High-affinity Uptake Research Articles

CPK28-mediated phosphorylation enhances nitrate transport activity of NRT2.1 during nitrogen deprivation.

Nitrate (NO3 -) serves as the primary inorganic nitrogen source assimilated by most terrestrial plants. The acquisition of nitrate from the soil is facilitated by NITRATE TRANSPORTERS (NRTs), with NRT2.1 being the key high-affinity nitrate transporter. The activity of NRT2.1, which has multiple potential phosphorylation sites, is intricately regulated under various physiological conditions. Here, we discovered that CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28) positively regulates nitrate uptake under nitrogen deprivation conditions. We found CPK28 as the kinase targeted by immunoprecipitation followed by mass spectrometry and examined the in-planta phosphorylation status of NRT2.1 in cpk28 mutant plants by employing quantitative MS-based phosphoproteomics. Through a combination of in vitro phosphorylation experiment and immunoblotting using phospho-specific antibody, we successfully demonstrated that CPK28 specifically phosphorylates NRT2.1 at Ser21. Functional analysis conducted in Xenopus oocytes revealed that co-expression of CPK28 significantly enhanced high-affinity nitrate uptake of NRT2.1. Further investigation using transgenic plants showed that the phosphomimic variant NRT2.1S21E, but not the nonphosphorylatable variant NRT2.1S21A, fully restored high-affinity 15NO3 - uptake ability in both nrt2.1 and cpk28 mutant backgrounds. This study clarifies that the kinase activity of CPK28 is promoted during nitrogen deprivation conditions. These significant findings provide valuable insights into the intricate regulatory mechanisms that govern nitrate-demand adaptation.

Whole cell affinity for 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) in the marine bacterium Candidatus Pelagibacter st. HTCC7211 explains marine dissolved HMP concentrations.

Vitamin B1 is a universally required coenzyme in carbon metabolism. However, most marine microorganisms lack the complete biosynthetic pathway for this compound and must acquire thiamin, or precursor molecules, from the dissolved pool. The most common version of Vitamin B1 auxotrophy is for thiamin's pyrimidine precursor moiety, 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP). Frequent HMP auxotrophy in plankton and vanishingly low dissolved concentrations (approximately 0.1-50 pM) suggest that high-affinity HMP uptake systems are responsible for maintaining low ambient HMP concentrations. We used tritium-labelled HMP to investigate HMP uptake mechanisms and kinetics in cell cultures of Candidatus Pelagibacter st. HTCC7211, a representative of the globally distributed and highly abundant SAR11 clade. A single protein, the sodium solute symporter ThiV, which is conserved across SAR11 genomes, is the likely candidate for HMP transport. Experimental evidence indicated transport specificity for HMP and mechanistically complex, high-affinity HMP uptake kinetics. Km values ranged from 9.5 pM to 1.2 nM and were dramatically lower when cells were supplied with a carbon source. These results suggest that HMP uptake in HTCC7211 is subject to complex regulation and point to a strategy for high-affinity uptake of this essential growth factor that can explain natural HMP levels in seawater.

A fungal endophyte increases plant resilience to low nutrient availabilities: a case of Fe acquisition in legumes.

Microbial inocula are considered a promising and effective alternative solution to the use of chemical fertilizers to support plant growth and productivity since they play a key role in the availability and uptake of nutrients. Here, the effect of a beneficial of a fungal root endophyte, Fusarium solani strain K (FsK), on nutrient acquisition efficiency of the legume Lotus japonicus was studied, and putative mode-of-action of the endophyte at a molecular level was determined. Plant colonization with the endophyte resulted in increased shoot and root fresh weight under Fe deficiency compared to control nutrient conditions. Plant inoculation with FsK was associated with a significant increase in macro- and micronutrient concentration in leaves at an early stage of endophyte inoculation and a replenishment of Fe content under prolonged iron starvation. The mechanistic basis of the plant growth promotion capabilities of the endophyte is exerted at the transcriptional level since we recorded changes in the expression levels of genes related to iron uptake in FsK-colonized plants under stress conditions compared to uninoculated plants. In addition, the observed changes in the ethylene biosynthesis-related genes suggest a possible implication of ethylene in the mode of action used by FsK to enhance plant response to nutrient stress conditions. Finally, we demonstrated that the endophyte possesses a reductive high-affinity Fe uptake system and identified a ferric reductase that was induced in planta under Fe deficiency conditions, indicating that this fungal Fe homeostasis mechanism may result in a benefit in nutrient acquisition for the plant as well.

