High-affinity Transporter Research Articles

Structural insights into the Oryza sativa cation transporters HKTs in salt tolerance.

The high-affinity potassium transporters (HKTs), selectively permeable to either Na+ alone or Na+/K+, play pivotal roles in maintaining plant Na+/K+ homeostasis. Although their involvement in salt tolerance is widely reported, the molecular underpinnings of Oryza sativa HKTs remain elusive. In this study, we elucidate the structures of OsHKT1;1 and OsHKT2;1, representing two distinct classes of rice HKTs. The dimeric assembled OsHKTs can be structurally divided into four domains. At the dimer interface, a half-helix or a loop in the third domain is coordinated by the C-terminal region of the opposite subunit. Additionally, we present the structures of OsHKT1;5 salt-tolerant and salt-sensitive variants, a key quantitative trait locus associated with salt tolerance. The salt-tolerant variant of OsHKT1;5 exhibits enhanced Na+ transport capability and displays a more flexible conformation. These findings shed light on the molecular basis of rice HKTs and provide insights into their role in salt tolerance.

Apoptosis signal-regulating kinase 1 (Ask1) deficiency alleviates MPP+-induced impairment of evoked dopamine release in the mouse hippocampus.

The dopaminergic system is susceptible to dysfunction in numerous neurological diseases, including Parkinson's disease (PD). In addition to motor symptoms, some PD patients may experience non-motor symptoms, including cognitive and memory deficits. A possible explanation for their manifestation is a disturbed pattern of dopamine release in brain regions involved in learning and memory, such as the hippocampus. Therefore, investigating neuropathological alterations in dopamine release prior to neurodegeneration is imperative. This study aimed to characterize evoked hippocampal dopamine release and assess the impact of the neurotoxin MPP+ using a genetically encoded dopamine sensor and gene expression analysis. Additionally, considering the potential neuroprotective attributes demonstrated by apoptosis signal-regulating kinase 1 (Ask1) in various animal-disease-like models, the study also aimed to determine whether Ask1 knockdown restores MPP+-altered dopamine release in acute hippocampal slices. We applied variations of low- and high-frequency stimulation to evoke dopamine release within different hippocampal regions and discovered that acute application of MPP+ reduced the amount of dopamine released and hindered the recovery of dopamine release after repeated stimulation. In addition, we observed that Ask1 deficiency attenuated the detrimental effects of MPP+ on the recovery of dopamine release after repeated stimulation. RNA sequencing analysis indicated that genes associated with the synaptic pathways are involved in response to MPP+ exposure. Notably, Ask1 deficiency was found to downregulate the expression of Slc5a7, a gene encoding a sodium-dependent high-affinity choline transporter that regulates acetylcholine levels. Respective follow-up experiments indicated that Slc5a7 plays a role in Ask1 deficiency-mediated protection against MPP+ neurotoxicity. In addition, increasing acetylcholine levels using an acetylcholinesterase inhibitor could exacerbate the toxicity of MPP+. In conclusion, our data imply that the modulation of the dopamine-acetylcholine balance may be a crucial mechanism of action underlying the neuroprotective effects of Ask1 deficiency in PD.

Structures and ion transport mechanisms of plant high-affinity potassium transporters

Plant high-affinity K+ transporters (HKTs) mediate Na+ and K+ uptake, maintain Na+/K+ homeostasis, and therefore play crucial roles in plant salt tolerance. In this study, we present cryoelectron microscopy structures of HKTs from two classes, class I HKT1;1 from Arabidopsis thaliana (AtHKT1;1) and class II HKT2;1 from Triticum aestivum (TaHKT2;1), in both Na+- and K+-bound states at 2.6- to 3.0-Å resolutions. Both AtHKT1;1 and TaHKT2;1 function as homodimers. Each HKT subunit consists of four tandem domain units (D1–D4) with a repeated K+-channel-like M–P–M topology. In each subunit, D1–D4 assemble into an ion conduction pore with a pseudo-four-fold symmetry. Although both TaHKT2;1 and AtHKT1;1 have only one putative Na+ ion bound in the selectivity filter with a similar coordination pattern, the two HKTs display different K+ binding modes in the filter. TaHKT2;1 has three K+ ions bound in the selectivity filter, but AtHKT1;1 has only two K+ ions bound in the filter, which has a narrowed external entrance due to the presence of a Ser residue in the first filter motif. These structures, along with computational, mutational, and electrophysiological analyses, enable us to pinpoint key residues that are critical for the ion selectivity of HKTs. The findings provide new insights into the ion selectivity and ion transport mechanisms of plant HKTs and improve our understanding about how HKTs mediate plant salt tolerance and enhance crop growth.

