Light-regulated protein kinases from plants

Rajnish Khanna, Purdue University

Abstract

To understand the role protein kinases play in photoregulated seedling development, a set of protein kinases and their genes from the garden pea were studied. The genes encoding these kinases, PsPK1 through PsPK5, are differentially expressed during de-etiolation of seedlings. I focused on PsPK3 and PsPK5 because transfer of etiolated seedlings to white light causes their mRNAs to reach minimum levels within apical buds in a few hours. The gene responses are unusual in that the mRNAs decline when etiolated seedlings are illuminated with continuous light, but not with single pulses of light. The spectral sensitivity of the gene responses shows that phytochrome mediates the decline in mRNA levels. The PsPK3 and PsPK5 catalytic domains belong to the AGC group VIII of protein kinases. PsPK3 is predicted to be located in the nucleus, whereas PsPK5 is closely related to NPH1 from Arabidopsis, the probable photoreceptor for phototropism. The catalytic domains of PsPK3 and PsPK5 were expressed in E. coli as glutathione S-transferase fusion proteins to be tested for catalytic activity and to serve as antigens for polyclonal antibody production. Both fusion proteins become highly labeled in autophosphorylation assays. Unexpectedly, point mutations in the catalytic domains that were expected to lack kinase activity were equally labeled. On protein gel blots of extracts from apical buds and hooks, the PsPK antisera detect polypeptides of molecular weights close to those predicted by the cDNA sequences. The levels of these polypeptides decline during greening in buds and hooks, as do the mRNA levels. To test whether the PsPK mRNAs were light-sensitive during seedling development, 3 to 7 day-old seedlings grown in darkness were exposed to white light for a day. The PsPK mRNAs are light-responsive in buds and hooks throughout pea seedling development, up to 7 days. Both PsPK3 and PsPK5 transcripts are more light-responsive in hooks than in buds. While PsPK3 mRNAs decline ∼6-fold, PsPK5 mRNAs decline ∼52-fold in response to white light in the hooks of 6 day-old seedlings. The rapid light-regulated expression and similarity with known second messenger-dependent kinases suggest that PsPK3 and PsPK5 may be involved in signal transduction during the early phases of photoregulated seedling development.

Degree

Ph.D.

Advisors

Watson, Purdue University.

Subject Area

Molecular biology|Botany

COinS