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Surprisingly, compared to the induction of MYB21 and MYB24 expression by JA
treatment which is detectable at 18 hrs post treatment (Figure 5D), GA induction
of the expression of these two MYB genes in ga1-3 gai-t6 rga-t2 rgl1-1 happens
much later and only became detectable at 72 hrs (Figure 7B). More interestingly,
GA induction of the expression of DAD1 is obviously detectable at 48 hrs which
is prior to GA-induced expression of MYB21 and MYB24 in the ga1-3 gai-t6
rga-t2 rgl1-1 quadruple mutant (Figure 7B). Our data suggest that GA might first
induce the expression of DAD1 to promoter JA production then via JA signaling
to regulate the expression of MYB21 and MYB24.
Expression of MYB21, MYB24, and MYB57 Is Necessary But
Insufficient for Normal Stamen Filament Elongation in ga1-3
gai-t6 rga-t2 rgl1-1
As shown in the above, MYB21, MYB24 and MYB57 act downstream of DEL-
LAs in controlling stamen filament elongation. Expression of MYB21, MYB24
and MYB57 was repressed and floral development was arrested in the ga1-3 gai-t6
rga-t2 rgl1-1 Q3 quadruple mutant (Figure 2B and 2C). Regarding the fact that
JA content is reduced in the young flower buds of Q3 we questioned whether res-
toration of expression of these MYBs by exogenous application of JA could rescue
the stamen development to the ga1-3 gai-t6 rga-t2 rgl1-1 Q3 plants. We analyzed
the flowers of JA-treated ga1-3 gai-t6 rga-t2 rgl1-1 plants and found that repeated
JA application was unable to rescue the stamen development (Figure 8) though
JA could restore the expression of the three MYB genes (Figure 5D), indicating
that expression of MYB21, MYB24 and MYB57 alone was insufficient for normal
stamen development in the ga1-3 gai-t6 rga-t2 rgl1-1 mutant. Furthermore, we
found that exogenous application of GA to the ga1-3 gai-t6 rga-t2 rgl1-1 plants
was able to induce the expression of MYB21, MYB24 and MYB57 (Figure 7B)
and recover normal floral development (Figure 8). Taken together, our results
demonstrate that besides these JA-inducible MYBs, other important GA-regulat-
ed JA-independent factors are needed for normal stamen filament development
in ga1-3 gai-t6 rga-t2 rgl1-1.
Overexpression of MYB21 Restored Stamen Filament
Elongation and Fertility to opr3 Flowers
To test our hypothesis that GA acts through JA to control expression of the MYB
genes to promote filament elongation, we fused MYB21 gene with the CaMV35S
promoter (pCAMBIA1301 vector) and this construct was used to generate trans-
genic plants in the opr3 mutant background. Semi-quantitative RT-PCR showed
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