Nutrient levels control root growth responses to high ambient temperature in plants.

Global warming will lead to significantly increased temperatures on earth. Plants respond to high ambient temperature with altered developmental and growth programs, termed thermomorphogenesis. Here we show that thermomorphogenesis is conserved in Arabidopsis, soybean, and rice and that it is linked to a decrease in the levels of the two macronutrients nitrogen and phosphorus. We also find that low external levels of these nutrients abolish root growth responses to high ambient temperature. We show that in Arabidopsis, this suppression is due to the function of the transcription factor ELONGATED HYPOCOTYL 5 (HY5) and its transcriptional regulation of the transceptor NITRATE TRANSPORTER 1.1 (NRT1.1). Soybean and Rice homologs of these genes are expressed consistently with a conserved role in regulating temperature responses in a nitrogen and phosphorus level dependent manner. Overall, our data show that root thermomorphogenesis is a conserved feature in species of the two major groups of angiosperms, monocots and dicots, that it leads to a reduction of nutrient levels in the plant, and that it is dependent on environmental nitrogen and phosphorus supply, a regulatory process mediated by the HY5-NRT1.1 module.

Genome-wide identification and expression-pattern analysis of sulfate transporter (SULTR) gene family in cotton under multiple abiotic stresses and fiber development.

Sulfate transporter (SULTR) proteins are in charge of the transport and absorption on sulfate substances, and have been reported to play vital roles in the biological processes of plant growth and stress response. However, there were few reports of genome-wide identification and expression-pattern analysis of SULTRs in Hibiscus mutabilis. Gossypium genus is a ideal model for studying the allopolyploidy, therefore two diploid species (G. raimondii and G. arboreum) and two tetraploid species (G. hirsutum and G. barbadense) were chosen in this study to perform bioinformatic analyses, identifying 18, 18, 35, and 35 SULTR members, respectively. All the 106 cotton SULTR genes were utilized to construct the phylogenetic tree together with 11 Arabidopsis thaliana, 13 Oryza sativa, and 8 Zea mays ones, which was divided into Group1-Group4. The clustering analyses of gene structures and 10 conserved motifs among the cotton SULTR genes showed the consistent evolutionary relationship with the phylogenetic tree, and the results of gene-duplication identification among the four representative Gossypium species indicated that genome-wide or segment duplication might make main contributions to the expansion of SULTR gene family in cotton. Having conducted the cis-regulatory element analysis in promoter region, we noticed that the existing salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA) elements could have influences with expression levels of cotton SULTR genes. The expression patterns of GhSULTR genes were also investigated on the 7 different tissues or organs and the developing ovules and fibers, most of which were highly expressed in root, stem, sepal, receptacel, ovule at 10 DPA, and fiber at 20 and 25 DPA. In addition, more active regulatory were observed in GhSULTR genes responding to multiple abiotic stresses, and 12 highly expressed genes showed the similar expression patterns in the quantitative Real-time PCR experiments under cold, heat, salt, and drought treatments. These findings broaden our insight into the evolutionary relationships and expression patterns of the SULTR gene family in cotton, and provide the valuable information for further screening the vital candidate genes on trait improvement.

Mitochondrial citrate transporters Ctp1-Yhm2 and respiratory chain: A coordinated functional connection in Saccharomyces cerevisiae metabolism.

The mitochondrial inner membrane contains some hydrophobic proteins that mediate the exchange of metabolites between the mitochondrial matrix and the cytosol. Ctp1 and Yhm2 are two carrier proteins in the yeast Saccharomyces cerevisiae responsible for the transport of citrate, a tricarboxylate involved in several metabolic pathways. Since these proteins also contribute to respiratory metabolism, in this study we investigated for the first time whether changes in citrate transport can affect the structural organization and functional properties of respiratory complexes. Through experiments in yeast mutant cells in which the gene encoding Ctp1 or Yhm2 was deleted, we found that in the absence of either mitochondrial citrate transporter, mitochondrial respiration was impaired. Structural analysis of the respiratory complexes III and IV revealed different expression levels of the catalytic and supernumerary subunits in the Δctp1 and Δyhm2 strains. In addition, Δyhm2 mitochondria appeared to be more sensitive than Δctp1 to the oxidative damage. Our results provide the first evidence for a coordinated modulation of mitochondrial citrate transport and respiratory chain activity in S. cerevisiae metabolism.

The molecular principles underlying diverse functions of the SLC26 family of proteins.

