5' upstream sequences from the wun1 gene are responsible for gene activation by wounding in transgenic plants.

A 1.2-kilobase pair fragment of the 5' upstream region of a potato wound-inducible gene (wun1) was fused to different marker genes (wun1-CAT, wun1-NPTII). Stable integration of a wun1-CAT chimeric gene into the tobacco genome led to a high wound-inducible chloramphenicol acetyltransferase activity in leaves. Transient expression experiments in potato protoplasts showed that wun1 carries a strong promoter sequence similar in strength to the 35S promoter. The same intensity of expression was also observed using wun1 constructs in transient experiments with rice protoplasts. wun1 mRNA was shown to accumulate to high levels in potato leaves collapsing as a result of infection with the phytopathogen Phytophthora infestans. The wun1 product might, therefore, play a role in a general physiological reaction to stress correlated with cell death.

UV-inducible transient expression in parsley protoplasts identifies regulatory cis-elements of a chimeric Antirrhinum majus chalcone synthase gene.

It was shown previously that parsley protoplasts retain differential responsiveness to external stimuli, e.g. UV light. This opened the way for the development of transient expression assays to identify DNA sequences acting as regulatory cis-elements in the transcriptional activation of UV-inducible plant genes. The chalcone synthase (chs) gene of Antirrhinum majus (Snapdragon) is inducible by visible and by UV light. We demonstrate that the kinetics of light induction of a chimeric A.majus chs-nptII gene observed during transient expression in the parsley protoplasts reflect the kinetics of UV-induced CHS expression in A.majus seedlings in vivo. We define three regulatory sequence regions, each differing qualitatively and quantitatively in their effects on gene expression. Immediately upstream of the TATA box (-34) a sequence, with coordinates -39 to -197, functions as an orientation independent UV-light responsive element. The complete 1.1 kb promoter or three tandem copies of this element are capable of rendering a heterologous minimal promoter UV responsive. The next upstream region (-197 to -357) contains sequences that do not by themselves cause UV-induced expression, but specifically potentiate the level of UV-induced expression when combined with the TATA-box proximal UV-responsive element. A third element (-661 to -564) has the properties of a general enhancer since it increases the level of both uninduced and UV-induced expression.

Parsley protoplasts retain differential responsiveness to u.v. light and fungal elicitor.

The differential response of cultured parsley cells to u.v. irradiation and elicitor treatment is a paradigm for analysis of specific plant defense responses. We demonstrate that freshly isolated parsley protoplasts, in the absence of detectable cell wall, maintain fully the ability to be activated by these important environmental factors. Stimulated protoplasts synthesize typical qualitative patterns and amounts of potentially protective flavonoid glycosides and coumarin phytoalexins following either u.v. irradiation or treatment with fungal elicitor, respectively. Induced accumulation of mRNAs and enzymes of the phenylpropanoid biosynthetic pathways is nearly identical in protoplasts and cells. Stimulation of protoplasts with elicitor requires only a short period of contact, which is not sufficient for cell wall regeneration. Importantly, there is no activation of these pathways during protoplast preparation. These results establish that parsley protoplasts respond appropriately to two physically distinct stimuli and might serve as an especially suitable system for the analysis of signal transduction and gene activation.