Genome-wide identification of endogenous viral sequences in alfalfa (Medicago sativa L.).

Endogenous viral elements (EVEs) have been for the most part described in animals and to a less extent in plants. The endogenization was proposed to contribute toward evolution of living organisms via horizontal gene transfer of novel genetic material and resultant genetic diversity. During the last two decades, several full-length and fragmented EVEs of pararetroviral and non-retroviral nature have been identified in different plant genomes, both monocots and eudicots. Prior to this work, no EVEs have been reported in alfalfa (Medicago sativa L.), the most cultivated forage legume in the world. In this study, taking advantage of the most recent developments in the field of alfalfa research, we have assessed alfalfa genome on the presence of viral-related sequences. Our analysis revealed segmented EVEs resembling two dsDNA reverse-transcribing virus species: Soybean chlorotic mottle virus (family Caulimoviridae, genus Soymovirus) and Figwort mosaic virus (family Caulimoviridae, genus Caulimovirus). The EVEs appear to be stable constituents of the host genome and in that capacity could potentially acquire functional roles in alfalfa's development and response to environmental stresses.

Multigenome analysis implicates miniature inverted-repeat transposable elements (MITEs) in metabolic diversification in eudicots.

Plants produce a plethora of natural products, including many drugs. It has recently emerged that the genes encoding different natural product pathways may be organized as biosynthetic gene clusters in plant genomes, with >30 examples reported so far. Despite superficial similarities with microbes, these clusters have not arisen by horizontal gene transfer, but rather by gene duplication, neofunctionalization, and relocation via unknown mechanisms. Previously we reported that two Arabidopsis thaliana biosynthetic gene clusters are located in regions of the genome that are significantly enriched in transposable elements (TEs). Other plant biosynthetic gene clusters also harbor abundant TEs. TEs can mediate genomic rearrangement by providing homologous sequences that enable illegitimate recombination and gene relocation. Thus, TE-mediated recombination may contribute to plant biosynthetic gene cluster formation. TEs may also facilitate establishment of regulons. However, a systematic analysis of the TEs associated with plant biosynthetic gene clusters has not been carried out. Here we investigate the TEs associated with clustered terpene biosynthetic genes in multiple plant genomes and find evidence to suggest a role for miniature inverted-repeat transposable elements in cluster formation in eudicots. Through investigation of the newly sequenced Amborella trichopoda, Aquilegia coerulea, and Kalanchoe fedtschenkoi genomes, we further show that the "block" mechanism of founding of biosynthetic gene clusters through duplication and diversification of pairs of terpene synthase and cytochrome P450 genes that is prevalent in the eudicots arose around 90-130 million years ago, after the appearance of the basal eudicots and before the emergence of the superrosid clade.

Investigation of terpene diversification across multiple sequenced plant genomes.

Plants produce an array of specialized metabolites, including chemicals that are important as medicines, flavors, fragrances, pigments and insecticides. The vast majority of this metabolic diversity is untapped. Here we take a systematic approach toward dissecting genetic components of plant specialized metabolism. Focusing on the terpenes, the largest class of plant natural products, we investigate the basis of terpene diversity through analysis of multiple sequenced plant genomes. The primary drivers of terpene diversification are terpenoid synthase (TS) "signature" enzymes (which generate scaffold diversity), and cytochromes P450 (CYPs), which modify and further diversify these scaffolds, so paving the way for further downstream modifications. Our systematic search of sequenced plant genomes for all TS and CYP genes reveals that distinct TS/CYP gene pairs are found together far more commonly than would be expected by chance, and that certain TS/CYP pairings predominate, providing signals for key events that are likely to have shaped terpene diversity. We recover TS/CYP gene pairs for previously characterized terpene metabolic gene clusters and demonstrate new functional pairing of TSs and CYPs within previously uncharacterized clusters. Unexpectedly, we find evidence for different mechanisms of pathway assembly in eudicots and monocots; in the former, microsyntenic blocks of TS/CYP gene pairs duplicate and provide templates for the evolution of new pathways, whereas in the latter, new pathways arise by mixing and matching of individual TS and CYP genes through dynamic genome rearrangements. This is, to our knowledge, the first documented observation of the unique pattern of TS and CYP assembly in eudicots and monocots.

Clustering of Pathogen-Response Genes in the Genome of Arabidopsis thaliana.

