NDP kinase 2 interacts with two oxidative stress-activated MAPKs to regulate cellular redox state and enhances multiple stress tolerance in transgenic plants

Abstract

NDP kinases (NDPKs) are multifunctional proteins that regulate a variety of eukaryotic cellular activities, including cell proliferation, development, and differentiation. However, much less is known about the functional significance of NDPKs in plants. We show here that NDPK is associated with H(2)O(2)-mediated mitogen-activated protein kinase signaling in plants. H(2)O(2) stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Proteins from transgenic plants overexpressing AtNDPK2 showed high levels of autophosphorylation and NDPK activity, and they have lower levels of reactive oxygen species (ROS) than wild-type plants. Mutants lacking AtNDPK2 had higher levels of ROS than wild type. H(2)O(2) treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6, two H(2)O(2)-activated A. thaliana mitogen-activated protein kinases. In the absence of H(2)O(2) treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the myelin basic protein phosphorylation activity of AtMPK3 in vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ. Thus, AtNDPK2 appears to play a previously uncharacterized regulatory role in H(2)O(2)-mediated MAPK signaling in plants.

Figure 1

The AtNDPK2 transcript accumulates during oxidative stress. Total RNA was prepared from 2-week-old Arabidopsis seedlings treated with 4 mM H₂O₂. Total RNA from each sample (10 μg) was electrophoresed on a formaldehyde agarose gel. The number above each lane indicates the time in hours the plants were exposed to H₂O₂. By prestaining the gels with ethidium bromide (Bottom), it was confirmed that an equal amount of RNA was applied to each lane. The RNA blot was probed with ³²P-labeled AtNDPK1, -2, and -3 cDNA.

Figure 2

Overexpression of AtNDPK2 in Arabidopsis plants enhances NDPK and autophosphorylation activity. (A) Western blot analysis. Crude protein from plants (1 μg) was resolved by SDS/12% PAGE and transferred to nitrocellulose. Membrane strips were immunoblotted with AtNDPK2-specific antibody. (B) Autophosphorylation assay. The samples used in A were incubated at 30°C for 15 min with 0.5 μCi [γ-³²P]ATP. The reaction mixtures then were subjected to electrophoresis on a 12% acrylamide gel and autoradiographed. (C) NDPK assay. The protein samples used in A were incubated with GDP and [γ-³²P]ATP, resolved on PEI cellulose TLC plates, and exposed to x-ray film. Con, control with only GDP and [γ-³²P]ATP; w.t., wild type; t.g., AtNDPK2-overexpressing transgenic plant.

Figure 3

AtNDPK2 regulates the cellular redox state. Intracellular ROS in Arabidopsis wild-type (w.t.), AtNDPK2-overexpressing transgenic (t.g.), and AtNDPK2-knockout mutant (k.o.) protoplasts were measured. (A) Microscopic analysis. Phase-contrast display (Upper) and the corresponding fluorescence data after incubation with DCFH-DA (Lower) are depicted. (B) Flow cytometric analysis. The intracellular ROS contents of wild-type and AtNDPK2-transgenic cells were measured by flow cytometry. (C) Spectrofluorometric analysis. The intracellular ROS abundance in the protoplasts used in A was measured by fluorescence spectrophotometry. Results represent the mean of three separate experiments.

Figure 4

AtNDPK2 is involved in H₂O₂-stimulated phosphorylation of two putative oxidative stress-activated proteins. Crude proteins (1 μg) from untreated wild-type cells (w.t.), wild-type cells treated with 0.2 mM H₂O₂ for 15 min (w.t. + H₂O₂₎, untreated AtNDPK2-overexpressing transgenic cells (t.g.), and AtNDPK2-knockout mutant (k.o.) were incubated at 30°C for 15 min with 0.5 μCi [γ-³²P]ATP. The reaction mixtures then were subjected to SDS/15% PAGE and autoradiographed. (A) Total protein stained with Coomassie brilliant blue. (B) Phosphorylase activity.

Figure 5

AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. (A) Yeast two-hybrid assay. Full-length Arabidopsis NDPK1, -2, and -3 (NK1, -2, and -3) cDNAs were cloned into pACT2 as in-frame fusions with the AD of GAL4. Full-length AtMPK1, -3, and -6 (MK1, -3, and -6) cDNAs were cloned into pAS2-1 as in-frame fusions with the BD of GAL4. Yeast cells (pJ69-4A) were transformed with the plasmids (depicted at Upper Left) and incubated at 30°C for 3 days to select for interactions. trp-leu, transformants selected on SD media lacking tryptophan and leucine; trp-leu-ade, transformants selected on SD media lacking leucine, tryptophan, and adenine; LacZ, β-galactosidase assay for the transformants. The interacting proteins, tumor suppressor p53, and SV40 large T-antigen, fused with the BD and the AD of GAL4, respectively, were used as a positive control. Cells transformed with pACT2 and pAS2-1 were used as a negative control. (B) In vitro protein pull-down assay. Approximately 1 μg of GST (lane a) or recombinant GST-tagged AtNDPK2 (lane b) was incubated with 1 μg of recombinant His-tagged AtMPK3 and pulled down with GST resin after preblocking with 1% BSA. After washing, the bound protein was fractionated by SDS/12% PAGE and subjected to Western blot analysis with anti-GST and anti-His monoclonal antibodies.

Figure 6

AtNDPK2 enhances the MBP phosphorylation activity of AtMPK3. Recombinant GST-AtNDPK2 (0.1 μg) alone or His-AtMPK3 (1 μg) with (+) or without (−) GST-AtNDPK2 (0.1 μg) was incubated with MBP (final concentration of 1.0 μg/μl) and 0.5 μCi [γ-³²P]ATP. The proteins were subjected to SDS/12% PAGE and autoradiographed to detect the phosphorylated proteins.

Figure 7

AtNDPK2-overexpressing transgenic plants are protected from multiple stress. The environmental stress tolerance of wild-type (w.t.), AtNDPK2-overexpressing transgenic (t.g.), and AtNDPK2-knockout mutant (k.o.) plants was investigated. (A) Tolerance to cold stress. Arabidopsis plants were frozen at −7°C for 1 h, returned to the original growth conditions (38), and photographed 1 week later. The surviving plants all showed green pigmentation. (B) Tolerance to salt stress. Arabidopsis seedlings were raised for 2 weeks on MS medium (Left) or for 3 weeks in MS medium containing 50 mM NaCl (Right) to assess their survival under salt stress. (C) Tolerance to MV. Fully expanded leaves from 3-week-old plants were transferred to MS liquid medium containing 1.0 μM MV for 7 days, and the percentage of plants that survived was recorded. Dying plants were albino and displayed a loss of chlorophyll content.