OsJAZ13 Negatively Regulates Jasmonate Signaling and Activates Hypersensitive Cell Death Response in Rice

Abstract

Jasmonate ZIM-domain (JAZ) proteins belong to the subgroup of TIFY family and act as key regulators of jasmonate (JA) responses in plants. To date, only a few JAZ proteins have been characterized in rice. Here, we report the identification and function of rice OsJAZ13 gene. The gene encodes three different splice variants: OsJAZ13a, OsJAZ13b, and OsJAZ13c. The expression of OsJAZ13 was mainly activated in vegetative tissues and transiently responded to JA and ethylene. Subcellular localization analysis indicated OsJAZ13a is a nuclear protein. Yeast two-hybrid assays revealed OsJAZ13a directly interacts with OsMYC2, and also with OsCOI1, in a COR-dependent manner. Furthermore, OsJAZ13a recruited a general co-repressor OsTPL via an adaptor protein OsNINJA. Remarkably, overexpression of OsJAZ13a resulted in the attenuation of root by methyl JA. Furthermore, OsJAZ13a-overexpressing plants developed lesion mimics in the sheath after approximately 30-45 days of growth. Tillers with necrosis died a few days later. Gene-expression analysis suggested the role of OsJAZ13 in modulating the expression of JA/ethylene response-related genes to regulate growth and activate hypersensitive cell death. Taken together, these observations describe a novel regulatory mechanism in rice and provide the basis for elucidating the function of OsJAZ13 in signal transduction and cell death in plants.

Keywords: OsJAZ13; cell death; hypersensitive response; jasmonate; rice; signal transduction.

Figure 1

Identification of three alternative splicing variants of OsJAZ13. (A) RT-PCR analysis of OsJAZ13 transcripts using RNAs isolated from MH86 at the grain filling stage. This experiment was performed for at least three independent biological repeats. (B) Schematic diagram of alternatively spliced transcripts OsJAZ13a, OsJAZ13b, and OsJAZ13c, respectively. Blank box represented exons. (C) Alternative splicing differentially affects domain distribution in the three OsJAZ13 splice variants.

Figure 2

Expression pattern analysis of OsJAZ13. (A) The relative expression of OsJAZ13 in various tissues. RNA was extracted from different organs of MH86, at the seedling and filling stage. (B) Expression patterns of OsJAZ13 indicated by β-glucuronidase (GUS) staining. Samples at seedling, flowering, and filling stages were collected, stained, and then photographed with a SONY camera. (a) Stem, (b) leaf, (c) root, (d) sheath, (e) panicle, and (f) seed. (C) Three-leaf-stage seedlings of MH86 were treated with 100 µM MeJA (0.1% DMSO dissolved and as mock treatment), 100 µM ethephon (ddH₂O dissolved and as mock treatment), 4 °C, 42 °C, mechanical wounding, and 200 mM NaCl. Various numbers in this figure represent different time points for sample treatment. Samples were collected at indicated times. ACTIN served as internal control. All experiments were performed for at least three independent biological repeats.

Figure 3

Subcellular localization of OsJAZ13 splice variants. OsJAZ13s-YFP fusion proteins (pAVA321/35S:: YFP, 35S:: OsJAZ13a/YFP, 35S:: OsJAZ13b/YFP, 35S:: OsJAZ13c/YFP, and 35S:: OsJAZ13d (amino acids 1 to 110 of OsJAZ13)/YFP) were transiently expressed in rice protoplast. YFP fluorescence was detected, using a confocal laser, after incubation for 24–48 h, in the dark, at 28 °C. Scale bars = 5 μm. This experiment was performed for three independent biological repeats.

Figure 4

Y2H assay of the interactions between OsJAZ13 and the key regulators of the JA signaling pathway. (A) Schematic diagram of OsJAZ13 splice variants and the truncated derivative. The diagram displays the highly conserved Jas (green) and TIFY (red) domains, as well as the weakly conserved sequence (gray). The indicated combinations of plasmids were cotransformed into the yeast reporter strain Y2H gold; successfully transformed colonies were identified on appropriate selective minimal agar. (B) Interaction of OsJAZ13a, OsJAZ13b, OsJAZ13c, and OsJAZ13d with OsMYC2 derivatives or OsNINJA in the Y2H system. (C) Coronatine-dependent interaction of OsJAZ13a with OsCOI1 in the Y2H system. (D) Interaction between OsNINJA and OsTPL in the Y2H system. (E) Y2H analysis of the dimerization of OsJAZ13 splice variants (OsJAZ13a, OsJAZ13b, OsJAZ13c) and the truncated derivative OsJAZ13d. The empty bait (pGBKT7) and prey (pGADT7) vectors were used as negative controls. This experiment was performed for at least three independent biological repeats.

