The biosynthesis of steroidal defense metabolites in plants

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The biosynthesis of steroidal defense metabolites in plants

The biosynthetic pathway of specific steroidal compounds in nightshade plants (such as potatoes, tomatoes and eggplants) starts with cholesterol. Several studies have investigated the enzymes involved in the formation of steroidal glycoalkaloids. Although the genes responsible for producing the scaffolds of steroidal specialized metabolites are known, successfully reconstituting of these compounds in other plants has not yet been achieved. The project group ‘Specialized Steroid Metabolism in Plants’ set out to find the missing piece of the puzzle. “With our research project, we particularly wanted to clarify which important component of the biosynthesis pathway has so far remained hidden from research and what role this gene or protein plays in the pathway. We also wanted to find out whether we could reconstitute the biosynthetic pathway after identifying the missing component. An important aspect of our study was also to find out more about the ecological role of steroidal saponins in plants,” says the senior author, describing the questions of the study.   

The research team used the wild plant Solanum nigrum (black nightshade) for the studies because it produces different steroidal compounds in different tissues, all of which are derived from the same precursor, cholesterol. In the leaves, the most important steroidal metabolite is a saponin called uttroside B, while in the berries the most important compounds are steroidal glycoalkaloids such as α-solasonine, α-solamargine and malonyl-solamargine.

The enzymes GAME6, GAME8 and GAME11 are involved in the formation of both types of compounds and are found in both leaves and berries. Confocal microscopy was used to determine where these enzymes are located in the cells.  Through biochemical and molecular biological analyses, the researchers identified a gene responsible for a protein called GAME15. Although GAME15 belongs to the family of cellulose synthase-like proteins, it has no function in cellulose production. Instead, it is important for the biosynthesis of steroidal compounds, even though it does not have a catalytic function like other enzymes.

“Our protein interaction experiments showed that GAME15 interacts with the enzymes GAME6, GAME8 and GAME11. These enzymes are responsible for the first steps in the hydroxylation of cholesterol, leading to the formation of the furostanol-aglycone (16,22,26-trihydroxycholesterol), a key branching point between the synthesis of steroidal saponins and glycoalkaloids. Using Solanum nigrum plants in which the GAME15 gene was knocked out, we were able to show that these plants were no longer able to produce steroidal glycoalkaloids and saponins,' explains the first author, one of the main findings of the study.

Steroidal saponins and steroidal glycoalkaloids are groups of compounds with promising medical applications. For example, recent studies show that certain saponins are highly effective in treating liver cancer. Steroidal glycoalkaloids also have anti-cancer properties, as well as antimicrobial and anti-inflammatory activities. "By identifying GAME15, we were able to reconstitute the metabolic pathway for steroidal compounds in heterologous hosts such as Nicotiana benthamiana, up to the scaffold furostanol, a precursor of steroidal saponins, and solasodine, an immediate precursor of steroidal glycoalkaloids," says the senior author.

Reconstituting medically interesting compounds in plants such as Nicotiana benthamiana is also known as 'pharming', a portmanteau of 'pharmaceutics' and 'farming'. Pharming involves the use of genetically modified plants with an integrated biosynthetic pathway for the production of medical compounds to produce drugs cost-effectively and on a large scale. The results of the study therefore open up possibilities for improved production of important steroid-based compounds. 

Steroidal glycoalkaloids are already known to be important plant defense substances. They are toxic compounds typical of solanaceous plants and are also found in potatoes, tomatoes and eggplants. However, their toxicity can be significantly reduced in crops by peeling, cooking or frying. In tomatoes, the glycoalkaloids are broken down during ripening, so they are barely detectable in the red fruit.  However, the ecological role of steroidal saponins in Solanum nigrum leaves was so far unknown.

A key clue came from the greenhouse team, who noticed that GAME15 knockout plants, which are unable to produce saponins, were more susceptible to insect herbivores than wild-type plants. Intrigued by this observation, the researchers carried out ecological experiments in the form of feeding trials with two natural Solanum pests. 

"In a first experiment, two herbivores, the leafhopper Empoasca decipiens and the Colorado potato beetle Leptinotarsa decemlineata, were given a choice between leaves of wild-type plants (which produce steroidal saponins) and leaves of Game15 knockout plants (which do not produce saponins due to the knockout of the GAME15 gene). After a week, we measured the damage caused by the insects. The results clearly showed that both herbivore species fed almost exclusively on the knockout leaves, showing a preference for them over the wild-type leaves. In a second experiment, we use a "forced-feeding” bioassay, specifically with Colorado potato beetles. In this test, we placed individual beetles with detached leaves from either wild-type or knockout plants. After just six hours, the beetles readily fed on the knockout leaves lacking steroidal saponins, while they largely avoided the wild-type leaves, apparently preferring to starve rather than feed," says another author, describing the first evidence for the role of steroidal saponins in plant defense.

One possible explanation for the different groups of compounds in leaves and berries is that these compounds are specialized to protect different plant tissues. Leaves are more likely to be attacked by herbivores, while berries are more susceptible to pathogens.

 “Our discoveries highlight how Solanaceae plants have adapted a cellulose-synthase like protein hijacked from a core metabolism role (such as cellulose biosynthesis) to a structural role, required for the biosynthesis of compounds specialized in the defense of the plants against pathogens. This discovery opens up new opportunities for engineering crops with enhanced resistance to pests and for the production of important steroid-based compounds to fight cancer and other diseases,” says another senior author of the study.

https://www.science.org/doi/10.1126/science.ado3409

https://sciencemission.ayurmatrika.com/A-scaffold-protein-manages-the-biosynthesis-of-steroidal-defense-metabolites-in-plants