ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 5, с. 651-661
ЭКСПЕРИМЕНТАЛЬНЫЕ ^^^^^^^^^^^^ СТАТЬИ
УДК 581.1
MAPKs AS A CROSS POINT IN H2O2 AND AUXIN SIGNALING UNDER COMBINED CADMIUM AND ZINC STRESS IN RICE ROOTS1 © 2014 F. Y. Zhao*, M. M. Han*, S. Y. Zhang**, J. Ren*, F. Hu*, X. Wang*
*College of Life Sciences, Shandong University of Technology, Zibo, Shandong Province, P.R. China **Shandong Rice Research Institute, Jining, P.R. China Received July 26, 2013
Previously, we have reported the role of MAPKs (mitogen-activated protein kinases) under cadmium stress. This work continue to explore the relationship between MAPKs, H2O2, auxin signaling, and OsHMA and OsZIP gene expression in rice (Oryza sativa L.) roots under combined cadmium (Cd) and zinc (Zn) stress. Compared with Cd, Cd+Zn reduced Cd levels but increased Zn accumulation in the roots. Three OsMAPK genes were negatively regulated, while two OsHMA and two OsZIP genes were positively regulated by MAPK pathways under Cd+Zn stress. Transgenic rice expressing DR5-GUS exhibited enhanced GUS activity in H2O2-, PD (MAPKK inhibitor PD98059)-, or (Cd+Zn)-treated roots, which also exhibited increased H2O2 concentrations, whereas GUS staining decreased in roots in response to Cd+Zn+PD, DMTU (N,N'-dimeth-ylthiourea, a H2O2 scavenger), or Cd+Zn+DMTU treatment, with reduced H2O2 levels. GUS levels were consistent with H2O2 levels, suggesting that MAPK pathway-mediated auxin redistribution occurs via H2O2, and H2O2 functions downstream of MAPK but upstream of auxin signaling pathways. Furthermore, MAPK pathways serve specific functions in regulating the expression of some key genes of auxin signaling (OsYUCCA, OsPIN, OsARF, and OslAA) under Cd+Zn stress. Overall, MAPK cascades function in the integration of metal transport, H2O2 generation, and auxin signaling in rice seedlings grown under Cd+Zn stress.
Keywords: Oryza sativa — auxin signaling — combined cadmium and zinc stress — H2O2 — MAPK
DOI: 10.7868/S001533031404023X
INTRODUCTION
Plant growth is severely affected by toxic concentrations of heavy metals. Cadmium (Cd) is the most toxic of them. Zinc (Zn) ions have both essential and toxic effects on plant cells. At low concentrations, Zn plays an important role in plant metabolic processes as well as cell proliferation and differentiation [1]; however, high concentrations of Zn retard plant growth and development [2]. The interaction between Cd and Zn exerts either protective or toxic effects on plant growth, depending on the concentration, treatment duration and pattern, and the type of plant exposed to the treatment [3]. Gene expression patterns change when plants encounter excessive amounts of heavy metals [4, 5]. Mitogen-activated protein kinase (MAPK) cascades are known to be one of the major pathways, by which extracellular signals, such as growth factors, hormones, and stress stimuli, are
1 This text was submitted by the authors in English.
Abbreviations'. JA — jasmonic acid; MAPK — mitogen-activated protein kinase; PD - PD 98059.
Corresponding author. Feng Yun Zhao. College of Life Sciences, Shandong University ofTechnology, Zibo, Shandong Province, 255049 P.R. China; fax. +86-533-317-8992; e-mail. zfy1226@126.com
transduced in plant cells, eliciting intracellular responses [6]. Experimental evidence from different plant species indicates that several MAPK pathways are activated in response to heavy metals. For example, OsBWMKl (blast- and wound-inducible MAPK gene) was found to be induced by diverse stressors, such as heavy metals, high salt and sucrose, and drought, suggesting that OsBWMKl causes diverse stress signals in rice to converge [4]. OsMAPK2, OsMSRMK3 (a multiple stress responsive gene), and OsWJUMKl (a wound- and JA-uninducible gene) were upregulated in rice upon exposure to multiple stresses, such as H2O2 and the heavy metals (Cd, Cu, and Hg) [7, 8]. In addition, Yeh et al. [9] suggested that plants respond to excess Cd2+ and Cu2+ through the induction of several distinct MAPK pathways. Lin et al. [5] found that ROS may function in the Zn-trig-gered MAPK pathways in rice roots. We have previously showed that the expression of OsMAPK2, OsMPK12, OsMAPK14, OsMAPK44, OsMSRMK3, and OsMSURPK2 were all down-regulated by Cd stress [10].