Integration of shoot-derived polypeptide signals by root TGA transcription factors is essential for survival under fluctuating nitrogen environments

Unlike plants in the field, which experience significant temporal fluctuations in environmental conditions, plants in the laboratory are typically grown in controlled, stable environments. Therefore, signaling pathways evolved for survival in fluctuating environments often remain functionally latent in laboratory settings. Here, we show that TGA1 and TGA4 act as hub transcription factors through which the expression of genes involved in high-affinity nitrate uptake are regulated in response to shoot-derived phloem mobile polypeptides, CEP DOWNSTREAM 1 (CEPD1), CEPD2 and CEPD-like 2 (CEPDL2) as nitrogen (N) deficiency signals, and Glutaredoxin S1 (GrxS1) to GrxS8 as N sufficiency signals. CEPD1/2/CEPDL2 and GrxS1-S8 competitively bind to TGA1/4 in roots, with the former acting as transcription coactivators that enhance the uptake of nitrate, while the latter function as corepressor complexes together with TOPLESS (TPL), TPL-related 1 (TPR1) and TPR4 to limit nitrate uptake. Arabidopsis plants deficient in TGA1/4 maintain basal nitrate uptake and exhibit growth similar to wild-type plants in a stable N environment, but are impaired in regulation of nitrate acquisition in response to shoot N demand, leading to defective growth under fluctuating N environments where rhizosphere nitrate ions switch periodically between deficient and sufficient states. TGA1/4 are crucial transcription factors that enable plants to survive under fluctuating and challenging N environmental conditions.

Regulation of potassium uptake in Caulobacter crescentus.

Potassium (K+) is an essential physiological element determining membrane potential, intracellular pH, osmotic/turgor pressure, and protein synthesis in cells. Here, we describe the regulation of potassium uptake systems in the oligotrophic α-proteobacterium Caulobacter crescentus known as a model for asymmetric cell division. We show that C. crescentus can grow in concentrations from the micromolar to the millimolar range by mainly using two K+ transporters to maintain potassium homeostasis, the low-affinity Kup and the high-affinity Kdp uptake systems. When K+ is not limiting, we found that the kup gene is essential while kdp inactivation does not impact the growth. In contrast, kdp becomes critical but not essential and kup dispensable for growth in K+-limited environments. However, in the absence of kdp, mutations in kup were selected to improve growth in K+-depleted conditions, likely by increasing the affinity of Kup for K+. In addition, mutations in the KdpDE two-component system, which regulates kdpABCDE expression, suggest that the inner membrane sensor regulatory component KdpD mainly works as a phosphatase to limit the growth when cells reach late exponential phase. Our data therefore suggest that KdpE is phosphorylated by another non-cognate histidine kinase. On top of this, we determined the KdpE-dependent and independent K+ transcriptome. Together, our work illustrates how an oligotrophic bacterium responds to fluctuation in K+ availability.IMPORTANCEPotassium (K+) is a key metal ion involved in many essential cellular processes. Here, we show that the oligotroph Caulobacter crescentus can support growth at micromolar concentrations of K+ by mainly using two K+ uptake systems, the low-affinity Kup and the high-affinity Kdp. Using genome-wide approaches, we also determined the entire set of genes required for C. crescentus to survive at low K+ concentration as well as the full K+-dependent regulon. Finally, we found that the transcriptional regulation mediated by the KdpDE two-component system is unconventional since unlike Escherichia coli, the inner membrane sensor regulatory component KdpD seems to work rather as a phosphatase on the phosphorylated response regulator KdpE~P.

