Evidence for functional interaction between brassinosteroids and cadmium response in Arabidopsis thaliana

Florent Villiers 3 4 Agne` s Jourdain 3 Olivier Bastien 3 Nathalie Leonhardt 2 Shozo Fujioka 0 Gabrielle Tichtincky 3 Francxois Parcy 3 Jacques Bourguignon 3 V eronique Hugouvieux 3 0 RIKEN Advanced Science Institute , Wako-shi, Saitama 351-0198, Japan 1 413), College Park , MD 20742, USA 2 CEA-CEN Cadarache, DEVM-LEMS Bat 156, F-13108 St Paul Lez Durance, France 3 CEA Laboratoire de Physiologie Cellulaire V ege tale, UMR5168 Commissariat a` l'Energie Atomique/CNRS/Universite Joseph-Fourier/ INRA, Institut de Recherches en Technologies et Sciences pour le Vivant , Commissariat a` l'Energie Atomique-Grenoble de Grenoble, 17 rue des Martyrs, F-38054 Grenoble cedex 9, France 4 Present address: Plant Signal Transduction laboratory (J. Kwak), University of Maryland, 0219 Bioscience Research Building ( Plant hormones, in addition to regulating growth and development, are involved in biotic and abiotic stress responses. To investigate whether a hormone signalling pathway plays a role in the plant response to the heavy metal cadmium (Cd), gene expression data in response to eight hormone treatments were retrieved from the Genevestigator Arabidopsis thaliana database and compared with published microarray analysis performed on plants challenged with Cd. Across more than 3000 Cd-regulated genes, statistical approaches and cluster analyses highlighted that gene expression in response to Cd and brassinosteroids (BR) showed a significant similarity. Of note, over 75% of the genes showing consistent (e.g. opposite) regulation upon BR and Brz (BR biosynthesis inhibitor) exposure exhibited a BR-like response upon Cd exposure. This phenomenon was confirmed by qPCR analysis of the expression level of 10 BR-regulated genes in roots of Cd-treated wild-type (WT) plants. Although no change in BR content was observed in response to Cd in our experimental conditions, adding epibrassinolide (eBL, a synthetic brassinosteroid) to WT plants significantly enhanced Cd-induced root growth inhibition, highlighting a synergistic response between eBL and the metal. This effect was specific to this hormone treatment. On the other hand, dwarf1 seedlings, showing a reduced BR level, exhibited decreased root growth inhibition in response to Cd compared with WT, reversed by the addition of eBL. Similar results were obtained on Brz-treated WT plants. These results argue in favour of an interaction between Cd and BR signalling that modulates plant sensitivity, and opens new perspectives to understand the plant response to Cd. - Plants are sessile organisms that have developed complex signalling networks to respond and adapt to adverse conditions such as biotic and abiotic stresses, including drought, cold or salt stress. Plants produce a wide variety of hormones that, in addition to regulating growth and development, are involved in biotic and abiotic stress responses (Bari and Jones, 2009; Gill and Tuteja, 2010). Soil pollution by metals is a major problem in many industrialized and Third World countries, and is responsible for a number of human maladies including cancer, bone fragility, and kidney dysfunction (Ishihara et al., 2001). Widely present in cultured soils due to applications of contaminated sewage sludge, it accumulates in plants from where it is disseminated along the whole food chain. In this The Author [2011]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: context, understanding the mechanisms for plant protection and toxic sequestration can be considered as one of the most important challenges in the coming decades in order to reduce the amount and effects of metal pollutants. The impact of heavy metals on plant physiology has, therefore, been extensively studied and the effect of cadmium (Cd) or zinc (Zn) as model heavy metals has led to the identification of some detoxification pathways in plants as well as in yeast. Several molecular components involved in plant Cd uptake, accumulation, and tolerance, have now been identified (di Toppi and Gabbrielli, 1999; Clemens, 2006a). In particular, low specificity ion (Fe2+, Ca2+, and Zn2+) transporters have been suggested to enable Cd to enter the plant cells (Thomine et al., 2000; Connolly et al., 2002; Perfus-Barbeoch et al., 2002). Once inside the cell, it interferes with many biological processes and mainly leads to reduced growth and leaf chlorosis. Nutrient uptake is disturbed and, in leaves, Cd triggers the degradation of the photosynthesis apparatus (Fagioni et al., 2009) and disturbs water status by inducing stomatal closure. Cd is also suggested to target several enzymes, especially those which require metallic ions as co-factors, particularly Zn binding proteins. Cd does not mediate the direct production of reactive oxygen species (ROS) via the Fenton and Haber Weiss reaction, but the increase of ROS it triggers via the deregulation of redox control mechanisms also explains part of its toxicity. Cd detoxification is mainly mediated through chelators such as phytochelatins (PCs), metallothioneins, and organic acids (Cobbett and Goldsbrough, 2002; Clemens, 2006a). In particular, PC chelation is one of the best characterized Cd detoxification process (Howden et al., 1995; Ebbs et al., 2002; Clemens, 2006b). Phytochelatins are thiol-rich peptides that are synthesized in the presence of heavy metals from glutathione (GSH) and related thiols by PC synthases, and facilitate the sequestration of heavy metals into vacuoles (Steffens et al., 1986; Vogeli-Lange and Wagner, 1990; Salt and Rauser, 1995; Clemens et al., 1999; Ha et al., 1999; Vatamaniuk et al., 1999, 2000; Romanyuk et al., 2006). Up to now very few data concern hormone signalling networks involved in the plant response to heavy metals. Large-scale transcriptomic, proteomic, and metabolomic analyses were undertaken in Arabidopsis challenged with Cd (Herbette et al., 2006; Sarry et al., 2006). They revealed that transcription of many genes involved in the sulphur assimilation pathway and GSH metabolism was enhanced in response to the metal, providing an adequate supply of GSH for PC production. Many other genes involved in oxidative stress and calcium signalling were also found to be regulated and a cross-talk between ROS, nitric oxide, and calcium was proposed to regulate the cellular response of pea plants to Cd (Rodriguez-Serrano et al., 2009). Nitric oxide was shown to be a key regulator of Cd-induced programmed cell death in Arabidopsis suspension cultures (De Michele et al., 2009). However, despite the increasing knowledge of the cellular Cd response, several key points remain unclear. Among them, the hormone signalling pathway occurring during this stress is still poorly characterized and some authors suggest that the specific response of the plant against toxic metal ions would probably implicate at least one hormone (Clemens, 2006a; Weber et al., 2006). Rodriguez-Serrano (...truncated)