Shoot chloride exclusion and salt tolerance in grapevine is associated with differential ion transporter expression in roots

BACKGROUND: Salt tolerance in grapevine is associated with chloride (Cl-) exclusion from shoots; the rate-limiting step being the passage of Cl- between the root symplast and xylem apoplast. Despite an understanding of the physiological mechanism of Cl- exclusion in grapevine, the molecular identity of membrane proteins that control this process have remained elusive. To elucidate candidate genes likely to control Cl- exclusion, we compared the root transcriptomes of three Vitis spp. with contrasting shoot Cl- exclusion capacities using a custom microarray. RESULTS: When challenged with 50 mM Cl-, transcriptional changes of genotypes 140 Ruggeri (shoot Cl- excluding rootstock), K51-40 (shoot Cl- including rootstock) and Cabernet Sauvignon (intermediate shoot Cl- excluder) differed. The magnitude of salt-induced transcriptional changes in roots correlated with the amount of Cl- accumulated in shoots. Abiotic-stress responsive transcripts (e.g. heat shock proteins) were induced in 140 Ruggeri, respiratory transcripts were repressed in Cabernet Sauvignon, and the expression of hypersensitive response and ROS scavenging transcripts was altered in K51-40. Despite these differences, no obvious Cl- transporters were identified. However, under control conditions where differences in shoot Cl- exclusion between rootstocks were still significant, genes encoding putative ion channels SLAH3, ALMT1 and putative kinases SnRK2.6 and CPKs were differentially expressed between rootstocks, as were members of the NRT1 (NAXT1 and NRT1.4), and CLC families. CONCLUSIONS: These results suggest that transcriptional events contributing to the Cl- exclusion mechanism in grapevine are not stress-inducible, but constitutively different between contrasting varieties. We have identified individual genes from large families known to have members with roles in anion transport in other plants, as likely candidates for controlling anion homeostasis and Cl- exclusion in Vitis species. We propose these genes as priority candidates for functional characterisation to determine their role in chloride transport in grapevine and other plants.Sam W Henderson, Ute Baumann, Deidre H Blackmore, Amanda R Walker, Rob R Walker and Matthew Gilliha

Measurement and differentiation of ligand-induced calmodulin conformations by dual polarization interferometry

In early drug discovery, knowledge about ligand-induced conformational changes and their influence on protein activity greatly aids the identification of lead candidates for medicinal chemistry efforts. Efficiently acquiring such information remains a challenge in the initial stages of lead finding. Here we investigated the application of dual polarization interferometry (DPI) as a method for the real-time characterization of low molecular weight (LMW) ligands that induce conformational changes. As a model system we chose calmodulin (CaM), which undergoes large and distinct structural rearrangements in response to calcium ion and small molecule inhibitors such as trifluoperazine (TFP). We measured concentration-dependent mass, thickness, and density responses of an immobilized CaM protein layer, which correlated directly with binding and conformational events. Calcium ion binding to CaM induced an increase in thickness (≤0.05 nm) and decrease in density (≤-0.03 g/cm(3)) whereas TFP induced an increase in both thickness (≤0.05 nm) and density (≤0.01 g/cm(3)). The layer measurements reported here show how DPI can be used to assess and differentiate ligands with distinct structural modes of action

Orally Active 7-Substituted (4-Benzyl-phthalazin-1-yl)-2-methyl-piperazin-1-yl]-nicotinonitriles as Active-site Inhibitors of Sphingosine-1-Phosphate Lyase for the Treatment of Multiple Sclerosis

Sphingosine-1-phosphate (S1P) lyase has recently been implicated as a therapeutic target for the treatment of multiple sclerosis (MS), based on studies in a genetic mouse model. Potent active-site directed inhibitors of the enzyme are not known so far. Here we describe the discovery of (4-benzyl-phthalazin-1-yl)-2-methyl-piperazin-1-yl]-nicotinonitrile 1 in a high-throughput screen using a biochemical assay, and its further optimization. This class of compounds was found to inhibit catalytic activity of S1PL by binding to the active site of the enzyme, as seen in the co-crystal structure of derivative 15 with the homodimeric human S1P lyase. 15 induces profound reduction of peripheral T cell numbers after oral dosage and confers pronounced protection in a rat model of multiple sclerosis . In conclusion, this novel class of direct S1P lyase inhibitors provides excellent tools to further explore the therapeutic potential of T cell-targeted therapies in multiple sclerosis and other autoimmune and inflammatory diseases