Protein and hordein fraction content in barley seeds as affected by sowing date and their relations to malting quality

Protein and hordein fraction content in barley seeds as affected by sowing date and their relations to malting quality 1 2 first-author 1 3 1 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zhanggp@zju.edu.cn †E-mail:zhanggp@zju.edu.cn zhanggp@zju.edu.cn Department of Agronomy, Zhejiang University, Hangzhou 310029, China Department of Agronomy, Zhejiang University, Hangzhou 310029, China Key Laboratory of Oasis Eco-agriculture of Xinjiang Bingtuan, Shihezi 832003, China Key Laboratory of Oasis Eco-agriculture of Xinjiang Bingtuan, Shihezi 832003, China Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China The effect of sowing date on grain protein, hordein fraction content and malting quality of two-rowed spring barley was investigated by using ten commercial cultivars with different grain protein content and the relationships among these traits were examined. The results showed that grain protein content and B hordein content increased as the sowing date postponed and were significantly affected by sowing date, while C and D hordein contents were less influenced by sowing date. There were significant differences in grain protein and hordein fraction content among the ten cultivars. The coefficient of variation of D hordein content was much larger than that of B and C hordein contents, suggesting its greater variation caused by different sowing dates. Beta-amylase activity and diastatic power were also significantly affected by sowing date, with malt extract being less affected. Significant differences in measured malt quality were found among the ten cultivars. Grain protein was significantly correlated with B hordein and malt extract positively and negatively, respectively. There was no significant correlation between beta-amylase activity or diastatic power and grain protein content. B hordein was negatively and significantly correlated with malt extract, but no significant correlations between C hordein, D hordein and malting quality traits. Barley ( Hordeum vulgare L.) Sowing date Protein Hordein Malting quality INTRODUCTION 1 Barley ( Hordeum vulgare L.) is widely used as food and feed, but its most economically important use is for malting and brewing. Many barley characters are involved in malt quality, of which protein content is the most important, as it has been observed to be related to malt qualities, such as extract and diastatic power (Weston et al., 1993 1993 ; Eagles et al., 1995 1995 ). Barley suitable for malting should have low grain protein content, as high protein content will not only reduce malt extract, but also deteriorate final beer quality. On the other hand, protein content has been observed to be correlated with diastatic power (Eagles et al., 1995 1995 ; Molina-Cano et al., 1997 1997 ). This means that protein content has dual effects on malt quality, negative effect by deceasing malt extract and Kolbach index, and positive effect by increasing diastatic power. Diastatic power is closely correlated to beta-amylase activity. For a given cultivar, protein content is positively correlated with beta-amylase activity (Yin et al., 2002 2002 ). However, no significant correlation was detected between beta-amylase activity and protein content among different cultivars planted in the same environment (Zhang et al., 2005 2005 ). Therefore, it is imperative to study the genotypic and environmental variations of protein components and their relationships to malt quality. Hordein, as the main storage protein fraction in barley seeds, accounts for up to half of the total protein in the mature grains, and may be classified into four groups named B, C, D and γ hordeins based on their electrophoretic mobilities (Shewry et al., 1985 1985 ). The B and C fractions account for 70%~80% and 10%~12%, respectively, of the total hordein, while the D and γ fractions are minor components. Molina-Cano et al.( 2001 2001 ) examined the genetic (G), environmental (E) and G×E effects on hordein factions in a mutant (TL43) and its parent cultivar, the malting barley cultivar Triumph, in various seasons at Dundee (E Scotland) and Lleida (NE Spain) and found that there was a G×E interaction for B hordein and no such interaction for C and D hordein contents. When studying the effect of grain protein on the malting quality, Howard et al.