Distribution and Variation of Indole Glucosinolates in Woad

Plant Physiol. (1971) 48, 498-503

Distribution and Variation of Indole Glucosinolatesin Woad (Isatis tinctoria L.)1

Received for publication April 15, 1971

MALCOLM C. ELLIOTT2 AND BRUCE B. STOWEDepartment of Biology, Yale University, New Haven, Conn1ecticuit 06520

ABSTRACT

The exceptionally high levels in woad (Isatis tinctoria L.) ofthree indolic goitrogens, namely glucobrassicin, neoglucobras-sicin, and glucobrassicin-l-sulfonate, permit the facile study oftheir distribution in the plant and their changes during its de-velopment. Woad seeds contain as much as 0.23% fresh weightof glucobrassicin but no other indole glucosinolate, while 1-week-old seedlings also contain substantial amounts of neogluco-brassicin and glucobrassicin-l-sulfonate in their shoots whethergrown in the light or dark. The sulfonate is not found in roots,and light depresses neoglucobrassicin levels in shoots. Sterileroot cultures synthesize glucobrassicin and neoglucobrassicin,and significant quantities of these were even found to be ex-creted by the roots of intact sterile seedlings in culture. Thismay explain the long known deleterious effect of woad andother cruciferous crops on subsequent plantings and the obser-vation could be of ecological importance. Long term changes inlevels of all three substances in the plant are similar and arecompatible with earlier suggestions that the compounds couldbe auxin precursors at the time of flower stem elongation. Sincesterile seedlings readily incorporate 3SO42- into indole glucos-inolates and relative specific radioactivities suggest that gluco-brassicin is the precursor of the other two compounds, pathwaysof goitrogen biosynthesis should be relatively easily determinedin this material.

In previous papers (9, 10) we have reported the isolationfrom woad (Isatis tinctoria L.) of crystalline salts of 3-indolyl-methylglucosinolate (glucobrassicin [14]), 1-methoxy-3-indolyl-methylglucosinolate (neoglucobrassicin [15]), and a new indoleglucosinolate which we identified as 1-sulfo-3-indolylmethyl-glucosinolate (glucobrassicin sulfonate [9]), (Fig. 1).

Indole glucosinolates have been found to be very widely dis-tributed among members of the Cruciferae (24, 33) and arealso detectable in some plants of the families Capparidaceae,Tovarinaceae, and Resedaceae (31). They are of interest be-cause neither their mode of biosynthesis nor their role in theplant has been resolved (11), although Kutacek and Kefeli(22) have suggested that glucobrassicin may be able to act asa precursor of indole auxins at certain stages in the life cycle

1 This work was supported by United States Public Health Serv-ice Research Grant GM-06921 from the National Institutes ofHealth to B. B. S.

2Present address: School of Biology, The University, LeicesterLE 1 7RH, England.

of the plant (e.g., during very rapid growth such as "bolting"of the flower shoot).Animal nutrition is influenced by indole glucosinolates due

to the fact that the enzyme myrosinase (which is released fromspecial cells in the plant tissues upon crushing) acts to releasethe goitrogenic thiocyanate ion from them (10, 41). In areaswhere large quantities of brassicaceous crops are eaten or fedto cows (which transfer part of the thiocyanate to their milk)these thiocyanate ions are at least partially responsible forendemic goiter in the human population (Ref. 28 and refer-ences therein).

Finally, the distribution of indole glucosinolates in the plantkingdom has provided useful information for chemotaxonomicconsiderations (1 1. 31, 33).As the woad plant has exceptionally high levels of three

indole glucosinolates, its study should cast light on these topics.We have examined the changes which occur in the levels ofeach of the indole glucosinolates in the shoots and roots ofwoad plants during germination, growth, and flowering. A sim-ilar study with the mustard plant (Sinapis alba L.) has recentlybeen published by Bergmann (3).

