Plant Physiol. (1992) 100, 225-2300032-0889/92/1 00/0225/06/$01 .00/0

Received for publication December 5, 1991Accepted March 12, 1992

Maturation Proteins and Sugars in Desiccation Tolerance ofDeveloping Soybean Seeds'

Sheila A. Blackman*, Ralph L. Obendorf, and A. Carl LeopoldBoyce Thompson Institute (S.A.B., A.C.L.) and Department of Soil, Crop, and Atmospheric Sciences,

Cornell University (R.L.O.), Ithaca, New York 14853

ABSTRACT

The desiccation-tolerant state in seeds is associated with highlevels of certain sugars and maturation proteins. The aim of thiswork was to evaluate the contributions of these components todesiccation tolerance in soybean (Glycine max [L.] Merrill cv Chip-pewa 64). When axes of immature seeds (34 d after flowering)were excised and gradually dried (6 d), desiccation tolerance wasinduced. By contrast, seeds held at high relative humidity for thesame period were destroyed by desiccation. Maturation proteinsrapidly accumulated in the axes whether the seeds were slowlydried or maintained at high relative humidity. During slow drying,sucrose content increased to five times the level present in the axesof seeds held at high relative humidity (128 versus 25 pg/axis,respectively). Stachyose content increased dramatically frombarely detectable levels upon excision to 483 pg/axis during slowdrying but did not increase significantly when seeds were incubatedat high relative humidity. Galactinol was the only saccharide thataccumulated to higher levels in axes from seeds incubated at highrelative humidity relative to axes from seeds that were slowlydried. This suggests that slow drying serves to induce the accumu-lation of the raffinose series sugars at a point after galactinolbiosynthesis. We conclude that stachyose plays an important rolein conferring desiccation tolerance.

The remarkable mechanism that allows most mature an-giosperm seeds to survive desiccation to extremely low watercontents is poorly understood. Among the protective com-ponents that have been proposed to be important in theacquisition of desiccation tolerance during seed developmentare proteins and soluble sugars. The group of proteins knownas Late Embryogenesis Accumulating, or Lea (13), proteinsinclude some that accumulate during the maturation dryingphase of seed development. Some of these maturation pro-teins have been correlated with the ability of the seed toprogress into seedling growth (29), whereas others have beencorrelated with desiccation tolerance (2, 4). However, someadditional process is apparently necessary for the develop-ment of desiccation tolerance. We have shown that matura-tion protein accumulation alone is not sufficient to conferdesiccation tolerance in developing soybean (4).Another important component of desiccation tolerance

may be the accumulation of a high level of soluble sugars, a

1 This work was supported by the International Board of PlantGenetic Resources.

characteristic of mature orthodox seeds (1). They have beenimplicated by correlation as adaptive agents for desiccationtolerance during seed development and germination (9, 18,19). In particular, cultivars of soybean (Glycine max) accu-mulate high levels of the raffinose series of oligosaccharides,particularly stachyose, in addition to sucrose (12, 18, 21, 31).Evidence for the protective role of soluble sugars has alsobeen inferred from model systems (7, 11). The soluble sugar,trehalose, protects cytosolic components in yeast against de-siccation-, frost-, and heat-induced damage in vivo (34). It isthought that the hydroxyl constituents of sugars may replacethe hydration shell around membranes and thus preventstructural damage as water is removed (10, 11). In addition,sucrose has been shown to protect soluble enzymes fromsalt-induced damage in vitro (32). The presence of a largeroligosaccharide along with sucrose may enhance protectionstill further by favoring vitrification rather than crystallization(6, 9, 17, 33, 35).Although oligosaccharides such as stachyose have been

recognized as protective agents in leaves against cold-induceddamage, they are often referred to as a carbon reserve forgermination rather than as a desiccation protectant in theaxes of nonendospermic legume seeds (12). Raffinose andstachyose are synthesized by the following reactions (12)catalyzed by galactinol synthase (UDP-galactose:inositolgalactosyltransferase) (Eq. 1), raffinose synthase (galactinol:sucrose 69'u-a-D-galactosyltransferase) (Eq. 2), and stachyosesynthase (galactinol:raffinose 69al-z-D-galactosyltransferase)(Eq. 3):

