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.
Highlights
Chlorophyll a (Chl) is an essential tetrapyrrole pigment in photosynthesis that is synthesized from glutamate through a complex pathway consisting of at least 15 enzymes[1,2,3]
Comparisons of ChlB amino acid sequences among various photosynthetic organisms that lack RNA editing processes indicated that the two amino acid residues in black pine ChlB that are generated by RNA editing (Leu[206] and Trp213) are highly conserved among plants and cyanobacteria (Fig. 1B)
To investigate whether these two amino acid residues are functionally important for Dark-operative protochlorophyllide oxidoreductase (DPOR) activity, and to evaluate the activity of the edited and pre-edited forms of ChlB, we tried to express chlB together with chlN encoding the other subunit of the NB protein of DPOR from P. thunbergii in Escherichia coli
Summary
Chlorophyll a (Chl) is an essential tetrapyrrole pigment in photosynthesis that is synthesized from glutamate through a complex pathway consisting of at least 15 enzymes[1,2,3]. Light is used for photosynthesis and in addition is a major environmental signal that regulates Chl biosynthesis In these plants, light is required for reduction of protochlorophyllide (Pchlide) to chlorophyllide a by the enzyme light-dependent Pchlide oxidoreductase (LPOR). The physiological significance of the presence of both LPOR and DPOR in various photosynthetic organisms remains largely unknown, light and oxygen appear to be key factors in their functional differentiation in cyanobacteria[16] and it has been reported that DPOR is required for Chl production under short-day conditions in the liverwort Marchantia polymorpha[17]. The site in chlN undergoes the conversion from the codon CCU (Pro285) to UCU (Ser), and the two sites in chlB undergo the conversion from codons CCG (Pro206) and CGG (Arg213) to CUG (Leu) and UGG (Trp), respectively These three amino acids in ChlN and ChlB are highly conserved among many oxygenic phototrophs[23] (Fig. 1B). No direct causal links among these amino acid substitutions and DPOR activity has been identified, in part, because DPOR is extremely sensitive to oxygen and can be assayed only under anaerobic conditions in vitro[16, 24, 25]
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