Genome-Specific Granule-Bound Starch Synthase I (GBSSI

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Genome specific GBSSI influences starch biochemical and functional characteristics in near-isogenic wheat (Triticum aestivum L.) lines Geetika Ahuja, Sarita Jaiswal, Pierre Hucl, and Ravindra Nath Chibbar J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 22 Nov 2013 Downloaded from http://pubs.acs.org on November 26, 2013

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Journal of Agricultural and Food Chemistry

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Genome specific GBSSI influences starch biochemical and functional characteristics in

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near-isogenic wheat (Triticum aestivum L.) lines

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Authors: Geetika Ahuja, Sarita Jaiswal, Pierre Hucl, Ravindra N. Chibbar*

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Address: Department of Plant Sciences & Crop Development Centre, College of Agriculture and

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Bioresources, 51 Campus Drive, University of Saskatchewan, Saskatoon SK S7N 5A8, Canada

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Corresponding author: Professor Ravindra N. Chibbar, Department of Plant Sciences & Crop

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Development Centre, College of Agriculture and Bioresources, 51 Campus Drive, University of

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Saskatchewan, Saskatoon SK S7N 5A8, Canada.

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E-mail: [email protected]

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Phone: +1-306-966-4969

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Fax: +1-306-966-5015

ACS Paragon Plus Environment


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ABSTRACT

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Near-isogenic wheat (Triticum aestivum L.) lines differing at the Waxy locus were studied for the

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influence of genome specific granule-bound starch synthase I (GBSSI/Waxy; Wx-A, Wx-B, Wx-

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D) on starch composition, structure and in vitro starch enzymatic hydrolysis. Grain composition,

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amylose concentration, amylopectin unit-chain length distribution and starch granule size

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distribution varied with the loss of functional GBSSI. Amylose concentration was more severely

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affected in genotypes with GBSSI missing from two genomes (double nulls) than from one

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genome (single nulls) lines. Unit glucan chains (DP 6-8) of amylopectin were reduced with the

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complete loss of GBSSI as compared to wheat starch with full complement of GBSSI. Wx-A

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and Wx-B had an additive effect towards short chain phenotype of waxy amylopectin. Loss of

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Wx-D isoprotein alone significantly (p < 0.05) reduced the C-type starch granules. However,

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absence of Wx-D in combination with Wx-A or Wx-B increased the B-type and C-type starch

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granules but decreased the volume of A-type starch granules. Rate of in vitro starch enzymatic

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hydrolysis was highest in completely waxy grain meal and purified starch. However, presence of

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Wx-D reduced wheat starch hydrolysis as it increased the large A-type starch granule content

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(volume %) and reduced short chains (DP 6-8) in amylopectin. Factors such as small C-granules,

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amylose concentration and long chains of amylopectin (DP 23-45) also influenced wheat starch

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hydrolysis.

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Keywords: near-isogenic wheat, waxy, amylopectin chain length distribution, starch hydrolysis


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INTRODUCTION

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Endosperm of a wheat grain is occupied mostly (~70%) by starch, which is a glucan homo-

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polymer, composed of one-quarter amylose (105–106 Da) and three-quarters amylopectin (107–

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109 Da). Amylose is a pre-dominantly linear molecule made up of α-1,4 linked glucose residues

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with a degree of polymerization of ~800 (in wheat) and few branches1. Amylopectin, on the

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other hand, is a highly branched glucan polymer with 4-5% α-1,6 branches and degree of

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polymerization (DP) of 105–107. The amylopectin branches form an organized structure and are

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categorized into: short A-chains (DP 6-12), intermediate B-chains (DP 13-24 up to 50) and a

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long inter-cluster C-chain, with one free reducing end per amylopectin molecule2.

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Amylose and amylopectin have different structural and physiological characteristics and hence

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exhibit different reactions within the body during digestion and subsequent release of glucose

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molecules for absorption3. Various studies have shown that amylose to amylopectin ratio, degree

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of polymerization and/or branching of glucan polymers are important determinants of the extent

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of starch enzymatic hydrolysis and hence digestibility of food3, 4, 5. Rate of starch enzymatic

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hydrolysis is reduced in starches with higher amylose concentration4. Relation between

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amylopectin chain length distribution and resistant starch has also been studied in different plant

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systems5. Changes in the amylopectin chain length distribution facilitate retrogradation to

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produce B- and V- type crystalline structures, leading to more resistant starch. It is generally

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believed that increased proportion of longer chains makes the starch more resistant to digestion6.

