Comparison of mitochondrial gene expression and polysome loading in different tobacco tissues.

BACKGROUND: To investigate translational regulation of gene expression in plant mitochondria, a mitochondrial polysome isolation protocol was established for tobacco to investigate polysomal mRNA loading as a proxy for translational activity. Furthermore, we developed an oligonucleotide based microarray platform to determine the level of Nicotiana tabacum and Arabidopsis thaliana mitochondrial mRNA. RESULTS: Microarray analysis of free and polysomal mRNAs was used to characterize differences in the levels of free transcripts and ribosome-bound mRNAs in various organs of tobacco plants. We have observed higher mitochondrial transcript levels in young leaves, flowers and floral buds as compared to fully expanded leaves and roots. A similar pattern of abundance was observed for ribosome-bound mitochondrial mRNAs in these tissues. However, the accumulation of the mitochondrial protein COX2 was found to be inversely related to that of its ribosome-bound mRNA. CONCLUSIONS: Our results indicate that the association of mitochondrial mRNAs to ribosomes is largely determined by the total transcript level of a gene. However, at least for Cox2, we demonstrated that the level of ribosome-bound mRNA is not reflected by the amount of COX2 protein.

LEA gene expression, RNA stability and pigment accumulation in three closely related Linderniaceae species differing in desiccation tolerance.

Desiccation-tolerant plants (Craterostigma plantagineum and Lindernia brevidens) evolved a highly efficient strategies to prevent dehydration-induced irreversible damage. The protection system involves synthesis of LEA proteins, decrease of photosynthetic activity and activation of antioxidant systems. The regulation of these processes requires joint action of multiple proteins. Here, we present comparative analyses of accumulation of transcripts encoding components of the protection machinery, such as selected LEA proteins, enzymes of the chlorophyll degradation pathway and anthocyanin biosynthesis enzymes in total and polysomal RNA pools. The analyses revealed that desiccation-tolerant plants recruit mRNAs to ribosomes with higher efficiency than the desiccation-sensitive species L. subracemosa. Desiccation-tolerant species accumulated high amounts of LEA transcripts during dehydration and precisely controlled the amounts of chlorophyll keeping it at a level sufficient to activate photosynthesis after rehydration. In contrast, mRNA of L. subracemosa was prone to dehydration-induced degradation, decomposition of the photosynthetic apparatus and degradation of free chlorophyll. Thus, the results of the studies point to differences in the control of gene expression and degradation of chlorophyll in desiccation-tolerant versus desiccation-sensitive species when the plants were subjected to dehydration.

Natural Variation of Cold Deacclimation Correlates with Variation of Cold-Acclimation of the Plastid Antioxidant System in Arabidopsis thaliana Accessions.

Temperature variations impact on the balance between photosynthetic electron transport and electron-consuming assimilation reactions and transiently increase generation of reactive oxygen species (ROS). Previous studies demonstrated that the expression of C-repeat binding factors (CBFs), which activate cold acclimation reactions, respond to chloroplast ROS signals and that cold deacclimation is partly halted for days after the transfer of acclimated plants to optimal growth conditions in four Arabidopsis accessions from cold-continental habitats. We hypothesized that these accessions differ from others in the regulation of the plastid antioxidant system (PAS). In the present study, we compared the expression intensity of the 12 most prominent PAS genes for peroxidases, superoxide dismutase and low molecular weight antioxidant regenerating enzymes in 10 Arabidopsis accessions with regulation of CBF and COR (cold regulated genes) transcript levels and cold-regulated metabolite levels prior to cold, after 2 week long cold acclimation and during the first 3 days of deacclimation. In the accessions with prolonged activation of cold responses, by trend, weaker induction of various cold-inducible PAS genes and stronger decreases in the expression of negatively cold-regulated PAS genes were observed. Low PAS gene expression delayed the post-cold decrease in H2O2 levels after transfer of the plants from cold to optimal growth conditions. We conclude that weaker expression of various PAS genes in the cold is an adapted strategy of the Arabidopsis accessions N14, N13, Ms-0, and Kas-1 to avoid full inactivation of cold-responses in the first days after the end of the cold period.

Time-dependent deacclimation after cold acclimation in Arabidopsis thaliana accessions.

During low temperature exposure, Arabidopsis thaliana and many other plants from temperate climates increase in freezing tolerance in a process termed cold acclimation. However, the correct timing and rate of deacclimation, resulting in loss of freezing tolerance and initiation of growth is equally important for plant fitness and survival. While the molecular basis of cold acclimation has been investigated in detail, much less information is available about deacclimation. We have characterized the responses of 10 natural accessions of Arabidopsis thaliana that vary widely in their freezing tolerance, to deacclimation conditions. Sugar, proline and transcript levels declined sharply over three days in all accessions after transfer of cold acclimated plants to ambient temperatures, while freezing tolerance only declined in tolerant accessions. Correlations between freezing tolerance and the expression levels of COR genes and the content of glucose, fructose and sucrose, as well as many correlations among transcript and solute levels, that were highly significant in cold acclimated plants, were lost during deacclimation. Other correlations persisted, indicating that after three days of deacclimation, plant metabolism had not completely reverted back to the non-acclimated state. These data provide the basis for further molecular and genetic studies to unravel the regulation of deacclimation.

