Effect of Chloramphenicol on Light Dependent Development of Seedlings ofPhaseolus vulgaris var. Black Valentine, With Particular
Reference to Development ofPhotosynthetic Activity 1 2, 8
Maurice M. MarguliesDivision of Radiation & Organisms, Smithsonian Institution, Washington, D.C.
IntroductionSeedlings of higher plants germinated in solutions
of chloramphenicol (D-threo-N-dichloroacetyl-1-p-nitrophenyl-2-amino-1,3-propanediol) have their de-velopnment inhibited. The most noticeable effect isthe inhibition of the formation of normal green colorof leaves (13, 16). Chloramphenicol is a specific in-hibitor of protein synthesis in bacteria where it in-hibits the incorporation into protein of amino acidsattached to soluble ribonucleic acid (3). Chloram-phenicol can also inhibit amino acid incorporationcatalyzed by a cell-free system obtained from higherplants ( 14).
The work presented in this paper was undertakento study light dependent chloroplast maturation, aprocess that is not yet fully understood. Chloram-phenicol inhibition of this process would be one wayof indicating participation of protein synthesis. Aninhibitory effect of antibiotic on the light dependentdevelopment of photosynthetic activity of Phaseolusvudlgaris var. Black Valentine has been found. Inaddition, a partial inhibition of chlorophyll synthesiswas observed. Other light dependent responses ofthe plant were not affected.
Materials & Methods- Growth of Plant Materials: Phaseolus vldgaris
(L.) var. Black Valentine was used throughout.Etiolated plants were grown as described by Wolffand Price (20), except that seeds were not sterilizedwith hypochlorite. Normal green plants were grownin a greenhouse and were subirrigated with nutrientsolution. Etiolated plants were used 6 days afterplanting, and primary leaves of the greenhouse grownplants were used 8 to 12 days after planting.0 Application of Chloramphenicol: Chlorampheni-col was a gift of Parke, Davis and Co. Unless men-tioned, solutions of antibiotic contained 4 nig,/ml.
1 Received Dec. 2, 1961.2 Published with the approval of the secretary of the
Smithsonian Institution.3 Research was supported in part by funds provided by
U.S. Atomic Energy Commission Contract AT (30-1) -2373.
Solutions were applied to leaves by either of twoprocedures which follow.
I. Leaves, which included the entire epicotyl, ashort piece of hypocotyl, and one cotyledon (see fig1) were placed in petri dishes which contained a discof filter paper and chloramphenicol solution. Foreach square centimeter of petri dish area 1/10th mlof solution was used, so that contact with the cotyledonof each of the leaves was provided, but the leavesthemselves were not submerged. This procedure wasused when the effects of concentration of antibioticwere examined.
II. Intact plants were sprayed with a solutionof chloramphenicol applied as a mist from an atom-izer. The spray was directed at the hypocotyl hooksfrom above. For every flat of approximately 200plants 50 ml of solution were used.
- Irradiation of Leaves & Plants: Leaves or intactplants were transferred to an irradiation chamberan hour after they had been treated with chloramphen-icol. Irradiation was with light from white fluor-escent lamps. The intensity of irradiation was about1,000 /w/cm2 and was measured with a thermopile.This corresponded to an intensity of illumination ofabout 1,000 ft-c. Leaves in petri dishes were incontact with test solution throughout the irradiationperiod. Unless mentioned, this period lasted for 24hours. Except during white light irradiation periods,live plant material was handled in dim green light(19).> Determination of Anthocyanin: The 2-cm por-tion of hypocotyl from between 1.5 and 3.5 cm belowthe point of attachment of the cotyledons was ex-tracted and the anthocyanin content of the extractdetermined (16). Extractions were done in tripli-cate and ten 2-cm portions of hypocotyl were usedfor each extraction. Only relative values of antho-cyanin are given. The anthocyanin content of hypo-cotyls of unirradiated plants was not determined sinceit was negligible (9).o Determination & Separation of Chlorophylls: Asample containing only leaves was pressed betweenabsorbent paper to remove adhering water. Thesample was weighed and was then heated in boilingwater for 30 seconds. Chlorophyll was extracted bygrinding leaves with 80 % acetone in a Virtis-type
homogenizer. Particulate material was removed byfiltration and the chlorophyll content of the solutionwas determined according to Arnon (1). Each leafsample weighed 0.5 to 2.0 g. Usually, chlorophyllcontent of a batch of leaves was determined by ex-tracting three replicate samples.
The chlorophyll of chloroplast suspensions wasextracted by mixing with 80 % acetone. Particulatematerial was removed by centrifugation and the chlor-ophyll content of the solution was determined asabove.
Carotenoids were removedl from petroleum ethersolutions of leaf pigments by chromatography onsugar (17). The chlorophyll band was removedlfrom the column and repeatedly extracted with ether,or the column was developed further to separate thechlorophylls, and the individual chlorophyll bandswvere removed andl extracte(l. The ether solutionswere evaporated to dryness in vacuo, and the residues(lissolved in acetone. The petroleum ether solutionsapplied to the columns were prepared by repeatedlyextracting 80 % acetone extracts of heat-killed leafmaterial. Acetone was removed from the petroleumether by washing with water and the petroleum ethervas dried with anhydrous sodium sulfate. Absorp-tion spectra were measured with a Cary recordingspectrophotometer.0- Measurement of Photosynthesis: Photosynthesiswas determined by measuring the light dependent fixa-tion of carbon dioxide-C'4. Leaf samples were ex-posed to carbon dioxidle-C14 in \Vkarburg flasks orspecially constructed irradiation chambers similar tothe one described by Aronoff (2). Each leaf sampleweighedl about 50 mg and consisted of leaf pieces fromat least four different plants that had been treate(li(lentically.
'When Warburg flasks were used as irradiationchambers, only relative rates of carbon dioxidle fixa-tion were determined. Each flask had a capacity ofabout 20 ml, two side arms, and a flat rectangularbottom. The sodium carbonate-CI4 was preparedlfrom barium carbonate-C'4 obtained from Oak RidgeNational Laboratory. Sodium carbonate-C'4 (20-30 mc/mmole) (5-10 ,uc) was placedl in a sidearm. Carbon dioxide was generated by adding 0.1ml 10 % (w: v) phosphoric acid through a ventingplug. The flasks were then shaken for 10 minutesin the (lark, followed by irra(diation from below for10 minutes. At the end of the irradiation period,0.1 ml of 20 % KOH (w: v) was added to the secondsidle arimi through a venting plug. After 10 minutesthe flasks were opened and the leaves killed in boil-ing 80 % ethanol. For samples exposed to carbondioxide-C'4 in the (lark, the procedure was the same,except that leaves were not irradiated during the30 minute incubation period. The gas in the flaskswvas air. The tenmperature of the water bath was20 C. Light was provided by a pair of white fluor-escent lamps for each line of manometers. Thelamps were submerged in the water bath and yielded1,000 ft-c at the level of the flasks.
Irradiation chambers were used so that a largenumber of samples could be exposed to the same at-mosphere containing carbon dioxide-C14 of knownspecific activity. Two test chambers were arrangedin parallel with a gas generator and vacuum line.The chamber and gas generator were evacuated, and250 Mmoles of carbon dioxide-C14 of known specificactivity were generated. The gas in the generatorwas flushed into the chambers with carbon (lioxidefree air till the pressure was nearly that of the at-mosphere. The volume of the system which con-tained radioactive carbon dioxide was about a liter.One chamber was covered with aluminuml foil an(dthe other w-as illuminated for 10 minutes. Light,supplied from white fluorescent lamps, had an in-tensity of about 1,000 ft-c at the level of the chamber.The ambient air temperature was about 20 C. Afterillumination, the chambers w-ere evacuated, flushedwith carbon dioxide free air, and the radioactivecarbon (lioxide trapped in sodium hydroxide. Thenthe chambers were opened andl the leaf samlples (Irop-ped into boiling 80 % ethanol.
Leaves and ethanol extract were grouindl in aTenbroeck homogenizer. Aliquot portions of thehomogenate, correspondling to 0.2 to 1.0 mg of freslhleaf material were plated on 2.4 cm diameter plain-chettes, spread with additional ethanol as necessary,acidified with- a (Irop of 0.1 N hydrochloric acid an(ddried at 90 to 100 C. Radioactivity was determinedwxith a thin windlow gas flow (letector operatedl as aGeiger counter.
Fixation per gram fresh weight for the total in-cubation period was calculated for illuminated andunilluminatedl leaf samples. The fixation dependlenton the 10 minute illumination period is the differencebetween these two values, and was used to calculatethe rate of fixation per unit chlorophyll. The chloro-phyll content of leaves was estimated by analysis ofsamples other than those used to test for carbondioxidle fixation.
- Hill Reaction: Each 25 g of leaves was groun(lfor 1 minute in a WVaring blendor at line voltage with200 ml of solution containing: 0.4 AI sucrose: 0.05 Mrodiuim phosphate; 0.01 al NaCl; 0.01 Mi Ver-
sene acidl; adljusted to pH 7.0. The resulting honmog-enate was filtered through cheesecloth, and wasthen centrifuged at 250 to 500 X g for 2 minutes.The resulting supernatant suspension was centrifugedat 10,000 X g for 30 minutes. The pellet from thiscentrifugation was suspended in 2 to 5 ml of solu-tion with the aid of a Tenbroeck homogenizer, andthe suspension was filtered through glass wool. Thesolution used for suspending the pellet was the sameas that used for grinding leaves, except that Versenewas omittedl.
The Hill reaction with quinone as oxidant wasmeasured manometrically (18), and that withl ferri-cyanidle as oxidant was measured by observing thedecrease in optical density at 400 mg. Each 3.0 mlof reaction mixture contained 1.5 Lmoles of ferri-cyani(le and 2.0 ml of the buffer used for suspension
of the plastid particles. The millimolar extinctioncoefficient used for calculation of rates of ferricyanidereduction was 0.96 (A. T. Jagendorf, personal com-munication). Irradiation for the quinone reactionwas with light from white fluorescent lamps whichgave an intensity of 1,000 ft-c at the level of themanometer flasks. The light for the ferricyanidereaction was obtained from a 300 watt photofloodbulb and was filtered through 10 cm of 1 % (w: v)copper sulfate. The light intensity at the 3 ml cu-vettes used as reaction vessels was 5,000 ft-c. Thequinone Hill reaction was conducted at 20 C and theferricyanide Hill reaction at about 25 C.
Results
Whether or not leaves are treated with chloram-phenicol, the only visible difference after 24 hoursof irradiation is in the pigmentation of the leaves(fig 1). The same is true for intact plants (fig 2).In both instances chloramphenicol-treated leaves areyellow-green, while untreated controls are deep green.In both treated and untreated intact plants, the leavesopen and expand, hooks open, and hypocotyls becomered with anthocyanin. Irradiation of intact plantsfor another 24 hours produces very marked and ap-proximately equal expansion of leaves of treated anduntreated plants (fig 3). The untreated plants,however, are somewhat taller. In addition, the leavesof untreated plants contain starch while those ofchloramphenicol-treated plants do not. Starch wastested for with I2-KI solution after extraction ofleaves with boiling 80 % ethanol.
The lack of inhibitory action of chloramphenicolon hook opening has been confirmed using excisedhooks (table I). The irradiated hooks were exposedto 400 "w/cm2 of red light (600-700 m/A) for 10minutes, and then incubated in the dark for 24 hours,at which time hook opening was measured (8).Measurement of the anthocyanin content of hypocotylsof chloramphenicol-treated and control plants showedno inhibitory action of antibiotic at the end of 24hours of irradiation. However, a 25 % inhibition
Fig. 1 (top). The effect of chloramphenicol and lighton leaf development. Reading from left to right, thepetri dishes contain: (1) water, (2) 4 mg/ml chloram-phenicol, and (3) water. Dish 1 had been kept in thedark for 24 hours, and dishes 2 and 3 exposed to light for24 hours.
Fig. 2 (center). The effect of chloramphenicol onplant development in the light. The plants on the left hadbeen treated with a solution of chloramphenicol (4 mg/ml) and those on the right were treated with water. Bothgroups of plants were then exposed to light for 24 hours.
Fig. 3 (bottom). The effect of chloramphenicol onplant development in the light. The plants on the left hadbeen treated with a solution of chloramphenicol (4 mg/ml) and those on the right were treated with water.Both groups of plants were then exposed to light for 48hours.
The effectiveness of chlorophyll of control leaves wasthe same at 4 and at 24 hours.
The rates of carbon dioxide fixation were thesame for etiolated leaves that had been irradiated for24 hours or for greenhouse grown leaves, 100 to 150Amoles/mg chlorophyll/hour ( 150-200 Amoles/gfr wt/hr). The rates for chloramphenicol-treatedleaves were 2 to 10 Amoles/mg chlorophyll/hour.
Although chloramphenicol results in an inhibitionof (levelopment of photosynthetic activity wlhen it ispresent during the period of chlorophyll accumula-tion, it has no effect on photosynthesis when chloro-phyll is already fornmed (table IV). Leaves wereirradiated for 24 hours without chloramphenicol andwere then returned to the dark. Some of thenm weretransferre(d to dishes containing chloramlpheinicol.The renmainder were left in water. At the endl of 4lhours, both antibiotic-treatedl an(I control leaves weretestedl for the abilitv to fix carbon dioxi(le in thelight. No significaint (lifference vas note(l.
Table IV
Effect of Chloramphenicol on Photosynthesis of LeavesGreened by 24 Hours of Irradiation
was observe(d at the end of 48 hours of irradiation(table II).
Leaves irradiated for 4 or 24 hours in contactwith chloramphenicol solution did not have detectablephotosynthetic activity, even though considerablequantities of chlorophyll had been formed (table III).When it was assunmed that doubling of carbon dioxidefixation in the light over that in the dark could havebeen detected, it was evident that at the end of 4hours of irradiation, the chlorophyll in chlorampheni-col-treated leaves was one-fifth as effective as chloro-phyll in control leaves. At the end of 24 hours, thechlorophyll fronm treated leaves was not more thana tenth as effective as chlorophyll in coIntrol leaves.
Table III
Effect of Chloramphenicol on Developmentof Photosynthesis
Treatmentofetiolated~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Treatment of etiolated
leaves
Chloram- Hr
(4 mg/ml) irradiated
+
0
4240
424
Photosyn-thesis/gfr wt*(relativevalues)
-0.713.73.0.21.7
-2.1
Chloro-phyll( m.g/gfr x\t)
< 0.0050.291.4
< 0.0050.200.54
Photo-synthe-
sis/chloro-phyll
45
52
<10**< 4**
* Corrected for a dark fixation of about 2.** It was assumed that a total fixation of 4 or greater
could have been distinguished from the dark fixationof 2.
Chloram-Experi- phenicolment (4mg/
I-l)
1
2
Photosyn-thesis/gfr wt*
(relativevalues)
- 37+ 29- 16+ 14
Chlorophyll(mg/gfr wt)
Photosyn-thesis/
chlorophyll
1.5 251.3 221.5 111.3 11
* In Experiment 1, corrected for a dark fixation of 2; inExperiment 2, for a dark fixation of 1.
The green pigments of both treated aindl untreate(dleaves appear to be the saame. Thus, the absence ofphotosynthetic activity is not (lue to a lack of clhloro-phylls. Absorption spectra of 80 % acetone extractsfrom both sources can be superimposed fromii 550 to700 m,nu but not fromii 400 to 500 mA (fig 4). In thisregion. extracts froml chloramphenicol-treate(d plantshave the greater relative absorption. The dlifferencespectrunm shows absorption mlaxima at 475, 445, 420mni. These data show that the ratios of clhlorophylla to chlorophyll b in extracts of treated an(d untreate(lplants are approximately equal. The ratio of clhloro-phylls to carotenoi(ds, however, is lower in treate(dthan untreated plants. The green pigments are read-ily trainsferred fromI 80 C6 acetone to petroleunm etlher.This eliminiates the possibility that the pigmlenits arechliorophyllides (20).
\When carotenoi(ds are renmoved from the extractsby chromiiatography on sugar, the absorption spectrabecomle nearly superimiiposable from 400 to 700 ill,(fig 5). Tlis confirnms tllat the mlixture of greenpigments in extracts from treate(d andl control leaves
is the same. If chromatograms of extracts fromchloramphenicol-treated or untreated leaves are de-veloped further, two main bands are found. One ofthese has absorption characteristics of chlorophyll a.The major absorption peak in the blue was at 430mIA, and the minor at 412 mn. The nlajor peak in
Table V
Effect of Light Intensity & Chloramphenicolon Chlorophyll Synthesis
* Irradiation period lasted 16 hours.** Standard deviation.
the red was at 663 myA and the minor at 617 mi. Theratio of the heights of the major red maximum tothat of the major blue maximum was 0.78 for thepigment from untreated leaves and 0.82 for that fromtreated leaves. These values are in good agreementwith those of Harris and Zscheile (7). The pigmentcontained in the second band, probably chlorophyll b,had peaks at 432, 457, 617, and at 645 mA in the case
D of material from untreated leaves, and at 650 myA formaterial from chloramphenicol-treated leaves. Theratio of heights of the major red maximum to themajor blue maximum (645 mA band/457 mn band)was 0.35 for the pigment from untreated leaves, and0.41 for that from chloramphenicol-treated leaves.This green pigment from chloramphenicol-treatedleaves was contaminated with chlorophyll a as indi-cated by a shoulder in the absorption spectrum at 663m,u. The location of the minor red peak from ma-terial obtained both from treated and untreated leavesdiffers from that of chlorophyll b (7).
The results of a typical experiment testing theeffect of chloramphenicol concentration on chlorophyllaccumulation are presented in figure 6. Leaves wereirradiated for 18 hours. No inhibition was observedat 5 jug/ml, but a significant 15 % inhibition at 10
v(<'1
Fig. 4 (top). Absorption spectra in 80 % acetone ofextracts of leaves treated with 4 mg/ml chloramphenicol(closed circles) and control leaves treated with water(opent circles). The difference between the two is repre-sented by the remaining curve (crosses).
Fig. 5 (center). Absorption spectra in acetone of ex-tracts of leaves treated with 4 mg/ml chloramphenicol(closed circles) and control leaves treated with water(open circles) after removal of carotenoids.
Fig. 6 (bottom). Effect of chloramphenicol concen-tration on chlorophyll synthesis. The concentration ofchlorophyll in leaves without chloramphenicol was 1.08mg/g fresh weight.
Table VIEffect of Chloramphenicol Concentration oIn Ability of
Chlorophyll to Catalyze Photosynthesis
Chloram-phenicol,g/ml
045810204050100400
2,0004,000
Expt.Series 110010095 ±23
108±23
84±3170-+-10
. . .
. . .
18± 1469+5
Photosynthesis/chlorophyll*%c of control
Expt. Series 2
Expt. 1
100+15
64± 15
62±1043-+-24
25 +-622±8
Expt. 2
100-+ 10. .
108- 18.
120± 1688±7
48±624±5
. . .
* See text for details.
ug/nml. In some experiments, significant inhibitionwas not observed at 10 but at 20 ig/ml. A 60 %inhibition was observed at a concentration of 4 mg/ml.Occasionally inhibition as great as 80 % was observedat this concentration. A variation of light intensityfrom 125 to 940 ft-c produced only small changes inthe quantity of chlorophyll synthesized both in theabsence and presence of chloramphenicol (table V).Light intensity was varied by covering petri dishescontaining leaves with cheesecloth, or cheesecloth andtyping paper.
