Amino Acid Permease Research Articles

Identification of an Acidic Amino Acid Permease Involved in d-Aspartate Uptake in the Yeast Cryptococcus humicola.

d-aspartate oxidase (DDO) catalyzes the oxidative deamination of acidic d-amino acids, and its production is induced by d-Asp in several eukaryotes. The yeast Cryptococcus humicola strain UJ1 produces large amounts of DDO (ChDDO) only in the presence of d-Asp. In this study, we analyzed the relationship between d-Asp uptake by an amino acid permease (Aap) and the inducible expression of ChDDO. We identified two acidic Aap homologs, named “ChAap4 and ChAap5,” in the yeast genome sequence. ChAAP4 deletion resulted in partial growth defects on d-Asp as well as l-Asp, l-Glu, and l-Phe at pH 7, whereas ChAAP5 deletion caused partial growth defects on l-Phe and l-Lys, suggesting that ChAap4 might participate in d-Asp uptake as an acidic Aap. Interestingly, the growth of the Chaap4 strain on d- or l-Asp was completely abolished at pH 10, suggesting that ChAap4 is the only Aap responsible for d- and l-Asp uptake under high alkaline conditions. In addition, ChAAP4 deletion significantly decreased the induction of DDO activity and ChDDO transcription in the presence of d-Asp. This study revealed that d-Asp uptake by ChAap4 might be involved in the induction of ChDDO expression by d-Asp.

The Amino Acid Transporter OsAAP4 Contributes to Rice Tillering and Grain Yield by Regulating Neutral Amino Acid Allocation through Two Splicing Variants

BackgroundAmino acids, which are transported by amino acid transporters, are the major forms of organic nitrogen utilized by higher plants. Among the 19 Amino Acid Permease transporters (AAPs) in rice, only a small number of these genes have been reported to influence rice growth and development. However, whether other OsAAPs are responsible for rice growth and development is unclear.ResultsIn this study, we demonstrate that OsAAP4 promoter sequences are divergent between Indica and Japonica, with higher expression in the former, which produces more tillers and higher grain yield than does Japonica. Overexpression of two different splicing variants of OsAAP4 in Japonica ZH11 significantly increased rice tillering and grain yield as result of enhancing the neutral amino acid concentrations of Val, Pro, Thr and Leu. OsAAP4 RNA interference (RNAi) and mutant lines displayed opposite trends compared with overexpresing (OE) lines. In addition, exogenous Val or Pro at 0.5 mM significantly promoted the bud outgrowth of lines overexpressing an OsAAP4a splicing variant compared with ZH11, and exogenous Val or Pro at 2.0 mM significantly enhanced the bud outgrowth of lines overexpressing splicing variant OsAAP4b compared with ZH11. Of note, the results of a protoplast amino acid-uptake assay showed that Val or Pro at different concentrations was specifically transported and accumulated in these overexpressing lines. Transcriptome analysis further demonstrated that OsAAP4 may affect nitrogen transport and metabolism, and auxin, cytokinin signaling in regulating rice tillering.ConclusionOur results suggested that OsAAP4 contributes to rice tiller and grain yield by regulating neutral amino acid allocation through two different splicing variants and that OsAAP4 might have potential applications in rice breeding.

Concurrent overexpression of amino acid permease

Using pea as our model crop, we sought to understand the regulatory control over the import of sugars and amino acids into the developing seeds and its importance for seed yield and quality. Transgenic peas simultaneously overexpressing a sucrose transporter and an amino acid transporter were developed. Pod walls, seed coats, and cotyledons were analysed separately, as well as leaves subtending developing pods. Sucrose, starch, protein, free amino acids, and endogenous cytokinins were measured during development. Temporal gene expression analyses (RT-qPCR) of amino acid (AAP), sucrose (SUT), and SWEET transporter family members, and those from cell wall invertase, cytokinin biosynthetic (IPT) and degradation (CKX) gene families indicated a strong effect of the transgenes on gene expression. In seed coats of the double transgenics, increased content and prolonged presence of cytokinin was particularly noticeable. The transgenes effectively promoted transition of young sink leaves into source leaves. We suggest the increased flux of sucrose and amino acids from source to sink, along with increased interaction between cytokinin and cell wall invertase in developing seed coats led to enhanced sink activity, resulting in higher cotyledon sucrose at process pea harvest, and increased seed number and protein content at maturity.

