Isolation and characterization of PbCS2 gene regulated by iron deficiency and auxin-based systemic signals in Pyrus betulifolia

• We cloned a gene encoding a citrate synthase from Pyrus betulifolia and designated it as PbCS2.
• The deduced PbCS2 protein contains a conservative WPNVDAHS sequence considered as a marker for CS proteins.
• The expression of PbCS2 was significantly upregulated in resistance to iron deficiency.
• The Fe-deprivation-activated systemic signals were involved in the regulation of the Fe-deficient responses.
• PbCS2 expression in the root was induced by IAA.

Iron (Fe) is an essential micronutrient for plants and citric acid acts as a chelate substance for the long-distance transmission of Fe. Citrate synthase (CS) protein is a key enzyme in the synthesis of citric acid. Using PCR amplification, a gene encoding a putative citrate synthase was isolated from Pyrus betulifolia, a widely-used rootstock native to China, and designated it as PbCS2. This gene was 1422 bp in length with an open reading frame encoding a protein of 473 amino acids. The deduced PbCS2 protein contains a conserved citrate synthase domain and conservative WPNVDAHS sequence existing in the PWPN-box. Phylogenetic analyses clearly demonstrated that PbCS2 had the highest homology with rosaceous CS proteins. The expression of PbCS2 was enriched in roots, leaves and phloem, but relatively weak in xylem. Fe deficiency induced substantial up-regulation of the PbCS2 expression in the roots. The split-root experiments showed that Fe deprivation in one part of the root system caused dramatic up-regulation of the PbCS2 expression in the Fe-sufficient portion, indicating that the PbCS2 expression was triggered by systemic signals. Furthermore, supplying auxin to the de-topped shoot could recover the Fe deficiency-caused up-regulation of the PbCS2 expression in the Fe-supplied part of the root system. By contrast, NPA application (an auxin transport inhibitor) to the shoot apex blocked up-regulation of the PbCS2 expression in the untreated portion. These results strongly indicated that Fe deficiency-caused alterations of the PbCS2 expression were mediated by IAA.