Distribution of Branches in Whole Starches from Maize Mutants

Article pubs.acs.org/JAFC

Distribution of Branches in Whole Starches from Maize Mutants Deficient in Starch Synthase III Fan Zhu,† Eric Bertoft,‡ and Koushik Seetharaman*,‡ †

School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Avenue, St. Paul, Minnesota 55108, United States

‡

ABSTRACT: An earlier study explored the possibility of analyzing the distribution of branches directly in native, whole starch without isolating the amylopectin component. The aim of this study was to explore if this approach can be extended to include starch mutants. Whole starches from du1 maize mutants deficient in starch synthase III (SSIII) with amylose content of ∼30− 40% were characterized and compared with the wild type of the common genetic background W64A. Clusters were produced from whole starch by hydrolysis with α-amylase of Bacillus amyloliquefaciens. Their compositions of building blocks and chains were analyzed further by complete α-amylolysis and by debranching, respectively, whereafter the products were subjected to gel permeation and anion exchange chromatography. The size and structure of the clusters were compared with those of their isolated amylopectin component. Whereas the whole starch of the wild type sample had a branched structure similar to that of its amylopectin component, the results showed that the du1 mutation resulted in more singly branched building blocks in the whole starch compared to the isolated amylopectin. This suggested that amylose and/or intermediate materials in whole du1 starches likely contributed to the composition of branches. This study explored an alternative procedure to characterize the composition of branches in the whole starch without fractionating the components. KEYWORDS: whole starch, starch synthase III, structure, cluster, building block

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isolated from flour as opposed to the fractionated starch components) without separating the amylose and amylopectin. In a previous study with normal and waxy maize,7 we explored the opportunity to understand starch structure without separating the amylose and amylopectin fractions from whole starch. We showed that the branching pattern in whole normal maize starch was comparable to that of waxy maize starch. One of the critiques of that research, however, was that the comparison of the whole normal starch was not with the amylopectin component from the same normal starch. In this study we therefore isolated the amylopectin component and compared it with the same whole maize starch of the genetic background W64A. We further used du1 mutants of maize with the same genetic background that are deficient in starch synthase III (SSIII)8,9 as a test material to study if analyses of the branching pattern in mutant whole starches are useful for the understanding of the effect of the mutation. The mutations were detailed by Lin et al.,10 and the molecular structure of the starches was reported previously by Zhu et al.11 Further studies on the cluster and building block structure of amylopectin from the same samples were also presented.12 It was shown that the du1 mutation altered the structure of starch and its amylopectin component and that it also increased the apparent amylose content. This study compares the cluster and building block structure of whole du1 maize starches with the data for clusters and building blocks in amylopectin presented previously.12

INTRODUCTION Starch is a complex mixture of diverse biopolymers of glucose ranging from linear to branched molecules.1 However, separation of these components in mutant starches can be a challenge. One of the most commonly employed methods for normal starches is based on butanol precipitation as originally developed by Schoch.2 By this method the starch components are fractionated into a butanol-precipitated fraction, referred to as amylose, and a butanol-non-precipitated fraction, commonly known as amylopectin. Variations in the butanol precipitation based methods have been developed over the years. For example, Klucinec and Thompson used a mixture of isoamyl alcohol and butanol to obtain a precipitate consisting of amylose and intermediate material, whereas the supernatant consisted of amylopectin.3 The amylose was then separated from the intermediate material by precipitation in butanol. When research is conducted on different mutant samples, however, the definitions and the isolation of the different starch components become more confounding. Analysis of starch samples from mutant plants with detailed genetics presents an opportunity to understand biosynthesis− structure relationships. However, the analysis of the branched structure of the starch components down to the cluster and building block levels4 requires a relatively large amount of sample. Whereas starches from plants most often are available in comparatively large amounts, starches from organisms such as algae or leaves5,6 are available only in small quantities and do not in practice permit a separation into amylopectin and other components. This seriously hinders further exploration of the structure of starch from interesting mutants or pursuing other research questions. A possible alternative could be to analyze whole starch samples (“whole” denotes the native starch © 2014 American Chemical Society

Received: Revised: Accepted: Published: 4577

February 10, 2014 March 31, 2014 March 31, 2014 March 31, 2014 dx.doi.org/10.1021/jf500697g | J. Agric. Food Chem. 2014, 62, 4577−4583

Journal of Agricultural and Food Chemistry

Article

Figure 1. Time course of α-amylolysis of whole starch from W64A by GPC on Sepharose CL 6B. wt %, weight-based percentage.

