Grain position in spike of wheat (Triticum aestivum L.) affects glutenin macropolymer particles distribution
La posición del grano en la espiga de trigo (Triticum aestivum L.) afecta la distribución de las partículas del macropolímero de glutenina
Zhongmin Dai1, 2, Yanping Yin2, Yong Li2, Li Cao2, Zhenlin Wang2*
1 Biology Department, Dezhou University, Dezhou 253023, China.
]]> 2 State Key Laboratory of Crop Biology, Agronomy College, Shandong Agricultural University, Tai'an 271018, China. * Author for correspondence (dzm66@126.com).
Received: August, 2013.
Approved: February, 2014.
Abstract
Glutenin macfopolymef (GMP), an important component of wheat (Triticum aestivum L.), consists of high weight (HMW-GS) and low-molecular-weight (LMW-GS) gutenin subunits. In wheat, the subunit composition and GMP characteristics are important factors affecting processing quality. The objective of this study was to characterize the effects of soil water status, cultivar and grain position in wheat spike, on GMP particles distribution. Wheat cultivars Jinan 17, Yannong 24 and Lumai 21 with different end-use quality were used in 2009-2011 growing season, and two water regimes (irrigated and rainfed): two irrigations (750 m3 ha-1 each at jointing and booting stage) for irrigated, and none for rainfed. The experimental design was a complete randomized block with three replicates; data were analyzed whith ANOVA and Tukey test (p ≤ 0.05) for treatment means, using SPSS. All determinations were replicated three times. Plot dimension was 3 mx3 m. At maturity, the heads flowering on the same date were sampled and detached as basal and distal grains according to the position on spikelets. GMP particles distribution and HMW-GS content were affected (p ≤ 0.05) by soil water, genotype and grain position. Basal grains on middle spikelets showed higher (p ≤ 0.05) HMW-GS and GMP content than those in distal grains at maturity. The volume and surface area percentage of >100 µm GMP particles in basal grains were higher (p ≤ 0.05) than that in distal grains, but those of <12 µm particles in basal grains were lower than that in distal grains. Compared with irrigation, percent volume and surface area of >100 µm GMP particles in basal and distal grains were significantly increased (p ≤ 0.05), and content of HMW-GS and GMP were also increased in rainfed condition. As a result, basal and distal grains have different quality characteristics and application. A promising breeding strategy would be to improve grain yield and quality by increasing average grain weight rather than grain number within a spikelet. When managing for wheat production, the water factor was the most important manipulation to regulate the yield and quality.
Key words: High-molecular-weight glutenin subunit, glutenin macropolymer, grain position, rainfed cultivation, wheat.
Resumen
]]> El macropolímero de glutenina (GMP), un componente importante del trigo (Triticum aestivum L.), se compone de sublimidades de glutenina con peso molecular alto (HMW-GS) y bajo (LMW-GS). En el trigo, la composición de subunidades y características de GMP son factores importantes que afectan la calidad del procesamiento. El objetivo de este estudio fue caracterizar los efectos del estado hídrico del suelo, los cultivares y la posición del grano en la espiga de trigo sobre la distribución de las partículas GMP. Los cultivares de trigo Jinan 17, Yannong 24 y Lumai 21, con diferente calidad en el uso final, fueron utilizados en la temporada de crecimiento 2009-2011 y se aplicaron dos regímenes hídricos (riego y temporal): dos riegos (750 m3 ha-1 cada uno en la etapa de elongación del tallo y formación de la espiga) para los de riego, y ninguno para los de temporal. El diseño experimental fue bloques completos al azar con tres repeticiones y los datos se analizaron con ANDEVA y la prueba de Tukey (p ≤ 0.05), para comparar de las medias de los tratamientos, usando SPSS. Todas las determinaciones se repitieron tres veces. La dimensión de la parcela fue de 3 mx3 m. En la madurez, las espigas floreciendo en la misma fecha se muestrearon y separaron como granos basales y distales según la posición en las espiguillas. La distribución de las partículas GMP y el contenido de HMW-GS fueron afectados (p ≤ 0.05) por el agua del suelo, el genotipo y la posición del grano. Los granos basales en las espiguillas de la parte media tenían más (p ≤ 0.05) HMW-GS y GMP que los distales en madurez. El volumen y porcentaje del área superficial de las partículas GMP >100 µm en granos basales fueron mayores (p ≤ 0.05) que en los distales, pero los de partículas de <12 µm en los granos basales eran menores que en los distales. En comparación con el riego, el porcentaje de volumen y área superficial de partículas GMP > 100 µm en los granos basales y distales aumentaron significativamente (p<0.05), y los contenidos de HMW-GS y GMP también aumentaron en condiciones de temporal. Como resultado, los granos basales y distales tienen características diferentes de calidad y aplicación. Una estrategia de mejora genética prometedora sería mejorar el rendimiento del grano y su calidad aumentando el peso medio del grano en lugar del número de granos dentro de una espiguilla. En la producción de trigo, el manejo del factor agua fue el más importante para regular el rendimiento y la calidad.Palabras clave: subunidad de glutenina de alto peso molecular, macropolímero de glutenina, posición del grano, cultivo de temporal, trigo.
