These authors contributed equally to this work.
Induction of jasmonate signalling regulators MaMYC2s and their physical interactions with MaICE1 in methyl jasmonate-induced chilling tolerance in banana fruit
Version of Record online: 21 JUN 2012
© 2012 Blackwell Publishing Ltd
Plant, Cell & Environment
Volume 36, Issue 1, pages 30–51, January 2013
How to Cite
ZHAO, M.-L., WANG, J.-N., SHAN, W., FAN, J.-G., KUANG, J.-F., WU, K.-Q., LI, X.-P., CHEN, W.-X., HE, F.-Y., CHEN, J.-Y. and LU, W.-J. (2013), Induction of jasmonate signalling regulators MaMYC2s and their physical interactions with MaICE1 in methyl jasmonate-induced chilling tolerance in banana fruit. Plant, Cell & Environment, 36: 30–51. doi: 10.1111/j.1365-3040.2012.02551.x
Research highlight: Two MYC2 genes, MaMYC2a and MaMYC2b, and one homolog of the inducer of the CBF gene, MaICE1 were isolated and characterized from banana fruit. MaMYC2a and MaMYC2b were induced rapidly following MeJA treatment during cold storage. MaMYC2s interacted with MaICE1, thus our work provides strong evidence that MaMYC2 is involved in MeJA-induced chilling tolerance in banana fruit through physically interacting and likely functionally coordinating with MaICE1, revealing a novel mechanism for ICE1 in response to cold stress as well as during development of induced chilling tolerance.
- Issue online: 3 DEC 2012
- Version of Record online: 21 JUN 2012
- Accepted manuscript online: 31 MAY 2012 07:09AM EST
- Received 26 January 2012; accepted for publication 25 May 2012
Figure S1. Photograph of chilling injury symptoms of MeJA-treated and control banana fruit during 5 days of storage at 7 °C. The arrow indicates banana fruit symptoms as pitting and brown patches presented in the peel.
Figure S2. Phylogenetic tree of the deduced amino acid sequences of MaMYC2a, MaMYC2b and other plant bHLH proteins. The phylogenetic tree was generated based on an alignment of the full length deduced amino acid sequences of 42 bHLH proteins, including, Vitis vinifera VvMYC1 (EU447172), VvMYCA1 (EF193002); Arabidopsis thaliana AtMYC1 (AtbHLH12; D83511), AtMYC2 (AtbHLH06; Q39204), TT8 (AtbHLH42; Q9FT81.2), GL3 (AtbHLH01; Q9FN69.1), EGL3 (AtbHLH02; Q9CAD0.1), bHLH92; Q9FIX5.1), AIB(ABA-Inducible bHLH-Type Transcription Factor; AtbHLH17; Q9ZPY8.2), SPT (Spatula; AtbHLH24; Q9FUA4.1), ALC (Alcatraz; AtbHLH73; Q9FHA2.1), AMS (Aborted Microspores; AtbHLH21; Q9ZVX2.2), ILR3 (IAA-Leucine Resistant 3; AtbHLH105; Q9FH37.1), ICE1 (Inducer of CBF Expression 1; AtbHLH116; Q9LSE2.1), ORG2(OBP3-Responsive Gene 2; AtbHLH38; Q9M1K1.1), PIF3 (Phytochrome Interacting Factor 3; AtbHLH08; O80536.1), PIL1 (Phytochrome Interacting Factor 3-Like 1; AtbHLH124; Q8L5W8.1), BEE1 (Brassinosteroids Enhanced Expression 1; AtbHLH44; Q8GZ13.1), RSL4 (Root Hair Defective 6-Like 4; AtbHLH54; Q8LEG1.1), RGE1 (Retarded Growth of Embryo 1; AtbHLH95; Q9FXA3.2), FIT (Fe-Deficiency Induced Factor 1; AtbHLH29; Q0V7X4.1), BPEp (Big Petal; AtbHLH31; Q0JXE7.1), SPCH (Speechless; AtbHLH98; Q700C7.1); Antirrhinum majus DELILA (AAA32663); Oryza sativa Rc (BAF42667); Petunia hybrida AN1 (AAG25928) and JAF13 (AAC39455); Zea mays B (CAA40544), Lc (AAA33504), and IN1 (AAB03841); Malus domestica bHLH33 (ABB84474); Gerbera hybrida MYC1 (CAA07614); Perilla frutescens MYC-RP (BAA75513), MYC-GP (BAA75514); Ipomoea purpurea Ivory Seed (BAD18982); Gentiana triflora GtbHLH1 (BAH03387), SN (X60706), b1 (NP_001105706), R1 (NP_001106073); Solanum lycopersicum JAMYC2 (CAF74710) and JAMYC10 (CAF74711). Alignments were made using CLUSTAL X multiple sequence software. The phylogenetic tree was constructed by the Neighbor-Joining method using the MEGA programme with default settings. MaMYC2a and MaMYC2b are shown in black circles. Numbers at the branchpoints indicated bootstrap values.
