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Two GCC boxes and AP2/ERF-domain transcription factor ORA59 in jasmonate/ethylene-mediated activation of the PDF1.2 promoter in Arabidopsis.

Zarei A, Körbes AP, Younessi P, Montiel G, Champion A, Memelink J - Plant Mol. Biol. (2011)

Bottom Line: Using the chromatin immunoprecipitation technique we were able to show that ORA59 bound the PDF1.2 promoter in vivo.Finally, we show that a tetramer of a single GCC box conferred JA/ethephon-responsive expression, demonstrating that the JA and ET signaling pathways converge to a single type of GCC box.Therefore ORA59 and two functionally equivalent GCC box binding sites form the module that enables the PDF1.2 gene to respond synergistically to simultaneous activation of the JA and ET signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biology, Sylvius Laboratory, Leiden University, P.O. Box 9505, 2300 RA, Leiden, The Netherlands.

ABSTRACT
Plant defense against microbial pathogens depends on the action of several endogenously produced hormones, including jasmonic acid (JA) and ethylene (ET). In defense against necrotrophic pathogens, the JA and ET signaling pathways synergize to activate a specific set of defense genes including PLANT DEFENSIN1.2 (PDF1.2). The APETALA2/Ethylene Response Factor (AP2/ERF)-domain transcription factor ORA59 acts as the integrator of the JA and ET signaling pathways and is the key regulator of JA- and ET-responsive PDF1.2 expression. The present study was aimed at the identification of elements in the PDF1.2 promoter conferring the synergistic response to JA/ET and interacting with ORA59. We show that the PDF1.2 promoter was activated synergistically by JA and the ET-releasing agent ethephon due to the activity of two GCC boxes. ORA59 bound in vitro to these GCC boxes and trans-activated the PDF1.2 promoter in transient assays via these two boxes. Using the chromatin immunoprecipitation technique we were able to show that ORA59 bound the PDF1.2 promoter in vivo. Finally, we show that a tetramer of a single GCC box conferred JA/ethephon-responsive expression, demonstrating that the JA and ET signaling pathways converge to a single type of GCC box. Therefore ORA59 and two functionally equivalent GCC box binding sites form the module that enables the PDF1.2 gene to respond synergistically to simultaneous activation of the JA and ET signaling pathways.

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Both GCC boxes are essential for JA- and ethephon-responsive expression of PDF1.2 promoter derivative SF in stably transformed Arabidopsis plants. Each bar represents average GUS activity values determined in pools of 10 T2 seedlings from 8 independent transformed lines for each construct corrected for protein concentration ± SE. Seedlings were control-treated (C) or treated with 50 μM JA, 1 mM of the ET-releasing agent ethephon (E) or both (EJA) for 24 h. The asterisk marks the only value that was different from any of the others in a one-way ANOVA (P < 0.05)
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Fig5: Both GCC boxes are essential for JA- and ethephon-responsive expression of PDF1.2 promoter derivative SF in stably transformed Arabidopsis plants. Each bar represents average GUS activity values determined in pools of 10 T2 seedlings from 8 independent transformed lines for each construct corrected for protein concentration ± SE. Seedlings were control-treated (C) or treated with 50 μM JA, 1 mM of the ET-releasing agent ethephon (E) or both (EJA) for 24 h. The asterisk marks the only value that was different from any of the others in a one-way ANOVA (P < 0.05)

Mentions: The expression of the PDF1.2 gene is synergistically induced by a combination of JA and ET (Penninckx et al. 1998). To study the contribution of the two GCC boxes to JA- and ET-responsive activity of PDF1.2 promoter derivative SF, we generated stably transformed plants containing the GUS fusion constructs shown in Fig. 1 via Agrobacterium-mediated transformation. T2 seedlings from eight independent transgenic lines for each construct were treated with JA, the ET-releasing agent ethephon or both for 24 h. Consistent with the accumulation of endogenous PDF1.2 mRNA (Penninckx et al. 1998), PDF1.2 promoter activity was relatively weakly induced by JA or ethephon alone, but strongly induced by the combination (Fig. 5). Mutation of either GCC box dramatically decreased PDF1.2 promoter activity in response to JA or JA/ethephon. Mutation of the GCC box at positions −262 to −257 reduced activity to the level observed with the wild-type promoter after control treatment, whereas mutation of the GCC box at positions −222 to −214 left a very low residual response to JA/ethephon. Mutation of both GCC boxes strongly reduced PDF1.2 promoter activity to levels below the level of the wild-type promoter after control treatment. Therefore in stably transformed plants the two GCC boxes were functionally equivalent and were both necessary for JA- and JA/ethephon-responsive activity of PDF1.2 promoter derivative SF.Fig. 5


Two GCC boxes and AP2/ERF-domain transcription factor ORA59 in jasmonate/ethylene-mediated activation of the PDF1.2 promoter in Arabidopsis.

