senescence: action of auxin and kinetin in control of ... · pole beans (phlascoluts zulqaris l.)...

Plant Physiol. (1967) 412. 1334-1342

Senescence: Action of Auxin and Kinetin in Control of RNA andProtein Synthesis in Subcellular Fractions of Bean Endocarpl

Joseph A. SacherDepartment of Botany, California State College at Los Angeles, Los Angeles, California 90032

Received April 24, 1967.

Sionnnary. A comiparative study was made of the efffects of auxin (a-naphthaleneacetic acid), kinetin (6-furfurylaminopurine) and a mixture of auxin and kinetin appliedin vivo on synthesis okf RNA and protein and the distribution of such synthesis am'ongstthe subcellular fractions of sections of endocarp from Kentucky Wonder pole beans(Phascolus vulgaris, L.). Auxin caused considerable enhancement of incorporation oflabeledl precursors into RNA and protein of all suibcellular fractions, and induced netsynthesis of RNA and protein. That auxin-induced net synthesis of protein is repressedby actinomycin D indicates that auxin acts primarily to stimulate synthesis of RNA. asa result of which synthesis of protein tis enhanced. The effect of kinetin alone onsynthesis of RNA, or of kinetin on auxin-induced synthesis of RNA was variable, witheither stimulation or inhibition observed in different experiments. Kinetin-enhancementof synthesis of both RNA and protein in subcellular fractions also varied, with enhance-ment of synthesis in 1 or all subcellular fractions amnong different experimenits. Thevariable effect of kinetin did not seem to be related to the amount of endogenous oradded auxin. The mlode of action of kine-tin is discussed.

Kinetin has a small but significant effect in retard-ing the rate of degradation df RNA in excised sectionsof bean endocarp during a 24-h>our period (13). Forexcised leaves or leaf sections kinetin delays senes-cence and stimulates synthlesis of nucleic acids and/orprotein (5, 6, 8, 17, 20), but its site of action has notbeen elucidated. It has been shown that kinetin getsincorporated unaltered into both RNA and DNA ofLei?nWa miinor (19) and that the effect of kinetin ongrowuth of cultures of tobacco pith tissue is correlatedwith its iincorporation into tran.sfer RNA (2). Fur-ther, extracts of serine transfer RNA from yeast,liver and Esclerichia coli have cytokinin activity inthe tobacco callus assay (16).

For bean endocarp tissue sections evidence hasbeen presented (11, 12, 13) that the primary actionof atuxin in preventing senescence is on the synthesisof RNA. Following are reported the resullts offurther stutdies of the effect of auxin, kinetin andmixttures of auxin and kinetin on synthesis of RNAandl protjin in bean endocarp tissue sections.

Materials and Methods

Illcitb(atio)l Procedu res. For some experiments(table I) seg-mlenits of the pods of KentuckyvW:onder

1 This investigation was suppor-ted by National Sci-ence Foulnidationl Grant GB-4088.

pole beans (Phlascoluts zulqaris L.) al)out 1.5 cm11 inlength were prepared anid aged as d;e-crih)ed previously(9, 11) in water or solutions containiing auxin(a-naphthalene acetic acid, \ A) aind/or kinetin(6-furfurylaminopurine) for 24 or 48 hours. Repli-cates (1.5 g fr wt) were removed from b)oth fresh(control) and aged segments, washed and( assayedfor RNA and protein as describedl following. Othlerreplicates of fresh andlaged endocarp were dried invacutum over concentrated sulfuric acid' to ascertainchanges in fresh wxeight (luring aging oNving to uptakeof wN-ater.

For other experiments 1.5 or 2.0 g fresh weightrep!1icates of 1.5 mnm thick sectionis of endlocarp wereprepare(ld, washled, blotted aintl incuibated at 29° insolutioins of 25 jg/ml streptomyvcin l)buffered to pH6.1 with phosp1hate bufifer. The solutions ve\re duial-labeled Wvith L-leucine-L-Tl4C ( 230 ic/mm ) orl l-Ieu-cine-U-Tr (2.3 C/nma) for assay of the rate ofprotein synthesis, and orotic-6-14C acid (34 milc/mm)from Volk Radiochemical Company. Burbank, Cali-fornia, or uridine-5-T ( 30 C/(im1) fromii NuclearClhicago Corporation, De. Plaines, Iliinois, for assayof RN.Ak synthesis. NAA, kinetini or actinomvcin Dwere adided to the solutionis as in(licatedl in the legendsto the tables. After incuLbationi tlle t ssu2 sectioniswere washed for 1 hour in runn:ng tap water toremove all traces of radioactivity f rolmi the freespace, blotted and placed into 4 volumes of 95 %ethanol, or frozen witl dry ice, priparat ry to grilld-ing, extractioon anid assay.

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SACHER-KINETIN AND AUXIN ACTION ON RNA AND PROTEIN SYNTHESIS

Analytical Procedure. For assay of total RNAand protein the tissue was ground in 4 volumes of95 % ethanol using a Potter-Elvehjem homogenizer,centrifuged and the supernatant fraction decantedand saved for radioassay. Using a modification ofa previously described method (6) the 'precipitate was

extracted su-ccessively twice each with 80 % ethanol(v/v), a solution of cold 5 % trichloroacetic acidand once each with 95 % ethanol, absolute ethanol,absolute ethanol-ether (1:1 v/v) and ether. Thefirst ethanol washing contained unlabeled solutes to0.0025 M each of the kinds used in the precedinglabeling experinment. The final' precipitate was dried,extracted with 0.3 N KOH at 370 for 18 hours,centrifuged and the supernatant fraction decantedand a;liquots taken for assay of protein by the methodof Lowry et al. (4), followed by acidification with10 % perchloric acid to pH 2 and chilling, for cen-

trifugal precipitation of lprotein, DNA and KCl'04.This final supernatant fraction was assayed at 260m,u with a Bausch and Lomlb model 505 recordingspectrophototmeter using a standlard curve preparedby use of a hydrolyzed preparation of purified yeastRNA for converting optical densities to jug of ribosenucleotides.

For assay of RNA and protein in subcellularfractions tissue sections were frozen with dry ice,powdered in a cold mortar, followed by addition of a

solution of 0.25 M sucrose, 0.002 M CaCIL (or 0.0015M MgCl) anid 0.025 M tris buffer, pH 7 for transferof the powdered tissue into centrifuge tubes. Thehomogenate was centrifuged at 20 at 30,000 X g for30 minutes to yield the heavy (fraction 1) precipi-tate; the supernatant fraction was centrifuged at110,000 X g for 80 minutes to yield the ribosomal(fraction II) 'precipitate. To the latter supernatantfraction 3 volumes of absolute ethanoll were addedfolqowed by chilling overnight and centrifugation at10,000 X g to yield a precipitate of -the soluble(fraction III) RNA and protein. The 3 precipitate-

fractions were extracted and assayed for RNA andprotein as described above.

Intact ribosomes may be isolated after similarfreezing and grinding procedures (3). The RNA/protein ratio of about 0.64 for the ribosomal fractionand the proportion of total RNA in this fraction(ca. 0.40) is similar to values reported (18) forplant tissues. Tissue frozen on dry ice powdered so

finelv in a mortar that it was sometimes not possibleto get a firm,precipitate with centri'fugation at3000 X g. Therefore in most experiments the initialcentrifugation was at 30,000 X g.

Radioassay. Assays of the rate of synthesis ofRNA and ,protein were based on the ratio of dpmincorporated/dpm total uptake (dpm incorporatedplus dpm in the ethandl soluble fraction). The accu-

racy of this method of assay has been reported pre-

viously (13). Aliquots of the ethanol soluble fractionwere counted in 10 mt volumes of a p-dioxane-ethyleneglycol-naphthalene solvent fluor system described byBray (1). Aliquots of the KOH-extracts were

counted in 10 ml volumes of a toluene solvent-fluor(4 g PPO and 50 mg POPOP/1 scointillation gradetoluene) system with addi'tion as needed of a solu-bilizer (Nuclear Chicago Corp. N'CS, a quaternaryammonium base in toluene). Initial counting ratesof tritium were carefully checked to make certainthere was no contribution from chemiluminescence.

Results

Effect of Kinetin and Autxin on Synthesis ofRNA and Protein. From the data in table I it maybe seen that during a 24-hour incubation of tissuesegments auxin caused an increase in the level oftotal RNA (see also 13), while from 15 to 22 % ofthe RNA underwent degradation in 'presence ofkinetin. Auxin enhanced substantially the net syn-thesis of total protein (ifrom 15-34 %), most of whichoccurred in the ribosomal and soluble (R-S) protein

Table I. Effect of Atxi7t and Kinetin on Level of RNA and Protein during Aging over a 24- or 48-hour PeriodDuplicate 1.5 g fr wt samples of endocarp were removed from fresh bean pod segments and from segments aged

24 or 48 hours in petri dishes in presence of water or solutions of NAA, kinetin or auxin plus kinetin, and assayedfor RNA and protein. NAA was 25 ,ug/ml; kinetin was 10 ,g/ml. Assays of aged tissue were corrected for increasein fr wt dtue to water uptake.

R-S proteini = ribosomal plus soluble protein (precipitate from 30,000 X g supernatant fraction of a tissue ho-mogenate by adding 1 volume of cold 14 % triclhloroacetic acid).

Expt I Expt II Expt IIIaged 24 hrs aged 24 hrs aged 48 hrs

Total Total Total Total RNAProtein (mg) RNA Protein (mg) RNA protein (% of

(% of (% of (mg) control)Treatimeiit Total R-S control) Total R-S control)

1. Zero time control 4.93±-0.20 1.67 100±-3 3.85±-0.15 1.28 100±-0.0 5.69±-0.06 100±4-5.72. NAA 5.64±0.12 2.91 108+4.3 5.16±0.12 2.85 112 ... 6.27±0.04 95±1.43. NAA + kinetin 5.36±0.06 2.31 100±0 4.7240.12 1.93 100±3.0 6.25 ±0.23 90±4.64. Kinetin 4.76±0.07 2.20 85 ±4.5 4.10±0.26 1.61 78±4.8 5.0&:0.08 66±4.05. H,O 3.90±0.0 1.58 71±2.8 3.55±0.23 1.38 64±3.5 4.86±0.20 63±4.9

1335

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Table 11. Effect of Auxin, Kinetin anid Actinzomycin D on the Rate of RNA and Protein Synthesis by BeantEndocarp Dutal-labeled with Orotic-6-'4C and L-leucine-U-T

Duplicate 400 mg fr wt samples of tissue incubated 20 hr in 1.0 ml solutions buffered to pH 6.1, containing25 Ag/ml streptomycin, 1.2 uc orotic-6-14C acid and 3 gc L-leucine-U-T. NAA was 5 ,ug/ml, kinetin 10 ,ug/ml,actinomycin D 10 Ag/ml.

