Author
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Turley, Rickie |
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Taliercio, Earl |
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Submitted to: Plant Physiology and Biochemistry
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 3/13/2008 Publication Date: 4/26/2008 Citation: Turley, R.B., Taliercio, E.W. 2008. Cotton Benzoquinon Reductase: Up-Regulation During Early Fiber Development and Heterologous Expresson and Characterization in Pichia Pastoris. Plant Physiology and Biochemistry. 46:780-785 Interpretive Summary: Cotton is a highly valued natural fiber which accounts for approximately $120 billion of business revenues per year in the USA. Yields of cotton lint production have reached a plateau in recent years which could threaten the continued success of the industry. We have been identifying proteins which may be involved in increasing yields of fiber. Ovule proteins have been compared between cotton lines which produce fiber and cotton mutants which do not produce fiber. Any differences in proteins, such as the appearance or disappearance of a protein could give clues as to what is occurring in the ovule during first production of fiber. The mutant line used is identical to the fiber producing ovules in everything except fiber production. We found two proteins which are only in the fiber producing line and only after the fiber begins to develop. These two proteins were identified as benzoquinone reductases which function in the destruction of dangerous compounds that the plants produce. We characterized cotton benzoquinone reductases and found that they occur in the stem and in the fibers at all ages. It has been reported that toxic compounds exist in ovule epidermal cells which disappear in cells prior to becoming fiber. Benzoquinone reductase could play a role in detoxifying these compounds. Understanding the role of this protein and other proteins in fiber development will allow us to develop strategies for increasing the number of fibers on each seed and lint percent. Technical Abstract: Benzoquinone reductase (BR) is an enzyme which catalyzes the bivalent redox reactions of quinones without the production of free radical intermediates. Using 2-D PAGE, two proteins were found to be up-regulated in wild-type cotton ovules during the fiber initiation stage. These proteins were excised from the gel, partially sequenced and identified to be BR isoforms. PCR was used to amplify the full length coding regions of 609 bp together and once cloned, the restriction enzyme HindIII was used to identify the clones encoding the BR1 (1 site) and BR2 (2 sites) isoforms. Both deduced protein sequences had 203 residues which differed at 14 residues. The molecular mass and pIs were similar between the measured protein (2D PAGE) and the theoretical protein (deduced). Heterologous proteins BR1 and BR2 were produced for further study by ligating the BR1 and BR2 clones in frame into the alpha-factor secretion sequence in pPICZ alpha A vector and expressed with Pichia pastoris. Six Pichia colonies were evaluated for expression of each BR isoform. Both BR1 and BR2 were approximately 26.5 kDa and did enzymatically reduce 2,6 dimethoxybenzoquinone similar to the fungal BR. |
