Activity in Oat Leaf Protoplasts - NCBI

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Received for publication December 16, 1977 and in revised form March 12, 1978 .... RNase activity was measured as described previously (1). For .... GABBAY EJ, RR SHIMSHAK 1968 Topography of nucleic acid helices in solutions. IV. Effect.
Plant Physiol. (1978) 62, 158-160

Short Communication

Dual Mechanisms in Polyamine-mediated Control of Ribonuclease Activity in Oat Leaf Protoplasts' Received for publication December 16, 1977 and in revised form March 12, 1978

RAVINDAR KAUR-SAWHNEY, ARIE ALTMAN,2 AND ARTHUR W. GALSTON Department of Biology, Yale University, New Haven, Connecticut 06520 ABSTRACT

MATERIALS AND METHODS

Dibasic amino acids and polyamies added to oat ( Avea sadvaL.) leaf protoplast isolation media decrease the RNase activity of extracted protoplasts relative to controls. This effect, wich is manifested even when the added polyamine Is removed by exhaustive dialysis prior to assay, is due to a prevention of the rise in RNase activity which usually folows protoplast Isolation. Polyanines, but not dibasic amino acids, also decrease RNase activity in diro. This hI vitro effect seems to result from electrovalent attachment of the polyamine to the RNA, because the greater the net positive charge on the polyane, the greater is its inhibitory effect ih ntro. The activity of dibasic amo acids when added during protoplast isolation probably results from their conversion to polyamines.

Chemicals. HCl salts of the dibasic amino acids, diamines, and polyamines were obtained from Sigma Chemical Co. Yeast RNA, also obtained from Sigma, was purified by the method of FrischNiggemeyer and Reddi (8). Cellulysin B was obtained from Calbiochem.

In previous investigations (1, 9, 1 1), we reported that the RNase activity of oat leaf protoplasts increases sharply during the several hr of their incubation. This rise in RNase is accompanied by decreased net incorporation of uridine into RNA, as well as by general deterioration and ultimate lysis of the protoplasts. We suggested that this increased RNase activity in protoplasts and leaves (24, 25) may be in part responsible for the limited survival and only rare cell division activity of cereal protoplasts in culture

(5, 19).

The rise in RNase activity is drastically inhibited if protoplasts

are incubated in the presence of L-arginine or if they are isolated from leaves pretreated with L-arginine, L-lysine, cadaverine, putrescine, cycloheximide, or kinetin (1, 11, 13). Protoplasts so treated are more stable to lysis and show higher RNA-synthetic

activity (1, 11, 13, 14) than controls. It appeared that an understanding of the mechanism by which polyamines inhibit the rise in RNase activity might help in obtaining more stable protoplasts which could be useful in cell culture experiments aimed at improving cereal crops. Although polyamines are known to affect the synthesis of DNA, RNA, and protein (2, 4, 6, 20), there are only a few reports, limited to bacterial and mammalian systems, dealing with the effect of polyamines on DNA and RNA degradation by nucleases (3, 17, 23). The main objective of this investigation was to determine the effects of L-argfinine and related diamines and polyamines on the degradation of RNA by RNase from oat leaf protoplasts.

' Aided by Grant AER #72-03387 from the RANN program of the National Science Foundation to A. W. G. 2 Permanent address: Department of Horticulture, The Hebrew University of Jerusalem, P.O. Box 12, Rohovot, Israel.

