improved method detecting auxin-induced hydrogen efflux ...hormone application 2 hours after...

Plant Physiol. (1980) 66, 561-5650032-0889/80/66/0561/05/$00.50/0

An Improved Method for Detecting Auxin-induced Hydrogen IonEfflux from Corn Coleoptile Segments'

Received for publication December 3, 1979 and in revised form April 17, 1980

MICHAEL L. EVANS AND MARY JO VESPER2Department of Botany, The Ohio State University, Columbus, Ohio 43210

ABSTRACT

Conditions necessary to detect maximal auxin-induced H+ secretionusing a macroelectrode have been investigated using corn coleoptile seg-ments. Auxin-induced H+ secretion is strongly dependent upon oxygenationor aeration when the tissue to volume ratio is high. Cuticle disruption orremoval is also necessary to detect substantial auxin-induced H' secretion.The auxin-induced decrease in pH of the external medium is stronger whenthe hormone is applied to tissue in which the cuticle has been disruptedwith an abrasive than when the hormone is applied to tissue from whichthe cuticle and epidermis have been removed by peeling. The lowerdetectable acidification ofthe external medium when using peeled segmentsappears to be due in part to the leakage of buffers into the medium and inpart to the removal of the auxin-sensitive epidermal cells.The sensitivity of corn coleoptile segments to auxin, as measured by H+

secretion, increases about 2-fold during the first 2 hours after excision.Tlhis change in apparent sensitivity to auxin as reflected by H' secretionis paralleled by a time-dependent change in the growth response to auxin.Under optimal conditions for detecting H+ efflux (oxygenation, abrasion,hormone application 2 hours after excision), the latent period in auxin-induced H+ efflux (about 7 or 8 minutes) is only half as great as the latentperiod in auxin-induced growth (about 18 to 20 minutes). These observa-tions are consistent with the acid growth hypothesis of auxin action.

According to the acid growth hypothesis of auxin action, auxinenhances growth by stimulating the secretion of H+ ions from thecytoplasm into the cell wall. The resultant acidification of the cellwall is thought to lead to wall loosening and accelerated growth(5, 8, 13, 14).

If auxin-induced H+ secretion acts as a mediator of auxin-induced growth, one would expect hormone-induced H+ efflux toprecede hormone-induced acceleration of growth. The most com-monly used method of measuring H+ efflux is to place a largenumber (e.g. 40) of 1-cm long tissue segments in a small volume(e.g. 5 ml) of stirred weak buffer and monitor the change in pH ofthe medium in the presence and absence of hormone (12, 14).Since the cuticle is said to offer substantial resistance to the entryand exit ofH+ ions (3), it is common practice to remove the cuticleeither by peeling the tissue segments with a pair of fine-tippedforceps or abrading them with emery powder. Using this method,auxin-induced H+ efflux appears to begin about 20 min or moreafter application of hormone (1, 12, 14) even in those tissues inwhich growth is maximally promoted within 15 min. This rather

'This research was supported by National Science Foundation GrantPCM 78-0581.

2Present address: Department of Biology, The University of NorthCarolina at Charlotte, UNCC Station, Charlotte, N. C. 28223.

long apparent latent period in auxin-induced H+ secretion seemsto be due, at least in part, to the large volume of the incubationsolution compared with the volume of the cell wall fluid in whichH+ would normally be accumulating. When the volume of theexternal medium is minimized, shorter latent periods are observed.Cleland (2), using a flat-tipped electrode resting on a row ofpeeledAvena coleoptile sections, reported an average latent period of 14min in induction of H+ secretion by auxin, whereas Jacobs andRay (9), using a pH microelectrode inserted into the free space,reported latent periods of 12 min in corn coleoptile segments andan average of 15 to 18 min in pea stem segments. Inasmuch as thelatent periods observed for auxin-induced H+ secretion in thesetissues were less than the corresponding latent periods for auxin-induced growth, these data may be viewed as consistent with theacid-growth hypothesis. However, in both studies, those authors(2, 9) reported considerable variability in the latent periods for H+efflux and, in both studies, there were instances in which no auxin-induced H+ efflux was detected (personal communications).Through slight modifications of previously reported methodol-

ogy, we have developed a simple procedure for measuring hor-mone-induced H+ efflux in corn coleoptile segments. Using thisprocedure, we can reproducibly detect auxin-induced H+ secretionin corn coleoptiles with a latent period of about 7 min. This isapproximately half the latent period associated with auxin-in-duced growth in this tissue.

