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, 98 (2), 771-6

The Arabidopsis-accelerated Cell Death Gene ACD2 Encodes Red Chlorophyll Catabolite Reductase and Suppresses the Spread of Disease Symptoms

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The Arabidopsis-accelerated Cell Death Gene ACD2 Encodes Red Chlorophyll Catabolite Reductase and Suppresses the Spread of Disease Symptoms

J M Mach et al. Proc Natl Acad Sci U S A.

Abstract

accelerated cell death 2 (acd2) mutants of Arabidopsis have spontaneous spreading cell death lesions and constitutive activation of defenses in the absence of pathogen infection. Lesion formation in acd2 plants can be triggered by the bacterial toxin coronatine through a light-dependent process. Coronatine-triggered and spontaneous lesion spreading in acd2 plants also requires protein translation, indicating that cell death occurs by an active process. We have cloned the ACD2 gene; its predicted product shows significant and extensive similarity to red chlorophyll catabolite reductase, which catalyzes one step in the breakdown of the porphyrin component of chlorophyll [Wüthrich, K. L., Bovet, L., Hunziger, P. E., Donnison, I. S. & Hörtensteiner, S. (2000) Plant J. 21, 189-198]. Consistent with this, ACD2 protein contains a predicted chloroplast transit peptide, is processed in vivo, and purifies with the chloroplast fraction in subcellular fractionation experiments. At some stages of development, ACD2 protein also purifies with the mitochondrial fraction. We hypothesize that cell death in acd2 plants is caused by the accumulation of chlorophyll breakdown products. Such catabolites might be specific triggers for cell death or they might induce cellular damage through their ability to absorb light and emit electrons that generate free radicals. In response to infection by Pseudomonas syringae, transgenic plants expressing excess ACD2 protein show reduced disease symptoms but not reduced growth of bacteria. Thus, breakdown products of chlorophyll may act to amplify the symptoms of disease, including cell death and yellowing. We suggest that economically important plants overexpressing ACD2 might also show increased tolerance to pathogens and might be useful for increasing crop yields.

Figures

Figure 1
Figure 1
Complementation of the acd2 mutant phenotype. acd2–2 plants with a control transgene containing the gene adjacent to ACD2 (AT4 g36990) (A) and the ACD2 (AT4 g37000) genomic transgene (B).
Figure 2
Figure 2
(A) Western blot of acd2 mutant plants with anti-ACD2 antiserum. Lanes contain protein from approximately equal weights of leaf tissue. Lanes: 1, Columbia; 2, acd2–2; 3, acd2–5; 4, acd2–6; 5, acd2–8; 6, acd2–12E13; 7, acd2–7. The doublet above the ACD2 band is not detected by antiserum from an independently inoculated rabbit or by affinity-purified antiserum (data not shown); thus, we conclude that it is not specific to the ACD2 protein. (B) ACD2 localizes to chloroplasts and mitochondria. Lanes: 1, crude extract; 2, purified mitochondria; 3, purified chloroplasts. M, mitochondrial form of ACD2; X, chloroplast form of ACD2; CHL, chlorophyll in mg/ml; MITO, mitochondria/microliter.
Figure 3
Figure 3
Western blot analysis of ACD2 protein abundance in senescence and pathogenesis. Each lane contains equal amounts of protein by Bradford assay (Bio-Rad). (A) ACD2 in senescence. Duplicate samples of the ninth true leaf of wild-type plants through its development. At day 3, the leaf was approximately 3 mm long. The chlorophyll measurement is the average of the two leaves. (B) ACD2 in the HR. Panels show wild-type leaves mock inoculated (MgSO4) or inoculated with 0.01 A600 of P. syringae containing vector only (pLAFR) or containing pLAFR with avrRpm1 or avrRpt2. (C) ACD2 in disease. As in B, infection with P. syringae containing pLAFR only compared with mock inoculation. Strong disease symptoms were seen at 48 h.
Figure 4
Figure 4
Effects of light, translation, and SA on acd2 cell death. (A) Light activates the acd2 mutant phenotype. The center of each indicated acd2–2 leaf was spotted with either 0.01% methanol as a control (M) or coronatine (D and L). The leaf marked D (dark) was covered. The other leaves (including L, light) remained in the light for 24 h. Similar treatment of the wild-type control did not induce any cell death or chlorosis. (B) The acd2 phenotype requires active translation. Wild-type (COL) or acd2–2 mutant (acd2) plants were spotted with either coronatine (C) or coronatine and cycloheximide (C + CHX) and were left in the light for 24 h. Depletion of SA exacerbates the acd2 mutant phenotype. acd2–2 (C) or acd2–2 nahG (D).
Figure 5
Figure 5
Overexpression of ACD2 protein suppresses symptoms of pathogen infection. (A) Western blot of plant extracts, with equal amounts of protein loaded. Lanes 1 and 2, wild type; lane 3, 35S-ACD2. (B) Ion leakage 48 h after inoculation with 0.003 A600 of P. syringae strain PsmDG3. White bars are wild type; shaded bars are 35S-ACD2. (C) Growth, in colony-forming units/cm2, of virulent PsmDG3 on wild type (○) or 35S-ACD2 (■).

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