Plant Physiol. (1990) 92,881-8850032-0889/90/92/0881 /05/$01 .00/0

Received for publication July 28, 1989and in revised form October 19, 1989

Molecular Characterization of the Brittle-2 Gene Effect onMaize Endosperm ADPglucose Pyrophosphorylase Subunits

Jack Preiss*, Stephanie Danner, Peter S. Summers, Matthew Moreill, Carolyn R. Barton, Limei Yang, andMatthew Nieder

Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824 (J.P., S.D., P.S.S., M.M.);and ESCAgenetics Corporation, San Carlos, California 94070 (C.R.B., L.Y., M.N.).

ABSTRACT

Activity of the enzyme ADPglucose pyrophosphorylase isknown to be reduced in maize (Zea mays L.) endosperm mutantsat two independent loci, Shrunken-2 (Sh2) and Brittle-2 (Bt2).Spinach leaf ADPglucose pyrophosphorylase has previously beenshown to comprise two subunits of 51 and 54 kilodaltons. Anti-bodies raised to each of the two subunits of spinach leaf ADP-glucose pyrophosphorylase were found to cross-react to differentbands on Western blots prepared from polyacrylamide gel elec-trophoresis separated wild-type maize endosperm proteins. Theanti-spinach leaf 51 kilodalton subunit antibody cross-reactedwith a 55 kilodalton maize endosperm protein and the anti-spinach leaf 54 kilodalton subunit antibody cross-reacted with a60 kilodalton maize endosperm protein. These immunologicalreactions were observed in maize endosperm extracts and witha highly purified preparation of maize endosperm ADPglucosepyrophosphorylase. Mutant bt2 endosperm lacked the 55 kilodal-ton subunit while mutant sh2 endosperm lacked the 60 kilodaltonsubunit on Western blots. These results suggest that the maizeendosperm ADPglucose pyrophosphorylase is made up of twoimmunologically dissimilar subunits and that the bt2 and sh2mutations cause reduction in ADPglucose pyrophosphorylaseactivity through the lack of one of these two subunits. An ADP-glucose pyrophosphorylase cDNA clone antigenically selectedfrom a rice seed cDNA expression library was found to hybridizestrongly with a cDNA corresponding to a maize endosperm tran-script which is absent in a W64A bt2 mutant. Thus, the bt2 mutantcauses the absence not only of the small subunit but of thecorresponding transcript. Bt2 is implicated as the structural genefor the small (54 kilodalton) subunit of maize endosperm ADP-glucose pyrophosphorylase.

ADPglucose pyrophosphorylase has been implicated as akey regulatory enzyme for starch biosynthesis in both leafandstorage tissues (14). Mutation at the independent lociShrunken-2 (Sh2) and Brittle-2 (Bt) results in dramatic re-duction of maize endosperm ADPglucose pyrophosphorylaseactivity (7, 17) as well as approximately 25% of normal starchcontent (4, 6, 17). However, the mechanism by which thesemutants cause reduction of enzyme activity had not previ-ously been established.

Present address: Plant Environmental Biology Group, ResearchSchool of Biological Sciences, Australian National University, Can-berra, A.C.T. 2601 Australia.

Those ADPglucose pyrophosphorylase enzymes which havebeen characterized to date from both plant and bacteria havenative molecular masses ofapproximately 200 kD (15). Maizeendosperm native enzyme is 230 kD by gel filtration analysis(13). Bacterial ADPglucose pyrophosphorylase have beenfound to be homotetramers of a single subunit having ap-proximately 50 kD molecular mass. On the other hand, theADPglucose pyrophosphorylase of plants appears to be madeup of two subunits. Thus, spinach leaf ADPglucose pyro-phosphorylase has been purified to homogeneity and has anative molecular weight of 206 kD (5). The purified enzymeconsists of large and small subunits of molecular mass 54 and51 kD as determined by SDS gel electrophoresis. Rabbitpolyspecific antibodies have been prepared to purified spinachleaf subunits (12). Antibodies to each subunit did not crossreact to the other subunit. These antibodies have been usedto demonstrate the presence of two subunits in the enzymefrom Arabidopsis thaliana leaf ( 10) and potato tuber (T Okita,private communication).

