phenylpropanoid metabolism in cotyledons of raphanus sativus and the effect of competitive in vivo...

Botanisches Institut, Universirat Koln, Federal Republic of Germany

Phenylpropanoid Metabolism in Cotyledons of Raphanus sativusand the Effect of Competitive in vivo Inhibitionof L-Phenylalanine Ammonia-Lyase (PAL)by Hydroxylamine Derivatives

DIETER STRACK, NORBERT TKOTZ and MARGRET KLUG

With 6 figures

Received 10 April 1978 . Accepted 6 May 1978

Summary

In the four genera of the Brassicaceae studied we have observed the following: if sinapin(sinapoylcholine) is present in the seed, it is degraded during early stages of germination andat the same time sinapoylglucose accumulates. The accumulation kinetics of sinapoylglucoseare different for each plant and the data also indicate a further metabolism of this compound.Only in the genus Raphanus have we observed the accumulation of a second sinapoylderivative and this is correlated with sinapoylglucose disappearance.

This paper provides evidence that the formation of sinapoylglucose in the cotyledons ofRaphanus sativus is an ultimate result of interconversions of the «seed-sinapoyl derivatives»,and our results indicate that PAL is not involved in sinapoyl derivative metabolism inRaphanus. In vivo inhibition of PAL activity with a-aminooxy-p-phenylpropionic acid (AOP)does not affect the accumulation of sinapoylglucose, whereas the formation of anthocyanins,flavonols and a feruloyl derivative is severely depressed. The K j value was found to be1.13 X 10-8 M (K/Km = 3.5 X 10-5). Administration of AOP to the intact seedling revealed150 values to be 0.14 mM for pelargonidin, 0.16 mM for kaempferol, and 0.18 mM for theferuloyl derivative.

Key words: Brassicaceae, germination, phenylpropanoid metabolism, high-pressure liquidchromatography, PAL inhibition, hydroxylamine derivatives.

Introduction

To date it has been shown for various members of the Brassicaceae that the seed

constituent, sinapin (sinapoylcholine), is rapidly degraded during early stages ofgermination (TZAGOLOFF, 1963; Bopp and LijDICKE, 1975; STRACK, 1977) and at the

Abbreviations: PAL = L-phenylalanine ammonia-lyase; TLC = thin-layer chromatography; CC = column chromatography; HPLC = high-pressure liquid chromatography;AOA = a-aminooxy acetic acid; AOP = a-aminooxy-p-phenylpropionic acid; BAOP =N-benzyl-oxycarbonyl-a-aminooxy-p-phenylpropionic acid.

Z. Pflanzenphysiol. Bd. 89. S. 343-353. 1978.

344 DIETER STRACK, NORBERT TKOTZ and MARGRET KLUG

same time another derivative (sinapoylglucose) is found to accumulate. Comparativekinetics suggest that an interconversion between these two compounds occurs,however, there are quantitative differences among the plants studied.

The Raphanus system, previously described (STRACK, 1977), is considerably morecomplex than those of the other genera studied. Besides sinapin there are two othermajor sinapoyl derivatives, one of which is rapidly degraded, whereas the other ismaintained at a constant level. Quantitative analyses of this system suggest that asimple interconversion does not occur and PAL mediated de novo synthesis of sinapicacid has never been completely excluded as a possible contributing factor.

AMRHEIN et al. (1976) and AMRHEIN and GODEKE (1977) reporteda-aminooxy-p-phenylpropionic acid (AOP) as being a potent and specific competitiveinhibitor of PAL and they suggested the use of this compound for studies ofphenylpropanoid metabolism. Therefore, we applied this inhibitor to Raphanus todetermine its effect on sinapoylglucose accumulation as well as the metabolism offlavonoid derivatives and a feruloyl derivative.

Material and Methods

1. Plant material and culture conditions

Seeds of Raphanus sativus L. var. sativus, cv. Saxa were purchased from Zwaan u. Co's u.Komp., Delfter Marktgartner-Samenzucht GmbH, Netherlands. Brassica oleracea capitata,Sinapis alba, and Lepidium sativum were purchased from local merchants. Plants were grownin a phytotron under fluorescent light (ca. 7000 lux) at 22°C and 70 % relative humidity.Plants to be harvested on or before the 3rd day of germination were grown on filter paper,the others in defined soil (type T, L. Keller, Cologne, West Germany), mixed 1:1 with peat.

