Ascorbate oxidase is the potential conductor of a symphony of signaling pathways.

The functional role of ascorbate oxidase (AO; EC 1.10.3.3) has never been fully explained so far, due to the difficulties in understanding the presence of an enzyme specifically oxidizing ascorbate with no obvious advantage, and the apparent disadvantage of lowering plant stress resistance as a consequence of ascorbate consumption. Here we suggest a complete change of perspective, by proposing an essential role of AO as a modulator of both ascorbate and oxygen content, with relevant implications related to signaling. By affecting the overall redox state, AO is actually involved in redox regulation in the extracellular matrix. In addition, AO can contribute to creating a hypoxic microenvironment, especially relevant in the maintenance of meristem identity and the establishment of mutualistic plant-microbe interactions. We also hypothesize the possible involvement of AO in the activation of a signaling cascade analogous to the mechanism of prolyl hydroxylases/Hypoxia Inducible Factors in animals.

An AM-induced, MYB-family gene of Lotus japonicus (LjMAMI) affects root growth in an AM-independent manner.

The interaction between legumes and arbuscular mycorrhizal (AM) fungi is vital to the development of sustainable plant production systems. Here, we focus on a putative MYB-like (LjMAMI) transcription factor (TF) previously reported to be highly upregulated in Lotus japonicus mycorrhizal roots. Phylogenetic analyses revealed that the protein is related to a group of TFs involved in phosphate (Pi) starvation responses, the expression of which is independent of the Pi level, such as PHR1. GUS transformed plants and quantitative reverse transcription PCR revealed strong gene induction in arbusculated cells, as well as the presence of LjMAMI transcripts in lateral root primordia and root meristems, even in the absence of the fungus, and independently of Pi concentration. In agreement with its putative identification as a TF, an eGFP-LjMAMI chimera was localized to the nuclei of plant protoplasts, whereas in transgenic Lotus roots expressing the eGFP-LjMAMI fusion protein under the control of the native promoter, the protein was located in the nuclei of the arbusculated cells. Further expression analyses revealed a correlation between LjMAMI and LjPT4, a marker gene for mycorrhizal function. To elucidate the role of the LjMAMI gene in the mycorrhizal process, RNAi and overexpressing root lines were generated. All the lines retained their symbiotic capacity; however, RNAi root lines and composite plants showed an important reduction in root elongation and branching in the absence of the symbiont. The results support the involvement of the AM-responsive LjMAMI in non-symbiotic functions: i.e. root growth.

Two putative-aquaporin genes are differentially expressed during arbuscular mycorrhizal symbiosis in Lotus japonicus.

BACKGROUND: Arbuscular mycorrhizas (AM) are widespread symbioses that provide great advantages to the plant, improving its nutritional status and allowing the fungus to complete its life cycle. Nevertheless, molecular mechanisms that lead to the development of AM symbiosis are not yet fully deciphered. Here, we have focused on two putative aquaporin genes, LjNIP1 and LjXIP1, which resulted to be upregulated in a transcriptomic analysis performed on mycorrhizal roots of Lotus japonicus. RESULTS: A phylogenetic analysis has shown that the two putative aquaporins belong to different functional families: NIPs and XIPs. Transcriptomic experiments have shown the independence of their expression from their nutritional status but also a close correlation with mycorrhizal and rhizobial interaction. Further transcript quantification has revealed a good correlation between the expression of one of them, LjNIP1, and LjPT4, the phosphate transporter which is considered a marker gene for mycorrhizal functionality. By using laser microdissection, we have demonstrated that one of the two genes, LjNIP1, is expressed exclusively in arbuscule-containing cells. LjNIP1, in agreement with its putative role as an aquaporin, is capable of transferring water when expressed in yeast protoplasts. Confocal analysis have demonstrated that eGFP-LjNIP1, under its endogenous promoter, accumulates in the inner membrane system of arbusculated cells. CONCLUSIONS: Overall, the results have shown different functionality and expression specificity of two mycorrhiza-inducible aquaporins in L. japonicus. One of them, LjNIP1 can be considered a novel molecular marker of mycorrhizal status at different developmental stages of the arbuscule. At the same time, LjXIP1 results to be the first XIP family aquaporin to be transcriptionally regulated during symbiosis.

