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Journal of Cereal Science 47 (2008) 407416 www.elsevier.com/locate/jcs

Winter wheat genotypes under different levels of nitrogen and water stress: Changes in grain protein composition
C. Saint Pierre, C.J. Peterson, A.S. Ross, J.B. Ohm1, M.C. Verhoeven, M. Larson, B. Hoefer
Oregon State University, Rm 107 Crop Science Bldg. Corvallis, OR 97331-3002, USA Received 20 December 2006; received in revised form 11 May 2007; accepted 23 May 2007

Abstract Hard white winter wheat with superior and consistent quality is preferable for Asian markets. This study investigated the combined inuences of moisture decit during grain-ll and N management on protein quality, dough rheological properties, and protein molecular weight distributions in soft and hard winter wheats. Genotypes were grown under an irrigation gradient and two N-fertilization levels. Grain polyphenol oxidase (PPO) activity, SDS sedimentation, and Mixograph analyses were evaluated. Flour protein composition was characterized using SE-HPLC. Moisture stress during grain-ll increased our protein content. N fertilization increased our protein content. No signicant correlation was found between our protein and PPO. Changes in protein composition were related to general increases in protein content, regardless if the result of reduced irrigation or increased fertilization rate. The percentage of monomeric proteins increased more than the polymeric proteins as our protein increased. Similarly, SDS sedimentation volume increased as a function of protein content. As expected, subunit GluD1 5+10 was associated with larger sedimentation volume and higher dough strength in genotypes as compared to those with subunit GluD1 2+12. Biplot analyses showed that genotypes of similar protein quality and composition responded similarly to N and irrigation treatments. r 2007 Elsevier Ltd. All rights reserved.
Keywords: Hard white wheat; Grain protein; SE-HPLC; Stress

1. Introduction Hard white wheat with low ash, moderate to high starch paste viscosity, moderately strong and extensible dough, and high color stability is required for making fresh noodles with acceptable color, texture, and bite characteristics. Desired protein content varies according to the type of noodle (Crosbie and Ross, 2004; Hou, 2001). Hard white wheat production targeting the Chinese yellow alkaline
Abbreviations: AMMI, additive main effects and multiplicative interaction; ET, evapotranspiration; HWW, hard white winter wheat; PPO, polyphenol oxidase; SWW, soft white winter wheat; SDS, sodium dodecyl sulfate; SE-HPLC, size-exclusion high performance liquid chromatography Corresponding author. International Maize and Wheat Improvement Centre (CIMMYT), Km 45 Carretera Mexico-Veracruz, El Batan, Texcoco, Edo. de Mexico 56130, Mexico. Tel.: +52 55 5804 2004; fax: +52 55 5804 7558. E-mail address: c.saintpierre@cgiar.org (C. Saint Pierre). 1 Present address: USDA-ARS-RRVARC, Wheat Quality Lab, 214 Harris Hall, NDSU, Fargo, ND 58105, USA. 0733-5210/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2007.05.007

noodle class requires a grain protein percent of 12.5% or higher (1012% our protein). Flours having high sodium dodecyl sulfate (SDS) sedimentation volumes are preferred for alkaline noodles, giving them a rm bite and their particular texture (Hou, 2001). SDS sedimentation volume has been shown to be positively related to noodle cutting force and sensory hardness (Baik et al., 1994; Huang and Morrison, 1988; Park et al., 2003; Ross et al., 1997). Wesley et al. (1999) studied Canadian hard spring wheats with a GluD1 5+10 background that were deliberately constructed to be variant at the Glu-A1 and Glu-B1 loci. Lines that expressed increased dough strength also exhibited increased cooked noodle cutting force and viscoelasticity. The trials were conducted at effectively equivalent our protein content (range 10.811.2%), so there seems little doubt that changes in cooked noodle texture were causally related to changes in gluten protein composition. Color is an important criterion of quality in Asian noodles (Asenstorfer et al., 2006). It is often the rst