Figure 2. Time course of α-amylolysis of whole starch from du1-M3 by GPC on Sepharose CL 6B. wt %, weight-based percentage. Dashed arrow indicates the boundary region between linear and branched fractions.

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determined on the basis of a previous description.13 The given activities of other enzymes were according to the supplier. Time Course Analysis of α-Amylolysis for Cluster Production from Whole Starch. Whole starch (20 mg) was dissolved in 400 μL of 90% dimethyl sulfoxide (DMSO) by gentle heating and then constant stirring for 3 days at room temperature (∼22 °C) to ensure the complete dissolution of starch before hot double-distilled water (1.4 mL) was added. After cooling, α-amylase (200 μL, 0.9 U/ mL) in NaOAc buffer (0.01 M, pH 6.5) was added to start the reaction in a water bath (25 °C) with magnetic stirring. The concentrations for substrate and α-amylase in the reaction system were 10 mg/mL and 0.09 U/mL. Samples (200 μL) were taken from 20 to 150 min. If not analyzed immediately, NaOH solution (4 μL, 5.0 M) was added and mixed to destroy the α-amylase, and then the sample was stored at −18 °C. Before analysis, the sample was diluted in water (200 μL), conditioned by NaOH solution (40 μL, 5.0 M), and analyzed by GPC

MATERIALS AND METHODS

Starches and Enzymes. The maize samples including one wild type (W64A) and two single dull1 mutants (du1-Ref and du1-M3) were the same specimens used in a previous study.10 They were of the W64A inbred genetic background. Both dull1 mutations caused complete loss of SSIII enzyme activity, but du1-Ref expressed the protein at low level, whereas du1-M3 produced no protein.10 The molecular structure of the starches and the cluster and building block structure of amylopectins were reported previously.11,12 α-Amylase of Bacillus amyloliquefaciens (EC 3.2.1.1), β-amylase of barley (EC 3.2.1.2, specific activity = 705 U/mg), isoamylase of Pseudomonas amyloderamosa (EC 3.2.1.68, specific activity = 210 U/ mg), and pullulanase of Klebsiella pneumoniae (EC 3.2.1.41, specific activity = 699 U/mg) were from Megazyme (Wicklow, Ireland). The activity of the α-amylase (413 U/mL at pH 6.5, 25 °C) was 4578

dx.doi.org/10.1021/jf500697g | J. Agric. Food Chem. 2014, 62, 4577−4583


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on Sepharose CL 6B as described below under Chromatographic Techniques. Production and Characterization of α-Dextrins and β-Limit Dextrins of Clusters from Whole Starch. Whole starch (160 mg) was dissolved in 3.2 mL of 90% DMSO by gentle heating and then constant stirring for 3 days at room temperature (∼22 °C) to ensure the complete dissolution of starch. Hot double-distilled water (11.2 mL) was added and, after cooling, α-amylase (1.6 mL, 0.9 U/mL) in NaOAc buffer (0.01 M, pH 6.5) was added to start the reaction in a water bath (25 °C) with magnetic stirring. The reaction was continued for 90 min before being stopped by the addition of NaOH (320 μL, 5.0 M) and standing at room temperature for 2 h to destroy the αamylase. Pure methanol (80 mL) was added to precipitate the αdextrins of clusters. The sample was left at 4 °C for 3 h before the precipitate was recovered by centrifugation (4000g, 20 min) at room temperature. The precipitate was washed twice with pure methanol (10 mL) and allowed to stand in a fume hood to remove the methanol through evaporation. The external chain segments of α-dextrins of clusters were removed by β-amylase to obtain the β-limit dextrins of clusters as previously described.7 Briefly, α-dextrins were dissolved in hot water to a carbohydrate concentration of 10 mg/mL and mixed with NaOAc buffer (0.01 M, pH 6.0) and β-amylase (1 μL/10 mg of dextrins). The β-amylolysis reaction was conducted at room temperature overnight, then boiled for 5 min, condensed by rotary evaporation at 50 °C, and purified through two coupled PD-10 columns (GE Healthcare Life Sciences, Piscataway, NJ, USA) to remove maltose and buffer salt. The β-amylolysis was repeated, and the resulting hydrolysate solution was run twice through the PD-10 columns to completely remove maltose and buffer salt from the β-LDs. The solution of β-LDs of clusters was condensed to a smaller volume for a suitable concentration for further analysis and stored in a freezer (−30 °C) before use. Debranching. Solutions of β-LDs and building blocks with a carbohydrate amount of 1 mg were diluted in hot water to make up a volume (225 μL). After cooling, NaOAc buffer (25 μL, 0.01 M, pH 5.5), pullulanase (1 μL), and isoamylase (1 μL) were added. The reaction was at room temperature for 20 h with stirring to completely break the α-(1,6)-linkages before analysis by high-performance anion exchange chromatography (HPAEC). The reaction was terminated by boiling for 10 min. Chromatographic Techniques. The molecular weight distributions of the clusters and building blocks were analyzed by gel permeation chromatography (GPC) of Sepharose CL 6B (Pharmacia, Uppsala, Sweden) and of Superdex 30 (Pharmacia), respectively, as described previously.12 The column was calibrated as described previously.14 The building block mixtures and also the debranched samples were analyzed by HPAEC as described previously.12 Statistical Analysis. Tests were conducted in triplicate and analyzed using SPSS version 19.0 software (IBM Corp., Armonk, NY, USA). Differences between means of data were compared by least significant difference at a significance level of p < 0.05.