Preparation of Protoplasts. Procedures for growth of plants and isolation of protoplasts have been described earlier (9). Briefly, for studies of RNase in protoplasts (in vivo), the lower epidermis of 6-day-old leaves of Avena sativa L. (var. Victory) was peeled off and the peeled leaves floated on a solution containing 0.5% (w/v) Cellulysin in 0.6 M mannitol and I mm phosphate buffer at pH 5.7, and incubated for 2 hr at 31 ± 1 C to extract protoplasts. Similar protoplast isolations were carried out in the presence of 10 mM L-arginine or 1 mm spermine. The released protoplasts were collected by centrifugation at 50g for 8 min, washed twice with 0.6 M mannitol (pH 5.7), resuspended in mannitol, counted in a hemocytometer, and adjusted to 1 x 106 protoplasts/ml. Oneml aliquots of suspension were used for determining RNase activity in the protoplasts. For studies of RNase in cell-free extracts (in vitro), protoplasts were similarly prepared in the absence of amines and were used for the preparation of the crude RNase, as described below. Extraction and Assay of RNase from Protoplasts. The enzyme was extracted by a procedure modified from Wyen et al. (26). Protoplast suspensions (30 ml), containing 106 protoplasts/ml, were centrifuged for 8 min at 50g. The resulting pellet was homogenized in 30 ml of cold Tris-HCl buffer (25 mM, pH 7.5) with a glass pestle and recentrifuged for 10 min at 12,000g at 4 C. The supernatant fraction was passed through Miracloth and used as the crude RNase preparation. RNase activity was measured as described previously (1). For experiments probing the effects of amines on RNase in protoplasts, the reaction mixture consisted of 0.4 ml of RNA solution (containing 375 ,ug of yeast RNA in 62.5 mm acetate buffer at pH 5.5), 0.1 ml of 1 mm phosphate buffer at pH 5.7, and 0.1 ml of the crude RNase. For studies of in vitro effects of applied amines, 0.1 ml of appropriate concentration of various amines in phosphate buffer was incubated with the yeast RNA prior to assay. The assay mixture was then incubated at 37 C for 30 min and the reaction terminated by adding 0.5 ml of 2.5% (w/v) trichloroacetic acid and 0.3% La(NO3)3. Each experimental sample was assayed in duplicate, in addition to the blank, in which 0.1 ml of the crude enzyme was added after the trichloroacetic acid. The mixture was cooled in ice for at least 1 hr and centrifuged. The supematant fraction was then diluted 3-fold with water and the acid-soluble nucleotides measured by recording A at 260 nm. RNase activities were expressed in corrected A units after subtraction of the blank values.

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RESULTS AND DISCUSSION Reduction in the RNase activity of protoplasts from oat leaves had been observed previously when leaves were pretreated with diamines prior to protoplast isolation (11, 13) or when protoplasts from untreated leaves were isolated and incubated in the presence of L-arginine (1). We now report that protoplast RNase activity is dramatically decreased when the diamines or polyamines are present only during the 2-hr incubation in Cellulysin needed for preparation of protoplasts. For example, when compared with control treatments, 10 mM L-arginine or I mm spermine in the protoplast isolation ihedium reduced RNase activity by 72 and 80%o, respectively. Concentrations of spermine higher than I mm caused protoplasts to aggregate, while higher arginine levels had no further effects. If the protoplasts are incubated for an additional 3 hr, the RNase activity in controls rises by more than 40%o, but remains constant in the polyamine-treated series (Table I). Since the crude RNase from the protoplasts was exhaustively dialyzed to remove excess polyamines prior to addition of the RNA substrate, this effect must be due to differences in enzyme activity. Compounds containing multiple amine groups, when added to the enzymic assays solutions in vitro along with yeast RNA, had various effects. With L-arginine and L-lysine no decrease in RNase activity was observed, with the diamines cadaverine and putrescine activity was reduced slightly, while with the polyamines spermidine and spermine marked decreases in the activity of protoplast RNase occurred (Table II). Spermine is more effective than spermidine in decreasing the activity of protoplast RNase and this effect becomes more pronounced with increasing concentrations of the polyamine (Table III). Increase in incubation time to 1 hr did not decrease RNase activity further, indicating that the action of polyamines does not involve further biological transformations, but is most likely the result of a direct reaction between amine and some reactant in the assay. Similar results were obtained in vitro with pancreatic RNase, indicating that the effect is not specific for plant or for oat leaf protoplast RNase. Polyamines, which are positively charged in the range of cellular pH values, are known to bind strongly to the acidic phosphate groups of RNA and DNA in bacteria, animals, and plants (2, 4, 6, 22). This electrovalent bonding makes the nucleic acids more compact spatially and hence less available to nucleases (18, 23). The in vitro reduction in the activity of protoplast RNase by polyamines is probably the result of such binding. Depending on the type of RNase involved, the presence of spermidine and spermine may render natural RNA and synthetic polyadenylic acid less susceptible to degradation by endonucleases (10, 15). The Table I. Inhibition by spermine of the rise in RNase activity in protoplasts incubated for three hours Excess exogenously fed spermine was removed by dialysis prior to addition of substrate RNA for the RNase assay. Treatment