In this report, some observations on improving methodologyfor detecting H+ efflux are presented and the use of the method todetect rapid auxin-induced H+ efflux is described. The system isalso used to examine time-dependent changes in the magnitude ofauxin-induced H+ efflux which closely parallel changes in tissuesensitivity to auxin as measured by enhancement of growth.

MATERIALS AND METHODS

Plant Material. Experiments were done using coleoptiles from4- to 5-day-old corn (Zea mays L., hybrid WF 9 x 38, Customaize,Inc., Momence, Ill.). Growing conditions were as described pre-viously (6).

Scanning Electron Microscopy. Scanning electron microscopeobservations were made of the surface of undisturbed, abraded,and peeled coleoptiles. Segments were fixed for 5 min in 1.5% (v/v) phosphate-buffered glutaraldehyde for 30 s followed by theaddition of aqueous OS04 to a final concentration of 1% (w/v).The total fixation time was 10 min. The segments then weredehydrated in a graded ethanol series and subjected to criticalpoint drying using liquid CO2. The segments then were coatedwith gold and observed at 20 kev using an Hitachi S-500 scanningelectron microscope.

Cuticle Removal. To facilitate H+ efflux from coleoptile seg-ments, the cuticle was removed either by peeling (12) or byrubbing the segments with an abrasive (4). Peeling was done byphysically removing strips of material from the surface of thecoleoptile before excising segments. This was done over an esti-

561

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Plant Physiol. Vol. 66, 1980

mated 80%o of the surface of the coleoptile. For abrasion, a 1:1mixture of emery No. 305 (Edmund Scientific Company, Barring-ton, N. J.) in water was used. The coleoptile was stroked gentlythree times from base toward the tip with the emery paste betweenthumb and forefinger. The coleoptile was rotated approximately600 between strokes. After the abrasion procedure, the coleoptileswere rinsed thoroughly with distilled H20 and floated on a largevolume of buffer until segments were cut and the leaves wereremoved. Although the purpose of the abrasion procedure ismerely to disrupt the cuticle, A. M. Jones and L. N. Vanderhoef(personal communication) found that the procedure causes cellu-lar damage in localized regions of abraded materials (Fig. 1). Thisis in large measure unavoidable since they determined that theemery particles are on the average some 35 times larger in diameterthan the thickness of the cuticle.Measurement of H+ Efflux. Forty coleoptile segments I cm in

length were placed in a glass vial containing 4 ml 1 mM K-phosphate (initial pH 6.2-6.3). The vial was 63 mm in height and17 mm in diameter with a total volume of 14 ml. A small Teflonmagnetic spin bar was used to stir the incubation medium. Thespin bar was separated from the coleoptile segments by a disc ofplastic screen mounted 5 mm above the bottom of the vial. AMarkson semimicro combination pH electrode (No. 2885) with aComing model 7 pH meter was used to measure pH. Output forthe pH meter was recorded on a Sargent-Welch model SRLGrecorder with a full scale sensitivity of 0.1 mv using a chart speedof 15.24 cm/h. The output from the pH meter was attenuated witha back voltage to allow adjustment of the recorder to a full scaledisplacement over the pH range of 5.7 to 6.4. In those experimentsin which oxygenation was used in addition to stirring, 02 wasbubbled at the rate of 26 liters/h through the incubation mediumusing a 22-gauge (0.4 mm i.d.) syringe needle.At the end ofsome experiments, the total amount ofH' released

after the addition ofauxin was determined by titrating the mediumback to the pH at the time of hormone addition. This was doneusing the same experimental set-up and titrating with 8 mm NaOHwith the segments remaining in the medium.The sensitivity of corn coleoptile segments to auxin has been

shown to increase with time after excision (15). Therefore, in thoseH' secretion experiments involving application of auxin, thehormone was added 90 min after excision of the tissue, unlessotherwise noted.Measurement of Buffering Capacity of Incubation Medium. To

determine the extent to which buffering substances were releasedinto the medium during the course of an experiment, the segmentswere removed from the medium and the medium was adjusted topH 4.0 using HCI. The medium then was titrated with 8 mmNaOH over the pH range 4.0 to 6.3. The titration was recordedusing the experimental setup for measuring H' secretion, exceptthat the recorder sensitivity was reduced to 0.5 mv full scale anda chart speed of 152.4 cm/h was used.Measurement of Growth. Growth experiments with corn co-

leoptile segments were done using the shadowgraphic growthrecording device described previously (7). Preparation of thesegments was as described by Vesper and Evans (15).