2. Extraction oj soluble phenylpropanoid compounds

Extractions were carried out by treatment with an Ultra Turrax homogenizer for 2 min.Derivatives of hydroxycinnamic acids and kaempferol were extracted with 10 ml 80 0/0

methanol and pelargonidin derivatives in 20 ml methanol, containing 1 % HCI (v/v). Thehomogenates were centrifuged at 3000 Xg for 10 min.

3. Chromatography

Polyamide CC and polyamide centrifugationDerivatives of hydroxycinnamic acids and kaempferol were fractionated on polyamide CC

6, grain size < 0.16 mm (7 g, bed dimension 2 X 12 cm) (STRACK, 1977). Fractionation bypolyamide centrifugation was done with disposable 10 nil-syringes, filled with 2 g polyamideCC 6, grain size < 0.07 mm, equilibrated with water. The syringes were hung in 25ml-centrifuge tubes, leaving a free space of ca. 5 ml at the tube bottom. Unbound water waseliminated by centrifuging 3 times at 1000 X g for 5 min. Extracts of 20 seedlings wereadjusted to 0.5 ml 80 % methanol and 200 ,ul was applied to each syringe column. Aftercentrifugation at 3,000 X g for 30 sec, sinapoylcholine, sinapoylglucose and a feruloylderivative were eluted at 3000 X g with 5 3-ml 40 % methanol washes. The kaempferolderivatives were eluted by 5 3-ml 80 % methanol aliquots.

TLC on microcrystalline cellulose (AVICEL)

Polyamide eluants containing the hydroxycinnamic acid derivatives were evaporated todryness under reduced pressure at 40°C and the residue was redissolved in 4 ml 50 0/0

methanol. 100 ,ul were chromatographed on AVICEL (20 X 5 cm) in CAW, CHCI3-gl. acetic

Z. Pjlanzenphysiol. Bd. 89. S. 343-353. 1978.

and the feruloyl derivative(8 : 1 : 2); hR f values:

PAL inhibition 345

acid-H20 (3 : 2, H 20 saturated). Separation of sinapoylglucosewas accomplished with BAW, n-butanol-gl.acetic acid-H20sinapoylglucose = 52; the feruloyl derivative = 61.

High-pressure liquid chromatography (HPLC)

The chromatographic system used was obtained from Spectra-Physics (Santa Clara,California, U.S.A.) and is described elsewhere (STRACK and KLUG, in press). Thechromatographic column, 25 X 4 mm, was prepacked with LiChrosorb RP-8 or RP-18 (5 ftm)(E. Merck, Darmstadt, West Germany) and was run at ambient temperature.

Separation was accomplished by gradient elution:Solvent A: methanol-water-acetic acid (5 : 90 : 5);Solvent B: methanol-water-acetic scid (90: 5: 5);Gradient profile: linear 0 to 70 % Bin 35 min or

o to 35 Ofo B in 45 min;Flow rate: 1.0 mllmin;Detection: 312 nm, 0.08 AUFS;Sample size: 10 ftl.Extracts of 20 pairs of cotyledons were adjusted to 4 ml 50 Ofo methanol and before being

applied to HPLC were centrifuged at 50,000 X g for 20 min. The precision of peak areas forearly and late peaks was 1-2 % relative standard deviation. Determination of free sinapicacid (2 N NaOH, 1 hr at room temperature, adjusted to pH 1 and extracted into ether) wascarried out with a linear gradient 0 to 50 Ofo B in 20 min.

