Figure 1.
Phenotype of the cea62 mutant.
OsAOS2 expression was strongly induced in the cea62 mutant (A). Two-month-old plants (B), tillers (C) and leaves (D) of the wild type (WT) and cea62 mutant. (E) The expression of several histochemical markers in cea62 and wild-type plants was analyzed. Leaves were stained with DAB, NBT or trypan blue at 21 or 30 days after sowing. And JA production (F) was much greater in the cea62 mutant than in the wild type. (G–I) Quantification of several agricultural traits, including tiller number (G), seed setting ratio (H), and relative pollen activity (I). Each bar is the mean ± SD of 30 replicate samples.
Figure 2.
Molecular identification of the cea62 mutant.
(A) Map-based cloning of cea62. The CEA62 gene (red arrow) was mapped to the short arm of chromosome 2 between markers S2-570 and S2-630 and was delimited to a 37-kb region with eight candidate genes. A single-base substitution (TAC→TGA) at the 1146th position of LOC_Os02g02000 was present in the cea62 mutant. (B) Functional complementation of the cea62 mutant. Three-month-old wild type, cea62 and complemented cea62 (cea62-C) plants are shown. (C) Expression of OsHPL3/CEA62 in the wild type (WT), cea62 mutant and cea62-C plants and (D) expression of OsHPL3/CEA62 in different tissues of the unwounded wild type, as analyzed by quantitative RT-PCR. Rice OsACT was used as an internal control. These data were obtained from three independent replicates. Each bar is the mean ± SD of three replicate samples.
Figure 3.
In vitro enzyme activity assays of OsHPL3 and quantification of C6-aldehydes and JA.
(A) OsHPL3 activity was monitored by the loss of absorbance of substrate at 234 nm in leaf extracts of the wild type (WT), cea62 mutant and cea62 complemented transgenic plant (cea62-C) using 13-hydroperoxylinolenic acid (13-HPOT) or (B) 13-hydroperoxylinoleic acid (13-HPOD) as substrate. Results were obtained from one experiment with three biological replicates, and similar results were obtained in three independent experiments. (C) The levels of n-hexanal and (E)-2-hexenal in the WT, cea62 and cea62-C. (D) The quantity of JA in the WT and cea62 mutant. Histograms are the average of triplicate assays and the bars indicate SD.
Figure 4.
Expression patterns of JA-related genes in the wild type and cea62 mutant.
OsLOX and OsAOS2 are JA biosynthesis genes in rice. OsJAZ6, OsJAZ8 and OsJAmyb are JA signaling genes in rice. OsPR1b and OsPR10 are pathogen-related genes in rice. OsNPR1 is a key regulator of an SA-dependent systemic resistance gene. Rice Actin1 was used as a reference control. Leaves of the wild type (WT), the cea62 mutant at two weeks (cea62) and three months (cea62/LM) after sowing and the cea62 complemented transgenic (cea62-C) plant were used to monitor the expression of these marker genes. In all panels, the mean is based on the average of three biological repeats. Each bar is the mean ± SD of three replicate samples.
Figure 5.
Expression patterns of OsHPL3 and JA-related maker genes in the wild type and cea62 mutant at different developmental stages.
The expression of JA biosynthesis genes, (A) OsLOX and (B) OsAOS2; JA signaling genes, (C) OsJAZ6 and (D) OsJAZ8; defense-related maker genes, (E) OsPR1b and (F) OsPR10; and (G) OsHPL3 were monitored at various time points. The enzyme activity of (H) OsHPL3 was also recorded. These data were obtained from three independent replicates. Each bar is the mean ± SD of three replicate samples.
Table 1.
Selected differentially expressed genes functionally classified in the cea62 mutant compared with the wild type by microarray analysis.
Figure 6.
Disease resistance test to rice bacterial blight Xanthomonasoryzaepvoryzae (Xoo) T1 strains.
Photographs were taken at 20 days after inoculation. (A) Lesion lengths on leaves of wild-type (WT) and cea62 plants at 20 days after inoculation. (B) Data represented here were obtained in one experiment with 30 biological replicates, and similar results were obtained in three independent experiments. Asterisks indicate significant differences in the cea62 mutant compared with WT plants. (*P<0.05; **P<0.01; Student's t test). Scale bars, 1 cm.
Figure 7.
The wounding response of cea62 plants.
The expression of OsJAmyb and OsJAZ6 were monitored in wound-treated wild type (WT) and cea62 plants before and after the LM phenotype appeared. The results were obtained from three independent experiments.
Figure 8.
The expression of SA-regulated genes and quantity of SA and JA in the leaves of the WT and cea62 mutant before and after the lesion mimic phenotype appeared.
The leaves of 2-week-old and 2-month-old wild-type (WT) and cea62 mutant plants were used to monitor the expression of the SA-regulated genes (A) OsEDS1 and (B) OsPAD4 and to quantify (C) SA and (D) JA levels. Each bar is the mean ± SD of three replicate samples.