Isolation and characterization of an endonuclease synthesized by barley (Hordeum vulgare L.) uninucleate microspores.
Few biochemical and molecular details are available on microspore growth and development. In this work, a nuclease was partially purified from diffusates of barley (Hordeum vulgare L.) microspores by using concanavalin-A as ligand. The chromatographic preparation contained a 34-kDa protein with nucleolytic activity; the enzyme (called BMN: barley microspore nuclease) was very stable at pH > 8.0 and temperatures below 50 degrees C. Activity was highest at pH 5.6 and increased almost exponentially with temperature until a breakpoint between activity and stability was reached at 70 degrees C. Although BMN was able to cleave RNA, the enzyme showed a remarkable preference for DNA, especially in the single-stranded form. The best homopolymeric substrates were poly(dA) and poly(A), whereas poly(dC), poly(G) and poly(I) were almost completely uncleaved. When incubated with intact nuclei, BMN caused a nucleosomal DNA ladder of approximately 200 bp. On the basis of DNA laddering, substrate specificity, Mg2+ -dependence and best performance at apoplastic pH, BMN can be referred to as a putative apoptotic nuclease involved in pollen development.
Marchetti
S
S
Dipartimento di Produzione Vegetale e Tecnologie Agrarie, University of Udine, Italy. stefano.marchetti@dpvta.uniud.it
Zaina
G
G
Chiabà
C
C
Pappalardo
C
C
Pitotti
A
A
D017209:Apoptosis; D018744:DNA, Plant / Q000032:analysis; D004706:Endodeoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D004722:Endoribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D001467:Hordeum / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes; D008540:Meiosis; D011058:Pollen / Q000166:cytology / Q000235:genetics / Q000254:growth & development; D013170:Spores / Q000502:physiology; D013379:Substrate Specificity
D018744:DNA, Plant; D007527:Isoenzymes; D004706:Endodeoxyribonucleases; D004722:Endoribonucleases