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A possible reason could be that longer chains form longer helices, which are further stabilized by

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hydrogen bonds, distributed over the entire crystalline region, hence decreasing digestibility6.



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Starch, on the basis of its digestion behaviour after consumption, can be divided into digestible

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and resistant starch7. The portion of starch which gets digested within 20-30 min of ingestion is

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called Readily Digestible Starch (RDS), and that which gets digested within 120 min of ingestion

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is called Slowly Digestible Starch (SDS). Resistant starch (RS) is referred to that portion of

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starch which resists digestion after 120 min8, escapes digestion in the small intestine/ upper

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gastrointestinal tract and is fermented in the large intestine by gut microflora. RS offers many

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positive physiological effects on human body such as reduction in glycemic index, production of

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short chain fatty acids, particularly butyrate, which prevents colorectal cancer; and better

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absorption of minerals like calcium and iron9.

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Amylopectin synthesis is a complex process and involves an array of enzymes. These include

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ADP-glucose pyrophosphorylase (AGPase), starch synthases (SSI, SSII, SSIII, and SSIV), starch

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branching enzymes (SBEI, SBEII) and starch debranching enzymes (DBE)10. Amylose synthesis

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and elongation, however, involves a single enzyme, Granule-bound starch synthase I (GBSSI),

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also known as ‘Waxy’ protein10. Plants lacking the waxy gene, which encodes GBSSI, produce

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starch without amylose, known as waxy starch. GBSSI has also been reported to be involved in

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the elongation of amylopectin chains, particularly for very long branches11.

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Wheat is an allohexaploid crop (Triticum aestivum L., 2n = 6x = 42; BBAADD), consisting of

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three genomes derived from three diploid parents. Therefore, wheat endosperm has three

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isoforms of GBSSI encoded by the Waxy (Wx) loci; Wx-A1, Wx-B1, Wx-D1, located on

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chromosomes 7AS, 4AL (translocated from 7BS) and 7DS respectively12. Absence of GBSSI in

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any of the three genomes would affect the concentration of amylose and amylopectin, starch

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structure and functional properties. The objective of this work was to study the effect of GBSSI


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from the three wheat genomes on starch composition, structure and in vitro starch hydrolysis.

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The terms ‘GBSSI’ and ‘Waxy’ have been used interchangeably.

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MATERIALS AND METHODS

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Material

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Plant material for this study included thirty-two Canadian western red spring (CWRS) near-

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isogenic waxy wheat (Triticum aestivum L.) lines, including four lines of each Wx-A+B+D+, A-

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B+D+, A+B-D+, A+B+D-, A-B-D+, A-B+D-, A+B-D- and A-B-D-. These lines (CDC Teal*7/ CDC

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Wx2) were derived by backcrossing CDC Wx2 (Wx-AIb, Wx-BIb, Wx-DIb null alleles; donor

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parent) to CDC Teal (complete complement of Wx alleles; recurrent parent) six times. Both the

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parents were developed at University of Saskatchewan13, 14. Four replicates of each of the near-

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isogenic line were grown in a Randomized Complete Block Design in 2010 in Kernen (52°09’

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N, 106°33’ W) and Goodale (52°03’ N, 106°29’ W) Crop Research Farms, University of

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Saskatchewan, Saskatoon, Canada. One thousand seeds from each genotype were weighed for

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thousand grain weight (TGW) determination. To prepare grain meal, 10 g of seeds were ground

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by UDY mill (UDY Corporation, Fort Collins CO, USA) equipped with a 0.5 mm sieve. White

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bread (Wonder bread, George Weston Limited, Toronto ON, Canada) was used as control for in

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vitro hydrolysis study and calculation of hydrolysis index.

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Analysis of GBSSI polypeptides

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Starch was purified from wheat seeds (7-10) using a modified method involving cesium chloride

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density gradient centrifugation15. Proteins were extracted from the starch granules by suspending

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10 mg starch in 300 µL denaturing electrophoresis buffer16. GBSSI was separated by

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polyacrylamide (10% v/v, 1 mm thick gel) electrophoresis (Protean II, Biorad, Hercules CA,



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USA) under denaturing conditions16. Separated polypeptides were visualised by silver staining17.

