Nitrogen sensing and regulatory networks: it's about time and space.

A plant's response to external and internal nitrogen signals/status relies on sensing and signaling mechanisms that operate across spatial and temporal dimensions. From a comprehensive systems biology perspective, this involves integrating nitrogen responses in different cell types and over long distances to ensure organ coordination in real time and yield practical applications. In this prospective review, we focus on novel aspects of nitrogen (N) sensing/signaling uncovered using temporal and spatial systems biology approaches, largely in the model Arabidopsis. The temporal aspects span: transcriptional responses to N-dose mediated by Michaelis-Menten kinetics, the role of the master NLP7 transcription factor as a nitrate sensor, its nitrate-dependent TF nuclear retention, its "hit-and-run" mode of target gene regulation, and temporal transcriptional cascade identified by "network walking." Spatial aspects of N-sensing/signaling have been uncovered in cell type-specific studies in roots and in root-to-shoot communication. We explore new approaches using single-cell sequencing data, trajectory inference, and pseudotime analysis as well as machine learning and artificial intelligence approaches. Finally, unveiling the mechanisms underlying the spatial dynamics of nitrogen sensing/signaling networks across species from model to crop could pave the way for translational studies to improve nitrogen-use efficiency in crops. Such outcomes could potentially reduce the detrimental effects of excessive fertilizer usage on groundwater pollution and greenhouse gas emissions.

Role of BASIC PENTACYSTEINE transcription factors in a subset of cytokinin signaling responses.

Cytokinin plays diverse roles in plant growth and development, generally acting by modulating gene transcription in target tissues. The type-B Arabidopsis response regulators (ARR) transcription factors have emerged as primary targets of cytokinin signaling and are required for essentially all cytokinin-mediated changes in gene expression. The diversity of cytokinin function is likely imparted by the activity of various transcription factors working with the type-B ARRs to alter specific sets of target genes. One potential set of co-regulators modulating the cytokinin response are the BARLEY B-RECOMBINANT/BASIC PENTACYSTEINE (BBR/BPC) family of plant-specific transcription factors. Here, we show that disruption of multiple BPCs results in reduced sensitivity to cytokinin. Further, the BPCs are necessary for the induction of a subset of genes in response to cytokinin. We identified direct in vivo targets of BPC6 using ChIP-Seq and found an enrichment of promoters of genes differentially expressed in response to cytokinin. Further, a significant number of BPC6 regulated genes are also direct targets of the type-B ARRs. Potential cis-binding elements for a number of other transcription factors linked to cytokinin action are enriched in the BPC binding fragments, including those for the cytokinin response factors (CRFs). In addition, several BPCs interact with a subset of type-A ARRs. Consistent with these results, a significant number of genes whose expression is altered in bpc mutant roots are also mis-expressed in crf1,3,5,6 and type-A arr3,4,5,6,7,8,9,15 mutant roots. These results suggest that the BPCs are part of a complex network of transcription factors that are involved in the response to cytokinin.

The Role of Cytokinin During Infection of Arabidopsis thaliana by the Cyst Nematode Heterodera schachtii.

Plant-parasitic cyst nematodes induce the formation of hypermetabolic feeding sites, termed syncytia, as their sole source of nutrients. The formation of the syncytium is orchestrated by the nematode, in part, by modulation of phytohormone responses, including cytokinin. In response to infection by the nematode Heterodera schachtii, cytokinin signaling is transiently induced at the site of infection and in the developing syncytium. Arabidopsis lines with reduced cytokinin sensitivity show reduced susceptibility to nematode infection, indicating that cytokinin signaling is required for optimal nematode development. Furthermore, lines with increased cytokinin sensitivity also exhibit reduced nematode susceptibility. To ascertain why cytokinin hypersensitivity reduces nematode parasitism, we examined the transcriptomes in wild type and a cytokinin-hypersensitive type-A arr Arabidopsis mutant in response to H. schachtii infection. Genes involved in the response to biotic stress and defense response were elevated in the type-A arr mutant in the absence of nematodes and were hyperinduced following H. schachtii infection, which suggests that the Arabidopsis type-A arr mutants impede nematode development because they are primed to respond to pathogen infection. These results suggest that cytokinin signaling is required for optimal H. schachtii parasitism of Arabidopsis but that elevated cytokinin signaling triggers a heightened immune response to nematode infection.

The Plasminogen-Apple-Nematode (PAN) domain suppresses JA/ET defense pathways in plants.

