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, 66 (22), 7045-59

Mutation of SPOTTED LEAF3 (SPL3) Impairs Abscisic Acid-Responsive Signalling and Delays Leaf Senescence in Rice

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Mutation of SPOTTED LEAF3 (SPL3) Impairs Abscisic Acid-Responsive Signalling and Delays Leaf Senescence in Rice

Seung-Hyun Wang et al. J Exp Bot.

Abstract

Lesion mimic mutants commonly display spontaneous cell death in pre-senescent green leaves under normal conditions, without pathogen attack. Despite molecular and phenotypic characterization of several lesion mimic mutants, the mechanisms of the spontaneous formation of cell death lesions remain largely unknown. Here, the rice lesion mimic mutant spotted leaf3 (spl3) was examined. When grown under a light/dark cycle, the spl3 mutant appeared similar to wild-type at early developmental stages, but lesions gradually appeared in the mature leaves close to heading stage. By contrast, in spl3 mutants grown under continuous light, severe cell death lesions formed in developing leaves, even at the seedling stage. Histochemical analysis showed that hydrogen peroxide accumulated in the mutant, likely causing the cell death phenotype. By map-based cloning and complementation, it was shown that a 1-bp deletion in the first exon of Oryza sativa Mitogen-Activated Protein Kinase Kinase Kinase1 (OsMAPKKK1)/OsEDR1/OsACDR1 causes the spl3 mutant phenotype. The spl3 mutant was found to be insensitive to abscisic acid (ABA), showing normal root growth in ABA-containing media and delayed leaf yellowing during dark-induced and natural senescence. Expression of ABA signalling-associated genes was also less responsive to ABA treatment in the mutant. Furthermore, the spl3 mutant had lower transcript levels and activities of catalases, which scavenge hydrogen peroxide, probably due to impairment of ABA-responsive signalling. Finally, a possible molecular mechanism of lesion formation in the mature leaves of spl3 mutant is discussed.

Keywords: Abscisic acid; MAPKKK; catalase activity; lesion mimic mutant; reactive oxygen species; rice; spotted leaf3..