This paper reports evidence that the maize endospermADPglucose pyrophosphorylase also has a subunit structuresimilar to that of spinach leaf. A small subunit of 55 kD cross-reacts to spinach small subunit (51 kD) antibody and a largesubunit of60 kD cross-reacts to antibody raised to the spinachleaf large subunit (54 kD). Furthermore, bt2 maize mutantlacks an immunologically detectable small subunit while sh2mutant apparently lacks the large subunit.

Barton et al. (2) have isolated cDNA clones correspondingto partial transcripts of poly(A) RNA absent in endosperm ofW64a sh2 and bt2 as compared to W64A wild type maize.These cDNA clones are identified as pES6-66 and pES6-75,respectively. Northern analyses of mutant and wild-typepoly(A) RNA established that both mutants are deficient inmRNA transcripts ofapproximately 2.3 kb. Moreover, hybridrelease translation results in individual protein transcripts of54 and 60 kD from pES6-75 and pES6-66, respectively.Protein bands corresponding to these molecular masses wereabsent in SDS gels prepared from endosperm protein extractsof bt2 and sh2 isogenic lines. Thus, these sh2 and bt2 allelesapparently cause the absence of individual maize endospermproteins through the lack of messenger RNA. In light ofimmunological evidence presented in this paper indicatingtwo subunits of 55 and 60 kD for maize endosperm ADP-glucose pyrophosphorylase, these data suggest that pES6-75and pES6-66 code for the two subunits. However, a close

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Plant Physiol. Vol. 92, 1990

correspondence in molecular mass is not sufficient proof ofidentity. For this it was necessary to provide evidence ofsequence homology.Anderson et al. (1) have isolated a rice endosperm ADP-

glucose pyrophosphorylase cDNA clone using antibodies topurified spinach leaf ADPglucose pyrophosphorylase with aXgt 11 expression library. In this paper we describe strongDNA:DNA hybridization between this rice ADPglucose py-rophosphorylase cDNA clone and the cDNA clone corre-sponding to a transcript absent in bt2 (pES6-75). The tran-script missing in bt2 is thus indicated as the messenger RNAfor the small 54 kD subunit of maize endosperm.

MATERIALS AND METHODS

Chemicals and Biological Material

All chemicals and biological reagents were purchased at thehighest purity available from either Sigma, Cal Biochem, orBoehringer-Mannheim. Bicinchoninic acid protein reagentwas obtained from Pierce Chemical Co. Protein molecularmass standards were from Pharmacia and nitrocellulose mem-branes (BA-85; 0.45 AM) was from Schleicher and Schull.Maize endosperm (13) and spinach leaf ADPglucose pyro-phosphorylase ( 12) were purified as described.

Seeds

W64A and isogenic lines ofmaize (Zea mays L.) containingsh, (ref) or bt2 (ret) were used. Developing maize seeds usedfor enzyme activity and western blotting studies were har-vested at 22 d post self-pollination, frozen with dry ice, andstored at -80°C until used. The sh2 and bt2 mutants were alsoharvested 22 d after self-pollination.

Preparation of Maize Endosperm Crude Extract forEnzyme Assay

Preparation of the crude extract was carried out at 4°C. Theendosperm was separated from the seed coat and embryobefore grinding. Single kernels were ground in chilled 1 mLRagnoti glass tubes and pestles. The homogenate was sus-

pended in 150 AL of grinding buffer detailed by Plaxton andPreiss (13) using DTT instead of DTE. The grinding bufferwas made just before grinding so that the added PMSF andDTT were fresh. The suspension was centrifuged at 3000g for20 min. The pellet was reextracted with 150 AL of grindingbuffer and centrifuged again at 3000g for 20 min. The super-natants were combined to be assayed.

