Understanding the Role of Chloroplast Signalling in Plant Development and Drought Tolerance
Date
2015
Authors
Phua, Su Yin
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Abstract
3’-phosphoadenosine 5’-phosphate (PAP) was recently proposed
as a new
chloroplast retrograde signal that accumulates during drought
stress. PAP is a
by-product of secondary sulfur assimilation, generated by
SULFOTRANSFERASES (SOTs) upon transferring of the sulfate group
from
3’-phosphoadenosine 5’-phosphosulfate (PAPS) to sulfate
acceptor molecules.
PAP is efficiently degraded by the SAL1 phosphatase into
inorganic
phosphate and adenosine monophosphate (AMP). Consequently, PAP
levels
are normally kept at very low during plant development.
Constitutive
over-accumulation of this retrograde signal resulted in plants
with pleiotropic
altered phenotypes as demonstrated by the sal1 mutant in
Arabidopsis,
namely improved drought tolerance, altered rosette morphology and
delayed
development. These multifaceted altered phenotypes of sal1
correlate well
with the many transcriptional changes, which are mainly due to
the inhibition
of 5’- 3’ EXORIBONUCLEASES (XRNs) by PAP. However, the
details of
downstream changes that links SAL1-PAP-XRN to the many altered
phenotypes of sal1 remains poorly understood. This thesis
investigates the
possibility of conferring drought tolerance to plants by
manipulating the
SAL1 gene expression with minimal negative effects on growth,
while
unraveling the signalling pathway(s) contributing to sal1 altered
development
and drought tolerance.
I hypothesised that PAP accumulation at early developmental
stages is
detrimental for plant growth and development while its
accumulation at later
stages of development or prior to drought stress is beneficiary
for conferring
plant drought tolerance. To test this hypothesis, two different
strategies for
easy manipulation of SAL1 expression were attempted: inducible
silencing of
SAL1 in wild-type Arabidopsis and inducible complementation of
SAL1 in
the sal1 mutant background. Surprisingly, efficient silencing of
SAL1 could
not be achieved even with a strong constitutive promoter driving
the
expression of SAL1-hair-pin RNA interference (hpRNAi) or
SAL1-artificial
microRNA (amiRNA) and inducible-silencing was similarly
inefficient. On
the other hand, inducible complementation of SAL1 in the sal1
mutant
background allowed for better manipulation of SAL1 expression and
PAP levels. This provides the platform for exploring the
engineering of drought
tolerant plants without compromising plant development by
manipulating
PAP levels via regulating SAL1 expression.
Since hormones are key regulators of plant development, the
possible
interaction between PAP and hormone signalling was investigated
to study
the basis of sal1 developmental phenotypes. Significantly, a
comprehensive
hormonal profiling showed reduced gibberellins (GAs) content in
sal1 and
improved growth rate was achieved when sal1 was treated with GAs
and
brassinosteroids (BRs), which are interdependent hormones that
promote
growth. Additionally, sal1 germination was hyper-responsive to
treatments
with abscisic acid (ABA) and/or GAs biosynthetic inhibitor -
paclobutrazol
(PAC). Furthermore, this observation can be reproduced by feeding
wild-type
seeds with PAP in addition to ABA and PAC, suggesting that PAP
could act
as secondary messenger for both GAs and ABA signalling.
Meanwhile, upon
taking into consideration both the findings of this thesis and
literature
available, it is proposed that SAL1-PAP-XRN stabilises DELLA
accumulation
by reducing transcription of key GA biosynthetic genes, allowing
the DELLA
protein to interact and affect the key transcription factors of
light and other
hormonal signalling pathways such as BRs, jasmonic acid (JA) and
ethylene.
It is likely that the overall interactions and feedback between
1) altered sulfur
metabolism, 2) altered hormonal homeostasis and 3) inhibitory
effects of PAP
on XRNs and other nucleotide-binding proteins eventually
culminate in the
pleitropic developmental phenotype and drought tolerance of
sal1.
The ultimate application from this project will be to engineer
drought
tolerance in Brassicaceae by manipulating the SAL1-PAP pathway.
This thesis
has generated the tools that will aid in the above engineering
and uncovered
the potential connections between the sal1 altered phenotypes
and
SAL1-PAP-XRNs, linking chloroplast signalling to plant hormonal
homeostasis in regulating plant development and drought
tolerance.
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Keywords
SAL1, PAP, transgenics, drought, germination, plant hormones
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