Effects of X- and gamma radiation on anthocyanin content in leaves of Rumex and other plant genera

Radiation

1968, Vol. 8, pp. 7 to 16. Pergamon

Botany,

Press. Printed in Great Britain.

EFFECTS OF X- AND GAMMA RADIATION ON ANTHOCYANIN CONTENT IN LEAVES OF RUMEX AND OTHER PLANT GENERA* A. H. SPARROW, M. FURUYAt and SUSAN S. SCHWEMMER Biology Department, Brookhaven National Laboratory, Upton, New York 11973, U.S.A. (Received

25 August

1967)

Abstract-The concentration of anthocyanin pigments in the leaves of seedlings of Rumex crispus L., R. Izydrolapathum Huds. and R. sanguineus L. was found to increase following exposure to X-rays or B°Co y-rays. The time-course of formation of and the increase in extractable anthocyanins were dependent upon the species, the exposure and the age of the leaves at the time of irradiation. Maximum increased content approached ZO-fold that of untreated plants. Large increases in anthocyanin content after irradiation were generally indicative of lethal or near lethal exposures. Chromatographic studies of the anthocyanin offered no evidence of a radiation-induced change in the structural integrity of the pigment molecules. A visual increase in red pigmentation occurred in the leaves of 16 species from other genera irradiated chronically and 21 species irradiated acutely. It thus appears that enhanced pigment (presumably anthocyanin) formation is a common response of higher plants to ionizing radiation. The exposure required to produce significant increases varied widely with the species. R&urn&La

concentration en pigments anthocyaniques des feuilles des plantules de L. R. Ivdrolapathum Huds. et R. sanguineus L. s’accroit aprts exposition aux rayons aux rayons y. Le temps de formation des anthocyanes extractibles ainsi que leur accroissement depend de l’espece, de la dose et de Page des feuilles au moment de l’irradiation. Le contenu maximal est voisin de 20 fois celui des plantes non traides. Des acroissements importants des contenus en anthocyane aprts irradiation indiquent gentralement des doses l&ales ou subletales. Des etudes par chromatographie des anthocyanes ne montrent pas de changements radio-induits de l’inttgritt structurale des molecules de pigments. Un accroissement visible de la pigmentation rouge survient dans les feuilles de 16 especes d’autres genres irradiees de man&e chronique et de 21 esptces irradites de man&e aigue. 11 apparait done que la formation accrue de pigments (probablement des anthocyanes) est une reponse commune des plantes suptrieures aux radiations ionisantes. La dose requise pour produire des accroissements significatifs varie largement d’une esptce a l’autre. Rumex X et

crirpus

Zusammenfassung-Nach Bestrahlung mit Rijntgenstrahlen oder s’JCo Gammastrahlen liess sich ein Ansteigen der Konzentration der Anthocyane in B&tern der Keimlinge von Rumex crispus L., R. hydrolapathum Huds. und R. sanguineus L. feststellen. Der Zeitpunkt der Bildung und die Zunahme von extrahierbaren Anthocyanen war abhangig von Art, Dosierung sowie Alter der Blatter zur Zeit der Bestrahlung. Der Maximal-Gehalt war annahernd 20 ma1 grosser als in unbehandelten Pflanzen. Starkes Ansteigen des Anthocyan-Gehalts *Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commission. tPresent address: Biological Institute, Faculty of Science, Nagoya University, Chikusa, Nagoya, Japan. 7

