Transcriptional activation of the parsley chalcone synthase promoter in heterologous pea and yeast systems.

Introduction by electroporation of different parsley (Petroselinum crispum) CHS-promoter/beta-glucuronidase(GUS)-reporter constructs into pea (Pisum sativum L.) protoplasts leads to a high constitutive GUS-expression and to the loss of the light-inducibility seen in the homologous parsley protoplast system. These results indicate that Unit 1 of the parsley CHS-promoter is only partly responsible for the GUS-expression detected. Instead, additional cis-elements, which are located downstream within 100 bp from the transcriptional start site, mediate the de-repression in pea protoplasts. In contrast, in yeast (Saccharomyces cerevisiae) cells, the GUS expression from the heterologous CHS/GUS construct is controlled by elements between Unit 1 and -100 bp. In both pea and yeast cells, transcription factors different from those regulating UV-responsiveness in parsley, are probably mediating the constitutive expression from the heterologous construct. The results with pea protoplasts imply that protoplastation of pea leaf cells itself induces de-repression as a result of stress to the protoplasts. This notion was strengthened by the finding that mRNA levels of the endogenous chalcone synthase were drastically increased as the result of the protoplastation procedure.

Ultraviolet-B-radiation-induced changes in nicotinamide and glutathione metabolism and gene expression in plants.

Pea (Pisum sativum L. cv. Greenfeast) plants were exposed to supplementary ultraviolet-B (UV-B) radiation (biologically effective dose rates normalised to 300 nm, UV-B[BE,300]: 0.18, 0.32 or 1.4 W m[-2]). Leaf nicotinamide, trigonelline, GSHtot (total glutathione) and GSSG (oxidised glutathione) levels remained unchanged after exposure to the lowest dose rates. 1.4 W m(-2) UV-B(BE,300) gave rise to 60-fold and 4.5-fold increases in GSSG and GSHtot, respectively. 3.5-fold and 9.5-fold increases were found in nicotinamide and trigonelline, respectively. cab (Chlorophyll-a/b-binding protein) transcript levels decreased and CHS (chalcone synthase) and PAL (phenylalanine ammonia-lyase) mRNA increased after shorter UV-B exposures (hours) to the higher dose rate of UV-B, and after exposure to the intermediate dose rate. CHS and PAL mRNAs also increased after prolonged exposure to the lowest dose rate. cab transcripts completely disappeared, whereas CHS and PAL mRNA levels rose by 60-fold and 17-fold, respectively, after 12 h exposure at the highest dose rate and 12 h of development. Our results indicate that nicotinamide or trigonelline do not function as signalling compounds for CHS and PAL gene expression. Elevated nicotinamide and trigonelline levels occur in response to UV-B, but only at UV-B doses high enough to cause oxidative stress.

UV-B- and oxidative stress-induced increase in nicotinamide and trigonelline and inhibition of defensive metabolism induction by poly(ADP-ribose)polymerase inhibitor in plant tissue.

Nicotinamide and trigonelline contents increased in Catharanthus roseus tissue culture after exposure to 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) or vanadylsulfate and in Pisum sativum leaves after exposure to UV-B radiation. Vanadylsulfate increased phenylalanine ammonia-lyase (PAL) activity and the content of reduced and oxidized gluthathione in C. roseus tissue culture. The increases in PAL activity caused by 2 mM AAPH or 0.2mM vanadylsulfate were prevented by 0.1 mM 3-aminobenzamide (3-AB), an inhibitor of poly(ADP-ribose)polymerase. Present results support the hypothesis [Berglund, T., FEBS Lett. (1994) 351, 145-149] that nicotinamide and/or its metabolites may function as signal transmittors in the response to oxidative stress in plants and that poly(ADP-ribose)polymerase has a function in the induction of defensive metabolism.