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Export of salicylic acid from the chloroplast requires the multidrug and toxin extrusion-like transporter EDS5 - PubMed

Export of salicylic acid from the chloroplast requires the multidrug and toxin extrusion-like transporter EDS5

Mario Serrano et al. Plant Physiol. 2013 Aug.

Abstract

Salicylic acid (SA) is central for the defense of plants to pathogens and abiotic stress. SA is synthesized in chloroplasts from chorismic acid by an isochorismate synthase (ICS1); SA biosynthesis is negatively regulated by autoinhibitory feedback at ICS1. Genetic studies indicated that the multidrug and toxin extrusion transporter ENHANCED DISEASE SUSCEPTIBILITY5 (EDS5) of Arabidopsis (Arabidopsis thaliana) is necessary for SA accumulation after biotic and abiotic stress, but so far it is not understood how EDS5 controls the biosynthesis of SA. Here, we show that EDS5 colocalizes with a marker of the chloroplast envelope and that EDS5 functions as a multidrug and toxin extrusion-like transporter in the export of SA from the chloroplast to the cytoplasm in Arabidopsis, where it controls the innate immune response. The location at the chloroplast envelope supports a model of the effect of EDS5 on SA biosynthesis: in the eds5 mutant, stress-induced SA is trapped in the chloroplast and inhibits its own accumulation by autoinhibitory feedback.

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Figures

Figure 1.
Figure 1.

Distribution of free and conjugated SA in leaves of Arabidopsis after stimulation of SA biosynthesis by UV exposure. Three-week-old Arabidopsis plants were exposed to UV, and SA was subsequently determined. A, Free and conjugated SA content in whole leaves. B, SA relative distribution to non-UV-induced plants in the cytosol and chloroplasts. Representative results of three independent experiments each with three replicates are shown. Significant differences (Student’s t test; P < 0.05) of means ±

sd

from noninduced wild-type plants are indicated by asterisks. FW, Fresh weight.

Figure 2.
Figure 2.

EDS5 localizes at the chloroplast envelope. A, CLSM images of mesophyll protoplasts from Arabidopsis expressing EDS5-YFP and 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 were carried out as described (Yoo et al., 2007). Bars = 10 μm.

Figure 3.
Figure 3.

EDS5 catalyzes the specific transport of SA. A, UV induction or constitutive overexpression of EDS5 enhances SA uptake into isolated chloroplasts. Chloroplasts were 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. Significant differences (Student’s t test; P < 0.05) of means ±

sd

(n = 4) from non-UV-induced wild-type (WT) plants are indicated by asterisks. B, Export of SA but not IAA from protoplasts is reduced by UV induction or constitutive overexpression of EDS5. Mesophyll protoplasts were simultaneously loaded with [14C]SA and the auxin IAA ([3H]IAA) as an unspecific control. Reduced SA export over the protoplast plasma membrane caused by UV induction or constitutive overexpression of EDS5 (35S::EDS5) is most probably due to chloroplast trapping of SA provided by EDS5; its absence in eds5 demonstrates indirectly the identity of EDS5 as an SA transporter. EDS5 is specific for SA, as chloroplast trapping by EDS5 was not observed with IAA, used as an unspecific control. Significant differences (ANOVA using the Tukey test for multiple comparisons; P < 0.05) of means ±

se

(n = 4) from noninduced wild-type plants are indicated by asterisks. Note that the chloroplast uptake directionalities reported here for EDS5 in A and B underline the expected bidirectionality of EDS5 transport activity and are most likely caused by the nonphysiological high concentration of labeled SA during the transport assays. C, Schematic representation of the protoplast export experiment. Protoplasts of Arabidopsis leaves prepared from wild-type, UV-treated wild-type, 35S::EDS5, and eds5 plants were incubated with [14C]SA (left). Protoplasts were then washed (middle) and incubated in fresh medium, allowing the quantification of export (right). When EDS5 is present (after UV treatment or in 35S::EDS5 plants; bottom panel), SA is transported into the chloroplast and will contribute to a lesser total export over the plasma membrane, as observed in B.

Figure 4.
Figure 4.

EDS5 catalyzes the specific transport of SA in yeast. A, EDS5-GFP is localized on small, punctate vesicles surrounding the plasma membrane of Saccharomyces cerevisiae. B, EDS5-HA comigrates with the plasma membrane marker H+-ATPase in continuous Suc gradients judged by western detection. Assay and marker enzymes are described by Kamimoto et al. (2012). C, EDS5 yeast show reduced export of SA. Whole yeast were loaded simultaneously with labeled SA ([14C]SA) and IAA ([3H]IAA), and net retention was quantified as described by Kamimoto et al. (2012). Altered SA but not IAA retention in EDS5-HA and EDS5-GFP yeast compared with the vector control (Control) demonstrate the identity of EDS5 as a specific SA transporter. A significant difference (Student’s t test; P < 0.05) of means ±

se

(n = 4) from vector control yeast is indicated by the asterisk. Note that negative retention (i.e. export) for SA not found for IAA argues for a strong background SA efflux activity by an endogenous transport system on the yeast plasma membrane (for details, see text).

Figure 5.
Figure 5.

Free and conjugated SA accumulate in the chloroplasts of eds5 mutants overproducing SA. SA content in 35S::EDS5 plants and eds5 mutants were both transformed with ALC::pSAS. A, Free and conjugated SA content in leaves 0 and 24 h after treatment with ethanol. Significant differences (Student’s t test; P < 0.05) of means ±

sd

(n = 4) from non-ethanol-induced plants are indicated by asterisks. FW, Fresh weight. B, Free and conjugated SA content in isolated chloroplasts 24 h after treatment with ethanol. The inset shows the expression of ALC::pSAS in leaves of transgenic plants after ethanol treatment. Significant differences (Student’s t test; P < 0.05) of means ±

sd

(n = 4) from 35S::EDS5 plants are indicated by asterisks. C, Model of EDS5 action. Left, the functional EDS5 (in either 35S::EDS5 or wild-type plants induced by biotic or abiotic stress) exports SA made in the chloroplast. Middle, in eds5 mutants, SA accumulates in the chloroplast and presumably shuts down its own biosynthesis by a negative feedback that has yet to be characterized in detail. Right, in eds5 mutants induced to express pSAS, SA accumulates in the chloroplasts.

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