During Senescence' Daphne J

Effect of Kinetin on Protein & Nucleic Acid Metabolism in Xanthium Leaves During Senescence' Daphne J. Osborne 2 Division of Biology, California Institute of Technology, Pasadena, California

The chemical changes that occur in leaves as they demonstrated that both chlorophyll degradation and grow old have been well characterized for many protein loss in detached wheat leaves are retarded species. The endogenous factors which control and if the blades are floated on solutions of benzimidazole regulate these changes in plant cells remain, to a at 50 mg/liter. In the same year, Richmond and great extent, obscure, and the problem of why the Lang (12) showed that similar effects could be ob- cell eventually dies is yet unsolved. A normal fea- tained if excised leaves of Xanthium are kept with ture of the ageing leaf blade is a continuous decline their petioles dipping into solutions of kinetin at 5 in protein level (1). The most rapid fall occurs mg/liter. Mothes and Engelbrecht (5) sprayed so- during senescence and is associated with irreversible lutions of kinetin directly onto leaves of Nicotiana yellowing, loss of chlorophyll, and the eventual death and reported (1959) that the retention of chlorophyll of the organ. When a mature leaf is excised from is localized to the areas of the blade to which kinetin the plant and the petiole kept in water, these same is supplied. They found that labelled amino acids symptoms of senescence occur and, provided the migrate to, and accumulate in, the treated parts of petiole does not form roots, the protein content of the tobacco leaves, and they suggest that kinetin retards blade may fall to less than half the original value leaf senescence by causing the treated areas to act within a few days. If, however, the petiole forms as loci for the accumulation of metabolites (5, 6). roots, the behavior of the excised leaf is quite differ- Extensive investigations have shown that both pro- ent. It will remain green and photosynthetically tein synthesis and ribonucleic acid synthesis is stimu- active, increasing in dry weight and total protein for lated in kinetin-treated parts of tobacco leaves. (15). periods extending, in some cases, to several years Mothes and Engelbrecht (7) conclude, however, that (2), much longer, in fact, than the leaf would survive "accumulation, . . . is not the consequence of syn- in situ. thesis, but mass synthesis of protein, for example, is The factors responsible for senescence and the the consequence of an accumulation of amino acids." decline of total protein in the leaf are not fully under- In 1959, (8) it was demonstrated that a number stood. Experiments with excised leaves have shown of auxins are markedly effective in retarding the that the decrease in protein content of the blade senescence of attached or detached autumn leaves of is not necessarily due to a lack of carbohydrate, ni- Prunus serrulata-senriko. Where ethanolic solutions trogen, or other nutrients (14, 4) or to an inability of esters of 2,4-dichlorophenoxyacetic acid are ap- of the cells themselves to synthesize amino acids plied to the blades, the cells retain their green color (13) but is due, rather, to a failing ability to in- for some 10 to 20 days longer than those in the sur- corporate these amino acids into protein (11). Pro- rounding untreated parts of the blade, or in the vided the leaf has a growing root system, the in- control leaves. The retention of green color in the corporation of amino acids proceeds normally, and treated areas is associated with an actively photo- it seems logical to assume that the roots metabolize synthetic chlorophyll and a maintenance of the and supply the blade with certain factors necessary initial protein level (9). Attempts to retard se- for the continued synthesis of protein. nescence by various treatments with kinetin proved During the last 4 years, several chemical sub- unsuccessful in this species. stances have been shown to retard senescence of leaf Precise information is still meagre concerning blades, and in this respect the compounds would ap- the part played by either auxins or kinins in the pear to substitute either