MATERIALS AND METHODS

Plant Material. Seeds of woad (l. tinctoria L.) were collectedfrom plants which had flowered in Yale University's MarshBotanic Garden in 1967 and had been stored at room tem-perature over the winter. The field-grown woad plants wereobtained from the crop produced by sowing these seeds in thespring of 1968.For sterile culture, seeds were dissected out of the fruits,

sterilized as described previously (10), and then transferred toautoclaved 14-cm Petri dishes (about 100 seeds per dish) con-taining 15 ml of the inorganic solution used by Danckwardt-Lilliestrom (7) modified to include an Fe-EDTA preparation(38), 11 ml/liter, instead of ferric tartrate. The germinatedseeds were allowed to grow in this solution in a constant tem-perature room at 25 C either in complete darkness or under16-hr photoperiods of approximately 1000 ft-c of mixed in-candescent and fluorescent light for 1 week. When 35S incor-poration experiments were carried out, part of the sulfate inthe inorganic solution was replaced by K235S04 to give an ac-tivity of 0.55 ,tc/ml.

For sterile culture of excised roots of the woad plant, sterileseeds as above were transferred to sterile Petri dishes contain-ing two Whatman No. 1 filter papers moistened with distilledwater. The Petri dishes were incubated at 25 C for 5 days andthen 1-cm apices were cut from the seedling roots and trans-ferred to 100-ml flasks containing 50 ml of root culture me-dium (7) with Fe-EDTA as the iron source as described above.Considerable variation was found in the rate of growth of rootsinitiated from seedlings; about 10% of the sample tested

498

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LEVELS OF GLUCOBRASSICINS IN WOAD

showed growth rates better than 4 mm/day and only thesewere used for initiation of clones. Clones were established andmaintained as described by Street and Henshaw (38) by alter-nation of "sector" and "tip" cultures. The passage length was 3weeks. Although not further studied by us, we can confirmDanckwardt-Liiliestrom's interesting observation (7) that someof these roots will spontaneously regenerate buds in culture.When 5S incorporation experiments were to be carried out,

part of the sulfate in the culture medium was replaced byK235SO4 to give a radioactivity of 0.18 ,uc/ml.

Extraction of Plant Material and Estimation of Indole Glu-cosinolate. The plant material was carefully cleaned andwashed with tap water and distilled water before extractionwith boiling methanol as previously described (10). The com-bined methanolic extracts were concentrated under reducedpressure at 30 C; the final volume was adjusted (in a volumetricflask) to be equivalent to 1 ml per 2 g fresh weight of originalplant material. Aliquots of 0.5 ml were applied as 8-cm streaksto washed (10) Whatman No. 3 MM chromatography paper.The chromatograms were double developed in 1-butanol-etha-nol-water (4:1:3, upper phase). The positions of indole glu-cosinolates were determined by spraying parallel strips withp-dimethylaminocinnamaldehyde reagent (10). The appropriateareas were cut out from the chromatogram and the compoundswere eluted with distilled water by the method of Dent (8). Thevolumes of the eluates were adjusted to 10 ml with distilledwater; 2-ml aliquots were removed, mixed with 1 ml of citratephosphate buffer (pH 7.0), 1 ml of myrosinase solution, and0.1 ml of 50 mm ascorbic acid solution, and then incubated at37 C for 2 hr. After incubation 2 ml of 96% (w/v) trichloro-acetic acid solution were added and the solution was diluted to20 ml with distilled water and centrifuged at 48,000g for 10min to precipitate the enzyme. Two-milliliter aliquots from thesupernatant were used for estimation of thiocyanate by themethod of Aldridge (1) as modified by Michajlovskij andLanger (27). The blank was prepared in exactly the same waybut the myrosinase solution was replaced by 1 ml of distilledwater. In each experiment an enzyme blank (lacking substrate)was run to check that thiocyanate had been completely re-moved from the enzyme during preparation.When this method was used to determine recoveries of glu-

cobrassicin added to the homogenate after filtration, values be-tween 83 and 85% were obtained.

Myrosinase Preparation. Myrosinase solution was preparedfrom mustard seeds (S. alba L.) by the method of Neuberg andWagner (29). SCN- originally present in the solution was re-moved by shaking the solution three times for 30 min withDowex 2-X4 in the chloride form (13).

Determination of Radioactivity. Zones of radioactivity onpaper chromatograms were located by a Vanguard paper stripcounter. The radioactivity of zones eluted with methanol-water(2: 1, v/v) was determined in Bray's solution (4) with an Ansi-tron liquid scintillation spectrometer. Solid residues werecounted in Bray's solution containing Cab-O-Sil (34 g/liter).Standard quench-correcting procedures were applied.

