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Comparison of proteomic responses to individual and combinatorial copper and iron deficiencies in Arabidopsis thaliana rosettes.

ABSTRACT: Purpose: plants exposed to multiple simultaneous adverse growth conditions trigger molecular responses that differ from the sum of those to individual stressors. Copper and iron are fundamental elements required for proper photosynthesis, energy production, DNA metabolism and hormone sensing, among all. Therefore, copper and iron deprivation limits plant yield. In natural environments, simultaneous deficiency to copper and iron can occur. As part of a multiple high-throughput study to identify combinatorial responses to both copper and iron deficiency, proteomic profiling of Arabidopsis thaliana rosette leaves exposed to copper and/or iron deficiencies have been conducted.

INSTRUMENT(S): impact II

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Rosette

SUBMITTER: Antoni Garcia-Molina

LAB HEAD: Dario Leister

PROVIDER: PXD013598 | Pride | 2020-06-22

REPOSITORIES: pride

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Project description:The transition metal copper (Cu) is an essential element for all living organisms. In plants, Cu plays key roles in electron transport chains of chloroplasts and mitochondria, as well as in cell wall metabolism, ethylene perception, molybdenum cofactor synthesis and oxidative stress protection. Because of its physiological importance, suboptimal concentrations of Cu trigger a re-organization of metabolism to economize on Cu, and pronounced Cu deficiency causes severe growth reduction and defects in male fertility. However, when present in excess, Cu can be highly toxic. Therefore, Cu uptake, utilization and cellular concentrations are strictly regulated. A number of components of the Cu-homeostatic network of Arabidopsis have already been identified. However, the mechanisms that control responses of plant gene expression to Cu-deficiency are only partly understood. In Chlamydomonas reinhardtii, the transcription factor Crr1 is required for activating and/or repressing the expression of a number of genes in response to Cu deficiency. This protein contains a plant-specific DNA-binding domain (SBP domain), ankyrin repeats and a C-terminal cysteine-rich region with similarity to a Drosophila metallothionein (MT). In Arabidopsis, there is a family of 16 proteins with SBP domains named SPL family (SBP-like) of which a subset of proteins have been implicated in flowering time control and floral development. Among all of them, AtSPL7 is the most similar to Crr1 (27 % identity), and AtSPL1 (25 % identity), AtSPL12 (24 % identity) and AtSPL14 (23 % identity) share a common protein architecture. The goal of this work was to obtain a complete account of the response of the Arabidopsis thaliana transcriptome to Cu deficiency, as well as to determine the role of AtSPL7 in the transcriptional response to Cu deficiency. For this purpose, three independent experiments were carried out including Col-0 wild-type and spl7-2 mutant plants grown in Cu-sufficient and Cu-deficient hydroponic media, respectively. Root and shoot transcriptomes were established by RNA-Seq for quantitative comparisons. Sampling of root and shoot tissues of wild-type (WT, Col-0) and spl7-2 mutant plants (the mutant is knock-down for the transcription factor SPL7, which plays a key role in the transcriptional regulation of the copper deficiency responses) cultivated hydroponically in a modified Hoagland's solution under Cu-sufficient (control) and Cu-deficient conditions.

Project description:Copper and iron are essential micronutrients for most living organisms because they participate as cofactors in biological processes including respiration, photosynthesis and oxidative stress protection. In many eukaryotic organisms, including yeast and mammals, copper and iron homeostases are highly interconnected; however such interdependence is not well established in higher plants. Here we propose that COPT2, a high-affinity copper transport protein, functions under copper and iron deficiencies in Arabidopsis thaliana. COPT2 is a plasma membrane protein that functions in copper acquisition and distribution. Characterization of the COPT2 expression pattern indicates a synergic response to copper and iron limitation in roots. We have characterized a knockout of COPT2, copt2-1, that leads to increased resistance to simultaneous copper and iron deficiencies, measured as reduced leaf chlorosis and improved maintenance of the photosynthetic apparatus. We propose that COPT2 expression could play a dual role under Fe deficiency. First, COPT2 participates in the attenuation of copper deficiency responses driven by iron limitation maybe aimed to minimize further iron consume. On the other hand, global expression analyses of copt2-1 mutants versus wild type Arabidopsis plants indicate that low phosphate responses are increased in copt2-1 plants. In this sense, COPT2 function under Fe deficiency counteracts low phosphate responses. These results open up new biotechnological approaches to fight iron deficiency in crops. Four biological replicates of Arabidopsis seedlings were generated for 2 genotypes, Col-0 and copt2-1 mutant; and 3 growth condictions; first one an iron(Fe) and copper(Cu) sufficient medium (+Fe + Cu), second one an Fe deficient and Cu sufficient medium (-Fe+Cu) and third one an Fe and Cu deficient medium (-Fe-Cu). For each growth one comparasion was made, copt2-1 mutant versus Col-0; in each comparasion four biological replicates were made, two replicas were labeled with Cy5 for the mutant sample and Cy3 for the Col-0 sample, while the other two replicas were reversed-labeled.