Nodding on and off: transcription factor cis-elements that regulate nitrate-dependent gene expression for root nodule symbiosis

Nitrogen is a crucial plant macronutrient, and yet often is in short supply in soils in a form, such as nitrate or ammonium, that plants can assimilate. Plants have developed a variety of mechanisms for enhancing nitrogen acquisition. One of the most fascinating and effective solutions is found in legumes, which develop a symbiotic relationship with nitrogen-fixing bacteria, housed inside specialized organs called root nodules, that allows the plants to grow in nitrogen-deficient soils. However, nodule development in nitrogen-sufficient soils can be harmful for plant growth, as carbon is needlessly diverted to nodule growth. Therefore, legumes need to maintain a balance between obtaining nitrogen and limiting the loss of energy during root nodule symbiosis. However, the molecular mechanisms that determine how legumes regulate nodulation in varying nitrate concentrations remain unclear. In this issue, Nishida et al. (2021) show that the cis-elements bound by two transcription factors (TFs) regulate nitrate-dependent gene expression to turn on and off nodulation in Lotus japonicus roots.

NODULE INCEPTION (NIN)-LIKE PROTEIN (NLP) TFs serve as important regulators of nitrate-inducible gene expression in plants (Konishi and Yanagisawa, 2013). A previous study used a forward genetic screen to identify an NLP in L. japonicus, LjNLP4, as a negative regulator of nodulation in the presence of nitrate (Nishida et al., 2018). In this study, the authors identified another mutant that continued to form nodules in the presence of high nitrate. Genome sequencing mapped the mutation to LjNLP1, an NLP TF related to LjNLP4. Real-time RT-PCR and transcriptome (RNA-seq) analyses demonstrated the genetic requirement of LjNLP4 and LjNLP1 for nitrate-inducible gene expression. NITRITE REDUCTASE 1 (LjNIR1) and CLAVATA3/ESR-related (CLE)-ROOT SIGNAL 2 (CLE-RS2) had reduced expression after nitrate treatment in Ljnlp4 and Ljnlp1 loss-of-function mutant plants compared with wild-type. Notably, the expression of these two nitrate-inducible genes was more greatly attenuated in the Ljnlp4/Ljnlp1 double mutant than in either single mutant alone. This suggests that LjNLP4 and LjNLP1 have an overlapping function in mediating nitrate-induced gene expression in roots.

To understand how LjNLP4 transcriptionally regulates genes involved in nitrate control of nodulation, the authors combined their RNA-seq data with DNA affinity purification (DAP)-seq to identify LjNLP4 DNA-binding sites in the genome. The analysis identified two conserved DNA sequence motifs that contained semi-palindromic structures. Interestingly, analysis of previously published chromatin immunoprecipitation (ChIP)-seq data revealed that LjNIN, an essential TF that targets genes with positive roles in nodulation, can also bind to these LjNLP4 target cis-elements. Further combinational analyses of the authors’ RNA-seq and the published ChIP-seq data of LjNIN revealed a key finding; LjNLP4 did not bind to the cis-elements of some LjNIN-target genes whose expression was upregulated by rhizobia, but downregulated by nitrate, such as LjNF-YB. Using an electrophoretic mobility shift assay (EMSA), the authors examined the similarities and differences of the cis-elements bound by LjNLP4 and LjNIN. Both LjNLP4 and LjNIN bound to cis-elements in the LjNIR1 and CLE-RS2 promoters, whereas only LjNIN bound to the cis-elements in the LjNF-YB promoter. They found that one of the two motifs in the promoter of LjNF-YB has a less perfect palindromic structure, preventing LjNLP4 to bind to the promoter of LjNF-YB and transcriptionally activate it.

Given that TFs form dimers when they bind to cis-elements with palindromic structures, the authors hypothesized that LjNLP4 and LjNIN might dimerize. Size exclusion chromatography coupled with multi-angle scattering analysis demonstrated that LjNLP4 and LjNIN act as homodimers to induce gene expression. The authors performed additional EMSAs to investigate how DNA-binding by LjNLP4 or LjNIN could be influenced by the presence of the other protein. These experiments revealed that LjNIN preferred to form heterodimers with LjNLP4 rather than homodimers with itself. Additionally, the LjNLP4 homodimer occupies the LjNIN-binding sites, reducing the ability of LjNIN to bind to its target genes. Given that LjNLP4 and LjNIN are negative and positive regulators of nitrate-induced effects on nodulation, respectively, the authors predicted that the LjNLP4–LjNIN heterodimer may play an important role in inducing and repressing symbiotic gene expression. Indeed, transactivation assays in L. japonicus protoplasts showed that LjNLP4 interfered with the induction of genes mediated by LjNIN and that the differences in cis-elements targeted by these two TFs affect nitrate-dependent gene expression (see Figure).

Figure.

Model for regulation of root nodule symbiosis gene expression in L. japonicus. A proposed model of how LjNLP4 (purple ellipses) and LjNIN (yellow rectangles) regulate gene expression for nodulation in a nitrate-dependent manner. In the absence of nitrate (upper panel), LjNIN homodimers bind to cis-elements of positive regulators for nodulation, outcompeting the negative regulators and promoting nodulation. In the presence of nitrate (lower panel), LjNIN forms heterodimers with LjNLP4. LjNLP4 homodimers and LjNLP4–LjNIN heterodimers can only bind to the most conserved motif (orange arrow), outcompeting the positive regulators and ultimately inhibiting nodulation. Adapted from Nishida et al. (2021, Figure 9).

This study advances our understanding in how legumes regulate nodulation. Through the use of TFs, plants can “nod on and off” depending on the concentration of nitrate in the environment.