Induction of the arginine decarboxylase ADC2 gene provides evidence for the involvement of polyamines in the wound response in Arabidopsis

Abstract

Polyamines are small ubiquitous molecules that have been involved in nearly all developmental processes, including the stress response. Nevertheless, no direct evidence of a role of polyamines in the wound response has been described. We have studied the expression of genes involved in polyamine biosynthesis in response to mechanical injury. An increase in the expression of the arginine decarboxylase 2 (ADC2) gene in response to mechanical wounding and methyl jasmonate (JA) treatment in Arabidopsis was detected by using DNA microarray and RNA gel-blot analysis. No induction was observed for the ADC1 gene or other genes coding for spermidine and spermine synthases, suggesting that ADC2 is the only gene of polyamine biosynthesis involved in the wounding response mediated by JA. A transient increase in the level of free putrescine followed the increase in the mRNA level for ADC2. A decrease in the level of free spermine, coincident with the increase in putrescine after wounding, was also observed. Abscisic acid effected a strong induction on ADC2 expression and had no effect on ADC1 expression. Wound-induction of ADC2 mRNA was not prevented in the JA-insensitive coi1 mutant. The different pattern of expression of ADC2 gene in wild-type and coi1 mutant might be due to the dual regulation of ADC2 by abscisic acid and JA signaling pathways. This is the first direct evidence of a function of polyamines in the wound-response, and it opens a new aspect of polyamines in plant biology.

Figure 1

DNA microarray gene expression analysis during wounding and MetJA treatments in Arabidopsis. A, Scatter plot of mRNA ratios from DNA microarray analysis of local wounded plants (top), systemic wounded plants (middle), and MetJA-treated plants (bottom) compared with control unwounded or untreated plants. mRNA ratios (mRNA level treatment versus control) correspond to average ratio of two slides hybridized with direct and reverse labeling, as indicated in “Materials and Methods.” RNS1, S-like RNase 1 At2g02990; ADC2, ADC2 At4g34710; TAT, Tyr amino transferase-like protein At2g24850; AOS, allene oxide synthase At5g42650; and Lox3, lipoxygenase 3 At1g17420. B, RNA gel analysis of genes induced during wounding and MetJA treatment according with the DNA microarray analysis. Lanes contain 10 μg of total RNA extracted from control leaves (C), local (L) and systemic (S) wounded leaves, or leaves treated with MetJA (J). Blots were hybridized sequentially with ³²P-labeled RNS1 (At2g02990; Taylor and Green, 1991), and AOS (expressed sequence tag [EST] nos. AA394958 and At5g42650). Ethidium bromide staining was used to ensure equal loading of all lanes in the gel.

Figure 2

RNA gel analysis of the expression of genes involved in polyamine biosynthesis during wounding and MetJA treatment. Lanes contain 10 μg of total RNA extracted from control leaves (C), local (L) and systemic (S) wounded leaves, or leaves treated with MetJA (J). Blots were hybridized sequentially with ³²P-labeled ADC2 (EST nos. T46784 and At4g34710), ADC1 (EST nos. H36915 and At2g16500), SPDS1 (EST nos. AA597626 and At1g70310)l SPDS2 (EST nos. T20920 and At1g23820), and S-adenosyl Met decarboxylase (EST nos. AA395848 and At3g02470). Ethidium bromide staining was used to ensure equal loading of all lanes in the gel.

Figure 3

RNA gel analysis of local (A) and systemic (B) wounded plants. One-half of the rosette leaves were wounded and harvested (A, local wounding) at different time points along with unwounded leaves from the same plants (B, systemic wounding) or leaves from unwounded plants (control). Lanes contain 10 μg of total RNA. Blots were hybridized sequentially with ³²P-labeled ADC2 (EST nos. T46784 and At4g34710) and ADC1 (EST nos. H36915 and At2g16500). Ethidium bromide staining was used to ensure equal loading of all lanes in the gel.

Figure 4

Changes in polyamine levels after local wounding. Samples from locally wounded leaves were collected at different time points and polyamines were analyzed as indicated in the “Materials and Methods.” A, Quantification of mRNA levels for ADC2 (from Fig. 3A; relative units); B, putrescine level (μg g⁻¹ fresh weight); C, spermidine level (μg g⁻¹ fresh weight); D, spermine level (μg g⁻¹ fresh weight). B, C, and D correspond to the mean of three independent samples ± se.

Figure 5

RNA gel analysis of JA-treated wild-type plants (A) and locally wounded coi1 mutant (B). Lanes contain 10 μg of total RNA extracted from wild-type plants treated with 50 μm JA (JA) or untreated control plants, or coi1 mutant wounded or unwounded, and harvested at different time points. Blots were hybridized sequentially with ³²P-labeled ADC2 (EST nos. T46784 and At4g34710) and ADC1 (EST nos. H36915 and At2g16500). Ethidium bromide staining was used to ensure equal loading of all lanes in the gel.

Figure 6

RNA gel analysis of wounded, 50 μm ABA, or 50 μm putrescine (A) and wounded, 1 μm DFMA-treated, or wounded and DFMA-treated (B) liquid-cultured plants. Lanes contain 10 μg of total RNA extracted from seedlings grown in liquid as described in the “Materials and Methods.” Blots were hybridized sequentially with ³²P-labeled ADC2 (EST nos. T46784 and At4g34710) and ADC1 (EST nos. H36915 and At2g16500). Ethidium bromide staining was used to ensure equal loading of all lanes in the gel.