Growth Inhibition by Amino Acids in Saccharomyces cerevisiae.

Amino acids are essential metabolites but can also be toxic when present at high levels intracellularly. Substrate-induced downregulation of amino acid transporters in Saccharomyces cerevisiae is thought to be a mechanism to avoid this toxicity. It has been shown that unregulated uptake by the general amino acid permease Gap1 causes cells to become sensitive to amino acids. Here, we show that overexpression of eight other amino acid transporters (Agp1, Bap2, Can1, Dip5, Gnp1, Lyp1, Put4, or Tat2) also induces a growth defect when specific single amino acids are present at concentrations of 0.5–5 mM. We can now state that all proteinogenic amino acids, as well as the important metabolite ornithine, are growth inhibitory to S. cerevisiae when transported into the cell at high enough levels. Measurements of initial transport rates and cytosolic pH show that toxicity is due to amino acid accumulation and not to the influx of co-transported protons. The amino acid sensitivity phenotype is a useful tool that reports on the in vivo activity of transporters and has allowed us to identify new transporter-specific substrates.

Chemical-Genetic Interactions with the Proline Analog L-Azetidine-2-Carboxylic Acid in Saccharomyces cerevisiae.

Non-proteinogenic amino acids, such as the proline analog L-azetidine-2-carboxylic acid (AZC), are detrimental to cells because they are mis-incorporated into proteins and lead to proteotoxic stress. Our goal was to identify genes that show chemical-genetic interactions with AZC in Saccharomyces cerevisiae and thus also potentially define the pathways cells use to cope with amino acid mis-incorporation. Screening the yeast deletion and temperature sensitive collections, we found 72 alleles with negative chemical-genetic interactions with AZC treatment and 12 alleles that suppress AZC toxicity. Many of the genes with negative chemical-genetic interactions are involved in protein quality control pathways through the proteasome. Genes involved in actin cytoskeleton organization and endocytosis also had negative chemical-genetic interactions with AZC. Related to this, the number of actin patches per cell increases upon AZC treatment. Many of the same cellular processes were identified to have interactions with proteotoxic stress caused by two other amino acid analogs, canavanine and thialysine, or a mistranslating tRNA variant that mis-incorporates serine at proline codons. Alleles that suppressed AZC-induced toxicity functioned through the amino acid sensing TOR pathway or controlled amino acid permeases required for AZC uptake. Further suggesting the potential of genetic changes to influence the cellular response to proteotoxic stress, overexpressing many of the genes that had a negative chemical-genetic interaction with AZC suppressed AZC toxicity.

The C2 domain of the ubiquitin ligase Rsp5 is required for ubiquitination of the endocytic protein Rvs167 upon change of nitrogen source.

Ubiquitination is a key signal for endocytosis of proteins on the plasma membrane. The ubiquitin ligase Rsp5 of Saccharomyces cerevisiae, which contains an amino-terminal membrane-binding C2 domain, three substrate-recognizing tryptophan-tryptophan (WW) domains and a carboxyl-terminal catalytic homologous to the E6-AP carboxyl terminus (HECT) domain, can ubiquitinate plasma membrane proteins directing them for endocytosis. Here, we examined the roles of the C2 domain in endocytosis for the downregulation of the general amino acid permease Gap1, which is one of nitrogen-regulated permeases in S. cerevisiae. First, we constructed several rsp5 mutants producing Rsp5 variants without the C2 domain or with amino acid changes of membrane-binding lysine residues. These mutants showed defects in endocytosis of Gap1 in response to a preferred nitrogen source. Intriguingly, we found that ubiquitination of Gap1 in these mutant cells was highly similar to that in wild-type cells during endocytosis. These results indicate that the C2 domain is essential for endocytosis but not for ubiquitination of substrates such as Gap1. Moreover, genetic and biochemical analyses showed that the endocytic protein Rvs167 was ubiquitinated via Rsp5 and the C2 domain was required for efficient ubiquitination in response to a preferred nitrogen source. Here, we propose a mechanism for the C2 domain-mediated endocytosis of plasma membrane permeases.

