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Citrus fruits come in many varieties and tastes; some are sweet, and some are sour. Extremely sour. Biologists have long sought for the mechanism that enables cells of, for example, lemons to become acidified to such an extreme extent. A team of biologists from the University of Amsterdam, supervised by prof. Ronald Koes and dr. Francesca Quattrocchio, has finally solved this riddle. Their findings are published in Nature Communications.

Citrus fruits come in many varieties and tastes, from sweet to extremely sour. Photo: Pixabay

Oranges, lemons, grapefruit, mandarins, pomelos; all citrus fruits belong to a single family. Yet they all taste different. For a large part this is caused by of the amount of acid within the fruits. Oranges contain little acid, and lemons a lot; they can have a pH as low as 2. That is quite extreme.  How is this possible?

Pumps

This question has been a long-standing enigma in biochemistry. Plant cells store acids and many other molecules in their vacuole. This is the fluid-filled space that makes up the largest part of the cells within citrus fruits, as you can clearly see  when you look at the flesh of the fruits. Molecules are pumped in and out of the vacuole by biological pumps in its circumference. Pumping H+ (or protons) into the vacuole makes the fluid acidic. Usually, plant cells cannot increase the acidity of their vacuoles below a pH of about 3.5. At this level the difference in the H+ concentration between the inside of the vacuole and the rest of the plant cell is so steep that it would cost a huge amount of energy to pump in more protons.

But clearly, citrus fruits somehow manage to do this. Biochemists have searched for decades for the elusive proton pump that is strong enough to make citrus fruits so extremely acidic. Now, finally, the team of prof. Ronald Koes and dr. Francesca Quattrocchio has identified this long-sought pump. In Nature Communications of 26 February they describe their quest.

Petunia

The team usually works on an entirely different plant: petunia. The cells in the petals of petunia flowers are also hyperacidified (though not as extreme as in citrus fruits), which gives the petals a red-violet colour. Using petunia varieties bearing flowers with a reduced acidity and a blue colour the team had previously discovered a number of genes that control the acidity of the vacuole in the flower petals, and thereby their colour. These findings gave Koes and Quattrocchio the idea to look for related genes in citrus plants. 

With success. The biologists analysed a range of citrus fruits, including sweet and sour lemons, oranges and pomelo’s. And they kept finding the same result: in the sour fruits, two genes called CitPH1 and CitPH5 were very active. But in the sweet fruits, they were not. This way they proved that these two genes encode for the elusive, extremely strong proton pump.

Useful for breeders

With their discovery, the biologists from the University of Amsterdam have not just solved a long-standing fundamental riddle; their findings are also very useful for citrus fruit breeders. ‘All citrus fruits we know today are the results of thousands of years of careful selection and breeding’, explains Koes. ‘Now that we have finally found the genes that are responsible for the acidity of the fruits, breeders can develop new varieties much more easily. They can now detect how acidic the fruits will be – and thus, what they will taste like – in very young plants, years before they will produce their first fruits.’

Koes adds: ‘Something else that is really interesting, is that genes similar to CitPH1 and CitPH5 are also active in other types of fruit, like, apples and grapes. Thus, it is very likely that these genes are also crucial for the the acidity, and therefore the taste, of these fruits and probably many others.’

Purple hints

A little bonus fact: as said before, in petunias you can easily detect the level of acidification because of the flower color. In citrus trees, something similar is the case. Koes: ‘There is a lemon tree, a variety called Faris, that has both branches that carry sweet fruits and branches that carry sour fruits. In the branches bearing sour lemons, the young leaves are purple, and the seeds within the lemons have purple spots. In the branches carrying sweet lemons, the young leaves are green and the seeds are not spotted. This is once again nicely linked with the activity of the genes we found.’  

Details of the publication

Pamela Strazzer, Cornelis E. Spelt, Shuangjiang Li, Mattijs Bliek, Claire T. Federici, Mikeal L. Roose, Ronald Koes  & Francesca M. Quattrocchio: ‘Hyperacidification of Citrus fruits by a vacuolar proton-pumping P-ATPase complex’, in: Nature Communications (26 February 2019). Link: https://www.nature.com/articles/s41467-019-08516-3