Assay for ADPglucose Pyrophosphorylase Activity

The assay used was that of Plaxton and Preiss in thepyrophosphorylase direction (13). Five ItL ofthe crude extractwas routinely used for assaying. Ten ,uL of 100 mM NaPPiinitiated the reaction. Concentrations of the components ofthe reaction mixture and controls for the assay were the sameas previously described (13). A unit of activity was defined as

I ,umol of glucose- 1-P formed in 1 min.

Crude Extract Preparation for Gel Electrophoresis

The grinding buffer was 15 mM Tris-HCl (pH 8.0), 2 mMDTT, 1 mM EDTA, and 1.5 mm PMSF. Three kernels wereground at once in 100 ,uL of grinding buffer as describedabove. Suspensions were centrifuged at 3000g for 20 min.The supernatant was spun in an Eppendorf microcentrifugefor 6 min. The resultant supernatant was removed for loadingon the gels. Protein concentrations for all crude extracts weredetermined using the procedures of Smith et al. (16) withbovine plasma albumin as the reference standard.

Electrophoresis of SDS Gels and Western Blotting

Electrophoresis was carried out based on the procedure ofPlaxton and Preiss (13). Gels wee electroblotted onto nitro-cellulose membranes for 1 h at 0.8 to 1.0 amp using aTransphor TE 42 electroblotting apparatus (Hoeffer ScientificInstruments). Transfer buffer used was that of Burnette (3).After electroblotting gels were stained for protein with Coo-massie blue R-250. Nitrocellulose membranes were incubatedfor 1 h at 37°C in a blocking buffer of 50 mm Tris-HCl (pH7.5), 150 mm NaCl, 0.1% NP-40, and 1% BSA. The blockingbuffer was discarded and replaced by a primary antibodydilution. Antibodies used were affinity purified rabbit anti-spinach leaf ADPglucose pyrophosphorylase IgG (13) andaffinity purified rabbit antibodies raised against spinachADPglucose pyrophosphorylase upper and lower subunits(12). The membranes were incubated with the antibody di-lution at 37°C for 1 h. Nitrocellulose membranes were thenwashed five times for 5 min with 50 mM Tris-HCl (pH 7.5),150 mm NaCl, and 0.1% NP-40. Following washes the mem-brane was incubated in secondary antibody of Affinity Puri-fied Goat anti-Rabbit IgG Horseradish Peroxidase Conjugate.The membrane was washed twice for 5 min with the previouswash buffer and three times for 5 min with 50 mM Tris-HCl(pH 7.5). Color was developed on each membrane using 12mg 4-chloro- l-naphthol in 4 mL methanol plus 50 mm Tris-HCI (pH 7.5), 150 mM NaCl, and 12 ,uL 30% H202. Mem-branes were shaken in this solution until color developed. Thesolution was poured off and membranes rinsed with water tostop development.

cDNA clones

The rice endosperm ADPglucose pyrophosphorylase cDNAclone (pRE7A) was obtained by screening of a XgtlI cDNAlibrary with antibody to spinach ADPglucose pyrophospho-rylase as described by Andersen et al. (1). This clone, whichwas ligated into the Eco R site of pUC 18, is identical to thedescribed AGP- 1 (1). Maize cDNA clones pES6-66 and pES6-75 were obtained by differential screening of a maize endo-sperm cDNA library with labeled cDNA probes synthesizedfrom mutant versus wild-type endosperm RNA (2). The pES6-66 clone did not hybridize to sh2 RNA while pES6-75 did nothybridize to bt2. Library and probe cDNA were constructedfrom poly(A) RNA isolated from W64A wild type or mutantendosperm 20 d postpollination.

Inserts were released from the cDNA clones by restrictionand subjected to Southern analysis (1 1). Probes were synthe-

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MAIZE ADPglucose PYROPHOSPHORYLASE SUBUNITS: THE BRITTLE-2 GENE

sized from purified inserts of pES6-66 and pES6-75 using therandom primer method (8). Hybridization was performed asdescribed by Maniatis et al. (1 1) except that the prehybridi-zation and hybridization fluid contained IX Denhardt's so-lution, 100 jg/mL calf thymus DNA, 20 Atg/mL poly(A), andneither salmon sperm DNA nor EDTA. Hybridization wasperformed overnight at 55TC.

