Identification of glutamine synthetase as a contryphan-Bt binding protein by His-tag pull-down

Background: Contryphan-Bt is a D-tryptophan-containing disulfide-constrained decapeptide recently isolated from the venom of Conus betulinus. The molecular targets of contryphans are controversial, and the identification of its interacting proteins may be of great importance. Methods: His-tag pull-down assays were performed to investigate intracellular binding proteins of contryphan-Bt from rat brain lysate. Bt-Acp-[His]6, a contryphan-Bt derivative containing hexahistidine tag, was synthesized and used as the bait. As a control, Acp-[His]6 was used to exclude nonspecific bindings. Results: Glutamine synthetase was identified as a potential contryphan-Bt binding protein by pull-down assays and subsequent LC-MS/MS. The binding of contryphan-Bt to glutamine synthetase was confirmed and determined using microscale thermophoresis, with a Kd of 74.02 ± 2.8 μM. The binding did not affect glutamine synthetase activity, suggesting that the interaction site was distinct from the catalytic center. Conclusions: Glutamine synthetase was identified as a novel contryphan-Bt binding protein. This is the first report in which the conopeptide binds to an intracellular protein.

Enlisting wild grass genes to combat nitrification in wheat farming: A nature-based solution

Active nitrifiers and rapid nitrification are major contributing factors to nitrogen losses in global wheat production. Suppressing nitrifier activity is an effective strategy to limit N losses from agriculture. Production and release of nitrification inhibitors from plant roots is termed “biological nitrification inhibition” (BNI). Here, we report the discovery of a chromosome region that controls BNI production in “wheat grass” Leymus racemosus (Lam.) Tzvelev, located on the short arm of the “Lr#3Nsb” (Lr#n), which can be transferred to wheat as T3BL.3NsbS (denoted Lr#n-SA), where 3BS arm of chromosome 3B of wheat was replaced by 3NsbS of L. racemosus. We successfully introduced T3BL.3NsbS into the wheat cultivar “Chinese Spring” (CS-Lr#n-SA, referred to as “BNI-CS”), which resulted in the doubling of its BNI capacity. T3BL.3NsbS from BNI-CS was then transferred to several elite high-yielding hexaploid wheat cultivars, leading to near doubling of BNI production in “BNI-MUNAL” and “BNI-ROELFS.” Laboratory incubation studies with root-zone soil from field-grown BNI-MUNAL confirmed BNI trait expression, evident from suppression of soil nitrifier activity, reduced nitrification potential, and N2O emissions. Changes in N metabolism included reductions in both leaf nitrate, nitrate reductase activity, and enhanced glutamine synthetase activity, indicating a shift toward ammonium nutrition. Nitrogen uptake from soil organic matter mineralization improved under low N conditions. Biomass production, grain yields, and N uptake were significantly higher in BNI-MUNAL across N treatments. Grain protein levels and breadmaking attributes were not negatively impacted. Wide use of BNI functions in wheat breeding may combat nitrification in high N input–intensive farming but also can improve adaptation to low N input marginal areas.

Diverse Understory Vegetation Alleviates Nitrogen Competition with Crop Trees in Poplar Plantations