Translatome and Transcriptome Analyses Reveal the Mechanism that Underlies the Enhancement of Salt Stress by the Small Peptide Ospep5 in Plants.

Salt stress significantly impedes plant growth and the crop yield. This study utilized de novo transcriptome assembly and ribosome profiling to explore mRNA translation's role in rice salt tolerance. We identified unrecognized translated open reading frames (ORFs), including 42 upstream transcripts and 86 unannotated transcripts. A noteworthy discovery was the role of a small ORF, Ospep5, in conferring salt tolerance. Overexpression of Ospep5 in plants increased salt tolerance, while its absence led to heightened sensitivity. This hypothesis was corroborated by the findings that exogenous application of the synthetic small peptide Ospep5 bolstered salt tolerance in both rice and Arabidopsis. We found that the mechanism underpinning the Ospep5-mediated salt tolerance involves the maintenance of intracellular Na+/K+ homeostasis, facilitated by upregulation of high-affinity potassium transporters (HKT) and Na+/H+ exchangers (SOS1). Furthermore, a comprehensive multiomics approach, particularly ribosome profiling, is instrumental in uncovering unannotated ORFs and elucidating their functions in plant stress responses.

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.

Exploring OsHAK Transporters for the Salt Responsiveness in Rice

Environmental stresses are the major constraint in crop growth and productivity, particularly in rice. Improving rice against major abiotic stressors such as salinity is one of the major thrust areas. In this direction, the trait of sodium exclusion was the primary focus among the rice breeders to enhance salt tolerance. Along similar lines, increased uptake of potassium, the counter ion of Na+, through high-affinity potassium transporters (HAK) assumes significance in the context of maintaining a favorable Na/K ratio, a low Na/K is an indicator of salt tolerance. The OsHAK family in rice includes 27 members and an insight into the evolution, structure, and expression through in silico approaches was attempted. Phylogenetic analysis exhibited four distinct groupings of the OsHAK family, and functional motif analysis revealed a characteristic consensus sequence among the OsHAK transporters. Relative gene expression analysis based on published microarray data put forth the differential regulation of OsHAK3 X2, OsHAK8, OsHAK14(X2), OsHAK15 and OsHAK26 between salt-tolerant landrace Pokkali and susceptible cultivar IR29 and their tissue-specific expression profiles predicted. This study narrowed down a set of putative OsHAKs from the larger family for their role in enhancing cellular potassium under salt stress thus paving the way for crop improvement.

Loss of the yeast transporter Agp2 upregulates the pleiotropic drug-resistant pump Pdr5 and confers resistance to the protein synthesis inhibitor cycloheximide.

The transmembrane protein Agp2, initially shown as a transporter of L-carnitine, mediates the high-affinity transport of polyamines and the anticancer drug bleomycin-A5. Cells lacking Agp2 are hyper-resistant to polyamine and bleomycin-A5. In these earlier studies, we showed that the protein synthesis inhibitor cycloheximide blocked the uptake of bleomycin-A5 into the cells suggesting that the drug uptake system may require de novo synthesis. However, our recent findings demonstrated that cycloheximide, instead, induced rapid degradation of Agp2, and in the absence of Agp2 cells are resistant to cycloheximide. These observations raised the possibility that the degradation of Agp2 may allow the cell to alter its drug resistance network to combat the toxic effects of cycloheximide. In this study, we show that membrane extracts from agp2Δ mutants accentuated several proteins that were differentially expressed in comparison to the parent. Mass spectrometry analysis of the membrane extracts uncovered the pleiotropic drug efflux pump, Pdr5, involved in the efflux of cycloheximide, as a key protein upregulated in the agp2Δ mutant. Moreover, a global gene expression analysis revealed that 322 genes were differentially affected in the agp2Δ mutant versus the parent, including the prominent PDR5 gene and genes required for mitochondrial function. We further show that Agp2 is associated with the upstream region of the PDR5 gene, leading to the hypothesis that cycloheximide resistance displayed by the agp2Δ mutant is due to the derepression of the PDR5 gene.

The Functional Diversity of the High-Affinity Nitrate Transporter Gene Family in Hexaploid Wheat: Insights from Distinct Expression Profiles.

