[Show abstract][Hide abstract] ABSTRACT: Elongation of the primary root during post germination of Medicago truncatula seedlings is a multigenic trait responsive to exogenous nitrate. Quantitative trait loci approach suggested the involvement of the nitrate transporter MtNPF6.8 in the inhibition of primary root elongation by high exogenous nitrate. In the present work, the inhibitory effect of nitrate on the elongation of primary roots via the inhibition of the elongation of root cortical cells was abolished in npf6.8 knockdown lines. Accordingly we propose that MtNPF6.8 mediates nitrate inhibitory effect on primary root growth in M. truncatula. pMtNPF6.8/GUS promoter-reporter fusions in Agrobacterium rhizogenes-generated transgenic roots showed the expression of MtNPF6.8 in the pericycle region of primary and lateral roots (LR) and in LR primordia and tips. MtNPF6.8 expression is insensitive to IAA and stimulated by ABA, which restored the inhibitory effect of nitrate in npf6.8 knockdown lines. It is then proposed that ABA acts downstream MtNPF6.8 in this nitrate-signaling pathway. Furthermore, MtNPF6.8 was shown to transport ABA in Xenopus oocytes suggesting an additional role of MtNPF6.8 in ABA root to shoot translocation. 15NO3--influx experiments showed that only the inducible component of the low-affinity transport system (iLATS) was affected in npf6.8 knockdown mutants. This indicates that MtNPF6.8 is a major contributor to the iLATS. The short-term induction (within 30 min) by nitrate of the expression of NR1 and NR2, genes that encode two nitrate reductase isoforms, was greatly reduced in the npf6.8 knockdown lines supporting a role of MtNPF6.8 in primary nitrate response in M. truncatula.
[Show abstract][Hide abstract] ABSTRACT: Nitrate transporters received little attention in legumes probably because these species are able to adapt to N starvation by developing biological N2 fixation. Still it is important to study nitrate transport systems in legumes because nitrate intervenes as a signal in regulation of nodulation probably through nitrate transporters. The aim of the present work was to achieve a molecular characterization of NRT2 and NAR2 (NRT3) families in order to allow further works that would unravel their involvement in nitrate transport and signaling. Browsing the latest version of the M. truncatula genome annotation (v4 version) revealed three putative NRT2 members that we have named MtNRT2.1 (Medtr4g057890.1), MtNRT2.2 (Medtr4g057865.1) and MtNRT2.3 (Medtr8g069775.1) and two putative NAR2 members we named MtNAR2.1 (Medtr4g104730.1) and MtNAR2.2 (Medtr4g104700.1). The regulation and the spatial expression profiles of MtNRT2.1, the coincidence of its expression with that of MtNAR2.1 and MtNAR2.2 and the size of the encoded protein with 12 TM spanning regions strongly support the idea that MtNRT2.1 is a nitrate transporter with a major contribution to the HATS, while a very low level of expression characterized MtNRT2.2. Unlike MtNRT2.1, MtNRT2.3 showed a lower level of expression in the root system but was expressed in the shoots and in the nodules thus suggesting an involvement of the encoded protein in nitrate transport inside the plant and/or in nitrate signaling pathways controlling post-inoculation processes that govern nodule functioning.
Physiologia Plantarum 11/2014; · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide (NO) production and amino acid metabolism modulation, in particular abscisic acid (ABA)-dependent proline accumulation, are stimulated in planta by most abiotic stresses. However, the relationship between NO production and proline accumulation under abiotic stress is still poorly understood, especially in the early phases of plant development. To unravel this question, this work investigated the tight relationship between NO production and proline metabolism under water-deficit stress during seedling establishment. Endogenous nitrate reductase-dependent NO production in Medicago truncatula seedlings increased in a time-dependent manner after short-term water-deficit stress. This water-deficit-induced endogenous NO accumulation was mediated through a ABA-dependent pathway and accompanied by an inhibition of seed germination, a loss of water content, and a decrease in elongation of embryo axes. Interestingly, a treatment with a specific NO scavenger (cPTIO) alleviated these water-deficit detrimental effects. However, the content of total amino acids, in particular glutamate and proline, as well as the expression of genes encoding enzymes of synthesis and degradation of proline were not affected by cPTIO treatment under water-deficit stress. Under normal conditions, exogenous NO donor stimulated neither the expression of P5CS2 nor the proline content, as observed after PEG treatment. These results strongly suggest that the modulation of proline metabolism is independent of NO production under short-term water-deficit stress during seedling establishment.
