Plant Physiol, April 2002, Vol. 128, pp. 1303-1312
Spermidine-Binding Proteins. Purification and Expression Analysis
in Maize1
Annalisa
Tassoni,
Richard M.
Napier,
Marina
Franceschetti,
Michael
A.
Venis, and
Nello
Bagni*
Dipartimento di Biologia Evoluzionistica Sperimentale and
Interdepartmental Center of Biotechnology, University of Bologna,
40126, Bologna, Italy (A.T., M.F., N.B.); and Department of Plant
Genetics and Biotechnology, Horticulture Research International,
Wellesbourne Warwick CV35 9EF, United Kingdom (R.M.N., M.A.V.)
Polyamine-binding proteins have been identified in a wide
range of organisms, including mammals, yeasts, and bacteria. In this
work, we have investigated specific spermidine binding to plant
membrane proteins purified from microsomes of etiolated maize
(Zea mays) coleoptiles. In the final purification step, specific spermidine-binding activity (Kd
6.02 10
7 M) was eluted from a HiTrapQ
fast-protein liquid chromatography column at about 0.25 M
NaCl, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of
the most active fraction showed a major polypeptide of about 60 kD and
another copurifying 18-kD protein. Competition experiments, performed
on HiTrapQ active fractions, confirmed the specificity of the binding.
Upon Sephadex G-100 gel filtration, spermidine binding was associated
almost exclusively with the 18-kD protein. On the basis of the
N-terminal sequences, degenerate oligonucleotide probes were designed
and used to isolate, by reverse transcriptase-polymerase chain reaction
and polymerase chain reaction, cDNA fragments of about 1 kb for the
60-kD protein, and 0.9 kb for the 18-kD protein. Northern-blot analysis
performed on etiolated coleoptiles and different tissues from 10-d-old
maize plants indicated the presence of two different mRNAs of 1.7 and 0.7 kb. Southern-blot analysis indicated that the genes encoding the
60- and 18-kD proteins are probably derived from differential processing of the same precursor mRNA. Using rabbit polyclonal antibodies raised against these proteins, affinity purification and
dot-blot experiments detected analogous membrane proteins in monocot
and dicot plants.
1
This work was supported by a Project of
Technological Priority Short Term Fellowship Advanced Molecular
Institute in Community Agriculture-Science-European Economic
Interest Grouping (contract no. PTP151), by the
Interdepartmental Center of Biotechnology (University of
Bologna), and by a Marie Curie Fellowship from the European Economic
Commission (IV Framework, Biotechnology Program, project no.
ERB4001GT980107), all to A.T. This work was also supported by the funds
of University of Bologna for selected research topics, special project
"Molecular Signals in Cell Differentiation."
*
Corresponding author; e-mail bagninel{at}kaiser.alma.unibo.it; fax
39-051-242576.
© 2002 American Society of Plant Physiologists
