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, 9, 1925
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The Rice SPOTTED LEAF4 ( SPL4) Encodes a Plant Spastin That Inhibits ROS Accumulation in Leaf Development and Functions in Leaf Senescence

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The Rice SPOTTED LEAF4 ( SPL4) Encodes a Plant Spastin That Inhibits ROS Accumulation in Leaf Development and Functions in Leaf Senescence

Giha Song et al. Front Plant Sci.

Abstract

Lesion mimic mutants (LMMs) are usually controlled by single recessive mutations that cause the formation of necrotic lesions without pathogen invasion. These genetic defects are useful to reveal the regulatory mechanisms of defense-related programmed cell death in plants. Molecular evidence has been suggested that some of LMMs are closely associated with the regulation of leaf senescence in rice (Oryza sativa). Here, we characterized the mutation underlying spotted leaf4 (spl4), which results in lesion formation and also affects leaf senescence in rice. Map-based cloning revealed that the γ ray-induced spl4-1 mutant has a single base substitution in the splicing site of the SPL4 locus, resulting in a 13-bp deletion within the encoded microtubule-interacting-and-transport (MIT) spastin protein containing an AAA-type ATPase domain. The T-DNA insertion spl4-2 mutant exhibited spontaneous lesions similar to those of the spl4-1 mutant, confirming that SPL4 is responsible for the LMM phenotype. In addition, both spl4 mutants exhibited delayed leaf yellowing during dark-induced or natural senescence. Western blot analysis of spl4 mutant leaves suggested possible roles for SPL4 in the degradation of photosynthetic proteins. Punctate signals of SPL4-fused fluorescent proteins were detected in the cytoplasm, similar to the cellular localization of animal spastin. Based on these findings, we propose that SPL4 is a plant spastin that is involved in multiple aspects of leaf development, including senescence.

Keywords: lesion mimic mutant; microtubule severing protein; reactive oxygen species; rice (Oryza sativa); senescence; spastin.