( 1996 1996 ) found that variation in growth conditions resulted in a wide range of grain protein contents and malt extract values, as well as obvious difference in the proportions of the individual B, C and D hordeins in grains. Great effort has been made to clarify the relationship between hordein fractions and malting quality. Baxter and Wainwright ( 1979 1979 ) found that B hordein influenced malting quality, while Shewry et al.( 1980 1980 ) and Riggs et al.( 1983 1983 ) reported contrasting results. Peltonen et al.( 1994 1994 ) studied the effect of B, C and D hordeins on malting quality of northern European barleys and found that the B fraction had some effect on malting quality through changing adjusting diastatic power. Howard et al.( 1996 1996 ) evaluated the relationship between D hordein and malting quality of barley under a Mediterranean-type climate in Australia and discovered that the D hordein fraction showed the strongest negative correlation with malt extract across seasons, treatments and cultivars, indicating the existence of a putative causal relationship between D hordein and malting quality. However, Brennan et al.( 1998 1998 ) utilized pairs of near-isogenic barley lines, with and without D hordeins, and did not find any relationship between D hordeins and malting quality. It is often difficult to keep protein content below the upper limit (11.5%), because protein synthesis is variable over the environmental conditions (Smith, 1990 1990 ). In general, higher available nitrogen in soil (Varvel and Severson, 1987 1987 ; Weston et al., 1993 1993 ; Eagles et al., 1995 1995 ), and abiotic stresses including drought (Morgan and Riggs, 1981 1981 ; Coles et al., 1991 1991 ; Grant et al., 1991 1991 ; Birch and Long, 1990 1990 ) or heat, in particular combination with water stress (Macnicol et al., 1993 1993 ; Savin and Nicolas, 1996 1996 ) may increase barley protein content. Hence, an understanding of agronomic factors affecting hordein fractions and the relationship between hordein fractions and malting quality is necessary for a producer to take optimal measures to improve grain quality and for breeders to utilize the relationship to boost breeding efficiency. Thus, the objectives of the current investigation were (a) to determine the effect of sowing date on the content of hordein fractions and malting quality; (b) to evaluate the relationship between hordein fractions and malting quality by changing expression of both traits due to changed environments caused by different sowing dates, so as to sort out some of the conflictive results available. MATERIALS AND METHODS 1 Cultivars 2 Ten commercial two-rowed spring malting barley cultivars planted currently in China were used, i.e. cv. Stark, cv. Logan, cv. Favorit, cv. B1202, cv. Xinpi 1, cv. Klages, cv. CA 2 -1, cv. CDC Thompson, cv. Celink and cv. ND 14636. These cultivars showed similar growth duration, and different grain protein content. Field experiment 2 The field experiment was conducted at the Agriculture Experimental Station of Shihezi University in 2004 and the previous crop was upland cotton. The soil type is grey desert soil with total N of 0.15%, available N, P 2 O 5 and K 2 O is 116.2 mg/g, 34.8 mg/kg and 234.2 mg/kg, respectively. A split-plot experimental design was used with three replicates, sowing date of main plot recorded and cultivar planted in the sub-plot. Sowing dates were 26 March (D1), 10 April (D2) and 25 April (D3). Each plot was 3.5 m×1.6 m, had 8 rows and 0.2 m between rows. The seeding rate was 350 seeds/m 2 . Before sowing, N as urea and P as super-phosphate were applied at the rate of 75 kg/ha and 30 kg/ha, respectively. Another 75 kg/ha of N was top-dressed in equal proportions during tillering and booting stages, respectively. Grain sampling and chemical analysis 2 At maturity, six rows in the middle of each plot were harvested and threshed. The obtained grains were passed over a series of sieves and only the size of fraction between 2.5 and 2.8 mm (about 80% of total sample) was used for subsequent analyses. This eliminated the possibility of influence of grain size on protein content, as observed by Swanston and Molina-Cano ( 2001 2001 ). Grain samples were dried at 80 °C in an oven for 2 d, ground in a Tecator Cyclotec sample mill (Tecator AB, Hoganas, Sweden), and then passed through a 0.5 mm sieve. Protein fractions were sequentially extracted according to Shewry et al.( 1983 1983 ). Isolation of hordeins was based on the method of Howard et al.