Transcription of damage-induced RNA in Arabidopsis was frequently initiated from DSB loci within the genic regions.

DNA double-strand breaks (DSBs) are the most severe DNA lesions and need to be removed immediately to prevent loss of genomic information. Recently, it has been revealed that DSBs induce novel transcription from the cleavage sites in various species, resulting in RNAs being referred to as damage-induced RNAs (diRNAs). While diRNA synthesis is an early event in the DNA damage response and plays an essential role in DSB repair activation, the location where diRNAs are newly generated in plants remains unclear, as does their transcriptional mechanism. Here, we performed the sequencing of polyadenylated (polyA) diRNAs that emerged around all DSB loci in Arabidopsis thaliana under the expression of the exogenous restriction enzyme Sbf I and observed 88 diRNAs transcribed via RNA polymerase II in 360 DSB loci. Most of the detected diRNAs originated within active genes and were transcribed from DSBs in a bidirectional manner. Furthermore, we found that diRNA elongation tends to terminate at the boundary of an endogenous gene located near DSB loci. Our results provide reliable evidence for understanding the importance of new transcription at DSBs and show that diRNA is a crucial factor for successful DSB repair.

Inducible Expression of the Restriction Enzyme Uncovered Genome-Wide Distribution and Dynamic Behavior of Histones H4K16ac and H2A.Z at DNA Double-Strand Breaks in Arabidopsis.

DNA double-strand breaks (DSBs) are among the most serious types of DNA damage, causing mutations and chromosomal rearrangements. In eukaryotes, DSBs are immediately repaired in coordination with chromatin remodeling for the deposition of DSB-related histone modifications and variants. To elucidate the details of DSB-dependent chromatin remodeling throughout the genome, artificial DSBs need to be reproducibly induced at various genomic loci. Recently, a comprehensive method for elucidating chromatin remodeling at multiple DSB loci via chemically induced expression of a restriction enzyme was developed in mammals. However, this DSB induction system is unsuitable for investigating chromatin remodeling during and after DSB repair, and such an approach has not been performed in plants. Here, we established a transgenic Arabidopsis plant harboring a restriction enzyme gene Sbf I driven by a heat-inducible promoter. Using this transgenic line, we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) of histones H4K16ac and H2A.Z and investigated the dynamics of these histone marks around the endogenous 623 Sbf I recognition sites. We also precisely quantified DSB efficiency at all cleavage sites using the DNA resequencing data obtained by the ChIP-seq procedure. From the results, Sbf I-induced DSBs were detected at 360 loci, which induced the transient deposition of H4K16ac and H2A.Z around these regions. Interestingly, we also observed the co-localization of H4K16ac and H2A.Z at some DSB loci. Overall, DSB-dependent chromatin remodeling was found to be highly conserved between plants and animals. These findings provide new insights into chromatin remodeling that occurs in response to DSBs in Arabidopsis.

Cryptic promoter activation occurs by at least two different mechanisms in the Arabidopsis genome.

In gene-trap screening of plant genomes, promoterless reporter constructs are often expressed without trapping of annotated gene promoters. The molecular basis of this phenomenon, which has been interpreted as the trapping of cryptic promoters, is poorly understood. Here, we found that cryptic promoter activation occurs by at least two different mechanisms using Arabidopsis gene-trap lines in which a firefly luciferase (LUC) open reading frame (ORF) without an apparent promoter sequence was expressed from intergenic regions: one mechanism is 'cryptic promoter capturing', in which the LUC ORF captured pre-existing promoter-like chromatin marked by H3K4me3 and H2A.Z, and the other is 'promoter de novo origination', in which the promoter chromatin was newly formed near the 5' end of the inserted LUC ORF. The latter finding raises a question as to how the inserted LUC ORF sequence is involved in this phenomenon. To examine this, we performed a model experiment with chimeric LUC genes in transgenic plants. Using Arabidopsis psaH1 promoter-LUC constructs, we found that the functional core promoter region, where transcription start sites (TSSs) occur, cannot simply be determined by the upstream nor core promoter sequences; rather, its positioning proximal to the inserted LUC ORF sequence was more critical. This result suggests that the insertion of the coding sequence alters the local distribution of TSSs in the plant genome. The possible impact of the two types of cryptic promoter activation mechanisms on plant genome evolution and endosymbiotic gene transfer is discussed.