Figure S3. Amino acid sequence alignment of the MaICE1 protein with other plant ICE1 proteins. MaICE1 was aligned with Arabidopsis AtICE1 (Q9LSE2.1), Raphanus sativus RsICE1 (ADY68771.1), Populus trichocarpa PtICE1 (ABN58427.1), and Brassica napus BnICE1 (AEL33687.1). Identical and similar amino acids were presented by black and gray shading, respectively. Gaps were introduced to optimize alignment. The bHLH domain and ACT-like domain were included in the rectangle.
Figure S4. Amino acid sequence alignment of the MYC2s proteins with ICE1 proteins. MYC2s proteins including MaMYC2a, MaMYC2b, AtMYC2 were aligned with AtICE1 and MaICE1. Identical and similar amino acids were presented by black and gray shading, respectively. Gaps were introduced to optimize alignment. The JAZ interaction domain (JID), acidic domain (AD) and bHLH domain and ACT-like domain were included in the rectangles respectively.
Figure S5. Changes of endogenous JA content (a) and expression of MaMYC2a (b), MaMYC2b (c) genes in MeJA-treated and control banana fruit during 5 days of storage at 22 °C. Expression levels of each gene are expressed as a ratio relative to the time 0 d, which was set at 1. Each value represents the means of three replicates, and vertical bars indicate the SE. An asterisk indicates a statistical difference at the 5% level between MeJA-treated and control fruit.
Figure S6. BiFC visualization of the interaction between JID domain of MaMYC2s and C-terminal portion of MaICE1 (373-509aa) in transiently co-expressed tobacco BY-2 protoplasts. (a) JID domain of MaMYC2s and C-terminal portion of MaICE1 were fused with the N- and C-terminus of YFP, respectively. (b) JID domain of MaMYC2s and C-terminal portion of MaICE1 were fused with the C- and N-terminus of YFP, respectively. Expressions of JID domain of MaMYC2s or C-terminal portion of MaICE1 alone were used as negative controls. YFP indicates fluorescence of YFP; Merge is digital merge of bright field and fluorescent images. The length of the bar (25 µm) is as indicated in the photographs.
Figure S7. Physical interaction between Arabidopsis AtMYC2 and AtICE1 detected in yeast two-hybrid assays and in the BiFC system. (a) Diagram of AtMYC2 and AtICE1 protein domains. (b) Interactions between AtMYC2 and AtICE1 in the yeast two-hybrid assay. Different truncated derivatives of AtMYC2 or AtICE1 were tested for interaction with AtICE1 or AtMYC2, respectively. Gold Y2H yeast strains were cotransformed with pGBKT7-AtMYC2 derivatives (bait) and pGADT7-AtICE1 (prey). The ability of yeast cells to grow on synthetic medium lacking tryptophan, leucine, histidine, and adenine (QDO), and QDO medium containing 125 µ M Aureobasidin A (QDO/A), and turn blue in the presence of the chromagenic substrate X-α-Gal (QDO/X/A), was scored as a positive interaction. Yeast cells transformed with pGBKT7 + pGADT7-AtICE1, or pGBKT7-AtMYC2 (JID) + pGADT7-T was included as negative controls. (c) BiFC visualization of the AtMYC2 and AtICE1 interaction in transiently co-expressed tobacco BY-2 protoplasts. MaMYC2s and MaICE1 proteins were fused with the N- and C-terminus of YFP, respectively. (b) MaMYC2s and MaICE1 proteins were fused with the N- and C-terminus of YFP, respectively. Expressions of AtMYC2 or AtICE1 alone were used as negative controls. YFP indicates fluorescence of YFP; Merge is digital merge of bright field and fluorescent images. The length of the bar (25 µm) is as indicated in the photographs.
Figure S8. Interaction between C-terminal portion of MaMYC2s and C-terminal portion of MaICE1 detected in yeast two-hybrid assays. Gold Y2H yeast strains were cotransformed with truncated C-terminal of MaMYC2s (including bHLH and ACT-like domain) in pGBKT7 (bait) and C-terminal of MaICE1 (including bHLH domain and ACT-like domain) in pGADT7 (prey). The ability of yeast cells to grow on synthetic medium lacking tryptophan, leucine, histidine, and adenine (QDO), and QDO medium containing 125 µ M Aureobasidin A (QDO/A), and turn blue in the presence of the chromagenic substrate X-α-Gal (QDO/X/A), was scored as a positive interaction.
Table S1. Primer sequences used for fusing GFP.
Table S2. Primer sequences used for subcloning into pGBKT7.
Table S3. Primer sequences used in quantitative real-time PCR analysis.
Table S4. Primer sequences used for Yeast Two-Hybrid and BiFC assays.
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