analysis of the rice adp-glucose transporter (osbt1 ...phology and amylopectin structure. the...

Analysis of the Rice ADP-Glucose Transporter (OsBT1)Indicates the Presence of Regulatory Processes in theAmyloplast Stroma That Control ADP-GlucoseFlux into Starch1[OPEN]

Bilal Cakir, Shota Shiraishi, Aytug Tuncel, Hiroaki Matsusaka, Ryosuke Satoh, Salvinder Singh,Naoko Crofts, Yuko Hosaka, Naoko Fujita, Seon-Kap Hwang, Hikaru Satoh*, and Thomas W. Okita*

Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164 (B.C., A.T., S.-K.H.,T.W.O.); Faculty of Agriculture, Kyushu University, Fukuoka 812–8581, Japan (S.Sh., H.M., R.S., H.S.);Department of Agricultural Biotechnology, Assam Agricultural University, Jorhat–785013, Assam, India (S.Si.);and Department of Biological Production, Akita Prefectural University, Akita City, Akita 010–01195, Japan(N.C., Y.H., N.F.)

ORCID IDs: 0000-0003-4610-0317 (B.C.); 0000-0002-4666-5679 (A.T.); 0000-0003-1439-412X (R.S.); 0000-0002-6792-2232 (N.C.);0000-0002-6792-2232 (N.F.); 0000-0001-9552-5341 (S.-K.H.); 0000-0002-2246-0599 (T.W.O.).

Previous studies showed that efforts to further elevate starch synthesis in rice (Oryza sativa) seeds overproducing ADP-glucose(ADPglc) were prevented by processes downstream of ADPglc synthesis. Here, we identified the major ADPglc transporter bystudying the shrunken3 locus of the EM1093 rice line, which harbors a mutation in the BRITTLE1 (BT1) adenylate transporter(OsBt1) gene. Despite containing elevated ADPglc levels (approximately 10-fold) compared with the wild-type, EM1093 grainsare small and shriveled due to the reduction in the amounts and size of starch granules. Increases in ADPglc levels in EM1093were due to their poor uptake of ADP-[14C]glc by amyloplasts. To assess the potential role of BT1 as a rate-determining step instarch biosynthesis, the maize ZmBt1 gene was overexpressed in the wild-type and the GlgC (CS8) transgenic line expressing abacterial glgC-TM gene. ADPglc transport assays indicated that transgenic lines expressing ZmBT1 alone or combined with GlgCexhibited higher rates of transport (approximately 2-fold), with the GlgC (CS8) and GlgC/ZmBT1 (CS8/AT5) lines showingelevated ADPglc levels in amyloplasts. These increases, however, did not lead to further enhancement in seed weights even whenthese plant lines were grown under elevated CO2. Overall, our results indicate that rice lines with enhanced ADPglc synthesis andimport into amyloplasts reveal additional barriers within the stroma that restrict maximum carbon flow into starch.

Cereal grains contribute a significant portion ofworldwide starch production. Unlike other plant tis-sue, starch biosynthesis in the endosperm storageorgan of cereal grains is unique in its dependence on

two ADP-Glc pyrophosphorylase (AGPase) isoforms(Denyer et al., 1996; Thorbjørnsen et al., 1996; Sikkaet al., 2001), a major cytosolic enzyme and a minorplastidial one, to generate ADP-glucose (ADPglc), thesugar nucleotide utilized by starch synthases in theamyloplast (Cakir et al., 2015). The majority of ADPglcin cereal endosperm is generated in the cytosol fromAGPase (Tuncel and Okita, 2013) as well as by Sucsynthase (Tuncel and Okita, 2013; Bahaji et al., 2014)and subsequently transported into amyloplasts by theBRITTLE-1 (BT1) protein located at the plastid envelope(Cao et al., 1995; Shannon et al., 1998).

The Bt1 gene, first identified in maize (Zea mays;Mangelsdorf, 1926) and isolated by Sullivan et al.(1991), encodes a major amyloplast membrane proteinranging from 39 to 44 kD (Cao et al., 1995). The BT1protein and its homologs belong to the mitochondrialcarrier family (Sullivan et al., 1991; Haferkamp, 2007),which has a diverse range of substrates (Patron et al.,2004; Leroch et al., 2005; Kirchberger et al., 2008). Theassignment of BT1 protein as the ADPglc transporter incereal endosperms was first proposed by Sullivan et al.(1991), and then it was characterized based on the in-creased ADPglc levels and reduced ADPglc import rate

1 This work was supported by grants from the Japan Society forthe Promotion of Science (to H.S.), the Division of Chemical Sciences,Geosciences, and Biosciences, Office of Basic Energy Sciences, U.S.Department of Energy (grant no. DE–FG02–12ER20216 to S.-K.H.and T.W.O.), the U.S. Department of Agriculture National Instituteof Food and Agriculture (project no. WNP00590), and the Agricul-tural Research Center, College of Agricultural, Human, and NaturalResource Sciences, Washington State University.

* Address correspondence to [email protected] and [email protected].

The authors responsible for distribution of materials integral to the find-ings presented in this article in accordance with the policy described in theInstructions for Authors (www.plantphysiol.org) is: Thomas W. Okita([email protected]) and Hikaru Satoh ([email protected]).

B.C., S.Sh., A.T., H.M., R.S. S.Si., N.C., Y.H. and S.-K.H. designedand performed the experiments and analyzed the data; N.F., S.-K.H.,H.S., and T.W.O. supervised the research; B.C., H.S., and T.W.O.wrote the article with critical inputs from the other authors.

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in endosperms of BT1-deficient maize and barley(Hordeum vulgare) mutants (Tobias et al., 1992; Shannonet al., 1996, 1998; Patron et al., 2004). Biochemical trans-port studies of the maize BT1 showed that it importedADPglc by counter exchanging with ADP (Kirchbergeret al., 2007). The wheat (Triticum aestivum) BT1 homologalso transports ADPglc but has similar affinities for ADPand AMP as the counter-exchange substrate (Bowsheret al., 2007).

Evidence from previous studies by our laboratory(Sakulsingharoj et al., 2004; Nagai et al., 2009) sug-gested the potential role of BT1 as well as otherdownstream processes as a rate-limiting step in starchbiosynthesis in the transgenic rice (Oryza sativa) GlgC(CS8) lines overexpressing an up-regulated AGPase(Escherichia coli glgC-TM). In GlgC (CS8) rice lines, grainweights (starch) are elevated up to 15% compared withwild-type plants, indicating that the AGPase-catalyzedreaction is a rate-limiting step in starch biosynthesisunder normal conditions. When transgenic GlgC (CS8)plants were grown under elevated CO2 levels, no fur-ther increases in grain weight were evident comparedwith those grown at ambient CO2. As Suc levels areelevated in leaf blades, leaf sheaths, culms (Rowland-Bamford et al., 1990), and peduncle exudates (Chenet al., 1994) in rice plants grown under elevated CO2,developing GlgC (CS8) grains were unable to convertthe increased levels of sugars into starch. This lack ofincrease indicated that the AGPase-catalyzed reaction(ADPglc synthesis) was no longer rate limiting and thatone or more downstream processes regulated carbonflux from source tissues in developing GlgC (CS8) en-dosperm (Sakulsingharoj et al., 2004). This view is alsosupported by a subsequent metabolite study in whichseveral GlgC (CS8) lines were found to contain up to46% higher ADPglc levels than wild-type plants (Nagaiet al., 2009). As this increase in ADPglc levels wasnearly 3-fold higher than the increase in grain weight,starch biosynthesis is saturated with respect to ADPglclevels and carbon flow into starch is restricted by one ormore downstream steps. Potential events thatmay limitthe utilization of ADPglc in starch in GlgC (CS8) linesare the import of this sugar nucleotide via the BT1transporter into amyloplasts and/or the utilization ofADPglc by starch synthases. Mutant analysis of the twomajor starch synthases indicated no significant impacton grain weight when one of these starch synthases wasnonfunctional, suggesting that this enzyme activity,contributed by multiple enzyme isoforms, is present atexcessive levels (Fujita et al., 2006, 2007). Therefore, wesuspected that BT1 is the likely candidate limiting car-bon flow into starch in GlgC (CS8) endosperms.

