Plant Physiol. (1971) 47, 623-628

Invertase Activity in Normal and Mutant MaizeEndosperms during Development"2

Received for publication October 5, 1970

T. A. JAYNES3 AND 0. E. NELSON4Department of Botany and Plant Pathology, Purdue University, Lafayette, Indiana 47907

ABSTRACT

A bound invertase and two soluble invertases are found inthe developing endosperm of maize (Zea mays L.). The twosoluble invertases can be separated on diethylaminoethyl-cellu-lose and Sephadex columns and distinguished by their kineticconstants. One soluble invertase, invertase I, is present fromthe 10- to 28-day stages of endosperm development with maxi-mal activity per normal endosperm at the 12-day stage. In twoendosperm mutant lines, shrunken-I and shrunken-2, there isa second increase in invertase I activity later in developmentwhich could be a secondary effect caused by the abnormalmetabolism in these lines. Another soluble invertase, invertaseII, is present in the embryo upon germination and is alsofound in the very young developing endosperm (6-day stage).The third form of invertase, bound invertase, is present in theendosperm by the 6-day stage, and its activity remains approx-imately constant during development.

Carbohydrate is translocated in the maize plant as sucrose(17). The initial reaction in the utilization of sucrose is gen-erally believed to be the invertase-catalyzed hydrolysis of it toglucose and fructose.

Invertase (fi-D-fructofuranoside fructohydrolase, E. C.3.2.1.26) exists in several forms in plant tissues. Invertase isattached to cell walls in yeast (11) as well as in higher plantsincluding corn coleoptiles (14). This bound invertase is tightlyassociated with the cell walls for it cannot be removed bytreatment with salt solutions or organic solvents (4, 9). Inaddition to the distinction of bound and soluble forms, iso-zymes of the soluble invertase have been found. Yeast has adifferent invertase external to the plasma membrane than theinvertase found in the cytoplasm (7). Light and heavy forms ofinvertase have been described in Neurospora. The light formmay be produced from the heavy form by various treatments(20). Some invertase isozymes have been associated with

'Supported by the Purdue Research Foundation and the Na-tional Science Foundation (GB-15104).

'Journal Paper 4193, Purdue University Agricultural Experi-ment Station.

'Present address: Department of Agronomy, University of Ne-braska, Lincoln, Neb. 68503. Submitted to Purdue University inpartial fulfillment of the requirements for the degree of Doctorof Philosophy.

'Present address: Department of Genetics, University of Wis-consin, Madison, Wis. 53706.

meristematic regions, e. g., immature sugar cane tissue hasan acid invertase whereas mature tissue has a neutral invert-ase (8). Similarly, an alkaline invertase is found in ungermi-nated bean seeds whereas an acid invertase is present inthe embryo upon germination (1).How the different forms of invertase relate to metabolism

is not clear. Experiments show hydrolysis of sucrose duringcellular uptake in yeast (12) and Neurospora (19) and somehigher plants (9) but not in others (15). In the radioactivelabeling experiments of Shannon (25) using whole corn plants,sucrose was hydrolyzed even before entering the endospermand then resynthesized in endosperm tissue. Invertases presentin the corn endosperm could be important in regulating theutilization of this resynthesized sucrose. The relationship ofinvertase to metabolism in the corn endosperm has been ex-amined here by the identification of three invertases in cornendosperm and the examination of their activities in normaland mutant endosperms during development.

MATERIALS AND METHODS

Collection of the Developing Seeds. The developing seedswere harvested at stated intervals following controlled self-pollination to assure genotypic purity. The normal corn (Zeamays L.) used in this study was a hybrid single cross, B37 XB14. The endosperm mutants, shrunken-i, shrunken-2, andsugary-2, were inbred stocks. Samples of corn were harvestedat 2-day intervals starting at 6 days (6-day stage) and con-tinuing to 28 days (28-day stage) after the date of pollination.At harvest the kernels were cut off the cob, immediately frozenon Dry Ice, and stored frozen (-20 C) until needed.