Figure 3. Development of average DP (degree of polymerization) of branched dextrins as a function of time during α-amylolysis.

Characterization of Clusters. Isolated clusters from whole starch were subjected to β-amylolysis to remove external chains that remained after α-amylolysis. The molecular weight distribution of the resulting β-limit dextrins (β-LDs) is shown in Figure 4. The average DP of clusters from W64A (64.6) was

Figure 4. Molecular weight distribution obtained by GPC on Sepharose CL 6B of β-limit dextrins of clusters. wt %, weight-based percentage.

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RESULTS Time Course Analysis of α-Amylolysis of Whole Starch. Hydrolysis kinetics of whole starches treated with αamylase of B. amyloliquefaciens by using GPC is presented in Figure 1 (W64A) and Figure 2 (du1-M3). The hydrolysis profile for du1-Ref was similar to that of du1-M3. The fractions prior to 125 are primarily branched dextrins, whereas those after that are mostly linear dextrins.15 These latter dextrins were likely the product of both amylose and the external and intercluster segments of amylopectin or intermediate materials. Figure 3 shows the kinetics of starch degradation over time. The hydrolysis was rapid in the initial stage and then became slower. The average DP of the branched dextrins leveled off around DP 150. On the basis of these observations, 90 min of α-amylolysis was chosen for large-scale cluster production for all whole maize starches.

smaller than that of du1-Ref (78.3) and du1-M3 (86.9) (Table 1). The same trend was also found for the peak DP (120 for W64A and 140 for du1 mutants). du1 mutant samples had a higher average number of chains per cluster (NC 13.2−15.2) than W64A (NC 10.9). Average chain length (CL 5.7−5.9), internal chain length (ICL 3.0−3.3), and total internal chain length (TICL 9.9−10.3) were similar for all genotypes. Unit chains of clusters can be categorized into a- (carrying no other chains) and b-chains (carrying other chains).16 These chains are different from “A” and “B” chains typically designated in amylopectin molecules.17 The a-chains are completely external chains and have DP 2 and 3 in the βLDs,18 whereas the b-chains represent the internal unit chain profiles of the clusters and are presented in Figure 5. The bchains can be further divided into different categories according 4579



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Table 1. Characterization of β-Limit Dextrins of Clusters from Whole Maize Starchesa genotype

DP

peak-DP

NC

peak-NC

CL

ICL

TICL

W64A du1-Ref du1-M3

64.6c 78.3b 86.9a

120b 140a 140a

10.9c 13.2b 15.2a

20.2b 23.7a 24.4a

5.9a 5.9a 5.7a

3.3a 3.2ab 3.0b

10.3a 10.1ab 9.9b

a

DP, average degree of polymerization (DP) estimated by GPC; NC, number of chains estimated as DP/CL; CL, average chain length estimated by HPAEC; ICL, internal chain length = (CL − ECL) × NC/(NC − 1) − 1, in which ECL is 2 for β-limit dextrin; TICL, total internal chain length = CL of b-chains in β-LD − 1.5. Different lower case letters within a column indicate significant differences (p < 0.05).