Figure 1. A and B, Seed morphology (A) and starch content (B) of thewild-type (WT; cv TC65) and EM1093. C, Scanning electron micro-graphs of isolated starch granules and the surface of wild-type andEM1093 endosperms. D, Effects of the shr3mutation on the chain length

distribution of amylopectin from EM1093 comparedwith thewild-type.The chain length distributions were characterized using 8-amino-1,3,6-pyrenetrisulfonic capillary electrophoresis. The data are repre-sentative of three different experiments Average starch contents werestatistically different based on one-way ANOVA with Tukey’s multiplecomparison test (P , 0.05).

1272 Plant Physiol. Vol. 170, 2016

Cakir et al.

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The aim of this study was to investigate the role ofBT1 in mediating the transport of ADPglc into amylo-plast and to determine whether this transport activity israte limiting in rice endosperm. In order to addressthese questions, we show that BT1 is the major trans-porter of ADPglc by analysis of the EM1093 rice line,which contains a mutation at the shrunken3 (shr3) locusand, specifically, in the OsBt1-1 gene. Second, weassessed the impact of the expression of the maizeZmBt1 gene in wild-type and GlgC (CS8) seeds to de-termine the potential limiting role of BT1 transport ac-tivity on starch biosynthesis. Our results indicate thatBT1 is essential for starch synthesis but is not rate lim-iting and that one or more stroma-localized processeslimit maximum carbon flow into starch.

RESULTS

Genetic and Biochemical Characterization of the shr3Mutant (EM1093)

EM1093 plants (shr3) produce seeds that have ashriveled phenotype due to the severe reduction in starchcontent (Fig. 1A). This phenotype is likely caused by adefect in one of the major enzymes involved in starchbiosynthesis (Kawagoe et al., 2005; Tuncel et al., 2014).Starch levels were significantly decreased around 80% inshr3 seeds compared with the wild-type (Fig. 1B). Themorphology of starch granules and the chain lengthdistribution of amylopectin in wild-type and shr3mutantendosperms were investigated by scanning electron mi-croscopy (SEM) and 8-amino-1,3,6-pyrenetrisulfoniccapillary electrophoresis, respectively. SEM imagingshowed that the starch granules in endosperms of shr3were smaller andmore spherical compared with those ofthe wild-type (Fig. 1C). Moreover, the shr3 mutationstrongly affected the chain length distribution of amylo-pectin, as the proportion of chains with degree of po-lymerization less than 13were elevated, whereas those ofchains with degree of polymerization greater than 14were decreased (Fig. 1D). These results clearly indicatethat mutation in the shr3 locus results in depressingstarch content as well as altering starch granule mor-phology and amylopectin structure.The shriveled grain phenotype of shr3 is similar to

that seen for the mutant lines EM541 (shr1) and EM22(shr2), which also produce shrunken seeds due to thelack of large (L2) and small (S2b) subunits, respec-tively, of the endosperm cytosolic AGPase (Kawagoeet al., 2005; Tuncel et al., 2014). To determine whether

EM1093 contained a mutation in one of the structuralgenes for the major cytoplasmic AGPase, the EM1093line was crossed with EM541 (Tuncel et al., 2014) orEM22 (Kawagoe et al., 2005), marker lines for the shr1and shr2 loci, respectively. F1 seeds derived from boththe EM1093 3 EM541 and EM1093 3 EM22 crosseswere normal in appearance, whereas F2 seeds demon-strated a ratio of wild-type to shrunken phenotype thatfitted well to the expected 9:7 ratio of independent geneinheritance (Table I). This F2 segregation pattern ver-ifies that the shr3 gene locus of EM1093 is indepen-dent of both shr1 and shr2 loci. In addition, EM1093wasalso crossed with the wild-type parental cv Taichung65 (TC65). F1 seeds exhibited a wild-type phenotype,while the segregation mode of normal to shrunkenseeds fitted well to the expected ratio of a single in-heritance, 3:1 (105:31), in the F2 progeny (Table I). Theseresults indicated that the shr3 mutation in EM1093 iscontrolled by a single gene locus and is unrelated to theshr1 and shr2 loci.

To further verify that the shr3 locuswas not anAGPasestructural gene, immunoblot studies were conducted.Total proteins, extracted from developing seeds ofEM1093, were subjected to immunoblot analysis usinganti-S2b (AGPase small subunit) and anti-L2 (AGPaselarge subunit) antibodies. The results demonstrated thatthe EM1093 line contains both S2b and L2 proteins (Fig.2A), suggesting that the shriveled phenotype is likely dueto one of the other major starch biosynthetic enzymes.

In order to identify the proximate chromosome lo-cation of shr3, linkage analysis was performed bycrossing EM1093 (japonica) with the indica rice cvKasalath. Mapping studies showed that the shr3 genecosegregatedwith three markers, RM1347, C10005, andC63223, on chromosome 2 (Fig. 2B; Supplemental TableS1). The F2 population derived from the cross betweenrice cv Kasalath and EM1093 indicated that the shr3mutation cosegregated with the RM1347 marker(Supplemental Table S1). This result clearly demon-strated that the shr3 locus is located on the short arm ofchromosome 2 (Fig. 2B).

Mutation in the Bt1 Gene Is Responsible for shr3

Analysis of the rice genomic map for genes locatednear the RM1347 marker implicated the OsBt1-1 as apotential candidate gene.Amutation in theOsBt1-1 gene,which was initially determined by targeting induced lo-cal lesions in genomes (TILLING; Suzuki et al., 2008),

Table I. Segregation analysis of F2 seeds derived from crosses of EM1093 with EM540 and EM22 and ofEM1093 with wild-type cv TC65 lines

x2 values are based on inheritance ratios of 9:7 for EM crosses and 3:1 for wild-type and EM crosses.

