Excess copper induced physiological and proteomic changes in germinating rice seeds

Abstract

Copper is an essential micronutrient for plants. Present at a high concentration in soil, copper is also regarded as a major toxicant to plant cells due to its potential inhibitory effects against many physiological and biochemical processes. The interference of germination-related proteins by heavy metals has not been well documented at the proteomic level. In the current study, physiological, biochemical and proteomic changes of germinating rice seeds were investigated under copper stress. Germination rate, shoot elongation, plant biomass, and water content were decreased, whereas accumulation of copper and TBARS content in seeds were increased significantly with increasing copper concentrations from 0.2 mM to 1.5 mM followed by germination. The SDS–PAGE showed the preliminary changes in the polypeptides patterns under copper stress. Protein profiles analyzed by two-dimensional electrophoresis (2-DE) revealed that 25 protein spots were differentially expressed in copper-treated samples. Among them, 18 protein spots were up-regulated and 7 protein spots were down-regulated. These differentially displayed proteins were identified by MALDI-TOF mass spectrometry. The up-regulation of some antioxidant and stress-related proteins such as glyoxalase I, peroxiredoxin, aldose reductase, and some regulatory proteins such as DnaK-type molecular chaperone, UlpI protease, and receptor-like kinase clearly indicated that excess copper generates oxidative stress that might be disruptive to other important metabolic processes. Moreover, down-regulation of key metabolic enzymes like alpha-amylase or enolase revealed that the inhibition of seed germinations after exposure to excess copper not only affects starvation in water uptake by seeds but also results in failure in the reserve mobilization processes. These results indicate a good correlation between the physiological and biochemical changes in germinating rice seeds exposed to excess copper.

Introduction

Heavy metals, which are among the major pollutants contaminating our environment, can severely restrict plant growth (Woolhouse, 1983). Nowadays, environmental contamination and exposure to heavy metals is a growing problem throughout the world. Soil pollution due to heavy metals currently involves about 235 million hectares (Giordani et al., 2005). The major anthropogenic activities that contaminate soil with heavy metals as well as copper include several industrial processes and agricultural practices (Xiong, 1998). Copper is an essential micronutrient for plants, but it can be toxic to the plants at higher concentrations (Xiong and Wang, 2005). In plants, copper interacts with a wide range of physiological and biochemical processes in cells, and elevated copper concentration inhibits the normal growth and development (Caspi et al., 1999).

It is known that metal sensitivity and toxicity to plants are influenced by not only the concentration and the toxicant types, but are also dependent to several developmental stages of the plants (Liu et al., 2005). Seed germination is one of the most highly sensitive physiological processes in plants and is regulated by several hormonal interaction and environmental factors (Iglesias and Babiano, 1996). In addition, seed germination is more sensitive to metal pollution because of a lack of some defense mechanisms (Liu et al., 2005, Xiong and Wang, 2005). It is well documented that germination process is highly disturbed by metal stress, however, there are not much explanation on the molecular mechanism of the inhibition of seed germination caused by metal stress. Gene or protein identification in response to biotic or abiotic stresses is the basic step for understanding of the molecular mechanism or to produce enhanced tolerance transgenic plants to that particular stress. Recently, molecular cloning of heavy metal-responsive genes and their over expression in model plants has been reported (DiDonato et al., 2004). However, little information is available in the field of plant proteomics related to heavy metals or copper toxicity.

Proteomics is one of the most recent high-throughput biotechnological approaches that are being used to address the biological function of plant proteins in different biotic or abiotic responses (Agrawal et al., 2002, Kim et al., 2001, Kim et al., 2004). Proteomics, or the systematic analysis of the proteins expressed by the genome, is not only a powerful molecular tool for describing complete proteomes at the organelle, cell, organ or tissue levels but also, for comparing proteomes as affected by different physiological conditions, such as those resulting from the exposure to heavy metal or other stressful environmental factors. However, inadequate reports have been published on seed proteome analysis subjected to copper stress. To the best of our knowledge, to date, there is no report of the proteomic analysis of copper stress-treated germinating rice seeds. Rice is a model plant for genomics and proteomics work because of its well established database system. It has been reported that among the all heavy metals, copper severely affected rice seed germination (Wang, 1994).

To investigate the copper stress-responsive proteins in germinating rice seeds and their possible function in response to metal stress, a proteomic approach has been adopted in combination with physiological analysis. The main goal of this study is to identify proteins or genes that are highly regulated by copper stress that may provide a new insight to generate enhanced metal–stress tolerant plants. Our results showed that a group of stress and antioxidant proteins were differentially expressed in germinating rice seeds when they exposed to copper stress. These results are in good correlation with the physiological analysis.