Figure 5. Fractionation by HPAEC of unit chains of debranched β-limit dextrins of clusters from whole starches. b0 = 4−6, b1 = 7−18, b2 = 19−27, b3 ≥ 28. wt %, weight-based percentage.

and b3-chains. However, du1 mutant samples had somewhat lower amounts of b2-chains (3.3−3.6%) and higher amount of b0-chains (11.8−12.2%) than W64A (b2 = 4% and b0 = 11.4%). Characterization of Building Blocks in Clusters. To further understand the organization of chains in clusters, the clusters were subjected to extensive hydrolysis to release the small and tightly branched building blocks. As expected, the building block profile exhibited two distinguishable peaks with the one around fraction 123 (DP ∼ 8) representing singly branched building blocks and the one around fraction 136 (DP ∼ 2) representing linear dextrins (Figure 6). The branched building blocks can generally be categorized into five groups (groups 2−6), of which groups 2−4 have a corresponding number of chains and groups 5 and 6 are more heterogeneous with ≥5 chains per block.19,20 The molecular structure of building blocks is presented in Table 3. The branched building blocks had an average DP of 11.0−11.2 and the NC ranged from 2.8 to 3.0 for all genotypes. The chain length parameters of the building blocks were also very similar. From the structures of clusters and their building blocks, the interconnection and composition of branched building blocks in clusters were calculated (Table 4). The interblock chain length (IB-CL) was similar for all of the genotypes (6.5−6.6). The number of blocks per cluster ranged from 5.5 (du1-M3) to

to their length and on the basis of their possible function as a part of the cluster structure (Table 2).16 The amounts of achains in the clusters of whole starch of all different genotypes were similar (59.6−60.3%), and this was also the case for b1Table 2. Relative Molar Composition (Percent) of Chain Categories in β-Limit Dextrins of Clusters from Whole Maize Starches in Comparison with That from the Amylopectina genotype

a

b0

b1

b2

b3

W64A du1-Ref du1-M3 W64AAP du1-RefAP du1-M3AP

60.3a 59.6a 61.0a 51.4 51.7 52.5

11.4b 12.2a 11.8ab 10.2 9.0 9.5

22.8a 23.0a 22.3b 19.6 19.2 19.4

4.0a 3.6b 3.3b 3.5 4.5 4.4

1.4a 1.5a 1.3a 1.4 2.1 2.3

a

For the whole starch of W64A, du1-Ref, and du1-M3, the DP range of a = 2−3, b0 = 4−6, b1 = 7−18, b2 = 19−27, and b3 ≥28 for β-LDs; for the amylopectin (W64AAP, du1-RefAP, and du1-M3AP), the composition was from the φ,β-LDs as detailed previously, the DP range of a = 2, b0 = 4−6, b1 = 7−18, b2 = 19−27, b3 ≥28 for φ,βLDs.12 Different lower case letters within a column of whole starches (W64A, du1-Ref, and du1-M3) indicate significant differences (p < 0.05). 4580



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Table 4. Composition of Building Blocks in β-Limit Dextrins of Clusters of Whole Maize Starchesa distribution of block categories (%) genotype

IBCL

DBbl

NBbl

Gr 2

Gr 3

Gr 4

Gr 5 and Gr 6

W64A du1-Ref du1-M3

6.6a 6.5a 6.5a

6.5a 6.5a 6.4a

4.2b 5.1a 5.5a

60.4a 58.0b 57.6b

21.3b 22.3a 22.7a

8.8b 9.1a 9.1a

9.5b 10.6a 10.5a

a IB-CL, interblock chain length per cluster = mol %linear × DPlinear/mol %branched + 4; DBbl, density of building blocks per cluster = NBbl/ DPcltr × 100, in which DPcltr is the average DP of φ,β-LDs of clusters; NBbl, number of building blocks per cluster = wt %Bbl/100 × DPcluter/ DPBbl, in which wt %Bbl is the weight percent of branched building blocks. Categorization of groups 2−6 of building blocks is based on their number of chains per block. Different lower case letters within a column indicate significant differences (p < 0.05).

