“Understanding the molecular basis of symbiotic nitrogen fixation is the key to more sustainable agriculture in the future”

Modern agriculture completely depends on synthetic nitrogen fertilizers, which are produced from non-renewable energy sources. These fertilizers currently support lives of ca. 7 billion people on Earth, but will be depleted within the next 150 years. Besides the non-sustainable nature of synthetic nitrogen fertilizers, they destroy soil and water ecosystems and contribute to the greenhouse effect (global warming). The natural alternative to synthetic nitrogen fertilizers is symbiotic nitrogen fixation, which exists in limited evolutionary branches of the plant kingdom, notably in the legume family. Legumes can fix atmospheric nitrogen in cooperation with bacteria called rhizobia, which live in so-called root nodules. Unfortunately, most plants that constitute the human diet have no ability to fix their own nitrogen. The project supported by the TÜBİTAK 1001 program started recently at Boğaziçi University for understanding this natural nitrogen fixation mechanism in the model legume Medicago truncatula. Dr. Igor Kryvoruchko from the Department of Molecular Biology and Genetics and his team will realize a systematic functional analysis of alternative proteins involved in symbiotic nitrogen fixation in the model legume. Understanding the molecular basis of this process and using that information in plant breeding programs will uncouple the agriculture from synthetic nitrogen fertilizers in the future. We conducted an interview with Dr. Kryvoruchko for the details of his project.
Kenan Özcan

The project titled Functional Analysis of Alternative Open Reading Frames (AltORFs) Involved in Symbiotic Nitrogen Fixation in the Model Legume Medicago truncatula will focus on alternative proteins (altProts) that represent the hidden dimension of genetic studies, because they are coded by the same RNA molecules as already known proteins, but in different reading frames, which are not taken into account by genome annotation projects. Some altProts have already been implicated in the control of symbiotic nitrogen fixation in the past, but no systematic large-scale analysis of their functions have been conducted so far. The improvement of nitrogen fixation in legumes and the development of non-legume crops that supply their own nitrogen require the full understanding of how altProts shape the symbiosis.

Could you please introduce yourself, your academic background and areas of interest? What were the reasons for choosing Boğaziçi University for your academic career?

Dr. Igor Kryvoruchko: I am from Kyiv, Ukraine. I graduated from Taras Shevchenko University in Kyiv. Then, I went to Italy to work in a plant molecular biology lab in Naples, which had great impact on my future career. Later I completed my MSc project in Germany with a fellowship from DAAD. My PhD study was conducted at Max Planck Institute for Molecular Plant Physiology in Potsdam-Golm, which is one of the top institutions in Germany. After that, I moved to the USA for four years and worked in the Samuel Roberts Noble Foundation, which is a private research center. The Noble Foundation is among the world’s leading institutions for legume biology and it made an enormous contribution to sequencing the genome of Medicago truncatula and to the development of indispensable resources for this important model legume.

I have been in Turkey for seven years and my preference for Boğaziçi University was driven by several factors. While I was working in the USA, I had many Turkish colleagues and one of them was the head of the department in Kafkas University in Kars. He invited me to establish an international research center in Kars to make an inventory of the natural diversity of legume species in Eastern Anatolia. When I joined Kafkas University, however, we could not realize this ambitious project because of bureaucratic reasons. Then, I have decided to apply to Boğaziçi University and joined the Department of Molecular Biology and Genetics in 2018.

“Based on the information gained from molecular analysis, we will be able to modify the plants that do not make nitrogen fixation”

Your research is mainly focused on fundamental and applied aspects of symbiotic nitrogen fixation. What are the effects of nitrogen on the productivity of crop plants and food production? What is symbiotic nitrogen fixation?

Synthetic nitrogen fertilizers can support very high yields, which are necessary to feed the growing world’s population. At the same time, they harm beneficial soil microorganisms and destroy water ecosystems. Organic fertilizers such as manure, on the other hand, are beneficial for the soil and water quality, but cannot support large-scale intensive agriculture. Symbiotic nitrogen fixation is the only sustainable alternative to the current agricultural practices, if we extend it to non-legume crops.

