Differential accumulation of sulfur-rich and sulfur-poor ... - PubAg - USDA

Journal of Cereal Science 44 (2006) 101–112 www.elsevier.com/locate/jnlabr/yjcrs

Differential accumulation of sulfur-rich and sulfur-poor wheat flour proteins is affected by temperature and mineral nutrition during grain development F.M. DuPont a,*, W.J. Hurkman a, W.H. Vensel a, R. Chan a, R. Lopez b, C.K. Tanaka a, S.B. Altenbach a a

United States Department of Agriculture, Agricultural Research Service, Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710, USA b Biosource-Invitrogen Corporation, 542 Flynn Road, Camarillo, CA 93012, USA Received 3 March 2006; accepted 15 April 2006

Abstract Hard red spring wheat (Triticum aestivum cv Butte86) was grown under controlled environmental conditions and grain produced under 24/17 8C, 37/17 8C or 37/28 8C day/night regimens with or without post-anthesis N supplied as NPK. Flour proteins were analyzed and quantified by differential fractionation and RP-HPLC, and endosperm proteins were assessed by two-dimensional gel electrophoresis (2-DE). High temperature or NPK during grain fill increased protein percentage and altered the proportions of S-rich and S-poor proteins. Addition of NPK increased protein accumulation per grain under the 24/17 8C but not the 37/28 8C regimen. However, flour protein composition was similar for grain produced with NPK at 24/17 8C or 37/28 8C. 2-DE of gluten proteins during grain development revealed that NPK or high temperature increased the accumulation rate for S-poor proteins more than for S-rich proteins. Flour S content did not indicate S-deficiency, however, and addition of post-anthesis S had no effect on protein composition. Although, high-protein flour from grain produced under the 37/28 8C regimen with or without NPK had loaf volumes comparable to flour produced at 24/17 8C with NPK, mixing tolerance was decreased by the high temperature regimen. q 2006 Elsevier Ltd. All rights reserved. Keywords: Gliadin; Glutenin; Two-dimensional gel electrophoresis; Nitrogen

1. Introduction Protein content and composition, key determinants of wheat flour breadmaking quality, are influenced by genetics, environment and crop management (DuPont and Altenbach, 2003; Fowler, 2003; Fowler et al., 1990; Gupta et al., 1992; Payne, 1987). Flour protein content is strongly influenced by the supply of N, as well as by environmental factors such as temperature during grainfill. High temperature affects the synthesis of protein and starch differentially, resulting in increased grain protein percentage yet lower grain weight. In contrast, N promotes the synthesis of additional protein per

Abbreviations: 2-DE, 2-dimensional gel electrophoresis; DTT, dithiothreitol; HMW-GS, high molecular weight glutenin subunit; LMW-GS, low molecular weight glutenin subunit; MS, mass spectrometry; NIR, near infrared spectrophotometry; RP-HPLC, reverse phase high pressure liquid chromatography; TFA, trifluoroacetic acid. * Corresponding author. Tel.: C1 510 559 5702; fax: C1 510 559 5818. E-mail address: [email protected] (F.M. DuPont). 0733-5210/$ - see front matter q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2006.04.003

grain (Altenbach et al., 2003; Bhullar and Jenner, 1985; Corbellini et al., 1998; Daniel and Triboi, 2000; Fowler et al., 1990; Zahedi et al., 2004). In field studies it is difficult to determine if environmental influences on flour protein content, composition and quality should be attributed to temperature, N, or interactions between the two. Thus there is a need for controlled environment studies that compare the effects of high temperature and N. Flour protein composition is complex, with over one hundred genes encoding the abundant proline- and glutamine-rich prolamins that influence mixing and baking. These proteins include the high molecular weight and low molecular weight glutenin subunits (HMW-GS and LMW-GS) as well as the monomeric a-, g- and u-gliadins. The HMW-GS and LMW-GS are linked by disulfide bonds to form the glutenin polymers responsible for the unique viscoelastic properties of wheat flour dough, while the a-, g- and u-gliadins contribute to dough extensibility. In addition, some albumins and globulins are relatively abundant although their role in flour quality is not known. One property that distinguishes the different groups of wheat flour storage proteins is S content, which is highest for the albumins and globulins, moderately high for g-gliadins and

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LMW-GS, intermediate for a-gliadins, low for the HMW-GS and close to zero for the u-gliadins (Anderson and Greene, 1997; D’ovidio and Masci, 2004; Gianibelli et al., 2001; Payne, 1987; Tatham and Shewry, 1995; Zhao et al., 1999). Tatham and Shewry (1995) refer to the LMW-GS, g- and a-gliadins as S-rich and the u-gliadins as S-poor. Studies of mRNA profiles during grain development revealed that temporal expression of the genes for the gliadins and glutenins is coordinately regulated (Altenbach et al., 2002). In contrast, studies of protein composition reported that applications of N and S influence the proportions of the different protein types (Daniel and Triboi, 2002; Gupta et al., 1992; Timms et al., 1981; Weiser et al., 2004; Wrigley et al., 1984), perhaps through transcriptional, post-transcriptional, translational or posttranslational mechanisms. In previous papers, we reported the effects of postanthesis temperature and mineral nutrients on grain fill (Altenbach et al., 2002, 2003; DuPont et al., 2006). Grains produced under a 24/17 8C cool days/cool nights temperature regimen were large, with high starch content. Protein per grain doubled when post-anthesis NPK was supplied from anthesis to maturity, but NPK had little effect on the pattern of grain development, rate and duration of grainfill, or rate and duration of starch accumulation. When grains were produced under a 37/28 8C hot days/warm nights temperature regimen, the duration of grain fill was greatly reduced, and post-anthesis NPK had little effect on protein accumulation. Effects of a 37/17 8C hot days/cool nights temperature regimen were intermediate, with moderate reductions of starch with or without NPK, and moderate

