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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.

Next Page | A genome dedicated to combating things that nibble on it

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Native Arabidopsis thaliana flowers in Brandenburg, Germany. (Image by Thomas Schoepke—www.plant-pictures.com.)



















“We do this famous trick of making the plants glow in the dark,” says Professor Steve Kay. Pictured here is a plant showing positive luciferase expression, as seen under a florescent lamp.