Kozak Sequence Acts as a Negative Regulator for De Novo Transcription Initiation of Newborn Coding Sequences in the Plant Genome.

The manner in which newborn coding sequences and their transcriptional competency emerge during the process of gene evolution remains unclear. Here, we experimentally simulated eukaryotic gene origination processes by mimicking horizontal gene transfer events in the plant genome. We mapped the precise position of the transcription start sites (TSSs) of hundreds of newly introduced promoterless firefly luciferase (LUC) coding sequences in the genome of Arabidopsis thaliana cultured cells. The systematic characterization of the LUC-TSSs revealed that 80% of them occurred under the influence of endogenous promoters, while the remainder underwent de novo activation in the intergenic regions, starting from pyrimidine-purine dinucleotides. These de novo TSSs obeyed unexpected rules; they predominantly occurred ∼100 bp upstream of the LUC inserts and did not overlap with Kozak-containing putative open reading frames (ORFs). These features were the output of the immediate responses to the sequence insertions, rather than a bias in the screening of the LUC gene function. Regarding the wild-type genic TSSs, they appeared to have evolved to lack any ORFs in their vicinities. Therefore, the repulsion by the de novo TSSs of Kozak-containing ORFs described above might be the first selection gate for the occurrence and evolution of TSSs in the plant genome. Based on these results, we characterized the de novo type of TSS identified in the plant genome and discuss its significance in genome evolution.

The mutation nrpb1-A325V in the largest subunit of RNA polymerase II suppresses compromised growth of Arabidopsis plants deficient in a function of the general transcription factor IIF.

The evolutionarily conserved 12-subunit RNA polymerase II (Pol II) is a central catalytic component that drives RNA synthesis during the transcription cycle that consists of transcription initiation, elongation, and termination. A diverse set of general transcription factors, including a multifunctional TFIIF, govern Pol II selectivity, kinetic properties, and transcription coupling with posttranscriptional processes. Here, we show that TFIIF of Arabidopsis (Arabidopsis thaliana) resembles the metazoan complex that is composed of the TFIIFα and TFIIFß polypeptides. Arabidopsis has two TFIIFß subunits, of which TFIIFß1/MAN1 is essential and TFIIFß2/MAN2 is not. In the partial loss-of-function mutant allele man1-1, the winged helix domain of Arabidopsis TFIIFß1/MAN1 was dispensable for plant viability, whereas the cellular organization of the shoot and root apical meristems were abnormal. Forward genetic screening identified an epistatic interaction between the largest Pol II subunit nrpb1-A325V variant and the man1-1 mutation. The suppression of the man1-1 mutant developmental defects by a mutation in Pol II suggests a link between TFIIF functions in Arabidopsis transcription cycle and the maintenance of cellular organization in the shoot and root apical meristems.

Evolution of Green Plants Accompanied Changes in Light-Harvesting Systems.

Photosynthetic organisms have various pigments enabling them to adapt to various light environments. Green plants are divided into two groups: streptophytes and chlorophytes. Streptophytes include some freshwater green algae and land plants, while chlorophytes comprise the other freshwater green algae and seawater green algae. The environmental conditions driving the divergence of green plants into these two groups and the changes in photosynthetic properties accompanying their evolution remain unknown. Here, we separated the core antennae of PSI and the peripheral antennae [light-harvesting complexes (LHCs)] in green plants by green-native gel electrophoresis and determined their pigment compositions. Freshwater green algae and land plants have high Chl a/b ratios, with most Chl b existing in LHCs. In contrast, seawater green algae have low Chl a/b ratios. In addition, Chl b exists not only in LHCs but also in PSI core antennae in these organisms, a situation beneficial for survival in deep seawater, where blue-green light is the dominant light source. Finally, low-energy Chl (red Chl) of PSI was detected in freshwater green algae and land plants, but not in seawater green algae. We thus conclude that the different level of Chl b accumulation in core antennae and differences in PSI red Chl between freshwater and seawater green algae are evolutionary adaptations of these algae to their habitats, especially to high- or low-light environments.

ppdb: plant promoter database version 3.0.