Retraction Note to: Molecular Cloning of an Amino Acid Permease Gene and Structural Characterization of the Protein in Common Bean (Phaseolus vulgaris L.).

The Editor-in-Chief has retracted this article [1]. Some concerns were raised after publication, and subsequent post publication peer review concluded that only a partial sequence of the AAP protein was used for bioinformatics analysis. As the amino acid binding domain of AAP was not completely covered in the truncated polypeptide the authors studied, the deduced structure has no biological significance. The Editor-in-Chief has invited the authors to submit a revised manuscript which will undergo peer review. All authors agree to this retraction.

Plasma membrane to vacuole traffic induced by glucose starvation requires Gga2-dependent sorting at the trans-Golgi network.

In the yeast Saccharomyces cerevisiae, acute glucose starvation induces rapid endocytosis followed by vacuolar degradation of many plasma membrane proteins. This process is essential for cell viability, but the regulatory mechanisms that control it remain poorly understood. Under normal growth conditions, a major regulatory decision for endocytic cargo occurs at the trans-Golgi network (TGN) where proteins can recycle back to the plasma membrane or can be recognized by TGN-localised clathrin adaptors that direct them towards the vacuole. However, glucose starvation reduces recycling and alters the localization and post-translational modification of TGN-localised clathrin adaptors. This raises the possibility that during glucose starvation endocytosed proteins are routed to the vacuole by a novel mechanism that bypasses the TGN or does not require TGN-localised clathrin adaptors. Here, we investigate the role of TGN-localised clathrin adaptors in the traffic of several amino acid permeases, including Can1, during glucose starvation. We find that Can1 transits through the TGN after endocytosis in both starved and normal conditions. Can1 and other amino acid permeases require TGN-localised clathrin adaptors for maximal delivery to the vacuole. Furthermore, these permeases are actively sorted to the vacuole, because ectopically forced de-ubiquitination at the TGN results in the recycling of the Tat1 permase in starved cells. Finally, we report that the Mup1 permease requires the clathrin adaptor Gga2 for vacuolar delivery. In contrast, the clathrin adaptor protein complex AP-1 plays a minor role, potentially in retaining permeases in the TGN, but it is otherwise dispensable for vacuolar delivery. This work elucidates one membrane trafficking pathway needed for yeast to respond to acute glucose starvation. It also reveals the functions of TGNlocalised clathrin adaptors in this process. Our results indicate that the same machinery is needed for vacuolar protein sorting at the GN in glucose starved cells as is needed in the presence of glucose. In addition, our findings provide further support for the model that the TGN is a transit point for many endocytosed proteins, and that Gga2 and AP-1 function in distinct pathways at the TGN.

Uptake of exogenous serine is important to maintain sphingolipid homeostasis in Saccharomyces cerevisiae.

Sphingolipids are abundant and essential molecules in eukaryotes that have crucial functions as signaling molecules and as membrane components. Sphingolipid biosynthesis starts in the endoplasmic reticulum with the condensation of serine and palmitoyl-CoA. Sphingolipid biosynthesis is highly regulated to maintain sphingolipid homeostasis. Even though, serine is an essential component of the sphingolipid biosynthesis pathway, its role in maintaining sphingolipid homeostasis has not been precisely studied. Here we show that serine uptake is an important factor for the regulation of sphingolipid biosynthesis in Saccharomyces cerevisiae. Using genetic experiments, we find the broad-specificity amino acid permease Gnp1 to be important for serine uptake. We confirm these results with serine uptake assays in gnp1Δ cells. We further show that uptake of exogenous serine by Gnp1 is important to maintain cellular serine levels and observe a specific connection between serine uptake and the first step of sphingolipid biosynthesis. Using mass spectrometry-based flux analysis, we further observed imported serine as the main source for de novo sphingolipid biosynthesis. Our results demonstrate that yeast cells preferentially use the uptake of exogenous serine to regulate sphingolipid biosynthesis. Our study can also be a starting point to analyze the role of serine uptake in mammalian sphingolipid metabolism.

