Transcriptome Profiling and Physiological Studies Reveal a Major Role for Aromatic Amino Acids in Mercury Stress Tolerance in Rice Seedlings

<div><p>Mercury (Hg) is a serious environmental pollution threat to the planet. The accumulation of Hg in plants disrupts many cellular-level functions and inhibits growth and development, but the mechanism is not fully understood. To gain more insight into the cellular response to Hg, we performed a large-scale analysis of the rice transcriptome during Hg stress. Genes induced with short-term exposure represented functional categories of cell-wall formation, chemical detoxification, secondary metabolism, signal transduction and abiotic stress response. Moreover, Hg stress upregulated several genes involved in aromatic amino acids (Phe and Trp) and increased the level of free Phe and Trp content. Exogenous application of Phe and Trp to rice roots enhanced tolerance to Hg and effectively reduced Hg-induced production of reactive oxygen species. Hg induced calcium accumulation and activated mitogen-activated protein kinase. Further characterization of the Hg-responsive genes we identified may be helpful for better understanding the mechanisms of Hg in plants.</p></div

Gene ontology (GO) analysis of 1,263 genes upregulated with Hg treatment.

<p>FDR, false discovery rate.</p

Comparative analysis of genes regulated by short- and long-term Hg exposure and encoding (A) receptor-like kinases (RLKs) and (B) transcription factors (TFs).

<p>In each functional category, the genes are grouped according to their regulation by short- or long-term Hg stress. In each protein kinase or TF family, light and dark orange bars represent the proportion of upregulated short- and long-term Hg-regulated genes (fold change ≥2; false discovery rate <0.05), respectively. Black bars indicate the proportion of genes per kinase or TF categories to total number of TFs or kinases in the genome. Fisher’s exact test was used to assess the significance of overrepresented protein kinase and transcription-factor families. Asterisks indicate families significantly overrepresented in the response group (P<0.05).</p

Induction of aromatic metabolites and effect of amino acid treatment to Hg-induced reactive oxygen species (ROS) production in rice roots.

<p>(A) Hg-responsive genes involved in the chorismate metabolic process and free aromatic amino acid accumulation in rice roots. Genes in red are upregulated in Hg-treated rice roots. Enzyme abbreviations: ASα anthranilate synthase α subunit; DAHPS 3-deoxy-D-arabino-heptulosonat-7-phosphate synthase; DHQS 3-dehydroquinate synthase; SK shikimate kinase; EPSPS 5-enolpyruvylshikimate 3-phosphate synthase; IGPS, indole-3-glycerol phosphate synthase; PDT, prephenate dehydratase; TSα, tryptophan synthase αsubunit; TSβ, tryptophan synthase βsubunit. (B) HPLC chromatograms of aromatic amino acids Phe, Tyr and Trp in rice root extracts with or without Hg treatment. Column, 250×4.6 mm I.D.; flow-rate, 2.0 ml/min; mobile phase, methanol–50 mM sodium phosphate buffer (pH 6.5) (3∶97, v/v). (C) Effect of aromatic amino acid (Phe, Trp) treatment on 25 µM Hg-induced ROS accumulation in rice roots. Root samples pretreated or not with 100 µΜ aromatic amino acid (Phe, Trp) for 30 min were treated with 25 µM Hg for 0 to 3 h. Superoxide anion and H₂O₂ levels were detected by treating roots with nitroblue tetrazolium and 3,3′-diamninobenzidine, respectively.</p

Large enzyme family, secondary metabolism and phytohormone genes were up- or downregulated with Hg stress.

<p>MapMan was used to visualize large enzyme family (A), secondary metabolism (B), and phytohormone (C) genes up- or downregulated with short- or long-term 25-µM Hg treatment. Each BIN or subBIN is represented as a block, with each transcript displayed as a square in red for transcripts upregulated or blue for transcripts downregulated.</p

Calcium accumulation and response of mitogen-activated protein kinase (MAPK) activity to Hg by dose and time in rice roots.

<p>(A) Root samples were labeled with 10 µM Oregon Green 488 BAPTA-1, a calcium indicator, for 30 min, then treated with 25 µM Hg for various times (1∼24 h). Green fluorescence indicates the presence of calcium. Five control and 5 treated roots showed similar results. Magnification for all images was ×100. Rice roots were treated with (B) 25 µM Hg for various times. A 15-µg aliquot of crude protein was separated by SDS-PAGE and analysed by in-gel kinase activity assay and western blot with anti-phospho-extracellular signal-regulated kinase (ERK) or anti-ERK antibodies. Arrows indicate kinase-active bands.</p

Alleviation of allelochemical juglone-induced phytotoxicity in tobacco plants by proline

<div><p>Juglone (5-hydroxy-1,4-naphthoquinone) is an important allelochemical in walnut trees (<i>Juglans nigra</i> L.). Its allelopathic potential has been reported in different plant species. We investigated the phytotoxic effects of the allelochemical juglone and the protective role of proline in tobacco seedlings. Juglone inhibited the growth of tobacco seedlings and increased reactive oxygen species content in tobacco roots. Moreover, juglone stress increased proline concentration. The expression of two proline synthesis genes, pyrroline-5-carboxylate synthetase and ornithine aminotransferase, was upregulated and that of a proline catabolism gene, proline dehydrogenase, was downregulated with juglone treatment in tobacco roots. Furthermore, plants pretreated with proline and then exposed to juglone showed attenuated toxic effects in roots. Proline was able to modulate allelochemical juglone-induced stress in tobacco. In summary, this study suggested that increased proline content in the tobacco seedlings treated with juglone may mitigate the deleterious effect of allelochemical stress in plants by inhibiting reactive oxygen species accumulation.</p></div