Spatiotemporal analysis identifies ABF2 and ABF3 as key hubs of endodermal response to nitrate.

Nitrate is a nutrient and a potent signal that impacts global gene expression in plants. However, the regulatory factors controlling temporal and cell type-specific nitrate responses remain largely unknown. We assayed nitrate-responsive transcriptome changes in five major root cell types of the Arabidopsis thaliana root as a function of time. We found that gene-expression response to nitrate is dynamic and highly localized and predicted cell type-specific transcription factor (TF)-target interactions. Among cell types, the endodermis stands out as having the largest and most connected nitrate-regulatory gene network. ABF2 and ABF3 are major hubs for transcriptional responses in the endodermis cell layer. We experimentally validated TF-target interactions for ABF2 and ABF3 by chromatin immunoprecipitation followed by sequencing and a cell-based system to detect TF regulation genome-wide. Validated targets of ABF2 and ABF3 account for more than 50% of the nitrate-responsive transcriptome in the endodermis. Moreover, ABF2 and ABF3 are involved in nitrate-induced lateral root growth. Our approach offers an unprecedented spatiotemporal resolution of the root response to nitrate and identifies important components of cell-specific gene regulatory networks.

A simple RNA preparation method for SARS-CoV-2 detection by RT-qPCR.

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.

Transcriptomic profiles reveal differences in zinc metabolism, inflammation, and tight junction proteins in duodenum from cholesterol gallstone subjects.

Cholesterol Gallstone Disease (GSD) is a common multifactorial disorder characterized by crystallization and aggregation of biliary cholesterol in the gallbladder. The global prevalence of GSD is ~10-20% in the adult population but rises to 28% in Chile (17% among men and 30% among women). The small intestine may play a role in GSD pathogenesis, but the molecular mechanisms have not been clarified. Our aim was to identify the role of the small intestine in GSD pathogenesis. Duodenal biopsy samples were obtained from patients with GSD and healthy volunteers. GSD status was defined by abdominal ultrasonography. We performed a transcriptome study in a discovery cohort using Illumina HiSeq. 2500, and qPCR, immunohistochemistry and immunofluorescence were used to validate differentially expressed genes among additional case-control cohorts. 548 differentially expressed genes between GSD and control subjects were identified. Enriched biological processes related to cellular response to zinc, and immune and antimicrobial responses were observed in GSD patients. We validated lower transcript levels of metallothionein, NPC1L1 and tight junction genes and higher transcript levels of genes involved in immune and antimicrobial pathways in GSD patients. Interestingly, serum zinc and phytosterol to cholesterol precursor ratios were lower in GSD patients. A significant association was observed between serum zinc and phytosterol levels. Our results support a model where proximal small intestine plays a key role in GSD pathogenesis. Zinc supplementation, modulation of proximal microbiota and/or intestinal barrier may be novel targets for strategies to prevent GSD.

Nitrate in 2020: Thirty Years from Transport to Signaling Networks.

Nitrogen (N) is an essential macronutrient for plants and a major limiting factor for plant growth and crop production. Nitrate is the main source of N available to plants in agricultural soils and in many natural environments. Sustaining agricultural productivity is of paramount importance in the current scenario of increasing world population, diversification of crop uses, and climate change. Plant productivity for major crops around the world, however, is still supported by excess application of N-rich fertilizers with detrimental economic and environmental impacts. Thus, understanding how plants regulate nitrate uptake and metabolism is key for developing new crops with enhanced N use efficiency and to cope with future world food demands. The study of plant responses to nitrate has gained considerable interest over the last 30 years. This review provides an overview of key findings in nitrate research, spanning biochemistry, molecular genetics, genomics, and systems biology. We discuss how we have reached our current view of nitrate transport, local and systemic nitrate sensing/signaling, and the regulatory networks underlying nitrate-controlled outputs in plants. We hope this summary will serve not only as a timeline and information repository but also as a baseline to define outstanding questions for future research.

Whole Genome Sequence, Variant Discovery and Annotation in Mapuche-Huilliche Native South Americans.

Whole human genome sequencing initiatives help us understand population history and the basis of genetic diseases. Current data mostly focuses on Old World populations, and the information of the genomic structure of Native Americans, especially those from the Southern Cone is scant. Here we present annotation and variant discovery from high-quality complete genome sequences of a cohort of 11 Mapuche-Huilliche individuals (HUI) from Southern Chile. We found approximately 3.1 × 106 single nucleotide variants (SNVs) per individual and identified 403,383 (6.9%) of novel SNVs events. Analyses of large-scale genomic events detected 680 copy number variants (CNVs) and 4,514 structural variants (SVs), including 398 and 1,910 novel events, respectively. Global ancestry composition of HUI genomes revealed that the cohort represents a sample from a marginally admixed population from the Southern Cone, whose main genetic component derives from Native American ancestors. Additionally, we found that HUI genomes contain variants in genes associated with 5 of the 6 leading causes of noncommunicable diseases in Chile, which may have an impact on the risk of prevalent diseases in Chilean and Amerindian populations. Our data represents a useful resource that can contribute to population-based studies and for the design of early diagnostics or prevention tools for Native and admixed Latin American populations.