The relative abilities of chlorophyll, formed inthe presence of different concentrations of chloram-phenicol, to catalyze photosynthesis are presented intable VI. The data presented under the hea(ling,Experiment Series 1, represent the pooled results offour individual experiments in which the ability tofix carbon dioxide was tested in manometer flasks.The value for chlorophyll content and for carbondioxide fixed/g fresh weight of leaf for the control(no chloramphenicol) in each experiment was setequal to 100, and other values adjusted accordingly.In each experiment the ratio of photos)ynthesis to
chlorophyll for each concentration of chloramlphenicolwas calculated. Then the average value of this ratiofor each concentration of chloramphenicol was cal-culated for the four experiments and is presented inthe table.
Each column under the heading, ExperinmenitSeries 2, represents an individual experinment in wliclhcarbon dioxide fixation was conducted in irradiationchambers. This procedure permitted uniform ex-posure to light and atmosphere containing C14O. ofthree replicates for eachl of six groups of leaves, eachtreate(l with a different concentration of chloramiiplheni-icol. An equal nunmber of samples were exposed tothe same atmosphere, at the sanme time, in the (lark.Ratios of photosynthesis to chlorophyll content foreach leaf sample were calculatedI and, then the aver-age ratio for each concentration of chloramiiphenicol.The ratio for zero concentration of anitibiotic \\ aset equal to 100 aln(l the values for other conicelntra-tions adjusted accordingly.
Significant inhibition canI be obtaine(d at a coni-centration as low as 5 ug/nml. Inhibitioni is alwaysobserved at a concentration of 40 ug/nl or lowx er.Inhibition of 90 %tc is consistently observed at 4 ng/ ml.
The possible causes of variability within experi-ments has not been investigated. One possible sourceis the procedure by which chlorophyll contenit is esti-mated. For this estimation, leaf samples other thanithose actually exposedlto carbon dioxide-C14 wereused.
Ferricyanide Hill reaction activity of greenparticles from leaves treated with chloranmlphelnicol andirradiated for 24 hours is only a tenth that of particlesfrom control leaves not treated with antibiotic (tableVII). Activities of particles from control leaveswere about 200 ,umoles ferricyanide reduced /nigchlorophyll/hour. The same value is obtained forchloroplasts from mature primary leaves (11). Thenearly inactive particles from chloramplhenicol-treate(dleaves did not have an inhibitory effect oIn activeparticles. With cluinone as oxidant, particles fromlcontrol leaves evolved oxygen in the light at the samiierate as particles fronm plants grown in a greenihouse.Thus, it would seem probable that reduction of ferri-cyanide is also accomipanied by oxygen evolutioni.
le VII
Effect of Illumination of Leaves With or Without Chloramphenicoloni Ferricyanide Hill Reaction Activity of Green Particles From Them
Source of particles Changes inoptical density ,umoles ferri-
Leaves treated Leaves treated at 400 mTT cyanide reduced/mgwith water with 4 mg/ml (0-5 min chlorophyll/hr
chloramphenicol illunlination),g chlorophyll/3.0 ml
DiscussionChloramphenicol inhibits only some of the light
dependent responses of bean seedlings. Responsesaffected by antibiotic include chlorophyll synthesis,and the development of photosynthetic activity. Theformer is only partially, and the latter completelyinhibited. The inhibition of photosynthetic activitycan also be demonstrated on a subcellular level bya failure of chloroplasts to develop the ability to carry
out the Hill reaction. These light dependent re-
sponses inhibited by chloramphenicol are associatedwith the maturation of the chloroplast. In contrast,
other light dependent responses (opening of the hypo-cotyl hook, leaf expansion, and anthocyanin formationin the hypocotyl), at most, are slightly affected.
The mechanism of action of chloramphenicol hasbeen most thoroughly investigated in bacteria whereit has been shown to be a specific inhibitor of proteinsynthesis (3). It seems reasonable to conclude thatchloramphenicol is inhibiting light dependent mat-uration of the chloroplasts of bean because it isinhibiting protein synthesis. However, conclusiveevidence to support this view has not been presented.