directly or indirectly for the retardation of the biochemical changes associated unknown so-called root factors. with senescence but it is clear that they must exert In 1957, Person, Samborski, and Forsyth (10) a control through some fundamental cellular processes. In this paper are described some effects of one kinin, kinetin, upon the metabolism of protein and nucleic 1 Received Jan. 15, 1962. acids during the senescence of excised leaves of 2 Present address: Agricultural Research Council The results evi- Unit of Experimental Agronomy, Department of Agri- Xanthium pennsylvanicumi. provide culture, Oxford. dence that kinetin may control senescence in these 595 596 PLANT PHYSIOLOGY leaves by substituting in some way for an essential with hot ethanol/ether (3: 1 v/v). The pellet was factor necessary for maintaining the synthesis of then suspended in 0.3 N KOH and incubated at 37 C ribonucleic acid. for 16 hours. After centrifuging, the supernate was collected, the residue was twice suspended in w\ater then & Methods and centrifuged; the three supernates were Materials combined and made up to volume. An aliquot of the solution was used for protein determinations (see - Plant Material. Plants of Xanithiurn pennsylzan- below). A second aliquot was adjusted to pH 1.5 icun11i Wall. were grown under controlled condlitions The to 2.0 with HClO, to precipitate deoxyribonucleic with supplementary light to prevent flowvering. acid (DNA). DNA and insoluble KCIO, were re- experiments wsere carried out with fully mature leaves movedl by centrifuging and the total ribonucleotides from the ninth or tenth no(le below the apical bud. in the supernate determiiine(d by nmeasuremenit of the The leaves were excised and with their petioles kept, spec- in beakers of Nater, in large Plexiglass chambers in optical (lensity at 260 m,u in a Carey recording 240 340 mit. (liffuse light and high humidity at 20 C. After 2 trophotometer scanning between andl aci(l. The residue con- to 3 days in these conditions there is some loss of C. Deoxvribonucleic (4 chlorophyll and a small decline in the content of taining DNA was washed twice in 5 TCA at 0 C, both protein and nucleic acid, which in(licates the once in absolute ethanol, once in etlhanol/ether (3: 1 first stages of senescence in the blade. Leaves were v/'v), suspended in 0.5 N HClO1, anid( then incubated then selected for uniformity of green color. The at 90 C for 7 nlinutes. After centrifuging, the super- kinetin treatment of whole leaves, andl the treatment nate was dlecante(l, the residue twice washed w\ith of (lumbell-shaped pieces of blade (See table II) are water andl the three supernates comlbined aind nmadec described in the appropriate sections. For all other up to volume. Excess perchlorate was removeed by experiments, leaf (liscs 1.4 cnm in (lianmeter were adding a measured volume of KOH and( eliminatiing punched from interveinal areas of the bladle and the insoluble KClI4 by centrifuging. The (leoxy- treated as follows (some 60 (liscs could be obtained ribonucleotide content of the supernate was (leter- from each leaf). Groups of seven discs were either minedl by mleasuremiient of the optical dlensity at 260 floated upon 0.8 ml of the appropriate aqlueous solu- mnu in the Carey recording spectrophotometer. tion in a 10 ml petri dish, or arrangedl directly upon D. Protein. Protein w-as (letermine(l by the the dry glass. In both cases, the snmall (lishes w\ere methodl of Biuret and the total protein values were enclosed in larger dislhes lined w-ith damp filter paper, obtaine(d froml a sumimiation of the protein in 1, the and the larger dlishes were stacked in enamel trays solution of nucleotides resulting from alkaline h1- paper an(l tinfoil. The linedl with damp covered ith (lrolysis of RNA, (wvhich contains up to half of were in darkness at 24 C. trays kept the total protein in soluble forml) and 2, the insolu- 0 Preparation of Solutions. Kinetin was mlade up ble residue left from the above hydrolysis. The as an solutioni and \ as generally usecl at aqueous a further ex- 40 mg/liter. Media for the incorporation studies latter protein was ren(lere(I soluble by 1 N NaOH at 100 C for 4' 2 minutes. containecl either 14-C-1-leucine (approx. 