Caractérisation du transporteur de nitrate à double affinité, MtNPF6.8 (MtNRT1.3), de Medicago truncatula : rôles dans le transport et la perception du signal nitrate

Nitrate, a major nitrogen source for most plants, is not only a nutrient but also a signaling molecule. However, there are contrasting responses to nitrate between different higher plants. In the model legume Medicago truncatula, nitrate has an inhibitory effect on the primary root growth in post-germination phase. A quantitative genetic study has shown that a nitrate transporter is localized at the peak of a QTL involved in the primary root growth. Functional characterization of the transporter, named MtNRT1.3 and renamed MtNPF6.8, showed that it encodes a dual affinity nitrate transporter. MtNPF6.8 is likely to participate in the nitrate influx in the plant. After obtaining three knockdown lines by RNA interference, experiments using K15NO3 showed that this transporter is effect involved in nitrate influx related to the inducible low affinity transport system (iLATS). However, mutation in MtNPF6.8 does not any effect on nitrogen metabolism. In addition, studies on the primary root growth have confirmed the involvement of the transporter on phenotypic trait. In wild-type plants, cortical cell size decreased after nitrate treatment, showing that primary root growth was due to this reduced cell elongation. The possibility that ABA also plays a role in mediating this nitrate dependent response is heavily favored. All these results, reinforced by a study of mutants expressing this transporter in A. thaliana, indicate that MtNPF6.8 is a nitrate sensor for Medicago in the post-germination phase, independently of its nitrate transport activity

Involvement of Medicago truncatula glutamate receptor-like channels in nitric oxide production under short-term water deficit stress

Early stages of plant development are highly susceptible to environmental cues, and seedlings have to develop sophisticated mechanisms to sense and respond to abiotic stresses. We have previously identified that abscisic acid (ABA), nitric oxide (NO) and modulation of nitrogen metabolism are involved in adaptive responses in Medicago truncatula seedlings under water deficit stress. Here, we investigated whether glutamate receptor-like channels (GLRs) played a role in the developmental physiological processes of Medicago seedlings during post-germination after a short-term water deficit stress. Twenty-nine independent MtGLR genes have been identified and then divided into four clades following a phylogenetic analysis; seventeen of them exhibited specific domains which are characteristic of animal ionotropic glutamate receptors. Under drought stress, ABA-induced NO accumulation was significantly reduced in presence of a GLR competitive antagonist, suggesting that this water deficit-induced endogenous NO production was mediated through a MtGLR-dependent pathway. Water deficit-induced inhibition of embryo axis elongation was strongly reduced whereas loss of water content was alleviated when MtGLRs were inhibited. These results suggest that glutamate receptors-like channels are required, through their involvement in NO production, in adaptive responses under short-term water-deficit stress during Medicago seedling establishment

Nitrate transporters : an overview in legumes

Main conclusion The nitrate transporters, belonging to NPF and NRT2 families, play critical roles in nitrate signaling, root growth and nodule development in legumes. Nitrate plays an essential role during plant development as nutrient and also as signal molecule, in both cases working via the activity of nitrate transporters. To date, few studies on NRT2 or NPF nitrate transporters in legumes have been reported, and most of those concern Lotus japonicus and Medicago truncatula. A molecular characterization led to the identification of 4 putative LjNRT2 and 37 putative LjNPF gene sequences in L. japonicus. In M. truncatula, the NRT2 family is composed of 3 putative members. Using the new genome annotation of M. truncatula (Mt4.0), we identified, for this review, 97 putative MtNPF sequences, including 32 new sequences relative to previous studies. Functional characterization has been published for only two MtNPF genes, encoding nitrate transporters of M. truncatula. Both transporters have a role in root system development via abscisic acid signaling: MtNPF6.8 acts as a nitrate sensor during the cell elongation of the primary root, while MtNPF1.7 contributes to the cellular organization of the root tip and nodule formation. An in silico expression study of MtNPF genes confirmed that NPF genes are expressed in nodules, as previously shown for L. japonicus, suggesting a role for the corresponding proteins in nitrate transport, or signal perception in nodules. This review summarizes our knowledge of legume nitrate transporters and discusses new roles for these proteins based on recent discoveries