UDP-D-galactose + myo-inositol -* galactinol + UDP (1)

galactinol + sucrose raffinose + myo-inositol (2)

raffinose + galactinol - stachyose + myo-inositol (3)

The committing step in this pathway is the synthesis ofgalactinol from myo-inositol and UDP-galactose.The aim ofthis work was to test the hypothesis that high levels of bothmaturation proteins and oligosaccharides are necessary forthe development of desiccation tolerance in maturing soy-bean seeds. We have previously shown that maturation pro-teins may be important components of desiccation tolerance(4). We report here that maturation proteins appear to beincapable of providing desiccation tolerance unless high lev-els of certain oligosaccharides, particularly stachyose, alsoaccumulate. Furthermore, the stachyose level is not correlatedwith the germination rate. Therefore, altemative forms of

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Plant Physiol. Vol. 100, 1992

reduced carbon appear to be as effective substrates as stach-yose during early germination. This suggests that the primaryneed of the axis for stachyose is not as a storage reserve butas a protectant during desiccation.

MATERIALS AND METHODSPlant Material

Soybean plants (Glycine max [L.] Merrill cv Chippewa 64)were greenhouse grown as previously described (24). Devel-oping pods were removed from plants and surface sterilizedin 10% bleach (0.5% hypochlorite) for 5 min and then rinsedfive times in sterile water. Seeds were excised from the podsaseptically and seeds of 34 DAF were selected on the basisof morphological and fresh weight criteria (30).

Premature Drying Treatments

Immature seeds, excised at 34 DAF, were dried slowlyduring the course of 6 d by transferring them daily to succes-sive sealed desiccators in which atmospheres of progressivelylower RHs (92.5, 87, 75, 51, 45, and 32.5%) were maintainedover saturated solutions of KNO3, Na2CO3, NaCl, Mg(NO3)2,K2CO3, and MgCl2, respectively (4). For the high RH controltreatment, seeds were placed in desiccators over water-satu-rated filter paper for 6 d (4).

Germination Tests

To test for rate and final extent of germination, 20 to 25seeds were placed aseptically in moist rolls of germinationpaper for 7 d at 25F8C. On each day, seeds were scored ashaving germinated if the axis had elongated and brokenthrough the seed coat.

Desiccation Tolerance Test

To test the ability of seeds to tolerate desiccation, samplesof 20 to 25 seeds were dried rapidly at 13% RH over asaturated solution of LiCl at 250C. After 6 d, seeds werehumidified for 24 h in an atmosphere of high RH above, butnot touching, moist paper before testing for germination (4).

Determination of Water ConcentrationWater concentration of three samples of five axes each was

determined by measuring the difference between fresh anddry weights and expressed as grams of H20 per gram of dryweight. Dry weights were determined after drying axes for24 h at 900C under reduced pressure. The dry weight of axesaveraged 1.63 mg/axis.

Heat-Stable Protein Extraction and AnalysisHeat-stable proteins were extracted from axes as previously

described (4) except that the extraction buffer contained 50mM Tris-HCl (pH 7.5), 5 mM MgCl2, 5 ,ug/mL each of antipainand leupeptin, and 1 mm PMSF. Proteins were analyzed bythe dye-binding assay (Bio-Rad) and by electrophoresis on10 to 18% polyacrylamide gradient gels containing SDS aspreviously described (4).

Sugar Extraction and AnalysisAfter designated periods of time during the slow drying or

