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Characterization of pedicel β‐glucuronidase activity in developing maize ( Zea mays ) kernels
Author(s) -
Muhitch Michael J.
Publication year - 1998
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1034/j.1399-3054.1998.1040318.x
Subject(s) - pedicel , biology , endogeny , biochemistry , incubation , botany
A conspicuous endogenous maize ( Zea mays L.) β‐glucuronidase (GUS) activity was observed in histochemical assays of non‐transformed maize kernels, confounding the use of Escherichia coli gusA as a reporter gene. Appearance of the endogenous activity was developmentally dependent and highly tissue‐specific, being localized to the upper pedicel (basal maternal kernel) tissues where the black layer forms in the latter stages of kernel development. Pedicel homogenates exhibited GUS activity using either p ‐nitrophenyl‐β‐ D ‐glucuronide or 4‐methylumbelliferyl‐β‐ D ‐glucuronide (MUG) as substrates. Pedicel GUS was apparently not the result of endophyte contamination of enzyme isolates since no endophytes could be cultured. The MUG‐based activity had a pH optimum of 4 to 5 and was separable into two isoforms by anion exchange chromatography with K m values for MUG of 2.2 and 2.7 µ M for the early‐ and late‐eluting forms, respectively. The pedicel GUS isoforms had very similar characteristics: native M r of approximately 32000, stimulation by assay at 60°C, inhibition at high ionic strength or in the presence of EDTA and relative insensitivity to the E. coli GUS inhibitor saccharic acid‐1,4‐lactone. Only the early‐eluting form, however, was capable of hydrolyzing the histocbemical GUS substrate 5‐bromo‐4‐chloro‐3‐indoyl‐β‐ D ‐glucuronide. Neither isoform exhibited antifungal activity against Fusarium moniliforme . In contrast to the in vitro activity, pedicel endogenous GUS measured histochemically was completely inhibited by saccharic acid‐1,4‐lactone, unaffected by EDTA and greatly decreased by incubation at elevated assay temperature. A modification of the standard histochemical GUS assay allowed complete suppression of endogenous GUS activity while enhancing E. coli ‐derived GUS activity in kernels transiently expressing the gusA gene. Possible roles of these endogenous GUS activities within the black layer region of the kernel pedicel are proposed.