Mammalian SLC26 proteins are membrane-based anion transporters that belong to the large SLC26/SulP family, and many of their variants are associated with hereditary diseases. Recent structural studies revealed a strikingly similar homodimeric molecular architecture for several SLC26 members, implying a shared molecular principle. Now a new question emerges as to how these structurally similar proteins execute diverse physiological functions. In this study, we sought to identify the common versus distinct molecular mechanism among the SLC26 proteins using both naturally occurring and artificial missense changes introduced to SLC26A4, SLC26A5, and SLC26A9. We found: (i) the basic residue at the anion binding site is essential for both anion antiport of SLC26A4 and motor functions of SLC26A5, and its conversion to a nonpolar residue is crucial but not sufficient for the fast uncoupled anion transport in SLC26A9; (ii) the conserved polar residues in the N- and C-terminal cytosolic domains are likely involved in dynamic hydrogen-bonding networks and are essential for anion antiport of SLC26A4 but not for motor (SLC26A5) and uncoupled anion transport (SLC26A9) functions; (iii) the hydrophobic interaction between each protomer's last transmembrane helices, TM14, is not of functional significance in SLC26A9 but crucial for the functions of SLC26A4 and SLC26A5, likely contributing to optimally orient the axis of the relative movements of the core domain with respect to the gate domains within the cell membrane. These findings advance our understanding of the molecular mechanisms underlying the diverse physiological roles of the SLC26 family of proteins.

Isolation of Saccharomycopsis species from plant material.

Saccharomycopsis species are natural organic sulphur auxotrophs. Their genomes do not encode genes for the uptake and assimilation of sulphate and thus these species cannot grow on media lacking e.g. methionine. Due to the similarity between sulphate and selenate, uptake and assimilation of selenate occurs through the same pathway starting from sulphate transporters encoded by the homologs of the SUL1 and SUL2 genes in S. cerevisiae. Lack of these transporters renders Saccharomycopsis species resistant to selenate levels that are toxic to other microorganisms. We used this feature to enrich environmental samples for Saccharomycopsis species. This led to the isolation of S. schoenii, S. lassenensis and a hitherto undescribed Saccharomycopsis species with limited by-catch of other yeasts, mainly belonging to Metschnikowia and Hanseniaspora. We performed growth and predation assays to characterize the potential of these new isolates as predacious yeasts. Most Saccharomycopsis species are temperature sensitive and cannot grow at 37°C; with the exception of S. lassenensis strains. Predation assays with S. schoenii and S. cerevisiae as prey indicated that predation was enhanced at 20°C compared to 30°C. We crossed an American isolate of S. schoenii with our German isolate using marker directed breeding. Viable progeny indicated that both strains are interfertile and belong to the same biological species. S. lassenensis is heterothallic, while S. schoenii and the new Saccharomycopsis isolate, for which we suggest the name S. geisenheimensis sp. nov., are homothallic.

Transceptor NRT1.1 and receptor-kinase QSK1 complex controls PM H+-ATPase activity under low nitrate.

NRT1.1, a nitrate transceptor, plays an important role in nitrate binding, sensing, and nitrate-dependent lateral root (LR) morphology. However, little is known about NRT1.1-mediated nitrate signaling transduction through plasma membrane (PM)-localized proteins. Through in-depth phosphoproteome profiling using membranes of Arabidopsis roots, we identified receptor kinase QSK1 and plasma membrane H+-ATPase AHA2 as potential downstream components of NRT1.1 signaling in a mild low-nitrate (LN)-dependent manner. QSK1, as a functional kinase and molecular link, physically interacts with NRT1.1 and AHA2 at LN and specifically phosphorylates AHA2 at S899. Importantly, we found that LN, not high nitrate (HN), induces formation of the NRT1.1-QSK1-AHA2 complex in order to repress the proton efflux into the apoplast by increased phosphorylation of AHA2 at S899. Loss of either NRT1.1 or QSK1 thus results in a higher T947/S899 phosphorylation ratio on AHA2, leading to enhanced pump activity and longer LRs under LN. Our results uncover a regulatory mechanism in which NRT1.1, under LN conditions, promotes coreceptor QSK1 phosphorylation and enhances the NRT1.1-QSK1 complex formation to transduce LN sensing to the PM H+-ATPase AHA2, controlling the phosphorylation ratio of activating and inhibitory phosphorylation sites on AHA2. This then results in altered proton pump activity, apoplast acidification, and regulation of NRT1.1-mediated LR growth.