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quality variable that the consumer will take into consideration when buying the product. Raw noodles must retain a stable color for 2448 h after preparation. The presence of the enzyme polyphenol oxidase (PPO) in the our is largely responsible for noodle darkening over time. A positive association between PPO activity and our protein content was reported by Baik et al. (1994). However, Habernicht et al. (2002) found little or no association between PPO activity and both grain protein and SDS sedimentation volume in early generation selections. The later result suggests potential to select for both high protein and low PPO activity. Flour protein content is signicantly increased under water decit (Guttieri et al., 2000; Ozturk and Aydin, 2004), mainly due to higher rates of accumulation of grain N and lower rates of accumulation of carbohydrates. Irrigation, on the other hand, may decrease our protein content by dilution of nitrogen with carbohydrates. Water decit has been demonstrated to affect wheat end-use quality (Guttieri et al., 2000, 2001, 2005). Ozturk and Aydin (2004) observed that late water stress increased grain protein, SDS sedimentation volume, and wet gluten content relative to the fully irrigated treatment in a redgrained winter bread variety. Increases in SDS sedimentation volumes were explained by the increases in grain protein content. Variations in our quality in a hardgrained winter cultivar were related to changes in protein composition from drought stress during grain lling (Gooding et al., 2003). Post-anthesis drought may reduce the duration of storage proteins accumulation, without changing the rate of accumulation of gliadins and glutenins in both spring and winter wheat cultivars (Panozzo et al., 2001; Triboi et al., 2003). N fertilization increases the total quantity of our proteins, resulting in an increase in both gliadins and glutenins (Dupont and Altenbach, 2003; Johansson et al., 2001, 2004; Martre et al., 2003; Triboi et al., 2000). Gliadins increase preferentially over glutenins as N accumulation in the grain increases (Triboi et al., 2000; Wieser and Seilmeier, 1998). Consequently, the ratio of gliadin to glutenin proteins was positively correlated with grain N content, even when both gliadins and glutenins increased (Wieser and Seilmeier, 1998). Those variations may lead to a decrease in gluten strength and mixing properties (Johansson et al., 2004). Other authors have reported that the relative amount of gliadins and glutenins was not affected by the increase in N fertilization. The inconsistency in responses could be a consequence of genotypic differences and variation in the allocation of N to protein subunits (Luo et al., 2000; Triboi et al., 2000; Wieser and Seilmeier, 1998). Dupont and Altenbach (2003) suggested that transcriptional regulation determines the response of o-gliadin genes to fertilizer. A number of studies have examined the inuence of either N or drought on grain and our quality, both in the eld or under controlled conditions (Daniel and Triboi, 2000; Flte et al., 2005; Gooding et al., 2003; Guttieri

et al., 2000; Habernicht et al., 2002; Johansson et al., 2001, 2004; Ozturk and Aydin, 2004). However, fewer studies have addressed the interaction of these main effects (Altenbach et al., 2003; Guttieri et al., 2005) and no information is available for hard white winter wheat genotypes. The rst objective of this study was to investigate the effects of moisture decit and N fertilization and their interactions on our protein content, quality, and protein molecular weight distributions of hard white winter wheat (HWW) genotypes. Considering the production of HWW as a priority to respond to the growing Asian market, the second objective was to evaluate HWW genotype response to water stress and N fertilization in terms of stability and end-use properties. Preferred genotypes would have acceptable protein content and end-use quality over a wide range of environmental conditions. A third objective was to identify management practices to maximize grain yield production while avoiding low protein concentration and/ or reduced quality. 2. Experimental 2.1. Materials Seven diverse HWW genotypes were grown in the study (Table 1). Genotypes included ve Oregon State University breeding program experimental lines with varying glutenin composition and quality characteristics (Table 1). NW97S277 is a Nebraska cultivar, sib of Antelope. It has good bread-baking properties, but relatively unadapted to the Pacic Northwest. IDO591 is an experimental line from the University of Idaho with potential for use in both bread and noodle applications. Two regionally adapted soft white winter wheat (SWW) cultivars, Stephens and Eltan, were included in the study. Stephens is major soft wheat cultivar that has been grown in the region for over 20 years. Eltan is a unique soft white cultivar that performs well in bread and Asian noodle applications at high grain protein levels. Eltan has been used as check variety in HWW development by USDAARS Western Wheat Quality Laboratory because it produces Asian noodles with superior color. Replicated trials were conducted at Hermiston (Hermiston Agricultural Research & Extension Center; 451490 N, 1191170 W) and Madras (Central Oregon Agricultural Research Center; 441400 N, 1211080 W), Oregon, USA, during 20022003 and 20032004. Grain from trials in Hermiston in 20032004 was damaged during storage, hence results from this environment are not reported. Average air temperature was 12.5 1C for Hermiston and 9.9 1C for Madras. Total recorded annual precipitation levels at each location were 192 and 266 mm (Hermiston; 20022003 and 20032004, respectively), and 238 and 310 mm (Madras; 20022003 and 20032004, respectively). Seed was planted in seven 0.15 m spaced rows at a rate of 110 kg seed ha1. Plot size was 2.7 1.2 m. Nine

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C. Saint Pierre et al. / Journal of Cereal Science 47 (2008) 407416 Table 1 Genotype, identication class, origin, and Glu-locus composition for genotypes grown in Oregon State Genotype OR941048 OR850513-19 OR953475 OR942496 OR943576 IDO591 NW97S277 Stephens Eltan ID OR048 OR319 OR475 OR496 OR576 IDO591 N277 Stpn Eltan Class HWW HWW HWW HWW HWW HWW HWW SWW SWW Origin OSU OSU OSU OSU OSU U. of Idaho USDA-ARS-U. Nebraska OSU WA State U. Pedigree ID80-628/3/CER/YMH/HYS/4/ CER/YMH/HYS RBS/ANZA/3/KVZ/HYS//YMH/ TOB/4/BOW S CER//YMH/HYS/3/PI372129 CEBECO148//CNO/INIA//LFN/3/ K//PET/RAF/4/ND/P101//AZT MRS/CI14482//YMH/HYS/3/ RONDEZVOUS MANNING/KARLs PRONGHORN/ARLIN Nord Desprez/CI13438 Luke//BR-70443-4, PI167822/ CI13438 Glu-A1 2* 2* 2* 1 2* NA 2* 2* 1 Glu-B1 7+9 7+9 7+9 6+8, 17+18, 7 6+8 NA 7+9 7+9 7+9 Glu-D1 2+12 2+12 5+10 5+10 2+12 NA 5+10 2+12 5+10 409