Journal of Experimental Botany 03/2014; · 5.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Stress associated proteins (SAP) have been already reported to play a role in tolerance acquisition of some abiotic stresses. In the present study, the role of MtSAP1 (Medicago truncatula) in tolerance to temperature, osmotic and salt stresses has been studied in tobacco transgenic seedlings. Compared to wild type, MtSAP1 overexpressors were less affected in their growth and development under all tested stress conditions. These results confirm that MtSAP1 is involved in the response processes to various abiotic constraints. In parallel, we have performed studies on an eventual link between MtSAP1 overexpression and proline, a major player in stress response. In an interesting way, the results for the transgenic lines did not show any increase of proline content under osmotic and salt stress, contrary to the WT which usually accumulated proline in response to stress. These data strongly suggest that MtSAP1 is not involved in signaling pathway responsible for the proline accumulation in stress conditions. This could be due to the fact that the overexpression of MtSAP1 provides sufficient tolerance to seedlings to cope with stress without requiring the free proline action. Beyond that, the processes by which the MtSAP1 overexpression lead to the suppression of proline accumulation will be discussed in relation with data from our previous study involving nitric oxide.
Journal of plant physiology 02/2013; · 2.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The impact of Medicago truncatula stress-associated protein gene (MtSAP1) overexpression has been investigated in Nicotiana tabacum transgenic seedlings. Under optimal conditions, transgenic lines overexpressing MtSAP1 revealed better plant development and higher chlorophyll content as compared to wild type seedlings. Interestingly, transgenic lines showed a stronger accumulation of nitric oxide (NO), a signaling molecule involved in growth and development processes. This NO production seemed to be partially nitrate reductase dependent. Due to the fact that NO has been also reported to play a role in tolerance acquisition of plants to abiotic stresses, the responses of MtSAP1 overexpressors to osmotic and salt stress have been studied. Compared to the wild type, transgenic lines were less affected in their growth and development. Moreover, NO content in MtSAP1 overexpressors was always higher than that detected in wild seedlings under stress conditions. It seems that this better tolerance induced by MtSAP1 overexpression could be associated with this higher NO production that would enable seedlings to reach a high protection level to prepare them to cope with abiotic stresses.
[Show abstract][Hide abstract] ABSTRACT: Effects of water deficit and/or abscisic acid (ABA) were investigated on early seedling growth of Medicago truncatula, and on glutamate metabolism under dark conditions. Water deficit (simulated by polyethylene glycol, PEG), ABA and their combination resulted in a reduction in growth rate of the embryo axis, and also in a synergistic increase of free amino acid (AA) content. However, the inhibition of water uptake retention induced by water deficit seemed to occur in an ABA-independent manner. Expression of several genes involved in glutamate metabolism was induced during water deficit, whereas ABA, in combination or not with PEG, repressed them. The only exception came from a gene encoding 1-pyrroline-5-carboxylate synthetase (P5CS) which appeared to be induced in an ABA-dependent manner under water deficit. Our results demonstrate clearly the involvement of an ABA-dependent and an ABA-independent regulatory system, governing growth and glutamate metabolism under water deficit.
