Plant hormone signaling I

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• Plant hormone signaling I
• 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
• 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.

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• 2) Biological functions enhance cell elongation,
  apical dominance, Promote lateral root formation,
  vascular tissue differentiation
• 3) Signaling how does auxin work?
• Two responses have been used as models to study
  auxin function cell elongation and gene
  expression
• 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).

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C) How does ABP regulate auxin-induced cell
elongation? Auxin has been shown to enhance cell
wall acidification by promoting H-ATPase
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.
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• D) From auxin to gene expression
• 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 - - -
- -
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Promoter GUS/reporter
IAA
-
Isolation of proteins bound to this
sequence Resulting in discovery of ARFauxin
response factor, a transcription factor
controling auxin-induced gene expression

TGTCTC --AuxRE
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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
Gene/DNA
Promoter
transcribed region
mRNA
IAA/Aux
OFF
ARF
Gene/DNA
Promoter
transcribed region
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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).
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The summary how does auxin regulate gene
expression?
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3. Gibberellins 1)overview gibberillic acid
discovered as a fungal compound that enhances
rice growth but reduce seed productionfolish
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.
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• 2) Signal transduction for GA responses
• 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 GA-
• response mutants (slender mutants).
• What are the genes ?
• GAIGA Insensitive a transcription
• factor that repress GA-induced gene
• expression
• RGARepressor of ga mutants
• (ga standing for GA-deficient).
• Similar to GAI.
• SPY (spindly)slender plant mutant
• Encodes glycosylation enzyme

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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
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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?
• 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
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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 Ca-independent 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
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• 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 alpha-amylase gene
  expression
• 9-10 alpha-amylase is synthesized at ER and
  secreted through the vesicular trafficking
• 11 A Ca-dependent pathway regulates the secretion.