Assay for Soluble Invertases. Total volume of reaction mix-tures for analysis of invertase activity during developmentwas 0.175 ml containing in part, 0.1 ml of a 0.01 M sucrosesolution, 0.01 ml of 0.2 M sodium acetate buffer (pH 5.0),and a variable quantity of an invertase-containing prepara-tion depending on the stage of endosperm development. Thereaction mixture was incubated 15 min at 37 C. Six-tenths ofa milliliter of the copper-containing Nelson's reagent (22) wasadded to stop the reaction. Next this mixture was heated at95 C for 15 min, cooled, and 0.6 ml of arsenomolybdate re-agent added (22). Absorbance was read at a wavelength of520 nm on a Bausch and Lomb Spectronic 20. Activity wasconverted to amount of sucrose hydrolyzed by measuring theabsorbance of a standard solution containing glucose andfructose.The dialyzed homogenate from the endosperm was frac-

tionated before measuring activity because in initial experi-ments with this extract invertase I activity was curvilinear witha change in protein concentration of the reaction mixture.This may be attributed to the invertase inactivator present atlater stages of development. The inactivator is discussed in

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JAYNES AND NELSON

0.3

IEa.2 E

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10 14Fraction Number

FIG. 1. Elution profile with DEAE-cellulose column fractiona-tion of a coextract made from normal endosperms at the 16-daystage and germinated embryos of normal corn. Two and one-halfgrams of endosperms and an equal weight of germinated embryoswere ground together in a mortar with 9 ml of 0.01 M tris-maleatebuffer (pH 8.0). After elution of 5 ml of this dialyzed homogenate,protein (0) and invertase activity (A) were measured as describedin "Materials and Methods".

the following paper (13). If care was taken to assay unfrac-tionated invertase I at a high buffer concentration, to add in-vertase I rather than sucrose at zero time, and to limit the reac-tion time to 15 min, then an activity per endosperm could bemeasured in the extract which was similar to that after frac-tionation on DEAE5-cellulose. If these precautions were nottaken, activity was reduced and was not linear with an in-crease in amount of extract in the reaction mixture. Afterfractionating the homogenate on DEAE-cellulose, these pre-cautions were not necessary.

Extraction and Fractionation of Invertase I. Invertase Iwas extracted from endosperms with 0.05 M tris-maleatebuffer (pH 8.0) containing 1 mm mercaptoethanol. Five gramsof endosperms (about 80 endosperms of 12-day or 20 endo-sperms of 22-day normal corn) were ground in 6.5 ml of bufferwith a mortar and pestle. The homogenate was centrifugedat 22,000g for 10 min at 0 C. The resulting supernatant frac-tion was dialyzed against 1 liter of 0.01 M tris-maleate buffer(pH 7.0) also containing 1 mm mercaptoethanol for 18 hr withone change of dialysate. Protein in the dialyzed homogenatewas measured by the Lowry method (18). Dialysis and sub-sequent fractionation procedures were run at 3 C.The DEAE-cellulose used to fractionate the dialyzed ho-

mogenate was equilibrated with 0.01 M tris-maleate buffer(pH 7.0) as specified by Peterson and Sober (23). A column,1 cm X 15 cm, was packed with DEAE-cellulose under at-mospheric pressure. Five milliliters of dialyzed homogenate,about 25 mg of protein, were added to the column. The pro-tein was eluted by a slightly concave NaCl gradient formedby a siphon-mixing of 150 ml of 0.01 M tris-maleate buffer(pH 7.0) containing NaCl (1.0 M) into 150 ml of 0.01 M tris-maleate buffer which was flowing to the column. Five-milli-liter fractions were collected. When the kinetic constants ofinvertase were studied, an invertase containing fraction wasused which was first dialyzed 5 hr against 1 liter of a 1 mmmercaptoethanol solution containing 3 drops of 0.2 M acetatebuffer (pH 5.0). The amount of invertase I in the endospermduring development was measured after fractionating theprotein extract on a DEAE-cellulose column. Each fraction

IaAbbrevytions:DEAE: diethylaminoethyl;shruuken: sl; sug-

ary: su.

was analyzed for invertase I activity, and the separate activ-ities were added to give a total.