Figures

Fig. 1.
Fig. 1.
Phenotypic characterization of the spl3 mutant. Whole plants (upper panels) and leaf blades (lower panels) of 50-, 80-, and 120-DAS WT and spl3 mutant grown in the paddy field. F, flag leaf; 1 to 5, 1st to 5th leaf blade from the top. Scale bar, 30cm.
Fig. 2.
Fig. 2.
ROS accumulation in the spl3 leaf blades. (A) WT and spl3 mutant were grown under SD or CL conditions at 30 °C for 1 month in the growth chamber. (B, C) The accumulation of H2O2)and O2 were detected by staining with 3,3′-diaminobenzidine (DAB) (B) and NBT (C), respectively. (D, E) H2O2 quantification was performed using Amplex Red. 2nd leaves from 30-DAS WT and spl3 mutant grown under SD and CL conditions (D), and 1st to 4th leaves from the main culm of 90-DAS WT and spl3 mutant grown in the paddy field (E). Mean and standard deviation values were obtained from more than three biological replicates. These experiments were repeated three times with similar results. (D, E) Statistical analysis using Student’s t-test, *P<0.05; **P< 0.01.
Fig. 3.
Fig. 3.
Map-based cloning of the spl3 locus. (A) Genetic mapping of the spl3 locus. The spl3 locus was initially narrowed down to the region between two SSR markers, RM14395 and RM14423, on the short arm of chromosome 3. The PCR primer sequences of SSR and STS markers are listed in Supplementary Table S1 at JXB online. (B, C) Fine physical mapping of the spl3 locus. The spl3 locus region was narrowed down to a 161.2-kb interval between S3015.2 and SSR-5 markers using six recombinants in F2 individuals. (D) The SPL3 gene structure. Thirteen exons and 11 introns are designated by black rectangles and lines, respectively; a 1-bp deletion occurs in the 1st exon, leading to a frameshift mutation.
Fig. 4.
Fig. 4.
The spl3 mutant shows a stay-green phenotype during leaf senescence. (A) Phenotype of WT and spl3 mutant at 40 DAH. (B–D) Changes of total Chl levels (B), chloroplast protein levels (C), and Fv/Fm ratios (D) in WT and spl3 mutant at 0 and 40 DAH. (E) Transcript levels of the three SAG genes in the WT and spl3 mutant at 0 and 40 DAH were measured by RT-qPCR. Transcript levels of OsNAP, NYC1, and SGR were normalized to the transcript levels of OsUBQ5. (F–H) The spl3 leaves stay green during dark-induced senescence. Changes of visible phenotype (F), total Chl levels (G), and ion leakage rates (H) in the WT and spl3 mutant before and after 4 DDI were examined. (G, H) Black, white, and grey bars indicate 0, 4, and 6 DDI, respectively. Statistical analysis using Student’s t-test, *P<0.05; **P<0.01. Mean and standard deviation values were obtained from more than three biological replicates. These experiments were repeated twice with similar results.
Fig. 5.
Fig. 5.
The spl3 mutant is less responsive to exogenous ABA and hypersensitive to drought stress. (A) Effect of different concentrations of ABA (0, 5, and 10 μM) on root growth is significantly reduced in the spl3 mutant (each square = 1.8×1.8cm2). (B, C) The root length (B) and number of adventitious roots (C) in 8-d-old plants were measured. (D, E) The spl3 mutant shows a sensitive phenotype to drought stress. Four-week-old WT and spl3 plants grown under LD conditions were dehydrated for 5 d (5 DDT). The phenotype (D) and ion leakage rate (E) before and after dehydration are shown, respectively. (F, G) Effect of ABA (10 μM) on stomatal closure is significantly reduced in the spl3 leaves (F), and stomatal apertures were measured (G). White bar, 5 μm. Statistical analysis using Student’s t-test, **P<0.01). Mean and standard deviation values were obtained from more than three biological replicates. These experiments were repeated three times with similar results. DT, days of treatment. DDT, days of drought treatment.
Fig. 6.
Fig. 6.
Expression of ABA signalling-related genes in the spl3 mutant after ABA treatment. The 10-d-old WT and spl3 seedlings were transferred to MS solution containing 10 μM ABA and were sampled after 6h for RT-qPCR analysis. RT-qPCR was used to measure the relative transcript levels of (A) OsABI1, (B) OsABI3, (C) OsABI4, (D) OsDSG1, (E) OsABI5, (F) OsAREB1, (G) OsbZIP23, (H) OsSAPK8, (I) OsSAPK9, (J) OsSAPK6, (K) OsRePRP2.1, and (L) OsDSR1 and transcript levels were normalized to the transcript levels of OsUBQ5. Mean and standard deviation values were obtained from more than three biological replicates. These experiments were repeated twice with similar results. Statistical analysis using Student’s t-test, *P<0.05; **P<0.01).
Fig. 7.
Fig. 7.
Catalase activity is reduced in the spl3 mutant after ABA treatment. (A–C) The leaf discs were taken from the 2nd leaves of 1-month-old WT and spl3 plants grown under LD conditions and were treated with 20 μM ABA for 12h, and then were sampled for RT-qPCR. RT-qPCR was used to measure the relative transcript levels of OsCatA (A), OsCatB (B), and OsCatC (C), which were normalized to the transcript levels of OsUBQ5. (D) Relative catalase activity in leaves from 1-month-old WT and spl3 seedlings treated for 6h with 20 μM ABA. Mean and standard deviation values were obtained from more than three biological replicates. These experiments were repeated three times with similar results. HT, hours of treatment. Statistical analysis using Student’s t-test, *P<0.05; **P<0.01.

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