Hydrogen peroxide activates cell death and defense gene expression in birch

Abstract

The function of hydrogen peroxide (H(2)O(2)) as a signal molecule regulating gene expression and cell death induced by external stresses was studied in birch (Betula pendula). Ozone (O(3)), Pseudomonas syringae pv syringae (Pss), and wounding all induced cell death of various extents in birch leaves. This was temporally preceded and closely accompanied by H(2)O(2) accumulation at, and especially surrounding, the lesion sites. O(3) and Pss, along with an artificial H(2)O(2) producing system glucose (Glc)/Glc oxidase, elicited elevated mRNA levels corresponding to genes encoding reactive oxygen species detoxifying enzymes, Pal, Ypr10, and mitochondrial phosphate translocator 1. In addition to the regulation of gene expression, Glc/Glc oxidase also induced endogenous H(2)O(2) production in birch leaves, accompanied by cell death that resembled O(3) and Pss damage. Wound-induced gene expression differed from that induced by O(3) and Pss. Thus, it appears that at least two separate defense pathways can be activated in birch leaves by stress factors, even though the early H(2)O(2) accumulation response is common among them all.

Figure 1

Lesion development and H₂O₂ accumulation in birch leaves. A, Birch leaf with lesions 10 h after the beginning of an 8-h ozone (150 nL L⁻¹) exposure. B, DAB staining reveals H₂O₂ accumulation at and around lesion sites (arrow) in a cleared leaf 10 h after the beginning of the O₃ exposure. C, A leaf treated similarly as in B but without DAB shows the location and shape of the necrotic lesions. D, Pss-infiltrated leaf at 2 h after the infiltration, showing no dead necrotic lesions. E, DAB-stained and -cleared Pss-infiltrated leaf at 2 h shows H₂O₂ accumulation at the infiltration sites. F, A leaf treated similarly as in E, but without inclusion of DAB. G, Pss-infiltrated leaf at 48 h after the infiltration showing dead necrotic lesions at injection sites (arrow). H, DAB-stained and -cleared Pss-infiltrated leaf at 48 h shows H₂O₂ accumulation around lesions (arrow). I, A leaf treated similarly as in H, but without inclusion of DAB. J, Wounded leaf at 2 h. K, DAB staining shows H₂O₂ accumulation at the wound surface 2 h after wounding in a cleared leaf. L, A leaf treated similarly as in K, but without inclusion of DAB. M, G/GO-treated leaf at 2 h. N, DAB-stained G/GO-treated leaf at 2 h showing H₂O₂ accumulation surrounding injection site (arrow). O, A leaf at 2 h, treated similarly as in N, but without inclusion of DAB. P, G/GO-treated leaf at 48 h showing lesions resembling those caused by O₃ treatment (Fig. 1A). Q, DAB-stained G/GO-treated leaf at 48 h showing H₂O₂ accumulation around lesions and in sites not showing visible damage (arrow). R, A leaf at 48 h treated similarly as in Q, but without inclusion of DAB. S, DAB staining shows H₂O₂ accumulation at the wound surface 2 h after wounding in a cleared leaf. T, A leaf treated similarly as in K and S, but with inclusion of CAT in the infiltration buffer. Removal of H₂O₂ by CAT indicates the specificity of the DAB staining for H₂O₂.

Figure 2

Subcellular localization of O₃-induced H₂O₂ accumulation in birch leaf cells with CeCl₃ staining and TEM. H₂O₂ precipitates CeCl₃ forming electron-dense cerium perhydroxide, visible as black spots. Individual precipitates are indicated by arrows in C and E. Samples were exposed to 150 nL L⁻¹ of O₃ for 8 h and kept in clean air for 15 min before infiltration with CeCl₃ essentially to visualize only H₂O₂ that is produced by the plant cells. Samples were collected before the beginning of treatment (A and B), 2 h after the beginning of the treatment (C and D), and 10 h after the beginning of the treatment (E and F). Samples are shown both with (+CeCl₃; A, C, and E) and without (−CeCl₃; B, D, and F) cerium chloride staining. cw, Cell wall; c, chloroplast; s, starch grain in chloroplast; a, (intercellular) air space. Scale bar = 200 nm.

Figure 3

O₃ induction of birch Pal, Mpt1, and Ypr10 at the transcriptional level. A, Plants were exposed to 150 nL L⁻¹ O₃ for 8 h, leaves were collected at the indicated time points after the beginning of the exposure, and transcript steady-state levels of Pal, Mpt1, and Ypr10 were determined. Bar represents O₃ exposure. B, To determine the duration of O₃ exposure required for changes in mRNA levels at 8 h, plants were exposed to 0, 2, 4, or 8 h of O₃ followed by 8, 6, 4, or 0 h of clean air, respectively (0/8, 2/6, 4/4, and 8/0), after which leaf samples were collected and transcript steady-state levels were determined. Black and white bars represent the duration of O₃ exposure and clean air, respectively. C, To study the re-inducibility of the genes, plants were exposed to two O₃ peaks, the second one taking place 48 h after the beginning of the first one, and samples were collected at indicated time points. Bars represent the duration of O₃ exposure. Methylene blue-stained RNA is shown as loading control.

Figure 4

O₃ induces birch Apx, Cat, Cu/ZnSod, and Gst at the transcriptional level. Saplings were exposed to 150 nL L⁻¹ O₃ for 8 h, and samples were collected at 0, 2, 8, 24, and 48 h. Bar represents O₃ exposure. Methylene blue-stained RNA is shown as loading control.

Figure 5

Pss and wound induction of gene expression in birch leaves. A, Leaves were infiltrated with an avirulent Pss strain J900 and collected at indicated time points, and the transcript accumulation of Pal, Ypr10, and Mpt1 was determined. B, Control infiltration with 10 mm MgSO₄ had no effect. C, Leaves were wounded throughout the leaf area and leaves were collected at indicated time points. Pal mRNA levels, but not Ypr10 or Mpt1, peak at 2 h and decline thereafter. Equal loading of RNA is visualized with hybridization with 18S rRNA.

Figure 6

H₂O₂ generating system G/GO induces birch Pal, Ypr10, Apx, Cat, Cu/ZnSod, Gst, and Mpt1 at the transcript level with differential timing patterns.