Naoki Fusada

Identification and light-induced expression of a novel gene of NADPH-protochlorophyllide oxidoreductase isoform in Arabidopsis thaliana

In Arabidopsis thaliana, we identified a novel gene of a NADPH‐protochlorophyllide oxidoreductase (POR) isoform, which catalyzes the light‐dependent protochlorophyllide a reduction in the chlorophyll (Chl) biosynthetic pathway. The deduced amino acid sequence of the novel POR isoform (PORC) showed significant identities (∼75%) with the previously isolated two POR isoforms of A. thaliana. Contrasting with these POR isoforms, the PORC transcript increased in etiolated seedlings by illumination, and was dominantly expressed in immature and mature tissues. The present results demonstrated that Chl biosynthesis and chloroplast biogenesis in A. thaliana are controlled by three POR isoforms, which are differentially controlled by light and development.

NADPH-protochlorophyllide oxidoreductase in cucumber is encoded by a single gene and its expression is transcriptionally enhanced by illumination.

NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the photoreduction of protochlorophyllide a in the chlorophyll biosynthetic pathway. Recently, two POR genes, the expressions of which were differently regulated by light, were isolated in barley and Arabidopsis, and it is suggested that in angiosperms, the chlorophyll synthesis is controlled by the differential action of the two distinct POR isozymes. Meanwhile, we have isolated POR cDNA from cucumber, and reported that its expression was enhanced by illumination. In this study, we analyzed the gene expression and organization of cucumber POR in more detail. Northern blot analysis with 5′- or 3′-noncoding gene-specific and full length probes and RNase protection assay revealed that cucumber POR mRNA is transcribed from a single gene. Run-on assay revealed that the transcript level of isolated nuclei from illuminated cotyledons was 6-fold that of dark control, indicating that the light-enhanced expression of cucumber POR is caused by transcriptional activation. Using degenerated primers and the genomic DNA of cucumber as template, PCR showed the presence of only one type of fragment encoding cucumber POR. The PCR product was hybridized with the genomic Southern blot from cucumber and only one band was detected under low stringency conditions. From these results, we conclude that there is a single POR gene in cucumber and PORs are organized by different gene families among higher plants, and in cucumber the single POR may play the roles of two POR genes, which are present in other higher plants such as Arabidopsis and barley.

Identification of two differentially regulated isoforms of protochlorophyllide oxidoreductase (POR) from tobacco revealed a wide variety of light- and development-dependent regulations of POR gene expression among angiosperms.

NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide a in the chlorophyll biosynthetic pathway. Here, we identified two distinct POR cDNAs from tobacco. Both POR isoforms are encoded by a respective single copy gene in tobacco genome. The overall deduced amino acid sequences of two tobacco cDNAs, designated here POR1 and POR2, displayed significant identities (∼75%), but showed different patterns of light and developmental regulation. In contrast to the previously isolated POR isoforms of Arabidopsis thaliana and barley, the expression of both tobacco POR isoforms were not negatively regulated by light and persisted in matured green tissues. Furthermore, the expression of both genes appeared to be regulated by a diurnal regulation. These results show a wide variety of light- and development-dependent regulations of POR gene expression among angiosperms. Furthermore, phylogenetic analysis including tobacco revealed that POR gene family is differentially represented by angiosperms, most of which is probably caused by independent gene duplication in individual plant. Present results imply a modification of the previous concept that chlorophyll biosynthesis and chloroplast differentiation in angiosperms are ubiquitously controlled by unique functions of two POR isoforms.