Effect of NAA,Ratio of* Kinetin or Act. D

dpm incorporated on ratio Protein* RNA*dpm total uptake Orotic as % of

Treatment Leucine Orotic Acid Leucine Acid 0 time controlsSE SE SE SE

1. None (0 time)2. H,O aged3. H90 + Act. D4. NAA5. NAA + Act. D6. Kinetin

0.712±0.0050.619±0.0360.807±0.0070.569±0.0400.782±0.008

0.552 ±0.0150.290±0.0150.751 ±0.0220.244+0.0270.389±0.018

-13 %+13 %-30 %+11 %

-4'±3i--61-31

... 100±4.2

... 100-+-0.8W7 % 89-4-3.96 % 117±0.48 % 101±5.4,0 % 115±2.0

100+± 1.889±40.071+±3.6106±0.485 ...

89± 1.2* Means and standard errors of duplicate samples of tissue.

fractioni; auxin induced a 74 to 122 % net synthesisof ths fraction.

Olf interest is the effect of kinetin on proteinsynthesis as, notwithstanding the occurnence of a lossof 15 to 22 % of total RNA, kinetin enhanced netsynthesis of R-S protein from 26 to 30 %. ALthoughthere was no significant difference iin the level oftotal protein between tissue aged in kinetin and thezero time control tissue, there was 15 to 22 % moreprotein in tissue aged in kinetin than in water.Finally, kinetin inhibited auxin-indticed synthesis ofR-S protein 21 to 32 % and total protein 5 to 9 %.Although in experiments I and II (table I) assaysfor R-S protein were made only with single samplesof tissue, the inhibitory dffect of kinetin on auxin-induced synthesis of R-S protein was essentially thesame in both exvperiments. During a 48-hour incuba-tion degradation of total RNA and protein occurredat about the same rate 1for tissue aged in water orkinetin (ca. 40 % for RNA and 12 % for protein),while auxin maintained the level of RNA and protein.

In a further experiment the effect of kinetin andauxin was assayed on the rate of incorporation oforotic acid and L-leucine and the level of total RNAand protein during an inicubation of tissue sectionsfor 20 hours. In order to provide for a comparisonof the mechanism of auxin- versus kinetin-inducedprotein synthesis, actinomycin D, a selective inhibitorof RNA synthesis (7), was used to repress aux.in-induced synthesis of RNA. Kinetin inhibited incor-poration of orotic acid 30 %, which was associatedwith an 11 % loss of RNA (table II). Despite thisdegradation of RNA kinetin enhanced incorporationof leucine 11 % and caused a 15 % increase in thele,vel of total protein. Auxin stimulated incorporationof orotic acid 36 % and leucine 13 % and caused anet synthesis of RNA (6 %) and protein (17 %).Actiinomycin D repressed auxin-induced synthesis ofRNA and, protein, as measured by inhibition of therate of incorporation of precursors and its effect in

causing, a decrease in the level of RNA and protein.Actinomvncin D caused reduction in RNA of about12 % as comnpared with the water- or auxin-controland caused a similar reduction in the level of protein.The results support the evidence from previousstudies (11,12, 13) that the primary action of auxinis on synthesis of RNA and that auxin-indtuced syn-thesis of protei.n is a consequence of enhancement ofRNA synthesis.

Assay of Effcct of Kinetin and Auxin on Synt-tlhcsis of RNA and P-roteini in Subcellular Fractions.During a short (4-hr) incubation of fresh tissuesections kinetin inhibited (19-43 %) incorporation oforotic acid into RNA of 3 subcellular (30,000 X g,110,000 X g and 110,000 X g supernatant) fractions(table III). Stimulation of incorporation of leucine(12 %) by kinetin occurred only in the soluble pro-tein ifraction.

The accuracy of assessments of the rate of syn-thesis of total RNA and protein based on measure-ments of the ratio of radioactivity incorporated/totaluptake of radioactivity' was demonstrated in a previousreport (13). The data in table III show that theuse of the ratio of dpm incorporated/dpm total uptakeprov,ides an accurate method also for assessing therate of synthesis in subcellular fractions, even whenthe amount of radioactivity incorporated is as low as2 % of the total uptake. Th-e statistical significanceoif the data in table III is shcown by the small size ofthe staindard errors of the means for duplicate samplesof tissue, averaging' -+ 2.18 % 'for all subcellularfractions and ± 1.07 % for total RNA and protein.The larger average standard error for the former isattributable to slight variability inherent in the com-bined grinding and difEferential centrifugation pro-cedures. The use of the ratio enables accuratemeasuremients of the rate of synthesis when totaluptake varies among duplicate samples, or whencomnparisons are made among experimental treatmentswhich may markedly affect the rate of uptake (13)

1336 PLANT PHYSIOLOGY




Table III. Effect of Kinetin on Rate of Substrate Incorporation in Subcellular Fractions by Bean EndocarpSections Dual-labeled wuith Orotic-6-14C Acid and L-leucine-U-T for 4 Hours

Duplicate 2.0 g fr wt samples of fresh endocarp sections were incubated for 4 hr in 5.0 ml solutions bufferedto pH 6.1 containing 25 /ig/ml streptomycin, 2.9 ,uc orotic-6-14C acid and 3.0 ,uc L-leucine-U-T. Kinetin was 10tLg/ml. Fraction I is the 30,000 X g precipitate of a tissue homogenate; Fraction II is the 110,000 X g precipitateof the 30,000 X g supernatant fraction; Fraction III is the precipitate from addition of 3 vol absolute ethanol tothe 110,000 X g supernatant fraction.

Ratio of dpm incorporated*

dpm total uptake Effect ofSubcellular Labeled H20 Kinetin kinetinfraction substrate (10 Atg/ml) on ratio

SE SEFraction I 14C orotic acid 0.0457±0.0002 0.0293±0.0003 -36 %

II " " " 0.0353±0.0003 0.0201±0.0005 -43 %"III " " " 0.0290±0.0015 0.0234±0.0015 -19 0,

Total (I, II, III) " " " 0.110 ±0.002 0.0728±0.0006 -33 /Fraction I 3H-leucine 0.360 +0.006 0.353 ±0.003 Qi

" II " " 0.0389±0.0016 0.0338±0.0 -13 %" III " " 0.389 ±0.015 0.435 ±+0.005 +12 %

Total (I, II, III) " " 0.788 ±+0.010 0.822 ± 0.002 + 4 %* Means and standard errors of duplicate samples of tissue.

In some experiments the use of the ratio has enableddemonstrating a 2 to 5 % stimulation of RNA orprotein synthesis in suibcellulhr fractions (e.g., tablesIII, IV).

Effect of Kinetin and Autxini on RNA and ProteinSynthesis dutring Long Incuibations. The effect ofkinetin and auxin on the levels of RNA and, proteinover a period of 24 hours is shown in table V andfigure 1. In this experiment assays were made alsoof the efifect of kinetin and auxin on incorporationof uridine and leucine (table IV).

The inhibitory effect of kinetin on incorporationof ur.idine-5-T into the 3 subcellular fractions andenhancement of leucine incorporation only in fractionIII (soltvlble protein) is very similar to the resultsobtained in a 4-hour incubation (conmpare tables IIIand IV), except that the enhancement of leurcineinconporation 'in frac.tion III protein is about twice

as great as it was durinig the sh,orter incubation.Auxin, in contrast, markedly stimulated the rate ofincorporation of labeled -precursors into the 3 sub-cellular fractions of both RNA and protein.

Consistent with the stimulating effect of auxinon incorporation of uridine is the substantial auxin-induced net synthesis of RNA in fraictions I and II

(table V). For fraction III there occurred a 32 %loss of RNA, but in the water-control. the loss ofRNA was 61 %. Thus the data on both the rate ofincorporat,ion of uridine and degradation df RNAindicate that the rate of turnover in fraction III ishighest among th,e suibcellular fractions. Auxin alsoinduced a net synthesis of protein in fractions I andII and maintained the level in fraction III.

Despite the inhibition of uridine incorporation bykinetin. there was slightly less degradation of RNAin tissue aged in presence of kinetin than in water-

Table IV. Effect of Auxin and Kinetin on the Rate of Incorporationl of L-leucine-U-14C and Uridine-5-T inSubcellular Fractions over a 24-houtr Period

Duplicate 1.5 g fr wt endocarp sections incubated 24 hours in 5.0 ml volumes of solutions buffered to pH 6.1with phosphate buffer containing 25 ,g/ml streptomycin and dual-labeled 3 ,uc uridine-5-T and 1.2 /JC L-leucine-U-14C.Subcellular fractions as in table III.

Ratio of dpm incorporated/dpm total uptake*

Subcellular Labeled NAA Effect Kinetin Effect offraction substrate H'0 10 ,g/ml of NAA 10 ug/ml kinetin

SE SE SEFractioni I Uridine 0.2035±0.0055 0.342 ±0.0050 +68 % 0.142 ±0.0015 -30 %

II " 0.111 ±0.010 0.190 ±0.016 +71 % 0.0630±0.0006 -43 %"III " 0.0363±0.0015 0.0687±0.0028 +89 % 0.0294±0.00015 -19 %

Total I, II, III " 0.351 ±0.017 0.601 ±0.00 +71 % 0.234 ±0.0020 -33 %Fraction 1 Leucine 0.344 ±0.00050 0.371 ±0.012 + 8 % 0.352 ±0.0033 + 2 %0

II " 0.0386±0.00015 0.0517±0.0016 +33 % 0.0343±0.0013 -11 ToIII " 0.275 ±0.0010 0.355 ±+0.009 +29 % 0.356 +0.015 +29 %

Total I, II, III " 0.657 ±0.0010 0.779 ±0.0010 +19 % 0.742 ±0.013 +13 %* Mean and standard errors for duplicate batches of tissue.

1337



PLANT PHYSIOLOGY

:.5 . .00 ,E11 ------------

13! Fraction I.w ~~Prot6inVr a II0 24 44

0.3

0.2

0.1

C

.3.5

3.0

CP

-a0

lb.

z4

0 24

Hours

FIG. 1. Effect of kinetin and auxin on level ofRNA and protein in 3 subcellular fractions, and thetotal of the 3 fractions. The values for 24 and 48 hourswere derived from somewhat different aging proceduresas described in footnote to table V (other data for thisexperiment shown in tables IV and V).

4I

aged tissue. Although degradation of RNA occurredin presence of kinetin there was a small amount ofnet -synthesis of protein in fract`on I, and moreprotein in fraction III -than in the water-aged -controls.

In this same experiment separate replEcates ofendocarp'sections were aged in Petri dishes for 48hours on f1ilter paper wetted with 5 ml of a solutionof 25 ug/ml stre.ptomycin plus or minus kinetin (10jug/ml), and then assayed for the amount of RNAand protein in the 3 subcellular fractions. Thereoccurred a large amount of degradation of RNA inpresence or absence of kinetin, although kinetin hada suibstantial ef,fect in preventing degradation ascompared with the water-aged tissue. Kinetin, how-ever, indticed a net svnthesis of protein in the heavyand riibosomal fractions. Degradlation occurred ofsoluble protein, but much less than in tissue aged inwater (table V and fig 1).

Effect of Kinietinz on RNA and Protein Synthesisafter Tissue Sections are Aged for 22 Hours inWater. Another phase of this series of experimentson the effect of .kinetin in replicates 'from 1 batch oftissue is illustrated in table VI. Four of the Tepli-cates of endocarip sections which' were aged in Petridishes on filter paper wetted with water were removedafter 22 hours, washed and incubated' fbr 4 hours insolutions dual-labeled with uridine and leucine inpresence and' absence of kinetin. Kinetin caused anincrease in the rate of incorporation of iboth labeledsubstrates, mostly in the rilbosomal and soluble 'frac-tions. Also, in these 2 fractions there was about30 % more 'RNA and protein in the tissue incuibatedin kinetin ior 4 hours than in tissue incubated inwater. The difference in the amount of RNA andprotein is -probably a composite of degradation inabsence and some net synthesis in presence of kinecin.These results indicate that there occurs a changeduring water-aging in some factor or condition whichaffects the response of the tisisue to ikinetin.

Interaction of A uxin and Kinetin on Synthesisof RNA and Protein int Subcellular Fractions. Thatvariability may occur in interaction among auxin andkinetin among different batches of beans is indicatedby the results following (table VII) as co.niparedwith those of experiment I and II in table I. Con-ditions of aging, however, were different in the 2experiments. In the experiments illustrated in table Ientire .pericarp segments (ca. 1.2 cm in length) wereaged in Petri dishes and the endocarp removed forassay at the end of 24 hours. In the experimentfollowing endocarp sections (1.5 mm thlick) wereprepared' and aged for 22 hours in dual-labeled solu-tion's in f'lasks in a shaker. During 22 hours agingauxin stimulated the rate of synthesis of RNA andprotein in all subcellular fractions except fraction Iprotein (table VII). Kinetin enhanced substantiallythe effect of auxin on the rate of incorporation ofleucine in all fractions, but kinetin-induced net syn-thesis of protein occurred only in the soluble fractionof protein. Also kinetin stimulated both the rate of

0

a.

NA,_̂__.X ~ KKnotin -------_sV.f.,- -;1

Fraction 11I

P~~~~~~froteina~~~

NAA __, I

I..., Kin'!tin

TotalProtein\

I

1338

2.0



SACHER-KINETIN AND AUXIN ACTION ON RNA AND PROTEIN SYNTHESIS 1339

Table V. Effect of AnAxini *nd Kiuieti,i oni Level of RNA and Protein in Snibcellular Fractionis fromii EndocarpSectionts Aged 24 and 48 Hours

Other data for this experiment showni in table IV and fig 1.

Level of RNA or protein expressed*as % of 0 time control

Subcellularfraction

I -RNAII - RNAIII - RNATotal I, II, III

I - ProteinII - ProteinIII - ProteinTotal I, II, III

Zero timepg RNAand

protein

155142140437

1404179

14223005

Expt I**aged 24 hrs

NAAH,O 10 pg/ml

85783971

1001137188

12612368110

122160100113

Expt II**aged 48 hrs

Kinetin Kinetin10 pg/ml H20 10 pAg/ml

98734678

107120***88

100

65532952

94854068

79905576

1091406791

* All values are the means of closely replicating duplicate samples of tissue. Average SE of the mean was±2.6 % of the mean value for protein and ±3.2 % for RNA.

** Experimienit I and II had same zero controls but in experiment I the tissue sections were aged in flasks in ashaker at 300, while in experiment II the tissue sections were aged at 250 in Petri dishes on filter paperswetted with water or a kinetin solution.

*** Differelnce from water-control no, significant at 5 % level.

incorporation of uridine and the net synthesis of RNAin the 3 subcellular fractions.

Effect of Kinetin Concentration on RNA andProteinz Switthesis. Duplicate samples of tissue wereinctubated for 22 hours in presence of 4 concentrationsof kinetin. Duplicate auxin- and water-4controls weresimilarlv incubated.

Tihe results (tables VIII and IX) showv that auxinenhanced greatly the incorporation of both substratesand caused an increase in RNA and protein in eachof the 4 sub,cellular fractions as compared with thewater-control.

Kinetin at 0.01 ,ug/ml had no effect. The higherconcentrations of kinetin enhanced inconporation ofuridine and leucine in the 5000 X g fraction. With,inthe range of concentrations of kinetin used the en-hancement of incorporation by kinetin increased withincrease in kinetin concentration. This enhancementwas more apparent on inconporation of uridine thanleucine. Only at 1 Ug/mli kinetIn was there somestimulation of incorporation of both precursors alsoin the ribosomal and soluble fractions.

The stimulat,ion of incorporation of uridine andleucine in the 5000 X g fraction was paralleled by a

Table V7I. Effect of Kinetini on RNA antd Protein Syntthesis After Aging of Eiidocarp Tissue Sections in Waterfor 22 Hours

After water-aging for 22 hours tissue sections were washed and incubated for 4 hours with or without kinetin.Incubation conditions and sulicellular fractions as in table III.

