Transceptor NRT1.1 and receptor-kinase QSK1 complex controls PM H+-ATPase activity under low nitrate.

NRT1.1, a nitrate transceptor, plays an important role in nitrate binding, sensing, and nitrate-dependent lateral root (LR) morphology. However, little is known about NRT1.1-mediated nitrate signaling transduction through plasma membrane (PM)-localized proteins. Through in-depth phosphoproteome profiling using membranes of Arabidopsis roots, we identified receptor kinase QSK1 and plasma membrane H+-ATPase AHA2 as potential downstream components of NRT1.1 signaling in a mild low-nitrate (LN)-dependent manner. QSK1, as a functional kinase and molecular link, physically interacts with NRT1.1 and AHA2 at LN and specifically phosphorylates AHA2 at S899. Importantly, we found that LN, not high nitrate (HN), induces formation of the NRT1.1-QSK1-AHA2 complex in order to repress the proton efflux into the apoplast by increased phosphorylation of AHA2 at S899. Loss of either NRT1.1 or QSK1 thus results in a higher T947/S899 phosphorylation ratio on AHA2, leading to enhanced pump activity and longer LRs under LN. Our results uncover a regulatory mechanism in which NRT1.1, under LN conditions, promotes coreceptor QSK1 phosphorylation and enhances the NRT1.1-QSK1 complex formation to transduce LN sensing to the PM H+-ATPase AHA2, controlling the phosphorylation ratio of activating and inhibitory phosphorylation sites on AHA2. This then results in altered proton pump activity, apoplast acidification, and regulation of NRT1.1-mediated LR growth.

A high-confidence Physcomitrium patens plasmodesmata proteome by iterative scoring and validation reveals diversification of cell wall proteins during evolution.

Plasmodesmata (PD) facilitate movement of molecules between plant cells. Regulation of this movement is still not understood. Plasmodesmata are hard to study, being deeply embedded within cell walls and incorporating several membrane types. Thus, structure and protein composition of PD remain enigmatic. Previous studies of PD protein composition identified protein lists with few validations, making functional conclusions difficult. We developed a PD scoring approach in iteration with large-scale systematic localization, defining a high-confidence PD proteome of Physcomitrium patens (HC300). HC300, together with bona fide PD proteins from literature, were placed in Pddb. About 65% of proteins in HC300 were not previously PD-localized. Callose-degrading glycolyl hydrolase family 17 (GHL17) is an abundant protein family with representatives across evolutionary scale. Among GHL17s, we exclusively found members of one phylogenetic clade with PD localization and orthologs occur only in species with developed PD. Phylogenetic comparison was expanded to xyloglucan endotransglucosylases/hydrolases and Exordium-like proteins, which also diversified into PD-localized and non-PD-localized members on distinct phylogenetic clades. Our high-confidence PD proteome HC300 provides insights into diversification of large protein families. Iterative and systematic large-scale localization across plant species strengthens the reliability of HC300 as basis for exploring structure, function, and evolution of this important organelle.

Comparison of path-based centrality measures in protein-protein interaction networks revealed proteins with phenotypic relevance during adaptation to changing nitrogen environments.

Plants must rapidly adapt to changes in nutrient conditions. Especially adaptations to changing nitrogen environments are very complex involving also major adjustments on the protein level. Here, we used a size-exclusion chromatography-coupled to mass spectrometry approach to study the dynamics of protein-protein interactions induced by transition from full nutrition to nitrogen starvation. Comparison of interaction networks established for each nutrient condition revealed a large overlap of proteins which were part of the protein-protein interaction network, but that same set of proteins underwent different interactions at each treatment. Network topology parameter betweenness centrality (BC) was found to best reflect the relevance of individual proteins in the information flow within each network. Changes in BC for individual proteins may therefore indicate their involvement in the cellular adjustments to the new condition. Based on this analysis, a set of proteins was identified showing high nitrogen-dependent changes in their BC values: The receptor kinase AT5G49770, co-receptor QSK1, and proton-ATPase AHA2. Mutants of those proteins showed a nitrate-dependent root growth phenotype. Individual interactions within the reconstructed network were tested using FRET-FLIM technology. Taken together, we present a systematic strategy comparing dynamic changes in protein-protein interaction networks based on their network parameters to identify regulatory nodes. SIGNIFICANCE: Protein-protein interactions are known to be important in cellular signaling events, but the dynamic changes in interaction networks induced by external stimuli are still rarely studied. We systematically analyzed how changes in the nutrient environment induced a rewiring of protein-protein interactions in roots. We observed small changes in overall protein abundances, but instead a rewiring of pairwise protein-protein interactions. Betweenness centrality was found to be the optimal network topology parameter to identify protein candidates with high relevance to the information flow in the (dynamic) network. Predicted interactions of those relevant nodes were confirmed in FLIM/FRET experiments and in phenotypic analysis. The network approach described here may be a useful application in dynamic network analysis more generally.