Research Article The calcium-dependent protein kinase RcCDPK2 phosphorylates sucrose synthase at Ser 11 in developing castor oil seeds Eric T. Fedosejevs 1, *, Suzanne A. Gerdis 1 , Sheng Ying 1, † , Michal Pyc 2 , Erin M. Anderson 2 , Wayne A. Snedden 1 , Robert T. Mullen 2 , Yi-Min She 3 and William C. Plaxton 1,4 1 Department of Biology, Queen’s University, Kingston, ON, Canada K7L 3N6; 2 Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1; 3 Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, 3888 Chenhua Road, Shanghai 201602, China; and 4 Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada K7L 3N6 Correspondence: William C. Plaxton (plaxton@queensu.ca) Imported sucrose is cleaved by sucrose synthase (SUS) as a critical initial reaction in the biosynthesis of storage end-products by developing seeds. Although SUS is phosphory- lated at a conserved seryl residue by an apparent CDPK (Ca 2+ -dependent protein kinase) in diverse plant tissues, the functions and mechanistic details of this process remain obscure. Thus, the native CDPK that phosphorylates RcSUS1 (Ricinus communis SUS1) at Ser 11 in developing COS (castor oil seeds) was highly purified and identified as RcCDPK2 by MS/MS. Purified RcSUS1-K (-kinase) and heterologously expressed RcCDPK2 catalyzed Ca 2+ -dependent Ser 11 phosphorylation of RcSUS1 and its corre- sponding dephosphopeptide, while exhibiting a high affinity for free Ca 2+ ions [K 0.5 (Ca 2+ ) < 0.4 mM]. RcSUS1-K activity, RcCDPK2 expression, and RcSUS1 Ser 11 phosphorylation peaked during early COS development and then declined in parallel. The elimination of sucrose import via fruit excision triggered RcSUS1 dephosphorylation but did not alter RcSUS1-K activity, suggesting a link between sucrose signaling and posttranslational RcCDPK2 control. Both RcCDPK2-mCherry and RcSUS1-EYFP co-localized throughout the cytosol when transiently co-expressed in tobacco suspension cells, although RcCDPK2-mCherry was also partially localized to the nucleus. Subcellular fractionation revealed that ∼20% of RcSUS1-K activity associates with microsomal membranes in developing COS, as does RcSUS1. In contrast with RcCDPK1, which catalyzes inhibitory phosphorylation of COS bacterial-type phosphoenolpyruvate carboxylase at Ser 451 , RcCDPK2 exhibited broad substrate specificity, a wide pH–activity profile centered at pH 8.5, and insensitivity to metabolite effectors or thiol redox status. Our combined results indicate a possible link between cytosolic Ca 2+ -signaling and the control of photosyn- thate partitioning during COS development. Introduction A pivotal initial step in the biosynthesis of storage end-products by developing seeds is the UDP- dependent conversion of imported photosynthate (i.e., sucrose) into UDP-glucose and fructose, cata- lyzed by SUS (sucrose synthase; E.C. 2.4.1.13). SUS has been linked to the channeling of sucrose into starch and cellulose biosynthesis [1–3], with SUS overexpression enhancing starch and fiber accumula- tion in maize (Zea mays) and cotton (Gossypium hirsutum) seeds, respectively [4,5]. Transcriptomic and proteomic studies together with SUS activity assays and immunolocalization indicate that SUS also supports the production of carbon skeletons, ATP, and reducing power (via glycolysis and oxida- tive pentose phosphate pathway) required for storage oil and protein synthesis in developing oil seeds [6–11]. However, comparatively little is known about the biochemical properties and control of oil *Present address: Department of Chemistry and Biochemistry, Mount Allison University, 63C York St., Sackville, NB, Canada E4L 1G8. †Present address: Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA. Accepted Manuscript online: 10 August 2016 Version of Record published: 11 October 2016 Received: 30 May 2016 Revised: 3 August 2016 Accepted: 10 August 2016 © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society 3667 Biochemical Journal (2016) 473 3667–3682 DOI: 10.1042/BCJ20160531