Nitric oxide (NO) is a signaling and defense molecule of major importance in living organisms. In the model legume Medicago truncatula, NO production has been detected in the nitrogen fixation zone of the nodule, but the systems responsible for its synthesis are yet unknown and its role in symbiosis is far from being elucidated. In this work, using pharmacological and genetic approaches, we explored the enzymatic source of NO production in M. truncatula-Sinorhizobium meliloti nodules under normoxic and hypoxic conditions. When transferred from normoxia to hypoxia, nodule NO production was rapidly increased, indicating that NO production capacity is present in functioning nodules and may be promptly up-regulated in response to decreased oxygen availability. Contrary to roots and leaves, nodule NO production was stimulated by nitrate and nitrite and inhibited by tungstate, a nitrate reductase inhibitor. Nodules obtained with either plant nitrate reductase RNA interference double knockdown (MtNR1/2) or bacterial nitrate reductase-deficient (napA) and nitrite reductase-deficient (nirK) mutants, or both, exhibited reduced nitrate or nitrite reductase activities and NO production levels. Moreover, NO production in nodules was found to be inhibited by electron transfer chain inhibitors, and nodule energy state (ATP-ADP ratio) was significantly reduced when nodules were incubated in the presence of tungstate. Our data indicate that both plant and bacterial nitrate reductase and electron transfer chains are involved in NO synthesis. We propose the existence of a nitrate-NO respiration process in nodules that could play a role in the maintenance of the energy status required for nitrogen fixation under oxygen-limiting conditions.

Excised maize (Zea mays) root tips were used to follow the effects of a prolonged glucose starvation. Respiration rate began to decrease immediately after excision, reaching 30 to 40% of its initial value after 20 hours, and then declined more slowly until death of the tissues, which occurred after 200 hours of starvation. During the whole process, respiration could be uncoupled by 2,4-dinitrophenol and the energy charge remained high. These results suggest that in excised maize root tips, respiration rate is essentially limited by the rate of biosyntheses (ATP-utilizing processes) rather than mitochondrial number. Carbohydrates are the main respiratory substrates for plants, furnishing the malate and the acetyl-CoA necessary for the functioning of the Krebs cycle. Fifteen years ago it was still considered that, during the day, photosynthesis allowed starch synthesis in such amounts that the plant, particularly the root system, was never deprived of sugars. However, it is now known that carbohydrate starvation is common in most higher plants. Indeed, microbial, insect, or herbivore attacks or reduction in light intensity or temperature may cause a substantial decrease in photosynthesis, and thus lead to starvation.Since the end of the seventies, carbohydrate starvation has been studied in a number of plant species: wheat (28, 29), maize (14, 18), barley (8), pearl millet (1), pea (20,26,27), soybean (13, 25), sycamore (7, 10, 12, 17), etc. These studies have shown that in most cases, sugar starvation triggers the following sequence in plant cells: (a) the depletion of intracellular carbohydrate content and the subsequent decrease of respiration (1,12,18,20,25); (b) the breakdown of lipids and proteins (1,7,12,28) and a decline in the respiratory quotient from 1 to 0.75 (18); (c) an increase in inorganic phosphate ( 12,17), phosphorylcholine (7, 17), and free amino acids (10), and a concomitant decline in nucleotides (17, 18) and glycolytic enzymatic activities (12); and (d) the more or less marked disappearance of some cell ultrastructures (1, 29).The origin of the respiratory decrease during starvation was first attributed to carbohydrate depletion, by way of limitation of the substrate either for respiration or for biosynthetic processes; however, some experiments showed that root respiration rate was not a simple function ofcarbohydrate supply (8). Journet and co-workers (12) reported that during starvation the decrease in uncoupled respiration of sycamore cells was attributable to a progressive decrease in the number of mitochondria per cell; these authors concluded that the availability of respiratory substrates for the mitochondria does not determine the respiration rate of starved cells.In the present work, we investigated changes in 02 consumption, different organic compounds (sugars, fatty acids, proteins, adenine nucleotides), enzyme activities, and physical parameters (fresh and dry weight, osmolarity) in excised maize (Zea mays) root tips from the beginning of glucose starvation to tissue dea...