RESULTS AND DISCUSSION

Seeds of another crucifer, Brassica oleracea L., have beenreported (12) to lack glucobrassicin and neoglucobrassicin. Butextracts of woad seeds, prepared by placing the seeds in boilingmethanol for 5 min and then grinding them in the still boilingmethanol, contained a compound which was indistinguishablefrom glucobrassicin on the basis of its RF values in five paperchromatographic solvents and the chromogenic tests used pre-viously (10). Neither neoglucobrassicin nor glucobrassicin sul-fonate was detected in the seed extract. Since the quantity of

.: N -OS 03*CH2- C

S- C6H1105

RFIG. 1. Molecular structure of indole glucosinolates. a: R = H,

glucobrassicin; b: R = OCH3, neoglucobrassicin; c: R = SO-s,glucobrassicin- 1-sulfonate.

Table I. Inidoleglucosinolate Content of Woad Seed and Seedlings

Seeds1-Week-old

seedlingsLight

grownShootsRootsPer plant

EtiolatedShootsRootsPer plant

Glucobrassicin

mg/ mg/100 g 100 gfresh dryweight weight230 ...

PI/part

2.74

10.1 175.62.3311 .51166.8 0.31

- 2.64

6.1 160.0 1.757.8 199.50.13- - 1.88

Neoglucobrassicin GlucobrassicinSulfonate

mg/100 gfreshweight0

0.215.4

5.110.0

mg/100g

dryweight

3.40222.9

132.5255.0

jUg/part

0

0.040.420.46

1.470.171.64

mg/ mg/100g 100gfresh dryweight weight

0 ...

2.90

0.40

50.20

11.00

sPg/partO0

0.6700.67

0.110~0.11

glucobrassicin present was very high (230 mg per 100 g ofseeds [Table I]) it was clearly not present merely as a contami-nant on the surface of the seed; this conclusion was reinforcedby the demonstration that several washes of the seed with50% aqueous methanol (room temperature) did not reduce thequantity of glucobrassicin extractable from the seeds.

In the hope of obtaining some idea of the fate of the glu-cobrassicin upon germination of the seeds, sterile 1-week-oldseedlings of woad were grown and the content of indole glu-cosinolates in roots and shoots was determined. Since manystudies of indole glucosinolates have been carried out in dark-grown tissue (3, 20, 23, 31-35), we have compared the indoleglucosinolate content of seedlings grown in a regime of 16 hrof light and 8 hr of darkness with that of seedlings grown incomplete darkness (Table I).

It is clear that the indole glucosinolate content of the rootexceeds that of the shoot whether the comparisons are madeupon a fresh weight or a dry weight basis and whether thetissue is grown in the light or the dark. The proportions of theindividual glucosinolates differ dramatically between the shootsand roots. In the light-grown seedlings the shoots have a highlevel of glucobrassicin, a very low level of neoglucobrassicin(glubr3/neo = 50.5), and an intermediate level of glucobras-sicin sulfonate. The roots of these plants have high levels ofglucobrassicin and neoglucobrassicin (glubr/neo = 0.74) andno glucobrassicin sulfonate. The situation in the etiolated plantsis very different; here the shoots have high levels of glucobras-sicin and neoglucobrassicin (glubr/neo = 1.21) and a low levelof glucobrassicin sulfonate whereas the roots (like the roots ofthe light-grown plants) have high levels of glucobrassicin andneoglucobrassicin (glubr/neo - 0.76) and no glucobrassicinsulfonate.

'Abbreviations: glubr: glucobrassicin; neo: neoglucobrassicin.

Plant Physiol. Vol. 48, 1971 499



ELLIOTT AND STOWE

z

0

LIGHT GRROWN SEEDLING SHOOTS

z0Li.I-

zw

0U)

11 I I I ( IGLUBR. GLUBR. NEOGLUBR.

SULPHONATE

FIG. 2. Scans of radioactivity on paper chromatograms (1-butanol-acetic acid-water, 4:1:2) of methanol extracts of 1. tinc-toria shoots and roots after incubation in 'SO42-. Loadings forlight-grown seedling shoots and etiolated seedling shoots and rootswere equivalent to 0.5 g fresh weight; for light-grown seedling rootsand excised roots loading was equivalent to 0.3 g fresh weight. Lo-cations of RF zones of authentic compounds were detected bycolor reagent indicated at base. Sulfate and glucobrassicin sul-fonate run together in this solvent. GLUBR.: glucobrassicin.