Cross Combination F1 Seeds Segregation in F2 Total x2

Wild-Type shr

EM540 (shr1) 3 EM1093 Normal 199 148 347 0.17EM22 (shr2) 3 EM1093 Normal 168 142 310 0.54Wild-type (cv TC65) 3 EM1093 Normal 105 31 136 0.35

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was verified by DNA sequencing of OsBt1-1 genomicDNA (GenBank accession no. NM_001052765). InEM1093, a G-to-A replacement at position 513 in thefirst exon was detected, resulting in a Trp codon beingconverted to a nonsense codon (Trp-171Stop; Fig. 2C).Such an error generates a truncated polypeptide (170amino acids) thatwould be subject to rapid degradationin the cell (Laughlin et al., 1998) or cause nonsense-mediated mRNA decay (Isshiki et al., 2001). In eitherinstance, loss of intact BT1 polypeptide was expected.

To verify the loss of the BT1 polypeptide, amylo-plast membrane fractions were prepared from devel-oping wild-type and EM1093 seeds, and extractedproteins were resolved by SDS-PAGE. Polyacrylamidegel slices containing polypeptide in the vicinity of 41kD, the size of OsBT1, were treated with trypsin, andeluted peptides were analyzed by high-resolution liquidchromatography-tandemmass spectrometry (LC-MS/MS).This analysis showed the presence of unique peptidesequences spanning 45% coverage of the intact BT1protein in wild-type membranes. By contrast, no BT1peptides were detected from EM1093 (Table II), indicat-ing that amyloplasts of EM1093 lack intact BT1 protein(Table II). Overall, the results from crossing experiments,proteomic analysis, and DNA sequencing verify that themutation at the shr3 locus is due to a disruption in theOsBt1-1 gene and a loss of BT1 protein in EM1093.

Plastids Isolated from shr3 Endosperm Fail to EfficientlyTransport ADPglc

Purified amyloplasts from the wild-type and EM1093were tested for their transport of ADP-[14C]glc. Anapproximate ADPglc import rate was determinedby measuring the uptake of ADP-[14C]glc and in-corporation into starch (Table III; Fig. 3A). Signifi-cantly higher levels of ADP-[14C]Glc (approximately245 nmol g21 fresh weight) were transported and in-corporated into wild-type (cv Kitaake and cv TC65)amyloplasts compared with EM1093 (shr3) endo-sperms (49 nmol g21 fresh weight) in the absence ofany counter-exchange adenylate nucleotides (TableIII; Fig. 3A). ADPglc import rates nearly doubledwhen wild-type amyloplasts were preincubated witheither ADP or AMP, whereas EM1093 amyloplastsfailed to exhibit such an increase in ADPglc import(Table III; Fig. 3A). ATP had no influence on ADPglctransport (Fig. 3A), a result similar to those from anearlier study of the wheat BT1 (Bowsher et al., 2007).Overall, these results showed that the loss of BT1protein in EM1093 results in poor ADPglc import andincorporation into starch by intact amyloplasts and, inturn, lower starch content (Figs. 1 and 3A; Table III).

The shr3 Mutant Accumulates Higher Levels of ADPglc

The loss of ADPglc transport activity into amyloplastwould likely elevate cytoplasmic ADPglc levels due totheir active synthesis by AGPase (Tuncel and Okita,2013) or by Suc synthase (Bahaji et al., 2014). Estimationof ADPglc levels from developing seed extracts showedthat EM1093 contained dramatically increased (ap-proximately 10-fold) ADPglc levels compared with thewild-type (Fig. 3B; Table IV). This result is consistentwith an earlier study in maize (Shannon et al., 1996),where bt1 kernels contained 13-fold more ADPglc thannormal kernels. Additionally, compartmentalization ofADPglc levels demonstrated that wild-type amylo-plasts contained around 52% of the total ADPglc,whereas most of the ADPglc in EM1093 existed in the

Figure 2. Characterization of the shr3 mutant (EM1093). A, Immuno-blot analysis of proteins extracted from developing (14-DAF) seeds ofthe wild-type (cv TC65) and endosperm mutant (EM) lines. EM1093contains both AGPase subunits, as assessed by immunoblotting usingantibodies raised against AGPase L2 and S2b. B, Linkage map of chro-mosome 2. shr3maps near the marker RM1347, whereOsBt1-1 closelyresides. C, Location of the nonsense mutation in the OsBT1-1 gene.A G-to-A mutation at the first exon results in a premature terminationcodon in EM1093. WT, Wild-type.


Cakir et al.



cytosol (Fig. 3B; Table IV). Thus, the loss of BT1 proteinresulted in dramatically decreased ADPglc transportinto amyloplasts and, in turn, higher ADPglc levels incytosol of EM1093 seeds.

Introduction of ZmBt1 into Wild-Type and GlgC (CS8)Transgenic Rice Lines

Results fromprevious studies suggested the possibilitythat the OsBt1 activity limited carbon flux into starch,especially in the starch-overaccumulatingGlgC (CS8) ricelines expressing an up-regulated bacterial AGPase(Sakulsingharoj et al., 2004; Nagai et al., 2009). This po-tential rate-limiting role of the BT1 transporter could betested directly by overexpressing the native BT1 trans-porter in wild-type and GlgC (CS8) transgenic rice lines.To verify the expression of native BT1 at the protein level,an antibody was required. Despite repeated attempts,however, we were unable to generate an antibody to theOsBT1 polypeptide or selected peptide fragments. A BT1antibody was available to the maize ortholog, ZmBT1.Unfortunately, the antibody to the maize transporter didnot cross act with the rice OsBT1 protein. In view of thesecircumstances, the maize ZmBt1 complementary DNA(cDNA) under the control of the Globulin promoter (Wu

et al., 1998)was transformedviaAgrobacterium tumefaciensinto the various rice lines.

The generated transgenic plants were screened for theexpression of both GlgC-TM (bacterial AGPase) andZmBT1 proteins by immunoblot analysis using anti-GlgC and anti-ZmBT1 antibodies. Transgenic plantsexpressing the ZmBt1 cDNA (AT5 lines) alone or coex-pressed with glgC-TM (CS8) during seed developmentwere identified by immunoblotting (Fig. 4). All ZmBt1-expressing transgenic plants showed two polypeptidebands corresponding to ZmBT1 at approximately 41 kD(Shannon et al., 1998), whereas transgenic plants con-taining glgC-TM expressed a single band at 48 kD (Fig. 4).These transgenic lines were further propagated untilhomozygous T3 lines were identified in order to char-acterize the role of BT1 on starch biosynthesis.

Higher ADPglc Uptake Is Readily Detected in AmyloplastsIsolated from ZmBT1 (AT5) Transgenic Plants

Transgenic Lines Harboring ZmBt1 Exhibit Higher ADPglcTransport Capacity

Intact amyloplasts were isolated from developing riceseeds (12–15 d after pollination [DAP]) to investigate the

Table III. Uptake and incorporation of ADP-[14C]glc into starch by purified intact amyloplasts in thepresence or absence of the counter-exchange ADP

Total starch synthase activity was estimated by lysing the amyloplasts and measuring the amount ofincorporation of ADP-[14C]glc into starch. Each value represents the mean 6 SE of three independentexperiments.