Dof1.7 and NIGT1 transcription factors mediate multilayered transcriptional regulation for different expression patterns of NITRATE TRANSPORTER2 genes under nitrogen deficiency stress.

Elucidating the mechanisms regulating nitrogen (N) deficiency responses in plants is of great agricultural importance. Previous studies revealed that decreased expression of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) transcriptional repressor genes upon N deficiency is involved in N deficiency-inducible gene expression in Arabidopsis thaliana. However, our knowledge of the mechanisms controlling N deficiency-induced changes in gene expression is still limited. Through the identification of Dof1.7 as a direct target of NIGT1 repressors and a novel N deficiency response-related transcriptional activator gene, we here show that NIGT1 and Dof1.7 transcription factors (TFs) differentially regulate N deficiency-inducible expression of three high-affinity nitrate transporter genes, NRT2.1, NRT2.4, and NRT2.5, which are responsible for most of the soil nitrate uptake activity of Arabidopsis plants under N-deficient conditions. Unlike NIGT1 repressors, which directly suppress NRT2.1, NRT2.4, and NRT2.5 under N-sufficient conditions, Dof1.7 directly activated only NRT2.5 but indirectly and moderately activated NRT2.1 and NRT2.4 under N-deficient conditions, probably by indirectly decreasing NIGT1 expression. Thus, Dof1.7 converted passive transcriptional activation into active and potent transcriptional activation, further differentially enhancing the expression of NRT2 genes. These findings clarify the mechanism underlying different expression patterns of NRT2 genes upon N deficiency, suggesting that time-dependent multilayered transcriptional regulation generates complicated expression patterns of N deficiency-inducible genes.

Low-CO2-inducible bestrophins outside the pyrenoid sustain high photosynthetic efficacy in diatoms.

Anion transporters sustain a variety of physiological states in cells. Bestrophins (BSTs) belong to a Cl- and/or HCO3- transporter family conserved in bacteria, animals, algae, and plants. Recently, putative BSTs were found in the green alga Chlamydomonas reinhardtii, where they are upregulated under low CO2 (LC) conditions and play an essential role in the CO2-concentrating mechanism (CCM). The putative BST orthologs are also conserved in diatoms, secondary endosymbiotic algae harboring red-type plastids, but their physiological functions are unknown. Here, we characterized the subcellular localization and expression profile of BSTs in the marine diatoms Phaeodactylum tricornutum (PtBST1 to 4) and Thalassiosira pseudonana (TpBST1 and 2). PtBST1, PtBST2, and PtBST4 were localized at the stroma thylakoid membrane outside of the pyrenoid, and PtBST3 was localized in the pyrenoid. Contrarily, TpBST1 and TpBST2 were both localized in the pyrenoid. These BST proteins accumulated in cells grown in LC but not in 1% CO2 (high CO2 [HC]). To assess the physiological functions, we generated knockout mutants for the PtBST1 gene by genome editing. The lack of PtBST1 decreased photosynthetic affinity for dissolved inorganic carbon to the level comparable with the HC-grown wild type. Furthermore, non-photochemical quenching in LC-grown cells was 1.5 to 2.0 times higher in the mutants than in the wild type. These data suggest that HCO3- transport at the stroma thylakoid membranes by PtBST1 is a critical part of the CO2-evolving machinery of the pyrenoid in the fully induced CCM and that PtBST1 may modulate photoprotection under CO2-limited environments in P. tricornutum.

SULTR2;1 Adjusts the Bolting Timing by Transporting Sulfate from Rosette Leaves to the Primary Stem.

Sulfur (S) is an essential macronutrient for plant growth and metabolism. SULTR2;1 is a low-affinity sulfate transporter facilitating the long-distance transport of sulfate in Arabidopsis. The physiological function of SULTR2;1 in the plant life cycle still needs to be determined. Therefore, we analyzed the sulfate transport, S-containing metabolite accumulation and plant growth using Arabidopsis SULTR2;1 disruption lines, sultr2;1-1 and sultr2;1-2, from seedling to mature growth stages to clarify the metabolic and physiological roles of SULTR2;1. We observed that sulfate distribution to the stems was affected in sultr2;1 mutants, resulting in decreased levels of sulfate, cysteine, glutathione (GSH) and total S in the stems, flowers and siliques; however, the GSH levels increased in the rosette leaves. This suggested the essential role of SULTR2;1 in sulfate transport from rosette leaves to the primary stem. In addition, sultr2;1 mutants unexpectedly bolted earlier than the wild-type without affecting the plant biomass. Correlation between GSH levels in rosette leaves and the bolting timing suggested that the rosette leaf GSH levels or limited sulfate transport to the early stem can trigger bolting. Overall, this study demonstrated the critical roles of SULTR2;1 in maintaining the S metabolite levels in the aerial part and transitioning from the vegetative to the reproductive growth phase.