Previously, we used heterologous expressed sequence tag (EST) mapping to generate a profile of 4 935 pathogen-response genes of Arabidopsis thaliana. In this work, we performed a computer analysis of this profile, revealing 1 594 non-homologous clustered genes distributed among all A. thaliana chromosomes, whose co-regulation may be related to host responses to pathogens. To supplement computer data, we arbitrarily selected two clusters and analyzed their expression levels in A. thaliana ecotypes Col-0 and C24 during infection with the yellow strain of Cucumber mosaic virus CMV(Y). Ecotype Col-0 is susceptible to CMV(Y), whereas C24 contains the dominant resistance gene RCY1. Upon infection with CMV(Y), all clustered genes were significantly activated in the resistant ecotype C24. In addition, we demonstrated that posttranslational histone modifications associated with trimethylation of histone H3 lysine 27 are most likely involved in regulation of several cluster genes described in this study. Overall, our experiments indicated that pathogen-response genes in the genome of A. thaliana may be clustered and co-regulated.

Mapping of heterologous expressed sequence tags as an alternative to microarrays for study of defense responses in plants.

BACKGROUND: Microarray technology helped to accumulate an immense pool of data on gene expression changes in response to different environmental factors. Yet, computer- generated gene profiling using expressed sequence tags (EST) represents a valuable alternative to microarrays, which allows efficient discovery of homologous sequences in evolutionarily different species and comparison of gene sets on the whole genome scale. In this study, we used publicly available EST database derived from different plant species infected with a variety of pathogens, to generate an expression profile of homologous genes involved in defense response of a model organism, Arabidopsis thaliana. RESULTS: EST-driven prediction identified 4,935 genes (16% of the total Arabidopsis genome) which, according to the origin of EST sets, were associated with defense responses in the reference genome. Profiles of defense-related genes, obtained by mapping of heterologous EST, represent putative Arabidopsis homologs of the corresponding species. Comparison of these profiles in pairs and locating common genes allowed estimating similarity between defense-related gene sets of different plant species. To experimentally support computer data, we arbitrarily selected a number of transcription factor genes (TF) detected by EST mapping. Their expression levels were examined by real-time polymerase chain reaction during infection with yellow strain of Cucumber mosaic virus, a compatible virus systemically infecting Arabidopsis. We observed that 65% of the designated TF were upregulated in accordance with the EST-generated profile. CONCLUSION: We demonstrated that heterologous EST mapping may be efficiently used to reveal genes involved in host defense responses to pathogens. Upregulated genes identified in this study substantially overlap with those previously obtained by microarrays.

The pattern of chromosome folding in interphase is outlined by the linear gene density profile.

Spatial organization of chromatin in the interphase nucleus plays a role in gene expression and inheritance. Although it appears not to be random, the principles of this organization are largely unknown. In this work, we show an explicit relationship between the intranuclear localization of various chromosome segments and the pattern of gene distribution along the genome sequence. Using a 7-megabase-long region of the Drosophila melanogaster chromosome 2 as a model, we observed that the six gene-poor chromosome segments identified in the region interact with components of the nuclear matrix to form a compact stable cluster. The six gene-rich segments form a spatially segregated unstable cluster dependent on nonmatrix nuclear proteins. The resulting composite structure formed by clusters of gene-rich and gene-poor regions is reproducible between the nuclei. We suggest that certain aspects of chromosome folding in interphase are predetermined and can be inferred through in silico analysis of chromosome sequence, using gene density profile as a manifestation of "folding code."

Up-regulation of the Ku heterodimer in Drosophila testicular cyst cells.

In Drosophila, developing germline cysts in testis are enveloped by two somatic cyst cells essential for germline development and male reproduction. The cyst cells continue development along with the germline. However, the mechanisms of somatic gene expression in testes are poorly understood. We report transcriptional up-regulation of the Ku heterodimer in cyst cells. The initial up-regulation is independent of germline, and transcription is further augmented during spermatogenesis. Abundance of Ku in the cyst cell cytoplasm suggests the role for Ku subunits in the regulation of sperm individualization.

Virus-induced gene silencing of the RPC5-like subunit of RNA polymerase III caused pleiotropic effects in Nicotiana benthamiana.

In eukaryotic cells, RNA polymerase III is highly conserved and transcribes housekeeping genes such as ribosomal 5S rRNA, tRNA and other small RNAs. The RPC5-like subunit is one of the 17 subunits forming RNAPIII and its exact functional roles in the transcription are poorly understood. In this work, we report that virus-induced gene silencing of transcripts encoding a putative RPC5-like subunit of the RNA Polymerase III in a model species Nicotiana benthamiana had pleiotropic effects, including but not limited to severe dwarfing appearance, chlorosis, nearly complete reduction of internodes and abnormal leaf shape. Using transcriptomic analysis, we identified genes and pathways affected by RPC5 silencing and thus presumably related to the cellular roles of the subunit as well as to the downstream cascade of reactions in response to partial loss of RNA Polymerase III function. Our results suggest that silencing of the RPC5L in N. benthamiana disrupted not only functions commonly associated with the core RNA Polymerase III transcripts, but also more diverse cellular processes, including responses to stress. We believe this is the first demonstration that activity of the RPC5 subunit is critical for proper functionality of RNA Polymerase III and normal plant development.