Understory vegetation plays a crucial role in nutrient turnover and cycling in plantations, but it also competes for nutrients with crop trees when only a single species is present due to its specific nutrient requirements. However, it remains unclear whether this competition can be alleviated when the species richness of understory vegetation increases. In this study, we tested different gradients of understory vegetation species richness, including understory vegetation removal (UR), the retention of a single main understory vegetation species (RS), and the retention of natural diverse understory vegetation (RD) as part of a poplar (Populus deltoides ‘Nanlin-3804′) plantation, to study their effects on poplar growth, and to evaluate nitrogen (N) usage and how this was affected by the interactions between the poplar and understory vegetation. The results showed a generally lower periodic growth, and a significant decline in the foliar chlorophyll content and glutamine synthetase activity of poplar under treatment with RS and RD compared to those under UR treatment conducted in July 2019, which clearly indicated N competition between the understory vegetation and poplar trees. However, no significant difference was detected in the foliar chlorophyll content and glutamine synthetase activity of poplar under RD and RS treatment; only the nitrate reductase activity in poplar leaves under RD treatment declined significantly, by 22.25%, in June 2019. On the contrary, the diameter at breast height (DBH) of the poplar under RD treatment showed an increase of 34.69% from July to August 2019, compared with that under RS treatment. Furthermore, the increase in the species richness of understory vegetation resulted in an increase in the δ15N values in the poplar leaves, which was strongly regulated by the NH4+-N pool in the 10–20 cm soil layer, indicating the effective coordination of N utilization between poplar and understory vegetation when diversified understory plant species were present. These findings demonstrate the essential role of understory vegetation species diversity in alleviating N competition with crop trees, and provide guidance for understory vegetation management in poplar plantations.

Nutrient uptake and assimilation in fragrant rosewood (Dalbergia odorifera T.C. Chen) seedlings in growing media with un-composted spent mushroom residue

The purpose of this study was to detect nutrient uptake and assimilation in woody plants subjected to growing media with some peat replaced by spent mushroom residue (SMR). Fragrant rosewood (Dalbergia odorifera T.C. Chen) seedlings were cultured in five types of growing media with SMR and peat in volumetric proportions of 0% (control), 25%, 50%, 75%, and 100%. With the increase of SMR proportion, ammonium- and nitrate-nitrogen (N) concentrations declined but available phosphorus (P) concentration and electrical conductance both increased. Seedlings in the full SMR substrates showed obvious mortality. Seedlings in substrates with SMR in proportions higher than 25% showed symptoms of excessive N and P toxicities. The utilization efficiency for P was highest in the 25% SMR growing-media. Mineral N in substrates had a positive relationship with growth and biomass but not with glutamine synthetase activity. Available P was negatively related with acid phosphatase activity in both leaves and roots. Un-composted SMR can replace 25% of peat in growing media for fragrant rosewood seedlings, benefitting P uptake and assimilation.

Pharmacological upregulation of GLT-1 alleviates the cognitive impairments in the animal model of temporal lobe epilepsy

It is known that hippocampal epileptogenesis is accompanied by hyperexcitability, glutamate-related neuronal dysfunctions and consequently cognitive deficits. However, the neuroprotective role of astrocytic glutamate uptake through the Glutamate Transporter-1 (GLT-1) remains to be unknown in these processes. Therefore, to assess the effect of glutamate uptake, pharmacological upregulation of GLT-1 using ceftriaxone administration (200 mg/kg/day, i.p, 5 days) was utilized in Li-PIL animal models of temporal lobe epilepsy (TLE). Glutamate concentration and glutamine synthetase activity were analyzed using biochemical assays. In addition, GLT-1 gene expression was assessed by RT-qPCR. Finally, cognitive function was studied using Morris water maze (MWM) test and novel object recognition task (NORT). Our results demonstrated that the acute phase of epileptogenesis (first 72 hours after Status Epilepticus) was accompanied by an increase in the hippocampal glutamate and downregulation of GLT-1 mRNA expression compared to controls. Ceftriaxone administration in epileptic animals led to a reduction of glutamate along with elevation of the level of glutamine synthetase activity and GLT-1 expression in the acute phase. In the chronic phase of epileptogenesis (4 weeks after Status Epilepticus), glutamate levels and GLT-1 expression were decreased compared to controls. Ceftriaxone treatment increased the levels of GLT-1 expression. Furthermore, impaired learning and memory ability in the chronic phase of epileptogenesis was rescued by Ceftriaxone administration. This study shows that astrocytic glutamate uptake can profoundly impact the processes of hippocampal epileptogenesis through the reduction of glutamate-induced excitotoxicity and consequently rescuing of cognitive deficits caused by epilepsy.