functions of heteromeric and homomeric isoamylase-type ... · functions of heteromeric and...

Functions of Heteromeric and HomomericIsoamylase-Type Starch-Debranching Enzymes inDeveloping Maize Endosperm1[W][OA]

Akiko Kubo2,3, Christophe Colleoni2,4, Jason R. Dinges5, Qiaohui Lin, Ryan R. Lappe, Joshua G. Rivenbark,Alexander J. Meyer, Steven G. Ball, Martha G. James, Tracie A. Hennen-Bierwagen, and Alan M. Myers*

Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011(A.K., C.C., J.R.D., Q.L., R.R.L., J.G.R., A.J.M., S.G.B., M.G.J., T.A.H.-B., A.M.M.); and Unite de GlycobiologieStructurale et Fonctionnelle, UMR8576 CNRS, Universite des Sciences et Technologies de Lille, Villeneuved’Ascq cedex 59655, France (S.G.B.)

Functions of isoamylase-type starch-debranching enzyme (ISA) proteins and complexes in maize (Zea mays) endosperm werecharacterized. Wild-type endosperm contained three high molecular mass ISA complexes resolved by gel permeationchromatography and native-polyacrylamide gel electrophoresis. Two complexes of approximately 400 kD contained both ISA1and ISA2, and an approximately 300-kD complex contained ISA1 but not ISA2. Novel mutations of sugary1 (su1) and isa2,coding for ISA1 and ISA2, respectively, were used to develop one maize line with ISA1 homomer but lacking heteromeric ISAand a second line with one form of ISA1/ISA2 heteromer but no homomeric enzyme. The mutations were su1-P, which causedan amino acid substitution in ISA1, and isa2-339, which was caused by transposon insertion and conditioned loss of ISA2. Inagreement with the protein compositions, all three ISA complexes were missing in an ISA1-null line, whereas only the twohigher molecular mass forms were absent in the ISA2-null line. Both su1-P and isa2-339 conditioned near-normal starchcharacteristics, in contrast to ISA-null lines, indicating that either homomeric or heteromeric ISA is competent for starchbiosynthesis. The homomer-only line had smaller, more numerous granules. Thus, a function of heteromeric ISA notcompensated for by homomeric enzyme affects granule initiation or growth, which may explain evolutionary selection forISA2. ISA1 was required for the accumulation of ISA2, which is regulated posttranscriptionally. Quantitative polymerase chainreaction showed that the ISA1 transcript level was elevated in tissues where starch is synthesized and low during starchdegradation, whereas ISA2 transcript was relatively abundant during periods of either starch biosynthesis or catabolism.

Starch biosynthesis is a central function in plantmetabolism that is accomplished by a multiplicity ofconserved enzymatic activities. Two known activitiesare starch synthase, which catalyzes the polymeriza-tion of glucosyl units into a(1/4)-linked “linear”chains, and starch-branching enzyme, which catalyzesthe formation of a(1/6) glycoside bond branches that

join linear chains. Acting together, the starch synthasesand starch-branching enzymes assemble the relativelyhighly branched polymer amylopectin, with approxi-mately 5% of the glucosyl residues participating in a(1/6) bonds, and the lightly branched molecule am-ylose. Amylopectin and amylose assemble into semi-crystalline starch granules, which in land plants andgreen algae are located in plastids.

A third activity necessary for normal starch biosyn-thesis is provided by starch-debranching enzyme(DBE), which hydrolyzes a(1/6) linkages. Two DBEclasses have been conserved separately in plants(Beatty et al., 1999). These are referred to here aspullulanase-type DBE (PUL) and isoamylase-typeDBE (ISA), based on similarity to prokaryotic enzymeswith particular substrate specificity. ISA function instarch production is implied from genetic observationsthat mutations typically result in reduced starch con-tent, abnormal amylopectin structure, altered granulemorphology, and accumulation of abnormally highlybranched polysaccharides similar to glycogen. Appre-ciable amounts of phytoglycogen have been detectedonly in plants with compromised ISA function, in-cluding maize (Zea mays), rice (Oryza sativa), andbarley (Hordeum vulgare) endosperm (James et al.,1995; Kubo et al., 1999; Burton et al., 2002), Arabidop-

1 This work was supported by the U.S. Department of Agriculture(grant no. 2002–35318–12646 to M.G.J. and A.M.M.).

2 These authors contributed equally to the article.3 Present address: Biochemical Research Laboratory, Ezaki Glico

Co., Ltd., 4–6–5 Utajima, Nishiyodogawa-ku, Osaka 555–8502, Japan.4 Present address: Unite de Glycobiologie Structurale et Fonc-

tionnelle, UMR8576 CNRS, Universite des Sciences et Technologiesde Lille, Villeneuve d’Ascq cedex 59655, France.

5 Present address: Foley, Lardner, LLP, 150 East Gilman Street,Madison, WI 53701.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Alan M. Myers ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.110.155259

956 Plant Physiology�, July 2010, Vol. 153, pp. 956–969, www.plantphysiol.org � 2010 American Society of Plant Biologists

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sis (Arabidopsis thaliana) leaves (Zeeman et al., 1998),and Chlamydomonas (Mouille et al., 1996). The primaryrole of PUL is in starch mobilization; however, abiosynthetic function also is indicated, because muta-tions affect starch level and structure in plants withcompromised ISA function (Kubo et al., 1999; Dingeset al., 2003; Wattebled et al., 2005, 2008; Streb et al.,2008). The specific biosynthetic function(s) of DBE isnot yet known, although data have been presentedconsistent with (1) editing preamylopectin structure toenable crystallization, (2) elimination of soluble glu-cans that compete with crystalline starch, and/or (3)regulation of granule initiation (Ball et al., 1996; Zeemanet al., 1998; Myers et al., 2000; Burton et al., 2002;Nakamura, 2002; Bustos et al., 2004).Four genes coding for DBE proteins are strictly

conserved in the Chlorophyta lineage, three for ISAproteins and one for a PUL protein (Deschamps et al.,2008). Evolutionary maintenance indicates functionalsignificance; however, specific roles for each DBEprotein are not fully understood. In potato (Solanumtuberosum) tuber, antisense down-regulation of eitherISA1 or ISA2 caused the same abnormal starch phe-notype (Bustos et al., 2004), and these two proteinsexist together in a heteromeric complex (Ishizaki et al.,1983; Hussain et al., 2003). Similarly, in Arabidopsisleaf, elimination of ISA1, ISA2, or both conditioned thesame phenotype of starch deficiency and phytoglyco-gen accumulation, and both proteins were required forthe presence of the same enzymatic activity (Delatteet al., 2005; Wattebled et al., 2005). Biochemical char-acterization of ISA in developing kidney bean (Pha-seolus vulgaris) seeds also identified a heteromericcomplex containing ISA1 and ISA2 (Takashima et al.,2007). Thus, it appears that in tuber, leaf, and cotyle-don, the ISA1 and ISA2 proteins function together in acomplex. ISA2 is likely regulatory, because it containsvariant amino acids at essential catalytic residues ofthe a-amylase superfamily that render it enzymati-cally inactive (Hussain et al., 2003). Such amino acidchanges are present in ISA2 of all species characterizedto date (Deschamps et al., 2008), indicating functionalselection for a noncatalytic protein beginning veryearly in the divergence of the Chlorophyta. Mutationalanalysis of ISA3 function in Arabidopsis indicated aprimary role in starch mobilization but also a partiallyoverlapping function with ISA1/ISA2 in biosynthesis(Streb et al., 2008; Wattebled et al., 2008).Endosperm tissue from rice contains multiple ISA

complexes (Fujita et al., 1999; Utsumi and Nakamura,2006). An ISA1/ISA2 heteromer is present, as in tuberand leaf, and in addition rice endosperm contains ahomomeric complex containing only ISA1. Whetherthe two classes of ISA complex have specific functionsin endosperm, and how these may differ from starchbiosynthesis in leaf or tuber, are not known. In thisstudy, homomeric ISA1 and heteromeric ISA1/ISA2complexes were identified in maize endosperm andfound to be analogous to the rice ISAs, and theirfunctions were investigated using mutations that spe-

cifically prevent the formation of each class of com-plex. The results showed that either heteromeric orhomomeric ISA is sufficient for near-normal starchsynthesis but also that both forms have a function(s)that is not fulfilled by the other.