Figure 5

OsJAZ13 functions in jasmonate signaling. (A) Overexpression of OsJAZ13a exhibited the increased JA-insensitivity phenotype, and OsJAZ13b showed in slight JA-insensitivity; OsJAZ13d and suppression of OsJAZ13 resulted in JA-sensitivity phenotype similar to wild-type plants. The photograph indicated the phenotypes of seven-day-old seedlings of wild-type (WT) and transgenic lines grown on MS medium with 20 μM MeJA or under normal conditions. Scale bars = 1 cm. (B,C) Quantification of MeJA-induced growth inhibition of transgenic and WT plants. Data are the mean ± SD (n = 10 seedlings per genotype). Asterisks indicate significant differences in root and shoot length (^** p < 0.01, Student’s t-test) in comparisons with WT grown in the absence or presence of MeJA. Oxa-28 and Oxa-35 represent overexpression lines of OsJAZ13a; Oxb-1 and Oxb-16 represent overexpression lines of OsJAZ13b; Oxd-2 and Oxd-14 represent overexpression lines of OsJAZ13d; and Ri-4 and Ri-7 represent antisense suppression lines of OsJAZ13. All experiments were performed for three independent biological repeats.

Figure 6

Stability of OsJAZ13 splice variants in vivo. Transgenic rice seedlings expressing Ubi-OsJAZ13a-YFP, Ubi-OsJAZ13b-YFP, and Ubi-OsJAZ13d-YFP grown in 1/2MS medium treated with water (−MeJA) or 50 μM MeJA (+MeJA) for 2 h, or 100 μM MG132 + 50 μM MeJA (+MG132/MeJA) for 2 h. A confocal laser scanning microscopy was used to observed the YFP signal in root tissue. This experiment was performed for three independent biological repeats. Scale bars = 100 µm.

Figure 7

Expression analysis of JA-signaling-related genes in OsJAZ13a-overexpression plants treated with MeJA by qRT-PCR. RNA samples were collected at the indicated time from three-leaf-stage seedlings of wild-type and OsJAZ13a-overexpression lines treated with 100 µM MeJA. ACTIN served as an internal control. The values are means ± SD (n = 3 replicates). Different lowercase letters indicate significant differences (p < 0.05). The y-axis represents the relative expression level. This experiment was performed for three independent biological repeats.

Figure 8

OsJAZ13a-overexpression plants develop spontaneous necrosis on the sheath, which accumulates ROS and high expression of hypersensitive response marker gene. (A) Necrotic spots on sheath of OsJAZ13a-overexpression plants indicated by WT (a) and Oxa (b). Scale bars = 5 cm. Magnified views of the WT (c) and Oxa (d) are shown in the right panels. The plants were grown in a greenhouse (25 °C, 55–65% humidity, 14 h light/10 h dark) for 30–45 days. (B) Detection of H₂O₂ content in the leaves of OsJAZ13a-overexpression lines while lesion appearing on the sheath, WT (above) and Oxa (bottom). The brown region staining by DAB on leaves indicates the H₂O₂ level. (C) Detection of PCD in the leaves of OsJAZ13a-overexpression lines. The blue spots on leaves by typan blue staining indicate cell death, WT (above) and Oxa (bottom). (D) Expression analysis of OsJAZ13 and hypersensitive response marker gene HSR203J by RT-PCR; RNA was extracted from leaves of OsJAZ13a-overexpression plants when lesion emerged in the sheath. ACTIN served as internal control. These experiments were performed for three independent biological repeats.

Figure 9

Expression analysis of necrosis-development-related genes in OsJAZ13a-overexpression lines. RNA was extracted from rice leaves of OsJAZ13a overexpression lines and wild type, and which were planted in greenhouse and lesion appeared on the sheath of transgenic plants. We performed qRT-PCR to analyze associated genes involved in JA/ET signaling pathway OsMYC2, OsERF1, OsEIL1, OsERF047, and OsERF077; pathogen-defense genes OsNPR1 and OsPR1b; and defense-related genes OsPR2, OsPR31, OsPR4, OsPR5-like, and OsPR10. ACTIN served as the internal control. The values are means ± SD (n = 3 replicates). Different lowercase letters indicate significant differences (p < 0.05). The y-axis represents the relative expression level. This experiment was performed for three independent biological repeats.

Figure 10

Phenotype analysis of OsJAZ13a-overexpression plants under paddy fields. OsJAZ13a-overexpression lines and the wild type were transplanted to paddy fields about 30–45 days till plants developed to the tillering stage. (a) Wild type, (b) one tiller died, (c) two tillers died, (d–g) comparation of the sheath between dead and normal tillers, (h) differential level of the water-soaked tiller, (i) the comparison of leaves, and (j) the cross-section of a dead tiller. This experiment was performed for three independent biological repeats. Scale bars = 10 cm (a–c) and 1 cm (d–j).