Periodic Reporting for period 1 - EvoNIN (Unraveling key genetic innovations behind the emergence of the root-nodule symbiosis)
Reporting period: 2017-09-01 to 2019-08-31
Massive use of human-made nitrogen fertiliser causes profound changes to the global nitrogen cycle, which are only comparable to the human impact on the global carbon cycle. Additionally, the primary raw material for nitrogen fertilizer is a non-renewable fossil fuel - natural gas. Over 5% of natural gas consumed in the US annualy is used solely for nitrogen fertilizer production. This industrial process also produces significant amounts of CO2 (about 3-5% of global carbon emissions), therefore having a vast impact on climate change. However, despite the negative environmental impact, mankind cannot stop using the fertiliser, because 48% of the world’s population can only be sustained through the application of the nitrogen fertilizer.
The aim of the proposed research was to contribute to the development of improved crops, which can use atmospheric nitrogen as a direct source of nitrogen and do not rely on fertilisers.
Atmospheric nitrogen (N2) is the largest reservoir of nitrogen on Earth, but it is inaccessible to most of the land plants, forcing farmers to use human-made fertiliser. However, a few plant species (including legumes) evolved an ability to form symbiosis with nitrogen-fixing bacteria and use atmospheric nitrogen as the primary source of nitrogen. The idea of transferring the ability to form symbiosis from legumes to other economically important plants, such as corn or wheat, has first appeared about 50 years ago, but remained to be an unfeasible goal due to a insufficient genetics/genomics technology development in the past.
In order to transfer this symbiosis to other important crops and enable them to fixate atmospheric nitrogen, we need to understand: how did this ability to form symbiosis evolve in the first place and what were the key genetic innovations behind it? The availability of multiple plant genomes sequenced recently (also in the host lab) allowed us to compare the genomic sequences of the plants capable of nitrogen-fixing symbiosis and those, which do not fixate nitrogen, and find the differences of the gene regulation in different species.
Conclusions:
Here we discovered – within the promoter of the Nodule Inception (NIN) gene – a cis-regulatory element (PACE), exclusively present in plants, capable of nitrogen-fixing symbiosis (also root-nodule symbiosis or RNS). Our data pinpoint the emergence of PACE as a key evolutionary invention which enabled bacterial uptake into infection threads, a unique and unifying feature of this symbiosis, and thus laid the foundation for the evolution of present day RNS.
The aim of the proposed research was to contribute to the development of improved crops, which can use atmospheric nitrogen as a direct source of nitrogen and do not rely on fertilisers.
Atmospheric nitrogen (N2) is the largest reservoir of nitrogen on Earth, but it is inaccessible to most of the land plants, forcing farmers to use human-made fertiliser. However, a few plant species (including legumes) evolved an ability to form symbiosis with nitrogen-fixing bacteria and use atmospheric nitrogen as the primary source of nitrogen. The idea of transferring the ability to form symbiosis from legumes to other economically important plants, such as corn or wheat, has first appeared about 50 years ago, but remained to be an unfeasible goal due to a insufficient genetics/genomics technology development in the past.
In order to transfer this symbiosis to other important crops and enable them to fixate atmospheric nitrogen, we need to understand: how did this ability to form symbiosis evolve in the first place and what were the key genetic innovations behind it? The availability of multiple plant genomes sequenced recently (also in the host lab) allowed us to compare the genomic sequences of the plants capable of nitrogen-fixing symbiosis and those, which do not fixate nitrogen, and find the differences of the gene regulation in different species.
Conclusions:
Here we discovered – within the promoter of the Nodule Inception (NIN) gene – a cis-regulatory element (PACE), exclusively present in plants, capable of nitrogen-fixing symbiosis (also root-nodule symbiosis or RNS). Our data pinpoint the emergence of PACE as a key evolutionary invention which enabled bacterial uptake into infection threads, a unique and unifying feature of this symbiosis, and thus laid the foundation for the evolution of present day RNS.
All plants that form nitrogen-fixing root-nodule symbiosis are monophyletic, meaning they have descended from one ancestor about 90 million years ago. The latter implies that the genetic innovations enabling symbiosis are shared by the nitrogen-fixers, but are absent from non-symbiotic plants. Previous knowledge obtained in our laboratory indicates that the emergence of the root-nodule symbiosis is most probably NOT associated with the emergence of the novel genes, specific for the nitrogen-fixing plants.Therefore in this project we tested the hypothesis that the gain of novel cis-regulatory elements could have been such a key event. Cis-regulatory elements are specific short DNA-sequences, located often in the promoter region, upstream of the ATG - the protein start codon. Such sequences are recognised and bound by the various regulatory proteins and therefore participate in the gene expression regulation. It has been shown that changes in gene regulation are important drivers of functional and morphological evolution. Emergence or loss of even a single cis-regulatory element can lead to dramatic phenotypic consequences, e.g. novel organ formation. Given the monophyletic origin of the symbiotic plants we have looked for the cis-elements which are specifically present and conserved in the symbiotic clade (and therefore, possibly inherited from the common ancestor) and are absent in non-symbiotic plants, belonging to other clades.