increases in rate and amount of protein accumulation with added NPK. In this paper, proteins were separated by differential fractionation, RP-HPLC and 2-DE to quantify the effects of NPK and temperature on albumin/globulin, gliadin and glutenin fractions and on individual flour and endosperm proteins. Relationships to protein S, Cys and Met content and to flour baking quality were analyzed. Effects of the 24/17 8C regimen, where NPK had large effects on protein accumulation, and the 37/28 8C regimen, where NPK had little effect on protein accumulation, were examined in detail to determine effects specific to temperature or NPK. Intermediate treatments at 37/17 8C and half-strength NPK were also analyzed. 2. Experimental 2.1. Plant material and growth conditions Plants of the US hard red spring wheat Triticum aestivum ‘Butte86’ were grown at 24 8C days, 17 8C nights with drip irrigation (24/17 8C regimen) as described in Altenbach et al. (2003). Grain was produced in the 13 experiments outlined in Table 1. Except for experiment 12, there was one set of pots per treatment. For the different NPK regimens, plants were watered with 0, 1 or 2 emitters (0!, 0.5! and 1! NPK) from anthesis until maturity and hand watered to maintain similar pot weights. For the high temperature regimens, pots were transferred at anthesis to a climate-controlled greenhouse maintained at 37 8C days and 17 or 28 8C nights (37/17 8C and

Table 1 Post-anthesis growing conditions and protein percentages for flour produced under various environmental regimens Flour protein percentagea,b

Expt c

Temp NPKd 1a 2a 3a 4a 5a 6b 7b 8b 9b,e 10b,e 11a 12a,f 13a Avgg

24/17 0! (%) 9.4C0.2 9.4C0.2 6.9C0.5

24/17 0.5! (%)

24/17 1! (%)

13.9C0.1 14.5C0.1

15.4C0.2

24/17 1!CS (%)

37/17 0! (%)

37/17 0.5! (%)

37/17 1! (%)

37/28 0! (%)

37/28 1! (%)

37/28 1!CS (%)

14.4C0.7 10.6C0.1 9.4C0.5

16.4C0.1 14.3C0.3 17.3 17.4

16.5C0.2 14.6C0.1 18.2 16.7

8.8

14.9 14.0 12.5

7.3C0.4 7.8C0.01 8.3C1.0

37/28 0.5! (%)

13.7C0.7

18.8 18.7 14.3C0.1 14.3C1.0 14.2C0.0 15.5C1.4

14.6C0.3

14.6

10.0C0.6

14.4

15.6C1.3

16.3C0.0 15.6C0.7

18.8C0.1

All plants were grown under the 24/17 8C regimen with 1! fertilizer until anthesis, except as indicated. a Flour protein percentageCSTD was determined by N analysis in triplicate of the single batch of flour. b Flour protein percentage was determined by NIR with a standard error of prediction of 0.13% of the single batch of flour. c Temperature regimen from anthesis to maturity. d NPK fertilizer regimen from anthesis to maturity. e Pots were supplied with 0.5! NPK from sowing until maturity. f Experiment 12 was divided into three separate replicates at 0! and three at 1! NPK, providing a total of six separate flour samples. g Average plus STD.

18.7C0.0

18.6C0.0

18.2C0.1 18.0C0.8

18.6


37/28 8C regimens). For experiment 11, pots for the 24/17 8C and 37/28 8C pots were divided into four treatment groups. S was supplied to one group as 500 ml of 2 mg/ml CaSO4 per week for 4 weeks, starting at anthesis while other plants received 500 ml H2O. For experiment 12, pots were divided into six treatment groups. Three groups received 0! NPK and three received 1! NPK from anthesis until maturity. For all experiments, mature grains from all pots in a treatment group were pooled. Average single kernel weight for mature grain was determined by sampling 100 grains from each pool. Samples of 100 g each were milled to flour. For experiment 13 grains were collected throughout development, and the endosperm was expressed and frozen in liquid nitrogen as previously described (Vensel et al., 2005). 2.2. Milling and baking Grain was tempered to 15% moisture and milled to flour in 100 g batches, using a Brabender Quadromat Jr. mill (Hackensack, NJ). Flour protein content was determined in triplicate by Dumas nitrogen combustion analysis of 15 mg samples, using a Model FP-428 LECO nitrogen analyzer with a 10 ml gas collection tube (LECO Corporation, St Joseph, MI), an EDTA standard, and a protein to N ratio of 5.7 (AACC Method 46-30, American Association of Cereal Chemists, 2000). Alternatively, flour N, C and S were determined by combustion analysis of 35 mg samples, in triplicate, using an Elementar Vario Macro Elemental Analyzer (Hanau, Germany) in CNS mode with a sulfadiazine standard. Protein was calculated as 5.7 times N. Flour protein content also was determined by NIR (AACC method 39-11, American Association of Cereal Chemists, 2000) using the NIR Systems 6500 (NIR Systems, Silver Springs, MD). Micro-bake tests using 10 g of flour were performed in duplicate at the Hard Winter Wheat Quality Laboratory (US Department of Agriculture, Agricultural Research Service, Manhattan, KS) according to previously described methods (DuPont et al., 2006). 2.3. Sequential extraction of albumins/globulins, gliadins and glutenins Proteins were sequentially extracted from flour by the method of DuPont et al., 2005. Briefly, flour samples were extracted twice with 1 ml of 0.3 M NaI, 7.5% 1-propanol (NaI– propanol) per 100 mg of flour. The extracts were centrifuged for 10 min at 4500 g to remove starch and insoluble protein. The glutenins were extracted from the starch pellet with 0.4 ml 2% SDS and 25 mM DTT in 25 mM TRIS, pH 8.0 per 100 mg of flour and centrifuged as above to remove the starch. The extraction was repeated and the extracts pooled. The two NaI– propanol supernatants were pooled and four volumes of 0.1 M ammonium acetate in 100% methanol added. Following incubation at K20 8C overnight, the gliadins were pelleted by centrifugation as above. Four volumes of 100% acetone were added to the supernatant, which was then stored at K20 8C for at least 2 days. The albumins and globulins were