RESULTS

Surface Effects of Peeling and Abrasion. Figure I shows scan-ning electron micrographs of the surface of undisturbed, abraded,and peeled corn coleoptile segments. In abraded specimens therewas occasionally an area where obvious cellular damage hadoccurred (Fig. IC), but the great majority of the epidermal cellsremained intact (Fig. ID). In peeled segments, large sheets ofepidermal cells were removed, revealing the underlying cells (Fig.IB). There were numerous tears in underlying cells, but thedamage at any one location was not as great as the isolated areasof damage found in abraded tissue.

Oxygen Dependence of Auxin-induced Ho Secretion. In mostof the published studies of hormone-induced H+ secretion usingsubmerged segments, the segments have been stirred or shaken,but no additional precautions have been taken to assure adequateaeration, (1, 10, 12). Parrish and Davies (11), in a study of H+efflux in pea stem segments, used aeration to agitate the segmentsand noted a strong dependence ofthe apparent rate ofH+ secretionon the rate of aeration. We have compared auxin-induced H+secretion in peeled corn coleoptile segments using stirring aloneversus stirring supplemented with oxygenation (Fig. 2). In theabsence of oxygenation, the pH of the medium equilibrates atabout 6.0, as compared with 6.3 to 6.4 when oxygenation is used.The lower equilibration pH in the absence of oxygenation is mostlikely due to the accumulation of respiratory CO2. There is amajor difference in the kinetics of auxin-induced H+ secretion inoxygenated versus nonoxygenated peeled tissue. Using oxygenatedtissue, the latent period is about 20 min, whereas, with nonoxy-genated tissue, the latent period is nearly 30 min. There is also adifference in the apparent rate of H+ secretion. With oxygenation,the pH drops from about 6.3 to 5.8 over an 80-min period afterthe initiation of auxin-induced H+ secretion, whereas, in nonox-ygenated tissue, the pH drop over a comparable time period isfrom about 6.0 to 5.8. Over that portion of the pH range traversedin both experiments (i.e. below about 6.0), the rate of drop in pHis about twice as great in oxygenated tissue as compared withnonoxygenated tissue. In similar experiments, it was noted thataeration was as effective as oxygenation in maximizing auxin-induced H+ secretion.

Auxin-induced H+ Secretion in Peeled versus Abraded Tissue.Figure 3 shows a comparison of apparent auxin-induced H+secretion in peeled versus abraded coleoptile tissue as well as intissue which has been neither abraded nor peeled. In all cases, thesolutions were oxygenated. Using intact tissue, no auxin-inducedH+ secretion is detectable during the first 90 min after addition ofhormone. After 90 min, weak acidification of the medium can bedetected in some experiments (not shown in Fig. 3). The lack ofsignificant auxin-induced H+ secretion in intact segments supportsthe conclusion drawn by others (3, 14) that abrasion or peeling isa prerequisite for detecting auxin-stimulated acidification.Of the two common methods for removing or disrupting the

cuticle, the abrasion method allows considerably stronger andmore rapid auxin-induced H+ secretion (Fig. 3). Using abradedtissue, auxin-induced H+ secretion is consistently detectable witha lag of about 7 min, whereas, in peeled tissue, the lag is about 15min. Similarly, the rate of auxin-induced H+ efflux appears to begreater in abraded tissue, the pH dropping from about 6.4 to 5.7in 50 min as compared with about 100 min in peeled tissue.

Release of Buffering Substances by Peeled and Abraded Tissue.Although the data of Figure 3 indicate that auxin-induced H+efflux is stronger in abraded than in peeled tissue, the indicationis an indirect one. The data show that the rate at which the pH ofthe medium drops is greater when using auxin-treated abradedtissue than when using auxin-treated peeled tissue, but they pro-vide no indication of the absolute amount of H+ released. Wehave noticed an accumulation of buffering substances in mediacontaining peeled or abraded tissue, with considerably morebuffering capacity accumulating in media containing peeled tissue(Fig. 4). Using the titration technique described under "Materialand Methods," it was found that approximately I Leq OH- wasrequired to titrate 4 ml 1 mm K-phosphate incubation buffer itselffrom pH 4.0 to 6.3 and that this medium tended to act as a buffernear pH 6 as expected. After the medium had held peeled coleop-tile segments for 2.5 h, approximately 3 ,ueq OH- were required toadjust the pH over the same range, and the shape of the titrationcurve was indicative of accumulation of substances adding buffer-ing capacity over the more acid range (pH 4 to 5.5), as might beexpected if organic acids were released into the medium from

562 EVANS AND VESPER



AUXIN-INDUCED H+ EFFLUX

wounded cells. The buffering capacity of medium containingabraded tissue was intermediate between that of medium withoutsegments and medium containing peeled segments, whereas me-dium which had held intact segments had only slightly greaterbuffering capacity than the phosphate buffer alone. This differ-ential release of buffering substances from peeled versus abradedtissue may arise from differences in the extent of tissue woundingduring peeling versus abrasion. In any event, the differentialleakage of buffering substances into the medium makes directcomparison of pH shifts an invalid measure of relative net H+efflux using these two methods.The data suggest the possibility that the apparent weaker in-

duction of H+ secretion by auxin in peeled tissue as comparedwith abraded tissue is due, at least in part, to the greater leakageof buffering substances from peeled tissue. To test this possibility

FIG. 1. Scanning electron micrographs of undisturbed, abraded, andpeeled corn coleoptile segments. A, Undisturbed; B, peeled; the epidermishas been peeled from the right hand portion, causing some cellular damagein underlying cells (arrows). A smaller strip of epidermis has been peelednear the left hand portion of the field of view; C, abraded. The field waschosen to include the area where obvious cellular disruption had occurred:D, abraded segment at lower magnification to show the isolated nature ofcellular damage. A, B, and C, x 100; D, x 40.

further, peeled coleoptile segments were placed in a large volume(1 liter) of 1 mm K-phosphate buffer which was oxygenated andstirred rapidly for 1 h. The segments then were placed in thespecially constructed vial for H' secretion measurement. Thewashing of the segments was included to disperse buffering sub-stances leaking from the wounded segments. Washing led to ameasurable increase in apparent auxin-induced H' secretion inpeeled segments, but the rate remained less than that shown bynonwashed abraded segments (data not shown). This indicatesthat the apparent reduced rate of H' secretion in peeled segmentsis only partly due to the greater leakage of buffering materialsfrom peeled tissue.That the differential release of buffering substances from peeled

versus abraded segments does not account totally for differencesin apparent H' secretion is not surprising. According to Figure 4,


! 31i"' t

fi

563




pH 6.1

6.0- 2

5.9

-20 0 20 40 60

TIME IN MINUTES

FIG. 2. Effect ofoxygenation on auxin-induced hydrogen ion secretion.Segments were placed in the measuring vial with moderate stirring of themedium. Upper curve: Stirring supplemented with oxygenation; lowercurve: no oxygenation. IAA (10 AM9) was added at the arrow in each curve.

Curves are from representative experiments. Each experiment was re-

peated at least 12 times.

abrade

62

571- IAA-20 0 20 40 60 80 100

TIME IN MINUTES

FIG. 3. Influence of the method of cuticle disruption on apparentauxin-induced H+ secretion. Segments were prepared by abrasion, peeling,or neither treatment ("intact"). IAA (10 Mm) was added at the arrow ineach curve. Medium was both oxygenated and stirred in all cases. Curvesare from representative experiments. Each experiment was repeated atleast 12 times except for the "intact" sample (three times).

the major buffering activity of the released material is from aboutpH 4 to about pH 5.5. The pH shift resulting from auxin treatmentranges from about 6.3 to 5.7 where the influence of these buffersshould be greatly reduced. Another factor which might be involvedin the weakening of apparent auxin-induced H+ secretion inpeeled tissue is that peeling removes cells which may contribute tototal H+ efflux. To assess the relative contribution of tissue re-moval to the apparent weakening of auxin-induced H+ effluxfrom peeled tissue, total auxin-induced H+ efflux from peeledversus abraded coleoptiles was determined using back titration toeliminate differences due to differential release of buffers (TableI). Clearly, the net amount of H+ secreted by abraded tissuesubstantially exceeds that by peeled tissue. These results suggestthat lesser apparent auxin-induced H+ efflux in peeled tissue isdue in part to the release of buffering substances from the tissueand in part to the removal of the auxin-sensitive epidermal layer.Time-dependent Changes in H' Secretion Response to Auxin.

