Polyphenoloxidase Silencing Affects Latex Coagulation in Taraxacum Species

Abstract

Latex is the milky sap that is found in many different plants. It is produced by specialized cells known as laticifers and can comprise a mixture of proteins, carbohydrates, oils, secondary metabolites, and rubber that may help to prevent herbivory and protect wound sites against infection. The wound-induced browning of latex suggests that it contains one or more phenoloxidizing enzymes. Here, we present a comprehensive analysis of the major latex proteins from two dandelion species, Taraxacum officinale and Taraxacum kok-saghyz, and enzymatic studies showing that polyphenoloxidase (PPO) is responsible for latex browning. Electrophoretic analysis and amino-terminal sequencing of the most abundant proteins in the aqueous latex fraction revealed the presence of three PPO-related proteins generated by the proteolytic cleavage of a single precursor (pre-PPO). The laticifer-specific pre-PPO protein contains a transit peptide that can target reporter proteins into chloroplasts when constitutively expressed in dandelion protoplasts, perhaps indicating the presence of structures similar to plastids in laticifers, which lack genuine chloroplasts. Silencing the PPO gene by constitutive RNA interference in transgenic plants reduced PPO activity compared with wild-type controls, allowing T. kok-saghyz RNA interference lines to expel four to five times more latex than controls. Latex fluidity analysis in silenced plants showed a strong correlation between residual PPO activity and the coagulation rate, indicating that laticifer-specific PPO plays a major role in latex coagulation and wound sealing in dandelions. In contrast, very little PPO activity is found in the latex of the rubber tree Hevea brasiliensis, suggesting functional divergence of latex proteins during plant evolution.

The polyphenol oxidase gene family in poplar: phylogeny, differential expression and identification of a novel, vacuolar isoform

“…Close examination revealed the presence of small extensions of the plastid envelope or stromules, further supporting their identity as the leucoplasts found in onion epidermis (Fig 6h, inset) (Kitajima et al 2009;Köhler and Hanson 2000). The ability of a classical TP to direct a Taraxacum oYcinale PPO to plastids was also shown using GFP fusions by Wahler et al (2009). We conclude that unlike most PPOs, PtrPPO13 is localized to the vacuole, and that amino acids 1-35 are suYcient for this localization.…”

Oxidative discolouration in whole-head and cut lettuce: biochemical and environmental influences on a complex phenotype and potential breeding strategies to improve shelf-life

“…PPO is a copper-requiring metallo-enzyme associated with (although not found to be intrinsic to) the thylakoid membranes of the chloroplast in many plants including lettuce (Sommer et al 1994;Chazarra et al 1996;Wahler et al 2009). PPO catalyses two distinct reactions; the o-hydroxylation of monophenols to produce o-diphenols (monophenolase or cresolase activity) and the subsequent oxidation of the o-diphenols to o-quinones (diphenolase or catecholase activity), both of which require the presence of molecular oxygen (López-Galvez et al 1996;Chazarra et al 2001;Toivonen and Brummell 2008).…”

Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases

“…However, in most cases, their individual physiological role and especially their natural substrates remain unknown. Beside aurone formation, their specific involvement in biosynthetic processes was shown only in a few cases (1,(7)(8)(9)(10)(11). Recently, Sullivan (7) wondered whether there exist more PPOs that are involved in the plant's secondary metabolism and whether the few so far known specialized PPOs represent only "the tip of the iceberg.…”