Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants

Abstract

Despite the recognition of H(2)O(2) as a central signaling molecule in stress and wounding responses, pathogen defense, and regulation of cell cycle and cell death, little is known about how the H(2)O(2) signal is perceived and transduced in plant cells. We report here that H(2)O(2) is a potent activator of mitogen-activated protein kinases (MAPKs) in Arabidopsis leaf cells. Using epitope tagging and a protoplast transient expression assay, we show that H(2)O(2) can activate a specific Arabidopsis mitogen-activated protein kinase kinase kinase, ANP1, which initiates a phosphorylation cascade involving two stress MAPKs, AtMPK3 and AtMPK6. Constitutively active ANP1 mimics the H(2)O(2) effect and initiates the MAPK cascade that induces specific stress-responsive genes, but it blocks the action of auxin, a plant mitogen and growth hormone. The latter observation provides a molecular link between oxidative stress and auxin signal transduction. Finally, we show that transgenic tobacco plants that express a constitutively active tobacco ANP1 orthologue, NPK1, display enhanced tolerance to multiple environmental stress conditions without activating previously described drought, cold, and abscisic acid signaling pathways. Thus, manipulation of key regulators of an oxidative stress signaling pathway, such as ANP1/NPK1, provides a strategy for engineering multiple stress tolerance that may greatly benefit agriculture.

Figure 1

Oxidative stress responses in Arabidopsis protoplasts. (A) H₂O₂ activated two oxidative stress-induced promoters. Arabidopsis protoplasts were transfected with GST6-LUC (GST6), HSP18.2-LUC (HSP18.2), RD29A-LUC (RD29A), or CaMV35S-LUC (35S) reporter constructs and incubated without (−) or with (+) 200 μM H₂O₂ for 3 h before the promoter activities were measured. Data are the results of triplicate samples and three independent experiments. (B) H₂O₂ induces two putative MAPKs. Arabidopsis protoplasts were treated with 200 μM H₂O₂ or water for 10, 15, and 30 min. The MAPK in-gel kinase activity assay was performed as described (37).

Figure 2

ANP1 initiates an oxidative stress-inducible MAPK cascade. (A) Expression of the ANP kinases. Arabidopsis protoplasts were transfected with five different ANP constructs expressing various HA-tagged kinases: the catalytic domain of ANP1 (ΔANP1), the catalytic domain of ANP2 (ΔANP2), the catalytic domain of ANP3 (ΔANP3), the catalytic domain of ANP1 with the ATP-binding site mutation K98M (ΔANP1m), and a full-length ANP1 (ANP1). The HA-tagged kinases were labeled by [³⁵S]methionine, immunoprecipitated with an anti-HA antibody, separated by SDS/PAGE (10%) and visualized by fluorography. The ANPs were expressed as double bands which might be caused by the presence of two initiation sites. (B) ANPs activate two endogenous MAPKs. Arabidopsis protoplasts were transfected with the same five ANP constructs or with vector DNA (control). Activation of endogenous MAPKs in the transfected cells was detected by an in-gel kinase activity assay. (C) ANP1 activates AtMPK3 and AtMPK6 in vivo. Arabidopsis protoplasts were transfected with a construct expressing one of the Arabidopsis MAPKs (AtMPK2 to 7) alone, or cotransfected with another construct expressing ANP1 catalytic domain (ΔANP1). Protein levels of the ectopically expressed ANP1 and MAPKs were detected after immunoprecipitation with an anti-HA antibody (Upper). Asterisks indicate nonspecific bands. Activity of the MAPKs was assayed in immunocomplex with MBP as a substrate (Lower). The ³²P-labeled MBP was separated by SDS/PAGE (15%) and visualized by autoradiography. (D) Stress activation of AtMPK3 and ANP1. Arabidopsis protoplasts were transfected with AtMPK3 construct alone (Left) or cotransfected with full-length ANP1 (AtMPK3 + ANP1) (Center) or active ANP1 (AtMPK3 + ΔANP1) as a positive control (Right). The transfected protoplasts were incubated for 4 h to allow protein expression before treatment with 200 μM H₂O₂, 4°C (cold), 1 μM 1-naphthaleneacetic acid (auxin), or 100 μM ABA for 15 min. The AtMPK3 activity was assayed in immunocomplex. All experiments presented were repeated at least three times with similar results.

Figure 3

The ANP pathway represents a molecular link between stress and auxin signaling. (A) ANP1 activates oxidative stress-inducible gene expression. Arabidopsis protoplasts were cotransfected with one of the reporter constructs: GST6-LUC (GST6), HSP18.2-LUC (HSP18.2), or RD29A-LUC (RD29A) and one of the ANP constructs as described in the legend of Fig. 2A. Other control kinase constructs were the catalytic domains of CTR1 (ΔCTR1), ASK1 (ΔASK1), and CK1-1 (CK1-1). Vector DNA was used as a control (control). The transfected protoplasts were incubated for 3 h to allow kinase expression before 200 μM H₂O₂ was added to induce the GST6 and HSP18.2 promoters. The cells were incubated for another 3 h before the promoter activities were measured. (B) ANPs repress the auxin response. Arabidopsis protoplasts were cotransfected with the GH3-LUC reporter construct and one of the kinase constructs as described in the legends of Figs. 2A and 3A. The transfected protoplasts were incubated for 3 h to allow kinase expression before 1 μM 1-naphthaleneacetic acid (auxin) was added to induce the GH3 promoter. The cells were incubated for another 3 h before the GH3 promoter activity was measured. (C) H₂O₂ suppresses the auxin-responsive GH3 promoter. Arabidopsis protoplasts were transfected with the GH3-LUC reporter construct and incubated in the absence (− auxin) or presence of 1 μM 1-naphthaleneacetic acid (+ auxin) and 200 μM H₂O₂, or 100 μM ABA for 3 h before activity of the GH3 promoter was measured. (D) ABA induces the RD29A promoter. Arabidopsis protoplasts were transfected with the RD29A-LUC reporter construct and incubated in the absence or presence of 100 μM ABA for 3 h before activity of the RD29A promoter was measured. All data presented on the figure are the results of triplicate samples and three independent experiments.

Figure 4

Stress tolerance of transgenic tobacco plants expressing constitutively active NPK1. (A) Normal vegetative growth of NPK1 plants. Wild-type (WT) and transgenic (2A, 3B, 4A) plants were germinated and grown on a ¼× MS medium for 3 weeks. (B) Tolerance to freezing temperature. Plants were grown on plates for 10 days before freezing temperature treatment (−10°C, 3 h). The photograph was taken 11 days after the treatment. (C) Tolerance to heat shock. Plants were grown for 10 days before heat treatment (48°C, 45 min). The photograph was taken 18 days after the treatment. (D) Tolerance to salt stress. Plants were grown for 6 days and then transferred to plates containing 300 mM NaCl for 3 days. The photograph was taken 11 days after the plants were returned to ¼× MS medium plates. The graphic data are results of 50 plants of each genotype for C and D.