Effect of preillumination with red light on photosynthetic parameters and oxidant-/antioxidant balance in Arabidopsis thaliana in response to UV-A.

The effect of preillumination with low intensity (10µmol quanta m(-2)s(-1), 10min) light of different wavelengths in the spectral range of 550-730nm on photosynthesis and activity of PSII, the content of photosynthetic pigments and H2O2, as well as the peroxidase activity in the leaves of 26-d-old Arabidopsis thaliana wild-type (WT) plants in response to UV-A radiation was studied. UV-A decreased the activity of the PSII, the content of Chl a, Chl b and carotenoids, as well as increased the peroxidase activity and H2O2 level in the WT leaves. Preillumination of the leaves with red light (RL, λmax=664nm) reduced the inhibitory effect of UV radiation on photosynthesis and activity of the PSII, indicated by delayed light emission as well as the H2O2 level, but increased the peroxidase activity in the leaves compared to illumination by UV radiation only. Illumination with RL alone and the subsequent exposure of plants to darkness increased the peroxidase activity and the transcription activity of genes of the transcription factors APX1 and HYH. Preillumination of leaves with RL, then far red light (FRL, λmax=727nm) partially compensated the effect of the RL for all studied parameters, suggesting that the active form of phytochrome (PFR) is involved in these processes. Preillumination with the wavelengths of 550, 594 and 727nm only did not have a marked effect on photosynthesis. The hy2 mutant of Arabidopsis with reduced synthesis of the phytochrome B chromophore showed decreased resistance of PSII to UV-A compared with the WT of Arabidopsis. UV radiation reduced Chl a fluorescence much faster in the hy2 mutant compared to the WT. Preillumination of the hy2 mutant with RL did not affect the PSII activity and H2O2 level in UV-irradiated leaves. It is assumed that the formation of the increased resistance of the photosynthetic apparatus of Arabidopsis to UV-A radiation involves PFR and the antioxidant system of plants, partly by inducing transcriptional activity of some antioxidant and transcription factors genes.

Are clustered genes in the genomes of Arabidopsis and Drosophila regulated differently?

In the eukaryotic genome, genes with similar functions tend to co-localize in close proximity. Such gene clusters together with non-clustered genes constitute a chromatin domain which is a higher order regulatory unit. On a lower level co-expressed genes are regulated by differential activity of transcription factors (TF). We compared genome-wide distributions of TF in gene clusters in the genomes of Drosophila melanogaster and Arabidopsis thaliana. This revealed a significant excess of TF genes in gene clusters of the Arabidopsis genome, whereas in the genome of Drosophila distribution of TF in gene clusters did not differ from stochastic. We speculate that these alternatives could lead to different pathways of regulation of clustered genes in two species and to evolutionary-progressive changes in architecture of regulatory networks, governing the activity of clustered genes in the animal kingdom.

Transcriptional regulation by Modulo integrates meiosis and spermatid differentiation in male germ line.

Transcriptional activation in early spermatocytes involves hundreds of genes, many of which are required for meiosis and spermatid differentiation. A number of the meiotic-arrest genes have been identified as general regulators of transcription; however, the gene-specific transcription factors have remained elusive. To identify such factors, we purified the protein that specifically binds to the promoter of spermatid-differentiation gene Sdic and identified it as Modulo, the Drosophila homologue of nucleolin. Analysis of gene-expression patterns in the male sterile modulo mutant indicates that Modulo supports high expression of the meiotic-arrest genes and is essential for transcription of spermatid-differentiation genes. Expression of Modulo itself is under the control of meiotic-arrest genes and requires the DAZ/DAZL homologue Boule that is involved in the control of G(2)/M transition. Thus, regulatory interactions among Modulo, Boule, and the meiotic-arrest genes integrate meiosis and spermatid differentiation in the male germ line.

Large clusters of co-expressed genes in the Drosophila genome.

Clustering of co-expressed, non-homologous genes on chromosomes implies their co-regulation. In lower eukaryotes, co-expressed genes are often found in pairs. Clustering of genes that share aspects of transcriptional regulation has also been reported in higher eukaryotes. To advance our understanding of the mode of coordinated gene regulation in multicellular organisms, we performed a genome-wide analysis of the chromosomal distribution of co-expressed genes in Drosophila. We identified a total of 1,661 testes-specific genes, one-third of which are clustered on chromosomes. The number of clusters of three or more genes is much higher than expected by chance. We observed a similar trend for genes upregulated in the embryo and in the adult head, although the expression pattern of individual genes cannot be predicted on the basis of chromosomal position alone. Our data suggest that the prevalent mechanism of transcriptional co-regulation in higher eukaryotes operates with extensive chromatin domains that comprise multiple genes.