1 2 3 4 5 67 8

RESULTS AND DISCUSSION

Enzyme Activity in Normal and Mutant EndospermExtracts

As seen in Table I the specific activity observed for ADP-glucose pyrophosphorylase with the bt, and sh, endospermextracts were about 3 and 6%, respectively. These results arein agreement with previous observations by Tsai and Nelson( 17) and Dickinson and Preiss (7).

Immunological Studies

Rabbit antibodies to spinach ADPglucose pyrophosphoryl-ase native, small subunit and large subunit were used tospecifically detect ADPglucose pyrophosphorylase subunits inWestern blots of wild-type, btr and sh, W64a maize endo-sperm as well as spinach leaf proteins (Figs. 1-3). Antispinachleaf holoenzyme antibody detects two bands in wild-typemaize endosperm extracts (Fig. 1, lane 5; see also in Fig. 4A,lane 1). The upper band observed for wild-type enzyme wasnot noted before in Western blots of maize enzyme with anti-spinach leaf ADPglucose pyrophosphorylase (13). The bandpositions correspond to peptide molecular masses of 55 and60 kD. Extracts from bt, endosperm show only the 60 kD(Fig. 1, lane 6) band while extracts from sh, endosperm showonly the 55 kD band with the same antiholoenzyme antibody(Fig. 1, lanes 7 and 8). Spinach leaf purified enzyme showsthe two spinach ADPglucose pyrophosphorylase subunits at51 and 54 kD (12). No bands were observed with pre-immuneserum (Fig. 1, lanes 1-4).

Antispinach leaf 51 kD subunit antibody detects a single55 kD band with Western blots of wild-type and sh, maizeendosperm extracts (Fig. 2, lanes 2, 3, 5, and 6). This antibodydoes not cross-react with bt2 endosperm extract (Fig. 2, lanes4 and 7). A control lane with the spinach enzyme shows the51 kD subunit as well as a small amount of cross-reactivityto the 54 kD upper subunit (Fig. 2, lanes 1 and 8).

Anti-spinach leaf 54 kD subunit antibody detects the larger60 kD band with Western blots of normal and bt2 endospermextracts (Fig. 3, lanes 2, 4, 5, and 7), while the sh2 endosperm

Table I. ADPglucose Pyrophosphorylase Activities in Normal andMutant Maize Endosperm Extracts

The results are the average of at least 3 assays. Standard devia-tions of specific activity were less than ±15%.

Endosperm Activity Specific Activity

units/mL units/mgNormal 2.13 0.16sh2 0.036 0.0094bt2 0.02 0.004

- _..

Figure 1. Western blot analysis of spinach leaf ADPglucose pyro-phosphorylase and maize endosperm extracts. Aliquots of the normalmaize (lanes 1 and 5, 100 mg), of bt2 (lanes 2 and 6, 100 Mg), and ofsh2 (lanes 3, 4, 7, and 8, 100 ,ug) extracts were electrophoresed andreacted with antiserum prepared with native spinach leaf ADPglucosepyrophosphorylase (lanes 5-8) and with preimmune serum (lanes 1-4) as described in "Materials and Methods."

...,...,...

Figure 2. Western blot analysis of spinach leaf ADPglucose pyro-phosphorylase and maize endosperm extracts with affinity purifiedanti-51 kD subunit spinach leaf ADPglucose pyrophosphorylase. Ali-quots of spinach leaf enzyme (lanes 1 and 8, 500 ng), normal maizeendosperm extract (lane 2, 100 Mg; lane 5, 200 ag), sh2 endospermextract (lane 3, 100 Mg; lane 6, 200 Mg), and bt2 endosperm extract(lane 4, 100 Mg; lane 7, 200 Mg) were electrophoresed and reactedwith antiserum as described in "Materials and Methods."

shows no cross-reacting bands with the 60 kD band (Fig. 3,lanes 3 and 6). The control lane with spinach leaf enzymeindicates antibody specificity to the 54 kD subunit (Fig. 3,lanes 1 and 8).