Three-week-old maize (Zea mays L.) plants were submitted to light/dark cycles and to prolonged darkness to investigate the occurrence of sugar-limitation effects in different parts of the whole plant. Soluble sugars fluctuated with light/dark cycles and dropped sharply during extended darkness. Significant decreases in protein level were observed after prolonged darkness in mature roots, root tips, and young leaves. Glutamine and asparagine (Asn) changed in opposite ways, with Asn increasing in the dark. After prolonged darkness the increase in Asn accounted for most of the nitrogen released by protein breakdown. Using polyclonal antibodies against a vacuolar root protease previously described (F. James, R. Brouquisse, C. Suire, A. Pradet, P. Raymond [1996] Biochem J 320: 283-292) or the 20S proteasome, we showed that the increase in proteolytic activities was related to an enrichment of roots in the vacuolar protease, with no change in the amount of 20S proteasome in either roots or leaves. Our results show that no significant net proteolysis is induced in any part of the plant during normal light/ dark cycles, although changes in metabolism and growth appear soon after the beginning of the dark period, and starvation-related proteolysis probably appears in prolonged darkness earlier in sink than in mature tissues.Carbohydrate deprivation is a fact of life for most higher plants. When the emergence of young seedlings and their transition to autotrophy is delayed (Elamrani et al., 1994b), when competition occurs between sink tissues such as roots, flowers, or fruits (Dejong and Grossman, 1995;Ho, 1996), or when under environmental constraints, the photosynthesis rate or the translocation of nutrients to sink tissues decreases (Amthor and McCree, 1990;Setter, 1990) and higher plants may experience carbon starvation. Similarly, in some types of senescence (Noodén, 1988) or in postharvest situations (King et al., 1990), the degradation of some tissue or cell structures is clearly related to the appearance of carbon-starvation symptoms, such as a decrease in sugar content, net lipid and protein breakdown, degradation of plastids, or loss of membrane selective permeability. The metabolic consequences of carbohydrate starvation have been studied in a number of plant models, and the situation may be summarized as follows: to survive, plant cells have to adapt to the lack of carbohydrates by substituting protein and lipid metabolism for sugar metabolism through autophagic processes (James, 1953;Thomas, 1978;Saglio and Pradet, 1980;Journet et al., 1986;Roby et al., 1987; Baysdorfer et al., 1988;Peoples and Dalling, 1988;King et al., 1990; Brouquisse et al., 1991;Tassi et al., 1992;Elamrani et al., 1994b).There are many reports of degradation and synthesis of proteins during carbon deprivation. Thus, enzymic activities related to sugar metabolism and respiration (Journet et al., 1986; Brouquisse et al., 1991;Irving and Hurst, 1993), nitrogen reduction and assimilation (Thomas, 1978;Peeters and Van Laere, 1992; Brouquisse ...

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A challenging question is to define more precisely when and where reactive species are generated and to develop adapted tools to detect their production in vivo. To investigate the role of Noxs and NRs in the production of H(2)O(2) and NO, respectively, the use of mutants under the control of organ-specific promoters will be of crucial interest. The balance between ROS and NO production appears to be a key point to understand the redox regulation of symbiosis.

Nitric oxide (NO) is a gaseous molecule that participates in numerous plant signalling pathways. It is involved in plant responses to pathogens and development processes such as seed germination, flowering and stomatal closure.Using a permeable NO-specific fluorescent probe and a bacterial reporter strain expressing the lacZ gene under the control of a NO-responsive promoter, we detected NO production in the first steps, during infection threads growth, of the Medicago truncatula–Sinorhizobium meliloti symbiotic interaction. Nitric oxide was also detected, by confocal microscopy, in nodule primordia.Depletion of NO caused by cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide), an NO scavenger, resulted in a significant delay in nodule appearance. The overexpression of a bacterial hmp gene, encoding a flavohaemoglobin able to scavenge NO, under the control of a nodule-specific promoter (pENOD20) in transgenic roots, led to the same phenotype. The NO scavenging resulting from these approaches provoked the downregulation of plant genes involved in nodule development, such as MtCRE1 and MtCCS52A. Furthermore, an Hmp-overexpressing S. meliloti mutant strain was found to be less competitive than the wild type in the nodulation process.Taken together, these results indicate that NO is required for an optimal establishment of the M. truncatula–S. meliloti symbiotic interaction.

Excised maize (Zea mays L.) root tips were used to monitor the effects of prolonged glucose starvation on nitrogen metabolism. Following root-tip excision, sugar content was rapidly exhausted, and protein content declined to 40 and 8% of its initial value after 96 and 192 h, respectively. During starvation the contents of free amino acids changed. Amino acids that belonged to the same "synthetic family" showed a similar pattern of changes, indicating that their content, during starvation, is controlled mainly at the level of their common biosynthetic steps. Asparagine, which is a good marker of protein and amino-acid degradation under stress conditions, accumulated considerably until 45 h of starvation and accounted for 50% of the nitrogen released by protein degradation at that time. After 45 h of starvation, nitrogen ceased to be stored in asparagine and was excreted from the cell, first as ammonia until 90-100 h and then, when starvation had become irreversible, as amino acids and aminated compounds. The study of asparagine metabolism and nitrogen-assimilation pathways throughout starvation showed that: (i) asparagine synthesis occurred via asparagine synthetase (EC 6.3.1.1) rather than asparagine aminotransferase (EC 2.6.1.14) or the β-cyanoalanine pathway, and asparagine degradation occurred via asparaginase (EC 3.5.1.1); and (ii) the enzymic activities related to nitrogen reduction and assimilation and amino-acid synthesis decreased continuously, whereas glutamate dehydrogenase (EC 1.4.1.2-4) activities increased during the reversible period of starvation. Considered together, metabolite analysis and enzymic-activity measurements showed that starvation may be divided into three phases: (i) the acclimation phase (0 to 30-35 h) in which the root tips adapt to transient sugar deprivation and partly store the nitrogen released by protein degradation, (ii) the survival phase (30-35 to 90-100 h) in which the root tips expel the nitrogen released by protein degradation and starvation may be reversed by sugar addition and (iii) the cell-disorganization phase (beyond 100 h) in which all metabolites and enzymic activities decrease and the root tips die.

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