Nitrogen is one of the most abundant elements on Earth, composing 78% of the atmosphere. Yet, despite its prevalence, its availability in soil is remarkably limited. Surface mineral soils contain only about 0.05% to 0.2% N, and of this, less than 5% exists in forms readily accessible to plants, primarily nitrate (NO₃⁻) and ammonium (NH₄⁺). The remainder is bound in organic compounds, necessitating gradual release through mineralization.

Nitrogen is a critical nutrient for plant growth, directly impacting structural development, metabolic processes, and crop productivity. Fluctuations in soil nitrogen levels, due to factors like climate, microbial activity, and soil composition, require plants to adapt continuously to secure an adequate supply.

Nitrogen deficiency is a major agricultural challenge, often leading to heavy fertilizer reliance. In response to nitrogen-limiting conditions, plants activate molecular networks that regulate nitrogen transport, assimilation, carbon metabolism, and hormone signaling. Adaptive responses, such as enhanced lateral root elongation, help optimize nitrogen uptake, but prolonged deficiency stunts above-ground growth and reduces crop yields.

A recent study by Bian and colleagues provides key insights into the transcriptional regulation of plant responses to nitrogen availability. The research focuses on NIN-LIKE PROTEIN 7 (NLP7) and its paralog, NLP6; critical transcription factors (TFs) that govern nitrate-induced gene expression in plants. Utilizing systems biology tools such as gene editing and protoplast-based assays, the authors identified specific TF binding motifs and validated in vivo interactions. Moreover, the study extends beyond Arabidopsis to Solanum lycopersicum (tomato), uncovering both conserved and species-specific features of this nitrogen responsive regulatory circuit.

RESULTS

Gene Circuit and Cis-Regulatory Elements

In Arabidopsis thaliana, AtNLP7 and AtNLP6 play a pivotal role in orchestrating transcriptional responses to nitrate fluctuations. Despite their central function, the regulatory mechanisms governing their own expression remain insufficiently understood. These TFs, along with other regulatory proteins, form complex transcriptional networks that maintain nitrogen homeostasis. These networks, which include autoregulatory (Figure 1a) and feedforward loops (Figure 1b), ensure robust gene expression and sustained activation of target genes (Figure 1c).

This preprint explores the upstream transcriptional regulation of NLP6/7 in Arabidopsis thaliana. More, specifically, the study explored a five-gene regulatory circuit involved in nitrogen metabolism. The authors expanded on previously identified cis-regulatory elements and TF binding sites that define gene interactions within this circuit. This led to the identification of a sub-network of 29 interactions, reduced to 8 key interactions with 15 cis- elements using the modified TARGET assay.

Interactions including between AtNLP6/ AtNLP7 and AtNIR1, AtNLP7 with itself, AtARF18 to AtANAC032 and AtDREB26; AtANAC032 to AtNLP7; and AtDREB26 to AtNIR1 were validated. New potential interactions, such as between AtNLP6 and AtNLP7 to AtARF18 and AtDREB26; AtANAC032 to AtDREB26; AtDREB26, AtARF18 and AtANAC032 to AtNIR1; and AtDREB26 and AtANAC032 to AtNLP6 were also identified. Potential autoregulation was identified for AtANAC032, AtARF18, AtDREB26 and AtNLP6.

These interactions were also validated in vivo which revealed that AtARF18 directly represses AtANAC032, which in turn represses AtNLP7, while AtNLP7 activates AtNIR1. AtDREB26, was shown to be activated by AtNLP7 and AtNLP6 and repressed by AtARF18. The resulting regulatory network demonstrated a coherent feedforward loop where AtANAC032 regulates AtNIR1 through AtNLP7, further refining our understanding of nitrogen regulatory networks in Arabidopsis (Figure 2).

Evaluating the conservation of regulatory network in Arabidopsis and tomato

The study examined the conservation of the nitrate response regulatory network between Arabidopsis and tomato, using Arabidopsis’ established network as a base. Transcriptional responses to nitrogen were assessed by creating mutant alleles in both Arabidopsis and tomato, targeting genes in the nitrogen regulatory circuit. Root system architecture analysis highlighted both conserved and divergent features in nitrate regulation across the two species. The authors further explore the upstream regulatory regions of the nitrate response network of tomato genes to identify candidate binding sites for ARF18/9, DREB26, and NLP6/7 TFs. Significant conservation of DNA-binding domains was observed between Arabidopsis and tomato. Notably, interactions between AtARF18 and SlNLP7A/B, as well as AtARF18 and SlDREB26, were conserved, but some interactions, like AtARF18 being able to bind to the promoters of SlNLP7A/B, were not observed with AtARF18 and AtNLP6/AtNLP7, highlighting species-specific differences in the regulatory network. While there is conservation in repression of DREB26 and activation of NIR1, there exists major differences including the repression of SlNLP7B by SlDREB26 in tomato, the lack of NAC032 interactions in tomato, and the absence of feedforward loops in tomato.

Why I like this article

Nitrogen is indispensable for plant growth and agricultural productivity, yet its excessive use in fertilizers has led to significant environmental concerns. This study provides crucial insights into the regulatory mechanisms governing nitrogen utilization in plants, offering potential pathways for improving nitrogen use efficiency. While synthetic fertilizers have revolutionized agriculture by enhancing crop yields, their overuse contributes to soil degradation, water contamination, and greenhouse gas emissions. A deeper understanding of how plants respond to nitrogen deficiency is essential for breeding crops that require less fertilizer while maintaining high productivity.

A key strength of this study lies in its investigation of the upstream transcriptional regulation of NLP6 and NLP7, central players in nitrate signalling. Previous studies using Yeast-1-Hybrid techniques identified potential TFs involved in regulating nitrogen metabolism but lacked the ability to determine their interactions or the specific cis-regulatory motifs they target. To address this, the authors employed advanced systems biology tools, including gene editing and protoplast-based assays, to pinpoint TF binding motifs and validate their interactions in vivo.

Moreover, by extending their analysis beyond Arabidopsis to Solanum lycopersicum (tomato), the study highlights both conserved and species-specific regulatory interactions in nitrogen metabolism. This comparative approach not only enhances our understanding of nitrogen- responsive networks but also lays the foundation for translational research in crop species. Identifying precise regulatory sequences and binding sites within promoter regions opens new avenues for engineering crops with optimized nitrate metabolism and improved nitrogen uptake efficiency.

Tags: gene network, nitrogen assimilation, sustainability

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