Sephadex G-200 used to fractionate the extract was pre-pared as recommended by the manufacturer. The Sephadexwas equilibrated with 0.01 M tris-maleate buffer (pH 7.0) andpacked to give a column 2.3 cm X 35 cm.

Extraction of Invertase II. Invertase II was extracted fromgerminated kernels of the B37 X B14 single cross. The kernelsgerminated in 3 days at room temperature on a moist filterpaper in a Petri dish. The endosperms were separated and dis-carded. Two and one-half grams of germinated embryos wereground with a mortar and pestle in 9 ml of 0.05 M tris-maleatebuffer (pH 8.0). Subsequent steps of extraction and columnfraction were identical to those for invertase I (however, in-vertase II eluted in a different position upon DEAE-cellulosefractionation).

Extraction and Assay of Bound Invertase. To extract thecellular debris, 5 g of endosperms were homogenized in 30 mlof cold acetone, and the mixture was centrifuged at 12,000gfor 5 min. Next the precipitate was homogenized in 30 mlof hexane, centrifuged, and then washed three times by a sim-ilar procedure with 30 ml of water each time. After the lastwater wash, the debris was homogenized in acetone, centri-fuged, and dried. The product could be ground slightly witha mortar and pestle, but no activity remained after extensivegrinding.

For assay of bound invertase, 15 mg of the cell debris weresuspended in 0.15 ml of a solution that contained sucrose at16.6 mm and acetate buffer (pH 5.0) at 34 mM. The reactionwas stopped with 0.2 ml of 0.1 N NaOH. Ten minutes afteraddition of NaOH, 2 ml of water was added, stirred, and themixture centrifuged. One milliliter of the supernatant fractionwas removed and added to one milliliter of Nelson's reagent.The subsequent steps of assay were identical to those for thesoluble invertases.

RESULTS

Distribution of Two Soluble Invertases in the Seed. Twosoluble invertases were present at different stages of seed de-velopment. One form of soluble invertase, designated invertase

120E

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80Effluent (ml)

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FIG. 2. Elution profile with Sephadex G-200 fractionation ofdialyzed homogenates from (A) endosperms at the 22-day stageand (B) embryos after germination. Five-milliliter portions of thedialyzed homogenate were added to the column. After elution,protein (0) and invertase activity (A\) were measured as describedin "Materials and Methods".

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INVERTASE ACTIVITY IN MAIZE ENDOSPERM

E

E

1001/S (M)

300

FIG. 3. Lineweaver-Burk plot of (A) invertase II extracted fromgerminated embryos, (B) invertase II extracted from endospermsat the six-day stage and (C) bound invertase extracted from endo-sperms at the 22-day stage. Extraction and assay conditions aregiven in "Materials and Methods".

I, was present only in the endosperm from 10 to 28 days afterpollination. Invertase I was not found in the embryo upongermination. On the other hand, invertase II was found ingerminated embryos and in endosperms harvested before the10-day stage of development. Neither invertase was detectedin the developing embryo.

Differences in Chromatographic Properties of the SolubleInvertases. The two soluble invertases separated after frac-tionation of homogenates on DEAE-cellulose. Invertase Ieluted at 0.3 M NaCI in the salt gradient (fraction No. 19-23),whereas invertase II eluted in earlier fractions (12 to 14). Inthe experiment of Figure 1, equal weights of 16-day endo-sperms (containing invertase I) and germinated embryos (con-taining invertase II) were ground together and the homogenatefractionated on DEAEcellulose. The two invertases eluted asthey did when fractionated separately. Activity in both peakswas about that expected. For this reason the different elutionpatterns of the two invertases on DEAE-cellulose was unlikelyto be an artifact due to differences in the tissues from whichthe extracts were made.The two soluble invertases were also separated by fractiona-

tion on Sephadex G-200. The elution profile of a homogenatefrom 22-day endosperms is diagrammed in Figure 2A. Invert-ase I eluted in the exclusion volume (at 50 ml for this frac-tionation). Since Sephadex G-200 has an exclusion limit ofapproximately 300,000, the molecular weight of invertase Iprobably exceeds this value. No invertase I activity was evidentin other fractions. When homogenates from germinated em-

bryos were fractionated on Sephadex G-200 (Fig. 2B), invert-ase activity (invertase II) eluted at a later position than invert-ase I, indicating a smaller molecular weight.