A. I-I. SPARROW,

M. FURUYA

and SUSAN

S. SCHWEMMER

nach Bestrahlung war im allgemeinen zurtickzufuhren auf letale oder nahezu letale DosieUntersuchungen des Anthocyans erbrachten keinen Anhalt rungen. Chromatographische ftir eine strahleninduzierte Veranderung der Struktur der Pigmentmolekiile. In den Blattern von 16 Arten anderer chronisch bestrahlter Gattungen und von 21 stark Es hat somit den bestrahlten Arten kam es ZLI einer sichtbar erhohten roten Pigmentierung. Anschein, dass erhohte Pigmentbildung (vermutlich Anthocyan) eine allgemeine Reaktion hoherer Pflanzen auf ionisierende Strahlung ist. Die fur ein merkliches Ansteigen benijtigten Dosen waren weitgehend von der jeweiligen Art abhingig. INTRODUCTION Trrr;, COLOR changes of leaves in the fall to brilliant shades of red are due in part to production of anthocyanin. Similar color changes in various plant parts often appear after exposure to ionizing radiation, with the most evident changes usually occurring in the leaves.t*JZ1) However, previous observations have been incidental to other radiobiological aspects under investigation and were limited in extent. The current investigation was an attempt to determine the nature of the pigments involved in the color changes in normal and irradiated Rumex leaves and to measure the pigment content during the course of development of the seedlings under the given experimental conditions, since so many environmental factors may change the rate of pigment synthesis. t2) An apparent enhanced anthocyanin formation based on visual observations only is also reported in several other genera treated with ionizing radiation.

MATERIALS AND METHODS (a) Plant materials Seeds of R. sanguineus L. and R. hydrolapathum Huds. were obtained from the Botanic Garden, University of Copenhagen, Denmark. Seeds of R. crispu~ L. were collected in Bellport, New York, U.S.A. All three species are weeds, commonly known as dock. The sources of the other genera reported on are too numerous to mention here but are available on request from the authors. (b) Culture ofpl an ts and irradiation procedures Seeds were germinated in soil and grown in a greenhouse. At a suitable size they were transplanted into soil in 2&in. square plastic pots. When the sixth seedling leaf appeared,

the seedlings were subjected to X- or y-radiation. The radiation exposures used for both the time-course and the exposure-response series were based on previous unpublished experiments by the authors. The source of X-rays was a G. E. Maxitron 250 X-ray machine. Plants were irradiated in the vertical position. Exposures were made at exposure rates varying from 675 to 716 R/min at 250 kVp and 30 mA using a 1 mm Al filter. Immediately after irradiation the potted seedlings were moved to a controlled-environment chamber and grown until harvest under an 18-hr day at 22”&-1°C with both incandescent and cool white fluorescent bulbs producing about 2200 ft-c. The wattage ratio was 1 : 12, incandescent to fluorescent. Gamma irradiation was done in the Brookhaven y-field where a 6oCo source is currently in operation. The potted seedlings were exposed for 16 hr at various distances from the source, hence the exposure rates varied with the total exposures. The following morning they were immediately returned to a controlledenvironment chamber operated under the same regime as described above for the X-rayed seedlings. Unirradiated (control) seedlings for the y-irradiated plants were set out overnight in a field comparable to the y-field with respect to variations in temperature, light intensity and humidity. For the experiments designed to follow changes in pigment over several weeks, two populations of potted seedlings were used. One population received 10 kR of X- or y-rays while the other was not treated. At periods of 1, 3, 7, 14, 2 1, 28 and 35 days postirradiation, leaves were collected from each of the two populations. For the exposure-response experiments, the plants were divided into groups, each group receiving a different amount of radiation. Leaves were collected from each exposure group

EFFECTS and from the untreated population at about 3 weeks postirradiation.

OF X- AND GAMMA only

once,

(c) Extraction procedures To determine the exposure-response relationship, leaves were harvested 3 or 4 weeks postirradiation, since preliminary experiments indicated that the greatest differences would be evident at this time. The fourth, fifth and sixth leaf blades (starting at the base of the seedling with the cotyledons, (Fig. 1) were

FIG. 1. Drawing of R. lyfrolapathum Huds. showing the alternate arrangement of leaves and the numhering system employed. removed from plants of each exposure group and from the untreated control group. Each blade was weighed, cut into thin strips to facilitate extraction and placed in a test tube containing 10 ml of 0.1 N HCl.(l”) The tubes were kept overnight in a vacuum jar under partial pressure at room temperature to remove the anthocyanin pigment from the leaves. The liquid was then decanted and the amount of pigment measured with a Klett-Summerson photoelectric calorimeter using a 540 my peak green filter. Measurements were made in terms of Klett units/ml of HCl/g fresh weight of leaf tissue.

