Minisymposium 22: Salinity

Abs # 43002: Hyperosmotic stress, a SnRK2b protein kinase and phospholipid signaling - connecting the dots.

Presenter:	Aghoram, Karthik , kaghoram@unity.ncsu.edu
Authors	Aghoram, Karthik  (A)   Goshe, Michael B (A)   Soderblom, Erik J (A)   Dewey, Ralph E (A)
Affiliations:	(A): North Carolina State University

Cellular signaling in response to water deficit is highly complex and involves an array of signaling molecules. Inositol-based compounds like PIP₂ and IP₃ accumulate or turn over in response to osmotic stress and are believed to mediate cellular stress-signaling processes. However, little is known about the early signaling events that transduce osmotic-stress perception into changes in polyphosphoinositide metabolism. We have discovered in plants two early components of a novel signaling pathway, which we hypothesize regulates polyphosphoinositide metabolism during hyperosmotic stress. First, we identified Ssh1p, a membrane-associated phosphatidyl inositol transfer protein. In vitro, Ssh1p can enhance the activity of PI-kinases, which catalyze the biosynthesis of phosphorylated PI species – precursors of PIP₂ and IP₃. Upon hyperosmotic stress, Ssh1p becomes rapidly phosphorylated. One consequence of phosphorylation is its dissociation from the membrane. The second component of the pathway is the protein kinase (SPK1) that phosphorylates Ssh1p. This Ser-Thr kinase belongs to the SnRK2b family, members of which are now emerging as key components of osmotic-stress responses in plants. Hyperosmotic stress mediates the activation of SPK1 in a Ca²⁺ and ABA-independent manner, leading to Ssh1p phosphorylation in vivo. This pathway is conserved in all plants tested, and can be reconstituted in yeast (Monks et al., 2001, Plant Cell 13: 1205). Our current efforts are focused on elucidating the mechanism of activation of the SPK1-Ssh1p pathway, and understanding its role in regulating whole-plant response to osmotic stress and phospholipid metabolism. We have purified active and inactive forms of recombinant SPK1 from yeast to assess structural differences that may explain the mechanism of its activation. Recent results indicated that SPK1 is activated by dephosphorylation. We are using tandem mass spectrometry and site-directed mutagenesis to pinpoint the sites of regulatory phosphorylation. Furthermore, we are using Arabidopsis genetics to understand the role of this pathway in whole-plant responses to osmotic stress. We have obtained insertion mutants in an Arabidopsis Ssh1p ortholog, and are initiating morphological and biochemical studies.