5 X 10 A traction witlh and 14-C- 106 counts/nml) or 14-C-orotic acid (approx. 2.5 X In the experiments in which 14-C-leucine 10 -t, 106' counts/i ml) and included 20,000 units of orotic acid were used, the nucleic acid ancl protein penicillin g/liter. contents were mieasure(l in separate sets of (liscs. - Chemical Analyses. Unless otherwise stated, RNA and DNA were (letermlined as described before. duplicate or triplicate biological samples were assay- The protein content of the (liscs incubated wvith ed, an(d each replicate consistedl of seven discs totalling leucine was determined directly on the 5 ;(4 TCA- approximately 250 mlg. insoluble residue whiclh had been suitably extracte(d A. Chlorophvll. All discs were extracted three with 80 (l ethanol, absolute ethanol, and hot ethanol/ tinmes in hot 80 c/c ethanol, the combined chlorophyll ether as described earlier. The pellet w%as suspendedl extracts were mlade up to volume, and the optical in 1 N NaOH at 100 C for 412 miinutes and protein density measured at the absorption maximum for levels determined in aliquots of the solubilize(d protein chlorophyll a (665 nmiA) in a Beckmian Model B spec- by Biuret analysis. trophotometer. - Determinations of Specific Activity. Duplicate B. Ribonucleic acid. (RNA) After three ex- of the solutions of ribonucleotides discs aliquots (0.2 ml) tractions in hot 80 % ethanol, the decolorize(l and deoxyribonucleotidles were plated onto sand- a homogenizer, were homogenized in Potter-Elvehjem aluminum and counted to 3 (4 ac- centrifuged, andl the residue resuspended and centri- blasted planchettes curacy under a Nuclear-Chicago end-window gas-flow fuged a further three times in 80 % ethanol. The with a window. Radio- was suspended in 5 trichloroacetic acid counter fitted AMicromil pellet then % incu- (TCA) at 0 C for 15 minutes, centrifuged, and this activity in the solutions of protein from discs process repeated twice more followed by one extrac- bated with 14-C-leucine was (letermined by counting tion of the pellet with 80 % ethanol, one extraction duplicate aliquots (0.1 ml) plated onto glass with absolute ethanol, and twvo further extractions planchettes. OSBORNE-KINETIN EFFECT ON PROTEIN & NUCLEIC ACID METABOLISM 597 Table I Chlorophyll, Protein, & Nucleic Acid Content of Discs of Xanthium Leaf Blade Floated on Kinetin Solution *

Sample Chlorophyll Protein (P) RNA DNA P/RNA RNA/DNA O.D. mg Ag MAg 0 days 74 8.42 263 85 0.032 3.10 1 day C 62 6.99 225 75 0.031 3.00 K 69 7.73 238 72 0.032 3.31 2 days C 36 5.54 169 64 0.032 2.65 K 58 7.18 213 72 0.033 2.96 3 days C 25 4.25 134 58 0.031 2.32 K 50 4.73 161 62 0.029 2.60 4 days C ...... K 35 4.64 149 60 0.031 2.48 SD 2.8 0.25 14.3 5.4 0.002 0.19 * 40 mg/liter (K) or Water (C) Incubated in darkness at 24 C.

Experimental Results the conditions described earlier, by which time the water-treated parts had yellowed while those parts - Effect of Kinetin Upon Levels of Protein & treated with kinetin were still green. Each dumbell Nucleic Acids. The changes in the levels of total was then cut into its two subtending discs and the protein, RNA, DNA, and chlorophyll were deter- strip of tissue connecting the two was discarded. mined in control leaf discs and discs which were Each group of discs was analyzed separately (sum- treated with kinetin during a period of senescence extending over 4 days. Suitably randomized discs were floated upon solutions of either kinetin (40 mg/l) or water, which were changed daily. At daily intervals triplicate samples of discs were analyzed. The results presented in table I and figure 1, show the continuous falls in the levels of protein and nucleic acid which are associated with the yellowing and loss of chlorophyll symptomatic of senescence. In the presence of kinetin the fall in these levels is initially slower, and senescence is retarded though not pre- vented. The ratio of total protein to total RNA ap- proaches a constant value for all samples, and is not affected by kinetin. In contrast, the ratio of RNA to DNA falls as senescence progresses, and this fall is retarded in the presence of kinetin. Similar conclusions