PHYSIOLOGIA PLANTARUM 91: 708-714, 1994Frirjted in Dcinfuirl. - (;// !̂,'/lr̂ jvicnrj

l Q FhysictSSS i

Growth and CO2 uptake for cladodes and fruit of theCrassulacean acid metabolism species Opuntia fkus-indica duringfruit development

Paolo Inglese, .Alvaro A, Israel and Park S, Nobel

Inglese. P. Israel, \ . .\. and Nobel, P. S. 1994. Growth and CO; nptake for cladodesand fruit of the Crassulacean aeid metabolism speeies Opuntia ficus-indica during fraildevelopment. - Physiol. Plant. 91: 708-714.

Growth and daily nel CO; uptake were measured for fruit of Opuntia ficusindica andfor its eladodes with 0. 5, 10 and 15 fruit and for cladodes afler fruit removal. Growthof individual fruit decreased but fruit dry weight per cladode increased as the numberof fruit per cladode increased. Removal of fruit decreased Ihe total daily net CO; uptakeof the bearing cladode by about 10%. From 15 lo 45 days afler flowering, nocturnalCO; uptake per unit area of the fruit averaged 19% of' that of cladodes and Ihendeclined, as did the chlorophyll content and the aetivity of the CO;-flxing enzymes.Fruil growth for O. ficus-indica was supported by the bearing eladode as well as othercladodes. espeeially for cladodes with more than 5 fruit.

Key words - Chlorophyll, CO; exchange. Crassuiacean acid metabolism. PEPCase.Rubisco. sink strenglh.

F. Inglese. ICLAF. Univ. di Keggio Calabria. 89061 Kcggio Calabria. Italy: .A. A. Israeland P. S. Nobel (corresponding author), Dept of Biology and UCLA-DOE iMboratory.Univ. of California. Uis Angeles. CA 90024-1606. USA.

Introduction

Mo.si of the dry matter produced anntiafiy by mature fruittrees is allocated to fruit growth. For instance. 709r ofannual dry matter prdduclion by apple and peach trees isincorporated into their fruit (Chalmers and van den Ende1975, Heim et al. 1979). Fruiting trees have increasedphotosynthetic capacity compared with trees without frnitor trees from which the fruit is removed (.Maggs 1963,Palmer 1992). Furthermore, the telative sink strength offruit affects dry matter partitioning, with fruit-bearirtgtrees having reduced vegetative and root growth (Maggs1963, Chalmers and van den Ende 1975, Heim et al.1979, Palmer 1992). Assimilate demand of growing fruitcan increase nel CO, uptake of leaves for some species(Wardlaw 1990) but not others (Daie 1985). Altering thesource:sink ralio by removing fruit may decrease leafCO; uplake. particularly dunng periods of rapid fruitgrowth (Hansen 1970, Chalmers et al, 1975, DeJong1986, Palmer 19921, but again not for all species (Plaut el

Received 8 November. 1993: revised 4 March. 1994.

708

al. 1987). How fruit growth modifies leaf CO; uptake isunclear, although effects on stomatal conductance (De-Jong 1986). photorespiration (Lenz 1979) and assimilatefeedback on photosynthesis (Gucci et al. 1991) are docu-mented. Fruit can also photosynthetically contribute totheir own carbon accumulation. The contribtilion, whichvaries with species (Hansen 1970, Bazzaz et al. 1979,Birkhold et al. 1992, Whiley et al. 1992) and environ-mental eonditions (Jones 1981, Pavel and DeJong1993a), generally deereases during fruit developmentfMoreshet and Green 1980, Jones 1981, 'Whiley e! al.1992, Pavel and DeJong 1993b).

The present study utilizes the highly productive Cras-sulacean aetd metabolism (CAM) speeies of prickly pearcactus, Opuntia fieus-indiea, whieh ean have a biomassgain of up to 50 Mg dry weight ha"' year-' and eanproduce 6 Mg dr> weight of fruit ha"' year"' (Gareia deCortazar and Nobel 1992, Nobel et al. 1992). Daily netCO; uptake for cladodes of O. ficu.s-indica grow ing underwell-watered conditions, optimal tetnperatures (day/tiight

Phv-siul. Planl. 91. 1

.air temperatures of 25/15°C) and saturating photosyn-thettc photon flux (PPF; 25 mol m -̂ day ' ) can be 344mmol m - (Nobel 1988), and its dry matter accumulationrate ean be about 7.0 g m -̂ day"' in the field (Aeevedo etal. 1983). Cladodes of O. fieti.s-indica assimilate CO:mostly during the rtight, although 2-week-o!d cladodesand flower buds have mostly daytime CO; uptake (Aee-vedo et al. 1983).

The outer pericarp of the truit of O. fkus-indica origi-nates from the receptacle that surrounds the inferior ovaryand so analomieally resembles the outer tissue of a ela-dode. This pericarp remains green until aboul 3 weeksbefore harvest, suggesting that net CO, uptake by a fruitmay oeeur during its development. Also, fruit do notdevelop on a cladode until its dry weight exceeds aminimum value for its surface area by at least 33 g, the'cladode excess drj' weight' (Gareia de Cortazar andNobel 1992). Our objective was to investigate CO:: up-take for eladodes in relation lo sink strength (fruit loadand growth) and for fruits from initiation lo maturation.Fruil were also removed from eiadodes with differentfruit loads soon after flowering and the effects on cladodenet CO; uptake rates were examined.

Abbreviations - PEPCase. phosphoenolpyruvate carboxylase:Ruhisco. 1 ibulose-1.5-bisphosphave carboxylase/oxygenase.

Materials and methods

Plant materia] and environmental conditluns

Measurements were made on 4-year-old Opuntia ficus-indica (L.) Miller from flowering on 12 May throughharvest of ripe fruit on 26 July 1993 at the AgriculturalResearch Station, University of California, Riverside.CA. The plants were spaced at 1.8-m intervals along rows3.5 m apart that were oriented easl-vvest to maximize PPFinterception (Nobel 1988). The approximately 2-m-tal]hedgelike rows were irrigated biweekly and weededmonthly.

Based on hourly temperatures monitored with fourthermocotjples, the average day/night air temperatureswere 24/l6°C, 3()/19°C, 35/23°C, 30/!9°C and 26/l6''Con 27 May, 11 June, 26 June, 1 I July and 26 July,respeetively (dates for rneasuremenl of CO; uptake). PPFwas measured hourly from dawn to dusk in the approxi-mately vertical planes of each of the 35 monitored cla-dodes with an LI-88 integrating quantum photometer(Li-Cor. Lincoln, NE, USA). For these unshaded eia-dodes, the total daily PPF averaged 39, 42, 38, 34 and 31mol m"-̂ day"' on 27 May, 11 June, 26 June, 11 July and26 July, respectively.

Cladode and fruit growth

Cladode width and length were mea.sured at floweringand at harvest. The area (cm-) of both sides of a eladodewas calculated from 1.54 x length (cm) x width (em)(Nobel 1988). Cladode dry weight at flowering was esti-

mated from samples removed with a cork borer 1.4 cm indiameter and then dried to constant weight in a forced-draft oven at 80°C. All cladodes were detaehed after fruitharvest and their fresh and dry weights were measured.Excess dry weight was determined as the difference be-tween actual dry weight and the dry weight obtained fromthe regression DW = 0.833 A + 0.278 A- (r= = 0.99),where DW is cladode dry weight in g and A is its surfacearea in cm- (Garcia de Cortazar and Nobel 1992).

Length and maximum diameter of ali fruit on thecladodes tJsed for CO. uptake measurements were deter-mined ever\ 2 weeks from flowering to harvest. Fruitfresh weight (FW in g) was determined from the regres-sion FW = 12.0 + 0.352 D -I- 5.07 D- (f = 0.95), and fruitdry weight was determined from DW = 11,4 - 8.29 D +1.73 D- (r- = 0.94), where D is the ma.\imum diameter ofthe fruit in cm. These regression equations were derivedfrom 30 fruit har.'ested ever>' 2 weeks, 10 fruit comingfrom cladodes with 5, 10 and 15 fruit eaeh. Fruit surfacearea A was calculated from the regression A = 23.7 -i-19.8 D -t- 1.96 D= (r- = 0.98); this regression was derivedfrom the areas of 30 fruit of differeni sizes the peels ofwhich were flattened and the areas of paper silhouettesdetermined with a Li-Cor LI-3KX) leaf area meter. Sto-matal frequencies were delennined for epidemnal peelsexamined at lOOx with a BH-2 phase-contrast miero-seope (Olympus, Lake Success, NY, USA). Ripe (edible)fruit were harvested on 26 July 1993. when the peelswere fully colored (yellow).

CO2 uptake measurements

Net COi uptake was measured on I -year-old, east-westfacing, unshaded, terminal eladodes with naturally oecur-ring loads of 0, 5, 10 or 15 fruil. Measurements weremade over 24-h periods ever\ 15 days on five eladodesper treatment (each on a different plant) beginning 2weeks after flowering (12 May 1993) and continuing tofruit harvest (26 July 1993) by u.sing a Li-Cor Li-6200portable photosynthesis system. The leaf chamber wasreplaced with an acrylic cylinder (3.4 cm in diameter, 2.5cm in length) with two layers of foam-rubber gasketattaehed al the distal end of the cylinder to fit onto awest-facing cladode surface (Cui et al. 1993). CO: uptakewas also measured on five unshaded fruit selected atflowering from three cladodes (eaeh on a different plant)with naturally occurring loads of 10 f'rtiit. To fit onto thefruit, the gasket was reduced in size to 1.5 cm in lengthand 1.2 ctn in w idth.

To estimate the carbon budgets of cladodes and fruit,daily net CO2 uptake per utiit area was multiplied by theorgan area to obtain the daily CO; uptake per organ. Thisuptake was then summed for the 75-day period fromflowering to fruit harvest. COi uptake was converted todry weight gain, assuming 30 g of dry matter per mole olCO; fixed (Cui et al. 1993). Finally, this dry wetght gainwas compared to the weight of the organs to estimateimport or export of photo-assimilate.

Phvsiol. Pianl. 91. 19')4 709

0 15 30 45 60

Time a f t e r f lcwer ing (days)

Fig, I. Time course of I'ruit dry weight accumulation for ela-dudes of Opuntia ficus-indica bearing 5 (O). 10 (i) and 15 (G)fruit. Dala are means ± SE (n = 5 plants), except when the errorbar is smaller than the symbol.

Chlorophyll

Chlorophyll content was determined on tissue samplesremoved with a eork borer 1.4 cm in diameter at 1200 hon 18 July 1993 from !5 flower buds, 15 flowers and 15fruits at each of three developmental stages. The ehlo-renehyma was excised with a razor blade, stored in liquidnitrogen, and then suspended in N,N-dimethylformamidefor 24 h at 6"C in the dark. .'Vbsorbance was determined at647 and 664 nm (Moran 1982) with a Beekman DU-64spectrophotometer (Beckman Instruments, Fullerton, CA.USA).

Enzyme activity

The aetivies of ribulose-l,5-bispbosphate carboxylase/o.xygenase (Rubiseo) and phosphoenolpyruvate earboxy-

lase (PEPCase) were determined for 15 eladodes, 15flower buds. 15 flowers and 15 fruiis at three devel-opmental stages. Samples oi the ehlorenehyma were col-lected with the eork borer at 1400 h on 18 July 1993 forRubiseo and at 2200 h for PEPCase. Samples were storedin liquid nitrogen until enzyme aetivity was measuredtadiometrically as '''CO; fixed into stable products (Beeret al. 1990).

Rubisco was extracted by grinding 0.5 g fresh weightof ehlorenehyma in an ice-cooled mortar containing 200mM Tris-HCl (pH 8.0), 10 mM MgCl; and 10 mMNaHCO,. Assays were condueted at 30°C in 4-ml plasticvials with O.I ml of plant extraet plus 0.4 ml of 50 mMTris-HCl (pH 8.0), 10 mM MgCl; and 5 mM dtthtothrei-tol. The vials were sealed with rubber stoppers and 0.025ml of 500 mM NaH'^CO, was injected, followed 10 minlater by 0.025 ml of 11 mM ribulose bisphosphate. Thereaction was terminated after 60 s by adding 0.1 ml of 6M HCl, and unfixed '^CO; was removed by flushing thevials with air for 8 h. After resuspension in 0.1 ml of 50'7r(v/v) ethanol. 2.5 ml Bio-Safe II scintillation liquid (Re-search Products International, Mount Prospect, IL, USA)were added before "C aetivity was measured with aBeekman 6000SC scintillation counter.

