Effect of OA-inhibitor of protein phosphatases PP1 and PP2A — on initiation of DNA replication and mitosis in Vicia faba root meristems

Abstract

Key words: car bo hy drate star va tion, con trol points of the cell cy cle, okadaic acid, pro tein phosphatases, Vicia faba var. mi nor Ab stractAc cord ing to the prin ci pal con trol point (PCP) hy poth e sis, exper i ments with ex cised, car bo hy drate-starved sta tion ary root meristems of Vicia faba var. mi nor have dem on strated that cells which pre vi ously di vided asynchronously were pref er entially blocked in G 1 (PCP1) and G 2 (PCP2) phases. When station ary phase meristems are sup plied with ex og e nous car bohy drate (2 % su crose), the G1-and G2-ar rested cells start out DNA rep li ca tion and mi totic di vi sions, re spec tively. The resump tion of DNA syn the sis and mi to sis is not im me di ate and the de lays of G 1 -and G 2 -ar rested cells are found dif fer ent. Using this model, we ex am ined the ef fects of 4 pulse in cu ba tions with okadaic acid (OA), a spe cific in hib i tor of PP1 and PP2A, on the du ra tion of in ter vals elaps ing be tween the pro vi sion of su crose and the first ap pear ance of S-and M-phase cells. We have found that de pend ing on the pe riod dur ing which OA had been ap plied, the re lease from G1 and G2 phase ar rest-points be comes pro longed, show ing dif fer ent time-course mod i fi cations. The ob tained data pro vide ev i dence that ac ti va tion of PP1 and PP2A is re quired to al low the cells for both PCP1→S and PCP2→M tran si tions in root meristems of V. faba.List of ab bre vi a tions: CDKs -cyclin-de pend ent kin ases, OA -okadaic acid, PCP: PCP1, PCP2 -prin ci pal con trol point: 1, 2, PPases: PP1, PP2A, PP2B, PP2C -serine/threonine pro tein phosphatases: 1, 2A, 2B, 2C, pRb -ret i no blas toma pro tein, PTPases -pro tein ty ro sine phosphatases In tro duc tion Pro gres sion through the cell di vi sion cy cle inEukaryotes is achieved by the op er a tion of a se ries of check points, each of them en sur ing the com pletion of one step of the cy cle prior to ini ti a tion of the next. The ex is tence of check point con trol sys tem in plants was first rec og nized 30 years ago by Van't Hof and Kovacs (1972). Their ex per i ments with ex cised, car bo hy drate-starved sta tion ary plant root meristems dem on strated, that cells which pre viously di vided asynchronously, be came pref er entially blocked in G1 and G2 phase. The stop page of the cell cy cle was re vers ible and the pro vi sion of ex cised roots with su crose stim u lated cell cy cle. Cells ar rested in G1 started out the S phase, while cells ar rested in G2 started out M phase. The reproducibility of this phe nom e non was the ba sis for the prin ci pal con trol point (PCP) hy poth e sis in plant cells. The first prin ci pal con trol point in G1 phase was termed PCP1, while the sec ond prin ci pal con trol point in G2 phase was termed PCP2 (reviewed by Van't Hof 1985). Pro tein phosphorylation and dephosphorylation -cat a lyzed by pro tein kin ases and phosphatases, re spec tively -are the key 303 ACTA PHYSIOLOGIAE PLANTARUM

In situ activities of hexokinase and fructokinase in relation to phosphorylation status of root meristem cells of Vicia faba during reactivation from sugar starvation

“…2E, H). Presence of protein phosphatases and kinases inhibitors during the last 3 h before revival of the cell cycle still caused the prolongation of cell cycle blockade, but at this time, this reaction was the weakest in comparison with other (Polit and Maszewski 2004, 2005). The presence of OA between 9 and 12 h of culture did not change HK activity, but the presence of 6‐DMAP at the same time had a marked effect (Fig.…”

“…Presence of protein phosphatases and kinases inhibitors (OA and 6‐DMAP) during the first 3 h of cell regeneration caused the most prolonged blockade of the cell cycle and decrease in the number of cells, which were able to start again (Polit and Maszewski 2005). When meristem cells were regenerated for 3 h in 2% sucrose, in the presence of OA, the activity of organelle‐associated HK was nearly absent, while relatively intensive staining of FK was observed (Figs 2F and 3F).…”

“…for 3 days. After starvation, the roots were transferred to the sterile White’s medium (WM) containing 1% (and 2 and 4%) glucose (WMG) or 2% sucrose (WMS) or 2% sucrose with 1 μ M OA or 2% sucrose with 3 m M 6‐DMAP (Sigma‐Aldrich, Poznań, Poland) and cultivated under the same conditions for 3 h, similar to Polit and Maszewski (2004, 2005). Some roots from the experimental series were fixed and the others were transferred to the fresh sterile WMG or WMS, respectively, and cultivated for the next 6 h (9‐h treatments, data not shown).…”

“…At the beginning of regeneration, in the presence of sucrose, meristematic cells are extremely sensitive to inhibitors of protein kinases [cyclin‐dependent kinases (CDK)] and protein phosphatases 1 or 2A (PP1/PP2A), which results in the prolonged blockade of cell cycle. This sensitivity decreases with time of cell regeneration (Polit and Maszewski 2004, 2005, Polit et al 2004).…”

An improved method for the cell cycle synchronization of Vicia faba root meristem cells

“…However, biochemical analysis of metabolism and molecular studies of gene expression in a given phase of cell division in planta are often difficult, because cell cycle progression in somatic tissues is asynchronous and only some cells are cycling in a given moment. Perfect synchronization would permit precise biochemical and molecular studies of regulatory mechanisms, which determine cell cycle regulation at the level of gene expression and protein phosphorylation in different phases of the cycle (Polit and Maszewski 2005;Polit and Kaźmierczak 2007).…”