Lines Intact Starch Synthase Activity

Buffer +ADP Buffer +ADP

nmol h21 g21 fresh wtWild-type (cv Kitaake) 235 6 33 437 6 45 145 6 13 140 6 11ZmBT1 (AT5-1) 445 6 43 850 6 47 153 6 11 152 6 10ZmBT1 (AT5-19) 450 6 34 860 6 39 150 6 9 154 6 11GlgC (CS8) 265 6 23 440 6 63 125 6 7 120 6 8GlgC/ZmBT1 (CS8/AT5-18) 466 6 41 881 6 70 147 6 12 141 6 16GlgC/ZmBT1 (CS8/AT5-27) 440 6 44 897 6 71 144 6 11 154 6 19GlgC/ZmBT1 (CS8/AT5-37) 454 6 50 985 6 72 149 6 13 139 6 17Wild-type (cv TC65) 245 6 33 480 6 49 125 6 18 135 6 15EM1093 (cv TC65) 49 6 7 51 6 6 43 6 5 41 6 3

Table II. Identification of proteins from the wild-type and EM1093

Seed extracts from the wild-type and EM1093 were resolved by SDS-PAGE and the polyacrylamide gelarea containing 41-kD polypeptides treated with trypsin and resulting peptides analyzed by LC-MS/MS.BT1 peptides were detected only in the wild-type and not in EM1093. Matched peptide values indicate thenumber of unique peptides matched. Values in parentheses signify the percentage of sequence coverageby peptide mass fingerprinting (PMF) using LC-MS/MS. ND, Not detected.

Identifier Protein Name Matched Peptides

Wild-Type EM1093

LOC_Os02g10800 ADPglc transporter (BT1) 7 (45) NDLOC_Os10g26060 Glutelin 2 (16) 3 (21)LOC_Os01g64700 Tubby-like F-box protein3 1 (9) 1 (12)LOC_Os05g33570 Pyruvate, phosphate dikinase1 1 (11) 1 (13)LOC_Os07g46460 Ferredoxin-dependent Glu synthase 1 (10) 1 (11)





role of the ZmBT1 transporter on ADPglc uptake ca-pacity. Amyloplasts isolated from the transgenic linescontaining ZmBt1 (AT5-1, CS8/AT5-18, CS8/AT5-27,and CS8/AT5-37) exhibited approximately 2-fold

higher ADP-[14C]glc transport-incorporation ratesthan the wild-type and GlgC (CS8; Fig. 5; Table III).Irrespective of the rice line, preincubation of the in-tact amyloplasts with ADP stimulated ADP-[14C]glctransport-incorporation rates by nearly 2-fold. By con-trast, ADP-[14C]glc incorporation into starch by rupturedamyloplasts from the various rice lines was considerablylower than the rates measured for intact amyloplasts.Moreover, the rates of ADP-[14C]glc incorporation byruptured amyloplasts were not stimulated by ADP, un-like the rates observed for intact amyloplasts (Table III),and did not vary from one rice line to another. Thus, theobserved increases in ADP-[14C]glc transport and incor-poration into starch by intact amyloplasts from the var-ious ZmBT1-expressing rice lines are due to enhancedtransport into the amyloplast stromal compartment andnot to differences in starch synthase activities. These re-sults are consistent with the view that overexpression ofZmBT1 leads to an increase in ADPglc uptake across theamyloplast membrane.

ADPglc Levels in Transgenic Seeds

The contribution of elevated ADPglc transport ca-pacity to ADPglc levels in transgenic rice seeds wasinvestigated. Transgenic ZmBT1 (AT5) plants express-ing the maize BT1 alone contained ADPglc levels sim-ilar to those observed for the wild-type. Consistent withour earlier results (Nagai et al., 2009), total ADPglclevels in GlgC (CS8) plants were elevated around40% compared with the wild-type (Table IV). ADPglclevels were also increased in the double transgenic lines(GlgC/ZmBT1) to the same extent (approximately 40%)observed in GlgC (CS8) compared with the wild-type(Table IV). Even though ZmBT1 (AT5) lines exhibitedhigher in vitro transport rates of ADPglc (Fig. 5),these transgenic plants failed to accumulate increasedADPglc levels in amyloplasts compared with thewild-type (Table IV). In both rice lines, about 50%of the total ADPglc was evident in isolated amylo-plasts. A much higher proportion (65%–69%) of totalADPglc was associated with the amyloplasts of GlgC(CS8) and GlgC/ZmBT1 (CS8/AT5) endosperms(Table IV).

Figure 3. ADPglc incorporation and levels in wild-type and EM1093 riceseeds.A,Uptakeand incorporationofADPglc into starchofpurifiedamyloplastsfrom thewild-type (WT) and EM1093 in the presence or absence of adenylates.Amyloplasts were preloadedwith buffer (black vertical lines), 20 mM ATP (bluehorizontal lines), 20 mM ADP (red checkered lines), or 20 mM AMP (greencrosswise lines) for 10 min. The reaction containing 100 mL of isolated amy-loplasts was started by adding 4 mM ADP-[14C]glc. Uptake experiments wereperformed at 30˚C for 30min. FW, Fresh weight. B, ADPglc levels in wild-typeandEM1093seedsweredeterminedbya reversedirectionassay.Data representmeans6 SE of three independent experiments.Blackvertical lines represent totalADPglc levels, and blue horizontal lines represent amyloplast ADPglc levels.

Table IV. ADPglc levels in total and amyloplast fractions of wild-type and transgenic lines

Each value represents the mean 6 SE of three independent experiments.

Lines

Total

(nmol/seed) Amyloplast

Percentage

of Amyloplast

nmol seed21

Wild-type (cv Kitaake) 0.87 6 0.07 0.46 6 0.02 52.9ZmBT1 (AT5-1) 0.91 6 0.06 0.45 6 0.04 49.5ZmBT1 (AT5-19) 0.89 6 0.04 0.44 6 0.02 49.5GlgC (CS8-18) 1.17 6 0.05 0.76 6 0.02 65.0GlgC/ZmBT1 (CS8/AT5-18) 1.15 6 0.08 0.80 6 0.02 69.6GlgC/ZmBT1 (CS8/AT5-27) 1.19 6 0.09 0.83 6 0.04 69.7GlgC/ZmBT1 (CS8/AT5-37) 1.16 6 0.01 0.78 6 0.03 67.2Wild-type (cv TC65) 0.92 6 0.09 0.47 6 0.05 51.1EM1093 (cv TC65) 10.3 6 0.19 0.16 6 0.03 1.6


Cakir et al.



Analysis of Amylose and Amylopectin Content fromWild-Type and Transgenic Endosperms

To test whether elevated ADPglc levels lead to amodified starch form, the amylose and amylopectincontents were investigated by gel filtration chroma-tography. GlgC (CS8) and GlgC/ZmBT1 (CS8/AT5)lines contained higher amylose contents (fraction 1)comparedwith wild-type endosperms (Table V). This ismost likely due to elevated ADPglc levels in thesetransgenic lines (Table IV), since granule-bound starchsynthase I (GBSSI), which is responsible for amyloseformation, possesses lower affinity toward ADPglcthan the other starch synthase isozymes (Clarke et al.,1999). By contrast, the amylose content from ZmBT1(AT5) lines was not significantly different from thatseen for wild-type endosperms (Table V). There werealso no significant changes in the fraction III-fraction IIratio between the transgenic lines and the wild-typeindicating that the chain length distribution of amylo-pectin was not affected (Table V).

