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Protein ubiquitylation regulates a broad variety of biological processes in all eukaryotes. Recent work identified a novel class of cullin-containing ubiquitin ligases (E3s) composed of CUL4, DDB1, and one WD40 protein, believed to act as a substrate receptor. Strikingly, CUL4-based E3 ligases (CRL4s) have important functions at the chromatin level, including responses to DNA damage in metazoans and plants and, in fission yeast, in heterochromatin silencing. Among putative CRL4 receptors we identified MULTICOPY SUPPRESSOR OF IRA1 (MSI1), which belongs to an evolutionary conserved protein family. MSI1-like proteins contribute to different protein complexes, including the epigenetic regulatory Polycomb repressive complex 2 (PRC2). Here, we provide evidence that Arabidopsis MSI1 physically interacts with DDB1A and is part of a multimeric protein complex including CUL4. CUL4 and DDB1 loss-of-function lead to embryo lethality. Interestingly, as in fis class mutants, cul4 mutants exhibit autonomous endosperm initiation and loss of parental imprinting of MEDEA, a target gene of the Arabidopsis PRC2 complex. In addition, after pollination both MEDEA transcript and protein accumulate in a cul4 mutant background. Overall, our work provides the first evidence of a physical and functional link between a CRL4 E3 ligase and a PRC2 complex, thus indicating a novel role of ubiquitylation in the repression of gene expression.

Mitochondria arose from the endosymbiosis of a bacterium related to contemporary members of Alphaproteobacteria (2). Despite a probable monophylogenetic origin and the conservation of most of the biological roles of mitochondria, mitochondrial (mt) genome organization and expression are extraordinarily diverse among eucaryotes. As exemplified in animals and higher plants, mt genome size can vary from 16 kbp to several hundreds of kilobase pairs, respectively. This large increase in plant mt genome size is not correlated with increasing coding capacity but is rather explained by the presence of large intergenic regions, which are virtually absent from animal mt genomes. Plant mt genomes also contain insertions of nuclear and plastid sequences as well as foreign DNA from viral and unknown origins. As a consequence of the diversity in genome organization, mechanisms controlling mt gene expression also differ markedly between organisms. Transcription of the human mt genome is a relatively straightforward process, as both strands are transcribed from single promoters lying in the noncoding regulatory region. By contrast, numerous promoters are scattered along both strands of plant mt genomes. Moreover, even multiple promoters for a single gene are a common feature in plant mitochondria (11, 14). The recent analysis of these promoter sequences in Arabidopsis thaliana also revealed a somehow relaxed promoter specificity, as multiple sequences are able to initiate transcription (11). In addition, there is so far no evidence for a transcription termination mechanism in plant mitochondria. For instance, large regions downstream of rrn genes are expressed, although they do not give rise to stable transcripts (4). The emerging picture is that transcription in plant mitochondria is a relaxed process which, in general, exhibits little control or modulation. Rather, posttranscriptional events such as processing and control of RNA stability account for proper control of gene expression in plant mitochondria (4, 7, 13). Exoribonucleases are major players in RNA 3′ processing and degradation processes. We have recently characterized an A. thaliana nuclear gene (At5g14580) that encodes a mitochondrial exoribonuclease belonging to the polynucleotide phosphorylase (PNPase) family (17). The mt PNPase is essential for viability in Arabidopsis, in contrast to the chloroplast PNPase-like protein encoded by the At3g03710 gene. Down-regulation of the mt PNPase results in the accumulation of unprocessed RNAs, such as atp9 and orfB mRNA and 18S rRNA precursors (16, 17). Moreover, RNA species that are quickly turned over in wild-type (WT) plants accumulate in the absence of PNPase. Such RNAs include, for instance, 18S rRNA degradation intermediates and the leader of the 18S rRNA, which is removed by an endonuclease from the primary transcript. These initial studies revealed that PNPase is essential for several aspects of mt RNA metabolism, including 3′ processing and degradation processes. Interestingly, all RNA substrates of PNPase that we have investigated so far are polyadenylated. Although mature RNAs are not constitutively polyadenylated in plant mitochondria, poly(A) tails trigger rapid exonucleolytic degradation by PNPase, similar to the situation reported first for Escherichia coli and later for chloroplasts (1, 3). To determine new substrates, and thus novel biological roles, of PNPase, we cloned about 300 polyadenylated mt RNAs from plants down-regulated for PNPase (PNP− plants). These sequences were considered degradation tags, as they allowed the identification of different classes of transcripts requiring PNPase for their degradation as judged by their accumulation in PNP− versus WT plants. Our results indicate that maturation by-products such as rRNA leaders or tRNA intergenic sequences are generally degraded by PNPase. In addition, we show that a major role for PNPase is the degradation of transcripts that are, in some cases, expressed to surprisingly high levels from regions lacking known genes. Some of these RNAs, which include transcripts of chimeric open reading frames (ORFs) created by mt DNA recombination events or antisense (AS) RNA transcribed from the opposite DNA strand of a known gene, could have a deleterious effect on mitochondrial function. These data show that the lack of tight transcriptional control in Arabidopsis mitochondria is counterbalanced by polyadenylation-mediated decay by PNPase.

