In contrast with the model Escherichia coli Clp protease, the ATP-dependent Clp protease in higher plants has a remarkably diverse proteolytic core consisting of multiple ClpP and ClpR paralogs, presumably arranged within a dual heptameric ring structure. Using antisense lines for the nucleus-encoded ClpP subunit, ClpP6, we show that the Arabidopsis thaliana Clp protease is vital for chloroplast development and function. Repression of ClpP6 produced a proportional decrease in the Clp proteolytic core, causing a chlorotic phenotype in young leaves that lessened upon maturity. Structural analysis of the proteolytic core revealed two distinct subcomplexes that likely correspond to single heptameric rings, one containing the ClpP1 and ClpR1-4 proteins, the other containing ClpP3-6. Proteomic analysis revealed several stromal proteins more abundant in clpP6 antisense lines, suggesting that some are substrates for the Clp protease. A proteolytic assay developed for intact chloroplasts identified potential substrates for the stromal Clp protease in higher plants, most of which were more abundant in young Arabidopsis leaves, consistent with the severity of the chlorotic phenotype observed in the clpP6 antisense lines. The identified substrates all function in more general housekeeping roles such as plastid protein synthesis, folding, and quality control, rather than in metabolic activities such as photosynthesis.

The identity and scope of chloroplast and mitochondrial proteases in higher plants has only started to become apparent in recent years. Biochemical and molecular studies suggested the existence of Clp, FtsH, and DegP proteases in chloroplasts, and a Lon protease in mitochondria, although currently the full extent of their role in organellar biogenesis and function remains poorly understood. Rapidly accumulating DNA sequence data, especially from Arabidopsis, has revealed that these proteolytic enzymes are found in plant cells in multiple isomeric forms. As a consequence, a systematic approach was taken to catalog all these isomers, to predict their intracellular location and putative processing sites, and to propose a standard nomenclature to avoid confusion and facilitate scientific communication. For the Clp protease most of the ClpP isomers are found in chloroplasts, whereas one is mitochondrial. Of the ATPase subunits, the one ClpD and two ClpC isomers are located in chloroplasts, whereas both ClpX isomers are present in mitochondria. Isomers of the Lon protease are predicted in both compartments, as are the different forms of FtsH protease. DegP, the least characterized protease in plant cells, has the most number of isomers and they are predicted to localize in several cell compartments. These predictions, along with the proposed nomenclature, will serve as a framework for future studies of all four families of proteases and their individual isomers.

The ATP-dependent Clp protease is one of the newly identified proteolytic systems in plant organelles that incorporate the activity of molecular chaperones to target specific polypeptide substrates and avoid inadvertent degradation of others. We describe new nuclear-encoded ClpC (ClpC1) and ClpP (ClpP3-5) isomers in Arabidopsis thaliana that raise the total number of identified Clp proteins to 19. The extra Clp proteins are localized within the stroma of chloroplasts along with the ClpD, -P1 and -P6 proteins. Potential differential regulation among these Clp proteins was analysed at both the mRNA and protein level. A comparison between different tissues showed increasing amounts of all plastid Clp proteins from roots to stems to leaves suggested the greatest abundance of proteins was in chloroplasts. The increases in protein were mirrored at the mRNA level for most ClpP isomers (ClpP1, -3, -4 and -6) but not for the three Hsp100 proteins (ClpC1, -C2 and -D) and ClpP5, which exhibited little change in transcript levels, suggesting post-transcriptional/translational regulation. Potential stress induction was also tested for all chloroplast Clp proteins by a series of brief and prolonged stress conditions. Short-term moderate and severe stresses (desiccation, high salt, cold, heat, oxidation, wounding and high light) all failed to elicit significant or rapid increases in any chloroplast Clp protein. However, increases in mRNA and protein content for ClpD and several ClpP isomers did occur during long-term high light and cold acclimation of Arabidopsis plants. These results reveal the great complexity of Clp proteins within the stroma of plant chloroplasts, and that these proteins, rather than being rapidly induced stress proteins, are primarily constitutive proteins that may also be involved in plant acclimation to different physiological conditions.

