The Effect of Two Amino acid Residue Substitutions via RNA Editing on Dark-operative Protochlorophyllide Oxidoreductase in the Black Pine Chloroplasts

Abstract

Dark-operative protochlorophyllide oxidoreductase (DPOR) is a key enzyme to produce chlorophyll in the dark. Among photosynthetic eukaryotes, all three subunits chlL, chlN, and chlB are encoded by plastid genomes. In some gymnosperms, two codons of chlB mRNA are changed by RNA editing to codons encoding evolutionarily conserved amino acid residues. However, the effect of these substitutions on DPOR activity remains unknown. We first prepared cyanobacterial ChlB variants with amino acid substitution(s) to mimic ChlB translated from pre-edited mRNA. Their activities were evaluated by measuring chlorophyll content of dark-grown transformants of a chlB-lacking mutant of the cyanobacterium Leptolyngbya boryana that was complemented with pre-edited mimic chlB variants. The chlorophyll content of the transformant cells expressing the ChlB variant from the fully pre-edited mRNA was only one-fourth of the control cells. Co-purification experiments of ChlB with Strep-ChlN suggested that a stable complex with ChlN is greatly impaired in the substituted ChlB variant. We then confirmed that RNA editing efficiency was markedly greater in the dark than in the light in cotyledons of the black pine Pinus thunbergii. These results indicate that RNA editing on chlB mRNA is important to maintain appropriate DPOR activity in black pine chloroplasts.

Fortuitous events in the evolution of Light-dependent Protochlorophyllide Oxidoreductase

“…It has been shown previously that ChlL gene of the gymnosperm Thuja standishii has high rate of non-synonymous substitution confirming its dispensability (Kusumi et al, 2006). Thus, greater selection pressure acted on LIPOR genes leading to its complete absence from higher gymnosperm, gnetophytes, and angiosperms (Walmsley et al, 1999;Yamamoto et al, 2017). Taken together these findings suggest that LPOR genes seem to be to have higher functionality and importance compared to LIPOR genes in the oxygenic world.…”

Transcriptome-Wide Characterization of Alkaloids and Chlorophyll Biosynthesis in Lotus Plumule

“…Chl is synthesized through a complex pathway derived from the biosynthesis of the 5-aminolevulinic acid (ALA) precursor. Of the 16 enzymes reported to be involved in Chl biosynthesis, the conversion of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) by the light-dependent Pchlide oxidoreductase (LPOR) is the only light-requiring reaction in angiosperms (Yamamoto et al, 2017 ). The LPOR encoding genes are nuclear-encoded and are distributed throughout oxygenic photosynthetic organisms.…”

Functional expression of an oxygen-labile nitrogenase in an oxygenic photosynthetic organism

“…On the other hand, oxygen production by photosystem II may be incompatible with oxygen-sensitive nitrogenase. Chloroplast genomes of many algae and plants such as gymnosperms (e.g., Pinus thunbergii ) 19 and mosses (e.g., Physcomitrella patens and Marchantia polymorpha ) 20 , 21 encode three subunits of a nitrogenase-like enzyme, dark-operative protochlorophyllide oxidoreductase (DPOR), that shows similar oxygen sensitivity as nitrogenase 20 . This suggests that chloroplasts have the potential ability to express and accommodate active nitrogenase 14 .…”