 + ) assimilation into free amino acids and total reduced nitrogen was monitored in both roots and shoots of 2-week-old tomato ( Lycopersicon esculentum Mill.) seedlings supplied with 5 mM 99% ( 15 NH 4 ) 2 SO 4 in hydroponic culture. Amino acid pools increased approximately 2-fold and 15 N concentrations averaged 1.5- and 2-fold higher in roots and shoots, respectively, in αKG-treated plants compared with the control. Free NH 4 + in tissues decreased sharply with αKG and was almost 90% lower in shoots of treated plants at the same time that the total 15 N assimilation increased 2-fold. This increase of 15 NH 4 + assimilation due to exogenous αKG clearly indicates that the availability of carbon skeletons is a key limiting factor for NH 4 + assimilation in tomato plants.

Influence of the form of nitrogen on ammonium, amino acids and N-assimilating enzyme activity in maize genotypes

Abstract An understanding of nitrogen (N) interactions with plant growth and metabolism is important in order to increase the efficiency of fertilizer amendments for crop production. This study was initiated to evaluate the effect of the form of N and buffering with calcium carbonate (CaCO3) on the activity of ammonium (NH4) assimilating enzymes in a maize hybrid, a GDH‐deficient mutant, and the N6 normal inbred the mutant was derived from. Plants were grown in a growth chamber in sand culture with 10 mM of the different N forms [nitrate (NO3), NH4, ammonium nitrate (NH4STO3), and NH4 plus CaCO3). Hybrid plants grown in the unbuffered NH4 were much smaller than those grown in NO3 as the only source of N, but had comparable growth with each source of nitrogen if the pH was controlled. In contrast, a glutamate dehydrogenase‐deficient mutant (GDH) inbred and its normal control grew almost as well on both NH4 treatments as on NO3. GDH activity was higher in plants grown with NH4 than NO3, and it was almost ni...

Maize growth and ammonium assimilation enzyme activity in response to nitrogen forms and pH control 1

Abstract Plant growth, glutamine synthetase and glutamine dehydrogenase activities of two maize genotypes were compared in the presence of NH4 + and NO3 ‐ forms of N in sand culture. Ammonium reduced growth of the P3732 genotype 64% and the B73 x Mol7 hybrid 59% as compared to NO3 ‐. Both glutamine synthetase and glutamate dehydrogenase activities in roots tended to be higher with NH4 as compared to NO3. As the pH in the medium was increased by adding CaCO3, glutamine synthetase and glutamate dehydrogenase activities in roots of both genotypes were reduced; however, glutamine synthetase activity in leaves of NH^‐treated plants increased at the higher pH of the growing medium.

Effects of supplemental nitrogen on nitrogen‐assimilation enzymes, free amino nitrogen, soluble sugars, and crude protein of rice

Abstract An upland rice variety IAC‐47 was grown in a greenhouse to determine the effect of foliar nitrogen (N) supplementation during grain development on the activity of the N assimilation enzymes, nitrate reductase (NR) and glutamine synthetase (GS), on free amino‐N content and leaf soluble sugars, and on grain crude protein content. At 10 and 20 days after anthesis (DAA), the leaves were fertilized with a liquid fertilizer containing 32% N as 12.8% urea, 9.6% ammonium (NH4), and 9.6% nitrate (NO3) in increasing rates corresponding to 0,20+20, 40+40, and 60+60 kg N ha‐1. Leaves were collected twice (at 12 DAA and 14 DAA for GS activity, sugar and amino‐N content, and at 11 and 13 DAA for NRA) after each application of leaf N. The late foliar application of N increased significantly grain crude protein without a corresponding decrease in grain weight. The NR activity (NRA) increased after the foliar application of N. In the flag leaf, 60+60 kg N ha‐1 (21 DAA) resulted in higher NRA (20x over the control...

Similarities in response of maize genotypes to water logging and ammonium toxicity 1

Abstract Plant growth, total nitrogen (N), and free ammonia (NH3) in tissue of different maize genotypes were compared in the presence of either nitrate (NO3)‐or ammonium (NH4)‐forms of ? at two oxygen (O2) levels in solution culture. Shoot and root growth was significantly less with ??4 than with NO3 under low O2 conditions; however, NH4 toxicity was significantly reduced by supplying maize plants with additional O2 in the solution culture, and root and shoot growth of NH4‐supplied plants were not significantly different than that of NO3‐treated plants. On the other hand, maize genotypes differed significantly in their response to ? forms and O2. The genotype, Saracura, selected for tolerance to water logging, had the highest tolerance to ??4 based on plant growth under low O2 pressure and the concentration of free NH4 in tissues. This research suggests there may be a common mechanism or an interaction with the mechanism for tolerance to NH4 and water logging.

Kinetics of 15NH4 + assimilation in tomato plants: evidence for 15NH4 + assimilation via GDH in tomato roots 1

Abstract The kinetics of 15NH4 + assimilation into free amino acids and total reduced nitrogen were monitored in both roots and shoots of two week old tomato seedlings supplied with 5 mM 99% (15NH4)2SO4 via the aerated root medium in hydroponic culture, in the presence and absence of a 2 h pre‐incubation with 1 mM methionine sulfoximine (MSX). The labeling kinetics of amino acids in roots of tomato plants in the presence of MSX show that continued assimilation of 15NH4 + can occur when the GS/GOGAT cycle is inhibited. In the presence of MSX, three amino acids [glutamate, alanine, and y‐amino butyrate (GABA)] of the root tissue continue to become labeled with 15N under conditions where labeling of the amino‐N moiety of glutamine is completely inhibited. This indicates primary ammonia assimilation via GDH, or alternatively, assimilation of ammonia into alanine via alanine dehydrogenase. Free ammonia accumulates rapidly in both shoots and roots of tomato in response to MSX. The labeled ammonia accumulated in...

Nitric oxide increases tolerance responses to moderate water deficit in leaves of Phaseolus vulgaris and Vigna unguiculata bean species.

Drought stress is one of the most intensively studied and widespread constraints, and nitric oxide (NO) is a key signaling molecule involved in the mediation of abiotic stresses in plants. We demonstrated that a sprayed solution of NO from donor sodium nitroprusside increased drought stress tolerance responses in both sensitive (Phaseolus vulgaris) and tolerant (Vigna unguiculata) beans. In intact plants subjected to halting irrigation, NO increased the leaf relative water content and stomatal conductance in both species. After cutting leaf discs and washing them, NO induced increased electrolyte leakage, which was more evident in the tolerant species. These leaf discs were then subjected to different water deficits, simulating moderate and severe drought stress conditions through polyethylene glycol solutions. NO supplied at moderate drought stress revealed a reduced membrane injury index in sensitive species. In hydrated discs and at this level of water deficit, NO increased the electron transport rate in both species, and a reduction of these rates was observed at severe stress levels. Taken together, it can be shown that NO has an effective role in ameliorating drought stress effects, activating tolerance responses at moderate water deficit levels and in both bean species which present differential drought tolerance.