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

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Summary

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 N2-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 T2 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.
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Keywords: PEP carboxylase (PEPC); Ser/Thr-kinases; in situ RT–PCR; nodule-enhanced PEPC-kinases (PpcKs); root nodules; soybean transformation

Document Type: Research Article

Affiliations: 1: Department of Biochemistry, 2: Center for Biotechnology,

Publication date: March 1, 2007

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