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Plant Physiol. Vol. 92, 1990

:2! 3 4 6 8 +

Figure 3. Western blot analysis of spinach leaf ADPglucose pyro-phosphorylase and maize endosperm extracts with affinity purifiedanti-54 kD subunit spinach leaf ADPglucose pyrophosphorylase. Ali-quots of spinach leaf enzyme (lanes 1 and 8, 500 ng), normal maizeendosperm extract (lane 2, 100 ,g; lane 5, 200 Ag), sh2 endospermextract (lane 3, 100 tg; lane 6, 200 Ag), and bt2 endosperm extract(lane 4, 100 Iug; lane 7, 200 Isg) were electrophoresed and reactedwith antiserum as described in "Materials and Methods."

Figure 4, A, B, and C show that antibodies prepared withnative spinach leaf ADPglucose pyrophosphorylase, and withthe separated spinach leaf ADPglucose pyrophosphorylasesubunits also gave reactions with the highly purified maizeendosperm ADPglucose pyrophosphorylase (13). As shownpreviously (13), the highly purified maize endosperm enzyme

C2-ethyl agarose fraction contained three major peptides of98, 60, and 55 kD molecular mass. As seen in Figure 4A thenative antispinach leaf ADPglucose pyrophosphorylase cross-reacted with the 55 and 60 kD protein bands. The reactionwas greater with the 55 kD subunit. However, antibodyprepared with the 54 kD spinach leaf subunit gave a morepositive reaction with the maize endosperm 60 kD subunitthan with the 55 kD subunit (Fig. 4B) and the anti-51 kDspinach leaf ADPglucose pyrophosphorylase gave a strongreaction with the maize endosperm ADPglucose pyrophos-phorylase lower molecular mass 55 kD subunit (Fig. 4C). Aslight reaction was observed with the 60 kD subunit and thespinach leaf anti-5 1 kD ADPglucose pyrophosphorylase. Noimmunological reactions were observed with the use ofpreim-mune serum.A previous study (13) did not detect an immunoreaction

of the native anti-spinach ADPglucose pyrophosphorylasewith the maize endosperm 60 kD subunit present in the crudeextract. However, further and more careful studies with anti-bodies prepared with the separated subunits and with thenative spinach leaf enzyme clearly indicated a reaction withthe 60 kD subunit of the highly purified maize endospermADPglucose pyrophosphorylase fraction (Figs. 2-4). The an-tibody to the native spinach leafenzyme gives a much strongerreaction with the maize endosperm enzyme 55 kD subunitthan with 60 kD subunit. This may suggest a stronger ho-mology between the lower molecular mass subunits (51 and55) than with the higher molecular mass subunits (54and 60).

DNA:DNA Hybridization

The above immunological data indicate that maize endo-sperm contains an ADPglucose pyrophosphorylase enzymehaving two subunits which are similar but slightly larger thanthose of spinach. The results also indicate that sh2 maizeendosperm lacks a large subunit while bt2 maize endospermlacks a small subunit. The fact that the cDNA clones corre-sponding to mRNA absent in bt2 and sh2 endosperm willhybridize to mRNAs which translate 54 and 60 kD proteins(2) suggests that these mRNAs code for the large and smallADPglucose pyrophosphorylase subunits, respectively. ADNA:DNA hybridization experiment further confirms thissuggestion in the case of the bt, maize endosperm mutation.