In an earlier report (15), it was noted that the sensitivity of the

C.

0

4 4.5 5 5.5 6 6.5

pH

FIG. 4. Titration curves of incubation medium after containing tissuesegments prepared by various methods. In each case, 40 1-cm segments

were placed in 4 ml stirred oxygenated buffer for 2.5 h. The segments thenwere removed and the medium was adjusted to pH 4.0 with HCI beforetitrating to pH 6.3 with 8 mm NaOH. Curves are from representativeexperiments. Each experiment was repeated three times.

Table I. Total Auxin-induced H' Secretion during First 60 Min AfterAddition of 10 pMM IAA to Intact, Peeled, or Abraded Corn Coleoptile

Tissue

Total H' secreted was determined by back-titration with 8 mM NaOHto the pH at the time of auxin addition. Each experiment was done withoxygenation plus stirring. The data are averages of4, 6, and 12 experimentsfor intact, peeled, and abraded tissue, respectively.

Total H' Secreted duringTissue Preparation First 60 minueq

Intact 0Peeled 0.79Abraded 2.04

growth response to auxin in corn coleoptile segments changes asa function of time after excision. It was found that, during thefirst 3 h following excision, the magnitude of the auxin responseincreases 2- to 3-fold, whereas the latent period associated withthe response decreases by about 3-fold. If auxin-induced H+secretion is closely tied to growth, one might expect similarchanges in the magnitude and latent period of auxin-induced H+secretion in the hours following excision. This was tested bymeasuring auxin-induced H+ efflux when the hormone was addedto abraded segments 30 min versus 2 h after excision (Fig. 5). Therate of drop in pH when auxin is added to tissue 2 h after excisionis about 3 times the rate of drop in pH when auxin is added 30min after excision. Total H+ secreted as measured by back titrationI h after exposure to auxin was found to be 2.04 ,ueq in the tissueused 2 h after excision as compared with 0.6 ,eq in the tissue used0.5 h after excision. Similarly, the latent period in auxin-inducedH+ secretion is about 3 times as long when the hormone is addedto tissue 0.5 h after excision versus 2 h after excision. These time-dependent changes in auxin-induced H+ secretion are paralleledclosely by time-dependent changes in the growth response asshown in the upper portion ofFigure 5. Although the latent periodin the growth response using tissue 0.5 h after excision is about 60min in the example shown in Figure 5, the average value for allexperiments was about 48 min.

564 EVANS AND VESPER



AUXIN-INDUCED H+ EFFLUX

E

-E 2-c

I

ui I

pH

AA

62 \ 5h6

2h

-10 0 20 40 60TIME IN MINUTES

FIG. 5. Parallel time-dependent changes in auxin sensitivity as mea-

sured by H+ secretion and growth. Segments were tested for their growthresponse to auxin (top) and their H+ secretion response to auxin (bottom)either 0.5 or 2.0 h after excision from seedlings. Auxin (0.5 ,AM) was addedat the arrow in each curve. The growth experiments were done with intactsegments. The pH experiments were done with abraded segments. Growthexperiments were repeated at least 12 times; pH experiments were repeated12 times (2 h) or four times (0.5 h).

DISCUSSION

The magnitude and latent period of auxin-induced H+ secretionin corn coleoptile tissue is strongly dependent on the extent ofoxygenation, the method of cuticle disruption, and the time ofhormone application relative to the time of excision of the seg-ments. Oxygenation (or aeration) is crucial for maximum hor-mone-induced H+ efflux. Stirring the medium is, by itself, insuf-ficient. Auxin-induced H+ efflux is stronger and more rapid insegments which have been abraded as compared with segmentswhich have been peeled. This appears to be due in part to thegreater release of buffering substances by peeled segments and inpart to the removal or disruption of the auxin-responsive epider-mal cells during peeling.The sensitivity ofcorn coleoptile segments to auxin, as measured