[Show abstract][Hide abstract] ABSTRACT: MtSAP1 (Medicago truncatula stress-associated protein 1) was revealed as a down-regulated gene by suppressive subtractive hybridization between two mRNA populations of embryo axes harvested before and after radicle emergence. MtSAP1 is the first gene encoding a SAP with A20 and AN1 zinc-finger domains characterized in M. truncatula. MtSAP1 protein shares 54% and 62% homology with AtSAP7 (Arabidopsis thaliana) and OsiSAP8 (Oryza sativa) respectively, with in particular a strong homology in the A20 and AN1 conserved domains. MtSAP1 gene expression increased in the embryos during the acquisition of tolerance to desiccation, reached its maximum in dry seed and decreased dramatically during the first hours of imbibition. Abiotic stresses (cold and hypoxia), abscisic acid and desiccation treatments induced MtSAP1 gene expression and protein accumulation in embryo axis, while mild drought stress did not affect significantly its expression. This profile of expression along with the presence of anaerobic response elements and ABRE sequences in the upstream region of the gene is consistent with a role of MtSAP1 in the tolerance of low oxygen availability and desiccation during late stages of seed maturation. Silencing of MtSAP1 by RNA interference (RNAi) showed that the function of the encoded protein is required for adequate accumulation of storage globulin proteins, vicilin and legumin, and for the development of embryos able to achieve successful germination.
Plant Physiology and Biochemistry 03/2011; 49(3):303-10. · 2.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The modulation of primary nitrogen metabolism by water deficit through ABA-dependent and ABA-independent pathways was investigated in the model legume Medicago truncatula. Growth and glutamate metabolism were followed in young seedlings growing for short periods in darkness and submitted to a moderate water deficit (simulated by polyethylene glycol; PEG) or treated with ABA. Water deficit induced an ABA accumulation, a reduction of axis length in an ABA-dependent manner, and an inhibition of water uptake/retention in an ABA-independent manner. The PEG-induced accumulation of free amino acids (AA), principally asparagine and proline, was mimicked by exogenous ABA treatment. This suggests that AA accumulation under water deficit may be an ABA-induced osmolyte accumulation contributing to osmotic adjustment. Alternatively, this accumulation could be just a consequence of a decreased nitrogen demand caused by reduced extension, which was triggered by water deficit and exogenous ABA treatment. Several enzyme activities involved in glutamate metabolism and genes encoding cytosolic glutamine synthetase (GS1b; EC 220.127.116.11.), glutamate dehydrogenase (GDH3; EC 18.104.22.168.), and asparagine synthetase (AS; EC 22.214.171.124.) were up-regulated by water deficit but not by ABA, except for a gene encoding Δ(1)-pyrroline-5-carboxylate synthetase (P5CS; EC not assigned). Thus, ABA-dependent and ABA-independent regulatory systems would seem to exist, differentially controlling development, water content, and nitrogen metabolism under water deficit.
Journal of Experimental Botany 10/2010; 62(2):605-15. · 5.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The modulation of primary nitrogen metabolism by hypoxic stress was studied in young Medicago truncatula seedlings. Hypoxic seedlings were characterized by the up-regulation of glutamate dehydrogenase 1 (GDH1) and mitochondrial alanine aminotransferase (mAlaAT), and down-regulation of glutamine synthetase 1b (GS1b), NADH-glutamate synthase (NADH-GOGAT), glutamate dehydrogenase 3 (GDH3), and isocitrate dehydrogenase (ICDH) gene expression. Hypoxic stress severely inhibited GS activity and stimulated NADH-GOGAT activity. GDH activity was lower in hypoxic seedlings than in the control, however, under either normoxia or hypoxia, the in vivo activity was directed towards glutamate deamination. (15)NH(4) labelling showed for the first time that the adaptive reaction of the plant to hypoxia consisted of a concerted modulation of nitrogen flux through the pathways of both alanine and glutamate synthesis. In hypoxic seedlings, newly synthesized (15)N-alanine increased and accumulated as the major amino acid, asparagine synthesis was inhibited, while (15)N-glutamate was synthesized at a similar rate to that in the control. A discrepancy between the up-regulation of GDH1 expression and the down-regulation of GDH activity by hypoxic stress highlighted for the first time the complex regulation of this enzyme by hypoxia. Higher rates of glycolysis and ethanol fermentation are known to cause the fast depletion of sugar stores and carbon stress. It is proposed that the expression of GDH1 was stimulated by hypoxia-induced carbon stress, while the enzyme protein might be involved during post-hypoxic stress contributing to the regeneration of 2-oxoglutarate via the GDH shunt.