4. Quantitative determination of phenylpropanoid compounds

TL chromatographed hydroxycinnamic acid derivatives were marked under UV, scrapedinto 10 ml-centrifuge tubes, eluted with 2 ml methanol, centrifuged at 3000 X g for 5 min andthe absorbance of the supernatant was measured at 330 nm. The polyamide CC eluantscontaining kaempferol derivatives were evaporated to dryness under reduced pressure at40°C, the residue redissolved in 2 ml 2 N HCI and heated at 100°C for 30 min. Kaempferolwas extracted into ethylacetate, evaporated to dryness and redissolved in 3 ml methanol,containing 1 Ofo AICla. After 15 min (formation time of the AI-complex) the absorbance wasmeasured at 425 nm. The quantities of pelargonidin derivatives were determined bymeasuring the absorbance of the centrifuged extracts at 515 nm. For pelargonidin derivativesthe absorbance coefficient at ). max was determined to be 1 X 104 cm2 /mmole, and forkaempferol and sinapoyl derivatives a value of 2 X 104 cm2 /mmole was used.

Identification of pelargonidin and kaempferol was done by comparison with authenticsamples.

With the exception of sinapin, the quantitative changes of sinapoyl derivatives in thefour genera of the Brassicaceae (fig. 1 and 2) were determined with HPLC.

5. PAL testExtraction of PAL50 seedlings were treated with an Ultra Turrax homogenizer for 2 min in 10 ml water free,

cold (-20°C) acetone and the homogenate was centrifuged at 3000 Xg for 10 min. The pelletwas washed 3 times with cold acetone and 2 times with cold ether then dried in a desiccatorin vacuo. The dried powder was suspended in a known volume of 0.05 M borate buffer, pH8.8, containing 1 mM 2-mercaptoethanol. Addition of 0.5 g Dowex 1 X 2 (Serva, Heidelberg,West Germany), equilibrated with borate buffer, was routinely used during PAL extraction(GROSS et aI., 1975). The suspension was centrifuged at 3000 X g for 10 min and thesupernatant was applied to Sephadex G-25 centrifugation.

Treatment with Dowex resulted in 70 % depression of the absorbance at 290 nm and 14 0/0

less protein content, however PAL activity was not affected.



Determination of PAL activity

PAL activity was determined spectrophotometrically by measuring the increase inabsorbance at 290 nm (PMQ II, Zeiss) (ZUCKER, 1965). Assay: 0.1-1.0 ml enzyme extractwas mixed with L-phenylalanine and adjusted to 3 ml, giving a substrate concentration of 5mM. The KM value was determined from double-reciprocal plots according to Lineweaverand Burk and found to be 3.2 X 10-4 M. The standard deviation of the activity determinationwas ± 2.9 pmoles/min/pair of cotyledons.

Determination of the reaction product

L-14C-phenylalanine was incubated with enzyme and the labelled product was identifiedby TLC as trans-cinnamic acid. The UV spectrum of the product was identical with that oftrans-cinnamic acid.

6. PAL inhibition experiments

PAL inhibitors

L-a-aminooxy acetic acid semihydrochloride (AOA) (Sigma, St. Louis, Mo.), N-benzyloxycarbonyl-L-a-aminooxy-p'-phenylpropionic acid dicyc1ohexylammonium salt (BAOP) andL-a-aminooxy-p-phenylpropionic acid (AOP) were a generous gift of Prof. Dr. N. AMRHEIN(Ruhr-Universitat Bochum, West Germany).

Administration of the PAL inhibitors

24 hr old intact seedlings (60) were placed into 5 ml aqueous solution of AOA, BAOP orAOP and were shaken for 6 hr. The seedlings were removed and grown on filter papermoistened with the inhibitor solution of the same concentration.

In vitro characterization of PAL inhibition by AOP

The nature of inhibition of PAL was determined from double-reciprocal plots according toLineweaver and Burk, from which K,[ values and K j• were determined ..• app

Results

1. Dynamics of sinapoyl derivatives

Comparisons of the quantitative changes of sinapoyl derivatives duringgermination of the 4 plants studied here reveal a marked difference betweenRaphanus and the other three members of the Brassicaceae. In Raphanus, sinapindegradation and sinapoylglucose accumulation is most rapid and show the greatestfluctuations in concentration. In the other three genera there is degradation ofsinapin but only low level accumulation of sinap::>ylglucose. Fig. 1 summarizes theresults of high-pressure liquid chromatographic analyses of the sinapoyl derivatives.Since sinapin could not be chromatographed with our HPLC system it had to beanalyzed with TLC.

