Chlorophyll synthesis in x-irradiated etiolated bean leaf tissue

Radiation

Botany,

1962,

pp.

CHLOROPHYLL

269

to 275.

Pergamon

SYNTHESIS IN BEAN LEAF L. PRICE

Division

Press Ltd.

of Radiation

and

Printed

in Great

Britain.

X-IRRADIATED TISSUE*t

ETIOLATED

and W. I-L KLEIN

Organisms, Smithsonian (Receiued 12 October

Institution, 1961)

Washington,

D.C.,

U.S.A.

Abstract-X-irradiated etiolated bean leaf tissue exhibits a marked reduction of chlorophyll synthesis when exposed to continuous white light of high intensity. However, chlorophyll synthesis is appreciably increased in this X-irradiated tissue if subjected to a few minutes of visible radiant energy either before or after X-irradiation, then placed in the dark for several hours, and subsequently exposed to high intensity white light. This light pretreatment, furthermore, appears to be much more effective in the red than in the blue region of the spectrum. The response to light pretreatment of X-irradiated leaves is markedly similar to that found in control samples of non-X-rayed tissue. Rather than an interaction of ionizing radiation and nonionizing radiant energy, light pretreatment appears to induce an independent photomorphogenic response related to the chlorophyll-synthesizing mechanism which is superimposed upon the damage resulting from X-ray dosages.

RCsum&Un tissu de feuilles etioltes de ftve irradie par les rayons X montre une reduction marquee de synthbe chlorophyllienne apres exposition a une lumitre blanche continue de forte inter&t. Cependant, la synthese chlorophyllienne est accrue de man&e appreciable lorsque ce tissu irradie est soumis pendant quelques minutes a l’energie radiante visible, soit avant, soit apres irradiation et place ensuite a l’obscuritt pendant plusieurs heurcs puis ulterieurement expose a une lumiere blanche de forte inter&e. Ce pretraitement lumineux apparait, en outre, ttre beaucoup plus efficace dans la region rouge que dans la region bleur du spectre. La rtponse au pretraitement lumineux des feuilles irradites est remarquablement similaire a celle obtenue dans des tchantillons controles de tissu non irradie. Plutot qu’une interaction entre les radiations ionisantes et l’tnergie radiante non ionisante, le prttraitement lumineux parait induire une rtponse photo-morphogene independante en relation avec le mecanisme de synthese de la chlorophylle qui se superpose au dommage cause par les doses de rayons X. etioliertes Bohnenbllttergewebe das mit anZusammenfassung-Rontgenbestrahltes, dauerndem hochintensen weissen Licht bestrahlt wird, zeigt eine bedeutende Verminderung der Chlorophyllsynthese. Falls aber dieses bestrahlte Gewebe entweder vor oder nach der Rontgenbestrahlung fur einige Minuten sichtbare Strahlencnergie erhalt, wird dann fur einige Stunden in die Dunkelheit gegeben, und danach dem hochintensen weissen Licht dargeboten, dann wird die Chlorophyllsynthese bedeutendlich erhoht. Ausserdem scheint diese Lichtvorbehandlung vie1 wirkungsvoller im roten als im blauen Bereich des Spektrums zu sein. Die Reaktion der bestrahlten Blatter zur Lichtvorbehandlung gleicht sehr der der nichtbestrahlten Kontrollen. Anstatt einer Wechselwirkung der ionisierenden Bestrahlung und nichtionisierenden Strahlenenergie scheint Lichtvorbehandlung eine unabhingige photomorphogenetische Reaktion hervorzurufen, die mit dem Chlorophyllsyntheseprozess zusammenhangt und die dem durch Rlintgenbestrahlung hervorgerufenen Schaden iiberlagert ist. *Published tThis work AT(30-l)-2373. B

with the approval was carried out

of the Secretary with the support

of the Smithsonian Institution. of the U.S. Atomic Energy 269

Commission

under

Contract

970

CHLOROPHYLL

SYNTHESIS

IN X-IRRADIATED

INTRODUCTION

dark-grown leaf tissue is placed in continuous light of relatively high intensity (1000 pW/cm2), three stages in pigment synthesis become apparent. First, there is the immediate conversion of protochlorophyllideo) to chlorophyllide. This is followed by the second stage, referred to as the lag or latent phase, in which the rate of chlorophyll synthesis rises slowly, and finally the third stage, in which there is a sustained high rate of pigment synthesis. TOLBERT and GAILEY(~) have shown that in 7-day-old Thatcher wheat seedlings, sustained chlorophyll synthesis begins about the third hour of illumination. Data for bean leaf tissue \vith one cotyledon attached is in agreement with this observation, although generally, the rate of pigment synthesis is somewhat higher even during the early hours. WITHROW et a1.c3) and VIRGIN(~) have reported that. the early period or lag phase in the synthesis of chlorophyll in etiolated leaves is affected by a pretreatment with visible radiant energy, such pretreatment, followed by a dark period, resulting in an almost complete elimination of the lag or latent phase in subsequent pigment synthesis under high light intensity (Fig. 3, curves 0 and O+L). In addition, red light has been found to be many times more effective than blue.c5v a) Recently, GAILEY and TOLBERT~‘) have shown that when etiolated wheat leaf tissue is subjected to gamma rays from a cobalt-60 source, the lag phase in chlorophyll synthesis is extended. A dose of 150 kiloroentgens (kr) increases the latent period to as much as 10 hr in continuous light. If, however, the gammairradiated etiolated leaves are subjected to 1 hr of high intensity white light immediately following the ionizing radiation and placed in the dark for several hours, then upon returning the tissue to continuous white light, there is a rise in chlorophyll-synthesizing ability accompanied by a shortening of the lag phase, suggesting that nonionizing radiation induces a recovery or reversal of the effects of ionizing energy. In view of these latter observations and those of Withrow et al. and Virgin, the interaction of light and ionizing energy as they affect chlorophyll synthesis was investigated. A medical WHEN

ETIOLATED

BEAN

LEAF

TISSUE

type X-ray unit was used for ionizing irradiation treatment and observations were primarily confined to the effects of light and Xrays. MATERIALS

AND

METHODS

Bean seeds (Phaseolus vulgaris Linn., “Great Northern” or “Burpee’s stringless green pod”) were planted in gravel beds and subirrigated with tap water. The tops of the dark-grown seedlings were excised just below the cotyledons six days after planting. The seedling tops with the attached pairs of primary leaves and one cotyledon removed from each were placed in a petri dish containing filter paper moistened with water. Although excised leaves on a substrate of carbohydrates can be used, the presence of the cotyledon results in more sharply defined responses assaying for chlorophyll when content.@) Generally, twenty plants were used per sample dish. All experimental pro:edures were carried out at 25°C. A Westinghouse medical type X-ray machine operating at 140 kilovolts and 8 mA was used for X-ray treatments. Samples were placed on a turntable 14 in. below the X-ray tube housing. A 60 min exposure gave a total dosage of 12 kr; a stationary exposure of 8 min resulted in an equivalent dose. Inasmuch as a comparison between filtered (1 mm aluminum) and unfiltered X-rays resulted in the same response, unfiltered X-irradiation was used in all experiments. Two sheets of black paper covered the tube housing aperture in order to shield the plants from any filament glow. In general, Xirradiation or light pretreatment was followed by an 18 hr development period in darkness, after which the samples were placed in high intensity white light (1000 pW/cm2) for various intervals. All preparation of tissue and manipulation were carried out under a green safelight.@) Pretreatment with light was accomplished with a fluorescent luminaire of 15 lamps ( 15 W “warm white”) with an irradiance of 1000 tJ.W/cm2 17 in. below the lamps. Exposures of l-10 min were used as pretreatments. The same luminaire was employed for the chlorophyll synthesis assay period, in which the exposure time was extended to as much as 24 hr. By periodically removing samples from under

L. PRICE

and W. H. KLEIN

the luminaire and extracting the chlorophyll, time course studies were obtained. Pigment concentration of aqueous-acetone extracts of leaf tissue was measured on a Beckman Model D.U. spectrophotometer. Chlorophyll content on a fresh weight or per plant basis was determined, using the methods described by ARNON.(~O) RESULTS

AND

intervening 18-hr dark period. The data (Fig. 1) for leaves subjected to various doses of X-irradiation and similarly analysed indicate that synthesis of protochlorophyllide in the dark was inhibited with increasing dosage. The greatest sensitivity was exhibited with dosages below 8 kr, whereas any additional X-irradiation had very little effect on the inhibition of the synthesis of pigment precursor. Samples subjected to the same dosages of X-ray and regimen as described above and placed under white light for 5 hr, indicated that the continued synthesis of chlorophyll had a similar radiosensitivity, in that most of the loss in chlorophyll-synthesizing ability occurs with dosages below 8 kr. An X-ray dosage of 12 kr was employed as a standard dosage, inasmuch as the slope of the

DISCUSSION

If dark-grown leaves are exposed to 1 min of high intensity light, the chlorophyll produced is an approximation of the protochlorophyllide content of the tissue. The net difference in the amount of chlorophyll formed between such a sample and another subjected to the same light treatment 18 hr later, reflects the amount of protochlorophyllide synthesized during the

0

2

4

271

6

6

X-DOSAGE

IO

,

I

12

14

(kd

FIG. 1. Effect of X-rays on synthesis of protochlorophylhde during an 18-hr dark period, as measured by its conversion to chlorophyll. A -etiolated control samples exposed to 1 min of white light at start of experiment. A-etiolated experiment.

control

x-x-rayed

samples allowed

1 min

of light.

samples

exposed

to

1 min

of white

light

to develop in the idark overnight

24 hr

after

start

of

and then exposed to

272

CHLOROPHLL Table

SYNTHESIS 1.

Eflect

IN

of X-irradiation

X-IRRADIATED

ETIOLATED

an the conversion

of protochlorophyllide

No light pretreatment Treatment

BEAN

LEAF

to chlorophyll

TISSUE a*

1 min. light pretreatment 12 kr X-ray

Freshly excised

0.59

--

18 hr dark period

0.99

Net gain

0.40

-

-

---

Net gain of light pretreatment

over dark

0.39

* pg/plant. radiosensitivity curves is at a minimum at this value. It is to be noted that even at this high dosage, there was little destruction of protochlorophyllide nor of its ability to be converted to chlorophyll. The data in Table 1 suggests that ionizing radiation reduces the chiorophyllsynthesizing potential of the plant by inhibiting the development of proplastids(11*12) inasmuch as no new protochlorophyllide is synthesized during the dark period in X-rayed tissue. The data also reveals a lack of interaction between light and X-rays, since the net gain was the same in chlorophyll content of both the lightpretreated, X-irradiated and unirradiated leaves over that of corresponding unpretreated controls. In a second set of experiments, leaf samples were subjected to X-ray treatment and immediately irradiated under the fluorescent lamp bank at approximately 1000 ,uW/cm2. Periodically, samples were removed and extracted. Time course data for X-irradiated and control tissues (Fig. 2) revealed that the X-ray treatment had reduced the rate of synthesis of cholorophyll to about one-half that of the control. An additional two sets of plants, one as control and the other receiving X-irradiation, were exposed to 10 min of white light as pretreatment, and then placed in the dark for 10 hr. When these samples were returned to the light for assaying, chlorophyll synthesis increased rapidly. The rate of chlorophyll synthesis in these light-pretreated leaves was so stimulated that in 12 hr of light, the chlorophyll content was equal to that of the unpretreated

control sets that were exposed to light continuously for 24 hr (Fig. 2). Although in leaves subjected to ionizing radiation the synthesis of chlorophyll had been reduced to approximately one-half that of the control, nevertheless, pigment synthesis exhibited a response LO light pretreatment which was similar in proportion to that found in unirradiated control sets. This further indicated an independence of action between ionizing and visible radiation without any evidence of any additional recovery or reversal from the damage due to ionizing radiation attributable to the light pretreatment. A time course study similar to that described above was made using a Van de Graaff accelerator, on leaves exposed to 100 kr equivalent x-rays. The results observed did not differ from those of the X-irradiated tissue, except that chlorophyll synthesis response to 100 kr dosage was reduced to one-tenth that of controls, as against one-half for X-irradiation. Again, the light pretreatment resulted in the same proportional stimulation in response in both controls and X-irradiated leaves. A change in experimental procedure was introduced in order to eliminate continuous sampling over a 24-hr period. Plants were prepared, X-irradiated and pretreated on one day, allowed to develop in the dark overnight, and assayed the following day. Following this regimen, an additional test was made to further demonstrate the independence of light and X-irradiation responses. Two sets of pretreated X-irradiated plants were used; one set was light-pretreated for several minutes prior to the X-ray exposure, while the

L. PRICE

and

H.

273

KLEIN

- 600

- 500

- 400

-300

- 200

- 100

uu 0

2

4

6 8 IO 12 LIGHT ASSAY TIME

14 (hr)

16

18

20

’

and light pretreatment on chlorophyll synthesis. Scale on the FIG. 2. Effect of X-ray left is for X-rayed tissues; the right hand scale is for controls. o -control samples placed under white light at start of experiment (0). A -X-rayed and placed under white light at start of experiment (X). e-control samples pretreated with 10 min of light, followed by 10 hr of dark and then returned to light (O+L). ~-X-rayed samples pretreated with 10 min of light, followed by 10 hr of dark and then returned to light (X+L).

other set was post-X-ray illuminated. All samples were then placed in the dark overnight, and the next morning all were placed under the assay luminaire for 8 hr. Again the independence of the light pretreatment reactions and that of the X-irradiation was demonstrated by the fact that the pre- and post-X-ray light-treated samples exhibited a negligible difference in pigment accumulation (Fig. 3). The net response to the light pretreatment by X-irradiated tissue is of the same magnitude as that of the non-Xirradiated tissue. In order to establish the photomorphogenic

nature of the light reaction, the response of chlorophyll synthesis to blue versus red energy pretreatment was examined. Two interference filter monochromators as described by %hTHROW were arranged to give monochromatic energy at 450 and 650 mp. For pretreatment, samples were exposed to 2.4 em3 at the red station and 9.6 em3, or four times as much endrgy, at the blue station. Control samples were extracted immediately after the pretreatment to obtain the initial protochlorophyllide conversion. Test plants were kept in the dark overnight and were then placed under the white light assay lumi-

274

CHLOROPHYLL

SYNTHESIS

IN X-IRRADIATED

ETIOLATED

BEAN

LEAF

TISSUE

FIG. 3. Effect ofX-ray and pre- or post-X-ray light treatment on subsequent chlorophyll synthesis. l -lo-rein white light pretreatment (1000 yW/cm?), 18-hr dark period, continuous white light (O+L). o--18-hr dark period, continuous white light (0). A-12 kr X-ray, 18-hr dark period, continuous white light (X). A-12 kr X-ray, IO-min white light pretreatment, 18-hr dark period, continuous light (X+L). X-IO-min white light pretreatment, 12 kr X-ray, 18-hr dark period, continuous white light (L+X).

naire for 5 hr, after which time they were extracted. As shown in Table 2, the red pretreatment in both controls and X-rayed samples gave significantly greater stimulation to the chlorophyll-synthesizing mechanism than did the blue. The accumulated experimental evidence indicates that a short light pretreatment stimulates subsequent chlorophyll synthesis in both X-rayed and control samples through a photomorphogenic response which manifests itself in enhanced proplastid maturation and/or increased proplastid number. This photomorphogenie response is independent of the protochlorophyllide conversion action spectrum, as well as the effects of ionizing radiation.