We have used RT-PCR and low-stringency cDNA library screening to isolate the coding sequence of the sunflower auxin-binding protein (ABP1). All the clones analysed contained the same nucleotide sequence, suggesting that ABP1 is encoded by a single-copy gene in sunflower. The deduced amino acid sequence shows a high degree of similarity with ABP1 proteins from other plant species. Most remarkably, the sunflower protein lacks two cysteine residues present in all other plant ABPs known to date and shown to be involved in a disulfide bridge in the maize protein. Genomic Southern hybridization data support the existence of a single copy of the ABP1 gene in the sunflower genome. Northern hybridization corroborated earlier observations indicating that the steady-state level of ABP1 transcript is higher in actively dividing and growing organs than in the rest of the plant: it is more abundant in the shoot apex, floral buds and immature embryos than in mature leaves, stem, roots and ray flowers. To characterize the tissular ABP1 transcript distribution in sunflower, various organ sections were analysed upon in situ hybridization. Localized accumulation of the ABP1 transcript suggests that its spatial expression is highly regulated at the tissue level. In addition, the transcript preferentially accumulates in tissues having a high rate of cellular division, such as shoot and root apical meristems, leaf primordia and pro-vascular tissues. The ABP1 expression pattern was also studied at a temporal scale during lateral root formation. Real time PCR showed an elevation of the steady state level of the ABP1 transcript in root axes after 36 h of seed germination. In situ hybridization revealed that this global increase is the result of local accumulation of the ABP1 transcript in lateral root primordia, which are known to develop under auxin action. The possibility that a high ABP1 expression level correlates with a high cellular sensitivity to auxin is discussed.

Degradation of mRNAs is usually initiated by deadenylation, the shortening of long poly(A) tails to oligo(A) tails of 12-15 As. Deadenylation leads to decapping and to subsequent 5' to 3' degradation by XRN proteins, or alternatively 3' to 5' degradation by the exosome. Decapping can also be induced by uridylation as shown for the non-polyadenylated histone mRNAs in humans and for several mRNAs in Schizosaccharomyces pombe and Aspergillus nidulans. Here we report a novel role for uridylation in preventing 3' trimming of oligoadenylated mRNAs in Arabidopsis. We show that oligo(A)-tailed mRNAs are uridylated by the cytosolic UTP:RNA uridylyltransferase URT1 and that URT1 has no major impact on mRNA degradation rates. However, in absence of uridylation, oligo(A) tails are trimmed, indicating that uridylation protects oligoadenylated mRNAs from 3' ribonucleolytic attacks. This conclusion is further supported by an increase in 3' truncated transcripts detected in urt1 mutants. We propose that preventing 3' trimming of oligo(A)-tailed mRNAs by uridylation participates in establishing the 5' to 3' directionality of mRNA degradation. Importantly, uridylation prevents 3' shortening of mRNAs associated with polysomes, suggesting that a key biological function of uridylation is to confer 5' to 3' polarity in case of co-translational mRNA decay.

