Messenger RNA degradation is initiated at the 5' end and follows sequence- and condition-dependent modes in chloroplasts.

Using reporter gene constructs, consisting of the bacterial uidA (GUS) coding region flanked by the 5' and 3' regions of the Chlamydomonas rbcL and psaB genes, respectively, we studied the degradation of mRNAs in the chloroplast of Chlamydomonas reinhardtii in vivo. Extending the 5' terminus of transcripts of the reporter gene by more than 6 nucleotides triggered rapid degradation. Placing a poly(G) tract, known to pause exoribonucleases, in various positions downstream of the 5' terminus blocked rapid degradation of the transcripts. In all these cases the 5' ends of the accumulating GUS transcripts were found to be trimmed to the 5' end of the poly(G) tracts indicating that a 5' → 3' exoribonuclease is involved in the degradation process. Several unstable variants of the GUS transcript could not be rescued from rapid degradation by a poly(G) tract showing that sequence/structure-dependent modes of mRNA breakdown exist in the Chlamydomonas chloroplast. Furthermore, degradation of poly(G)-stabilized transcripts that accumulated in cells maintained in the dark could be augmented by illuminating the cells, implying a photo-activated mode of mRNA degradation that is not blocked by a poly(G) tract. These results suggest sequence- and condition-dependent 5' → 3' mRNA-degrading pathways in the chloroplast of C. reinhardtii.

Mutational analysis of eggplant latent viroid RNA processing in Chlamydomonas reinhardtii chloroplast.

Viroids of the family Avsunviroidae, such as eggplant latent viroid (ELVd), contain hammerhead ribozymes and replicate in the chloroplasts of the host plant through an RNA-based symmetrical rolling-circle mechanism in which oligomeric RNAs of both polarity are processed to monomeric linear RNAs (by cleavage) and to monomeric circular RNAs (by ligation). Using an experimental system consisting of transplastomic lines of the alga Chlamydomonas reinhardtii, a mutational analysis of sequence and structural elements in the ELVd molecule that are involved in transcript processing in vivo in a chloroplastic context was carried out. A collection of six insertion and three deletion ELVd mutants was created and expressed in C. reinhardtii chloroplast. All mutants cleaved efficiently except for the control with an insertion inside the hammerhead ribozyme domain, supporting the prediction that this domain is necessary and sufficient to mediate transcript cleavage in vivo. However, two deletion mutants that cleaved efficiently showed ligation defects, indicating that during RNA circularization, other parts of the molecule are involved in addition to the hammerhead ribozyme domain. This is probably a quasi double-stranded structure present in the central part of the molecule which contains the ligation site in an internal loop. However, the mutations prevented the viroid from infecting its natural host, eggplant, indicating that they affected other essential functions in ELVd infectious cycle. The insertion in the terminal loop of the right upper hairpin of ELVd did not have this effect; it was tolerated and partially maintained in the progeny.

Specific roles of 5' RNA secondary structures in stabilizing transcripts in chloroplasts.

RNA secondary structures, e.g. stem-loops that are often found at the 5' and 3' ends of mRNAs, are in many cases known to be crucial for transcript stability but their role in prolonging the lifetime of transcripts remains elusive. In this study we show for an essential RNA-stabilizing stem-loop at the 5' end of rbcL gene transcripts in Chlamydomonas that it neither prevents ribonucleases from binding to the RNA nor impedes their movement along the RNA strand. The stem-loop has a formative function in that it mediates folding of a short sequence around its base into a specific RNA conformation, consisting of a helical and single-stranded region, i.e. the real structure required for longevity of rbcL transcripts in chloroplasts. Disturbing this structure renders transcripts completely unstable, even if the sequence of this element is not altered. The requirement of a specific 5' sequence and structure for RNA longevity suggests an interaction of this element with a trans-acting factor that protects transcripts from rapid degradation in chloroplasts.

Influence of cooking conditions on organoleptic and health-related properties of artichokes, green beans, broccoli and carrots.

Colour, pigments, total phenolic content and antioxidant activity were investigated in artichokes, green beans, broccoli and carrots cooked under different conditions. Domestic induction hobs with temperature control were used to evaluate the effect of boiling, sous-vide cooking and water immersion cooking at temperatures below 100°C on the properties of each vegetable. Sous-vide cooking preserved chlorophyll, carotenoids, phenolic content and antioxidant activity to a greater extent than boiling for all of the vegetables tested and retained colour better, as determined by a(∗). A reduction of only 10-15°C in the cooking temperature was enough to improve the properties of the samples cooked by water immersion, except for green beans. Artichokes and carrots suffered pronounced losses of antioxidant activity during boiling (17.0 and 9.2% retention, respectively), but the stability of this parameter significantly increased with sous-vide cooking (84.9 and 55.3% retention, respectively).

Processing of RNAs of the family Avsunviroidae in Chlamydomonas reinhardtii chloroplasts.

The family Avsunviroidae comprises four viroid species with the ability to form hammerhead ribozymes that mediate self-cleavage of the multimeric plus and minus strands resulting from replication in the chloroplast through a symmetric rolling-circle mechanism. Research on these RNAs is restricted by their host range, which is limited to the plants wherein they were initially identified and some closely related species. Here we report cleavage and ligation in transplastomic Chlamydomonas reinhardtii expressing plus- and minus-strand dimeric transcripts of representative members of the family Avsunviroidae. Despite the absence of viroid RNA-RNA transcription, the C. reinhardtii-based system can be used to address intriguing questions about viroid RNA processing and, in particular, about the cellular factors involved in cleavage and ligation.