may be drawn for the relationship of RNA content to protein and DNA levels in the data presented in table II. This experiment was designed to determine if the speed and pattern of senescence in the individually treated kinetin and control tissues was mutually altered if the two treatments were arranged in adjoining portions of blade. Accordingly, dumbell-shaped pieces of tissue were cut from the interveinal areas of the leaves in such a way that each pair of discs was connected by a nar- row strip of tissue 2 mm wide and 3 mm long. Either water or kinetin solution (0.2 ml) (40 mg/l) was applied uniformly to the upper surface of the discs so that either both discs of each dumbell were treated - DAYS with water (H20)-(H20), or both discs were treat- Fig. 1. Protein, RNA, DNA, and chlorophyll con- ed with kinetin (K)-(K), or one of each pair was tent of discs of Xanthium leaf blade floated on solutions treated with water and the other with kinetin (H9O) - of kinetin, 40 mg/liter (K) or water (C). See table I (K). The discs were kept for 3 days on glass under for standard deviations. 598 PLANT PHYSIOLOGY Table II Chlorophyll, Protein, & Nucleic Acid Content of Joined Discs of Xanthium Leaf Blade * Chlorophyll Protein (P) RNA DNA P/RNA RNA/DNA Sample O.D. mg PLg lg 0 days 67 6.90 266 105 0.025 2.53 3 days 23 5.07 205 95 0.024 2.16 (H,O) - (H20) 59 6.74 271 96 0.024 2.82 (K) - (K)

C)3 2.32 (H20) - 21 5.02 200 86 0.025

-(K) 6.89 288 100 0.023 2.88 SD 0.002 0.18 * In which either one or both the discs were treated with kinetin solution, 40 mg/l (K) or water (C) Incubated in darkness at 24 C for 3 days.

marized in table II). It is evident fronm the data kinetin, leaving the opposite half as a control. At that in this kind of experiment there is no measurable daily intervals, the incorporation of labelled leucine influence of either a kinetin-treated or a control area and orotic acid into the protein and nucleic acid frac- upon either the speed or pattern of senescence of the tions, respectively, was compared in the two halves. other. The retardation of senescence in a treated At the start of the experiment, one disc was cut from part is apparently due only to the presence of kinetin, either side of the main vein of each leaf and duplicate and is not enhanced by any metabolites which may sets of these discs were incubated in darkness at 24 C move from a control to a treated tissue under the in- for 6 hours with either labelled leucine or orotic acid. fluence of the kinetin. This is referred to as the 0 day sample in table III. 0 Effect of Kinetin Upon Incorporation of 14-C- One-half of the upper surface of each leaf was then Leucine Into Protein & 14-C-Orotic Acid Into painted with either an aqueous solution of kinetin Nucleic Acids. (40 mg/1) or with distilled water, andl thenceforth I. Kinetin treatmizents cn whole leaves. In these the leaves were kept in darkness. On the next. and experiments one-half of each leaf Nwas treated with the subsequent 2 days, two discs were cut froim both

Table III Incorporation of 14-C-Leucine Into Protein & of 14-C-Orotic Acid Into RNA in Discs of Xanthium Leaf Blade Cut From Kinetin-treated (K) & Control (C) Halves of Detached Leaves * Total P cts. RNA Days after Protein (P) RNA DNA Total P mg X 10-2 treatment mg Total cts. Sp. Act. ,Lg Total Sp-Act. lag Total RNA ,g Total RNA ,ug DNA X 10-2 X 10-2 cts. 0 day 4.63 343.2 74.2 168 3,035 18.1 91 0.027 2.04 1.85 1 day C 3.92 293.0 74.7 134 2,595 19.5 79 0.029 2.18 1.69 K 5.11 510.4 99.9 183 4,935 27.0 99 0.027 2.79 1.85 2 days C 3.13 204.6 65.3 120 1,725 14.3 i2 0.026 1.71 1.66 K 5.01 476.2 95.0 201 3,580 17.9 88 0.025 2.36 2.28 3 days C 2.28 91.0 39.9 75 879 11.7 48 0.030 1.21 1.56 K 4.90 379.8 77.5 203 2,850 14.0 92 0.024 1.87 2.21 SD 6.28 1.05 0.10 * Incubation period 6 hours. OSBORNE-KINETIN EFFECT ON PROTEIN & NUCLEIC ACID METABOLISM 599 the kinetin-treated and control half of each leaf and also precludes the possibility of effects due to bac- duplicate sets of the discs from the two treatments terial contaminations. were incubated as before with leucine or orotic acid. Calculation of the ratio of total protein to total The leaves were selected