high RH treatments, seeds were removed from desiccators,

and the axes were dissected out, frozen in liquid nitrogen,and stored at -800C until extraction. Sugars were extractedfrom the axes as follows (3). Samples of five axes wereweighed and then homogenized in a microfuge tube with aplastic homogenizer in 500 uL of 50% ethanol containing100 gg/mL of melezitose-dihydrate (Sigma) as the internalstandard. The slurry was incubated at 750C for 30 min andthen centrifuged in a microfuge at 16,000g. The supematantwas removed, and the pellet was washed in 500 ,uL of 50%ethanol containing melezitose as before. The combined su-pernatants were dried under a stream of nitrogen gas. Thedried extract, after dissolution in 1 mL of distilled H20, wasfiltered through a 0.8- x 4-cm column containing 0.5 mL ofAmberlite IRA-94 ion exchange resin, 1.0 mL of Dowex5OWx4 ion exchange resin, and 0.5 mL of polyvinylpolypyr-rolidone (all from Sigma) at a flow rate of 0.5 mL/min (5).After the collected aqueous eluate was lyophilized, the driedresidue was resuspended in 1 mL of trimethylsilylimidazole(Trisil-Z; Pierce) and heated at 750C for 0.5 h, and 2 juL ofderivatized sugars were analyzed by GC (Hewlett-Packard-5890) on a 15-m SPB-1 (Supelco) column (0.53 mm i.d.) withdimethylpolysiloxane (0.5-,um film thickness) as the station-ary phase and He (2 mL/min) as the carrier gas. The injectorwas operated at 2750C and the flame ionization detector at3500C. Detector gases were 30 mL/min H2, 400 mL/min air,and 25 mL/min makeup gas (He). The column temperaturewas preprogrammed for 10 min at 1800C, followed by a10°C/min increase to a final temperature of 3200C for 15min. Sugars were identified by comparison of retention timeswith known standards (Sigma, except for galactinol, whichwas a kind gift from Dr. Robert Turgeon). Under theseconditions, the major soybean saccharides were well resolvedwith retention times of 19.4 (sucrose), 21.8 (galactinol), 24.5(raffinose), 25.1 (melezitose), and 35.9 min (stachyose). Sac-charides were quantified by reference to a standard curve ofthe ratio of the peak area of the sugar to the internal standardversus micrograms of known sugar. Peak areas were com-puted by a Hewlett-Packard-3380S integrator.

RESULTS

Slow Drying and High RH TreatmentsPromote Germination

As maturing soybean seeds gradually dry in situ, theyadapt to lower and lower water concentrations until they arecapable of withstanding desiccation to less than 0.10 g ofH20/g of dry weight at 44 DAF (4). To study the adaptationsthat allow seeds to tolerate desiccation, we modified theartificially imposed drying regimen detailed by Rosenbergand Rinne (27) so that comparable desiccation tolerance wasachieved in developing excised seeds of 34 DAF (4). Thistreatment was compared with a control treatment in whichexcised seeds were maintained for the duration of the exper-iment in a water-saturated chamber (high RH controls). Thewater concentration of the axes in seeds undergoing slowdrying began to change significantly after day 2 (75% RHand below), declining from 1.78 g of H20/g of dry weightupon excision to 0.06 g of H20/g of dry weight after 6 d at32.5% RH (Fig. 1). In contrast, the axes of high RH seedsslightly increased in water content until day 3 of treatment,

226 BLACKMAN ET AL.

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PROTEINS AND SUGARS IN DESICCATION TOLERANCE OF SOYBEAN SEEDS

after which they returned to their original level of about 2.00p < °Slow-dried g of H20/g of dry weight after 5 d.

---- High RH We tested seeds undergoing these treatments to ensure that) .4', germination was not adversely affected before desiccation

,,-.400 (Fig. 2A). Germination of freshly excised seeds was only 43%under the conditions of our germination test. One day after

)- ^ t~"'0-Q excision, germination was greater than 80% and was nearly100% after 2 d for both regimens. Seeds undergoing bothtreatments also had comparable rates of germination (Fig.2B). The time to 50% germination decreased to a minimumafter 3 d during both treatments. Thus, both treatmentspromoted the final percentage and the rate of germination in

0 1 2 3 4 5 6 7 excised developing seeds.

Days After ExcisionFigure 1. Water content (dry weight basis) of axes from seedsundergoing slow drying (0) or high RH control (0) treatments for 1to 6 d. Values represent the means ± SE of the mean of threesamples of five axes each.

00

°100ff X0- zID__

E 60

40

De 20 -v High RH

0 o

Ln~~~~~~

6 5

X E 5 -2 . .0 1 2 3 4 5 6 7

Days After ExcisionFigure 2. Germination percentage (A) and rate (B) for exciseddeveloping soybean seeds undergoing slow drying (0) or high RHcontrol (0) treatments for 1 to 6 d. Samples of 20 to 25 seeds were

removed on successive days of each treatment and placed on moistgermination paper. A, Percentage of germination (i.e. radicle emer-gence) after 7 d on moist germination paper. B, Days required toreach 50% germination.

Slow Drying Promotes Desiccation Tolerance

In contrast to germination results, the treatments had mark-edly different effects on desiccation tolerance (Fig. 3). Rapiddesiccation at 13% RH destroyed germination unless theseeds had experienced more than 2 d of the slow dryingregimen. The percentage of seeds that were desiccation tol-erant increased between 2 and 6 d of slow drying as axiswater concentration declined from 2.10 to 0.06 g of H20/gof dry weight. In contrast, seeds held at high RH, whichmaintained greater than 1.80 g of H20/g of dry weight, never

acquired desiccation tolerance.

Maturation Proteins Accumulate during Slow Drying

We previously reported that a set of seven maturationproteins were consistently present in desiccation-tolerantseeds and, hence, may contribute to desiccation tolerance (4).However, apparently identical maturation protein levels wereachieved after 6 d of either slow drying or high RH treatmentsof young excised seeds (34 DAF) (4). Here we show that thelevel of these proteins increased at approximately the same

rate in both treatments (Fig. 4). Maturation proteins (indicatedby arrows) were not detectable in the axes of freshly excisedseeds (34 DAF) but accumulated to high levels within 2 dafter excision in both the slow drying and high RH treat-

I-o0

.-

cn

0

_o0

0 1 2 3

Day After4 5 6

Excision7

Figure 3. Development of desiccation tolerance during slow drying(0) or high RH control (0) treatments. Samples of 20 to 25 seedswere desiccated rapidly at 13% RH and then tested for germination.

.4-i

3:

-ao

N.0C14

c,)

4.0C

3.OC

2.OC

1.Oc

O.oc

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Plant Physiol. Vol. 100, 1992

Figure 4. SDS-PAGE analysis of heat-stable proteins from devel-oping soybean seeds (34 DAF) either freshly excised (lanes 1 and8), slowly dried for 1, 2, or 3 d (lanes 2, 4, 6, 9, 1 1, and 13) or heldat high RH for 1, 2, or 3 d (lanes 3, 5, 7, 10, 12, and 14). Protein inlanes 1 through 7 was loaded on an equivalent axis basis, whereasthat in lanes 8 through 14 was loaded on a per microgram of proteinbasis. Molecular masses (kilodaltons) of standard proteins (lane 15)are indicated. Arrows indicate positions of migration of heat-stableproteins previously identified as being associated with maturationand desiccation tolerance (3).

ments. Thus, not only did maturation proteins accumulate inhigh RH seeds (which do not achieve desiccation tolerance)but they also reached their maximum level in slow-driedseeds well before the seeds achieved desiccation tolerance(Fig. 3). This supports our previous conclusion (4) that a highlevel of maturation proteins alone cannot confer desiccationtolerance.

Slow Drying Induces the Accumulationof Oligosaccharides

Because the presence of maturation proteins in the highRH control seeds was not sufficient to confer desiccation

Figure 5. Content of sucrose (A), stachyose (B),raffinose (C), and galactinol (D) in axes of ex-cised developing soybean seeds undergoingslow drying (0) or high RH control treatment(0). Values are the means F1 SE of the mean offive samples of five axes each. Note the differ-ent scales on the y axis for each panel. U)

x

0)

l-.0)

L.m

L.)L')

0

tolerance, we predicted that soluble saccharides might be anadditional requirement. Soluble sugars were extracted fromaxes of seeds undergoing high RH or slow drying treatmentsand analyzed by GC. Stachyose and, to a lesser extent,raffinose accumulated in axes from seeds undergoing slowdrying but not in those maintained at high RH. Galactinolcontent increased substantially during the high RH treatment(Fig. 5). Sucrose content in the axes of excised seeds decreaseddramatically from its initial value of 211 to 54 and 62 jig/axis in slow dried and high RH seeds, respectively, within 1d after excision (Fig. 5A). In subsequent days, the axes fromslowly dried seeds accumulated sucrose. After day 6, thesucrose level in axes from slow-dried seeds (128 jig/axis) wasabout 5 times that in axes from high RH control seeds (25jig/axis). This higher sucrose level was associated with theincreased desiccation tolerance of the slow-dried seeds (Fig.3). However, because freshly excised seeds contained evenmore sucrose and yet were not desiccation tolerant, it seemsthat sucrose was not the determining factor in desiccationtolerance.