We have focused our attention on the plant NODULE INCEPTION (NIN) gene. Nodule is a special plant organ, within which the nitrogen-fixing bacteria are accommodated and the symbiotic nutrient exchange is taking place. Development of root nodules is a multistep program mediated by exchange of signal molecules between the nitrogen- fixing bacteria and the plant roots. On the molecular level bacterial signal molecules trigger the signalling cascade of the host plant, leading to activation of the NIN gene expression. NIN is a central transcriptional regulator; it orchestrates the root nodule development program and the symbiont uptake via activation of the expression of several downstream target genes. We have compared the promoter regions of 27 various NIN genes, originating from the nitrogen-fixing clade, with 10 outliers, coming from more distant species, which do not belong to the nitrogen-fixing clade and are incapable of symbiosis. After the comprehencive analysis we were able to identify only one novel cis-regulatory element - Predisposition-Associated Cis-regulatory Element (PACE) - exclusively present in the nitrogen-fixing clade. Moreover, we have observed that the evolutionary loss or mutation of PACE is associated with loss of this symbiosis, further supporting our hypothesis.
Interestingly, PACE encompasses binding site of the transcription factor Cyclops, long known to be a part of the transcriptional network, regulating symbiosis. We further tested whether the ability of the certain NIN promoter to be activated by Cyclops correlates with the ability of the corresponding plant to form symbiosis. Indeed, we found that the transactivation by Cyclops was restricted to NIN promoters from nitrogen-fixing species. Importantly, PACE was necessary and sufficient for the activation of the NIN promoter by Cyclops. Together these results are in line with the hypothesis that the regulatory connection link between Cyclops and the NIN promoter was established in the last common ancestor of this symbiotic clade and could have served as an initiating event for the establishment of the nitrogen-fixing symbiosis.
We have focused our attention on the plant NODULE INCEPTION (NIN) gene. Nodule is a special plant organ, within which the nitrogen-fixing bacteria are accommodated and the symbiotic nutrient exchange is taking place. Development of root nodules is a multistep program mediated by exchange of signal molecules between the nitrogen- fixing bacteria and the plant roots. On the molecular level bacterial signal molecules trigger the signalling cascade of the host plant, leading to activation of the NIN gene expression. NIN is a central transcriptional regulator; it orchestrates the root nodule development program and the symbiont uptake via activation of the expression of several downstream target genes. We have compared the promoter regions of 27 various NIN genes, originating from the nitrogen-fixing clade, with 10 outliers, coming from more distant species, which do not belong to the nitrogen-fixing clade and are incapable of symbiosis. After the comprehencive analysis we were able to identify only one novel cis-regulatory element - Predisposition-Associated Cis-regulatory Element (PACE) - exclusively present in the nitrogen-fixing clade. Moreover, we have observed that the evolutionary loss or mutation of PACE is associated with loss of this symbiosis, further supporting our hypothesis.
Interestingly, PACE encompasses binding site of the transcription factor Cyclops, long known to be a part of the transcriptional network, regulating symbiosis. We further tested whether the ability of the certain NIN promoter to be activated by Cyclops correlates with the ability of the corresponding plant to form symbiosis. Indeed, we found that the transactivation by Cyclops was restricted to NIN promoters from nitrogen-fixing species. Importantly, PACE was necessary and sufficient for the activation of the NIN promoter by Cyclops. Together these results are in line with the hypothesis that the regulatory connection link between Cyclops and the NIN promoter was established in the last common ancestor of this symbiotic clade and could have served as an initiating event for the establishment of the nitrogen-fixing symbiosis.
Our data pinpoint the acquisition of PACE as a key event during the evolution of nodulation. Together with our discovery that multiple independent losses of PACE are associated with multiple losses of root-nodule symbiosis within the symbiotic clade, our data underpin the essential position of PACE in the evolutionary gain and loss of root-nodule symbiosis. The next step will be to transfer the L. japonicus NIN gene under the regulation of PACE-containing promoter into the non-symbiotic plant and explore whether such modification provides certain phenotypic features, characteristic for the symbiotic plants. These experiments, however, lay outside the scope of the “EvoNIN” project. We assume that our findings lay the foundations for future development of the rationally designed sustainable crops, independent of human-made nitrogen fertiliser. Our research will, therefore, contribute to further implementation of sustainable agriculture in Europe and reducing the human impact on the environment, which are two of the top European priorities.