The DEMEtRA project was organized in three Work Packages (WPs).
The first WP consisted in a deep biochemical characterization of the Arabidopsis thaliana protein NADKc1, a NAD kinase whose activity is totally dependent on calmodulin (CaM) and calcium (Ca2+). Previously, it was shown that this protein is involved in increasing NADP in response to pathogen elicitors and is pivotal for triggering the pathogen-induced oxidative burst.
To this end, we successfully built a phylogenetic analysis of NADKc-like proteins in the whole tree of life. This showed that NADKc-like proteins are present in all groups of plants, from basal bryophytes like Marchantia polymorpha, to angiosperms, like A. thaliana and Solanum lycopersicum. Similar proteins are present also in both major groups of green algae - Streptophyta and Chlorophyta – although in the latter the presence of the gene is more scattered. To extend these findings, we performed affinity assays with CaM, and enzymatic assays on NADKc proteins from various plant/algal groups, thus reconstructing the evolutionary origin of the CaM binding site and NAD kinase activity.
We then focused on a deeper characterization of NADKc mechanism of action, using site-directed mutagenesis, crystallography and in silico simulations with AlphaFold. Results so far suggest that CaM acts by causing a deep conformational rearrangement of the protein that would allow either the access of substrates or the catalysis itself.
The second WP aimed at investigating the role of NADKc in biotic and abiotic stress with the help of genetically encoded fluorescent indicators for calcium, ROS and NAD(P)/H.
To this end, we generated transgenic lines of A. thaliana wild-type, nadkc mutants as well as NADKc overexpressors expressing indicators for calcium, hydrogen peroxide (H2O2), the glutathione redox status, as well as the NADH/NAD and the NADPH/NADP redox couples (the latter with help from our collaborator M. SchwarzlanderSchwarzländer, University of Münster). All lines displayed good levels of indicators expression and were therefore suitable for studies.
We then built up a protocol for and in vivo time course of ROS production in response to avirulent bacterial strains. Leaf infection with an avirulent Pseudomonas syringae strain revealed an impairment in the local ROS response in nadkc1 mutants. However, differences with wild-type plants are mild, and are not linked to differences in bacterial growth. Since a gene expression analysis showed that NAKDc1 is mostly present in roots and young seedlings, we have decided to establish a protocol for root analysis and infection tests with root-infecting bacteria, thanks to the help of our colleagues and international collaborators working with pathogenic bacteria and fungi. Experiments with this new setup are ongoing.
Meanwhile, the discovery of two NADKc homologous genes specifically localized in the pollen raised the question of the importance of NAD metabolism for plant reproduction. We have generated a line with reduced expression levels of one of the two homologous proteins and abolished expression of the second and we are in the process of obtaining full knock out mutants with the CRISPR/Cas9 technology. Preliminary results highlight reduced seeds levels in the downregulated line in normal growth conditions and drastic reduction in seeds production at high temperatures.
WP3 was originally intended for the in vivo testing of improved NADKc variants (i.e. with higher CaM affinity or higher NAD turnover). However, since improved variants were not found in nature and no clues were found in order to build synthetic variants, we reoriented WP3 to a physiological analysis of NADKc variants from basal plants and algae. We overexpressed in nadkc A. thaliana mutants the NADKc variants from the basal plant M. polymorpha and the green alga B. plumosa (the latter with lower CaM affinity and deeper sequence divergence to angiosperms). These lines will be used to confirm whether the NADKc versions selected are able to revert the nadkc mutant phenotype in vivo.
A manuscript about the results found in WP1 and 3 is currently being assembled. A second manuscript about WP2 will be drafted at the end of physiological experiments. Further manuscripts are expected in the future about the role of NADP in plant reproduction.