Impact of culture media on primary human corneal endothelial cells derived from old donors.

Corneal endothelial dysfunction is a major indication for corneal transplantation. However, a global shortage of donor corneal tissues and risks associated with corneal surgeries have prompted exploration of alternative options, including tissue-engineered grafts or cell injection therapy. Nonetheless, these approaches require a controlled culture of primary human corneal endothelial cells (HCEnCs). Although HCEnCs established from young donors are generally more proliferative and maintain a better phenotype, corneas from old donors are more frequently accessible from eye banks due to a lower corneal endothelial cell count than the necessary threshold required for transplantation. In this study, we investigated various culture media to evaluate which one is the most appropriate for stimulating the proliferation while maintaining cell morphology and function of HCEnCs derived from old donors (age >65 years). All experiments were performed on paired research-grade donor corneas, divided for the conditions under investigation in order to minimize the inter-donor variability. Cell morphology as well as expression of specific markers were assessed at both mRNA (CD166, SLC4A11, ATP1A1, COL8A1, α-SMA, CD44, COL1A1, CDKN2A, LAP2A and LAP2B) and protein (ZO-1, α-SMA, Ki67 and LAP2) levels. Results obtained showed how the Dual Media formulation maintained the hexagonal phenotype more efficiently than Single Medium, but cell size gradually increased with passages. In contrast, the Single Medium provided a higher proliferation rate and a prolonged in vitro expansion but acquired an elongated morphology. To summarize, Single medium and Dual media preserve morphology and functional phenotype of HCEnCs from old donor corneas at low passages while maintenance of the same cell features at high passages remains an active area of research. The new insights revealed within this work become particularly relevant considering that the elderly population a) is the main target of corneal endothelial therapy, b) represents the majority of corneal donors. Therefore, the proper expansion of HCEnCs from old donors is essential to develop novel personalised therapeutic strategies and reduce requirement of human corneal tissues globally.

Structural and functional insights into the lipid regulation of human anion exchanger 2.

Anion exchanger 2 (AE2) is an electroneutral Na+-independent Cl-/HCO3- exchanger belongs to the SLC4 transporter family. The widely expressed AE2 participates in a variety of physiological processes, including transepithelial acid-base secretion and osteoclastogenesis. Both the transmembrane domains (TMDs) and the N-terminal cytoplasmic domain (NTD) are involved in regulation of AE2 activity. However, the regulatory mechanism remains unclear. Here, we report a 3.2 Å cryo-EM structure of the AE2 TMDs in complex with PIP2 and a 3.3 Å full-length mutant AE2 structure in the resting state without PIP2. We demonstrate that PIP2 at the TMD dimer interface is involved in the substrate exchange process. Mutation in the PIP2 binding site leads to the displacement of TM7 and further stabilizes the interaction between the TMD and the NTD. Reduced substrate transport activity and conformation similar to AE2 in acidic pH indicating the central contribution of PIP2 to the function of AE2.

SLC26 Anion Transporters.

Solute carrier family 26 (SLC26) is a family of functionally diverse anion transporters found in all kingdoms of life. Anions transported by SLC26 proteins include chloride, bicarbonate, and sulfate, but also small organic dicarboxylates such as fumarate and oxalate. The human genome encodes ten functional homologs, several of which are causally associated with severe human diseases, highlighting their physiological importance. Here, we review novel insights into the structure and function of SLC26 proteins and summarize the physiological relevance of human members.

Phosphoregulation in the N-terminus of NRT2.1 affects nitrate uptake by controlling the interaction of NRT2.1 with NAR2.1 and kinase HPCAL1 in Arabidopsis.