water and cooled. In this aggregated physical form it would become resistant to α-amylolysis. Nevertheless, these remnants were hydrolyzed by subsequent dissolution and β-amylolysis as shown by their absence in the purified cluster fractions (Figure 4). Another noticeable difference was the larger amount of dextrins produced by the α-amylase at the boundary between the branched and linear dextrins15 (fractions 120−125) with whole starches compared to amylopectin, especially for the du1M3 sample toward later stages of α-amylolysis (Figure 2). This may be attributed to extensive hydrolysis of both amylose and intermediate fractions22 to DP ∼40. The large production of this material from the du1 samples may be attributed to the higher amounts of amylose (36.5 and 40.5% for mutants vs 29.6% for normal maize) with altered molecular structure as a result of SSIII deficiency as shown previously.11 The external chain segments that remain in the clusters after the action of α-amylase can largely be removed either with βamylase or sequentially with phosphorylase a and then βamylase.23 The external chain lengths (ECL) of a- and b-chains in β-LDs are slightly different from that of φ,β-LDs: In β-LDs a-chains have DP 2 or 3 and the ECL of the b-chains is 1 or 2, whereas in φ,β-LDs all a-chains have DP 2 and the ECL of all b-chains is 1.23 The internal structure of the LDs is, however, identical in β-LDs and φ,β-LDs. In this study, β-LDs of the clusters were produced as opposed to φ,β-LDs in our previous study on the purified amylopectin,12 as this saves 2 days of analysis time. A comparison of the clusters from the two protocols is presented in Figures 7 and 9. The differences in the profiles in the region of the short chains (15 (where the small differences in ECL of β-LDs and φ,βLDs diminish) of clusters from whole normal maize were very

Figure 6. Fractionation of building blocks in β-limit dextrins of clusters of whole maize starches on Superdex 30. Groups (Gr) of building blocks are shown. wt %, weight-based percentage.

4.2 (W64A). All genotypes had a density of blocks per cluster around 6.4−6.5. Clusters in the du1 mutant samples had somewhat less of group 2 blocks (57.6−58.0%) compared with W64A (60.4%), but more of groups 3−6 blocks.

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DISCUSSION In a previous study, we highlighted that the cluster and building block attributes were similar between whole normal and waxy maize starches.7 Results from this study, in which the amylopectin was isolated from normal starch (W64A) and compared with the same starch, confirmed that the cluster and building block data from whole normal starch are reflective of the structure of the corresponding amylopectin (Figure 7). This study also takes this approach further with mutant starch that affects the structure of the amylopectin component. Results in this study will be compared with the fine structure of amylopectin from these same samples reported in a previous publication.12 The pattern of α-amylolysis of samples in this study agreed with the previous result for normal maize starch with an apparent amylose content of 23%.7 However, there remained large dextrins (fractions 45−70) toward the end of hydrolysis (Figures 1 and 2) that are typically not observed with pure amylopectin samples7,15 and even from these samples12 as compared in Figure 8. This may therefore be attributed to the amylose and/or the intermediate components of starch. Hayashi et al.21 reported that amylose could reassociate and precipitate when whole starch in DMSO solution is diluted in

Table 3. Characterization of Building Blocks in β-Limit Dextrins of Clusters from Whole Maize Starchesa linear

branched

genotype

mol %

DP

wt %

mol %

DP

CL

ICL

TICL

NC

W64A du1-Ref du1-M3

56.4a 56.1a 55.1b

2.0a 2.0a 2.1a

80.8b 81.2ab 81.5b

43.6b 43.9b 44.9a

11.0a 11.2a 11.1a

3.8ab 3.7b 4.0a

1.7b 1.6b 2.1a

4.3ab 4.2b 4.5a

3.0a 3.0a 2.8a

Linear dextrins have DP 1, 2, and 3, and branched building blocks have DP ≥ 5. DP, average DP estimated by GPC; CL, average chain length estimated by HPAEC; ICL, internal chain length = (CL − ECL) × NC/(NC − 1) − 1, in which external chain length (ECL) is estimated as 2; TICL, total internal chain length; NC, number of chains = DP/CL. Different lower case letters within a column indicate significant differences (p < 0.05).

a

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Figure 7. Unit chain length distribution comparison of clusters from whole starch (β-limit dextrins) and its amylopectin (φ,β-limit dextrins) of W64A. Data for amylopectin are from a previous paper.12 wt %, weight-based percentage.

