effect of kinetin-naphthaleneacetic interaction upon … phyll content (wlhich we use as the primary...

Plant Physiol. (1968) 43, 1271-1278

Effect of the Kinetin-Naphthaleneacetic AcidInteraction Upon Total RNA and Protein in

Senescing Detached Leaves1G. J. VonAbrams and Harlan K. Pratt

Department of Vegetable Crops, University of California, Davis, California 95616

Received March 18, 1968.

Abstract. The interaction between kinetin and naphthaleneacetic acid in the regulation ofsenescence of excised tissue of mature broccoli leaves has been used to examine theextent of synchrony between changes in chlorophyll, RNA, and protein. Kinetin increasedthe net uptake of 14C-labeled orotic acid and leucine. Naphthaleneacetic acid decreased theeffect of kinetin on net uptake after long treatment, but in short-time treatments theauxin increased the effect of kinetin on net uptake. Results of long (24 hr) treatmentsindicated a general synchrony between the loss of RNA, protein, and chlorophyll. Naphthalene-acetic acid reduced the stabilizing effect of kinetin upon chlorophyll content and upon thecontent and synthesis of RNA. In short-time experiments, however, RNA content andsynthesis were transiently increased by kinetin, and further increased by kinetin plusnaphthaleneacetic acid, while chlorophyll content decreased in the presence of kinetin anddecreased further in the presence of kinetin pluis naphthaleneacetic acid. Actinomycin-Daccelerated the loss of chlorophyll, RNA and protein and strongly depressed the rate of RNAsynthesis. In the presence of actinomycin-D the stabilizing effect of kinetin upon RNA wassubstantially reduced. In contrast, the chlorophyll and protein contents remained higher thanin the control. Actinomycin-D did not nullify the basal incorporation of orotic acid into RNA,nor did it negate the effect of kinetin upon incorporation. The failure of synchrony betweenchanges in chlorophyll and RNA does not substantiate the proposal that kinetin regulatessenescence by a direct effect upon DNA-dependent RNA synthesis.

The senescence of detached leaves is character-ized by loss of chlorophyll, associated with declininglevels of RNA and protein, and the rate of senescencemay be regulated by kinetin or other exogenousgrowth substances (5, 12,21,25). It is not knownhow the regulation is accomplished. Kinetin retardsthe loss of chlorophyll, RNA, and protein, and en-hances synthesis of RNA and protein in detachedleaves and leaf tissue of several species. Inhibitionof the effect of kinetin by actinomvcin-D has led tothe proposal that the hormone influences the rate ofsenescence by regulating DNA-dependent RNA syn-thesis (13, 28). Changes in permeability and freespace are associated with senescence and have beenshown to be susceptible to hormone regulation in thefleshy leaves of Rhoeo. A substantial loss of proteinoccurred prior to detectable increase in free space(15). However, even the most subtle alteration ofan interface might well be the predominant event inthe control of senescence. Attempts to correlate thefunction of kinetin in the regulation of senescencewith the induction of specific enzymes have been

1 This investigation was supported in part by a re-search grant (UI-00102) from the National Center forUrban and Industrial Health, United States Public HealthService.

12'71

inconclusive ( 1). A\lothes has shown that kinetinis able to cause directed transport and localizedaccumulation of amino acids. From the results ofa series of investigations, he has concluded that thisis the primary function of kinetin in the regulationof leaf senescence (10). There is evidence w-hiclhis inconsistent with this proposal ( 12, 23, 27), butnone by whiclh it is conclusively contradicted.

WN e have previously reported that a-naphthalene-acetic acid (NAA) strongly depresses the effect ofkinetin, but does not by itself influence the senescenceof detached leaves of broccoli or Xanthliim (22).This being so, the regulation of senescence bykinetin may be experimentally altered by the presenceor absence of NAA. and the degree of synchronyamong the effects of kinetin on the various param-eters of senescence may be observed. The inter-action between kinetin and NAA was used subse-quently to examine the relationship between theregulation of senescence and the directed transportand accumnulation of substrate (23). The auxin byitself had little effect, but when present with kinetinit simultaneously reduced the effect of kinetin uponsenescence and increased directed transport andaccumulation in the treated locus.

In the present report we describe experiments inwhich the kinetin-NAA interaction was used toexamine the relationship between changes in chloro-

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1272

phyll content (wlhich we use as the primary senes-cence indicator) and changes in the content andsynthesis of total RNA and protein in excised tissueof broccoli leaf.