These results indicate differences in glucosinolate synthesisby shoots and roots and particularly large effects of the lighttreatment on shoots. The effects of light noted by Bergmann(3) in mustard hypocotyls were much less marked. In contrast,the roots of the light- and dark-grown plants tend to be verysimilar in glucosinolate pattern although the dark-grown rootshave a higher content on a dry matter basis.

This variation in glucosinolate production by different or-gans and under different environmental conditions could beimportant in chemosystematic surveys and gives emphasis tothe warning by Ettlinger and Kjaer (I 1) on this point.We have commented previously (9) that an unidentified com-

pound detected by Schraudolf (33) in a wide variety of cruci-fers might be glucobrassicin sulfonate. Although he failed todetect his compound in I. tinctoria he used etiolated tissuewhich, as shown here, contains much less sulfonate than thegreen tissue which we used in our original observations. Thesolvent Bergmann (3) used would not have separated it fromthe 3oSO,2- given to his mustard seedlings. Dr. Schraudolf now

confirms (personal communication) that the two compoundsare likely to be the same. Hence this goitrogen is probablywidely distributed among plants of the family Cruciferae, in-cluding edible species.One intriguing problem posed by these results is the fate of

glucobrassicin in the seed. Table I also includes calculatedlevels of glucosinolate present on a per seed, per shoot, perroot, and per plant basis. There is no net increase of glucobras-sicin during the change from the nonimbibed seed to the 1-week-old green seedling, and there is actually a net loss ofglucobrassicin during the growth of the etiolated seedling.No glucosinolates could be detected in the culture solution

which had contained the 1-week-old seedlings (in contrast tothe situation discussed later when much longer growth periodswere used). This posed the problem of the metabolic fate ofseed glucobrassicin. Either the glucobrassicin from the seed iscompletely degraded and all the glucosinolates of the seedlingsare the products of de novo synthesis, or the seed glucobras-sicin is wholly or partially incorporated into seedling gluco-sinolates. While it is not known whether the 1-N methoxylationreaction resulting in the formation of neoglucobrassicin occursafter the complete glucobrassicin molecule has been synthe-sized or at some earlier stage, the work of Mahadevan andStowe (26) shows that the sulfonation leading to the formationof glucobrassicin sulfonate occurs after the formation of indoleacetaldoxime in the biosynthetic pathway and probably occursby direct sulfonation of glucobrassicin.

In the hope of obtaining further information on these points,sterile woad seeds were germinated in the inorganic nutrientsolution used previously but modified to contain part of thesulfate as wSO2-. The Petri dishes were kept for 7 days eitherin 16-hr photoperiods or in complete darkness as before. Meth-anolic extracts of the shoots and roots were prepared in theusual way; when chromatographed in 1-butanol-acetic acid-water (4:1:2) the peaks of radioactivity were as shown in Fig-ure 2. The peak of radioactivity due to glucobrassicin sulfonatecoincides with that due to free sulfate in this solvent but it maybe separated from the free sulfate by development in 1-penta-nol-pyridine-water (7:7:6) when the glucobrassicin sulfonatemigrates to RF 0.42 and the free sulfate remains near the ori-gin. The glucobrassicin sulfonate peak in the latter solvent con-ceals an unidentified component which can readily be separatedby rechromatographing the eluate from this region in the first(butanol-acetic acid-water) solvent, where the contaminant mi-grates to RF 0.46 while glucobrassicin sulfonate remains at RF0.18.

Table II shows the distribution of 3S label form aaSO2-. Thetotal amount of activity taken up by green seedlings is higher

Table II. Distributionz of I5S from Sulfate in Woad Seedlintgsafter 1-Week Inzcuibation

1-Week-Old ToethanolExtract IndoleSeedlings Mehnl xrc G lucosinclates

,sc/g, fresl: weight

Light grownShootRoot

EtiolatedShootRoot

2.214.76

Aclg dry lcIgweight freih

weighit

38.35 0.5669.06 1.12

1.11 29.31 0.162.29 58.43 0.33I II~~~~~~~~~~~~~~

%o ofTotalActiv-

Residue ity inIndoleGlucosi-nolate

pc/g dry etC g .tlgweight tres,h dryI weight weight- -O--"

