February 7, 2003

Chua lab discovers protein that regulates early growth arrest

An infant plant sleeps peacefully within its seed, entirely shielded from drought and other harsh conditions that might otherwise threaten its well-being. When the time comes at last to wake up and stretch its budding leaves, the young seedling must do so very carefully; once it decides to enter the unpredictable world outside, there's no turning back to the safe haven of the seed. Yet, growing up may not be quite as risky for a young plant as scientists once believed. According to Rockefeller University research, newborn plants have a second chance to hold off on growth after breaking through their seed coats.

In the presence of the plant hormone ABA, young Arabidopsis plants will hold off on growth until outside conditions become favorable (top). Mutant plants overproducing the newly identified AFP protein, on the other hand, lack this developmental arrest and blossom without delay (bottom). These and other experiments indicate that AFP helps to restore growth in normal plants by terminating the developmental arrest.

 This developmental arrest, or checkpoint, offers protection against the possibility that a plant accidentally sprouts or "germinates" when conditions are poor, for example in times of drought.

"A cool summer rain might fool a winter plant into germinating too early, when there's not enough water
in the soil," says Nam-Hai Chua, Ph.D., head of the Laboratory of Plant Molecular Biology at Rockefeller. "This growth arrest would give that plant the chance to salvage its mistake by essentially
freezing growth for up to 30 days."

The findings may have applications in the food biotechnology industry, because they suggest new
genetic strategies for creating drought-resistant crops.

Now, new research from the Chua lab, reported in the Feb. 1 issue of Genes & Development, identifies a novel protein in the experimental plant Arabidopsis that helps terminate this developmental arrest,
thereby indirectly reinitiating growth. Like a police officer restoring the flow of traffic by removing a barricade, this protein, called AFP, re-establishes growth by eliminating the primary protein, called
ABI5, in charge of executing the arrest.

"Previously, we had determined that ABI5 is essential for this early growth arrest to occur," says Luis
Lopez-Molina, Ph.D., first co-author of the paper and, until last September, a postdoctoral fellow at
Rockefeller. "Now, we have identified AFP as contributing to the removal of ABI5.

"In other words, ABI5 triggers the delay and AFP helps terminate it," says Lopez-Molina.

In the United States and all over the world, drought increasingly plagues farmlands, resulting in decreased food productivity. By better understanding how plants naturally tolerate drought and other
environmental stressors, Chua and colleagues hope to genetically enhance this ability in crop plants.

In addition, the latest research may lead to improvements in methods for seed storage. In every bag of
stored seeds, a certain percentage goes to waste because some seeds prematurely germinate and begin to grow — a costly loss for poor, developing countries. Because the Rockefeller scientists now know the identity of two of the molecular wardens in charge of this developmental delay, it may be possible to genetically control when plants initiate growth, and save a lot of otherwise squandered seeds.

Other scientists involved in this research include co-author and equal contributor S颡stien Mongrand,
Ph.D., another former postdoctoral associate at Rockefeller who is now at Centre National de la R飨
erche Scientifique/ Université Bordeaux 2, France; and Natsuko Kinoshita, a former guest student at
Rockefeller now studying at City University of New York (CUNY). Lopez-Molina has joined the
faculty of CUNY, where he is starting his own plant biology laboratory.

A pause before blossoming

The Rockefeller researchers first uncovered this novel developmental arrest in 2001 while studying abscisic acid (ABA), the primary plant hormone responsible for safeguarding both newborn and adult plants against environmental stress. In newborns, ABA blocks premature growth; in adults, it actively regulates a plant's response to stress, for example by closing a plant's pores in times of drought to prevent the "sweating out" of much-needed water. At the time, scientists believed that ABA's specific job in newborns was to stall germination until conditions became ripe for growth.

L-R: Nam-Hai Chua, Sébastien Mongrand and Luis Lopez-Molina

But the Rockefeller team changed this notion when they discovered that the hormone was in fact better at keeping young plants in an arrested state of development for up to 30 days — after they had left the comfort of their seeds.

Further exploring this early growth arrest, the researchers hit upon the key protein behind it, called
ABI5. They showed that for ABA to be able to pause growth, ABI5 must be present.

"We found that mutant plants lacking ABI5 could not arrest growth in response to drought and
subsequently died, whereas normal plants survived under the same conditions," says Lopez-Molina.

Together, these experiments suggest that ABA, through the activity of ABI5, gives newborn plants a
second opportunity to hold off on growth until auspicious conditions arise.

Devious partner

In the new Genes & Development paper, the story continues to unfold with the discovery of AFP, a
novel ABI5-interacting protein. After the scientists had characterized the role of ABI5 in the
developmental arrest, they set out to find proteins that regulate ABI5. Using a technique called the
"yeast two-hybrid screen," they fished out a novel protein-binding partner of ABI5, and later named it
AFP for "ABI five-binding protein."

The researchers then discovered that when they presented a newly germinated plant with high amounts of ABA, AFP levels rose in tandem with those of ABI5. In addition, mutant plants lacking AFP showed abnormally high ABI5 protein levels, resulting in hypersensitivity to ABA. This provided evidence that AFP negatively regulates ABI5 in normal plants. Further studies with enzymes called "proteasome inhibitors" indicated that AFP promotes the removal of ABI5 by the proteasome; in basic terms this means that AFP signals the cell to chop up ABI5 into little bits.

"AFP is like a soldier that sits and waits until the right time, then — boom — eliminates ABI5," says
Lopez-Molina, adding, "this makes sense because you have to get rid of ABI5 somehow so the plant
will grow when favorable conditions appear."

Filling out the picture even more, the researchers showed how AFP might target ABI5 for degradation. It turns out that AFP and ABI5 travel to the same spots in a cell's nucleus — spots that contain a known enzyme, called COP1, thought to promote the chopping up of proteins. "AFP takes ABI5 by the hand and carries it to protein-destruction factories located in the cell's nucleus. From this moment on, the plant can resume growth," says Mongrand.

Furthermore, the discovery that AFP is produced in tandem with ABI5 in response to stress exclusively during a short time window further supports the existence of a second developmental checkpoint.

Indeed, it would seem that a newly sprouted plant unexpectedly faced with a drought can thank ABA,
ABI5 and AFP for its survival.

The researchers acknowledge the expert help of the Rockefeller Bio-imaging Resource Center directed by Alison North.

Founded by John D. Rockefeller in 1901, The Rockefeller University was this nation's first biomedical research university. Today it is internationally renowned for research and graduate education in the biomedical sciences, chemistry, bioinformatics and physics. A total of 21 scientists associated with the university have received the Nobel Prize in medicine and physiology or chemistry, 16 Rockefeller scientists have received Lasker Awards, have been named MacArthur Fellows and 11 have garnered the National Medical of Science. More than a third of the current faculty are elected members of the National Academy of Sciences.