The tricalbin family of membrane contact site tethers is involved in the transcriptional responses of Saccharomyces cerevisiae to glucose

Cellular organelles maintain areas of close apposition with other organelles at which the cytosolic gap in between them is reduced to a minimum. These membrane contact sites (MCS) are vital for organelle communication and are formed by molecular tethers that physically connect opposing membranes. Although many regulatory pathways are known to converge at MCS, a link between MCS and transcriptional regulation-the primary mechanism through which cells adapt their metabolism to environmental cues-remains largely elusive. In this study, we performed RNA-sequencing on Saccharomyces cerevisiae cells lacking tricalbin proteins (Tcb1, Tcb2, and Tcb3), a family of tethering proteins that connect the endoplasmic reticulum with the plasma membrane and Golgi, to investigate if gene expression is altered when MCS are disrupted. Our results indicate that in the tcb1Δ2Δ3Δ strain, pathways responsive to a high-glucose environment, including glycolysis, fermentation, amino acid synthesis, and low-affinity glucose uptake, are upregulated. Conversely, pathways crucial during glucose depletion, such as the tricarboxylic acid cycle, respiration, high-affinity glucose uptake, and amino acid uptake are downregulated. In addition, we demonstrate that the altered gene expression of tcb1Δ2Δ3Δ in glucose metabolism correlates with increased growth, glucose consumption, CO2 production, and ethanol generation. In conclusion, our findings reveal that tricalbin protein deletion induces a shift in gene expression patterns mimicking cellular responses to a high-glucose environment. This suggests that MCS play a role in sensing and signaling pathways that modulate gene transcription in response to glucose availability.

DarA-the central processing unit for the integration of osmotic with potassium and amino acid homeostasis in Bacillus subtilis.

Cyclic di-adenosine monophosphate (c-di-AMP) is a second messenger involved in diverse metabolic processes including osmolyte uptake, cell wall homeostasis, as well as antibiotic and heat resistance. This study investigates the role of the c-di-AMP receptor protein DarA in the osmotic stress response in Bacillus subtilis. Through a series of experiments, we demonstrate that DarA plays a central role in the cellular response to osmotic fluctuations. Our findings show that DarA becomes essential under extreme potassium limitation as well as upon salt stress, highlighting its significance in mediating osmotic stress adaptation. Suppressor screens with darA mutants reveal compensatory mechanisms involving the accumulation of osmoprotectants, particularly potassium and citrulline. Mutations affecting various metabolic pathways, including the citric acid cycle as well as glutamate and arginine biosynthesis, indicate a complex interplay between the osmotic stress response and metabolic regulation. In addition, the growth defects of the darA mutant during potassium starvation and salt stress in a strain lacking the high-affinity potassium uptake systems KimA and KtrAB can be rescued by increased affinity of the remaining potassium channel KtrCD or by increased expression of ktrD, thus resulting in increased potassium uptake. Finally, the darA mutant can respond to salt stress by the increased expression of MleN , which can export sodium ions.IMPORTANCEEnvironmental bacteria are exposed to rapidly changing osmotic conditions making an effective adaptation to these changes crucial for the survival of the cells. In Gram-positive bacteria, the second messenger cyclic di-AMP plays a key role in this adaptation by controlling (i) the influx of physiologically compatible organic osmolytes and (ii) the biosynthesis of such osmolytes. In several bacteria, cyclic di-adenosine monophosphate (c-di-AMP) can bind to a signal transduction protein, called DarA, in Bacillus subtilis. So far, no function for DarA has been discovered in any organism. We have identified osmotically challenging conditions that make DarA essential and have identified suppressor mutations that help the bacteria to adapt to those conditions. Our results indicate that DarA is a central component in the integration of osmotic stress with the synthesis of compatible amino acid osmolytes and with the homeostasis of potassium, the first response to osmotic stress.

The high-affinity tryptophan uptake transport system in human cells.

The L-tryptophan (Trp) transport system is highly selective for Trp with affinity in the nanomolar range. This transport system is augmented in human interferon (IFN)-γ-treated and indoleamine 2,3-dioxygenase 1 (IDO1)-expressing cells. Up-regulated cellular uptake of Trp causes a reduction in extracellular Trp and initiates immune suppression. Recent studies demonstrate that both IDO1 and tryptophanyl-tRNA synthetase (TrpRS), whose expression levels are up-regulated by IFN-γ, play a pivotal role in high-affinity Trp uptake into human cells. Furthermore, overexpression of tryptophan 2,3-dioxygenase (TDO2) elicits a similar effect as IDO1 on TrpRS-mediated high-affinity Trp uptake. In this review, we summarize recent findings regarding this Trp uptake system and put forward a possible molecular mechanism based on Trp deficiency induced by IDO1 or TDO2 and tryptophanyl-AMP production by TrpRS.