ppdb (http://ppdb.agr.gifu-u.ac.jp) is a plant promoter database that provides information on transcription start sites (TSSs), core promoter structure (TATA boxes, Initiators, Y Patches, GA and CA elements) and regulatory element groups (REGs) as putative and comprehensive transcriptional regulatory elements. Since the last report in this journal, the database has been updated in three areas to version 3.0. First, new genomes have been included in the database, and now ppdb provides information on Arabidopsis thaliana, rice, Physcomitrella patens and poplar. Second, new TSS tag data (34 million) from A. thaliana, determined by a high throughput sequencer, has been added to give a ∼200-fold increase in TSS data compared with version 1.0. This results in a much higher coverage of ∼27,000 A. thaliana genes and finer positioning of promoters even for genes with low expression levels. Third, microarray data-based predictions have been appended as REG annotations which inform their putative physiological roles.

Pentatricopeptide repeat proteins involved in plant organellar RNA editing.

C-to-U RNA editing has been widely observed in organellar RNAs in terrestrial plants. Recent research has revealed the significance of a large, plant-specific family of pentatricopeptide repeat (PPR) proteins for RNA editing and other RNA processing events in plant mitochondria and chloroplasts. PPR protein is a sequence-specific RNA-binding protein that identifies specific C residues for editing. Discovery of the RNA recognition code for PPR motifs, including verification and prediction of the individual RNA editing site and its corresponding PPR protein, expanded our understanding of the molecular function of PPR proteins in plant organellar RNA editing. Using this knowledge and the co-expression database, we have identified two new PPR proteins that mediate chloroplast RNA editing. Further, computational target assignment using the PPR RNA recognition codes suggests a distinct, unknown mode-of-action, by which PPR proteins serve a function beyond site recognition in RNA editing.

Transcriptome analysis of respiration-responsive genes in Chlamydomonas reinhardtii: mitochondrial retrograde signaling coordinates the genes for cell proliferation with energy-producing metabolism.

Plant organelles are not only the recipients of signals from the nucleus, but also elicit signals to regulate nuclear genes; the latter process is called retrograde regulation. We previously reported a novel mitochondrial retrograde regulation in Chlamydomonas reinhardtii; nuclear photosynthesis genes are regulated in response to mitochondrial respiratory electron transport (RET). However, the physiological roles of this retrograde regulation are not yet fully understood. In this study, we performed a genome-wide transcriptome analysis of this alga to reveal what kinds of genes are responsive to this RET signal, using Chlamydomonas macroarrays containing 10,368 expressed sequence tag clones. From the analysis, we identified 147 inducible and 35 repressive genes based on a couple of criteria: induction/repression by activated respiration caused by exogenously added acetate, and the cancellations of these responses by treatment with antimycin A, an inhibitor of RET. Interestingly, genes for respiration, photosynthesis, glycolysis/gluconeogenesis, protein biosynthesis, cell wall biogenesis and flagella were significantly induced by RET-derived signals. From these findings, we discuss the physiological role of mitochondrial retrograde signaling in this unicellular alga, in terms of the coordination of cell proliferation with energy-producing metabolism.

Preculture in an enriched nutrient medium greatly enhances the Agrobacterium-mediated transformation efficiency in Arabidopsis T87 cultured cells.

The Arabidopsis T87 cell line has been widely used in both basic and biotechnological plant sciences. Agrobacterium-mediated transformation of this cell line was reported to be highly efficient when precultured in Gamborg's B5 medium for a few days. However, because we could not obtain the expected efficiency in our laboratory, we further examined the preculture conditions of Arabidopsis T87 cells in the Agrobacterium-mediated transformation. As a result, we found that preculture in an excess amount of Murashige and Skoog (MS) macronutrients before cultivation in the B5 medium enhanced the transformation efficiency up to 100-fold, based on the transformed callus number on selective gellan gum plates. In this study, transformants were labeled with green fluorescent protein (GFP), and we found multiple fluorescent spots on individual transgenic calli. Therefore, the actual number of transgenic clones seems much more than that of transgenic calli. In our MS macronutrient-rich culture condition, T87 cells tended to aggregate and formed bigger cell clumps, a change that might be related to the enhancement of transformation efficiency. Based on these results, we report an improved protocol of Agrobacterium-mediated transformation of Arabidopsis T87 cells with high efficiency.

