VOLUME 12, NUMBER 21 • MAY 4, 2001

Chua Lab Uncovers Second Line of Defense in Plants

Resting peacefully inside its seed, a newborn plant is completely safeguarded against drought and other harsh conditions. Toss a handful of seeds onto a parched patch of sandy land, for example, and the plants will remain happily asleep. Only when the seedlings sense that the soil is ripe for growth will they break through their seed coats and blossom into full-grown plants.

Professor Nam-Hai Chua, Sébastien Mongrand and Luis Lopez-Molina (left to right) identified a protein in plants that confers resistance to drought.

But what if a hibernating plant is accidentally triggered to germinate by an unusually cold night in the midst of a hot summer? New research in the laboratory of Professor Nam-Hai Chua suggests that newly sprouted plants may have a second opportunity to defend themselves against drought, once they have left the safety of their seeds. The work shows that a well-known plant hormone delays the growth of experimental plants in order to give them one last chance to monitor their environment for signs of dryness before initiating growth. Furthermore, the researchers have identified a specific protein as a key player in the process.

"You have a seed that’s asleep, but when it wakes up it looks around and asks: do I have enough water?"says Chua, Andrew W. Mellon Professor and head of the Laboratory of Plant Molecular Biology.

The findings, reported in the April 3 issue of the Proceedings of the National Academy of Sciences (Early Edition #14), are of immediate interest to agricultural and biotechnology industries, because they suggest that crops potentially could be genetically modified to be more resistant to drought. Dry, salty lands in developing countries tend to depress food productivity, hence tougher crops that are less sensitive to arid conditions might prove beneficial.

"Our work reveals a novel level of complexity in the early growth process and suggests that it may be possible to manipulate plants so that they can better cope with stressful conditions, such as dry or high salt soils," says Luis Lopez-Molina, one of two lead authors of the paper.

Lopez-Molina and Sébastien Mongrand, both postdoctoral fellows, show that ABA——a plant hormone known to inhibit germination——also arrests growth of newly germinated Arabidopsis plants for up to 30 days. Moreover, they provide evidence that ABA activates a recently isolated Arabidopsis protein called ABI5, and demonstrate that this protein is essential to the newborn plant’s ability to protect itself against drought during this developmental delay.

Arabidopsis, a well-studied weed in the mustard family, is a model system for the study of plant development because of a number of factors, including its small size and rapid generation time.

The protein ABI5 may protect Arabidopsis plants from drought by arresting growth. Above: Normal strains (left) exhibit a developmental delay in the presence of the plant hormone ABA, whereas mutant strains lacking the ABI5 protein do not.

ABA plays a role in both germination in young plants and stress responses in adult plants. Its levels rise during germination, and it has been shown to have an inhibitory effect on growth. Furthermore, when adult plants are under environmental stress, such as drought, this hormone will induce the stomata——a plant’spores——to close. In essence, it prevents the plant from sweating so that it doesn’t lose precious water.

ABA’s ability to delay both germination and early growth was discovered when Lopez-Molina and Mongrand realized that seeds would in fact germinate after a certain period of time when grown in the presence of the hormone. They noticed, however, that the germinated plants did not green right away, and they later demonstrated that ABA could effectively block growth for up to 30 days.

"One of the messages of this paper is that ABA delays germination, but is more efficient at keeping germinated embryos in a resting, protective state," says Lopez-Molina.

Because previous studies demonstrated that mutant Arabidopsis plants lacking the ABI5 protein grew without interruption after germination, the researchers wondered how this protein was linked to ABA’s ability to maintain arrested germinated em-bryos. To study more precisely the role of ABI5, they genetically engineered strains of Arabidopsis to produce an excess of the protein and observed their behavior.

The transgenic plants were found to exhibit a developmental delay only when ABA was present. Therefore, the researchers concluded, ABA must turn on, or activate, ABI5. Next, Lopez-Molina and Mongrand showed that mutant strains lacking the ABI5 protein, when grown in the presence of ABA, had lower survival rates than their normal counterparts if faced with drought conditions. Whereas normal plants survived, on average, after 36 hours of drought treatment, mutants survived after only 12.

But perhaps the most intriguing finding of all was that adult Arabidopsis plants overproducing the ABI5 protein lost less water than average, implying that they were more resistant to drought.

"A normal plant will lose water. A transgenic line overproducing ABI5 loses water less rapidly, probably because it is oversensitive to ABA," says Lopez-Molina.

At present, the researchers are exploring the question of how ABA activates ABI5. Preliminary studies show that ABI5 is phosphorylated in the presence of ABA, but it is not known whether this phosphorylation actually results in an increase in ABI5 activity. To answer this question, the researchers are collaborating with the Laboratory for Mass Spectrometry and Gaseous Ion Chemistry, headed by Professor Brian T. Chait.

Derek McLachlin, a postdoctoral associate in Chait’s lab, is using mass spectrometry to locate sites in the ABI5 protein that are phosphorylated in living Arabidopsis plants. Once these sites are identified, Lopez-Molina and Mongrand plan to make mutant versions of ABI5 that either contain no phosphorylation sites, or sites that are permanently turned on, in the hopes of ascertaining whether ABI5 is indeed activated via phosphorylation.

The researchers are also searching for "suppressor mutants" as a means to identify proteins, in addition to ABA, that regulate the activity of ABI5. Lopez-Molina says that if their hypothesis about ABI5 being activated via phosphorylation is right, then this technique might allow them to identify the protein kinase responsible for the job.

Lopez-Molina’s research was supported by the Swiss National Science Foundation and the Human Frontier Science Program Organization.