High-affinity nitrate transporters (NRT) are key components for nitrogen (N) acquisition and distribution within plants. However, insights on these transporters in wheat are scarce. This study presents a comprehensive analysis of the NRT2 and NRT3 gene families, where the aim is to shed light on their functionality and to evaluate their responses to N availability. A total of 53 NRT2s and 11 NRT3s were identified in the bread wheat genome, and these were grouped into different clades and homoeologous subgroups. The transcriptional dynamics of the identified NRT2 and NRT3 genes, in response to N starvation and nitrate resupply, were examined by RT-qPCR in the roots and shoots of hydroponically grown wheat plants through a time course experiment. Additionally, the spatial expression patterns of these genes were explored within the plant. The NRT2s of clade 1, TaNRT2.1-2.6, showed a root-specific expression and significant upregulation in response to N starvation, thus emphasizing a role in N acquisition. However, most of the clade 2 NRT2s displayed reduced expression under N-starved conditions. Nitrate resupply after N starvation revealed rapid responsiveness in TaNRT2.1-2.6, while clade 2 genes exhibited gradual induction, primarily in the roots. TaNRT2.18 was highly expressed in above-ground tissues and exhibited distinct nitrate-related response patterns for roots and shoots. The TaNRT3 gene expression closely paralleled the profiles of TaNRT2.1-2.6 in response to nitrate induction. These findings enhance the understanding of NRT2 and NRT3 involvement in nitrogen uptake and utilization, and they could have practical implications for improving nitrogen use efficiency. The study also recommends a standardized nomenclature for wheat NRT2 genes, thereby addressing prior naming inconsistencies.

Genome-wide transcriptome profiling of mulberry (Morus alba) response to boron deficiency and toxicity reveal candidate genes associated with boron tolerance in leaves

Mulberry (Morus alba) is an essential plant with countless economic benefits; however, its growth and metabolic processes are hampered by boron (B) stresses. Very little research has been performed to elucidate boron tolerance and detoxification mechanisms in this species. The M. alba cultivar, Yu-711, was exposed to five different concentrations of boric acid (H3BO3), including deficient (T1; 0 mM) moderate B deficiency (T2; 0.02 mM), sufficient (CK; 0.1 mM) and toxic (T3 and T4; 0.5 and 1 mM) levels for 18 days of growth in pots experiment. Transcriptome analysis of B deficiency and toxicity treatments was performed on mulberry leaves. The transcriptome data reveal that a total of 6114 genes were differentially expressed (DEGs), of which 3830 were up-regulated and 2284 were down-regulated. A comparative analysis between treatment groups CK-vs-T1 (deficiency) and CK-vs-T4 (toxicity) indicates that 590 and 1383 genes were down-regulated in both deficiency and B toxicity, respectively. The results show that 206 genes were differentially expressed in all treatments. B deficiency and toxicity significantly altered the expression of the key aquaporins (PIP2-1, PIP2-7, PIP2-4 and NIP3-1) and high-affinity boron transporter genes (BOR1 and BOR7). In addition, boron stress also altered the expression of antioxidants and photosynthesis-related genes. B stresses were found to alter several transcription factors including ERF1B, which is associated with the regulation of boron uptake and the synthesis and signaling of phytohormones. Unravelling the mechanisms of B tolerance and detoxification is important and would give us further insight into how B stresses affect mulberry plants.

The Mechanisms of Molybdate Distribution and Homeostasis with Special Focus on the Model Plant Arabidopsis thaliana.

This review article deals with the pathways of cellular and global molybdate distribution in plants, especially with a full overview for the model plant Arabidopsis thaliana. In its oxidized state as bioavailable molybdate, molybdenum can be absorbed from the environment. Especially in higher plants, molybdenum is indispensable as part of the molybdenum cofactor (Moco), which is responsible for functionality as a prosthetic group in a variety of essential enzymes like nitrate reductase and sulfite oxidase. Therefore, plants need mechanisms for molybdate import and transport within the organism, which are accomplished via high-affinity molybdate transporter (MOT) localized in different cells and membranes. Two different MOT families were identified. Legumes like Glycine max or Medicago truncatula have an especially increased number of MOT1 family members for supplying their symbionts with molybdate for nitrogenase activity. In Arabidopsis thaliana especially, the complete pathway followed by molybdate through the plant is traceable. Not only the uptake from soil by MOT1.1 and its distribution to leaves, flowers, and seeds by MOT2-family members was identified, but also that inside the cell. the transport trough the cytoplasm and the vacuolar storage mechanisms depending on glutathione were described. Finally, supplying the Moco biosynthesis complex by MOT1.2 and MOT2.1 was demonstrated.