De novo activated transcription of inserted foreign coding sequences is inheritable in the plant genome.

The manner in which inserted foreign coding sequences become transcriptionally activated and fixed in the plant genome is poorly understood. To examine such processes of gene evolution, we performed an artificial evolutionary experiment in Arabidopsis thaliana. As a model of gene-birth events, we introduced a promoterless coding sequence of the firefly luciferase (LUC) gene and established 386 T2-generation transgenic lines. Among them, we determined the individual LUC insertion loci in 76 lines and found that one-third of them were transcribed de novo even in the intergenic or inherently unexpressed regions. In the transcribed lines, transcription-related chromatin marks were detected across the newly activated transcribed regions. These results agreed with our previous findings in A. thaliana cultured cells under a similar experimental scheme. A comparison of the results of the T2-plant and cultured cell experiments revealed that the de novo-activated transcription concomitant with local chromatin remodelling was inheritable. During one-generation inheritance, it seems likely that the transcription activities of the LUC inserts trapped by the endogenous genes/transcripts became stronger, while those of de novo transcription in the intergenic/untranscribed regions became weaker. These findings may offer a clue for the elucidation of the mechanism by which inserted foreign coding sequences become transcriptionally activated and fixed in the plant genome.

Chloroplast acquisition without the gene transfer in kleptoplastic sea slugs, Plakobranchus ocellatus.

Some sea slugs sequester chloroplasts from algal food in their intestinal cells and photosynthesize for months. This phenomenon, kleptoplasty, poses a question of how the chloroplast retains its activity without the algal nucleus. There have been debates on the horizontal transfer of algal genes to the animal nucleus. To settle the arguments, this study reported the genome of a kleptoplastic sea slug, Plakobranchus ocellatus, and found no evidence of photosynthetic genes encoded on the nucleus. Nevertheless, it was confirmed that light illumination prolongs the life of mollusk under starvation. These data presented a paradigm that a complex adaptive trait, as typified by photosynthesis, can be transferred between eukaryotic kingdoms by a unique organelle transmission without nuclear gene transfer. Our phylogenomic analysis showed that genes for proteolysis and immunity undergo gene expansion and are up-regulated in chloroplast-enriched tissue, suggesting that these molluskan genes are involved in the phenotype acquisition without horizontal gene transfer.

Three novel subunits of Arabidopsis chloroplastic NAD(P)H dehydrogenase identified by bioinformatic and reverse genetic approaches.

Chloroplastic NAD(P)H dehydrogenase (NDH) plays a role in cyclic electron flow around photosystem I to produce ATP, especially in adaptation to environmental changes. Although the NDH complex contains 11 subunits that are homologous to NADH:ubiquinone oxidoreductase (complex I; EC 1.6.5.3), recent genetic and biological studies have indicated that NDH also comprises unique subunits. We describe here an in silico approach based on co-expression analysis and phylogenetic profiling that was used to identify 65 genes as potential candidates for NDH subunits. Characterization of 21 Arabidopsis T-DNA insertion mutants among these ndh gene candidates indicated that three novel ndf (NDH-dependent cyclic electron flow) mutants (ndf1, ndf2 and ndf4) had impaired NDH activity as determined by measurement of chlorophyll fluorescence. The amount of NdhH subunit was greatly decreased in these mutants, suggesting that the loss of NDH activity was caused by a defect in accumulation of the NDH complex. In addition, NDF1, NDF2 and NDF4 proteins co-migrated with the NdhH subunit, as shown by blue native electrophoresis. These results strongly suggest that NDF proteins are novel subunits of the NDH complex. Further analysis revealed that the NDF1 and NDF2 proteins were unstable in the mutants lacking hydrophobic subunits of the NDH complex, but were stable in mutants lacking the hydrophilic subunits, suggesting that NDF1 and NDF2 interact with a hydrophobic sub-complex. NDF4 protein was predicted to possess a redox-active iron-sulfur cluster domain that may be involved in the electron transfer.

Heterogeneity of Arabidopsis core promoters revealed by high-density TSS analysis.

