Involvement of components of the phospholipid-signaling pathway in wound-induced phenylpropanoid metabolism in lettuce (Lactuca sativa) leaf tissue

11 pages, 8 figures.In plant tissue, a wound signal is produced at the site of injury and propagates or migrates into adjacent tissue where it induces increased phenylalanine ammonia lyase (PAL, EC 4.3.1.5) activity and phenylpropanoid metabolism. We used excised mid-rib leaf tissue from Romaine lettuce (Lactuca sativa L., Longifolia) as a model system to examine the involvement of components of the phospholipid-signaling pathway in wound-induced phenolic metabolism. Exposure to 1-butanol vapors or solutions inhibited wound-induced increase in PAL activity and phenolic metabolism. Phospholipases D (EC 3.1.4.4), an enzyme involved in the phospholipid-signaling pathway is specifically inhibited by 1-butanol. Re-wounding tissue, in which an effective 1-butanol concentration had declined below active levels by evaporation, did not elicit the normal wound response. It appears the 1-butanol-treated tissue developed resistance to wound-induced increases in phenylpropanoid metabolism that persisted even when active levels of 1-butanol were no longer present. However, a metabolic product of 1-butanol, rather than 1-butanol itself, may be the active compound eliciting persistence resistance. Inhibiting a subsequent enzyme in the phospholipid-signaling pathway, lipoxygenase (LOX; EC 1.13.11.12) with 1-phenyl-3-pyrazolidinone (1P3P) or reducing the product of LOX activity with diethyldithio-carbamic acid (DIECA) also inhibited wound-induced PAL activity and phenolic accumulation. The effectiveness of 1-butanol, DIECA, and 1P3P declined as the beginning of the 1-h immersion period was delayed from 0 to 4 h after excision. This decline in effectiveness is consistent with involvement of the inhibitors in the production or propagation of a wound signal. The wound signal in lettuce moves into adjacent tissue at 0.5 cm h−1, so delaying application would allow the signal to move into and induce the wound response in adjacent tissue before the delayed application inhibited synthesis of the signal. Salicylic acid (SA) inhibits allene oxide synthase (AOS, EC 4.2.1.92), another enzyme in the phospholipid-signaling pathway. Exposure to 1 or 10 mM SA for 60 min reduced wound-induced phenolic accumulation by 26 or 56%, respectively. However, 1 mM SA lost its effectiveness if applied 3 h after excision, while 10 mM SA remained effective even when applied 4 h after excision. At 1 mM, SA may be perturbing the wound signal through inhibition of AOS, while at 10 mM it appears to have some generally inhibitory effect on subsequent phenolic metabolism. These data further implicate the phospholipid-signaling pathway in the generation of a wound signal that induces phenolic metabolism in wounded leaf tissue.Peer reviewe

Heat shock treatments delay the increase in wound-induced phenylalanine ammonia-lyase activity by altering its expression, not its induction in Romaine lettuce (Lactuca sativa) tissue

Wounding lettuce (Lactuca sativa L., var. Longifolia) leaves induced an eight-fold increase in the activity of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), and the subsequent accumulation of phenolic compounds and tissue browning at 10degreesC. PAL is a key enzyme in the synthesis of phenolic compounds. A PAL cDNA was previously isolated by reverse-transcription PCR using total RNA from wounded lettuce leaves. RNA gel blots showed that maximum accumulation of both PAL mRNA and PAL enzyme activity occurred 24 h after wounding. A 2-min heat shock at 45degreesC administered within 5 min of wounding delayed the wound-induced increase in PAL activity, but did not delay the increase in wound-induced PAL mRNA. Changes in the content of PAL protein were also followed by immunoblot using anti-PAL antibody raised against the bacterially expressed protein from the cDNA. Immunoblots showed that the level of PAL protein in wounded lettuce tissue was significantly reduced by the heat shock treatment. These data suggest that heat shock reduces the rise in wound-induced PAL enzyme activity by reducing the translation of wound-induced PAL mRNA, or by increasing the turnover of the induced PAL protein