Variants in ABCG8 and TRAF3 genes confer risk for gallstone disease in admixed Latinos with Mapuche Native American ancestry.

Latin Americans and Chilean Amerindians have the highest prevalence of gallstone disease (GSD) and gallbladder cancer (GBC) in the world. A handful of loci have been associated with GSD in populations of predominantly European ancestry, however, they only explain a small portion of the genetic component of the disease. Here, we performed a genome-wide association study (GWAS) for GSD in 1,095 admixed Chilean Latinos with Mapuche Native American ancestry. Disease status was assessed by cholecystectomy or abdominal ultrasonography. Top-10 candidate variants surpassing the suggestive cutoff of P < 1 × 10-5 in the discovery cohort were genotyped in an independent replication sample composed of 1,643 individuals. Variants with positive replication were further examined in two European GSD populations and a Chilean GBC cohort. We consistently replicated the association of ABCG8 gene with GSD (rs11887534, P = 3.24 × 10-8, OR = 1.74) and identified TRAF3 (rs12882491, P = 1.11 × 10-7, OR = 1.40) as a novel candidate gene for the disease in admixed Chilean Latinos. ABCG8 and TRAF3 variants also conferred risk to GBC. Gene expression analyses indicated that TRAF3 was significantly decreased in gallbladder (P = 0.015) and duodenal mucosa (P = 0.001) of GSD individuals compared to healthy controls, where according to GTEx data in the small intestine, the presence of the risk allele contributes to the observed effect. We conclude that ABCG8 and TRAF3 genes are associated with GSD and GBC in admixed Latinos and that decreased TRAF3 levels could enhance gallbladder inflammation as is observed in GSD and GSD-associated GBC.

SMZ/SNZ and gibberellin signaling are required for nitrate-elicited delay of flowering time in Arabidopsis thaliana.

The reproductive success of plants largely depends on the correct programming of developmental phase transitions, particularly the shift from vegetative to reproductive growth. The timing of this transition is finely regulated by the integration of an array of environmental and endogenous factors. Nitrogen is the mineral macronutrient that plants require in the largest amount, and as such its availability greatly impacts on many aspects of plant growth and development, including flowering time. We found that nitrate signaling interacts with the age-related and gibberellic acid pathways to control flowering time in Arabidopsis thaliana. We revealed that repressors of flowering time belonging to the AP2-type transcription factor family including SCHLAFMUTZE (SMZ) and SCHNARCHZAPFEN (SNZ) are important regulators of flowering time in response to nitrate. Our results support a model whereby nitrate activates SMZ and SNZ via the gibberellin pathway to repress flowering time in Arabidopsis thaliana.

The Calcium Ion Is a Second Messenger in the Nitrate Signaling Pathway of Arabidopsis.

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 Ca(2+) concentration. We found that nitrate also induces cytoplasmic concentration of inositol 1,4,5-trisphosphate. Increases in inositol 1,4,5-trisphosphate and cytoplasmic Ca(2+) 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 Ca(2+) 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 Ca(2+) channel blockers or phospholipase C inhibitors. These results indicate that Ca(2+) 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 Ca(2+) in response to nitrate required for changes in expression of prototypical nitrate-responsive genes.

Systems approach identifies TGA1 and TGA4 transcription factors as important regulatory components of the nitrate response of Arabidopsis thaliana roots.

Nitrate acts as a potent signal to control global gene expression in Arabidopsis. Using an integrative bioinformatics approach we identified TGA1 and TGA4 as putative regulatory factors that mediate nitrate responses in Arabidopsis roots. We showed that both TGA1 and TGA4 mRNAs accumulate strongly after nitrate treatments in roots. Global gene expression analysis revealed 97% of the genes with altered expression in tga1 tga4 double mutant plants respond to nitrate treatments, indicating that these transcription factors have a specific role in nitrate responses in Arabidopsis root organs. We found TGA1 and TGA4 regulate the expression of nitrate transporter genes NRT2.1 and NRT2.2. Specific binding of TGA1 to its cognate DNA sequence on NRT2.1 and NRT2.2 promoters was confirmed by chromatin immunoprecipitation assays. The tga1 tga4 double mutant plants exhibit nitrate-dependent lateral and primary root phenotypes. Lateral root initiation is affected in both tga1 tga4 and nrt1.2 nrt2.2 double mutants, suggesting TGA1 and TGA4 regulate lateral root development at least partly via NRT2.1 and NRT2.2. Additional root phenotypes of tga1 tga4 double mutants indicate that these transcription factors play an important role in root developmental responses to nitrate. These results identify TGA1 and TGA4 as important regulatory factors of the nitrate response in Arabidopsis roots.

Systems approaches map regulatory networks downstream of the auxin receptor AFB3 in the nitrate response of Arabidopsis thaliana roots.

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.