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.

Effects of time-space conversion on microflora structure, secondary metabolites composition and antioxidant capacity of Codonopsis pilosula root.

In order to study the relationship between medicinal plant Codonopsis pilosula phenotype, secondary metabolites, antioxidant capacity and its rhizosphere soil nutrients, root-related microorganisms under seasonal and geographical changes, high-throughput sequencing technology was used to explore the bacterial community structure and variation in rhizosphere soil and root endosphere from six regions of Dingxi City, Gansu Province during four seasons. Secondary metabolites composition and antioxidant capacities of C. pilosula root collected successively from four seasons were determined. The chemical properties, nutrient content and enzyme activities of rhizosphere of C. pilosula were significantly different under different temporal and spatial conditions. All soil samples were alkaline (pH 7.64-8.42), with water content ranging from 9.53% to 19.95%, and electrical conductivity varied widely, showing obvious time-scale effects. Different time scales were the main reasons for the diversity and structure of rhizosphere bacterial community of C. pilosula. The diversity and richness of rhizosphere bacterial community in autumn and winter were higher than those in spring and summer, and bacterial community structure in spring and summer was more similar to that in autumn and winter. The root length and diameter of C. pilosula showed significant time gradient difference under different spatiotemporal conditions. Nutrition and niche competition lead to significant synergistic or antagonistic interactions between rhizosphere bacteria and endophytic bacteria, which invisibly affect soil properties, abundance of functional bacteria and even yield and quality of C. pilosula. Soil properties, rhizosphere bacteria and endophytic bacteria directly promoted root phenotype, stress resistance and polysaccharide accumulation of C. pilosula.

Structures and mechanisms of the Arabidopsis auxin transporter PIN3.

The PIN-FORMED (PIN) protein family of auxin transporters mediates polar auxin transport and has crucial roles in plant growth and development1,2. Here we present cryo-electron microscopy structures of PIN3 from Arabidopsis thaliana in the apo state and in complex with its substrate indole-3-acetic acid and the inhibitor N-1-naphthylphthalamic acid (NPA). A. thaliana PIN3 exists as a homodimer, and its transmembrane helices 1, 2 and 7 in the scaffold domain are involved in dimerization. The dimeric PIN3 forms a large, joint extracellular-facing cavity at the dimer interface while each subunit adopts an inward-facing conformation. The structural and functional analyses, along with computational studies, reveal the structural basis for the recognition of indole-3-acetic acid and NPA and elucidate the molecular mechanism of NPA inhibition on PIN-mediated auxin transport. The PIN3 structures support an elevator-like model for the transport of auxin, whereby the transport domains undergo up-down rigid-body motions and the dimerized scaffold domains remain static.

Differences in geological conditions have reshaped the structure and diversity of microbial communities in oily soils.

High-throughput sequencing was used to study the microbial community structure diversity changes in oil-contaminated soils under different spatial distances and environmental conditions. 239 Phyla, 508 Classes, 810 Orders, 1417 Families, 2048 Genera, 511 Species of microbial communities were obtained from 16 samples in three regions. The physicochemical properties of the soil, microorganisms' community structure has been changed by Petroleum hydrocarbon (PHA). Alpha diversity results showed that the soil contained high bacterial diversity, especially in Qingyang's loess soil. The bacterial abundance was in the order of loess soil > black soil > sandy soil. Beta diversity revealed that spatial distance limitation and random variation of repeated samples may be the main factors leading to soil heterogeneity and microbial community structure differences. The dominant bacteria phyla with broad petroleum hydrocarbon degradation ability such as Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria were identified. Pseudomonas, Bacillus, Nocardioides, Oceanobacillus, Sphingomonas, Alkanindiges and Streptomyces were identified as functional microbial for the PHA degradation. The microbial communities manifested the co-exclusion under different geological conditions, and played the key role in the soil PHA degradation through amino acid metabolism, energy metabolism and carbohydrate metabolism. The correlation results of amos structural equation showed that the diversity and abundance of soil microorganisms in different regions were controlled by soil PHA content and environmental factors. Altitude, annual average temperature and annual rainfall were positively correlated with microbial diversity. Annual rainfall and soil physical and chemical factors exhibited the most significant influence on it. Microbial diversity indirectly affected the PHA content in different type soil. We believe that reshape the structure and diversity of microbial communities in soil could be changed and reshaped by different geological conditions, pollutants and soil type. This study can provide helps for understanding the ecological effect of geomicrobiology formation under the driving force of geographic environment and other factors.

Structural basis of ALMT1-mediated aluminum resistance in Arabidopsis.

