ac and · pounds which might effect the greening process. in separate experiments, ... whereas, in...

CARBON DIOXIDE FIXATION BY ETIOLATED PLANTS AFTEREXPOSURE TO WHITE LIGHT1 2N. E. TOLBERT AND F. B. GAILEY 3

BIOLOGY DIVISION, OAK RIDGE NATIONAL LABORATORY, OAK RIDGE, TENNESSEE

Upon exposure of an etiolated plant to light, photo-chemical, biochemical, and physiological changes occur.Total chlorophyll synthesis and the conversion ofprotochlorophyll to chlorophyll a, have been studiedextensively (10, 13). Gross physiological changes andcessation of elongation have been the subject of manyinvestigations in plant physiology. Some of theseprocesses are independent of chlorophyll synthesis andphotosynthesis (22). A few changes in enzymatic ac-tivity during the greening of an etiolated plant havebeen reported (1, 8, 17).

Measurements of the formation of chlorophyll bythe etiolated plant after exposure to light have beenextensively investigated. Oxygen evolution by etio-lated barley plants after they were placed in the lightindicated that this process did not commence until theseedlings had been in the light 30 minutes (14). Ex-periments with etiolated oats also showed oxygen evo-lution after chlorophyll formation (6). Irving (9)reported that greening etiolated barley and bean seed-lings did not fix CO until they had developed a largepart of their chlorophyll. Observations have now beenmade on the fixation of C1402 and the productsformed during increasing time intervals after an etio-lated wheat plant has been placed in the light. Theresults indicate that for many hours after the start ofillumination, there is a slow and irregular formationof some of the reactions in CO2 fixation during photo-synthesis.

PROCEDUREEtiolated Thatcher wheat plants were grown at

240 C in sand with Hoagland nutrient solution. Onthe seventh day the plants were illuminated with be-tween 600 and 1000 fc of continuous white light from150-watt reflector flood bulbs. Water filters removedthe heat. Fifteen leaves were taken for chlorophyllanalyses (22) at hourly intervals after illuminationbegan. At the same intervals but with differentbatches of wheat, the ability of the leaves to fix CO2was measured. For this latter test, one or two leaveswere exposed in a small-scale photosynthesis chamber(7) for 10 minutes at 1000 fc of light to an atmos-phere of air containing C1402. The volume of thechamber was 100 ml into which was released 25 /Ac ofC14 from NaC1403 of a specific activity of 25 % C14so that the final CO2 concentration did not increaseappreciably. During this period the temperature waskept at 230 C by submerging the apparatus in a waterbath.

1 Received May 12, 1955.2 Work was performed under contract no. W-7405-

eng-26 for the Atomic Energy Commission.3 Research Participant during Summer 1954. Present

address: Biology Department, Berea College, Berea,Kentucky.

At the end of the 10-minute period, the C14-ex-posed leaves were killed by maceration in boiling 30 %methanol. An aliquot was counted for total C14 fixa-tion in the soluble extract. Another portion wassubjected to paper chromatographic analysis, water-saturated phenol being used as the first solvent andbutanol-propionic acid-water for the second develop-ment (3). Autoradiographs were made of each chro-matogram, with No-Screen x-ray film to locate theradioactive compounds; the total counts per spotwere determined with a G-M tube. This activity wasthen expressed as a percentage of the total activity onthe chromatogram or as actual counts per second ofthe compound in one leaf. Since it was not practica-ble to start with an exact quantitative aliquot on eachpaper chromatogram, counts of radioactivity in thecompounds among different experiments may be inerror by as much as 15 %. The percentage distribu-tion of radioactivity within one experiment should notbe more variable than the counting errors.

In later experiments etiolated plants were exposedto light after first being sprayed with various com-pounds which might effect the greening process. Inseparate experiments, plants were sprayed with water,0.3 M glucose, 0.3 M ribose, 0.1 TM mixture of 20 %sedoheptulose and 80 % sedoheptulosan (designatedsedoheptulose after its physiologically active compo-nent), or 0.1 AI glycolic acid. The plants of each-experiment were sprayed, while still in the dark, at24, 22, 19, 16, and 0 hour intervals before illumination.Bottles for spraying indicator solutions on paperchromatogframs were used. These conditions werechosen since the same procedure with glycolic acid hasbeen shown to activate glycolic acid oxidase (17).Glycolic acid at pH 2.3 was used since these previousexperiments have shown that the sodium salt of theacid when sprayed on leaves was not effective in acti-vating glycolic acid oxidase. After exposure to lightand just before the 10-minute photosynthesis test withC1402, the leaf blades were cut off and washed brieflyunder running, distilled water to remove excess re-agent. The leaves appeared normal and contained novisible bacterial or mold contamination from thistreatment.

RESULTS AND DISCUSSIONSeveral light intensities were used in trial experi-

ments to ascertain the most advantageous conditionsfor chlorophyll synthesis. These qualitative resultsindicated that light intensities around 1000 fc producethe most rapid greening; whereas, in very high lightintensity from a photoflood bulb or sunlight, theplants greened very slowly. Of equal importance,etiolated leaves cut off at their base and placed inwater turned green and fixed C1402 at much slowerrates than in experiments where the plant remained

491

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PLANT PHYSIOLOGY

intact during the greening period. Racusen andAronoff have shown that decapitated leaves incorpo-rate amino acids into protein more slowly than in theintact plant (11), and thus the slower greening proc-ess in decapitated etiolated leaves in the present ex-periments may reflect less rapid protein synthesis forthe formation of the chloroplast. However, cuttingoff the leaf for the final 10-minute photosynthesis ex-periment with C1402 did not seem to affect the C1402fixation. Thus there are several differences betweenthe present experiments and those of Irving (9):1) The use of the whole etiolated plant rather than acut-off top has been found necessary for satisfactoryresults; 2) more controlled and somewhat higher lightintensities have been used; and 3) radioactive C1402has permitted measurements of rate of fixation andproducts formed. It has not been possible to ascer-tain the effect of unlabeled CO2 produced inside thecell by respiration on the rate of fixation of externalradioactive C1402. However, the absence of C14 inthe products of the path of carbon in photosynthesisfor several hours of greening would indicate no photo-synthetic fixation, and permits qualitative measure-ments on the formation of this process.

