The spines and bloom of cucumber (Cucumis sativus L.) fruit are two important quality traits related to fruit market value. However, until now, none of the genes involved in the formation of cucumber fruit spines and bloom trichomes has been identified. Here, the characterization of trichome development in wild-type (WT) cucumber and a spontaneous mutant, glabrous 1 (csgl1) controlled by a single recessive nuclear gene, with glabrous aerial organs, is reported. Via map-based cloning, CsGL1 was isolated and it was found that it encoded a member of the homeodomain-leucine zipper I (HD-Zip I) proteins previously identified to function mainly in the abiotic stress responses of plants. Tissue-specific expression analysis indicated that CsGL1 was strongly expressed in trichomes and fruit spines. In addition, CsGL1 was a nuclear protein with weak transcriptional activation activity in yeast. A comparative analysis of the digital gene expression (DGE) profile between csgl1 and WT leaves revealed that CsGL1 had a significant influence on the gene expression profile in cucumber, especially on genes related to cellular process, which is consistent with the phenotypic difference between csgl1 and the WT. Moreover, two genes, CsMYB6 and CsGA20ox1, possibly involved in the formation of cucumber trichomes and fruit spines, were characterized. Overall, the findings reveal a new function for the HD-Zip I gene subfamily, and provide some candidate genes for genetic engineering approaches to improve cucumber fruit external quality.

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BackgroundP2X3 and P2X2/3 purinergic receptor-channels, expressed in primary sensory neurons that mediate nociception, have been implicated in neuropathic and inflammatory pain responses. The phospholipids phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) are involved in functional modulation of several types of ion channels. We report here evidence that these phospholipids are able to modulate the function of homomeric P2X3 and heteromeric P2X2/3 purinoceptors expressed in dorsal root ganglion (DRG) nociceptors and in heterologous expression systems.ResultsIn dissociated rat DRG neurons, incubation with the PI3K/PI4K inhibitor wortmannin at 35 μM induced a dramatic decrease in the amplitude of ATP- or α,β-meATP-evoked P2X3 currents, while incubation with 100 nM wortmannin (selective PI3K inhibition) produced no significant effect. Intracellular application of PIP2 was able to fully reverse the inhibition of P2X3 currents induced by wortmannin. In Xenopus oocytes and in HEK293 cells expressing recombinant P2X3, 35 μM wortmannin incubation induced a significant decrease in the rate of receptor recovery. Native and recombinant P2X2/3 receptor-mediated currents were inhibited by incubation with wortmannin both at 35 μM and 100 nM. The decrease of P2X2/3 current amplitude induced by wortmannin could be partially reversed by application of PIP2 or PIP3, indicating a sensitivity to both phosphoinositides in DRG neurons and Xenopus oocytes. Using a lipid binding assay, we demonstrate that the C-terminus of the P2X2 subunit binds directly to PIP2, PIP3 and other phosphoinositides. In contrast, no direct binding was detected between the C-terminus of P2X3 subunit and phosphoinositides.ConclusionOur findings indicate a functional regulation of homomeric P2X3 and heteromeric P2X2/3 ATP receptors by phosphoinositides in the plasma membrane of DRG nociceptors, based on subtype-specific mechanisms of direct and indirect lipid sensing.

Platelet-activating-factor receptor (PAFR) responds to platelet-activating factor (PAF), a phospholipid mediator of cell-to-cell communication that exhibits diverse physiological effects. PAFR is considered an important drug target for treating asthma, inflammation and cardiovascular diseases. Here we report crystal structures of human PAFR in complex with the antagonist SR 27417 and the inverse agonist ABT-491 at 2.8-Å and 2.9-Å resolution, respectively. The structures, supported by molecular docking of PAF, provide insights into the signal-recognition mechanisms of PAFR. The PAFR-SR 27417 structure reveals an unusual conformation showing that the intracellular tips of helices II and IV shift outward by 13 Å and 4 Å, respectively, and helix VIII adopts an inward conformation. The PAFR structures, combined with single-molecule FRET and cell-based functional assays, suggest that the conformational change in the helical bundle is ligand dependent and plays a critical role in PAFR activation, thus greatly extending knowledge about signaling by G-protein-coupled receptors.

The indel in the promoter of CsHDZIV11 co-segregates with fruit spine density and could be used for molecular breeding in cucumber. Fruit spine density is an important quality trait for marketing in cucumber (Cucumis sativus L.). However, the molecular basis of fruit spine density in cucumber remains unclear. In this study, we isolated a mutant, few spines 1 (fs1), from CNS2 (wild type, WT), a North China-type cucumber with a high density of fruit spines. Genetic analysis showed that fs1 was controlled by a single recessive Mendelian factor. Bulked segregant analysis combined with genome resequencing were used for mapping fs1 in the F2 population derived from a cross between the fs1 mutant and WT, and it was located on chromosome 6 through association analysis. To develop more polymorphic markers to locate fs1, another F2 population was constructed from the cross between fs1 and 'Chinese long' 9930. Then, fs1 was narrowed down to a 110.4-kb genomic region containing 25 annotated genes. A fragment substitution was identified in the promoter region of Csa6M514870 between fs1 and WT. This fragment in fs1 was also present in wild cucumber. Csa6M514870 encodes a PDF2-related protein, a homeodomain-leucine zipper IV transcription factor (CsHDZIV11/CsGL3) sharing high identity and similarity with proteins related to trichome formation or epidermal cell differentiation. Quantitative reverse-transcription PCR revealed a higher expression level of CsHDZIV11 in young fruits from fs1 compared to WT. A molecular marker based on this indel co-segregated with the spine density. This work provides a solid foundation not only for understanding the molecular mechanism of fruit spine density, but also for molecular breeding in cucumber.