Comparison of Seed Weight and Seed Morphologybetween Wild-Type and Transgenic Plants

The effect of ZmBt1 introduction on starch biosyn-thesis was examined by measuring the average weight

of mature seeds from transgenic lines grown underambient and elevated CO2 conditions. Under ambientconditions, ZmBT1 (AT5-1 andAT5-19) transgenic linesdid not demonstrate any significant changes in grainweight compared with the wild-type (Table VI). Bycontrast, as shown earlier (Nagai et al., 2009), the GlgC(CS8) transgenic lines exhibited about an 11% increasein grain weight, an increase that was also reflected ingrain size (Table VI). GlgC/ZmBT1 (CS8/AT5) linesproduced heavier seeds but were nearly identical inweight to those from the CS8 line (Table VI).

As sugars provided by photosynthetic source leavesmay be limiting under ambient conditions, transgenicplantswere grownunder elevatedCO2 levels (1000ml L

21)during the reproductive stage to investigate whetherthese plants could utilize the extra Suc and incorporate itinto starch. Similar to the results obtained under ambientCO2 levels, ZmBT1 (AT5) transgenic lines exhibited thesame grainweight as those from thewild-type (Table VI).Although GlgC (CS8) lines produced heavier seeds, theincreases in seed weights were similar to those producedunder ambient CO2 conditions (Table VI). Likewise,GlgC/ZmBT1 (CS8/AT5) lines also showed a similarincrease in grain weight, varying 11% to 13% from thewild-type, but this increase was nearly identical to thoseobtained under ambient conditions. Overall, ZmBT1(AT5) and GlgC/ZmBT1 (CS8/AT5) lines failed to incor-porate the excess assimilate generated by higher photo-synthesis under elevated CO2 into starch compared withthe wild-type and CS8 lines, respectively.

Starch Turnover Does Not Account for the Lack ofIncreased Starch Synthesis When ADPglc Is in Excess

The expression of a plastidial targeted bacterialAGPase gene in Russet Burbank potato (Solanumtuberosum) tubers resulted in a dramatic elevation ofstarch content (Stark et al., 1992). A comparable study(Sweetlove et al., 1996) in the potato cv Desiree, how-ever, failed to observe enhanced starch content. Meta-bolic labeling analysis indicated that the failure of cvDesiree tubers to accumulate more starch was likelydue to increased starch turnover (Sweetlove et al.,1996). To directly assess whether starch turnover is amajor factor limiting carbon flux into starch in rice,

Figure 4. Immunoblot analysis of proteins extracted from developing(14-DAF) seeds of the wild-type (WT) and GlgC (CS8) and ZmBT1 (AT5)transgenic lines. CS8 lines, harboring the E. coli glgC-TM gene and AT5lines, expressing the ZmBt1 gene, were analyzed using antibodiesraised against GlgC-TM and ZmBT1.

Figure 5. Uptake and incorporation of ADP-[14C]glcinto starch by purified amyloplasts from wild-type(WT) and transgenic rice seeds. ADPglc import intoamyloplasts is shown for the wild-type, GlgC (CS8),ZmBT1 (AT5), and GlgC/ZmBT1 (CS8/AT5) lines inthe presence or absence of adenylates. Amyloplastswere preloaded with buffer (black vertical lines),20 mM ATP (blue horizontal lines), 20 mM ADP (redcheckered lines), or 20 mM AMP (green crosswiselines) for 10 min. Uptake experiments were per-formed at 30˚C for 30 min. Data represent means6 SE

of three independent experiments. FW, Fresh weight.





ADP-[14C]glc pulse-chase labeling studies were con-ducted (Fig. 6). Purified amyloplasts were incubatedwith ADP-[14C]glc for 20 min and then chased withexcess nonradioactive ADPglc. No obvious loss of ra-dioactivity from starch was detected in both wild-typeand CS8 amyloplasts up to 60min of incubation. Hence,starch turnover rates are very low and not responsiblefor the inability of CS8 rice lines to increase starchproduction when ADPglc is in excess.

DISCUSSION

In cereals, the majority of the endosperm ADPglc issynthesized in the cytosol. Therefore, cereals are de-pendent on the BT1 transporter to import cytosolicADPglc into amyloplast, which is subsequently incor-porated into starch (Denyer et al., 1996; Thorbjørnsenet al., 1996; Sikka et al., 2001). This view is supported byBT1-deficient mutants of maize kernels (Shannon et al.,1998) and barley seeds (Patron et al., 2004), which haddrastically reduced starch content. Until now, however,a detailed analysis of BT1 function in starch biosyn-thesis in rice endosperms was lacking.

In this study, EM1093 seeds contained dramaticallydecreased starch content due to mutation in the shr3locus. OsBt1-1 was initially identified as a potentialcandidate gene underlying the shr3 locus by TILLING.Subsequent DNA sequencing studies showed the

presence of a nonsense mutation in the OsBt1-1 genefrom EM1093 plants, resulting in a premature stop co-don and, in turn, a loss of protein (Fig. 2C). Proteomicanalysis confirmed the absence of tryptic fragmentscorresponding to intact OsBT1 from amyloplast mem-branes from EM1093 (Table II). Moreover, amyloplastsfrom EM1093 (shr3) exhibited very low rates of ADPglctransport into amyloplast (Fig. 3A; Table III), which isconsistent with the decreased seed weight. The severereduction in grain weight also indicates that the hexosesugar (e.g. Glc-1-P and Glc-6-P) transporters, located inthe plastidial membrane, as well as the amyloplast-localized AGPase are unable to compensate for theloss of a functional BT1 transporter in shr3 endosperm.Although EM1093 plants accumulated dramaticallyincreased ADPglc levels in the endosperm comparedwith wild-type plants (Fig. 3B), their seeds only con-tained around 20% of normal starch content due to theloss of BT1 protein and its transport activity. Overall,BT1 is required for a normal rate of starch synthesisduring rice seed development.

ZmBT1 is targeted to the amyloplast via the presenceof a plastid-targeting leader sequence. Expression ofZmBT1 produced a polypeptide doublet at approxi-mately 41 kD in developing rice seeds as viewed byimmunoblotting (Fig. 4). An identical polypeptidedoublet was also observed in developing maize ker-nels by Shannon et al. (1998), who suggested that the

Table V. Composition of carbohydrate in endosperm fractions of transgenic lines

Three fractions (I, II, and III) were detected by refractive index detectors. Fraction I is enriched withamylose, whereas fractions II and III are enriched with amylopectin chains. Total carbohydrate content is100%. Each value represents the mean 6 SE of three independent experiments.