Southern hybridization experiments were performed usinglabeled probes synthesized from purified PstI inserts ofcDNAclones, pES6-75 (RNA not present in bty) and pES6-66 (RNAnot present in she). These probes were hybridized understringent conditions against PstI digests of themselves andEcoR 1 digests of ADPglucose pyrophosphorylase cDNAclones from rice endosperms (pRE7A). As shown in FigureSA, the probe corresponding to mRNA absent in Bt, (pES6-75) hybridizes to itself (lane 3) and to the rice endosperminsert (lane 1) but not to the pES6-66 insert (lane 2). In FigureSB, the probe corresponding to mRNA absent in Sh2 (pES6-66) is seen to hybridize to the pUC 18 and pBR 322 plasmids(lanes 1-3) and to itself (lane 2) but not to the insert for therice ADPglucose pyrophosphorylase cDNA clone (lane 1) orto the pES6-75 insert (lane 3). Molecular masses calculated

Figure 4. Western blot analysis of spinach leaf ADPglucose pyro-phosphorylase and maize endosperm enzyme with antiserum pre-pared with native spinach leaf ADPglucose pyrophosphorylase (A),with affinity-purified anti-54 kD subunit spinach leaf ADPglucosepyrophosphorylase (B) and with affinity-purified anti-51 kD subunitspinach leaf ADPglucose pyrophosphorylase (C). A, Lanes 1, maizeextract, 100 Asg; 2, 3, 4, purified maize endosperm ADPglucosepyrophosphorylase, 2, 0.5, and 0.2 Ag, respectively; 5, purified spin-ach leaf ADPglucose pyrophosphorylase, 0.5 lug. B, Lanes 1, 500 ngof purified spinach leaf ADPglucose pyrophosphorylase; 2, 3, 4, 5,0.2, 0.5, 2, and 5 jig of purified maize endosperm ADPglucosepyrophosphorylase, respectively. C, Lanes 1, 2, 3, 4, purified maizeendosperm ADPglucose pyrophosphorylase, 5, 2, 0.5 and 0.2 lug,respectively; 5, 0.5 lg of purified spinach leaf ADPglucose pyrophos-phorylase.

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MAIZE ADPglucose PYROPHOSPHORYLASE SUBUNITS: THE BRITTLE-2 GENE

A B

Kb 23 2 3

6 5-

2 3-

a

4-

O 8 -

O 5 -

a

-RE7A

<-ES6-66

<-ES6 -75

Figure 5. Test of hybridization to rice ADPglucose pyrophosphoryl-ase cDNA (lane 1) with probe corresponding to message absent inmaize bt2 (A) or sh2 (B) mutant. Rice ADPglucose pyrophosphorylase(pRE7A), maize pES6-66 (mRNA absent in sh2), and maize pES6-75(mRNA absent in bt2) cDNA clones (lanes 1-3, respectively) werepredigested with restriction enzymes to excise the inserts (Eco R1or Pst I) electrophoresed and were blotted as described in the text.Replicate filters were hybridized against probe synthesized fromcDNA inserts from pES6-75 (mRNA absent in bt2) in A lanes, pES6-66 (mRNA absent in sh2) in B lanes. Positions of ethidium bromidedetected inserts and molecular mass markers on the agarose gel are

indicated. In the B lanes at greater than 6.5 kb cDNA probe (ES-6-66) hybridizes to both pUC18 (lane 1) and pBR322 (lanes 2 and 3)vectors as well as residual undigested plasmid (lane 2).

using DNA standards corresponded to previously publishedvalues for these plasmids and inserts (data not shown).

These results indicate a clear homology between mRNAabsent in bt2 and rice endosperm ADPglucose pyrophospho-rylase mRNA. The bt2 mutation thus results not only inabsence of the smaller 55 kD subunit of maize endospermADPglucose pyrophosphorylase protein but also in absenceof the corresponding transcript. The immunological evidenceindicates that the sh, mutation results in absence of the larger60 kD subunit. Since we have yet to characterize the actualBt2 gene the data are not sufficient to distinguish between thepossibilities that either Bt2 is a structural gene for the enzymeor regulates transcription of such a gene. The Sh2 gene hasbeen sequenced and appears to be a structural gene (personalcommunication, Dr. C Hannah, University of Florida,Gainesville). Thus, the data are consistent with the postula-tion of Hannah and Nelson (9) that both Sh2 and Bt2may be structural genes for maize endosperm ADPglucosepyrophosphorylase.