Stachyose was not present in axes of freshly excised seedsand accumulated in the axes only during slow drying of seeds(Fig. 5B). After 2 d of slow drying, the stachyose level beganto increase dramatically to reach 483 ,jg/axis (almost 4 timesthe level of sucrose) after 5 d. Stachyose did not accumulateto a significant extent in axes of high RH control seeds. Thus,stachyose accumulation correlated well with increasing desic-cation tolerance, and this oligosaccharide appears to be agood candidate for an important component in desiccationtolerance of soybeans.

Neither raffinose (Fig. 5C) nor galactinol (Fig. 5D) accu-mulated to the same extent as did stachyose and sucrose inthe axes of seeds undergoing slow drying. The raffinose levelremained between 0 and 5 jig/axis through the first 4 d ofslow drying and then accumulated modestly to 11 jig/axisafter day 5 (Fig. 5C). The galactinol content increased after 1d of excision during slow drying, but as stachyose began toaccumulate after day 2 (Fig. 5B), galactinol content declined.Thus, both galactinol and raffinose appear to have beenrapidly converted to stachyose during slow drying. In con-trast, galactinol was the only saccharide that accumulated inaxes of seeds maintained at high RH to a level higher thanthat in axes from slow-dried seeds (Fig. 5D). Galactinol

Sucrose A Raffinose C1 2

8

En\nO94s9

Stachyose B Galactinol D 8 D

< 4 A - -~~~~~~~

1 2 3 4 5 6fl2st,2A f f a %r Jvi4 i 44

228 BLACKMAN ET AL.

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PROTEINS AND SUGARS IN DESICCATION TOLERANCE OF SOYBEAN SEEDS

increased to 6 ,ug/axis after 3 d of the high RH controltreatment. Because galactinol accumulated, whereas raffinoseand stachyose did not, galactinol conversion to the highermol wt oligosaccharides appears to have been blocked in theseeds that were held at high RH.

Soluble sugars other than sucrose and stachyose were onlya minor proportion (in total, <10%) of the total soluble sugarpool of axes from slow-dried seeds, and their levels did notdiffer significantly between slow-dried and high RH controlseeds (data not shown). It is, therefore, unlikely that saccha-rides other than stachyose and sucrose were determiningfactors in the development of desiccation tolerance in slow-dried seeds.

Preliminary evidence gained from visual inspection of freshsections stained with potassium iodide suggests that theaccumulation of the soluble saccharides in slow-dried seedsproceeds at the expense of starch (data not shown). At 34DAF, starch was detected in the axes of freshly excised seedsbut not in axes from seeds after 6 d of slow drying. Incontrast, axes from high RH-treated seeds retained starchafter 6 d.

DISCUSSION

A number of functional changes occur during naturalsoybean seed maturation including the ability to germinaterapidly and to tolerate desiccation (2, 4, 22, 23, 25). Immatureseeds of certain plants when detached and slowly driedacquire traits, including germination and desiccation toler-ance, that mimic those of mature seeds (15, 27, 28).At 34 DAF (mid-pod fill), soybean seeds can germinate

only at a very slow rate and are not desiccation tolerant. Ifmoisture is withdrawn gradually during the course of 6 d(Fig. 1) such that the final water concentration (0.06 g ofH20/g of dry weight) is equivalent to that of the mature seed,they become functionally similar to mature seeds in at leasttwo respects. First, the rate of germination is accelerated (Fig.2B) and, second, the ability to tolerate desiccation is acquired(Fig. 3). These traits are acquired during natural seed devel-opment at 38 and 48 DAF, respectively (4, 23).

Germination (Fig. 2), but not desiccation tolerance (Fig. 3),was also promoted in seeds that were simply maintained ator above their preexcision water content rather than beingslow dried. Promotion of germination in excised, immatureembryos (either zygotic or somatic) by similar treatments hasalso been documented elsewhere (16, 26, 27).We examined the protein profiles and soluble sugar con-

tents of these seeds to identify components that might un-derlie different functional properties, particularly desiccationtolerance. Neither the rate nor final extent of maturationprotein accumulation differed between axes of seedsundergoing slow drying and high RH control treatment (Fig.4). Therefore, accumulation of maturation proteins is prob-ably induced directly or indirectly in response to excision orto changes in water relation parameters undetectable by ourmethods. Rosenberg and Rinne (27) also observed that twoproteins accumulated in whole immature soybean seeds afterthey were excised and incubated at high RH for 24 h.

