Molecular cloning, nuclear gene structure, and developmental expression of NADPH: protochlorophyllide oxidoreductase in pea (Pisum sativum L.)

Abstract

Complementary DNA clones and a corresponding nuclear gene (lpcr) encoding the NADPH-dependent protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) have been characterized from pea (Pisum sativum L.). The pea lpcr gene encodes a 43,118 Da precursor polypeptide comprised of a transit peptide of 64 amino acids and a mature protein of 336 amino acids. The coding portion of the gene is interrupted by four introns, two of which are located within the transit peptide coding portion of the gene. The deduced primary structure for the pea protein is similar to those reported for Arabidopsis and two monocot species. Northern blot analysis revealed little to no decrease in steady-state levels of mRNA encoding the enzyme in etiolated leaves illuminated with continuous white light for up to 48 h. In contrast, western blot analysis showed that the major immunoreactive species present in whole leaf extracts decreased to nearly undetectable levels during this same 48 h period. These results suggest that pchlide reductase activity in pea is primarily regulated post-transcriptionally, most likely at the level of translation initiation/elongation or protein turnover.

Structure and Expression of Chloroplast-Localized Porphobilinogen Deaminase from Pea (Pisum sativum L.) Isolated by Redundant Polymerase Chain Reaction

“…At the junction of the NH2-terminal extension and the mature protein is the sequence IRA S (open triangle in Fig. 2), which conforms with the consensus cleavage site recognized by the stromal processing peptidase (Gavel and von Heijne, 1990) and is similar to the conserved tetrapeptide motif (V/IRA S/Q/E) recognized in precursors of other Chl biosynthetic enzymes, for example ALA dehydratase (Boese et al, 1991) and Pchlide reductase (Spano et al, 1992a(Spano et al, , 1992b). Similarity to Other Known PBG Deaminases Figure 3 shows the alignment of the amino acid sequence of pea PBG deaminase with those from other organisms, where completely conserved residues are indicated with an asterisk (*) and conservative substitutions with an apostrophe (').…”

The Regulation of Enzymes Involved in Chlorophyll Biosynthesis

“…Also, there are cDNA clones available for POR proteins from both angiosperms, such as barley (Schulz et al, 1989;Holtorf et al, 1995), wheat (Teakle and Griffiths, 1993), oat (Darrah et al, 1990) (Fig. 6), pea (Spano et al, 1992b), Arabidopsis (Benli et al, 1991), and gymnosperms, such as pine (Spano et al, 1992a;Forreiter and Apel, 1993). In cyanobacteria, such as Synechocystis sp.…”

“…In gymnosperms, both light-dependent and light-independent versions of the Pchlide-reducing enzymes seem to exist (Selstam et al, 1987;Ou et al, 1990;Spano et al, 1992a;Forreiter and Ape], 1993). In mountain pine (Pinus mugo), Forreiter and Ape1 (1 993) detected two immunologically related POR polypeptides using an antibody raised against a barley pPORA-p-galactosidase fusion protein expressed in E. coli (Schulz et al, 1989).…”

Nucleotide distribution in gymnosperm nuclear sequences suggests a model for GC-content change in land-plant nuclear genomes

“…CpG suppression is found to varying extents in different plant genomes (Hepburn et al 1987;Quigley et al 1989;Campbell and Gowri 1990;. Lack of suppression has been attributed to both protection against methylation (Bird 1986) as well as specific G-T mismatch repair (Hepburn et al 1987); for the purpose of our model, a distinction between these (Brinkmann et al 1987) and tobacco (Shih et al 1986), GapC from maize (Brinkmann et al 1987) and tobacco (Shih et al 1986), Lhcbl from rice (Lhcbl*l or 2120, Matsouka 1990) and tomato (Lhcbl*2 or Cab-lB, Pichersky et al 1985), Lhcb2 from rice (Lhcb2*l or 2123, Matsouka 1990) and tomato , Lhcb5 from barley (SCrensen et al 1992) and tomato , Pal from rice (Minami et al 1989) and tomato (EMBL M90692), Pcr from barley (Schultz et al 1989) and pea (Spano et al 1992a), and…”