Even if we accept the high environmental costs of synthetic nitrogen fertilizers, they cannot be used infinitely because the huge amount of energy necessary for their production comes from non-renewable energy sources, namely coal, oil, natural gas, and uranium, all of which will be depleted within the next 40 to 150 years. It means that without an urgent solution, no intensive agricultural production will be possible in the future. In this respect, the finite nature of fossil fuels is not the only problem, because even if an infinite energy source is found, synthetic fertilizers will continue to destroy the natural ecosystems. Therefore, biologists have to develop a conceptually different strategy. The international consortium, a part of which is our lab, currently explores a possibility of modifying existing plants in such a way that they undergo symbiosis with nitrogen-fixing organisms to obtain their own nitrogen. This highly ambitious task is not too far from practical realization in economically-important non-legume plants like rice, because some genetic components necessary for the symbiosis are already present in most land plants. In addition to genes that are important for the symbiosis in all plants, there are also species-specific genes that have been recruited for the nitrogen fixation from other biological processes. Our lab is interested in both “shared” symbiotic genes and species-specific symbiotic genes. The nitrogen-fixing symbiosis is basically a trade in which plants use the currency of photosynthetic products (carbohydrates) to pay for biologically-available nitrogen supplied by bacteria.

Your recent project granted by TÜBİTAK will initiate a systematic functional analysis of novel altORFs involved in Symbiotic Nitrogen Fixation in the legume Medicago truncatula. How did this project begin and what are your research objectives?

My main training and career are in the area of plant functional genomics. In our routine work, we disrupt candidate genes and observe the effect of this genetic change on symbiotic nitrogen fixation and other biological processes. This way we establish links between genes and phenotypes and deduce functions of uncharacterized genes. The accuracy of establishing these functional links depends on whether the same mutation affects one protein or more than one protein. It is known that in human and other organisms at least 25% of genes overlap with each other in genomic DNA. But in addition to overlapping genes, there are also mini genes embedded in known genes, but in different reading frames. These are so-called alternative open reading frames (altORFs) and their corresponding translation products are called alternative proteins (altProts). To me as a biologist involved in functional genomics studies, it is very important to make sure that a single mutagenesis event affects only one protein. However, in recent times, it became gradually clear that alternative proteins that reside in known genes challenge the interpretation of mutagenesis based experiments. Namely, from now on, we and all other researchers have to study separately the effect of a given mutation on the main protein and on any altProt that is disabled by the same mutation. The existence of alternative proteomes revolutionized our knowledge about the true coding potential of genomes. It adds an extra dimension to the complexity of genetic studies. Within the last few years, the number of altProts with symbiosis-related functions have been described. In our project, we initiate the first comprehensive effort to detect altProts in the model legume M. truncatula and to reveal their functions in symbiotic nitrogen fixation.

Without the full inventory of translated altORFs, the progress in molecular genetics of symbiotic nitrogen fixation will be impossible, because observed mutant phenotypes will continue to be erroneously assigned to main proteins whenever the mutation coincides with an altORF. Alternative proteins can be highly conserved among different species. However, many of them are unique to species in which they are found. We think that altProts as very likely candidates for the species-specific genetic component of symbiotic nitrogen fixation I mentioned earlier. Together with genes re-specialized for the symbiosis from non-symbiotic functions, non-conserved altORFs should be the main target of engineering the symbiosis in non-legume plants, possibly with the minimal need for genetic transformation.

“We will create a database for alternative proteins with a potential function in symbiotic nitrogen fixation”

Why did you choose a legume as the plant model in your project? What are the characteristics of legumes regarding this?

Legume family is not the only evolutionary lineage that developed the ability to fix nitrogen in cooperation with bacteria. However, legumes are the largest and the most diverse of such lineages. Most legume crops, such as soybean, faba bean, garden pea etc., are too difficult to use for research directly because of the number of limiting factors, such as low amenability to genetic transformation, large genome size, large plant size, long generation time, outcrossing nature etc. For that reason, easy-to-study model legumes have been chosen to represent the whole legume family. Among the two well-established legume models, Lotus japonicus and M. truncatula, Medicago has more resources that can be useful for our work. Fundamental principles of nitrogen fixation discovered in Medicago will help us understand the mechanisms that control nitrogen fixation in crop legumes. This knowledge is important for our ability to improve nitrogen fixation in existing symbioses and to enable nitrogen fixation in non-legume plants. Needless to say that the global food security depends on the success of these research efforts.

