1. Volume 5, Issue 1, Pages 73-84 (January 2012)
Tobacco Transcription Factors NtMYC2a and NtMYC2b Form Nuclear Complexes with the NtJAZ1 Repressor and Regulate Multiple Jasmonate-Inducible Steps in Nicotine Biosynthesis 
Zhang Hong-Bo , Bokowiec Marta T. , Rushton Paul J. , Han Sheng-Cheng , Timko Michael P.  
Molecular Plant 
Volume 5, Issue 1, Pages (January 2012)
DOI: /mp/ssr056
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

2. Figure 1
Alignment of Tobacco and Arabidopsis MYC2 Homologs. Shown is an alignment of the AtMYC2 (GenBank Accession: AT1G32640), NtMYC2a (GenBank Accession: HM466974), NtMYC2b (GenBank Accession: HM466975), and NtMYC2c (GenBank Accession: HM466976) amino acid sequences. Black highlighted residues indicate 100% identity; gray highlighted indicate similarity values of 75% or higher. The dotted residues indicate the putative acidic activation domain; the bHLH–Zip domain is included in the rectangle.
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3. Figure 2
MeJA-Induced Changes in NtMYC2a and NtMYC2b Transcript Levels.
(A) Semiquantitative RT–PCR analysis of NtPMT and NtActin control transcripts in BY-2 cells at various times following MeJA treatment. This was used as internal control. JA indicates tobacco BY-2 cells with MeJA treatment and Control indicates BY-2 cells harvested at the same time points without treatment.
(B) Quantitative RT–PCR analysis of NtMYC2a/b expression at various times after MeJA treatment of tobacco BY-2 cells. Average value ± SD (n = 3).
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4. Figure 3
Analysis of NtMYC2 Binding to the G-Box in the GAG Motif of the NtPMT1a Gene Promoter.
(A) EMS assay using α-32P–dCTP-labeled wild-type GAG motif fragments and fragments (gAG, GaG, and GAg) containing mutations and purified His–NtMYC2aΔN protein. The asterisk indicates free probe.
(B) ChIP (chromatin immunoprecipitation) assay. Ppmt, amplification of GAG motif in NtPMT promoter; NtActin, amplification of negative control NtActin; Control, sGFP expressing BY2 cells; MYC2a, NtMY2a–YFP expressing BY2 cells; MYC2b, NtMYC2b–YFP expressing BY2 cells; IP, immunoprecipitation; Mock, immunoprecipitation without antibody; Input, samples before immunoprecipitation.
(C) Transient assay with BY2 cell protoplasts. Relative GUS/LUC activities are expressed relatively to the value of control transformation (set arbitrarily as 1) that was transformed with reporter vector, control effector vector, and internal control vector. Error bars indicate ±SE. Data represent the average of at least three independent experiments. Schematic drawings of reporter and effector vectors are shown over the histograms of transient assay. TATA indicates CaMV 35S TATA box. GAG, reporter is four GAG-controlled GUS; gAG, reporter is four gAG-controlled GUS; Control, effector is control effector vector; MYC2a, effector is vector expressing NtMYC2a; MYC2b, effector is vector expressing NtMYC2b.
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5. Figure 4
Sub-Cellular Localization of NtMYC2s and the In Vivo Interaction of NtMYC2s and NtJAZ1.
(A) Sub-cellular localization of NtMYC2s. BY2 cells stably expressing fusion protein NtMYC2a–YFP and NtMYC2b–YFP were used to visualize the sub-cellular localization of NtMYC2a and NtMYC2b. Wild-type (WT) and sGFP-expressing (sGFP) cells were used as controls. Nucleus was visualized by DAPI staining. FP indicates fluorescence protein sGFP/YFP.
(B) The in vivo interaction of NtMYC2s and NtJAZ1. Protoplasts of BY2 cells were co-transformed with a combination of vectors transiently expressing the indicated fusion or non-fusion proteins. cYFP and nYFP indicate the C-terminal and N-terminal part of YFP protein, respectively; NtJAZ1–nYFP, NtMYC2a–cYFP, and NtMYC2b–cYFP indicate vectors expressing these fusion proteins; YFP indicates fluorescence of YFP; Merge is digital merge of bright field and fluorescent images.
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6. Figure 5
Nicotine Biosynthetic Gene Expression in Wild-Type (WT) BY-2 Cells and Independently Derived Transgenic BY-2 Overexpressing NtMYC2a- and NtMYC2b. The relative expression values were generated with the histogram function of Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA) by calculating the values of luminosity of each blot in the inverted picture of RNA gel blot (i.e. the blot spot is inverted to light spot) over the corresponding luminosity value of loading control rRNA visualized by EtBr staining. –JA indicates cells untreated with MeJA; +JA indicates cells treated with MeJA for 24 h; OEmyc2a indicates cells expressing NtMYC2a; OEmyc2b indicates cells expressing NtMYC2b; the independently derived transgenic BY-2 overexpressing NtMYC2a- and NtMYC2b are labeled 1–4. Numbers over the bars indicate the actual relative expression values, of which ‘0’ indicates no detectable signal in the untreated cells.
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7. Figure 6
Effects of RNAi Knockdown of NtMYC2s Expression on JA-Induced Expression of Nicotine Biosynthetic Genes. (A) qRT–PCR analysis of NtMYC2s transcription in wild-type (WT) and NtMYC2–RNAi BY2 cells treated with MeJA. The NtMYC2s transcription in WT was arbitrarily set as 1.
(B) qRT–PCR analysis of nicotine biosynthetic gene transcription in wild-type (WT) and NtMYC2–RNAi BY2 cells treated with MeJA for 24 h. For each gene, the relative transcription values were generated by comparing its expression level in the indicated lines to that in the untreated WT BY2 cells. Error bar indicates ±SD (n = 3). Asterisks indicate P-values of Student's t-test: one asterisk for P < 0.05 and two asterisks for P < 0.005.
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8. Figure 7
Model for Regulation of Nicotine Biosynthetic Gene Expression by NtMYC2a/b. In the absence of JA, NtMYC2 forms a complex with NtJAZ repressors and no nicotine biosynthetic gene expression occurs. The presence of JA leads to the formation of JA-Ile, which promotes interaction between JAZ proteins and SCFC°I1 ubiquitin ligase, which leads to JAZ degradation via the 26S proteasome, then frees NtMYC2 transcription factors to activate the expression of JA-inducible TFs through binding to the G-box-like elements in their promoters, and these TFs cooperate with NtMYC2 to regulate NtPMT genes and other nicotine biosynthetic genes.
Molecular Plant 2012 5, 73-84DOI: ( /mp/ssr056)
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions