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.