Transcript Q: How does GA induce gene expression in the aleurone cells?

Plant hormone signaling I
1.
What is a plant hormone? How to determine if a molecule
functions as a hormone or not? Several criteria:
1) produced by plant and has profound effect on growth
and development 2) transportable (from one site to another
inside the plant) 3) functions at a low level (often
micromolar or less) 4) level regulated tightly by
biosynthesis and degradation.
Currently accepted families: auxin, GA, cytokinin, ethylene,
abscisic acid (ABA), steroids, jasmonic acid, peptides
2. Auxin
1) Overview: IAA is the major endogenous form of auxin,
produced mainly at rapidly growing tissues like meristems,
young leaves, transported polarly from top to bottom of
the plants.
2) Biological functions: enhance cell elongation, apical
dominance, Promote lateral root formation, vascular tissue
differentiation…
3) Signaling: how does auxin work?
A) Two responses have been used as models to study auxin
function: cell elongation and gene expression
B) Auxin receptor?
One candidate is ABP (auxin-binding protein): identified by
photoaffinity labeling technique (auxin-photoactive group
that will be attached to an amino acid group in a protein when
this protein gets very close to the auxin molecule). ABP is a
small protein about 200 AA long and has a KDEL sequence
at the C-terminus. Considering its location in the cell, how
could this protein be auxin receptor?
What is the evidence for ABP function? Essential gene for
embryo development (knockout embryo cannot develop to
mature stage).
C) How does ABP regulate auxin-induced cell elongation?
Auxin has been shown to enhance cell wall acidification by promoting HATPase activity.
Some studies showed
That ABP regulates
H-pump activity.
BUT it is not clear if
ABP is a true receptor
for auxin. Certainly,
ABP is not the ONLY
Receptor.
D) From auxin to gene expression
a) Auxin-induced genes and promoter analysis
Gene/DNA
Promoter
transcribed region
mRNA
Detect gene expression at mRNA level: northern blot
An typical pattern of auxin-induced gene expression
0
1h
2h
3h
4h
IAA +
+
+
+
+
Promoter
GUS/reporter
IAA
+
+
TGTCTC
--AuxRE
Isolation of proteins bound to this sequence
Resulting in discovery of ARF—auxin
response factor, a transcription factor
controling auxin-induced gene expression
b) Auxin-induced genes:
early genes encoding inhibitors of ARF. These proteins are called IAA/Aux.
They were first discovered in Theologis lab as they worked on “rapid”
auxin-induced genes. The IAA proteins form dimers with other IAA proteins
and with ARF. When they bind ARF, ARF activity is suppressed so that gene
expression is shut down.
ON
ARF
Promoter
Gene/DNA
transcribed region
mRNA
IAA/Aux
ARF
OFF
Promoter
Gene/DNA
transcribed region
c) The genetics analysis further revealed how auxin, the hormone, may
regulate the gene expression process
Several auxin-insensitive mutants were identified and they all represent
genes encoding IAA/Aux or components in the ubiquitin-dependent
protein degradation pathway.
For example, axr2 and axr3 (auxin-resistant mutants) have mutation in the
IAA/Aux genes. Tir1and ask1 are mutants that have mutation in genes for
the ubiquitin pathway (the E3 enzyme complex).
The summary: how does auxin regulate gene expression?
3. Gibberellins
1)overview: gibberillic acid discovered as a fungal compound that
enhances rice growth but reduce seed production—”folish seedling
disease. Later on similar compounds were identified in plants and
defined as a growth hormone with many functions such as
promoting stem growth, seed germination, flowering etc.
2) Signal transduction for GA responses
A) In the arabidopsis seedlings: mutant selection for plants that are more
or less sensitive to GA. Several categories have been isolated:
--GA-insensitive dwarf mutants,
--suppressors of GA-deficient
dwarf, and constitutive GAresponse mutants (slender mutants).
What are the genes ?
GAI—GA Insensitive: a transcription
factor that repress GA-induced gene
expression
RGA—Repressor of ga mutants
(ga standing for GA-deficient).
Similar to GAI.
SPY (spindly)—slender plant mutant
Encodes glycosylation enzyme
B) How does GA work to regulate a gene expression?
Again protein degradation pathway is the key control point! It turns out that
GA works to degrade GAI/RGA repressors.
Evidence for the involvement of protein degradation: the gai and rga
mutants all inhibited the GA-induced degradation of RGA/GAI proteins.
Recent studies identified a ubiquitin-ligase for GAI/RGA proteins. It is
likely that GA signaling may follow a similar paradigm as IAA
signaling
C) GA signaling in germination of monocot plants
--a story from studies of aleurone cells
aleurone
Germination: GA is produced in the embryo,
released to the endosperm, and diffused to the
Aleurone cells where GA induces gene
expression for alpha-amylase synthesis.
The enzyme is then secreted into endosperm
to digest the starch to produce sugar for
the growth of embryo into a seedling.
Q: How does GA induce gene expression in
the aleurone cells?
a)
Where is the receptor for GA? Some
evidence indicates that it is located at the
cell surface (plasma membrane) but not
identified at molecular level.
b) A number of components may serve as
intermediates that pass the GA signal to
the nucleus. These include G-proteins,
calcium, protein kinases/phosphatases,
embryo
endosperm
c) Common signaling components found between germination and stem growth
For example, the RGA/GAI proteins also prove to be repressors for alpha-amylase
expression in aleurone cells; the GA-Myb type of transcription factors required for
aleurone gene expression is also positive regulator of stem growth.
d) Transcription factor genes like GA-Myb are activated before the alpha-amylase
gene to establish a gene expression cascade:
e) calcium-dependent pathway is
required for alpha-amylase secretion
through vesicular transport and Caindependent pathway (involves
cGMP) is essential for GA-induced
gene expression cascade.
f) Genetic and biochemical evidence
for involvement of G protein: rice
dwarf1 mutant has mutation in the
gene encoding the G alpha subunit; G
protein inhibitors block while
activators enhance aleurone gene
expression
1.
2.
GA binding to receptor
GA receptor may interact
with G protein that leads to
two branches of signaling,
one is calcium-dependent
and other is Ca-independent
3-6 A second messenger
(possibly cGMP) may work
in the calcium independent
pathway to lead to
degradation of repressor
proteins (RGA/GAI) and
turns on GA-Myb
expression.
7-8 GA-myb activate alphaamylase gene expression
9-10 alpha-amylase is
synthesized at ER and
secreted through the
vesicular trafficking
11 A Ca-dependent pathway
regulates the secretion.