Spatial distribution of cytosolic NADP+-isocitrate dehydrogenase in pine embryos and seedlings

Abstract

Cytosolic NADP(+)-linked isocitrate dehydrogenase (NADP(+)-IDH) is considered the main enzyme catalyzing the production of 2-oxoglutarate for amino acid biosynthesis in plants. We characterized a full-length cDNA encoding the cytosolic NADP(+)-IDH in the gymnosperm Pinus pinaster Ait. The deduced amino acid sequence exhibited high homology with available sequences in angiosperms. Genomic analysis indicated that only one gene, or two genes with a high degree of homology, encodes the protein in P. pinaster. Cytosolic NADP(+)-IDH is up-regulated during embryo germination concomitantly with glutamine synthetase. Immunohistochemical analysis of germinating seeds and young seedlings showed a broad spatial pattern of NADP(+)-IDH expression. The protein was detected in vascular tissues of germinating embryo and seedling organs, as well as in other cellular types and tissues, including parenchyma and epidermal cells. The spatial pattern of NADP(+)-IDH expression in the embryo and seedling organs did not coincide with the reported spatial patterns for other key enzymes of nitrogen metabolism. Treatment of seedlings with phosphinothricin, an inhibitor of glutamine synthetase (GS), differentially affected GS and NADP-IDH in cotyledons. In response to herbicide treatment, GS was up-regulated in 0.5-cm-long cotyledons, whereas NADP(+)-IDH was up-regulated in 1.5-cm-long cotyledons, suggesting that 2-oxoglutarate is required to overcome the herbicide effect in tissues with a high demand for glutamate. The results indicated that cytosolic NADP(+)-IDH is a housekeeping enzyme that has not undergone functional specialization during evolution. Its spatial pattern in pine tissues suggests that it facilitates metabolism in different ways depending on the characteristics of the particular tissue and cellular type.

Characterization and developmental expression of a glutamate decarboxylase from maritime pine

“…Thus, GAD activity, protein, and mRNA levels during hypocotyl development are similar to the expression levels of cytosolic NADP + -IDH, an enzyme associated with hypocotyl development and vascular tissue (Figs. 4, 5;Palomo et al 1998;Pascual et al 2008), and these genes also exhibited a similar expression pattern during the induction of reaction wood in pine (Fig. 6).…”

“…The study of GAD expression was carried out during the development of pine seedlings, a model that has proven to be useful in understanding the role of enzymes involved in nitrogen and carbon metabolism enzymes in gymnosperms (Pascual et al 2008). Low GAD levels were observed in dry (Fig.…”

“…b Northern-blot analysis showing expression of GAD and other genes in control (non-bent) plants and in the compression, and lateral zone of bent plants. Pascual et al 2008). Accordingly, it could be suggested that GAD does not have a primordial role at the early stages of pine seedling development.…”

“…For histochemical analysis by optical microscopy, pine hypocotyl was Wxed in 1.25% glutaraldehyde in 100% methanol by the freeze substitution method (Regan et al 1999). Samples were embedded in paraYn, and 10-mthick sections were prepared as described previously (Pascual et al 2008). Staining with hematoxylin and eosin was carried out following standard procedures (Charboneau et al 2004) with minor modiWcations.…”

“…4e), suggesting that GAD activity is the major regulator of hypocotyl GABA levels during seedling development. To further characterize GAD in pine, we compared GAD expression with the expression of other genes encoding enzymes associated with seedling diVerentiation, including cytosolic NADP + -IDH (Palomo et al 1998;Pascual et al 2008), ascorbate peroxidase (APX), and oxidase (AOX) (Pignocchi and Foyer 2003;de Pinto and De Gara 2004), enzymes linked with ligniWcation and signaling, including laccase (LAC, Gavnholt and Larsen 2002), and 1-aminocyclopropane-1-carboxylic acid synthase (ACS), and oxidase (ACO), enzymes involved in ethylene synthesis that has been described to be connected to ligniWcation of plant stem (Klintborg et al 2002). Northern blots hybridized with speciWc probes indicated that these genes were diVerentially expressed in organs of pine seedlings (Fig.…”