Wound-induced expression of the FAD7 gene is mediated by different regulatory domains of its promoter in leaves/stems and roots

Abstract

The FAD7 gene is expressed preferentially in the chlorophyllous tissues of unwounded plants. Wounding activates the expression of the FAD7 gene not only in chlorophyllous tissues, but also in nonchlorophyllous tissues of stems and roots. Our previous study suggested that wound-responsive transcriptional activation by the FAD7 promoter in leaves/stems and roots is brought about by a jasmonic acid (JA)-independent and JA-dependent signaling pathway, respectively. In this paper, we show that a specific region (from -259 to -198) in the FAD7 promoter is required for wound-activated expression of this gene in leaves and stems, while another region (from -521 to -363) is necessary not only for wound-activated but also for JA-responsive expression of this gene in roots. Thus, different regulatory regions of the FAD7 promoter mediate distinct wound-induced expression of this gene in leaves/stems and roots. Gel mobility shift assays revealed the wound-inducible DNA-binding activity to the -242/-223 region in both stem and leaf nuclear extracts. In fact, deletion of this region abolished wound response of the FAD7 promoter, suggesting the in vivo role of this site. Furthermore, we detected root nuclear factors interacting with the region from -433 to -363 of this promoter. Wounding and methyl jasmonate treatments induced differently these DNA-binding activities. These results suggest that different regulatory mechanisms mediate the wound-induced expression of the FAD7 gene in aerial and subterranean organs.

Figure 1

5′-Deletion analysis of the FAD7 promoter concerning wound responsiveness in each organ. Tobacco plants carrying a GUS gene under the control of derivatives of the FAD7 promoter were grown on soil at 26°C under continuous illumination for about 3 months. Wounding treatments were carried out as described in “Materials and Methods.” GUS activities (n = 5) were determined in both unwounded and wounded young portions of leaves (A), stems (B), and roots (C) in each FAD7 promoter-GUS transgenic line. For each construct, two to six independent transgenic lines were investigated. C and W indicate, respectively, average values of GUS activities (nmol methylumbelliferone min⁻¹ mg⁻¹ protein) in unwounded and wounded tissues of the transgenic lines examined. Induction of GUS activity by wounding is expressed by the ratio of the average GUS activity in wounded tissues to that in unwounded tissues. Each bar indicates the GUS activity of an individual transgenic line. The horizontal line represents the position where the value of the ratio is 1.0.

Figure 2

5′-Deletion analysis of the FAD7 promoter concerning MeJA responsiveness in roots. As described in “Materials and Methods,” MeJA solution was hydroponically applied to tobacco plants carrying a series of the 5′-deleted FAD7 promoter-GUS fusion genes that had been grown for about 3 months. GUS activities (n = 5) were determined in both untreated and MeJA-treated roots in each FAD7 promoter-GUS transgenic line. Induction of GUS activity by MeJA application is expressed by the ratio of the average GUS activity in MeJA-treated tissues to that in untreated tissues. Each bar represents an individual transgenic line. The horizontal line represents the position where the value of the ratio is 1.0.

Figure 3

A wound-inducible nuclear factor in stems binds to the −262 to −203 fragment of the FAD7 promoter. The labeled DNA probe was incubated with or without 10 μg of tobacco nuclear proteins from unwounded or wounded stems. The unlabeled −262/−203 fragment was added as a competitor DNA to the binding reaction mixture at 10- and 50-fold molar excesses. The specific complexes S1 and S2 are indicated by arrows. The position of nonspecific bands is marked by an asterisk.

Figure 4

Identification of the binding site of a wound-inducible nuclear factor in stems. A, Competitive binding assays using the −262 and −223 radiolabeled probes of the FAD7 promoter. An unlabeled 20-bp fragment (−242/−223) was added to the binding reaction at at 10- or 50-fold molar excesses before the addition of 10 μg of tobacco nuclear protein from wounded leaves. The specific complex S3 is indicated by an arrow. The position of nonspecific bands is marked by an asterisk. B, Binding assays of stem nuclear factor to a labeled −242/−223 fragment of the FAD7 promoter. The −242/−223 probe was incubated with 10 μg of tobacco nuclear proteins from unwounded or wounded stems. The unlabeled DNA fragment was added to each reaction mixture at 10- or 50-fold molar excesses. The specific complexes S4 and S5 are indicated by arrows.

Figure 5

Wound-inducible DNA-binding activity in leaves. The labeled DNA probe (−242 to −223) was incubated with 5 μg of tobacco nuclear protein from unwounded or wounded leaves. Competitor DNAs were added at 100-fold molar excesses of the labeled probe. The specific complexes L1 and L2 are indicated by arrows.

Figure 6

Wounding and MeJA application modulated the DNA-binding activity of root nuclear factors interacting with a region (−433 to −363) of the FAD7 promoter. The −433/−363 fragment (A) of the FAD7 promoter and an as-1 element (B) were used as probes of mobility shift assays. Binding reaction was performed with or without of 10 μg of tobacco nuclear protein from untreated, wounded, and MeJA-treated roots. The unlabeled −433/−363 fragment was added to each binding reaction mixture at 10- and 50-fold molar excesses. The specific complexes R1 and R2 are indicated by arrows.