Plast envelope membrane and catalyzes the export of SA from the chloroplast towards the cytoplasm.Plant Physiology? August 2013, Vol. 162, pp. 1815?821, plantphysiol.org ?2013 American Society of Plant Biologists. All Rights Reserved.Serrano et al.Benefits AND DISCUSSIONFree and conjugated SA accumulate in the leaves of Arabidopsis right after stimulation with UV light (Fig. 1A), as described previously (Nawrath et al., 2002; Fragni e et al., 2011). Surprisingly the raise in free and conjugated SA will not be measurable inside the chloroplast fraction (Fig. 1B). This may well be explained by rapid export of SA from the chloroplast for the cytoplasm. Therefore, we predict that (1) EDS5 is positioned in the chloroplast envelope; (2) mutants with impaired EDS5 function are unable to export SA; and hence (three) in these mutants, SA is trapped in the chloroplast and inhibits its synthesis by adverse feedback. To test this hypothesis, EDS5 was localized by the stable expression of fluorescent EDS5-yellow fluorescent protein (YFP) under the control with the constitutive cauliflower mosaic virus 35S (35S) promoter (35S::EDS5-YFP) in transgenic Arabidopsis plants that were cotransformed with all the chloroplast marker 35S::RecA-CFP (see “Materials and Methods”). Confocal laser scanning microscopy (CLSM) of mesophyll protoplasts resulted in RecA-cyan fluorescent protein (CFP) fluorescence overlapping together with the autofluorescence of chlorophyll in plastids (Fig. 2A). In contrast, the fluorescence ofFigure 1. Distribution of cost-free and conjugated SA in leaves of Arabidopsis right after stimulation of SA biosynthesis by UV exposure. Threeweek-old Arabidopsis plants were exposed to UV, and SA was subsequently determined. A, Absolutely free and conjugated SA content material in complete leaves. B, SA relative distribution to non-UV-induced plants within the cytosol and chloroplasts.Price of 2-(3-Bromopyridin-4-yl)acetonitrile Representative benefits of three independent experiments each and every with 3 replicates are shown. Important variations (Student’s t test; P , 0.05) of means six SD from noninduced wild-type plants are indicated by asterisks. FW, Fresh weight.Figure two. EDS5 localizes in the chloroplast envelope. A, CLSM photos of mesophyll protoplasts from Arabidopsis expressing EDS5-YFP along with the chloroplast marker RecA-CFP. B, Mesophyll protoplasts from Arabidopsis expressing EDS5-YFP transfected with the inner envelope marker prCIA5-TM2-RFP (CIA5-RFP; Cerutti et al., 1992). The protoplast isolation and transfection have been carried out as described (Yoo et al., 2007). Bars = 10 mm.2-chloro-5-(methylthio)pyrimidine Order Plant Physiol.PMID:23672196 Vol. 162,EDS5-Mediated Export of Salicylic AcidFigure 3. EDS5 catalyzes the precise transport of SA. A, UV induction or constitutive overexpression of EDS5 enhances SA uptake into isolated chloroplasts. Chloroplasts had been incubated with labeled SA ([14C]SA), and SA uptake was quantified by scintillation counting. Enhanced SA uptake caused by UV induction or constitutive overexpression of EDS5 (35S::EDS5) is absent in eds5, demonstrating the identity of EDS5 as an SA transporter. Substantial variations (Student’s t test; P , 0.05) of suggests 6 SD (n = four) from non-UV-induced wild-type (WT) plants are indicated by asterisks. B, Export of SA but not IAA from protoplasts is decreased by UV induction or constitutive overexpression of EDS5. Mesophyll protoplasts were simultaneously loaded with [14C]SA plus the auxin IAA ([3H]IAA) as an unspecific control. Decreased SA export over the protoplast plasma membrane triggered by UV induction or constitutive overexpression of EDS5 (35S::EDS5) is mo.
