Genome-Wide Identification and Characterization of Ammonium Transporter (AMT) Genes in Rapeseed (Brassica napus L.).

Ammonium transporters (AMTs) are plasma membrane proteins mediating ammonium uptake and transport. As such, AMTs play vital roles in ammonium acquisition and mobilization, plant growth and development, and stress and pathogen defense responses. Identification of favorable AMT genotypes is a prime target for crop improvement. However, to date, systematic identification and expression analysis of AMT gene family members has not yet been reported for rapeseed (Brassica napus L.). In this study, 20 AMT genes were identified in a comprehensive search of the B. napus genome, 14 members of AMT1 and 6 members of AMT2. Tissue expression analyses revealed that the 14 AMT genes were primarily expressed in vegetative organs, suggesting that different BnaAMT genes might function in specific tissues at the different development stages. Meanwhile, qRT-PCR analysis found that several BnaAMTs strongly respond to the exogenous N conditions, implying the functional roles of AMT genes in ammonium absorption in rapeseed. Moreover, the rapeseed AMT genes were found to be differentially regulated by N, P, and K deficiency, indicating that crosstalk might exist in response to different stresses. Additionally, the subcellular localization of several BnaAMT proteins was confirmed in Arabidopsis protoplasts, and their functions were studied in detail by heterologous expression in yeast. In summary, our studies revealed the potential roles of BnaAMT genes in N acquisition or transportation and abiotic stress response and could provide valuable resources for revealing the functionality of AMTs in rapeseed.

Genome-wide identification and analysis of high-affinity nitrate transporter 2 (NRT2) family genes in rapeseed (Brassica napus L.) and their responses to various stresses.

BACKGROUND: High-affinity nitrate transporter 2 (NRT2) genes have been implicated in nitrate absorption and remobilization under nitrogen (N) starvation stress in many plant species, yet little is known about this gene family respond to various stresses often occurs in the production of rapeseed (Brassica napus L.). RESULTS: This report details identification of 17 NRT2 gene family members in rapeseed, as well as, assessment of their expression profiles using RNA-seq analysis and qRT-PCR assays. In this study, all BnNRT2.1 members, BnNRT2.2a and BnNRT2.4a were specifically expressed in root tissues, while BnNRT2.7a and BnNRT2.7b were mainly expressed in aerial parts, including as the predominantly expressed NRT2 genes detected in seeds. This pattern of shoot NRT expression, along with homology to an Arabidopsis NRT expressed in seeds, strongly suggests that both BnNRT2.7 genes play roles in seed nitrate accumulation. Another rapeseed NRT, BnNRT2.5 s, exhibited intermediate expression, with transcripts detected in both shoot and root tissues. Functionality of BnNRT2s genes was further outlined by testing for adaptive responses in expression to exposure to a series of environmental stresses, including N, phosphorus (P) or potassium (K) deficiency, waterlogging and drought. In these tests, most NRT2 gene members were up-regulated by N starvation and restricted by the other stresses tested herein. In contrast to this overall trend, transcription of BnNRT2.1a was up-regulated under waterlogging and K deficiency stress, and BnNRT2.5 s was up-regulated in roots subjected to waterlogging. Furthermore, the mRNA levels of BnNRT2.7 s were enhanced under both waterlogging stress and P or K deficiency conditions. These results suggest that these three BnNRT2 genes might participate in crosstalk among different stress response pathways. CONCLUSIONS: The results presented here outline a diverse set of NRT2 genes present in the rapeseed genome that collectively carry out specific functions throughout rapeseed development, while also responding not just to N deficiency, but also to several other stresses. Targeting of individual BnNRT2 members that coordinate rapeseed nitrate uptake and transport in response to cues from multiple stress response pathways could significantly expand the genetic resources available for improving rapeseed resistance to environmental stresses.

Comprehensive Curriculum for Internal Medicine Residents on Primary Care of Patients Identifying as Lesbian, Gay, Bisexual, or Transgender.

