Wheat quality related differential expressions of albumins and globulins revealed by two-dimensional difference gel electrophoresis (2-D DIGE)
Introduction
Common or bread wheat (Triticum aestivum L.) is one of the most important food sources in the world. Improvement of grain quality has been a major objective of wheat breeding. The protein composition of mature wheat seeds is important in determining bread-making quality [1]. Recently, there has been an apparent increase in the number of studies on protein synthesis and accumulation during seed development [2], [3], [4].
Wheat proteins are divided into two major categories: prolamins including gliadins and glutenins, and non-prolamins consisting of water-soluble albumins and salt-soluble globulins. The genetic control of non-prolamins appears to be complex with genes assigned to different chromosomes [5]. Compared to gliadins and glutenins, few studies on non-prolamins have been carried out so far. In fact, non-prolamins possess multiple functions during growth and development of wheat. For instance, albumins and globulins include enzymes and inhibitors of enzymes that regulate development at different stages. The relative amounts of essential amino acids such as aspartate, threonine, lysine and tryptophan for humans are more abundant in albumins and globulins, but less than adequate in storage proteins. Furthermore, the compositions of the amino acids in albumins and globulins are relatively well balanced and have highly nutritional value. On the other hand, non-prolamins can lead to some health problems, such as allergy, asthma, diarrhea, and vomiting [6], [7]. For example, WP5212 putative protein, with a high amino acid sequence homology to wheat storage globulin Glb1, might be a diabetes-inducing protein [8].
Non-prolamins also influence the processing and rheological properties of wheat flour [9], [10]. In general, poor quality wheat flours are readily improved by certain enzyme addition such as amylases and/or xylanases. Pentosanase activity is reported to improve gluten elasticity and other bread-making criteria including rheological properties and/or water distribution [11]. Primo-Martin et al. investigated changes in the quantity, quality, and viscoelastic properties of the glutenin macropolymer by the addition of enzymes—pentosanase, glucoseoxidase, laccase, and their combinations. They found that glucoseoxidase gave the least extensible and most resistant dough, and pentosanase/glucoseoxidase resulted in dough with improved extensibility [12]. In recent years, the benefits of the use of endoxylanases, enzymes which are able to hydrolyse the xylan backbone of arabinoxylan has stimulated further interest in the bread-making industry [13], [14]. Further research revealed that endoxylanases attack the arabinoxylan xylan backbone in a random manner, causing a decrease in the degree of polymerization of the substrate and liberating oligomers, xylobiose and xylose with retention of their configuration. Moreover, endoxylanases can decrease the degree of cross-linking of the water-unextractable arabinoxylan to bring arabinoxylan fragments in solution, thus increasing viscosity of the aqueous phase. In bread-making, endoxylanases are almost routinely used in flour mixtures to improve dough handling properties such as oven spring and loaf volume [13].
Some high molecular mass albumins (HMW albumins) and certain globulins (triticins) also have functions of storage proteins by forming part of the gluten protein complex through disulfide bonds [15]. Gupta et al. [16] demonstrated that some HMW albumins disappeared rapidly during seed germination and early seedling growth, suggesting that they might serve as nutritional sources in the early growth stages of wheat. These proteins were not detected in the reduced protein extracted from 3-day-old roots, undifferentiated shoots and 5-day-old leaf tissue, implying that they might be seed-specific as other wheat storage proteins.
Recently, with the development and progress of protein separation technology, more and more non-prolamins have been studied. For example, Wong et al. [17] identified 23 thioredoxin targets in the starchy endosperm of mature wheat seeds using a thiol-specific probe, monobromobimane, with proteomics and enzyme assays. Wong et al. investigated other 68 thioredoxin targets from total KCl-soluble extracts of endosperm and flour and separated by 2-DE in developing wheat seeds in 2004 [18] and further separated by KCl-soluble, albumin/globulin fraction of wheat (T. aestivum L.) starchy endosperm into a methanol-insoluble fraction that contained metabolic proteins and a methanol-soluble fraction [19]. More recently, Vensel et al. [20] performed a 2-DE/MS proteomics study to identify the non-prolamins of wheat endosperm during two developmental stages (10 days post-anthesis (dpa) and 36 dpa), and identified over 250 proteins. Although considerable work of investigating albumins and globulins has been performed, little is known about their proteomic profiles during different grain development stages.
In this study we examined the accumulations of albumin and globulin of the developing wheat grain using a two-dimensional difference gel electrophoresis (2D-DIGE) and MS. 2D-DIGE, a new approach for comparative proteome analysis, is faster than conventional 2-DE analyses and is effective for comparing paired protein samples directly on the same 2-D gel. Its application in wheat grain proteomics is very limited. Differentially expressed non-prolamins from five developmental stages in the post-anthesis period of two bread wheat cultivars with clearly different quality properties were analyzed by 2D-DIGE, and their synthesis and accumulation characteristics were investigated. Our results should be useful for further understanding the expression profiles and functional properties of wheat non-prolamins related to specific synthetic, metabolic, regulatory or protective roles, and processing qualities.
Section snippets
Plant materials
Two bread wheat cultivars Jing 411 and Sunstate were planted at the experimental station of CAAS, Beijing, during 2005–2006. Jing 411 is a poor quality Chinese wheat cultivar that is not suitable for a wide range of wheat end-products and is often used as a control germplasm representing low quality; Sunstate is a prime-hard grade wheat variety from Australia that possesses good bread-making properties and a range of superior flour processing parameters such as extensibility. According to the
Protein expression profiles during grain development
The 2D-DIGE procedure for study of the expression profiles and comparative proteomic analyses of albumins and globulins was successfully optimised. A representative image from a 2-D DIGE gel is shown in Fig. 1A–D. The samples were also separated by preparative gels for protein identification (Fig. 2).
A total of 2500 spots were obtained in each developmental stage using DIGE software (v.5.0.1, GE Healthcare). In general, the proteome profiles were similar between Jing 411 and Sunstate over all
Discussion
In the past thirty years, many studies have focused on the genetic basis of wheat quality. Phenotypic differences among wheat cultivars with regard to grain and dough quality traits result from the action of many different genes. In addition, the effects of climatic conditions during plant growth and development on quality often exceed those of genotypes [23], [24]. For example, it is well documented that temperature is an important environmental factor affecting dough quality. In the present
Acknowledgements
We are grateful to Professor Robert McIntosh for constructive suggestions in reviewing the manuscript. This research was financially supported by grants from the National Natural Science Foundation of China (30830072 and 30771334), the Ministry of Science and Technology of China (2002CB111300 and 2006AA10Z186) and the Key Developmental Project of Science and Technology, Beijing Municipal Commission of Education (KZ200910028003).
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These authors contributed equally to this work.