BackgroundThe advent of genomics-based technologies has revolutionized many fields of biological enquiry. However, chromosome walking or flanking sequence cloning is still a necessary and important procedure to determining gene structure. Such methods are used to identify T-DNA insertion sites and so are especially relevant for organisms where large T-DNA insertion libraries have been created, such as rice and Arabidopsis. The currently available methods for flanking sequence cloning, including the popular TAIL-PCR technique, are relatively laborious and slow.ResultsHere, we report a simple and effective fusion primer and nested integrated PCR method (FPNI-PCR) for the identification and cloning of unknown genomic regions flanked known sequences. In brief, a set of universal primers was designed that consisted of various 15-16 base arbitrary degenerate oligonucleotides. These arbitrary degenerate primers were fused to the 3' end of an adaptor oligonucleotide which provided a known sequence without degenerate nucleotides, thereby forming the fusion primers (FPs). These fusion primers are employed in the first step of an integrated nested PCR strategy which defines the overall FPNI-PCR protocol. In order to demonstrate the efficacy of this novel strategy, we have successfully used it to isolate multiple genomic sequences namely, 21 orthologs of genes in various species of Rosaceace, 4 MYB genes of Rosa rugosa, 3 promoters of transcription factors of Petunia hybrida, and 4 flanking sequences of T-DNA insertion sites in transgenic tobacco lines and 6 specific genes from sequenced genome of rice and Arabidopsis.ConclusionsThe successful amplification of target products through FPNI-PCR verified that this novel strategy is an effective, low cost and simple procedure. Furthermore, FPNI-PCR represents a more sensitive, rapid and accurate technique than the established TAIL-PCR and hiTAIL-PCR procedures.

Summary Survival of trees growing in temperate zones requires cycling between active growth and dormancy. This involves growth cessation in the autumn triggered by a photoperiod shorter than the critical day length. Variations in GIGANTEA (GI)‐like genes have been associated with phenology in a range of different tree species, but characterization of the functions of these genes in the process is still lacking. We describe the identification of the Populus orthologs of GI and their critical role in short‐day‐induced growth cessation. Using ectopic expression and silencing, gene expression analysis, protein interaction and chromatin immunoprecipitation experiments, we show that PttGIs are likely to act in a complex with PttFKF1s (FLAVIN‐BINDING, KELCH REPEAT, F‐BOX 1) and PttCDFs (CYCLING DOF FACTOR) to control the expression of PttFT2, the key gene regulating short‐day‐induced growth cessation in Populus. In contrast to Arabidopsis, in which the GI‐CONSTANS (CO)‐FLOWERING LOCUS T (FT) regulon is a crucial day‐length sensor for flowering time, our study suggests that, in Populus, PttCO‐independent regulation of PttFT2 by PttGI is more important in the photoperiodic control of growth cessation and bud set.

BackgroundIn all domains of life, transfer RNA (tRNA) molecules contain modified nucleosides. Modifications to tRNAs affect their coding capacity and influence codon-anticodon interactions. Nucleoside modification deficiencies have a diverse range of effects, from decreased virulence in bacteria, neural system disease in human, and gene expression and stress response changes in plants. The purpose of this study was to identify genes involved in tRNA modification in the model plant Arabidopsis thaliana, to understand the function of nucleoside modifications in plant growth and development.ResultsIn this study, we established a method for analyzing modified nucleosides in tRNAs from the model plant species, Arabidopsis thaliana and hybrid aspen (Populus tremula × tremuloides). 21 modified nucleosides in tRNAs were identified in both species. To identify the genes responsible for the plant tRNA modifications, we performed global analysis of the Arabidopsis genome for candidate genes. Based on the conserved domains of homologs in Sacccharomyces cerevisiae and Escherichia coli, more than 90 genes were predicted to encode tRNA modifying enzymes in the Arabidopsis genome. Transcript accumulation patterns for the genes in Arabidopsis and the phylogenetic distribution of the genes among different plant species were investigated. Transcripts for the majority of the Arabidopsis candidate genes were found to be most abundant in rosette leaves and shoot apices. Whereas most of the tRNA modifying gene families identified in the Arabidopsis genome was found to be present in other plant species, there was a big variation in the number of genes present for each family.Through a loss of function mutagenesis study, we identified five tRNA modification genes (AtTRM10, AtTRM11, AtTRM82, AtKTI12 and AtELP1) responsible for four specific modified nucleosides (m1G, m2G, m7G and ncm5U), respectively (two genes: AtKTI12 and AtELP1 identified for ncm5U modification). The AtTRM11 mutant exhibited an early-flowering phenotype, and the AtELP1 mutant had narrow leaves, reduced root growth, an aberrant silique shape and defects in the generation of secondary shoots.ConclusionsUsing a reverse genetics approach, we successfully isolated and identified five tRNA modification genes in Arabidopsis thaliana. We conclude that the method established in this study will facilitate the identification of tRNA modification genes in a wide variety of plant species.

Plant secondary growth derives from the meristematic activity of the vascular cambium. In Arabidopsis thaliana, cell divisions in the cambium are regulated by the transcription factor WOX4, a key target of the CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (ESR)-RELATED 41 (CLE41) signaling pathway. However, function of the WOX4-like genes in plants that are dependent on a much more prolific secondary growth, such as trees, remains unclear. Here, we investigate the role of WOX4 and CLE41 homologs for stem secondary growth in Populus trees. In Populus, PttWOX4 genes are specifically expressed in the cambial region during vegetative growth, but not after growth cessation and during dormancy, possibly involving a regulation by auxin. In PttWOX4a/b RNAi trees, primary growth was not affected whereas the width of the vascular cambium was severely reduced and secondary growth was greatly diminished. Our data show that in Populus trees, PttWOX4 genes control cell division activity in the vascular cambium, and hence growth in stem girth. This activity involves the positive regulation of PttWOX4a/b through PttCLE41-related genes. Finally, expression profiling suggests that the CLE41 signaling pathway is an evolutionarily conserved program for the regulation of vascular cambium activity between angiosperm and gymnosperm tree species.

BackgroundAlthough a number of leucine-rich repeat receptor-like kinase-encoding genes (LRR-RLKs) have been identified in plants, a functional role has been determined for only a few. Recent studies have demonstrated that an LRR-RLK, PXY/TDR, is important for the process of secondary vascular development. Other studies have indicated that PXY/TDR is unlikely to be the sole LRR-RLK involved in this complex process.ResultsIn this study, in silico analyses led to the identification of three Arabidopsis LRR-RLK genes (PXY-correlated; PXC1, 2, 3) with transcript accumulation profiles that correlated strongly with several key regulators of vascular development, including PXY/TDR, HB-8, REV, and CLE41. Expression profiling using qPCR and promoter:reporter lines indicated that all three PXC genes are associated with the vasculature. One in particular, PXC1 (At2g36570), had a strong correlation with PXY/TDR. Shifting pxc1 mutants from long-days to short-days showed that loss of the gene led to a dramatic reduction in secondary wall formation in xylem fibers. Transcript analysis of mutants for a variety of secondary cell wall-associated genes, including PXY/TDR indicated that the pathways mediated by PXC1 connect with those mediated by the TDIF-PXY/TDR-WOX4 system.ConclusionsThe data indicate that the LRR-RLK, PXC1 is involved in secondary cell wall formation in xylem fibers. Whereas further study is needed to identify the ligands and mode of action of the PXC1 protein, it is clear from this work that similarly to the shoot apical meristem (SAM), secondary vascular development requires contributions from a number of LRR-RLKs.