A newly discovered subunit coded by the Glu-A1 locus which had a marginally faster mobility than subunit 2 but a slower mobility than subunit 3 had to be described as subunit 2*. OSU: Oregon State University.

wheat genotypes were planted as strips at 901 to the sprinkler lines. Genotypes were randomized within four replications. Plots were uniformly irrigated to replace 100% crop evapotranspiration (ET) until 1 week prior to anthesis. After that date, a central line-source sprinkler system was used to impose a continuous irrigation gradient. The water gradient was divided to represent four irrigation levels. Plots received 10080% (treatment I3), 8050% (treatment I2), 5030% (treatment I1), and less than 30% of measured ET replacement (I0). Grain from I0 was not included in this study because of grain yield limitations. Irrigation was scheduled using climatic data and ET measurements. ET was measured by AgriMet, Pacic Northwest Cooperative Agricultural Weather Network, using the 1982 KimberlyPenman ET model (Wright, 1982). Water was collected in cans during irrigation to determine the amount of water applied throughout the water gradient. The amount of water applied to fully irrigated plots was 200 and 190 mm at Hermiston in 20022003 and 20032004, respectively, and 380 and 480 mm at Madras in 20022003 and 20032004, respectively. Irrigation was continued until full canopy senescence. Alleys of 1 m between irrigation treatments were cut just prior to harvest to minimize border effects. Two soil nitrogen rates were imposed based on yield potential and desired grain protein level at full irrigation. All plots received a single fertilization of 170 kg N ha1 (N1) in early March (Feekes 4). A blend of urea and ammonium sulfate was used to provide 22.4 kg ha1 of sulfur. For the high N level (N2), there was a second application of 170 kg N ha1 in May (Feekes 7), prior to the last uniform irrigation. Fertilization treatments were randomly assigned to the four eld replications. Plots were harvested with a small-plot combine at maturity.

PPO activity was measured in grain as an indicator of noodle color stability (Anderson and Morris, 2001). After threshing and cleaning the grain, subsamples were taken for PPO determinations. The four eld replications were kept for those analyses. Two PPO activity determinations were run for each sample following AACC 22-85 (American Association of Cereal Chemists, 2000). The solution used for the color reaction consisted of 10 mM L-DOPA (L-Dihydroxyphenylalanine) in 50 mM MOPS [3-(N-morpholino) propanesulfonic acid], pH 6.5. A 1.5-mL aliquot of this solution was added to ve undamaged kernels in a 2 ml siliconized microfuge tube. The tubes were shaken by vortex (Fisher Scientic Vortex Genie 2, Mixer Shaker, Fisher Scientic Company L.L.C., Pittsburgh, PA) for 30 min at room temperature. Samples were then placed on ice to arrest the PPO reaction. Absorbance was read on 200 ml of the solution at 492 nm with a Fisher Scientic Multiskan photometric reader (Fisher Scientic Company L.L.C., Pittsburgh, PA). 2.2. Milling procedure A subsample of 600 g of grain from eld replication 1 was combined with 600 g of grain from eld replication 2 for milling. The goal was to increase the amount of our obtained for each treatment. Same procedure was followed with eld replications 3 and 4. Soft and hard wheats were milled on a Brabender Quadrumat Senior Mill (C.W. Brabender Instruments, NJ) after tempering to 14% and 15% moisture, respectively, following a modied AACC 26-50 procedure. Roll temperature was kept constant at 31 1C. Grain ow rate into the break rolls was adjusted to 130135 g min1 and 140145 g min1 for SWW and HWW genotypes, respectively. Bran, shorts, break our, and reduced our fractions were recovered after 16 min from the moment all grain passed through break rolls. Flour