Differences in Kinetic Properties of the Soluble Invertases.The two soluble invertases could also be distinguished by theirreaction kinetics. Invertase I had a Km of approximately 2 mM

at pH 5.0 while under the same conditions the Km of invertaseII was 10.5 mm (Fig. 3A). When the Km and Vmax of invertaseI were plotted as a function of pH (Fig. 4) little change wasnoted between pH 4.0 and pH 6.0. However, activity of in-vertase II decreased with the same change in pH (Fig. 5). At apH greater than 6.0 the V... of invertase I decreased and theKm increased. Invertase I after fractionation on DEAE-cellu-lose was evid.ntly denatured at neutral pH, for activity couldnot be recovered after preincubation at a neutral pH. Bothinvertases were stable at pH 7.0 in the dialyzed homogenate.

It was mentioned previously that some invertase II activitywas detected in endosperms at very early stages of develop-ment. The endosperm was small at the 6-day stage and wasembedded in maternal nucellus. To remove contaminating ma-ternal tissue, the endosperms were rinsed in cold water. Invert-ase activity after fractionating the extract from 6-day endo-sperms on DEAE-cellulose was present in the same fractionsas invertase II from germinated embryos. The Km of 9.7 mM(Fig. 3B) was also similar to that of invertase II.Bound Invertase. A third form of invertase present in corn

endosperm was bound to cellular debris after extraction. Starch

7

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FiG. 4. Plot of Km (0) and Vm. (A) of invertase I as a func-tion of the pH of reaction. Invertase I was reacted for 15 minwith two sucrose concentrations at each pH. Volume of reactionmixture was 0.15 ml containing: invertase I preparation from nor-mal endosperms at 14-day stage, 4 Itg of protein; acetate buffer(pH 4.0, 4.5, 5.0, and 5.5) and MES buffer (pH 5.5, 6.0, 6.5 and7.0), 16.7 mM; sucrose, 1.3 mm and 13.3 nM. Invertase I activityin MES buffer was corrected at pH 5.5 to correspond to activityin acetate buffer.

4

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4.0 4.5 5.0 5.5 6.0 6.5 7.0pH

FiG. 5. The pH optimum of invertase II. Invertase II was re-acted for 15 min with two concentrations of sucrose to calculatethe Vm... Reaction temperature was 37 C. Volume of reactionmixture was 0.175 ml containing: invertase II preparation fromgerminated normal kernels, 5 /Ag of protein; acetate buffer (pH4.0, 4.5, 5.0 and 5.5) and potassium phosphate buffer (pH 5.5,

6.0, 6.5, and 7.0), 14 mM; sucrose, 5.7 mm and 11.4 mM. InvertaseHI activity in phosphate buffer was corrected at pH 5.5 to cor-

respond to activity in acetate buffer.

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(3B)

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625Plant Physiol. Vol. 47, 1971

(4) A

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0- 0

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JAYNES AND NELSON

10 18 26 10 18 26

Days After Pollination

FIG. 6. Invertase I activity in normal corn and three mutantendosperm lines, shi/shi, sh2/sh2 and SU2/SU2. Assay conditionsare given in "Materials and Methods".

granules, selectively separated from the cell walls by passagethrough four layers of cheesecloth, had little bound invert-ase activity, so the enzyme was considered cell wall-bound.Bound invertase was not solubilized by salt or organic solvents.The acetone and hexane washes in the extraction were neces-sary for recovery of any bound invertase activity in endo-sperms at later stages of development.Bound invertase activity was assayed using 15 mg of cellular

debris suspended in the reaction mixture. With this method,replicates were more variable than those with the soluble in-vertases. The standard deviation for bound invertase activityin six replicates was calculated at 13.5% of the mean whilethat for invertase I in assay was 1.5% of the mean activity.Bound invertase activity was linear in an assay changing thetime of reaction from 10 to 30 min or changing the amountof cellular debris from 5 to 15 mg. The Km of bound invertasein one experiment (Fig. 3C) was 8 mM.

