Evaluation of the physiological action of coronatine relative and
derivatives. (a) Structures of coronatine (1) relatives
and derivatives: 7-epi-jasmonoyl-l-isoleucine
(2), coronatine (1), and related compounds
(ent1, 3-ent4, ent3-4, 3, and 4). (b) Effect of 1 derivatives on reopening (measured as size of aperture) of Arabidopsis thaliana (Col-0) abaxial stomata by abscisic
acid (ABA). Indole-3-acetic acid (IAA), which induces stomatal reopening,
was used as a positive control. Dashed line indicates the mean stomatal
aperture taken from a control setting, in which Arabidopsis leaf peels with closed stomata were incubated in MES buffer (pH
6.2) containing 2% EtOH. Bars represent mean stomatal aperture and
SE (n = 20 stomata). Letters a–d indicate
significant differences between the means (ANOVA: P < 0.05). (c) A stoma in the epidermis of Arabidopsis
thaliana. The width of the red bar was measured as the stomatal
aperture. (d) Yeast two-hybrid (Y2H) assay of compound-induced COI1-JAZ9
coreceptor formation. Lane A: Yeast cells cotransformed with pDEST22-JAZ9
and pDEST32-COI1. Lane B: Yeast cells cotransformed with pDEST22-RalGDS-m1
and pDEST32-Krev1 as a positive control. Lane C: Yeast cells cotransformed
with pDEST22-RalGDS-m2 and pDEST32-Krev1 as a negative control. (e)
Pull-down assay of purified GST-COI1 (5 nM) with recombinant E. coli expressed MBP-JAZ9 (approximately 40 nM) in the
presence of COR derivatives (100 nM). GST-COI1 bound to MBP-JAZ9 were
pulled down with amylose resin, and analyzed by immunoblotting. HRP-conjugated
anti-GST antibody was used for detection of GST-COI1. Anti-MBP antibody
and HRP-conjugated rat IgG antibody was used for showing the amounts
of MBP-JAZ9 as the input materials. (f, g) Relative transcript levels
of AOS and VSP2 in A. thaliana treated with 1 derivatives. Each relative transcript
level was assessed by quantitative RT-PCR. “Cont.” indicates
the mean AOS or VSP2 transcript
level in a buffer containing 2% EtOH only. Bars represent the mean AOS or VSP2 transcript levels of the test
samples (100 μM). Bars represent mean and SE (n = 3). Letters a–c indicate significant differences between
the means (ANOVA: P < 0.05).