9.80 0.2916.41 0.42

4.31 0.088.64 0.13

5.20 18.36.12 18.0

2.30 12.03.47 12.2

r - -. - _ i500 Plant Physiol. Vol. 48, 1971

-, -O --"




than that for the etiolated seedlings; also, the proportion of theradioactivity converted to indole glucosinolates is higher in thegreen (18%) than in the etiolated (12%) seedlings. It is interest-ing to find that, although the total activity in the root is muchhigher than in the shoot for both green and etiolated seedlings,the proportion of the activity contained in indole glucosinolatesis about the same (18%) for shoots and roots of light-grownplants and is also the same (12%) for shoots and roots ofetiolated plants.No attempt was made to determine the fate of the 'S taken

up by the plant and not incorporated into indole glucosinolates,but nonmetabolized 3SO42- would be expected in both the meth-anolic extract and in the residue in addition to any metabolitescontaining 3S (42).For determination of the specific radioactivities of the indole

glucosinolates, the methanolic extracts were first chromato-graphed in 1-pentanol-pyridine-water; the band of glucobras-sicin sulfonate (R, 0.42) and the double band of glucobrassicinand neoglucobrassicin (centered at R, 0.6) were eluted and re-chromatographed in distilled water (glucobrassicin sulfonate,RF 0.81; glucobrassicin and neoglucobrassicin, double band atR, 0.76); and the eluates from this chromatogram were re-chromatographed in 1-butanol-ethanol-water (4:1:3, upperphase) when glucobrassicin sulfonate had R,, 0.2, glucobras-sicin had Rr 0.35, and neoglucobrassicin had RF 0.42. Thecompounds thus purified yielded single peaks of radioactivitycoinciding with the p-dimethylaminocinnamaldehyde-positivezone when rechromatographed in each of the five solvents usedin this investigation (10).The eluates from the 1-butanol-ethanol-water chromatogram

were used for determination of the glucosinolate present byrelease of thiocyanate ions and for determination of radioiso-tope content by counting in Bray's solution. Table III presentsthe specific radioactivities and dilution values for each of theindole glucosinolates synthesized in the green and etiolatedseedlings. For both groups of seedlings the specific radioactivi-ties decrease (and dilution values increase) in the order gluco-brassicin -- neoglucobrassicin -e glucobrassicin sulfonate.

This is compatible with a conversion of the unlabeled glu-cobrassicin from the seedling to either neoglucobrassicin orglucobrassicin sulfonate before a high rate of biosynthesis ofglucobrassicin has commenced in the germinating seedling. Thelow specific radioactivities (and high dilution values) of theindole glucosinolates in the etiolated material are compatiblewith this suggestion, but in the light-grown material the highspecific radioactivities show that if the same process is oc-

curring there also must be rapid turnover occurring.Demonstrations of indole glucosinolate biosynthesis have

usually involved the use of leaf or hypocotyl tissue (3, 22, 23,31, 32, 35). The results discussed above suggested that the rootitself was capable of carrying out indole glucosinolate biosyn-thesis. For unequivocal confirmation of this, a clone of sterileroot cultures of I. tinctoria was developed as described in "Ma-terials and Methods." Culture vessels containing 1 liter of me-

dium were inoculated with thirty 1-cm "tip" cultures of woadroots (37) and the cultures were incubated at 25 C for 6 weeks.Then the roots (about 3 g per liter of media) were harvestedand extracted as before. The culture medium was passedslowly through an acid alumina column; this retained the in-dole glucosinolates and other anions but allowed the sucrose

to pass through. The column was washed and then the gluco-sinolates were eluted from it by 1 % K2SO, as described pre-

viously (10). The eluate was evaporated to dryness under re-

duced pressure, the indole glucosinolates were removed fromthe residue by warm absolute methanol, and the methanolicextract was concentrated and used for chromatography in theusual way.

Table III. Specific Radioactivity anid Dilutioni of Inidole Gliucosinio-lates Labeled with 355 from Sulfate

Glucobrassicin -Neoglucobrassicin GlucobrassicinGlucobrassicin ~~~Sulfonate1-Week-Old Seedlings

Specific Dilu- Specific Dilu- Specific Dilu-radio- tion radio- tion radio- tion

activity value' activity value activity value

Ac/lzinole cA!lmmole Lc/minmole

Light grownShoot 2134 1.12 1802 1.32 1409 1.7Root 2186 1.1 1764 1.34

EtiolatedShoot 715 3.35 618 3.87 245 9.75Root 938 2.55 840 2.85 ... ...

