A direct interaction of the Arabidopsis thaliana immunophilin ROF1 with phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate was identified using a phosphatidylinositol-phosphate affinity chromatography of cell suspension extracts, combined with a mass spectrometry (nano LC ESI-MS/MS) analysis. The first FK506 binding domain was shown sufficient to bind to both phosphatidylinositol-phosphate stereoisomers. GFP-tagged ROF1 under the control of a 35S promoter was localised in the cytoplasm and the cell periphery of Nicotiana tabacum leaf explants. Immunofluorescence microscopy of Arabidopsis thaliana root tips verified its cytoplasmic localization and membrane association and showed ROF1 localization in the elongation zone which was expanded to the meristematic zone in plants grown on high salt media. Endogenous ROF1 was shown to accumulate in response to high salt treatment in Arabidopsis thaliana young leaves as well as in seedlings germinated on high salt media (0.15 and 0.2 M NaCl) at both an mRNA and protein level. Plants over-expressing ROF1, (WSROF1OE), exhibited enhanced germination under salinity stress which was significantly reduced in the rof1− knock out mutants and abolished in the double mutants of ROF1 and of its interacting homologue ROF2 (WSrof1−/2−). Our results show that ROF1 plays an important role in the osmotic/salt stress responses of germinating Arabidopsis thaliana seedlings and suggest its involvement in salinity stress responses through a phosphatidylinositol-phosphate related protein quality control pathway.

Context: Phytopharmacology is a complex but very promising research area. The different plant parts and extraction methods may result in opposed effects. Phlomis species have been reported for both anti-inflammatory and tonic properties. Objective: The effect of Phlomis lanata Willd. (Lamiaceae) protein extracts on immune cell reactivity was studied in the experimental mouse model. Materials and methods: Protein extracts from P. lanata aerial parts were fractionated by Q-sepharose ion-exchange chromatography and applied to whole spleen cells or T-cell subsets at 5 mg/ml. Cell growth and cytokine production were evaluated after 4 and 2 d of culture using 3 H-thymidine-uptake and ELISA techniques, respectively. Results: Among the protein fractions tested, column wash proteins (W1) and the fraction eluted using 600 mM NaCl (F6) reduced by 76% and increased by 78% spleen cell proliferation, respectively. W1 suppressed proliferation of effector T-cells, but stimulated the growth of suppressor/regulatory cells by 62-148%. Although W1 stimulated IL-2 and IL-10 production from total spleen cells, it significantly increased IL-10 (50%) and reduced IL-2 (30-50%) production from T-cells, while TNF-a release was enhanced in CD25 + CD4 + by 92% and reduced by 50% in CD25 + CD8 + cells. F6 stimulated whole spleen cell growth, reduced proliferation of CD8 + and CD25 + cells by approximately 50%, while decreasing by 60-80% TNF-a production from CD25À and CD25 + CD8 + cells. Discussion and conclusion: The suppressive activity of W1 could be attributed to IL-10 and TNF-a, while the stimulatory effect of F6 could be attributed to the inhibition of T-regulatory cells. In the same plant, coexisting protein fractions induce both immunostimulatory and immunosuppressive activities.