Analysis of Invertase I Activity During Endosperm Develop-ment. To relate invertase I activity to metabolism in the endo-sperm, activity was measured at different stages of normal andmutant endosperm development. However, it should be notedthat other factors in the endosperm may change considerablywith development and variably influenev invertase activity dur-ing extraction. This is true with invertase I where an inacti-vating protein is present at later stages of development but notat early stages (13).The amount of invertase I activity per endosperm of normal

corn (+/+) and the endosperm mutant lines, sh,/sh1, sh2/sh,and SU2/SU2, that have abnormal carbohydrate metabolism andaccumulat2 sugars is plotted in Figure 6. The values are theresult of one fractionation of a 5-g sample of endosperms fromeach stage. The material used in this analysis was harvested in1968, but an analysis of invertase I activity in normal corn

harvested in 1966 and 1967 showed a similar loss of activityafter the 12-day stage. To give some measure of the variability,invertase I activity in four samples of normal endosperms atboth the 14- and 22-day stages was measured, and the standarddeviation calculated. If this standard deviation is expressed as

a percentage of the activity at each stage, then the standarddeviation at 14 days was 13% and that at 22 days was 34%.The decrease in invertase I activity per endosperm during de-velopment was also observed when measuring activity in thedialyzed homogenate. Activity was always greatest in normalendosperms 12 days after pollination and decreased on a per

endosperm basis afterward. The endosperm at the 12-day stagecontained 37 times more invertase I activity than at the 28-daystage.Two patterns of invertase I activity during development

were evident in the mutant endosperm lines. The endosperms

of the su2/su2 line had the same invertase I activity profile asnormal corn but a reduced enzyme activity at all stages of de-velopment. Part of this low invertase content can be attributedto the size of the su2/su2/Su2 endosperm which was two-thirdsof normal. The invertase I activity profiles in the +1 +-/ + andSU2/SU2/SU2 endosperms differed from those in sh,/sh,/sh, andsh2/sh2/sh2 endosperms where invertase I activity continuedto increase per endosperm from 12 to 16 days with a secondincrease 22 days after pollination. Analysis of activity in addi-tional samples of sh2/sh2/sh2 endosperms at the 18- and 22-daystages showed the same increase in activity by the later stage.Generally, soluble protein content in the endosperm increaseduntil 22 days after pollination and then was lower by the 28-day stage (Table I).The large difference in activity between normal and sh2l

sh2/sh2 endosperms at the 22-day stage was examined in a genedosage study following the appropriate pollinations. The ker-nels were harvested at the 22-day stage and invertase I activitywas measured. If invertase I was expressed as specific activityper endosperm, then the +/+/ + endosperms hydrolyzed 4.5;+/+/sh2, 5.24; +/sh2/sh2, 6.18, and sh2/sh2/sh2 endosperms,12.8 m,tmoles of sucrose per min per mg of protein. Withhomozygosity for the sh2 mutation, invertase activity increasedgreatly.

After invertase I activity in the mutant endosperm lines wasdetermined, the technique of enzyme extraction was checkedby coextracting endosperms of different genotypes or fromdifferent stages of development. Different endosperm samplesof equal weights were ground together in buffer with a mortarand pestle. The homogenate was centrifuged, and the resultingsupernatant was dialyzed and fractionated on DEAE-celluloseby the normal procedure. The result of a typical coextractionexperiment using sh2/sh2/sh, and +/+/+ endosperms at the22-day stage is given in Table II where the value for the co-extracted sample, 4.34, was considerably less than the inter-mediate value expected, 9.68. A decrease in invertase I ac-tivity after coextraction was also observed if the coextract wasdeionized on Bio-Gel P-2 rather than dialyzed before frac-tionation. However, if the endosperms were ground separatelyand then combined, activity was close to the intermediate value(Table II). Since the cellular debris was present in both thecoextract and the combined extract, it was not considered re-sponsible for the decrease in invertase I activity during coex-traction. This decrease following coextraction may indicate amechanism regulating the level of invertase.

