Novel roles for the polyphenol oxidase enzyme in secondary metabolism and the regulation of cell death in walnut

Abstract

The enzyme polyphenol oxidase (PPO) catalyzes the oxidation of phenolic compounds into highly reactive quinones. Polymerization of PPO-derived quinones causes the postharvest browning of cut or bruised fruit, but the native physiological functions of PPOs in undamaged, intact plant cells are not well understood. Walnut (Juglans regia) produces a rich array of phenolic compounds and possesses a single PPO enzyme, rendering it an ideal model to study PPO. We generated a series of PPO-silenced transgenic walnut lines that display less than 5% of wild-type PPO activity. Strikingly, the PPO-silenced plants developed spontaneous necrotic lesions on their leaves in the absence of pathogen challenge (i.e. a lesion mimic phenotype). To gain a clearer perspective on the potential functions of PPO and its possible connection to cell death, we compared the leaf transcriptomes and metabolomes of wild-type and PPO-silenced plants. Silencing of PPO caused major alterations in the metabolism of phenolic compounds and their derivatives (e.g. coumaric acid and catechin) and in the expression of phenylpropanoid pathway genes. Several observed metabolic changes point to a direct role for PPO in the metabolism of tyrosine and in the biosynthesis of the hydroxycoumarin esculetin in vivo. In addition, PPO-silenced plants displayed massive (9-fold) increases in the tyrosine-derived metabolite tyramine, whose exogenous application elicits cell death in walnut and several other plant species. Overall, these results suggest that PPO plays a novel and fundamental role in secondary metabolism and acts as an indirect regulator of cell death in walnut.

Figure 1.

PPO protein activity in leaf protein extracts from wild-type walnut (CR1) and transgenic walnut lines. Lines 40-1-1 and 78-2-8 were transformed with a jrPPO1 overexpression vector, and all other transgenic lines were transformed with a jrPPO1 silencing vector. PPO activity was measured spectrophotometrically by monitoring dopachrome accumulation at 490 nm using l-DOPA as a substrate. For each line, the mean activity from three biological replicates (± sem) is presented.

Figure 2.

Substrate specificity of JrPPO1. A, Polarimetric PPO activity assay using native o-diphenols as substrates. One unit (U) of activity is equivalent to 1 µmol of O₂ consumed per minute (Stewart et al., 2001). B, Spectrophotometric PPO activity assay using native monophenols as substrates. In all experiments, leaf protein extracts from wild-type walnut (CR1) and PPO-silenced walnut lines (PPO-Sil; lines 9-5-1, 5-10-2, and 1-2-15) were assayed. For each sample, the mean PPO activity from two to three replicates (± sem) is shown.

Figure 3.

Silencing of jrppo1 induces a lesion mimic phenotype. A, Wild-type walnut leaf. B, PPO-silenced line, early season (June). Inset, magnified view of necrotic lesions from abaxial side of leaf. C, PPO-silenced line, late season (September). The development of the necrotic lesions was not associated with the presence of any detectable pathogen. Bars = 4 cm.

Figure 4.

Silencing of jrPPO1 alters activity of the phenylpropanoid pathway in walnut. Metabolites are colored to represent changes in abundance in the PPO-silenced plant lines relative to the wild type: red indicates increased abundance, blue indicates decreased abundance, black indicates no change, and gray indicates not detected. Quantitative values representing fold change (PPO-silenced lines/wild type) are denoted in bold. The same color scheme is used to depict enzymes, with corresponding mRNA abundance ratios (PPO silenced/wild type) denoted in bold. Dashed arrows represent multiple enzyme-catalyzed reactions. PAL, Phe ammonia lyase; C4H, cinnamic acid 4-hydroxylase (walnut contigs 25466, 65385); 4HS, 4-hydroxybenzaldehyde synthase; 4CL, 4-coumarate-CoA ligase (walnut contig 19023); HCT, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (walnut contigs 12875, 58137, 17482); C3′H, 4-coumaroylester 3′-hydroxylase (walnut contigs 81814, 06713, 81815, 68910, 06709).

Figure 5.

Silencing of jrPPO1 has direct effects on Tyr metabolism in walnut. Metabolites are colored to represent changes in abundance in the PPO-silenced plant lines relative to the wild type: red indicates increased abundance, blue indicates decreased abundance, black indicates no change, and gray indicates not detected. Quantitative values representing fold change (PPO-silenced lines/wild type) are denoted in bold. Dashed arrows represent multiple enzyme-catalyzed reactions. Proposed reactions catalyzed by JrPPO1 are denoted in orange.

Figure 6.

Staining HCAAs in walnut leaf sections. A, Wild-type walnut leaf showing HCAA staining (blue-white fluorescence) in the vasculature. B, Leaf from a PPO-silenced line displaying pattern of HCAA staining similar to the wild type. C, HCAA staining in a region of a PPO-silenced leaf displaying lesion development. D, Light micrograph of the leaf section shown in C. The brown areas are necrotic lesions. All fluorescence micrographs were taken with identical illumination conditions. Bars = 100 µm.

Figure 7.

Treatment of wild-type walnut leaves with exogenous tyramine leads to the development of necrotic lesions. A, Detached wild-type leaf incubated in water for 4 d. B, Detached wild-type leaf incubated in a 5 mM solution of tyramine for 2 d. C, Detached wild-type leaf incubated in a 5 mM solution of tyramine for 4 d. Bars = 2 cm.