Micro Sculptors

Tiny RNAs mold the development of leaves

Although you may not feel like admitting it as you rake them into trash bags, leaves are works of art. Their brilliant colors and elegant shapes have attracted and inspired artists. Each leaf assumes its appearance and operations through a finely balanced process of cell division and specialization. Yet leaves of some plants don’t conform to popular notions of leaf beauty. They may, for example, scrunch up where other leaves of the same species lie flat. Oddly shaped leaves and the plants that yield them have intrigued botanists and cell biologists for more than a century because they provide clues into how normal plants take shape.

FRILLS. When a normal snapdragon leaf (left) lacks the cincinnata gene, cells along the outside of the leaf divide uncontrollably and form wavy edges (right). Coen
FOILED FLOWERS. When scientists add extra copies of a microRNA to a normal Arabidopsis plant (left), its petals are replaced by additional reproductive organs (right). This design is similar to what happens when the apetala gene is malfunctioning (center), suggesting that the microRNA normally acts on apetala. Chen/Science

For botanists studying development of plant structures, “this is probably the most exciting time,” says Nancy Dengler of the University of Toronto. “There have been hundreds of leaf-shape mutants identified during the 20th century, but it is really only in the last decade that the molecular identity of a number of [shape-changing] genes has been established.”

In their search for what makes leaf growth and other aspects of plant development go right or wrong, scientists have found both traditional genes and some surprising ones. In the oddball category, plants contain stretches of DNA that are influential even though they don’t encode any protein. Instead, they result in snippets of RNA that researchers call microRNAs. These snippets control biochemical reactions by squelching the creation of specific proteins (SN: 1/12/02, p. 24: Biological Dark Matter), just as some proteins do. Research over the past few years indicates that microRNAs play an essential role in the delicate control over when and where genes turn on and off in the developing organism, be it a mammal, fly, or plant.

“MicroRNAs control some really interesting biological functions and address long-standing questions in developmental biology,” says James C. Carrington of Oregon State University in Corvallis. “What microRNAs do is provide a totally novel layer of genetic regulation.”

“So many people have jumped into this field. The pace of discovery is so rapid, that literally every few days a breakthrough paper is published,” Carrington says. Many of these advances are coming from research on plants, where identification of the molecular targets of microRNAs is relatively straightforward.

This side up

In the first years after their initial identification in worms in 1993, microRNAs appeared to be a molecular rarity. When scientists began systematically searching through bits of RNA in various organisms, however, an explosion of microRNA discoveries occurred in worms, flies, fish, mammals, and plants.

“The numbers [of discoveries] went from two to a couple of hundred,” says Carrington. By 2002, for example, researchers had found about 20 different microRNAs in the commonly studied mustard plant called Arabidopsis.

By then, scientists had proposed that plant microRNAs control protein production by glomming onto messenger RNAs, an essential intermediate in the creation of a protein. The RNA snippets, each containing 21 nucleotides, attach wherever the longer RNA contains a chemical sequence complementary to that of the microRNA. This prevents the messenger RNA from forming a protein. In many cases, when microRNA binds, the larger RNA fragments.

In 2002, a group led by David P. Bartel at the Whitehead Institute in Cambridge, Mass., scanned the newly decoded Arabidopsis genome to identify genes that the species’ microRNAs should be able to stick to. The team identified 49 possible gene targets for 14 microRNAs. “Virtually all of them were important in Arabidopsis development,” says Bartel.

One family of target genes that immediately caught researchers’ attention plays an important role in early leaf development. Before a leaf pokes out from the shoot, the cells destined to form that leaf must determine whether they’ll end up on the top or bottom. If they’re top-leaf cells, they have to build in the proper proteins for catching photons. Bottom-surface cells need equipment for exchanging carbon dioxide and oxygen with the surrounding air.

Two genes that play a role in the up-down directive are phabulosa and phavoluta. In 2001, a group led by M. Kathy Barton, now at the Carnegie Institution of Washington in Stanford, Calif., identified topsy-turvy plants that harbor mutations in the DNA sequence of a region found in both the phabulosa and phavoluta genes. Whereas the genes are normally active in cells on a leaf’s top surface, these mutations somehow caused the encoded proteins to appear in cells that line the bottom of the leaf, transforming them into toplike cells.

In fact, Bartel’s team identified messenger RNA from phabulosa and phavoluta as potential targets of microRNAs. The researchers have proposed that mutations in phabulosa and phavoluta can render those genes invisible to a microRNA that would normally destroy their messenger RNA in leaf-bottom cells. This would blur the distinction between the two sides of a leaf.

Another lab later showed that in a slurry of plant extracts, microRNAs do cause phavoluta messenger RNA to be chopped up. At a June 2003 Arabidopsis meeting in Madison, Wis., Bartel and Barton’s groups presented evidence that microRNAs regulate these genes in whole plants, as well.

Flat or crinkly?

The influence of microRNAs extends beyond early leaf development. Once leaves start growing, the cells must divide and expand in a specific pattern to create a characteristic shape.

Organisms “go through a lot of trouble to make things flat,” says Enrico Coen of the John Innes Centre in Norwich, England. “Flatness is a specific outcome of genetic control ensuring that things come out a certain way.”

The curvature of leaves depends on the speed and duration of cell division. If cells in the middle replicate too much, the leaf bulges. If extra cells crowd the leaf edges, they fashion a crinkly perimeter.