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samples resulted from mixing break and reduction our streams. 2.3. SDS sedimentation SDS sedimentation assay was conducted as a measure of protein quality following AACC method 5660. A our subsample of 0.5 g was mixed with 4 ml distilled water for 20 s. After a resting interval of 10 min, samples were again mixed for 10 s. Once rested for 5 min, 12 ml lactic acid/SDS solution was added. Samples were gently mixed and rested for 2 min. SDS sedimentation volume was registered as height (mm) of the solidliquid interface line after 15-min sedimentation time. 2.4. Mixogram analyses Mixogram analyses were conducted to characterize dough rheological properties following a modied AACC 54-40A procedure. A Mixograph device (National Manufacturing Division, TMCO, Lincoln, NE) was used for mixing 10 g our and water. For each sample, the amount of water added for dough hydration was adjusted by visual comparison with the expected Mixograph curve for low, medium, and high protein levels (USDA/ARS, Western Wheat Quality Laboratory, Washington State University, Pullman, WA; Baenziger et al., 2001). Mixograph curves were analyzed using Mixsmart software (Version 3.4, 1992, National Manufacturing Division, TMCO, Lincoln, NE). Mixograph peak time was the time at which the curve reached the maximum height. Mixing stability was measured as the slope of the Mixogram midline curve 2 min after Mixograph peak time. Mixing tolerance was measured as width of the Mixograph curve 2 min after Mixograph peak time. 2.5. Flour protein content and composition Flour protein content was measured as percentage using a LECO CNS 2000 C, N, S analyzer (Leco Corporation, St. Joseph, MI). Protein was calculated as N 5.7 as indicated in AACC method 46-30. Proteins were extracted from wheat our using a two step extraction method according to Morel et al. (2000) with slight modication. SDS-extractable proteins were separated after dispersing 0.16 g our with 20 ml of 1% SDS, 01 M sodium phosphate buffer (pH 6.9). Samples were agitated for 30 min at 98 1C to reduce enzyme activity. After cooling down to room temperature, samples were sonicated for 180 s at 30% (5W) power setting to extract the SDS-unextractable proteins, i.e. proteins soluble only after sonication. Samples were centrifuged (Eppendorf Centrifuge 5413) for 30 min at 18,000 rpm. The supernant was ltered (0.45 mm HV Millipore DuraPore membrane lter), and 20 ml was injected on to the column. Proteins were characterized by size-exclusion high performance liquid chromatography (SE-HPLC) using a Phenomenex

BIOSEP SEC S4000 column (600 7.5 mm) and a guard column (75 7.5 mm) (Batey et al., 1991). The eluting buffer was 50% acetonitrile (v/v) in water with 0.1% Triuroacetic acid (v/v). Two repeated measures of each our sample were conducted. Resulting chromatograms were divided into three components of decreasing molecular size: polymeric proteins (high and low molecular weight glutenins), large monomeric proteins (gliadins), smaller monomeric proteins (soluble proteins, non-gluten forming albumins and globulins) (Borneo and Khan, 1999; Kuktaite et al., 2004). Limit times for integration purposes were 11.918.6, 18.621.6, and 21.624.8 min for glutenins, gliadins and soluble protein, respectively. The percentage of polymeric protein in the total protein was calculated as polymeric protein area/area total 100. Similarly, monomeric protein area/area total 100 and soluble protein area/area total 100 accounted for percentage of monomeric protein and soluble protein in the total protein, respectively. 2.6. Statistical analysis Data were analyzed by mixed effects analysis of variance with PROC MIXED in SAS (SAS Institute, 2001). Years and locations were combined as environments for the analysis, thus each year-location combination represents a different environment: Hermiston 20022003, Madras 20022003, and Madras 20032004. The effects of genotype, irrigation, N fertilization, and environment were considered as xed effects. Replications and the interactions with genotype, irrigation, N fertilization, and environment were considered random effects. Tests of signicance for xed effects and interactions of xed effects were conducted by combining the appropriate linear combination of mean squares (Hanks et al., 1980; Johnson et al., 1983; McIntosh, 1983). Replications were considered nested within environments. Values of kernel size, which were determined using the Single Kernel Characterization System model 4100 (Perten Instruments, Springeld, IL), were used as covariate in the PPO statistical analysis. Additive main effects and multiplicative interaction (AMMI) analyses and biplots were conducted according to Vargas and Crossa (2000) to explain genotype responses to treatment combinations. For this analysis, all genotypes were plotted against the six treatment combinations applied in the eld, i.e. two N fertilization levels times three irrigation levels. Gollob test was used for testing the signicance of each AMMI term (Vargas and Crossa, 2000). Only analyses with signicant AMMI terms were then used for biplots. Biplot of rst and second AMMI components (axes) for the nine genotypes (points) and the six treatment combinations (irrigation-N level combination, vectors) were obtained for SDS sedimentation volume from Madras in 20022003 and Mixograph tolerance data from Hermiston and Madras in 20022003. The rst AMMI component was plotted in the x-axis and the second AMMI component was plotted in the y-axis.

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Phenotypic correlation coefcients were calculated for the relationships between traits with SAS. 3. Results 3.1. Main effects and interactions Considerations on grain yield, grain properties, and grain protein content were discussed by Saint Pierre et al. (Unplublished Data). Nitrogen fertilization had no effect on grain yield. The main effect of the additional N treatment was to increase grain protein content. As expected, grain protein and our protein of both HWW and SWW genotypes signicantly increased with the second application of N. There was no signicant difference on average grain yield when the replacement of ET was reduced from 100% to 80%. However, average grain yield was reduced by 16% when irrigation was 5030% of ET replacement. Reducing irrigation from 100% to 5030% of ET replacement increased average grain protein content from 116.4 to 123 g kg1. The increase in grain protein was likely a function of yield reduction induced by water stress. Similarly, single kernel weight and diameter decreased as water stress increased. Genotype, N fertilization, and irrigation each had large effects on our protein content, protein composition, and dough mixing properties (Table 2). Interactions of environments with genotype, irrigation and N fertilization treatments were highly signicant for some variables, impacting the magnitude of treatment main effects. Large differences were observed among test sites in terms of climatic factors and soil properties that contributed to variation in plant responses. For example, precipitation and relatively greater heat stress at Hermiston in

