|Hurkman ii, William|
Submitted to: International Gluten Workshop
Publication Type: Abstract only
Publication Acceptance Date: 12/3/2003
Publication Date: 7/1/2004
Citation: Hurkman, W.J., Tanaka, C.K., Vensel, W.H., Wong, J.H., Balmer, Y., Buchanan, B.B. 2004. Analysis of wheat endosperm proteins during grain development and in response to high temperature using proteomics. International Gluten Workshop. p. 169-172 Interpretive Summary:
Technical Abstract: To gain a better understanding of the genetic basis for wheat grain development, a detailed picture of major events and the specific proteins involved must be established. Environmental interactions during grain-fill alter the time course for grain development and influence final grain weight, protein content, and starch content (Altenbach et al. 2003 J. Cereal Sci. 37: 9). Identification of molecular events influenced by environment would provide new insights into mechanisms that determine grain yield and quality. We are using a proteomics approach to identify wheat endosperm proteins, establish profiles for their accumulation during grain development, and characterize the effects of high temperatures on these profiles. We are focusing on three proteomes, the gluten proteins, the soluble proteins, and a subset of the soluble proteins, the thiol proteins. The gluten proteins are important to the value of the wheat crop, because they impart the rheological properties of elasticity and extensibility to wheat doughs that are essential for the production of baked products. The far less abundant soluble proteins are involved in biosynthetic and regulatory processes that are central to grain development. Disulfide bonds are not only important to the functionality of the gluten proteins, but they are emerging as a primary mechanism for altering the activity and structure of metabolic proteins. Wheat (Triticum aestivum L. cv. Butte 86) plants were grown in a climate-controlled greenhouse that had an average maximum daytime temperature of 24oC and nighttime temperature of 17oC. At various times after anthesis, plants were transferred to a second greenhouse with identical growing conditions except that the average maximum daytime temperature was 37oC and nighttime temperature was 28oC. Grain was harvested throughout development, endosperm collected, and soluble and storage proteins analyzed by 2-D gel electrophoresis. Proteins were quantified and developmental profiles were created using computer-based image analysis. Over 600 profiles were created for the soluble proteins and 400 for the gluten proteins. The identities of the storage proteins were confirmed by mass spectrometry. A map of over 100 soluble proteins identified by mass spectrometry was established; the majority function in protein synthesis, starch metabolism, and defense. Of these, 35 were identified as disulfide-containing proteins using a fluorescent thiol-specific probe, monobromobimane, coupled with 2D gel electrophoresis (Yano, H. et al. 2001 PNAS 98: 4794). The identification of thiol proteins opens a new door to studies on the identification of regulatory mechanisms, such as those based on phosphorylation and thiol redox status, in metabolic processes in wheat endosperm. The gluten proteins accumulated coordinately and all were present by mid development. High temperatures shortened this developmental program. The accumulation patterns for the soluble proteins were complex. However, some proteins accumulated during early, middle, or late stages of development and thus may be indicators of developmental age. The response of the soluble proteins to high temperature was similarly complex and varied protein by protein. This proteomics approach has provided a detailed picture of endosperm development at the protein level and the complex nature of the endosperm's response to high temperatures during grain-fill.