CLONING OF THE CDNA FOR GLUTAREDOXIN, AN ABUNDANT SIEVE-TUBE EXUDATE PROTEIN FROM RICINUS-COMMUNIS L AND CHARACTERIZATION OF THE GLUTATHIONE-DEPENDENT THIOL-REDUCTION SYSTEM IN SIEVE TUBES

Sieve-tube exudate protein (STEP) from Ricinus communis L. seedlings c onsists of a characteristic set of more than 100 different polypeptide s, against which a complex antiserum was raised. This antiserum cross- reacted with dominant protein species (molecular weights 10-30 kDa) pr esent in the sieve-tube exudate and, to a lesser extent, with proteins in tissue extracts of Ricinus and a wide range of other plant species . For further elucidation of the nature of individual STEPs in the sie ve tubes the anti-STEP serum was used to screen a cDNA expression libr ary constructed from Ricinus cotyledon mRNA. Two clones that differed in the 3' untranslated region encoded a protein of 11 kDa which showed striking homology to bacterial and eucaryotic glutaredoxin sequences. Glutaredoxin activity was confirmed for the recombinant protein after overexpression in Escherichia coli and characterised in detail in sie ve-tube exudate. Michaelis Menten constants (K-m) for reduced glutathi one and cysteine were 2 mM and 50 mu M, respectively. Besides L-cystei ne, dehydroascorbate and protein disulphides were also reduced by the activity present in the sieve-tube exudate. Glutathione, which is the obligate donor of reduced thiols for glutaredoxin, was present in siev e-tube sap in millimolar concentrations (up to 3 mM) with a ratio of t otal to oxidised glutathione of 3:1. It is suggested that glutaredoxin and glutathione in sieve tubes prevent oxidative damage and may be in volved in redox regulation of sieve-tube proteins.