Quantification of superoxide and hydrogen peroxide in leaves.

Reactive oxygen species (ROS) are produced in plants under both non-stressful and stressful conditions. Various histochemical staining methods have been developed and are widely used to visualize ROS accumulation sites. In contrast to qualitative analysis, quantification of ROS has been time- and labor consuming. As a consequence, the number of samples, which could be analyzed in parallel, has been limited. To overcome this problem, we introduce an improved semiquantitative method, in which ROS levels are quantified after histochemical staining in plant organs with the digital image analysis package ImageJ.

The strength of the miR398-Csd2-CCS1 regulon is subject to natural variation in Arabidopsis thaliana.

miR398 links expression of the three major chloroplast copper proteins, plastocyanin, CCS1 and Csd2, to copper availability. miR398 abundance was stronger plastocyanin-controlled in accessions from cold and continental habitats (Kas-1, Ms-0, WS) than in Cvi-0 and Col-0. Target gene regulation was broken for Csd2 in Cvi-0 upon cold-treatment. Comparison of miR398 levels, target gene regulation as well as Ago1 and miR168 expression demonstrated that the miR398 regulon can be overwritten by accession specific transcriptional regulation in Cvi-0. It is concluded that the escape from the miRNA control of copper homeostasis is linked to adaptation of Cvi-0 to its harsh natural habitat.

Natural genetic variation in the expression regulation of the chloroplast antioxidant system among Arabidopsis thaliana accessions.

Photosynthesis is the predominant source of reactive oxygen species in light. In order to prevent the negative influence of reactive oxygen species (ROS) on cell functionality, chloroplasts have evolved a highly efficient antioxidant protection system. Here, we present the first study on natural variation in this system. Comparison of temperature and developmental responses in seven accessions of Arabidopsis thaliana from northern habitats showed that the regulation is widely genetically manifested, but hardly correlates with geographic parameters. Transcript, polysomal RNA (pRNA) and protein data showed that the ecotypes use different strategies to adjust the chloroplast antioxidative defense system, either by regulating transcript abundance or initiation of translation. Comparison of mRNA and pRNA levels showed that Col-0 invests more into transcript accumulation, while Van-0, WS and C24 regulates the chloroplast antioxidant protection system more on the level of pRNA. Nevertheless, both strategies of regulation led to the expression of chloroplast antioxidant enzymes at sufficient level to efficiently protect plants from ROS accumulation in Col-0, WS, C24 and Van-0. On the contrary, Cvi-0, Ms-0 and Kas-1 accumulated high amounts of ROS. The expression of copper/zinc superoxide dismutase (Csd2), ascorbate peroxidases and 2-Cys peroxiredoxins was higher in Cvi-0 on the transcriptional level, while Csd2, peroxiredoxin Q, type II peroxiredoxin E and glutathione peroxidase 1 were induced in Ms-0 on the mRNA level. Similar to Kas-1, in which mRNA levels were less than or similar to Col-0 gene, specific support for translation was observed in Ms-0, showing that the ecotypes use different strategies to adjust the antioxidant system.

Identification and characterization of high molecular weight complexes formed by matrix AAA proteases and prohibitins in mitochondria of Arabidopsis thaliana.

We identify and characterize two matrix (m)-AAA proteases (AtFtsH3 and AtFtsH10) present in the mitochondria of Arabidopsis thaliana. AtFtsH3 is the predominant protease in leaves of wild type plants. Both proteases assemble with prohibitins (PHBs) into high molecular weight complexes (approximately 2 MDa), similarly to their yeast counterparts. A smaller PHB complex (approximately 1 MDa), without the m-AAA proteases, was also detected. Unlike in yeast, stable prohibitin-independent high molecular weight assemblies of m-AAA proteases could not be identified in A. thaliana. AtFtsH3 and AtFtsH10 form at least two types of m-AAA-PHB complexes in wild type plants. The one type contains PHBs and AtFtsH3, and the second one is composed of PHBs and both AtFtsH3 and AtFtsH10. Complexes composed of PHBs and AtFtsH10 were found in an Arabidopsis mutant lacking AtFtsH3 (ftsh3). Thus, both AtFtsH3 and AtFtsH10 may form hetero- and homo-oligomeric complexes with prohibitins. The increased level of AtFtsH10 observed in ftsh3 suggests that functions of the homo- and hetero-oligomeric complexes containing AtFtsH3 can be at least partially substituted by AtFtsH10 homo-oligomers. The steady-state level of the AtFtsH10 transcripts did not change in ftsh3 compared with wild type plants, but we found that almost twice more of the AtFtsH10 transcripts were associated with polysomes in ftsh3. Based on this result, we assume that the AtFtsH10 protein is synthesized at a higher rate in the ftsh3 mutant. Our results provide the first data on the composition of m-AAA and PHB complexes in plant mitochondria and suggest that the abundance of m-AAA proteases is regulated not only at the transcriptional but also at the translational level.