Overexpression of NtLHT1 affects the development of leaf morphology and abiotic tolerance in tobacco

LYSINE HISTIDINE TRANSPORTER1 (LHT1) is a crucial broad-specificity and high-affinity amino acid transporter affecting the uptake of nitrogen and probably the tolerance to abiotic stress in plants. However, little is known about the phenotypic functions of LHT1 in plant growth and development and abiotic stress tolerance. In this study, we identified the NtLHT1 gene from the tobacco variety Honghuadajinyuan (HD) and determined its important roles in leaf morphological development and plant resistance to abiotic stress. Comprehensive functional analyses using knockout and overexpression transgenic lines (ntlht1 and OE) revealed overexpression of NtLHT1 accelerated leave senescence and increased plant height, leaf number and plant tolerance under cold, salt and drought stresses. In addition, NtLHT1 overexpression significantly decreased the leaf elongation of HD, causing the leaves to change from a long-elliptical shape to an elliptical shape. However silencing NtLHT1 decreased the seed germination rate under NaCl and PEG stresses. Moreover, NtLHT1 significantly affected the contents of various amino acids, such as the neutral, acidic, non-polar and aromatic amino acids, ethylene precursor (ACC), GA3 and IAA in tobacco. These results suggested that the amino acid and ethylene precursor ACC transport activities of NtLHT1 provide fine regulatory function for plant growth and development and plant tolerance to abiotic stress.

Enhancing cognitive function in chronic TBI: The Role of α7 nicotinic acetylcholine receptor modulation

Traumatic brain injury (TBI) results in several pathological changes within the hippocampus that result in adverse effects on learning and memory. Therapeutic strategies to enhance learning and memory after TBI are still in the early stages of clinical development. One strategy is to target the α7 nicotinic acetylcholine receptor (nAChR), which is highly expressed in the hippocampus and contributes to the formation of long-term memory. In our previous study, we found that AVL-3288, a positive allosteric modulator of the α7 nAChR, improved cognitive recovery in rats after moderate fluid-percussion injury (FPI). However, whether AVL-3288 improved cognitive recovery specifically through the α7 nAChR was not definitively determined. In this study we utilized Chrna7 knockout mice and compared their recovery to wild-type mice treated with AVL-3288 after TBI. We hypothesized that AVL-3288 treatment would improve learning and memory in wild-type mice, but not Chrna7−/− mice after TBI. Adult male C57BL/6 wild-type and Chrna7−/− mice received sham surgery or moderate controlled cortical impact (CCI) and recovered for 3 months. Mice were then treated with vehicle or AVL-3288 at 30 min prior to contextual fear conditioning. At 3 months after CCI, expression of α7 nAChR, choline acetyltransferase (ChAT), high-affinity choline transporter (ChT), and vesicular acetylcholine transporter (VAChT) were found to be significantly decreased in the hippocampus. Treatment of wild-type mice at 3 months after CCI with AVL-3288 significantly improved cue and contextual fear conditioning, whereas no beneficial effects were observed in Chrna7−/− mice. Parietal cortex and hippocampal atrophy were not improved with AVL-3288 treatment in either wild-type or Chrna7−/− mice. Our results indicate that AVL-3288 improves cognition during the chronic recovery phase of TBI through modulation of the α7 nAChR.

Integrated genomics provides insights into the evolution of the polyphosphate accumulation trait of Ca. Accumulibacter

Candidatus Accumulibacter, a prominent polyphosphate-accumulating organism (PAO) in wastewater treatment, plays a crucial role in enhanced biological phosphorus removal (EBPR). The genetic underpinnings of its polyphosphate accumulation capabilities, however, remain largely unknown. Here, we conducted a comprehensive genomic analysis of Ca. Accumulibacter-PAOs and their relatives within the Rhodocyclaceae family, identifying 124 core genes acquired via horizontal gene transfer (HGT) at its least common ancestor. Metatranscriptomic analysis of an enrichment culture of Ca. Accumulibacter revealed active transcription of 44 of these genes during an EBPR cycle, notably including the polyphosphate kinase 2 (PPK2) gene instead of the commonly recognized polyphosphate kinase 1 (PPK1) gene. Intriguingly, the phosphate regulon (Pho) genes showed minimal transcriptions, pointing to a distinctive fact of Pho dysregulation, where PhoU, the phosphate signaling complex protein, was not regulating the high-affinity phosphate transport (Pst) system, resulting in continuous phosphate uptake. To prevent phosphate toxicity, Ca. Accumulibacter utilized the laterally acquired PPK2 to condense phosphate into polyphosphate, resulting in the polyphosphate-accumulating feature. This study provides novel insights into the evolutionary emergence of the polyphosphate-accumulating trait in Ca. Accumulibacter, offering potential advancements in understanding the PAO phenotype in the EBPR process.

