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.

Structural basis for sugar perception by Drosophila gustatory receptors.

Insects rely on a family of seven transmembrane proteins called gustatory receptors (GRs) to encode different taste modalities, such as sweet and bitter. We report structures of Drosophila sweet taste receptors GR43a and GR64a in the apo and sugar-bound states. Both GRs form tetrameric sugar-gated cation channels composed of one central pore domain (PD) and four peripheral ligand-binding domains (LBDs). Whereas GR43a is specifically activated by the monosaccharide fructose that binds to a narrow pocket in LBDs, disaccharides sucrose and maltose selectively activate GR64a by binding to a larger and flatter pocket in LBDs. Sugar binding to LBDs induces local conformational changes, which are subsequently transferred to the PD to cause channel opening. Our studies reveal a structural basis for sugar recognition and activation of GRs.

Structures and mechanisms of the Arabidopsis cytokinin transporter AZG1.

Cytokinins are essential for plant growth and development, and their tissue distributions are regulated by transmembrane transport. Recent studies have revealed that members of the 'Aza-Guanine Resistant' (AZG) protein family from Arabidopsis thaliana can mediate cytokinin uptake in roots. Here we present 2.7 to 3.3 Å cryo-electron microscopy structures of Arabidopsis AZG1 in the apo state and in complex with its substrates trans-zeatin (tZ), 6-benzyleaminopurine (6-BAP) or kinetin. AZG1 forms a homodimer and each subunit shares a similar topology and domain arrangement with the proteins of the nucleobase/ascorbate transporter (NAT) family. These structures, along with functional analyses, reveal the molecular basis for cytokinin recognition. Comparison of the AZG1 structures determined in inward-facing conformations and predicted by AlphaFold2 in the occluded conformation allowed us to propose that AZG1 may carry cytokinins across the membrane through an elevator mechanism.

Ligand activation mechanisms of human KCNQ2 channel.

The human voltage-gated potassium channel KCNQ2/KCNQ3 carries the neuronal M-current, which helps to stabilize the membrane potential. KCNQ2 can be activated by analgesics and antiepileptic drugs but their activation mechanisms remain unclear. Here we report cryo-electron microscopy (cryo-EM) structures of human KCNQ2-CaM in complex with three activators, namely the antiepileptic drug cannabidiol (CBD), the lipid phosphatidylinositol 4,5-bisphosphate (PIP2), and HN37 (pynegabine), an antiepileptic drug in the clinical trial, in an either closed or open conformation. The activator-bound structures, along with electrophysiology analyses, reveal the binding modes of two CBD, one PIP2, and two HN37 molecules in each KCNQ2 subunit, and elucidate their activation mechanisms on the KCNQ2 channel. These structures may guide the development of antiepileptic drugs and analgesics that target KCNQ2.

Polyamine-producing bacteria inhibit the absorption of Cd by spinach and alter the bacterial community composition of rhizosphere soil.

Polyamines (PAs) are small aliphatic nitrogenous bases with strong biological activity that participate in plant stress response signaling and the alleviation of damage from stress. Herein, the effects of the PA-producing bacterium Bacillus megaterium N3 and PAs on the immobilization of Cd and inhibition of Cd absorption by spinach and the underlying mechanisms were studied. A solution test showed that strain N3 secreted spermine and spermidine in the presence of Cd. Both strain N3 and the PAs (spermine+spermidine) immobilized Cd and increased the pH of the solution. Untargeted metabolomics results showed that strain N3 secreted PAs, N1-acetylspermidine, 3-indolepropionic acid, indole-3-acetaldehyde, cysteinyl-gamma-glutamate, and choline, which correlated with plant growth promotion and Cd immobilization. A pot experiment showed that rhizosphere soil inoculation with strain N3 and PAs improved spinach dry weight and reduced spinach Cd absorption compared with the control. These positive effects were likely due to the increase in rhizosphere soil pH and NH4+-N and PA contents, which can be attributed primarily to Cd immobilization. Moreover, inoculation with strain N3 more effectively inhibited the absorption of Cd by spinach than spraying PAs, mainly because strain N3 enabled a better relative abundance of bacteria (Microvirga, Pedobacter, Bacillus, Brevundimonas, Pseudomonas, Serratia, Devosid, and Aminobacter), that have been reported to have the ability to resist heavy metals and produce PAs. Strain N3 regulated the structure of rhizosphere functional bacterial communities and inhibited Cd uptake by spinach. These results provide a theoretical basis for the prevention of heavy metal absorption by vegetables using PA-producing bacteria.

Structural mechanisms of TRPV2 modulation by endogenous and exogenous ligands.