Lines Fraction I Fraction II Fraction III Fraction III:II Ratio

weight %Wild-type (cv Kitaake) 15.1 6 0.19 22.6 6 0.37 62.3 6 0.42 2.76 6 0.06GlgC (CS8-3) 16.9 6 0.70a 22.0 6 0.90 61.1 6 0.30 2.79 6 0.13ZmBT1 (AT5) 15.7 6 0.33 22.2 6 0.27 62.1 6 0.34 2.79 6 0.04GlgC/ZmBT1(CS8/AT5) 16.1 6 0.16a 21.9 6 0.08 62.0 6 0.15 2.84 6 0.01

aSignificant differences between transgenic lines and the wild-type by Student’s t test at P , 0.05.

Table VI. Average weights of individual seeds from various rice lines

Mean weight values (mg) were measured from five plants per line. Asterisks indicate that average seedweights were statistically different at the 95% confidence level compared with the wild-type. Percentagevalues indicate the percentage of seed weight compared with wild-type cv Kitaake, with the exception ofEM1093, which was compared with wild-type cv TC65. ND, Not determined.

Lines Ambient CO2 High CO2

Seed Weight Percentage Seed Weight Percentage

Wild-type (cv Kitaake) 23.3 6 0.6 100 24.4 6 0.8 100ZmBT1 (AT5-1) 23.5 6 0.7 101 25.3 6 0.9 103ZmBT1 (AT5-19) 23.6 6 0.6 101 24.9 6 0.7 102GlgC (CS8-18) 25.8 6 0.4* 111 27.1 6 0.9* 111GlgC/ZmBT1 (CS8/AT5-18) 26.1 6 0.7* 112 27.4 6 1.0* 112GlgC/ZmBT1 (CS8/AT5-27) 26.3 6 0.5* 113 27.6 6 1.4* 113GlgC/ZmBT1 (CS8/AT5-37) 25.7 6 0.5* 110 27.1 6 0.8* 111Wild-type (cv TC65) 22.9 6 0.7 100 ND NDEM1093 (cv TC65) 5.7 6 0.3 25 ND ND


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ZmBT1 precursor contained two peptide leader pro-cessing sites.In vitro transport and starch incorporation studies

demonstrated that ADPglc transport was stimulated bythe presence of ADP or AMP (Figs. 3A and 5). This re-sult is consistent with an earlier study from wheat(Bowsher et al., 2007), in which ADPglc transportwas enhanced by ADP and AMP as counter-exchangesubstrates. Interestingly, ATP had no impact on theADPglc uptake rate. As shown earlier (Bowsher et al.,2007), ATP is the primary substrate for ADP/ATPcarriers on the plastid membranes but not for BT1.Under physiological conditions, ADPglc is most likelyexchanged with ADP, since this adenylate nucleotide isgenerated by the action of starch synthases during thepolymerization of Glc residues.The transgenic lines, ZmBT1 (AT5) and GlgC/

ZmBT1 (CS8/AT5), had significantly higher ADP-[14C]glc transport and starch incorporation rates by in-tact amyloplasts than those isolated from the wild-typeand GlgC rice lines lacking the maize BT1 transporter(Fig. 5; Table III). Despite the significantly elevated

ADP-[14C]glc transport and starch incorporation ratesseen for amyloplasts from the ZmBT1 (AT5) transgeniclines, these did not translate into higher amyloplastcontent of ADPglc and higher seed weights, whichwere no different from the wild-type values (Table VI).Hence, ADP-[14C]glc transport and starch incorpora-tion rates by purified amyloplasts are poor indicators ofthe potential for seedweight increases. Better indicatorsof increased carbon flux into starch and, in turn, higherseed weights are total ADPglc levels per seed andamounts in amyloplasts. While ADPglc levels in theZmBT1 rice line were no different from the quantitiesmeasured in the wild-type, significantly elevated levelsof total ADPglc and amounts in amyloplasts werereadily evident in GlgC and GlgC/ZmBT1, rice linesthat bore seeds of 11% to 13% higher weight. Overall,these results indicate that OsBT1 is not a limiting factorin controlling carbon flux into starch.

The measured levels of ADPglc in developing riceseeds were 0.87 nmol seed21 (38.8 nmol g21 freshweight) for thewild-type and up to 1.19 nmol seed21 forthe GlgC transgenic lines (Table IV). These estimated

Figure 6. Pulse-chase studies using ADP-[14C]glc toassess starch turnover in intact amyloplast. Wild-type(WT; A) and GlgC (CS8; B) amyloplasts, preloadedwith ADP, were incubated in the presence of 0.1 mM

ADP-[14C]glc. After 20 min of incubation, a 100-foldexcess (10 mM) of unlabeled ADPglc was added to theamyloplast mixture, and the extent of radioactivity instarch was assessed. Data represent means 6 SE ofthree independent experiments. FW, Fresh weight.





amounts of ADPglc in developing rice seed (approxi-mately 40 nmol g21 fresh weight) are about 3- to 4-foldlower than the amounts (146 nmol g21 fresh weight)enzymatically estimated in developing barley grains(Tiessen et al., 2012). Moreover, the majority of ADPglc(approximately 80%) was located in the cytosol ofbarley endosperms, whereas approximately 50% of to-tal ADPglc was found in the nonamyloplast fraction indeveloping rice seeds (Table IV). It should be pointedout that measured ADPglc levels in this study weresimilar to the amounts estimated by LC-MS/MS anal-ysis in an earlier study from this laboratory (Nagaiet al., 2009). For example, the wild-type was estimatedto contain about 0.72 nmol seed21 by LC-MS/MS, anamount similar to the 0.87 nmol seed21 measured in thisstudy. Hence, the large 3- to 4-fold differences in ADPglclevels between developing rice and barley grains likelyrepresent the innate properties of each plant speciesand are not due to differences in the way the metab-olite samples were prepared or measured between thetwo studies.

The lack of further enhancement in seed weights byCS8 or ZmBT1/CS8 transgenic rice lines grown underelevated CO2 conditions compared with those grownunder ambient conditions (Table VI) clearly indicatethat BT1 transport or AGPase activities do not limitcarbon flux into starch. The expression of maize sh2AGPase genes in maize (Hannah et al., 2012), rice(Smidansky et al., 2003), and wheat (Smidansky et al.,2002) increased crop yields by increasing the number ofgrains produced per plant. Analyses of the variousGlgC and/or ZmBT1 transgenic plants cultured ingrowth chambers indicate no significant increases intotal seed number or total number of panicles per plant

(data not shown). We should emphasize, however, thatsuch yield studies require field-grown plants to elimi-nate limitations in photosynthesis and root growthfrom attaining maximum seed yields per plant.

Since the increases in ADPglc amounts in CS8 trans-genic lines (Table IV) did not result in correspondingincreases in grain weight (Table VI), these results pointout that one or more processes located in the stromacontrol starch synthesis when ADPglc is in excess (Fig.7). If ADPglc transport activity and levels are not bar-riers, what is responsible for limiting carbon flow intostarch in the GlgC (CS8) rice lines? One possible factorlimiting the carbon flux into starch could be simulta-neously occurring degradation of starch coupled withsynthesis, as observed inGlgC-overexpressing cvDesireetubers (Sweetlove et al., 1996). Similarly, down-regulationof glucan, water-dikinase, an enzyme that facilitatesstarch degradation, resulted in enhanced wheat grainyields and increases in biomass (Ral et al., 2012). Hence,increases in starch turnover, which may counterbalanceincreases in starch synthesis, may be responsible for theabsence of any further increases in starch synthesis andaccumulation, especially when rice plants are grownunder elevated CO2 conditions. Results from the ADP-[14C]glc pulse-chase study using purified amyloplastsindicate, however, that very little, if any, starch turnoveroccurs in the CS8 line (Fig. 6). Hence, starch turnover isunlikely to be the causal basis for the inability of CS8 linesto increase starch production.

