The CRISPR Technique Helps Reveal How Bean Genes Could Improve Nitrogen Fixation

A UCO team has applied the genome editing technique, which garnered a Nobel Prize in Chemistry in 2020, to unravel the functions of two genes involved in the metabolism of beans and that could not be characterized using other methodologies

Beans are a key food at the dietary level, boasting high nutritional value and constituting the most directly consumed legume. They also have a "superpower" in the field: they are able to fix nitrogen to the soil, so they reduce the need for nitrogen fertilizers (and their associated pollution) for them and the crops with which they are planted. However, in-depth studies of bean plants are hampered by a problem: it is highly resistant to genetic transformation. Thus, when research teams want to understand the functions of their genes, or how they behave, they cannot generate mutants or modified plants in which they silence or express the genes they want to study.

This was the situation the team that the University of Cordoba's Molecular Physiology and Plant Biotechnology Group faced as it set out to discern the metabolism of purine nucleotides, such as adenine, in this crop. Adenine is one of the 5 nitrogenous bases always present in DNA and RNA. Although decisive in all organisms, these are even more vital in beans, as they play a crucial role in the assimilation of the nitrogen fixed in the nodules. Thus, the researchers Josefa Muñoz Alamillo and Cristina María López focused on the study of the enzyme dedicated to the recycling of adenine in the nitrogen fixation cycle (adenine phosphoribosyl transferase - APRT) and found that, although this enzyme has a single function (adenine recycling), there are 4 different copies of the gene that encodes it.

"So, our goal was to figure out what the specific functions of those four copies of the gene that synthesize that enzyme were," explained researcher Josefa Muñoz. However, to ascertain the different functions of four gene copies that were so similar, almost identical, it was necessary to run tests in plants in which the rest of the genes were silenced, except for the one whose function they wanted to explore, or vice versa, something that was very difficult due to the beans resistance to transformation. "So we decided to use the CRISPR/Cas9 tool with which we achieved the genetic editing of two genes related to the recycling of nucleotides in their roots," continued the researcher.

Thus, the development of a system based on the CRISPR/Cas9 genetic scissors, which garnered a Nobel Prize in Chemistry in 2020, made possible the creation of two functional mutants of two of the copies of the genes that encode this enzyme that recycles adenine, and to characterize their functions separately. "We discovered that one of them does affect the recycling of adenine, but the other plays an essential role in the regulation of cytokinins, one of the hormones responsible for the growth of the plant's roots and nodules," Muñoz said.

Thus, gene expression analysis and metabolomic analysis reveal that, although the two copies of genes have redundant functions, they also have specific differentiated ones, something that was unknown until now. And, due to their similarity, and beans' resistance to being transformed by other traditional methods, the use of CRISPR/Cas9 (hardly used in legumes) was essential.

The analyses also revealed that the location of the genes was different, and involved in the specialization of their functions; while one was expressed in the chloroplast (the organs of the cell responsible for photosynthesis) the other was expressed in the cytosol (the aqueous part in which the organelles and the nucleus are submerged).

These advances constitute the most in-depth study ever of the metabolism of adenine in beans, revealing a differentiation of functions between two of the genes that produce the enzyme key to the recycling of adenine and that affect the formation of nodules and the metabolism of nitrogen, using, also as a novelty, the CRISPR/Cas system. According to the researchers, a first step is to characterize the functions of the other two genes involved and that could, apparently, have other functions, such as the regulation of root growth, and drought resistance, and to continue applying this powerful tool.

Reference:

Cristina María López, Saleh Alseekh, Félix J Martínez Rivas, Alisdair R Fernie, Pilar Prieto, Josefa M Alamillo, CRISPR/Cas9 editing of two adenine phosphoribosyl transferase coding genes reveals the functional specialization of adenine salvage proteins in common bean, Journal of Experimental Botany, Volume 76, Issue 2, 10 January 2025, Pages 346–362, https://doi.org/10.1093/jxb/erae424