Plant Genomics:

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Lecture 16 Circadian Rhythms
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What are circadian rhythms? Some definitions
Various metabolic processes (oxygen evolution,
respiration) cycle alternately through high and
low activity phases with a regular periodicity of
24 hrs. These rhythmic changes circadian
rhythms from the Latin circa diem
approximately a day Oscillator internal
pacemaker - coupled to variety of
physiological processes - unaffected by
temperature Light modulator of rhythms in
plants and animals
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Circadian rhythms exhibit characteristic features
Circadian rhythms arise from cyclic phenomena
that are defined by three parameters Period of
a rhythm time between comparable points in the
repeating cycle - measured
as the time between consecutive
maxima (peaks) or minima (troughs) Phase any
point in the cycle that is recognizable by its
relationship to the rest of the cycle - most
obvious phase points are the peak and trough
positions Amplitude distance between peak and
trough - can vary while period remains
unchanged
Entrainment Under natural conditions, the
endogenous oscillator is entrained
(synchronized) to a true 24-h period by
environmental signals
- phytochromes and chryptochromes entrain the
clock
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Characteristics of circadian rhythms
A typical circadian rhythm.
A circadian rhythm entrained to a 24 h light-dark
(L-D) cycle and its reversion to the free-running
period (here 26 h) following continuous darkness.
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Characteristics of circadian rhythms
Suspension of a circadian rhythm in continuous
bright light and the release or restarting of the
rhythm following transfer to darkness.
Typical phase-shifting response to a light pulse
given shortly after transfer to darkness. The
rhythm is rephased (delayed) without its period
being changed.
Beginning of subjective day
Beginning of subjective night
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Day/night cycling of gene expression is called a
diurnal rhythm, which is achieved by two
mechanisms
Diurnal regulation Diurnal Relating to or
occurring in a 24-hour period daily Diurnal
rhythms may persist when the organism is placed
in an environment devoid of time cues, such as
constant light or constant darkness. Therefore,
diurnal variations can be either light-driven or
clock-driven. Day/night variation in light
intensity can directly regulate genes via
photoreceptors such as phytochrome of
cryptochrome. Circadian clocks Cells have
internal oscillators that operate independently
of light stimulus (free-running). However, the
circadian clock can be reset by light.
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Circadian clock consists of 3 components
inputs, oscillator, outputs
elf3 mutations cause no circadian rhythm in
light, but rhythm is maintained in
constant dark toc1 mutations shorten
photoperiod ztl, fkf mutations lengthen
photoperiod CO flowering time regulator LHY,
CCA1 MYB-related putative transcription factors
The oscillator must be trained by inputs. A
complete mechanistic model for the oscillator
does not yet exist, but  genes encoding some of
the components have been identified.
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Which genes are regulated by a circadian clock?
Schaffer et al. 2001 Plant Cell 13 113 performed
2-label  gene array experiments using 11,521
Arabidopsis ESTs (Expressed Sequence Tags)
representing 7800 unique genes.  Of these, 1152
ESTs showed a difference in expression from day
to night.
R1 etc. - Individual experiments.
Green Red
All plants were trained on one of 3 light
regimes, and then an experimental regime was
tested. RNA was sampled at the end of the
experimental regime.
DD, continuous dark LD, 12-hr light/dark
cycles LL, continuous light
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206 ESTs showed both diurnal cycling and
differential expression in the dark.
green - stronger expression in the
morning red - stronger expression in the
afternoon black -  Less than 2-fold
difference between morning and
afternoon. cyc dark - cycling was seen in
continuous dark cyc light - cycling was seen in
continuous light
Complete clusters can be viewed at
http//www.prl.msu.edu/circl
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5 distinct patterns emerged
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5 distinct patterns emerged
61 circadian genes with high expression in the
morning
78 circadian genes with high expression in
afternoon
Longer time of expression
Shorter time of expression
23 genes induced in light and repressed in dark
44 genes repressed in light and induced in dark
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Categories of genes that cyclea
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Orchestrated transcription of key pathways by
the circadian clock
- Hamer et al. (2000) Science 290 2110 used
oligonucleotide microarray representing 8200
genes - 6 of these genes showed circadian
changes on steady-state mRNA level
1. Genes in the light-harvesting reactions of
photosynthesis are under clock control.
