Physiological and Transcriptomic Responses to Nitrogen Deficiency in Neolamarckia cadamba

Nitrogen (N) is one of the abundant and essential elements for plant growth and development, and N deficiency (ND) affects plants at both physiological and transcriptomic levels. Neolamarckia cadamba is a fast-growing woody plant from the Rubiaceae family. However, the physiological and molecular impacts of ND on this species have not been well investigated. Here, we studied how N. cadamba responds to ND under hydroponic conditions. In a physiological aspect, ND led to a reduction in biomass, chlorophyll content, and photosynthetic capacity. ND also impaired the assimilation of N as the activities of glutamine synthetase (GS) and nitrate reductase (NR) were decreased in the root. Interestingly, the lignin content of stem increased progressively during the ND stress. The main transcription factors, the transcription factors that are important to N regulation has been found to be upregulated, including Nodule inception-like protein 7 (NLP7), TGACG motif-binding factor 1 (TGA1), basic helix-loop-helix protein 45 (BHLH45), NAM, ATAF1,2, CUC2 (NAC) transcription factor 43 (NAC43), and basic leucine zipper pattern 44 (bZIP44). The expression of N transporters, such as nitrate transporter 2.4 (NRT2.4), ammonium transporter 3 (AMT3), and amino acid transporter protein 3 (AAP3), was also upregulated. In addition, phosphorus- and calcium-related genes such as phosphate starvation response 2 (PHR2) and cyclic nucleotide-gated ion channel 15 (CNGC15) were expressed more abundantly in response to ND stress. Our results reveal the physiological and molecular mechanisms by which woody plants respond to ND.

Comparative Transcriptomic and Proteomic Analysis of Exopalaemon carinicauda in Response to Alkalinity Stress

Saline-alkaline waters are stressful environments where most aquatic animals can’t survive normally, and alkalinity is one of the key limited environmental factors. Due to strong adaptability to environment, the ridgetail white prawn Exopalaemon carinicauda is a potential good species suitable for large-scale culture in saline-alkaline waters. Exploring its alkaline adaptability mechanism will help to guide more marine crustaceans to saline-alkaline culture. In this study, an integrative analysis of the gill-specific transcriptome and proteome at 0, 12, and 36 h after alkalinity stress was performed to identify important regulators and pathways involved in alkalinity adaption of E. carinicauda. A total of 3,157 differentially expressed genes (DEGs) and 443 differentially expressed proteins (DEPs) were identified at 12 and 36 h compared with 0 h. Base on the transcriptome analysis, the Gene Ontology (GO) enriched terms were mainly related to ion transport, including “calcium-transporting ATPase activity,” “ATPase coupled ion transmembrane transporter activity,” “divalent inorganic cation transmembrane transporter activity,” etc., and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways mainly refer to the processes of endocrine system at both 12, and 36 h. Based on the proteomic analysis, KEGG pathways related to lipolysis and amino acids metabolism were significantly enriched at 12 h, and carbohydrate metabolism and immune response were significantly enriched at 36 h. There were significantly up-regulated expressions of ion transport related genes including aquaporin, carbonic anhydrase, ammonium transporter Rh type A-like, Na+/H+-exchanger, etc., as well as ion transport proteins including V-type proton ATPase 116 kDa subunit a-like isoform X1, sodium-potassium ATPase beta, vesicle associated membrane protein, etc. after alkalinity exposure, which indicating their important roles in response to alkalinity stress. The results of integrated analysis between proteome and transcriptome showed that up-regulated DEG/DEP (aldehyde dehydrogenase) was significantly enriched at 12 h and the up-regulated DEG/DEP (peptidylglycine alpha) was significantly enriched at 36 h, suggesting the two molecules may be critical in response to alkalinity change. This study reveals the first time-course, gill-specific, combined transcriptomic and proteomic profiling associated with alkalinity adaption of E. carinicauda and provides new insights into the mechanisms underlying the molecular response to alkalinity stress in shrimp.

Investigating the Lipid Selectivity of the Ammonium Transporter AmtB in Heterogeneous Nanodiscs

The structure and function of membrane proteins can be significantly impacted by the surrounding lipid environment, but membrane protein-lipid interactions in lipid bilayers are often difficult to study due to their transient and polydisperse nature. Here, we used two native mass spectrometry (MS) approaches to investigate how the Escherichia coli ammonium transporter (AmtB) selectively remodels its local lipid environment in heterogeneous lipoprotein nanodiscs. First, we used gas-phase ejection to isolate AmtB with bound lipids from heterogeneous nanodiscs with different combinations of lipids. Second, we used solution-phase detergent flash extraction as an orthogonal approach to study AmtB remodeling with native MS. Flash extraction of AmtB showed that Triton X-100 retains lipid selectivity, but C8E4 distorts preferential lipid interactions. Both approaches reveal that AmtB has a few tight binding sites for PC, is selective for binding PG over-all, and is nonselective for PE, providing a detailed picture of how AmtB binds different lipid head groups in the context of mixed lipid bilayers.

