Atilio J. Barneix

The effect of inoculation with Azospirillum brasilense on growth and nitrogen utilization by wheat plants

Azospirillium brasilense is a rhizosphere bacteria that has been reported to improve yield when inoculated on wheat plants. However, the mechanisms through which this effect is induced is still unclear. In the present work, we have studied the effects of inoculating a highly efficient A. brasilense strain on wheat plant grown in 5 kg pots with soil in a greenhouse, under three N regimes (0, 3 or 16 mM NO3−, 50 ml/pot once or twice-a -week), and in disinfected or non-disinfected soil. At the booting stage, the inoculated roots in both soils showed a similar colonization by Azospirillum sp. that was not affected by N addition. The plants grown in the disinfected soil showed a higher biomass, N content and N concentration than those in the non-disinfected soil, and in both soils the inoculation stimulated plant growth, N accumulation, and N and NO3− concentration in the tissues.At maturity, the inoculated plants showed a higher biomass, grain yield and N content than the uninoculated ones in both soils, and a higher grain protein concentration than the uninoculated. It is concluded that in the present experiments, A. brasilenseincreased plant growth by stimulating nitrogen uptake by the roots.

Cytokinin-induced changes of nitrogen remobilization and chloroplast ultrastructure in wheat (Triticum aestivum).

Nitrogen (N) remobilization in wheat (Triticum aestivum) plants is crucial because it determines the grain protein concentration and the baking quality of flour. In order to evaluate the influence of cytokinins on N remobilization during N starvation, we analyzed various N remobilization parameters in wheat plants that were watered with 6-benzylaminopurine (BAP) either with or without KNO(3). Besides, the effects of BAP on protein synthesis were evaluated, and the size and ultrastructure of chloroplasts of BAP-treated plants were studied. BAP supply inhibited N remobilization of plants independently of N supply as shown by the increase in protein, Rubisco, chlorophyll, sugar and starch concentrations in the older leaves, the decrease in amino acid and sugar export to the phloem, and the decrease in protein, Rubisco and chlorophyll concentrations in the younger leaves. Besides, BAP supply increased nitrate reductase activity and decreased nitrate concentration, thus suggesting an increased assimilatory capacity. The increase in protein concentration could be explained mainly by a significant decrease in protein degradation and, to a lesser extent, by an increase in protein synthesis. Finally, an increase both in the size of the chloroplast and in the plastoglobuli and starch contents in BAP-supplied plants was observed. We propose that cytokinins retain the sink activity of the older leaves by inhibiting amino acid and sugar export to the phloem and stimulating assimilate accumulation in the chloroplasts of the older leaves. Besides, BAP may increase protein concentration of the older leaves both by decreasing protein degradation and maintaining protein synthesis even under stress conditions.

Regulation of NH4+ uptake in wheat plants: Effect of root ammonium concentration and amino acids

When N deficient 10-day-old wheat plants were supplied with 1.5 mol m−3 NH4+, net NH4+ uptake rapidly decreased during the first 6h, while root free-NH4+ and free amino acids concentration increased. However, after 24 h the NH4+ uptake rate increased again, as the internal NH4+ concentration decreased. When plants were pretreated during 40 h with different external NH4+ concentrations, net uptake measured on 1.0 mol m−3 NH4+ decreased with the increasing ion concentration during the pretreatment. This decrement coincided with both root free-NH4+ and total free amino acids levels. When N-starved and NH4+ fed plants were treated during 0, 3 or 6 h with 1.0 mol m−3 NH4+ in the presence of 1.0 mol m−3 MSX2, net uptake (measured without MSX) decreased with the length of the inhibitor treatment. In both groups, MSX significantly increased root free-NH4+ concentration, while the level of total free amino acids was only increased in N-starved plants. When N-starved plants were externally supplied with 1.0 mol m−3 of different amino acids or amides, net NH4+ uptake was only strongly inhibited in the presence of glutamine or asparagine. It is concluded that rapid changes in the concentration of certain amino acids during NH4+ nutrition might regulate the ion absorption, though at high endogenous levels of free NH4+ net uptake could be suppressed independently of the root concentration of free amino acids.