Introduction: Significant gaps remain in the training of health professionals regarding the care of individuals who identify as lesbian, gay, bisexual, and transgender (LGBT). Although curricula have been developed at the undergraduate medical education level, few materials address the education of graduate medical trainees. The purpose of this curriculum was to develop case-based modules targeting internal medicine residents to address LGBT primary health care. Methods: We designed and implemented a four-module, case-based, interactive curriculum at one university's internal medicine residency program. The modules contained facilitator and learner guides and addressed four main content areas: understanding gender and sexuality; performing a sensitive history and physical examination; health promotion and disease prevention; and mental health, violence, and reproductive health. Knowledge, perceived importance, and confidence were assessed before and after each module to assess curricular effectiveness and acceptability. General medicine faculty delivered these modules. Results: Perceived importance of LGBT topics was high at baseline and remained high after the curricular intervention. Confidence significantly increased in many areas, including being able to provide resources to patients and to institute gender-affirming practices (p < .05). Knowledge improved significantly on almost all topics (p < .0001). Faculty felt the materials gave enough preparation to teach, and residents perceived that the faculty were knowledgeable. Discussion: This resource provides an effective curriculum for training internal medicine residents to better understand and feel confident addressing LGBT primary health care needs. Despite limitations, this is an easily transferable curriculum that can be adapted in a variety of curricular settings.

A Qualitative Analysis of Resident Adverse Event Reporting: What's Holding Us Back.

This study utilized focus groups of residents, who report adverse events at differing rates depending on their hospital site, to better understand barriers to residents' reporting and identify modifiable aspects of an institution's culture that could encourage resident event reporting. Focus groups included residents who rotated at 3 hospitals and represented 4 training programs. Focus groups were audio recorded and analyzed using qualitative methods. A total of 64 residents participated in 8 focus groups. Reporting behavior varied by hospital culture. Residents worried about damage to their professional relationships and lacked insight into the benefits of multiple reports of the same event or how human factors engineering can prevent errors. Residents did not understand how reporting affects litigation. Residents at other academic institutions likely experience similar barriers. This study illustrates that resident reporting is modifiable by changing hospital culture, but hospitals have only a few opportunities to mishandle reporting before resident reporting attitudes solidify.

Encouraging Resident Adverse Event Reporting: A Qualitative Study of Suggestions from the Front Lines.

INTRODUCTION: Little is known about what motivates residents to report adverse events. The goals of the qualitative study were to: (1) better understand facilitators to residents' event reporting and (2) identify effective interventions that encourage residents to report. METHODS: The authors conducted focus groups of upper-level residents from 4 training programs (2 internal medicine, a pediatric, and a combined medicine-pediatric) who rotated at 3 institutions within a large healthcare system in 2016. Quantitative data on reporting experience were gathered. Focus groups were audio recorded and transcribed. Two coders reviewed transcripts using the editing approach and organized codes into themes. RESULTS: Sixty-four residents participated in 8 focus groups. Residents were universally exposed to reportable events and knew how to report. Residents' reporting behavior varied by site according to local culture, with residents filing more reports at the pediatric hospital compared to other sites, but all groups expressed similar general views about facilitators to reporting. Facilitators included familiarity with the investigation process, reporting via telephone, and routine safety educational sessions with safety administrators. Residents identified specific interventions that encouraged reporting at the pediatric hospital, including incorporating an attending physician review of events into sign-out and training on error disclosure. CONCLUSIONS: This study provides insight into what motivates resident event reporting and describes concrete interventions to increase reporting. Our findings are consistent with the Theoretical Domains Framework of behavioral change. These strategies could prove successful at other pediatric hospitals to build a culture that values reporting and prepares residents as patient safety champions.

Adaption of Roots to Nitrogen Deficiency Revealed by 3D Quantification and Proteomic Analysis.