( 1996 1996 ). Protein content was assayed by Kjeldahl method according to AACC ( 2000 2000 ) and multiplied by 6.25. The B, C and D hordeins can bind Coomassie blue to almost the same extent, suggesting that their contents may be estimated accurately by scanning the relevant stained bands (Rahman et al., 1982 1982 ). The measurement of the hordein band (fraction) was carried out using BandScan 2.0, and their contents were calculated using the following formula. $${\text{Content of hordein fraction (mg/g) = }}\frac{{V{\text{1}} \times {\text{1000/}}V{\text{2}} \times S}}{{W \times {\text{1000}}}}$$ V 1 , the total volume of solution for extraction, ml; V 2 , the volume of sample for SDS-PAGE (sodium dodecyl sulphate polyacrylaminde gel electrophoresis), μl; S , the total value of D, C or B hordeins determined by BandScan 2.0, μg; W , the sample weight for extraction. Beta-amylase activity was measured using the commercial kits (Megazyme Ltd. Ireland) according to McCleary and Codd ( 1989 1989 ). Malt analysis 2 Grain samples (200 g) were micro-malted in Joe White Micromalting System using the following schedule; steeping: 6:14:8:14:4 h at 16 °C (wet:dry:wet:dry:wet), germination: 96 h at 15 °C, kilning: 24 h at 65 °C. Malt extract (EX) and diastatic power (DP) were measured according to EBC ( 1975 1975 ). Statistical analysis 2 Differences among means of both sowing dates and cultivars were evaluated using least significant difference (LSD) and the data analysis was performed with SPSS Version 7.5 (SPSS, Michigan Avenue, Chicago, IL, USA). Coefficients of correlation between grain protein and hordein fraction contents, beta-amylase activity, malt extract and diastatic power were calculated. RESULTS 1 Effect of sowing dates on grain protein and hordein fraction contents 2 Significant differences were found in grain protein and B hordein content among sowing dates, although there was no the difference in C hordein and D hordein contents (Table 1 1 ). Averaged over ten cultivars, the D3 and D1 treatments had the highest and lowest grain protein and B hordein contents, respectively (Table 2 2 ), indicating that the postponed sowing dates led to increased grain protein and B hordein contents. Moreover, the effect of cultivar on grain protein and B hordein contents was much larger than that of sowing date, as reflected by the mean square value. Of the ten cultivars, Klages and Logan had the highest (14.57%) and lowest (12.56%) grain protein content, a highly significant difference. The coefficient of variation (CV) of grain protein content over 3 sowing dates among the 10 cultivars ranged from 7.52% for ND14636 to 2.01% for Klages. Similarly, a dramatic difference in B hordein contents was also found among the ten cultivars (Table 2 2 ), with Favorit and CA 2 -1 being the highest (127.44 mg/g) and the lowest (34.47 mg/g), respectively. Correspondingly CV variation ranged from 10.18% for Stark to 0.94% for Klages, indicating a distinct difference among the cultivars in their environmental response. GP: Grain protein; BH: B hordein; CH: C hordein; DH: D hordein Significant at 95% probability level Significant at 99% probability level 2 4 F -value GP BH CH DH Sowing date ( D ) 6.98 * * 8.50 * * 0.44 0.62 Cultivar ( V ) 19.67 ** ** 18.34 ** ** 28.18 ** ** 13.56 ** ** D × V 1.39 1.49 3.69 ** ** 4.47 ** ** GP: Grain protein; BH: B hordein; CH: C hordein; DH: D hordein The same letter within the same column represents no significant difference at 95% probability level s: Significant difference at 95% probability level; ns: No significant difference at 95% probability level 2 Factor 2 GP 2 BH 2 CH 2 DH Mean (%) CV (%) Mean (mg/g) CV (%) Mean (mg/g) CV (%) Mean (mg/g) CV (%) Sowing date ( D ) D 1 13.19b * * 6.37 62.84b 13.34 9.60a 7.87 0.87a 15.46 D 2 13.82a 6.11 63.85b 11.68 9.91a 6.84 0.80a 16.97 D 3 13.83a 6.89 75.17a 12.92 9.44a 8.45 0.91a 17.99 Cultivar ( V ) Stark 13.16d 3.69 80.78c 10.18 6.44f 6.51 0.83bcd 16.21 Logan 12.56e 5.15 75.79c 10.15 8.36de 5.38 1.03b 11.73 Favorit 14.71a 3.75 127.44a 7.53 10.00c 6.31 1.02bc 3.05 B1202 13.96b 4.46 90.49b 6.06 8.45de 3.58 0.79bcde 14.88 Xinpi 1 13.65bc 4.96 65.74d 2.47 9.61cd 3.79 1.82a 7.13 Klages 14.57a 2.01 42.19f 0.93 8.88cde 7.98 0.96bc 11.11 CA 2 -1 13.77bc 4.76 34.47g 4.34 15.13a 2.88 0.70cde 10.88 CDC thompson 12.58e 2.50 35.19g 6.73 7.97e 4.74 0.51ef 22.78 Celink 13.36cd 5.84 51.29e 9.14 12.58b 3.46 0.29f 14.60 ND14636 13.81bc 7.52 69.49d 2.11 9.08cde 3.84 0.63de 25.85 Interaction of D by V ns ns s s The variation of C and D hordein contents was mainly attributed to cultivars. In terms of C hordein content, CA 2 -1 ranked the highest (15.13 mg/g) and Stark the lowest (6.44 mg/g), with the difference between them being significant. The CV of C hordein content over sowing dates ranged from 7.98% for Klages to 2.88% for CA 2 -1. For D hordein content, Xinpi 1 and Celink