Ratio of*dpm incorporateddpm total uptake

KinetinH O 10 /g/ml0.03230.01030.01240.0551

0.3620.01970.2330.615

0.03760.01950.02450.0816

0.3800.02750.2920.700

Effect ofkinetinon ratio

+16%+89%+97%+47%

+ 5%+40%+25 %+14 %

,ug RNA and*protein

H.O Kii

1257972

276

ineti

12812494346

1601 1657189 247764 969

2554 2873

Effect ofkinetin on

level of RNAand proteinexpressed as% of water

controln

100157130125

100130127112

* Av-g SE of the means of ratio dpm incorporated/dpm total uptake was + 2.7 % of the mean value for subcellularfractions and + 1.0 % for the totals of the 3 fractions; for the amount of protein and RNA in subcellular frac-tions 2.1 % and + 3.1 % respectively.

Subcellularfractions

Labeledsubstrate

IIIIIITotal

IIIIIITotal

Uridine

Leucine,,



Table VII. Effcct of Aux.in and a Mixture of Auxin and Kinietini ont Syiitlicsis of RNA anld Proteinin Subcellular Fractionis

Duplicate 2.0 g fr wt of endocarp sections incubated 22 hours in 7.0 ml volumles of solutions buffered to pH 6.1containing 25 ,ug/ml streptomycin and dual-labeled with 3 uc uridiine-5-T and 1.2 Iuc L-leucine-L-14C. NAA andkinetin were 10lug/inl. Subcellular fraction as in table III.

Ratio dpm inicorporated/dpntotal uptake*

Subcellular Labeledfraction substrate

Fraction 1" II," III

Total I, II, III

Fraction I" II", III

Total I, II, III

Uridinc

Leucine,,

,,

,,

NAA +H20 NAA kinetin

0.201

0.1750.08730.463

0.348

0.04330.3680.760

0.2250.2380.1030.566

0.35400.05400.4080.812

0.2570.2930.1390.689

0.348

0.06110.4500.869

a Effect on

ratio

NA.NAA kine

+12% +28+36 % +67+18% +47+22 % +48

0 O

+25 % +41+11 % +22+ 7% +14

Level of RNA anid( protein*expresse(l as % of0 time controls

ZeroA + timiie 'NAA +tin ,ug* H,O NAA kinetin

16013959

358

1116193844

2153

RNA1007411693

Pn-otei11582121114

121104130116

132*:k126145137

132113159129

124126159138

* Means of closely replicating duplicate samples of tissue. The average of the stalndard errors of the means forduplicate samples, expressed as percent of the mean value, is -+- 1.6 % for the ratios dpm incorporated/dpmtotal uptake; for the amount of RNA and protein, ± 3.9 % for all RNA fractions and 4-+ 1.2 % for total RNA,+ 1.4 % for protein fractions and ± 0.7 % for total protein.

** Difference from water-control or NAA + K not siginificalnt at S % level.

large increase in the level of both RNA and proteinwith increasing concentration of kinetin. In 'fact,the higher concentrations of kinetin had a substan-tially greater effect than auxin in increasing theamount of RNA and protein.

Effect of Inhibitors oni the Action of Kinetin.A concentration of 15 /Agg/ml of actinomyvin Drepressed kinetin-induced stimulation of uridine in-coriporation albout 95 % and of leucine about 49 %in 3 subcellu'lar fractions. Similarly, in anotlher

experimlent DL P-fluorophenvlalanine ( X 1(-3 M)

inhi'bited comipletely kinetin-induced incorporation ofboth uridine and leucine. Therefore the results ofthese studies are of 11o value for determining whetherthe primary action of kinietin is on synthesiis of RNAor protein. This may be expected when usinlg suchinlhibitors in intact tissue, in view of the dependencyof protein synthesis oIn svtnthesis of RNA an(l therev erse. The resuilts imlav indicate, however, thatkinetin i!s acting to enlhaince sy nthesis rather thanpreventing breakldown of RNA and protein.

Table VIII. Effect of Kinctin Coicentratioi On Incorporation of Uiridiiie-5-7(T id -Lcncinc1C-i4C in Bcanl EndocarpTissue sections incubated 22 hours; conditions as in table VII.

Effect of NAA and kinetin on1 ratio d(pm incorporated/dpm total uptake*

Kinietin (.g/nml)Labeled H,0 NAA

Fraction substrate control 10 ,ug/ml 0.01 0.1 1 10

IIIIIIV

IIIIIIV

5000 X g30,000 X gRibosomalSolubleTotal

5000 X g30,000 X gRibosomalSolubleTotal

Uridine,,

Leucine,.9

to

I.,

,,9

0.1620.03640.1920.07400.4640.3140.02140.04070.3680.743

0.2760.07660.3220.1360.8110.3890.02680.06060.4370.913

+70

+110+68+84+75+24+25+49+19+23

0.1550.04050.1850.07020.4510.3150.02250.04010.3610.739

0

0

-30

0

0

0

0

0

0

0.2090.03120.1920.06570.4980.3560.01850.04280.3740.791

+29140

0

+ 8+13

0

0

0

+ 6

(0.2660.03860.2140.07610.5950.3540.02360.04550.3810.805

+650

+11+ 3+29+ 13

0

+15+ 3+ 8

0.3030.03900.1880.07250.6020.4060.02450.04370.3770.85 1

+870

0

0

+30

+29

0

0

0

+15

* Average standard error of the means expressed as percent of the mealn values was ± 1.04 % for RNA and+2.8 % for protein in subcellular fractions; ± 0.6 % for total RNA and -4- 1.5 % for total protein. All effectsnoted are significant at better than the 5 or 1 % level.