CHLOROPHYLL FORMATION AND CO, FIXATIONRATES: Figure 1 shows chlorophyll content and totalC1402 fixation at various times after etiolated leaveswere first exposed to light. A very small amount ofchlorophyll was present after the plants had been inthe light for 1 hour, owing to the rapid light-catalyzedconversion of protochlorophyll to chlorophyll, butlarge amounts were not produced for 2 to 3 hours.The rate of chlorophyll production was most rapidfrom about the third to the twelfth hour of light.After this period, the amount of chlorophyll in theplant increased more slowly, on wet weight basis, forabout 20 hours. After 1.5 days of continuous light,the now fully green plant contained a normal amountof chlorophyll.

The rate of C140, fixation wats very slow for about1 to 2 hours after rapid chlorophyll production hadstarted, i.e., after abouit 5 hours of continuous light.The amount fixed before this time, as measured accu-rately by the isotope method, amounted to less than

c/s C40, FIXEDRATO

q OF CHLOROPHYLL

FiG. 1. Totalduring greening.

DURATION OF EXPORE TO LIGHT (hr)

chlorophyll and riate of C'402 fixation

0.1 % of that fixed by the leaves after greening. Thuswhen these etiolated plants were placed in the light,several hours were required for the rate of chlorophyllsynthesis to become significant, and then 1 to 2 morehours of light before photosynthesis commenced, asmeasured by C1402 fixation. Total fixation of C1402began to increase at a rapid, linear rate after photo-synthetic fixation began, around the fifth hour of light,and so continued to increase during the next 24 hoursor longer. After about 32 hours the plant was fullygreen and fixed CO2 at a rate equal to that of a nor-mal plant. Almost all the C14 was fixed into the solu-ble extract during the 10-minute photosynthesis testperiod with C1402 by the greening plants and thedata reported are values for the activity in the solublefraction only.

Smith (14) noted that oxygen evolution duringgreening in etiolated barley seedlings began within 30minutes which indicated that a Hill reaction was fune-tioning in the whole leaves. At that time the chloro-phyll present was chiefly the small amounts formedby the initial photochemical conversion of protochloro-phyll to chlorophyll. Evolution of oxygen from oatleaves soon after exposure to light has also been re-ported (6). CO2 fixation, however, appears to com-mence later if comparison between these differentmonocotyledonous plants is reasonable. Such a com-parison seems valid in that the rate and time ofchlorophyll synthesis were similar.

The ratio of the rate of C14O2 fixation to totalchlorophyll is given across the top of figure 1 for eachperiod of analysis after the etiolated plants wereplaced in the light. Data on amount of chlorophyllformed has been reported as microgram per gram offresh tissue weight, in conformance with similar fig-ures in the literature. The average weight of one leaf(< 100 mg) for each analysis was determined forcalculating this ratio, since the leaves for C1402 fixa-tion experiments were not weighed, in order to facili-tate rapid killing for subsequent chromatographicanalysis of the products. The ratios indicate thatwith increasing time in 1000 fc of light the chlorophyllpresent in the greening wheat became much more effi-cient in promoting CO2 fixation. This ratio, calcu-lated for these particular experimental conditions, andC14 specific activity could vary with changes in lightintensity, temperature, and C02 partial pressure. In-creasing light intensity and CO., pressure might tendlto increase the difference in this ratio since the verysmall fixation rate in the slightly green plant shouldnot be limited by these factors. However, furtherstudies based on Blackman's principle of a rate-limit-ing step in the multiple process of photosynthesisshould aid in determining which portions of photosyn-thesis are rate limiting during various times in thegreening process.

PRODUCTS OF C1402 FIXATION: The productsformedI by fixation of C1402 for a 10-minute periodat the end of each hour after the plants had beenplaced in white light were analyzed by paper chroma-tographv. Figures 2 A and B are autoradiographs of

492



TOLBERT AND GAILEY-CO2 FIXATION

AL ANINE

GLUTAM ICAGOlD

GLUTAMINE

C °20 ATION BYETIOLATED HATCHER WHEATAFTER i FlH JrFLJGHT

SUCROSE

PHENOL-WATER

W.

AFTF~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

...2H

.:.ATFXTN BY'

ETIOLAICOr THiATTER W!HEATAFTE-R it PRA .OF LIGHtj

PHENOL- WATER

etiolated plants were placed in the light, the onlyM-ALIO three products to be labeled by C14O2 in significantAGID02isinfct

< amounts were malic, aspartic, and glutamic acids. Thecurve for percentage of C'4 in glutamic acid (not

c shown) was similar to that for aspartic acid; however,c there was only about one-half as much label in glu-a tamic acid. These products represent respiratory fix-

- ation. As will be shown later, small amounts of C14ASPARTIC ACID in phosphoglyceric acid (PGA) and sugar phosphates

were also detectable by the isotope method after 4hours of light. But the amount of this photosyntheticfixation was not significant even though the plants

PHOSPHATE were in the light and had formed appreciable amountsESTERS of chlorophyll by this time......._ MG_ As the plant greened during 2 to 8 hours of light,

t the total C14 incorporated into malic, aspartic, and¢ glutamic acids increased manyfold as chlorophyll was

produced (table I). The great increase in the amountof total C14 in the respiratory products was perhapsstimulated by the photosynthetic reducing power be-

ACL rTli

t, TABLE IZI RELATIVE AMOUNTS OF C1 INCORPORATED IN MALIC,cT ASPARTIC, AND GLUTAMIC ACIDS2

PGA...

'Fw ROP

.cI.x ...

tIDPG ': :'.

OR ISIN

FIG. 2. Products from C"402 fixation: A (above),after 4 his of light; B (below), after 12 hrs of light.

the clhromatograms of leaves which had had 4 hoursand 12 hours of light, respectively. C14 activity ineach compound was counted after detection on thechromatograms by autoradiography, and is expressedin figure 3 as a percentage of the total C14 fixed dur-ing that period. During the first 4 hours after the

ALANINE

A

0

A SERINE

A ^ t

4 8 12 16 2DURATION OF EXPOSURE TO LIGHT (hr)

FIG. 3. Percentage of total C'4 fixed in certain com-

pounds with increasing lumination periods.

DURATION TOTAL FIXATION PER LEAF PER 10 MINOF LIGHT * MALIC ASPARTIC GLUTAMIC

hr c/s c/s c/s2 >1 >1 <14 4 10 36 7 29 68 22 71 319 54 97 4512 56 50 3824 164 232 15832 268 334 46

* For chlorophyll concentration atperiods, see figure 1.

each of these

ing produced by the newly formed chlorophyll. Theearly increase was probably not caused by an increasein the pool size of newly formed intermediate com-pounds in the photosynthetic cycle, since these com-pounds had not yet accumulated in appreciableamounts.

The first product, other than the respiratory acids,to become labeled with C14 after the etiolated wheatplants had been placed in the light was a smallamount of PGA after about 4 hours of light (tablesIV, V, VI). At the fifth hour, PGA and alanine con-tained an abnormally high percentage of the total C14fixed. Figure 3 shows the percentage of C14 in alanineat this time. The C14 activity in alanine was proba-bly derived from PGA through pyruvic acid, thoughthe intermediate C3 compounds did not accumulate inappreciable amounts. It appears that the plants, atthis stage of greening, were able to reduce little of thePGA to the aldehyde level and then convert it tocarbohydrate. With plants exposed to white light forlonger periods of time, the percentage of the total C14fixed in the alanine dropped to a smaller, btut there-

9-

7.

5

z

x

fi40'

g 30'

20

10^-

493



PLANT PHYSIOLOGY

after constant, amount. However, the total radio-activity in the C3 compounds continued to increasewith increasing rate of C'402 fixation.

These results suggest that after 5 hours of light,the plant was able to commence slow photosyntheticcarboxylation to form PGA, of which part was con-

verted to alanine. Several explanations are then pos-

sible to explain the accumulation of alanine and PGA,with little C14 activity appearing in the sugars. Atthat stage, the plant may not have had sufficient re-

ducing power or active enzymes to reduce rapidlyPGA to hexose. On the other hand, saturation of thealanine and PGA pools may have been necessary forequilibria favoring synthesis of sucrose. In eithercase, this condition seemed to exist for an interval of1 to 2 hours at about the fifth hour after etiolatedThatcher wheat had been placed in white light.

After exposure of the plant to light for longer than5 hours, quantities of C14 began to appear in thesugar phosphates and sucrose. The percentage of C14going into sucrose and sugar phosphates, as reflectedby sucrose (fig 3), increased rapidly at this time, toreach a high and relatively constant maximum per-

centage value after the plants had been illuminatedfor 9 to 12 hours. Longer periods of greening seemedto have no further effect on the percentage of thefixed C14 which appeared in sucrose. However, it isimportant to note that after this period of light, thetotal C14 fixed was still only 15 to 20 % of that of a

fully greened plant after 32 hours of light (fig 1).The data indicate that by this time the reactions inthe reduction of PGA were functioning at about theequilibrium conditions of normal steady-state photo-synthesis. The increase in total fixation rate after 12hours must be the result of the increasing rate ofother, more slowly developing processes; such as for-mation of chlorophyll and of the CO2 acceptor, whichis ribulose diphosphate (RDP) or related to it. Onthe basis of the increasing chlorophyll efficiency ratiofor total CO2 fixation with further greening, still otherfactors probably exist wlhich limit photosynthesis afteronly 12 hours of greening.

FORMATION OF SERINE AND GLYCINE: Serine andglycine acquired C14 from C1402 during greening ofthe etiolated plant on a different time sequence fromthat of alanine (fig 3). PGA and alanine becameradioactive during the 10-minute C1402 fixation period

in leaves that had greened for 4 and 5 hours but gly-cine and serine were not labeled. In fact, about 20hours of light were required for a large amount of C14to be incorporated into serine. During photosyntheticfixation experiments with C1402 by green barley or

wheat plants, glycine and serine are always labeledwith a large portion of the C14 fixed (5, 16). It hasbeen assumed that these two amino acids were labeledquickly during short-time photosynthesis experimentsbecause they were end products of a short side reac-

tion from the carbon cycle of photosynthetic fixation(12, 18, 20, 21). At least two pathways have beenproposed for glycine and serine formation in plants;namely, the formation of serine from PGA, or their

formation from glycolic and glyoxylic acids. Glycolicacid is readily converted to glycine and serine inplants (12, 18) and should arise from the two carbonfragments by the action of transketolase on the sugarphosphates formed during photosynthesis. Further-more, it has been shown that a key enzyme, glycolicacid oxidase, of this side reaction is activated in etio-lated plants during the greening process in order toestablish this pathway (17). For Thatcher wheat,this activation period required about 20 hours of light.In the present experiments, again with Thatcherwheat, a rapid movement of C14 into serine was alsonot established until after 20 hours of light, whichimplies that the major pathway for the in vivo incor-poration of C14 into serine during photosynthesis inthis plant is via glycolic acid. Serine did not appearto be formed directly from triose and PGA, as hasbeen proposed for soybeans (20), since it was not sub-stantially labeled during the first 20 hours of greening,while alanine and PGA were being prodluced withlarge amounts of C14.

CO2 FIXATION BY LEAVES SPRAYED WITH VARIOUSCOMPOUNDS: A more detailed analysis has been madeof the organic phosphate esters of the path of carbonin photosynthesis during the first hours the etiolatedplant acquired the ability to fix C1402 photosyntheti-cally. Also, amounts of several of these intermediateswere raised artificially by spraying them on the etio-lated plant before exposure to light. Since the per-centage incorporation of C14 in sucrose had not variedsignificantly after the plants had been in the light for9 to 12 hours, in these experiments data on plants inthe light for longer than 8 hours were not kept exceptfor one 25-hour exposure.

Total C14 fixed by approximately equal leaf sam-ples sprayed with water in the dark 24 hours (used ascontrols) did not vary significantly from that fixed byunsprayed plants (table II). Feeding glucose to theetiolated plant prior to the experiment gave some in-crease in the rate of chlorophyll syntlhesis but a sub-

TABLE IIINFLUENCE OF SPRAYING VARIOUS SUBSTRATES ON ETIO-

LATED WHEAT LEAVES PRIOR TO ILLUMINATIONON RATE OF PHOTOSYNTHESIS

SOLUTION SPRAYED ON LEAVES PRIOR TOLIGHT EXPOSUJRE

DURATIONOF LIGHT SEDO- GLYCOLIC

WATER * GLUCOSE RIBOSE HEPTU- ACIDLOSE

hr c/s c/s c/s c/s c/s2 13 4 8 3 103 8 48 75 4 124 54 92 350 11 235 103 274 253 57 1516 238 230 172 180 3747 300 527 303 290 2508 725 510 478 370 550

25 1042 1800 1471 1350 1400

* Total C14 fixed during a 10-min test period withC`40, into the water soluble extract from 2 leaves.

494




stantially higher rate of CO2 fixation during the criti-cal time after 3 to 5 hours of light. Ribose fed to theetiolated plant stimulated CO2 fixation even more.

For both sugars, there still remained a 2-hour lag inlight before CO2 fixation began. In both cases, butnot for the water-sprayed control, after 6 hours oflight there was a great falter in the rate of increaseof CO2 fixation by the plant as if some precursor or

factor had been exhausted, and its synthesis was ratelimiting for a few hours thereafter. One explanationfor this may be that the presence of the free sugars

in large amounts in the leaf resulted in an unusualdemand on adenosine triphosphate (ATP) for theirphosphorylation before they could be used for theplant's growth. Thus much of the ATP produced bythe photosynthesis process (15) may not have beenavailable at this time for regeneration of the inter-mediates for photosynthesis.

Sedoheptulose sprayed on the etiolated leaves inthe dark inhibited CO2 fixation rather than stimu-lating it. These results were not expected since sedo-heptulose phosphate is intimately involved in themetabolic processes of photosynthesis (4). Since 80 %of this sugar was present in the spray as its anhydride,and since this anhydride is not metabolized by barleyleaves (19), there is a possibility that the inhibitionof the path of carbon of photosynthesis during green-

ing may be due to this unnatural form of the seven

carbon sugar.

Glycolic acid had no effect on the rate of CO2 fixa-tion during the first hours the leaves were in the light.Glycolic acid, when applied in exactly the same man-

ner, has been shown to activate glycolic acid oxidasein this plant (17). Therefore, this enzyme does notappear to be rate limiting in photosynthesis duringgreening, but rather is a side reaction from thisprocess.

After illumination for about 1 day, all the plantssprayed with the various compounds had a higher rateof C140., fixation than the water-sprayed control.

Compounds sprayed on the plant were then perhapsbeing utilized to stimulate growth of the plant.

In table III are tabulated the amounts of radio-active carbon fixed in sucrose and the organic phos-phate esters located on the chromatograms betweenPGA and the origin. These compounds represent amajor fraction of the total C14 fixed and include allthe sugar phosphates and PGA which are the activeconstituents of the path of carbon in photosynthesis.Glucose-, and especially ribose-treated plants, had astimulated sucrose synthesis after 4 hours and againafter 25 hours of illumination, in comparison to thewater controls. The data indicate that the increasedtotal fixation (table II) was a result of photosynthesisand not of greatly enhanced respiratory fixation.

COMPOSITION OF ORGANIC PHOSPHATE ESTERS:The first product of photosynthetic C1402 fixation iscarboxyl-labeled PGA and the immediate precursorfor this carboxylation is probably the pentose sugar,ribulose diphosphate (2). In the greening, etiolatedleaf, the pool size of this precursor should be vanish-ingly small or nonexistent. Analysis of the plant tis-sue by the chromatographic solvents employed gavepoor separation of the phosphate esters. These areason the chromatogram have been designated accordingto their major component as PGA, HMP (hexosemonophosphate), UDPG (uridine diphosphate-glu-cose), and RDP (fig 2B). A more detailed analysisof these phosphate ester areas has been made byeluting each phosphate area separately and hydro-lyzing the ester with a crude phosphatase preparation(Polidase S). Rechromatography of the organic moi-ety containing the C14 gave good separation and pro-vided identification of the original phosphate estersin each area. In tables IV, V, and VI, the yields ofsuch dephosphorylated products have been given rela-tive scores to indicate the order in which these keyesters acquire C14 from C1402 during the greeningperiod.

Results in table IV are for the Thatcher wheat

TABLE IIISUCROSE AND ORGANIC PHOSPHATE ESTERS FORMED DURING PHOTOSYNTHESIS

BY GREENING WHEAT LEAVES

REAGENT SPRAYED ON LEAVES BEFORE EXPERIMENT

DURATION WATER GLUCOSE R-BOSE SEDOHEPTULOSE GLYCOLIC ACIDOF LIGHT

SUCROSE PHOS- SUCROSE PHOS- SUCROSE PHOS- SUCROSE PHOS- PHOSPHATES ~PHATES PHATES PHATES SUCROSE PHATES

hX C/S C/S C/S C/S C/S C/S C/S C/S C/S C/S2 Trace < 1 0 Trace Trace 1 0 Trace Trace <13 Trace < 1 4 8 6 14 Trace Trace <1 < 14 4 10 8 13 1 3 2 45 8 25 42 79 42 84 4 13 20 346 50 54 46 80 36 37 11 31 38 887 42 68 94 157 34 67 33 61 49 568 140 131 136 181 88 112 33 77 107 120

25 217 143 705 337 601 302 334 138 443 296

* Phosph1ates include the C'4 in PGA, HMP, UDPG, and RDP. A more detailed analysis of these areas is givenin tables IV, V and VI.

495



PLANT PHYSIOLOGY

TABLE IVCOMPOSITION OF PHOSPHATE ESTERS FORMED DURING1O-MIN PHOTOSYNTHESIS BY ETIOLATED WHEAT LEAVESAFTER 24-Ha SPRAYINC WITH WATER AND THEN EXPOSURE

TO WHITE LIGHT

DURATION HYDROLYSIS PRODUCTS *OF LIGHTAND GLY- GLU- FRuc- SEDO- RIBU- Ri-

PHOSPHATE CERICCS TOEHEPTU-LOEBSARES AIDCOSE TOSE LOE LOSE BOSEAREAS ACID LOSE

2 hrTotalarea Trace Trace 0 0 0 0

3 hrPGA ... + 0 0 0 0 0HMP ... O Trace Trace 0 0 0UDPG ... O Trace 0 0 0 0

4 hrPGA .... ++ Trace Trace 0 0 0HMP .... Trace ++ + Trace 0 0UDPG...0 + 0 0 0 0RDP ..0. Trace Trace Trace 0 0

5 hrPGA .... ++ Trace Trace 0 0 0HMP .... Trace ++ + Trace 0 0UDPG ... 0 + 0 0 0 0RDP ....0 Trace + Trace Trace Trace

6 hrPGA.-II-I- Trace 0 0 0 0HMP.... Trace ++ + Trace 0 0UDPG ... 0 + 0 0 0 0RDP ....0 Trace + Trace Trace Trace

25 hrPGA.-H-IITrace + 0 0 0HMP.... + +++ ++ ++ 0 0UDPG ... 0 ++ 0 0 0 0RDP .... Trace Trace Trace Trace Trace Trace

* The C1 labeled PGA, HMP, UDPG and RDP areaseach were eluted separately from the original paperchromatogram, enzymatically hydrolyzed with PolidaseS, the products rechromatographed, and autoradiogramsmade of new chromatograms. These autoradiographswere scored by visual observation while comparingwhole sets of chromatograms. The total C14 in the com-bined organic phosphate areas before hydrolysis aregiven in table III.

leaves that had been sprayed with water and serve ascontrols for tables V and VI. After the etiolatedplants had been in the light for 3 hours, detectable C14activity was present in PGA, though this was but asmall percentage of the total fixed into respiratoryproducts. A vanishingly small trace of activity (toosmall to count) could be detected by autoradiographyin glucose and fructose from the HMP and UDPGareas. The results indicate that the PGA pool hadbuilt up first. After 4 hours of light the plant canreduce a little C1409 to the hexose sugar phosphatesbut the predominant fixation was still into malic,aspartic, and glutamic acids. The appearance of C14in the hexoses indicates some reversal of the Embden-Meyerhof pathway at this stage.

The 10-minute exposure of C1402 used in theseexperiments would be considered so long a labelingperiod in photosynthesis with a fully green leaf thatabout uniform distribution of C14 in the carbon atomsof all the sugars would be expected. Therefore the

appearance of significant amounts of C14 in PGAalone over a 10-minute period represents a trenien-dous change in rate.

Only traces of C14 activity were present in RDPfrom the 5th- to the 25th-hour analyses after illumi-nation of the leaves. Sedoheptulose phosphate didnot appear to accumulate significant amounts of labeluntil almost a day after the etiolated plants wereplaced in the light. This suggests that one reason forthe lag in CO2 fixation during the greening period andfor the low efficiency of the newly formed chlorophyllwas the failure of the plant to drive the photosyn-thetic cycle at a rate sufficiently high to regenerateenough RDP. The reversal of the Embden-Meyerhofpathway seemed to have been functioning satisfac-torily, probably because many of its enzymes hadbeen involved in the growth of the etiolated seedling.

It is impossible to determine by these data alonewhether the absence of active enzymes in the re-generation of RDP may have been responsible forslow recycling of the C14 to ribulose diphosphate.The very short lag phase in CO2 fixation (secondsto a few minutes) by a green plant, when transferredfrom dark to light, should be compared to the lag inphotosynthesis of over 24 hours during the greeningof the etiolated plant. The length of the inductionperiod in photosynthesis by a fully green plant maybe a measure of the rapidity with which the plant can

TABLE VCOMPOSITION OF PHOSPHATE ESTERS FOR-MED DURING1O-MIN PHOTOSYNTHESIS BY ETIOLATED M\ HEAT LEAVESAFTER 24-HR SPRAYING WITH GLUCOSE AND THEN Expo-

SURE TO WHITE L,JGHT

DURATIONOF LIGHTAND

PHOSPHATEAREAS

HYDROLYSIS PRODUCTS

GLY- GLU-CERIC COSEACID

2 hrTotal areas Trace 0

3 hrPGA ... -+ TraHMP .... Trace +4UDPG... O +

4 hrPGA ....+4+ 0HMP .... Trace +iUDPG ... O+0

5 hrPGA. -H±++ TraHMP.... + +4UDPG andRDP ... Trace +-

6 hrPGA .....- TraHMP .... + +4UDPG ... 0 -HRDP ..... O Tre

25 hrPGA. ±4+- TrmHMP + +-UDPG ... Trace +RDP. Trace +

iee

ace4-+

+-

acet-+

ace

ace

4+

FRUC- SEDO- RIBU- RI-TOSE H LOSE BOSE

LOSE

0 0 0 0

+ 0

+ TraceTrace Trace

Trace 0

+ TraceTrace Trace

Trace 0

+

Trace Trace

Trace 0

++- +

Trace 0

Trace Ti-ace

Trace 0

+

0 0

Tracc Trace

0 00 00 0

0 00 00 0

0 00 0

Trace Trace

0 00 00 0+ Trace

o 00 00 0t-+- +

496

+




TABLE VICOMPOSITION OF PHOSPHATE ESTERS FORMED DURING1O-MIN PHOTOSYNTHESIS BY ETIOLATED WHEAT LEAVESAFTER 24-HR SPRAYING WITH RIBOSE AND THEN EXPOSURE

TO WHITE LIGHT

DURATION HYDROLYSIS PRODUCTSOF LIGHTAND GLY- GL-Fu-SEDO-RIu RiPHOSPHATE CERIC GLI. FRUC- HEPTU- RIBU- RI-PHOPHAE CRICCOSE TOSE LET-[OSE BOSEAREA ACID LOSE

2 hrPGA ..... + + Trace 0 0 0UDPG ...0 Trace 0 0 0 0

3 hrPGA ....± Trace + Trace 0 0HMP .... Trace ± + + 0 0UDPG ... O + Trace 0 0 0RDP .... 0 Trace Trace Trace 0 0

4 hrPGA ..... Tirace + Trace 0 0HMP .... Trace ++ + Trace 0 0UDPG andRDP0... + 0 0 0 0

5 hrPGA ..... ++1 0 0 0 0 0HMP.... + +tl- +1- + 0 0UDPG andRDP . Trace -i--i- + Trace Trace 0

6 hrPGA. -i-H 0 0 0 0 0HMP.... O ++ ±+ + 0 0UDPG... O + 0 0 0 0RDP ... O Trace + Trace Trace Trace

25 hrPGA .....I + ±± + 0 0HMP + ±±++ + ++ 0 0UDPG Trace-Oi- 0 0 0 0RDP ... Trace + Trace Trace ±+ +

build up the pool size of carbon compounds in photo-synthesis provided all the enzymatic processes arepresent. The 24-hour lag in photosynthesis and pro-duction of significant RDP by a greening etiolatedplant, therefore, can hardly be explained by the slowaccumulation of significant pools of these compounds.Rather, it would appear that synthesis of adequateamounts of enzymes to catalyze some of these reac-tions might be a limiting factor.

In the area of the sugar phosphate esters there wasone unknown with an Rf of 0.2 in both the solventsused in chromatography. This unknown was nothydrolyzed by Polidase S. Its pool size with respectto total C14 became constant after the etiolated leaveshad had only 4 hours of light. Thus the rate of label-ing of this unknown was like that for PGA and ala-nine at the fifth hour of greening, except that it waspresent in rather small amounts and as the plantgreened further, more total C14 was not incorporatedinto the compound. In these respects it was uniqueamong the compounds being labeled by the C1402.

When the etiolated leaves were sprayed with glu-cose prior to illumination, the rate and amount ofC1402 incorporation into PGA and the hexose werestimulated (table V). However, detectable label inpentose phosphates did not occur in the leaves until

after they had been exposed to the same long periodof light as required by the water-sprayed controls.Leaves sprayed with ribose prior to the light exposurehad an even faster and more diversified buildup ofthe organic phosphate esters of photosynthesis (tableVI). In these leaves, there were measurable amountsof PGA and hexose phosphates after only 2 hours oflight; after 3 hours of light, considerable sedoheptu-lose phosphate was present, although it did not ap-pear with the water controls until the fifth hour, andnot in this amount until the twelfth hour of light.Nevertheless, RDP still showed the same small poolsize at these times. This may be caused by somelimiting step unaffected by the excess ribose present;or the large pool of fed and unlabeled pentose mayprevent, by mass action, rapid recycling of the fixedC1402 through RDP. This would have resulted in allthe newly formed C14-labeled PGA being used forhexoses and sucrose svnthesis. However, after theplants had been in the light for 1 day, substantialRDP was present, which probably reflects the stimu-lated total C02 fixation by plants fed this sugar.

In table VII are summarized the relative amountsof C14 appearing during the 10-minute photosynthesisperiod with C1402 in the products of an etiolatedThatcher wheat plant after exposure to 3 hours oflight. At this time, C14 label could be detected in thefirst photosynthetic fixation products, although theamount fixed in them was not an appreciable percent-age of the total fixation. Prior spray feeding of eitherglucose or ribose solutions to the etiolated leaves inthe dark stimulated C14 fixation into PGA, glucoseand fructose phosphates, and sucrose, but not intoRDP.

Since the unique carboxylation reaction of photo-synthesis utilizes RDP, the production of C'4-labeledPGA, hexoses, and sucrose without producing detecta-ble label in the pentoses or RDP during the earlyphases of the greening process indicate that the pen-toses were present in exceedingly small and rate-limit-ing amounts. As expected, feeding ribose to the etio-lated plant resulted in a large increase in photosyn-thetic fixation at these times, since the plant should beable to convert it to RDP. In an etiolated plant not

TABLE VIIRELATIVE AMOUNTS OF C14-LABELED PRODUCTS FROM1O-MIN PHOTOSYNTHESIS BY ETIOLATED WHEAT

AFTER EXPOSURE TO 3 HR OF LIGHT

SOLUTIONS SPRAYED ON LEAVESPRIOR TO LIGHT EXPOSURE

PRODUCTS SEDO- GLY-WATER GLu- RI- HEPTULOSE- COLIC

SEDOSAN ACID

Aspartic acid -H. +l++ 4-f--H- -H-+ +++Malic acid -H-+--+++II ±-1 -++ ++PGA area + ± ++ 0 +HMP area ... Trace -iH 1--f Trace +UDPG area .. Trace + + 0 TraceSucrose ...... Trace -IH ++± Trace +

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PLANT PHYSIOLOGY

sprayed with ribose, other sources for the RDP mustbe available for the first carboxylations, such as fromthe alternate glucose metabolism pathway. After-ward, the pool size of RDP remained very small, withthe result that none had been detected in these experi-ments. An explanation has not been found for theformation of a large pool of hexoses and sucrose bythe etiolated plants after 12 hours in the light whilenot converting them to RDP. At this time the plantswere quite green, but the total photosynthetic CO2fixation was still relatively low. One explanationwhich needs further testing is that of slow enzymaticadaptation for RDP synthesis.

