How does light control leaf

1
How does light control leaf plastid development?

• 3 parts to this model
• photoreceptor(s)
• signal transduction chain (2nd messengers)
• molecular responses (gene activation or
  de-repression)

2
Identifying 2nd Messengers for Phy Promotion of
Plastid Development

• Strategy
• Microinjection of a tomato aurea mutant
• Aurea lacks Phy A (important Phy) shows poor
  leaf/plastid development even in light
• Protocol
• Aurea cells are injected with possible 2nd
  messengers (cyclic nucleotides and Ca2 /CaM) or
  a non-hydrolyzable GTP analogue (G-protein
  activator).
• Accumulation of chloroplast proteins monitored
  with fluorescent antibodies, and pigments
  (anthocyanins) by their intrinsic fluorescence.

3
Phytochrome Signal Transduction Pathways
4
cGMP

• Cyclic GMP (a cyclic nucleotide)
• derived from GTP by guanylate cyclase
• Destroyed by a cGMP diesterase
• The cyclase is probably stimulated by the
  G-protein.

5
Ca2 and Calmodulin

• Ca2 activation typically mediated by Calmodulin
• Calmodulin is inactive unless bound to 4 Ca2
• Free Ca2 levels in the cytosol are low, so can
  be regulatory
• Free Ca2 levels can change rapidly by
• being released from subcellular stores (vacuole
  in plants)
• or by an influx of extracellular Ca2
• The G-protein could stimulate one or both sources
  of Ca2.

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Trimeric G-proteins

• Mediate receptor activation of downstream
  effectors (e.g., protein kinases).
• Have different conformational states with
  different affinities for receptor versus target
  proteins.
• ? subunit is a GTPase
• hydrolysis of GTP ? GDP important for G-protein
  to dissociate from target and recycle (target
  goes back to uninduced state).

7
G-protein activation of a phospholipase, which
produces a 2nd signal (a lipid). Receptor doesnt
bind to G-protein until activated by the ligand
(or light in the case of Phytochrome).
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Phytochrome Signal Transduction Pathways
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Problems (?) with the Model

• Arabidopsis has only one trimeric G protein
• Knockouts of the G? (and G?) genes did not
  inhibit red and far-red shoot/leaf development
• Over-expression of the G? gene, however, made
  plants hypersensitive to red and far- red light
• Conclusion this has not been resolved to many
  peoples satisfaction

10
How does light control leaf plastid development?

• 3 possible parts to this regulatory system
• photoreceptor(s)
• signal transduction chain (2nd messengers)
• molecular responses (gene activation or
  de-repression)

11
How is specific gene transcription controlled by
light?

• examine 5' regions upstream of light-regulated
  genes
• comparative sequence analysis of rbcS gene family
  showed some conserved 5 sequences
• Candidates for light-responsive elements (LRE)

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Analysis of the rbcS3a promoters cis-acting
elements
Chimeric gene
5rbcS3a CAT 3NOS

• 5'rbcS3a - 1000 bp (-1000 to 0) upstream of
  rbcS3a gene (pea)
• CAT - reporter gene
• 3'NOS - 3' region of the nopaline synthase gene
• When expressed in tobacco, chimeric gene showed
  light and Phytochrome-dependent expression, like
  rbcS3a
• Conclusion the upstream region is primarily
  responsible for the pattern of rbcS3a expression.

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• Deletion analysis of the 1000 bp promoter was
  performed to find smallest piece that would
  confer light-regulated expression on a reporter
  (chimeric) gene.
• Region of -330 to -50 conferred light-dependent
  regulation on a chimeric gene containing a
  minimal (constitutive) promoter

-330 to -50 35M CAT 3NOS
35SM - small portion of the 35S viral
promoter (CAAT box and TATA box) If -330 to -50
region was inverted, still conferred
light-dependent expression. Suggests this region
is an enhancer.
14
Other regions of the rbcS3a promoter also
important ______________________________________
III II I II III VI IV V -330
-50 1

• Boxes I -VI are conserved among pea rbcS
  promoters
• Box IV - TATA box, required for transcription
• Boxes I,II,III II,III - enhancer
• Boxes II and III (and II, III)- contain a GT
  motif
• GT motif important for light regulation
• - found in other light-regulated gene
  promoters, but also in non-light-regulated
  promoters!