1340 PLANT PHYSIOLOGY

I%




Table IX. Effcct of Kinetin Concentration on RNA antd Protein Synthesis in Bean EntdocarpOther data for this experiment shown in table VI.

1341

RNA and protein expressed as percent** of water-controlH20 control (mean and SE of duplicate samples)

Subcellular ,ug RNA and NAA Kinetin (,g/ml)fraction protein (10 ,g/ml) 0.01 0.1 1.0 10.0

I 5000 X g RNA 130 ± 6 130 ± 2.9 100 119 + 2.5 139 ± 3.3 152 ± 2.5II 30,000 X g " 23 + 0.8 163 3.1 100 100 100 100III Ribosomal " 102 ± 0.2 142 ± 1.4 100 100 100 100I\N Soluble " 64 + 5 155 ± 1.0 100 100 100 100

Total " 320 ±12 140 + 0.7 100 100 112 ± 0.6 119 + 3.0

I 5000 X g Protein 1014 ± 2.6 143 ± 0.0 92 + 1.9 166 + 5.E, 160 + 3.7 192 ± 0.9II 30,000 X g " 125 ± 5 111 ± 0.0 100 100 100 100III Ribosomal " * * * * * *IV Soluble " 1066 ± 0.0 129 ± 2.9 100 100 100 100

* Protein assays failed owing to an unexplained development of turbidity during colorometric assays using theFolin-phenol reagent.

** All values of 100 indicate no difference from water-control at 5 % level.

Discussion

In earlier work (10) on hormonal control ofsenescence of excised bean endocarp tissue it was

observed that kinetin alone had no measurable efifectin retarding the rate of senescence of bean endocarptissue over a period of 3 to 7 days, while auxin main-tained the tissue in a healthy condition for up to 14days. The ipresent results indicate that the lack ofan eniduring ef'fect of kinetin in preventing senescencewas probably attributable to the degradation of RNAin presence of kinetin.

In presence of auxin there is no requirement foradded kinetin. Auxin enhances incorporation of pre-cursors into RNA and protein in all subcellullarfractions and indcuces a net synthesis of total RNAfrom 6 to 26 % and of total protein from 13 to 34 %,and R-S protein from 74 to 122 % as shown by datain this paper and a recent report (13). Also auxindoes maintain the level of nucleic acids and proteinin excised tissue for up to 5 days (11).

Actinomycin D inhibited auxin-induced incorpora-tion of orotic acid and caused a 15 % loss of RNAas compared with the zero time controls (talble II).Attend,ing this there occurred a similar repression ofauxin-induced incorporation of leucine and net syn-thesis of protein. The evidence supports the con-clusion (12, 14) that in bean endocarp auxin-inducedsynthesis of protein is a consequence of a more

primary action of auxin in enhancing synthesis ofRNA. The results of 4 short-term (2-hr) experi-ments (Sacher, unpublished data) are consistent withthis view in that auxin-enihancement of incorporationof leucine in a suibcellular fraction never occurredexcept following auxin-enhancement of uridine in-corporation in that fraction.

The variability in response to kinetin occurred inexperiments done with different batches of tissue, foreach of which auxin enhanced incorporation of pre-cursors of RNA and protein and caused a net increasein RNA and protein. The data seem to indicate that

different batches of tissue vary in some factor orcondition which affects the capacity of the tissue torespond to kinetin. This factor does not appear tobe auxin as kine.tin stimulated sy.nthesis of RNA andprotein in tissue sections (a) which were water-aged,and thus probably depleted of auxin and (b) whenpresent along with a concentration of auxin whichcaused a large stimulation of RNA and protein syn-thesis.

A similar variability in response to kinetin hasbeen observed in Rhoeo discolor leaif sections. Thiswas clearly related to leaf-age as kinetin enhancedsynthesis of RNA and protein and prevented senes-cence in leaves which have just completed fulil exkpan-sion, but not in the more mature leaves (15). Auxin,in contrast, greatly stimulated synthesis of RNA andprotein in both the younger and older Ileaves. Alsoin the just-expanded leaves kinetin greatly augmentedthe stimulating, effect of auxin on synthesis of ,bothRNA and protein.

From the dififerent responses of bean endocar.p tokinetin i.t is not clear whether the primary action ofkinetin 'is on synthesis of RNA or !protein. Theexperimental results 'which ishow stimulation of pro-tein ;synthesis despite a degradation of RNA couldindicate that kinetin acts primarily on protein syn-thesis. This -possibility (for the mode of action shouldbe considered, particularly in view of evidence (2)showing a relationship between incorporation ofkinetin unaltered into transfer RNA and its effecton growth of plant tissue cultures. Also, transferRNA from other organisms has cytokinin activity inthe tobacco callus assay, although it is recognizedthat no functional role has been established for in-corporation of kinetin into RNA (16). The vari-ability in response to kinetin in different batches oftissue could be explicable, as suggested by Fox (2),in terms of kinetin acting in place of certain substi-tuted 'bases in tissue which has lost the capacity tosynthesize the latter.