20022003 as compared with Madras in both 20022003 and 20032004 reduced the response to the irrigation treatments at Hermiston. Variability at Madras 20022003 and Madras 20032004 was largely due to irrigation main effects and irrigation fertilization interactions. High initial N levels at Madras 20032004 resulted in a lower N response. N fertilization inuenced both protein quantity and quality (Table 3). As expected, increasing N rates increased our protein from an average of 94.2 g kg1 to 117.7 g kg1. The percentages of both monomeric and polymeric protein fractions signicantly increased when N rate was increased. The ratio of monomeric to polymeric protein fractions increased from 1.29 to 1.34 with increasing N. Soluble protein fraction decreased as N rate increased. Higher soil N availability increased SDS sedimentation volume by 33%. This result concurred with signicant positive correlations between SDS and total our protein and polymeric protein in all the environments. N fertilization responses were less evident at Madras 20032004, probably as consequence of a high initial soil N concentration. Dough rheological properties were evaluated after adjusting water absorption according to grain protein concentration in order to optimize dough development. Once differences due to protein content are removed, changes due to protein composition may become more evident. Values for Mixograph peak time, stability, and tolerance decreased as N fertilization rate increased (Table 3). That may be consequence of the higher ratio of monomeric to polymeric proteins observed at the higher N level. Irrigation treatments had less impact on protein composition than the N fertilization treatment. As averaged over

Table 2 Mean squares from analysis of variance for our protein content, our protein composition, SDS sedimentation volume, SDS sedimentation volume corrected for our protein concentration (cSDS), Mixograph properties, and polyphenol oxidase (PPO) activity from plants grown in Hermiston during 20022003, and Madras during 20022003 and 20032004
Source of variation df Flour protein Protein fraction Polymeric Genotype (G) Irrigation (I) Nitrogen (N) Environment (E) GI GN GE IN IE NE GIN GIE GNE INE GINE 8 2 1 2 16 8 16 2 4 2 16 32 16 4 32 707** 1780** 43773** 8334ns 12ns 117* 131ns 182* 1119* 6926* 19ns 17ns 50ns 10ns 14ns 21.0** 0.1ns 10.4** 51.9ns 0.4ns 1.1ns 0.9ns 1.3ns 0.4ns 1.3ns 0.3ns 0.3ns 1.1ns 0.2ns 0.4ns Monomeric 25.4** 15.7** 409* 18.8ns 0.4ns 2.4ns 4.4ns 1.4ns 10.6* 94.0ns 0.4ns 0.6ns 1.1ns 0.6ns 0.4ns Soluble 11.1* 12.8* 550** 122ns 0.3ns 1.5ns 2.9ns 0.6ns 12* 116* 0.3ns 0.6ns 1.3ns 0.7ns 0.4ns 0.083** 0.015** 0.198* 0.030ns 0.001ns 0.005ns 0.007ns 0.005ns 0.008* 0.053ns 0.001ns 0.001ns 0.003ns 0.001ns 0.001ns 1966** 738** 7803** 1258ns 7ns 28* 100** 53* 272* 1848* 9* 5ns 18ns 19ns 6ns 0.1395** 0.0174** 0.0263* 0.1504* 0.0005ns 0.0016ns 0.0060** 0.0001ns 0.0036ns 0.0151ns 0.0005* 0.0004ns 0.0006ns 0.0005ns 0.0004* Protein ratio SDS SDSc Mixograph Peak time 34.30** 0.31ns 22.57* 63.27* 0.18ns 0.49ns 1.86** 0.04ns 0.29ns 5.21ns 0.18ns 0.17ns 0.24ns 0.19ns 0.23ns Stability 49.7** 1.0ns 167.1** 69.4* 5.3* 8.4* 7.3ns 1.7ns 1.7ns 27.8ns 1.8ns 3.7ns 4.3ns 2.1ns 1.8ns Tolerance 212.5** 33.6ns 259.3* 0.9ns 18.3* 37.6* 21.6* 2.7ns 19.5ns 50.2ns 6.6ns 13.2ns 17.9ns 9.5ns 8.0ns 0.109** 0.003ns 0.0002ns 0.009ns 0.005ns 0.009ns 0.004ns 0.001ns 0.002ns 0.007ns 0.003ns 0.005ns 0.004ns 0.003ns 0.003ns PPO

*, ** Signicant at pp0.05, and po0.01, respectively; ns, not signicant, p40.05.