1 Specific radioactivity of sulfate supplied, specific radioactivityof indole glucosinolate.

The cultured excised roots were found to contain glucobras-sicin and neoglucobrassicin at 9.8 and 16.8 mg per 100 g freshweight, respectively, while they had released into the medium0.15 and 0.38 mg per 100 g fresh weight of roots of the samesubstances.

Thus, the excised roots are remarkably similar in indoleglucosinolate content to the roots of light-grown seedlingplants and they resemble these roots in morphology veryclosely also. When excised roots were grown for 1 week inculture medium containing 3SO4' the chromatogram of themethanolic extract of the roots yielded the radioisotope peaksshown in Figure 2. Hence roots synthesize indole glucosinol-ates, and in view of their similarity in indole glucosinolate con-tent to the roots of the light-grown woad seedlings it seems un-likely that any significant translocation of indole glucosinolatesbetween root and shoot or vice versa occurs.The discovery of small quantities of indole glucosinolates in

the culture medium is not entirely unexpected since the releaseof a variety of metabolites from excised root cultures of anumber of species has previously been reported (reviewed inRefs. 5 and 37). Three possible modes of release into the me-dium can be envisaged: the compounds may be actively se-creted by the roots into the medium, they may be releasedfrom dead cells which are sloughed off into the medium, orthey may be released from the cut end of the excised root.This latter possibility was eliminated by supporting sterilewhole woad seedlings on cheesecloth over the culture medium(like the excised root culture medium but lacking sucrose andvitamins) so that only their roots were immersed. The plantletswere kept in 16-hr photoperiods for 6 weeks; at the end of thisperiod the culture medium was found to contain glucobrassicinand neoglucobrassicin. Raj Bhandary et al. (30) used a similartechnique to demonstrate the release of alkaloids into theirculture medium by sterile seedlings of Atropa belladonna L.

This demonstration of the release of indole glucosinolatesby woad roots can provide the basis of some intriguing specula-tions. It is well known that yields of grass and clover grown onfields where Brassica had previously been cultivated are low(2, 6, 21, 36). It is also known that when wheat is grown in afield which has previously been cropped with woad, the colorof the wheat is much modified (16). In the middle ages re-strictions on woad growing were often imposed because of itseffect on later crops (18), and for this reason woad growerswere often forced into an itinerant existence long after the dis-appearance of other nomadic agriculture (16). Kutacek (21)has proposed that one of the factors acting in the Brassica ef-fect may be indole glucosinolates (or their degradation prod-

Plant Physiol. Vol. 48, 1971 501



Plant Physiol. Vol. 48, 1971

4-

80-inL-

uE 60-0O9 40-0)E 20-

':)A -

200-d

May June July Aug. Sept.

0160 \

in

iL120- *

0J)0

E 40-

May June July Aug. Sept.

0-0 Glucobrassicin0- -0 Neoglucobrassicin

0-0 Glucobrassicin sulphonate

FIG. 3. Changes in concentration of indole glucosinolates inshoots (A) and roots (B) of 1. tinctoria during first growing sea-son.

Table IV. Indoleglucosinolate Content of Bolting Woad Plants

2nd Year Plants

Glucobrassicin Neogluco- Glucobrassicinbrassicin SulfonateDate Hihharvested Height

cnt

litgl mg/ mg /mg/1Otg nglOigcmcr g fresli shoot g fresh0 shoot g fresi shootweight weighit weighitsApril 10 17 (rosette) 86 4.9 11 0.63 55 3.1Mayl 22 72 4.6 8 0.51 47 3.0May10 29 60 4.0 4 0.26 32 2.1May 17 42 (with 19 2.8 1 0.14 12 1.7

flowerbuds)

ucts) released from parts of the plants left in the soil. Thepresent studies suggest that not only decaying plant parts butalso actively growing, healthy roots may release indole gluco-sinolates (and possibly other important compounds such as thepolyphenols studied by Turetskaya and Kefeli [40]). The possi-ble ecological significance of such release for the large cruciferfamily need not be further stressed.