The plant aluminum (Al)-activated malate transporter ALMT1 mediates the efflux of malate to chelate the Al in acidic soils and underlies the plant Al resistance. Here we present cryo-electron microscopy (cryo-EM) structures of Arabidopsis thaliana ALMT1 (AtALMT1) in the apo, malate-bound, and Al-bound states at neutral and/or acidic pH at up to 3.0 Å resolution. The AtALMT1 dimer assembles an anion channel and each subunit contains six transmembrane helices (TMs) and six cytosolic α-helices. Two pairs of Arg residues are located in the center of the channel pore and contribute to malate recognition. Al binds at the extracellular side of AtALMT1 and induces conformational changes of the TM1-2 loop and the TM5-6 loop, resulting in the opening of the extracellular gate. These structures, along with electrophysiological measurements, molecular dynamic simulations, and mutagenesis study in Arabidopsis, elucidate the structural basis for Al-activated malate transport by ALMT1.

Molecular basis for ligand activation of the human KCNQ2 channel.

The voltage-gated potassium channel KCNQ2 is responsible for M-current in neurons and is an important drug target to treat epilepsy, pain and several other diseases related to neuronal hyper-excitability. A list of synthetic compounds have been developed to directly activate KCNQ2, yet our knowledge of their activation mechanism is limited, due to lack of high-resolution structures. Here, we report cryo-electron microscopy (cryo-EM) structures of the human KCNQ2 determined in apo state and in complex with two activators, ztz240 or retigabine, which activate KCNQ2 through different mechanisms. The activator-bound structures, along with electrophysiology analysis, reveal that ztz240 binds at the voltage-sensing domain and directly stabilizes it at the activated state, whereas retigabine binds at the pore domain and activates the channel by an allosteric modulation. By accurately defining ligand-binding sites, these KCNQ2 structures not only reveal different ligand recognition and activation mechanisms, but also provide a structural basis for drug optimization and design.

Expression of Aspergillus niger glucose oxidase in Pichia pastoris and its antimicrobial activity against Agrobacterium and Escherichia coli.

The gene encoding glucose oxidase from Aspergillus niger ZM-8 was cloned and transferred to Pichia pastoris GS115, a transgenic strain P. pastoris GS115-His-GOD constructed. The growth curve of P. pastoris GS115-His-GOD was consistent with that of Pichia pastoris GS115-pPIC9K under non-induced culture conditions. Under methanol induction conditions, the growth of the GOD-transgenic strain was significantly lowered than P. pastoris GS115-pPIC9K with the induced-culture time increase, and the optical densities of GOD-transgenic strain reached one-third of that of the P. pastoris GS115-pPIC9K at 51 h. The activity of glucose oxidase in the cell-free supernatant, the supernatant of cell lysate, and the precipitation of cell lysate was 14.3 U/mL, 18.2 U/mL and 0.48 U/mL, respectively. The specific activity of glucose oxidase was 8.3 U/mg, 6.52 U/mg and 0.73 U/mg, respectively. The concentration of hydrogen peroxide formed by glucose oxidase from supernatant of the fermentation medium, the supernatant of the cell lysate, and the precipitation of cell lysate catalyzing 0.2 M glucose was 14.3 µg/mL, 18.2 µg/mL, 0.48 µg/mL, respectively. The combination of different concentrations of glucose oxidase and glucose could significantly inhibit the growth of Agrobacterium and Escherichia coli in logarithmic phase. The filter article containing supernatant of the fermentation medium, supernatant of the cell lysate, and precipitation of cell lysate had no inhibitory effect on Agrobacterium and E. coli. The minimum inhibitory concentration of hydrogen peroxide on the plate culture of Agrobacterium and E. coli was 5.6 × 103 µg/mL and 6.0 × 103 µg/mL, respectively.

Cooperative transport mechanism of human monocarboxylate transporter 2.

Proton-linked monocarboxylate transporters (MCTs) must transport monocarboxylate efficiently to facilitate monocarboxylate efflux in glycolytically active cells, and transport monocarboxylate slowly or even shut down to maintain a physiological monocarboxylate concentration in glycolytically inactive cells. To discover how MCTs solve this fundamental aspect of intracellular monocarboxylate homeostasis in the context of multicellular organisms, we analyzed pyruvate transport activity of human monocarboxylate transporter 2 (MCT2). Here we show that MCT2 transport activity exhibits steep dependence on substrate concentration. This property allows MCTs to turn on almost like a switch, which is physiologically crucial to the operation of MCTs in the cellular context. We further determined the cryo-electron microscopy structure of the human MCT2, demonstrating that the concentration sensitivity of MCT2 arises from the strong inter-subunit cooperativity of the MCT2 dimer during transport. These data establish definitively a clear example of evolutionary optimization of protein function.