Journal of Experimental Botany 02/2008; 59(9):2325-35. · 5.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The major purpose of the cytosolic enzyme nitrate reductase (NR, EC 126.96.36.199) is to catalyse the reduction of nitrate to nitrite
at the expense of NAD(P)H. In addition to that, however, NR also catalyses, though with much lower capacity, two side reactions:
1) the reduction of nitrite to nitric oxide (NO) and 2) the reduction of molecular oxygen to superoxide (Ruoff and Lillo,
1990, Barber and Kay 1996), which can both react chemically to give the highly toxic compound peroxynitrite (Figure 1, Yamasaki
and Sakihama 2000). The production of such potentially toxic products (of which NO may also act as a signalling molecule in
plants) is probably one reason why NR expression and activity are so tightly controlled.
For regulation of NR expression and activity by light see chapter 6, Lillo (1994) and Lillo and Appenroth (2001). In leaves,
NR becomes more active upon illumination within minutes, and is as rapidly inactivated in the dark. Based on these short response
times it was suggested that NR activity would not be modulated via changes in transcription or translation, but via modulation
of the catalytic activity of the existing NR protein. More recently, in plants expressing NR under the control of the constitutive
35S-promoter, rapid light/dark changes in extractable NR activity from leaves were still observed, although mRNA levels expectedly
did not vary in dark/light (Vincentz et al. 1993). Such observations strongly confirm that the existing NR molecule undergoes a reversible modulation in catalytic activity.
In the past decade, the mechanism of the post-translational modulation has been investigated in detail.
[Show abstract][Hide abstract] ABSTRACT: There is now general agreement that nitric oxide (NO) is an important and almost universal signal in plants. Nevertheless, there are still many controversial observations and opinions on the importance and function of NO in plants. Partly, this may be due to the difficulties in detecting and even more in quantifying NO. Here, we summarize major pathways of NO production in plants, and briefly discuss some methodical problems.
[Show abstract][Hide abstract] ABSTRACT: A hypersensitive response (HR) was induced in tobacco leaves and cell suspensions by the fungal elicitor cryptogein, and NO production was followed by chemiluminescence and occasionally by diaminofluorescein (DAF)-fluorescence. Results from both methods were at least partly consistent, but kinetics was different. NO emission was not induced by cryptogein in leaves, whereas in cell suspensions some weak NO emission was observed, which was nitrate reductase (NR)-dependent, but not required for cell death. Nitric oxide synthase (NOS) inhibitors did not prevent cell death, but PR-1 expression was weakened. In conclusion, neither NR nor NOS appear obligatory for the cryptogein-induced HR. However, a role for NO was still suggested by the fact that the NO scavenger cPTIO prevented the HR. Unexpectedly, cPTI, the reaction product of cPTIO and NO, also impaired the HR but without scavenging NO. Thus, prevention of the HR by cPTIO is not necessarily indicative for a role of NO. Further, even a 100-fold NO overproduction (over wild type) by a nitrite reductase-deficient mutant did not interfere with the cryptogein-induced HR. Accordingly, the role of NO in the HR should be reconsidered.
Plant Cell and Environment 02/2006; 29(1):59-69. · 5.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Because of controversies in the literature on nitric oxide (NO) production by plants, NO detection by the frequently used diaminofluorescein (DAF-2 and DAF-2DA) and by chemiluminescence were compared using the following systems of increasing complexity: (i) dissolved NO gas; (ii) the NO donor sodium nitroprusside (SNP); (iii) purified nitrate reductase (NR); and (iv) tobacco cell suspensions. Low (physiological) concentrations (< or =1 nM) of dissolved NO could be precisely quantified by chemiluminescence, but caused no DAF-2 fluorescence. In contrast to NO gas, SNP, NR, or cell suspensions produced both good DAF fluorescence and chemiluminescence signals which were completely (chemiluminescence) or partly (DAF fluorescence) prevented by NO scavengers. Signal strength ratios between the two methods were variable depending on the NO source, and eventually reflect variable NO oxidation. DAF fluorescence in cell suspension cultures was also increased by an as yet unidentified compound(s) released from cells into the medium. These compounds gave no chemiluminescence signal and were not produced by NR-free mutants. Their production was stimulated by anoxia, by inhibitors of mitochondrial electron transport, and by the fungal elicitor cryptogein. Thus, changes in DAF fluorescence are not necessarily indicative for NO production, but may also reflect NO oxidation and/or production of other DAF-reactive compounds.