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then recovered by centrifugation. All protein fractions were freeze-dried and stored at K80 8C. Protein content was determined by combustion analysis of 10–15 mg samples. 2.4. RP-HPLC Freeze-dried proteins were dissolved at a concentration of 1 mg mlK1 in 6 M guanidine HCl adjusted to pH 8.0 with TRIS, plus 50 mM DTT, and alkylated with vinyl pyridine prior to HPLC. Five hundred micro-litre of protein was applied to a Jupiter (Phenomenex, Torrance CA) C18 semi-preparative RP-HPLC column or a Nucleosil (Ansys, Lake Forest, CA) C8 analytical column. Proteins were eluted using a Hewlet Packard Series 1100 HPLC (Wilmington, DE). Peptide bond absorbance was measured at 210 nm. Albumins/globulins were eluted from the Jupiter column using a gradient that incorporated a 10 min delay for sample loading, followed by an increase from 10 to 65% acetonitrile in 0.05% trifluoroacetic acid (TFA) at a rate of 1.5 ml minK1 for 90 min. Gliadins and glutenins were separated on a Nucleosil column using a similar gradient with a 10 min delay but at 0.8 ml minK1 for 60 min. HPLC peak areas were used to estimate the relative amount of protein in each peak. Individual peaks of u-gliadins or HMWGS were resolved, the area under each peak was calculated, and peak areas were summed to estimate total u-gliadins or HMWGS. All samples were analyzed in triplicate. 2.5. Two-dimensional gel analysis of gluten proteins and identification of proteins The methods for extraction and analysis of the gluten proteins by 2-DE are described in detail in Hurkman and Tanaka (2004). Protein spots were excised and identified by mass spectrometry as described in Vensel et al. (2005). Computer software (Non-linear Dynamics Limited, Newcastle Upon Tyne, UK) was used to calculate normalized spot volumes (individual spot volume/total spot volume!100). Average and standard deviation for normalized volumes for individual spots in three replicate gels were determined for each developmental time point. The total amount of that protein per kernel for each spot was estimated by multiplying the relative spot volume by the amount of gluten protein recovered per kernel. 3. Results 3.1. Flour protein content Flour was produced under three levels of post-anthesis fertilizer and three temperature regimens and flour protein percentage determined (Table 1). Both NPK and temperature regimen influenced final flour protein percentage. Under the 24/17 8C regimen, average flour protein content increased 1.9-fold with NPK, from 8.3 to 13.7 to 15.5% at 0!, 0.5! and 1! NPK, respectively. In the absence of NPK, flour protein percentage increased 1.2-fold at 37/17 8C and 1.9-fold at 37/ 28 8C, from 8.3 to 10.0 to 15.6% at 0! NPK. Under the

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37/17 8C regimen, average flour protein increased 1.6-fold, from 10.0 to 14.4 to 15.6% at 0!, 0.5! and 1! NPK, respectively. Under the 37/28 8C regimen, average flour protein increased only 1.2-fold, from 15.6 to 18.8 and 18.0 at 0!, 0.5! and 1! NPK respectively. Flour from plants that received S plus 1! NPK was similar in protein content to flour from plants that received only 1! NPK at 24/17 or 37/28 8C (experiment 11). 3.2. Quantitative fractionation and RP-HPLC of flour proteins The effects of the two extremes of NPK and temperature on protein composition were analyzed in detail using flour from experiments 11–13. Proteins were separated into albumin/globulin, gliadin and glutenin fractions and equal amounts of protein were analyzed by RP-HPLC (Figs. 1 and 2). Proteins in the HPLC peaks were previously identified by mass spectrometry and/or Edman sequencing (DuPont et al., 2005). Under the 24/17 8C regimen, NPK had little effect on the proportions of the proteins in the albumin/globulin fraction (Fig. 1A). In the gliadin fraction, the u-gliadin and two

a-gliadin peaks increased with 1! NPK (Fig. 1B). In the glutenin fraction, the HMW-GS peaks increased with 1! NPK, whereas NPK had little effect on the LMW-GS peaks (Fig. 1C). When proteins from grain produced under the 24/17 and 37/28 8C regimens with NPK were compared, fewer differences were detected. Temperature had little effect on the proportions of the proteins in the gliadin and glutenin fractions (Fig. 2B and C). However, the albumin/globulin fraction peak containing LTP increased under the 37/28 8C regimen (Fig. 2A). NPK had little effect on protein proportions under the 37/28 8C regimen; the HPLC traces for the three protein fractions from grain produced under the 37/28 8C regimen without NPK were similar to those for the protein fractions produced under the 37/28 8C regimen with 1! NPK (not shown). In these three experiments, average flour protein content increased from 7.4% without NPK to 14.3% with NPK at 24/17 8C and from 16.3 to 18.5% at 37/28 8C (Table 2). The albumin/globulin fraction decreased by half, from 9.0% of total flour protein under the 24/17 8C regimen without NPK to 4.5% with NPK and was 5.5% of total flour protein at 37/28 8C

Fig. 1. Effect of NPK on proteins in (A) albumin/globulin, (B) gliadin and (C) glutenin fractions. Grain was produced at 24/17 8C with NPK (—) or without postanthesis NPK (- - -) (experiment 13). Flour protein fractions were separated by RP-HPLC. Major protein peaks in panel A were identified as PURO, puroindoline; LTP, lipid transfer protein; GSP, grain softness protein; AMI, alpha-amylase inhibitor 0.19; CM3, alpha-amylase trypsin inhibitor CM3; AV, avenin-like protein. In panel B, peaks corresponding to chromosome 1B encoded u-gliadin (1Bu), chromosome 1D encoded u-gliadin (1Du), and chromosome 1A encoded u-gliadin (1Au) are indicated as well as HPLC peaks enriched in a-gliadins and g-gliadins. In panel C, individual peaks corresponding to the Dy10, Dx5, By9, Bx7, and Ax2* HMW-GS are indicated, as well as those HPLC peaks corresponding to LMW-GS.