NRT2.1, the major high affinity nitrate transporter in roots, can be phosphorylated at five different sites within the N- and the C-terminus. Here, we characterized the functional relationship of two N-terminal phosphorylation sites, S21 and S28, in Arabidopsis. Based on a site-specific correlation network, we identified a receptor kinase (HPCAL1, AT5G49770), phosphorylating NRT2.1 at S21 and resulting in active nitrate uptake. HPCAL1 itself was regulated by phosphorylation at S839 and S870 within its kinase domain. In the active state, when S839 was dephosphorylated and S870 was phosphorylated, HPCAL1 was found to interact with the N-terminus of NRT2.1, mainly when S28 was dephosphorylated. Phosphorylation of NRT2.1 at S21 resulted in a reduced interaction of NRT2.1 with its activator NAR2.1, but nitrate transport activity remained. By contrast, phosphorylated NRT2.1 at S28 enhanced the interaction with NAR2.1, but reduced the interaction with HPCAL1. Here we identified HPCAL1 as the kinase affecting this phospho-switch through phosphorylation of NRT2.1 at S21.

Protein Phosphorylation Orchestrates Acclimations of Arabidopsis Plants to Environmental pH.

Environment pH (pHe) is a key parameter dictating a surfeit of conditions critical to plant survival and fitness. To elucidate the mechanisms that recalibrate cytoplasmic and apoplastic pH homeostasis, we conducted a comprehensive proteomic/phosphoproteomic inventory of plants subjected to transient exposure to acidic or alkaline pH, an approach that covered the majority of protein-coding genes of the reference plant Arabidopsis thaliana. Our survey revealed a large set-of so far undocumented pHe-dependent phospho-sites, indicative of extensive post-translational regulation of proteins involved in the acclimation to pHe. Changes in pHe altered both electrogenic H+ pumping via P-type ATPases and H+/anion co-transport processes, putatively leading to altered net trans-plasma membrane translocation of H+ ions. In pH 7.5 plants, the transport (but not the assimilation) of nitrogen via NRT2-type nitrate and AMT1-type ammonium transporters was induced, conceivably to increase the cytosolic H+ concentration. Exposure to both acidic and alkaline pH resulted in a marked repression of primary root elongation. No such cessation was observed in nrt2.1 mutants. Alkaline pH decreased the number of root hairs in the wild type but not in nrt2.1 plants, supporting a role of NRT2.1 in developmental signaling. Sequestration of iron into the vacuole via alterations in protein abundance of the vacuolar iron transporter VTL5 was inversely regulated in response to high and low pHe, presumptively in anticipation of associated changes in iron availability. A pH-dependent phospho-switch was also observed for the ABC transporter PDR7, suggesting changes in activity and, possibly, substrate specificity. Unexpectedly, the effect of pHe was not restricted to roots and provoked pronounced changes in the shoot proteome. In both roots and shoots, the plant-specific TPLATE complex components AtEH1 and AtEH2-essential for clathrin-mediated endocytosis-were differentially phosphorylated at multiple sites in response to pHe, indicating that the endocytic cargo protein trafficking is orchestrated by pHe.

Enhanced Rice Resistance to Sheath Blight through Nitrate Transporter 1.1B Mutation without Yield Loss under NH4+ Fertilization.

Nitrogen fertilization can promote rice yield but decrease resistance to sheath blight (ShB). In this study, the nitrate transporter 1.1b (nrt1.1b) mutant that exhibited less susceptibility to ShB but without compromising yield under NH4+ fertilization was screened. NRT1.1B's regulation of ShB resistance was independent of the total nitrogen concentration in rice under NH4+ conditions. In nrt1.1b mutant plants, the NH4+ application modulated auxin signaling, chlorophyll content, and phosphate signaling to promote ShB resistance. Furthermore, the findings indicated that NRT1.1B negatively regulated ShB resistance by positively modulating the expression of H+-ATPase gene OSA3 and phosphate transport gene PT8. The mutation of OSA3 and PT8 promoted ShB resistance by increasing the apoplastic pH in rice. Our study identified the ShB resistance mutant nrt1.1b, which maintained normal nitrogen use efficiency without compromising yield.

The Beneficial Fungus Mortierella hyalina Modulates Amino Acid Homeostasis in Arabidopsis under Nitrogen Starvation.

Non-mycorrhizal but beneficial fungi often mitigate (a)biotic stress-related traits in host plants. The underlying molecular mechanisms are mostly still unknown, as in the interaction between the endophytic growth-promoting soil fungus Mortierella hyalina and Arabidopsis thaliana. Here, abiotic stress in the form of nitrogen (N) deficiency was used to investigate the effects of the fungus on colonized plants. In particular, the hypothesis was investigated that fungal infection could influence N deficiency via an interaction with the high-affinity nitrate transporter NRT2.4, which is induced by N deficiency. For this purpose, Arabidopsis wild-type nrt2.4 knock-out and NRT2.4 reporter lines were grown on media with different nitrate concentrations with or without M. hyalina colonization. We used chemical analysis methods to determine the amino acids and phytohormones. Experimental evidence suggests that the fungus does not modulate NRT2.4 expression under N starvation. Instead, M. hyalina alleviates N starvation in other ways: The fungus supplies nitrogen (15N) to the N-starved plant. The presence of the fungus restores the plants' amino acid homeostasis, which was out of balance due to N deficiency, and causes a strong accumulation of branched-chain amino acids. We conclude that the plant does not need to invest in defense and resources for growth are maintained, which in turn benefits the fungus, suggesting that this interaction should be considered a mutualistic symbiosis.