Coen and his colleagues investigated one crinkly-leaved snapdragon line first described in Germany in the 1930s. In the Feb. 28 Science, they report that disruptions in a gene called cincinnata can convert normal snapdragons to plants with similarly wavy, rounded leaves.

In a growing leaf that will end up flat, cells keep dividing until they get a signal to stop. Cells at the leaf tip stop dividing first, then the stop signal progresses to the base of the leaf. The signal goes slower in the center of the leaf and faster along the edges. Normally, the cincinnata gene is turned on where cells stop dividing, especially toward the perimeter of the leaf.

In the crinkly snapdragons and plants engineered to lack cincinnata, however, the progression of the stop signal is disturbed. The cells divide for a longer time than they should, especially along the leaf edges, resulting in the wavy leaves.

“This made us realize that normally the plant is doing something really carefully,” says Coen. He suggests that cincinnata expression influences how cells respond to arrest signals.

What controls the critical spacing of cincinnata expression that determines whether a plant will have crinkly or flat leaves? MicroRNAs make convincing candidates.

FOILED FLOWERS. When scientists add extra copies of a microRNA to a normal Arabidopsis plant (left), its petals are replaced by additional reproductive organs (right). This design is similar to what happens when the apetala gene is malfunctioning (center), suggesting that the microRNA normally acts on apetala.

Until recently, Detlef Weigel of the Max Planck Institute for Developmental Biology in Tübingen, Germany, had been puzzled by a mutant Arabidopsis plant line he described a few years ago. The plants–dubbed jaw because the leaves appear serrated like dinosaur teeth–resulted from the overexpression of a messenger RNA that didn’t make a protein. That didn’t make sense to Weigel until other scientists started describing the apparent functions of microRNAs.

“When I read these papers, it occurred to me that [jaw] might be a microRNA mutant,” says Weigel. He also realized that the jaw and cincinnata genes could be linked. Perhaps jaw mutants produce too much of a microRNA that chops up cincinnata, producing the same leaf defect seen when cincinnata isn’t expressed in the first place.

“What [Coen] found was a key controller of cell division during development,” says Carrington, who collaborated with Weigel. “What we found was a key controller of the controller.”

Weigel and Carrington’s group made their discovery in a series of experiments with an Arabidopsis engineered to overexpress jaw. The team reports in the Sept. 18 Nature that extra copies of jaw caused plants to have unusually low concentrations of a group of proteins related to cincinnata. These important developmental proteins share a structure called TCP–an acronym for the names of the three proteins in which it was discovered.

In one experiment, the researchers replaced a TCP-containing gene with a slightly altered version. The variation makes messenger RNA that’s resistant to microRNA binding but still produces a functional protein. The resulting plant line had multiple developmental problems and stopped growing at the seedling stage. Because there was no shortage of the cell-division-controlling protein, the researchers attributed these defects to the lack of microRNA binding to messenger RNA. This indicated that regulation by microRNA is required for normal plant growth.

The researchers hypothesized that in jaw mutants, a microRNA is repressing the normal TCP genes more than it usually does. The team then tested what would happen if it put enough TCP genes into the mutant plant so that even the abundant jaw microRNA couldn’t overcome them. When the scientists inserted excess copies of another TCP gene into jaw-mutant plants, the leaves were flatter and less crinkly than those in typical jaw mutants.

“It’s a pretty nice demonstration” of microRNA action, says plant geneticist Robert Martienssen of Cold Spring Harbor (N.Y.) Laboratory, who envisions more and more researchers using a similar set of experiments to demonstrate the actions of other microRNAs.

Two recent papers support this trend. In the Oct. 14 Current Biology, John Bowman’s group at the University of California, Davis shows that microRNAs control revoluta, a cousin of the phabulosa and phavoluta genes. When the revoluta messenger RNA sequence was changed so that microRNAs couldn’t bind to it, the cells in the plant’s stem developed abnormally.

It appears that flowers can depend on microRNAs, as well. In an upcoming Science paper, Xuemei Chen of Rutgers University in Piscataway, N.J., shows that a microRNA controls proper formation of plant reproductive organs and flower petals.

“We’re just scratching the surface in terms of the roles that [microRNAs] have in development,” says Bartel.

Mini mysteries

In addition to verifying targets of microRNAs, scientists are studying the basics of their action. While some microRNAs, such as those from jaw, appear to cause messenger RNA to be chopped up, others leave the RNA intact but use as yet undetermined methods to prevent protein formation. And while microRNAs appear to be potent gene regulators, little is known about how other plant molecules control the expression of the individual microRNAs.

These snippets of RNA might possess beneficial properties that, in some situations, would make them preferable to proteins for developmental tasks. For example, microRNAs could play a role when cells switch from an undifferentiated state, in which they could conceivably turn into a variety of cell types, to the state in which cells have a definite function. In plants, this happens when stem cells in the shoot take the plunge and start becoming leaf cells.

“In order to do that, [a cell] needs to turn on new genes, and turn off some of the old ones,” says Bartel.

When a cell shuts genes down, however, the messenger RNA produced earlier might still lurk in the cell, making unnecessary proteins. MicroRNAs might provide way to clear out these messages out quickly and efficiently, says Bartel.

Whatever their benefits or limitations, microRNAs represent a new class of molecules for scientists to explore in their attempt to understand the complexity that underlies the development of something as simple and elegant as a leaf.


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