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Fig. 2. Effect of temperature on proteins in (A) albumin/globulin, (B) gliadin and (C) glutenin fractions. Grain was produced with post-anthesis NPK at 24/17 8C (—) and 37/28 8C (- - -) (experiment 13). RP-HPLC analysis and labelling as in Fig. 1.

without NPK or 4.4% with NPK (Table 2). Because the treatments had little effect on the proportions of individual albumin/globulin peaks, only total albumins/globulins are presented in Table 2. The gliadin fraction increased from 42.8% without NPK to 46.7% of total flour protein with NPK at 24/17 8C. Similarly, under the 37/28 8C regimen the proportion of gliadins increased from 42.1% without NPK to 45.4% with NPK. At 24/17 8C the u-gliadins increased almost 2-fold, from 5.2 to

9.7% with addition of NPK, and increased from 7.7 to 9.8% with NPK at 37/28 8C. Since, the a- and g-gliadins were not clearly resolved, quantitative data for these proteins were combined. The a- and g-gliadins accounted for 29.1–30.4% of total flour protein at 24/17 and 37/28 8C, with or without NPK (Table 2). At 24/17 8C there was no significant effect of NPK on glutenin, which was approximately 47% of total flour protein, with or without NPK and increased to 49–50% at 37/28 8C.

Table 2 Effects of post-anthesis NPK and temperature on flour protein composition Temperature regimena

Fertilizer regimenb

Repsc

Flour proteind (%)

Albumins– globulinse (%)

Gliadinse (%)

a-,g-gliadinse (%)

u-gliadinse (%)

Gluteninse (%)

HMW-GSe (%)

LMW-GSe (%)

24/17 24/17 37/28 37/28 Pf

0! 1! 0! 1!

4 5 1 2

7.4C0.4 14.3C0.7 16.3 18.5C0.4 !0.01

9.0C0.5 4.5C0.1 5.5 4.4C0.4 !0.02

42.8C0.9 46.7C1.5 42.1 45.4C2.1 !0.02

30.0C0.3 30.1C0.9 29.1 30.4C1.2

5.2C0.4 9.7C0.6 7.7 9.8C1.0 !0.01

47.7C1.4 46.7C1.5 49.4 49.9C2.6

12.7C0.5 15.4C0.4 15.2 15.4C0.2 !0.01

33.1C0.7 30.9C0.8 32.2 32.1C2.2

Data are from experiments 11(-S), 12 and 13. a Temperature regimen from anthesis to maturity. b NPK fertilizer regimen from anthesis to maturity. c Number of replicate treatments; one each for experiments 11 and 13 and three for each treatment from experiment 12. d Total flour protein as percentage of dry weight (14% moisture). e As percent of total flour protein. f p Determined by single factor ANOVA.

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Table 3 Effects of post-anthesis NPK and fertilizer on estimated amount of protein type per kernel Treatmenta,b

Single kernel weight (mg)

Milling yield (%)

Flour per kernelc (mg)

Flour protein per kerneld (mg)

Albumins/globulinse (mg)

Gliadine (mg)

a-,g-gliadinse (mg)

u-gliadinse (mg)

24/17, 0! 24/17, 1! 37/28, 0! 37/28, 1!

47.1C1.5 53.1C2.8 22.8 24.5C0.7

67.8C1.1 68.7C0.5 61.7 57.5

31.9C1.0 36.5C2.0 14.1C0.3 14.1_0.3

2.4C0.2 5.2C0.4 2.3 2.6C0

0.22C0.03 0.23C0.01 0.13 0.12C0.01

1.03C0.09 2.44C0.26 0.96 1.18C0.05

0.72C0.06 1.59C0.14 0.67 0.79C0.03

0.12C0.01 0.50C0.06 0.18C 0.25C0.03

Treatmenta

Glutenine (mg)

HMW-GSe (mg)

LMW-GSe (mg)

HMW/LMW ratio

Gliadin/glutenin ratio

Total S-rich proteinf (mg)

Total S-poor proteing (mg)

S-rich/S-poor ratio

24/17, 0! 24/17, 1! 37/28, 0! 37/28, 1!

1.14C0.08 2.54C0.22 1.13 1.30C0.06

0.30C0.03 0.80C0.08 0.35 0.40C0.01

0.79C0.06 1.61C0.13 0.74 0.83C0.06

0.38 0.50 0.47 0.48

0.90 0.96 0.85 0.91

1.73C0.14 3.41C0.27 1.53 1.74C0.08

0.43C0.04 1.31C0.13 0.53 0.65C0.02

4.0C0.05 2.6C0.1 2.9 2.7C0.04

Data are from experiments 11 (-S), 12 and 13 with the same number of replicate treatments as in Table 2. a Temperature regimen from anthesis to maturity. b NPK fertilizer regimen from anthesis to maturity. c Amount of flour per kernel for each experiment was estimated by multiplying flour milling yield times the average single kernel weight. Since milling does not recover all of the floury endosperm, the protein amounts per kernel are slightly underestimated. d Flour protein per kernel for each experiment was estimated by multiplying flour percent protein times flour per kernel. e Amount of each protein type per kernel for each experiment was determined by multiplying flour protein per kernel times the percentage of the protein type in the flour. f Total of albumins/globulins, aCg-gliadins and LMW-GS. g Total of u-gliadins and HMW-GS.