Rapeseed (Brassica napus) is an important oil crop worldwide. However, severe inhibition of rapeseed production often occurs in the field due to nitrogen (N) deficiency. The root system is the main organ to acquire N for plant growth, but little is known about the mechanisms underlying rapeseed root adaptions to N deficiency. Here, dynamic changes in root architectural traits of N-deficient rapeseed plants were evaluated by 3D in situ quantification. Root proteome responses to N deficiency were analyzed by the tandem mass tag-based proteomics method, and related proteins were characterized further. Under N deficiency, rapeseed roots become longer, with denser cells in the meristematic zone and larger cells in the elongation zone of root tips, and also become softer with reduced solidity. A total of 171 and 755 differentially expressed proteins were identified in short- and long-term N-deficient roots, respectively. The abundance of proteins involved in cell wall organization or biogenesis was highly enhanced, but most identified peroxidases were reduced in the N-deficient roots. Notably, peroxidase activities also were decreased, which might promote root elongation while lowering the solidity of N-deficient roots. These results were consistent with the cell wall components measured in the N-deficient roots. Further functional analysis using transgenic Arabidopsis (Arabidopsis thaliana) plants demonstrated that the two root-related differentially expressed proteins contribute to the enhanced root growth under N deficiency conditions. These results provide insights into the global changes of rapeseed root responses to N deficiency and may facilitate the development of rapeseed cultivars with high N use efficiency through root-based genetic improvements.

Genome-Wide Identification and Expression Analysis of NRAMP Family Genes in Soybean (Glycine Max L.).

The NRAMP (natural resistance-associated macrophage protein) family of genes has been widely characterized in organisms ranging from bacteria to yeast, plants, mice, and humans. This gene family plays vital roles in divalent metal ion transport across cellular membranes. As yet, comprehensive analysis of NRAMP family genes has not been reported for soybean. In this study, bioinformatics analysis was conducted to identify 13 soybean NRAMP genes, along with their gene structures, phylogenetic relationships, and transmembrane domains. Expression analysis suggests that GmNRAMP genes function in numerous tissues and development stages. Moreover, soybean NRAMP genes were differentially regulated by deficiencies of N, P, K, Fe, and S, along with toxicities of Fe, Cu, Cd, and Mn. These results indicate that GmNRAMP genes function in many nutrient stress pathways, and might be involved in crosstalk among nutrient stress pathways. Subcellular localization analysis in Arabidopsis protoplasts confirmed the tonoplast or plasma membrane localization of selected soybean NRMAP proteins. Protein-protein interaction analysis found that the networks of three GmNRAMP proteins which putatively interact with nodulin-like proteins, almost distinct from the network that is common to the other 10 soybean NRAMP proteins. Subsequent qRT-PCR results confirmed that these three GmNRMAP genes exhibited enhanced expression in soybean nodules, suggesting potential functions in the transport of Fe or other metal ions in soybean nodules. Overall, the systematic analysis of the GmNRAMP gene family reported herein provides valuable information for further studies on the biological roles of GmNRAMPs in divalent metal ion transport in various soybean tissues under numerous nutrient stresses and soybean-rhizobia symbiosis.

Characterization of soybean ß-expansin genes and their expression responses to symbiosis, nutrient deficiency, and hormone treatment.

Expansins are plant cell wall-loosening proteins encoded by a superfamily of genes including α-expansin, ß-expansin, expansin-like A, and expansin-like B proteins. They play a variety of biological roles during plant growth and development. Expansin genes have been reported in many plant species, and results primarily from graminaceous members indicate that ß-expansins are more abundant in monocots than in dicots. Soybean [Glycine max (L.) Merr] is an important legume crop. This work identified nine ß-expansin gene family members in soybean (GmEXPBs) that were divided into two distinct classes based on phylogeny and gene structure, with divergence between the two groups occurring more in introns than in exons. A total of 887 hormone-responsive and environmental stress-related putative cis-elements from 188 families were found in the 2-kb upstream region of GmEXPBs. Variations in number and type of cis-elements associated with each gene indicate that the function of these genes is differentially regulated by these signals. Expression analysis confirmed that the family members were ubiquitously, yet differentially expressed in soybean. Responsiveness to nutrient deficiency stresses and regulation by auxin (indole-3-acetic acid) and cytokinin (6-benzylaminopurine) varied among GmEXPBs. In addition, most ß-expansin genes were associated with symbiosis of soybean inoculated with Rhizobium or abuscular mycorrhizal fungi (AMF). Taken together, these results systematically investigate the characteristics of the entire GmEXPB family in soybean and comprise the first report analyzing the relationship of GmEXPBs with rhizobial or AMF symbiosis. This information is a valuable step in the process of understanding the expansin protein functions in soybean and opens avenues for continued researches.

Genome-wide identification of soybean microRNAs and their targets reveals their organ-specificity and responses to phosphate starvation.