Knockout of the sugar transporter OsSTP15 enhances grain yield by improving tiller number due to increased sugar content in the shoot base of rice (Oryza sativa L.).

Sugar transporter proteins (STPs) play critical roles in regulating plant stress tolerance, growth, and development. However, the role of STPs in regulating crop yield is poorly understood. This study elucidates the mechanism by which knockout of the sugar transporter OsSTP15 enhances grain yield via increasing the tiller number in rice. We found that OsSTP15 is specifically expressed in the shoot base and vascular bundle sheath of seedlings and encodes a plasma membrane-localized high-affinity glucose efflux transporter. OsSTP15 knockout enhanced sucrose and trehalose-6-phosphate (Tre6P) synthesis in leaves and improved sucrose transport to the shoot base by inducing the expression of sucrose transporters. Higher glucose, sucrose, and Tre6P contents were observed at the shoot base of stp15 plants. Transcriptome and metabolome analyses of the shoot base demonstrated that OsSTP15 knockout upregulated the expression of cytokinin (CK) synthesis- and signaling pathway-related genes and increased CK levels. These findings suggest that OsSTP15 knockout represses glucose export from the cytoplasm and simultaneously enhances sugar transport from source leaves to the shoot base by promoting the synthesis of sucrose and Tre6P in leaves. Subsequent accumulation of glucose, sucrose, and Tre6P in the shoot base promotes tillering by stimulating the CK signaling pathway.

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.

Physiological and Transcriptomic Analysis Provides Insights into Low Nitrogen Stress in Foxtail Millet (Setaria italica L.).

Foxtail millet (Setaria italica (L.) P. Beauv) is an important food and forage crop that is well adapted to nutrient-poor soils. However, our understanding of how different LN-tolerant foxtail millet varieties adapt to long-term low nitrogen (LN) stress at the physiological and molecular levels remains limited. In this study, two foxtail millet varieties with contrasting LN tolerance properties were investigated through analyses of physiological parameters and transcriptomics. The physiological results indicate that JG20 (high tolerance to LN) exhibited superior biomass accumulation both in its shoots and roots, and higher nitrogen content, soluble sugar concentration, soluble protein concentration, zeatin concentration in shoot, and lower soluble sugar and soluble protein concentration in its roots compared to JG22 (sensitive to LN) under LN, this indicated that the LN-tolerant foxtail millet variety can allocate more functional substance to its shoots to sustain aboveground growth and maintain high root activity by utilizing low soluble sugar and protein under LN conditions. In the transcriptomics analysis, JG20 exhibited a greater number of differentially expressed genes (DEGs) compared to JG22 in both its shoots and roots in response to LN stress. These LN-responsive genes were enriched in glycolysis metabolism, photosynthesis, hormone metabolism, and nitrogen metabolism. Furthermore, in the shoots, the glutamine synthetase gene SiGS5, chlorophyll apoprotein of photosystem II gene SiPsbQ, ATP synthase subunit gene Sib, zeatin synthesis genes SiAHP1, and aldose 1-epimerase gene SiAEP, and, in the roots, the high-affinity nitrate transporter genes SiNRT2.3, SiNRT2.4, glutamate synthase gene SiGOGAT2, fructose-bisphosphate aldolase gene SiFBA5, were important genes involved in the LN tolerance of the foxtail millet variety. Hence, our study implies that the identified genes and metabolic pathways contribute valuable insights into the mechanisms underlying LN tolerance in foxtail millet.

Stereoselective Inhibition of High- and Low-Affinity Organic Cation Transporters.

