Article Figures & Data
Figures
- Figure 1.
Early Senescence Phenotype of Autophagy-Defective Mutants under Nutrient-Rich Conditions.
(A) The wild type, atg2, and atg5 mutant Arabidopsis were grown on vermiculite at 22°C with 16-h-light/8-h-dark cycles supplied with standard nutrient solution for 6 weeks.
(B) Schematic diagrams showing the onset of visible senescence in wild-type, atg2, and atg5 mutant plants grown under long-day (16 h light/8 h dark) and short-day (8 h light/16 h dark) conditions. Senescence on the first and second leaves started around the time point shown by the arrowheads in our experimental conditions. Results were reproduced in at least five independent experiments using four or more plants in each experiment.
- Figure 2.
Expression Patterns of Senescence- or Pathogen-Related Genes in Wild-Type and Autophagy-Defective Mutant Plants.
Total RNAs from leaves of wild-type, atg2, and atg5 plants grown on rockwool supplied with a rich nutrient solution for 3 to 4 weeks under long-day conditions were isolated and subjected to cycle-optimized RT-PCR using gene-specific primers and 18S rRNA as an internal control. SYBR-green was used for staining the gels. Gel pictures were rearranged for presentation purposes. Results were reproduced in three independent experiments.
- Figure 3.
Early Senescence and Excessive Immunity-Related PCD Phenotypes of Autophagy-Defective Mutants Suppressed by Inactivation of the SA Signaling Pathway.
(A) The NahG gene was introduced into atg5 by crossing. Photographs of 6-week-old plants of the indicated genotypes grown on vermiculite supplied with a rich nutrient solution under long-day conditions.
(B) The phenotype of the atg5 double mutants with sid2, npr1, coi1, jar1, and ein1. Photographs of 5-week-old plants grown on rockwool supplied with a rich nutrient solution under long-day conditions.
(C) The fifth to eighth leaves of each plant grown under short-day conditions for 8 weeks were infected with Pst-avrRpm1 (2 × 107 colony-forming units/mL) or 10 mM MgCl2 (mock). Photographs were taken 9 d after infection. Results were reproduced in at least three independent experiments using four or more plants in each experiment.
- Figure 4.
The atg-Dependent Phenotypes That Are Not Suppressed by Inactivation of the SA Signaling Pathway.
(A) Dark-induced early senescence phenotype of atg5 mutants is not suppressed by overexpression of the NahG gene. Seedlings of wild-type, atg5, and NahG atg5 were grown under long-day conditions for 1 week, after which they were maintained in the dark. The photographs were taken 1 week after the beginning of the dark treatment.
(B) Reduced growth rate of the primary root of atg mutants under nitrogen-depleted conditions is not suppressed by NahG.
(C) Statistical evaluation of primary root length. Seeds of wild-type, atg5, NahG, and NahG atg5 plants were sown on a nitrogen-free medium and, after 14 d, primary root length was measured using ImageJ. Error bars represent sd. All measurements were made on at least 10 individual plants. Asterisks indicate a significant difference from the wild type (P < 0.01; Student's t test).
(D) Phenotypes during artificially induced senescence. The first to fourth leaves of 2-week-old plants were detached and floated on 3 mM MES buffer, pH 5.7, at 22°C in the dark. The leaves were photographed at 0 d and after 2, 4, and 7 d of incubation. Representative leaves are shown.
- Figure 5.
Phenotypes of BTH-Treated Wild-Type, NahG, NahG atg5, sid2, atg5 sid2, npr1, and atg5 npr1 Plants.
(A) Mock-treated (left) and BTH-treated (right) wild-type, NahG, and NahG atg5 plants 7 d after treatment. BTH (100 μM) was sprayed on 6-week-old plants grown under long-day conditions, and after 4 d it was repeated. Photographs were taken 3 d after the second BTH treatment.
(B) and (C) BTH-treated sid2, atg5 sid2, npr1, and atg5 npr1 plants 10 d after treatment. BTH (100 μM) was sprayed on 7-week-old plants grown under short-day conditions, and after 4 d it was repeated. Then, after 6 d, photographs were taken. Results were reproduced in at least three independent experiments using four or more plants in each experiment.
- Figure 6.
DAB Staining of 8-Week-Old Plants Showing Sporadic Accumulation of Hydrogen Peroxide in Control Plants, atg2 and atg5 Mutants, and Lines Derived from Crosses with Mutants Defective in the SA Signaling Pathway.
Sixth or seventh leaves from 8-week-old plants grown under short-day conditions were detached and used for DAB staining. Representative leaves are shown. Numbers represent quantification of DAB staining as intensity per area from five leaves per genotype measured using ImageJ in arbitrary units with the mean ± 2 sd. Results were reproduced in three independent experiments using three plants in each experiment. Asterisks indicate a significant difference from the wild type (P < 0.01; Student's t test).
- Figure 7.
Negative Regulation of SA Signaling by Plant Autophagy.
(A) Autophagy is induced by BTH treatment. Roots of 7-d-old seedlings stably expressing GFP-ATG8a were excised and transferred to MS liquid medium with (right) or without (left) BTH (100 μM) for 8 h and then observed by fluorescence microscopy. Bars = 20 μm.
(B) Quantification of autophagosome-related structures. Numbers of autophagosome-related structures per root section were counted and the average number was determined for seven seedlings per treatment. Error bars indicate the sd. Results were reproduced in three independent experiments. An asterisk indicates a significant difference (P < 0.01; Student's t test).
(C) Hypothetical model for the role of autophagy during aging and immunity-related PCD. From this study, the following hypothetical model is proposed. During senescence and pathogen infection, SA signaling is accelerated by induction of SA biosynthesis, making an amplification loop through EDS1. Autophagy is induced by this SA signaling via NPR1 to operate a negative feedback loop modulating SA signaling that limits senescence and pathogen-induced chlorotic cell death. Based on Hofius et al. (2009) (*).
Tables
Hormones (ng/gFW) Wild Type atg5 SA 53.3 ± 3.85 159.6 ± 32.2 JA 68.2 ± 28.1 141.9 ± 40.6 JA-Ile 5.50 ± 2.32 10.3 ± 4.13 GA1 nd nd GA4 0.11 ± 0.05 0.17 ± 0.09 IAA 5.00 ± 0.04 6.25 ± 0.25 ABA 5.28 ± 0.22 7.00 ± 0.21 tZ 0.43 ± 0.05 0.30 ± 0.01 DHZ nd nd iP 0.05 ± 0.002 0.08 ± 0.001 tZR 1.44 ± 0.27 1.12 ± 0.15 iPR 0.75 ± 0.11 0.94 ± 0.13 Data represent the mean ± sd of three experiments. nd, not detected; FW, fresh weight.
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