BACKGROUND: Phosphorus (P) plays important roles in plant growth and development. MicroRNAs involved in P signaling have been identified in Arabidopsis and rice, but P-responsive microRNAs and their targets in soybean leaves and roots are poorly understood. RESULTS: Using high-throughput sequencing-by-synthesis (SBS) technology, we sequenced four small RNA libraries from leaves and roots grown under phosphate (Pi)-sufficient (+Pi) and Pi-depleted (-Pi) conditions, respectively, and one RNA degradome library from Pi-depleted roots at the genome-wide level. Each library generated ~21.45-28.63 million short sequences, resulting in ~20.56-27.08 million clean reads. From those sequences, a total of 126 miRNAs, with 154 gene targets were computationally predicted. This included 92 new miRNA candidates with 20-23 nucleotides that were perfectly matched to the Glycine max genome 1.0, 70 of which belong to 21 miRNA families and the remaining 22 miRNA unassigned into any existing miRNA family in miRBase 18.0. Under both +Pi and -Pi conditions, 112 of 126 total miRNAs (89%) were expressed in both leaves and roots. Under +Pi conditions, 12 leaf- and 2 root-specific miRNAs were detected; while under -Pi conditions, 10 leaf- and 4 root-specific miRNAs were identified. Collectively, 25 miRNAs were induced and 11 miRNAs were repressed by Pi starvation in soybean. Then, stem-loop real-time PCR confirmed expression of four selected P-responsive miRNAs, and RLM-5' RACE confirmed that a PHO2 and GmPT5, a kelch-domain containing protein, and a Myb transcription factor, respectively are targets of miR399, miR2111, and miR159e-3p. Finally, P-responsive cis-elements in the promoter regions of soybean miRNA genes were analyzed at the genome-wide scale. CONCLUSIONS: Leaf- and root-specific miRNAs, and P-responsive miRNAs in soybean were identified genome-wide. A total of 154 target genes of miRNAs were predicted via degradome sequencing and computational analyses. The targets of miR399, miR2111, and miR159e-3p were confirmed. Taken together, our study implies the important roles of miRNAs in P signaling and provides clues for deciphering the functions for microRNA/target modules in soybean.

Functional characterization of 14 Pht1 family genes in yeast and their expressions in response to nutrient starvation in soybean.

BACKGROUND: Phosphorus (P) is essential for plant growth and development. Phosphate (Pi) transporter genes in the Pht1 family play important roles in Pi uptake and translocation in plants. Although Pht1 family genes have been well studied in model plants, little is known about their functions in soybean, an important legume crop worldwide. PRINCIPAL FINDINGS: We identified and isolated a complete set of 14 Pi transporter genes (GmPT1-14) in the soybean genome and categorized them into two subfamilies based on phylogenetic analysis. Then, an experiment to elucidate Pi transport activity of the GmPTs was carried out using a yeast mutant defective in high-affinity Pi transport. Results showed that 12 of the 14 GmPTs were able to complement Pi uptake of the yeast mutant with Km values ranging from 25.7 to 116.3 µM, demonstrating that most of the GmPTs are high-affinity Pi transporters. Further results from qRT-PCR showed that the expressions of the 14 GmPTs differed not only in response to P availability in different tissues, but also to other nutrient stresses, including N, K and Fe deficiency, suggesting that besides functioning in Pi uptake and translocation, GmPTs might be involved in synergistic regulation of mineral nutrient homeostasis in soybean. CONCLUSIONS: The comprehensive analysis of Pi transporter function in yeast and expression responses to nutrition starvation of Pht1 family genes in soybean revealed their involvement in other nutrient homeostasis besides P, which could help to better understand the regulation network among ion homeostasis in plants.

Identification of soybean purple acid phosphatase genes and their expression responses to phosphorus availability and symbiosis.

