Polyamine-induced DNA Synthesis and Mitosis in Oat Leaf Protoplasts

Abstract

Freshly isolated protoplasts from leaves of oat seedigs (var. Victory) which do not divide when cultured on a wide range of media are capable of incorporating tritiated leucine, uridine, and thymidine into trichloroacetic acid-insoluble macromolecules. Over 70% of the leucine and uridine incorporated over an 18-hour period are found in protein and RNA, respectively, as shown by hydrolysis of the macromolecular products with a specific protease or RNase. In contrast, little or none of the tritiated thymiine is incorporated into macromolecules hydrolyzable by DNase over an 18-to 96-hour period. Incorporation of thymidine into trichloroacetic acid-insoluble material declines sharply with increasing time of culture after 18 hours. However, addition of diamines or polyamines to the medium not only prevents the decline, but actually increases net thymidine incorporation, including a fraction going into DNA. A significant increase in mitoses and binucleate protoplasts is also observed in 72-to 168-hour cultures.The inability of oat leaf protoplasts to synthesize significant quantities of DNA suggests that they are arrested at the G1 phase of the celi cycle.Treatment with polyamines appears to promote both DNA synthesis and the inception of mitotic activity in oat protoplasts, as in numerous animal and microbial cells.Protoplasts obtained from leaves of certain plant species, especially members of the Solanaceae like tobacco and potato, can synthesize new walls, undergo nuclear and cellular division, and ultimately regenerate entire plants (4, 22). Protoplasts from leaves of cereals do not undergo sustained mitotic division when cultured under similar conditions on a wide range of media (6, 17). These nondividing protoplasts show a time-dependent decrease of incorporation of precursors into presumptive protein and nucleic acids (8) and an increase in potentially detrimental hydrolases such as RNase and Protease (10). This decrease in net synthetic activity and lack ofmitosis in cereal protoplasts may result from a complex of senescence-induced changes (1) or injury caused by plasmolysis (19) that must precede protoplast isolation. These stresses can produce severe metabolic alterations, contributing to a blockage of the cell cycle and a consequent lack of mitosis.Much evidence indicates that the naturally occurring diamines and polyamines are involved in regulating nucleic acid synthesis and cell division in microorganisms and animals (2); similar evidence is far less extensive for plants (3,23 of RNase and protease activity in oat leaves (16).Recently, substantial evidence has accumulated suggesting an important role of polyamines in regulating DNA synthesis (5, 7) and an orderly progression through the animal cell cycle (9, 13, 24). For example, specific inhibitors of polyamine synthesis have been shown to prevent certain mammalian cells from entering mitosis (12, 24), while addition of spermidine or spermine to such cells results in rapid resumption of cell division (7, 21). Because of these facts, we conside...

Polyamine Oxidase in Oat Leaves: A Cell Wall-Localized Enzyme

“…In addition, putrescine or spermidine (1 mM) was added to the cultures to stabilize the protoplasts against lysis and to increase their macromolecular biosynthetic activity (1). The protoplast suspensions were cultured as hanging drops (15) or as thin layers of 2 to 3 ml in 15 x 60 mm Petri dishes and incubated in moist chambers in the dark at room temperature. All manipulations were performed aseptically in a laminar flow hood.…”

“…Protoplasts were prepared by a procedure (15) involving surface sterilization of the leaves, stripping of the lower epidermis and exposure of the peeled side of leaf tissue to cell wall-degrading enzyme (Cellulysin, Calbiochem) in 0.6 M mannitol. The isolated protoplasts were washed by centrifugation and resuspension in the osmoticum and cultured in B5 medium (10) containing additives (16) to promote cell wall regeneration.…”

“…In addition to stabilizing protoplasts against lysis, they promote the synthesis of macromolecules including DNA, increase the frequency of mitotic divisions (15) and inhibit the senescence-linked rise in RNase, protease, and Chl breakdown activities in protoplasts and leaves (8,13,14). If the polyamines act in vivo to promote mitosis and prevent senescence, then it might be expected that polyamine titer would fall as cells cease division and enter senescence.…”

Effects of Exogenous 1,3-Diaminopropane and Spermidine on Senescence of Oat Leaves

“…This suggested that Dap, like other PAs, may be involved in the control of senescence. Although other PAs have been implicated in processes controlling cell growth (5,6,8,13) and senescence (2,3,9,16,18), virtually nothing has been reported about the biological activity of Dap in plant cells.…”

Métabolisme et rôle des polyamines dans le développement de la plante

“…Les polyamines semblent impliquees dans Ia replication de I'ADN et dans Ia regulation de Ia transcription de l'ARN (Marton et Morris, 1987). In vitro, Basu et al (1991) (Kaur-Sawhney et al, 1980). Sur Ia mcme plante, les polyamines controlent aussi l'activite des RNases et des proteases KaurSawhney et al, 1978).…”