With the expansion of the RNA world, antisense strategies have become widespread to manipulate nuclear gene expression but organelle genetic systems have remained aside. The present work opens the field to mitochondria. We demonstrate that customized RNAs expressed from a nuclear transgene and driven by a transfer RNA-like (tRNA-like) moiety are taken up by mitochondria in plant cells. The process appears to follow the natural tRNA import specificity, suggesting that translocation indeed occurs through the regular tRNA uptake pathway. Upon validation of the strategy with a reporter sequence, we developed a chimeric catalytic RNA composed of a specially designed trans-cleaving hammerhead ribozyme and a tRNA mimic. Organelle import of the chimeric ribozyme and specific target cleavage within mitochondria were demonstrated in transgenic tobacco cell cultures and Arabidopsis thaliana plants, providing the first directed knockdown of a mitochondrial RNA in a multicellular eukaryote. Further observations point to mitochondrial messenger RNA control mechanisms related to the plant developmental stage and culture conditions. Transformation of mitochondria is only accessible in yeast and in the unicellular alga Chlamydomonas. Based on the widespread tRNA import pathway, our data thus make a breakthrough for direct investigation and manipulation of mitochondrial genetics.

Terminal restriction fragment length polymorphism (T-RFLP) is used to monitor the structural diversity of complex microbial communities in terms of richness, relative abundance, and distribution of the major subpopulations and individual members. However, discrepancies of several nucleotides between expected and experimentally observed lengths of terminal restriction fragments (T-RFs), together with the difficulty of obtaining DNA sequence information from T-RFLP profiling, often prevent accurate phylogenetic characterization of the microbial community of interest. In this study, T-RFLP analysis of DNA from an artificial assembly of five bacterial strains was carried out with a combination of two size markers with different fluorescent tags. Precise sizing of T-RFs in the 50- to 500-nucleotide range was achieved by using the same dye for both samples and size markers. Phylogenetic assignment of the component microbial strains was facilitated by coupling T-RFLP to denaturing high-performance liquid chromatography (D-HPLC) of 16S RNA gene fragments followed by direct sequencing. The proposed coupling of D-HPLC and T-RFLP provides unambiguous characterization of microbial communities containing less than 15 microbial strains.

Plant growth involves the integration of many environmental and endogenous signals that together with the intrinsic genetic program determine plant size. At the cellular level, growth rate is regulated by the combined activity of two processes: cell proliferation and expansion. Gibberellins (GA) are plant-specific hormones that play a central role in the regulation of growth and development with respect to environmental variability [1], [2] and [3]. It is well established that GA promote growth through cell expansion by stimulating the destruction of growth-repressing DELLA proteins (DELLAs) [1], [4], [5] and [6]; however, their effects on cell proliferation remain unknown. Kinematic analysis of leaf and root meristem growth revealed a novel function of DELLAs in restraining cell production. Moreover, by visualizing the cell cycle marker cyclinB1:â-glucuronidase in GA-signaling mutants, we show that GA modulate cell cycle activity in the root meristem via a DELLA-dependent mechanism. Accordingly, expressing gai (a nondegradable DELLA protein [4]) solely in root meristem reduced substantially the number of dividing cells. We also show that DELLAs restrain cell production by enhancing the levels of the cell cycle inhibitors Kip-related protein 2 (KRP2) and SIAMESE (SIM). Therefore, DELLAs exert a general plant growth inhibitory activity by reducing both cell proliferation and expansion rates, enabling phenotypic plasticity.