Some effects of nitrate abundance and starvation on metabolism and accumulation of nitrogen in barley (Hordeum vulgare L. cv Sonja)

Nitrate and nitrite reductases were both induced by adding three concentrations of nitrate to the nutrient supply of nitrate-starved barley seedlings. Enzyme induction was not proportional to the amount of nitrate introduced. Glutamine synthetase also increased above a high endogenous activity but the increase did not differ significantly between any of the three nitrate treatments. Nitrate accumulated rapidly in leaves of plants given 4.0 mM or 0.5 mM nitrate but not with 0.1 mM nitrate. In all treatments, amino acids in leaves increased for 2 d, chiefly attributable to glutamine, then declined. Transferring plants from the three nitrate treatments to nitrate-free nutrient produced an immediate decline in nitrate reductase but nitrite reductase continued to increase for 2 d, before declining. Glutamine-synthetase activity was not affected by withdrawal of nitrate, nor did nitrate withdrawal retard plant growth during the 9-d period of the experiment. The disparity between accumulated nitrate and nitrate-reducing capacity and the rapid decrease in leaf nitrate when nutrient nitrate supply was removed, indicated the presence of a nitrate-storage pool that could be called upon to maintain amino-acid production in times of nitrogen starvation.

Plant Growth Regulators and Induction of Leaf Senescence in Nitrogen-Deprived Wheat Plants

The sequence of events and the signals that regulate the remobilization of nitrogen (N) reserves during senescence induced by N starvation were studied in leaf 3, the last fully expanded leaf, in 17-day-old wheat (Triticum aestivum L.) plants. The first event observed was a rapid decrease in the isopentenyl adenosine (iPA) concentration during the first 24 h of N starvation. No differences in t-zeatin riboside and dihydrozeatin riboside concentrations were observed until the end of the assay. During the following 6 days, a decrease in soluble amino acids, chlorophyll, and protein, as well as an increase in soluble sugar concentration and endoproteolytic activity, could be observed. At day 3 of the experiment, the abscisic acid (ABA) concentration in the leaves of N-deprived plants started to increase. After 6 days of N deprivation there was a rise in oxidative stress, as indicated by the increase in malondialdehyde concentration, as well as a decrease in the activities of antioxidant enzymes catalase and ascorbate peroxidase. To analyze interactions with leaf development, the first, second, third, and fourth leaves were studied. iPA concentration decreased in all the leaf stages, including leaf 4, which was not fully expanded. A linear correlation between iPA and protein concentration was determined. These results suggest that the sharp fall in iPA could be the earliest event that induces protein degradation during the development of senescence induced by N deficiency, and that only later is ABA accumulated and oxidative stress developed.

The effect of amino acids and amides on the regulation of nitrate uptake by wheat seedlings

Abstract Wheat plants grown hydroponically increased their nitrate uptake rate more than two‐fold after three days of N starvation. Exogenously supplied amino acids and amides had no effect on the nitrate uptake rate of plants well nourished in N. After three days of N starvation, however, some of the amino acids and amides supplied to plants inhibited up to 50% of the nitrate uptake rate. The most effective inhibitor was aspartic acid. Asparagine, glutamine or phenylalanine did not show any inhibitory effect. The percentage of inhibition was not increased by increasing the amino acid concentration, nor did the addition of mixed amino acids and amides increase the inhibition exerted by one amino acid alone. During the three days of N starvation, there was a decrease in the concentration of endogenous amino acids in the roots, but not all amino acids decreased their concentration at the same rate. It is suggested that the endogenous levels of some amino acids may repress the nitrate uptake system in plants...