Increase in levels of the photosynthetic enzyme,
triphosphopyridine nucleotide-linked glyceraldehyde-3-phosphate dehydrogenase, have been observed whenetiolated plants of bean (10) or pea (6) are irradi-ated. This would indicate that light dependent mat-uration of chloroplasts of higher plants is accom-
panied by protein synthesis. Therefore, chloroplastdevelopment should be sensitive to chloramphenicolif protein synthesis in these organisms can be in-hibited by the antibiotic.
Almost complete inhibition of protein synthesisof bacteria can be obtained at a concentration ofchloramphenicol of 10 iug/ml (3). In contrast, theincorporation of amino acids into protein by a cell-free system from maize endosperm is inhibited only60 % by 0.4 mg/ml (14). Thus the requirement forconcentrations of 0.4 to 4.0 mg/ml to obtain nearlycomplete inhibition of light dependent developmentof photosynthetic activity of bean leaves can be con-
sistent with action through a specific inhibition ofprotein synthesis.
The concentration of chloramphenicol needed with-in the plant to prevent development of photosyntheticactivity cannot be determined from the data pre-sented. It is probable that this concentration is lessthan that of the solution applied, since it has beenobserved that the concentration of chloramphenicolwithin higher plants can vary from 100th to 1/3 thatof the solution applied (4, 5, 12). These variationsdepend, in part, on the plant species used and on themode of application of the antibiotic.
The lack of Hill reaction activity of plastid ma-
terial from chloramphenicol-treated leaves can fullyaccount for the lack of photosynthetic activity ofthose leaves. It can be concluded that etiolatedleaves lack a substance, other than the photosyntheticpigments, which is needed for photosynthetic activity.
When present in photosynthetically functional leaves,this substance, possibly a protein, is intimately boundto the chloroplast. The lack of Hill reaction activityin plastids from antibiotic-treated leaves does notmean that this reaction is the only point at which acomponent necessary for photosynthesis is rate-limiting.
Like the inhibitory effect of chloramphenicol onthe development of photosynthetic activity, the in-hibitory effect of the antibiotic on chlorophyll ac-cumulation can also be interpreted in terms of aninhibition of protein synthesis. Two possibilities areconsidered: A, That the inhibition of chlorophyllformation is a result of increased photooxidation ofleaf pigments because of the inability of antibiotic-treated leaves to photosynthesize. The pigments ofleaves treated with inhibitors of photosynthesis aremore susceptible to the photooxidation than are thephotosynthetically active pigments (15). B, Thatchloramphenicol inhibits light induced formation ofenzymes necessary for chlorophyll synthesis. Suchadaptive enzyme formation has not been demonstrated,but might occur since the maximum rate of chlorophyllaccumulation is not achieved for 2 to 4 hours afterthe start of irradiation of etiolated bean leaves (21).This second explanation is more probable. The firstpossibility is improbable since an eightfold variationin light intensity has almost no effect on chlorophyllaccumulation whether or not antibiotic is present.
Summary
Etiolated plants that have been treated with chlor-amphenicol do not develop photosynthetic activitywhen irradiated. Synthesis of chlorophyll is marked-ly, but only partially, inhibited. Light dependentleaf expansion, opening of the hypocotyl hook, andanthocyanin formation are not inhibited by the anti-biotic. The inhibitory action of chloramphenical onchlorophyll synthesis is not affected by large changesin the intensity of irradiation. The inhibition of thedevelopment of photosynthetic activity is not due toan inhibitory action of chloramphenicol on photo-synthesis, nor to a lack of chlorophylls. Green par-ticles from photosynthetically inactive leaves, ob-tained by irradiation in the presence of chloramphen-icol, do not carry out the Hill reaction. This lackof Hill reaction activity can completely account forthe lack of photosynthetic activity of the leaves.The inhibitory action of chloramphenicol on the de-velopment of photosynthesis and on chlorophyll for-mation has been interpreted in terms of the knowninhibitory effect of the antibiotic on protein synthesisin bacteria.
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