Many drugs have chiral centers and are therapeutically applied as racemates. Thus, the stereoselectivity in their interactions with membrane transporters needs to be addressed. Here, we studied stereoselectivity in inhibiting organic cation transporters (OCTs) 1, 2, and 3 and the high-affinity monoamine transporters (MATs) NET and SERT. Selectivity by the inhibition of 35 pairs of enantiomers significantly varied among the three closely related OCTs. OCT1 inhibition was nonselective in almost all cases, whereas OCT2 was stereoselectively inhibited by 45% of the analyzed drugs. However, the stereoselectivity of the OCT2 was only moderate with the highest selectivity observed for pramipexole. The (R)-enantiomer inhibited OCT2 4-fold more than the (S)-enantiomer. OCT3 showed the greatest stereoselectivity in its inhibition. (R)-Tolterodine and (S)-zolmitriptan inhibited OCT3 11-fold and 25-fold more than their respective counterparts. Interestingly, in most cases, the pharmacodynamically active enantiomer was also the stronger OCT inhibitor. In addition, stereoselectivity in the OCT inhibition appeared not to depend on the transported substrate. For high-affinity MATs, our data confirmed the stereoselective inhibition of NET and SERT by several antidepressants. However, the stereoselectivity measured here was generally lower than that reported in the literature. Unexpectedly, the high-affinity MATs were not significantly more stereoselectively inhibited than the polyspecific OCTs. Combining our in vitro OCT inhibition data with available stereoselective pharmacokinetic analyses revealed different risks of drug-drug interactions, especially at OCT2. For the tricyclic antidepressant doxepine, only the (E)-isomer showed an increased risk of drug-drug interactions according to guidelines from regulatory authorities for renal transporters. However, most chiral drugs show only minor stereoselectivity in the inhibition of OCTs in vitro, which is unlikely to translate into clinical consequences.

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.

Identification of the High-Affinity Potassium Transporter Gene Family (HKT) in Brassica U-Triangle Species and Its Potential Roles in Abiotic Stress in Brassica napus L.

Members of the high-affinity potassium transporter (HKT) protein family regulate the uptake and homeostasis of sodium and potassium ions, but little research describes their roles in response to abiotic stresses in rapeseed (Brassica napus L.). In this study, we identified and characterized a total of 36 HKT genes from the species comprising the triangle of U model (U-triangle species): B. rapa, B. nigra, B. oleracea, B. juncea, B. napus, and B. carinata. We analyzed the phylogenetic relationships, gene structures, motif compositions, and chromosomal distributions of the HKT family members of rapeseed. Based on their phylogenetic relationships and assemblage of functional domains, we classified the HKT members into four subgroups, HKT1;1 to HKT1;4. Analysis of the nonsynonymous substitutions (Ka), synonymous substitutions (Ks), and the Ka/Ks ratios of HKT gene pairs suggested that these genes have experienced strong purifying selective pressure after duplication, with their evolutionary relationships supporting the U-triangle theory. Furthermore, the expression profiles of BnaHKT genes varies among potassium, phytohormone and heavy-metal treatment. Their repression provides resistance to heavy-metal stress, possibly by limiting uptake. Our results systematically reveal the characteristics of HKT family proteins and their encoding genes in six Brassica species and lay a foundation for further exploration of the role of HKT family genes in heavy-metal tolerance.

Integrated BSA-seq and RNA-seq analysis to identify candidate genes associated with nitrogen utilization efficiency (NUtE) in rapeseed (Brassica napus L.)

Rapeseed (Brassica napus L.) is one of the important oil crops, with a high demand for nitrogen (N). It is essential to explore the potential of rapeseed to improve nitrogen utilization efficiency (NUtE). Rapeseed is an allotetraploid crop with a relatively large and complex genome, and there are few studies on the mapping of genes related to NUtE regulation. In this study, we used the combination of bulk segregant analysis sequencing (BSA-Seq) and RNA sequencing (RNA-Seq) to analyze the N-efficient genotype ‘Zheyou 18’ and N-inefficient genotype ‘Sollux’, to identify the genetic regulatory mechanisms. Several candidate genes were screened, such as the high-affinity nitrate transporter gene NRT2.1 (BnaC08g43370D) and the abscisic acid (ABA) signal transduction-related genes (BnaC02g14540D, BnaA03g20760D, and BnaA05g01330D). BnaA05g01330D was annotated as ABA-INDUCIBLE bHLH-TYPE TRANSCRIPTION FACTOR (AIB/bHLH17), which was highly expressed in the root. The results showed that the primary root length of the ataib mutant was significantly longer than that of the wild type under low N conditions. Overexpression of BnaA5.AIB could reduce the NUtE under low N levels in Arabidopsis (Arabidopsis thaliana). Candidate genes identified in this study may be involved in the regulation of NUtE in rapeseed, and new functions of AIB in orchestrating N uptake and utilization have been revealed. It is indicated that BnaA5.AIB may be the key factor that links ABA to N signaling and a negative regulator of NUtE. It will provide a theoretical basis and application prospect for resource conservation, environmental protection, and sustainable agricultural development.