Polyphenol oxidase from hybrid poplar. Cloning and expression in response to wounding and herbivory

Abstract

The inducible expression of polyphenol oxidase (PPO), a presumed antiherbivore enzyme, was examined in hybrid poplar (Populus trichocarpa x Populus deltoides). Following mechanical wounding simulating insect damage, PPO activity increased dramatically in wounded and unwounded leaves on wounded plants beginning at 24 and 48 h, respectively. A hybrid poplar PPO cDNA was isolated and its nucleotide sequence determined. On northern blots, PPO transcripts were detected within 8 h of wounding, and reached peak levels at 16 and 24 h in wounded and unwounded leaves, respectively. Methyl jasmonate spray and feeding by forest tent caterpillar also induced PPO expression. The induction of PPO was strongest in the youngest four leaves, which were generally avoided by caterpillars in free feeding experiments. This wound- and herbivore-induced expression of PPO in hybrid poplar supports the defensive role of this protein against insect pests.

Figure 1

Wound induction of PPO in hybrid poplar leaves. Young hybrid poplar saplings were wounded as described in “Material and Methods,” and wounded leaves (○) and unwounded leaves on wounded plants (●) were harvested at the indicated times for PPO assays. Each point represents the means (±se), determined independently from samples of three wounded plants.

Figure 2

Nucleotide and predicted protein sequences of hybrid poplar PtdPPO cDNA. The thylakoid transit peptide is double underlined, and the copper-binding domains are single underlined. Within the copper-binding domains, the primers used to amplify the PPO fragment employed as a probe are double underlined, and conserved His residues are marked with an asterisk. The PtdPPO cDNA has been deposited in GenBank (accession no. AF 263611).

Figure 3

Southern analysis of PPO in poplar. The entire PtdPPO cDNA was used as a probe. H11-11 is the TD hybrid used in this study, whereas 53-246 is a related TD hybrid; 93-968 is the P. trichocarpa parent of 53-246. E, EcoRI; H, HindIII; X, XbaI.

Figure 4

Northern analysis of PPO mRNA accumulation following wounding. Wounded and unwounded (systemic) leaves on wounded plants were harvested at the indicated times for RNA extraction. A, Top panel, X-ray film image of blots hybridized with PPO probe; bottom panel, the ethidium bromide-stained gel as a loading control. B, Graphical representation of PPO transcript intensity as determined from phosphor imager. □, Wounded leaves; ●, unwounded leaves.

Figure 5

Northern analysis of PPO mRNA induction by MeJa. Saplings were treated with MeJa or mock-sprayed, and analyzed after 24 h. A, Top panel, x-ray film image of blots hybridized with PPO probe; bottom panel, the ethidium bromide-stained gel as a loading control. A through D refer to individual plants tested. Y, Young leaves; O, old leaves.

Figure 6

FTC feeding preference and induction of PPO activity in hybrid poplar saplings. Top panel, Percentage of material consumed from individual leaves after 6 d of FTC feeding. Bottom panel, PPO activity in remaining leaf material. Hatched and black bars represent individual plants.

Figure 7

Northern analysis of PPO wound induction in leaves of different ages. A range of leaves of a young poplar sapling were mechanically wounded as described in “Materials and Methods,” and analyzed after 24 h. Leaves 2, 9, and 16 were used as controls, and harvested immediately prior to the wounding treatment. A, Top panel, X-ray film image of blots hybridized with PPO probe. Control and treatment panels were from the same blot; however, the control panel was exposed for 68 h to visualize very low PPO mRNA levels, whereas the wound treatment panel was exposed for 6.5 h. Bottom panel, The ethidium bromide-stained gel as a loading control. B, Graphical representation of PPO transcript intensity as determined from phosphor imager.