SUMMARYEtiolated Thatcher wheat plants were exposed to

1000 fc of white light and at hourly intervals after-ward determinations were made of total chlorophyll,rate of C1402 fixation, and the C14 products whichwere formed. Continuous, rapid chlorophyll synthesisbegan after 2 to 3 hours of light. During the first 4hours of light, C14 was fixed at a low rate into malic,aspartic, and glutamic acids. After 4 and 5 hours oflight, PGA and alanine were labeled, and after 5 to 6hours of light C'4 appeared in hexose phosphates andsucrose at an inereasing rate. At the beginning ofphotosynthetic fixation there was a period duringwhich C14 accumulated in PGA and alanine withoutbeing further reduced to hexoses in large amounts.After 9 to 12 hours of light, the percentage of thetotal C14 fixed which was incorporated among PGA,sucrose, and hexose phosphates, was constant, butonly at one-quarter the rate of a normally greenplant.

A limiting factor during this greening process wasthe availability of RDP, which may have been par-tially limited by slow enzymatic adaptation in theplant. The effect on the subsequent greening processof spraying glucose, ribose, sedoheptulose-sedoheptu-losan, and glycolic acid on the etiolated plants in thedark prior to the exposure to light was studied. Ri-bose, and to a lesser extent, glucose, stimulated CO,fixation during the first hours of greening, but theamount of RDP present still appeared to be a rate-limiting factor. Glycolic acid was without effect andsedoheptulose-sedoheptulosan was inhibitory to theformation of an active photosynthetic cycle.

Label did not appear in large amounts in serineuntil after the period for activation of glycolic acidoxidase. This indicated that the major pathway forserine synthesis during photosynthesis was via theglycolic acid pathlway.

The authors wish to acknowledge the capable tech-nical assistance of Miss Patricia Kerr during thisinvestigation.

LITERATURE CITED1. APPLEMAN, D. Catalase-chlorophyll relationship in

barley seedlings. Plant Physiol. 27: 613-621. 1952.2. BASSHAM, J. A., BENSON, A. A., KAY, L. D., HARRIS,

A. Z., WILSON, A. T., and CALVIN, M. The pathof carbon in photosynthesis. XXI. The cyclic re-generation of carbon dioxide acceptor. Jour.Amer. Chem. Soc. 76: 1760-1770. 1954.

3. BENSON, A. A., BASSHAM, J. A., CALVIN, M., GOOD-ALE, T. C., HAAS, V. A., and STEPKA, W. The pathof carbon in photosynthesis. V. Paper chroma-tography and radioautography of the products.Jour. Amer. Chem. Soc. 72: 1710-1718. 1950.

4. BENSON, A. A., BASSHAM, J. A., CALVIN, M., HALL,A. G., HIRSCH, H. E., KAWAGUCHI, S., LYNCH, V.,and TOLBERT, N. E. The path of carbon in photo-synthesis. XV. Ribulose and sedoheptulose. Jour.Biol. Chem. 196: 703-716. 1952.

5. BENSON, A. A. and CALVIN, M. The path of carbonin photosynthesis. VII. Respiration and photo-synthesis. Jour. Exptl. Bot. 1: 63-68. 1949.

6. BLAAUW-JANSEN, G., KOMEN, J. G., and THOMAS,J. B. On the relation between the formation ofassimilatory pigments and the rate of photosyn-thesis in etiolated oat seedlings. Biochim. Bio-phys. Acta 5: 179-185. 1950.

7. BURRIS, R. H., WILSON, P. W., and STUTZ, R. E.Incorporation of isotopic carbon into compoundsby biosynthesis. Bot. Gaz. 111: 63-69. 1949.

8. GALSTON, A. W., BONNER, J., and BAKER, R. S.Flavoprotein and peroxidase as components of theindoleacetic acid oxidase system of peas. Arch.Biochem. 42: 456-470. 1953.

9. IRVING, A. A. The begining of photosynthesis andthe development of chlorophyll. Annals Bot. 24:805-819. 1910.

10. KOSKI, V. M., FRENCH, C. S., and SMITH, J. H. C.The action spectrum for the transformation ofprotochlorophyll to chlorophyll a in normal andalbino corn seedlings. Arch. Biochem. 31: 1-17.1951.

11. RACUSEN, D. W. and ARON'OFF, S. Metabolism ofsoybean leaves. VI. Exploratory studies in pro-tein metabolism. Arch. Biochem. Biophys. 51:68-78. 1954.

12. SCHOU, L., BENSON, A. A., BASSHAM, J. A., andCALVIN, M. The path of carbon in photosynthesis.XI. The role of glycolic acid. Physiol. Plantarum3: 487-495. 1950.

13. SMITH, J. H. C. Protochlorophyll, precursor ofchlorophyll. Arch. Biochem. 19: 449-454. 1948.

14. SMITH, J. H. C. The development of chlorophylland oxygen-evolving power in etiolated barleyleaves when illuminated. Plant Physiol. 29: 143-148. 1954.