Our limited understanding of plant promoters does not allow us to recognize any core promoter elements for the majority of plant promoters. To understand the promoter architecture of Arabidopsis, we used the combined approach of in silico detection of novel core promoter elements and large-scale determination of transcription start sites (TSSs). To this end, we developed a novel methodology for TSS identification, using a combination of the cap-trapper and massively parallel signature sequencing methods. This technique, CT-MPSS, allowed us to identify 158 237 Arabidopsis TSS tags corresponding to 38 311 TSS loci, which provides an opportunity for quantitative analysis of plant promoters. The expression characteristics of these promoters were analyzed with respect to core promoter elements detected by our in silico analyses, revealing that Arabidopsis promoters contain two main types of elements with exclusive characteristics, the TATA type and the GA type. The TATA-type promoters tend to be associated with the Y Patch and the Inr motif, and cause high expression with sharp-peak TSS clusters. By contrast, the GA type produces broad-type TSS clusters. Unlike mammalian promoters, plant promoters are not associated with CpG islands. However, plant-specific GA-type promoters share some characteristics with mammalian CpG-type promoters.

ppdb: a plant promoter database.

ppdb (http://www.ppdb.gene.nagoya-u.ac.jp) is a plant promoter database that provides promoter annotation of Arabidopsis and rice. The database contains information on promoter structures, transcription start sites (TSSs) that have been identified from full-length cDNA clones and also a vast amount of TSS tag data. In ppdb, the promoter structures are determined by sets of promoter elements identified by a position-sensitive extraction method called local distribution of short sequences (LDSS). By using this database, the core promoter structure, the presence of regulatory elements and the distribution of TSS clusters can be identified. Although no differentiation of promoter architecture among plant species has been reported, there is some divergence of utilized sequences for promoter elements. Therefore, ppdb is based on species-specific sets of promoter elements, rather than on general motifs for multiple species. Each regulatory sequence is hyperlinked to literary information, a PLACE entry served by a plant cis-element database, and a list of promoters containing the regulatory sequence.

Two RNA editing sites with cis-acting elements of moderate sequence identity are recognized by an identical site-recognition protein in tobacco chloroplasts.

The chloroplast genome of higher plants contains 20-40 C-to-U RNA editing sites, whose number and locations are diversified among plant species. Biochemical analyses using in vitro RNA editing systems with chloroplast extracts have suggested that there is one-to-one recognition between proteinous site recognition factors and their respective RNA editing sites, but their rigidness and generality are still unsettled. In this study, we addressed this question with the aid of an in vitro RNA editing system from tobacco chloroplast extracts and with UV-crosslinking experiments. We found that the ndhB-9 and ndhF-1 editing sites of tobacco chloroplast transcripts are both bound by the site-specific trans-acting factors of 95 kDa. Cross-competition experiments between ndhB-9 and ndhF-1 RNAs demonstrated that the 95 kDa proteins specifically binding to the ndhB-9 and ndhF-1 sites are the identical protein. The binding regions of the 95 kDa protein on the ndhB-9 and ndhF-1 transcripts showed 60% identity in nucleotide sequence. This is the first biochemical demonstration that a site recognition factor of chloroplast RNA editing recognizes plural sites. On the basis of this finding, we discuss how plant organellar RNA editing sites have diverged during evolution.

Differentiation of core promoter architecture between plants and mammals revealed by LDSS analysis.

Mammalian promoters are categorized into TATA and CpG-related groups, and they have complementary roles associated with differentiated transcriptional characteristics. While the TATA box is also found in plant promoters, it is not known if CpG-type promoters exist in plants. Plant promoters contain Y Patches (pyrimidine patches) in the core promoter region, and the ubiquity of these beyond higher plants is not understood as well. Sets of promoter sequences were utilized for the analysis of local distribution of short sequences (LDSS), and approximately one thousand octamer sequences have been identified as promoter constituents from Arabidopsis, rice, human and mouse, respectively. Based on their localization profiles, the identified octamer sequences were classified into several major groups, REG (Regulatory Element Group), TATA box, Inr (Initiator), Kozak, CpG and Y Patch. Comparison of the four species has revealed three categories: (i) shared groups found in both plants and mammals (TATA box), (ii) common groups found in both kingdoms but the utilized sequence is differentiated (REG, Inr and Kozak) and (iii) specific groups found in either plants or mammals (CpG and Y Patch). Our comparative LDSS analysis has identified conservation and differentiation of promoter architectures between higher plants and mammals.