A second process that may limit carbon flow intostarch centers on the starch synthases. We had earlierdiscounted this possibility and focused on the ADPglctransporter, as genetic mutant studies showed that lossof action of the major starch synthase isoforms, SSI and

Figure 7. Summary of ADPglc compartmentalization in the wild-type and transgenic lines. A, ADPglc levels are present at nearlyequal amounts in the cytosol and amyloplasts in wild-type (WT) and ZmBT1 (AT5) lines. B, ADPglc levels inGlgC (CS8) andGlgC/ZmBT1 (CS8/AT5) lines are increased significantly (approximately 80%) in amyloplasts while decreased slightly (approximately9%) in the cytosol compared with the wild-type and ZmBT1 (AT5) lines. This nearly doubling of ADPglc levels, however, onlytranslated into an approximately 11% increase (on average based on three independent transgenic lines) in starch content,indicating that the maximum flux of carbon into starch is tightly regulated. This slowing in net starch synthesis, mediated byelevated ADPglc levels, is probably not caused by starch turnover but more likely by a reduction in one or more starch synthase(SS) isoform enzyme activities.


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SSIII, did not have a significant impact on starch syn-thesis and accumulation. Mutant lines that lack one ofthese major starch synthase isoforms had starch levelssimilar to that seen for the wild-type (Fujita et al., 2006,2007). The loss or reduction in catalytic activity of one ofthese enzymes is apparently compensated by one ormore of the other starch synthase isoforms.Although genetic studies (Fujita et al., 2006, 2007) imply

that themajor starch synthases (SSI andSSIIIa) do not limitstarch synthesis in endosperm under normal conditions,their catalytic activities may be rate determining in thetransgenic riceGlgC (CS8) lines,whereADPglc is in excess(Fig. 7). Indeed, this view is supported by the intracellulardistribution of ADPglc in the GlgC (CS8) transgenic lines(Table IV). Irrespective of the presence or absence ofmaizeBT1, the transgenic GlgC (CS8) lines showed elevatedamounts of ADPglc in the amyloplast compared with thewild-type or ZmBT1 (AT5) lines (Table IV). This resultsupports the view that the catalytic activity of one ormoreof the starch synthase isoforms is insufficient to accom-modate the excess ADPglc present in the amyloplast.A significant reduction in starch synthase activity in

the GlgC (CS8) lines may be the result of the plant’sresponses to excess ADPglc. Earlier metabolite analy-sis showed that CS8 lines not only contain elevatedlevels of ADPglc but also proportional increases inupstreammetabolites, UDPglc, Fru, Glc-6-P, andGlc-1-P,and possibly Glc and Suc (Nagai et al., 2009). Thehexose sugars and Suc are well-documented signalingmolecules that control gene and protein expression(Tognetti et al., 2013; Sheen, 2014), and the elevation ofthese sugars will likely lead to the reprogramming ofgene expression. Indeed, preliminary RNA sequencingstudies of the wild-type and CS8 lines show significantalterations in the steady-state RNA levels for severalstarch biosynthetic enzymes, including significant de-pression of SSIII expression and an increase in invertaseRNA sequences. Moreover, elevated expression of astarch-binding domain protein (SBDP) was observed.This SBDP gene has only been identified by computa-tional analysis of the rice genome; hence, nothing isknown about whether it has a role in starch synthesis.Other studies have demonstrated that nonenzymaticcarbohydrate-binding proteins playmajor roles in starchbiosynthesis. A novel gene, FLOURY ENDOSPERM6(FLO6), encodes for a CBM48-containing protein, whichinteracts with ISOAMYLASE1 and presumably facili-tates its debranching activity in rice (Peng et al., 2014).Transgenic plants lacking the FLO6 protein have lowerstarch content and modified starch granules. Addition-ally, a novel protein containing a CBM48 domain local-izes the GBSS to starch granules (Seung et al., 2015). Theabsence of this starch-targeting protein resulted in amylose-free starch in Arabidopsis (Arabidopsis thaliana) leaves.Therefore, other carbohydrate-bindingmodule-containingproteins may take part in starch biosynthesis, either asaccessory proteins to known starch biosynthetic en-zymes, which normally lack carbohydrate-binding do-mains, or potentially as regulators controlling the flux ofcarbon into starch.

Overall, our results demonstrated that ADPglctransport by BT1 is essential for the normal rate ofstarch synthesis in rice endosperm. On the other hand,enhanced ADPglc import did not further increase thesink strength of developing rice seeds, indicating thatone or more biochemical processes in the amyloplaststroma control carbon flux into starch. Ongoing effortsby this laboratory are directed at identifying theselimiting factors and elucidating their roles in regulatingstarch biosynthesis as a means to increase the yields ofthis cereal grain that feeds a large proportion of theworld’s population.

MATERIALS AND METHODS

Generation and Screening of the Endosperm Mutant

The sh3 mutation in the rice (Oryza sativa) EM1093 line was generated byN-methyl-N-nitrosourea treatment of independent fertilized egg cells of thejaponica cv TC65, as described previously (Satoh andOmura, 1979). Segregationanalysis was performed by crossing shr3 with cv TC65 (wild type), EM541(shr1), and EM22 (shr2). RFLP analysis was performed by crossing the indica cvKasalath with EM1093. The rice plants were grown under natural conditions atthe Kyushu University experimental field plots. Amutation in theOsBt1-1 geneof EM1093 was initially identified by TILLING (Suzuki et al., 2008) and sub-sequently confirmed by DNA sequencing of the OsBt1-1 gene.

Plasmid Construction and Rice Transformation

The plant expression vector (pAT5) was generated by cloning the ZmBt1cDNA under the control of the rice endosperm-specific Globulin promoter(Zheng et al., 1993) into pCAMBIA 1303. pAT5was then transformed into wild-type cv Kitaake and the transgenic CS8 line via Agrobacterium tumefaciensAGL1(Toki, 1997). Identification of the transgenic plants expressing both the glgC-TM(Sakulsingharoj et al., 2004) and ZmBT1 genes was carried out by histochemicalGUS staining. These plants were further screened for the expression of bothGlgC-TM and ZmBT1 proteins by immunoblot analysis using anti-GlgC andanti-ZmBT1 antibodies as described previously (Cao et al., 1995; Sakulsingharojet al., 2004). T3 lines homozygous for both transgenes were used in this study.

Plant Growth under the Elevated CO2 Condition

Wild-type, single transgenic (ZmBT1overexpressing), anddouble transgenic(ZmBT1 and GlgC-TM overexpressing) rice plants were grown in a controlledchamber under a 12-h photoperiod at 26°C during the day and 22°C at night.The relative humidity and photosynthetic flux density was set to 70% and2,000 mmol m22 s21, respectively. The plants were grown at ambient CO2 levels(400 ml L21) until the reproductive stage and then were subjected to elevatedCO2 levels (1000 ml L21) to maturity.