RADIATION

9

The methods of handling and acutely irradiating the other genera for which observations are reported herein (Table 3) were approximately the same as for Rumex except that observations were carried out under field conditions rather than in controlled-environment growth chambers. The methods used for the chronic exposures of certain genera listed in Table 4 are given elsewhere. (d)i Paper chromatografihic procedures The following methodc5) was used fol the detection and examination of Rumex pigments both prior to and after irradiation with X- and y-rays. Fresh leaves of both untreated and irradiated groups were placed in a Sohxlet reflux condenser with methanol and refluxed three times. These successive extracts were then combined and flash-evaporated to gummy precipitates, portions of which were placed on the chromatographic paper (Whatman No. 1) and then completely dried. The chromatograms were developed in two dimensions with n-butanol, glacial acetic acid, distilled water (4 : 1: 2.2v/v) and50/b acetic acid. The chromatograms were examined in white light and with U.V. light. Rf values were calculated on the basis of at least two duplicate runs, the values appearing in Table 1 being the averages obtained. (d)ii S’ectrophotometric determinations Selected colored areas from chromatograms of untreated and irradiated material were cut from the sheet and extracted with 2 ml of 50% aqueous methanol. The solution was then decanted and its absorption spectrum measured.

RESULTS

(a) Partial characteriZation of unirradiated and irradiated pigment Table 1 summarizes the results of the chromatographic and spectrophotometric tests. The red-colored pigments from both untreated and irradiated Rumex leaves were characterized as anthocyanin glycosides based on data from U.V. and visual absorption spectra, color reactions

A. H. SPARROW,

10 Table

Species R. sanguineus

1.

Rf values and spectro&hotometric

Treatment none y-rays

R. crispus

none X-rays

R. hydrolapathum

M. FURUYA

none X-rays

Properties

and SUSAN

S. SCHWEMMER

of anthocyanin

compounds

Average Rf values k S.D. n-BuOH :HOAc :H,O 5%HOAc (4:1:2.2) Compound

A : 0.35 a 0.01 B : 0.38 f 0.03 A’: 0.33 f 0.01 B’: 0.34+ 0.01 C : 0.32 f 0.02 C’: 0.33 f 0.01 D : O-24+ 0.01 D’: 0.23 + 0.01

and the Rf values, although final identification of the chemical structure of the pigment has not been made. Extracts of both R. hydrolapathum and R. crisp21s showed only one main anthocyaninon thechromatograms. R.sanguineus, however, showed two anthocyanins of different RI values. It was evident that no new anthocyanin structures appeared on the chromatograms studied as a result of X- or y-irradiation. The Rf values for R. hydrolapathum and R. crispus did not change within the normal range of error when X-irradiated. Irradiation of R. sanguineus withy-rays did not change the Rf values of either compound when compared with the unirradiated control. The absorption spectra measurements were essentially the same for all three speciesin both untreated and irradiated samples.

0.24+ 0.01 0.39 + 0.02 0.25 + 0.01 0.39 * 0.01 0.20 f 0.01 0*21* 0.01 0.25 + 0.01 0.23 f 0.01