INTRODUCTION
Wheat (Triticum aestivum L.) is the most consumed food crop in the world, being processed for a range of breads, baked goods, pasta, and noodles. In wheat, glutenin macropolymer (GMP) is a major component of grain and an important factor affecting the processing quality of wheat (Don et al., 2005). GMP consists of high molecular weight glutenin subunits (HMW-GS) linked with low molecular weight glutenin subunits (LMW-GS) through disulfide bonds (Goesaert et al., 2005). HMW-GS play an important role in determining the glutenin protein network structure (Don et al., 2006), and LMW-GS may also have a specific effect on glutenin aggregation (Dupont and Altenbach, 2003). GMP that consists of a higher ratio of HMW-GS to LMW-GS is correlated with improved wheat flour quality (Pechanek et al., 1997).
There is a significant correlation between amount of GMP in wheat grains and flour quality variables, such as loaf and physical dough properties (Weegels et al., 1996; Tarekegne and Labuschagne, 2005). GMP quantity is an indicator of wheat flour quality and the rigidity of the GMP gel-layer, expressed as the G' (Pa) value obtained from a dynamic rheological measurement, correlates with bread-making quality (Pritchard, 1993). There is also a strong positive correlation between dough development time and G' plateau values of GMP isolated from flour (Bekkers et al., 2000). GMP particle distribution size is another important factor for wheat bread-making quality (Dupont and Altenbach, 2003). According to Don et al. (2006), GMP particle size strongly correlates with dough development time. Both the quantity and composition of HMW-GS correlates with dough properties and bread-making quality. Besides, subunits 2+12 and 5+10 are strongly correlated with weak and strong gluten properties, respectively. However, total amount of HMW-GS and its relative amount to total grain protein play more important roles than HMW-GS composition in determining flour quality (Vasil and Anderson, 1997; Sliwinski et al., 2004). Thus, accumulation of HMW-GS in grain could be important for wheat quality development.
Content and size distribution of GMP in wheat grains are both genetically and environmentally controlled; individual grains of wheat cultivars show significant variation in grain mass and nutrient concentration depending on their position within the spike (Stoddard, 1999). Grains in distal positions on the spikelet have lower N, macronutrient and micronutrient concentrations than those in proximal positions on basal-central spikelets (Calderini and Ortiz-Monasterio, 2003). Environmental factors, such as soil water content, have also significant effect on GMP characteristics. Moisture stress during grain-filling increases flour protein content and sodium dodecyl sulfate (SDS) sedimentation volume (Saint Pierre et al., 2008). Drought promote HMW-GS accumulation at early grain filling stage, whereas the case is just opposite at late grain filling stage (Jiang et al., 2009). According to Li et al. (2011), increased N levels promote accumulation of HMW and LMW-GS, GMP content, and proportion of the larger particle of GMP under irrigated condition; besides, protein content also increased under rainfed condition. Both dough development time and stability time are longest with single post-anthesis irrigation, while the second irrigation lead to shortened dough development and stability time and weakened gluten strength, as well as decreased glutenin polymerization index and average size of GMP (Yao et al., 2010). Hence, a better understanding of size distribution of GMP particles in wheat grains could help determine suitable management practices and design wheat breeding strategies aimed at increasing grain yield without affecting grain quality.
In the north of China wheat is traditionally irrigated two or three times, at jointing and booting stages (or grain filling stage). The booting stage is the critical period of water requirement. However, frequent soil water stress has influenced both the dry matter production and the grain formation of wheat (Ma et al., 2007). Water-saving irrigation technology more and more important in recent years. Hence, a better understanding of formation of glutenin in wheat grains under different irrigation patterns could help determine suitable management practices.
The objectives of this study were to assess granule size distribution of GMP in grains set at different positions within spike in three winter wheat cultivars, and to determine the role played by soil water status in determining GMP and HMW-GS contents.
