Inhibitors of oxidative enzymes affect water uptake and vase life of cut Acacia holosericea and Chamelaucium uncinatum stems

Abstract

Cut Acacia holosericea (Velvet Leaf Wattle) foliage has a short vase life, possibly because of blockage in xylem vessels. We indirectly investigated a hypothesised role for peroxidase and phenoloxidase enzyme activities in xylem occlusion of Acacia stems by using their inhibitors. We also tested these inhibitors with cut Chamelaucium uncinatum (Geraldton waxflower), another woody stemmed cut flower.

The peroxidase inhibitors used were 3-amino-1,2,4-triazole (AT), catechol (CH), hydroquinone (HQ), p-phenylene diamine (PD) and copper sulphate (CS, Chamelaucium only). The catechol oxidase inhibitors were tropolone (TP), 4-hexylresorcinol (HR) and 2,3-dihydroxynaphthalene (DN). A laccase inhibitor, cetyltrimethylammonium bromide (CM), was also used. Other phenoloxidase inhibitors tested were p-chlorophenol (CP), p-nitrocathechol (NC), p-nitrophenol (NP) and sodium metabisulphite (SM). 2-Mercaptoethanol (ME), phenyl hydrazine (PH) and salicylhydroxamic acid (SH) were used as inhibitors of both peroxidase and phenoloxidase.

Twelve inhibitors (CH, HQ, DN, HR, TP, CM, CP, NC, NP, SM, PH and SH) significantly improved water uptake, maintained relative fresh weight and increased vase life of Acacia at least once in two experiments. In Chamelaucium, six inhibitors had significant positive effects on water relations (CH, PD, CS, CM, CP and PH) and vase life (AT, CH, PD, CS, ME and PH), while four of them (DN, TP, NC and NP) were phytotoxic at applied concentrations. Only one of the 46 inhibitor treatments inhibited transpiration and increased fresh weight, suggesting that the inhibitors mainly acted by increasing water uptake. Overall, results indicate that oxidative enzyme activities, potentially through phenolic deposition, contribute to xylem occlusion in woody cut stems of Acacia and Chamelaucium.

Introduction

Acacia holosericea (Velvet Leaf Wattle) has cut foliage potential due to its silvery green silky phyllodes (leaves). However, it has a short vase life due to insufficient water uptake causing early wilting and/or desiccation of phyllodes (Damunupola et al., 2010). Water uptake by cut flower stems, including A. holosericea, decreases with time after being stood into water, possibly due to occlusions in the xylem (Van Doorn, 1996). Physical blockages of xylem due to air embolism (cavitation), small particles or bacteria and bacterial products are well known (Van Doorn, 1996). Physiological plugging as a result of defensive metabolic responses to wounding has also been reported (Van Doorn and Cruz, 2000, Van Doorn and Vaslier, 2002, Vaslier and van Doorn, 2003, Loubaud and van Doorn, 2004, He et al., 2006).

Wounding plant tissues activates mechanisms for defence and healing (Leon et al., 2001). Deposition of materials such as suberin, lignin and tannin in the xylem as a physiological reaction to wounding was suggested long ago (Dean and Kolattukudy, 1976, Halevy and Mayak, 1981, Van Doorn, 1996). Williamson et al. (2002) considered that hydrophobic suberin formation was an early (<12 h) response to wounding that led to cavitation and water deficit stress. Peroxidase is involved in suberin deposition in wound healing potato tuber tissue (Espelie et al., 1986). The wound signal is transmitted over a distance of at least 4 cm through pea stem tissue (Peck and Kende, 1998). With no delay in response intensity, a hydraulic or electric signal is possibly responsible for spread of the wound response.

Inhibition by antioxidants of wound-induced xylem occlusion has been reported to occur in Chrysanthemum both during dry and wet storage (Van Doorn and Cruz, 2000). Catechol oxidase and laccase are major phenoloxidases (Walker, 1995). Xylem blockage in Chrysanthemum was delayed by the catechol oxidase inhibitors, tropolone and 4-hexylresorcinol (Van Doorn and Vaslier, 2002). Both peroxidase and catechol oxidase were involved in physiological blockage in the lowermost 5 cm of dry stored Bouvardia flower stems (Vaslier and van Doorn, 2003). Similar physiological blockage also occurs in Astilbe flowers (Loubaud and van Doorn, 2004).

S-carvone is a monoterpene known to inhibit phenylalanine ammonia-lyase (PAL) activity and synthesis of wound-induced compounds, like suberin (Oosterhaven et al., 1995a). However, it also has antimicrobial activity (Oosterhaven et al., 1995b). In vase solution, S-carvone extended the vase life of flowers from members of the families Proteaceae [viz. Hakea francisiana (Williamson et al., 2002) and Grevillea ‘Crimson Yul-lo’ (He et al., 2006)] and Myrtaceae [viz. Baeckea frutescens and Chamelaucium uncinatum (Damunupola et al., 2010)]. However, there were no positive effects on vase life of A. holosericea and Chrysanthemum (Damunupola et al., 2010). Like S-carvone, treatment with a catechol oxidase inhibitor, 4-hexylresorcinol, delayed stem end blockage in Grevillea ‘Crimson Yul-lo’ inflorescences (He et al., 2006). Thus, blockage in cut Grevillea stems was suggested to be physiologically mediated.

Peroxidase, polyphenoloxidase and PAL activities after wounding are associated with lignin and phenol metabolism (Okey et al., 1997). As a rapid response to wounding in wheat roots, peroxidases from cell surface release into the apoplast where they can display both oxidative and peroxidative activities (Minibayeva et al., 2009).

In the present study, inhibitors of peroxidase and phenoloxidase (viz. catechol oxidase, laccase, etc.) enzymes were tested for woody A. holosericea and C. uncinatum stems. It was hypothesised that these inhibitors of oxidative enzymes would suppress enzyme activity in response to wounding and thereby prevent or reduce physiological occlusion of xylem vessels for these species.