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Nitrogen (N)-based fertilizers increase agricultural productivity but have detrimental effects on the environment and human health. Research is generating improved understanding of the signaling components plants use to sense N and regulate metabolism, physiology, and growth and development. However, we still need to integrate these regulatory factors into signal transduction pathways and connect them to downstream response pathways. Systems biology approaches facilitate identification of new components and N-regulatory networks linked to other plant processes. A holistic view of plant N nutrition should open avenues to translate this knowledge into effective strategies to improve N-use efficiency and enhance crop production systems for more sustainable agricultural practices.

• High-efficiency methods for DNA assembly have enabled the routine assembly of synthetic DNAs of increased size and complexity. However, these techniques require customization, elaborate vector sets or serial manipulations for the different stages of assembly. • We have developed Loop assembly based on a recursive approach to DNA fabrication. The system makes use of two Type IIS restriction endonucleases and corresponding vector sets for efficient and parallel assembly of large DNA circuits. Standardized level 0 parts can be assembled into circuits containing 1, 4, 16 or more genes by looping between the two vector sets. • The vectors also contain modular sites for hybrid assembly using sequence overlap methods. - Loop assembly enables efficient and versatile DNA fabrication for plant transformation. We show the construction of plasmids up to 16 genes and 38 kb with high efficiency (> 80%). We have characterized Loop assembly on over 200 different DNA constructs and validated the fidelity of the method by high-throughput Illumina plasmid sequencing. • Our method provides a simple generalized solution for DNA construction with standardized parts. The cloning system is provided under an OpenMTA license for unrestricted sharing and open access.

One of the most striking examples of plant developmental plasticity to changing environmental conditions is the modulation of root system architecture (RSA) in response to nitrate supply. Despite the fundamental and applied significance of understanding this process, the molecular mechanisms behind nitrate-regulated changes in developmental programs are still largely unknown. Small RNAs (sRNAs) have emerged as master regulators of gene expression in plants and other organisms. To evaluate the role of sRNAs in the nitrate response, we sequenced sRNAs from control and nitratetreated Arabidopsis seedlings using the 454 sequencing technology. miR393 was induced by nitrate in these experiments. miR393 targets transcripts that code for a basic helix-loop-helix (bHLH) transcription factor and for the auxin receptors TIR1, AFB1, AFB2, and AFB3. However, only AFB3 was regulated by nitrate in roots under our experimental conditions. Analysis of the expression of this miR393/AFB3 module, revealed an incoherent feed-forward mechanism that is induced by nitrate and repressed by N metabolites generated by nitrate reduction and assimilation. To understand the functional role of this N-regulatory module for plant development, we analyzed the RSA response to nitrate in AFB3 insertional mutant plants and in miR393 overexpressors. RSA analysis in these plants revealed that both primary and lateral root growth responses to nitrate were altered. Interestingly, regulation of RSA by nitrate was specifically mediated by AFB3, indicating that miR393/AFB3 is a unique N-responsive module that controls root system architecture in response to external and internal N availability in Arabidopsis.

Auxin is a key phytohormone regulating central processes in plants. Although the mechanism by which auxin triggers changes in gene expression is well understood, little is known about the specific role of the individual members of the TIR1/AFB auxin receptors, Aux/IAA repressors, and ARF transcription factors and/or molecular pathways acting downstream leading to plant responses to the environment. We previously reported a role for AFB3 in coordinating primary and lateral root growth to nitrate availability. In this work, we used an integrated genomics, bioinformatics, and molecular genetics approach to dissect regulatory networks acting downstream of AFB3 that are activated by nitrate in roots. We found that the NAC4 transcription factor is a key regulatory element controlling a nitrate-responsive network, and that nac4 mutants have altered lateral root growth but normal primary root growth in response to nitrate. This finding suggests that AFB3 is able to activate two independent pathways to control root system architecture. Our systems approach has unraveled key components of the AFB3 regulatory network leading to changes in lateral root growth in response to nitrate.

An increase in nutrient dose leads to proportional increases in crop biomass and agricultural yield. However, the molecular underpinnings of this nutrient dose–response are largely unknown. To investigate, we assayed changes in the Arabidopsis root transcriptome to different doses of nitrogen (N)—a key plant nutrient—as a function of time. By these means, we found that rate changes of genome-wide transcript levels in response to N-dose could be explained by a simple kinetic principle: the Michaelis–Menten (MM) model. Fitting the MM model allowed us to estimate the maximum rate of transcript change (V max), as well as the N-dose at which one-half of V max was achieved (K m) for 1,153 N-dose–responsive genes. Since transcription factors (TFs) can act in part as the catalytic agents that determine the rates of transcript change, we investigated their role in regulating N-dose–responsive MM-modeled genes. We found that altering the abundance of TGA1, an early N-responsive TF, perturbed the maximum rates of N-dose transcriptomic responses (V max), K m, as well as the rate of N-dose–responsive plant growth. We experimentally validated that MM-modeled N-dose–responsive genes included both direct and indirect TGA1 targets, using a root cell TF assay to detect TF binding and/or TF regulation genome-wide. Taken together, our results support a molecular mechanism of transcriptional control that allows an increase in N-dose to lead to a proportional change in the rate of genome-wide expression and plant growth.