15. STREHLER, B. L. Photosynthesis-energetics and phos-phate metabolism. In: Phosphorus Metabolism.WV. D. McElroy and B. Glass, ed. Vol. II, Pp.491-502. Johns Hopkins Press, Baltimore. 1952.

16. TOLBERT, N. E. Formic acid metabolism in barleyleaves. Jour. Biol. Chem. 215: 27-34. 1955.

17. TOLBERT, N. E. and COHAN, M. S. Activation ofglycolic acid oxidase in plants. Jour. Biol. Chem.204: 639-648. 1953.

18. TOLBERT, N. E. and COHAN, M. S. Products formedfrom glycolic acid in plants. Jour. Biol. Chem.204: 649-654. 1953.

19. TOLBERT, N. E. and ZILL, L. P. Metabolism of sedo-heptulose-C'4 in plant leaves. Arch. Biochem. Bio-phys. 50: 392-398. 1954.

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20. VERNON, L. P. and ARONOFF, S. Metabolism of soy-bean leaves. II. Amino acids formed during short-term photosynthesis. Arch. Biochem. 29: 179-186.1950.

21. WEISSBACH, A. and HORECKER, B. L. The formationof glycine from ribose-5-phosphate. A symposiumon amino acid metabolism, W. D. McElroy and

B. Glass, ed. Pp. 741-742. Johns Hopkins Press,Baltimore. 1955.

22. WITHROW, R. B., KLEIN, XV. H., PRICE, L., andELSTAD, V. Influence of visible and near infraredradiant energy on organ development and pigmentsynthesis in bean and corn. Plant Physiol. 28:1-14. 1953.

PHOSPHORUS AND SULFUR COMPOUNDS IN PLANT XYLEM\I SAP'

N. E. TOLBERT AND H. WIEBE 2,3

BIOLOGY DIVISION, OAK RIDGE NATIONAL LABORATORY,OAK RIDGE, TENNESSEE

Since radioactive phosphorus and sulfur have be-come available, numerous investigators have deter-mined the rate and conditions for the movement ofthese two essential materials from the soil through theroots to the leaves. The work has involved detectionof the radioactivity as a measure of phosphorus orsulfur movement. It has been assumed that inorganicphosphate and sulfate were the major if not the onlyforms in which these elements moved in the upward-flowing xylem of the plant stem. Reports on phos-phorus compounds in this plant sap are conflicting;Pierre and Pohlman (4) reported some phosphoruspresent as organic esters, while Lundeg'ardh (3) foundno organic phosphates in the exudation of roots. Thepresent investigation with radioactive tracers andpaper chromatographic analyses indicates that, thoughinorganic phosphate is a major form of phosphorusbeing transported, there are in the xylem sap twoother phosphorus-containing compounds one of whichmay account for a substantial portion of the trans-ported phosphorus. Inorganic sulfate appears to bethe only form of sulfur being transported.

PROCEDURESTests have been made with plants which produce

a relatively large amount of exudate (bleeding) whentheir stems or leaf bases were cut off. Sacramentobarley has been used in most of the experiments. Theplants were grown in sand or soil in the greenhouse orin Hoagland's nutrient solution in a constant environ-ment room with about 800 fc of light on a 16-hourday and at 75 to 770 F. In some experiments, theplants in sand or soil have been used as grown; inothers, the sand or soil was carefully washed off theroots before placing them in 0.1 or 0.01 strengthHoagland's nutrient solution during the experiment towhich p32 or S35 was added. Plants grown in sandor soil into which p32 phosphate was poured or in-jected yielded poorer results than those grown inaerated nutrient solution with p32. The sand or soil

1 Received May 23, 1955.2 Work done while Dr. Wiebe was an Oak Ridge In-

stitute of Nuclear Studies Research Participant at theUniversity of Tennessee-Atomic Energy CommissionAgricultural Research Program.

3 Present address: Department of Botany and PlantPathology, Utah State Agricultural College, Logan, Utah.

bound the phosphate so tightly that the rate of ap-pearance of p32 in the plant stem was more erratic.Flooding the sand or soil with the solution containingthe tracer caused poor exudation. Polyethylene beakerswere used for holding the p32 nutrient solution sincethe radioactive phosphate did not adhere tightly tothe side wall of these containers. Plants were sus-pended by glass grids or by corks.

The radioactive phosphate or sulfate was placed inthe dilute nutrient solution and at the same time orlater the plant tops were removed near the base witha sharp razor blade. As the exudate appeared on thecut stump, it was removed completely by touchingthe origin point of a paper chromatogram to thestump. Thus for a given plant, the exudate couldbe transferred quantitatively, making it simple tocarry out time-course studies. The first collectionsafter cutting off the plant top utilized all the exudatethat appeared. When collection times were furtherapart than 1/2 hour, there was often an excess of itwhich dripped from the stump or which was removedby blotting .with a piece of filter paper. For theselater collections the stump was blotted with filterpaper 1/2 hour before the final sampling and all theexudate appearing after that was used for the chroma-tographic analysis.

Chromatograms were run on unwashed Whatman#1 paper. They were developed in the first directionwith water-saturated phenol and in the second direc-tion with butanol-propionic acid-water (1). Afterdevelopment, the chromatograms were exposed to no-screen x-ray film to establish the exact location of thep32. These spots were cut into small pieces for count-ing with an end-window Geiger-Miiller tube.

RESULTS AND DISCUSSIONOn chromatograms of the exudate collected from

the stump of decapitated barley seedlings, tomato,sunflower, bean, and willow there were 3 radioactivephosphorus spots which had Rf characteristics givenin table I. The discovery of only 3 phosphorus-con-taining compounds in the exudate was surprising,since the phosphorus was neither all inorganic phos-phate nor was there the multiplicity of known organo-phosphorus compounds associated with respirationwhich would be found in whole tissue. The two phos-phorus-containing compounds besides inorganic phos-

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ac and · pounds which might effect the greening process. in separate experiments, ... whereas, in...

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