(Dedicated to Professor Klaus Hahlbrock on the occasion of his 60th birthday) SUMM.^RY Epidermal tissue was isolated from Scots pine {Pinus sylvestris L.) needles by enzymatic digestion in order to study tissue distribution of u.v,-B-screening pigments. Up to 90 "o ofthe needle content of a group of diacylated flavonoi glycosides tbat were structurally closely related was found in the epidermal layer. Among these metabolites. 3",6"-di-para-coumaroyl-isoquerfitrin and 3",6"-di-para-coum3roy]-astragal in were the main u.v.-B-induced compounds in cot\ ledons and primary needles, respectively. However, catechin and astragalin (kaempferol 3-glucoside), two non-acylated fla\'onoid metabolites, were only observed in total needle extracts, and at levels independent of u.v.-B treatment. According to this metabolite distribution, tbe mRNA of chalcone syntbase, the key enzyme to flavonoids, was found in epidermal and mesophyll as well as vascular tissues. The major alkaliextractable wall-hound phenolic metabolites, astragalin, 4-coumaric acid, and ferulic acid, a minor component of tbe cell wall, were also found exclusively in the epidermal layer. These compounds were not stimulated by u.v.-B irradiation within the experimental period. Staining of needle cross sections and epidermal layer preparations with NaturstofTreagenz A confirmed the specific localization of wall-bound astragalin in the outer wall of the epidermal layer. Model calculations of u.v.-B absorptions at 300 nm of soluble and cell-wall-bound metabolites of the epidermal layer revealed an almost complete shielding of the mesophyll tissue from u.v.-B radiation.

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SummaryStress ethylene emission is positively correlated with ozone sensitivity in various plant species, indicating that ethylene may be involved in the control of ozone damage. This study shows that ozone exposure of tomato plants for 5 h at 85 nl 1-1 and above leads to leaf injury within 24 h. 1-aminocyclopropane-l-carboxylic acid (ACC) content and ACC synthase activity were accordingly elevated within 1-2 h. Pre-treatment of leaves with inhibitors of ACC synthase and ACC oxidase significantly inhibited the evolution of ethylene and reduced ozone-induced visible damage. Transcript levels for only one out of three SadenosyI-L-methionine (SAM) synthetase genes (SAM3), and one out of four ACC synthase genes (LE-ACS2) were induced by ozone (maximum at 2 h). Treatment with protein kinase (K-252a) and phosphatase inhibitors (calyculin A) revealed that ACC synthase activity was additionally regulated by protein phosphorylation/dephosphorylation. Transcripts of ACC oxidase (pTOM13 cDNA probe) displayed the fastest response of the parameters tested (maximum at 30 min), suggesting a regulatory role for ACC oxidase in ethylene formation of ozone-exposed plants. The results demonstrate a highly selective ozone response by ethylene biosynthetic genes which resembles that of plant-pathogen interactions.

Parsley (Petroselinum crispum 1.) is known to respond to ultraviolet irradiation by the synthesis of flavone glycosides, whereas fungal or elicitor stress leads to the synthesis of furanocoumarin phytoalexins. We tested how these defensive pathways are affected by a single ozone treatment (200 nL 1-'; 10 h). Assays were performed at the levels of transcripts, for enzyme activities, and for secondary products. The most rapid transcript accumulation was maximal at 3 h, whereas flavone glycosides and furanocoumarins were maximally induced at 12 and 24 h, respectively, after the start of ozone treatment. Ozone acted as a cross-inducer because the two distinct pathways were simultaneously induced. These results are consistent with the previously observed ozone induction of fungal and vira1 defense reactions in tobacco, spruce, and pine.

The S-adenosyl-l-methionine:pinosylvin-O-methyltransferase (PMT) 2 gene was sequenced from Scots pine (Pinus sylvestris). The open reading frame is arranged in two exons spaced by one 102-bp intron. Promoter regulatory elements such as two "CAAT" boxes and one "TATA" box were identified. Several cis-regulatory elements were recognized: stress-responsive elements (Myb-responsive elements) as well as G, H, and GC boxes. Moreover, elicitor-responsive elements (W boxes) and a sequence resembling the simian virus 40 enhancer core were found. In phloem and needles of control trees, the transcripts of stilbene synthase (STS) and PMT were hardly detectable. Increased ozone fumigation up to 0.3 L L Ϫ1 enhanced the transcript level of STS and PMT in needles but not in healthy phloem. Wounding, e.g. mock inoculation, of stem-phloem was characterized by a transient increase in STS and PMT transcripts, which was more pronounced in the case of fungal inoculation. Combination of fungal-challenge or mock treatment with ozone resulted in a positive interaction at 0.3 L L Ϫ1 . Scots pine stilbene formation appeared to be induced via STS and PMT gene expression upon ozone and fungal stress as well as wounding. The broad stress-responsiveness is in agreement with the range of various cis-acting elements detected in the STS and PMT promoters.