Two other major differences within the Brassicaceae studied are that onlyRaphanus seeds contain a second major sinapoyl derivative which is degradedrapidly and a second sinapoyl derivative which accumulates during germination. Thisnewly synthesized compound reaches its highest level after the 10th day ofgermination. A 3rd accumulating sinapoyl derivative previously reported (STRACK,


PAL inhibition 347

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Time (days)

Fig. 1: Sequential quantitative changes of sinapic acid esters in cotyledons of Brassicaceaeseedlings grown under a 14-hr day. Each point represents the mean of several high-pressureliquid chromatographic determinations of 2 extracts from 20 pairs of cotyledons.Sinapoylcholine was determined with TLC. - 0 -, -. -, - 6 -, sinapic acid estersdesignated with the symbols B4• B3 and B5•

1977) was not detected in the present study. Whether this discrepancy is a result ofthe different analytical system employed (HPLC) or a different seed source is not yetclear.

The complexity of the metabolism in Raphanus compared with the other threegenera studied is further demonstrated in fig. 2. These high-pressure liquidchromatograms, taken from 5 day old seedlings, show "fingerprints» of thephenylpropanoid pattern (STRACK and KLUG, in press). Peak 1, sinapoylglucose,appears in all 4 plants, whereas the 2 other major sinapoyl derivatives of Raphanuscotyledons (peaks 2 and 3) are not detectable in the other plants. Peak 2 representsthe 2nd accumulating sinapoyl derivative and peak 3 the Raphanus «seed-sinapoylderivative», which exhibits a fairly constant level during germination.



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Fig. 2: High-pressure liquid chromatograms of 5 day old Brasscicaceae seedlings grown undera 14-hr day. Extracts of 20 pairs of cotyledons were adjusted to 4 ml 50 Ofo methanol and 10,ulapplied to HPLC (see «Materials and Methods»). 1, sinapoylglucose; 2 and 3, sinapic acidesters. Peak 2 represents the 2nd accumulating sinapoyl derivative (B5 ) in Raphanuscotyledons, and peak 3 represents the «seed-sinapoyl derivative» which maintains a fairlyconstant level (see fig. 1).

2. PAL activity and accumulation of phenylpropanoid compounds

Determination of PAL activity in Raphanus seedlings was done at 6 hr intervalsbetween the 24 th and 72 nd hr of germination.

Cotyledons of light-grown seedlings exhibit typical PAL-kinetics (see fig. 3): i.e.activity rises rapidly, reaches a sharp maximum and decreases thereafter. At 24 hrthe activity is 10 pmoles/min/pair of cotyledons, the maximal activity is 90pmoles/min at 45 hr and by 66 hr the activity has fallen to 10 pmoles/min. Thehighest accumulation rates of phenylpropanoid compounds are: 18pmoles/min/cotyledon pairs for kampferol derivatives, 55 pmoles for pelargonidinderivatives and 140 pmoles for sinapoylglucose.


PAL inhibition 349

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Fig. 3: Time-course of PAL activity in cotyledons of light-grown Raphanus sativus seedlingsand the accumulation of phenylpropanoid compounds. - -. - -, sinapoylglucose; ···0"',pelargonidin dervatives; . - .•. - " kaempferol. Each point represents the mean of 4l!xperiments. The PAL determination was made on extracts from 50 cotyledon pairs,determination of phenylpropanoid compounds .on extracts from 20.

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Fig. 4: Time-course of PAL activity in cotyledons of dark-grown Raphanus sativus seedlingsand the accumulation of phenylpropanoid compounds. See legend to fig. 3.



In dark-grown cotyledons there is no marked increase in PAL activity (see fig. 4),anthocyanins are undetectable and kaempferol derivatives are found in only lowconstant levels. Only sinapoylglucose shows a high accumulation rate and reaches thesame level in both light- and dark-grown tissue.

The activity in the root shows a linear increase of about 4 pmoles per hour, and by72 hr reaches an activity of 100 pmoles/min/root. No difference was observedbetween light- and dark-grown seedlings.

PAL activity in the hypocotyl shows a maximum of 32 pmoles/min at 48 hr andan accumulation rate of pelargonidin derivatives of 36 pmoles/min and kaempferolderivatives of 2 pmoles/min. In 48 hr dark-grown hypocotyls PAL activity is lessthan 10 pmoles/min, anthocyanins are undetectable and flavonols are only found intrace amounts.