Suppression of immune response is a phenomenon that enables biological processes such as gamete fertilization, cell growth, cell proliferation, endophyte recruitment, parasitism, and pathogenesis. Here, we show for the first time that the Plasminogen-Apple-Nematode (PAN) domain present in G-type lectin receptor-like kinases is essential for immunosuppression in plants. Defense pathways involving jasmonic acid and ethylene are critical for plant immunity against microbes, necrotrophic pathogens, parasites, and insects. Using two Salix purpurea G-type lectin receptor kinases, we demonstrated that intact PAN domains suppress jasmonic acid and ethylene signaling in Arabidopsis and tobacco. Variants of the same receptors with mutated residues in this domain could trigger induction of both defense pathways. Assessment of signaling processes revealed significant differences between receptors with intact and mutated PAN domain in MAPK phosphorylation, global transcriptional reprogramming, induction of downstream signaling components, hormone biosynthesis and resistance to Botrytis cinerea . Further, we demonstrated that the domain is required for oligomerization, ubiquitination, and proteolytic degradation of these receptors. These processes were completely disrupted when conserved residues in the domain were mutated. Additionally, we have tested the hypothesis in recently characterized Arabidopsis mutant which has predicted PAN domain and negatively regulates plant immunity against root nematodes. ern1.1 mutant complemented with mutated PAN shows triggered immune response with elevated WRKY33 expression, hyperphosphorylation of MAPK and resistant to necrotrophic fungus Botrytis cinerea . Collectively, our results suggest that ubiquitination and proteolytic degradation mediated by the PAN domain plays a role in receptor turn-over to suppress jasmonic acid and ethylene defense signaling in plants.

Core cysteine residues in the Plasminogen-Apple-Nematode (PAN) domain are critical for HGF/c-MET signaling.

The Plasminogen-Apple-Nematode (PAN) domain, with a core of four to six cysteine residues, is found in > 28,000 proteins across 959 genera. Still, its role in protein function is not fully understood. The PAN domain was initially characterized in numerous proteins, including HGF. Dysregulation of HGF-mediated signaling results in multiple deadly cancers. The binding of HGF to its cell surface receptor, c-MET, triggers all biological impacts. Here, we show that mutating four core cysteine residues in the HGF PAN domain reduces c-MET interaction, subsequent c-MET autophosphorylation, and phosphorylation of its downstream targets, perinuclear localization, cellular internalization of HGF, and its receptor, c-MET, and c-MET ubiquitination. Furthermore, transcriptional activation of HGF/c-MET signaling-related genes involved in cancer progression, invasion, metastasis, and cell survival were impaired. Thus, targeting the PAN domain of HGF may represent a mechanism for selectively regulating the binding and activation of the c-MET pathway.

Validation of a high-confidence regulatory network for gene-to-NUE phenotype in field-grown rice.

Nitrogen (N) and Water (W) - two resources critical for crop productivity - are becoming increasingly limited in soils globally. To address this issue, we aim to uncover the gene regulatory networks (GRNs) that regulate nitrogen use efficiency (NUE) - as a function of water availability - in Oryza sativa, a staple for 3.5 billion people. In this study, we infer and validate GRNs that correlate with rice NUE phenotypes affected by N-by-W availability in the field. We did this by exploiting RNA-seq and crop phenotype data from 19 rice varieties grown in a 2x2 N-by-W matrix in the field. First, to identify gene-to-NUE field phenotypes, we analyzed these datasets using weighted gene co-expression network analysis (WGCNA). This identified two network modules ("skyblue" & "grey60") highly correlated with NUE grain yield (NUEg). Next, we focused on 90 TFs contained in these two NUEg modules and predicted their genome-wide targets using the N-and/or-W response datasets using a random forest network inference approach (GENIE3). Next, to validate the GENIE3 TF→target gene predictions, we performed Precision/Recall Analysis (AUPR) using nine datasets for three TFs validated in planta. This analysis sets a precision threshold of 0.31, used to "prune" the GENIE3 network for high-confidence TF→target gene edges, comprising 88 TFs and 5,716 N-and/or-W response genes. Next, we ranked these 88 TFs based on their significant influence on NUEg target genes responsive to N and/or W signaling. This resulted in a list of 18 prioritized TFs that regulate 551 NUEg target genes responsive to N and/or W signals. We validated the direct regulated targets of two of these candidate NUEg TFs in a plant cell-based TF assay called TARGET, for which we also had in planta data for comparison. Gene ontology analysis revealed that 6/18 NUEg TFs - OsbZIP23 (LOC_Os02g52780), Oshox22 (LOC_Os04g45810), LOB39 (LOC_Os03g41330), Oshox13 (LOC_Os03g08960), LOC_Os11g38870, and LOC_Os06g14670 - regulate genes annotated for N and/or W signaling. Our results show that OsbZIP23 and Oshox22, known regulators of drought tolerance, also coordinate W-responses with NUEg. This validated network can aid in developing/breeding rice with improved yield on marginal, low N-input, drought-prone soils.