RESULTS

Complete Maize Gene Set Coding for DBE Proteins

Three genes coding for ISA proteins and one codingfor a PUL protein are evident in the maize genomicdatabases (Yan et al., 2009). ISA1 and Zea mays PUL1(ZPU1) had been previously characterized fromcDNA and genomic sequences (GenBank accessionnos. EU970890, AF030882, and AF080567; James et al.,1995; Beatty et al., 1999). In this study, PCR primersbased on rice ISA gene sequences were used to amplifyfull-length cDNAs coding for ISA2 and ISA3 (GenBankaccession nos. AY172633 and AY172634). Each of thefour proteins is greater than 30% identical only to theother three members of the group among all predictedmaize polypeptides. Thus, ISA1, ISA2, ISA3, andZPU1 constitute the complete set of DBEs present inmaize. The genes that code for these proteins aredenoted sugary1 (su1; James et al., 1995), isa2, isa3, andzpu1 (Dinges et al., 2003), respectively. Summary in-formation about maize DBE genes and proteins ispresented in Supplemental Table S1.

Phylogenetic analysis showed that each maize DBEsequence falls into one of the four conserved familiespreviously identified in green algae and land plantspecies (Hussain et al., 2003; Deschamps et al., 2008;data not shown). Like ISA2 of potato, Arabidopsis, andother species, maize ISA2 exhibits amino acid varia-tion at six of the eight residues that are normallystrictly conserved in the a-amylase superfamily andare deemed critical for activity (Jespersen et al., 1991,1993; Hussain et al., 2003; Supplemental Table S2).

DBE Expression at the Level of mRNA Accumulation

Previous reverse transcription (RT)-PCR results in-dicated that ISA2 and ISA3 transcripts are present inendosperm tissue throughout development (Yan et al.,2009). To provide further insight into the potentialfunctions of each DBE, the relative levels of ISA1, ISA2,ISA3, and ZPU1 mRNA were measured using quan-titative PCR (qPCR). Expression was first measured inwhole kernels harvested from 4 to 40 d after pollina-tion (DAP), which includes the period of maximalstarch deposition (Prioul et al., 1994). The abundanceof all four DBE transcripts was highest at 20 DAP (Fig.1A), thus correlating with maximal starch deposition.ISA1 was present in the highest relative abundance,followed by ISA2 and ZPU1, and then by ISA3 at avery low level. Analyses of separated endosperm andembryo tissue from kernels harvested 20 DAP showedthat the great majority of the transcripts detected in

Roles of Multiple Isoamylase-Type DBEs in Maize Endosperm

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whole kernels are from endosperm (Supplemental Fig.S1A).

Transcripts were quantified in germinating seeds toexamine for potential functions in starch mobilization(Fig. 1B). Dry kernels were incubated at 30�C on moistfilter paper, and qPCR was conducted over 8 d. ISA2was the only mRNA detected in mature kernels (0 d ofgermination). The most abundant transcript through-out germination was ZPU1, which accumulated 4 to 6d post germination to levels 2- to 3-fold higher thanany of the other three mRNAs. ISA2, ISA3, and ZPU1were prevalent during the most active starch degra-dation period, from 4 to 7 d of germination, while ISA1was present only at trace levels.

Adult leaf tissue harvested at midday from field-grown plants was analyzed (Supplemental Fig. S1A) tocompare transient starch metabolism with storage

starch synthesis. All four transcripts were present inleaf, with the distinctions compared with endospermthat (1) ISA2 rather than ISA1 was in highest abun-dance, (2) appreciable amounts of ISA3 accumulated,and (3) ZPU1 was present at a low level. Transcriptlevels also were measured over a 16-h-light/8-h-darkphotoperiod in seedling leaves grown in a controlled-environment chamber (Supplemental Fig. S1B), againwith the result that ISA2was present in excess of ISA1,in this instance throughout the diurnal period.

Mutations Affecting ISA1 or ISA2

Mutations of su1 and isa2 were characterized andbackcrossed into the W64A inbred background. Twopreviously characterized su1- alleles were used: su1-4582 results from a Mutator (Mu) transposon in exon1 and is a null allele that fails to produce any ISA1(James et al., 1995; Rahman et al., 1998); su1-Ref is anucleotide substitution that changes the Trp residue atposition 578 to an Arg (Dinges et al., 2001; Remingtonet al., 2001; GenBank accession no. AAB97167). Twoother alleles were characterized in this study, su1-am(Mangelsdorf, 1947) and su1-P (Garwood and Creech,1972). Neither mutation by itself conditions a notice-able kernel phenotype, yet they failed to fully com-plement su1-Refwith regard to kernel morphology andwater-soluble polysaccharide (WSP) accumulation(Cameron, 1947; Mangelsdorf, 1947; Garwood andCreech, 1972). The su1 genomic locus of su1-am orsu1-P homozygotes was sequenced after PCR amplifi-cation. Identical sequences were observed, with asingle substitution compared with the wild type thatchanges the Arg residue at position 308 to an Ile. Thus,su1-am and su1-P appear to be the same allele, andsubsequent analyses utilized the latter mutation. Theobservation that su1-P by itself supports apparentlynormal kernel development yet fails to fully comple-ment the point mutant su1-Ref is most likely explainedby quaternary structure interactions of the two abnor-mal proteins. This conclusion is supported by theobservation that su1-P is dominant to the null allelesu1-4582 (i.e. su1-P/su1-4582 compound heterozygotesexhibit normal kernel morphology identical to su1-Phomozygotes; data not shown).

Transposon mutagenesis using the Trait Utility Sys-tem for Corn (Bensen et al., 1995) was used to obtain anisa2- null mutation. A large population was screenedby PCR for individuals containing Mu located withinisa2. Positive PCR signals generated using the Mu-endprimer 9242 and the isa2 gene-specific primer Ak2ridentified the allele isa2-339 (Fig. 2A). The sequence ofa junction fragment containing Mu and genomic DNArevealed that the transposon was located betweennucleotides 880 and 881 of the ISA2 coding sequence.During backcrosses into the W64A background, isa2-339 was identified by PCR amplification with primerpair 9242/Ak2r, and wild-type Isa1 was identified byprimer pair Ak1f/Ak2r that flanks the transposoninsertion site (Fig. 2, A and B).

Figure 1. Transcript levels in developing and germinating kernels.qPCR results are shown in arbitrary, relative units and are means6 SE ofthree biological replicates. Tissues are from wild-type inbredW64A. A,Kernel development. mRNA from whole kernels harvested at theindicated DAP was analyzed. B, Seed germination. mRNA from wholekernels was analyzed after the indicated times in germination condi-tions.

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Gene expression from the mutant alleles was ana-lyzed at the level of mRNA accumulation in 20-DAPendosperm (Fig. 2C). ISA1 was undetectable by RT-PCR analysis of a su1-4582 homozygote, in agreementwith previous RNA-blot hybridization data (Jameset al., 1995). Similarly, ISA2 transcript was not detectedin a homozygous isa2-339mutant. The point mutationssu1-Ref and su1-P both produced ISA1 transcriptdetected by RT-PCR with results indistinguishablefrom the wild type.

Accumulation of ISA1 and ISA2 proteins was alsomeasured. Antisera were generated using syntheticpeptides with unique sequences from either ISA1 orISA2 as the antigen. The same peptides were also usedto bind IgG from crude sera and generate affinity-purified fractions designated as aISA1 and aISA2.Escherichia coli strains expressing recombinant ISA1 orISA2 fused to glutathione S-transferase (GST) pro-vided a test to demonstrate that each IgG fraction isisoform specific (Fig. 3A). Soluble endosperm extractswere then probed with aISA1 or aISA2 in immunoblotanalyses (Fig. 3B). ISA1 was not detected in su1-4582mutants, whereas reduced amounts of the proteincompared with the wild type were observed in bothsu1-P and su1-Ref endosperm. Homozygous isa2-339endosperm lacked detectable ISA2. The protein andmRNA expression data together confirmed that su1-4582 and isa2-339 are null alleles and that su1-P andsu1-Ref produce abnormal forms of ISA1 protein.