Growth stimulation and nitrogen supply to wheat plants inoculated with Azospirillum brasilense

Abstract Twelve Azospirillum brasilense strains isolated from wheat (Triticum aestivum L.) roots were compared for root colonization, growth stimulation, and nitrogen (N) supply to young wheat plants cv. Klein Chamaco grown in sterile nutrient solutions without N. All the strains inoculated colonized both the root surface and interior, and most strains stimulated root and shoot growth, although the degree of stimulation was different for the different strains. Some strains increased the total N content of roots and tops at the end of the experiment, in one case up to 80% of the uninoculated plants, while others produced no effect on N content. No correlation could be found between growth stimulation or the amount of N supplied to the plant with the degree of root colonization. When the most efficient strain for N fixation was inoculated to different wheat cultivars, it stimulated growth and supplied N to the five cultivars tested, although the degree of root colonization, growth stimulation and N supply s...

Cellular envelopes and tolerance to acid pH in Mesorhizobium loti.

Abstract. Changes in the cell envelopes in response to acidity were studied in two strains of Mesorhizobium loti differing in their tolerance to pH. When the less acid-tolerant strain LL22 was grown at pH 5.5, membrane phosphatidylglycerol decreased and phosphatidylcholine increased, compared with cells grown at pH 7.0. On the other hand, when the more acid-tolerant strain LL56 was grown at pH 5.5, phosphatidylglycerol, phosphatidylethanolamine, and lysophospholipid decreased 25%, 39%, and 51% respectively, while phosphatidyl-N-methylethanolamine and cardiolipin increased 26% and 65% respectively compared with cells grown at pH 7.0. The longest-chain fatty acids (19:0 cy and 20:0) increased in both strains at pH 5.5, while in LL56 these fatty acids increased still further at pH 4.0. Variations in other wall and membrane properties such as cell hydrophobicity, lypopolysaccharides, and protein composition of the outer membrane in relation to acid pH are also discussed.

Regulation of glutamine synthetase 1 and amino acids transport in the phloem of young wheat plants.

The possible regulation of amino acid remobilization via the phloem in wheat (Triticum aestivum L.) by the primary enzyme in nitrogen (N) assimilation and re-assimilation, glutamine synthetase (GS, E.C. 6.3.1.2) was studied using two conditions known to alter N phloem transport, N deficiency and cytokinins. The plants were grown for 15 days in controlled conditions with optimum N supply and then N was depleted from and/or 6-benzylaminopurine was added to the nutrient solution. Both treatments generated an induction of GS1, monitored at the level of gene expression, protein accumulation and enzyme activity, and a decrease in the exudation of amino acids to the phloem, obtained with EDTA technique, which correlated negatively. GS inhibition by metionine sulfoximide (MSX) produced an increase of amino acids exudation and the inhibitor successfully reversed the effect of N deficiency and cytokinin addition over phloem exudation. Our results point to an important physiological role for GS1 in the modulation of amino acids export levels in wheat plants.

Pep‐carboxylase activity during ammonium‐assimilation in wheat plants

Abstract When 14‐day old wheat plants were transferred from a medium containing nitrate as the sole N‐source to one containing ammonium, the root NH+ 4‐concentration increased rapidly. This was followed by increases in root phosphoenol pyruvate carboxilase (PEP‐carboxylase) activity, concentration of glutamine, and total free amino acids. Eventually the PEP‐carboxylase activity reached a plateau and free‐NH+ 4 concentration increased thereafter. Similar plants transferred from NO3 ‐ to a mixed N‐source (NH+ 4 + NO3 ‐) also showed an increase in root PEP‐carboxylase activity. The enhancement was slower but reached the same final value as for the plants transferred to NH4 + ‐only. The concentrations of glutamine and free amino acids increased, but to a smaller extent than in the plants transferred to all ammonium. In shoots the treatments produced no detectable changes in PEP‐carboxylase activity. It is concluded that high PEP‐carboxylase activity in roots helps to prevent accumulation of ammonium to toxic ...