How phenology influences physiology in deciduous forest spring ephemerals

The protein phosphatase inhibitor cantharidin activates defense responses in rice leaves when applied exogenously at concentrations ranging from 100 to 500 μ M . Responses include the accumulation of the major rice phenolic phytoalexin sakuranetin and the lactone phytoalexin momilactone A. Accumulation of sakuranetin was preceded by an induction of phenylalanine ammonia lyase (PAL) activity and an increase in the activity of naringenin 7- O -methyltransferase (NOMT), the key enzyme in sakuranetin biosynthesis. Cantharidin also strongly induced accumulation of the probenazole (PBZ)-inducible protein (PBZ1) and two novel, related proteins named PBZ2 and PBZ3. Endothall, a herbicide and potent protein phosphatase inhibitor, but not its inactive analog (1,4-dimethylendothall) also induced sakuranetin accumulation, increased activity of NOMT and accumulation of the 3 PBZ proteins. In contrast, two other protein phosphatase inhibitors, calyculin A and microcystin LR, did not activate these defense responses. Induction of NOMT and PAL activity, and sakuranetin accumulation, was completely blocked by cycloheximide. Leaf segments treated with cantharidin and endothall showed brownish and orange colored lesions, respectively, similar to the lesion mimic mutants of rice. These results indicate a direct role for protein phosphorylation/dephosphorylation events in the activation of defense responses in rice, in particular on the accumulation of antifungal phytoalexins and the PBZ proteins.