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412 C. Saint Pierre et al. / Journal of Cereal Science 47 (2008) 407416 Table 3 Means and Duncan grouping for our protein content (g kg1), our protein composition (%), SDS sedimentation volume (cm3), SDS sedimentation volume corrected for our protein concentration (cSDS), Mixograph properties, and polyphenol oxidase (PPO) activity from plants grown in Hermiston during 20022003, and Madras during 20022003 and 20032004 Flour protein Genotypes Eltan IDO591 N277 Stephens OR048 OR319 OR475 OR496 OR576 Irrigation level I1 I2 I3 N fertilization N1 N2 Environment Hermiston 20022003 Madras 20022003 Madras 20032004 Protein fraction Polymeric Monomeric Soluble Protein ratio SDS cSDS Mixograph Peak time Stability Tolerance PPO

107.2b 104.3bc 105.0bc 98.8c 103.6bc 106.3b 115.3a 109.3b 102.9bc 109.8a 106.3a 101.6b 94.2a 117.7b 105.1a 97.5a 115.0a

35.6d 37.1a 36.1cd 34.7e 36.8ab 36.8ab 35.6d 36.4bc 35.7d 36.1a 36.1a 36.2a 35.9a 36.3b 36.3a 35.3a 36.7a

47.9b 46.2c 47.9b 47.9b 46.4c 46.9bc 48.9a 47.6b 47.6b 47.9a 47.5ab 47.1b 46.4a 48.6b 47.9a 47.0a 47.6a

16.5abc 16.7ab 16.1bc 17.4a 16.8ab 16.3bc 15.5c 15.9bc 16.6ab 16.1a 16.4ab 16.7b 17.7a 15.1b 15.8a 17.6a 15.8a

1.35ab 1.25d 1.33bc 1.38a 1.26d 1.27d 1.37a 1.31c 1.33bc 1.33a 1.32b 1.30c 1.29a 1.34b 1.32a 1.33a 1.30a

37cd 43a 40bc 20g 33e 36d 37cd 41ab 26f 38a 35b 32c 30a 40b 39a 32a 33a

0.34c 0.41a 0.38b 0.20f 0.32d 0.34c 0.32d 0.38b 0.25e 0.34a 0.33b 0.32b 0.32a 0.34b 0.37a 0.33ab 0.29b

3.71b 5.01a 4.94a 2.31d 2.62d 2.72d 3.23c 3.64b 2.69d 3.51a 3.35a 3.42a 3.69a 3.16b 3.69a 4.03a 2.54b

8.48cde 6.40ab 5.95a 9.32de 9.30de 8.26cde 7.81bcd 7.47abc 9.67e 7.96a 8.23a 8.09a 7.20a 8.98b 7.51a 7.67a 9.09b

11.02de 19.01a 17.94a 10.56e 11.43de 12.37cd 15.86b 13.36c 11.69de 14.34a 13.97a 12.85a 14.85a 12.64b 13.54a 13.96a 13.64a

0.28e 0.31d 0.56a 0.35c 0.42b 0.29de 0.44b 0.55a 0.35c 0.39a 0.40a 0.39a 0.40a 0.39a 0.37a 0.42b 0.39c

Within columns, different letters indicate signicant differences (po 0.05) between mean (Duncan test).Irrigation increased from I1 to I3. N-fertilization increased from N1 to N2.

the three locations, our protein, monomeric protein fraction, and the ratio of monomeric to polymeric proteins increased as water stress increased. The percentage of polymeric protein, on the other hand, was not affected by water stress. The percentage of soluble protein was reduced as water stress increased. Those responses, however, were not consistent over the 3 environments. A signicant increase in our protein content as the amount of irrigation was reduced was only observed at Madras in 20022003. Flour protein, polymeric, monomeric, and soluble protein fractions were not affected by water stress at Hermiston in 20022003. Shallow soils, lower heat stress, and lower precipitation in Madras than in Hermiston likely contribute to the magnitude of the irrigation effect on grain properties. Signicant variation in SDS sedimentation volume was attributed to irrigation treatments. Water stress increased SDS volume by 5.23 cm3 when irrigation was reduced from 10080% to 5030% of measure ET replacement. The effect of irrigation on SDS was associated in part with the increase in our protein concentration and polymeric protein percentage as water stress increased. Mixograph peak time, stability, and tolerance were relatively unchanged over irrigated treatments at all locations. A signicant irrigation fertilization interaction for our protein was observed. Increases in our protein

concentration due to N fertilization were higher under water stress than under well-watered situations. Similarly, increased SDS sedimentation volume as a result of N fertilization was higher under water stress than under wellwatered conditions. No signicant irrigation fertilization interactions were observed for either Mixograph parameters or PPO when evaluating the three environments. Linear increases were observed in the percentages of gliadin and glutenin proteins as our protein increased (Fig. 1). As our protein increased, the percentage of monomeric proteins (gliadins) increased more rapidly (po0.01) than the polymeric proteins (glutenins). The rate of changes in protein composition did not depend on whether the protein increase was related to increased soil N or water stress. Concentrations of albumin and globulin proteins increased with increasing protein content, but their relative proportions compared to the other protein fractions slightly decreased. SDS volume also increased as protein content was increased, both as a consequence of a reduced irrigation or N fertilization (Fig. 2). 3.2. Responses of genotypes Wheat genotypes evaluated in this study were genetically diverse for both agronomic and end-use quality traits. Signicant genotype main effects were found for our protein content, protein composition, and dough mixing

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Fertilization Irrigation Low . Low Medium High ... High Low Medium High

413

55 % of total flour protein 50 45 40 35 30 5.0

% Polymeric protein 2 y = 0.2x +33.6 R = 0.12 % Monomeric protein 2 y =0.8x + 38.7 R = 0.57

10.0 Flour protein (%)

15.0

Fig. 1. Relationship between % our protein and % of monomeric (gliadins) and polymeric (glutenins) proteins from eld experiments in Hermiston during 20022003, and Madras during 20022003 and 20032004.