in three species of

Rumex

U.V. and visible absorption, h4 _ Amax Amin 277 280 276 279 278 279 280 280

258 259 259 259 257 257 258 260

exposure (Figs. 2 and 3). The responsevaried with the species and with the age of the leaf, the matured leaf IV and the youngest leaf VI showing a higher concentration of anthocyanin at the higher exposures than rapidly growing leaf V possibly because of less dilution by cell enlargement. The results obtained for R. hydroLapathum with X-radiation showed a maximum effect in leaf IV, with leaf VI exhibiting the smallest increase (Fig. 3). For leaf IV of this species, increases continued up to 24 kR but leveled off at much lower exposures in leaves V and VI. Rumex hydrolapathum had initially a greater amount ofanthocyanin per g fresh weight of leaf tissue than R. sanguineus. This is particularly evident in Figs. 2 and 3. However, the increase of anthocyanin with increasing exposure was greater for R. sanguineus, in which the amount of anthocyanin per g of leaf tissue increased at least 16-fold in leaf IV and almost (b) Relation between exposure to ionizing radiation 20-fold in leaf VI. and amount of extractable anthocyanin The exposure ranges for R. sanguineus and Changes in anthocyanin content are accom- R. hydrolapathum were selected to include the panied by fluctuations in leaf weight and the lethal range of exposure for each speciesbased latter affect to some extent the magnitude of on previous experiments (Table 2). Thus the change in anthocyanin content for a few of the large increase in anthocyanin content in the exposures. However, the trends and con- leaves of R. sanguineus above 8 kR was in the clusions are basically the same whether Klett lethal or near lethal exposure range. The units/leaf or/g fresh weight are used. Because response in R. hydrolapathum was particularly of the variation in leaf weight with time and evident in leaf IV. Here also the largest among exposures we have used Klett units/g increases were found at the higher exposure fresh weight. levels which were in the lethal or supra lethal The amount of extractable anthocyanin range. Thus large increases in anthocyanin per g of leaf tissue increased with increasing content would seemto be indicative of eventual

EFFECTS

OF X- AND GAMMA

RUMEX

RADIATION

11

SANGUINEUS

Y RADIATION

z 6000 E z 700E w =

LEAF

H

LEAFP

ivJ

T 600-

I

5 500 2 e 2 4000 z 5 300s 2 54 zoo-

s

/

IO

I2

EXPOSURE

14

(kR)

FIG. 2. Plot of the data for leaves IV, V and VI of R. sanguineus L. irradiated with y-rays, exposure in kR vs. anthocyanin content in mean Klett units/g fresh weight f S.D. Table 2. Survival data (R) for three Rumex species afkr 16-hr exposures to y-radiation(lO ) Species

L’Jlo

LD6o

LDloo

R. hydrolapathum R. cri~@s R. sanguineus

7950 10,250 10,460

10,070 11,580 13,490

12,700 14,000 16,000

to have no effect on the exposure at which there was the first noticeable increase in anthocyanin content. (c) Time-course study Seedlings of R. crispus were exposed to 10 kR

of X-rays delivered at a rate of 675 R/min and leaves were collected at various periods after irradiation. The results are shown in Fig. 4. lethality in those plants which had received The treatment caused an immediate response the higher radiation exposures. Smaller in- in leaf IV one day postirradiation. There was creasescan and do occur, however, in the sub- a general increase in leaves IV and V up lethal range and these increasesare not indicative through the 14-day period shown in Fig. 4. of imminent death in most cases. The increasesin anthocyanin were not regular In both speciesthere was very little increase in pattern after 14 days and hence are not given in anthocyanin content below 6 kR. Rumex here. At 4 and 5 weeks, there was an additional sanguineus was irradiated with y-rays and R. complication: the leaves did not show regular hydrolapathum with X-rays, but this appeared differences between the untreated and irradi-

A. H. SPARROW,

12 900,

M. FURUYA

and SUSAN S. SCHWEMMER

--,--

fi, HYDROLAPATHUM X RADIATION

0

4

S

12

I6

20

24

26

32

0

4

6

L&l-LLl.-&;1L,,l-l-LI 12 16 20 24 EXPOSURE

26

32

0

4

6

12

-I 16 20

I 24

I -1.. :lJ 32

_ .~

fkR)

FIG. 3. Plot of the data for leaves IV, V and VI of R. hydrolapathum Huds. irradiated with X-rays, exposure in kR vs. anthocyanin content in mean Klett units/g fresh weight + SD.

