1. Volume 10, Issue 5, Pages 695-708 (May 2017)
Transporter-Mediated Nuclear Entry of Jasmonoyl-Isoleucine Is Essential for Jasmonate Signaling 
Qingqing Li, Jian Zheng, Shuaizhang Li, Guanrong Huang, Stephen J. Skilling, Lijian Wang, Ling Li, Mengya Li, Lixing Yuan, Pei Liu 
Molecular Plant 
Volume 10, Issue 5, Pages (May 2017)
DOI: /j.molp
Copyright © 2017 The Authors Terms and Conditions

2. Figure 1
Identification of AtJAT1/AtABCG16 as a JA Exporter in the Heterologous Yeast Cells.
(A) Yeast cells expressing AtJAT1/AtABCG16 were diluted (OD = 1.0, 0.1, 0.01, and 0.001) and grown on SD-URA plates supplemented with 6 mM exogenous JA for 5 days. Yeast cells with empty vector pDR195 were used as the control.
(B) Time-dependent 3H-JA loading (relative to the initial loading) in AtJAT1/AtABCG16 and PDR yeast cells at 2, 5, 10, 20, and 60 min of incubation with 8 nM 3H-JA.
(C) 3H-JA export (relative to the initial loading) in AtJAT1/AtABCG16 and PDR yeast cells 1 min after induction of export in the cells preloaded with 8 nM 3H-JA.
(D) Inhibition of AtJAT1/AtABCG16-mediated 3H-JA export by pretreatment of yeast cells with 25 μM glibenclamide (GC), 25 μM vanadate (VD), and 25 μM verapamil (VP) (relative to the control without inhibitor pretreatment).
Error bars represent SD; n = 3. *P < 0.05, **P < 0.01, ***P < by ANOVA (B and D) or Student’s t-test (C).
Molecular Plant  , DOI: ( /j.molp )
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3. Figure 2 AtJAT1/AtABCG16 Is Essential for JA Signaling.
(A) The promoter activities of AtJAT1/AtABCG16 in the vascular tissues of cotyledons and roots (indicated by arrows) of seedlings were enhanced by exogenous JA (2 μM) treatment (+JA). Scale bars, 1 mm.
(B) Time-dependent induction of AtJAT1/AtABCG16 transcripts by exogenous MeJA (0.03%) treatment (n = 3).
(C) Compared with Col, the atjat1-1 plants show markedly larger cell size of roots which was reverted in JAT1R (atjat1-1/35S::AtJAT1-GFP) plants, while coi1-30 and atjat1-2 displayed a slight increase in plant stature and cell size (n ≥ 23).
(D) Root growth inhibition (relative to control without JA treatment) induced by 2 μM exogenous JA, JA-Ile, or 1 μM COR treatment was decreased in coi1-30 and atjat1/atabcg16 plants (n ≥ 10). See also Supplemental Figure 4E.
(E and F) Time-dependent induction of the primary JA-responsive marker genes in JA synthesis (OPR3) (E) and signaling (JAZ5 and JAZ7) (F) by exogenous MeJA (0.03%) treatment in Col and atjat1-1 plants (n = 3).
(G) Compared with Col, atjat1/atabcg16 plants showed reduced resistance, while JAT1OX plants showed enhanced resistance, to B. cinerea (indicated by lesion area) (n = 3). See also Supplemental Figure 5.
Error bars represent SD. *P < 0.05, ***P < denote post-test significance compared with the control/Col by ANOVA.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Authors Terms and Conditions

4. Figure 3
Dual Localization of AtJAT1/AtABCG16 in Nuclear Envelope and Plasma Membrane.
(A and B) The AtJAT1-GFP signal were detected in the plasma membrane (PM) of the epidermal pavement cells, but in both PM and nucleus (indicated by arrows) in the stomatal guard cells (A) and root cells (B) of JAT1OX plants. Scale bar, 25 μm.
(C) Localization of AtJAT1-GFP in the PM and NE in epidermal pavement cells of tobacco. Scale bar, 25 μm.
(D) Localization of AtJAT1-GFP (driven by AtJAT1/AtABCG16 native promoter) florescence signal in the PM and NE (indicated by white arrows) of root cells. Scale bar, 25 μm.
(E–H) AtJAT1-GFP protein (driven by the AtJAT1/AtABCG16 promoter) was detected in PM (E) (spots indicated by blue circles) and NE (F) (spots indicated by red circles) by immunogold localization with anti-GFP antibody incubation, while no signal was detected in either PM (G) or NE (H) in controls without anti-GFP incubation. Insets show the magnification of cellular sections in the boxes. Scale bars, 500 nm.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Authors Terms and Conditions

