Arbuscular mycorrhizal fungi (AMF) occur in the rhizosphere and in plant tissues as obligate symbionts, having key roles in plant evolution and nutrition. AMF possess endobacteria, and genome sequencing of the endobacterium Candidatus Glomeribacter gigasporarum revealed a reduced genome and a dependence on the fungal host. To understand the effect of bacteria on fungal fitness, we used next-generation sequencing to analyse the transcriptional profile of Gigaspora margarita in the presence and in the absence of its endobacterium. Genomic data on AMF are limited; therefore, we first generated a gene catalogue for G. margarita. Transcriptome analysis revealed that the endobacterium has a stronger effect on the pre-symbiotic phase of the fungus. Coupling transcriptomics with cell biology and physiological approaches, we demonstrate that the bacterium increases the fungal sporulation success, raises the fungal bioenergetic capacity, increasing ATP production, and eliciting mechanisms to detoxify reactive oxygen species. By using TAT peptide to translocate the bioluminescent calcium reporter aequorin, we demonstrated that the line with endobacteria had a lower basal intracellular calcium concentration than the cured line. Lastly, the bacteria seem to enhance the fungal responsiveness to strigolactones, the plant molecules that AMF perceive as branching factors. Although the endobacterium exacts a nutritional cost on the AMF, endobacterial symbiosis improves the fungal ecological fitness by priming mitochondrial metabolic pathways and giving the AMF more tools to face environmental stresses. Thus, we hypothesise that, as described for the human microbiota, endobacteria may increase AMF innate immunity.

The implication of calcium as intracellular messenger in the arbuscular mycorrhizal (AM) symbiosis has not yet been directly demonstrated, although often envisaged. We used soybean (Glycine max) cell cultures stably expressing the bioluminescent Ca 21 indicator aequorin to detect intracellular Ca 21 changes in response to the culture medium of spores of Gigaspora margarita germinating in the absence of the plant partner. Rapid and transient elevations in cytosolic free Ca 21 were recorded, indicating that diffusible molecules released by the mycorrhizal fungus are perceived by host plant cells through a Ca 21 -mediated signaling. Similar responses were also triggered by two Glomus isolates. The fungal molecules active in generating the Ca 21 transient were constitutively released in the medium, and the induced Ca 21 signature was not modified by the coculture of germinating spores with plant cells. Even ungerminated spores were able to generate the signaling molecules, as proven when the germination was blocked by a low temperature. The fungal molecules were found to be stable to heat treatment, of small molecular mass (,3 kD), and, on the basis of extraction with an organic solvent, partially lipophilic. Evidence for the specificity of such an early fungal signal to the AM symbiosis is suggested by the lack of a Ca 21 response in cultured cells of the nonhost plant Arabidopsis (Arabidopsis thaliana) and by the up-regulation in soybean cells of genes related to Medicago truncatula DMI1, DMI2, and DMI3 and considered essential for the establishment of the AM symbiosis.

Calcium (Ca2+) is among the most important intracellular messengers in living organisms. Understanding of the players and dynamics of Ca2+ signalling pathways in plants may help to unravel the molecular basis of their exceptional flexibility to respond and to adapt to different stimuli. In the present review we focus on new tools that have recently revolutionized our view of organellar Ca2+ signalling as well as on the current knowledge regarding the pathways mediating Ca2+ fluxes across intracellular membranes. The contribution of organelles and cellular subcompartments to the orchestrated response via Ca2+ signalling within a cell is also discussed, underlining the fact that one of the greatest challenges in the field is the elucidation of how influx and efflux Ca2+ transporters/channels are regulated in a concerted manner to translate specific information into a Ca2+ signature.

Higher plants share with animals a responsiveness to the Ca 2؉ mobilizing agents inositol 1,4,5-trisphosphate (InsP3) and cyclic ADP-ribose (cADPR). In this study, by using a vesicular 45 Ca 2؉ flux assay, we demonstrate that microsomal vesicles from red beet and cauliflower also respond to nicotinic acid adenine dinucleotide phosphate (NAADP), a Ca 2؉ -releasing molecule recently described in marine invertebrates. NAADP potently mobilizes Ca 2؉ with a K 1/2 ‫؍‬ 96 nM from microsomes of nonvacuolar origin in red beet. Analysis of sucrose gradient-separated cauliflower microsomes revealed that the NAADP-sensitive Ca 2؉ pool was derived from the endoplasmic reticulum. This exclusively nonvacuolar location of the NAADP-sensitive Ca 2؉ pathway distinguishes it from the InsP3-and cADPR-gated pathways. Desensitization experiments revealed that homogenates derived from cauliflower tissue contained low levels of NAADP (125 pmol͞mg) and were competent in NAADP synthesis when provided with the substrates NADP and nicotinic acid. NAADP-induced Ca 2؉ release is insensitive to heparin and 8-NH2-cADPR, specific inhibitors of the InsP3-and cADPR-controlled mechanisms, respectively. However, NAADP-induced Ca 2؉ release could be blocked by pretreatment with a subthreshold dose of NAADP, as previously observed in sea urchin eggs. Furthermore, the NAADP-gated Ca 2؉ release pathway is independent of cytosolic free Ca 2؉ and therefore incapable of operating Ca 2؉ -induced Ca 2؉ release. In contrast to the sea urchin system, the NAADPgated Ca 2؉ release pathway in plants is not blocked by L-type channel antagonists. The existence of multiple Ca 2؉ mobilization pathways and Ca 2؉ release sites might contribute to the generation of stimulus-specific Ca 2؉ signals in plant cells.