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When a pathogen invades a plant, it releases various molecules and proteins into the plant’s extracellular environment. In response, the plant secretes its own proteins into the extracellular space to fight the infection. To better understand these types of processes, Nam-Soo Jwa, Ganesh Agrawal, and colleagues at Sejong University (South Korea), the Research Laboratory for Biotechnology and Biochemistry (Nepal), and the National Institute of Advanced Industrial Science and Technology, Tsukuba West (Japan) examined the rice secretome. In JPR (DOI 10.1021/pr8005149), they describe a new extraction method for the study of secreted proteins in rice seedlings and provide two reference maps of the rice secretome. Because isolating proteins from established plants (in planta) is a difficult task, investigators often analyze seed callus suspension-cultured cells (SCCs) grown in vitro. In this cell-culture system, seeds are grown for a few weeks on a solid callus-induction medium, then the resulting cell aggregate (callus) is moved to a liquid medium. The isolation of an SCC secretome is relatively easy because these calli secrete proteins directly into the surrounding medium. Only a few studies report the extraction of extracellular fluid (sap) from the leaves of seedlings grown in soil. In these cases, the scientists harvest sap by submerging leaves or other plant tissues in water or a buffer, then placing the container in a vacuum chamber. During this vacuum infiltration step, the risk of damaging the tissues is high, says Agrawal. “Therefore, there is a risk of contamination from proteins other than secreted proteins,” such as cytoplasmic or transmembrane proteins, he explains. In addition, researchers usually spin the tissues at high speeds, and this centrifugation step also can damage tissues and result in contamination. Unsatisfied with the vacuum infiltration technique, Jwa, Agrawal, and col-
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leagues developed their own isolation protocol. With the gravity extraction method (GEM), rice seedlings are sprayed with a detergent solution to allow water to stick to the leaves to elicit secretion. The next day, leaves are removed from the plants and placed in a 50 mL plastic tube. Inside the tube near the bottom is a plastic
GANESH AGRAWAL
Unlocking the secrets of the rice secretome
A GEM of a technique. (Left) Researchers used a new method called GEM, as well as (right) a conventional in vitro method to study the rice secretome.
sheet with 2-mm-diam holes. Leaves are placed in the tube above the sheet. The leaves are spun at low speed, which reduces the chance of cell breakage, and sap is collected at the bottom conical tip of the tube beneath the sheet. Next, the sap containing secreted proteins is spun at high speed to remove impurities. When proteins were run on a 2D gel at this point in the process, the researchers had a difficult time distinguishing low-abundance proteins because of a high level of background staining. “It took a long time to develop a new method to solve the background problem,” says Jwa. Eventually, they found that cetyltrimethylammonium bromide precipitation removed interfering compounds such as carbohydrates and allowed clear visualization of protein spots. According to Jwa, the entire GEM approach results in
Journal of Proteome Research • Vol. 7, No. 12, 2008
purer secretome preparations than the vacuum infiltration technique, but at a cost. “One disadvantage [of GEM] is the low recovery of secreted proteins,” he says. Biochemical and western blot analyses showed that the in planta (GEM) and in vitro (cell culture) samples contained few or no intracellular proteins. The samples were analyzed by 2DE, and protein spots on the reference maps were identified. Many fewer protein spots were visible on the gels of the in vitro secretome than on the gels of the in planta secretome, and only six of these proteins were common to both types of samples. Jwa explains, “A callus is an undifferentiated cell aggregate, and protein synthesis and secretion can be different from normal tissue in growing plants.” Although Jwa says that in planta analyses are more physiologically relevant, he points out that both systems have their merits and should be studied simultaneously. “Both methods can be used as complementary systems for complete secretome analysis under different conditions,” he states. Only 27% of the in planta secreted proteins were predicted to have a signal peptide, whereas 76% of the in vitro secreted proteins had a signal peptide. Typically, proteins with signal peptides travel through the Golgi apparatus and endoplasmic reticulum before being secreted. However, because the seedling secretome includes a large percentage of proteins lacking a signal peptide, they may secrete proteins via an alternative pathway, says Jwa. Of the secreted proteins identified with GEM and in vitro approaches, Jwa is most interested in proteases because they could play a role in destroying proteins that are secreted by pathogens. However, he points out that the 2DE reference maps generated in this study represent only the “first step toward unraveling the secret of the rice secretome” and that many more experiments still must be performed to compile a comprehensive atlas of the secretome. —Katie Cottingham
10.1021/pr800848k
© 2008 American Chemical Society
