The original objectives of the research were: i. To study the role of GA in
anther development, ii. To manipulate GA and/or GA signal transduction
levels in the anthers in order to generate male sterility. iii. To
characterize the GA signal transduction repressor, SPY. Previous studies
have suggested that gibberellins (GAs) are required for normal anther
development. In this work, we studied the role of GA in the regulation of
anther development in petunia. When plants were treated with the
GA-biosynthesis inhibitor paclobutrazol, anther development was arrested.
Microscopic analysis of these anthers revealed that paclobutrazol inhibits
post-meiotic developmental processes. The treated anthers contained pollen
grains but the connective tissue and tapetum cells were degenerated. The
expression of the GA-induced gene, GIP, can be used in petunia as a
molecular marker to: study GA responses. Analyses of GIP expression during
anther development revealed that the gene is induced only after
microsporogenesis. This observation further suggests a role for GA in the
regulation of post-meiotic processes during petunia anther development. Spy
acts as a negative regulator of gibberellin (GA) action in Arabidopsis. We
cloned the petunia Spy homologue, PhSPY, and showed that it can complement
the spy-3 mutation in Arabidopsis. Overexpression of Spy in transgenic
petunia plants affected various GA-regulated processes, including seed
germination, shoot elongation, flower initiation, flower development and the
expression of a GA- induced gene, GIP. In addition, anther development was
inhibited in the transgenic plants following microsporogenesis. The
N-terminus of Spy contains tetratricopeptide repeats (TPR). TPR motifs
participate in protein-protein interactions, suggesting that Spy is part of
a multiprotein complex. To test this hypothesis, we over-expressed the SPY's
TPR region without the catalytic domain in transgenic petunia and generated
a dominant- negative Spy mutant. The transgenic seeds were able to germinate
on paclobutrazol, suggesting an enhanced GA signal. Overexpression of PhSPY
in wild type Arabidopsis did not affect plant stature, morphology or
flowering time. Consistent with Spy being an O-GlcNAc transferase (OGT), Spy
expressed in insect cells was shown to O-GlcNAc modify itself. Consistent
with O-GlcNAc modification playing a role in GA signaling, spy mutants had a
reduction in the GlcNAc modification of several proteins. After treatment of
the GA deficient, gal mutant, with GA3 the GlcNAc modification of proteins
of the same size as those affected in spy mutants exhibited a reduction in
GlcNAcylation. GA-induced GlcNAcase may be responsible for this
de-GlcNAcylation because, treatment of gal with GA rapidly induced an
increase in GlcNAcase activity. Several Arabidopsis proteins that interact
with the TPR domain of Spy were identified using yeast two-hybrids screens.
One of these proteins was GIGANTEA (GI). Consistent with GI and Spy
functioning as a complex in the plant the spy-4 was epistatic to gi. These
experiments also demonstrated that, in addition to its role in GA signaling,
Spy functions in the light signaling pathways controlling hypocotyl
elongation and photoperiodic induction of flowering. A second Arabidopsis
OGT, SECRET AGENT (SCA), was discovered. Like SPY, SCA O-GlcNAc modifies
itself. Although sca mutants do not exhibit dramatic phenotypes, spy/sca
double mutants exhibit male and female gamete and embryo lethality,
indicating that Spy and SCA have overlapping functions. These results
suggest that O-GlcNAc modification is an essential modification in plants
that has a role in multiple signaling pathways.
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