Over-expression of Caj1, a plasma membrane associated J-domain protein in Saccharomyces cerevisiae, stabilizes amino acid permeases

Hsp70: J-domain protein (JDP) machines, along with the cellular protein degradation systems play a central role in regulating cellular proteostasis. An equally robust surveillance system operates at the plasma membrane too that affects proper sorting, stability as well as the turnover of membrane proteins. Although plausible, a definitive role of the Hsp70: JDP machine in regulating the stability of plasma membrane proteins is not well understood in Saccharomyces cerevisiae. Here we show that a moderate over-expression of Caj1, one of the thirteen JDPs residing in the nucleo-cytosolic compartment of S. cerevisiae reduced the cold sensitivity of tryptophan auxotrophic yeast cells by stabilizing tryptophan permeases, Tat1 and Tat2 in a J-domain dependent manner. Concomitantly, higher Caj1 levels also caused slow growth and increased plasma membrane damage at elevated temperatures possibly due to the stabilization of thermolabile plasma membrane proteins. Finally, we show that although majorly cytosolic, Caj1 also co-localizes with the membrane dye FM4-64 at the cellular periphery suggesting that Caj1 might interact with the plasma membrane. Based on the results presented in this study, we implicate the Hsp70: Caj1 chaperone machine in regulating the stability or turnover of plasma membrane proteins in budding yeast.

Designing and developing a high-resolution melting technique for accurate identification of Leishmania species by targeting amino acid permease 3 and cytochrome oxidase II genes using real-time PCR and in silico genetic evaluation

Discrimination, accurate identification, and reliable techniques are required for accurate identification of Leishmania parasites. High-resolution melting (HRM) is recognized as an authentic and exact method. The main objective of this research was optimizing HRM analysis for detecting and screening Leishmania major, Leishmania tropica and mix infections. Thirty-six DNA samples of Leishmania parasite were prepared and analyzed. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were targeted and six pairs of specific new primers were designed. Bioinformatics analysis was employed to predict DNA temperature resolution for each species and compared with in-vitro results. The genetic diversity of the selected gene regions was analyzed using PCR-sequencing method and DnaSP 5.10.01 software. They were submitted in GenBank (KU680818- KU680821 and KY041643- KY041649). The haplotype diversity for both AAP3 and COII genes was 96% and 87%, respectively. Tajima's D index was 0.65 for AAP3 and 0.36 for COII. CLC Genomics Workbench 11 software predictions were significant and close to these findings. The designed primers could be able to identify at least two Leishmania species. Temperature variations in HRM technique separated Iranian Leishmania parasites of L. major, L. tropica and mix infections. The target genes and our modified HRM method proved this technique could be useful in both clinical and experimental settings. Also, it can be effective for detecting Leishmania parasites in different hosts such as humans, reservoir hosts and vectors. Indeed, HRM can be used as a technique in Leishmania identification as well as for ecological and epidemiological research.

Evolutionary significance of amino acid permease transporters in 17 plants from Chlorophyta to Angiospermae

BackgroundNitrogen is an indispensable nutrient for plant growth. It is used and transported in the form of amino acids in living organisms. Transporting amino acids to various parts of plants requires relevant transport proteins, such as amino acid permeases (AAPs), which were our focus in this study.ResultsWe found that 5 AAP genes were present in Chlorophyte species and more AAP genes were predicted in Bryophyta and Lycophytes. Two main groups were defined and group I comprised 5 clades. Our phylogenetic analysis indicated that the origin of clades 2, 3, and 4 is Gymnospermae and that these clades are closely related. The members of clade 1 included Chlorophyta to Gymnospermae. Group II, as a new branch consisting of non-seed plants, is first proposed in our research. Our results also indicated that the AAP family was already present in Chlorophyta and then expanded accompanying the development of vasculature. Concurrently, the AAP family experienced multiple duplication events that promoted the generation of new functions and differentiation of sub-functions.ConclusionsOur findings suggest that the AAP gene originated in Chlorophyta, and some non-seed AAP genes clustered in one group. A second group, which contained plants of all evolutionary stages, indicated the evolution of AAPs. These new findings can be used to guide future research.

