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Title: Is there a signal transduction pathway that links events at the plasma membrane to the phosphorylation state of the mitochondrial pyruvate dehydrogenase complex?

item Miernyk, Jan
item Szurmak, Blanka
item Tovar-mendez, Alejandro
item Randall, Douglas
item Muszynska, Grazyna

Submitted to: Physiologia Plantarum
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/10/2006
Publication Date: 12/15/2006
Citation: Miernyk, J.A., Szurmak, B., Tovar-Mendez, A., Randall, D.D., Muszynska, G. 2006. Is there a signal transduction pathway that links events at the plasma membrane to the phosphorylation state of the mitochondrial pyruvate dehydrogenase complex?. Physiologia Plantarum. 129:104-113.

Interpretive Summary: Respiration is the use of energy by living cells to do work. Both growth and reproductive success are directly coupled to rates of respiration. As a result, respiration must be carefully controlled, or wasted energy would cause decreased crop yields and reduced agricultural productivity. The control of respiration in plant cells is a subject of ongoing study. Plant cells contain multiple forms of a protein thought to be important in overall control of respiration. A method was developed to precisely measure low levels of this protein. The method additionally allows measurement of a form of the protein that has been modified by addition of a phosphorous molecule. The ratio of the plus and minus phosphorous forms was shown to differ under different plant growth conditions. This information will be important to researchers in their attempts to increase agricultural productivity by altering the control of plant cell respiration, and to other plant scientists who will try to design more efficient crop plants through either classical breeding or biotechnology.

Technical Abstract: Monoclonal antibodies against the E1a subunit were used to quantify the mitochondrial pyruvate dehydrogenase complex (mtPDC) by enzyme-linked immunosorbent assay (ELISA). Recombinant Arabidopsis thaliana pyruvate dehydrogenase (E1) was used to calibrate the ELISA. Antibodies against a synthetic phospho-peptide corresponding to phosphorylation site one of E1alpha were used in an ELISA to quantify phospho-PDC (P-PDC). For calibration of the second ELISA, recombinant E1 was phosphorylated in vitro with recombinant A. thaliana E1-kinase. The two ELISA were used to quantify mitochondrial total- and P-PDC in clarified homogenates from Nicotiana tabacum BY-2 suspension cells. The level of mtPDC remained constant throughout the 7-day growth cycle at 25 + or - 1 ng/gfw. During the lag (day 0 to 2) and stationary (day 7) stages of the growth cycle the mtPDC was completely phosphorylated (inactive), while during the log-growth stage it was completely dephosphorylated (active). Exposure of 3- or 7-day post transfer suspension cells to osmotic stress significantly decreased proportion of P-PDC. A series of pharmacological studies were undertaken to gain insight into the signal-transduction pathways coupling osmotic stress perception with control of mitochondrial respiration. Results from these studies indicate a signal transduction pathway linking stress perception to control of mitochondrial respiration that includes protein kinases and phospho-protein phosphatases.