Elsevier

Journal of Proteomics

Volume 73, Issue 2, 1 December 2009, Pages 279-296
Journal of Proteomics

Wheat quality related differential expressions of albumins and globulins revealed by two-dimensional difference gel electrophoresis (2-D DIGE)

https://doi.org/10.1016/j.jprot.2009.09.014Get rights and content

Abstract

Comparative proteomics analysis offers a new approach to identify differential proteins among different wheat genotypes and developmental stages. In this study, the non-prolamin expression profiles during grain development of two common or bread wheat cultivars (Triticum aestivum L.), Jing 411 and Sunstate, with different quality properties were analyzed using two-dimensional difference gel electrophoresis (2-D DIGE). Five grain developmental stages during the post-anthesis period were sampled corresponding to the cumulative averages of daily temperatures (°C: 156 °C, 250 °C, 354 °C, 447 °C and 749.5 °C). More than 400 differential protein spots detected at one or more of the developmental stages of the two cultivars were monitored, among which 230 proteins were identified by MS. Of the identified proteins, more than 85% were enzymes possessing different physiological functions. A total of 36 differential proteins were characterized between the two varieties, which are likely to be related to wheat quality attributes. About one quarter of the proteins identified expressed in multiple spots with different pIs and molecular masses, implying certain post-translational modifications (PTMs) of proteins such as phosphorylations and glycosylations. The results provide new insights into biochemical mechanisms for grain development and quality.

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).

References (53)

  • L. Tamás et al.

    Heterologous expression and protein engineering of wheat gluten proteins

    J Cereal Sci

    (2006)
  • W.J. Hurkman et al.

    Effect of high temperature an albumin and globulin accumulation in the endosperm proteome of the developing wheat grain

    J. Cereal Sci

    (2009)
  • D.T. McLachlin et al.

    Analysis of phosphorylated proteins and peptides by mass spectrometry

    Curr Opin Chem Biol

    (2001)
  • S.B. Altenbach et al.

    Temperature, water and fertilizer influence the timing of key events during grain development in an US spring wheat

    J Cereal Sci

    (2003)
  • M.A. Ali-Benali et al.

    Comparative expression of five Lea genes during wheat seed development and in response to abiotic stresses by real-time quantitative RT-PCR

    Biochem Biophys Acta

    (2005)
  • Y. Ikeda et al.

    Specific binding of a 14-3-3 protein to autophosphorylated WPK4, an SNF1-related wheat protein kinase, and to WPK4-phosphorylated nitrate reductase

    J Biol Chem

    (2000)
  • E.A. MacGregor et al.

    Structural models of limit dextrinase inhibitors from barley

    J Cereal Sci

    (2000)
  • Y.G. Zhao et al.

    Hsp90 phosphorylation is linked to its chaperoning function

    J Biol Chem

    (2001)
  • F.M. Dupont et al.

    Differential accumulation of sulfur-rich and sulfur-poor wheat flour proteins is affected by temperature and mineral nutrition during grain development

    J Cereal Sci

    (2006)
  • F.M. Dupont et al.

    Protein accumulation and composition in wheat grains: effects of mineral nutrients and high temperature

    Euro J Agro

    (2006)
  • W.N. Sun et al.

    Small heat shock proteins and stress tolerance in plants

    Biochim Biophys Acta

    (2002)
  • L. Wang et al.

    Overexpression of chloroplast-localized small molecular heat-shock protein enhances chilling tolerance in tomato plant

    J Plant Physio Mol Biol

    (2005)
  • E. Johansson et al.

    Effects of wheat cultivar and nitrogen application on storage protein composition and breadmaking quality

    Cereal Chem

    (2001)
  • B.C. Clarke et al.

    Genes active in developing wheat endosperm

    Funct Integr Genomics

    (2000)
  • E. Johansson et al.

    Protein polymer build-up during wheat grain development: influences of temperature and nitrogen timing

    J Sci Food Agric

    (2005)
  • W. Weiss et al.

    Identification and characterization of wheat grain albumin/globulin allergens

    Electrophoresis

    (1997)
  • Cited by (79)

    • Proteomics as a tool for analyzing plant responses to abiotic and biotic stresses

      2023, Genomics, Transcriptomics, Proteomics and Metabolomics of Crop Plants
    • Differential expression of albumins and globulins of wheat flours of different technological qualities revealed by nanoUPLC-UDMS<sup>E</sup>

      2018, Food Chemistry
      Citation Excerpt :

      The enzyme is involved in the carbohydrate metabolism, preventing starch degradation by inhibiting α-amylase activity (Debiton et al., 2011). These results are in agreement with analytical experimentation studies that carried out a mapping of metabolic wheat protein using proteomic approaches (Gao et al., 2009; Merlino et al., 2009). However, in these works, the mentioned proteins did not appear as differentially expressed, probably due to the relatively low number of identified proteins.

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text