Effects on ISA2 of mutations that alter ISA1, and visaversa, were analyzed similarly. Absence of ISA2 inisa2-339 mutants had no effect on the accumulation of

Figure 2. su1- and isa2- mutations. A, Mutations in the ISA1 and ISA2mRNA sequences. Solid arrows represent coding regions, with theshaded area indicating the predicted plastid transit peptide and thewhite area indicating the mature protein. Numbers above the solidarrows indicate codon numbers. Dotted lines indicate 5# and 3#untranslated regions. The positions of Mu insertions and missensemutations are indicated. Small arrows show positions and orientationsof PCR primers used for genotype determination and RT-PCR. Thefigure is drawn to scale. B, isa2 genotype determination. Leaf genomicDNA fragments were PCR amplified using the indicated primer pairs.C, mRNA accumulation. cDNA fragments were amplified by RT-PCRfrom endosperm RNA collected 20 DAP. The ISA1 primer pair is Ql1f/Ql2r, and the ISA2 primer pair is Ak1f/Ak2r.

Figure 3. Immunoblot detection of ISA1 and ISA2. A, Recombinantproteins. Total E. coli soluble extracts (20 mg) from cells expressingGST-ISA1 or GST-ISA2 were fractionated by SDS-PAGE in triplicate andeither stained with Coomassie Brilliant Blue or probed with theindicated IgG fraction. B, Protein accumulation in isa1 and isa2mutants. Soluble extracts from 20-DAP endosperm of the indicatedgenotype (40 mg) were analyzed as in A.





ISA1 mRNA or protein (Figs. 2C and 3B). In contrast,loss of ISA1 in su1-4582 endosperm secondarily pre-vented ISA2 protein accumulation (Fig. 3B). This is aposttranscriptional effect, because the ISA2 mRNAlevel was not altered in a su1-4582 line whenmeasuredeither by standard RT-PCR (Fig. 2C) or qPCR (Supple-mental Fig. S1C). The point mutations su1-P and su1-Ref were distinct from the null allele because they didnot affect the ISA2 protein level (Fig. 3B). Homozygoussu1-Ref did condition a change in ISA2, however,because in addition to the normal protein a variantof higher apparentMr was also observed (Fig. 3B). Thiseffect was reproducible in multiple biological repli-cates (data not shown).

Characterization of Maize ISA Complexes

Multiple ISA complexes had been previously ob-served in maize endosperm total extracts; however,these were partly obscured by other starch-modifyingactivities (Dinges et al., 2001, 2003). To improve theresolution, total extracts were first fractionated byanion-exchange chromatography (AEC) on MonoQ

resin as described previously (Colleoni et al., 2003).Proteins in each fraction were separated by native-PAGE and then electroblotted to starch-containinggels. After incubation and staining with I2/KI solution,these zymogram analyses revealed three blue-coloredbands in a specific elution peak, which based on theircolor can be presumed as ISA activities (Fig. 4A). Thebands are referred to as ISA complexes I, II, and III, inorder from highest to lowest mobility in native-PAGE.The same analysis applied to su1-4582 endospermfailed to generate any blue bands, confirming theiridentities as ISAs and indicating that ISA1 is requiredfor all three activities (Fig. 4A; Dinges et al., 2001;Colleoni et al., 2003).

ISA protein compositions were characterized usingisoform-specific IgG fractions. The complexes wereconcentrated from wild-type 20-DAP endosperm byAEC on Q Sepharose. Immunoblots showed that ISA1and ISA2 bound completely to the column in lowNaClconcentration and eluted in the same sharp peak (datanot shown). Those fractions were pooled, concen-trated, and analyzed in zymograms. Duplicate gelsrun simultaneously in the same apparatus were trans-

Figure 4. Identification of ISA activities in 20-DAP endosperm extracts. A, Zymogram activitygels following MonoQ separation. Equal volumesof each fraction were analyzed. B, Correspon-dence of ISA proteins with ISA enzyme activities.Extracts were concentrated by AEC on Q Sephar-ose and separated by native-PAGE. ISA activitieswere visualized in zymograms, and ISA proteinswere identified by immunoblot analysis. Zymo-grams are from a single gel, and immunoblots arefrom a second gel run simultaneously in the sameapparatus. Numbers below each lane indicatemicrograms of protein loaded. C, Independentrepeat analysis of ISA activities in wild-type andisa2-339 endosperm. Details are as in B. D,Correspondence of ISA proteins with ISA enzymeactivities in su1-P endosperm. Details are as in B.

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ferred to nitrocellulose and probed with aISA1 oraISA2 in immunoblots. Three ISA activities were againvisualized in the zymogram, and immunoblot resultsshowed that ISA1 comigrates with all three complexes,whereas ISA2 comigrates with complexes II and III butis not present in form I (Fig. 4, B and D).ISA complexes were further purified by gel perme-

ation chromatography (GPC) of the proteins concen-trated by AEC. ISA1 and ISA2 in the GPC fractionswere detected by SDS-PAGE and native-PAGE fol-lowed by immunoblot. The two proteins eluted indistinct but overlapping GPC peaks at volumes indic-ative of molecular mass between approximately 200and 400 kD, with ISA2 in the larger complex (Fig. 5A).Neither ISA1 nor ISA2 was detected as a monomer.Native-PAGE revealed which ISA complexes werepresent in the two GPC peaks (Fig. 5B). The lowermolecular mass peak (concentrated in fractions C3–C5) contained predominantly a complex that migratedin native-PAGE at the position of ISA complex I andthat contained ISA1 but lacked ISA2. The highermolecular mass GPC peak (concentrated in fractionsC1–C3) contained a complex with both ISA1 and ISA2that migrated at the position of ISA form II. After longexposure, a weak immunoblot signal was also ob-served in the higher molecular mass GPC peak using

aISA1 IgG at the position expected for ISA complex III(data not shown).

Taken together, these data indicate that ISA1 andISA2 sort into multiple high Mr complexes in maizeendosperm. ISA complex I contains only ISA1 and isthe smallest of the complexes. This form is a homo-multimer, at least concerning the DBE protein present.ISA complexes II and III are heteromultimers contain-ing both ISA1 and ISA2 and are larger than form I, asjudged by GPC. These properties match closely withthe ISA complexes previously characterized from riceendosperm (Utsumi and Nakamura, 2006); however,the maize complexes have not yet been purified tohomogeneity, so at this point the presence of proteinsin addition to the ISAs cannot be ruled out.

Effects of ISA1 and ISA2 Mutations on ISA Activities

The composition of the ISA complexes was con-firmed by determining the effects of su1- or isa2- muta-tions that cause known deficiencies in the ISA1 and/orISA2 proteins. Total soluble extracts of 20-DAP endo-sperm from wild-type W64A and congenic lines homo-zygous for su1-4582, su1-P, or isa2-339were fractionatedby AEC. Immunoblot analysis of the fractions showedthat the ISA1 and ISA2 elution profiles were the same insu1-P and the wild type and that the ISA1 elutionprofile was the same in isa2-339 and the wild type, thusconfirming that no complexes were lost in the mutantsas the result of altered AEC behavior (data not shown).The ISA-containing fractions or their equivalents werepooled and concentrated and then analyzed by native-PAGE for DBE activity and the presence of ISA1 andISA2 in each complex. The su1-4582 control lacked anydetectable ISA activities in the zymograms, and immu-noblot signal was not detected for ISA1 or ISA2 at theposition of any of the three ISA complexes (Fig. 4B). Theisa2-339 line exhibited ISA complex I in both the zymo-gram and ISA1 immunoblot (Fig. 4B). ISA complexes IIand III weremissing in the isa2-339mutant, as indicatedby lack of ISA1 or ISA2 immunoblot signal at thosenative gel positions even on long exposure (Fig. 4B) andloss or major reduction of the blue activity bands of ISAcomplexes II and III (Fig. 4, B and C). A slight activity atthe position of ISA complex II was detected, however,in the isa2-339 mutant, despite the absence of ISA2protein (Fig. 4, B and C). These data confirm that ISA1 isa component of all three forms of the enzyme and ISA2is a component of forms II and III but not the form Icomplex.

