The Arabidopsis BELL1 and KNOX TALE homeodomain proteins interact through a domain conserved between plants and animals.

Interactions between TALE (three-amino acid loop extension) homeodomain proteins play important roles in the development of both fungi and animals. Although in plants, two different subclasses of TALE proteins include important developmental regulators, the existence of interactions between plant TALE proteins has remained unexplored. We have used the yeast two-hybrid system to demonstrate that the Arabidopsis BELL1 (BEL1) homeodomain protein can selectively heterodimerize with specific KNAT homeodomain proteins. Interaction is mediated by BEL1 sequences N terminal to the homeodomain and KNAT sequences including the MEINOX domain. These findings validate the hypothesis that the MEINOX domain has been conserved between plants and animals as an interaction domain for developmental regulators. In yeast, BEL1 and KNAT proteins can activate transcription only as a heterodimeric complex, suggesting a role for such complexes in planta. Finally, overlapping patterns of BEL1 and SHOOT MERISTEMLESS (STM) expression within the inflorescence meristem suggest a role for the BEL1-STM complex in maintaining the indeterminacy of the inflorescence meristem.

Chloroplast PNPase exists as a homo-multimer enzyme complex that is distinct from the Escherichia coli degradosome.

In Escherichia coli, the exoribonuclease polynucleotide phosphorylase (PNPase), the endoribonuclease RNase E, a DEAD-RNA helicase and the glycolytic enzyme enolase are associated with a high molecular weight complex, the degradosome. This complex has an important role in processing and degradation of RNA. Chloroplasts contain an exoribonuclease homologous to E. coli PNPase. Size exclusion chromatography revealed that chloroplast PNPase elutes as a 580-600 kDa complex, suggesting that it can form an enzyme complex similar to the E. coli degradosome. Biochemical and mass-spectrometric analysis showed, however, that PNPase is the only protein associated with the 580-600 kDa complex. Similarly, a purified recombinant chloroplast PNPase also eluted as a 580-600 kDa complex after gel filtration chromatography. These results suggest that chloroplast PNPase exists as a homo-multimer complex. No other chloroplast proteins were found to associate with chloroplast PNPase during affinity chromatography. Database analysis of proteins homologous to E. coli RNase E revealed that chloroplast and cyanobacterial proteins lack the C-terminal domain of the E. coli protein that is involved in assembly of the degradosome. Together, our results suggest that PNPase does not form a degradosome-like complex in the chloroplast. Thus, RNA processing and degradation in this organelle differ in several respects from those in E. coli.

The restorer Rfo gene acts post-translationally on the stability of the ORF138 Ogura CMS-associated protein in reproductive tissues of rapeseed cybrids.

This paper describes the analysis of the effect of the restorer gene Rfo on the expression of the ORF138 protein associated with Ogura cytoplasmic male sterility (CMS) which has been engineered in rapeseed by protoplast fusion. We show that the presence of the Rfo gene in the genome of the plants decreases the amount of ORF138 protein in floral buds, this effect being the most dramatic in anthers at the stage of development when the sterile phenotype is normally expressed. However, the amount of orf138 transcripts is not affected by the Rfo gene in the same organs at the same stages. Total polysome analyses of buds and anthers show that the orf138 transcripts are translated with the same efficiency in sterile and restored plants. From these results we infer that the Rfo gene product acts on the post-translational stability of the ORF138 protein, leading to a decrease in the accumulation of the protein and a restoration of fertility.

Low-copy-number molecules are produced by recombination, actively maintained and can be amplified in the mitochondrial genome of Brassicaceae: relationship to reversion of the male sterile phenotype in some cybrids.

A PCR analysis of mitochondrial (mt) genomes of cybrid rapeseed plants revealed substoichiometric concentrations of molecules bearing different configurations of the gene (orf138) responsible for Ogura cytoplasmic male sterility (CMS). These substoichiometric molecules are also present in plants bearing the unmodified Ogura cytoplasm. In one cybrid family, which shows reversion of the male sterile phenotype, we observed changes in the respective proportions of these molecules. The phenotypic (sterility-fertility) reversion occurs as a result of a modification of the equilibrium state between the different forms of the orf138 gene and is very probably determined by the level of expression of this gene. Stable situations are always characterized by one predominant form; the others, when present, exist in substoichiometric amounts. We report results indicating that the different forms of the orf138 gene are continuously interconverted by recombination and that an active mechanism is involved in the maintenance of some substoichiometric molecules.

The steady-state level of mRNA from the Ogura cytoplasmic male sterility locus in Brassica cybrids is determined post-transcriptionally by its 3' region.

We have investigated the control of the expression of three different configurations of the mitochondrial gene orf138, whose expression is correlated with Ogura cytoplasmic male-sterility in rapeseed cybrids. These configurations, termed Nco2.5/13S, Nco2.7/13F and Bam4.8/18S, specific to the 13S (sterile), 13F (fertile) and 18S (sterile) cybrids respectively, have the same 5' regions but different 3' regions. The orf138 transcript from Bam4.8/18S is 10-fold more abundant than the one from Nco2.5/13S, while no orf138 transcript from Nco2.7/13F accumulates. However, transcriptional activity measurements show that the rate of transcription is equivalent for the three configurations. These results strongly suggest that the steady-state level of mRNA from the orf138 locus is determined post-transcriptionally, most likely by its 3' region. To determine the role of these 3' regions, we have established an in vitro decay and processing system. In the presence of rapeseed mitochondrial lysate, synthetic RNAs corresponding to the 3' region of the Nco2.7/13F transcript are, as expected, less stable than RNAs corresponding to the 3' regions of the Nco2.5/13S and Bam4.8/18S transcripts. We have also observed in vitro processing of synthetic RNAs at the sites corresponding to the 3' ends of the natural mRNAs from Nco2.5/13S and Bam4.8/18S. Further analysis of the role of these 3' regions in in vitro RNA stability should help us to better understand post-transcriptional control in plant mitochondria.