similar to those of the clusters from amylopectin isolated from the same starch (Figure 7). The amounts of a-chains in the clusters of whole starch of different genotypes were, however, significantly higher than the amount of a-chains in clusters of amylopectin (Table 2). This can be attributed to the fact that chains with DP 3 in the β-LDs are mixtures of a- and b-chains24 and therefore overestimate the number of a-chains in the β-LDs, whereas in the φ,β-LDs, all achains have DP 2. In general, the du1 mutation resulted in an elevated amount of shorter b-chains around DP 5−15 and a decreased amount of intermediate-length b-chains around DP 15−30 (Figure 9). This did not agree with the previous results on the cluster structure of amylopectin of the same samples (Table 2). The effect of du1 mutation on the cluster size and NC (Table 1) was consistent with the previous result on the φ,β-LDs of clusters from amylopectin,12 although minor differences in the exact values for the du1 mutant starches were noted. The pattern of the influence of du1 mutation on the CL, ICL, and TICL of whole starch clusters was partially inconsistent with the results of amylopectin.12 Apparently, this might be attributed to clusters from branched components in either

Figure 8. Comparison of α-dextrins of whole starch and its amylopectin from du1-M3 during α-amylolysis at 120 min. Data for amylopectin are from a previous paper.12 wt %, weight-based percentage.

Figure 9. Unit chain length distribution comparison of clusters from whole starch (WS) (β-limit dextrins) and its amylopectin (φ,β-limit dextrins) of du1-M3. Data for amylopectin are from a previous paper.12 wt %, weight-based percentage. 4582



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(5) Ball, S.; Colleoni, C.; Cenci, U.; Raj, J. N.; Tirtiaux, C. The evolution of glycogen and starch metabolism in eukaryotes gives molecular clues to understand the establishment of plastid endosymbiosis. J. Exp. Bot. 2011, 62, 1775−1801. (6) Smith, A. M. Starch in the Arabidopsis plant. Starch/Staerke 2012, 64, 421−434. (7) Zhu, F.; Bertoft, E.; Seetharaman, K. Characterization of internal structure of maize starch without amylose and amylopectin separation. Carbohydr. Polym. 2013, 97, 475−481. (8) Gao, M.; Wanat, J.; Stinard, P. S.; James, M. G.; Myers, A. M. Characterization of dull1, a maize gene coding for a novel starch synthase. Plant Cell 1998, 10, 399−412. (9) Keeling, P. L.; Myers, A. M. Biochemistry and genetics of starch synthesis. Annu. Rev. Food Sci. Technol. 2010, 1, 271−303. (10) Lin, Q.; Huang, B.; Zhang, M.; Zhang, X.; Rivenbark, J.; Lappe, R. L.; James, M. G.; Myers, A. M.; Hennen-Bierwagen, T. A. Functional interactions between starch synthase III and isoamylasetype starch-debranching enzyme in maize endosperm. Plant Physiol. 2012, 158, 679−692. (11) Zhu, F.; Bertoft, E.; Källman, A.; Myers, A. M.; Seetharaman, K. Molecular structure of starches from maize mutants deficient in starch synthase III. J. Agric. Food Chem. 2013, 61, 9899−9907. (12) Zhu, F.; Bertoft, E.; Seetharaman, K. Composition of clusters and building blocks in amylopectins from maize mutants deficient in starch synthase III. J. Agric. Food Chem. 2013, 61, 12345−12355. (13) Bertoft, E.; Manelius, R.; Qin, Z. Studies on the structure of pea starches. Part 1: initial stages in α-amylolysis of granular smooth pea starch. Starch/Staerke 1993, 45, 215−220. (14) Bertoft, E.; Spoof, L. Fractional precipitation of amylopectin alpha-dextrins using methanol. Carbohydr. Res. 1989, 189, 169−180. (15) Bertoft, E. Composition of clusters and their arrangement in potato amylopectin. Carbohydr. Polym. 2007, 68, 433−446. (16) Bertoft, E.; Koch, K.; Åman, P. Building block organisation of clusters in amylopectin from different structural types. Int. J. Biol. Macromol. 2012, 50, 1212−1223. (17) Peat, S.; Whelan, W. J.; Thomas, G. J. Evidence of multiple branching in waxy maize starch. J. Chem. Soc., Chem. Commun. 1952, 4546−4548. (18) Summer, R.; French, D. Action of β-amylase on branched oligosaccharides. J. Biol. Chem. 1956, 222, 469−477. (19) Bertoft, E. Composition of building blocks in clusters from potato amylopectin. Carbohydr. Polym. 2007, 70, 123−136. (20) Bertoft, E.; Koch, K.; Åman, P. Structure of building blocks in amylopectins. Carbohydr. Res. 2012, 361, 105−113. (21) Hayashi, A.; Kotani, Y.; Cho, C. H. The rate of amylose precipitation from a dilute solution. Agric. Biol. Chem. 1984, 48, 949− 954. (22) Poutanen, K.; Lauro, M.; Suortti, T.; Autio, K. Partial hydrolysis of gelatinized barley and waxy barley starches by alpha-amylase. Food Hydrocolloids 1996, 10, 269−275. (23) Bertoft, E. Partial characterisation of amylopectin alpha-dextrins. Carbohydr. Res. 1989, 189, 181−193. (24) Bertoft, E. On the nature of categories of chains in amylopectin and their connection to the super helix model. Carbohydr. Polym. 2004, 57, 211−224. (25) Fujita, N.; Yoshida, M.; Kondo, T.; Saito, K.; Utsumi, Y.; Tokunaga, T.; Nishi, A.; Satoh, H.; Park, J. H.; Jane, J. L.; Miyao, A.; Hirochika, H.; Nakamura, Y. Characterization of SSIIIa-deficient mutants of rice: the function of SSIIIa and pleiotropic effects by SSIIIa deficiency in the rice endosperm. Plant Physiol. 2007, 144, 2009−2023. (26) Szydlowski, N.; Ragel, P.; Hennen-Bierwagen, T. A.; Planchot, V.; Myers, A. M.; Mérida, A.; d’Hulst, C.; Wattebled, F. Integrated functions among multiple starch synthases determine both amylopectin chain length and branch linkage location in Arabidopsis leaf starch. J. Exp. Bot. 2011, 62, 4547−4559.