white

SUMMARY On the basis of the experimental data presented, the independence of chlorophyll synthesis in its response to X-irradiation and light pretreatment has been demonstrated. Ionizing radiation “damage” reduces the chlorophyll-synthesizing potential of the plant. Whatever protochlorophyllide is present before the X-irradiation is not destroyed and can be photochemically converted to chlorophyll. However, depending upon dosage, new synthesis of additional protochlorophyllide is inhibited. An exposure to a short light pretreatment, on the other hand, can stimulate the subsequent chlorophyll synthesis through an independent photomorphogenic type response.

L. PRICE Table

2.

EJect

of monochroma&ic

and light

W.

H.

pretreatment

KLEIN

275

on subsequent

! , I

No X-ray I

Treatment

i Chlorophyll

Ratio:

Light

after pretreatment, 18 hr 5 hr light (1200 VW/cm*)

/

pretreated/unpretreated

*pg/g fresh weight. tTota1 chlorophyll less pretreatment *Blue=450 rnp, 9.6 mJ. SRed =650 ml+ 24 mJ. Acknowledgements-The ledge the technical

None

i 0

at end of pretreatment

Chlorophyll-l dark and

Light

249 -

pretreatment Blue* 4.80

chlorophyll*

Red3 6.81

synthesis 12 kr X-ray Light

I

None

)

0

pretreatment Blue* 4.55

Reds 7.05

I 266

411 1.07

I 1.65

28.4 -

31.6 1.11

48.4 1.70

values.

authors gratefully acknowassistance of Dr. J. V. SLADEK.

REFERENCES 1. WOLFF J. B. and PRICE L. (1957) Terminal steps of chlorophyll a biosynthesis in higher plants. Arch. Biochem. Biophys. 72, 293-30 1. 2. TOLBERT N. E. and GAILEY F. B. (1955) Carbon dioxide fixation by etiolated plants after exposure to white light. Plant Physiol. 30, 491499. 3. WITHROW R. B., WOLFF J. B. and PRICE L. (1956) Elimination of the lag phase of chlorophyll synthesis in dark-grown bean leaves by a pretreatment with low irradiance of monochromatic energy. Plant Physiol. 31 (Suppl.) : xiii-xiv. 4. VIRGIN H. I. (1957) Chlorophyll a content and transpiration of etiolated wheat leaves after pretreatment with a short light impulse followed by dark periods of varying lengths. Physiol. Plant.

10, 445-453. 5. VIRGIN H. I. (1958) Studies on the formation of protochlorophyll and chlorophyll a under varying light treatments. Physiol. Plant. 11, 347-362.

6. WOLFF J. B., PRICE L. and WITHROW R. B. (1957) Stimulation of protochlorophyll synthesis in dark-grown bean leaves by irradiation with low energy. Plant Physiol. 32 (Suppl.): ix. 7. GAILEY F. B. and TOLBERT N. E. (1958) Effect of ionizing radiation on the development of photosynthesis in etiolated wheat leaves. Arch. Biochem. Biophys. 76, 188-195. 8. WOLFF J. B. and PRICE L. (1960) The effects of sugars on chlorophyll biosynthesis in higher plants. J. Biol. Chem. 235, 1603-1608. 9. WITHROW R. B. and PRICE L. (1957) A darkroom safelight for research in plant physiology. Plant Physiol. 32, 244-248. 10. ARNON D. I. (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1-15. 11. BRESLAVIT~ L. B. (1960) Plants and X-rays. Translated from Russian and published by American Institute of Biological Sciences, Washington, D.C. 12. M~~HLETHALER K. (1955) Untersuchungen tiber die Struktur und Entwicklung der Proplastiden. Protoplasma 45, 264-279. 13. WITHROW R. B. (1957) An interference filter monochromator system for the irradiation of biological materials. Plant Physiol. 32, 355-360.