Proteomic identification of small, copper-responsive proteins in germinating embryos of Oryza sativa

Abstract

Background and aims: Although copper (Cu) is an essential micronutrient for plants and algae, excess Cu is toxic to most plants and can cause a wide range of deleterious effects. To investigate the response of rice (Oryza sativa) to Cu stress, a proteomic approach was used to analyse Cu stress-induced changes in the expression of low molecular-weight proteins in germinating rice seed embryos.

Methods: Rice seeds were germinated in the presence or absence of 200 microm Cu for 6 d, and embryos, including newly formed shoots and radicles, were isolated. After proteins were extracted from the germinating embryos and separated by two-dimensional PAGE, 16 proteins in the 6- to 25-kDa range were identified using MALDI-TOF mass spectrometry.

Key results and conclusions: Thirteen of the proteins identified, including metallothionein-like protein, membrane-associated protein-like protein, putative wall-associated protein kinase, pathogenesis-related proteins and the putative small GTP-binding protein Rab2, were up-regulated by Cu stress. Three proteins, a putative small cytochrome P450 (CYP90D2), a putative thioredoxin and a putative GTPase, were down-regulated by Cu stress. As far as is known, this study provides the first proteomic evidence that metallothionein and CYP90D2 are Cu-responsive proteins in plants. These findings may lead to a better understanding of plant molecular responses to toxic metal exposure.

Fig. 1.

Effect of Cu on rice radicle elongation. Rice seeds were treated with the indicated concentrations of Cu for 4, 6 or 8 d. Values shown represent means ± s.e. (n = 3) for three different experiments. Means denoted by the same letter did not differ significantly (P ≥ 0·05 according to Duncan's multiple range test).

Fig. 2.

Effect of Cu treatment on the content of (A) Cu, (B) total protein and (C) protein thiol (PT) in germinating rice embryos. Rice seeds were treated with the indicated concentrations of Cu for 6 d. Values shown represent means ± s.e. (n = 3) for three different experiments. Means denoted by the same letter did not differ significantly (P ≥ 0·05 according to Duncan's multiple range test).

Fig. 3.

SDS–PAGE of germinating rice embryo extracts from seeds treated with the indicated concentrations of Cu for 6 d. Equal amounts of protein (20 µg) were loaded in each gel lane. Arrows indicate protein bands altered by Cu treatment. Experiments were performed three times with similar results.

Fig. 4.

Two-dimensional PAGE of germinating rice embryo extracts from seeds treated with 0 (Con) or 200 µm Cu for 6 d. Equal amounts of protein (1 mg) were loaded on each gel. Arrows indicate the proteins identified.

Fig. 5.

Alignment of the deduced amino acid sequences of MT-like protein (spot 1 in Fig. 4) and RicMT (a member of the rice MT gene family; NCBI accession no. BAA19661).