The transient receptor potential vanilloid 2 (TRPV2) ion channel is a polymodal receptor widely involved in many physiological and pathological processes. Despite many TRPV2 modulators being identified, whether and how TRPV2 is regulated by endogenous lipids remains elusive. Here, we report an endogenous cholesterol molecule inside the vanilloid binding pocket (VBP) of TRPV2, with a 'head down, tail up' configuration, resolved at 3.2 Å using cryo-EM. Cholesterol binding antagonizes ligand activation of TRPV2, which is removed from VBP by methyl-ß-cyclodextrin (MßCD) as resolved at 2.9 Å. We also observed that estradiol (E2) potentiated TRPV2 activation by 2-aminoethoxydiphenyl borate (2-APB), a classic tool compound for TRP channels. Our cryo-EM structures (resolved at 2.8-3.3 Å) further suggest how E2 disturbed cholesterol binding and how 2-APB bound within the VBP with E2 or without both E2 and endogenous cholesterol, respectively. Therefore, our study has established the structural basis for ligand recognition of the inhibitory endogenous cholesterol and excitatory exogenous 2-APB in TRPV2.

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.

Crystal structure of the INTS3/INTS6 complex reveals the functional importance of INTS3 dimerization in DSB repair.

SOSS1 is a single-stranded DNA (ssDNA)-binding protein complex that plays a critical role in double-strand DNA break (DSB) repair. SOSS1 consists of three subunits: INTS3, SOSSC, and hSSB1, with INTS3 serving as a scaffold to stabilize this complex. Moreover, the integrator complex subunit 6 (INTS6) participates in the DNA damage response through direct binding to INTS3, but how INTS3 interacts with INTS6, thereby impacting DSB repair, is not clear. Here, we determined the crystal structure of the C-terminus of INTS3 (INTS3c) in complex with the C-terminus of INTS6 (INTS6c) at a resolution of 2.4 Å. Structural analysis revealed that two INTS3c subunits dimerize and interact with INTS6c via conserved residues. Subsequent biochemical analyses confirmed that INTS3c forms a stable dimer and INTS3 dimerization is important for recognizing the longer ssDNA. Perturbation of INTS3c dimerization and disruption of the INTS3c/INTS6c interaction impair the DSB repair process. Altogether, these results unravel the underappreciated role of INTS3 dimerization and the molecular basis of INTS3/INTS6 interaction in DSB repair.

Developing a highly efficient hydroxytyrosol whole-cell catalyst by de-bottlenecking rate-limiting steps.

Hydroxytyrosol is an antioxidant free radical scavenger that is biosynthesized from tyrosine. In metabolic engineering efforts, the use of the mouse tyrosine hydroxylase limits its production. Here, we design an efficient whole-cell catalyst of hydroxytyrosol in Escherichia coli by de-bottlenecking two rate-limiting enzymatic steps. First, we replace the mouse tyrosine hydroxylase by an engineered two-component flavin-dependent monooxygenase HpaBC of E. coli through structure-guided modeling and directed evolution. Next, we elucidate the structure of the Corynebacterium glutamicum VanR regulatory protein complexed with its inducer vanillic acid. By switching its induction specificity from vanillic acid to hydroxytyrosol, VanR is engineered into a hydroxytyrosol biosensor. Then, with this biosensor, we use in vivo-directed evolution to optimize the activity of tyramine oxidase (TYO), the second rate-limiting enzyme in hydroxytyrosol biosynthesis. The final strain reaches a 95% conversion rate of tyrosine. This study demonstrates the effectiveness of sequentially de-bottlenecking rate-limiting steps for whole-cell catalyst development.

[Community Structure of Heavy Metal Immobilized Bacteria in the Lettuce (Lactuca sativa L.) Rhizosphere in Soil Polluted by Heavy Metals and Its Effects on Reducing Heavy Metal Accumulation in Lettuce].

To investigate the diversity of culturable bacteria and heavy metal-immobilizing bacteria in vegetable rhizosphere soil with high concentrations of heavy metals and explore these microbial resources, two samples of Italian lettuce rhizosphere soil with high heavy metal concentration (HY) and low heavy metal concentration (DK) were collected from Xinxiang, Henan Province. The diversity of culturable bacteria and heavy metal-immobilizing bacteria in the rhizosphere soil of lettuce was compared by culturable separation technology and a solution adsorption experiment. The enhancement of Cd and Pb immobilization and lettuce growth by the strains was also investigated in a hydroponic experiment. The results showed that 400 strains belonging to 3 phyla and 14 genera were isolated from the HY sample, with ß-Proteobacteria being the dominant phylum. Meanwhile, 400 strains belonging to 4 phyla and 30 genera were isolated from the DK sample, with Firmicutes being the dominant phylum. A total of 146 strains had a strong ability to immobilize heavy metal and the Cd and Pb removal rates were greater than 80% in the HY sample; Brevundimonas, Serratia, Arthrobacter, and Pseudarthrobacter were the main genera. However, 44 strains had a strong ability to immobilize heavy metal and the Cd and Pb removal rates were greater than 80% in the DK sample, with Bacillus being the main genus. Compared with the control, inoculation with Serratia liquefaciens HY-22, Bacillus thuringiensis HY-53, and Acinetobacter lwoffii HY-157 significantly increased the dry weight of roots (7.5%-77.6%) and shoots (15.4%-67.2%) of the Italian lettuce and cauliflower lettuce and reduced the contents of Cd (38.7%-66.6%) and Pb (34.7%-62.5%) in roots and shoots of Italian lettuce. In addition, the contents of Cd and Pb in the fresh shoots of Italian lettuce and cauliflower lettuce in the presence of Bacillus thuringiensis HY-53 were lower than the Cd and Pb limits set by national food safety standards. Thus, the results provided strain resources and a theoretical basis for the remediation of Cd-and Pb-contaminated farmlands for the safe production of crops.

