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Initiation of Runaway Cell Death in an Arabidopsis Mutant by Extracellular Superoxide

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Science  27 Sep 1996:
Vol. 273, Issue 5283, pp. 1853-1856
DOI: 10.1126/science.273.5283.1853

Figures

  • Fig. 1.

    Superoxide generation in leaf tissue bordering lsd1 lesions. Leaves of wild-type control (Ws-0; A, B, G, and H) or lsd1 (C through F, I, and J) plants were treated with initiators of the lsd1 phenotype (shift to LD conditions, with or without SOD in the NBT staining or spraying with INA) and assayed for generation of superoxide (8) 48 hours after treatment. (A), (C), (E), (G), and (I) are images made with differential interference contrast microscopy; (B), (D), (F), (H), and (J) are ultraviolet-stimulated autofluorescence images (cell death marker) of the same leaf whole mount (3).

  • Fig. 2.

    Inhibition of lsd1 lesion initiation through inhibition of plasma membrane NAD(P)H oxidase. Leaves in SD conditions were treated with DPI (14) and scored for spreading lesions 40 hours after shift to LD conditions. Values are means of at least 17 treated leaves per point from three independent experiments.

  • Fig. 3.

    Induction of marker gene expression mirrors the requirement for superoxide in lesion initiation and spread. (A) Identification of candidate marker genes. The time course over 72 hours after shift to LD is shown at bottom; 72s is RNA from leaves kept in SD conditions for the duration. (B) Induction of marker gene expression upon generation of superoxide with X-XO. (C) Down-regulation of marker gene expression upon inhibition of NAD(P)H-oxidase. (D) Marker gene induction after various stimuli (listed at top; superoxide is X-XO treatment) in 1, wild-type Ws-0 leaves; 2, lsd1 leaves; 3, lsd1/phx21 leaves; and 4, lesion minus lsd3 leaves (3) as a control for nonspecific gene induction. Samples in (B) through (D) were harvested 48 hours after treatment.

Tables

  • Table 1.

    Generation of superoxide radicals is sufficient to induce lsd1 lesions. lsd1 leaves were treated (8) as shown, plants were kept in SD conditions, and spreading lesions were scored at 24 hours. Six independent experiments are pooled. When wild-type Ws-0 control leaves were treated with X-XO, 0 of 59 were lesioned.

    TreatmentsLesioned leaves/total leaves (n)
    XOXSOD
    0/70
    +1/74
    ++0/14
    +2/23
    0.02 U/ml4/31
    0.02 U/ml+84/104
    0.02 U/ml++18/87
    0.50 U/ml3/17
    0.50 U/ml+25/25
    0.50 U/ml++6/28
    Embedded ImageEmbedded Image
    0/10
    +0/10
    250 U/ml+0/17
    H2O2
    0.001 to 10 mM0/217
  • Table 2.

    Evolution of superoxide precedes onset of cell death in lsd1 leaves. Leaves (three to six per time point) were treated (8) and assayed for either onset of superoxide production measured through NBT staining (symbol at left in each column) or cell death measured by trypan blue uptake (symbol at right in each column) at the indicated time points. INA-treated (17) and X-XO-inoculated plants (8) were kept in SD conditions (6). Symbols for NBT staining are as follows: -, none on any leaf; +/-, light staining on some leaves; +, small, dark foci on all leaves with <50 cells; ++, larger foci on all leaves with >100 cells, surrounding lesion margin if present; and +++, intense staining (spreading beyond inoculation site in X-XO experiments). Symbols for trypan blue staining (3) are as follows: -, none; +/-, single dead cells on some leaves; +, foci of 1 to 10 cells on each leaf; and ++, large foci of >50 cells on each leaf. ND, not done.

    TreatmentGeno-typeMeasurement of NBT or trypan blue (hours)
    0481624324048
    Shift to LDIsd1−,−−,−−,−++,−++,+/−++,+++,++++,++
    Ws-0−,−−,−+/−,−−,−−,−+/−,−+−,−−,−
    X-XOIsd1++,−++,+/−++,+/−+++,+++++,++NDNDND
    Ws-0++,−+,−+,−+,−+/−,−NDNDND
    INAIsd1−,−−,−+,−++,+++,++++,++++,++++,++
    Ws-0−,−−,−−,−+/−,−+/−,−−,−−,−−,−

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