No abstract

Proteins entering the secretory pathway of eukaryotic cells are folded into their native structures in the endoplasmic reticulum (ER). Disruption of protein folding causes ER stress and activates signaling cascades, designated the unfolded protein response (UPR), that restore folding capacity. In mammals and yeast, the protein disulfide isomerases (PDIs) are key protein folding catalysts activated during UPR. However, little is known about the response of PDI genes to UPR in plants. In Arabidopsis thaliana, we identified 12 PDI genes that differed in polypeptide length, presence of signal peptide and ER retention signal, and the number and positions of thioredoxin and transmembrane domains. AtPDI gene expression was investigated in different tissues, in response to chemically induced UPR, and in null mutants of UPR signaling mediators (AtIRE1-2 and AtbZIP60). The expression of six AtPDI genes was significantly up-regulated by UPR and sharply attenuated by the transcription inhibitor, actinomycin D, indicating UPR induced AtPDI gene transcription. AtPDI and BIP2 (Binding protein) gene expression was not affected in the Atire1-2 mutant exposed to UPR, however, the expression of four AtPDI genes was decreased in the Atbzip60 mutant. We proposed that additional UPR signaling factors complement AtbZIP60 in the activation of AtPDI gene expression during ER stress in plants.

We studied the effects of spedral quality and fluence on the expression of several chloroplast-encoded photosynthesis genes and on the stability of their protein products in barley (Hordeum vulgare). During light-dependent chloroplast maturation, mRNA levels for psbD-psbC and psbA were maintained at higher levels compared with mRNAs encoding proteins for other photosynthesis functions (atpB, rbcL). Maintenance of psbD-psbC mRNA levels was accounted for by differential activation of the psbD-psbC light-responsive promoter by high-irradiance blue light and, secondarily, ultraviolet A (UV-A) radiation. Promoter activation was fluence dependent and required continuous illumination for 2 h at threshold fluences of 1.3 (blue light), 7.5 (white light), or 10 (UV-A) wmol m-* s-'. From immunoblot analysis experiments, we showed that the psbD-psbC gene products D2 and CP43 undergo light-mediated turnover similar to light-labile D1. Other photosynthesis proteins such as the í3 subunit of ATP synthase and the large subunit of ribulose-1,5-bisphosphate carboxylase were relatively stable. In the absence of protein synthesis, D2 degradation paralleled the degradation of D1 (relative half-lives, 9.5-10 h). CP43 decay was about half of D2 and D1 decay. In contrast with activation of the light-responsive promoter, the fluence-dependent degradation of D1, D2, and CP43 required 50-to 100-fold higher fluences of photosynthetically active white, red, blue, or UV-A irradiation. We interpret the different fluence and wavelength requirements to indicate that separate photosensory systems regulate activation of psbD-psbC transcription and turnover of D1, D2, and CP43. We propose that a blue light/UV-A photosensory pathway activates the psbD-psbC light-responsive promoter, differentially maintaining the capacity of mature chloroplasts to synthesize D2 and CP43, which are damaged and turned over in illuminated plants.

The synthesis of reaction center protein D2 and mRNAs which encode this protein are differentially maintained at high levels in mature barley chloroplasts. To understand the differential maintenance of psbD mRNA abundance, we have studied the transcription and the RNAs produced from the psbD‐psbC operon in plastids of light and dark‐grown barley seedlings. Ten psbD‐psbC RNAs synthesized in dark‐grown barley share four different 5′‐ends, two of which arise by transcription initiation, and one of which is generated by 5′‐processing of longer psbD‐psbC transcripts. Illumination of dark‐grown barley causes the decline of these ten transcripts, and the accumulation of two different psbD‐psbC RNAs. Capping assays, in vitro transcription and RNA processing experiments and treatment of plants with tagetitoxin (a selective inhibitor of chloroplast transcription), indicate that the light‐induced transcripts arise by transcription initiation. Run‐on transcription and RNA quantitation experiments provide evidence that both light‐induced transcription and RNA stability play roles in the accumulation of the light‐induced RNAs. These data document a novel mechanism for regulating plastid gene expression involving a light‐induced switch in psbD‐psbC promoter utilization.

The turnover of RNAs encoded by seven different barley chloroplast genes was analyzed after treatment of barley shoots with tagetitoxin, a selective inhibitor of chloroplast transcription. Changes in RNA stability were examined during chloroplast development using basal and apical leaf sections of 4.5-day-old dark-grown seedlings and apical leaf sections of 4.0-day-old dark-grown seedlings which had been illuminated for 12 h. Of the RNAs examined, a 2.6 kb unspliced precursor of tRNA(lys) exhibited the shortest half-life, which was estimated to be 3 h. The 16S rRNA and psbA mRNA had the longest estimated half-lives, which were greater than 40 h. Among mRNAs, half-lives were estimated to range from 6 h for psaA mRNA, to over 40 h for psbA mRNA. Therefore, barley chloroplast mRNAs have long half-lives relative to bacterial mRNAs. The stability of atpB mRNA and the unspliced precursor of tRNA-lys was not altered during chloroplast development, while the stability of psaA mRNA decreased 2-fold. In contrast, the stability of the 16S rRNA and mRNAs for rpoA, psbA and rbcL increased during chloroplast development. The stability of 16S rRNA increased markedly during chloroplast development in the dark and this increase was maintained in illuminated seedlings. The stability of rbcL mRNA increased 2.5-fold during chloroplast development in the dark, and then decreased 2-fold in chloroplasts of light-grown plants. The initial increase in rpoA and psbA mRNA stability was also light-independent, with total increases in stability of at least 5-fold. In the case of rpoA, the stability of 2 of the 13 polycistronic rpoA transcripts that were detected in dark-grown plants was selectively increased during chloroplast development. In conclusion, the stability of some transcripts is selectively increased and further modulated during chloroplast development in barley. We propose that the selective stabilization of chloroplast mRNA, which occurred independent of light, is an indication that non-light regulated developmental signals are involved in barley chloroplast mRNA stability.