Control

Spermine,

lmM

RNase activity 0 hr 3 hr Absolute Relative Absolute Relative (A at 260 nm) 0.632 100 0.910 144 0.130 20 0.134 21

Table II. Effect of various compounds containing amine groups (10 mM) on in vitro activity of oat leaf protoplast RNase Compounds were incubated with yeast RNA for 0 or 1 hour periods before the addition of the enzyme. Treatment

Relative RNase activity

(A Control L-arginine L-lysine Putrescine Cadaverine Spermidine

Spermine

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POLYAMINES AND PROTOPLAST RNase ACTIVITY

at

0 hr 100 102 107 100 105 70 54

260 nm) X 1 hr 100 100 105 90 91 70 47

Table III. Effect of various concentrations of compounds containing amine groups on in vitro activity of oat leaf protoplast RNase Compounds were added to yeast RNA isnediately before addition of the enzyme. Relative RNase activity

Conc. of

amines (mM)

Arginine

0 0.1 1.0 10.0

100 99 100 100

Spermidine

Spermine

(A at 260 nm) 100 98 88 70

100 98 70 54

RNase of oat leaves is an endoribonuclease, specific for linkages involving purines (24, 25) and its action is probably affected in this way by polyamines. Recently (7, 12, 16) polyamines have been shown to promote RNase activity in bacteria and animals but these RNase's have specificity for bonds involving pyrimidines rather than purines. No such effects have yet been noted for plant ribonucleases. Thus, both dibasic amino acids and polyamines decrease the RNase activity of oat leaf protoplasts relative to controls when added in vivo during protoplast isolation. This effect is due to an interference with RNA synthesis, since it involves the prevention of the rise in RNase activity seen during protoplast isolation and aging. In vitro, only polyamines are effective in decreasing RNase activity, probably through direct combination with substrate. Since the dibasic amino acids are known to be rapidly metabolized to polyamines in plant tissues (21), it is probably the polyamines which are primarily effective in reducing RNase activity in the in vivo experiments as well. LITERATURE CITED 1. ALTMAN A, R KAUR-SAWHNEY, AW GALSTON 1977 Stabilization of oat leaf protoplasts through polyamine-mediated inhibition of senescence. Plant Physiol 60: 570-574 2. BACHRACH U 1973 Function of Naturally Occurring Polyamines. Academic Press, New York 3. BACHRACH U, G EILON 1969 The effect of spermine and oxidized spermine on the enzymic degradation of DNA. Biochim Biophys Acta 179: 494-496 4. BAGNI N, DS FRACASSINI 1973 The role of polyamines as growth factors in higher plants and their mechanism of action. In Plant Growth Substances. Hirokawa Publishing Co, Tokyo, pp 1205-1217 5. BRENNEMAN F, AW GALSTON 1975 Experiments on the cultivation of protoplasts and calli of agriculturally important plants. 1. Oat (Avena sativa L.). Biochem Physiolog Pflanzen 168: 453-471 6. COHEN SS 1971 Introduction to the Polyamines. Prentice-Hall, Englewood Cliffs, NJ 7. FRANK JJ, IA HAWK, CC LEVY 1976 Peptides isolated from Enterobacter nuclease as potential polyamine binding sites. Biochim Biophys Acta 432: 369-380 8. FRISCH-NIGGEMEYER W, KK REDDI 1957 Studies on ribonuclease in tobacco leaves. 1. Purification and properties. Biochim Biophys Acta 26: 40-46 9. FUCHS Y, AW GALSTON 1976 Macromolecular synthesis in oat leaf protoplasts. Plant Cell Physiol 17: 475-482 10. GABBAY EJ, RR SHIMSHAK 1968 Topography of nucleic acid helices in solutions. IV. Effect of polyamines on RNase-catalyzed hydrolysis of polyadenylic acid. Biopolymers 6: 255-268 11. GALSTON AW, A ALTMAN, R KAUR-SAWHNEY 1978 Polyamines, ribonuclease and the improvement of oat leaf protoplasts. Plant Sci Lett I1: 69-79 12. IGARASHI K, H KUMAGAI, Y WATANABE, N TOYODA, S HIROSE 1975 Change of substrate specificity by polyamines of ribonucleases which hydrolyze ribonucleic acid at linkages attached to pyrimidine nucleotides. Biochem Biophys Res Commun 67: 1070-1077 13. KAUR-SAWHNEY R, WR ADAMS JR, J TSANG, AW GALSTON 1977 Leaf pretreatment with senescence retardants as a basis for oat protoplast improvement. Plant Cell Phyiol 18:

1309-1317 14. KAUR-SAWHNEY R, M RANCILLAC, B. STASKAWICZ, WR ADAMS JR, AW GALSTON 1976 Effect of cycloheximide and kinetin on yield, integrity and metabolic activity of oat leaf protoplasts. Plant Sci Lett 7: 57-67 15. KEDRACKI R, W SZER 1967 A note on the effect of spermine on degradation of pyrimidine polynucleotides by pancreatic ribonuclease. Acta Biochim Polon 14: 163-168 16. KUMAGAI H, K IGARASHI, M YOSHIKAWA, S HIROSE 1977 Effects of polyamines on the activities of Escherichia coli ribonuclease I and II. J Biochem 81: 381-388 17. LEVY CC, PA HIETER, SM LEGENDRE 1974 Evidence for the direct binding of polyamines to a ribonuclease that hydrolyzes ribonucleic acid at uridylic acid residues. J Biol Chem 249:

6762-6769 18. MITRA S, P KAESBERG 1963 Interaction of polyamines with turnip yellow mosaic virus RNA. Biochem Biophys Res Commun. I1: 146-151 19. POTRYKUS I, T CH HARMS, H LORz 1976 Problems in culturing cereal protoplasts. In D Dudits, GL Farkas, P Maliga, eds, Cell Genetics in Higher Plants. Akademiai Kiad6, Budapest, pp 129-140 20. SAKAI IT, SS COHEN 1976 Effects of polyamines on the structure and reactivity of tRNA. Prog Nucleic Acid Res 17: 15-42 21. SMITH TA 1975 Recent advances in the biochemistry of plant amines. Phytochemistry 14:

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865-890 22. STEVENS L 1969 The binding of spermine to the ribosomes and ribosomal ribonucleic acid from Bacillus stearothermophilus. Biochem J 113: 117-121 23. STEVENS L 1970 The biochemical role of naturally occurring polyamines in nucleic acid synthesis. Bol Rev 45: 1-27

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24. UDVARDY J, GL FARKAS, E MARRE 1969 On RNase and other hydrolytic enzymes in excised A vena leaf tissue. Plant Cell Physiol 10: 375-386 25. WILSON CM 1975 Plant nucleases. Annu Rev Plant Physiol 26: 187-208 26. WYEN NV, S ERDEI, J UDVARDY, G BAGi, GL FARKAS 1972 Hormonal control of nuclease level in excised A vena leaf tissue. I Exp Bot 23: 37-44