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The gel-electrophoresis separated polypeptides were transferred on a nitrocellulose membrane at

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10 V for 4 h18. Membrane with transferred polypeptides was processed and incubated with

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primary antibodies raised against wheat GBSSI19. The antigen-antibody complex was detected

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with goat anti-rabbit alkaline phosphate conjugate using BCIP/NBT immunoscreening color

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development kit (Biorad, Hercules CA, USA).

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Grain constituents concentration determination

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Total starch concentration was determined using the AACC approved method20, where ground

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meal samples (100 mg in duplicate) were hydrolyzed to dextrins and further D-glucose using α-

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amylase and amyloglucosidase respectively. Total starch concentration was determined as free

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glucose by determining the absorbance at 510 nm21 and calculated on a percent dry weight

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basis22. Protein concentration was determined by combustion method with the FP-528

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Protein/Nitrogen Analyzer (LECO Corporation, St. Joseph MI, USA). Percent protein

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concentration was obtained by the formula: %P = %N x C, where C is 5.7 for wheat20. Crude

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lipid concentration was determined by ANKOMXT15 extractor using petroleum ether as

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extraction solvent20. Percent lipid was expressed as weight of lipid per gram dry weight of the

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initial material used. Beta-glucan concentration was determined using enzymatic method20.

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Ground wheat meal (100 mg in duplicate) was digested with lichenase and β-glucosidase. Mixed

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linkage β-glucan concentration was calculated as free glucose by determining absorbance at 510

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nm23.

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Amylose concentration determination


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High performance size exclusion chromatography (HPSEC) was used to determine the amylose

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concentration19. Starch samples were debranched using isoamylase, freeze-dried, suspended in

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dimethyl sulfoxide (DMSO), and injected (40 µL) into a column (PL gel MiniMix 250 x 4.6 mm

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ID column, Polymer Laboratories Inc., Amherst MA, USA). Amylose and amylopectin were

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separated using HP-SEC (Waters 600 Controller, Waters 610 Fluid Unit, Waters 717 plus

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Autosampler, Waters 410 Differential Refractometer, Waters Corporation, Milford, MA). Data

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was collected and analyzed using Empower 1154 chromatography software (Waters Corporation,

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Milford MA, USA).

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Amylopectin chain length distribution analysis

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Glucan chain length distribution of amylopectin molecules was determined by fluorophore

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assisted capillary electrophoresis (FACE)24 using Proteome Lab PA800 (Beckman Coulter,

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Fullerton CA, USA) equipped with a 488 mm laser module. A modified starch debranching

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protocol25 was used to obtain unit amylopectin chains, which were labeled using 8-aminopyrene

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1, 2, 6-trisulfonate (APTS) in the presence of sodium cyanoborohydride/ tetrahydrofuran. The N-

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CHO (PVA) capillary with pre-burned window (50 µm ID, 50.2 cm total length) was used to

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separate debranched samples.

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Starch granule size distribution analysis

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Granule size distribution (by volume) in starch slurries was determined using a laser diffraction

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particle size analyser (Hydro 2000S, Malvern Instruments, Malvern WR, UK)25. Volume percent

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particle size distribution values were obtained using Malvern Mastersizer 2000 software

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(Malvern Instruments, Malvern WR, UK).

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In vitro kinetics of enzymatic starch hydrolysis



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Wheat grain meal and pure starch samples were enzymatically hydrolyzed in vitro for kinetic

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analysis, following a modified AACC approved method20. Meal and pure starch samples (100

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mg) were mixed with pancreatic α-amylase (10 mg/mL) and amyloglucosidase (3 U/mL,

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Megazyme International Ltd., Wicklow, Ireland) for starch hydrolysis. Reaction mixtures were

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incubated for 0, 30, 60, 90, 120 and 240 min at 37 °C, during which starch was hydrolyzed to D-

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glucose. A 1 mL aliquot was taken from the reaction mixture after every incubation time and

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reaction was terminated by adding an equal volume of 99% (v/v) ethanol and processed

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following the AACC approved method with modifications. Resistant starch and soluble starch

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concentrations were measured as free glucose by determining its absorbance at 510 nm22. Rate of

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starch hydrolyzed was expressed as percent of total starch at the end of each interval. Hydrolysis

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index was calculated using a non-linear model described previously26, 27.

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Statistical analyses

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Analysis of variation (ANOVA) of the means, multiple means comparisons using Tukey’s

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multiple range tests at p < 0.05 and Pearson’s bivariate correlations were performed with SPSS

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V. 19.0 software (SPSS Inc., Chicago IL, USA). Dendrograms were obtained by Minitab V.