ated plants. However, the higher levels of anthocyanin attained in the treated plants over the untreated plants generally were retained for the entire 5-week period. After 5 weeks, the plants which had been irradiated were near death, the deterioration in the irradiated leaves being much greater than in the untreated plants. The natural process of aging, however, was also occurring in the untreated plants as indicated by the increased anthocyanin content with the progression of time. Similar results were obtained for the other two species. (d) General observations on redpigmentation increase and radiation exposure The results shown in Tables 3 and 4 summarize the observations made by the senior

author over a period of years on various species of plants which had been acutely or chronically irradiated. These observations were incidental to the main purpose of the experiments and hence should be considered only as a general indication of the relationships between the increase in red pigmentation and the amount of radiation exposure given for the species listed. It should be noted that it was not definitely determined that the increased pigmentation evident in these irradiated leaves was due specifically to anthocyanins. In nearly all cases for the chronic data (Table 4), the leaf coloration (red or purple) preceded the death of the plants by a few weeks. Plants at lower exposure rates not showing this enhanced coloration usually went on to grow for the full season.

RPFECTS

OF

X- AND

GAMMA

13

RADIATION

II-1+-lI"I

1

!-T-T--

AUMEX CRISPUS -0 CONTROL 0 10,000 A (X RAYS) 0

0

0

1

LEAF

LEAFPT

P

i0 L

-..JI

2

4

-_I .&Il.-&,&L 6 8 IO

I I2

14

0

I 4

2

DAYS

FIG. 4. X-rays

Plot of the data for leaves IV, and control, days postirradiation

Table

3.

Minimum

Genus

I 6

I 8

I IO

I

I

I2

14

/,&l-L-LI-LL_L.L 0 2

Acer rubrum & saccharum Chrysanthemumyeroen & arcticum Cyanotis somaliensis Mentha aquaticur var. crispa M. arvensis M. pipe&a & rotund$olia Sedum aljircdii S. nevii & oryyzr~oliufn S. rupifagum S. tricarpum lii~olium repens
6

s

IO

I2

14

POSTIRRADIATION

V and VI of R. crisjus L. irradiated vs. anthocyanin content in mean weight + S.D.

ucute e.u,bosure required to yield observable pigmentation in Ihe leaves of various y-irradiated

and species

4

16-hr exuosure. ‘kR

a 12 4.5 30 15

18 10 12.5 5 30 25 7

’

increase @ants

with 10,000 R of Klett units/g fresh

of red or pur/de

Observation leaves red leaves purplish underside of leaves purplish leaves purplish leaves purplish red spots on red spots on red spots on red spots on leaves reddish underside of

all leaves

reddish

leaves leaves leaves leaves leaves

purple

purple

14

A. H. SPARROW,

M. FURUYA

and SUSAN S. SCHWEMMER

Table 4. Esbosure rate, duration of e°Co y-irradiation to yield observable increase of red pigmentation

Genus and species .4cer rubrum Acer saccharum C/nysanthenrum C. frutescens C. indicum

arclicurn

C. sonare C. yezoense Graptopetalum macdougalli G. paraguayewe Luzula acuminata Oenothera glauca fraseri Quercus rubra Sedum album &S.ellocambianum S. glauco~hyllum (U-330) S. guatamalense S. kamtschaticum S. 0ryziJolium S. pacttyphy~llum S. rupestre Sen2pervivum leclorum

R/day 50 50 300 600 1000 750 235 4000 500 6000

150 50 475

1250 1250 750 2000 1250 2000

1000 1500

DISCUSSION ?‘he present work clearly shows that the biosynthesis, or possibly the rate of degradation, of anthocyanins in Rumex leaves is influenced by suitable exposures of X- and y-radiation. The biosynthesis of C6-Cs-Cs types of plant pigments has been known to be affected by light, t3,‘) by plant growth hormones such as gibberellinc5) and naphthalene acetic acid,(i) and by metal-complexing agents and metabolic inhibitors.(17) It was demonstrated that ribonuclease inhibits anthocyanin formation in Spirodela and corn leaf-discs and that deoxyribonuclease does not,(13) which suggested to the authors that RNA is the limiting factor in the synthesis of anthocyanin, not DNA. However, in all cases mentioned above, it proved dilhcult to discover the exact roles played by these physical and chemical agents in the synthesis of anthocyanins, and the same seems to be true in the present case with ionizing radiation.