]]> MATERIALS AND METHODS
Plant materials and experiment description
The experiment was conducted at the experimental farm of the Research Institute of Agricultural Science (37 °N, 116 °E), Dezhou, PR China, during 2009-2010 and 2010-2011 growing seasons. Treatments were three wheat cultivars and two irrigation systems. Three winter wheat cultivars with different end-use quality were used: Jinan 17, Yannong 24 and Lumai 21. Yannong 24 and Lumai 21 were sown on October 15, 2010 and the 0-20 cm soil layer contained 83.6 mg kg-1 available N, 18.2 mg kg-1 available phosphate and 95.2 mg kg-1 available K. Jinan 17 and Lumai 21 were sown on October 17, 2009; in the soil the content of available N, P and K was 81.5, 17.6 and 93.6 mg kg-1. Irrigation systemsa were: 1) two irrigations (750 m3 ha-1 each at jointing and booting stages) and, 2) rainfed (no irrigation). The moisture content in soil after anthesis is shown in Figure 1. Plot dimension was 3 mx3 m and each experimental unit consisted of 10 rows of wheat with 25-cm spacing. Regional crop farming practices were implemented to minimize pest, disease, and weed incidence.
Plant sampling
Heads fully flowering on the same date were labeled with thread. At maturity, the labeled heads were hand-harvested on June 14, 2010 and June 12, 2011. Each sample was oven-dried at 70 °C for 72 h and used for measuring GMP particles distribution and content of GMP and HMW-GS. From the basal five to 12 spikelets on spikes of these cultivars, the first and second basal grains on each spikelet were detached as basal grains, whereas the most distal grain on the same spikelet was detached as distal grains. The basal and distal grains were respectively detached and then ground into flour to perform determinations.
GMP content analysis
A quantity of 0.05 g of flour was dispersed, mixed with 1 mL of SDS (98.5 %, Sigma) and centrifuged at 15,500 x g for 15 min using an Allegra X-64R centrifuge (Beckman, San Francisco, CA). Glutenin macropolymer content was measured using TU-1901 dual-wavelength spectrophotometer (Persee Instruments, Peking, China). Glutenin macropolymer content was calculated with a set of Kjeldahl protein values.
Isolation of glutenin macropolymer
Glutenin macropolymer-gel was isolated by dispersing 1.4 g of defatted flour into 0.05 mol L-1 SDS (28 mL) and then centrifuged at 80 000 x g for 30 min at 20 °C using an ultracentrifuge Beckman L-60 (Beckman, San Francisco, CA) as described by Graveland et al. (1985). The GMP gel-layer was collected on the top of the supernatant.
Coulter laser particle size analysis
]]> One gram of GMP-gel was added to 8 mL of 0.05 mol L-1 SDS solvent, the tube was sealed and placed on a roller-bank for 3 h at 23 °C and analyzed with a Coulter Laser LS13320 (Beckman Coulter Instruments, San Francisco, CA). From the pattern, the GMP surface area distribution and volume distribution was measured and calculated.HMW-GS content analysis
The quantification of the HMW-GS was performed according to Liang et al. (2002). HMW-GS were first separated by SDS-polyacrylate gel electrophoresis (SDS-PAGE), the gel was stained with 0.05 % Coomassie Brilliant Blue B250 (Sigma) for 24 h, and destained in distilled water for 48 h; then each band was separately cut off from the gel and placed in an Eppendorf tube. Depending on the intensity of each band, 1 mL of 50 % isopropyl alcohol containing 3 % SDS was added to the tube which was incubated at 37 °C for 24 h until the gel cleared. The extraction was monitored at 595 nm with a UV-2401 Shimadzu spectrophotometer (Shimadzu Corporation, Kyoto, Japan).
Experimental design and statistical analysis
The experimental design was a complete randomized block with three replicates, data were analyzed using ANOVA and Tukey test (p ≤ 0.05) for treatment means, with SPSS software. General correlation coefficients were calculated between diameter of GMP particles and content of GMP and HMW-GS.
RESULTS AND DISCUSSION
Content of total HMW-GS in grains
In the three cultivars, the HMW-GS total content in basal grains was higher (p ≤ 0.05) than in distal grains in the two irrigation treatments (Figure 2). Irrigated and rainfed conditions had different influence on HMW-GS content. Under irrigation, the HMW-GS content in basal and distal grains decreased when compared with those under rainfed. The results indicated that rainfed was beneficial for the accumulation of HMW-GS in wheat.
These results are similar to those of Li et al.( 2011), who indicated that rainfed could increase the contents of HMW-GS and LMW-GS and H/L values in wheat, although the proportions of single subunits varied under the irrigation treatments.
]]> GMP content in grainsChanges of GMP content in basal grains were higher than in distal grains (Figure 3), indicating that more glutenin is accumulated in basal grains. Besides, under rainfed GMP content in basal and distal grains were significantly increased by 9.318.7 % and 17.2-24.8 %, as compared to irrigation.