Seed Weight Measurements

At maturity, all seeds from individual plants were harvested, dried, andmeasured for total yield and average grain weight as described previously(Nagai et al., 2009). Briefly, panicles from transgenic plants were grown in agrowth chamber for two consecutive periods and then harvested. The averagegrain weight of each transgenic plant was measured by weighing 100 seedsfrom five panicles.

Amyloplast Isolation from Developing Rice Endosperm

Amyloplasts were isolated from developing rice seeds (12–15 DAP) as de-scribed previously (Sikka et al., 2001). After removal of the seed hull, the ends ofdeveloping seeds (approximately 2 g) were carefully removedwith forceps andtransferred into 5 mL of extraction buffer (0.8 M sorbitol, 50 mM HEPES-KOH,pH 7.5, 1 mM EDTA, 2 mM MgCl2, 10 mM KCl, 5 mM dithiothreitol, and0.1% (w/v) bovine serum albumin) for plasmolysis. After incubation in theextraction buffer on ice for 30 min, the seeds were gently chopped with a razor





blade. The homogenatewasfiltered through four layers ofMiracloth (Calbiochem),and the filtrate was kept on ice for 3 h to sediment amyloplasts via gravity. Thepellet, which consists of enriched amyloplasts, was resuspended in extractionbuffer. To check the intactness of the plastids, one-half of the isolated amyloplastswere disrupted by the addition of 0.5% Triton X-100 (v/v), agitated for 1 min, andcentrifuged at 20,000g for 10 min. The yield of intact amyloplasts was estimated atabout 25%, based on the relative amounts of the plastid enzymemarker, inorganicpyrophosphatase, recovered in the amyloplast fraction, as described previously(Sikka et al., 2001).

Analysis of Starch Granules of cv TC65 andEM1093 Endosperms

Starch granule morphology was investigated by SEM as described previ-ously (Wong et al., 2003). The chain length distribution of amylopectin isolatedfromwild-type and EM1093 endosperms was performed by amodifiedmethod(O’Shea et al., 1998) as described previously (Nakamura et al., 2002).

Size Fractionation of Debranched Starch and AmyloseContent Analysis from Wild-Type and Transgenic Lines

Powderedmature rice endosperm sampleswere gelatinized anddebranchedby Pseudomonas spp. isoamylase (Hayashibar) as described previously (Croftset al., 2012). The samples were then analyzed using a Toyopearl HW55S gelfiltration column (3003 20 mm) connected to three Toyopearl HW50S columns(300 3 20 mm; Fujita et al., 2007). The eluted glucans were detected with arefractive index detector (Tosoh RI-8020).

Synthesis of ADP-[14C]glc

ADP-[14C]glc was prepared from [14C]Glc-1-P as described previously(Preiss and Greenberg, 1972), except that the product was separated by thin-layer chromatography (TLC) instead of paper chromatography. Briefly, 25 mCiof [14C]Glc-1-P was converted to ADP-[14C]glc using purified potato (Solanumtuberosum) AGPase as described (Hwang et al., 2004). The final reaction mixturewas resolved by TLC for 7 h in solvent containing 95% ethanol:1 M ammoniumacetate, pH 7.5 (5:2). Hexose sugars were detected by spraying the TLC platewith 10% (v/v)H2SO4 inmethanol and thendrying at 120°C for 10min to visualizethe sugars. The top of the chromatogram containing ADPglc was collected andthen washed with absolute ethanol for 3 h to remove residual ammonium acetate.After drying, ADP-[14C]glc was eluted with water. Based on the initial startingamount of [14C]Glc-1-P, the yield ofADP-[14C]glcwas about 70%, as determined byabsorption at 260 nm and liquid scintillation spectrometry.

ADP-[14C]glc Transport Assay

The transport of ADPglc by intact amyloplasts was performed as describedpreviously (Shannon et al., 1998). Isolated amyloplasts (100 mL) were trans-ferred into a reaction mixture containing 100 mM Bicine, 0.5 M sorbitol, 12.5 mM

EDTA, 50 mM potassium acetate, 10 mM glutathione, and 4 mM ADP-[14C]glc(specific activity of 150 cpm nmol21). When evaluating the counter-exchangesubstrates, amyloplasts were preloaded with 20 mM AMP, ADP, or ATP beforecarrying out uptake-starch incorporation assays. The reactionwas carried out at30°C for 30 min and terminated by the addition of 2 mL of 75% methanolcontaining 1% (w/v) KCl. The insoluble starch was collected by centrifugationat 2,000g for 10 min and washed twice with methanol/KCl to remove excesssubstrate. The pellet was then resuspended in water, and the amount of ADP-[14C]glc converted into starch was quantified by liquid scintillation spectrometry.

Starch turnover was investigated by performing pulse-chase studies. Briefly,amyloplasts (100 mL), preincubated with 20 mM ADP, were used for the in-corporation assay in the presence of 0.1 mM ADP-[14C]glc (specific activity of900 cpm nmol21). After 20 min of uptake and incorporation, a 100-fold excess ofunlabeled ADPglc was added, and the reaction was incubated for up to anadditional 60 min.

Measurement of ADPglc Levels

Purified amyloplasts were isolated from 50 developing rice seeds (10–13DAP), lysed by the addition of 0.5% [v/v] Triton X-100, and then boiled for2 min to inactivate native AGPase activity. ADPglc content was then estimatedfor the purified amyloplast fraction and total seed extract by performing the

reverse AGPase reaction coupled with phosphoglucomutase and Glc-6-P de-hydrogenase reactions as described previously (Seferoglu et al., 2014). The re-action was initiated by the addition of excess amounts of near homogenouslypure potato AGPase and performed at 37°C for 20 min in reaction buffer con-taining 100mMHEPES, pH 7, 5mM dithiothreitol, 5 mM 3-phosphoglyceric acid,10 mM MgCl2, 0.6 mM NAD+, 0.4 mg mL21 bovine serum albumin, 0.5 units ofGlc-6-P dehydrogenase, and 0.5 units of phosphoglucomutase. The reactionwas terminated at 100°C and clarified by centrifugation, and the amount ofNADH was determined by measuring the A340. A standard curve for deter-mining the amounts of ADPglcwas generated by incubating known amounts ofADPglc in the coupled enzyme reaction mixtures. The total amount of ADPglcin amyloplasts was estimated by factoring in the percentage recovery of intactamyloplasts using inorganic pyrophosphatase as a plastid marker enzyme asdescribed earlier.

Supplemental Data

The following supplemental materials are available.

Supplemental Table S1. Linkage analysis of shr3 with three marker geneson chromosome 2.

ACKNOWLEDGMENTS

We thank Thomas D. Sullivan (University of Wisconsin School of Medicine)for providing the maize ZmBt1 cDNA and BT1 antibody.

Received December 17, 2015; accepted January 7, 2016; published January 11,2016.

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analysis of the rice adp-glucose transporter (osbt1 ...phology and amylopectin structure. the...

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