Towards the unveiling of the molecular interactions connecting subcellular Molybdenum and Iron homeostasis

Molybdenum (Mo) is a transition metal and micronutrient, which is essentially required in the form of molybdate by nearly all organisms including plants [1]. Molybdate is complexed by a Mo-binding pterin to form the biologically functional molybdenum cofactor (Moco), which is inserted into the so-called molybdo-enzymes. Mo is therefore involved in essential or important metabolic processes such as nitrogen assimilation, ABA synthesis, and purine catabolism and impaired Mo metabolism has dramatic effects on plant growth and yield. Most recently, a significant crosstalk between the metabolisms of iron (Fe) and Mo has been exposed [2]. Our goal is to investigate the mutual regulation of basic mechanisms underlying uptake and subcellular compartmentalization of Mo and Fe in Cucumis sativus (cucumber) plants grown under different Mo and Fe nutritional conditions. For that, cucumber plants have been grown in the following conditions: control, Fe deficiency, Mo deficiency or both deficiencies. The ionomes of whole tissues (root and leaves) as well as of root mitochondria have been profiled. Moreover, a high-throughput proteomic approach named “MudPIT” (Multidimentional Protein Identification Technology) [3], has been applied to identify novel proteins involved in Mo and/or Fe metabolism (such as molybdate storage proteins) and to focus on changes occurring in chloroplasts and mitochondria under the nutritional stresses indicated above. Fe content decreases in Fe deficient plants roots and Mo content decreases in Mo deficient plants roots. In addition, Fe content decreases in mitochondria purified from Fe-deficient roots and Mo content decreases in mitochondria purified from Mo-deficient roots, indicating that the above described treatments are effective in modifying Fe and Mo homeostasis at whole plant level and at the subcellular level. Alteration of Mo nutritional supply does not dramatically affect physiological parameters (chlorophyll content, O2 evolution, O2 consumption) relative to what has been observed under Fe deficiency in ten-days-old cucumber plants. Yet, striking changes are observed at protein and ion levels in plants grown under these nutritional stress conditions. Such results indicate that our experimental set-up is appropriate for uncovering not only early responses to Mo deficiency but also Mo and Fe molecular interactions. Most interesting, we observed a perturbation of mitochondrial Fe distribution under Mo deficiency. Furthermore, we identified more than hundred proteins whose expression levels were significantly altered in at least one of the reported nutritional stresses. Remarkably, among the various proteins, MudPIT also identified the Fe-storage and Fe-responsive protein ferritin only in mitochondria purified from control roots. This result nicely supports what we recently demonstrated in [4], namely that ferritin is functional in root mitochondria of Fe-sufficient cucumber plants, albeit at low levels relative to the accumulation observed under Fe excess [4]. These findings thus confirm that our proteomics approach is truly sensitive and can identify low-abundant proteins. Results obtained will be commented and potentiality of such approach will be illustrated. [1]Bittner and Mendel (2010) Springer-Verlag, Plant Cell Monogr 17:119-143. [2]Bittner (2014) Front. Plant Sci. 5:28. [3] Wolters et al (2001)Anal Chem. 73:5683-5690. [4] Vigani et al (2013) Front. Plant Sci 316:1-8.