An international team of scientists from the Czech Republic and the UK has succeeded in locating the gene responsible for the production of the polyphenol oxidase (PPO) enzyme in the genome of the faba bean. The enzyme plays an important role in the protection of the plant’s seed and its dormancy, i.e. the ability to wait in a dormant state for an external signal to trigger germination. On the other hand, it causes dark pigmentation and a bitter taste. This undesirable property of the PPO enzyme seems to have played an important role in the gradual breeding of not only faba beans. Growers selected seeds without pigmentation from plants in which this gene was not functional. The results of research aimed at decoding the genetic information of the largest genome of a diploid organism to date, involving experts from the UP Faculty of Science and the University of Reading in the UK, have been published in the prestigious journal Nature.
A group of scientists from the Palacký University Olomouc Faculty of Science used the previous knowledge gained in their search for the gene responsible for PPO production in wild peas to investigate the larger genome of the faba bean. The presence of characteristic phenolic precursors, together with sufficient activity of the PPO defense enzyme, led to the typical dark colouration of the hilum in pea seeds, a vestige of the connection with the mother plant. Cultivated pea varieties, in which PPO production is suppressed, lack pigmentation. “Even in the case of faba beans, there are seeds with different hilum pigmentation. In 2020, when we already partly knew how the PPO gene behaves in peas, we turned our joint attention to the search for the PPO gene in faba beans,” said Prof Petr Smýkal from the Department of Botany at the UP Faculty of Science, who heads the research team.
Scientists from the Departments of Botany and Analytical Chemistry, however, had their detective work cut out for them. The giant faba bean genome is largely (80%) composed of retrotransposon and transposon sequences and 34,000 protein-coding genes. While in peas only one gene is responsible for the production of the PPO enzyme, the faba bean has 10 copies of this gene side by side in its genome. “So our goal was to find the right gene, which we finally did,” said Smýkal. In the case of peas, the non-functionality of the protein involved in the production of the PPO enzyme is caused by a point mutation, whereas in faba beans the process preventing the production of PPO is more complex. “There is an insertion of a MITE retrotransposon in the promoter region, which leads to the switching off of the given PPO gene. This results in a pale beam hilum,” Smýkal described. Experts from the Department of Analytical Chemistry also participated in the research, specifically those from the group of Assoc Prof Petr Bednář. “They studied the differences in the distribution of metabolites in the seed hilum area and the surrounding tissue using mass spectrometry imaging,” added the botanist.
The non-functionality of a gene important for the production of the PPO enzyme has been repeatedly preferred by growers during the gradual domestication of agricultural crops, for example in rice and partly also in barley and foxtail millet. “This has been done despite the fact that this non-functionality may be associated with a certain disadvantage in plant defense against pathogens, since polyphenolic substances are closely related to seed protection,” Smýkal pointed out. Scientists are thus left to find a satisfactory answer to the question of why humans preferred to grow faba beans and other crops with a non-functional gene responsible for the production of the protective PPO enzyme.
One possible explanation, according to Smýkal, is that the formation of the PPO enzyme produces polymerized phenolic substances belonging to tannins, which are bitter and negatively affect the taste of the seeds. Ancient farmers avoided seeds with dark pigmentation, apparently because human culture at the time preferred light colours as a symbol of purity. “In the case of the faba bean, the pigmentation of the seed’s hilum was used in the past as a visual trait in the breeding of this crop. Growers knew that the dark colouring was related to the anti-nutritional content of the seed, which results in its bitter taste,” says Smýkal.
The faba bean has gradually almost disappeared from domestic fields and replaced by imported soybeans, which have lower yields. “Nevertheless, faba beans are a globally important crop and it is interesting that their wild ancestor is still unknown,” adds Smýkal. Although the browning of tissues and food is perceived by humans as a negative characteristic, phenolic substances can also have a beneficial impact on health. “Now that we know which gene is involved in the defense of seeds against negative influences, we have the possibility of targeted breeding of more resistant crops,” explained the scientist.
The faba bean represents a classic cytogenetic model with size-differentiated chromosomes. It was used, for example, in the first analyses of the plant genome using flow cytometry and subsequently also in the development of the chromosome sorting technique at the Olomouc workplace of Prof Jaroslav Doležel at the Institute of Experimental Botany of the Czech Academy of Sciences (CAS). Together with scientists from the Institute of Plant Molecular Biology of the CAS in České Budějovice, this group played a vital role in decoding and assembling the sequence of such a giant 13 Gb genome.
Olomouc scientists have previously discovered that the function of the PPO enzyme in the seeds of wild peas was significantly weakened during its domestication. Although the gradual breeding of peas increased their utility value for humans, it also had a negative impact on the seeds’ resistance to adverse external influences. “The first study on peas represents an initial milestone on the way towards a general understanding of the nature of PPO gene activity in connection with plant domestication. At the same time, it has opened up a number of questions that are a stimulus for further research,” added Smýkal.