were the highest (1.82 mg/g) and the lowest (0.29 mg/g), respectively. In addition, D hordein content had much larger CV of both cultivars and sowing dates than the other three parameters, suggesting its relative instability over environments and genotypes. Effect of sowing date on beta-amylase activity, malt extract and diastatic power 2 There was a significant effect of sowing date on beta-amylase activity and diastatic power, but such effect was not found for malt extract (Table 3 3 ). Beta-amylase activity and diastatic power increased as sowing date was postponed (Table 4 4 ). Thus, D1 and D3 treatments had the lowest and highest beta-amylase activity and diastatic power, respectively. The mean square value of the cultivar was much greater than that of the sowing date for malt extract, indicating that cultivar was the major factor affecting malt extract. The interactions between sowing date and cultivar were significant for all three malt quality traits. AM: Beta-amylase; DP: Diastatic power; EX: Malt extract Significant at 99% probability level 2 3 Mean squares 3 F -value AM DP EX AM DP EX Sowing date ( D ) 1032477 3038.40 0.58 163.24 ** ** 46.86 ** ** 1.22 Cultivar ( V ) 623825 3576.72 122.43 6.48 ** ** 18.23 ** ** 58.98 ** ** D × V 786207.6 3751.34 236.93 4.08 ** ** 9.56 ** ** 57.07 ** ** AM: Beta-amylase; DP: Diastatic power; EX: Malt extract The same letter within the same column represents no significant difference at 95% probability level s: Significant difference at 95% probability level 2 Factor 2 AM 2 DP 2 EX Mean (U/g) CV (%) Mean (WK) CV (%) Mean (%) CV (%) Sowing date ( D ) D 1 999.9c * * 18.03 340.3c 11.28 76.2a 2.15 D 2 1158.3b 12.73 365.2b 15.96 76.1a 4.05 D 3 1260.2a 13.72 381.6a 11.27 76.0a 2.06 Cultivar ( V ) Stark 1047.9e 22.69 333.4g 16.43 76.5b 1.36 Logan 1197.6abc 10.55 376.9c 13.13 76.4b 1.85 Favorit 1280.1a 18.34 368.7d 12.90 78.2a 5.71 B1202 1157.9bcd 14.15 323.2h 12.02 76.6b 2.07 Xinpi 1 1112.5cde 11.59 390.3b 14.86 75.6cd 1.43 Klages 1188.6abc 12.81 392.2b 18.65 73.3e 1.41 CA 2 -1 1022.0e 15.62 329.8g 16.79 73.4d 1.29 CDC Thompson 1076.0de 12.36 361.6e 14.87 76.5b 1.97 Celink 1066.8de 22.86 348.7f 12.14 76.5b 2.02 ND14636 1245.3ab 19.53 399.2a 14.61 76.0c 2.77 Interaction of D by V s s s There were significant differences in three malt quality parameters among ten cultivars (Table 4 4 ). Favorit had the highest beta-amylase activity (1280.1 U/g) and CA 2 -1 the lowest (1022.0 U/g). The CV of beta-amylase activity over sowing dates ranged from 22.86% for Celink to 10.55% for Logan among the ten cultivars. In terms of diastatic power, ND14636 (399.2 WK) ranked the highest and CA 2 -1 (329.8 WK) the lowest. The CV of diastatic power over sowing dates ranged from 12.02% for B1202 to 2.90% for Favorit. Concerning malt extract, Favorit and Klages were the highest (78.2%) and lowest (73.3%), with CV ranging from 1.29% (CA 2 -1) to 5.71% (Favorit). Relationships between protein and hordein fraction contents, and malt quality 2 The coefficients of correlation between grain protein and hordein fraction contents, beta-amylase, malt extract and diastatic power are given in Table 5 5 . Grain protein content was positively correlated with B hordein, but not correlated with C hordein and D hordein contents, suggesting that B hordein is the main component affecting grain protein content. There was a negative correlation between grain protein content and malt extract. No correlation was found between beta-amylase activity, diastatic power and grain protein content. However, beta-amylase activity was positively correlated with diastatic power. B hordein was negatively correlated with malt extract, whereas C and D hordein were not correlated with any malt quality parameter. GP: Grain protein; BH: B hordein; CH: C hordein; DH: D hordein; AM: Beta-amylase; DP: Diastatic power; EX: Malt extract Significant at 95% probability level Significant at 99% probability level GP BH CH DH AM DP EX GP 1 BH 0.3827 * * 1 CH 0.1479 0.1126 1 DH 0.0267 −0.0645 −0.5573 ** ** 1 AM 0.2589 0.2655 0.1095 −0.0182 1 DP 0.1004 −0.1337 −0.3201 0.3560 0.4859 * * 1 EX −0.4458 * * −0.4440 * * 0.0970 0.1710 −0.2776 0.0648 1 DISCUSSION 1 It is well documented that protein content in barley grains is genetically controlled, but easily affected by the environmental conditions (Smith, 1990 1990 ; Kaczmarek et al., 1999 1999 ). The current results showed that barley protein content varied with sowing date and cultivar, which accords with the findings of Yin et al.( 2002 2002 ) and Wang et al.( 2003 2003 ). Moreover, a distinct interaction between sowing date and cultivar was not found. Yin et al.