Journal of Experimental Botany 02/2006; 57(12):3043-55. · 5.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Quantitative data on nitric oxide (NO) production by plants, and knowledge of participating reactions and rate limiting factors are still rare. We quantified NO emission from tobacco (Nicotiana tabacum) wild-type leaves, from nitrate reductase (NR)- or nitrite reductase (NiR)-deficient leaves, from WT- or from NR-deficient cell suspensions and from mitochondria purified from leaves or cells, by following NO emission through chemiluminescence detection. In all systems, NO emission was exclusively due to the reduction of nitrite to NO, and the nitrite concentration was an important rate limiting factor. Using inhibitors and purified mitochondria, mitochondrial electron transport was identified as a major source for reduction of nitrite to NO, in addition to NR. NiR and xanthine dehydrogenase appeared to be not involved. At equal respiratory activity, mitochondria from suspension cells had a much higher capacity to produce NO than leaf mitochondria. NO emission in vivo by NiR-mutant leaves (which was not nitrite limited) was proportional to photosynthesis (high in light +CO(2), low in light -CO(2), or in the dark). With most systems including mitochondrial preparations, NO emission was low in air (and darkness for leaves), but high under anoxia (nitrogen). In contrast, NO emission by purified NR was not much different in air and nitrogen. The low aerobic NO emission of darkened leaves and cell suspensions was not due to low cytosolic NADH, and appeared only partly affected by oxygen-dependent NO scavenging. The relative contribution of NR and mitochondria to nitrite-dependent NO production is estimated.
The Plant Journal 04/2005; 41(5):732-43. · 6.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Wild type (WT), and nitrate reductase (NR)- and nitrite-reductase (NiR)-deficient cells of Chlorella sorokiniana were used to characterize nitric oxide (NO) emission. The NO emission from nitrate-grown WT cells was very low in air, increased slightly after addition of nitrite (200 microM), but strongly under anoxia. Importantly, even completely NR-free mutants, as well as cells grown on tungstate, emitted NO when fed with nitrite under anoxia. Therefore, this NO production from nitrite was independent of NR and other molybdenum cofactor enzymes. Cyanide and inhibitors of mitochondrial complex III, myxothiazol or antimycin A, but not salicylhydroxamic acid (inhibitor of alternative oxidase) inhibited NO production by NR-free cells. In contrast, NiR-deficient cells growing on nitrate accumulated nitrite and emitted NO at very high equal rates in air and anoxia. This NO emission was 50% inhibited by salicylhydroxamic acid, indicating that in these cells the alternative oxidase pathway had been induced and reduced nitrite to NO.
[Show abstract][Hide abstract] ABSTRACT: The mechanism of the post-translational modulation of nitrate reductase activity (NR, EC 188.8.131.52) is briefly summarized, and it is shown that by this mechanism nitric oxide production through NR is also rapidly modulated. New and partly unexpected details on the modulation mechanism have been obtained by using immunological techniques. The phosphorylation state of NR has been assessed with peptide antibodies raised against the serine phosphorylation motive of spinach NR. By co-immunoprecipitation experiments, 14-3-3 binding to phospho-NR and the function of Mg(2+) in that process has been elucidated. Conflicting data on the role of NR phosphorylation and 14-3-3 binding in controlling NR proteolysis are discussed. A possible role of other NR inactivating proteins is also briefly considered and the regulation of NR of Ricinus communis is described as an interesting special case that differs from the 'normal' mechanism in several important aspects.
Journal of Experimental Botany 05/2002; 53(370):875-82. · 5.79 Impact Factor