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.