( 2002 2002 ) and Wang et al.( 2003 2003 ) reported no interactions between cultivar and seeding rate, and between cultivar and location, respectively. In contrast, Molina-Cano et al.( 1997 1997 ; 2001 2001 ) found existence of interactions between genotype and location, between cultivar and environment (including location and year). A significant difference was observed among the cultivars in B, C and D hordein contents, which confirms the results reported by Molina-Cano et al.( 2001 2001 ). However, we did not find significant relation between cultivar and sowing date (environment) in B hordein content, as reported by Molina-Cano et al.( 2001 2001 ). There was considerable difference among cultivars and environments in beta-amylase activity (Swanston, 1980 1980 ; Ahokas and Naskali, 1990 1990 ). Arends et al.( 1995 1995 ) reported that the variation of beta-amylase activity among 11 cultivars ranged from 501 U/g to 1100 U/g and among 4 sites from 389 U/g to 1290 U/g, with environmental effect being greater than genotypic effect. Georg-Kraemer et al.( 2001 2001 ) examined beta-amylase activity of 10 Brazilian barley cultivars and found that there was a significant difference among cultivars, ranging from 716.72 U/g to 1470.55 U/g. Macnicol et al.( 1993 1993 ) studied the changes of beta-amylase activity under the controlled environments and found that water stress in the middle of the grain filling stage enhanced accumulation of beta-amylase, while heat stress had little influence. Similarly the present study showed great variation of beta-amylase activity among sowing dates and cultivars, and the existence of interaction between sowing date and cultivar. In addition, our finding of significant differences among cultivars in the aspects of malt extract and diastatic power confirmed the previous reports (Arends et al., 1995 1995 ; Bishop and Day, 1993 1993 ; Eagles et al., 1995 1995 ; Gibson et al., 1995 1995 ; Molina-Cano et al., 1997 1997 ) of similar findings. It may be concluded that the enhancement of beta-amylase activity and diastatic power due to delaying of sowing is closely associated with increased grain protein content, resulting in decreased malt extract. Thus, the complex interaction of grain protein content and malt quality traits should be fully taken into account during making the decision of sowing date for a given cultivar. The relationships among grain protein, hordein fractions and malt quality need to be defined for efficient malting barley breeding and production. The negative and significant correlation between grain protein content and malt extract found in the present study accords with similar findings in other investigations (Howard et al., 1996 1996 ). The positive correlation between beta-amylase and diastatic power observed in this study accorded with several similar findings (Arends et al., 1995 1995 ; Delcour and Verschaeve, 1987 1987 ; Gibson et al., 1995 1995 ). Swanston ( 1980 1980 ) reported a positive correlation between beta-amylase activity and grain protein content. However we did not detect significantly positive correlation between these two factors, as previously reported by Wang et al.( 2003 2003 ), indicating the possibility of developing cultivars with the desirable combination of high beta-amylase activity but low grain protein content. The negative correlation between B hordein and malt extract can be explained by the fact that B hordein is the main factor affecting grain protein content. 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Variation of beta-amylase activity in barley as affected by cultivar and environment and its relation to protein content and grain weight GP Zhang , JM Wang , JX Chen , FB Wu , SR Ding . The effect of cultivar and environment on beta-amylase activity is associated with the change of protein content in barley grains . J Agron Crop Sci , 2005 . in Press . The effect of cultivar and environment on beta-amylase activity is associated with the change of protein content in barley grains The authors wish to express their gratitude to P. Wang and Z.Z. Jin for skilled technical assistance and the two anonymous reviewers for their valuable suggestions and comments. 10.1631/jzus.2005.B1069 Project supported by the National Natural Science Foundation of China (Nos. 30270779 and 30471022) and Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP) (No. 20020335028), China 2005-07-29 2005-09-09 2005-10-28 2022-01-05 Copyright © 2005, Journal of Zhejiang University Science 2005 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 11 6B