Seasonal Variations in Indole Glucosinolates. The dramaticeffect of the light regime on indole glucosinolate content sug-gested a study of seasonal changes in field-grown woad plants.Some previous studies have been carried out but these have re-ferred either to glucobrassicin alone (25) or to total thiocya-nate-yielding glucosinolates (19).

Swain (39) and Hillis and Swain (17) have discussed the pos-sibility that seasonal changes in structurally related compoundscan reflect biosynthetic relationships and thus might reveal glu-cobrassicin's metabolic roles. Also, since Kutavcek and Kefeli(22) have speculated that glucobrassicin may be a precursor ofindole auxins at certain times in the life cycle of cruciferousplants, e.g., during "bolting" of the flowering stalk, a knowl-edge of the changes in indole glucosinolate levels at this timeis particularly desirable.

Figure 3 shows the seasonal changes in levels of the indoleglucosinolates in plants developing from seed sown at the be-ginning of April in the year of harvest. The changes of thethree indoles reasonably parallel each other and are similar tothose observed for glucobrassicin by Kutacek et al. (25) andfor total thiocyanate-yielding glucosinolates by Josefsson (19).However, Bergmann (3) did not find a later increase in mustardglucosinolates. The sharp increase in each of the compoundsfound in the September harvest of shoots is a consequence ofthe death of the old leaves at this time, so that the harvestedmaterial contains a higher proportion of young material whichis richer in indole glucosinolates (22). There are no changes inrelative levels which might permit any speculations to be madeabout the biosynthetic relationships between the three com-pounds.The leaves of the 1st year woad plants are killed by the

winter frosts; then new leaves are produced in the spring andthe rosette form is lost as the plant "bolts" and flowers. TableIV lists the changes in indole glucosinolate levels which occurduring bolting. There are significant decreases in each of theindole glucosinolates both on a concentration and per plantbasis, but there is no evidence of a dramatic change in theirrelative proportions during bolting. The decreases are in accordwith Kutacek and Kefeli's suggestion (22), and further work todemonstrate unequivocal conversion of glucobrassicin to indoleauxins is now indicated.

LITERATURE CITED

1. AsIDRIDGE, W. N. 1944. A new method for the estimation of micro quan-tities of ecanide and thiocyanate. Analyst 69: 262-265.

2. BENTLEY, J. A. AND A. S. BICKLE. 1932. Stuidies on plant growth hor-mones. II. Further biological properties of 3-indolylacetonitrile. J. Exp.Bot. 3: 406-423.

3. BERGAN-N. F. 1970. Die Glucosinolat-Biosynthese im Verlatuf der Onto-genese von Sinapis alba L. Z. Pflanzenphysiol. 62: 362-375.

4. GsiAY, G. A. 1960. A simple efficient liquid scintillator for counting aqueoussolutions in a liquiid scintillation counter. Anal. Biochem. 1: 279-285.

5. BC-TCHIER, D. N. ANKD H. E. STREET. 1964. Excised root culture. Bot. Rev.30: 513-586.

6. CAMPBELL, A. G. 1959. A germination inhibitor and root-growth retarderin Chou NMoellier (Brassica oleracea var.). Nature 183: 1263-1264.

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8. DENT, C. E. 1947. The amino-aciduria in Falconi syndrome. A study mak-ing extensive use of techniques based on paper partition chromatography.Biochem. J. 41: 240-253.

9. ELLIOTT, NI. C. AND B. B. STOWE. 1970. A novel sulfonated natural indole.Pliytochemistry 9: 16291632.

10. ELLIOTT, NI. C. AND B. B. STOWE. 1971. Indole compounds related to auxinsand goitrogens of woad. Plant Physiol. 47: 366-372.

11. ETTLTNGER, WI. G. AND A. KJAER. 1968. Sulfur compounds in plants. RecentAdvan. Phytochem. 1: 59144.

12. GMELIN, R. 1964. Occurrence, isolation, and properties of glucobrassicin andneoglucobrassicin. Colloq. Int. Centre Nat. Rech. Sci. 123: 159-168.

13. GMELN, R. AND A. I. VIRTANEN. 1960. The enzymic formation of thiocyanate(SCN-) from a precursor(s) in Brassica species. Acta Chem. Scand. 14:507-510.

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Plant Physiol. Vol. 48, 1971



Documents

Distribution and Variation of Indole Glucosinolates in Woad