Structural basis for DNA unwinding at forked dsDNA by two coordinating Pif1 helicases.

Pif1 plays multiple roles in maintaining genome stability and preferentially unwinds forked dsDNA, but the mechanism by which Pif1 unwinds forked dsDNA remains elusive. Here we report the structure of Bacteroides sp Pif1 (BaPif1) in complex with a symmetrical double forked dsDNA. Two interacting BaPif1 molecules are bound to each fork of the partially unwound dsDNA, and interact with the 5' arm and 3' ss/dsDNA respectively. Each of the two BaPif1 molecules is an active helicase and their interaction may regulate their helicase activities. The binding of BaPif1 to the 5' arm causes a sharp bend in the 5' ss/dsDNA junction, consequently breaking the first base-pair. BaPif1 bound to the 3' ss/dsDNA junction impacts duplex unwinding by stabilizing the unpaired first base-pair and engaging the second base-pair poised for breaking. Our results provide an unprecedented insight into how two BaPif1 coordinate with each other to unwind the forked dsDNA.

Effect of copper tolerant Elsholtzia splendens on bacterial community associated with Commelina communis on a copper mine spoil.

Facilitation, or positive plant-plant interaction, has received increasing concern from ecologists over the last two decades. Facilitation may occur through direct mitigation of severe environments or indirect mediation by a third participant from the same or different trophic levels. The copper (Cu) tolerant species Elsholtzia splendens facilitates the establishment and growth of co-occurring Commelina communis through indirect enrichment of microbial activity. However, whether and how E. splendens impacts the microbial community that is associated with C. communis is less known. We characterized the soil bacterial community in the rhizosphere of C. communis in the absence and presence of E. splendens using PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) and sequencing. The result showed that the richness of the bacterial community increased, but diversity and evenness remained similar, in the presence of E. splendens. Chloroflexi, Acidobacteria and Proteobacteria were the most dominant bacteria. The relative abundance of dominant and minor bacterial groups showed distinctly different responses to E. splendens. Principal component analysis and redundancy analysis indicated that variation of the bacterial community was determined by multiple factors and might be driven by the tested soil parameters collectively, or alternatively changed through plant root exudates or other microorganisms. Our results enhance the understanding of how the bacterial community associated with a beneficiary plant responds to a benefactor plant and suggests that the changes of bacterial community composition may have far-reaching influence on plant-soil feedback and the aboveground plant community in the long run.

[Residue and Degradation of Roxarsone in the System of Soil-Vegetable].

The field experiment was developed for simulating the residues, transformation and degradation in soil-vegetable system of Roxarsone contained in organic fertilizer. Under the treatment, the yield of Brassica chinensis decreased in low Roxarsone concentration with a decline by 15% to 32% compared with the control group; there had an accumulating role of vegetables to arsenic, and the root was the main part; total content of arsenic in the soil was positively correlated with the dose of the applied Roxarsone; total arsenic in soil first increased and then decreased with the planting time prolonged and peaked at 12.94 mg x kg(-1), while the level of inorganic arsenic in the soil stabilized after 30 d, which accounting for 66.75% to 98.56% of the total arsenic; there existed a positively significant correlation of total arsenic content between the Brassica chinensis and the soil as well as the arsenic enrichment factor of vegetables; the degradation rate of Roxarsone in soil was slow, there was still some Roxarsone remained in soil after 45 d when added the organic fertilizer which containing Roxarsone with the dose higher than 5 000 kg x hm(-2); Roxarsone could significantly increase the number of bacteria in the soil, and low concentration showed an inhibited role in the growth of fungi and actinomyces, while high concentration of Roxarsone promoted the growth.

[Effects of copper stress on the function of arbuscular mycorrhizal fungi community].

The functional differences of arbuscular mycorrhizal fungi (AMF) isolates from different sources have been extensively investigated in the last two decades. However, previous studies were mostly based on individual AMF species and the community level comparison was not addressed properly. Furthermore, many studies did not distinguish the difference between the effects of AMF source and community structure on their function, let alone concerned the significance of host plant. This study evaluated the effects of copper (Cu) stress on AMF community structure and compared the differences of AMF communities from Cu contaminated and uncontaminated substrates on performance of Zea mays through two short-term greenhouse pot culture experiments. The results showed that spore abundance and composition of AMF communities were changed dramatically under Cu stress compared with the control. The communities dominated by Rhizophagus intraradices and Claroideoglomus etunicatum from Cu contaminated soils conferred more benefits on Z. mays in terms of plant growth and physiological properties relative to that from control governed by Funneliformis mosseae.