Molecular characterization of a novel, nuclear-encoded, NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase in plastids of the gymnosperm Pinus sylvestris L.

Abstract

Angiosperms and algae possess two distinct glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes, an NAD(+)-dependent tetramer involved in cytosolic glycolysis and an NADP(+)-dependent enzyme of the Calvin cycle in chloroplasts. We have found that the gymnosperm Pinus sylvestris possesses, in addition to these, a nuclear-encoded, plastid-specific, NAD(+)-dependent GAPDH, designated GapCp, which has not previously been described from any plant. Several independent full-size cDNAs for this enzyme were isolated which encode a functional transit peptide and mature subunit very similar to that of cytosolic GAPDH of angiosperms and algae. A molecular phylogeny reveals that chloroplast GapCp and cytosolic GapC arose through gene duplication early in chlorophyte evolution. The GapCp gene is expressed as highly as that for GapC in light-grown pine seedlings. These findings suggest that aspects of compartmentalized sugar phosphate metabolism may differ in angiosperms and gymnosperms and furthermore underscore the contributions of endosymbiotic gene transfer and gene duplication to the nuclear complement of genes for enzymes of plant primary metabolism.

A nuclear gene of eubacterial origin in Euglena gracilis reflects cryptic endosymbioses during protist evolution.

“…Previous work indicated that genes for eukaryotic GAPDH enzymes are descendants of an ancient gene family which existed in the common ancestor of extant eubacteria (4,19,26). This view is supported by the GAPDH gene phylogeny in Fig.…”

Thioredoxins: structure and function in plant cells

“…Chloroplastic GAPDH uses preferentially NADPH and can be activated either by reduction or by metabolites (for a recent paper on the subject, see Baalmann et al, 1995). The situation with chloroplastic GAPDH is very complex, since the protein can use either NADH or NADPH as a substrate, both activities being present in the chloroplast, as evidenced recently in Pinus sylvestris (Meyer-Grauen et al, 1994). The NADPH-dependent enzyme is up-regulated by the thioredoxin system and is a tetramer composed of either the 4A subunits or 2A and 2B subunits.…”

Molecular characterization of transketolase (EC 2.2.1.1) active in the Calvin cycle of spinach chloroplasts

“…In vitro transcription, translation and import pTK39 was linearized with SacI, purified, transcribed in the presence of 1 mM 7mG(5')ppp(5')G (New England Biolabs) with T7 RNA polymerase (Pharmacia) and translated as described [27]. Preparation of intact chloroplasts from 80 g of 14-day old pea leaves and import were performed according to Clausmeyer et al [6] with a protocol kindly provided by R.-B.…”