Materials and Methods

Platt MViaterial. Broccoli (Brassica oleracea L.,-ar. italica, cv. Coastal) was grown in the green-house under prevailing natural light. Selected ma-ture leaves were harvested from plants bearing from10 to 15 fully expanded leaves. The detached leaveswvere washed in 0.01 % Tween-20, swabbed withcotton, immersed for 2 minutes in 0.5 % NaOClsolution, and rinsed repeatedly in sterile distilledwater. They were then placed in a 200 room undera light regime of 6 hours of dim green light alter-nating with 18 hours of darkness. Each leaf waskept with its petiole in an individual flask of steriledistilled water; the water was changed and the baseof the petiole was re-cut daily. Tissue samples wereexcised after 4 to /7 days, when the rate of chloro-phyll loss was near maximum. Except as noted,eaclh sample consisted of 8 mm or 12 mm disks,

disk from each of 25 leaves. Replicate samplesfor any 1 experiment were taken from the same setof leaves. The tissue disks were weighed and placedon filter paper saturated with a solution of penicillin('60 jzg/mIl) and mannitol (0.15 M) in covered petridishes. The tissue was preincubated on this mediumfor 30 hours and was then transferred to a petri dishcontaining filter paper saturated with a measuredv-olume of the test medium. For some experiments,particularly for brief incubation, the tissue wasv-acuunm infiltrated with the test medium beforetransfer to a petri dish. All manipulation of thetissue was performed aseptically, under dim greenlight. Incubation was at 200 in darkness.

Inicubation .Mledia. All media included Tween-20(0.01 %) and penicillin (60 ,ug/ml), and weres-terilized by filtration or by a combination of filtra-tion and autoclavinig. Kinetin and NAA were usedat equal concentrations, either 5 ju-r or 10 uM.

Actinom-ycin-D (Act-D) (20 ug/ml) was intro-duced for specified experiments. To reduce differ-enices of solute efflux in the presence of the active.substances, 0.15 AM mannitol was included as anosimoticum. During a specified period of the incu-hation sequence, orotic acid-6-14C (37 mc/mM) wasintroduced as a precursor of RNA, or L-leucine-U-1C (40 1ic/mM) as a precursor of protein. Label-ing of the media was in the range of 1 to 3 ptc per ml.

Bacterial Co/itaminatioan. After incubation, platecouinits were made of the media and of holmogenatesof parallel tissue samples for each experiment.Penicillin generally provided only 10 to 20 % coIn-trol of the increase in bacterial population, but itwas effective in preventing the rapid increase whichsometimes occurred in unprotected samples. Unlikechloramplhenicol and thiouracil, penicillin did not

PHYSIOLOGY

alter uptake of precursors and did not increase therate nor decrease the uniformity of chlorophyll loss.The number of bacteria within the tissue was notinfluenced by kinetin nor by NAA, and differencesof incorporation induced by the regulating substancescould not be correlated with differences in thebacterial count.

Chlorophyll Assay. Frozen tissue disks wereextracted for 5 minutes in each of 5 separate volumesof boiling 80 % ethanol. The extract was acidifiedas in the procedure of Wickliff and Aronoff (24),the combined extract was filtered, the volume ad-justed, and the optical density determined at 665 acnd653 m,u. In some experiments the frozen tissue washomogenized in acetone and the pigment transferredto ether by a procedure similar to that described bvSmith and Benitez (18). The optical densities weredetermined at 662 and 644 mu. To obtain statisti-cally valid measurements of changes in chlorophyllcontent during short-term experiments, it was neces-sary to increase the number of replications to 10 pertreatment. All procedures in the chlorophyll assavwere done at 40 under dim green light.RNA Assay. If not previously homogenized for

the extraction of chlorophyll, the chilled or frozentissue was homogenized in glass in cold 95 % ethanolcontaining 12C-orotic acid (0.1 mivm). A modifica-tion of the procedure described by Smillie and Krot-kov (1/7) was used to estimate total RNA. Thepellet was suispended for 10 miinutes in 7 % (tri-chloroacetic acid) at 00 and then washed twice incold 5 % TCA. The residue was washed witlorganic solvents, including a 30 miniute wash withether :ethanol :chloroformi (2:3 :1 ) at 370. The color-less de-fatted residue was digested in 0.3 N KOTfor 18 hours on a shaker at 370. After centriftuga-tion, DNA aind protein were precipitated fromii thesupernatant by the addition of HClO1 to a conicen-tration of 0.3 N and chilling to 40 for 3 hours. Aftercentrifugation and washing, absorhance of an alioltnotof the supe;rnatant Nwas mieasuired at 260 nmfu. Forconversion of absorbance values to weiglht equiva-lents, standards of l)urified yeast RNA wvere proc-essed in the samiie manner. In somie experimiienitsRNA analysis wlas performed according to tllemodification suggested by Key and Shannon (7).

Proteini Assay. The tissue was hlomogenized anda TCA-insoluble residue was prepared by the pro-cedure described for RNA. The residue was di-gested for 15 miinutes at 370 in N NaOlI conltainiingleucine (mnvi). After centrifugatioin and washlinig,aliquots of the sutpernatent were assayed for proteinby the Folin-phenol metlhod (9).

Measuremnet of Uptakc (1iid Jlncorporatioii.After incubatioin with 1'C-labeled precursor, thetissue was blotted. placed in a cheeseclotlh haig, rinsecl1 minute in deionized water, aind gently waslhed for30 minutes in a large volume of 0.1 M mainniitolsolution. The tissue vas rinsed, blotted, aind ex-tracted according to the previously described aina-lytical procedures. All fractions w\ere assayed for



VON ABRAMIS AND PRATT-HORMONE INTERACTION- ON RNA

radioactivity, and the total activity was used as anestimate of the net uptake of labeled substrate bythe tissue. Radioactivity was determined by dryingduplicate aliquots on glass-paper circles and countingin a toluene-fluor mixture in a liquid scintillationspectrometer. Quench correction was calculatedfrom internal standard series prepared for eachfraction. In some experiments, net uptake was veri-fied in parallel samples by a combustion method(23). A "corrected incorporation" value was cal-culated by dividing cpm incorporated by the ratioof the net uptake (total cpm remaining after wash-ing) of the treated sample to the net uptake of thecontrol sample. Sacher (14) has discussed the needfor a similar correction.

Table I. Effect of Pre-Treatment with Kinetin andNAA on Net Uptake of l4C-Orotic Acid and

14C-Leucine by Leaf DisksLeaf disks were incubated 10 hours with hormone

(10 AM), then 4 hours with the labeled precursor (1 pc/ml). The disks were washed, oven-dried, and combusted.The 14(CO2 evolved was trapped and counted. Meanand SE of six 16 mm disks.

Net uptakeTreatment 14C-Orotic acid 14C-Leucine

cpm cpmControl 58144 ± 2123 82560 ± 1735NAA 59048 ± 2407 80983 ± 2497Kinetin 65763 ± 945 86312 ± 1344Kinetin + NAA 69992 ± 1838 89864 ± 1062

Results

Effect of Excision. It was observed in pre-

liminarv experiments that the initial rate of senes-

cence of excised tissue was markedly different fromthat of the leaves from which the tissue was excised.Chlorophyll content remained nearly constant for as

long as 30 hours. Protein increased slightly for a

shorter period. RNA increased for 8 to 16 hoursafter excision, and then decreased sharply. Toavoid this irregularity in the course of senescence.

the excised disks were routinely preincubated("aged") for 30 hours before use.

LTptake. Pre-treatment of excised leaf tissue withkinetin anid NAA differentially influenced the netuptake of the labeled substrates subsequentlv pre-

sented (table I). Tissue disks were incubated inthe presenice of hormone for 10 hours. The tissuewas then rinsed, blotted, and incubated in the presence

of 1"C-letcine or '4C-orotic acid for 4 hours. Aftera 30-minute wash in 0.1 M mannitol. the disks were

blotted, oven-dried, and combusted as previouslydescribed (23). The 14CO2 produced by the com-

plete combustion was trapped in Hyamine-hydroxideand counted in a toluene-fluor mixture by liquidscintillationi spectrometry. The net uptake of bothprecursors wN-as increased significantly by kinetin,and increased further by kinetin plus NAA.

In eaclh of the incorporation experiments, netuptake w as estimated as the sum of the radioactivi-

ties of the analytical fractions. The results of longtreatments conflict with the data shown in table Iand with the results of experiments in which thetissue was treated for shorter periods of time (tableIVJ). The net uptake of precursor after 36 hours ofexposure to the hormones is shown in table III.

The net uptake of label from orotic acid, and to alesser extent from leucine, was markedly greater intissue preincubated with kinetin than in the controltissue, but when NAA was present with kinetinduring the preincubation, the subsequent net uptakeof label was less than that of tissue treated withkinetin alone.

Effect of Long Treatmetnt. The results shown intables II and III are typical of 5 separate experi-ments in which the incubation periods were longerthan 24 hours. Kinetin reduced the rate of net lossof chlorophyll. RNA, and protein, and partiallymaintained the rates of incorporation of the pre-cursors into RNA and protein. Although NAA byitself had little or no effect, the protective influenceof kinetin was severely depressed in the presence ofthe auxin. For all treatments, changes in the con-tent and synthesis of RNA and protein were reason-ably well correlated w-ith the change in chlorophyllcontent.

Effect of Brief Treatmtenlt. The short-time re-

sponse to the regulating substances was markedlydifferent from that observed after extended treat-

Table II. Effect of Long Treatmient WVith Kinetin and NAA on Chlorophyll, R.VA, and Protein Contentof Leaf Disks

The total treatment period was 40 hours. The concentration of each hormone was 5 /um. These data are fromthe same samples as those in table III. Chlorophyll: means and SE from all 10 replicate samples. RNA: meansand SE from the 5 replicate samples which received 14C-orotic acid from the last 4 hours of incubation. Protein:means and SE from the 5 replicate samples which received 14C-leucine.

Treatment Chlorophyll RNA Protein

7ng per g fr zwtInitial 1 78 ± 0.09 2.183 + 0.018 20.35 ± 0.44Control 1.12 ± 0.04 1.490 ± 0.008 14.08 ± 0.23NAA 1.16 ± 0.08 1.486 + 0.026 14.21 ± 0.96Kinetin 1.59 ± 0.05 1.892 ± 0.004 19.42 ± 0.07Kinetin + 'NAA 1.35 + 0.07 1.724 + 0.019 17.65 ± 0.51

1273



Table III. Effect of Long Treatmnent with Kinetin and NAA on Subsequent Net Uptake and Incorporationof Labeled Precursors into RNA an1d Protein

Leaf disks were incubated 36 hours (dark, 200) with 5 StM hormone media, then 4 hours with I4C-orotic acid or14C-leucine (1 A.c/ml). Means and SE of 5 replicate samples, each 25 disks. Values were adjusted to equate freshweight of samples to 360 mg. The "corrected incorporation" was calculated as the cpm incorporated divided by theratio of the Pet uptake of the treated sample to the net uptake of the control.

14C-Orotic acid 14C-Leucine

Net Incorporated Corrected Net Incorporated CorrectedTreatment uptake in RNA incorporation uptake in protein incorporation

ControlNAAKinetinKini + NAA

1456 ± 1041498 931877 - 681615 ± 64

cpM X 10-2208 + 12219 ± 17421 + 8293 ± 11

208213327264

2092 ± 942182 -+ 1242458 ± 632321 ± 56

ct/r X 10-2606 + 18645 ± 27963 -± 24817 ± 25

606618820737

ment. When treatment was terminated at 3 hours.the enhancement of incorporation of precursors intoRNA and protein was as great or greater in responseto kinetin + NAA as to kinetin alone (table IV).Changes in the measured content of chlorophyll,RNA, and protein during the 3-hour period werenot statistically significant, but the results of the6-hour treatments show that the chlorophyll level

was not inevitably stabilized during predominiianltlyanabolic RNA metabolism. After 6 hollrs in thepresence of kinetin, increases in RNA anid proteinabove the 0-time values were detected (table V),as well as continued enhancement of incorporation(table IV). Both the transient increase in contentof RNA and protein and the enhancement of in-corporation were greater in response to kinetin +

Table IV. Short Term Effect of Kinetin and NAA on Incorporation of 14C-Orotic Acid into RNA and14C-Leucine into Protein

Leaf disks were vacuum infiltrated with hormones (10 #WI) and labeled precursor (1.7 Ac/ml) concurrently andwxere then incubated (dark, 200) for 3 or 6 hours. Values are the means and SE of 5 replicate samples. Sam-pling for the 2 times of treatment was from different sets of leaves. Each sample was 25 disks (8 mm). Valuesare adjusted to equate fresh weight of samples to 360 mg. The "corrected incorporation" was calculated a; intable III.

14C-Orotic acid

Incorporated Correctedin RNA incorporation

ctrn X 10-2249 -+- 18262 ± 25343 ± 10398 -+- 18

993 ±1002 +1427 ±1536 ±

1124923

249261315339

Netuptake

1804 -t 351819 + 712003 ± 319098 ± 42

993 5535 ± 87 2322 +997 5597 ±103 2384 +1270 5832 -+ 43 2548 +1315 5991 l 72 2631 H-

14C-Leucine

Incorporated Correctedin protein inicorporation

cpm X 10-2753 + 19751 ± 31915 ± 25972 4- 15

29481235

Table V. Slhort Tcrm Effect of Kinetin and NAA on Content of Chlorophy1ll, RNA, and ProteinValues shown are from the same leaf disks used for the 6-hour treatment of table IV, and the same conditionis

apply. The chlorophyll values are the means and SE for all 10 replicate samples. The RNA values are the means forthe 5 replicates which received 14C-orotic acid, and the protein values are the means for the 5 replicates wlichreceived 14C-leucine.

RNA

mtg per g2.245 ±2.103 ±2.110 H-2.2772.302 ±

fyr wt0.0170.0090.0150.0060.007

Protein

21.72 0.1120.85 + 0.1420.94 +- 0.2621.98 + 0.1322.13 +- 0.06

NetuptakeTreatment

3-HrControlNAAKinetinKin + NAA6-Hr

ControlNAAKinetinKin + NAA

1025 ±1031 ±1117 -4-1203 +-

3884 4-3907 +4366 ±4539 -4-

23392752

22372428

753745824836

2322235824202432

Treatment Chlorophyll

InitialControlNAAKinetinKin + NAA

1 97 ± 0.021.77 ± 0.031.75 + 0.051.90 _- 0.031.82 - 0 02

1274 PLANT PHYSIOLOGY



VON ABRAM-IS AND PRATT-HORMONE INTERACTION ON R-NA

NAA than to kinetin alone. In contrast, the chloro-phyll content decreased in the presence of kinetin,and decreased even further in the presence ofkinetin + NAA (table V). The transient increaseof RNA was not restricted to the cut edge of thetissue, since removal of the outer rim after the incu-bation period showed that the same response hadoccurred in the center of the disk.

The (luration and intensity of the kinetin-inducedsurge of RNA and protein varied between crops ofleaves, but in most experiments there was no in-crease after 12 hours, and in 24 hours the contentof RN X\. and protein had fallen below the initial(0-time) values. Figure 1 shows the temporalrelationship between change in RNA content and

the chlange in rate of incorporation of orotic acidinto RNA over the 24-lhour period. Samiiples intriplicate were infiltrated with kinetin or with coni-

trol medium at 0-time, and 14C-orotic acid was adde(dat the intervals indicated in the figure. Eachi samplewvas incubated with the labeled precursor for 2 hours.

In the controls, both the synthesis and content ofRNA decreased gradually. In the presence ofkinetin the rate of incorporatioln increased duirlillngthe first three 2-hour periods, but decreased duringthe sixth 2-hour period and thereafter. The en-

hancement of incorporation by kine.in during thefirst 2-hour period was very slight, and this pre-sumablv reflects the lag period of kinetin functioiunder the conditions of the experiment.

Effect of Actinomycin-D. Table VI shows tlweeffect of act-D after a 12-hour inicubation l)eriod.At this time the kinetin-induced surg-,e of RNA,k wasslightly past its maximum, and RXNA synthesis c

more enhanced by kinetin + NAA thani bv kineLin

alone. Act-D accelerated the loss of chlorophyll.RNA, and protein, either witlh or without kinetin,but did not nullify the stabilizing influence of kinetin.The effect of act-D on RNA content was muclhgreater than on either chlorophyll or protein. Inthe presence of kinetin the chlorophyll level as

C)

0

0

Incorporotion

Kinetin Control

Hours

Fic. 1. Effect of kinetin on the quantity of RNAand on the rate of incorporation of 14C-orotic acid intoRNA in senescing leaf disks over a 24-hour period. Thedisks were infi'trated with kinetin (10 /sI) or withcontrol medium at 0-time, and 14C-orotic acid was added(2.2 Muc/mi) at the start of each 2-hour incubation period.Incorporation values have beeni corrected for differencesin net uptake. Each entry is the meani value from trip-licate samples, each sample 25 X 12 mm disks from25 leaves. Quanitity of RNA w-ith kinetin (*---*)witlhout kinietin (Q ---Q). The open portion of thevertical bars indicates 2-hour incorporation of 14C-oroticacid inlto RNA w\Nithout kinetin; the shaded portion in-dicates the increase in incorporation in the pr-esenice ofkilnetin.

Table VI. Effect of Actinonzycin-D, Kinctin, and NAA on the Content of Chlorophlyll, RNA, and Protein and onthe Incorporation of Label fri-om l4C-Orotic .4cid into RNA

Leaf disks were vacuum infiltrated with the test media at 0-time and incubated in the dlark, at 200, for a total of12 hours. 14C-Orotic acid (2.0 /hc/ml) was adde(d for the last 3 hours. Concentrations were 10 uNi for eaclh hor-mone, and 20 ug/ml for actinomycin-D. Means and SE for 5 replicates, 25 X 8 mm disks per sample. Values wereadjusted to equate fresh weight of samples to 365 mg, and incorporationi was corrected for differences in net uptakeas in table III.

% Of initial content Incorporation into RNA

Treatment ClhloroplivIl R N A Protein cpm X 10-2 As % control valuie

CoiLtro& 81.5 + 2.8 85.3 ± 2.1 88.6 ± 2.5 623 ...N A A 80.7 - 2.2 86.1 ± 1.7 87.2 + 2.1 627 100.6 + 3.4Kinetin 94.7 H 2.0 108.3 ± 0.7 98.5 ± 1.8 874 140.3 ± 2.8Kinetin + NAA 88.2 0.9 111.7 ± 1.4 95.7 ± 1.1 912 146.4 ± 3.1Act-D 73.4 ± 3.2 67.5 ± 4.0 79.4 + 3.5 189 30.3 ± 4.6NAA + Act-D 72 2 +- 2 5 68.4 ± 3.3 79.1 ± 3.1 185 29.7 ± 5.2Kin + Act-D 86.1 ± 1.3 80.2 ± 1.8 91.0 ± 2.0 427 68.5 ± 2.7Kin+NAA+Act-D 81.8 ± 2.9 83.9 ± 1.9 88.6 ± 2.3 436 70.0 ± 3.5

1275

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PLANT PHYSIOLOGY

reduced nearly as mnuch by NAA as byact-D, butwhen both NAA and act-D were present their com-

bined effect on the influence of kinetin was muchless than additive.

Act-D strongly depressed the basal rate of in-corporation of orotic acid label into RNA, but didnot nullify it. Even with higher concentrations ofact-D (to 100 ,ug/ml) and longer incubation periods,there was a persistent incorporation equal to ap-

proxilmately 30 % of the control value. In thepresenice of kinetin, the inhibitor reduced incorpora-tionI to a level far below that of the control but didnot negate the effect of kinetin.

Discussion

The decision to "age" (preincubate) the excisedtissue used in this study was based upon the diffi-cultx of interpreting the effect of exogenous regula-tors sul)erimposed upon the transient increase andsubsequent decrease of RNA following excision ofthe tissue. Similar transient interruption in thecourse of seniescence may be inferred from data ofother reports (16, 20). Mothes and Engelbrecht(11 ) have described the directed transport of sub-strate from the lamina to the petiole of the senescingleaf. Excision of the tissue from the leaf eliminatesdirected transport, and it is possible that this may

be the cause of the transient interruption of senes-

cence. The interruption is not a "wounid effect",since it is incdependent of the ratio of wound area

to tissue mass.

Although enterinig a period of rapid senescence,

this tissuie wvas clearly capable of physiologicalregulation of uiptake or retention. The net uptakeof both orotic acid and leucine was strongly influ-ence(l by the regulating substances used in the study.and the ,lncorporation values were corrected accord-ingly. For lack of more satisfactory criteria, we

has-e mlade the assumption that the label which didnot diffuse into ambient 0.1 -.r mannitol solutionidurinig a 30 mintiLe wash constitutes a mleasure ofthe amllotunt of labeled precursor wIhiclh wN-as availablefor sxynthesis froml intracellular pools. There al-eother assumptions which must be made if one is toaccept these values as nmeaniin;gful. We have no

information concernling replenishlmenit of the activ-eprecursor pools in response to incorporation, nor

are w-e able to estimate the rate of recycling of

unstable RNA. Tf interfaces and membranes of thissenesciing tissue are degenerate, as w-e believe, it i.possible that termiinial washing after lolg incub-ationperiods miiight obscure the concentration.s of precuirsor

xwhich wxere available for synthesis.The effect of NAA in the presence of kiinetini

chaniged fromii early enhancement of niet uptake tosubsequetnt depression of net uptake. It is unlikelythat this temlporal difference represelits a reversal indirect response to the hormones. We haNve showivthat NA k depressed the abilitv of kinetin to retardsenescencce. It folloxws that the chiange in net uptake

over a long incubation period in the presence ofkinetin + NAA is the difference between the netuptake of a mature tissue and that of a deeplysenescent tissue. The net uptake is a function ofboth influx and efflux. Mothes (10) has empha-sized the possible significance of retention as acorollary of accumulation in the function of kinetin.

The net loss of chlorophyll and protein whichoccurred during long treatment with kinetin andNAA appeared to parallel the change in content andsynthesis of RNA. This is consistent with the viewthat senescence mav be controlled by the regulationof RNA metabolism, but in fact these results serveonly to compare tissues which have already reachedquite different stages of senescence as a result ofthe action of the regulating substances.

The results of short incubation periods revealeda markedly different response. RNA content andsyinthesis increased under the influence of kinetin,while chlorophyll decreased. Under the influence ofkinetin + NAA. the increase of RNA and thedecrease of chlorophyll were greater still. Precisesynchrony therefore is lacking between loss ofchlorophyll and both the loss of RNA and thereduction of sviythesis of RNA in response to theregulating substances. This may reflect inadequacyof total RNA as a measure of the RNA metabolismwhich is relevant to senescence, or it may suggestthat loss of chlorophyll is only indirectly related toRNA metabolism. There is some indication thatkinetin exerts its effect in senescing leaf tissue uipoIna specific fraction of RNA (4,6,21) which may beobscured in the determination of total RNA. \V oll-giehn did not find stuch specificity, however, andhas recently reported experiments in which the re-sponse to kinetin was very slow aand occurredl non-specifically- in all RNA fractions (26). An incon-sistency in the correlation betweeni RNA andsenescence has been noted also by Beevers (2), andthe data of Srivastava and Ware (20) indicate aloss of chlorophyll concurrent w-ith a gain in RN.\after incubation in kinetin for 1 dLay.

It is remarkable that the effect of kinietin onlRNA was augmenited by N\TAA during brief treat-ment, but depressed after longer treatmiielnt. Wespeculate that NAA mlight increase the early rate ofuiptake of kinetin. or otherwise nmake kinetin moorecluickly available to its site of action. Over longerperiods, suclh acceleration wotuld be of nio iml)ortance.

Since there w-as ani actual inlcrease in RNA\conteint above the 0-timiie level, it is probable thlatkinetin not onlty miiaintainied the initial rate of svnI-thesis, but increased it. XVe are unable to evaluiatethe possibility that kinietin miglht also reduce tllerate of degradatioln of RNA.

Presumably act-D adnd NAA act upon quiitedlifferent componieints of the system which is re(oniredfor expressioni of thle effect of kilnetini upon clhloro-)hyll. Althouglh it is inmprobable that these 2 regti-lating substances funiiction competitivelv, their effectwas clearly non-additive. NTAA bv itself haid on1v

l 276)



VON ABRAMS AND PRATT-IIORMONE INTERACTION oN- RNA

slight effect, whereas act-D reduced the chlorophyllcontent severely. It has been reported that the lossof chlorophyll was retarded, rather than accelerated,by actinomycin in barley leaves (19) and in kale (8).

Act-D must be assumed to influence the chloro-phyll level via the inhibition of DNA-template RNAsynthesis. No other reported function of the in-hibitor appears to be relevant. It is most unlikelythat there is a significant turnover of chlorophvll inthis senescing tissue (e.g., 24), particularly underthe controlled light used for the study. Withoutevidence for active synthesis of chlorophyll, thereis no justification for assuming that act-D acts byblocking the production of an enzyme required forchlorophyll synthesis. Alternatively, it is suggestedthat the inhibitor might block the replacement oflabile chlorophyll-protecting structural protein or

galactolipid of the chloroplast.Although the protective influence of kinetin upon

RNA was strongly reduced by act-D, chlorophylland protein contents were relatively less effected,and remained higher than in the control tissue. Theprotective influence of kinetin upon chlorophyll andprotein may be not strictly dependent upon RNA. or

may be dependent upon only a stable or actinomvcin-

insensitive fraction of RNA. Act-D did not nullifythe basal incorporation of orotic acid into RNA, and

the residual synthesis may correspond to the actino-mvcin-resistant RNA synthesis reported to occur inspinach chloroplasts (3).

It is surprising that R'NA synthesis was clearlyenhanced by kinetin in the presence of act-D. WVehave meticulously monitored bacterial populationsafter these treatments, and have found no indicationof correlation between differences in the number otbacteria and differences in the incorporation ofprecursor into RNA. Since there appears to be a

fraction of RNA synthesis which persists in thepresence of act-D, it is possible that kinetin is ableto enhance the act-D resisLanit svnthesis. It is alsopossible that kinetin function results in the protectionof a fraction of DNA. presumably chloroplasticDNA, from association with act-D, and that a

corresponding fraction of RNA svnthesis remainsuninhibited.

Demonstration of the inhibition by act-D ofhormone-enhanced RNA synthesis has encouragedthe opinion that the regulation of senescence operatesbv a direct effect of the hormones upon DNA-dependent RNA synthesis (5, 13, 28). In the presentinvestigation, the results of long treatment withkinetin and NAA were consistent with this proposedmechanism. Shorter treatments, however, have re-

vealed a failure of synchrony between changes inchlorophyll content and both the content and syn-

thesis of total RNA. At the present time there isno conclusive evidence that RNA synthesis is thedirect and primary response to kinetin in senescingleaves, nor that failure to sustain RNA is the cause

of senescence. The observation that act-D inhibitskinetin-induced RNA synthesis indicates onlv that

the DN'A enmplate-RNA mechanismii miiust be fuinc-tional at the time RNA synthesis is influenced bykinetin. It does not prove that kinetin acts directlyupon D.NA-dependent RNA synthesis. Further,such correlation as exists between the supression byact-D of kinetin-induced RNA synthesis and ofkinetin-sustained chlorophyll levels suggests only thatactinomycin-sensitive RNA synthesis is required forfull expression of the effect of kinetin upon chloro-phyll. It does not demonstrate that kinetin indirectlyinfluences chlorophyll as a result of directly en-

hancing RNA synthesis.

Literature Cited

1. AN-DERSON, J. WV. AND K. S. ROWAN. 1966. Ac-tivity of aminoacyl-transfer-ribonucleic acid syn-thetases in tobacco-leaf tissue in relation to sen-escence and to the action of 6-furfurylaminopurine.Biochem. J. 101: 15-18.

2. BFEVERS, L. 1966. Effect of gibberellic acid onthe senescence of leaf disks of nasturtium (Tro-pacolum majus). Plant Physiol. 41: 1074-76.

3. B[SWAS, S. AND B. B. BISWAS. 1965. Effect ofpolyribonucleotides on amino acid incorporation bychloroplast ribosomes. Experientia 21: 251-53.

4. BURDETT, A. N. AND P. F. WVVAREING. 1966. Theeffect of kinetin on the incorporation of labeledorotate into various fractions of RNA of excisedradish leaf disks. Planta 71: 20-26.

5. FLETCHER. R. A. AND D. J. OSBORNE. 1966. Gib-berellin, as a regulator of protein and ribonucleicacid synthesis during senescence in leaf cells ofTaraxacuni officinale. Canadian J. Botany 44:739-45.

