STRESS RESPONSES IN ALFALFA (MEDICAGO-SATIVA L) .6. DIFFERENTIAL RESPONSIVENESS OF CHALCONE SYNTHASE INDUCTION TO FUNGAL ELICITOR OR GLUTATHIONE IN ELECTROPORATED PROTOPLASTS

Protoplasts derived from cell suspensions of alfalfa (Medicago sativa L.) responded to treatment with fungal elicitor (FE) by an increase in endogenous chalcone synthase (CHS) activity but were unresponsive to reduced glutathione (GSH). Preexposure of protoplasts to polyethylene glycol and electroporation resulted in strong responsiveness to GSH but little change in responsiveness to FE. Protoplasts from suspension cultures which had been subcultured more than 12 times lost responsiveness to GSH, but not FE, as assessed by measuring expression of a chimeric gene containing a bean CHS promoter linked to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. In protoplasts in which putative cis-acting CHS promoter sequences had been coelectroporated in trans with the intact CHS promoter-CAT construct, the extent of CAT expression depended upon the elicitor used (FE or GSH), the age (number of times subcultured) of the cells from which the protoplasts were isolated, and the nature of the coelectroporated CHS promoter sequence. For example, a region of the CHS promoter from -326 to -141 behaved as a trans-activator when coelectroporated with the CAT construct into unelicited protoplasts isolated from newly initiated cell suspensions, but the same region acted as a trans-silencer in the same protoplasts in the presence of FE. This silencer activity was much reduced in GSH-treated protoplasts. The results suggest that there are differences in the signal transduction pathways for elicitation of CHS transcription by Fe and GSH, which involve previously identified cis-elements in the CHS promoter.