The plant hormone auxin activates many early response genes that are thought to be responsible for diverse aspects of plant growth and development. The
involvement of MAPK cascades in plant hormone signaling has been previously reported [11]. Results from several studies indicate that MAPK cascades are negative regulators of auxin signaling in plants [12, 13]. By contrast, other studies provided evidence for the induction of kinases in plants by low levels of auxin [14, 15]. However, Tena and Renaudin [16] showed that auxin at high concentrations induces myelin basic protein (MBP) kinase activity in tobacco cell lines. Results from our previous DR5-GUS transgenic rice study indicate that MAPKs are negatively involved in the auxin response in Cd-free rice roots, but it is positively involved in the auxin response in Cd-stressed roots. Moreover, under Cd-stressed conditions, some auxin genes (e.g., OsPINlc) were negatively regulated, while others (e.g., OsYUCCA4) were positively regulated by MAPK pathways. These results suggest that the relationship between MAPKs and auxin signaling is complex [10]. However, whether MAPKs take part in the regulation of auxin signaling in response to Cd+Zn stress is unknown.
We have previously demonstrated that Cd triggers a wide range of physiological and cellular responses, including changes in plant growth, antioxidant systems, and auxin- and cell cycle-related gene expression [17]. We have also reported changes in plant growth and an-tioxidant systems in response to Cd and the combination of Cd and Zn in rice [18]. Previously, our laboratory have investigated the link between MAPKs, auxin signaling, and cell cycle-related gene expression in Cd-stressed rice roots [10]. However, it has not been determined whether the MAPKs are involved in the plant response to combined Cd and Zn stress.
The purpose of the present study was to analyze the relationship between MAPKs, H2O2 and auxin signaling, and the expression of OsHMA and OsZIP genes in response to combined Cd and Zn stress in rice roots. Specifically, we investigated the role of MAPKs in (Cd+Zn)-stressed roots by blocking MAPK transduction pathways using the MAP kinase kinase (MAPKK) inhibitor PD98059 (PD), a specific inhibitor ofMAPKK, which can disrupt MAPK signaling cascades by preventing the activation of MAPK [19].
MATERIALS AND METHODS
Plant materials and treatments. In our previous studies, the growth of whole rice (Oryza sativa L. cv. Zhonghua No. 11) seedlings was inhibited by 0.2 mM Cd, but not by 0.3 mM Zn (in Hoagland nutrient solution); however, when the plants were treated with 0.2 mM Cd plus 0.3 mM Zn, the growth of whole rice seedlings was inhibited. Although environmental actual concentrations of Cd and Zn are low, Cd and Zn ions are taken up by roots and accumulate to toxic concentrations in many plant species throughout the relatively long life-span of plants. To analyze the toxic effects of combined Cd and Zn stress on rice plants over shorter (7 days) periods of time, 0.2 mM
Cd(NO3)2 and 0.3 mM Zn(SO4)2 were used in this study.
Rice seeds were germinated on agar-solidified MS medium with or without 0.2 mM Cd(NO3)2 (Cd), 0.3 mM Zn(SO4)2 (Zn), and 0.2 mM Cd(NO3)2 plus 0.3 mM Zn(SO4)2 (Cd+Zn) in a growth chamber under 200 |mol/(m2 s) illumination, a 14-h photoperi-od, with day/night temperatures of 26/20°C, and a relative humidity levels of 60/80% for 7 days. The seedlings were then transferred onto Hoagland nutrient solution containing the same concentrations of Cd and/or Zn with or without 15 |M PD (PD 98059, a MAPKK inhibitor, "Invitrogen", United States) for 3 days under the same conditions. At the end of the treatments, the roots of the seedlings were used for further analyses. Each treatment was performed at least in triplicate using at least four 100-mL containers with 50 seedlings per container.
Determination of Cd and Zn concentrations. For
determination of Cd and Zn concentrations, rice seeds were germinated on agar-solidified MS medium with or without Cd and/or Zn for 7 days under the aforementioned conditions. The harvested roots were washed first in distilled water and then in 0.01 mM EDTA solution, and dried at 80°C until the materials reached constant weights. The dried root samples were ground using a stainless agate mortar, and 0.1 g of each sample was then digested with a mixture of HNO3 and HClO4 (4 : 1, v/v) in a microwave system. The Cd and Zn concentrations in the tissue extracts were measured using inductively coupled plasma spectroscopy (ICPS). The results were based on the average of three replicate determinations [20].
Determination of H2O2. After treatment as aforementioned, the localization of H2O2 was quantified using DAB (3,3-diaminobenzidine) staining method; the roots (at least 60 roots for each treatment) were stained in DAB solution for 10 h at room temperature, cleared with distilled water [21], and then boiled in 85% (v/v) ethanol. Micrographs were taken using an SMZ1500 dissecting microscope ("Nikon", Japan) with a Nikon D5000 digital camera. In the further analysis, the relationship between H2O2 and auxin distribution, the DR5-GUS transgenic rice (in an O. sativa L. cv. Zhonghua No. 11 background) was used for quantitative determination of H2O2. Briefly, the seeds of DR5-GUS transgenic rice were germinated in agar-solidified MS medium for 7 days under the aforementioned conditions. The seedlings were then transferred onto Hoagland nutrient solution supplemented with 0.06% H2O2, Cd+Zn, 15 mM DMTU (N,N'-dimeth-ylthiourea, a H2O2 scavenger) with or without Cd+Zn and 15 |M PD with or without Cd+Zn, respectively, for 6 h, with the PD pre-added for 2 h (for effectively
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