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16.0 software (Minitab Inc., State College PA, USA).

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RESULTS

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Screening of the genotypes

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Gel electrophoresis analysis of starch granule proteins showed that the most prominent

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polypeptide was GBSSI (mol. wt. ~60 kDa), which showed three bands, one originating from

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each genome (Figure 1). The waxy A, D and B isoproteins have apparent molecular masses of

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62.7, 58.7 and 56.7 kDa respectively28. Although GBSSI-A was clearly distinct, however


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GBSSI-B and –D were difficult to separate owing to their similar relative mobility on denaturing

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polyacrylamide gel electrophoresis, which has also been observed previously16. Three resolved

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polypeptides were observed at 100-115 kDa which could be SSIIa, as previously identified by

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immunoblotting antibodies against recombinant SSIIa-118. In wheat, mature SBEI has been

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predicted to have a molecular mass of 87 kDa29, which was also observed in the present study. It

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has also been shown that SBE1 gene produces alternatively spliced transcripts, which gives an

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87.4 kDa mature protein30. A larger variant of starch branching enzyme, SBEIc (152 kDa)31,

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however was not observed. At ~77 kDa, a polypeptide was observed in all genotypes which

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could be starch synthase I (SSI)32. In wheat the N- terminal sequence of 75 kDa polypeptide

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shows homology to rice soluble starch synthase33. In addition, various other lower molecular

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weight proteins such as puroindolines, 15 kDa34 were observed, which showed some differences

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between genotypes.

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Carbohydrates concentration

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Thousand grain weight (TGW) in Wx-A+B+D+ genotype was 40.1 g, while it varied significantly

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(p < 0.05) between 39.3 – 43.6 g for the other waxy null genotypes (Table 1). Lowest grain

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weight (39.3 g) was observed for Wx-A-B-D- and Wx-A-D- genotypes, whereas highest grain

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weight was observed for Wx-D- genotype (43.6 g).

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The total starch concentration in Wx-A+B+D+ was 63.1%, while it ranged from 57.7 to 64.8% in

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the partial and completely waxy genotypes (Table 1). Wx-A-B-D- showed significantly (p
15 µm)36. Wx-A-B-

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D- starch showed the highest volume percentage of C-granules and lowest volume percentage of

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A-granules among non-waxy and waxy null starches (Figure 4 C). Wx-A-B- and Wx-D- starches

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showed similar results i.e. reduced and increased volume percentage of C-granules and A-

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granules, respectively. This suggests a comparable role of Wx-D individually, and Wx-A, Wx-B

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together, in influencing the granule size distribution. Presence of Wx-D, either individually or in

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combination with Wx-A and/or Wx-B showed an increase in the volume percentage of A-

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granules (Figure 4 C). However, absence of Wx-D along with Wx-A or Wx-B increased the B-

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and C-granule volume (%) and reduced A-granule volume (%) (Figure 4 C). Normal distribution

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of number percent of starch granules can be divided into three portions; (i) d(0.1) – 10% of

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granules smaller than diameter set-point, (ii) d(0.5) – 50% of granules smaller than diameter set-

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point, and (iii) d(0.9) – 90% of granules smaller than diameter set-point. Values for d(0.1) in

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Wx-A-B-D- starch were significantly higher than those for Wx-A+B+D+ (data not shown),

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suggesting the influence of waxy allele on starch granule size distribution.

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In vitro starch hydrolysis


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The kinetics of hydrolytic studies in meal and pure starch showed a rapid increase in starch

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hydrolysis till 30 min, which is considered as Readily Digestible Starch (RDS). From 30-120

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min, there was a slow increase in the rate of starch hydrolysis, which is considered as Slowly

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Digestible Starch (SDS). After 120 min, starch hydrolysis in pure starch reached saturation and

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was completely digested by 240 min. This portion of starch is known as Resistant Starch (RS)8.

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In meal samples, however, the rate of starch hydrolysis was slower than in purified starch

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(Figure 5).

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Percent soluble starch in white bread reached ~90% within 30 min as compared to non-waxy and

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partial waxy genotypes, which reached ~25% (meal) and 40-45% (pure starch) in the same time

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period (Figure 5). Completely waxy genotype showed a significantly higher rate of starch

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hydrolysis than all partial waxy genotypes, reaching ~40% and ~60% in meal and pure starch

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samples respectively within 30 min of incubation with hydrolytic enzymes. For meal samples of

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partial waxy genotypes, percent soluble starch increased linearly throughout the experiment

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duration, however reaching saturation at 120 min in the completely waxy genotype. For pure

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starch samples, the rate of hydrolysis was significantly higher than meal. In the completely

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waxy, pure starch samples reached saturation in 90 min compared to 120 min in meal samples

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(Figure 5).