by auxin-induced H+ efflux, increases about 3-fold during the first2 h following excision. This is reflected both by a strong increasein the magnitude of auxin-induced H+ secretion and by a largereduction in the latent period preceding auxin-induced H+ efflux.These time-dependent changes in the magnitude and latent period

of auxin-induced H+ efflux are closely paralleled by time-depend-ent changes in the growth response to auxin. Although thisindicates a close correlation between growth and H+ efflux, it doesnot address the question of whether or not the rate of H+ effluxoccurring after auxin treatment is sufficient to account for theobserved growth response (16).Under optimal conditions, i.e. abraded tissue, stirring supple-

mented with oxygenation, and hormone addition 90 to 120 minfollowing tissue excision, strong auxin-induced H+ secretion canconsistently be observed beginning about 7 or 8 min after additionof hormone. The latent period of the growth response at a corre-sponding time after excision is nearly 20 min. Using the method-ology described here, we can consistently detect auxin-induced H+secretion with a latent period only half as great as that associatedwith growth. This observation as well as the closely parallel time-dependent changes in sensitivity to auxin as measured by both H+secretion and growth is consistent with the H+ secretion hypothesisof auxin action.

Acknowledgments-The authors thank Gary Floyd. Harold Hoops. and DavidStutes for their assistance in the scanning electron microscopy portion of this study.

LITERATURE CITED

1. CLELAND RE 1973 Auxin-induced hydrogen ion excretion from A vena coleoptiles.Proc Natl Acad Sci USA 70: 3092-3093

2. CLELAND RE 1976 Kinetics of hormone-induced H+ excretion. Plant Physiol 58:210-213

3. CLELAND RE, DL RAYLE 1978 Auxin. H' excretion. and cell elongation. BotMag Tokyo (special issue) 1: 125-139

4. DOWLER MJ, DL RAYLE, WZ CANDE, PM RAY. H DURAND. MH ZENK 1974Auxin does not alter the permeability of pea segments to tritium-labeled water.Plant Physiol 53: 229-232

5. EVANS ML 1974 Rapid responses to plant hormones. Annu Rev Plant Physiol25: 195-233

6. EVANS ML, R HOKANSON 1969 Timing of the response of coleoptiles to theapplication and withdrawal of various auxins. Planta 85: 85-95

7. EVANS ML, PM RAY 1969 Timing of the auxin response in coleoptiles and itsimplications regarding auxin action. J Gen Physiol 53: 1-20

8. HAGER A, H MENZEL, A KRAUSS 1971 Versuche und Hypothese zur Primarwir-kung des Auxins beim Streckungswachstum. Planta 100: 47-57

9. JACOBS M. PM RAY 1976 Rapid auxin-induced decrease in free space pH and itsrelationship to auxin-induced growth in maize and pea. Plant Physiol 58: 203-209

10. MARRi E. P LADO, F CALDOGNO. R COLOMBO 1973 Correlation between cellenlargement in pea internode segments and decrease in the pH of the mediumof incubation. I. Effects of fusicoccin, natural and synthetic auxins, andmannitol. Plant Sci Lett 1: 179-184

11. PARRISH DJ. PJ DAVIES 1977 On the relationship between extracellular pH andthe growth of pea stem segments. Plant Physiol 59: 574-578

12. RAYLE DL 1973 Auxin-induced hydrogen ion secretion in A vena coleoptiles andits implications. Planta 114: 63-73

13. RAYLE DL, RE CLELAND 1970 Enhancement of wall loosening and elongationby acid solutions. Plant Physiol 46: 250-253

14. RAYLE DL, RE CLELAND 1977 Control of plant cell enlargement by hydrogenions. Curr Topics Dev Biol 11: 187-214

15. VESPER MJ, ML EVANS 1978 Time-dependent changes in the auxin sensitivity ofcoleoptile segments. Apparent sensory adaptation. Plant Physiol 61: 204-208

16. VESPER MJ. ML EVANS 1979 Nonhormonal induction of H' efflux from planttissues and its correlation with growth. Proc Natl Acad Sci USA 76: 6366-6370

Plant Physiol. Vol. 66, 1980 565



improved method detecting auxin-induced hydrogen efflux ...hormone application 2 hours after...

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