BACKGROUND AND AIMS: Purple acid phosphatases (PAPs) are members of the metallo-phosphoesterase family and have been known to play important roles in phosphorus (P) acquisition and recycling in plants. Low P availability is a major constraint to growth and production of soybean, Glycine max. Comparative studies on structure, transcription regulation and responses to phosphate (Pi) deprivation of the soybean PAP gene family should facilitate further insights into the potential physiological roles of GmPAPs. METHODS: BLAST searches were performed to identify soybean PAP genes at the phytozome website. Bioinformatic analyses were carried out to investigate their gene structure, conserve motifs and phylogenetic relationships. Hydroponics and sand-culture experiments were carried out to obtain the plant materials. Quantitative real-time PCR was employed to analyse the expression patterns of PAP genes in response to P deficiency and symbiosis. KEY RESULTS: In total, 35 PAP genes were identified from soybean genomes, which can be classified into three distinct groups including six subgroups in the phylogenetic tree. The expression pattern analysis showed flowers possessed the largest number of tissue-specific GmPAP genes under normal P conditions. The expression of 23 GmPAPs was induced or enhanced by Pi starvation in different tissues. Among them, nine GmPAP genes were highly expressed in the Pi-deprived nodules, whereas only two GmPAP genes showed significantly increased expression in the arbuscular mycorrhizal roots under low-P conditions. CONCLUSIONS: Most GmPAP genes are probably involved in P acquisition and recycling in plants. Also we provide the first evidence that some members of the GmPAP gene family are possibly involved in the response of plants to symbiosis with rhizobia or arbuscular mycorrhizal fungi under P-limited conditions.

The MetaCyc Database of metabolic pathways and enzymes and the BioCyc collection of Pathway/Genome Databases.

MetaCyc (MetaCyc.org) is a universal database of metabolic pathways and enzymes from all domains of life. The pathways in MetaCyc are curated from the primary scientific literature, and are experimentally determined small-molecule metabolic pathways. Each reaction in a MetaCyc pathway is annotated with one or more well-characterized enzymes. Because MetaCyc contains only experimentally elucidated knowledge, it provides a uniquely high-quality resource for metabolic pathways and enzymes. BioCyc (BioCyc.org) is a collection of more than 350 organism-specific Pathway/Genome Databases (PGDBs). Each BioCyc PGDB contains the predicted metabolic network of one organism, including metabolic pathways, enzymes, metabolites and reactions predicted by the Pathway Tools software using MetaCyc as a reference database. BioCyc PGDBs also contain predicted operons and predicted pathway hole fillers-predictions of which enzymes may catalyze pathway reactions that have not been assigned to an enzyme. The BioCyc website offers many tools for computational analysis of PGDBs, including comparative analysis and analysis of omics data in a pathway context. The BioCyc PGDBs generated by SRI are offered for adoption by any interested party for the ongoing integration of metabolic and genome-related information about an organism.

Modelling applicability of fractal analysis to efficiency of soil exploration by roots.

BACKGROUND AND AIMS: Fractal analysis allows calculation of fractal dimension, fractal abundance and lacunarity. Fractal analysis of plant roots has revealed correlations of fractal dimension with age, topology or genotypic variation, while fractal abundance has been associated with root length. Lacunarity is associated with heterogeneity of distribution, and has yet to be utilized in analysis of roots. In this study, fractal analysis was applied to the study of root architecture and acquisition of diffusion-limited nutrients. The hypothesis that soil depletion and root competition are more closely correlated with a combination of fractal parameters than by any one alone was tested. MODEL: The geometric simulation model SimRoot was used to dynamically model roots of various architectures growing for up to 16 d in three soil types with contrasting nutrient mobility. Fractal parameters were calculated for whole roots, projections of roots and vertical slices of roots taken at 0, 2.5 and 5 cm from the root origin. Nutrient depletion volumes, competition volumes, and relative competition were regressed against fractal parameters and root length. KEY RESULTS: Root length was correlated with depletion volume, competition volume and relative competition at all times. In analysis of three-dimensional, projected roots and 0 cm slices, log(fractal abundance) was highly correlated with log(depletion volume) when times were pooled. Other than this, multiple regression yielded better correlations than regression with single fractal parameters. Correlations decreased with age of roots and distance of vertical slices from the root origin. Field data were also examined to see if fractal dimension, fractal abundance and lacunarity can be used to distinguish common bean genotypes in field situations. There were significant differences in fractal dimension and fractal abundance, but not in lacunarity. CONCLUSIONS: These results suggest that applying fractal analysis to research of soil exploration by root systems should include fractal abundance, and possibly lacunarity, along with fractal dimension.