Regulation of Plant NADPH Oxidase

Abstract

The production of Reactive Oxygen Species (ROS) is one of the key events occurring during the response of plants to environmental changes, and contributing to establish adaptive signaling pathways. A plasma membrane bound NADPH oxidase enzyme has been evidenced as the ROS producing system in various plant-microorganisms interactions. We very recently reported, that a protein of the 14-3-3 family was able to interact directly with the C-terminus part of this NADPH oxidase, and that modification of its expression in tobacco cells led to reduced amount of ROS production upon elicitation. In this addendum, we summarize this work, present additional results, and propose an hypothetic model of regulation of this oxidase in a plant defense context.

Introduction

Because of the key role played by ROS in the orchestration of plant defense, the mechanisms of their biosynthesis have been extensively studied using whole plants or suspension cells treated with various elicitor molecules. If it has been clearly established that a plasma membrane NADPH oxidase produces ROS in planta^1,2 or in elicited cells³ during incompatible interaction, its particular regulation is at the moment quite poorly known. Only general lines of signal transduction, such as calcium influx, phosphorylation, nitrate efflux or small G proteins, have been reported as regulating ROS production mediated by this oxidase. Furthermore, this ROS production occurs not only during plant-pathogens interactions, but also in ABA signaling during stomatal closure, or in response to an abiotic stress such as ozone exposure.⁴ This renders all the more necessary the elucidation at the molecular level of the different steps of the signaling cascades involved, in each of these physiological situations.

Identification of Regulators of Plant NADPH Oxidase

Cryptogein is a protein secreted by the oomycete Phytophthora cryptogea, able to induce an hypersensitive-like response and an acquired resistance in tobacco.⁵ We identified a plasma membrane NADPH oxidase, NtrbohD, as responsible for ROS production triggered on tobacco cells by this elicitor³ and demonstrated that this oxidase was regulated by a small G protein of the Rac family, without direct interaction between the two proteins.⁶ In order to further characterize the regulation of this enzyme, we used a two-hybrid screen in yeast to find proteins able to specifically interact with NtrbohD.

The use of the C-teminal part of NtrbohD as a bait, in a two-hybrid screen, led to the isolation of a cDNA encoding a protein belonging to the family of 14-3-3 proteins, Nt14-3-3h, which has already been demonstrated as specifically induced during the incompatible interaction occurring upon inoculation of tobacco (NN) with the TMV.⁷ When BY2 cell lines are transformed with antisense constructs of Nt14-3-3h, the expression of the transgene is correlated with a strong inhibition of ROS accumulation following elicitation with cryptogein. This demonstrated the role of a 14-3-3 protein in the direct regulation of an oxidase during an incompatible interaction.⁸ Furthermore, the extracellular alkalinization induced by cryptogein after 100 minutes is about 40% lower in the four transgenic lines accumulating the antisense transcripts of Nt14-3-3h compared to untransformed cells (Fig. 1A). We cheked that the activity of the plasma membrane H⁺-ATPase is not affected by the presence of antisense transcripts of Nt14-3-3h in these lines (data not shown). Nitrate efflux has been demonstrated as an early event of the cryptogein signalling pathway leading to extracellular alkalinization and ROS production.⁹ However, its direct effect on the extracellular alkalinization has been excluded by the authors. A large K⁺ efflux was also observed from tobacco cells treated with cryptogein¹⁰ and, more recently, works from two different laboratories clearly indicated a positive regulation of outward rectified K⁺ channels by 14-3-3 proteins.^11,12 It could thus be hypothesized that the K⁺ efflux induced by cryptogein could be counterbalanced by an influx of proton across the lipid bilayer, therefore generating part of the intra cellular acidification and extra-cellular alkalinization previously reported. The presence of antisense transcripts of Nt14-3-3h could thus prevent the activation of the outward K⁺ channel, consequently reducing the extracellular alkalinization. Preliminary results seem to confirm this hypothesis since the efflux of K⁺ triggered by cryptogein is strongly reduced in tobacco cells transformed with antisense constructs of Nt14-3-3h compared to wild type cells (Fig. 1B).

Figure 1.

(A) Extra-cellular pH changes in transgenic tobacco cells transformed with antisense construct of Nt14.3.3h cDNA. pky, cell line transformed with the empty binary vector pKY; 14.1 to 14.4, cell lines transformed with the antisense construct of Nt14.3.3h cDNA showing a drastic decreased in ROS production upon elicitation as evidenced in reference 8; C, control cells; T, cells treated with 50 nM cryptogein. Extra-cellular ΔpH measured after 100 min of treatment by cryptogein are expressed as percent taking the DpH obtained with ‘pkyT’ cells (1 unit pH) as the 100%. Results represent the mean of three independent experiments. (B) Extra-cellular K⁺ efflux triggered by cryptogein in the ‘pky’ cell line and in the ‘14.1’ cell line transformed with antisense construct of Nt14-3-3h. After 30, 60 or 120 minutes of treatment with 50 nM cryptogein, samples were harvested, and filtrated. The potassium content of the filtrate was quantified using HPLC. For each cell line the results are expressed as a percentage of the corresponding value at T = 0, immediately after cryptogein addition and each point represents the mean of three independent experiments; C, control cells; T, cells treated with cryptogein.

