Arbuscular mycorrhiza-induced shifts in foliar metabolism and photosynthesis mirror the developmental stage of the symbiosis and are only partly driven by improved phosphate uptake.

In arbuscular mycorrhizal (AM) plants, the plant delivers photoassimilates to the arbuscular mycorrhizal fungus (AMF), whereas the mycosymbiont contributes, in addition to other beneficial effects, to phosphate (PO4(3-)) uptake from the soil. Thereby, the additional fungal carbon (C) sink strength in roots and improved plant PO4(3-) nutrition may influence aboveground traits. We investigated how the foliar metabolome of Plantago major is affected along with the development of root symbiosis, whether the photosynthetic performance is affected by AM, and whether these effects are mediated by improved PO4(3-) nutrition. Therefore, we studied PO4(3-)-limited and PO4(3-)-supplemented controls in comparison with mycorrhizal plants at 20, 30, and 62 days postinoculation with the AMF Rhizophagus irregularis. Foliar metabolome modifications were determined by the developmental stage of symbiosis, with changes becoming more pronounced over time. In a well-established stage of mature mutualism, about 60% of the metabolic changes and an increase in foliar CO2 assimilation were unrelated to the significantly increased foliar phosphorus (P) content. We propose a framework relating the time-dependent metabolic changes to the shifts in C costs and P benefits for the plant. Besides P-mediated effects, the strong fungal C sink activity may drive the changes in the leaf traits.

High specificity in plant leaf metabolic responses to arbuscular mycorrhiza.

The chemical composition of plants (phytometabolome) is dynamic and modified by environmental factors. Understanding its modulation allows to improve crop quality and decode mechanisms underlying plant-pest interactions. Many studies that investigate metabolic responses to the environment focus on single model species and/or few target metabolites. However, comparative studies using environmental metabolomics are needed to evaluate commonalities of chemical responses to certain challenges. We assessed the specificity of foliar metabolic responses of five plant species to the widespread, ancient symbiosis with a generalist arbuscular mycorrhizal fungus. Here we show that plant species share a large 'core metabolome' but nevertheless the phytometabolomes are modulated highly species/taxon-specifically. Such a low conservation of responses across species highlights the importance to consider plant metabolic prerequisites and the long time of specific plant-fungus coevolution. Thus, the transferability of findings regarding phytometabolome modulation by an identical AM symbiont is severely limited even between closely related species.

The signal peptide of the Medicago truncatula modular nodulin MtNOD25 operates as an address label for the specific targeting of proteins to nitrogen-fixing symbiosomes.

The nodule-specific MtNOD25 gene of the model legume Medicago truncatula encodes a modular nodulin composed of different repetitive modules flanked by distinct N- and C-termini. Although similarities are low with respect to all repetitive modules, both the N-terminal signal peptide (SP) and the C-terminus are highly conserved in modular nodulins from different legumes. On the cellular level, MtNOD25 is only transcribed in the infected cells of root nodules, and this activation is mediated by a 299-bp minimal promoter containing an organ-specific element. By expressing mGFP6 translational fusions in transgenic nodules, we show that MtNOD25 proteins are exclusively translocated to the symbiosomes of infected cells. This specific targeting only requires an N-terminal MtNOD25 SP that is highly conserved across a family of legume-specific symbiosome proteins. Our finding sheds light on one possible mechanism for the delivery of host proteins to the symbiosomes of infected root nodule cells and, in addition, defines a short molecular address label of only 24 amino acids whose N-terminal presence is sufficient to translocate proteins across the peribacteroid membrane.

Antisense repression of the Medicago truncatula nodule-enhanced sucrose synthase leads to a handicapped nitrogen fixation mirrored by specific alterations in the symbiotic transcriptome and metabolome.

We analyzed the role of the sucrose (Suc) synthase MtSucS1 during nodulation of the model legume Medicago truncatula, integrating data for the developmental, transcriptional, and metabolic processes affected downstream of an impaired Suc cleavage in root nodules. To reduce carbohydrate supply to nodule tissues, transgenic plants expressing a p35S-driven MtSucS1-antisense fusion were constructed. These plants displayed an up to 90% reduction of MtSucS1 proteins in roots and nodules. Phenotypic studies of two independent MtSucS1-reduced lines demonstrated that only under conditions depending on nodulation, these plants appeared to be impaired in above-ground growth. Specifically plant height, shoot weight, leaf development, flowering, as well as seed maturation were reduced, and the efficiency of photosynthesis was affected. Concomitantly, a significantly enhanced root to shoot ratio with a marked increase in root tip numbers was observed. Root nodule formation was found retarded and the impaired nodulation was accompanied by a less efficient nitrogen (N) acquisition. The decreased total N content of MtSucS1-antisense lines and an enhanced carbon to N ratio in roots, nodules, and shoots correlated with the extent of MtSucS1 knockdown. On the level of transcription, effects of an MtSucS1 reduction were evident for genes representing important nodes of the nodule carbon and N metabolism, while metabolite profiling revealed significantly lower levels of amino acids and their derivatives particularly in strongly MtSucS1-reduced nodules. Our results support the model that nodule-enhanced Suc synthase 1 of the model legume M. truncatula is required for the establishment and maintenance of an efficient N-fixing symbiosis.

Identification and expression regulation of symbiotically activated legume genes.

Legume plants are able to enter two different endosymbioses with soil prokaryotes and soil fungi, leading to nitrogen-fixing root nodules and to arbuscular mycorrhiza (AM), respectively. We applied in silico and microarray-based transcriptome profiling approaches to uncover the transcriptome of developing root nodules and AM roots of the model legume Medicago truncatula. Several hundred genes were found to be activated in different stages of either symbiosis, with almost 100 genes being co-induced during nodulation and in arbuscular mycorrhiza. These co-induced genes can be associated with different cellular functions required for symbiotic efficiency, such as the facilitation of transport processes across the perisymbiotic membranes that surround the endosymbiotic bacteroids in root nodules and the arbuscules in AM roots. To specify promoter elements required for gene expression in arbuscule-containing cells, reporter gene fusions of the promoter of the Vicia faba leghemoglobin gene VfLb29 were studied by loss-of-function and gain-of-function approaches in transgenic hairy roots. These analyses specified a 85-bp fragment that was necessary for gene expression in arbuscule-containing cells but was dispensible for gene activation in root nodules. In contrast to promoters mediating gene expression in the infected cells of root nodules, the activation of genes in AM appears to be governed by more complex regulatory systems requiring different promoter modules.