Grape Notes in an Orchestra of Fruit Flavors

What makes a strawberry taste and smell like a strawberry? In this guest blog, Kevin M. Folta discusses his research, recently published in BMC Plant Biology, which examines the final step in the synthesis of methyl anthranilate (MA) in cultivated strawberries, a compound found naturally in many fruits as they ripen

The grape flavor in your cough medicine makes it go down easier. Grape soda tastes the way it does because of the same sweet molecule.  That familiar gooey-sticky-sweet sensation has an interesting origin in food chemistry, and now we’re unraveling its genetic basis as a naturally-occurring contributor to the flavors of fruits, even fruits that we don’t think of as grape tasting-  like strawberries.

The familiar “grape” notes in foods are imparted by a compound called methyl anthranilate (MA).  It was first synthesized and used as an artificial grape flavor, and is the familiar component of fruity drinks, perfumes and other common consumer products.  It is the flavor of purple. It also is a bird repellent.

The familiar “grape” notes in foods are imparted by a compound called methyl anthranilate.

Methyl anthranilate is also found naturally in many fruits as they ripen.  It is the conspicuous flavor signature of Concord grapes (Vitis labrusca).   In other fruits MA plays a supporting role as one small note in the orchestra of compounds that emanate from fruit at the first stage of digestion—mechanical disruption in the mouth.  Sugars and acids lay out a foundation of sensation to the tongue, and a constellation of volatilized compounds migrate up behind the palate to the olfactory receptors where they trigger nerve impulses that reassemble the sensory experience in the brain milliseconds later.

MA is just one part of a mind-brain connection that triggers thoughts of summer, desserts and enjoying healthy snacks.  This is why the flavors of fruits are so important—and if scientists can make them taste better, it might help inspire healthier choices for consumers and better profits for growers.

But flavors have not always been a priority in modern varieties. Breeders did a stellar job selecting varieties that could withstand disease, and produce high yields of large fruits that could be shipped across a continent and arrive in reasonable shape.  Over decades, intensive annual selection of fruits like strawberries led to erosion of sensory quality.

In fact, MA is present in wild strawberries and has been completely lost from just about all modern varieties. It exists only in a few older varieties and some new ones specifically bred for the grape aroma.

Our laboratory is interested in identifying the genes required for synthesis of important flavor volatiles so that they can be re-introduced to new varieties for the very first time. If we identify the genes required for the synthesis, it enables the development of DNA signatures that could hasten the breeding process.

Our work published in BMC Plant Biology by Pillet et al. started with a sophisticated approach to find associated genes, and identified the gene catalyzing the final step in MA synthesis.  The work married genomics, genetics, gene expression analyses, and strawberries poked with syringes.

First an MA-producing cultivar was crossed with a non-producer.  Their progeny were found to segregate for the aroma compound; some offspring had it, others didn’t.  Fruit from each resulting plant were analyzed for a snapshot of active genes (as inferred from RNA levels) that corresponded with the presence of MA.

Then all of the sequences of expressed genes from MA producers were combined into a list. All of the genes expressed in non-MA producers were combined into a separate list.  The two lists were compared computationally, and only five genes were obviously turned on in the producers and not in the non-producers.

One of them encodes an enzyme known as a methyltransferase.  A methyltransferase transfers methyl groups, and methyl anthranilate has “methyl” right in the name.  Essentially what this candidate enzyme might do is grab anthranilate and attach a methyl to it, making methyl anthranilate.

This hypothesis was tested in several ways.  First, we turned off the gene in MA-producing fruits using a process called RNA interference.  The fruit is literally injected with bacteria that transfer a backwards copy of the gene to the cells.  This new reverse genetic information suppresses the methyltransferase gene.  When we did this, the fruits did not produce MA.  That’s pretty strong evidence that the gene is driving synthesis.  When our methyltransferase gene is turned off, the fruits did not produce MA.

The second test was to produce the enzyme in bacteria, purify it, and determine if the synthesized enzyme could take a methyl donor compound, strip off the methyl group, and move it onto an anthranilate molecule to make methyl anthranilate.  It didn’t work.  There are lots of technical reasons for such failures.

However, if bacteria made the enzyme internally and were fed anthranilate in the culture medium, they could produce methyl anthranilate.  In another test we took frozen fruit powders and reconstituted them into a chemical soup containing anthranilate and a methyl donor.  They also were able to synthesize the grape aroma from the component parts!   When any of the components were left out, the fruit powders did not produce the MA.

In this work we used genomics and genetics approaches to identify a candidate gene that might perform the last step in MA synthesis.  If the gene was turned off, no MA was detected.  The corresponding enzyme could manufacture MA from its component parts in bacteria, and we could cold generate MA when we fed the right precursors to strawberry fruit components.  These data together make a compelling argument that the methyltransferase was indeed the correct gene in the process!

In this work we used genomics and genetics approaches to identify a candidate gene that might perform the last step in MA synthesis.

The final step was to identify traceable differences in the gene between MA producers and non-producers.  A part of the control region of the gene can be amplified in producers, but not non-produces.  This DNA sequence can serve as a “molecular marker”.  Now anyone wanting to breed strawberries for high levels of MA should test their plants first for the active version of the gene.  The progeny will have a higher likelihood of being MA producers.

There is a lot more to the story of MA in strawberry.  The study also showed how the levels are dependent on environmental conditions and stage of fruit ripening.  This is good news because it means there are more genes that contribute to the process, so the hunt continues.

In the future, the identification of genes contributing to individual flavor notes will allow plant breeders to combine genetics and get and get all of them into one great farmable strawberry.  The resulting flavorful berries will reclaim the lost sensory excitement of heirloom varieties, once again reunited in elite strawberries compatible with modern production demands.

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