Human ATP-binding cassette transporter 6 of subfamily B (ABCB6) is an ABC transporter involved in the translocation toxic metals and anti-cancer drugs. Using cryo-electron microscopy, we determined the molecular structure of full-length ABCB6 in an apo state. The structure of ABCB6 unravels the architecture of a full-length ABCB transporter that harbors two N-terminal transmembrane domains which is indispensable for its ATPase activity in our in vitro assay. A slit-like substrate binding pocket of ABCB6 may accommodate the planar shape of porphyrins, and the existence of a secondary cavity near the mitochondrial intermembrane space side would further facilitate substrate release. Furthermore, the ATPase activity of ABCB6 stimulated with a variety of porphyrin substrates showed different profiles in the presence of glutathione (GSH), suggesting the action of a distinct substrate translocation mechanism depending on the use of GSH as a cofactor.

Purinergic signaling contributes significantly to pain mechanisms, and the nociceptor-specific P2X3 ATP receptor channel is considered a target in pain therapeutics. Recent findings suggesting the coexpression of metabotropic P2Y receptors with P2X3 implies that ATP release triggers the activation of both ionotropic and metabotropic purinoceptors, with strong potential for functional interaction. Modulation of native P2X3 function by P2Y receptor activation was investigated in rat dorsal root ganglia (DRG) neurons using whole cell patch-clamp recordings. Application of the selective P2Y receptor agonist UTP decreased peak amplitudes of a,b-meATP-evoked homomeric P2X3-mediated currents, but had no effect on heteromeric P2X2/3-mediated currents. Treatment with phospholipase C inhibitor U73122 significantly reversed P2X3 current inhibition induced by UTP-sensitive P2Y receptor activation. We previously reported the modulation of P2X receptors by phospholipids in DRG neurons and injection of exogenous phosphatidylinositol-4,5-bisphosphate (PIP 2 ) fully reverses UTP-mediated regulation of P2X3 channel activity. Pharmacological as well as functional screening of P2Y receptor subtypes indicates the predominant involvement of P2Y2 receptor in P2X3 inhibition, and immunolocalization confirms a significant cellular coexpression of P2X3 and P2Y2 in rat DRG neurons. In summary, the function of P2X3 ATP receptor can be inhibited by P2Y2-mediated depletion of PIP 2 . We propose that expression of P2Y2 purinoceptor in nociceptive sensory neurons provides an homeostatic mechanism to prevent excessive ATP signaling through P2X3 receptor channels.

BackgroundIncreased neuronal excitability and spontaneous firing are hallmark characteristics of injured sensory neurons. Changes in expression of various voltage-gated Na+ channels (VGSCs) have been observed under neuropathic conditions and there is evidence for the involvement of protein kinase C (PKC) in sensory hyperexcitability. Here we demonstrate the contribution of PKC to P2X-evoked VGSC activation in dorsal root ganglion (DRG) neurons in neuropathic conditions.ResultsUsing the spinal nerve ligation (SNL) model of neuropathic pain and whole-cell patch clamp recordings of dissociated DRG neurons, we examined changes in excitability of sensory neurons after nerve injury and observed that P2X3 purinoceptor-mediated currents induced by α,β-meATP triggered activation of TTX-sensitive VGSCs in neuropathic nociceptors only. Treatment of neuropathic DRGs with the PKC blocker staurosporine or calphostin C decreased the α,β-meATP-induced Na+ channels activity and reversed neuronal hypersensitivity. In current clamp mode, α,β-meATP was able to evoke action-potentials more frequently in neuropathic neurons than in controls. Pretreatment with calphostin C significantly decreased the proportion of sensitized neurons that generated action potentials in response to α,β-meATP. Recordings measuring VGSC activity in neuropathic neurons show significant change in amplitude and voltage dependence of sodium currents. In situ hybridization data indicate a dramatic increase in expression of embryonic Nav1.3 channels in neuropathic DRG neurons. In a CHO cell line stably expressing the Nav1.3 subunit, PKC inhibition caused both a significant shift in voltage-dependence of the channel in the depolarizing direction and a decrease in current amplitude.ConclusionNeuropathic injury causes primary sensory neurons to become hyperexcitable to ATP-evoked P2X receptor-mediated depolarization, a phenotypic switch sensitive to PKC modulation and mediated by increased activity of TTX-sensitive VGSCs. Upregulation in VGSC activity after injury is likely mediated by increased expression of the Nav1.3 subunit, and the function of the Nav1.3 channel is regulated by PKC.