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In wheat grain meal, endogenous components like protein, lipid, β-glucan are present in addition

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to starch. Completely waxy genotype showed highest rate of hydrolysis, reaching a point of 50%

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hydrolysis in ~45 – 50 min, while other genotypes reached similar stage after ~120 min. RDS in

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completely waxy genotype was significantly (p < 0.05) higher (24.8%) as compared with non-

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waxy (12.9%) and partial waxy genotypes (11.0 – 16.0%) (Figure 6). RS in completely waxy

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genotype was significantly (p < 0.05) lower (3.1%) than non-waxy (13.4%) and partial waxy



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genotypes (11.7 - 27.6%). SDS values in different genotypes ranged from 61.4 to 74.5%, with no

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specific pattern. Wx-B-D- genotypes showed lowest concentration of RDS and highest

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concentration of RS.

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To investigate the influence of GBSSI on starch hydrolysis, purified starch was used to remove

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the confounding effects of other endogenous grain constituents. Hydrolysis curve indicated that

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50% of pure starch was hydrolyzed between 30-60 min in all genotypes. Similar to meal,

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completely waxy genotype showed highest amount of RDS (60.9%), followed by non-waxy and

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partial waxy genotypes (Figure 6). RS concentration was lowest in completely waxy genotype

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(0.2%). Consistent with the pattern of hydrolysis in meal, highest RS concentration was also

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found in Wx-B-D- genotypes in pure starch hydrolysis assay.

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Hydrolysis index (HI) for meal was highest in completely waxy genotype (94.3). Non-waxy

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genotype showed HI of 42.7. HI (pure starch) was also highest in completely waxy genotype

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(175.0), while it ranged from 117.1 to 139.3 in non-waxy and partial waxy genotypes (Figure 6).

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DISCUSSION

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GBSSI in relation with grain components accumulation

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Wheat flour composition influences its properties and end-use. Amylose concentration and

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amylopectin structure are integral part of starch functionality. In bread baking substitution of

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non-waxy wheat flour with ~10% waxy wheat flour results in higher loaf volume, while more

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than 30% substitution can lead to loss of granule rigidity and fusion of starch granules37. In the

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present study, GBSSI double null genotypes showed significantly higher total starch

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concentration (62.9 – 64.8%) than single null genotypes (57.7 – 60.5%) (Table 1) without

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significantly affecting TGW. A recent study in Sorghum, GBSSI gene associated single


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nucleotide polymorphic (SNP) markers affected starch content, independent of amylose

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concentration38. In addition to starch, dose dependent effect of Wx protein on amylose

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concentration was observed (Table 1), concurring with a previous report19. The gradual reduction

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in amylose concentration from wild type to two Wx gene null genotypes can be explained by the

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ability of the remaining functional gene to partially compensate for the missing Wx allele, as

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suggested previously39. However, over-expression of Wx genes does not affect amylose content,

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despite the increase in the amount of Wx protein 40. Thus it suggests evolutionary limitation of

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amylose threshold, above which it becomes inert to Wx gene dosage. However, considering

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previous reports and present results it can be suggested that in wheat, genome specific GBSSI

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activity is compensatory for amylose synthesis.

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On the basis of GBSSI catalytic potential for amylose synthesis, Wx-A1a was lowest, whereas

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Wx-B1a and Wx-D1a had similar activity41. Wx-A1 and Wx-B1 proteins have been reported to

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be additive in action, while Wx-D1 is independent and strongest among all the GBSSI

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isoproteins in wheat endosperm42. Various reports have suggested a non-additive behaviour of

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Wx proteins, with interaction action being the dominating character influencing amylose

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concentration43. Although order of contribution towards amylose synthesis is reported to be Wx-

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B1 ≥ Wx-D1 > Wx- A1 41 and/or Wx-D1 > Wx-B1 > Wx-A142, but no such pattern was observed

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in the present study (Table 1). Variants of Wx alleles have been studied in the past for their

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amylose synthesis ability and the postulated order is as: Wx-A1b (null) = Wx-A1e < Wx-A1c

Genome-Specific Granule-Bound Starch Synthase I (GBSSI

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