Nonsense-mediated mRNA decay of metal-binding activator MAC1 is dependent on copper levels and 3'-UTR length in Saccharomyces cerevisiae.

The nonsense-mediated mRNA decay (NMD) pathway was initially identified as a surveillance pathway that degrades mRNAs containing premature termination codons (PTCs). NMD is now also recognized as a post-transcriptional regulatory pathway that regulates the expression of natural mRNAs. Earlier studies demonstrated that regulation of functionally related natural mRNAs by NMD can be differential and condition-specific in Saccharomyces cerevisiae. Here, we investigated the regulation of MAC1 mRNAs by NMD in response to copper as well as the role the MAC1 3'-UTR plays in this regulation. MAC1 is a copper-sensing transcription factor that regulates the high-affinity copper uptake system. MAC1 expression is activated upon copper deprivation. We found that MAC1 mRNAs are regulated by NMD under complete minimal (CM) but escaped NMD under low and high copper conditions. Mac1 protein regulated gene, CTR1 is not regulated by NMD in conditions where MAC1 mRNAs are NMD sensitive. We also found that the MAC1 3'-UTR is the NMD targeting feature on the mRNAs, and that MAC1 mRNAs lacking 3'-UTRs were stabilized during copper deprivation. Our results demonstrate a mechanism of regulation for a metal-sensing transcription factor, at both the post-transcriptional and post-translational levels, where MAC1 mRNA levels are regulated by NMD and copper, while the activity of Mac1p is controlled by copper levels.

The Transcription Factors AcuK and AcuM Influence Siderophore Biosynthesis of Aspergillus fumigatus.

The mold Aspergillus fumigatus employs two high-affinity uptake systems, reductive iron assimilation (RIA) and siderophore-mediated iron acquisition (SIA), for the acquisition of the essential trace element iron. SIA has previously been shown to be crucial for virulence in mammalian hosts. Here, we show that a lack of AcuK or AcuM, transcription factors required for the activation of gluconeogenesis, decreases the production of both extra- and intracellular siderophores in A. fumigatus. The lack of AcuM or AcuK did not affect the expression of genes involved in RIA and SIA, suggesting that these regulators do not directly regulate iron homeostasis genes, but indirectly affect siderophore production through their influence on metabolism. Consistent with this, acetate supplementation reversed the intracellular siderophore production defect of ΔacuM and ΔacuK. Moreover, ΔacuM and ΔacuK displayed a similar growth defect under iron limitation and iron sufficiency, which suggests they have a general role in carbon metabolism apart from gluconeogenesis. In agreement with a potential role of the glyoxylate cycle in adaptation to iron starvation, transcript levels of the malate synthase-encoding acuE were found to be upregulated by iron limitation that is partially dependent on AcuK and AcuM. Together, these data demonstrate the influence of iron availability on carbon metabolism.

Transport mechanism of presynaptic high-affinity choline uptake by CHT1.

Choline is a vital nutrient and a precursor for the biosynthesis of essential metabolites, including acetylcholine (ACh), that play a central role in fetal development, especially in the brain. In cholinergic neurons, the high-affinity choline transporter (CHT1) provides an extraordinarily efficient reuptake mechanism to reutilize choline derived from intrasynaptical ACh hydrolysis and maintain ACh synthesis in the presynapse. Here, we determined structures of human CHT1 in three discrete states: the outward-facing state bound with the competitive inhibitor hemicholinium-3 (HC-3); the inward-facing occluded state bound with the substrate choline; and the inward-facing apo open state. Our structures and functional characterizations elucidate how the inhibitor and substrate are recognized. Moreover, our findings shed light on conformational changes when transitioning from an outward-facing to an inward-facing state and establish a framework for understanding the transport cycle, which relies on the stabilization of the outward-facing state by a short intracellular helix, IH1.

Novel endolithic bacteria of phylum Chloroflexota reveal a myriad of potential survival strategies in the Antarctic desert.