.A similar procedure was used for PEPCase. exeept that200 mM N-2-hy'droxyethylpiperazine-N'-2-ethanesul-fonie aeid (HEPE!?, pH 7.2) replaced Tris-HC! and 0.2%(v/v) bovtne serum albumin was added to the extractionmedium. The assay solution contained 100 mM HEPES(pH 8.0), 10 mM MgCl;, 6 units of malic dehydrogenase,25 \xM redueed nicotinamide adenine dinueleotide(NADH) and 10 mM NaH'VO,. The PEPCase reaction at25''C was initiated by adding 1 roM phosphoenolpyruvateand vvas terminated after 180 s by adding 6 M HCl.

Statistical analysis

Data were analyzed by one-way ANOV.4 according to arandomized complete hloek design, using 5 plants asreplicates. Differenees were eompared by Tukey's test.

Tah. 1. Cladnde area and cladode dry weighl at flov\ering and fruit harvest. Dry weight vi-as calculated hy a regression equation atflowering (12 May 1993) and by drying harvested eladodes 75 days later at fruit harvest (26 July 1993). Data are means ± SE (n = 5plantsl.

Initially

I)

510101515

710

Fruit(number per cladode 1

.Afler fruil removal

0050

100

15

Cladodesurface area

(cm-)

in.'i9± 91UO9±l04I125±l171244+124IO83± 66l2.10±li91 l()5± 91

CladodeIgl

Flowering

69..S±6.784.5±6.893.1 ±8.298.8±9.()89.5±7.095.8±6.59O.8±5.6

D'W'

Harvesl

84.()±6.694.6±9.499.6±6.7

1IO.8±6.697.3±9.5

108.4±5.998.3±9.7

Cladode

Flowering

27.4+2.242.7±3.l46.1 ±2.842.5±2.946.6±3.342.0±l.745.8±2.6

excess DW(g)

Harvesl

41.9±3.452.8±5.352.2±6.854.6±5.654.9±6.054.2±5.l.53.5±5.l

Physiul. Ptanl. 91. im4

IE 16 20 0 4 8

Solar time (h)

Fig. 2. Daily pattern of net CO; uptake for cladodes of 0.fieus-mdica with 10 fruil (O) and with fruit (10) removed (A).Measurements were made 60 days after flowering on 12 May1993 and the solid area indicates night. Data are means ± ST. (n =5 planls).

Results

Growth

Dry weight per fruit decreased as the number of fruil percladode inereased (P<0.01; Fig. 1). Compared with ela-dodes bearing 5 fruit, individual fruit dry weight at matu-rity was 16% lower for eladodes with 10 fruit and 25"??lower for eiadodes with 15 fruit. Individual fruit on cla-dodes with the fewest fruil had the highest growth rate,with an average of 0.156, 0.128 and 0.112 g dry weightday ' for eladodes with 5. 10 and 15 fruit, respectively.Fruit growth rate was highest during the last 30 days offruit developmetit (Eig. I). Total fruit dry weight pereladode inereased 'with fruit number and at harvest was68% higher for eladodes with 10 fruit and 122% higherfor cladodes with 15 fruit eompared with cladodes with 5

o

a.

0.5 -

'=; 0.0

12 t s 20 0 4Solar t i m e (h)

Fig. 3. Daily pattern of net CO, uptake for fruits of O. fiitidica measured 15 (O). 30 (A). 45 (D), 60 (V) and 75days after flowering. Data are means ± SE (n = 15 fruit).

fruit. The average increment of lotal fruit dry weight percladode from flowering to harvest was 0.78, 1.22 and1.68 g dry weight day"' for eladodes with 5, 10 and 15fruit, respeetively.

At flowering, the excess dry weight for eladodes with-out flowers was less than 33 g, whereas all eladodes withflowers exceeded 33 g excess dry weight (Tah. I). Cla-dode dry weight increased during fruit development. Cla-dodes with no fruil and those from whieh the fruit wereremoved accumulated 100 and 599c more dry weight,respeetively, than cladodes with fruit (P<0.05; Tab. 1).Eruit dry matter accounted for 91. 92 and 949c of the totaldry matter accumulated from flowering to harvest oncladodes with 5, 10 and 15 fruit, respectively. Stomatalfrequency was 33 + I mm - for cladodes, 14+1 mm - forflowers and 6 ± 0 mm - for ripe fruii at 75 days afterflowering (mean ± SE for n = L5).