At 24/17 8C the HMW-GS increased from 12.7% to 15.4% with NPK, and to approximately 15% of total flour protein at 37/28 8C with or without NPK. Like the a- and g-gliadins, individual LMW-GS were not clearly resolved and quantitative data were for the combined peaks. At 24/17 8C the LMW-GS decreased from 33.1 to 30.9% with NPK, and comprised approximately 32% of total flour protein at 37/28 8C, with or without NPK. The effects of NPK and temperature on the proportions of flour proteins were also determined by estimating the amount of each protein type per kernel (Table 3). Total flour protein per kernel increased 2.1-fold at 24/17 8C with NPK, but did not increase significantly at 37/28 8C. At 24/17 8C there was no effect of NPK on albumin/globulin content per kernel, but albumins/globulins decreased 40–50% at 37/28 8C. At 24/ 17 8C total gliadin increased 2.4-fold with NPK, but not at 37/ 28 8C. The aCg-gliadins increased 2-fold with NPK at 24/17 8C, but changed little at 37/28 8C. The u-gliadins increased over 4-fold with NPK at 24/17 8C, and increased approximately 1.4-fold at 37/28 8C. Total glutenin increased 2.3-fold with NPK at 24/17 8C, but not at 37/28 8C. HMW-GS increased 2.7-fold with NPK at 24/17 8C but not at 37/28 8C. LMW-GS increased 2.0-fold with NPK at 24/17 8C, but not at 37/28 8C. The ratio of HMW-GS to LMW-GS increased somewhat with NPK at 24/17 8C but did not change at 37/ 28 8C. The ratio of gliadins to glutenins was similar with all treatments. At 24/17 8C total S-rich proteins increased 2-fold, total S-poor proteins increased 3-fold, and the ratio of S-rich to S-poor proteins decreased with NPK. At 37/28 8C the ratio of S-rich to S-poor protein was similar to that at 24/17 8C with NPK, mainly due to the decrease in S-rich albumins/globulins and small increases in S-poor HMW-GS and u-gliadins.

To further elucidate the relationship between the proportions of gliadins and glutenins to flour protein content, a set of 12 flour samples including those with intermediate protein contents was evaluated (Fig. 3). Grain was produced under the moderate 37/17 8C temperature regimen as well as at 24/17 8C with 0!, 0.5! and 1! NPK. The temperature regimens are not indicated in the figure, but the results were similar whether plants were grown at 24/17 or 37/17 8C. Flour proteins were extracted and their amounts estimated from the intensity of RPHPLC peaks (Fig. 3). The relative amount of protein in each of the u-gliadin peaks increased with flour protein percentage (Fig. 3A) and total u-gliadin increased from 10.5 to 23.2% of the gliadin fraction, a 2.2-fold increase (Fig. 3B). The percentage of a- plus g-gliadins declined from 63.6 to 53.4% of the gliadin fraction as flour protein percentage increased (Fig. 3C). LMW-GS comprised 60.0–65.6%, and HMW-GS 22.4–25.4% of the glutenin fraction. The ratio of LMW-GS to HMW-GS protein amount varied from 2.3 to 2.7 but was not correlated with flour protein percentage (Fig. 3D). 3.3. 2-DE of gluten proteins during grain fill The effects of the treatments on the accumulation of individual gliadins and glutenins during grain development was analyzed by 2-DE. 2-DE clearly resolved the HMW-GS and u-gliadins. In addition, 2-DE resolved many individual aand g-gliadins and LMW-GS (Fig. 4). Proteins were identified by mass spectrometry of tryptic digests (Supplementary Table A). Spots selected for further analysis included three a-gliadins, three g-gliadins, three chromosome 1B-encoded u-gliadins, three LMW-GS and all five HMW-GS. Because gliadins and LMW-GS contain few tryptic digestion sites, some identifications were based on a single peptide fragment.


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Fig. 3. Relationship between gliadin and glutenin amounts as a function of flour protein content. Grain was produced under the 24/17 and 37/17 8C regimens with 0!, 0.5! and 1.0! NPK (experiments 1–5). Protein extracts were analyzed by RP-HPLC as in Fig. 1. Gliadins were calculated as percentage of total peak area in the gliadin fraction, and HMW-GS and LMW-GS were calculated as percentage of total peak area in the glutenin fraction. Individual u-gliadin peaks were distinguished as encoded by chromosomes 1A (1A), 1B (1B1and 1B2) and 1D (1D). Linear regression analysis gave r2 values of 0.79 for 1A, 0.61 for 1B1, 0.72 for 1B2, 0.82 for 1D, 0.87 for total u-gliadins, -0.25 for total aCg-gliadins, 0.03 for LMW-GS and 0.18 for HMW-GS.

Analysis of gene sequence data showed that these identifications were sufficient to distinguish between a- and g-gliadins and LMW-GS but not between individual genes. In fact, identical tryptic fragments were found for the three g-gliadin spots. MS did not detect tryptic fragments from the u-gliadins, but the chromosome 1B u-gliadins were previously located by 2-DE of the purified proteins (DuPont et al., 2004). Because gliadins and glutenins have been classified in the literature based on N-terminal sequence (D’Ovidio and Masci, 2004), the predicted N-terminal sequence is also indicated in Supplementary Table A. MS identification of HMW-GS Bx7 and By9 was problematic, with identification scores of only 10K2, but the identification agrees with their known mobility in SDS PAGE. The amount of each protein spot per kernel was estimated and plotted as a function of thermal 8C days (Fig. 5). Since, the estimate of total gluten per seed was based on the Lowry assay, which underestimates gluten protein, this analysis provides a relative rather than a absolute comparison of protein amounts. Because the HMW-GS were resolved into several spots each, probably as charge trains, the relative spot volumes for the two largest spots was used to calculate HMW-GS amount. The greatest amount of protein per kernel accumulated under the 24/17 8C regimen in the presence of NPK, with three

exceptions. Unlike the other proteins, similar amounts of a-c, g-a and LMW-a accumulated with or without NPK (Fig. 5K, L, and O). Under the 37/28 8C regimen with or without NPK the amount of each protein per kernel was similar to that accumulated at 24/17 8C without NPK, except for a-b, g-a