70 60 50 SDS (cm3) 40 30 20 10 0 5

Fertilization Irrigation Low . Low Medium High High ... Low Medium High y = 4.0076x - 7.5591 R2 = 0.4509

10 Flour protein (%)

15

Fig. 2. Relationship between % our protein and SDS sedimentation volume (cm3) from eld experiments in Hermiston during 2003, and Madras during 20022003 and 20032004.

properties. Duncan groups for those variables are showed in Table 3. The SWW Stephens (Glu-D1: 2+12) was clearly distinguished from the SWW Eltan (Glu-D1: 5+10) and the HWW group of genotypes, since it had the lowest our protein content and polymeric protein percentage. Stephens also had the highest soluble protein fraction and the highest monomeric to polymeric ratio. As expected for a SWW, Stephens had the lowest SDS sedimentation volume, Mixograph peak time, stability, and tolerance. In contrast, the other SWW genotype, Eltan, was ranked intermediate for our protein content, protein composi-

tion, and dough mixing properties. Eltan also showed the lowest PPO value. The good performance of this SWW could be related to its glutenin composition. Eltans glutenin composition at both Glu B1 (7+9) and GluD1 (5+10) locus is similar to the one found in NW97S277, which had the best Mixograph performance together with IDO591. IDO591 not only performed in the top group for Mixograph peak time, stability, and tolerance evaluations, but also had the highest SDS sedimentation values. The line OR942496, which also has subunit 5+10, was grouped with the high SDS genotypes. Within the HWW genotypes, OR943576 (Glu-D1: 2+12) had the lowest our protein and SDS sedimentation volume. Glutenin composition at Glu-D1 locus was correlated with both SDS and Mixograph peak time performance. Duncans grouping for Mixograph peak time discriminated genotypes having subunit 5+10 and 2+12 at the Glu-D1 locus. Genotypes with 5+10 subunit, i.e. NW97S277, Eltan, OR942496, and OR953475, had longer Mixograph peak times. Conversely, genotypes with 2+12 subunits, i.e. OR850513-19, OR943576, OR941048, and Stephens, were clustered in a different group with lower Mixograph peak times. Similar results were obtained for SDS, although one of the 2+12 linesOR85051319was not discriminated from the 5+10 group. Genotypes having 2+12 subunit had the lowest Mixograph stabilities, though Duncans grouping did not clearly identify different groups according to glutenin composition. Genotype response to irrigation nitrogen treatment interaction was explained using AMMI biplot analyses. For this analysis, all genotypes were plotted against the six treatment combinations applied in the eld (2 N 3 irrigation levels). Only AMMI analyses with signicant components are reported. For SDS sedimentation volume at Madras in 20022003 (Fig. 3a), the rst two AMMI components accounted for 86% of the interaction sum of squares between genotype and treatment. Stephens and OR943576 were clustered in the biplot, suggesting similar response to treatments. These genotypes also had the lowest average SDS sedimentation volume and lower levels of polymeric proteins, Mixograph peak time, stability, and tolerance. Eltan, NW97S277, and OR850513-19 represent a cluster distinct from Stephens and OR943576, suggesting a different pattern of responses to treatments. For Mixograph tolerance (Fig. 3b), the rst two AMMI components accounted for 85% of the interaction sum of squares. Stephens and OR943576 again had similar response and were distinct from other genotypes in the study. Genotypes with higher average mixing performance, such as Eltan, OR942496, and NW97S277, tended to cluster, indicating a similar response to treatments. Mixograph tolerance values were most stable to changes in N fertility in genotypes with good bread-baking properties such as OR942496, Eltan, IDO591, NW97S277, and OR85051319 than in genotypes with poorer bread-baking properties such as Stephens, OR943576, OR953475, and OR941048.

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3 N: high Irrigation: high

N: low Irrigation: high

Eltan IDO591 comp 1 OR319 -2.5 N277 N: high Irrigation: medium N: high Irrigation: low OR496 OR048 N: low Irrigation: low Stpn

IDO591 OR319 N: high Irrigation: high comp 1 Stpn

OR576

N: low OR475N: low Irrigation: Irrigation: high medium OR576

2.5 -2.5 N:high Irrigation: Eltan N277 mediumOR496 N:high Irrigation:low OR048

2.5 N: low Irrigation: medium N: low Irrigation: low OR475

-3

SDS Madras 2003

comp 2

comp 2

-3

Mixograph tolerance Hermiston and Madras 2003

Fig. 3. Biplot of rst and second AMMI components (axes) for nine genotypes (points) and six treatment combinations (irrigation-N level combination, vectors) for: (a) SDS sedimentation volume in Madras during 20022003; (b) Mixograph tolerance in Hermiston and Madras during 20022003.

