Purification and Characterization of Polyphenol Oxidase from Glandular Trichomes of Solanum berthaultii

Abstract

Type A glandular trichomes of the wild potato (Solanum berthaultii Hawkes) entrap insects by rapidly polymerizing the trichome contents after breakage by insect contact. Polymerization of trichome exudate appears to be driven by a soluble polyphenol oxidase (PPO). PPO constitutes up to 70% of the protein in individually collected trichomes and reaches a concentration approaching 200 gM in these organs. Trichome PPO has been purified and shown to be a monomeric copper metalloprotein with an isoelectric point of 5.5, possessing only o-diphenol oxygen oxidoreductase activity, and is larger than most other reported PPOs, with relative molecular weight of 59,000. Chlorogenic and caffeic acid were the most readily oxidized of 14 phenolic substrates tested. Polyclonal antibodies raised against the relative molecular weight 59,000 S. berthaultii trichome PPO were used to show that S. tuberosum L. trichomes express low levels of a cross-reactive protein that lacks detectable PPO activity.The selection process that has given rise to the modem cultivated potato (Solanum tuberosum L.) has drastically narrowed its genetic base. A few genetically similar varieties now account for nearly all of the acreage grown in the United States. Selection for horticultural quality may have been accompanied by the inadvertent removal or reduction in levels of important pest-resistance factors (e.g. steroidal glycoalkaloids) (34), and until recently, relatively little success has been achieved toward breeding for resistance to insects that have adapted to cultivated potato (e.g. the Colorado potato beetle, Leptinotarsa decemlineata). Hence, it is not surprising that the cultivated potato is susceptible to attack from a wide array of arthropod herbivores; little inherent insect resistance remains in S. tuberosum. There is, however, a broad diversity of genetic traits available in wild germplasm. Wild potato species exhibit substantial resistance to insect pests. The wild Bolivian potato species Solanum berthaultii Hawkes displays a high degree of resistance to aphids, leafhoppers, flea beetles, spider mites, and the Colorado potato beetle (11). This resistance is conferred by high densities of foliar glandular trichomes (8,9,11).Glandular trichomes are modified epidermal cells that fre-

Import, Targeting, and Processing of a Plant Polyphenol Oxidase

“…This was suggested to be the precursor of the 59-kD purified trichomal PPO (Kowalski et al, 1992) and was further supported by the structure of cDNA clones isolated from potato (Hunt et al, 1993). Cloned tomato PPO genes appeared to code for 66.1-to 70.6-kD precursors of 57.0-to 61.6-kD mature products (Shahar et al, 1992;Yu, 1992;Newman et al, 1993).…”

“…The primary antibody was polyclonal rabbit anti-Solanum bertaultii 59-kD trichome PPO (Kowalski et al, 1992). ['251]Protein A (Amersham, 30 mCi ng-' at 1:lOOO dilution) and autoradiography were used for visualization.…”

“…They are located in plastids, although reports of their occurrence in other cell compartments are quite abundant Mayer, 1987). PPO is often described as tightly bound to thylakoids but has also been observed in plastid envelopes or the thylakoid lumen (Mayer, 1987;Vaughn et al, 1988;Kowalski et al, 1992).…”

“…Furthermore, a11 of these polypeptides possess amino-terminal extensions with properties typical of nuclear-coded plastid proteins Cary et al, 1992;Shahar et al, 1992;Hunt et al, 1993;Newman et al, 1993). In vitro translation of potato trichome poly(A+) RNA produces a 67-kD polypeptide, whereas the native enzyme is 59 kD Kowalski et al, 1992;Hunt et al, 1993). The recent isolation of the entire gene family of tomato PPO ) presents a good opportunity for studying the import, targeting, and processing of PPO in higher plants.…”

Distribución, Localización E Inhibidores De Las Polifenol Oxidasas en Frutos Y Vegetales Usados Como Alimento

“…Diversas evidencias apuntan hacia la localización concreta de las PPOs de plantas superiores en las membranas tilacoidales (Mayer, 1987;Vaughn et al, 1988;Kowalski et al, 1992), aunque existen trabajos que describen la existencia de algunas PPOs vegetales en forma soluble en el lumen de los tilacoides (Sommer et al, 1994;Murata et al, 1997).…”