PSI, PSII reaction center
LHC
(A) LHCA genes are in blue, LHCB genes are in
pink. (B) Photosystem I genes are in red.
Photosystem II genes are in green. (C) Model for
function of photosynthesis gene products in
photosystems II (left) and I (right).
Colors of proteins match colors of corresponding
gene traces.
Some photosynthesis-related genes anticipate
subjective dawn and dusk. Note in the figure
above that many of the photosystem I II genes
begin to decrease in expression prior to dusk,
and increase in expression prior to dawn.
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Phenylpropanoid biosynthesis genes are also
activated before dawn
Enzymes in the phenylpropanoid pathway govern the
production of a wide range of phenolic compounds.
Included in these are flavonoid pigments, many of
which absorb UV light, thereby protecting plants
from UV damage (phenolic sunscreen).
Phenylpropanoid biosynthetic pathways
Red lines phenylpropanoid biosynthesis
genes Blue line PAP1, MYB
transcription factor involved in biosynthesis of
anthocyanins and lignins PAP1 perhaps a master
regulator of clock-controlled transcription of
these genes
Genes in red are all clock-controlled
Harmer et al. 2000
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Genes implicated in cell elongation are
circadian-regulated
Genes peaking toward the end of the day auxin
efflux carriers PIN3 and PIN7 (red), putative
expansin (green), putative polygalacturonase
(light blue), aquaporin (dark blue) Genes
peaking toward the end of the night Three cell
wall synthesis genes (gold)
Proposed mode of action of the products of these
clock-controlled genes in cell wall remodeling.
Harmer et al. 2000
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A novel clock-controlled promoter element
A cluster of 31 clock-controlled genes containing
an AAAATATCT promoter evening element. Promoters
of clock-controlled genes were scanned for
over-represented elements using alignment
software. The evening element was not
over-represented in any other alignment cluster.
Mutations of AAAATATCT greatly reduced the
ability of a promoter to confer circadian
rhythmicity on a luciferase reporter gene in
plants.
Harmer et al. 2000
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A novel clock-controlled promoter element
CCR2 promoter, which contains four evening
elements, was mutated and fused to the luciferase
reporter gene.
Fusions to the firefly luciferase gene consisted
of 450 bp (WT450) 130 bp (WT130, mut130_1,
mut130_2, mut130_1,2) 85 bp (WT85) of the CCR2
promoter upstream of the putative transcriptional
start site. Site 1 was replaced by gagcagctgc in
mut130_1 and mut130_1,2 Site 2 was replaced by
gagcagctgc in mut130_2 and mut130_1,2 Constructs
were introduced in Arabidopsis plants via
Agrobacterium-mediated gene transfer. Luciferase
assays were performed. Twelve lines examined for
each construct.
Harmer et al. 2000
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Firefly Bioluminescence The Luciferase gene from
firefliescan be used to make other organisms
glow in the dark
Bioluminescence from a firefly is created by an
enzymatic reaction that takes place in its
abdomen. The enzyme responsible for this
light-producing reaction is called luciferase.
Luciferase uses oxygen, ATP and luciferin to
generate light.
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Firefly Bioluminescence The Luciferase gene from
firefliescan be used to make other organisms
glow in the dark
The DNA encoding theluciferase gene can
beintroduced into the genetic makeup of the
other organisms.For example, scientists have
introduced thisgene into fruit flies,plants and
bacteria. Once they contain the gene for
luciferase, these geneticallyaltered organisms
can glow in the dark.
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Wheat seedlings The level of expression of the
CAB gene family, but not the RBCS gene family, is
regulated by a circadian rhythm in wheat.
CAB
CAB mRNA is primarily produced when needed i.e.
during daylight hours. Note, though, it comes on
a few hours before "sunrise".   RBCS is on
all the time.
RBCS
Nagy et al. (1988) Genes and Development 2 376
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Phytochrome interacts with the circadian clock to
control the level of expression of the CAB1 gene
within the rhythm.
12 h dark /12 h light
24 h dark
24 h Red light
24 h Far red light