Fig. 4. Two-dimensional gel of a KCl-insoluble protein fraction from wheat endosperm. Grain produced under the 24/17 8C regimen (experiment 13) was collected at 37 DPA. HMW 2a and 2b, HMW-GS Ax2*; HMW 5a and 5b, HMW-GS Dx5; HMW 7a and 7b, HMW-GS Bx7; HMW 9a and 9b, HMW-GS By9; HMW 10a and 10b, HMW-GS Dy10; LMW-a, -b, -c, LMW-GS; a-a, -b, c, a-gliadins, g-a, -b, -c, g-gliadins; and u-a, -b, -c, u-gliadins.

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Fig. 5. Time course for accumulation of individual gliadins and glutenins. Grain was produced under the 24/17 8C regimen (C B) (blue) and the 37/28 8C regimen (% $) (red), in the presence (C %) or absence (B $) of post-anthesis NPK (experiment 13). Estimated protein per kernel is plotted as a function of thermal time above 0 8C, starting at anthesis. Protein types are arranged in order of sulfur content. Estimated amount of each protein per kernel was calculated by multiplying the relative spot volume times the estimated amount of KCl-insoluble gluten recovered per kernel. Proteins are identified as in Fig. 4. (For interpretation of the reference to colour in this legend, the reader is referred to the web version of this article).

and LMW-a (Fig. 5J and O). For a-b (Fig. 5J) more protein accumulated and for LMW-a and g-a (Fig. 5O) less protein accumulated at 37/28 8C than at 24/17 8C without NPK. Rates of accumulation for this set of proteins were also calculated (Table 4). The rate of accumulation for the total gluten per kernel at 24/17 8C increased from 2.1

to 3.9 mg kernel 8C dayK1 with addition of NPK. Rates at 37/ 28 8C with or without NPK were 2.2 or 2.1 mg kernel 8C dayK1, identical to those at 24/17 8C without NPK. Rates of accumulation for individual proteins at 24/17 8C also increased with NPK, with the exception of a-c and g-a. With NPK the rates for the u-gliadins increased the most


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Table 4 Estimated rates of accumulation of individual gliadins and glutenins (mg kernel 8C dayK1) under the 24/17 and 37/28 8C temperature regimens in the presence or absence of NPK Protein spot

Total glutene a-a a-b a-c g-a g-b g-c u-a u-b u-c HMW-2 HMW-5 HMW-7 HMW-9 HMW-10 LMWa LMWb LMWc

Ratea

Ratio

24/17KNPK

24/17CNPK

37/28KNPK

37/28CNPK

24/17bC NPK:KNPK

KNPKc37/ 28:24/17

37/28dC NPK:KNPK

2.1 0.01 0.01 0.04 0.04 0.06 0.05 0.02 0.02 0.02 0.03 0.05 0.08 0.03 0.06 0.11 0.05 0.04

3.9 0.02 0.02 0.04 0.04 0.07 0.12 0.08 0.10 0.07 0.08 0.12 0.19 0.08 0.15 0.13 0.07 0.06

2.2 0.02 0.02 0.04 0.04 0.06 0.08 0.04 0.06 0.04 0.06 0.08 0.14 0.06 0.09 0.08 0.06 0.06

2.1 0.02 0.03 0.07 0.03 0.07 0.06 0.05 0.06 0.05 0.07 0.08 0.15 0.07 0.12 0.08 0.06 0.06

1.9 2.0 2.0 1.0 1.0 1.2 2.4 4.0 5.0 3.5 2.4 2.4 2.4 2.7 2.5 1.2 1.4 1.5

1.0 2.0 2.0 1.0 1.0 1.0 1.6 2.0 3.0 2.0 2.0 1.6 1.8 2.0 1.5 0.7 1.2 1.5

1.0 1.0 1.5 1.8 0.8 1.2 0.8 1.3 1.0 1.3 1.2 1.0 1.1 1.2 1.3 1.0 1.0 1.0

Protein spot identifications are from Fig. 4. a Rates were calculated for the linear period of protein accumulation, from 262 to 666 8C days under the 24/17 8C regimen and from 192 to 394 8C days under the 37/28 8C regimen. Rates were determined from linear regression analysis of the plots. b Ratio of rate at 24/17 8C with NPK to that at 24/17 8C without NPK. c Ratio of rate at 37/2 8C to that at 24/17 8C. d Ratio of rate at 37/28 8C with NPK to that at 37/28 8C without NPK. e Total KCl-insoluble gluten recovered from endosperm at each time point was plotted as in Fig. 5 and rates of protein accumulation calculated.

(3.5 to 5.0-fold), followed by the HMW-GS (2.4 – 2.7-fold). Except for g-c (2.4-fold) the rates for the g-gliadins and the LMW-GS had smaller increases (1.2 to 1.5-fold). The highest rates were for HMW-7, HMW-10, LMW-a, HMW-5 and g-c (0.19, 0.15, 0.13, 0.12 and 0.12 mg kernel 8C dayK1, respectively). Compared to rates at 24/17 8C without NPK, at 37/28 8C without NPK rates of accumulation of u-gliadins increased 1.6 to 3.0-fold, and rates for HMW-GS increased from 1.5- to 2.0fold. Effects on a- and g-gliadins were more variable, increasing for a-a and a-b but not a-c, and for g-c but not ga and g-b. Rates for LMW-GS at 37/28 8C decreased for LMW-a but increased somewhat for LMW-b and LMW-c. No rates at 37/28 8C were as high as those at 24/17 8C with NPK except for the a-gliadins, g-a and g-b and LMW-c. At 37/ 28 8C, rates were similar with or without NPK, except for a-b and a-c, which increased 1.5-fold or more, and g-a, which decreased somewhat.

