Abstract
To better understand the mechanisms involved in the heavy metal stress response and tolerance in plants, a proteomic approach was used to investigate the differences in Cu-binding protein expression in Cu-tolerant and Cu-sensitive rice varieties. Cu-binding proteins from Cu-treated rice roots were separated using a new IMAC method in which an IDA-sepharose column was applied prior to the Cu-IMAC column to remove metal ions from protein samples. More than 300 protein spots were reproducibly detected in the 2D gel. Thirty-five protein spots exhibited changes greater than 1.5-fold in intensity compared to the control. Twenty-four proteins contained one or more of nine putative metal-binding motifs reported by Smith et al., and 19 proteins (spots) contained one to three of the top six motifs reported by Kung et al. The intensities of seven protein spots were increased in the Cu-tolerant variety B1139 compared to the Cu-sensitive variety B1195 (p<0.05) and six protein spots were markedly up-regulated in B1139, but not detectable in B1195. Four protein spots were significantly up-regulated in B1139, but unchanged in B1195 under Cu stress. In contrast, two protein spots were significantly down-regulated in B1195, but unchanged in B1139. These Cu-responsive proteins included those involved in antioxidant defense and detoxification (spots 5, 16, 21, 22, 28, 29 and 33), pathogenesis (spots 5, 16, 21, 22, 28, 29 and 33), regulation of gene transcription (spots 8 and 34), amino acid synthesis (spots 8 and 34), protein synthesis, modification, transport and degradation (spots 1, 2, 4, 10, 15, 19, 30, 31, 32 and 35), cell wall synthesis (spot 14), molecular signaling (spot 3), and salt stress (spots 7, 9 and 27); together with other proteins, such as a putative glyoxylate induced protein, proteins containing dimeric alpha-beta barrel domains, and adenosine kinase-like proteins. Our results suggest that these proteins, together with related physiological processes, play an important role in the detoxification of excess Cu and in maintaining cellular homeostasis.
Highlights
Copper (Cu) is an essential micronutrient for plant growth and development, but excess Cu is extremely toxic and interferes with numerous physiological processes, such as photosynthesis, pigment synthesis, oxidative stress, nitrogen and protein metabolism and mineral uptake
Rice seedlings were exposed to Kimura B nutrient solution containing 8 μM Cu2+ for 3 days, while control plants were grown in normal nutrient solution containing 0.32 μM Cu
The Cu-immobilized metal affinity chromatography (IMAC) plus iminodiacetic acid (IDA)-Sepharose pre-chromatography method was used for the separation and isolation of Cu-binding proteins extracted from the roots of rice seedlings exposed to excess Cu, and six novel Cu-binding proteins were identified [14]
Summary
Copper (Cu) is an essential micronutrient for plant growth and development, but excess Cu is extremely toxic and interferes with numerous physiological processes, such as photosynthesis, pigment synthesis, oxidative stress, nitrogen and protein metabolism and mineral uptake. Proteins contain cysteine (Cys), methionine (Met), and histidine residues (His), which have high affinity to divalent metal ions, and play a key role in maintaining intracellular copper homeostasis and tolerance [3, 4]. A wide range of proteins involved in Cu detoxification and homeostasis has been identified in plants [2, 5]. Identification of the metal-binding proteins involved in plant responses to heavy metal toxicity aids understanding of the molecular mechanisms of metal tolerance. Immobilized metal affinity chromatography (IMAC) combined with mass spectrometry [6] has been employed to identify putative metal-binding proteins in bacterial [7], mammalian [8, 9], and plant cells [10, 11]
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