Up to now, about 200 hundred genes are known to be involved in nitrogen fixation in legumes. Twenty-five of these genes are alternative ORFs. Our approach will create a candidate list of symbiosis-related altORFs in a form of a public database, which will later be extended to other legume species. Nitrogen-fixation research community can use our database to target various altORFs by mutagenesis. Once we finish the identification of altORFs in Medicago, we will conduct a similar study in chickpea. Chickpeas have large economic importance in Turkey since they are regional export products. Our study will contribute to establishing a bridge between a legume model and a legume crop, because we will compare the evolutionary conservation, transcription, and translation of altORFs between the two legume species.

What are altORFs and why is detecting altORFs in the model plants important? How will they be detected in this project?

Our strategy combines bioinformatic analysis with publicly deposited transcriptomic and proteomic data for Medicago. This analysis is currently run by my master student, Umut Çakır, who is a very talented computational biologist graduated recently from our department. In fact, Umut is my old student from the bioinformatics class I teach here. The first stage of our project is prediction of all possible altORFs in the whole transcriptome of Medicago. Then we find evidence for translation of altORFs using three types of data: presence of corresponding peptides in public mass-spectrometry based proteomic libraries, evolutionary conservation, and codon usage optimization signature. Because our final goal is to study altORFs involved in symbiotic nitrogen fixation, another domain of our project focuses on identification of Medicago genes that are upregulated during the symbiosis. We use public RNA-seq and microarray data to find such symbiosis-related genes. This work is close to completion. It has been run by another member of our team, Taha Bumin Aydos, who graduates from our department this year. Finally, we are interested to learn which altORFs exist only in legumes, because legume-specific genes are more likely to have functions in symbiotic nitrogen fixation. This analysis is based on the global sequence similarity search and has been completed recently by Serhat Beyaz, who is my former student of bioinformatics too.

Generation of candidate altORF list using our in-silico approach is supported by a scholarship to Umut from the budget of TÜBİTAK 1002 program (fast support). Using this candidate list, we will conduct mutagenesis-based wet-lab experiments on a subset of the most promising altORFs. This work will be supported by our recently approved TÜBİTAK 1001 program budget. A PhD student will be employed for this experimental domain. Our study will discriminate between the effects of disrupting alternative proteins and their main proteins. It will also show us whether both proteins are involved in the symbiosis. In the end, we will be able to separate the function of one protein from another protein, even though they derive from the same genomic space.

Because it is difficult to demonstrate individual functions of overlapping genes, most alternative proteins characterized so far are coded by 5’-untranslated regions of messenger RNA and transcripts previously annotated as non-coding, including the long non-coding RNA type and two special types of regulatory RNA: pre-micro RNA and pre-small interference RNA. In contrast, there is only one report on a plant altORF that overlaps with its main protein-coding gene. For that reason, our primary focus will be on this poorly documented, but extremely relevant type of altORFs. Another novel aspect of our work is inclusion of ribosomal RNA and transfer RNA in the list of potentially protein coding molecules. Our preliminary data indicate that both RNA types contain regions highly similar to protein coding genes. Thus any translated altORF found on these transcripts will be very interesting to study, even if they have non-symbiotic functions.

“Our remote goal is to improve nitrogen fixation in chickpea on zinc-deficient soils, which is very important for Turkey”

What will be the future directions according to the information received from this project?

Our altORF database will be very useful for the progress in research on symbiotic nitrogen fixation in legumes. Using our database, researchers will be able to see if their gene-of-interest to be targeted by mutagenesis contains an overlapping altORF. If yes, they will have to use a different methodology specific to the situation where two genes are affected by the same mutagenesis event instead of one. This will help avoid misinterpretation of mutant phenotypes, which is crucial to all areas of plant biology.

Besides our main focus, alORFs, our study has one fundamental goal: to address the possibility of reverse complement translation of known transcripts. According to the modern view on genome evolution, this process used to be essential for the function of RNA-based proto-genomes, because their single-stranded RNA “chromosomes” coded information in both directions. We hypothesize that this mechanism may exist also in modern DNA-based genomes. This subject is currently a black box in the modern literature, since no one has proved its existence.

For functional characterization of alternative protein, we will involve our old collaborators from the USA and Spain, to increase the throughput of our studies. Later we will extend this type of analysis to chickpeas. By switching to a crop legume, I would like to contribute to the improvement of chickpea cultivation on Zn-depleted soils, because symbiotic nitrogen fixation in this crop is highly sensitive to Zn deficiency. Most areas of chickpea cultivation in Turkey suffer from low availability of Zn in soils. We hope that our efforts will coin to the economic stability in this region.