3. PAL inhibition

In the PAL inhibition experiments, we employed 3 different hydroxylaminederivatives (see «Materials and Methods»). In vivo experiments, at 0.5 mMconcentration, all derivatives severely depressed the feruloyl and flavonoidmetabolism (table 1). To determine secondary effects of the inhibitors we examined 4parameters: 1. gross morphology, 2. fresh weight, 3. chlorophyll content, and 4.degradation of sinapoylcholine. Table 1 shows that application of AOP did not result

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Fig. 5: Effects of in vivo inhibition of PAL activity by different AOP concentrations. AOPwas applied in 5 ml aqueous solutions for 6 hr to 60 24-hr old seedlings, which were thengrown for 18 hr on filter paper moistened with the inhibitor solutions.


PAL inhibition 351

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Fig. 6: Lineweaver-Burk plots of PAL activities for the determination of the K j value forAOP. At different AOP concentrations (M) the apparent KM values were determined.K j = (I) X KM X (KM -KMt 1 = 1.13 X 10-8 M.app

in major secondary effects, yet the formation of pelargonidin, kaempferol and theferuloyl derivative was depressed to the greatest extent. Accumulation ofsinapoylglucose remained unaffected.

BAOP also exhibited a strong inhibition effect but the sinapoylcholine degradationwas inhibited slightly.

AOA adversly affected the development of the seedlings from all four parametersmeasured. Fresh weight was depressed by 60 % , the chlorophyll content by 80 %,

and the degradation of sinapoylcholine by 75 %. Therefore AOA was the leastsuitable, since it has a broad inhibition capability.

With AOP, the most specific and active inhibitor, we conducted a dose-responseexperiment. The results are shown in fig. 5. It can be seen that inhibition is a function

Table 1: Effects of hydroxylamine derivatives on intact seedlings of Raphanus sativus.24 hr old seedlings were subjected to a 6 hr application in 0.5 mM inhibitor solutions andharvested after growth (18 hr) on filter paper moistened with the inhibitor solution. Valuesexpressed as % of control.

Newly appearing compounds Indicators for secondary effectsInhibitors

B2 BlO Pelargo- Kaempfe- B1 Fresh Chloro-nidin rol weight phyW-)

AOA 81 59 22 - 403 42 17BAOP 102 47 21 53 162 99 102AOP 103 34 13 18 103 98 104

B2 = Sinapoylglucose; BlO = Feruloyl derivative; B1 = Sinapoylcholine.,:") Determination according to BRUINSMA (1961).

z. Pjlanzenphysiol. Bd. 89. S. 343-353. 1978.


Table 2: PAL activity in AOP inhibited and untreated Raphanus seedlings. 24 hr old seedlings were subjected to a 6 hr treatment with water or 1 mM AOP and harvested aftergrowth (21 hr) on filter paper.

Incubation medium Mean of 3 determinations of PALactivity (pmoles cinnamate/minlpair of cotyledons)

Water 871 mM AOP 85

of AOP concentration. Inhibition of pelargonidin formation reaches 50 % at 0.14mM, kaempferol at 0.16 mM, and the feruloyl derivative at 0.18 mM. However, noeffect was observed on the formation of sinapoylglucose.

The PAL activity extracted from seedlings treated with the highest concentrationof AOP (1 mM) was found to be equal to that of the control (table 2).

Competitive inhibition by AOP is demonstrated in fig. 6. From 5 different AOPconcentrations, KMapp values were determined and the Kj value was found to be1.13 X to-8 M; K/KM = 3.5 X 10-5

•

Discussion

Raphanus sativus shows a distinct and different metabolism of sinapic acidderivatives, when compared with the other genera studied here (fig. 1 and 2). Thequantitative changes are the most rapid and it is likely that a complex series ofinterconversions occurs during germination. The PAL inhibition experiments haveshown clearly that flavonoid and ferulic acid synthesis in Raphanus sativus isseverely depressed, yet sinapic acid metabolism is unaffected. Since PAL is aprerequisite to both flavon·aid and hydroxycinnamic acid synthesis, its inhibitionshould be reflected by quantitative changes in both groups of compounds. Since thesinapoyl derivatives exhibit no response to inhibitors, this suggest that existing C6-C3

carbon skeletons are interconverted via degradative and biosynthetic reactions. If thisis indeed the case, then in PAL inhibition experiments with the other genera mightyield a similar result.