Plants have evolved robust mechanisms to respond and adapt to unfavorable environmental conditions, such as low temperature. The C-repeat/drought-responsive element binding factor CBF1/DREB1b gene encodes a transcriptional activator transiently induced by cold that controls the expression of a set of genes responding to low temperature (the CBF regulon). Constitutive expression of CBF1 confers freezing tolerance but also slows growth. Here, we propose that low temperature–induced CBF1 expression restrains growth at least in part by allowing the accumulation of DELLAs, a family of nuclear growth-repressing proteins, the degradation of which is stimulated by gibberellin (GA). We show that cold/CBF1 enhances the accumulation of a green fluorescent protein (GFP)–tagged DELLA protein (GFP-RGA) by reducing GA content through stimulating expression of GA-inactivating GA 2-oxidase genes. Accordingly, transgenic plants that constitutively express CBF1 accumulate less bioactive GA and as a consequence exhibit dwarfism and late flowering. Both phenotypes are suppressed when CBF1 is expressed in a line lacking two DELLA proteins, GA-INSENSITIVE and REPRESSOR OF GA1-3. In addition, we show that DELLAs contribute significantly to CBF1-induced cold acclimation and freezing tolerance by a mechanism that is distinct from the CBF regulon. We conclude that DELLAs are components of the CBF1-mediated cold stress response.

We have used RT-PCR and low-stringency cDNA library screening to isolate the coding sequence of the sunflower auxin-binding protein (ABP1). All the clones analysed contained the same nucleotide sequence, suggesting that ABP1 is encoded by a single-copy gene in sunflower. The deduced amino acid sequence shows a high degree of similarity with ABP1 proteins from other plant species. Most remarkably, the sunflower protein lacks two cysteine residues present in all other plant ABPs known to date and shown to be involved in a disulfide bridge in the maize protein. Genomic Southern hybridization data support the existence of a single copy of the ABP1 gene in the sunflower genome. Northern hybridization corroborated earlier observations indicating that the steady-state level of ABP1 transcript is higher in actively dividing and growing organs than in the rest of the plant: it is more abundant in the shoot apex, floral buds and immature embryos than in mature leaves, stem, roots and ray flowers. To characterize the tissular ABP1 transcript distribution in sunflower, various organ sections were analysed upon in situ hybridization. Localized accumulation of the ABP1 transcript suggests that its spatial expression is highly regulated at the tissue level. In addition, the transcript preferentially accumulates in tissues having a high rate of cellular division, such as shoot and root apical meristems, leaf primordia and pro-vascular tissues. The ABP1 expression pattern was also studied at a temporal scale during lateral root formation. Real time PCR showed an elevation of the steady state level of the ABP1 transcript in root axes after 36 h of seed germination. In situ hybridization revealed that this global increase is the result of local accumulation of the ABP1 transcript in lateral root primordia, which are known to develop under auxin action. The possibility that a high ABP1 expression level correlates with a high cellular sensitivity to auxin is discussed.

Summary Being sessile organisms, plants need rapid and finely tuned signaling pathways to adapt their growth and survival over their immediate and often adverse environment. Abscisic acid (ABA) is a plant hormone crucial for both biotic and abiotic stress responses. In this study, we highlight a function of six Arabidopsis MATH-BTB proteins in ABA signaling. MATH-BTB proteins act as substrate-binding adaptors for the Cullin3-based ubiquitin E3 ligase. Our genetic and biochemical experiments demonstrate that the MATH-BTB proteins directly interact with and target for proteasomal degradation the class I homeobox-leucine zipper (HD-ZIP) transcription factor ATHB6, which was previously identified as a negative regulator of ABA responses. Reducing CUL3 BPM function leads to higher ATHB6 protein accumulation, reducing plant growth and fertility, and affects stomatal behavior and responses to ABA. We further demonstrate that ABA negatively regulates ATHB6 protein turnover, a situation reminiscent to ABI5, another transcription factor involved in ABA signaling.