Deciphering the nitrate signaling pathway leading to a reduction of primary root growth in Medicago truncatula

In the model legume Medicago truncatula, nitrate has been shown to inhibit primary root growth through the reduction of root cell elongation. Nitrate, as an essential nutrient, also acts as a signal molecule that is sensed and transduced through a nitrate transporter MtNPF6.8, with RNAi mutants deficient in MtNPF6.8 being insensitive to nitrate [1, 2]. We tested here whether reactive oxygen species (ROS) could be downstream mediators of the nitrate signal since ROS are able to transduce ABA signal in other contexts and also govern the primary root growth. Thus, we analyzed the distribution of ROS (H2O2, O2•−, •OH) and peroxidase activity all along the primary root of seedlings sensitive or insensitive to nitrate using different genotypes of M. truncatula, three wild types and a npf6.8RNAi mutant grown with or without nitrate, to determine whether nitrate modifies ROS and peroxidase patterns. We found that nitrate modified the morphology of the root tip, induced an increase in H2O2, and a decrease in O2•− and •OH in seedlings sensitive to nitrate (R108, A17, and DZA315-16), but not in seedlings insensitive to nitrate (npf6.8RNAi mutant). These results suggest that ROS and peroxidases are downstream mediators in the nitrate signaling pathway. The origin of the change in ROS accumulation in response to nitrate was further investigated following the activity of major enzymes (peroxidase, SOD, Nox) able to interfere with ROS accumulation

Effects of secondary compounds from cactus and acacias trees on rumen microbial profile changes performed by Real-Time PCR

Plant rich secondary compounds had antimicrobial effects by acting against different rumen microbial populations. The current study investigated the influence of spineless cactus (Opuntia ficus indica f. inermis), Acacia nilotica and A. saligna on rumen microbial fermentation, using in vitro gas production technique, and microbial population profile changes, using a molecular-based technique (Real-Time PCR). The acacias and Opuntia reduced significantly total gas production (p<0.01), rumen CH4 production (p?0.01) and ammonia concentration (p<0.001). At 24h of incubation, Fungi population was 0.30- and 0.03 -fold reduced with A.nilotica and Opuntia as compared to 0h, but 2-and 1.24- fold higher with A.cyanophylla .Increases in the abundance of F.succinogenes were observed in all substrates; however, the tanniferous plants and Opuntia reduced the relative abundance of R.flavefaciens. Methanogenic population was increased with all substrates, except for Opuntia (0. 90- fold lower than the control). There was a significant reduction (p<0.05) in rumen protozoa count with A.cyanophylla, Opuntia and A.nilotica (3.68; 5.59 and 5.34 times, respectively). Results suggested that tannin sources from A.nilotica and A.cyanophylla had an indirect effect on methanogenesis. This study showed an antimicrobial activity of oxalates content of O. ficus indica

cDNA-AFLP profiling in the embryo axes during common bean germination

cDNA-AFLP fingerprinting was used to identify genes with modulated expression during germination in common bean (Phaseolus vulgaris L.). The analysis was performed on the embryo axes. Nine time points covering the whole germination were considered and 800 transcript-derived fragments (PvTDFs) were scored. Among them, 80 % showed no changes during germination. The 97 PvTDFs showing differential expressions during germination were sequenced along with 14 constant transcripts that were randomly chosen. The expression of seven variable PvTDFs was confirmed by real-time RT-PCR. We observed that 92 % of the transcript changes, including 35 % of appearing mRNAs, took place before radicule protrusion, 0–17 h after imbibition (HAI). A major shift in gene expression was observed between 9 and 14 HAI, suggesting a key moment of cell re-programming. Sequence homologies were found for 52 % of the sequenced PvTDFs. The identified transcripts encode proteins belonging to several functional groups including transcription factors, proteins involved in storage compound hydrolysis, cell elongation or oxidative stress protection