The Oxoglutarate Binding Site and Regulatory Mechanism Are Conserved in Ammonium Transporter Inhibitors GlnKs from Methanococcales

Protein inhibition is a natural regulatory process to control cellular metabolic fluxes. PII-family signal-transducing effectors are in this matter key regulators of the nitrogen metabolism. Their interaction with their various targets is governed by the cellular nitrogen level and the energy charge. Structural studies on GlnK, a PII-family inhibitor of the ammonium transporters (Amt), showed that the T-loops responsible for channel obstruction are displaced upon the binding of 2-oxoglutarate, magnesium and ATP in a conserved cleft. However, GlnK from Methanocaldococcus jannaschii was shown to bind 2-oxoglutarate on the tip of its T-loop, causing a moderate disruption to GlnK–Amt interaction, raising the question if methanogenic archaea use a singular adaptive strategy. Here we show that membrane fractions of Methanothermococcus thermolithotrophicus released GlnKs only in the presence of Mg-ATP and 2-oxoglutarate. This observation led us to structurally characterize the two GlnK isoforms apo or in complex with ligands. Together, our results show that the 2-oxoglutarate binding interface is conserved in GlnKs from Methanococcales, including Methanocaldococcus jannaschii, emphasizing the importance of a free carboxy-terminal group to facilitate ligand binding and to provoke the shift of the T-loop positions.

Characterization of the scavenger cell proteome in mouse and rat liver

The structural-functional organization of ammonia and glutamine metabolism in the liver acinus involves highly specialized hepatocyte subpopulations like glutamine synthetase (GS) expressing perivenous hepatocytes (scavenger cells). However, this cell population has not yet been characterized extensively regarding expression of other genes and potential subpopulations. This was investigated in the present study by proteome profiling of periportal GS-negative and perivenous GS-expressing hepatocytes from mouse and rat. Apart from established markers of GS+ hepatocytes such as glutamate/aspartate transporter II (GLT1) or ammonium transporter Rh type B (RhBG), we identified novel scavenger cell-specific proteins like basal transcription factor 3 (BTF3) and heat-shock protein 25 (HSP25). Interestingly, BTF3 and HSP25 were heterogeneously distributed among GS+ hepatocytes in mouse liver slices. Feeding experiments showed that RhBG expression was increased in livers from mice fed with high protein diet compared to standard chow. While spatial distributions of GS and carbamoylphosphate synthetase 1 (CPS1) were unaffected, periportal areas constituted by glutaminase 2 (GLS2)-positive hepatocytes were enlarged or reduced in response to high or low protein diet, respectively. The data suggest that the population of perivenous GS+ scavenger cells is heterogeneous and not uniform as previously suggested which may reflect a functional heterogeneity, possibly relevant for liver regeneration.

The Plant Growth-Promoting Fungus MF23 (Mycena sp.) Increases Production of Dendrobium officinale (Orchidaceae) by Affecting Nitrogen Uptake and NH4+ Assimilation

Dendrobium officinale Kimura et Migo is a traditional and scarce medicinal orchid in China. Mycorrhizal fungi could supply nitrogen (N) to orchids for seed germination and seedling recruitment. However, the N transport mechanism between orchids and the fungus is poorly understand. Early studies found that the fungus MF23 (Mycena sp.) could promote the growth of D. officinale. To better dissect the molecular interactions involved in N transport between D. officinale and MF23, transcriptome and metabolome analyses were conducted on conventional and mycorrhizal cultivations of D. officinale. Moreover, validation tests were carried out in the greenhouse to measure net fluxes of NO3− and NH4+ of roots by a non-invasive micro-test technology (NMT), determine N assimilation enzyme activity by the ELISA, and analyze the expression level of differentially expressed genes (DEGs) of N transporters and DEGs involved in N metabolism by RT-qPCR. Combined transcriptome and metabolome analyses showed that MF23 may influence N metabolism in D. officinale. The expression of DoNAR2.1 (nitrate transporter-activating protein), DoAMT11 (ammonium transporter), DoATFs (amino acid transporters), DoOPTs (oligopeptide transporters), and DoGDHs (glutamate dehydrogenases) in symbiotic D. officinale was upregulated. NMT results showed a preference for NH4+ in D. officinale and indicated that MF23 could promote the uptake of NO3− andNH4+, especially for NH4+. ELISA results showed that MF23 could increase the activity of glutamine synthetase (GS) and glutamate dehydrogenase. This study suggested that MF23 increases the production of D. officinale by affecting N uptake and NH4+ assimilation capacity.