for their uniform green RNA gives a value for all samples which is little color at the start of the experiment; by the 3rd day, affected by the kinetin treatment. However, the in- the control halves were markedly yellow while the corporation of labelled precursor into protein/unit color of the kinetin-treated halves appeared un- RNA falls strikingly during the senescence of both changed. the controls and the tissues treated with kinetin, al- The results of the analyses of total protein and though initially it is considerably increased by kinetin RNA are shown in table III, together with the above the original values. values for the incorporation of the labelled precursors A fall in the ratio of total RNA to total DNA into these fractions expressed both as total counts is again observed in the control tissue which is un- and as specific activities. During the experiment, dergoing rapid senescence, (cf. tables I & II) but the levels of total protein and RNA in the control in the kinetin treatments, where senescence is re- halves of the blades fell to less than half of the orig- tarded, the value rises above that in the initial inal values. In the kinetin-treated halves there is by samples. In whole leaves, therefore, kinetin treat- contrast a significant increase in the level of both ment results in a net increase in the level of both protein and RNA and this is coupled initially with a RNA and protein per unit DNA. If the values for higher net incorporation of labelled material into these incorporation of leucine and orotic acid are a true fractions (higher specific activities) compared with reflection of synthesis, then kinetin would appear to the original values. It is also seen that half the DNA stimulate the synthesis of both protein and RNA. is lost from the controls during senescence, while the II. Treatment of leaf discs with kinetin. The level remains unchanged following treatment with pattern of incorporation of 14-C-leucine into protein, kinetin. Counts of the radioactivity in the DNA do and 14-C-orotic acid into RNA, was determined in not differ significantly from background, showing discs of Xanthium leaf which were treated with either that little or no synthesis of DNA had occurred dur- kinetin or water after they had been punched from a ing the experiment. This result accords with the leaf blade. The initial values for incorporation were normal absence of cell division in mature leaves, but obtained from samples of discs 1.4 cm in diameter.

Table IV Incorporation of 14-C-Leucine Into Protein & of 14-C-Orotic Acid Into RNA Into Discs Cut From Xanthium Leaves * Total Cts. Incuba- Protein (P) RNA DNA Total P mg P X 10-2 RNA tion SampleSape time, mg Total Sp.Act. ,g Total Sp.Act. jg Total RNA Total RNA DNA hr Avg cts. X 10-2 Avg cts. //Ag /Ag X 102- 0 day 1 126.6 18.0 208 0.86 0.51 2 296.6 42.0 672 2.57 1.20 6.89 247 108 0.027 2.28 4 605.0 88.0 1,780 7.45 2.45 1 day 1 C 84.8 13.7 158 0.75 0.40 K 264.6 37.3 202 0.75 1.02 C 6.29 C 211 C 97 0.029 2.17 2 C 202.0 33.0 382 1.85 0.96 K 496.4 70.2 676 2.68 1.91 K 7.05 K 260 K 106 0.027 2.44 4 C 502.2 77.2 1,450 6.70 2.38 K 921.8 132.0 1,990 7.70 3.55 2 days 1 C 63.4 11.4 136 0.61 0.29 K 200.0 27.3 154 0.56 0.74 C 5.69 C 218 C 96 0.026 2.28 2 C 197.2 32.6 475 2.17 0.90 K 425.8 61.5 630 2.30 1.57 K 6.81 K 271 K 109 0.025 2.49 4 C 434.8 79.2 1.490 6.90 1.99 K 721.2 116.5 1,995 7.32 2.66 SD 1.32 0.13 0.11 * Discs pre-treated with either kinetin, 40 mg/l (K) or water (C) for 0, 1, or 2 days. 600 PLANT PHYSIOLOGY These were cut directly fronm a batch of suitable aged not pre-treated with kinetini but were incubated leaves an(l incubated with leucine or orotic acid for directly for 4 hours in solutions of labelled leucine periods of 1, 2, andl 4 hours. Numbers of larger or orotic acid with or without the addition of kinetin discs, 1.6 clm in (liameter, were also cut from the (25 mg/1) to the medlium. The presence of kinetin same samlple of leaves andl dividled into two com- increased the incorporation of labelled precursor into parable groups. To one group, 0.02 ml of distilled both protein and RNA and the specific activities of water was applied uniformly to the upper surface; both