These results were similar to those of Stoddard (1999), who concluded that grains from distal florets were always smaller and had lower nitrogen contents than those from the two proximal florets on each spikelet. There were differences in grain mass and N between florets within spikelets, and between spikelets within head varied according to cultivar.
There are some discrepancy between the results of the present study and those of Li et al. (2011), who found that the GMP contents increased as N rates increased under irrigation. But under the rainfed, the GMP contents showed difference with the N level. Some of these differences may be related mainly to the water-N interaction.
Particle volume distribution
The volume percentages of particles >100 µm in basal grains were 19.6-34.3 % and higher (p ≤ 0.05) than in distal grains (11.1-17.4 %) under irrigation and rainfed, whereas volume percentages of 12100 µm and <12 µm particles in distal grains were higher than those in basal grains (Table 1).
Under rainfed, volume percentages of >100 µm in basal and distal grains were increased (except Jinan 17 distal grains in 2009-2010), whereas those of <12 µm were decreased compared to irrigation. These results indicate that soil water deficit (rainfed condition) would increase volume percentages of large particles. Results also show that particle size distribution in basal and distal grains was significantly different, which might be due to differences in development time of floret and structure of vascular bundle in individual grains on a wheat spike. In basal grains, the development time of floret was earlier and the number and cross-sectional area of vascular bundles were higher than those in distal grains, which was beneficial for transportation and accumulation of N. As a result, basal grains have a stronger capacity of protein synthesis than distal grains, which might promote the development and growth of large GMP particles (Ru et al., 2006; Xiong et al., 2009).
Surface area distribution
Under irrigated and rainfed conditions, surface area percentages of > 100 µm in basal grains were 1.2-2.9 %, i.e. higher than those in distal grains (0.61.1 %), whereas surface area percentages <12 µm in basal grains were 74.1-85.8 % and lower than in distal grains (80.2-88.9 %) (Table 2). Compared with irrigation, the surface area percentages of >100 µm increased by 12.0-50.0 % and 0.0-83.3 % in basal and distal grains under rainfed, indicating that drought could increase surface area percentage of large particles in the three wheat cultivars.
Environment has a strong effect on the size of GMP particles and on GMP content in wheat grains (Yao etal., 2010; Li etal., 2011). Results of the present study confirmed that GMP particles size was affected by irrigation regime, since volume percentage and surface area percentage of large particles (>100 /mm) increased under rainfed, indicating that soil water deficit led to a change in percentage volume and surface area of GMP particles.
]]> According to Don et al. (2003), GMP consists of spherical glutenin particles and originates from protein bodies observed in developing grain. Van Herpen et al. (2008) suggest that protein bodies were the building blocks for the formation of larger glutenin particles during the desiccation phase of kernel development. A close correlation was found between accumulation of GMP and rapid loss of water during desiccation (Carceller and Aussenac, 2001). The premature desiccation of wheat grain induces SDS-insoluble polymer formation and percentage of SDS-insoluble polymers as a proportion of total polymers can increase from less than 10 % at the end of kernel ripening to 50 % in as little as 10 d. In the present study, the volume and surface area percentages of large GMP particles under rainfed conditions were significantly increased, suggesting that water-deficit treatment is probably beneficial for desiccation of grains and thus promote the formation of large GMP particles.Relationships between GMP size distribution and content of GMP and HMW-GS
Correlation between GMP size distribution and HMW-GS content with volume percentage <12 µm GMP particles was negative, but it was positive with >100 µm GMP particles (Table 3), suggesting that large GMP particles have high GMP content.
CONCLUSIONS
There is a higher volume and surface area percentages of large particles in basal grains than in distal grains, whereas those of small particles were the opposite. High-molecular-weight gutenin subunit and GMP content in basal grains were higher than that in distal grains, indicating that more GMP particles were accumulated in basal grains than in distal grains, and then the small ones assembled larger ones. The soil water deficit enhanced accumulation of HMW-GS and GMP, and was prone to forming larger GMP particles in basal and distal wheat grains; thus, irrigation levels could influence both glutenin biosynthesis and HMW-GS content, and eventually influence wheat flour quality. Regarding wheat production, irrigation was the most important factor for regulating yield and quality.
ACKNOWLEGEMENTS
This research was supported by the National Natural Science Foundation of China (31271667, 30871477), the Natural Science Foundation of Shandong Province, China (ZR2010CM044), the National Basic Research Program of China (973 Program 2009CB118602), and State Key Laboratory of Crop Biology (Grant no. 2012KF01) at Shandong Agricultural University, China.
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