Cell division and DNA topisomerase I activity in root meristems of pea seedlings during water stress

ABSTRACT

Low water potential, generated by PEG addition to the liquid medium of hydroponically grown pea seedlings, induces a fall in moisture content in the roots, followed by the arrest of elongation. This water stress reduces the mitotic index of root meristems during the treatment and induces the appearance of a peak of mitosis at 12 hours from the beginning of recovery. This peak suggests that during water stress the cell cycle is blocked in G2 or late S phase. In a first attempt to understand the biochemical events leading to cell cycle arrest, we tested the in vitro activity of DNA topoisomerase I extracted from stressed or control root meristems. The activity of this enzyme in extracts from stressed seedlings was lower than in controls, whereas it was higher in extracts from seedlings which had recovered from water stress for a few hours. The highest specific activity was observed with seedlings at 24 hours from the start of recovery. The fact that during stress treatments and recovery there was no variation in the synthesis of a 45 kDa protein, indicated as DNA topoisomerase I, suggested that the activity of this enzyme could be posttranslationally regulated. The hypothesis that variations in the concentration of unknown endogenous regulators of the activity of this enzyme may take place during water loss or uptake in the cytosol of meristematic cells is discussed.     

Nuclear Proteins During the Onset of Cell Proliferation in Pea Root Meristems

The DNA: proteins ratio in nuclei of root meristems of pea (Pisumsativum L. cv. Lincoln) changed considerably during germinationas cells moved from a quiescent to an actively proliferatingstate with a higher protein content in nuclei of the latter.Electrophoretic patterns of nuclear proteins extracted at differenttimes of germination were used to examine qualitative changes.The various patterns presented a substantial similarity butthere were some proteins whose content increased or decreasedand others which disappeared or appeared during germination.The bulk of these variations occurred between 24 and 48 h ofgermination, suggesting that they might be correlated to thetransition from quiescence to the proliferating state. The patternof nuclear proteins obtained from adult differentiated roottissue was also examined. We tried to purify five differentnuclear protein components from intact nuclei by a multi-stepextraction procedure using a series of different buffers toascertain the nature of proteins presenting major interestingvariations. Most of these proteins purified with the nuclearsap or ribosomal components. Key words: Cell proliferation, electrophoresis, Pisum sativum L, root meristems     

DNA Topoisomerase in Nuclei Purified from Root Meristems of Pisum sativum

DNA topoisomerase is present in nuclei purified from the rootmeristems of Pisum sativum seedlings. The DNA topoisomeraseis solubilized from nuclei by 500 mol m^–3 NaCl and relaxessupercoiled pBR322 DNA forming a series of DNA topoisomers whichmigrate electrophoretically between the supercoiled and opencircular forms. The presence of ATP in the incubation mixtureincreases the number of DNA topoisomers migrating electrophoreticallyin the region with slightly greater mobility than the open circularform. The formation of topoisomers with different linking numbersmight be the result of the activation of a different DNA topoisomerasewhich has a peculiar relaxing activity or introduces supercoilsinto the open circular form of pBR322 DNA. A low unknottingactivity with knotted P4 DNA is also present in the same nuclearpreparation. The hypothesis is made that both DNA topoisomerase I and IImight be present contemporaneously in these nuclei. The DNArelaxing activity seems to be stable and is activated by KC1.Partial purification by ion-exchange chromatography is not sufficientto separate these two DNA topoisomerases. Key words: Pisum sativum, pea, DNA topoisomerase, nuclei, cell proliferation