Characterization of spliced leader trans-splicing in a photosynthetic rhizarian amoeba, Paulinella micropora, and its possible role in functional gene transfer.

Paulinella micropora is a rhizarian thecate amoeba, belonging to a photosynthetic Paulinella species group that has a unique organelle termed chromatophore, whose cyanobacterial origin is distinct from that of plant and algal chloroplasts. Because acquisition of the chromatophore was quite a recent event compared with that of the chloroplast ancestor, the Paulinella species are thought to be model organisms for studying the early process of primary endosymbiosis. To obtain insight into how endosymbiotically transferred genes acquire expression competence in the host nucleus, here we analyzed the 5' end sequences of the mRNAs of P. micropora MYN1 strain with the aid of a cap-trapper cDNA library. As a result, we found that mRNAs of 27 genes, including endosymbiotically transferred genes, possessed the common 5' end sequence of 28-33 bases that were posttranscriptionally added by spliced leader (SL) trans-splicing. We also found two subtypes of SL RNA genes encoded by the P. micropora MYN1 genome. Differing from the other SL trans-splicing organisms that usually possess poly(A)-less SL RNAs, this amoeba has polyadenylated SL RNAs. In this study, we characterize the SL trans-splicing of this unique organism and discuss the putative merits of SL trans-splicing in functional gene transfer and genome evolution.

Identification of plant promoter constituents by analysis of local distribution of short sequences.

BACKGROUND: Plant promoter architecture is important for understanding regulation and evolution of the promoters, but our current knowledge about plant promoter structure, especially with respect to the core promoter, is insufficient. Several promoter elements including TATA box, and several types of transcriptional regulatory elements have been found to show local distribution within promoters, and this feature has been successfully utilized for extraction of promoter constituents from human genome. RESULTS: LDSS (Local Distribution of Short Sequences) profiles of short sequences along the plant promoter have been analyzed in silico, and hundreds of hexamer and octamer sequences have been identified as having localized distributions within promoters of Arabidopsis thaliana and rice. Based on their localization patterns, the identified sequences could be classified into three groups, pyrimidine patch (Y Patch), TATA box, and REG (Regulatory Element Group). Sequences of the TATA box group are consistent with the ones reported in previous studies. The REG group includes more than 200 sequences, and half of them correspond to known cis-elements. The other REG subgroups, together with about a hundred uncategorized sequences, are suggested to be novel cis-regulatory elements. Comparison of LDSS-positive sequences between Arabidopsis and rice has revealed moderate conservation of elements and common promoter architecture. In addition, a dimer motif named the YR Rule (C/T A/G) has been identified at the transcription start site (-1/+1). This rule also fits both Arabidopsis and rice promoters. CONCLUSION: LDSS was successfully applied to plant genomes and hundreds of putative promoter elements have been extracted as LDSS-positive octamers. Identified promoter architecture of monocot and dicot are well conserved, but there are moderate variations in the utilized sequences.

Recognition of RNA editing sites is directed by unique proteins in chloroplasts: biochemical identification of cis-acting elements and trans-acting factors involved in RNA editing in tobacco and pea chloroplasts.

RNA editing in higher-plant chloroplasts involves C-to-U conversions at specific sites. Although in vivo analyses have been performed, little is known about the biochemical aspects of chloroplast editing reactions. Here we improved our original in vitro system and devised a procedure for preparing active chloroplast extracts not only from tobacco plants but also from pea plants. Using our tobacco in vitro system, cis-acting elements were defined for psbE and petB mRNAs. Distinct proteins were found to bind specifically to each cis-element, a 56-kDa protein to the psbE site and a 70-kDa species to the petB site. Pea chloroplasts lack the corresponding editing site in psbE since T is already present in the DNA. Parallel in vitro analyses with tobacco and pea extracts revealed that the pea plant has no editing activity for psbE mRNAs and lacks the 56-kDa protein, whereas petB mRNAs are edited and the 70-kDa protein is also present. Therefore, coevolution of an editing site and its cognate trans-factor was demonstrated biochemically in psbE mRNA editing between tobacco and pea plants.