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The Molecules are the Message
Plants use the blue light photoreceptor, cryptochrome, to
measure the overall intensity of daylight and activated cryptochromes
transmit this information throughout the organism. They have
another set of receptors for red light, the phytochromes,
that work in the same way. And Arabidopsis uses the
ratio of activated red:blue photoreceptors to tell them how
crowded they are in their microenvironment.
The number of phytochromes and cryptochromes that are transported
into cell nuclei influences how nascent transcription factors
will respond to changing light conditions by altering genomic
expression to accommodate the updated environment and provide
the correct response.
Arabidopsis plants also produce phenylpropanoids
to protect them from harmful UV radiation, analogous to how
the phenyl compounds in commercial sunscreens work. Production
of these phenylpropanoids peaks just prior to dawn.
Chlorophyll-binding proteins are also turned on in the early
morning in anticipation of the coming sun. Lipid desaturation
enzymes and other molecules that confer cold and frost resistance
throughout the night are not needed throughout the day, and
so by mid-day, the expression of various transcription factors
that down-regulate these cold resistance genes peak.
Arabidopsis also down-regulates the synthesis of
enzymes that store, transport, and break down the sugars the
plant produces throughout the day until they are needed. Nitrogen
and sulfur assimilation and amino acid production is timed
so that these energy-intensive reactions can be performed
in the morning when the plants have the most energy available
to them.
Cell elongation genes are expressed in the late afternoon
and early evening and then put to rest by nighttime so that
other enzymes can begin to use the available energy to synthesize
the polysaccharides needed to reinforce the newly elongated
cell walls, working through the night to finish.
All and all, Kay and his colleagues found that of the 8,200
genes they surveyed, some 453—about six percent—moved
to a circadian beat. The expression of these 453 ebbed and
flowed with a period of 24 hours. About one quarter of the
circadian genes they identified had no known function. “It’s
really rather cool,” says Kay.
With so many uncharacterized circadian genes, we must assume
that much of the biochemical detail of the clock story is
not yet known in exhaustive detail.
Biology in Broad Strokes
Kay and his colleagues look at which genes are turned on
and off by using DNA chips made available through the Novartis
Agricultural Discovery Institute (now a part of Syngenta),
a collaboration which Kay values. “NADI has done a lot
to enhance plant research in academic labs,” he says.
“Those [DNA chips] allow us to do many things.”
DNA “chips” are glass or silicon wafers onto which
are deposited arrays of nucleic acid oligos, sometimes to
concentrations of tens of thousand per square centimeter.
One can quite easily put all or most of the oligos that correspond
to known genes in an organism on one of these so-called affinity
matrices and can then look for expression of every known gene
in a genome for entire tissues at a time.
There are limitations to the method. Post-translational
modification of expressed proteins into multiple isozymes—phosphorylated,
glycosylated, and enzyme modified variants, for instance—cannot
be observed. Also genes that are not annotated, or only expressed
in tiny amounts but nevertheless play a role in the clockworks,
may be missed. But the chips are perfect for looking at total
mRNA expression of a tissue at a time, which is exactly what
Kay is doing.
Kay also uses more traditional though no less enlightening
methods of looking at the pattern of expression for single
genes. He employs Agrobacterium tumefaciens as a vector
to attach markers onto genes in Arabidopsis without
disturbing the plant’s other functions. “We do this
famous trick of making the plants glow in the dark,”
he says.
By attaching a luminescent enzyme from fireflies, called
luciferase, to specific genes in various tissues in the Arabidopsis
plants, Kay and his colleagues can monitor when those genes
are turned on or off and how they respond to environmental
stimuli. They vary the amount of light a plant receives, for
instance, and observe how the gene expression and flowering
response of the plant varies accordingly.
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