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Trans-acting factors that bind to the pea rbcS3a
regulatory sequences

• N-H Chua lab (Rockefeller U.), identified a
  protein that binds to the GT motif, GT-1
• GT-1 binding activity present in both light and
  dark-grown plants
• Another identified factor binds to box VI, AF-1
• AF-1 activity present in light and dark-grown
  plants
• AF-1 also present in leaves and roots

16
Electrophoretic mobility shift assay (EMSA) of
TFII factors D,A,B,F on the Adenovirus major
late promoter.
Conclusions 1. D and B must form a complex on
DNA for Pol II or F to bind. 2. F must bind along
with Pol Pol cant bind w/o F.
Fig. 11.2a in Weaver
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Footprinting TFIID,A,B on a Class II Promoter
with phenanthrolineCu2 and with DNAse I.
D makes footprint, which is enhanced by A. B does
not expand the footprint, but makes 10 nt more
reactive.
Fig. 11.3b Weav.
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Factor Phosphorylation-Dephosphorylation

• At least 3 other proteins bind the rbcs3a
  promoter, 2 bind near box III (AF2 and AF3) 1
  (AT-1) binds upstream of the enhancer (at -500).
• These proteins are all phosphorylated, probably
  by kinase CK2.
• Effect is factor-specific
• Phosphorylation inhibits DNA binding by AT-1
• Phosphorylation promotes DNA binding by AF3
• Could regulate factor activity in vivo!

19
Factor regulation by subcellular localization?

• Another LRE, the G-box, also appears in many
  light-regulated promoters.
• Bound by a type of bZIP protein factor, called
  GBFs.
• Arabidopsis GBFs found in cytoplasm and nucleus.
• GBF2 distribution shifts from 5050 nucleuscyt
  in the dark to 8020 in the light.

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bZIP Transcription Factors - Basic DNA binding
domain - Leucine zipper that dimerizes the
proteins Can form homodimers or heterodimers.
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Genetic Analysis of Leaf Plastid Development

• Long hypocotyl mutants of Arabidopsis continued
• Hy5 mutant is a bZIP DNA-binding protein that
  promotes transcription from a number of genes
  with LREs
• It also responds to blue (Cry) and red light
  (Phy) receptors

22
More genetics Pleiotropic repressors of
leaf/plastid development

• Pleiotropy- one gene affects many processes
• J. Chory identified a mutant DET1 that shows
  photomorphogenesis in darkness
• leaves are not green but otherwise look like
  leaves
• plastids develop, look like chloroplasts
• rbcS and cab genes are on in the dark in DET1
• DET1 gene encodes a nuclear protein that
  interacts with hy5
• Conclusion DET1 acts to repress leaf development
  and gene expression, (possibly by inactivating
  hy5 ?)
• Light must shut-off/inactivate DET1 to allow
  photomorphogenesis (but not in all tissues)
• root plastids abnormal in DET1
• Suggests DET1 has role in dark to keep photogenes
  off in roots

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Light-grown
Dark-grown
Cop9 mutant
An Arabidopsis cop (constitutive
photomorphogenesis) mutant (it is similar to Det
and fusca mutants)
Wild-type
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Plastid ultrastructure
Dark-grown wild-type
Dark-grown Det1
Light-grown Det1
25
More genetics Pleiotropic repressors of
leaf/plastid development

• Pleiotropy- one gene affects many processes
• J. Chory identified a mutant DET1 that shows
  photomorphogenesis in darkness
• leaves are not green but otherwise look like
  leaves
• plastids develop, look like chloroplasts
• rbcS and cab genes are on in the dark in DET1
• DET1 gene encodes a nuclear protein that
  interacts with hy5
• Conclusion DET1 acts to repress leaf development
  and gene expression, (possibly by inactivating
  hy5 ?)
• Light must shut-off/inactivate DET1 to allow
  photomorphogenesis (but not in all tissues)
• root plastids abnormal in DET1
• Suggests DET1 has role in dark to keep photogenes
  off in roots

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COP9 Signalosome

• COP (constitutive photomorphogenesis) and FUSCA
  mutants are similar to DET mutants (i.e.,
  photomorphogenetic processes on in the dark).
• 10 of these genes were cloned, 8 of them encode
  subunits of a large complex, COP9 signalosome.
• Several genes have been identified that promote
  photomorphogenesis by inhibiting this
  signalosome.
• The COP9 signalosome is also involved in plant
  hormonal (auxin) signaling.
• There is also a counterpart complex in animal
  cells.

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The Sanger or dideoxy method of DNA sequencing.
1. 4 replication reactions, same primer but each
with a limited quantity of a different
dideoxynucleotide (ddNTP). 2. Incorporation of
the ddNTP terminates the reaction. 3. The 4
reactions are electrophoresed side-by-side on a
denaturing polyacrylamide gel. 4. Sequence is
read from bottom to top.
Figs 5.18, 5.19
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Automated DNA Sequencing
M. Hunkapillar L. Hood
1. The dideoxynucleotides are tagged with a
fluorescent group. 2. Each type of dideoxy
fluoresced a different color. 3. Only 1 reaction
needed, and only 1 lane of a gel to sequence a
stretch of DNA. 4. As DNA fragments reach the
bottom of the gel, a laser excites the dideoxy
and sensors detect the fluoroscence. 5. A
computer converts it to a linear sequence.