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Subcellular localization of luteolin glucuronides and related enzymes in rye mesophyll

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Abstract

Vacuoles were isolated by osmotic rupture of mesophyll protoplasts from the primary leaves of 4-d- and 7-d-old plants of rye (Secale cereale L.). Their content of two flavones, luteolin 7-O-[β-d-glucuronosyl-(1→2)β-d-glucuronide] (R2) and luteolin 7-O-[β-d-glucuronosy 1 (1→2) β-d-glucuronide]-4′-O-β-d-glucuronide (R1), as well as that of three specific flavone-glucuronosyltransferases involved in their biosynthesis and of a specific β-glucuronidase was determined in comparison to the parent protoplasts. The two flavonoids were found to be entirely located in the vacuolar fraction, together with 70% of the activity of UDP-glucuronate: luteolin 7-O-diglucuronide-4′-O-glucuronosyl-transferase (LDT; EC 2.4.1.), the third enzyme of the sequence of three transferases in the anabolic pathway. The activities of the first and second anabolic enzymes, UDP-glucuronate: luteolin 7-O-glucuronosyltransferase (LGT; EC 2.4.1.) and UDP-glucuronate: luteolin 7-O-glucuronide-glucuronosyltransferase (LMT; EC 2.4.1.) could not be found in the vacuolar fraction in appreciable amounts. The specific β-glucuronidase (EC 3.2.1.), catalyzing the deglucuronidation of luteolin triglucuronide to luteolin diglucuronide, was present with 90% of its activity in the digestion medium after isolation of mesophyll protoplasts, indicating an apoplastic localization of this enzyme. The data presented indicate a directed anabolic and subsequent catabolic pathway for the luteolin glucuronides in the mesophyll cells of rye primary leaves. This includes two cytosolic and a last vacuolar step of glucuronidation of luteolin, and the vacuolar storage of the luteolin triglucuronide. We propose the transport of the latter into the cell wall, after which the triglucuronide is deglucuronidated, this being the first step for further turnover.

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Abbreviations

LDT:

UDP-glucuronate: luteolin 7-O-di-glucuronide-4′-O-glucuronosyltransferase

LGT:

UDP-glucuronate: luteolin 7-O-glucuronosyltransferase

LMT:

UDP-glucuronate: luteolin 7-O-glucuronide-glucuronosyltransferase

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Authors and Affiliations

  1. Botanisches Institut der Universität zu Köln, Gyrhofstrasse 15, W-5000, Köln 41, Germany

    Stephan Anhalt & Gottfried Weissenböck

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  1. Stephan Anhalt
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  2. Gottfried Weissenböck
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Dedicated to Professor Ludwig Bergmann, Botanisches Institut der Universität zu Köln, on the occasion of his 65th birthday

Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged. We thank Professor J. Willenbrink (Köln) for critical reading of the manuscript.

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Anhalt, S., Weissenböck, G. Subcellular localization of luteolin glucuronides and related enzymes in rye mesophyll. Planta 187, 83–88 (1992). https://doi.org/10.1007/BF00201627

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