and approximate accumulated in leaves of irradiated plants

Duration of exposure, days 67 67 66 45 38 67 74 48 32 57 57 60 47 36

44 42 37 58 22 37 167

exposure

Approximate accumulated exposure, kR 3.4 3.0 19.8 27.0 38.0 50.3 17.4

192.0 16.0 342.0 8.6 3.0 22.3 45.0 5590 31.5 74.0 72.5 44.0 37.0 250.5

Our results indicate that Rumex leaves apparently always produce the same kinds of anthocyanin pigments irrespective of exposure to and amount of ionizing radiation, but that radiation markedly changes the amount of pigment which can be extracted. Similarly, with Spirodela, light and gibberellin cause large changes in the amounts of anthocyanin and flavonol pigments, but are without a qualitative effect on the pattern of hydroxylation of these C,--C,-C, compounds.(5) Also, in pea leaves, the same four flavonoid complexes were detected after various light treatments although the concentration of these compounds was markedly affected by light.(“) It is therefore suggested that certain external factors including X- and y-irradiation produce a quantitative change of the C,-C,-C, compounds in plant tissues but do not produce a qualitative change in their skeleton or pattern of hydroxylation. A further indication of the validity of this suggestion is that a pigment ‘mutation’ in Coleus leaves

EFFECTS

OF

X- AND

produced by fast neutron irradiation has been shown to be a change not in the structure of the pigment molecule itself, but rather in the quantity of this pigment present per leaf.(ll) Our chromatographic results showed that R. crispus and R. lydrolapathum leaves apparently contain only one anthocyanin whiie R. sazguineus has two. Rrcmex satzguineus is the only diploid of the three species used, the other two species being hexaploid and about 20-ploid. On the basis of NYBOM'S(~~) investigation of the number of different anthocyanidins to be found in species of various ploidies, the two polyploid RZUULK species here would be expected to exhibit more than one anthocyanin. Apparently these Rumex polyploids are exceptions to the general conclusions stated by NYBOM.(~~) The effect of X- and y-radiation on anthocyanin content has been shown to be highly dependent on the age of the leaves at the time of exposure and on postirradiation time. In untreated plants, anthocyanin pigments often become more evident in either juvenile or senescent leaves rather than in actively metabolizing or mature ones. These facts suggest that ionizing radiation does not affect anthocyanin synthesis directly, but rather has an indirect effect by causing a progressive change in the physiological age of the Rumex leaves, i.e. enhanced aging is associated with an increased amount of visible and extractable anthocyanin. Since enzymes are required for the formation and destruction of anthocyanin compounds,(2) the possibility exists that the ionizing radiation may damage directly these enzymes. However, although there is a marked increase in anthocyanin content after exposure to doses which are in the lethal or sublethal range for Rumex, these exposures are not of the magnitude generally associated with enzyme destruction (Table 4 of Ref. 9). GILLET and RAMAUT(~) have shown that very high exposures to X-rays can produce complete destruction of anthocyanin pigment in Maranta leuconeura. They found the minimum exposure required to produce this result was 300 kR and to get the effect in about one day required an exposure of ii60 kR. Exposures of this magnitude, though they produce gross changes in

