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Plant Physiol, July 2001, Vol. 126, pp. 1150-1161
Inhibition of Proteasome Activity Strongly Affects Kiwifruit
Pollen Germination. Involvement of the Ubiquitin/Proteasome Pathway as
a Major Regulator1
Anna
Speranza,*
Valeria
Scoccianti,
Rita
Crinelli,
Gian Lorenzo
Calzoni, and
Mauro
Magnani
Dipartimento di Biologia, Università di Bologna, via Irnerio
42, I-40126 Bologna, Italy (A.S., G.L.C.); and Istituto Botanico
(V.S.) and Istituto Chimica Biologica (R.C., M.M.), Università di
Urbino, via Bramante 28, I-61029 Urbino, Italy
The 26S proteasome is a multicatalytic complex that acts as primary
protease of the ubiquitin-mediated proteolytic pathway in eukaryotes.
We provide here the first evidence that the proteasome plays a key role
in regulating pollen tube growth. Immunoblotting experiments revealed
the presence of high levels of free ubiquitin and ubiquitin conjugates
in rehydrated and germinating pollen of kiwifruit [Actinidia
deliciosa var. deliciosa (A. Chev)
C. F. Liang et A. R. Ferguson]. Proteasome activity, assayed
fluorometrically, accompanied the progression of germination. Specific
inhibitors of proteasome function such as
benzyloxycarbonyl-leucinyl-leucinyl-leucinal (MG-132),
clasto-lactacystin  -lactone, and epoxomicin
significantly decreased tube growth or altered tube morphology.
High-molecular mass, ubiquitinated proteins accumulated in MG-132- and
-lactone-treated pollen, indicating that proteasome function was
effectively impaired. The inhibitors were also able to decrease in
vitro proteasome activity in pollen extracts. Because MG-132 can
inhibit calpains, as well as the proteasome, trans-epoxy
succinyl-L-leucylamido-(4-guanidino) butane (E-64), an
inhibitor of cysteine proteases, was investigated. Some reduction in
tube growth rate was observed, but only at 80 µM E-64,
and no abnormal tubes were produced. Furthermore, no inhibition of tube
growth was observed when another inhibitor of cysteine proteases,
leupeptin, or inhibitors of serine and aspartic proteases
(phenylmethylsulfonyl fluoride and pepstatin) were used. Our results
indicate that protein turnover during tube organization and elongation
in kiwifruit pollen is important, and our results also implicate the
ubiquitin/26S proteasome as the major proteolytic pathway involved.
1
This work was supported by the Italian Ministero
per l'Università e la Ricerca Scientifica e Tecnologica.
*
Corresponding author; e-mail asperanza{at}mail.cib.unibo.it; fax
39-051-242576.
© 2001 American Society of Plant Physiologists
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