Arsenic stress activates MAP kinase in rice roots and leaves

Abstract

The toxic metalloid arsenite has become a potential threat to rice growing regions leading to serious contamination in food chain. In the present study effect of different physiological concentration of arsenite that is toxic and triggers the molecular events were evaluated in rice seedlings. Along with severe effect on the growth of rice seedling, production of reactive oxygen species (ROS) and nitric oxide (NO) in arsenite treated rice roots was also observed. Activation of a 42 kDa mitogen activated protein kinase (MAPK/MPK) by arsenite was observed in rice leaves and 42 and 44 kDa in roots in dose dependent manner. The activated MAPK could be immunoprecipitated with anti-phospho-tyrosine antibody, 4G10. The kinetic of MAPK activation by arsenite was found to be dose dependent. Transcript analysis of MAPK family and immunokinase assay in arsenite treated rice seedling revealed significant level of induction in OsMPK3 transcripts in leaves and OsMPK3, OsMPK4 transcripts in roots. Among MAPK kinase (MKKs) gene family, OsMKK4 transcripts were found to be induced in arsenite treated rice leaves and roots. In-silico homology modeling and docking analysis supported OsMPK3–OsMKK4 interaction. The data indicates that arsenite stress is transduced through MAPK signaling cascade in rice.

Introduction

Heavy metal toxicity is one of the major environmental health problems in modern society, with potentially dangerous bioaccumulation through the food chain. Rapid industrialization and urbanization have enhanced the levels of toxic heavy metals in the environment, posing a potential health hazard for all living organisms [1], [2]. When accumulated in excess in plant tissues these metals can function as stressors causing physiological constraints and alterations in various vital growth processes [3]. The metalloid arsenic is a toxic pollutant ubiquitous in the environment. It is present both as arsenite (AsIII) and arsenate (AsV) in the environment, with arsenate being more prevalent than arsenite in soils [4], [5]. In response to toxic levels of heavy metals, plants synthesize Cys-rich, metal-binding peptides including phytochelatins and metallothioneins. Heavy metals are detoxified by chelation and sequestration by Cys rich metal-binding peptides in the vacuole [6], [7] while, various membrane transport systems play an important role in metal ion homeostasis and tolerance [8].

Gene expression patterns change when plants encounter excessive amounts of heavy metals [9]. Heavy metals are also reported to activate MAPKs in higher plants [10], [11]. Exposure of Medicago seedlings to excess copper or cadmium ions resulted in a complex activation pattern of four distinct MAPKs: SIMK, MMK2, MMK3 and SAMK [10] Agrawal et al. [12], [13] reported that the amount of OsMSRMK2, OsMSRMK3 and OsWJUMK1 transcripts increased in response to Cu and Cd treatments in rice seedlings. It was reported that OsMAPK2 transcripts increased within 12 h upon Cu treatment in rice suspension cells [14]. Further, activation of MAPK³ by different heavy metals like Cd and Cu [11], [15] and Zn [16] mostly in the range of 40 and 42 kDa was reported from rice. Tsai and Huang [17] showed that iron at higher concentrations activated a 42 kDa MAPK. Using different inhibitors it was demonstrated that MAPK, reactive oxygen species, protein phosphatase, and H⁺-ATPase might function in the plant iron-triggered signaling pathway in rice roots. Studies from mammalian system reveals that non-cytotoxic concentrations of As³⁺, V¹⁺ and Zn²⁺ induced a rapid phosphorylation of MAPK in BEAS cells [18]. They suggested that metal-induced activation of MAPK was due to inhibition of phosphatase activity. Similarly, trivalent As³⁺ reportedly activates JNK and P38 in HeLa cells. In case of plants, arsenic (As) toxicity and its biochemical effects have been mostly evaluated in ferns and a few higher plants. In a genome wise comparative study, Chakrabarty et al. [19] has shown that the different sets of genes are activated by arsenate an arsenite stress in rice. Arsenite has also shown to activate MAPK activity in Brassica juncea [20]. However a complete analysis of MAPK gene family involved in arsenic stress signal transduction has not been reported so far.

In the present investigation we have studied the effect of arsenite on the growth of rice seedling and its photosynthetic capability. To gain further insight into early events of arsenic stress signaling, involvement of ROS, NO, activation of MAPK and its correlation with increasing arsenite concentration in leaf and roots were also analyzed. Transcript analysis of all the members of MAPK and MKK gene family in rice under arsenic stress revealed the genes involved in this heavy metal signaling.