Heme regulates expression of phycobiliprotein photogenes in the unicellular rhodophyte, Cyanidium caldarium

R.F Troxler,S Lin,G.D Offner

doi:10.1016/s0021-9258(19)47104-4

Abstract

Allophycocyanin and phycocyanin in the red alga (Cyanidium caldarium) are chloroplast-encoded, light-harvesting accessory pigments composed of alpha and beta subunit polypeptides (17-19 kDa) to which 1 or more residues of the heme-derived bile pigment chromophore phycocyanobilin are attached by cysteinyl thioether linkages (Offner, G.D., and Troxler, R.F. (1983) J. Biol. Chem. 258, 9931-9940). Western blot experiments utilizing phycobiliprotein antisera revealed that immunoreactive allophycocyanin and phycocyanin apoproteins were absent in cells grown in the dark and present in cells exposed to light. Northern blot experiments using genomic DNA hybridization probes indicated that phycobiliprotein mRNAs were absent in the dark, whereas cells exposed to light contained two allophycocyanin mRNA transcripts, 1.4 and 1.6 kilobases in length, and one phycocyanin mRNA transcript, 3.0 kilobases in length, providing evidence that phycobiliproteins are encoded in photogenes which are only transcriptionally active in the light. Northern and Western analyses demonstrated that cells incubated in the dark with the heme precursor delta-aminolevulinate contained allophycocyanin and phycocyanin mRNAs and apoproteins, indistinguishable in size, number, and quantity from those made in the light. Cells incubated in the dark with delta-aminolevulinate, protoporphyrin IX, or heme, but not biliverdin or phycocyanobilin, synthesized allophycocyanin and phycocyanin alpha and beta apoproteins, suggesting a role for heme in the control phycobiliprotein gene expression. Cells incubated with heme in the dark produced allophycocyanin and phycocyanin mRNA transcripts, but did not produce mRNAs for four other photogenes coding for a P-700 reaction center protein, a 32-kDa herbicide-binding protein, and the large and small subunits of ribulose-bisphosphate carboxylase. These results show, for the first time, that heme is a regulatory factor specifically involved in transcriptional regulation of chloroplast genes for phycobiliproteins.

Highlights

(Cyanidium caldariuma) re chloroplast-encoded, light- bilisomes,which functionaslight-harvestingantenna pigharvesting accessory pigments composed of (Y and,8 ments located on the outer surfacesof thylakoid membranes subunit polypeptides (17-19 kDa) to which 1or more (2-4)
In the unicellular rhodophyte, C. caldarium, heme can substitutefor light in promoting the appearanocfe of nuclear encoded, nonpigmented linker polypeptides (2-4)
We2 and others (7) have showpnu,lsein-labeling experiments, that cycloheximide inhibitsincorporation of radiolabeled amino acids intolinker polypeptides, butnotintoPBPs, mRNA transcriptsfor APC and PC and that heme apparenwtlyhereas chloramphenicol inhibits incorporation of radioladoes not have any effect on regulation of mRNA transcripts beled amino acids into PBPs, but not into linpkoleyrpeptides

Summary

RESULTS

When cells grown heterotrophically in the dark were transferred to thelight, APC,PC,and chlorophyll a were detectable after 2 h, and thekinetics of this process displayed a 6-h lag RegulatioPnhoPtohgyecnoebiliprotein phase, followed by a period of more rapid pigment synthesis. This questionwas addressed in Northern analysewshere total to light (Fig. 4C) This provides indirect evidence that ALA, RNA from dark- and light-grown cells was electrophoresed or more probably a derived tetrapyrrole, is involved in proand blotted ontonitrocellulose, and the blotswere hybridized moting transcriptionof P B P genes. Southern analyses of the PBP DNA probes were performed, batedwith ALA, protoporphyrinIX, heme,biliverdin, and and itwas found that the APCprobe does not recognize the phycocyanobilin in the dark, and total soluble protein was PC probe, and vice versa, under the hybridization and wash isolated and examined on Western blots probedwith anticonditions used (Fig. 4.4). The mRNAs for P B P apoproteins PBP antisera These experimenstshowed that cells incubated were not detectable in dark-grown cells In all lanes containing immunoreactivPeC, the n subunit appears to partially split into a doublet, and althouwghe have no explanation for this result, it was considered to he an electrophoretic artifact

Phycobiliprotein Photogene Regulation

DISCUSSION

The presentweight of evidence strongly suggests that heme

The observed effect of heme on PBPs is unlikely to result