are higher when kinetin is presenit (table V). the other group was treated in a similar way with Furthermore, the total incorporation of leucine per solution (40 ). WVhen the unit RNA is stimulated by kinetin (luring the 4- 0.02 nml of kinetin mg/1 w surface was dry, the discs were arranged in petri hour-period. It ould appear therefore, that the (lislies andl store(d in (larkness at 24 C as (lescribed increase in the rate of incorporation of labelle(d in- before. After 1, an(d again after 2 (lays, batches of termediate into protein anid RNA is rapidly. and discs -were selected fronm each group. and a snmaller- probably directly, stimulate(d by kinetin, and( is likely sized (lisc, 1.4 cm in diameter, was punche(d from to be a true reflection of an increasedl synthesis of each one, thus remov-ing the (lamlage(l cells at the protein and RNA. former cut edges. Duplicate sets of kinetin andl control (liscs were then incubatedl with either leucine or orotic acid for 1, 2, andl 4 hours as before. (table Discussion IV). The (lecrease in total protein. RNA, ancd DNA It is evident froml the results that kinetiln retards during senescence of the controls, and the retardation the processes of senescence not only in whole leaves of these changes in the presence of kinetin. bear out but also in isolate(d discs of leaf bla(le. In this the general conclusions reached in the experiments respect therefore, the action of kinetin miust be (lirect alrea(ly (lescribecd. Again, the ratio of total protein an(l is not dependent uponl the utilization or accumula- to total RNA is approximlately constant, witlh or tion of nmaterials or mletabolites which may moxve into witlhout kinetiln. Furthermiiore, a comparisoni of the the kinetin-treate(d cells froml other untreated parts data in tables III aln(d IV shoxx s that the values for of the leaf. In experiments xvitlh leaf discs the effect the incorporation of 14-C-leucine into protein per of kinetin is not enhanced by the presence of anl ad- unit RNA are, in all cases, considerably higher in joining untreated portion of leaf blade. It is never- the presence of kinetin. These data, together with theless possible that the retardation of senescence by the evidence of the higlher specific activities of the kinetin is prolonged if movement of metabolites into protein and RNA following treatment writh kinetin the treated parts takes place as, for instanice, when suggest that kinetin stimulaltes the rates of synthesis whole leaves are treated locallv. But it is clear that of both RNA and proteini. this is not the prinmary action of kinetin in retarding Direct incitbation of le(af discs with kinetini. In senescence in Xanthium. the incorporation experinments (lescribed so far, the Certain changes in the tissue may be correlate-l tissues wvere pre-treated withl water or kinetin for a \vith the progress of senescence. These include a fall minimum perio(I of 24 hours before incubation with in the level of extractable chlorophyll, a (lecrease in labelled material andI it mlay be implied first, that the level of nucleic acid, both RNA and DNA, and the action of kinetin in stimulating incorporation is a reduction in the total content of protein. Treat- a secondary effect resulting from relatively long term ment with kinetin, however, results in a considerable, changes which are induce(d in the metabolism of the although not a total retention of chloroplhyll; a re- kinetin-treated cells. Second, the falling values for tention, and in certain cases, a small net synthesis incorporation in the control discs might be attributed of protein and RNA and a retention, but no meas- to a (lilution of the applie(l isotope by the endogenous urable synthesis, of DNA. These retentions induced pools of amino-aci(ds an(d nucleic acid precursors by kinetin are temporary and are later followed by wlhiclh normally increase (lurinig cellular senescence. decreases in the levels of chlorophyll, nucleic acid, In the following experiment, therefore., the discs were an(l protein which are symptomatic of senescent cells. Table V Incorporation of 14-C-Leucine Into Protein & of 14-C-Orotic Acid Into RNA by Discs of Xanthium Leaf Blade * ota