The su1-P mutant was unique because it lackedhomomeric ISA complex I but contained heteromericenzyme (Fig. 4D). The prominent blue band of ISAcomplex I activity was absent from su1-P endosperm,as was ISA1 immunoblot signal at that position on thenative gel. Heteromeric ISA complex II also was ap-parently missing or defective in su1-P endosperm,again as judged by both zymogram and immunoblotanalysis. ISA complex III was detected in su1-P ex-tracts when gels were loaded with a relatively large

Figure 5. GPC separation of ISA complexes. AEC fractions containingISA1 and ISA2 were concentrated and purified further by GPC. ISA1and ISA2 in the GPC eluate were detected by immunoblot analysis withaISA1 or aISA2. Equal volumes of each fraction were loaded. Theelution positions of standards of known molecular mass are indicated.A, SDS-PAGE. B, Native-PAGE. Lanes probed for ISA1 and ISA2 arefrom the same gel.





amount of protein, and immunoblot signals for bothISA1 and ISA2 comigrated with that activity. In addi-tion, blue color indicating ISA activity was evident as adiffuse signal migrating between the wild-type posi-tions of complexes II and III. Thus, the amino acidsubstitution at ISA1 residue 308 apparently preventsthe assembly of homomeric ISA (complex I) and theheteromeric complex II. The second heteromeric com-plex, form III, does assemble into an active enzyme insu1-P endosperm, and the diffuse region of ISA activ-ity in the zymogram may indicate abnormal assemblystates that occur only in the mutant.

Absence of a functional ISA1 homomeric complex inthe su1-P line suggested that the mutant ISA1 proteinin su1-P endosperm requires ISA2 in order to supportstarch biosynthesis. This was tested by self-pollinatinga su1-P/+, +/isa2-339 heterozygote. Approximatelyone-sixteenth of the kernels on the ears exhibited theshrunken, translucent appearance typical of ISA1-nullplants (24 mutant kernels, 358 normal kernels), andPCR analysis and DNA sequencing confirmed thatthese kernels were homozygous for both su1-P andisa2-339 (Supplemental Fig. S2). These data supportthe conclusion that the abnormal ISA1 produced fromsu1-P functions only as part of a heteromeric complexcontaining ISA2.

Effects of Changes in ISA Complexes onCarbohydrate Content

Carbohydrate content in the endosperm was com-pared between the wild type and lines lacking eitherthe homomeric or heteromeric ISA complexes. Specif-ically, comparisons were made between (1) the wildtype containing all three ISA complexes, (2) isa2-339mutants containing only homomeric ISA complex I,and (3) su1-P mutants that contain heteromeric ISAcomplex III. Endosperm tissue collected 20 DAP wasanalyzed from multiple biological replicates usingplants grown in the 2009 summer nursery (Fig. 6A;Supplemental Table S3). Granular starch content quan-tified on a dry weight basis did not vary significantlybetween wild-type, su1-P, and isa2-339 lines. Thus,either heteromeric or homomeric ISA is sufficient toproduce normal quantities of starch at mid develop-ment. One significant difference was noted betweenthe wild-type and isa2-339 lines, specifically an ap-proximately 10-fold increase in WSP in the mutant(Fig. 6A). Elevated WSP in isa2-339 lines comparedwith the wild type was also observed in two indepen-dent plants grown in the 2008 summer nursery (datanot shown). This effect of the ISA2-null mutation onWSP, however, is far less severe than that resultingfrom the ISA1-null mutation su1-4582. In the latterinstance, in agreement with previous results (Dingeset al., 2001), the starch content was reduced approxi-mately 6-fold compared with the wild type and WSPwas elevated 100-fold (Fig. 6A). Furthermore, theapproximately 10-fold elevation in Suc typical of theISA1-null mutant was not observed in isa2-339 lines.

The only other statistically significant difference com-pared with the wild type was slightly elevated Glccontent in isa2-339 endosperm (Fig. 6A).

WSP that accumulates at mid development in isa2-339 endosperm was analyzed by GPC on SepharoseCL-2B to estimate its molecular mass. Chromatogra-phy standards were amylopectin from wild-typestarch granules, phytoglycogen from su1-4582 endo-sperm, and free Glc. WSP from 20-DAP isa2-339 en-dosperm eluted from the column at a volume thatoverlapped the phytoglycogen peak (Fig. 7). Thus, theWSP accumulating during isa2-339 kernel develop-ment is a polysaccharide rather than a collection ofmaltooligosaccharides, and the size of the polymermatches the smaller range of phytoglycogenmoleculesfrom su1-4582 endosperm.

Carbohydrates were also quantified in endospermtissue from mature, dried kernels (Fig. 6B). At thisstage, the WSP content in isa2-339 was comparable tothe wild type, in contrast to mid development, whenWSP in the mutant was significantly elevated (Fig.6A). This is again distinct from su1-4582, where thehigh WSP content and low abundance of granularstarch persisted through kernel maturity. The simple

Figure 6. Carbohydrate content. Values are means6 SE, except for su1-4582, which are means of two biological replicates. Raw data areshown in Supplemental Table S3. Asterisks indicate significant differ-ences compared with the wild type at confidence levels of P, 0.01. A,Endosperm harvested 20 DAP. TheWSP result for isa2-339 is plotted onboth scales for ease of comparison. B, Endosperm from mature, driedkernels. DW, Dry weight.

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sugar content in mature kernels was comparable in thewild type, isa2-339, and su1-P. Again the ISA1-nullendosperm carbohydrate content was clearly distinctfrom the ISA2-null or su1-P mutants, in the sense thatsimple sugar contents were highly elevated at matu-rity. Finally, there appears to be a slight reduction intotal starch content in mature endosperm in the isa2-339 and su1-P lines, but this effect is far less severethan the starch deficiency in the ISA1 null mutant su1-4582 (Fig. 6B).Summary points from the carbohydrate content data

are as follows. First, the levels of simple sugars andstarch, either at mid development or maturity, are notdrastically altered by loss of ISA2 or the su1-P muta-tion of ISA1. Second, loss of ISA2 conditions an ab-normal accumulation of WSP at mid development thatis not observed at maturity. Third, the phenotypescaused by loss of ISA1 compared with loss of ISA2 areclearly distinct.

Effects of Changes in ISA Complexes on Starch Structure

Starch structure was characterized to determinewhether the ISA complexes provide specific functionsin the determination of amylose-amylopectin ratio,granule morphology, or amylopectin chain length

distribution. To determine amylose-amylopectin ratio,granular starch from mature kernels was dissolved indimethyl sulfoxide (DMSO), precipitated with etha-nol, suspended in 10 mM NaOH, and separated byGPC on Sepharose CL-2B. Quantification of Glc in thefractions revealed the amylose and amylopectin peaks.The amylopectin content forW64A, isa2-339, and su1-Pgranules was 75.1%, 71.4%, and 73.1%, respectively.Thus, either the heteromeric or homomeric ISA com-plex(es) functions to generate the wild-type amylose-amylopectin ratio in mature kernels.

Granule morphology from 20-DAP or mature endo-sperm starch was analyzed by scanning electronmicroscopy. At 20 DAP, the isa2-339 granules werenoticeably smaller than those from the wild type orsu1-P, although their overall spherical morphologywas not altered (Fig. 8). ISA2-null granules appeareddistinct from ISA1-null starch, which displayed dis-tinctive misshapen, flattened granules. Similar effectswere observed at maturity, with isa2-339 granulessmaller than the wild type and distinct in form fromsu1-4582 granules. The su1-P mutant has nearly nor-mal granule morphology at 20 DAP or maturity (Fig.8). These effects were observed in independent repli-cates from different growing seasons (data not shown).

The linear chain length distribution in 20-DAP am-ylopectin was compared between the four genotypes.As a control, su1-4582 starch exhibited the severechange in amylopectin structure observed previously(Dinges et al., 2001), with significant increases in thefrequencies of degree of polymerization (DP) 5 to 12chains compared with the wild type and decreases inDP 16 to 25 chains (Supplemental Fig. S3). Amylopec-tin from su1-P endosperm showed a small but consis-tent change from the wild type, reproducible in fourbiological replicates. In this instance, chains of DP 11 to13 were moderately increased in abundance, such thattheir frequency as a percentage of all chains analyzedwas 0.5% to 0.75% higher than the correspondingwild-type value (Supplemental Fig. S3). These valuesare well above background scatter for biological rep-licates. This is a far smaller effect, however, than thechanges in su1-4582 amylopectin (Supplemental Fig.S3; Dinges et al., 2001). Amylopectin chain lengthfrequency distribution from six biological replicatesof isa2-339 starch varied compared with each other,with differences of up to 0.4% of total for given chainlengths (Supplemental Fig. S4). The same degree ofvariation was observed in comparison between isa2-339 and the wild type (Supplemental Fig. S4). Thus,loss of ISA2 does not condition an obvious change inchain length distribution.

DISCUSSION

Distinct ISA Complexes in Maize Endosperm

This study characterized roles of conserved ISAproteins in maize endosperm starch biosynthesis and

Figure 7. GPC analysis of WSP. The W64A amylopectin standard wasisolated from whole starch granules. WSP from isa2-339 and su1-4582was precipitated from the soluble phase of 20-DAPendosperm extracts.





found differences from previously identified func-tions in dicots. In earlier studies, ISA1 and ISA2 wereseparately eliminated from Arabidopsis leaves orpotato tubers, and the resultant essentially identicalphenotypes indicated that the two proteins worktogether at the same step in amylopectin synthesis,presumably in a heteromeric complex (Bustos et al.,2004; Delatte et al., 2005; Wattebled et al., 2005, 2008).The roles of ISAs appeared to be different in maize andrice endosperm based on two lines of evidence. First,the presence of both ISA1 homomeric and ISA1/ISA2heteromeric complexes in rice suggested that ISA1functions in some instances independent of ISA2.Second, spontaneous su1 alleles have been detectedrepeatedly in maize, owing to a distinctive kernelphenotype, yet mutations affecting ISA2 have not

appeared. Thus, ISA2 most likely is not necessary fora wild-type kernel phenotype, which implies normalor near-normal starch granules. In this study, ISAcomplexes in maize endosperm were characterized,and their functions separated by genetic means, to testdirectly whether ISA1 and ISA2 requirements differbetween specific tissues and/or species.

Independent lines of evidence demonstrated thatISA1 and ISA2 are components of the same enzymecomplexes: (1) both proteins comigrated with specificISA activities in AEC, GPC, and native-PAGE; (2)functional su1 and isa2 were both required for thesame ISA activities; (3) coupling the isa2-339 and su1-Pmutations conditioned a synthetic phenotype indica-tive of the ISA-null condition; and (4) ISA2 failed toaccumulate, owing to a posttranscriptional effect,when ISA1 was completely absent. The evidence isalso clear that some ISA activity in maize endospermdoes not require ISA2. First, in wild-type tissue, ISA2did not comigrate with ISA1 in the most active ISAband (complex I). Second, complex I activity wasunaffected by loss of ISA2. Thus, the composition ofendosperm ISA complexes is similar in maize and rice(Utsumi and Nakamura, 2006). In both species, threeISA complexes were detected in zymograms, withISA1 and ISA2 both in the lower mobility complexes(II and III) and ISA1 exclusively in the highest mobilityform (complex I). The heteromeric maize ISA com-plexes are larger than the homomeric form, as judgedby GPC and as also seen in rice. The explanation fortwo different heteromeric complexes is not known.Possibilities include different subunit compositionsand/or posttranslational modification of one or morecomponents.

Coordinate Regulation of Protein Abundance

A likely explanation for the fact that ISA2 accumu-lation requires ISA1 is that free ISA2 is readily prote-olyzed. This predicts that all ISA2 is present as part ofa complex, and this was shown by the fact that nomonomeric ISA2 was detected in wild-type extracts byGPC and SDS-PAGE. A corollary is that the ISA2accumulating in su1-P or su1-Ref mutants, which pro-duce aberrant ISA1 proteins, should be found in a highMr complex, and this was demonstrated by GPCanalysis (data not shown). In addition to stability,molecular interactions between ISA1 and ISA2 appearto influence posttranslational modification of thelatter. This is suggested by the fact that in su1-Refendosperm, ISA2 is reproducibly detected both at itspredicted size and as a second band with lowermobility in SDS-PAGE. Both ISA2 bands were ob-served in total extracts and also in purified ISAcomplexes from su1-Ref endosperm (T.A. Hennen-Bierwagen, unpublished data). A possible explanationis that the conformation of ISA2 in complex with ISA1affects its posttranslational modification state.

In contrast to the instability of ISA2 in the absence ofISA1, the latter protein accumulated to normal levels

Figure 8. Granule morphology. Starch granules from 20-DAPor matureendosperm of the indicated genotypes were washed in acetone, airdried, and visualized by scanning electron microscopy.

Kubo et al.




in isa2-339mutant endosperm. In other words, ISA2 inmaize endosperm requires ISA1 in order to accumu-late, but ISA1 does not require ISA2. This relationshipis different from that of Arabidopsis leaf, where ISA1was found to be severely reduced in abundance in anAtisa2 mutant (Delatte et al., 2005), even though ISA2transcript was present. A second difference was notedwith regard to transcriptional regulation in potato,where ISA1 mRNA failed to accumulate when ISA2was specifically targeted by antisense transcription,and visa versa (Bustos et al., 2004). Such regulationwas not evident in maize endosperm, because loss ofISA1 had no effect on ISA2 mRNA accumulation, asdemonstrated by qPCR, and standard RT-PCR indi-cated that loss of ISA2 had no effect on the ISA1mRNA level.

Requirements for Homomeric and Heteromeric ISA

The study proceeded to test for effects on starchbiosynthesis resulting from loss of one or the other ofthe ISA complexes. The isa2-339 allele completelyeliminated ISA2, and accordingly, there were noISA1/ISA2 heteromeric complexes in this mutantendosperm. The resultant phenotype was distinctfrom the effects of mutations affecting ISA2 inArabidopsis. Atisa2 mutations caused decreasedleaf starch content to 20% to 30% of the wild type,identical to the effect of an ISA1-null mutation(Delatte et al., 2005; Wattebled et al., 2005). In con-trast, in isa2-339 endosperm, no decrease in starchcontent was observed at mid development and therewas only a minor effect at maturity. A second resultof the Atisa2 mutations was accumulation of phyto-glycogen to about 60% of total leaf carbohydrate,again identical to the ISA1 mutation. In maize en-dosperm, a slight increase in phytoglycogen to about7% of total carbohydrate resulted from the ISA2-nullmutation at mid development, about 10-fold higherthan the wild type. At maturity, however, the WSPcontent in isa2-339 was the same as in the wild type,less than 1% of total carbohydrate. This is in contrastto phytoglycogen accumulation in the maize ISA1-null line at about 60% of total carbohydrate at eithermid development or maturity. Also, the ISA1 andISA2 mutations in Arabidopsis had identical, severeeffects on amylopectin chain length distribution.Loss of ISA1 in maize endosperm had similar effects,but loss of ISA2 did not significantly alter this aspectof amylopectin structure.These observations are summarized as follows. Mu-

tation of either ISA1 or ISA2 in Arabidopsis leafconditions identical, severe effects. Loss of ISA1 inmaize endosperm has effects similar to those seen inthe dicot leaf, but these are not observed in the ISA2-null line. Thus, the ISA1/ISA2 heteromeric complexesII and III are not required in maize endosperm fornormal starch content and structure, and the ISA1homomeric complex is sufficient for most ISA func-tions.

The converse experiment was provided by su1-P,which allows the formation of an ISA1/ISA2 hetero-mer but not the ISA1 homomer. The remaining ISAactivity in su1-P plants was conclusively identified as aheteromeric complex by comigration with both ISA1and ISA2 and the fact that su1-P, isa2-339 doublemutants possess no ISA activity, as judged by kernelphenotype. Detailed characterization of carbohydratesin the su1-P mutant indicated normal metabolism (i.e.no phytoglycogen), and starch content and granulemorphology were both nearly normal. A small changein amylopectin chain length distribution was ob-served; however, this effect was much less severethan the alteration in ISA1-null plants. Thus, just as theheteromeric ISA was not required for most functions,neither was the homomeric ISA. Taken together, theseanalyses revealed that either heteromeric or homo-meric ISA in maize endosperm is able to support near-normal starch biosynthesis.

Two possibilities can be considered to explain whyonly heteromeric ISA is present in the su1-P mutant.One is that the mutation at residue 308 results inaltered protein-protein interactions such that the mu-tant ISA1 has lost a critical contact that allows forma-tion of the homomultimer. Modeling of the ISA1structure by comparison with the known structure ofPseudomonas isoamylase using the Phyre program(Katsuya et al., 1998; Kelley and Sternberg, 2009)showed residue 308 located on the protein surfacewithin an exposed pocket, consistent with the sugges-tion that the su1-Pmutation could affect protein-proteininteractions (Supplemental Fig. S5). According to thishypothesis, interactions of the mutant ISA1 with ISA2do not require Arg-308, allowing formation of one of theheteromeric complexes. The second possibility is thatthe mutant ISA1 in su1-P endosperm is present atabnormally low abundance. If ISA1were to have higheraffinity for ISA2 than for itself, heteromeric ISA wouldbe the only complex to form. The observation that su1-Pisa2-339 double mutants display a kernel phenotypesimilar to that of the ISA1-null mutant suggests that themutant ISA1 protein cannot productively interact withitself even in the absence of ISA2, thus favoring theformer hypothesis.

These conclusions raise the question of why ISA2has been retained in evolution, notably as a nonenzy-matic protein, if ISA1 alone can support amylopectinbiosynthesis. One hypothesis is based on the observa-tion of altered granule morphology in the isa2-339mutant. At mid development, the starch level is nor-mal in the mutant but granules are smaller, indicatingincreased granule number. Analogous effects wereobserved in potato tuber and barley endosperm, andfrom those data ISA activity was proposed to directlyrepress granule initiation (Burton et al., 2002; Bustoset al., 2004). Alternatively, a reduced rate of crystalli-zation of preamylopectin in the absence of ISA mayslow granule growth. Granules would thus be smallerand more precursor glucan would be available toallow the as yet uncharacterized initiation event to





occur more frequently than normal. Here, we suggestthat a function provided specifically by an ISA1/ISA2heteromer affects granule number and size, and thiscannot be fully compensated by the ISA1 homomericenzyme. Determination of granule number in partthrough the activity of the ISA1/ISA2 enzyme mayhave provided a selective advantage during evolutionof the Chlorophyte lineage, thus explaining why ISA2is so highly conserved. Maintaining an appropriatenumber of granules through activity of the hetero-meric complexes may be particularly important inleaves, because starch forms de novo in a regulardiurnal pattern and the deposition period is a singledaylight phase.

The presence of the ISA1 homomer in monocotendosperm may have arisen owing to selection forbulk starch synthesis as granules enlarge. This mayhave occurred during the cultivation of rice and maizeand selection for large, starchy seeds, or potentiallyfrom a selective advantage in monocot seeds in gen-eral. Quantification of maize transcript abundanceshowed the ISA1 level in endosperm to be 2- to3-fold higher than ISA2, whereas in leaf the ratio isreversed such that ISA2 predominates. Assuming thatprotein level is coordinate with transcript abundance,excess ISA1 protein in endosperm could explain thepresence of both heteromeric and homomeric ISA, incontrast to leaf, where all of the available ISA1 maybe committed to the heteromeric form. Accordingto this hypothesis, a change in tissue-specific activityof the su1 promoter could have provided an evolu-tionary advantage to cereals by introducing the homo-meric ISA in endosperm. Another possibility is thatsequence drift in the monocot lineage introducedchanges in ISA1 structure that allowed it to alsofunction as a homomer. For example, variation inISA1 sequence may have resulted in stability of theprotein in the absence of ISA2, in contrast to Arabi-dopsis, where ISA1 is unstable in the absence of ISA2(Delatte et al., 2005).

Transcript Levels

Predictions can be made from the transcript leveldata regarding the roles of ISA1 and ISA2 in starchcatabolism. Unlike developing endosperm, whereISA1 predominates, in germinating seed the ISA1transcript level is low compared with ISA2 and ISA3.ISA1 transcript is also present at comparatively verylow levels in leaves during the dark phase (Supple-mental Fig. S1B). Starch is rapidly degraded both ingerminating seeds and leaves at night, so it appearsthat ISA1 is not involved in catabolism or has a minorrole. ISA2, in contrast, is present in conditions of bothstarch biosynthesis and degradation in endosperm, inthe latter condition expressed at the same level asISA3. The ISA3 protein in Arabidopsis leaves is knownto be required for starch degradation (Wattebled et al.,2005; Delatte et al., 2006). These observations areconsistent with a proposed function of ISA2 in starch

degradation, in addition to its known biosyntheticrole. These suggestions presume that the transcriptlevels are indicative of protein accumulation, whichmay not be the case, so further investigation is neces-sary to test for a catabolic function of ISA2.

Finally, elimination of ISA1/ISA2 heteromeric com-plexes in isa2-339 plants allowed resolution of a minor,previously unidentified form of ISA activity seen inzymograms. The ISA proteins in that complex are notknown at this time. One possibility is that the minorform is a different type of ISA1 homomeric complexvisible only when heteromeric form II is absent andthat the low abundance of this assembly preventeddetection in aISA1 immunoblots. Another possibilityis that the activity could involve ISA3, because ISA3transcript is present in endosperm, even though atmuch lower relative abundance than ISA1 or ISA2.

CONCLUSION

Isoamylase-type DBE in maize endosperm functionsto support starch synthesis either as a heteromericmultisubunit complex containing both ISA1 and thenoncatalytic protein ISA2 or as a homomeric complexcontaining only ISA1. This is in contrast to Arabidop-sis leaf, where ISA2 is required and only a heteromericcomplex can support normal starch production. Rela-tively slight alterations from the wild type suggest thatheteromeric ISA in maize endosperm suppresses gran-ule initiation or is required for normal granule growthand that the ISA1 homomer cannot provide this func-tion. Homomeric ISA has specific functions that de-termine amylopectin structure that are not providedby heteromeric ISA. Tissue-specific changes in relativelevels of ISA1 and ISA2 transcripts, or functionalchanges in the ISA1 protein, could explain how maizeendosperm acquired the homomeric enzyme.

MATERIALS AND METHODS

Plant Materials

Maize (Zea mays) plants were field grown in summer nurseries at Iowa

State University. Developing kernels from self-pollinated ears were collected

at specified DAP, frozen in liquid N2, and stored at280�C. All lines used were

generated by backcrossing at least five times into the W64A inbred genetic

background.

cDNA Cloning

Total RNAwas isolated from W64A maize endosperm harvested 20 DAP

using the Concert Plant RNA Extraction Reagent (Invitrogen 12322-012).

Approximately 1 mg of RNA was treated with DNase (Promega RQ1) in a

volume of 10 mL for 30 min at 37�C, followed by heat inactivation at 65�C for

10 min. Random hexamer-primed cDNA synthesis of the DNase-treated RNA

was performed using the TAQman Reverse Transcription Reagents (Applied

Biosystems N808-0234). RT-PCR was performed using the SuperScript One-

Step system (Invitrogen 10928-034). Primers JD37 (5#-AAAGGACCTTATT-

TYCCNTAYCA-3#) and JD42 (5#-CCACATTCATCTCCCATRTTNAG-3#)amplified the central portion of the ISA2 cDNA. Primers JD21 (5#-CATCAC-

GACCACCTTCTCCATTTG-3#) and JD24 (5#-GAACTTGGCGTTAAYGCNG-

TNGARC-3#) amplified the central portion of the ISA3 cDNA.

Kubo et al.




5# and 3# cDNARACEwas performed using the GeneRacer Kit (Invitrogen

L1502-01). RACE of ISA2 at the 5# end was performed with the oligonucle-

otide JD62 (5#-CATTCTTCAAGGACGCGAGACACCTTA-3#), and 3# RACE

was performed using JD48 (5#-TCAAACCAGGCTAAGACAGATAC-3#). Sim-

ilarly, the 5# end of ISA3 was obtained using primer JD28 (5#-AGCCT-

CATTTGTATGGTTGTAAA-3#). The 3# sequence information for ISA3 was

contained in an EST clone provided by Pioneer Hi-Bred International.

qPCR, RT-PCR, and Genotype Determination by PCR

Real-time qPCR was performed with oligonucleotide primers specific for

each maize DBE cDNA or for the cDNA corresponding to 18S rRNA (Supple-

mental Table S4) using the Applied Biosystems ABI PRISM 5700 sequence

detection system. cDNAs from various tissues used as template were prepared

as described for cDNA cloning. Reactions were in 50 mL including cDNA, 100

nM each oligonucleotide primer, and 13 SYBRGreenPCRMasterMix (Applied

Biosystems 4309155). PCR cycling conditions were 50�C for 2 min, 95�C for 10

min, and 50 cycles of 94�C for 15 s and 60�C for 1 min. Fluorescence data were

collected using the Sequence Detector Software (version 1.3). Amplification

plots of the logarithmic increase in fluorescence (DRn) versus cycle number

were generated by subtracting the baseline fluorescence signals (collected

between cycles six and 15) from the total fluorescence measurements at each

cycle. The threshold cycle (CT) for each sample was calculated by determining

the point at which fluorescence exceeded the threshold limit, corresponding to

the exponential phase of the PCR (DRn = 0.1).

Gene-specific oligonucleotides were designed using Primer Express soft-

ware (Applied Biosystems). Primers were selected in regions of the sequences

that are specific to each isoform. To verify the efficiency of each primer pair, a

standard curve was generated with cDNA template diluted at 2-fold intervals.

Plotting CT versus log[cDNA] verified that the amplification with each primer

pair was linear with respect to the template concentration (data not shown).

For each gene-specific primer pair, the slope of the standard curve equaled

that of the 18S rRNA standard, allowing direct comparisons between the DCT

values for each individual gene (comparative CT method). All DBE gene-

specific primer sets used for analyzing the cDNA samples typically produced

a CT value between 20 and 35 cycles. cDNA samples were diluted 1,000-fold

prior to detection of 18S rRNA sequences, which then typically produced a

CT value between 14 and 20 cycles.

The CT values for three replicate assays of the same cDNA sample,

performed on a single reaction plate, were averaged. Those values obtained

from three biological replicates were then averaged to determine the relative

mRNA levels expressed in arbitrary units as follows: mRNA level = (22DCT)31,000, where DCT = (CT (target) – CT (18S rRNA)). All data are shown as relative

mRNA levels 6 SE.

Standard RT-PCR for detection of transcripts was as follows. Primers used

to detect ISA1 were Ql1f (5#-CACTCCACTCGAACGCACTA-3#) and Ql2r

(5#-CCTGGGTGTTTTGTCTTGCT-3#), and those used for ISA2 were Ak1f

(5#-GGCTGTTCGCAATGTTTGGC-3#) and Ak2r (5#-CAGGATCAAATGC-

TATGGCTTCC-3#). Total RNA was extracted from whole kernels harvested

20 DAP using the RNeasy Plant Mini Kit (Qiagen 74903) and reverse

transcribed using the Invitrogen SuperScript III First-Strand Synthesis System

(Invitrogen 18080-051). Reaction conditions for amplification of ISA1 were as

follows: 25 mL total volume, 5% DMSO, 1.5 mM MgCl2, 0.025 units of GoTaq

DNA polymerase (Promega M8298), 0.2 mM each deoxyribonucleotide tri-

phosphate, 1 mM each primer, 13 GoTaq buffer, and 0.5 mL of cDNA reaction

mix. Thermocycling conditions were 95�C for 2 min; 45 cycles of 95�C for 30 s,

64.5�C for 30 s, and 72�C for 60 s; and 72�C for 10 min. The PCR to detect ISA2

was the same except the reaction contained 4 mM MgCl2, no DMSO, and the

annealing temperature was 57�C.The isa2 genotype was determined by PCR amplification of genomic DNA

from leaf or kernel tissue. Isa2was detected by amplificationwith primersAk1f

and Ak2r, and isa2-339with theMu end primer 9242 and Ak2r. Genomic DNA

was prepared as described previously (Saghai-Maroof et al., 1984; Steiner et al.,

1995). Primers used to amplify the region of the su1 genomic locus contain-

ing su1-P were SU1PF (5#-CCTGCATAAACTCCCTTCAC-3#) and SU1PR

(5#-CCTTAGGTGCAAGCATGTAG-3#). PCR conditions for amplification of

genomic DNAwere the same as those used to generate ISA1 cDNA.

Immunological Methods

Polyclonal antisera were raised in rabbits against synthetic peptides with

sequences unique to either ISA1 or ISA2, following standard procedures

(Harlow and Lane, 1988; Hermanson, 2008). The antigen for ISA1 was peptide

ZmIsa1-a (5#-CDHYVNYFRWDKKEEE-3#). The Cys residue is not part of theISA1 sequence but was added to allow conjugation to the carrier protein

Keyhole limpet hemocyanin. Two peptides were used simultaneously as the

antigen for ISA2, ZmISA2-a (5#-KDRSSRLADNAAGTC-3#) and ZmIsa2-b

(5#-CLHINAELDEKLPDS-3#). In both instances, the Cys residue is not part of

the ISA2 sequence. Rabbits were immunized on day 0 with 200 mg of antigen

in Complete Freund’s adjuvant and boosted with 100 mg in Incomplete

Freund’s adjuvant on days 14, 28, 42, and 56. Sera were collected on days 49

and 63.

IgG fractions were purified from crude sera using the peptides as affinity

reagents. Peptide disulfides were reduced using Immobilized TCEP Disulfide

Reducing Gel (Pierce 77712) and covalently linked to SulfoLink Coupling

Resin (Pierce 20401). For ISA2-reactive IgG purification, a mixture of both

ZmIsa2-a and ZmIsa2-b was used. Crude sera were applied to the column,

and IgG was purified as described previously (Hennen-Bierwagen et al.,

2008). The IgG fractions raised against ISA1 and ISA2 are referred to as aISA1

and aISA2, respectively.

Native-PAGE and SDS-PAGE were performed on Criterion precast 7.5%

polyacrylamide Tris-HCl gels (Bio-Rad 345-0006). Proteins were transferred to

nitrocellulose filters and probed with affinity-purified IgG fractions diluted

1:1,000 as described (Ausubel et al., 1989). Signals were detected by chemi-

luminescence (GE Healthcare RPN2209).

Protein Extraction and Partial Purification

Extraction and purification steps were performed on ice or at 4�C.Endosperm tissue (2–10 g wet weight) was dissected from frozen kernels

and ground in a mortar and pestle under 0.5 to 1.0 mL g21 tissue extraction

buffer (50 mM Tris-acetate, pH 7.5, 20 mM dithiothreitol [DTT], 10 mM EDTA,

and 13 protease inhibitor cocktail; Sigma P-2714). Lysates were centrifuged at

5,000 rpm for 10 min. Supernatants were transferred to thick-wall polypro-

pylene tubes and centrifuged for 30 min at 40,000 rpm. These supernatants

were used as total soluble endosperm extract after determining protein

concentration (Ausubel et al., 1989).

