Figure 1.
Amino acid sequence alignment and phylogenetic tree of COI1 and OsCOIs.
A, Deduced amino acid sequences of COI1 and OsCOIs were aligned using the ClustalW program. Gray shading indicates identical residues. Approximately 55% identity was shown between COI and OsCOIs. Solid boxes indicate residues involved in the COI-coronatine complex. Dashed boxes indicate residues involved in the COI-JAZ interaction. Asterisks indicate amino acid residues involved in the COI-coronatine complex or COI-JAZ interaction, which shows the difference between coronatine and JAZ interactions. B, The phylogenetic tree of COI1 and OsCOIs. These results were drawn with DNAstar software.
Figure 2.
Relative expression levels of OsCOI1a, OsCOI1b and OsCOI2, as shown by qRT-PCR. Expression level of OsCOI1a was set to 1 arbitrarily and the relative expression levels of OsCOI1b and OsCOI2 are shown. Data represent mean values of three measurements and error bars represent standard deviation. Total RNA was isolated from 2-week-old seedlings and equal amounts of RNA were used for qRT-PCR analysis.
Figure 3.
Molecular modeling of the OsCOI-coronatine complex.
A, OsCOI1a-coronatine complex. B, OsCOI2-coronatine complex. COI1, OsCOI1a, and OsCOI1b have Tyr-386, Tyr-389, or Tyr-391 residues, respectively, but OsCOI2 has a His-391 residue at the interaction point. Hydrogen bonds are shown as yellow dotted lines. COI1, OsCOI1a and OsCOI1b form a 3.4 Å hydrogen bond but OsCOI2 forms a 4.45 Å hydrogen bond.
Table 1.
Summary of the Y2H assay of OsJAZs.
Figure 4.
Morphological phenotype of complemented coi1-1.
The coi1-1 mutant was complemented with OsCOI1a (#1134), OsCOI1b (#2124), OsCOI2(H391Y) (#H3159) or COI1 (#A0088), respectively, at the T2 generation. A, Siliques of 6-week-old plants grown in soil. The asterisks indicate developing siliques. B, Flowers of 6-week-old plants grown in soil. C, Fully developed siliques. D, Developing seeds in the silique.
Table 2.
Segregation of coi genotypes in transformants and their complementation frequency.
Figure 5.
Inducible expression of JA responsive genes by complementation.
Response of AOS and JR2 is shown in the coi1-1 mutant transformed with OsCOI1a (#1134), OsCOI1b (#2124), OsCOI2(H391Y) (#H3159) or COI1 (#A0088), respectively, at T2 generation. Plants were sprayed with 50 µM MeJA and total RNA was isolated at the indicated times after MeJA treatment and analyzed by Northern blot. rRNA was visualized by ethidium bromide staining to show an equal loading. No complemented plants were obtained using OsCOI2 in this experiment.
Figure 6.
Restoration of root growth inhibition phenotype by complementation.
Col-0, coi1-1 heterozygote, and complemented T3 homozygous lines were grown vertically on MS media containing 50 µM MeJA (left) or without MeJA (right). The asterisk indicates coi1-1 homozygote which was tested by PCR and XcmI enzyme digestion.