3.4. Amino acid composition Amino acid compositions were calculated for known protein sequences that were the best match to the spots in Fig. 5, to evaluate the relationship between their amino acid composition and the effects of NPK and temperature on their accumulation (Supplementary Table B). The mole percent of Cys plus Met differed between the protein classes, and was 0–0.01% for the u-gliadins, less than 0.5–1.7% for the HMWGS, 1.8–2.9% for the a-gliadins, 3.6–4.6% for the LMW-GS, 5.0% for the single g-gliadin sequence and 10.5% for a representative albumin, alpha-amylase Inhibitor 0.19. The values might be somewhat different if the exact sequence were known for each spot. Glutamine and proline content also differed between the protein classes. All gliadins and glutenins are categorized as prolamins, proteins that are rich in Gln plus Pro. The content of Gln plus Pro was 42–54% for all gliadins

Table 5 Effects of post-anthesis S and temperature on flour C, N and S composition Treatment

Flour proteina (%)

Ca,b (%)

Na,b (%)

Sa,b (%)

N:S

24/17 24/17CS 37/28 37/28CS

14.3C0.1 14.6C0.3 18.7C0.0 18.6C0.0

40.8C0.3 40.3C0.1 40.9C0.2 40.8C0.0

2.51C0.00 2.56C0.01 3.28C0.01 3.27C0.00

0.18C0.01 0.18C0.00 0.22C0.00 0.22C0.00

14:1 14:1 15:1 15:1

a b

Values are average plus standard deviation for flour samples from experiment 12. C, N and S as percent of dry matter.

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and glutenins, except for the u-gliadins, which ranged from 64 to 73% Gln plus Pro due to their 2-fold higher content of Pro. In contrast, the alpha-amylase Inhibitor 0.19 was only 14% Gln plus Pro. The calculated elemental composition of each protein (not shown) revealed that there was little difference in C, N, O or H content of any of the proteins (not shown), but the amount of S ranged from as low as 0.2% for HMW-GS Ax2* and the chromosome 1B encoded u-gliadins, to 3% for a typical low molecular weight albumin, the alpha-amylase inhibitor AMI 0.19. Among the gliadins and glutenins, the g-gliadins and LMW-GS had the greatest amounts of S and the HMW-GS and u-gliadins the least. 3.5. Effect of added S Because the response of the protein types to the NPK regimen appeared to correlate with the amount of S-containing amino acids, plants were grown under the 24/17 and 37/28 8C regimens in the presence of post-anthesis NPK with or without added S to test whether the amount of S was limiting (experiment 11). There was no effect of added S on yield or average single kernel weight (not shown). Proportions of C, N and S in flour were unaffected by the addition of S (Table 5). Analysis of additional flour samples indicated that percentage of S and the N to S ratio increased as flour protein increased, regardless of whether the grain was produced at 24/17 or 37/ 28 8C. (Supplementary Fig. A). 3.6. Baking quality Flour samples from seven experiments with a range of protein contents were evaluated for baking quality (Fig. 6). Under the 24/17 8C temperature regimen average flour protein content increased from 7.8 to 15.8% as application of post-anthesis NPK was increased from 0 to 1! NPK. Loaf volume increased from 56 to 94 ml, mixing tolerance scores increased from 2 to 3.5, and mix times decreased from 2.6 to 2.4 min (not significant). Compared to the 24/17 8C regimen, average flour protein content was higher at 0 and 0.5! NPK and similar at 1! NPK under the 37/28 8C temperature regimen. At 37/28 8C under all NPK regimens loaf volumes were similar to those at 24/17 8C with 1! NPK. Mixing tolerance at 0! NPK was similar for the 24/17 and 37/28 8C regimens, but it declined significantly at 0.5 and 1! NPK at 37/28 8C. Mixing times, however, were similar at 24/17 and 37/28 8C. An additional experiment (11) performed to test the effect of added S at 24/17 and 37/28 8C with 1! NPK revealed that protein content, loaf volume, and mixing tolerance were identical with or without added S (not shown). 4. Discussion High protein flours with nearly identical protein compositions were produced by growing plants with post-anthesis NPK or exposing plants to high daytime and nighttime temperatures during grain fill. At 24/17 8C with NPK, high

Fig. 6. Effect of temperature and NPK on baking quality. Grain was produced under the 24/17 8C (C) (blue) or 37/28 8C (B) (red) temperature regimens at three levels of post-anthesis NPK (experiments 6–10, 12, and 13). Mixing tolerance was calculated as in Dupont et al. (in press) and rated on a score of 0– 6, where 0 is unsatisfactory, 2 is questionable, 4 is satisfactory, 5 is excellent and 6 is outstanding. The number of replicates were nZ3 at 24/17 8C with no NPK; nZ2 at 24/17 8C with 0.5 NPK; nZ4 at 24/17 8C with 1.0 NPK; nZ2 at 37/28 8C with no NPK; nZ2 at 37/28 8C with 0.5 NPK; nZ3 at 37/28 8C with 1.0 NPK. Variance within groups for protein, 14.3; mix time, 2.5; mixing tolerance, 0.17; loaf volume, 750; between groups for protein, 206; mix time, 0.41; mixing tolerance, 10.1; loaf volume, 3293. Probability of difference between treatments for protein, mixing tolerance, and loaf vol, p!0.01. (For interpretation of the reference to colour in this legend, the reader is referred to the web version of this article).

