MicroRNAs (miRNAs) are a class of small RNAs that regulate gene
expression in eukaryotes. In plants, many miRNA families mediate
silencing of target genes, which are involved in plant
development and plant defence. For my thesis, I have been
investigating the miR159-GAMYB pathway, which appears conserved
from basal vascular plants to angiosperms. GAMYB transcription
factors have been demonstrated to have conserved roles of
programmed cell death (PCD) in both the seed aleurone and the
anther tapetum in a number of different plant species. However,
what the functional role of GAMYB is in vegetative tissues
remains unknown. In Arabidopsis, miR159 is critical for proper
growth, as its inhibition has a strong negative impact on
vegetative growth, due to deregulated GAMYB expression. However,
gamyb loss-of-functional mutants display a wild-type phenotype,
as their expression is silenced to phenotypically inconsequential
levels by miR159 in vegetative tissues. This raises two
questions: (1) how is GAMYB so strongly silenced; (2) why is
GAMYB strongly and widely transcribed in vegetative tissues for
it to be then completely repressed by miR159? These two questions
were the focus of my thesis.
Firstly, how the Arabidopsis MYB33 and MYB65 are so strongly
silenced in the model plant Arabidopsis was investigated. Both
genes were predicted to contain a distinctive RNA secondary
structure abutting the miR159 binding site, composed of two
stem-loop (SL) structures; whereas such SL structures were not
predicted to form in other GAMYB-like genes that are targeted
less efficiently by miR159 for expression regulation. Functional
analysis found that the RNA structure in MYB33 correlated with
strong silencing efficacy; introducing mutations to disrupt
either SL attenuated miR159 efficacy, while introducing mutations
to form an artificial stem-loop structure adjacent to a
miRNA-binding site restored strong miR159-mediated silencing.
Although how these predicted structures promote miR159-mediated
silencing are not determined, we speculate that the stem-loop
structures in the vicinity of the miR159 binding site promotes
accessibility of the binding site, where if adjacent sequences
form strong stem structures, they are less likely to base-pair
with binding site nucleotides, maintaining high accessibility of
the binding site. Interestingly, the RNA SL structures are
predicted to reside in GAMYB-like homologues of numerous
angiosperm and gymnosperm plant species, arguing that these
structures have been integral in the miR159-GAMYB regulatory
relationship over a long period of time. In addition, these
structures are not present in the Arabidopsis GAMYB-like
homologues that are not transcribed in vegetative tissues,
suggesting that selection for such structures only occurs for
homologues transcribed in vegetative tissues as to prevent their
expression and demarcating them as sensitive targets of miR159.
Secondly, to investigate the functional role of the miR159-GAMYB
pathway, target MIMIC159 (MIM159) transgenes, which can inhibit
endogenous miR159 activity, were expressed in a number of
Arabidopsis ecotypes, as well as in tobacco and rice. Inhibiting
miR159 in all three plant species resulted in similar phenotypic
outcomes, predominantly stunted growth and irregular leaf shape.
This implies that the function and expression of the miR159-GAMYB
pathway is strongly conserved in distant plant species. This
raises several questions: why is GAMYB widely transcribed if its
expression is strongly silenced by miR159 throughout the plant to
result in little to no phenotypic impact; and why has this been
strongly conserved across multiple plant species. When miR159
activity is inhibited in MIM159 tobacco leaves, pathways related
to plant defence response are most up-regulated. This included
PATHOGENESIS-RELATED PROTEIN (PR) mRNA levels that were 100-1000s
fold up-regulated compared to wild type, and correlated with
deregulated GAMYB expression. This finding suggests that the
miR159-GAMYB pathway is involved in the plant defence response to
biotic stress. However, PR expression is not up-regulated in
Arabidopsis or rice when miR159 is inhibited, suggesting that
despite the conserved nature of the miR159-GAMYB pathway, there
are species-specific differences in its function