6. GUNNIN G, B. E. S. AND W. K. BARKLEY. 1963.Kinin-induced directed transport and senescence indetached oat leaves. Nature 199: 262-65.

7. KEY, J. L. AND J. C. SHANNON. 1964. Enhance-ment by auxin of ribonucleic acid synthesis in ex-cised soybean hypocotyl tissue. Plant Physiol.39: 360-64.

8. KNYPL, J. S. 1967. Coumarin. phosphoin-D andCCC-the inhibitors of chlorophyll and proteindegradation in senescing leaf tissue of kale. Flora(Abt. A) 158: 230-40.

9. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR,AND R. J. RANDALL. 1951. Protein measurementwith the Folin phenlol reagenit. J. Biol. Chem.193: 265-75.

10. MOTHES, K. 1963. The role of kinietini in plantregulation. In: Regulateurs Naturels de la Crois-sance Vegetale. Centre Natl. Rech. Sci.. Paris. p131-40.

11. MOTHES, K. AND L. ENGELBREC1IT. 1961. Kinetinand its role in nitrogen metabolism. In: RecentAdvances in Botany. Univ. of Toronto Press. p996-1003.

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

13. OSBORNE, D. J. 1965. Interactions of hormona!substances in the growth and developniment of plants.J. Sci. Food Agr. 16: 1-413.

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14. SACHER, J. A. 1967. Dual effect of auxi:inihibi-tioni of ulptake and stimulation of RNA anid pro-tein synthesis: Assessment of synthesis. Zeit.Pflanzenphysiol. 56: 410-26.

15. SA\CHER, J. A. 1967. Control of sxniitlhesis of R-NAanid proteini in subcellular fractionis of Rhoeo dis-color leaf sections by auxin and kinetin duringseniescenice. Exptl. Geront. 2: 261-78.

16. SH \V, M., P. K. BHATT\CHARYA\, AND W. A.QUIcK. 1965. Chlorophyll, protein, and nucleicacid levels in detached, senescinlg whleat leaves.Caniadiani J. Botany 43: 739-46.

17. SMILLIE. R. M. .\ND G. KROTKOV. 1960. The esti-milatioin of nucleic acids in some algae and higherplants. Canadian J. Botany 38: 31-49.

18. SMITH, J. H. C. \ND A. BENITEZ. 1955. Chloro-plbx llS: analysis in plant materials. In ModernMethods of Planit Analysis. K. Paech and M. V.Tracey, eds. Springer-Verlag, Berlin. 4: 142-96.

19. SRIVA.ST.'WAR, B. I. S. 1967. Effect of kinietin onbiochemical changes in excised barle- leaves andin tobacco pitlh tissue culture. Anini. N. Y. Acad.Sci. 144: 260-78.

'20. SRIVASTAVA, B. I. S. AND G. \VARE. 1965. Tlle ef-fect of kinietini onl niucleic acids and nucleases ofexcised barley leaves. Plant Plhxysiol. 40: 62-64.

21. SI-GIURA, M., K. UMEIMURA, AND Y. OOTA. 1962.The effect of kiinetini on protein level of tobaccoleaf disks. Plivsiol. Plantarum 15: 457-64.

22. VONABRAMS;, G. J. AND H. K. PRATT. 1966. In-teraction of naplhthaleneacetic acid and kinetin inthe senescenlce of detached leaves. Plant Phv siol.41: 1425-1530.

23. VONABRAMS, G. J. .\ND H. K. PR.\TT. 1967. Theeffect of kinetin and naphthaleneacetic acid uponlocalized accunmulation as related to seinescenice indetached leaves. Planta 76: 306-08.

24. \VICKLIFF, J. L. AND S. ARONOFF. 1962. Evidencefor ab-,ence of diurnal variationi of chlorophy11content in mature leaves of sovbeain. Planit Phx -siol. 37: 590-94.

25. \OLLGIEHN, R. 1961. Untersuchungen iiber denEinfluss des Kinetins auf den Nucleinsaure- unidProteinistoffx-echsel isolierter Blatter. Flora 151411-37.

26. WVOLLGIEHN, R. 1967. Nucleic acid anid protein me-tabolism of excised leaves. Sxmp). Soc. Exptl.Biol. 21: 231-46.

27. VOl.LGIEHN, R. AND B. PARTHIE.R. 1964. Der Ein-fluss des Kinetins auf den RNTS unid Protein-stof fwechsel in abgeschnittenen, mit Hemmstof-ften behanidelten Tabakblattern. Phytochemistry 3:241-48.

28. WVOOLHOUSE, H. NV. 1967. The nature of selne-escence in plants. Symp. Soc. Exptl. Biol. 21:179-214.

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effect of kinetin-naphthaleneacetic interaction upon … phyll content (wlhich we use as the primary...

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