GAMMA

RADIATION

15

anthocyanin production, also induce sevcrc changes in the other vital processes of the plant since they far exceed the lethal exposure. Even the lowest exposure used by these authors would produce fairly quick death in the plants irradiated. Gillet and Ramaut indicated that, with the highest exposures, necrosis of the leaves becomes apparent within a few days after the irradiation, thus making it impossible to follow carefully the progress of anthocyanin decrease. Thus their results have relatively little bearing on the interpretation of our data. Having investigated a few species of Runs with regard to the relationship between anthocyanin production and ionizing radiation, it becomes evident that the red pigmentation which appears in the leaves of numerous other species under chronic and acute y-radiation is most likely of the anthocyanin group, although the particular pigment may vary from species to species. Since increased red or purplish pigment has been observed by the authors in at least 35 different species of higher plants after exposure to ionizing radiation, it can be concluded that this is a common response of higher plants to radiation stress. Acktloruledgrtletrts-The authors’ thanks are due to Miss BARBARA CARTWRIGHT and Mr. LLOYD SCIINRER for their technical assistance, and to Dr. H. W. SIEGELMAN and Miss VIRGINIA POND for critical reading of the manuscript.

REFERENCES I.

ARNOLD A. Chemical tion and segments 307-312.

2.

W. and ALBERT L. S. (1964) factors affecting anthocyanin formamorphogenesis in cultured hypocotyl of Imf~atiet~s Iralsamitia. PI. Physioi. 39,

BOCORAD L. (1958)

The biogenesis of flavonoids. A. Rev. PI. Plysiol. 9, 4171148. 3. DOWNS R. J. and SIEGELMAN I-I. W. (1963) Photocontrol of anthocyanin synthesis in milo seedlings. Pl. Plysiol. 38, 25-30. 4. FURUYA M. and GALSTON A. W. (1965) Flavonoid complexes in Pisunz sativunz L. I. Nature and distribution of the major components. P&o-

cttem. 4, 285-296.

16 5.

6.

7.

8.

9.

IO.

A. H. SPARROW,

M.

FURUYA

FURUYA M. and THIMANN K. V. (1964). The biogenesis of anthocyanin. XI. Effects of gibberellic acid in two species of Spirodela. AYCh5 Biochem. Biofihys. 108, 109-116. GILLET C. and RAMAUT J. (1964) Induction prtcoce par les rayons X de la decoloration des taches foliaires de Maranla leuconeura. Bull. Acad. r. Belg. Cl. Sci. 50, 1061-1066. GRILL R. and VINCIZ D. (1964) Anthocyanin formation in turnip seedlings (Brassica rupa L.): Evidence for two light steps in the biosynthetic pathway. Planta 63, 1-12. GUNCKEL J. E. and SPARROW A. H. (1954) Aberrant growth in plants induced by ionizing radiation. Brookhaven Symp. Biol. 6, 252-279. GUNCKEL J. E. and SPARROW A. H. (1961) Ionizing radiations: biochemical, physiological and morphological aspects of their effects on plants. In: Encyclopedia of Planl Physiology, Vol. 16. Springer-Verlag, Berlin, pp. 555-611. ICHIKAWA S. and SPARROW A. H. Unpublished results.

and

SUSAN

S. SCHWEMMER

11. LOVE J. E. and MALONE B. B. (1967) Anthocyanin pigments in mutant and non-mutant Coleus plants. Radiation Botany 7, 549-552. 12. NYBOM N. (1964) Thin-layer chromatographic analysis of anthocyanidins. Physiologia PI. 17, 157-164. 13. RADNER B. S. and THIMANN K. V. (1963) The biogenesis of anthocyanins. IX. The effect of ribonuclease on anthocyanin formation in Spirodela oligorrhira. Arch-s Biochem. Biophys. 102,

92-95. 14. SPARROW A. H. Unpublished results. 15. SPARROW A. I-I. (1966) Research uses of the gamma field and related radiation facilities at Brookhaven National Laboratory. Radialion Butajg 6, 377-405. 16. THIMANN K. V. and EDMONDSON Y. H. (1949) The biogenesis of the anthocyanins. I. General Nutritional conditions leading to anthocyanin formation. Archs Biochem. 22, 33-53. 17. THIMANN K. V. and RADNER B. S. (1962) The biogenesis of anthocyanins VII. The yequirement for both purines and pyrimidines. Archs Biochem. Biophys. 96, 270-279.