Combining -Omics to Unravel the Impact of Copper Nutrition on Alfalfa (Medicago sativa) Stem Metabolism

Abstract

Copper can be found in the environment at concentrations ranging from a shortage up to the threshold of toxicity for plants, with optimal growth conditions situated in between. The plant stem plays a central role in transferring and distributing minerals, water and other solutes throughout the plant. In this study, alfalfa is exposed to different levels of copper availability, from deficiency to slight excess, and the impact on the metabolism of the stem is assessed by a non-targeted proteomics study and by the expression analysis of key genes controlling plant stem development. Under copper deficiency, the plant stem accumulates specific copper chaperones, the expression of genes involved in stem development is decreased and the concentrations of zinc and molybdenum are increased in comparison with the optimum copper level. At the optimal copper level, the expression of cell wall-related genes increases and proteins playing a role in cell wall deposition and in methionine metabolism accumulate, whereas copper excess imposes a reduction in the concentration of iron in the stem and a reduced abundance of ferritins. Secondary ion mass spectrometry (SIMS) analysis suggests a role for the apoplasm as a copper storage site in the case of copper toxicity.

Keywords: ATX1; Cellulose synthase; Copper deficiency; Copper excess; Ionomics; Stem.

Fig. 1

Leaf phenotype of plants cultivated at 10 µM Cu, 7 months after germination. Note the presence of interveinal chlorosis.

Fig. 2

PCA of alfalfa mineral content. The mineral content of the 10 elements Mn, Fe, Co, Cu, Zn, Mo, Na, Mg, K and Ca is analyzed in four replicates and in four organs (fine roots, taproot, stem and leaves). Six conditions of copper availability are tested (3 nM, 30 nM, 300 nM, 1 µM, 3 µM and 10 µM). Black dots represent each sample (96 in total). Squares represent the barycenter of the 24 samples per organ.

Fig. 3

PCA of alfalfa mineral content carried out by plant organ (A, fine roots; B, taproot; C, stem; D, leaves). The mineral content of the 10 elements Mn, Fe, Co, Cu, Zn, Mo, Na, Mg, K and Ca is analyzed in four replicates. Six conditions of copper availability are tested [(Cu1) 3 nM, (Cu2) 30 nM, (Cu3) 300 nM, (Cu4) 1 µM, (Cu5) 3 µM and (Cu6) 10 µM]. Black dots represent each sample (24 in total). Squares represent the barycenter of the four replicates per copper condition.

Fig. 4

Copper content measured in the fine roots according to the concentration of copper in the medium. The blue frame refers to the range of copper concentration in which the content of copper can be modeled by the function: Copper in plant (in µg g^–1 DW) = 10^1.73 × [Copper in the medium (in µM)]^0.54.

Fig. 5

PCA of the gene expression data. Red dots refer to basal samples, and black dots refer to apical samples. Green and yellow circles represent the apical stem individuals from plants grown at Cu1 and Cu2, respectively. Triangles refer to the barycenter of the latter. The expression profile of 14 cell wall-related genes analyzed on 48 samples is represented.

Fig. 6

Principal component analysis (PCA) of the gene expression data carried out on (A) the apical samples and (B) the basal samples. Squares represent the barycenter of the four replicates per copper condition. Represented is the expression profile of 14 cell wall-related genes analyzed on 24 samples each. (Cu1) 3 nM Cu, (Cu2) 30 nM Cu, (Cu3) 300 nM Cu, (Cu4) 1 µM Cu, (Cu5) 3 µM Cu and (Cu6) 10 µM Cu.

Fig. 7

PCA of the differentially regulated proteins identified in (A) the apical samples and (B) the basal samples. Squares represent the barycenter of the four replicates per copper condition. Represented is the expression profile of 51 and 35 proteins (apex and base) analyzed on 24 samples each. (Cu1) 3 nM Cu, (Cu2) 30 nM Cu, (Cu3) 300 nM Cu, (Cu4) 1 µM Cu, (Cu5) 3 µM Cu and (Cu6) 10 µM Cu. Red arrows link the barycenter of the samples grown under copper deprivation (Cu1), optimal copper (Cu4) and copper excess (Cu6).

Fig. 8

PCA showing the distribution of the individuals based on the set of 91 proteins differentially regulated by the treatment. Squares represent the barycenter of the eight replicates per copper condition and (B) the barycenter of the 24 replicates per stem region. Represented is the expression profile of 91 proteins analyzed on 48 samples. In (A) (Cu1) 3 nM Cu, (Cu2) 30 nM Cu, (Cu3) 300 nM Cu, (Cu4) 1 µM Cu, (Cu5) 3 µM Cu and (Cu6) 10 µM Cu; in (B) red dots refer to apical samples and black dots to basal samples.

Fig. 9

NanoSIMS analysis of the peripheral region to the central region of a basal stem section prepared by freezing followed by freeze substitution. (A) ¹²C¹⁴N^– image in green scale color from low to high intensity, (B) copper distribution ⁶³Cu^– in red scale color, (C) color overlay for CN and Cu. Scale bar = 5 µm. In the red square is a focus of the cambial region of the stem.