This two-hybrid screen also led to the isolation of a cDNA (accession number AJ309007), encoding a protein phosphatase 2C, which is identical to NtPP2C1 gene which is downregulated upon oxidative stress.¹³ Northern blot analysis indicated that the corresponding transcript was very abundant in flowers, apex and mature leaves (data not shown) and that, upon elicitation of excised leaves with cryptogein, its level decreased as a function of time (Fig. 2A). In tobacco cells transformed with sense constructs of NtPP2C1 under a strong constitutive promoter, ROS production upon cryptogein treatment was significantly decreased in most lines tested (Fig. 2B). This suggests a negative regulation of NtrbohD activity by NtPP2C1. Many data exist concerning the intervention of PP2C in signaling pathways connected to ROS production and the possible regulation of PP2C by the cellular redox balance.¹⁴ To this respect, it is noteworthy that the closest Arabidopsis isoform of NtPP2C1 is AtPP2CA (At3g11410), which belongs to the group of PP2C genes associated with ABA signaling. Our data represent, the first evidence of a direct interaction between a PP2C and a ROS producing system. This result is all the more interesting, that a recent quantitative phospho-proteomic approach of early elicitor signaling in Arabidopsis indicated that AtrbohD is very tightly regulated by phosphorylation, since, in response to a 10 minute incubation with flagelline 22 or xylanase, phosphorylation of seven different residues of this protein was observed.¹⁵ This makes quite evident, the necessity to keep rboh proteins dephosphorylated in resting cells, and thus quite plausible their negative regulation by phosphatases.

Figure 2.

(A) Expression of NtPP2C1 in tobacco leaves treated with cryptogein. Tobacco mature leaves were treated by cryptogein during 0, 7, 15, 24 and 28 hours (10 ml of 0.1 µg/mL cryptogein was applied to the petiole of excised tobacco leaves). Total RNAs (10 µg) were extracted from mature leaves and analyzed by northern blot, probed with a ³²P-labeled restriction fragment corresponding to the NtPP2C1 cDNA. The autoradiograph of the hybridized blot is shown above the corresponding gels stained with ethidium bromide. (B) ROS production in transgenic tobacco cells transformed with sense construct of NtPP2C1 cDNA. BY2 tobacco cells transformed with the empty binary vector pKY or with sense construct of NtPP2C1 cDNA (12A lines) were treated with 50 nM of cryptogein. Every 10 min, ROS production was measured using chemiluminescence. Results represent the mean of four independent experiments. C, control cells; T, treated cells by cryptogein. ROS production values measured during 100 min of cryptogein treatment were summed and expressed as percent taking ROS production in pKY cell line as the 100%.

Conclusions

Although the core of plant and animal ROS producing system has been conserved, the regulation of these proteins in the two kingdoms are quite different. In particular, no homologs of the regulatory proteins known to regulate gp91^phox through a direct interaction, could be identified in Arabidopsis genome. Here, we provide some elements of understanding of the complex regulation of NtrbohD occurring during elicitation of tobacco cells, summarized in Figure 3.

Figure 3.

Hypothetic model of regulation of NtrbohD during elicitation of tobacco cells. Nt14-3-3h and PP2C1 have been evidenced as direct interactors of the C-terminus part of NtrbohD in a two-hybrid screen. PP2C1 appears as a negative regulator of NtrbohD activity, and its down regulation upon cryptogein treatment could promote NtrbohD phosphorylation, allowing ROS production. On the opposite, Nt14-3-3h, which expression is enhanced by cryptogein, seems necessary for NtrbohD activation. Moreover, this protein could act as scaffold between the oxidase, and proteins mediating ionic fluxes responsible for pH variation of intra- and extra-cellular compartments. Finally, the small G protein NtRac5 has also been proved to regulate NtrbohD, but without direct interaction with the protein.⁶ It is noteworthy that NtrbohD, Nt14-3-3h and NtRac5 have been identified in plasma membrane microdomains.⁸,¹⁶

Addendum to: Elmayan T, Fromentin J, Riondet C, Alcaraz G, Blein JP, Simon-Plas F. Regulation of Reactive Oxygen Species Production by a 14-3-3 Protein in Elicited Tobacco Cells. Plant Cell Environ. 2007;30:722–732. doi: 10.1111/j.1365-3040.2007.01660.x.