For all the genotypes it was observed that the percentages of monomeric proteins (gliadins) increased as our protein increased. Greatest slopes (1.2 and 1.1) for the increment in gliadins as our protein increased were found for Stephens and OR943576, both genotypes with low bread-baking properties. On the other hand, the greatest slopes for the increment in polymeric proteins (glutenins) as our protein increased were observed in cultivars with good mixing properties as IDO591 (slope 0.47), OR850513-19 (slope 0.46), and NW97S277 (slope 0.40). All genotypes also showed an increment in SDS volume as protein content was increased. The lowest slope for the increment in SDS volume was observed in OR941048 and Stephens. 4. Discussion N fertilization showed a positive contribution to protein quantity, without affecting PPO levels. Soil N level had no effect on PPO activity at any location. Moreover, no signicant correlation was found between our protein and PPO. Park et al. (1997) had reported a negative relation between those variables among hard red and hard white wheats. In contrast, Baik et al. (1994) found a positive relationship between our protein and PPO activity. Our results, agree with Habernicht et al. (2002) who suggested a weak relationship between protein and PPO and that high proteinlow PPO combinations are possible. Similar conclusions were obtained in both hard white and red spring cultivars (Guttieri et al., 2005), suggesting the potential of using N management as strategy for increasing protein quantity while improving bread and noodle enduse properties.

PPO values were generally unaffected by irrigation treatments. It was previously reported that hard white and red spring cultivars increased PPO activity when exposed to water stress (Guttieri et al., 2005), mainly as consequence of increased our extraction rates under low irrigated treatments (Baik et al., 1994). It has also been reported that small kernels, which are expected under water stress, contained less PPO than large kernels in a single cultivar (Baik et al., 1994; Demeke et al., 2001). Further analyses are needed to explain the relationship, if any, between irrigation and PPO when milling yield remains constant. Nevertheless, the high magnitude of the genotype mean square in relation to the other sources of variation at each environment reinforces the idea that substantial progress for PPO could be achieved through appropriate selection of genotypes. Glutenin composition at Glu-D1 locus was related to SDS and Mixograph values. Subunit 5+10 had been previously associated with larger sedimentation volume and higher dough strength than subunit 2+12 (Luo et al., 2001; Payne, 1987). Accordingly with previous studies, our results support the use of glutenin composition as markers for the selection of genotypes with superior dough properties (Gianibelli et al., 2001). Dough properties have been also related to the gliadin/ glutenin balance (Pena et al., 2005). In our study, the percentage of monomeric proteins (gliadins) increased more rapidly than the polymeric proteins (glutenins) as our protein increased, resulting in an increased ratio of monomeric to polymeric proteins. Similar results were found by Triboi et al. (2000). Maximum rate of synthesis of glutenins was reported to occur later than the maximum rate of gliadins (Panozzo et al., 2001), so a late water stress

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could have been more detrimental to the synthesis of glutenins than the gliadins. However, the rate of changes in protein composition did not depend on whether the protein increase was related to increased soil N or water stress. Similarly, SDS volume increased as protein content was increased, both as a consequence of a reduced irrigation or N fertilization. The results support previous research (Guttieri et al., 2005) which suggested that the two strategies to elevate grain protein (N and irrigation management) have similar effects on protein quality as measured by Mixograph or loaf volume. Grouping of genotypes in the biplots indicated that genotypes responded to treatment combinations similarly within quality groups. Since similar patterns of response were demonstrated for wheat quality groups, same management strategies could be recommended when targeting specic nal end-uses, independently of genotype differences. N fertilization and irrigation treatments had a large inuence on our protein content, protein composition, and dough mixing properties. Changes in protein composition were related to general increases in protein concentration, regardless of whether increased protein concentration was the result of reducing irrigation or increasing fertilization rate. Concentrations of polymeric and monomeric proteins increased in response to increasing total our protein. As our protein increased, the percentage of monomeric proteins (gliadins) increased more rapidly than the polymeric proteins (glutenins) when evaluated by SEHPLC analysis. Albumin and globulin proteins were the least responsive to changing protein content. Higher slopes for the increment of gliadins as protein content increased were found for genotypes with low mixing properties. Contrary, higher slopes for the increment of glutenin fraction as protein content increased were observed in genotypes with good Mixograph performance. Signicant interactions of genotype with N and irrigation were observed for protein quality, protein composition, and dough mixing properties. However, genotypes of similar protein quality and composition responded similarly to the N and stress treatments. This reinforces the importance of crop management strategies to reach desired marketing targets for our quality and end-product performance. Both N fertilization and irrigation management could be considered as strategy for increasing protein quantity without a negative impact on end-use properties of hard white winter wheats as evaluated by SDS and Mixograph properties. Variation in PPO activity was mainly related to differences in the genotypes. Genotype selection for low PPO seems to be the best strategy to avoid undesirable discoloration in noodles. References
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