The amino acid transporter AAP1 mediates growth and grain yield by regulating neutral amino acid uptake and reallocation in Oryza sativa.

Nitrogen (N) is a major element necessary for crop yield. In most plants, organic N is primarily transported in the form of amino acids. Here, we show that amino acid permease 1 (AAP1) functions as a positive regulator of growth and grain yield in rice. We found that the OsAAP1 gene is highly expressed in rice axillary buds, leaves, and young panicles, and that the OsAAP1 protein is localized to both the plasma membrane and the nuclear membrane. Compared with the wild-type ZH11, OsAAP1 overexpression (OE) lines exhibited increased filled grain numbers as a result of enhanced tillering, while RNAi and CRISPR (clustered regularly interspaced short palindromic repeat; Osaap1) knockout lines showed the opposite phenotype. In addition, OsAAP1-OE lines had higher concentrations of neutral and acidic amino acids, but lower concentrations of basic amino acids in the straw. An exogenous treatment with neutral amino acids promoted axillary bud outgrowth more strongly in the OE lines than in the WT, RNAi, or Osaap1 lines. Transcriptome analysis of Osaap1 further demonstrated that OsAAP1 may affect N transport and metabolism, and auxin, cytokinin, and strigolactone signaling in regulating rice tillering. Taken together, these results support that increasing neutral amino acid uptake and reallocation via OsAAP1 could improve growth and grain yield in rice.

The Transcription Factor Stp2 Is Important for Candida albicans Biofilm Establishment and Sustainability

The fungal pathogen Candida albicans forms polymorphic biofilms where hyphal morphogenesis and metabolic adaptation are tightly coordinated by a complex intertwined network of transcription factors. The sensing and metabolism of amino acids play important roles during various phases of biofilm development – from adhesion to maturation. Stp2 is a transcription factor that activates the expression of amino acid permease genes and is required for environmental alkalinization and hyphal growth in vitro and during macrophage phagocytosis. While it is well established that Stp2 is activated in response to external amino acids, its role in biofilm formation remains unknown. In addition to widely used techniques, we applied newly developed approaches for automated image analysis to quantify Stp2-regulated filamentation and biofilm growth. Our results show that in the stp2Δ deletion mutant adherence to abiotic surfaces and initial germ tube formation were strongly impaired, but formed mature biofilms with cell density and morphological structures comparable to the control strains. Stp2-dependent nutrient adaptation appeared to play an important role in biofilm development: stp2Δ biofilms formed under continuous nutrient flow displayed an overall reduction in biofilm formation, whereas under steady conditions the mutant strain formed biofilms with lower metabolic activity, resulting in increased cell survival and biofilm longevity. A deletion of STP2 led to increased rapamycin susceptibility and transcriptional activation of GCN4, the transcriptional regulator of the general amino acid control pathway, demonstrating a connection of Stp2 to other nutrient-responsive pathways. In summary, the transcription factor Stp2 is important for C. albicans biofilm formation, where it contributes to adherence and induction of morphogenesis, and mediates nutrient adaption and cell longevity in mature biofilms.

Proline Homeostasis in Saccharomyces cerevisiae: How Does the Stress-Responsive Transcription Factor Msn2 Play a Role?

Overexpression of MSN2, which is the transcription factor gene in response to stress, is well-known to increase the tolerance of the yeast Saccharomyces cerevisiae cells to a wide variety of environmental stresses. Recent studies have found that the Msn2 is a feasible potential mediator of proline homeostasis in yeast. This result is based on the finding that overexpression of the MSN2 gene exacerbates the cytotoxicity of yeast to various amino acid analogs whose uptake is increased by the active amino acid permeases localized on the plasma membrane as a result of a dysfunctional deubiquitination process. Increased understanding of the cellular responses induced by the Msn2-mediated proline incorporation will provide better comprehension of how cells respond to and counteract to different kinds of stimuli and will also contribute to the breeding of industrial yeast strains with increased productivity.