Presymbiotic factors released by the arbuscular mycorrhizal fungus Gigaspora margarita induce starch accumulation in Lotus japonicus roots.

* Nutrient exchange is the key symbiotic feature of arbuscular mycorrhiza (AM). As evidence is accumulating that plants sense presymbiotic factors from AM fungi and prepare for colonization, we investigated whether modifications in plant sugar metabolism might be part of the precolonization program. * Inoculation of Lotus japonicus roots in a double Millipore sandwich with the AM fungus Gigaspora margarita prevented contact between the symbionts but allowed exchange of signal molecules. Starch content was used as a marker for root carbohydrate status. * Mycorrhizal colonization of L. japonicus roots led to a decrease in starch concentration. In roots inoculated in the double sandwich, the polysaccharide accumulated after 1 wk and persisted for at least 4 wk. The response was absent in the castor myc(-) mutant, sym4-2, while transcript levels of both CASTOR and POLLUX were slightly enhanced in the wild-type L. japonicus roots, suggesting a requirement of the corresponding proteins for the starch-accumulation response. Exudates obtained from fungal spores germinated in the absence of the plant also induced starch accumulation in wild-type L. japonicus roots. * We conclude that factors released from germinating AM fungal spores induce changes in the root carbon status, possibly by enhancing sugar import, which leads to starch accumulation when colonization is prevented.

A mycorrhizal-specific ammonium transporter from Lotus japonicus acquires nitrogen released by arbuscular mycorrhizal fungi.

In mycorrhizal associations, the fungal partner assists its plant host by providing nitrogen (N) in addition to phosphate. Arbuscular mycorrhizal (AM) fungi have access to inorganic or organic forms of N and translocate them via arginine from the extra- to the intraradical mycelium, where the N is transferred to the plant without any carbon skeleton. However, the molecular form in which N is transferred, as well as the involved mechanisms, is still under debate. NH(4)(+) seems to be the preferential transferred molecule, but no plant ammonium transporter (AMT) has been identified so far. Here, we offer evidence of a plant AMT that is involved in N uptake during mycorrhiza symbiosis. The gene LjAMT2;2, which has been shown to be the highest up-regulated gene in a transcriptomic analysis of Lotus japonicus roots upon colonization with Gigaspora margarita, has been characterized as a high-affinity AMT belonging to the AMT2 subfamily. It is exclusively expressed in the mycorrhizal roots, but not in the nodules, and transcripts have preferentially been located in the arbusculated cells. Yeast (Saccharomyces cerevisiae) mutant complementation has confirmed its functionality and revealed its dependency on acidic pH. The transport experiments using Xenopus laevis oocytes indicated that, unlike other plant AMTs, LjAMT2;2 transports NH(3) instead of NH(4)(+). Our results suggest that the transporter binds charged ammonium in the apoplastic interfacial compartment and releases the uncharged NH(3) into the plant cytoplasm. The implications of such a finding are discussed in the context of AM functioning and plant phosphorus uptake.

Genome-wide reprogramming of regulatory networks, transport, cell wall and membrane biogenesis during arbuscular mycorrhizal symbiosis in Lotus japonicus.

* Arbuscular mycorrhizas (AMs) contribute significantly to soil nutrient uptake in plants. As a consequence of the fungal colonization and of the deep reorganization shown by arbusculated cells, important impacts on root transcriptome are expected. * An Affymetrix GeneChip with 50,000 probe-sets and real-time RT-PCR allowed us to detect transcriptional changes triggered in Lotus japonicus by the AM fungus Gigaspora margarita, when arbuscules are at their maximum (28 d postinoculation (dpi)). An early time (4 dpi) was selected to differentiate genes potentially involved in signaling and/or in colonization of outer tissues. * A large number (75 out of 558) of mycorrhiza-induced genes code for proteins involved in protein turnover, membrane dynamics and cell wall synthesis, while many others are involved in transport (47) or transcription (24). Induction of a subset (24 genes) of these was tested and confirmed by qRT-PCR, and transcript location in arbusculated cells was demonstrated for seven genes using laser-dissected cells. * When compared with previously published papers, the transcript profiles indicate the presence of a core set of responsive genes (25) that seem to be conserved irrespective of the symbiotic partner identity.