Elevator-like movements of prestin mediate outer hair cell electromotility.

The outstanding acuity of the mammalian ear relies on cochlear amplification, an active mechanism based on the electromotility (eM) of outer hair cells. eM is a piezoelectric mechanism generated by little-understood, voltage-induced conformational changes of the anion transporter homolog prestin (SLC26A5). We used a combination of molecular dynamics (MD) simulations and biophysical approaches to identify the structural dynamics of prestin that mediate eM. MD simulations showed that prestin samples a vast conformational landscape with expanded (ES) and compact (CS) states beyond previously reported prestin structures. Transition from CS to ES is dominated by the translational-rotational movement of prestin's transport domain, akin to elevator-type substrate translocation by related solute carriers. Reversible transition between CS and ES states was supported experimentally by cysteine accessibility scanning, cysteine cross-linking between transport and scaffold domains, and voltage-clamp fluorometry (VCF). Our data demonstrate that prestin's piezoelectric dynamics recapitulate essential steps of a structurally conserved ion transport cycle.

Finding Balance in Adversity: Nitrate Signaling as the Key to Plant Growth, Resilience, and Stress Response.

To effectively adapt to changing environments, plants must maintain a delicate balance between growth and resistance or tolerance to various stresses. Nitrate, a significant inorganic nitrogen source in soils, not only acts as an essential nutrient but also functions as a critical signaling molecule that regulates multiple aspects of plant growth and development. In recent years, substantial advancements have been made in understanding nitrate sensing, calcium-dependent nitrate signal transmission, and nitrate-induced transcriptional cascades. Mounting evidence suggests that the primary response to nitrate is influenced by environmental conditions, while nitrate availability plays a pivotal role in stress tolerance responses. Therefore, this review aims to provide an overview of the transcriptional and post-transcriptional regulation of key components in the nitrate signaling pathway, namely, NRT1.1, NLP7, and CIPK23, under abiotic stresses. Additionally, we discuss the specificity of nitrate sensing and signaling as well as the involvement of epigenetic regulators. A comprehensive understanding of the integration between nitrate signaling transduction and abiotic stress responses is crucial for developing future crops with enhanced nitrogen-use efficiency and heightened resilience.

Genome-wide identification and expression analysis of the NRT genes in Ginkgo biloba under nitrate treatment reveal the potential roles during calluses browning.

Nitrate is a primary nitrogen source for plant growth, and previous studies have indicated a correlation between nitrogen and browning. Nitrate transporters (NRTs) are crucial in nitrate allocation. Here, we utilized a genome-wide approach to identify and analyze the expression pattern of 74 potential GbNRTs under nitrate treatments during calluses browning in Ginkgo, including 68 NITRATE TRANSPORTER 1 (NRT1)/PEPTIDE TRANSPORTER (PTR) (NPF), 4 NRT2 and 2 NRT3. Conserved domains, motifs, phylogeny, and cis-acting elements (CREs) were analyzed to demonstrate the evolutionary conservation and functional diversity of GbNRTs. Our analysis showed that the NPF family was divided into eight branches, with the GbNPF2 and GbNPF6 subfamilies split into three groups. Each GbNRT contained 108-214 CREs of 19-36 types, especially with binding sites of auxin and transcription factors v-myb avian myeloblastosis viral oncogene homolog (MYB) and basic helix-loop-helix (bHLH). The E1X1X2E2R motif had significant variations in GbNPFs, indicating changes in the potential dynamic proton transporting ability. The expression profiles of GbNRTs indicated that they may function in regulating nitrate uptake and modulating the signaling of auxin and polyphenols biosynthesis, thereby affecting browning in Ginkgo callus induction. These findings provide a better understanding of the role of NRTs during NO3- uptake and utilization in vitro culture, which is crucial to prevent browning and develop an efficient regeneration and suspension production system in Ginkgo.