Figures

FIGURE 1
FIGURE 1
Phenotypic characterization of the spl4-1 mutant. The pictures of N8 and spl4-1 plants and leaves were taken from plants grown in the paddy field at 87 days after sowing (DAS) (A,B), 118 DAS (C,D), and 162 DAS (E,F). (A,C,E) Plant phenotype of wild-type Norin 8 (N8, left) and the spl4-1 mutant (right). White scale bars = 6 cm. (B,D,F) Representative leaves of N8 and spl4-1 plants. The Roman numerals I, II, III, and IV represent the first, second, third, and fourth leaves from the top of the plants, respectively. Black triangles and boxes in (B) indicate the location and enlargement of autonomous lesions. Black scale bars = 2 cm. (G) The plant height was measured at 162 DAS. (H) Total chlorophyll concentration was determined from the first leaves (I) of N8 and spl4-1 plants shown in (F). Mean and standard deviations were obtained from more than three biological replicates. Asterisks indicate a statistically significant difference between N8 and spl4-1 plants according to Student’s t-test (∗∗∗P < 0.001). These experiments were repeated twice with similar results.
FIGURE 2
FIGURE 2
Internode length of the spl4-1 mutant. The length of panicles and internodes was measured in wild-type Norin 8 (N8) and the spl4-1 mutant grown in the field for 160 d after sowing. The picture (A) and the length (B) of panicles and internodes. Mean and standard deviations of all agronomic traits were obtained from twenty plants. Asterisks indicate statistically significant differences between N8 and spl4-1 plants according to Student’s t-test (∗∗P < 0.01, ∗∗∗P < 0.001). The plant photos shown are representative of three independent observations.
FIGURE 3
FIGURE 3
The leaves of spl4-1 mutant temporarily rolled before lesion formation. The pictures of the N8 and spl4-1 plants and leaves were taken of plants grown in the field at 56 days after sowing (DAS) (A–C) and 74 DAS (D–F). (A,D) Plant phenotype of wild-type Norin 8 (N8, left) and the spl4-1 mutant (right). White scale bars = 5 cm. (B,E) Representative leaves of N8 and spl4-1 plants. The Roman numerals I, II, and III represent the first, second, and third leaves from the top of the plants, respectively. Black scale bars = 2 cm. (C,F) The leaf width was measured in N8 and spl4-1 plants. Mean and standard deviations were obtained from more than three biological replicates. Asterisks indicate a statistically significant difference between N8 and spl4-1 plants according to Student’s t-test (∗∗∗P < 0.001). The plant photos shown are representative of three independent observations.
FIGURE 4
FIGURE 4
ROS accumulation in the spl4-1 mutant. (A) Visualization of singlet oxygen (1O2) detected by the SOSG fluorescent probe. The leaves of wild-type Norin 8 (N8) and the spl4-1 mutant were sampled at 133 d after sowing (DAS). The fluorescence of SOSG is green and chlorophyll (Chl) auto-fluorescence is red. Scale bars =5 μm. (B) DAB staining for hydrogen peroxide (H2O2) (dark brown). The leaves of N8 and spl4-1 plants were obtained at 60, 70, 80, and 125 DAS. NT, not treated. These experiments were repeated twice with similar results.
FIGURE 5
FIGURE 5
Map-based cloning of the SPL4 locus. (A) Physical mapping of the SPL4 locus. The SPL4 locus was initially mapped to a 1.7-Mb region between two markers, STS1 and RM587, on the short arm of chromosome 6. (B) Fine mapping of the SPL4 locus. The locus was further mapped within a 77-kb region between the STS8 and STS9 markers. Numbers below the line indicate the number of F3 recombinants at the marker regions. (C) Candidate genes in the 77-kb region. (D) The G to C substitution in SPL4 in the spl4-1 mutant. Black and white bars represent the exon and untranslated region, respectively. The black line represents the intron. The black arrow indicates the G to C substitution position in the spl4-1 mutant. (E) SPL4 nucleotide sequence. The black shading represents the SPL4 mRNA. Red arrows and the red character indicate the splicing sites and the G to C substitution, respectively. (F) Domain structure of SPL4 containing the MIT_spastin and AAA-type ATPase domains. Numbers indicate the amino acid position of SPL4.
FIGURE 6
FIGURE 6
Phenotype of the T-DNA insertion spl4-2 mutant. (A) Schematic diagram depicting the position of the T-DNA insertion in the SPL4 locus (Os06g03940). Black and white bars represent the exon and untranslated region, respectively. The black line represents the introns. The black arrow and open triangle indicate the location of the spl4-1 mutation and the SPL4 T-DNA insertion (spl4-2, PFG_3A-16679), respectively. (B) The mutation of SPL4 was verified in the leaves of 2-month-old wild-type plants (N8 and DJ) and spl4 mutants. The transcripts levels of SPL4 were determined by RT-qPCR analysis and normalized to that of OsUBQ5. Mean and standard deviations were obtained from more than three biological replicates. (C) Comparison of the formation of autonomous lesions between wild-type plants (N8 and DJ) and spl4 mutants grown in the field at 118 days after sowing. These experiments were repeated twice with similar results.
FIGURE 7
FIGURE 7
The spl4 mutants exhibit delayed leaf yellowing during dark-induced senescence. (A,B) Detached leaves of 2-month-old wild-type Dongjin (DJ) grown in the field were incubated on 3 mM MES (pH 5.8) buffer at 28°C with the abaxial side up in complete darkness. The leaf yellowing phenotype (A) and expression profile of SPL4 (B) were determined at 0, 2, and 4 days after dark incubation (DDI). The transcript levels of SPL4 were determined by RT-qPCR analysis and normalized to that of OsUBQ5. Different letters indicate significant differences according to one-way ANOVA and Duncan’s least significant range test (P < 0.05). (C–E) Detached leaves of Norin 8 (N8), DJ, and the spl4 mutants (spl4-1 and spl4-2) grown in the field for 2 months were subjected to dark conditions as shown in (A). The leaf yellowing phenotype (C) and total chlorophyll contents (D) were observed at 0 and 4 DDI. (E) An immunoblot assay was performed using antibodies against photosynthetic proteins (Lhca2, Lhca3, Lhcb2, Lhcb6, PsaA, and PsbD) and the large subunit of rubisco (RbcL). Mean and standard deviations were obtained from more than three biological replicates. Different letters indicate significant differences according to one-way ANOVA and Duncan’s least significant range test (P < 0.05). Asterisks indicate statistically significant differences between the wild-type plants and spl4 mutants according to Student’s t-test (∗∗P < 0.01, ∗∗∗P < 0.001). These experiments were repeated twice with similar results. Chl, chlorophyll; FW, fresh weight.
FIGURE 8
FIGURE 8
Subcellular localization of SPL4. (A–C) SPL4 fused with fluorescence proteins was transiently expressed in rice protoplasts (A) and onion epidermal cells (B,C) and observed by fluorescence microscopy. (A) The upper and lower panels show the localization of GFP and GFP-SPL4 in a rice protoplast, respectively. Chl, auto-fluorescence of chlorophyll. Scale bars = 5 μm. (B) The upper panels show the localization of yellow fluorescent protein (YFP) as a control and the lower panels show the localization of YFP-SPL4 in onion cells. DAPI, blue-fluorescent stain of DNA. Scale bars = 100 μm. (C) Enlargement of the DAPI-merged YFP-SPL4 signal shown in (B). Scale bar = 10 μm. The confocal images shown are representative of three independent observations. (D) Sequence analysis of putative NESs derived from plant spastin proteins. Red characters indicate the conserved hydrophobic amino acids, Φ = L, I, F, V, or M. x = any amino acid.
FIGURE 9
FIGURE 9
Agronomic traits of the spl4-1 mutant. Agronomic traits were compared between wild-type Norin 8 (N8) and the spl4-1 mutant grown in the field for 160 d after sowing. (A) 500-grain weight. (B) Number of panicles per plants. (C) Picture of fertile and sterile seeds from N8 and spl4-1 plants. The seed photo shown is representative of twenty independent measurements. (D) Yield per plants. (E) Seed setting rate. (F) Length of main panicle. (G) Number of spikelets per main panicle. Mean and standard deviations of all agronomic traits were obtained from twenty plants. Asterisks indicate a statistically significant difference between N8 and spl4-1 plants according to Student’s t-test (P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

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