The ice-free McMurdo Dry Valleys of Antarctica are dominated by nutrient-poor mineral soil and rocky outcrops. The principal habitat for microorganisms is within rocks (endolithic). In this environment, microorganisms are provided with protection against sub-zero temperatures, rapid thermal fluctuations, extreme dryness, and ultraviolet and solar radiation. Endolithic communities include lichen, algae, fungi, and a diverse array of bacteria. Chloroflexota is among the most abundant bacterial phyla present in these communities. Among the Chloroflexota are four novel classes of bacteria, here named Candidatus Spiritibacteria class. nov. (=UBA5177), Candidatus Martimicrobia class. nov. (=UBA4733), Candidatus Tarhunnaeia class. nov. (=UBA6077), and Candidatus Uliximicrobia class. nov. (=UBA2235). We retrieved 17 high-quality metagenome-assembled genomes (MAGs) that represent these four classes. Based on genome predictions, all these bacteria are inferred to be aerobic heterotrophs that encode enzymes for the catabolism of diverse sugars. These and other organic substrates are likely derived from lichen, algae, and fungi, as metabolites (including photosynthate), cell wall components, and extracellular matrix components. The majority of MAGs encode the capacity for trace gas oxidation using high-affinity uptake hydrogenases, which could provide energy and metabolic water required for survival and persistence. Furthermore, some MAGs encode the capacity to couple the energy generated from H2 and CO oxidation to support carbon fixation (atmospheric chemosynthesis). All encode mechanisms for the detoxification and efflux of heavy metals. Certain MAGs encode features that indicate possible interactions with other organisms, such as Tc-type toxin complexes, hemolysins, and macroglobulins.IMPORTANCEThe ice-free McMurdo Dry Valleys of Antarctica are the coldest and most hyperarid desert on Earth. It is, therefore, the closest analog to the surface of the planet Mars. Bacteria and other microorganisms survive by inhabiting airspaces within rocks (endolithic). We identify four novel classes of phylum Chloroflexota, and, based on interrogation of 17 metagenome-assembled genomes, we predict specific metabolic and physiological adaptations that facilitate the survival of these bacteria in this harsh environment-including oxidation of trace gases and the utilization of nutrients (including sugars) derived from lichen, algae, and fungi. We propose that such adaptations allow these endolithic bacteria to eke out an existence in this cold and extremely dry habitat.

Zinc Starvation Induces Cell Wall Remodeling and Activates the Antioxidant Defense System in Fonsecaea pedrosoi.

The survival of pathogenic fungi in the host after invasion depends on their ability to obtain nutrients, which include the transition metal zinc. This essential micronutrient is required to maintain the structure and function of various proteins and, therefore, plays a critical role in various biological processes. The host's nutritional immunity limits the availability of zinc to pathogenic fungi mainly by the action of calprotectin, a component of neutrophil extracellular traps. Here we investigated the adaptive responses of Fonsecaea pedrosoi to zinc-limiting conditions. This black fungus is the main etiological agent of chromoblastomycosis, a chronic neglected tropical disease that affects subcutaneous tissues. Following exposure to a zinc-limited environment, F. pedrosoi induces a high-affinity zinc uptake machinery, composed of zinc transporters and the zincophore Pra1. A proteomic approach was used to define proteins regulated by zinc deprivation. Cell wall remodeling, changes in neutral lipids homeostasis, and activation of the antioxidant system were the main strategies for survival in the hostile environment. Furthermore, the downregulation of enzymes required for sulfate assimilation was evident. Together, the adaptive responses allow fungal growth and development and reveals molecules that may be related to fungal persistence in the host.

Inactivation of siderophore iron-chelating moieties by the fungal wheat root symbiont Pyrenophora biseptata.