Activity of Bound Invertase During Development. The ac-tivity of bound invertase per endosperm of +/+/+ and shkIsh2/sh2 endosperms at several stages of development is givenin Table III (activity in s1i12/su2/su2 endosperms was similar tonormal). However, bound invertase activity varied as much as50% between samples from the same ear. A similar variationin bound invertase activity of tissue culture lines has beennoted by Straus (26). Attempts to reduce this variability in cornendosperm with different extraction methods were unsuccess-ful. Since no major trend in the amount of activity duringdevelopment of these endosperm lines was found, bound in-vertase activity per endosperm probably changes little duringdevelopment. This suggests that the decrease in invertase I ac-tivity noted during development is not due to a binding of in-vertase I to some particulate fraction.The 6-day stage of the endosperm was examined for bound

invertase activity. Due to the small size of the endosperm atthis stage, bound invertase could not be extracted using thenormal procedure. The endosperms were ground and washedwith water and acetone directly in centrifuge tubes. Afterwashing and drying the debris, buffer and sucrose were added

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INVERTASE ACTIVITY IN MAIZE ENDOSPERM

to initiate the reaction. High activity for bound invertase waspresent, but it was not possible to analyze it quantitatively.

DISCUSSION

Two soluble invertases are present in the corn kernel at dif-ferent stages of development. The two enzymes can be physi-cally separated and distinguished by their kinetic constants.Though the two molecules differ considerably in molecularweight, no interconversion of them has been observed.

Invertase I has its greatest activity in normal corn 12 daysafter pollination. Activity per endosperm decreases markedlyduring the period of greatest net starch synthesis. For thisreason, one may question whether the first step in the synthe-sis of starch is the invertase-catalyzed hydrolysis of sucrose.Turner and Turner (29), De Fekete and Cardini (6), Murataet al. (21), Pressey (24), and De Fekete (5) have all suggestedthat the first step is the transfer of glucose to a nucleoside di-phosphate catalyzed by UDP-sucrose glucosyltransferase. Forthe synthesis of ADP-glucose, ADP can replace UDP in thereaction. However, the affinity of the enzyme for UDP is muchgreater than for ADP, and the utilization of ADP is much in-hibited by the presence of uridine nucleotides (6). Further, themuch reduced starch formation in the shrunken-2 and brittle-2mutants of maize which have very low levels of ADP-glucosepyrophosphorylase activity (2, 27) makes it unlikely that themajor portion of ADP-glucose utilized in starch synthesis issynthesized via the UDP(ADP)-sucrose glucosyltransferase.The pathway suggested by Turner and Turner (29) in which(a) UDP-glucose is formed via the UDP-sucrose glucosyltrans-ferase and (b) glucose 1-P and UTP are formed from UDP-glucose and pyrophosphate by UDP-glucose pyrophosphoryl-ase, seems increasingly probable. ADP-glucose can then besynthesized from glucose 1-P and ATP by ADP-glucose pyro-phosphorylase. However, UDP-sucrose glucosyltransferase ac-tivity is not detectable in developing endosperms until the 12-day stage, after starch synthesis has commenced (28). It ispossible that the hydrolysis of sucrose catalyzed by invertase Iis the first step in the conversion of sucrose to starch early inendosperm development (prior to 12 days after pollination) aswell as in the first step in glycolysis in the developing endo-sperm.The pattern of invertase I activity during the development

of sh,/sh1/sh1 and sh2/sh2/sh2 endosperms is different fromthat in normal or su2/su2/su2 endosperms. This new patterncould be caused by an accumulation of metabolic intermedi-ates. Laughnan (16) noted that sugars of the endosperm (par-ticularly sucrose) show a large increase in the sh2/sh2/sh2 endo-sperm. In the sh2 dosage study, invertase I activity also increasedthe most in the sh2/sh2/sh2 endosperms. However, the level ofinvertase at the 28-day stage of sh2/sh2/sh2 endosperms approaches that in normal corn despite the continued high levelof sucrose in the endosperm.The su2/su2/su2 endosperm differs from the other two endo-