TV rng- IT ot I1.s'. IDr iu- -2 Protein (P) RNA Total P mg 'I'otal (As P X 1U--., Sample mg Total counts Sp.Act. /.g Total Sp.Act. Total RNA ,ug Total RNA Lg X 10-2 X 102 counts 432.2 57.7 275 1,600 5.82 0.027 1.57 Control 7.48 (-+0.02) (+-0.5) 1.95 Kinetin 7.06 523.6 74.2 269 1,830 6.80 0.026 (4-2.6) ( _0.18) * During a 4 hour period of incubation, with and wvithout the addlition of kinetin at 25 mg, liter to the nmediumn. OSBORNE-KINETIN EFFECT ON PROTEIN & NUCLEIC ACID METABOLISM 601 Where the chemical determinations have both It is of interest to compare the present results on been made on the same samples of discs (tables I & Xanthium leaf tissues with those obtained by other II), the relationship between the total protein and workers on tobacco. Using excised leaf discs of total RNA in both control and kinetin-treated tissues Nicotiana rustica, Wollgiehn (15) has shown that in various stages of senescence is constant, and this kinetin enhances the incorporation of 14-C-glycine is not surprising in view of the now well-established into protein and of P3204 into RNA, provided the role of RNA in protein synthesis. This fact, and leaves are sprayed with kinetin at least 24 hours be- the fact that the ratio of RNA (or protein) to DNA fore discs are incubated in solutions of the labelled falls as senescence progresses, suggests that the de- precursors. No stimulation of incorporation of cline in protein level is due not to the potential for P3204 into RNA was apparent, however, if incorporation of amino acids discs into protein becoming were cut from unsprayed leaves and incubated direct- impaired during senescence, but rather it is due ly with kinetin (0.003-30 mg/I) and to a reduction in the number of effective sites where Na,HP3204 for amino acids are so assembled. The addition of 8 hours. kinetin maintains the RNA(or protein)/DNA ratio The present author has also found no measurable and in certain conditions increases it. One inter- stimulation of incorporation of labelled precursors pretation of these data is that a primary action of into protein or RNA when leaf discs of Nicotiana kinetin might be to regulate RNA synthesis with a ruistica are incubated in media containing kinetin and resultant regulation of one or more of the steps precursor for periods extending to 7 hours, although leading to protein synthesis. significant stimulations can be measured in Xanthium The results for the incorporation of 14-C-orotic leaf discs within this time. (table V). acid into the kinetin-treated parts of whole leaves This evidence from Xanthium and Nicotiana, to- and isolated discs, seemingly could support this in- gether with that from Prunus (9) and Phaseolus (3), terpretation. The incorporation of precursor into suggests that there is considerabl variation in the RNA declines during senescence, but in the presence biochemical responses of different plant species to of kinetin, the original value is maintained (table kinins, and it seems not unlikely that natural kinins IV) or surpassed (tables III & V). The RNA iso- will prove to be equally species-specific in their lated from the kinetin-treated parts has a higher action. specific activity than the controls both when the The results of the tissue is pre-treated with kinetin (tables III & IV) present investigation point to or when the kinetin is added at the same time as an impairment of nucleic acid synthesis as a possible orotic acid (table V). These values for the incor- prinmary feature of cellular senescence and a cause poration of labelled orotic acid into RNA, and the of net protein loss in Xanthium leaves. The role corresponding specific activities, imply that the syn- of the kinin, kinetin, in retarding senescence in this thesis of RNA is stimulated by kinetin. species would appear to be closely linked to its ability The parallel experiments with 14-C-leucine to maintain the synthesis of RNA. (tables III, IV, & V) demonstrate that the specific activity of the protein is also significantly higher following treatment with kinetin. The incorporation Summary of leucine into protein per unit RNA is considerably greater in the presence of kinetin, though the protein/ RNA ratio