Genome-wide identification of the amino acid permease genes and molecular characterization of their transcriptional responses to various nutrient stresses in allotetraploid rapeseed

BackgroundNitrogen (N), referred to as a “life element”, is a macronutrient essential for optimal plant growth and yield production. Amino acid (AA) permease (AAP) genes play pivotal roles in root import, long-distance translocation, remobilization of organic amide-N from source organs to sinks, and other environmental stress responses. However, few systematic analyses of AAPs have been reported in Brassica napus so far.ResultsIn this study, we identified a total of 34 full-length AAP genes representing eight subgroups (AAP1–8) from the allotetraploid rapeseed genome (AnAnCnCn, 2n = 4x = 38). Great differences in the homolog number among the BnaAAP subgroups might indicate their significant differential roles in the growth and development of rapeseed plants. The BnaAAPs were phylogenetically divided into three evolutionary clades, and the members in the same subgroups had similar physiochemical characteristics, gene/protein structures, and conserved AA transport motifs. Darwin’s evolutionary analysis suggested that BnaAAPs were subjected to strong purifying selection pressure. Cis-element analysis showed potential differential transcriptional regulation of AAPs between the model Arabidopsis and B. napus. Differential expression of BnaAAPs under nitrate limitation, ammonium excess, phosphate shortage, boron deficiency, cadmium toxicity, and salt stress conditions indicated their potential involvement in diverse nutrient stress responses.ConclusionsThe genome-wide identification of BnaAAPs will provide a comprehensive insight into their family evolution and AAP-mediated AA transport under diverse abiotic stresses. The molecular characterization of core AAPs can provide elite gene resources and contribute to the genetic improvement of crop stress resistance through the modulation of AA transport.

Genome-wide identification, characterization and expression analysis of the amino acid permease gene family in tea plants (Camellia sinensis)

Amino acid permeases (AAPs) are involved in transporting a broad spectrum of amino acids and regulating physiological processes in plants. In this study, 19 AAP genes were identified from the tea plants genome database and named CsAAP1–19. Based on phylogenetic analysis, the CsAAP genes were classified into three groups, having significantly different structures and conserved motifs. In addition, an expression analysis revealed that most of CsAAP genes were specifically expressed in different tissues, especially CsAAP19 was expressed only in root. These genes also were significantly expressed in the Baiye 1 and Huangjinya cultivars. Nitrogen treatments indicated that the CsAAPs were obviously expressed in root. CsAAP2, −6, −12, −13 and − 16 were significantly expressed at 6 d after the glutamate treatment, while the expression trend at 24 h after contained the ammonium. These results improve our understanding of the CsAAP genes and their functions in nitrogen utilization in tea plants.

Molecular Cloning of an Amino Acid Permease Gene and Structural Characterization of the Protein in Common Bean (Phaseolus vulgaris L.).

Plants synthesize amino acids by collateral metabolic pathways using primary elements carbon and oxygen from air, hydrogen from water in soil and nitrogen from soil. Following synthesis, amino acids are immediately used for metabolism, transient storage or transported to the phloem. Different families of transporters have been identified for import of amino acids into plant cells. The first identified amino acid transporter, amino acid permease 1 (AAP1) in Arabidopsis belongs to a family of eight members and transports acidic, neutral, and basic amino acids. Legumes fix atmospheric nitrogen through a symbiotic relationship with root nodules bacteria. Following fixation, nitrogen is reduced to amino acids and is exported via different amino acid transporters. However, information is lacking about the structure of these important classes of amino acid transporter proteins in plant. We have amplified AAP from Phaseolus vulgaris, an economically important leguminous plant grown all over the world, and sequenced. The sequence has been characterized in silico and a three-dimensional structure of AAP has been predicted and validated. The information obtained not only enhances the knowledge about the structure of an amino acid permease gene in P. vulgaris, but will also help in designing protein-ligand studies using this protein as well.

Overexpression of GmAAP6a enhances tolerance to low nitrogen and improves seed nitrogen status by optimizing amino acid partitioning in soybean.

