Mutation Types of CYP71P1 Cause Different Phenotypes of Mosaic Spot Lesion and Premature Leaf Senescence in Rice

Abstract

Lesion mimic mutants (LMMs) are ideal materials for studying programmed cell death and defense response in plants. Here we report investigations on two LMMs (msl-1 and msl-2) from the indica rice cultivar JG30 treated by ethyl methyl sulfone. Both of the mutants showed similar mosaic spot lesions at seedling stage, but they displayed different phenotypes along with development of the plants. At tillering stage, larger orange spots appeared on leaves of msl-2, while only small reddish-brown spots exhibit on leaves of msl-1. At heading stage, the msl-2 plants were completely dead, while the msl-1 plants were still alive even if showed apparent premature senility. For both the mutants, the mosaic spot lesion formation was induced by light; DAB and trypan blue staining showed a large amount of hydrogen peroxide accumulated at the lesion sites, accompanied by a large number of cell death. Consequently, reactive oxygen species were enriched in leaves of the mutants; SOD and CAT activities in the scavenging enzyme system were decreased compared with the wild type. In addition, degraded chloroplasts, decreased photosynthetic pigment content, down-regulated expression of genes associated with chloroplast synthesis/photosynthesis and up-regulated expression of genes related to senescence were detected in the mutants, but the abnormality of msl-2 was more serious than that of msl-1 in general. Genetic analysis and map-based cloning revealed that the lesion mimic and premature senescence traits of both the mutants were controlled by recessive mutated alleles of the SL (Sekiguchi lesion) gene, which encodes the CYP71P1 protein belonging to cytochrome P450 monooxygenase family. The difference of mutation sites and mutation types (SNP-caused single amino acid change and SNP-caused early termination of translation) led to the different phenotypes in severity between msl-1 and msl-2. Taken together, this work revealed that the CYP71P1 is involved in regulation of both premature senescence and cell death in rice, and its different mutation sites and mutation types could cause different phenotypes in terms of severity.

Keywords: rice; cell death; leaf senescence; lesion mimic mutants; mutation types; reactive oxygen species.

FIGURE 1

Phenotypic characterization of wild type and mutants. (A) Phenotypes at seedling stage (30 days after sowing). (B) Phenotypes at the young panicle differentiation stage. (C) Leaves at seedling stage. (D) Leaves at tillering stage. (E) Plant phenotypes at heading stage. (F) Formation of mosaic spot lesion is light dependent. From left to right: leaf of wild type, leaf of msl-1 covered with aluminum foil for 7 days, leaf of msl-1 recovered to light for 7 days, leaf of msl-2 covered with aluminum foil for 7 days, leaf of msl-2 recovered to light for 7 days. (G) Diaminobenzidine (DAB) staining showing accumulation of H₂O₂ at mosaic lesions. (H) Trypan blue staining, showing cell death at the mosaic lesions. Bar = 1 cm (A); Bar = 10 cm (B,E).

FIGURE 2

Premature senescence identification of mutant leaves. (A–F) Ultrastructural analysis of chloroplasts in wild type and mutants by transmission electron microscopic. C, chloroplast; CE, chloroplast membrane; CW, cell wall; OG, osmiophilic plastoglobuli. (G) Determination of photosynthetic pigment content. (H) Expression assay of chloroplast synthesis-related genes. (I) Expression assay of photosynthetic-related genes. (J) Expression assay of senescence-related genes. Error bars represent the standard deviations of three biological replicates. * and **, significant differences at P < 0.05 and P < 0.01, respectively (Student’s t-test).

FIGURE 3

Analysis of ROS accumulation and relative expression of antioxidant enzymes in wild type and mutants. (A) H₂O₂ contents. (B) Malondialdehyde (MDA) contents. (C) SOD enzyme activity. (D) CAT enzyme activity. Error bars represent the standard deviations of three biological replicates. **, significant differences at P < 0.01 (Student’s t-test).

FIGURE 4

Map-based cloning of msl-1 and msl-2. (A) The msl-1 and msl-2 loci were fine-mapped to 58kb region between Indel markers S1 and L4 on chromosome 12. (B) Gene structure of LOC_Os12g16720 and the mutation site in msl-1. (C) Gene structure of LOC_Os12g16720 and the mutation site in msl-2. The black rectangles represent exons and the red inverted triangle represents the mutation sites. (D) Analysis of SL gene expression in different tissues of WT. Error bars represent the standard deviations of three biological replicates.

FIGURE 5

Bioinformatics analysis of target genes. (A) Protein sequence alignment of the CYP71P1 protein homologies among multiple species, both the mutation sites of msl-1 (at 73nd residue) and msl-2 (at 414th residue) are highly conserved. (B) Protein three-dimensional structure of CYP71P1, msl-1 and msl-2. (C) Phylogenetic tree analysis of the CYP71P1 homologies.

FIGURE 6

Schematic structure of CYP71P1 protein and the mutation sites in msl-1 and msl-2. The black dotted bordered rectangle indicates CYP71P1 the highly conserved P450 superfamily domain of wild type CYP71P1. (A) The mutation site of ell1 and sl-MH-1, ell1 had a single base substitution at 275th nucleotide position (C275A) and sl-MH-1 also had a single base substitution at 1,205th nucleotide position (G1205T). (B) The mutation site of msl-1, with a base change from T to C at the 218th position. (C) The mutation site of msl-2, with the G-to-A single-base alteration, leading to premature termination of the protein translation.

FIGURE 7

Proposed working model for SL-involved rice leaf senescence and cell death. The conversion of tryptamine to serotonin is catalyzed by CYP71P1 encoded by SL (Sekiguchi lesion) gene. Mutations of SL affect or block the catalytic process, accompanied with accumulated tryptamine and the low level of serotonin. Exogenous applied serotonin enhances resistance to Magnaporthe grisea (M. grisea) in the sl mutant. Low level of serotonin causes excessive reactive oxygen species (ROS) accumulation in the sl mutants, triggering programmed cell death (PCD)-mediated cell apoptosis, resulting in lesion formation. Excessive ROS also contribute to activation of pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) to plant pathogens like M. oryzae and Xanthomonas oryzae pv. oryzae. ROS can function as signaling messengers to induce chloroplast degradation directly or by regulating the changes of senescence-associated genes (SAGs), resulting in the premature leaf senescence phenotype.