Zarei A, Körbes AP, Younessi P, Montiel G, Champion A, Memelink J - Plant Mol. Biol. (2011)

Both GCC boxes are essential for JA- and ethephon-responsive expression of PDF1.2 promoter derivative SF in stably transformed Arabidopsis plants. Each bar represents average GUS activity values determined in pools of 10 T2 seedlings from 8 independent transformed lines for each construct corrected for protein concentration ± SE. Seedlings were control-treated (C) or treated with 50 μM JA, 1 mM of the ET-releasing agent ethephon (E) or both (EJA) for 24 h. The asterisk marks the only value that was different from any of the others in a one-way ANOVA (P < 0.05)
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3044237&req=5

Fig5: Both GCC boxes are essential for JA- and ethephon-responsive expression of PDF1.2 promoter derivative SF in stably transformed Arabidopsis plants. Each bar represents average GUS activity values determined in pools of 10 T2 seedlings from 8 independent transformed lines for each construct corrected for protein concentration ± SE. Seedlings were control-treated (C) or treated with 50 μM JA, 1 mM of the ET-releasing agent ethephon (E) or both (EJA) for 24 h. The asterisk marks the only value that was different from any of the others in a one-way ANOVA (P < 0.05)
Mentions: The expression of the PDF1.2 gene is synergistically induced by a combination of JA and ET (Penninckx et al. 1998). To study the contribution of the two GCC boxes to JA- and ET-responsive activity of PDF1.2 promoter derivative SF, we generated stably transformed plants containing the GUS fusion constructs shown in Fig. 1 via Agrobacterium-mediated transformation. T2 seedlings from eight independent transgenic lines for each construct were treated with JA, the ET-releasing agent ethephon or both for 24 h. Consistent with the accumulation of endogenous PDF1.2 mRNA (Penninckx et al. 1998), PDF1.2 promoter activity was relatively weakly induced by JA or ethephon alone, but strongly induced by the combination (Fig. 5). Mutation of either GCC box dramatically decreased PDF1.2 promoter activity in response to JA or JA/ethephon. Mutation of the GCC box at positions −262 to −257 reduced activity to the level observed with the wild-type promoter after control treatment, whereas mutation of the GCC box at positions −222 to −214 left a very low residual response to JA/ethephon. Mutation of both GCC boxes strongly reduced PDF1.2 promoter activity to levels below the level of the wild-type promoter after control treatment. Therefore in stably transformed plants the two GCC boxes were functionally equivalent and were both necessary for JA- and JA/ethephon-responsive activity of PDF1.2 promoter derivative SF.Fig. 5

Bottom Line: Using the chromatin immunoprecipitation technique we were able to show that ORA59 bound the PDF1.2 promoter in vivo.Finally, we show that a tetramer of a single GCC box conferred JA/ethephon-responsive expression, demonstrating that the JA and ET signaling pathways converge to a single type of GCC box.Therefore ORA59 and two functionally equivalent GCC box binding sites form the module that enables the PDF1.2 gene to respond synergistically to simultaneous activation of the JA and ET signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biology, Sylvius Laboratory, Leiden University, P.O. Box 9505, 2300 RA, Leiden, The Netherlands.

ABSTRACT
Plant defense against microbial pathogens depends on the action of several endogenously produced hormones, including jasmonic acid (JA) and ethylene (ET). In defense against necrotrophic pathogens, the JA and ET signaling pathways synergize to activate a specific set of defense genes including PLANT DEFENSIN1.2 (PDF1.2). The APETALA2/Ethylene Response Factor (AP2/ERF)-domain transcription factor ORA59 acts as the integrator of the JA and ET signaling pathways and is the key regulator of JA- and ET-responsive PDF1.2 expression. The present study was aimed at the identification of elements in the PDF1.2 promoter conferring the synergistic response to JA/ET and interacting with ORA59. We show that the PDF1.2 promoter was activated synergistically by JA and the ET-releasing agent ethephon due to the activity of two GCC boxes. ORA59 bound in vitro to these GCC boxes and trans-activated the PDF1.2 promoter in transient assays via these two boxes. Using the chromatin immunoprecipitation technique we were able to show that ORA59 bound the PDF1.2 promoter in vivo. Finally, we show that a tetramer of a single GCC box conferred JA/ethephon-responsive expression, demonstrating that the JA and ET signaling pathways converge to a single type of GCC box. Therefore ORA59 and two functionally equivalent GCC box binding sites form the module that enables the PDF1.2 gene to respond synergistically to simultaneous activation of the JA and ET signaling pathways.

Show MeSH
Related in: MedlinePlus