5. Figure 4 AtJAT1/AtABCG16 Mediates Nuclear Entry of JA-Ile.
(A) Time-dependent uptake of 3H-JA and 3H-JA-Ile (32 nM) by the nuclei isolated from calli induced from root segments of Col and atjat1-1 plants (n = 4).
(B) Concentration-dependent JA-Ile uptake rate (VJA-Ile-uptake) mediated by AtJAT1/AtABCG16 was calculated from JA-Ile retention data in Supplemental Figure 6A and the Km value was estimated after fitting the data to the Michaelis–Menten equation (n = 3).
(C) Inhibition of AtJAT1/AtABCG16-mediated 3H-JA-Ile uptake by pretreatment of the isolated nuclei with 80 μM glibenclamide (GC), 200 μM vanadate (VD), and 80 μM verapamil (VP) (relative to the control without inhibitor pretreatment) (n = 4).
(D) AtJAT1/AtABCG16-mediated 3H-JA-Ile uptake by the isolated nuclei in the presence (+) and absence (−) of 10 mM ATP (n = 3).
(E) 3H-JA-Ile uptake of the isolated nuclei was decreased in atjat1-1, but enhanced in JAT1OX cells (n = 4).
(F) Relative nuclear 3H-JA-Ile (32 nM) uptake mediated by AtJAT1/AtABCG16 in the presence of unlabeled 256 nM JA-Ile, COR, JA, MeJA, LA, OPDA, SA, ABA, and 4-MTB (n = 4).
Error bars represent SD. **P < 0.01, ***P < denote post-test significance compared with the control by ANOVA.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Authors Terms and Conditions

6. Figure 5
AtJAT1/AtABCG16 Is Essential to Maintain a Critical Nuclear JA-Ile Concentration to Activate JA Signaling.
(A and B) Electron microscopic autoradiographs of cultured Col cells with (A) or without (B) 3H-JA feeding. Scale bars, 2 μm.
(C and D) Electron microscopic autoradiographs of cultured jar1 (C) and atjat1-1 (D) cells 2 hours after feeding with 0.4 μM 3H-JA. The plasma membrane (PM) and nuclear envelope (NE) are indicated by arrows. Silver particles in the nucleus (Nu) and cytoplasm are indicated by red and blue circles, respectively. Insets show the magnification of the cellular sections in the boxes. Scale bars, 2 μm.
(E) The abundance of silver particles in the nucleus, cytoplasm, and the whole cell of Col (A), jar1 (C), and atjat1-1 (D) cells was compared (n = 4).
(F) Visualization of JAZ1-GFP in the nuclei of primary root tips of Col or atjat1-1 plants in the presence (+JA) (5 min treatment) or absence (−JA) of 2 μM exogenous JA. Scale bars, 100 μm.
(G) The jar1 and atjat1-1 mutant plants were largely normal in late stamen development, whereas the jar1;atjat1-1 homozygous plants exhibited male sterility due to short anther filaments and non-dehiscent anthers in stage 13 (lower panel) but not stage-12 flowers (top panel), which could be rescued by exogenous JA treatment (+JA). Scale bars, 500 μm.
Error bars represent SD. *P < 0.05, ***P < denote post-test significance compared with the control by ANOVA within the groups indicated by lower lines.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Authors Terms and Conditions

7. Figure 6
AtJAT1/AtABCG16 Also Mediates Cellular Export of JA across the PM.
(A) Time-dependent 3H-JA and 3H-JA-Ile (16 nM) accumulation by suspension cells derived from root segments of Col and atjat1-1 plants.
(B) Concentration-dependent AtJAT1/AtABCG16-mediated JA export rate (VJA-export) was calculated from JA retention data in Supplemental Figure S6B and the Km value was estimated after fitting the data to the Michaelis–Menten equation.
(C) 3H-JA accumulation was increased in atjat1-1 but decreased in JAT1OX cells.
(D) Inhibition of AtJAT1/AtABCG16-mediated 3H-JA export by pretreatment of the suspension cells with 80 μM glibenclamide (GC), 200 μM vanadate (VD), and 80 μM verapamil (VP) (relative to the control without inhibitor pretreatment).
(E) Relative nuclear 3H-JA (16 nM) export mediated by AtJAT1/AtABCG16 in the presence of unlabeled 160 nM JA, JA-Ile, COR, MeJA, LA, OPDA, SA, ABA, and 4-MTB.
Error bars represent SD; n = 4. **P < 0.01, ***P < denote post-test significance compared with the control/Col by ANOVA.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Authors Terms and Conditions

8. Figure 7
A Model Showing the Roles of AtJAT1/AtABCG16 in Regulating JA Signaling.
AtJAT1/AtABCG16 controls the subcellular distribution of JA-Ile and JA in the nucleus and cytoplasm, respectively, by mediating the cellular export of JA and nuclear import of JA-Ile. JA and JA-Ile are interconvertible in the cytosol catalyzed by JAR1 and JIH1, respectively, and JA-Ile can also be converted to 12OH-JA-Ile by CYP94B3. The imported JA-Ile in the nucleus binds with SCFCOI1-JAZ coreceptor, recruiting JAZ transcriptional repressors for ubiquitylation and degradation by 26S proteasome. The degradation of JAZ proteins relieves their repression on the activity of transcription factors (TFs), initiating the transcriptional cascades essential for JA signaling. The integration of a dual function by AtJAT1/AtABCG16 in mediating nuclear entry of JA-Ile and cellular export of JA enables plants to adapt constantly to the growth–defense dynamic by prompt activation and deactivation of JA signaling.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Authors Terms and Conditions