Nature is inventive: Plants have developed a variety of substances with which they protect themselves from voracious pests or pathogens. As it now turns out, some of these defense substances are surprisingly produced by different plants using very different synthetic pathways and using different enzymes. Since the plants are not closely related, the researchers assume that their metabolic pathways have evolved independently several times, but with the same result.
Individual species from very different plant families produce special defense substances, so-called benzoxazinoids, which are poisonous to many herbivorous insects and animals. The plants also use it to repel microorganisms. In addition, benzoxazinoids are involved in the interaction between individual plants, where they support relatives and damage non-specific plants. The synthesis route of these indole-derived chemical compounds involves at least eight steps catalyzed by as many enzymes and has been known since the 1990s - but only for monocotyledonous grasses such as corn, wheat and rye.
However, benzoxazinoids occur in many other plant species that are evolutionarily far apart. Until now, it was not known how these plants produce the defensive substances; several attempts to elucidate their metabolic pathways were unsuccessful. However, it is assumed that they differ from the biosynthesis in corn.
Why can such different plants produce the same substances?
A research team led by Matilde Florean from the Max Planck Institute for Chemical Ecology has now investigated the synthesis pathway of benzoxazinoids in two of these very distantly related plant species: the zebra plant (Aphelandra squarrosa) and the common golden nettle (Lamium galebodolon). While the first is a houseplant and produces benzoxazinoids primarily in the roots, the second grows on the edges of forests and produces these substances in all parts of the plant. For both plant species, the researchers analyzed their ingredients or metabolic (intermediate) products and the entirety of their active genes in all parts of the plant.
The scientists compared these data between the various organs of the zebra plant and with closely related species of golden nettle, which do not produce benzoxazinoids. From this, they identified 90 or 57 genes that could be involved in the formation of these compounds. They then introduced these into tobacco plants (Nicotiana benthamiana) and investigated whether the genes are really involved in the formation of benzoxazinoids and which enzymes are produced from them.
The comparison showed that completely different enzymes are responsible for the formation of benzoxazinoids in the two plants examined than in corn. “In maize, a number of closely related cytochrome P450 enzymes carry out specific steps in the metabolic pathway. In the other two plant species, however, various other classes of enzymes as well as other cytochrome P450 enzymes are active,” explains senior author Tobias Köllner from the Max Planck Institute for Chemical Ecology. For example, the species studied use a dual-function flavin-containing monooxygenase for two consecutive oxidation steps instead of two different cytochrome P450 enzymes as in grasses.
Parallel evolution of metabolic pathways
The researchers report that such a variety of enzymes that carry out the same reactions is surprising. They conclude that the biosynthesis of these substances has developed independently several times over the course of plant evolution. This is unusual in botany, as conserved groups of substances such as defensive substances usually have the same evolutionary origin: a prehistoric plant usually developed a way through which it produces these substances. Subsequently, their descendants and the species developed from them further developed and modified this technology.
In this case, golden nettle and zebra plants have reinvented the proverbial wheel in the form of their defensive substances - this is rather an exception in the plant kingdom. “Our work shows how flexible plant metabolism can be: they can independently invent very different strategies to produce the same chemical compounds,” says co-author Sarah O'Connor from the Max Planck Institute for Chemical Ecology. This has happened at least three times in the evolutionary history of benzoxazinoids, as their study shows. In the future, the team wants to elucidate the synthesis of these compounds in other plant families.
Source: Matilde Florean (Max Planck Institute for Chemical Ecology) et al., Proceedings of the National Academy of Sciences (PNAS), doi: 10.1073/pnas.2307981120