LITERATURE CITED

1. Anderson JM, Hnilo J, Larson R, Okita TW, Morell M, PreissJ (1989) The encoded primary sequence of a rice seed ADP-glucose pyrophosphorylase subunit and its homology to thebacterial enzyme. J Biol Chem 264: 12238-12242

2. Barton C, Yang L, Galvin M, Sengupta-Gopalan C, Borelli T(1986) Isolation of the Shrunken-2 and Brittle-2 genes frommaize. In JC Shannon, DP Knievel, CD Boyer, eds, Regulationof Carbon and Nitrogen Reduction and Utilization in Maize.American Society of Plant Physiologists, Rockville, MD, pp363-365

3. Burnette WN (1981) "Western blotting" electrophoretic transferof proteins from sodium dodecyl sulfate-polyacrylamide gelsto unmodified nitrocellulose and radiographic detection withantibody and radioiodinated protein A. Anal Biochem 112:195-203

4. Cameron JW, Teas HJ (1954) Carbohydrate relationships indeveloping and mature endosperms ofbrittle and related maizegenotypes. Am J Bot 41: 50-55

5. Copeland L, Preiss J (1981) Purification of spinach leaf ADP-glucose pyrophosphorylase. Plant Physiol 68: 996-1001

6. Creech RG (1965) Genetic control of carbohydrate synthesis inmaize endosperm. Genetics 52: 1175-1186

7. Dickinson DB, Preiss J (1969) Presence of ADPglucose pyro-phosphorylase in Shrunken-2 and Brittle-2 mutants of maizeendosperm. Plant Physiol 44: 1058-1062

8. Feinberg AP, Vogelstein B (1983) A technique for labeling DNArestriction endonuclease fragment to high specific activity.Anal Biochem 132: 6-13

9. Hannah LC, Nelson OE (1976) Characterization of ADPglucosepyrophosphorylase from Shrunken-2 and Brittle-2 mutants ofMaize. Biochem Genet 14: 547-560

10. Lin T-P, Caspar T, Somerville CR, Preiss J (1988) A starch-deficient mutant ofArabidopsis thaliana with low ADPglucosepyrophosphorylase activity lacks one of two subunits of theenzyme. Plant Physiol 88: 1175-1181

11. Maniatis T. Fritsch EF, Sambrook J (1982) Molecular Cloning:A Laboratory Manual. Cold Spring Harbor Laboratory, ColdSpring Harbor, NY

12. Morell MK, Bloom M, Knowles V, Preiss J (1987) Subunitstructure ofspinach leafADPglucose pyrophosphorylase. PlantPhysiol 85: 182-187

13. Plaxton WC, Preiss J (1987) Purification and properties ofnonproteolytic degraded ADPglucose pyrophosphorylase frommaize endosperm. Plant Physiol 83: 105-112

14. Preiss J (1988) Biosynthesis of starch and its regulation. In JPreiss, ed, The Biochemistry of Plants, Vol 14. Academic Press,New York, pp 181-254

15. Preiss J, Bloom M, Knowles VL, Plaxton WC, Okita TW, LarenR, Harmon AC, Putnum-Evans C (1987) Regulation of starchsynthesis: enzymological and genetic studies. In G Bruening, JHarada, T Kosuge, A Hollaender, eds, Tailoring Genes forCrop Improvement: An Agricultural Perspective. PlenumPress, New York, pp 133-152

16. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH,Provenzani MD, Fujimoto EK, Goeke NM, Olson BJ, KlenkDC (1985) Measurement of protein using bicinchoninic acid.Anal Biochem 150: 76-85

17. Tsai C-Y, Nelson OE (1966) Starch-deficient maize mutantlacking adenosine diphosphate glucose pyrophosphorylase ac-tivity. Science 151: 341-343

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