Since in Brassica, Sinapis and Lepidium the only rapidly degraded seed constituentis sinapin, we might assume, as suggested for Sinapis by Bopp and LUDICKE (1975),that this compound is the precursor of sinapoylglucose. In Raphanus however thiscannot be clearly decided, since a second «seed-sinapoyl derivative» (B4) might alsobe involved.

In 24 hr old Raphanus seedlings there is a marked drop ofthe major seed constituents(fig. 1), however, the sinapoylglucose at that time is detectable only in small amounts.Preliminary measurements have shown that the total amount of sinapic acid does notchange, thus it is difficult to explain the rapid, early decrease in derivatives and thedelayed appearance of sinapoylglucose. It might be expected that free sinapic acid


PAL inhibition 353

would accumulate during derivative degradation and then be consumed in subsequentsinapoylglucose formation. We have examined the free sinapic acid content at eachstage of development but no evidence has been found to indicate a transient pool.Therefore this interconversion is not a simple one, but might involve intermediatesnot yet identified.

Determination of PAL activity in Raphanus cotyledons, grown in dark and light,show the expected correlation with flavonoid accumulation (fig. 3 and 4), but there isno correlation with the accumulation of sinapoylglucose. This does not exclude thepossibility that some sinapoylglucose formation is mediated through PAL, however,it is unlikely that de novo synthesis of sinapic acid c·ontributes significantly to thesinapoylglucose pool, since in PAL inhibited cotyledons, the accumulation of thiscompound is not affected.

Of the 3 PAL inhibitors used, AOA proved to be unsuitable, because it severelydamaged the seedlings. BAOP and AOP showed no major secondary effects on theseedlings, however BAOP did affect the degradation of sinapin slightly. A detailedstudy of these inhibitors has been done by AMRHEIN et al. (1976) and AMRHEIN andGODEKE (1977). Their results with Fagopyrum esculentum showed a PAL affinity forAOP of K j = 1.4 X 10-9 M and we found a value of Ki = 1.13 X 10-8 M withRaphanus.

It was also observed that AOP does not affect the total amount of PAL (table 2),thus it is likely that the level of this enzyme is not controlled by the endproducts. ENGELSMA (1968)suggested repression of PAL by endproducts of cinnamic acid metabolism.Our results however, which are in agreement with those reported for Fagopyrum(AMRHEIN et aI., 1976), make feedback repression as a control mechanism of PAL inRaphanus unlikely.

Acknowledgement

We are grateful to Prof. N. AMRHEIN (University of Bochum) for a generous gift ofhydroxylamine derivatives and his interest in this work.

References

AMRHEIN, N., K.-H. GODEKE, and V. 1. KEFELI: Ber. Deutsch. Bot. Ges. 89, 247 (1976).AMRHEIN, N. and K.-H. GODEKE: Plant Sci. Lett. 8, 313 (1977).Bopp, M. and W. LUDICKE: Z. Naturforsch. 30 C, 663 (1975).BRUINSMA, J.: Biochim. Biophys. Acta 52, 576 (1961).ENGELSMA, G.: Planta (Bed.) 82, 355 (1968).GROSS, G. G., R. L. MANSELL, and M. H. ZENK: Biochem. Physio!. Pflanzen 168, 41 (1975).STRACK, D.: Z. Pflanzenphysiol. 84,139 (1977).STRACK, D. and M. KLUG: Z. pflanzenphysio!. 88, 279 (1978).TZAGOLOFF, A.: Plant Physio!' 38, 202 (1963).ZUCKER, M.: Plant Physio!. 40, 779 (1965).

Dr. D. STRACK, Botanisches Institut der Universitat Koln, Gyrhofstra6e 15, D-5000 Koln41, Federal Republic of Germany.

Z. Pflanzenphysiol. Ed. 89. S. 343-353. 1978.

phenylpropanoid metabolism in cotyledons of raphanus sativus and the effect of competitive in vivo...

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