SummaryAmino acid transport via phloem is one of the major source‐to‐sink nitrogen translocation pathways in most plant species. Amino acid permeases (AAPs) play essential roles in amino acid transport between plant cells and subsequent phloem or seed loading. In this study, a soybean AAP gene, annotated as GmAAP6a, was cloned and demonstrated to be significantly induced by nitrogen starvation. Histochemical staining of GmAAP6a:GmAAP6a‐GUS transgenic soybean revealed that GmAAP6a is predominantly expressed in phloem and xylem parenchyma cells. Growth and transport studies using toxic amino acid analogs or single amino acids as a sole nitrogen source suggest that GmAAP6a can selectively absorb and transport neutral and acidic amino acids. Overexpression of GmAAP6a in Arabidopsis and soybean resulted in elevated tolerance to nitrogen limitation. Furthermore, the source‐to‐sink transfer of amino acids in the transgenic soybean was markedly improved under low nitrogen conditions. At the vegetative stage, GmAAP6a‐overexpressing soybean showed significantly increased nitrogen export from source cotyledons and simultaneously enhanced nitrogen import into sink primary leaves. At the reproductive stage, nitrogen import into seeds was greatly enhanced under both sufficient and limited nitrogen conditions. Collectively, our results imply that overexpression of GmAAP6a enhances nitrogen stress tolerance and source‐to‐sink transport and improves seed quality in soybean. Co‐expression of GmAAP6a with genes specialized in source nitrogen recycling and seed loading may represent an interesting application potential in breeding.

A Novel Resistance Pathway for Calcineurin Inhibitors in the Human-Pathogenic Mucorales Mucor circinelloides.

Mucormycosis is an emerging lethal fungal infection in immunocompromised patients. Mucor circinelloides is a causal agent of mucormycosis and serves as a model system to understand genetics in Mucorales. Calcineurin is a conserved virulence factor in many pathogenic fungi, and calcineurin inhibition or deletion of the calcineurin regulatory subunit (CnbR) in Mucor results in a shift from hyphal to yeast growth. We analyzed 36 calcineurin inhibitor-resistant or bypass mutants that exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked cnbRΔ mutant background without carrying any mutations in known calcineurin components. We found that a majority of the mutants had altered sequence in a gene, named here bycA (bypass of calcineurin). bycA encodes an amino acid permease. We verified that both the bycAΔ single mutant and the bycAΔ cnbRΔ double mutant are resistant to calcineurin inhibitor FK506, thereby demonstrating a novel mechanism of resistance against calcineurin inhibitors. We also found that the level of expression of bycA was significantly higher in the wild-type strain treated with FK506 and in the cnbRΔ mutants but was significantly lower in the wild-type strain without FK506 treatment. These findings suggest that bycA is a negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucor and that calcineurin suppresses expression of the bycA gene at the mRNA level to promote hyphal growth. BycA is involved in the Mucor hypha-yeast transition as our data demonstrate positive correlations among bycA expression, protein kinase A activity, and Mucor yeast growth. Also, calcineurin, independently of its role in morphogenesis, contributes to virulence traits, including phagosome maturation blockade, host cell damages, and proangiogenic growth factor induction during interactions with hosts.IMPORTANCEMucor is intrinsically resistant to most known antifungals, which makes mucormycosis treatment challenging. Calcineurin is a serine/threonine phosphatase that is widely conserved across eukaryotes. When calcineurin function is inhibited in Mucor, growth shifts to a less virulent yeast growth form, which makes calcineurin an attractive target for development of new antifungal drugs. Previously, we identified two distinct mechanisms through which Mucor can become resistant to calcineurin inhibitors involving Mendelian mutations in the gene for FKBP12, including mechanisms corresponding to calcineurin A or B subunits and epimutations silencing the FKBP12 gene. Here, we identified a third novel mechanism where loss-of-function mutations in the amino acid permease corresponding to the bycA gene contribute to resistance against calcineurin inhibitors. When calcineurin activity is absent, BycA can activate protein kinase A (PKA) to promote yeast growth via a cAMP-independent pathway. Our data also show that calcineurin activity contributes to host-pathogen interactions primarily in the pathogenesis of Mucor.