We investigated the ability of four plant and soil-associated fungi to modify or degrade siderophore structures leading to reduced siderophore iron-affinity in iron-limited and iron-replete cultures. Pyrenophora biseptata, a melanized fungus from wheat roots, was effective in inactivating siderophore iron-chelating moieties. In the supernatant solution, the tris-hydroxamate siderophore desferrioxamine B (DFOB) underwent a stepwise reduction of the three hydroxamate groups in DFOB to amides leading to a progressive loss in iron affinity. A mechanism is suggested based on the formation of transient ferrous iron followed by reduction of the siderophore hydroxamate groups during fungal high-affinity reductive iron uptake. P. biseptata also produced its own tris-hydroxamate siderophores (neocoprogen I and II, coprogen and dimerum acid) in iron-limited media and we observed loss of hydroxamate chelating groups during incubation in a manner analogous to DFOB. A redox-based reaction was also involved with the tris-catecholate siderophore protochelin in which oxidation of the catechol groups to quinones was observed. The new siderophore inactivating activity of the wheat symbiont P. biseptata is potentially widespread among fungi with implications for the availability of iron to plants and the surrounding microbiome in siderophore-rich environments.

The superior growth of Kluyveromyces marxianus at very low potassium concentrations is enabled by the high-affinity potassium transporter Hak1.

The non-conventional yeast Kluyveromyces marxianus has recently emerged as a promising candidate for many food, environment, and biotechnology applications. This yeast is thermotolerant and has robust growth under many adverse conditions. Here, we show that its ability to grow under potassium-limiting conditions is much better than that of Saccharomyces cerevisiae, suggesting a very efficient and high-affinity potassium uptake system(s) in this species. The K. marxianus genome contains two genes for putative potassium transporters: KmHAK1 and KmTRK1. To characterize the products of the two genes, we constructed single and double knock-out mutants in K. marxianus and also expressed both genes in an S. cerevisiae mutant, that lacks potassium importers. Our results in K. marxianus and S. cerevisiae revealed that both genes encode efficient high-affinity potassium transporters, contributing to potassium homeostasis and maintaining plasma-membrane potential and cytosolic pH. In K. marxianus, the presence of HAK1 supports growth at low K+ much better than that of TRK1, probably because the substrate affinity of KmHak1 is about 10-fold higher than that of KmTrk1, and its expression is induced ~80-fold upon potassium starvation. KmHak1 is crucial for salt stress survival in both K. marxianus and S. cerevisiae. In co-expression experiments with ScTrk1 and ScTrk2, its robustness contributes to an increased tolerance of S. cerevisiae cells to sodium and lithium salt stress.

Sugar Transport in Thermophiles: Bridging Lignocellulose Deconstruction and Bioconversion.

Biomass degrading thermophiles play an indispensable role in building lignocellulose-based supply chains. They operate at high temperatures to improve process efficiencies and minimize mesophilic contamination, can overcome lignocellulose recalcitrance through their native carbohydrate-active enzyme (CAZyme) inventory, and can utilize a wide range of sugar substrates. However, sugar transport in thermophiles is poorly understood and investigated, as compared to enzymatic lignocellulose deconstruction and metabolic conversion of sugars to value-added chemicals. Here, we review the general modes of sugar transport in thermophilic bacteria and archaea, covering the structural, molecular, and biophysical basis of their high-affinity sugar uptake. We also review how thermophilic sugar transporters are identified and characterized, including recent genetic studies on their function. With this understanding of sugar transport, we discuss strategies for how sugar transport can be engineered in thermophiles, with the potential to enhance the conversion of lignocellulosic biomass into renewable products.

Zinc acquisition and its contribution to Klebsiella pneumoniae virulence.

Klebsiella pneumoniae is a World Health Organization priority pathogen and a significant clinical concern for infections of the respiratory and urinary tracts due to widespread and increasing resistance to antimicrobials. In the absence of a vaccine, there is an urgent need to identify novel targets for therapeutic development. Bacterial pathogens, including K. pneumoniae, require the d-block metal ion zinc as an essential micronutrient, which serves as a cofactor for ~6% of the proteome. During infection, zinc acquisition necessitates the use of high affinity uptake systems to overcome niche-specific zinc limitation and host-mediated nutritional immunity. Here, we report the identification of ZnuCBA and ZniCBA, two ATP-binding cassette permeases that are highly conserved in Klebsiella species and contribute to K. pneumoniae AJ218 zinc homeostasis, and the high-resolution structure of the zinc-recruiting solute-binding protein ZniA. The Znu and Zni permeases appear functionally redundant with abrogation of both systems required to reduce K. pneumoniae zinc accumulation. Disruption of both systems also exerted pleiotropic effects on the homeostasis of other d-block elements. Zinc limitation perturbed K. pneumoniae cell morphology and compromised resistance to stressors, such as salt and oxidative stress. The mutant strain lacking both systems showed significantly impaired virulence in acute lung infection models, highlighting the necessity of zinc acquisition in the virulence and pathogenicity of K. pneumoniae.