Tah. 2. Daily nel COi uptake for cladodes and fruit of O. ficusindica during fruit development. Data are means ± si (n = 5 lorcladodes and 15 for fruil).

Initially

055

10101515

Fruit

Fruil(numher per cladode)

.After fruit removal

0050

100

15

15

228±9198+5218+8201 ±4224±6208+4236±5

42+2

30

I6I±5144+2156+3146±6[63±8148±3165±5

3!±l

CO- uptake (mmol m"- dav"':Time after flowering (daysl

45

125+^IIO±2118+3107±412O±3115±212S±5

23±l

1

60

I76±3152±5178±6I5S±3I87±4156±4I91±6

L^±l

75

244±9226+723.1+82^1+'i239±6229+7242±7

7±0

Phvsiol Plant, 91. IW4 711

3 0 0

„ 150 -

250 -

200 -

100

50

11

T

-10 0 30 60 75Time after flowering (days)

Fig. 4. Time course of chlorophyll conleni in the ehlorenehymaof flower buds (-10 daysl, flowers (0 daysl and fruil of O.ficus-indica. Data are means ± SE (n = 15 truit).

CO, uptake

Removal of fruil just after flowering redueed eladodeCO; uptake rates (Eig. 2). Total daily net CO, uptakedeerea.sed 10% (P<0.05) for 15 to 45 days after flower-ing when 5, 10 or 15 fruit were removed (Tab. 2). At 60days after flowering, the deerease averaged 17'7f (P<0,01), but at harvest (75 days), no significant differ-enees in total daily net CO, uptake occurred (Tab. 2).

Cladode total daily net CO, uptake was maximal at 15days (27 May) and at 75 days (26 July) after flowering(Tab. 2), when day/night air temperatures were about25/16°C. CO, uptak'e was 30'7r lower at 30 days (11 June)and 60 days (I I July) after flowering, when day/night airtemperatures were higher by 5/3 °C, and 509c lower at 45days (26 June), when temperatures were higher by10/7°C. The PPE intereepted by the cladodes alwaysaveraged al least 31 mol m"- day"'.

Net CO; uptake by fruit oeeurred mainly at night(Fig. 3). Instantaneous rates never exceeded L5 jjmol nr-'s"' and decreased with fruit age. Total daily net CO;uptake per unit area for fruit up to 45 days after floweringaveraged 199c of that for eiadodes and then declined toonly 3% for ripe fruit at 75 days after flowering (Tab. 2).

Substaniial decreases in fruit CO; tjptake (Tab. 2) oc-curred sooner than decreases in chlorophyll eontent(Eig. 4) Chlorophyll a and b both decltned during I'ruitdevelopment: their ratio was 2.82 in flowers and 2.43 inripe fruit. Thirty days after flowering, the total chlo-rophyll eonlent per unit area of fruit was 17% lower thanin flowers, and when the peel eolor change began 60 daysafter flowering it was 40'^ lower (Fig. 4), at a time whendaily net CO; had deeeased 69% (Tab. 2). PEPCase andRubisco aetivities in the fruit ehlorenehyma (Fig. 5) wereapproximately eonstant until 30 days after flowering and

per unit area represented 50% of the PEPCase aetivityand 60% of the Rubiseo aetivity of the eladodes (data notshown). The CO; f'txing enzyme activity of the fruitsharply declined at 60 days after flowering and nearlydisappeared by fruit harvest at 75 days (Eig. 5).

Discussion

Cladodes of Opuntiaf'tcus-itidica had lO'̂ r lower net CO;uptake when fruit were removed. When day/night airtemperatures inereased from 25/16°C to 35/23°C, totaidaily net CO; uptake decreased 50%, consistent withprevious results for this species (Nobel 1988). Based onan average total daily net CO; uptake of 250 mmol m"-day"' and the eladode surfaee area (0.11 m-), a dry weightaeeumulation rale of 0.83 g day"' eladode"' occurredduring fruit development. This dry matler production met95% of the photo-assimilate requirements for cladodesbearing 5 fruit. 'When the number of fruit per eladodeincreased to 10 and 15, the contribution of cladode photo-synthesis to the daily gain in fruit dry weight declined to60 and 47%, respectively. Extensive import of photo-assimilate from neighboring cladodes is hence necessaryto support fruit growth for eladodes with many fruit.Compartmental analysis of carbon partitioning in O. fi-cu.s-indica indicates that a considerable amount of photo-assimilate is exported from basal eladodes to newly initi-ated daughter eladodes and that the flux increases withsink demand (Luo and Nobel 1992).