Charting a temporal path in gene networks requires linking early transcription factor (TF)-triggered events to downstream effects. We scale-up a cell-based TF-perturbation assay to identify direct regulated targets of 33 nitrogen (N)-early response TFs encompassing 88% of N-responsive Arabidopsis genes. We uncover a duality where each TF is an inducer and repressor, and in vitro cis-motifs are typically specific to regulation directionality. Validated TF-targets (71,836) are used to refine precision of a time-inferred root network, connecting 145 N-responsive TFs and 311 targets. These data are used to chart network paths from direct TF 1 -regulated targets identified in cells to indirect targets responding only in planta via Network Walking. We uncover network paths from TGA1 and CRF4 to direct TF 2 targets, which in turn regulate 76% and 87% of TF 1 indirect targets in planta , respectively. These results have implications for N-use and the approach can reveal temporal networks for any biological system. Temporal control of transcriptional networks enables organisms to adapt to changing environment. Here, the authors use a scaled-up cell-based assay to identify direct targets of nitrogen-early responsive transcription factors and validate a network path mediating dynamic nitrogen signaling in Arabidopsis .

The technique RT-qPCR for viral RNA detection is the current worldwide strategy used for early detection of the novel coronavirus SARS-CoV-2. RNA extraction is a key pre-analytical step in RT-qPCR, often achieved using commercial kits. However, the magnitude of the COVID-19 pandemic is causing disruptions to the global supply chains used by many diagnostic laboratories to procure the commercial kits required for RNA extraction. Shortage in these essential reagents is even more acute in developing countries with no means to produce kits locally. We sought to find an alternative procedure to replace commercial kits using common reagents found in molecular biology laboratories. Here we report a method for RNA extraction that takes about 40 min to complete ten samples, and is not more laborious than current commercial RNA extraction kits. We demonstrate that this method can be used to process nasopharyngeal swab samples and yields RT-qPCR results comparable to those obtained with commercial kits. Most importantly, this procedure can be easily implemented in any molecular diagnostic laboratory. Frequent testing is crucial for individual patient management as well as for public health decision making in this pandemic. Implementation of this method could maintain crucial testing going despite commercial kit shortages.

Understanding how plants sense and respond to changes in nitrogen availability is the first step toward developing strategies for biotechnological applications, such as improvement of nitrogen use efficiency. However, components involved in nitrogen signaling pathways remain poorly characterized. Calcium is a second messenger in signal transduction pathways in plants, and it has been indirectly implicated in nitrate responses. Using aequorin reporter plants, we show that nitrate treatments transiently increase cytoplasmic Ca²⁺ concentration. We found that nitrate also induces cytoplasmic concentration of inositol 1,4,5-trisphosphate. Increases in inositol 1,4,5-trisphosphate and cytoplasmic Ca²⁺ levels in response to nitrate treatments were blocked by U73122, a pharmacological inhibitor of phospholipase C, but not by the nonfunctional phospholipase C inhibitor analog U73343. In addition, increase in cytoplasmic Ca²⁺ levels in response to nitrate treatments was abolished in mutants of the nitrate transceptor NITRATE TRANSPORTER1.1/Arabidopsis (Arabidopsis thaliana) NITRATE TRANSPORTER1 PEPTIDE TRANSPORTER FAMILY6.3. Gene expression of nitrate-responsive genes was severely affected by pretreatments with Ca²⁺ channel blockers or phospholipase C inhibitors. These results indicate that Ca²⁺ acts as a second messenger in the nitrate signaling pathway of Arabidopsis. Our results suggest a model where NRT1.1/AtNPF6.3 and a phospholipase C activity mediate the increase of Ca²⁺ in response to nitrate required for changes in expression of prototypical nitrate-responsive genes.

Development of crops with improved nitrogen use efficiency (NUE) is essential for sustainable agriculture. However, achieving this goal has proven difficult since NUE is a complex trait encompassing physiological and developmental processes. We thought to tackle this problem by taking a systems biology approach to identify candidate target genes. First, we used a supervised machine-learning algorithm to predict a NUE gene network in Arabidopsis (Arabidopsis thaliana). Second, we identified BT2, a member of the Bric-a-Brac/Tramtrack/Broad gene family, as the most central and connected gene in the NUE network. Third, we experimentally tested BT2 for a role in NUE. We found NUE decreased in plants overexpressing BT2 gene compared to wild-type plants under limiting nitrate conditions. In addition, NUE increased compared to wild-type plants under low nitrate conditions in double mutant plants in bt2 and its closely related homolog bt1, indicating a functional redundancy of BT1 and BT2 for NUE. Expression of the nitrate transporter genes NRT2.1 and NRT2.4 increased in the bt1/bt2 double mutant compared to wild-type plants, with a concomitant 65% increase in nitrate uptake under low nitrate conditions. Similar to Arabidopsis, we found that mutation of the BT1/BT2 ortholog gene in rice (Oryza sativa) OsBT increased NUE by 20% compared to wild-type rice plants under low nitrogen conditions. These results indicate BT gene family members act as conserved negative regulators of nitrate uptake genes and NUE in plants and highlight them as prime targets for future strategies to improve NUE in crops.

Understanding the factors that modulate bacterial community assembly in natural soils is a longstanding challenge in microbial community ecology. In this work, we compared two microbial co-occurrence networks representing bacterial soil communities from two different sections of a pH, temperature and humidity gradient occurring along a western slope of the Andes in the Atacama Desert. In doing so, a topological graph alignment of co-occurrence networks was used to determine the impact of a shift in environmental variables on OTUs taxonomic composition and their relationships. We observed that a fraction of association patterns identified in the co-occurrence networks are persistent despite large environmental variation. This apparent resilience seems to be due to: (1) a proportion of OTUs that persist across the gradient and maintain similar association patterns within the community and (2) bacterial community ecological rearrangements, where an important fraction of the OTUs come to fill the ecological roles of other OTUs in the other network. Actually, potential functional features suggest a fundamental role of persistent OTUs along the soil gradient involving nitrogen fixation. Our results allow identifying factors that induce changes in microbial assemblage configuration, altering specific bacterial soil functions and interactions within the microbial communities in natural environments.