In contrast to these abundant maturation proteins, solublesaccharide levels (especially stachyose) were markedly differ-

ent in axes of seeds undergoing these two treatments (Fig. 5).Stachyose and sucrose levels increased to approximately 270and 80 mg/g of dry weight, respectively-2.7 times higherthan the values reported for naturally matured axes (100 mgof stachyose/g of dry weight and 30 mg of sucrose/g of dryweight) (18). This differential may reflect a real differencebetween seeds that are matured precociously and those thatmature in vivo but may also be due to the different cultivarused. Levels of soluble sugar differ among soybean cultivarswith some cultivars having even higher levels than thosereported here (21).Our results show a correlation between stachyose level and

desiccation tolerance (r2 = 0.835) when immature soybeanseeds are artificially matured by slow drying. Similar associ-ations between high levels of saccharides and desiccationtolerance have been reported during natural Brassica seeddevelopment (19), during germination of pea, soybean, andcorn seeds (18), and during slow drying of corn and Penni-setum pollen (14). Taken together, results of these studiesprovide strong support for the hypothesis that saccharidesare important in conferring tolerance to the stress of desic-cation. Soluble saccharides can protect isolated cellular com-ponents from desiccation- or osmotic stress-induced damagein vitro (7, 11, 32) and can protect cytosolic components inyeast against desiccation-, frost-, and heat-induced damagein vivo (34). The mechanism of saccharide protection hasbeen proposed to be either through sugar-hydroxyl replace-ment of the hydration sites of susceptible intracellular com-ponents (10, 11) or formation of a high-viscosity vitreousstate wherein deleterious reactions are suppressed (6, 17,20, 35).

Because germination of seeds with axes that lack stachyose(i.e. high RH control seeds) does not differ from seeds withaxes that contain stachyose (i.e. slow-dried seeds), we caninfer that the high level of stachyose is not necessary toprovide nutrients to hasten radicle emergence. Sufficientreduced carbon for early germination can apparently bederived from starch or other soluble saccharides present inaxes from high RH-treated seeds as well as from stachyose.In contrast, our data do support an important role for stach-yose in the development of desiccation tolerance.

Furthermore, our data suggest that the accumulation ofraffinose series sugars and, hence, desiccation tolerance canbe induced in axes of seeds during slow drying but not duringincubation at high RH. Axes of high RH seeds do not accu-mulate these higher mol wt sugars despite the build up ofgalactinol (Fig. 5B). Galactinol synthase has been implicatedas an important regulator of carbon partitioning betweensucrose and raffinose saccharides in developing soybeanseeds (21, 31). Galactinol synthase activity and galactinolcontent actually increase before maximum stachyose synthe-sis and subsequently decline as the seeds mature and losewater (present study and ref. 21). Similarly, raffinose syn-thase activity is present as early as 5 DAF in developingsoybean and does not increase dramatically during the re-mainder of development (8). Thus, although galactinol andraffinose synthase activity may be important in the regulationof carbon flow into the raffinose family biosynthetic path-way, some other factor may be required for the accumulationof these sugars to high levels. It is possible that stachyose

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Plant Physiol. Vol. 100, 1992

synthase activity is limiting or that intracellular conditionsare not conducive to the accumulation of stachyose when theseeds are incubated at high RH.

In summary, the evidence from the present work suggestsan important role for the oligosaccharides that accumulateduring maturation of most orthodox seeds-to provide pro-

tection from the stress of severe desiccation. The role ofoligosaccharides in desiccation tolerance cannot be replacedby maturation proteins during severe desiccation. The mat-uration proteins may be an essential part of an 'early responsesystem" that protects against the stresses imposed at the onsetof desiccation before saccharides reach high levels, or theymay work in concert with oligosaccharides to function in thedevelopment of desiccation tolerance.

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