Page 213 - PC2019 Program & Proceedings
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PLANT CANADA 2019

               S211. Autophosphorylation inhibits the Ca2+-dependent protein kinase RcCDPK1 from developing
               castor oil seeds
                          *
               Kilburn, R. ; W. Snedden; W. Plaxton
               Queen's University

               Phosphoenolpyruvate carboxylase (PEPC) is a tightly-regulated enzyme that plays diverse roles in plant
               metabolism, particularly the anaplerotic replenishment of Krebs’ cycle intermediates withdrawn for
               biosynthesis. An unusual PEPC isozyme known as ‘bacterial-type PEPC’ (BTPC) is highly expressed as a
               catalytic and regulatory subunit of a novel Class-2 PEPC heteromeric complex in developing castor oil
               seeds (COS). The ‘allosterically-desensitized’ Class-2 PEPC dynamically associates with mitochondria in
               vivo, and was hypothesized to mediate a large anaplerotic flux of PEP to oxaloacetate in support of COS
               storage oil and protein synthesis, while simultaneously recycling respired CO2. During COS development
                                                                                                          2+
               BTPC is subject to in vivo inhibitory phosphorylation at Ser451, catalyzed by RcCDPK1, a soluble Ca -
               dependent protein kinase. Although CDPK autophosphorylation has been well established, its functions
               remain elusive. Most CDPK transphosphorylation assays have employed non-physiologically relevant
               protein or synthetic peptide substrates, whereas the influence of autophosphorylation on CDPK activity
                                           2+
               may be substrate-dependent. Ca -stimulated autokinase activity of recombinant RcCDPK1 was readily
               detected and multiple autophosphorylated Ser, Thr, and Tyr residues were mapped via nanoHPLC-
                                                 32
               MS/MS. Quantitative assays using [γ- P]ATP demonstrated that prior autophosphorylation markedly
               inhibits RcCDPK1’s ability to transphosphorylate its BTPC substrate at Ser451. Ongoing research
                                                                                                  2+
                                                                                2+
               includes assessing the impact of autophosphorylation on RcCDPK1’s Ca -sensitivity, and Ca -
               dependent interaction with BTPC. Results will provide insights into the link between plant C-metabolism,
                  2+
               Ca -dependent signaling, and the biological relevance of CDPK autophosphorylation.
               Ryan Kilburn (12rjk2@queensu.ca)




               S212. Recent advances in plant ubiquinone (Coenzyme Q) biosynthesis and engineering
                                                      1
                              1
                                          1
               Soubeyrand, E. ; T. Johnson ; S. Latimer ; A. Bernert ; M. Kelly ; J. Kim ; T. Colquhoun ; A.
                                                                  1
                                                                                                    1
                                                                                     1
                                                                            1
                                1
               Block ; G. Basset
                     2
               1 University of Florida
               2 USDA
               Ubiquinone is a liposoluble and redox-active molecule that is made up of benzenoid and prenyl moieties.
               It serves as a vital electron carrier in the respiratory chain of mitochondria and some bacteria, and doubles
               as a potent lipid and protein antioxidant. Recent evidence from our laboratory indicates that land plants
               have evolved the unprecedented ability to derive the benzenoid ring of ubiquinone from the metabolism
               of phenylpropanoids (Plant Cell 26: 1938-1948). I will present data from gene network modeling
               combined with reverse genetics and isotopic tracer experiments in Arabidopsis and tomato that
               demonstrate that the cognate metabolic architecture is split into two branches, the first one originating
               from the β-oxidation of p-coumarate in peroxisomes, while the second one stems from the peroxidative
               cleavage of a flavonol, called kaempferol, in the cytosol (Plant Cell 30: 2910-2921). Having dissected the
               molecular determinants of such a cleavage, I will show that using a synthetic biology approach it is
               possible to capture this catabolic branch to re-route kaempferol towards the accumulation of ubiquinone
               in Arabidopsis leaves and tomato fruits. I will briefly discuss how this paradigm shift regarding the
               functional significance of flavonols in plant tissues offers new opportunities for increasing the nutritional
               value and stress resistance of crops.

               Eric Soubeyrand (esoubeyrand2@ufl.edu)





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