One-year-old, terminal eladodes of O. fleus-itidieawithout fruit are a souree of photo-assimilate for growingorgans, such as roots, developing eladodes and fruit.They can have net CO; uptake rates similar to those ofeladodes with fruit but they allocated only 25% of theirphoto-assimilates to their own dry weight gain. Photo-assimilate import for cladodes with fruit increased during

30 -

' >

20 -3

e .2Ou"3Ea.

0 -II) PEPCase

1 Rubisco

n-10 0 30 60 75

Time after flowering (days)

Fig. 5. Time eourse of PEPCase and Rubisco acdvity in theehliirenchyma of flower buds (-10 days), flowers {0 days) andfruit of O. fuits-indicu. Dala are means ± SE (n = \5 fruit).

712 . Pliini 91. I

the last 30 days of fruit growth, when the flesh rapidlydevelops and accumulates sugars (Barbera and Inglese1993). At this stage, the import of photo-assimilateranged from 30 to 70% of the dry weight gain for cla-dodes with 5 and 15 fruit, respectively. On the otherhand, during the early stages of fruit growth, net CO;uptake by eladodes ean nearly support fruit growth. In-deed, no more than 6 fruit should be left on a eladode toattain a large commercial size (120 g fresh weighl perfruit: Barbera and Inglese 1993). Further investigation isnecessary to determine whether carbon fluxes to cladodeswith fruit involve newly synthesized assimilate or siorednonstructural earbohydrates, which can represent 209f oftlie eladode dry matter for this speeies (Nerd and Nobel1991).

Altering souree:sink relationships by removing fruitsoon after flowering decreased the total daily net CO;uptake by the cladodes of O. ficu.s-indica. The effectdisappeared at fruit ripening, most likely beeause of asmall assimilate demand then. Similar effects oeeur forPrunus persica (DeJong 1986) and P. dorrtestica (Gucciet al. 1991). CO; uptake is also reduced by sink removalor girdling for the leafy CAM speeies Kalanehoe pinnata(Mayoral et al. 1991). The accompanying increase ofglucan in source leaves can eause feedback inhibition ofphotosynthesis (Hansen 1970, Chalmers et al. 1975,Gucci et al. 1991).

Unlike flower btids and young eladodes (Aeevedo etal. 1983), fruit of O. ficus-indica showed typical CAMbehavior. Net CO; uptake of fruit decreased from 0.18mmot day"' at 15 days after flowering to 0.06 mmol day"for ripe fruit al 75 days. The photosynthetic contributionof fruit to their carbon demand is thus greatest during theearly stages of fruit development (9'3f of dry' weight gainfor the first 30 days), .similar to peaeh (Pavel and DeJong1993b), apple (Jones 1981), orange (Moreshet and Green1980) and avocado (Whiley et al. 1992). The deereases instomatal frequency, chlorophyll content and enzyme ae-tivity with frtiil age help account lor the lower CO;uptake by fruit compared with that of cladodes. Thechlorophyll contenl in fruit ehlorenehyma of 0. ficu.s-indica was 50% less than that of its cladodes (Nobel andHartsock 1983).

In summary, more than 50 fruil nr" of eladode surfaeearea leads to substantial com]>etition lor photo-assimi-lates, reducing fruit growth rate and harvest size. Fruitsize can hence be controlled by fruit thinning and bypruning of terminal eladodes. Shading of cladodes shouldbe avoided to ensure maximal light availability and hencemaximal CO, uptake (Nobel 1988). Eruit CO; uptakeeontributes only partially to fruit earbon demand, partic-ularly during ripening when stjgars aeeumulate rapidly.

Aekmmtedgments - This reseaieh was supported hy the ItalianResearch Council for (CNR) Fellowship contract 203.06.49/031931 and by the Environmental Sciences Division. Office ofHealth and Environmental Research. U.S. Department ofEnergy contract DE-FCO3-87-ER60615.

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