The PEP-carboxylase kinase gene family in Glycine max (GmPpcK1-4):: an in-depth molecular analysis with nodulated, non-transgenic and transgenic plants

Phosphoenolpyruvate carboxylase (PEPC) is a widely distributed metabolic enzyme among plant and prokaryotic species. In vascular plants, the typical PEPC is regulated post-translationally by a complex interplay between opposing metabolite effectors and reversible protein phosphorylation. This phosphorylation event is controlled primarily by the up-/down-regulation of PEPC-kinase (PpcK), an approximately 31-kDa Ser/Thr-kinase. As a sequel to earlier investigations related to PEPC phosphorylation in N-2-fixing nodules of Glycine max, we now present a detailed molecular analysis of the PpcK multigene family in nodulated soybeans. Although the GmPpcK1-4 transcripts are all expressed throughout nodule development, only the nearly identical GmPpcK2/3 homologs are nodule-enhanced and up-/down-regulated in vivo by photosynthate supply from the shoots. In contrast, GmPpcK1 is a 'housekeeping' gene, and GmPpcK4 is a highly divergent member, distantly removed from the legume PpcK subfamily. Real-time qRT-PCR analysis indicates that GmPpcK2/3 are overwhelmingly the dominant PpcKs expressed and up-/down-regulated throughout nodule development, mirroring the expression properties of nodule-enhanced PEPC (GmPpc7). In situ RT-PCR investigation of the spatial localization of the GmPpcK1-4 and GmPpc7 transcripts in mature nodules is entirely consistent with this view. Complementary histochemical and related RNA gel-blot findings with nodulated, GmPpcK1/3 promoter::GUS-expressing T-2 plants provide direct experimental evidence that (i) PpcK gene expression is controlled primarily at the transcriptional level; and (ii) the contrasting expression properties of GmPpcK1/3 are conferred largely by regulatory element(s) within the approximately 1.4-kb 5'-upstream region. As a result of our multifaceted analyses of GmPpcK1-4, GmPpc7 and PEPC-phosphorylation in the soybean nodule, it is proposed that the GmPpcK2/3 homologs and GmPpc7 together comprise the key molecular 'downstream players' in this regulatory phosphorylation system within the mature nodule's central zone.