PLANT PHYSIOLOGY , Vol 113, Issue 3 873-880, Copyright © 1997 by American Society of Plant Biologists
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DEVELOPMENT AND GROWTH REGULATION |
Proteinase Activity during Tracheary Element Differentiation in Zinnia Mesophyll Cultures
E. P. Beers and T. B. Freeman
Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0327
The zinnia (Zinnia elegans) mesophyll cell culture tracheary element (TE)
system was used to study proteinases active during developmentally
programmed cell death. Substrate-impregnated gels and single-cell assays
revealed high levels of proteinase activity in differentiating TEs compared
with undifferentiated cultured cells and expanding leaves. Three
proteinases (145, 28, and 24 kD) were exclusive to differentiating TEs. A
fourth proteinase (59 kD), although detected in extracts from all tissues
examined, was most active in differentiating TEs. The 28- and 24-kD
proteinases were inhibited by thiol proteinase inhibitors, leupeptin, and
N-[N-(L-3-trans-carboxirane-2-carbonyl)-L-leucyl]-agmatine (E-64). The 145-
and 59-kD proteinases were inhibited by the serine proteinase inhibitor
phenylmethylsulfonyl fluoride (PMSF). Extracts from the TE cultures
contained sodium dodecyl sulfate-stimulated proteolytic activity not
detected in control cultures. Sodium dodecyl sulfate-stimulated proteolysis
was inhibited by leupeptin or E-64, but not by PMSF. Other tissues,
sucrose-starved cells and cotyledons, that contain high levels of
proteolytic activity did not contain TE-specific proteinases, but did
contain higher levels of E-64-sensitive activities migrating as 36- to
31-kD enzymes and as a PMSF-sensitive 66-kD proteinase.