sperm mutant lines in that it accumulates slightly less starchthan normal, and this starch has a higher amylose percentagethan normal (3). Even though the su2 line has a low invertaseI activity at 12 days compared to normal endosperms, activitystill decreases during development similar to that in normalendosperms. This suggests that the mechanism controlling theamount of invertase operates to decrease the level of invertasepresent at 12 days rather than maintain a given level of in-vertase at various stages of development.

Invertase II was present in the germinated embryo as well asin the endosperm at very early stages of development. Invert-ase II could be the soluble form of the bound invertase. The

Table I. Soluble Proteinz Content per Endosperm of Normal andThtree Endosperm Mutant Lines of Corn, shi/shl, sh2/sh2, and

SU2/SU2, dutring Development

Protein/EndospermStage of

Development+/+/+ sk/shl/sh shs/sh2/sl, sU2/SU2/sU2

days mg

10 0.16 0.10 0.1512 0.45 0.36 0.39 0.5614 1.27 0.96 1.12 0.9316 1.79 1.20 1.54 1.0618 2.23 1.80 1.80 _20 2.26 - - -22 2.52 2.37 2.47 1.2824 2.42 - 2.19 1.4926 2.56 2.14 2.60 1.2028 2.30 1.87 2.33 1.23

Table II. Invertase I Activity inz a Coextract anzd Combined Extractof sh2/sh2/sh2 antd +/+/+ Enidosperins at the 22-day StageThe samples were coextracted by grinding 2.5 g of both sh2/sh2/

sh2 and +/+/+ endosperms in buffer with a mortar and pestle.In the combined extraction 2.5 g of each were ground separatelyand then combined and dialyzed.

ExtractActivity/mg of

Protein FractionatedGenotype Treatment

mjimoles/min+/+/+ 1.99sh2/sh2/sh2 17.38++1/+ and sh2/sh2s/sh2 Coextracted 4.43+/+/+ and sh2/sh2/sh2 Combined 8.43

Table III. Bound Invertase Activity per Endosperm of +1+/+ andsh2lsh2/sh2 during Development

Genotype Stage of Cellular Activity/Development Debris Endosperm

days mg/endosperm mjmoles/min

+/+/+ 14 8 2316 24 722 62 828 125 17

sh2/sh2/sh2 14 9 1716 10 1422 25 2228 44 11

Km values of the two invertases were approximately the sameand the soluble invertase II was present along with bound in-vertase at early stages of development. Possibly invertase II issynthesized in endosperms and attached to the cell walls onlyat very early stages of development. This suggestion is sup-ported by experiments of Hellebust and Forward (10) whichshowed a high level of invertase in the young cell elongationregion of the corn root. Their experiments showed that totalinvertase activity (bound and soluble) decreased in older re-gions of the root.The role of the bound invertase is not clear. The investiga-

tions of Shannon (25) indicate that sucrose is hydrolyzed priorto or upon entry into the endosperm and then resynthesized to

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628 JAYNES A

serve as a storage form prior to utilization in glycolysis orstarch synthesis. It is possible that the bound invertase func-tions in sucrose hydrolysis during entry into the endosperm.Bound invertase may also function in a pathway leading to thesynthesis of cell wall components.

LITERATURE CITED

1. COOPER, R. A. AND R. N. GREENSHIELDS. 1964. The partial purification andsome properties of two sucrases of Phaseolus vulgaris. Biochem. J. 92: 357-364.

2. DICKENSONx, D. B. AND J. P. PREISS. 1969. Presence of ADP-glucose pyrophos-phorylase in shrunken-2 and brittle-2 mutants of maize endosperm. PlantPhvsiol. 44: 1058-1062.

3. Du-N-N. G. M., H. H. KRANTER, AND R. L. WN HISTLER. 1953. Gene closage effectson coiln endosperm carbohydiates. Agron. J. 45: 101-104.

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