The history of wound responses of plants has been marked by several milestones including the discovery by the late Clarence ‘Bud’ Ryan (my former postdoc supervisor) of herbivore-inducible proteinase inhibitors. Bud often told the ‘open window’ story relating how he noticed that tomato plants near open windows accumulated proteinase inhibitors. This led to his classic 1972 Science paper.
Bud Ryan's very thorough 2004 interview with Julie Kerssen. Interesting reading from today’s perspective.
More from an interview that Bud gave to his university : WSU.
Experiments leading to the discovery that jasmonate (JA) is a key signal in plant defense
Bud's lab was a very attractive venue for visiting postdocs. The lab had a very good reputation, Bud's team was strong and many interesting techniques were available to attract visitors. Among these was a very simple and powerful immunodiffusion assay for the measurement of proteinase inhibitor proteins. This made the lab a very attractive place for visitors interested in joining Bud to learn about the induction of proteinase inhibitor (PI) gene expression. Indeed, as soon as it became feasible to study signal transduction in the wound response (in the late 1980's), postdocs came to the lab to exploit this assay. Bud had submitted a research proposal on this theme around 1987 and around this time EEF approached Bud with the idea to find a putative ‘second messenger’ involved in PI induction in wounded tomato leaves. But where should one start in such a project? Fortunately, Bud had an almost photographic memory of previous experiments conducted in his lab and knew where to find them in old laboratory books. An early unpublished experiment by Mary Kay Walker-Simmons had indicated that linolenic acid treatment could stimulate proteinase inhibitor production but that linoleic acid was ineffective. This was one the early clues found in old laboratory notebooks that a linolenic acid-based signal pathway might somehow control part of the wound response. Other clues came after many weeks of reading the literature. Brady Vick and Don Zimmerman, researchers in Fargo, ND, had previously reported the biosynthetic route of an interesting fatty acid derivative called "jasmonic acid" (JA). They had shown that his substance was synthesized from linolenic acid. At that time, JA's function was unknown but experiments being conducted in Japan, Germany and the USA all suggested that it was biologically active. Indeed, JA was starting to be known as a "senescence-promoting substance", as a "growth inhibitor" and also as a stimulator of the accumulation of proteins of unknown function called "jasmonate-inducible proteins". This was clearly a substance worth testing in the proteinase inhibitor (PI) assay.
The first sample of JA we got was probably furnished by John Everard from the late Vince Franceschi's laboratory. The sample was in the form of methyl jasmonate (MeJA) and this was suspended in water and sprayed onto plants. Two days later the levels of induced proteinase inhibitor proteins were spectacular - so high that it was at first hard to see what had happened. Firstly, the amount of proteinase inhibitor protein induced was larger than that resulting from any other treatment – we quickly established a laboratory record for proteinase inhibitor levels. We were sure that we’d found a defense signal molecule and that a key role of JA was to control defense gene expression. That turned out to be correct, but this simple experiment was to yield yet more information. Interestingly, methyl jasmonate "spread" from the sprayed plant to nearby controls. This was due to its volatility and this immediately raised the exciting possibility that plants able to produce methyl jasmonate might be able to stimulate proteinase inhibitor gene expression in tomato plants placed in their vicinity.
Pullman is not far East of the Columbia Basin – an area colonized by a remarkable shrub, the Great Basin sagebrush (Artemisia tridentata). The idea to test this plant as a source of volatile methyl jasmonate came from an early Japanese publication (by J. Ueda and J. Kato) demonstrating the presence of methyl jasmonate in another Artemisia species. With haste (and on a Sunday), EEF drove out towards Walla Walla and collected twigs of the sagebrush. These were returned immediately to the laboratory and placed in an incubation box with tomato plants to see if PI induction in the tomatoes occurred. This experiment worked ; proteinase inhibitor proteins accumulated in the tomato plants. In a final step, the leaf surfaces of sagebrush growing in the field were rinsed with ethanol and this ethanol was returned to the laboratory, fractionated and then each fraction tested for activity. The final active fraction was highly enriched in methyl jasmonate that was absent in all inactive fractions.
Throughout all this Bud was highly enthusiastic and discussions with him were compelling. The work carried on at a fast pace and the support of other members of the Ryan laboratory was considerable. The atmosphere that Bud created at this period made the laboratory an exceptional place to be. Not only was jasmonate being identified as a wound signal but the structure of systemin was being revealed…