Protein phosphorylation in Vicia faba root meristem cells during the first steps of leaving principal control points after sucrose application

Abstract

Before Vicia faba root meristem cells stopped by carbohydrate starvation in principal control points (PCP1 and PCP2) start sucrose induced replication and division they go through a phase of metabolic regeneration. This interval is characterised st great sensitivity to the inhibitors of cyclin-dependent protein kinases and protein phosphatases (PPs). In the present research, changes of phosphoprotein levels in the nucleolus, nucleus and cytoplasm were analysed using okadaic acid and 6-dimethylaminopurine (6-DMAP) during the first period of cell regeneration in sucrose (0-3 h). It was established that when the cells start to leave checkpoints, the balance between protein phosphorylation and dephosphorylation shifts towards the intensified activity of PPs. Furthermore, it was also established that the structures appearing during cell regeneration, which were located around cell nuclei and which contained large amounts of phosphorylated proteins, were plastids. The reactions of protein phosphorylation which took place in the plastids were directly correlated with starch synthesis and were stopped by inactivation of protein phosphatases (PP1 and/or PP2A).

Ultrastructural changes associated with the induction of premature chromosome condensation in Vicia faba root meristem cells

“…Also in the studies carried out by Polit (2009), attempting to elucidate the causes of diversified sensitivity of cells to the inhibitors of protein kinases and phosphatases used during G1-S and G2-M transitions, it has been shown that the induced phosphothreonine-positive areas are plastids accumulating starch grains (after the treatment of V. faba cells with 6-DMAP but not with OA). Polit (2009) has confirmed the results obtained by ultrastructural examinations that revealed the around-nucleus location of starch-rich plastids in control cells, and has confirmed their lack in the carbohydrate-starved cells. The carbohydrate-starved cells were strongly vacuolizated, while the plastids containing no starch grains were pushed into peripheral regions of the cell.…”

“…After 3 h of regeneration in sucrose, the irregular plastids, often with elongated shapes, returned close by the nucleus, showing then the presence of newly synthesized fine starch gains that just after 12 h of regeneration completely filled the interior of plastids. Ultimately, it has been concluded that the around-nucleus location of plastids can be connected with the still not settled plastid-to-nucleus signaling in meristem cells (Polit 2009). Given the above results, the observed phosphorylation of threonine (in HU/CF-treated cells) and its location in plastids may have contributed to the mobilization of sugars in the cell, necessary for CF-induced transitions in the cell life cycle.…”

“…Cytochemical detection of starch was performed on the preparations from Feulgen-stained root meristem cells using a standard procedure of staining with Lugol’s solution (I in KI) for 5 min (Polit et al 2002; Polit 2009). Briefly, 1.5-cm-long apical fragments of primary roots of V. faba were fixed in cold Clarke’s mixture (absolute ethanol/glacial acetic acid; 3:1, v/v ) for 1 h (according to Bruni et al 1976), washed three times with 96 % ethanol, rehydrated (70–30 % ethanol, distilled water), hydrolyzed in 4 M HCl (2 h), and stained with Schiff’s reagent (pararosaniline).…”

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

“…from the cotyledons during seed germination), meristematic cells of primary roots divide continuously, which results in root persistent growth. Plastids directly surrounding nuclei in these cells are filled with big starch grains (Polit 2009). In sugar‐starved, and recovered, meristematic cells of primary roots of V. faba , the metabolic activity of plastids is connected with sugar transformations, as was indicated by visualization of active HK and FK located closely to these organelles (or inside); the colocalization of the plastids and the products of enzyme reaction were also shown (Figs 2–4).…”

“…Our previous results suggest that after a 3‐day carbohydrate malnutrition of root tips isolated from cotyledons, the meristematic cells used up nutrients (including the starch pool stored in plastids) and the cell cycle was stopped in G1 and G2 phases by expression of the PCP1 and PCP2, respectively (Polit 2009, Polit et al 2004). In this study, we used similar experimental model and time‐course and showed that the consumption of sugar pool led to the considerable decrease in the activity of HK and FK (Figs 2C–3C).…”