Coordinated phosphate uptake by extracellular alkaline phosphatase and solute carrier transporters in marine diatoms.

Marine diatoms express genes encoding potential phosphate transporter and alkaline phosphatase (APase) under phosphate-limited (-P) condition. This indicates that diatoms use high-affinity phosphate uptake system with organic phosphate hydration. The function of molecules playing roles for Pi uptake was determined in this study. Pi uptake and APase activity of two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana, were monitored during acclimation to -P condition. The transcript levels of Pi transporter were analyzed, and Pi transporters were localized with GFP tagging in diatom cells. KO mutants of plasma membrane solute carrier proteins (SLC34s) or APase were established, and their phenotype was evaluated. Some Na+ /Pi transporter candidates, SLC34s in P. tricornutum and T. pseudonana, increased transcript under -P condition. Whole-cell Pi transport was specifically stimulated by sodium ion but independent of potassium, lithium, or proton. Genome-editing KO of PtSLC34-5 and APase (Pt49678) in P. tricornutum was highly inhibitory for Pi uptake, and KO of TpSLC34-2 was also highly inhibitory for Pi uptake in T. pseudonana. SLC34s and APase were co-expressed under -P conditions in marine diatoms. SLC34s play a major role in the initial acclimation stage of diatom cells to -P condition and APase plays an increasing role in the prolonged Pi-starved condition.

PsNRT2.3 interacts with PsNAR to promote high-affinity nitrate uptake in pea (Pisum sativum L.)

Nitrate, the primary form of nitrogen absorbed by plants, supplies essential compounds for plant growth and development. Peas are frequently used as rotation crops to improve and stabilize soil fertility. However, the determinants of nitrate uptake and transport in peas remain largely unclear, primarily due to the pea genome's complexity and size. In this study, we utilized the complete genomic information of peas to identify three PsNRT2 family genes within the pea genome. We conducted a comprehensive examination of their protein conserved domains, physicochemical properties, gene structure, and phylogenetic evolution, revealing PsNRT2.3 as the potential key gene for high-affinity nitrate transport in peas. Subcellular localization studies indicated that PsNRT2.3 resides on the plasma membrane. Using hairy root transformation, we noted the predominant expression of PsNRT2.3 in the root stele, which is inducible by nitrate. Our experiments involving overexpression and silencing methods further confirmed that PsNRT2.3 plays a key role in enhancing nitrate uptake in peas. Additionally, our work showed that PsNAR could interact with PsNRT2.3, modulating pea nitrate uptake. After silencing PsNAR, even with the normal expression of PsNRT2.3, the ability of peas to absorb nitrate was significantly reduced. In conclusion, this study identifies the high-affinity nitrate transport gene PsNRT2.3 in peas and clarifies its critical role and regulatory network in nitrate transport, contributing to a new understanding of nitrate utilization in peas.

Tryptophan-Starved Human Cells Overexpressing Tryptophanyl-tRNA Synthetase Enhance High-Affinity Tryptophan Uptake via Enzymatic Production of Tryptophanyl-AMP.

Our previous study demonstrated that L-tryptophan (Trp)-depleted cells display a marked enhancement in Trp uptake facilitated by extracellular tryptophanyl-tRNA synthetase (TrpRS). Here, we show that Trp uptake into TrpRS-overexpressing cells is also markedly elevated upon Trp starvation. These findings indicate that a Trp-deficient condition is critical for Trp uptake, not only into cells to which TrpRS protein has been added but also into TrpRS-overexpressing cells. We also show that overexpression of TrpRS mutants, which cannot synthesize tryptophanyl-AMP, does not promote Trp uptake, and that inhibition of tryptophanyl-AMP synthesis suppresses this uptake. Overall, these data suggest that tryptophanyl-AMP production by TrpRS is critical for high-affinity Trp uptake.