protein flour was mainly the result of increased protein synthesis per grain, but at 37/28 8C it was mainly the result of decreased starch accumulation (Altenbach et al., 2003; DuPont et al., 2006). A major effect of either treatment was to increase the proportions of S-poor proteins. Our results and those of others indicate that at least three different environmental conditions may increase the proportion of S-poor proteins in flour. These are S-deficiency, abundant N,


and exposure to high temperature during grain fill. S-deficient grains were reported to have S-levels of 1.2 mg/g or less, or an N to S ratio of 17 or greater (Zhao et al., 1999), and S-deficient flours were reported to have less than 0.1% S or N to S ratios from 22 to 33 (Wieser et al., 2004). S-deficiency was correlated with large increases in S-poor u-gliadins and HMW-GS and increased resistance to mixing (MacRitchie and Gupta, 1993; Moss et al., 1981; Shewry et al., 2001; Wieser et al., 2004; Wrigley et al., 1984; Zhao et al., 1999). In a detailed quantitative study Wieser et al. (2004) reported increases in LMW-GS and g-gliadins, but little change in a-gliadin proportions when plants were supplied with increased S. Similar changes in the proportions of the flour protein types in response to increased N have been reported (Timms et al., 1981). Pence et al. (1954) first reported that relative amounts of the S-rich albumins and globulins decreased as flour protein increased, possibly because of increased N-fertilization. In a detailed study, Wieser and Seilmeier (1998) reported that proportions of u-gliadins and HMW-GS increased with N fertilization whereas proportions of LMWGS and g-gliadins decreased, similar to the effects of S deficiency. Daniel and Triboi (2000) also reported that the proportions of u-gliadins increased with N, although they reported an increase in proportion of g-gliadins not observed in the other studies. In our experiments, flour S of 0.2% was well within the range of S-sufficient flours. Flour S increased with flour N content and there was no effect of added S, as previously reported by Randall et al. (1990). Nonetheless, addition of post-anthesis N greatly increased the rate of accumulation of S-poor u-gliadins and HMW-GS. The proportions of S-poor proteins also increased when grain was produced under the 37/ 28 8C regimen. There are other reports of increased proportions of u-gliadins when grain is exposed to high temperature during grain fill (Corbellini et al., 1998; Daniel and Triboi, 2000). Similar to our results, Daniel and Triboi also reported that a-gliadins increased somewhat and g-gliadins decreased. Unlike our results, Don et al. (2005) reported a decrease in the ratio of HMW-GS to LMW-GS with a heat treatment. Regulation of gene expression for gliadins and glutenins is influenced by several factors. In general, transcription for all glutenin and gliadin types is coordinately regulated (Altenbach et al., 2002), but transcript levels may be sensitive to N in wheat (DuPont et al., 2005) and barley (Mu¨ller and Knudsen, 1993; Shewry et al., 2001). It is not yet clear whether the level of N available in the wheat plant alters the pattern of protein accumulation through effects on transcription, message stability, translation, size and composition of the amino acid pool, or protein turnover. In wheat endosperm, regulatory mechanisms may direct surplus N towards synthesis of increased amounts of Gln and Pro, thus favoring synthesis of the u-gliadins and HMW-GS, proteins rich in these amino acids. In contrast, the supply of Cys and Met in the endosperm may limit the rate of synthesis of the S-rich proteins. For instance, it is known that Cys and Met influence the rate of synthesis of S-rich storage proteins in seeds of dicotyledonous plants (Demidov et al., 2003; Krishnan

111

et al., 2005; Tabe et al., 2002). It is also possible that high temperatures interfered with transport or production of amino acids. For example, Zahedi et al. (2004) reported that adding post-anthesis N increased amino acid transport to the grain under a low but not under a high temperature regimen. In addition, many of the enzymes for amino acid synthesis in the endosperm appear to be located in the amyloplasts (Balmer et al., 2006). Starch synthesis in the amyloplasts is greatly reduced by high temperature. One might speculate that amino acid biosynthesis might also be reduced by high temperature. Further research is needed to explain the actual basis for the similar effects of decreased S, increased N and high temperature on differential accumulation of S-rich and S-poor proteins in the endosperm. Protein percentage, composition, loaf volume and mixing time were similar for flour from grain produced at 24/17 8C with NPK or at 37/28 8C without or with NPK. Mixing tolerance was low at 37/28 8C, however, indicating that some critical property of the flour differed from that produced at 24/17 8C. Deleterious effects of high temperature on flour quality could be mainly due to effects on polymer formation, rather than on proportions of gliadins and or glutenin subunits (Blumenthal et al., 1995; Don et al., 2005). A 42 kDa LMW-GS is implicated in flour quality (D’ovidio and Masci, 2004; Maruyama-Funatsuki et al., 2005) so it is of interest that this type of LMW-GS (LMW-a) decreased at 37/ 28 8C. It is possible that effects of temperature on this major LMW-GS influenced polymer characteristics, but more research on polymer composition and structure is needed to determine this. It is also of interest that there were greater ratios of HMW-GS to LMW-GS at 24/17 8C with NPK and at 37/28 8C with or without NPK. A greater ratio of HMW-GS is reported to correlate with larger polymer size, but it has been suggested that deleterious effects of heat on quality are related to a decrease in polymer size (Corbellini et al., 1998; Don et al., 2005). Although it is not trivial to obtain reliable measures of actual polymer size and structure, such measurements might improve understanding of the different effects of N and temperature on flour quality, despite their similar effects on flour composition. Acknowledgements This research was funded by USDA ARS CRIS#532543000-025. We would like to thank Margo Shellenberger Caley and Brad Seaborne, USDA/ARS Grain Marketing and Production Research Center/Hard Winter Wheat Quality Laboratory, Manhattan, KS for the milling and microbreadmaking. Mention of a specific product name by the United States Department of Agriculture does not constitute an endorsement and does not imply a recommendation over other suitable products. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jcs.2006.04.003.

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