remains constant (cf. tables I & II). A decline in levels of protein, RNA, DNA, and This suggests that an increase in the synthesis of chlorophyll mark the progress of senescence in de- protein reflects the stimulation of RNA synthesis. tached Xanthium leaves. It is possible that the higher specific activities of Addition of kinetin to detached leaves and to the protein and RNA, which are maintained in the excised discs temporarily arrests the senescent presence of kinetin, could be ascribed to an increase changes and maintains a relatively high ratio of in the synthesis of particular fractions of protein RNA (or protein) to DNA. The kinetin effect ap- and RNA. It is possible also that abnormal types pears to operate directly and is not dependent upon of of protein and RNA may be included. Results the accumulation of metabolites from untreated experiments on the metabolism of another kinin tissues. (benzyladenine) have indicated that hydrolysates of Incorporation of 14-C-leucine into protein and RNA from treated leaf discs of Xanthium (but not 14-C-orotic acid into RNA is increased by kinetin of Phaseolus), contain small quantities of a substance which has chromatographic characteristics similar to indicating a stimulation of both RNA and protein that of benzyladenylic acid (3). In the absence of synthesis. more direct evidence for the presence in Xanthium It is suggested that the effect of kinetin in retard- of an RNA of abnormal composition induced by ing senescence in Xanthium leaf cells is mediated kinins, these suggestions must remain largely spec- through its action in sustaining nucleic acid and ulative. protein synthesis. 602 PLANT PHYSIOLOGY Acknowledgments 7. MOTHES, K. & L. ENGELBRECHT. 1961. Kinetin- induced directed transport of substances in excised The author is indebted to Professor James Bonner leaves in the dark. Phytochemistry 1: 58-62. for his advice and counsel during the investigation. She 8. OSBORNE, D. J. 1959. Control of leaf senescence wishes also to acknowledge the assistance of Dr. Maria Marquinez, Miss Sally Madge, and Mr. Alan Huber and by auxins. Nature 183: 1459-1460. to thank Prof. G. E. Blackman of the Department of 9. OSBORNE, D. J. & M. HALLAWAY. 1960. Auxin Agriculture, Oxford, for his interest and encouragement. control of protein levels in detached autumn leaves. Nature 188: 240-241. Literature Cited 10. PERSON, C., D. J. SAMIBORSKI, & F. R. FORSYTH. 1957. Effect of benzimidazole on detached wheat 1. CHIBNALL, A. C. & G. H. \WILTSHIRE. 1954. A leaves. Nature 180: 1294-5. study with isotopic nitrogen of protein metabolism 11. RACUSEN, D. W. & S. ARANOFF. 1954. Metabolism in detached runner bean leaves. New Phytol. 53: of soybean leaves. VI. Exploratory studies in 38-43. protein metabolisnm. Arch. Biochem. Biophys. 51: 2. GREGORY, F. G. & B. SAMIAN. TARAI. 1950. Factors 68-78. concerned in rooting responses of isolated leaves. 12. RICHMOND, A. E. & A. LANG. 1957. Effect of J. Exptl. Botany 1: 159-193. kinetin on protein content & survival of detached 3. MCCALLA, D. M., D. J. MORRE, & D. J. OSBORNE. Xanthium leaves. Science 125: 650-1. 1962. Metabolism of a kinin, benzyladenine. 13. ROGERS, B. J. 1955. Incorporation of radioactive Biochim. Biophys. Acta. 55: 522-528. acetate & sucrose into amino acids & protein of 4. MOTHES, K. & L. ENGELBRECHT. 1956. tber den excised organs of red kidney bean. Plant Physiol. Stickstoffumsatz in Blattstecklingen. Flora 143: 30: 377-379. 428-472. 14. NVICKERY, H. B., G. W. PUCHER, A. J. WAKEMAN, 5. MOTHES, K. & L. ENGELBRECHT. 1959. The role & C. S. LEAVENWORTH. 1946. Chemical investi- of kinetin in accumulation processes of excised gationis of the metabolism of plants. I. The nitro- leaves. Proc. IX Internatl. Botain. Congress, gen nutrition of Narcissius pocticlls. Bull. Conn. Montreal. 273. Agric. Expt. Sta. No. 496. 6. MOTHES, K., L. ENGELBRECHT, & H. R. SCHUTTE. 15. WOLlGIE.HN, R. 1961. Untersuchungen iiber den 1961. tTber den Akkumulation von a-aminoiso- Einfliiss des Kinetins auf den Nucleinsauire und buttersauire im Blattgewebe unter dem Einfliiss von Proteinstoffwechsel isolierter Blatter. Flora 151: Kinetin. Physiol. Plantarum 14: 72-76. 41 1-437.