For AEC, a 1-mL HiTrapQ HP column (GE Health Sciences) was equili-

brated in buffer A (50 mM Tris-acetate, pH 7.5, with 10 mM DTT added

immediately prior to use). Total soluble extract was applied to the column in a

5-mL volume of extraction buffer at a flow rate of 1 mL min21. The column

was washed with 10 mL of buffer A, and bound proteins were eluted in a 20-

mL linear gradient of 0 to 1 M NaCl in the same buffer while collecting 1-mL

fractions. Two fractions in the conductivity range of approximately 28 to 38mS

cm21 were pooled and concentrated 5- to 10-fold by centrifugal filtration.

For GPC, a Superdex 200 10/300 column was equilibrated in buffer A

containing 150 mM NaCl. Concentrated AEC fractions were applied in a

volume of 0.5 mL, and the column was eluted in the same buffer at a flow rate

of 0.25 mL min21 while collecting 0.4-mL fractions.

Zymogram Analyses

Native-PAGE gels were run in standard Laemmli gel buffer (25 mM Tris

and 192 mM Gly) adjusted to pH 8.0, with 1 mM DTT added prior to use. Gels

were run with the apparatus on ice at 60 V for approximately 5 h. Proteins on

native gels were electroblotted at room temperature overnight at 10 V to a

7.5% acrylamide gel in 375 mM Tris-HCl, pH 8.8, containing 0.375% potato

starch (Sigma S4251). After transfer, the starch-containing gels were stained

for 5 min in iodine solution (1% KI and 0.1% I2.). Isoamylase-type DBE

activities were identified by light blue color on a purple background. Dupli-

cate gels run in the same apparatus were transferred to nitrocellulose and

analyzed by immunoblot to allow identification of specific proteins comigrat-

ing with each ISA activity.

Expression and Immunoblot Analysis ofRecombinant Proteins

Recombinant GST-ISA2 fusion protein was expressed in Escherichia coli

from plasmid pEXP15-ZmISA2, which was constructed as follows. ISA2

nucleotides 139 to 2,537 were amplified by PCR and cloned into pDONR221

using Gateway technology (Invitrogen). This region contains codons 35

through the native C terminus at codon 799 and the native stop codon. The

first 34 codons, predicted to specify a plastid transit peptide, were omitted.

The 5# PCR primer included six additional codons preceding the ISA2 coding





region that specify a thrombin cleavage site. The resultant Gateway entry

clone was used to transfer the modified ISA2 coding region to expression

plasmid pDEST15, producing pEXP15-ZmISA2. This plasmid contains a

recombinant gene driven by the T7 promoter that codes for a fusion protein

comprising GSTat the N terminus, followed by the thrombin site, followed by

the ISA2 cDNA sequence. The relevant regions of the plasmid were sequenced

to confirm that all recombination events had occurred as expected. Recom-

binant GST-ISA1 was expressed similarly from plasmid pEXP15-ZmISA1. In

this instance, ISA1 nucleotides 235 to 2,457 were included in the expression

plasmid. The recombinant gene contained codons 50 to 789, omitting the first

49 codons predicted to code for a transit peptide.

E coli strain Rosetta DE3-pLysS (Novagen) was used for protein expression.

Cultures of 15 mL of Luria-Bertani medium containing 50 mg L21 carbenicillin

(Sigma), in 50-mL flasks, were inoculated with 0.3 mL of an overnight

preculture. After growth at 37�C for 3.5 h, expression was induced by the

addition of isopropylthio-b-galactoside to 0.1 mM, and the cultures were

grown at 16�C overnight. Cells were collected by centrifugation, suspended in

one-tenth volume of sonication buffer (13 phosphate-buffered saline, pH 7.2,

5 mM DTT, and 0.1% Triton X-100), and lysed by sonication. Lysates were

centrifuged at 39,000g at 4�C for 30 min, and the supernatant was filtered

through a 0.45-mm filter. Protein concentration was determined, and 20 mg of

total soluble lysate was analyzed by SDS-PAGE and immunoblot.

Carbohydrate Characterization

Starch, Glc, Fru, Suc, and WSP content as a function of endosperm dry

weight was determined as described previously (Dinges et al., 2001). Briefly,

endosperm harvested 20 DAP was dissected from five kernels that had been

stored at 280�C and ground together in water in a mortar and pestle. All

manipulations were performed on ice or at 4�C until subsequent boiling steps.

A 1-mL sample of the crude lysate was dried to allow determination of the dry

weight of tissue analyzed. The remainder of the crude lysate was centrifuged

for 10 min at 10,000g to separate soluble and granular glucans. The pellet was

washed once in water, and the supernatant was pooled with that from the

initial centrifugation. The soluble fraction was boiled for 30 min to inactivate

enzymes. A portion of the starch pellet was dissolved by boiling for 30 min in

100% DMSO and then diluted 100-fold in water for analysis. Glc, Suc, Fru, and

glucan polymer in each fraction was quantified as described (Dinges et al.,

2001). Multiple biological replicates were analyzed for each genotype, and

statistical significance was determined by a P value of less than 0.01 in

Student’s t test. Analysis of mature kernel glucans was performed similarly. In

this instance, dried seeds were incubated at 50�C for 24 h in 0.3% sodium

metabisulfite and 1% lactic acid. Endosperm was then removed from two

kernels, and carbohydrates were extracted as described for 20-DAP endo-

sperm.

Size separation of glucans by GPC utilized a Sepharose CL-2B column (1.5

cm diameter 3 75 cm length) run in 10 mM NaOH. Amylose and amylopectin

from granules were solubilized by boiling starch in 100% DMSO, then 5-mg

Glc equivalents were precipitated with 5 volumes of 100% ethanol overnight

at 220�C, suspended in 1 mL of 10 mM NaOH, and applied to the column.

Glucans were eluted at a flow rate of 0.5 mL min21 while collecting 1.3-mL

fractions. Glc equivalents were measured following complete hydrolysis with

amyloglucosidase (Megazyme E-AMGDF) using a colorimetric Glc oxidase/

peroxidase method (Sigma GAGO-20). WSP from isa2-339 or su1-4582 endo-

sperm harvested 20 DAP were analyzed similarly after precipitation from the

soluble phase by the addition of 5 volumes of 100% ethanol.

Linear chain length distributions in whole starch debranched with

commercial isoamylase (Megazyme E-ISAMY) were determined by fluores-

cence-assisted carbohydrate electrophoresis using a Beckman P/ACE

capillary system. Whole starch was solubilized by boiling in 100% DMSO,

then debranched, derivatized, and analyzed as described previously (O’Shea

et al., 1998; Dinges et al., 2003).

Starch granules were prepared for scanning electron microscopy by

washing with 100% acetone and air drying. They were sputter coated with

gold palladium and examined with a JSM-5800LV microscope at 10 kV.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession numbers AY172633 and AY172634.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. DBE transcript levels determined by qPCR.

Supplemental Figure S2. Kernel phenotype of su1-P, isa2-339 double

mutants.

Supplemental Figure S3. Differences in amylopectin chain length

distribution (I).

Supplemental Figure S4. Differences in amylopectin chain length

distribution (II).

Supplemental Figure S5. Predicted structure of ISA1.

Supplemental Table S1. Maize DBE genes and predicted proteins.

Supplemental Table S2. Sequence variation in ISA2.

Supplemental Table S3. Carbohydrate content data.

Supplemental Table S4. Primers for qPCR.

ACKNOWLEDGMENTS

Scanning electron microscopy analysis was performed by the Iowa State

University Microscopy and NanoImaging Facility with the expert assistance

of Randall Den Adel. Fluorescence-assisted carbohydrate electrophoresis

analysis was performed at the Iowa State University Metabolomics Facility.

Received February 22, 2010; accepted May 5, 2010; published May 6, 2010.

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