PLANT PHYSIOLOGY

The inhibition of uri,dlne incorporation is difficultto account for, but could be consistent with the fore-going if kinetin wxere sometimes to Icause an increasein riboniuclease. An alternative explanation for theinhibition of uridine incorporation by kinetin couldlreside in its getting incorporated into DN'A unaltered,which has been reported for Lemlna miiinor (19).

Other experimental results based *on comparativestudies of the effect of auxin and kinetin in frtiit andleaf tissues may inidicate that the primary action ofkinetin is on synthesis of RNA. The effect ofkinetin on synthesis of RNA and protein is \verysimilar in sections of bean endocarp and Rlioco leaves.In the first iplace kinetin greatly augments the effectof auxin on RNA and 'protein synthesis in bothtissues. In the second place 5 experiments withRlioco lealf isections (15) showed that kinetin neverstimulated protein synthesis in a subcellular fractionexcept 1when it stimulated RNA synithesis in thatfraction. This appears to be true for bean endocarp(;tables VII and IX), although verification bv moreexperiments of shorter duration is needed to assist inlocalizing the initial effect of kinetin.

Acknowledgment

I thanik 'Mrs. Elizabeth Anderson and M\rs. AnneJacobson for a valuable technical assistance; and Merck,Sharpe, and Dohme Research Laboratory for sanmples ofactinomycin D.

Literature Cited

1. BRAY, G. A. 1960. A simple efficient liquid scin-tillator for counting aqueous solutions in a liquidscintillation counter. Analyt. Biol. 1: 279-85.

2. Fox, J. E. 1966. Incorporation of a kinin, N ,6-benzvladenine into soluble RNA. Plant Physiol.41: 75-82.

3. Ku, L. AND R. J. ROMANI. 1966. Ribosonmes froImipear fruit. Science U.S. 154: 408-10.

4. LOW\RY, 0. H., J. ROSEBROUGH, A. L. FARR, ANDR. J. RANDALL. 1951. Protein measurementw-ith a Folin phenol reagent. J. Biol. Chem. 193:265-75.

5. MOTHES, K., L. ENGELBRECHT, AND 0. KULAJEWA.1959. t'ber die Wirkung des Kinetins auf Stick-stuiffverteiluing und Eiweiss-synthese in isolierteBlitterni. Flora (Jeina) 147: 445-64.

6. OSBORNE, D. 1962. Effect of kinetin on proteinand nucleic acid metabolism in Xanthiuni leavesduring senescence. Plant Physiol. 37: 595-602.

7. REiCH, E., R. M. FRANKLIN, A. J. SHATKJN, ANDE. L. TATUM. 1961. Effect of actinomycin Din cellular nucleic acid synthesis and virus pro-duction. Science U.S. 134: 556-57.

8. RiCHMOND, A. E. AND A. LAND. 1957. Effect ofkinetin on protein content and survival of de-tached Xanthiuioi leavXes. Science 125: 650-51.

9. SACHER, J. A. 1957. Relationship between auxinand memnbrane-integrity in tissue senescence andabscission. Science 125: 1199-1200.

10. SACHER, J. A. 1959. Studies on auxin-membranepermeability relations in fruit and leaf tissues.Plant Physiol. 34: 365-72.

11. SACHER, J. A. 1963. Senescence: hormone actionand metabolism of nucleic acids and proteins in

plaint tissue. Life Science 1: 866-71.12. SACHER, J. A. 1965. Senescence: hormonal coIn-

trol of RNA and protein synthesis in excisedbean podl tissue. Am. J. Botany 52: 841-48.

13. SACHER, J. A. 1967. Dual effect of auxin: inhi-bitiori of uptake and stimulation of RNA andprotein syinthesis: assessment of synthesis. Z.Pflanzeinphysiol. 56: 410-26.

14. SACHER, J. A. 1967. Studies of permeability,RNA and protein turnover during ageing of fruitand leaf tissues. In: Some Aspects of the Bi-ology of Ageing. H. WV. WVoolhouse, ed. 21stSymp. Brit. Soc. Exptl. Biol. Cambridge Uni-versity Press. p 269-303.

15. SACHER, J. A. 1967. Conitrol of synthesis ofRNA and protein in subcellular fractions of Rhocodiscolor leaf sections by auxin and kinetin duringsenescence. J. Exptl. Geront. In Press.

16. SKooG, F., D. J. ARMSTRONG, J. D. CHERAYIL, A. E.HAMPEL, AND R. AM. BOCK. 1966. Cytokininactivity: localization in transfer RNA prepara-tions. Science U.S. 154: 1354-56.

17. SUGIURA, AI., K. U-MEMURA, AND Y. OOTA. 1962.The effect of kinetin on protein level of tobaccoleaf disks. Physiol. Plantarum 15: 457-64.

18. T'so, P. 0. P. AND C. S. SATO. 1959. Synthesisof ribonucleic acid in plants I. Distribution ofribonucleic acid and of protein among subcellularcomponents of pea epicotyls. Exptl. Cell Res.17: 227-36.

19. VAN EYK, J. 1963. Investigatioin of the mode ofaction of kinetin with Lemnia minor (L.). Ph.D.Dissertation, University Leiden.

20. W$VOLLGIEHN, R. 1961. Untersuchungen iiber denEinfluiss des Kinetinis auf den Nucleinsaure andProteinstoffwechsel isolierter Bliitter. Flora 151411-37.

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senescence: action of auxin and kinetin in control of ... · pole beans (phlascoluts zulqaris l.)...

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