intermediate materials or branched amylose molecules, which likely had relatively higher amounts of shorter chains. When the building blocks from whole starch (Table 3) were compared to those from amylopectin,12 their structures were similar, but there were compositional differences in the small building blocks. Group 2 building blocks was more abundant in whole starch compared to clusters from purified amylopectin, and group 3 building blocks were correspondingly lower. The difference in the composition and structure of clusters and building blocks from whole starch and isolated amylopectin, although small, do reflect the influence of amylose and intermediate components. The deficiency in SSIII probably resulted in altered levels of other enzymes related to biosynthesis of amylose, as suggested by Fujita et al. and Szydlowski et al.25,26 This likely influenced the branching pattern of several starch components, which ultimately gave rise to different structural characteristics of the clusters when whole starch was analyzed as test material. In conclusion, the cluster and building block structures of whole starches deficient in starch synthase III were compared with those of isolated amylopectin from a previous study. Structural and compositional similarities were observed but with some minor differences. The structural differences included a higher proportion of singly branched building blocks (group 2) in clusters of whole starch with a corresponding reduction of group 3 building blocks compared to isolated amylopectin. This difference was likely due to changes in the structural features in the amylose and/or intermediate components as affected by the deficiency in SSIII. On the basis of these results, it is apparent that the use of whole starches to explore branching patterns cannot replace the analysis of purified amylopectins, especially in mutant samples. However, it could be used as a complement and will provide useful information in the analysis of samples obtained in only small quantities like from leaf starches, from which purified amylopectin cannot be obtained in large enough quantities.

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AUTHOR INFORMATION

Corresponding Author

*(K.S.) E-mail: [email protected]. Funding

The financial support of the Natural Sciences and Engineering Research Council of Canada (NSERC) is gratefully acknowledged. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Maize starch samples were kindly provided by Professor Alan M. Myers at the Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, USA.

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REFERENCES

(1) Buléon, A.; Colonna, P.; Planchot, V.; Ball, S. Starch granules: structure and biosynthesis. Int. J. Biol. Macromol. 1998, 23, 85−112. (2) Schoch, T. J. Fractionation of starch by selective precipitation with butanol. J. Am. Chem. Soc. 1942, 64, 2957−2961. (3) Klucinec, J. D.; Thompson, D. B. Fractionation of high-amylose maize starches by differential alcohol precipitation and chromatography of the fractions. Cereal Chem. 1998, 75, 887−896. (4) Pérez, S.; Bertoft, E. The molecular structures of starch components and their contribution to the architecture of starch granules: A comprehensive review. Starch/Staerke 2010, 62, 389−420. 4583


Distribution of Branches in Whole Starches from Maize Mutants

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