Ultraconserved elements between the genomes of the plants Arabidopsis thaliana and rice

Ultraconserved elements, sequences with 100% identity with no insertions or deletions between genomes, have been found in both vertebrate and invertebrate genomes; whether plant genomes contain ultraconserved elements, however, is unknown. We consequently compared the genomes of Arabidopsis thaliana and rice, which diverged about 200 million years ago, and identified 25 ultraconserved elements that are longer than 100 bp. Similar to those previously found, ultraconserved elements in plants tend to occur in clusters and locate at noncoding regions; nevertheless, they have many distinct features. For instance, the longest ultraconserved element between the 2 plant genomes is 1491 bp, much longer than the longest one (779 bp) between the human and rodent genomes. Some biological implications are discussed, but the functions of these plant ultraconserved elements and the reasons why they are practically frozen during the evolution of millions of years remain a mystery.     

Identification and analysis of homoeologous segments of the genomes of rice and Arabidopsis thaliana.

Using contiguous genomic DNA sequences of Arabidopsis thaliana, we were able to identify a region of conserved structure in the genome of rice. The conserved, and presumptive homoeologous segments, are 194 kb and 219-300 kb in size in Arabidopsis and rice, respectively. They contain five homologous genes, distinguished in order by a single inversion. These represent the first homoeologous segments identified in the genomes of a dicot and a monocot, demonstrating that fine-scale conservation of genome structure exists and is detectable across this major divide in the angiosperms. The conserved framework of genes identified is interspersed with non-conserved genes, indicating that mechanisms beyond segmental inversions and translocations need to be invoked to fully explain plant genome evolution, and that the benefits of comparative genomics over such large taxonomic distances may be limited.     

Distinct modes of adventitious rooting in Arabidopsis thaliana

The literature describes different rooting protocols for Arabidopsis thaliana as models to study adventitious rooting, and results are generally perceived as comparable. However, there is a lack of investigations focusing on the distinct features, advantages and limitations of each method in the study of adventitious rooting with both wild-type (WT) ecotypes and their respective mutants. This investigation was undertaken to evaluate the adventitious rooting process in three different experimental systems, all using A. thaliana, analysing the same rooting parameters after transient exposure to auxin (indole-3-acetic acid) and control conditions: excised leaves, de-rooted plants and etiolated seedlings. The founding tissues and sites of origin of roots differed depending on the system used, whereas all rooting patterns were of the direct type (i.e., without callus formation). None of the systems had an absolute requirement for exogenous auxin, although rooting was enhanced by this phytohormone, with the exception of de-rooted plants, which had adventitious rooting strongly inhibited by exogenous auxin. Root elongation was much favoured in isolated leaves. Auxin-overproducing mutants could not be used in the detached leaf system due to precocious senescence; in the de-rooted plant system, these mutants had a WT-like rooting response, whereas the expression of the 'rooty' phenotype was only evident in the etiolated seedling system. Adventitious rooting of etiolated WT seedlings in the presence of exogenous auxin was inhibited by exogenous flavonoids, which act as auxin transport inhibitors; surprisingly, the flavonoid-deficient mutant chs had a lower rooting response compared to WT. Although Arabidopsis is an excellent model system to study adventitious rooting, physiological and developmental responses differed significantly, underlining the importance of avoiding data generalisation on rooting responses derived from different experimental systems with this species.     

Expression of Distinct Self-Incompatibility Specificities in Arabidopsis thaliana

The interplay of balancing selection within a species and rapid gene evolution between species can confound our ability to determine the functional equivalence of interspecific and intergeneric pairs of alleles underlying reproduction. In crucifer plants, mating specificity in the barrier to self-fertilization called self-incompatibility (SI) is controlled by allele-specific interactions between two highly polymorphic and co-evolving proteins, the S-locus receptor kinase (SRK) and its S-locus cysteine rich (SCR) ligand. These proteins have diversified both within and between species such that it is often difficult to determine from sequence information alone if they encode the same or different SI specificity. The self-fertile Arabidopsis thaliana was derived from an obligate outbreeding ancestor by loss of self-incompatibility, often in conjunction with inactivation of SRK or SCR. Nevertheless, some accessions of A. thaliana can express self-incompatibility upon transformation with an SRK–SCR gene pair isolated from its self-incompatible close relative A. lyrata. Here we show that several additional and highly diverged SRK/SCR genes from A. lyrata and another crucifer plant, Capsella grandiflora, confer self-incompatibility in A. thaliana, either as intact genes isolated from genomic libraries or after manipulation to generate chimeric fusions. We describe how the use of this newly developed chimeric protein strategy has allowed us to test the functional equivalence of SRK/SCR gene pairs from different taxa and to assay the functionality of endogenous A. thaliana SRK and SCR sequences.MATING reactions in plants, fungi, and animals are strongly influenced by molecular recognition machineries that act as gauges of genetic relatedness (Brown and Casselton 2001; Nasrallah 2005; Yamazaki and Beauchamp 2007). Many plants with hermaphroditic flowers have evolved inbreeding avoidance mechanisms, known as self-incompatibility (SI) systems. These systems are based on the ability of the female reproductive apparatus (the pistil) to discriminate among genetically distinct pollen grains, resulting in the failure of self-pollination despite functional female and male reproductive structures. In the Brassicaceae (crucifers), specific recognition of pollen by the epidermal cells of the stigma (a structure located at the tip of the pistil) is controlled by haplotypes of the S locus, and activation of the SI response leading to inhibition of pollen tube growth occurs if pollen and stigma are derived from plants that express the same S-locus haplotype (S haplotype). Within self-incompatible crucifer species, the number of S haplotypes and corresponding SI specificities is usually high, with >50 reported in some species (Watanabe et al. 2000), and SI dictates that self-incompatible plants are typically heterozygous and carry two S haplotypes. Each S haplotype is composed of two highly polymorphic genes that are the determinants of SI specificity in stigma and pollen (Stein et al. 1991; Schopfer et al. 1999). The S-locus receptor kinase (SRK) gene encodes a single-pass transmembrane serine/threonine kinase localized on the surface of stigma epidermal cells, and the S-locus cysteine-rich protein (SCR) gene encodes a small peptide localized in the pollen coat. SCR is the ligand for SRK and will bind to the extracellular domain of SRK (hereafter eSRK) only if both proteins are encoded by the same S-locus haplotype (Kachroo et al. 2001; Takayama et al. 2001; Chookajorn et al. 2004). The binding of SCR to its cognate eSRK triggers an intracellular phosphorylation cascade that results in pollen rejection by a poorly understood mechanism.A mechanistic understanding of the recognition phase of SI requires detailed structure–function analyses of SRK and SCR aimed at identifying the amino acid residues that determine their allele-specific interaction and explaining the puzzling dominance/recessive interactions exhibited by different SRK alleles in the heterozygous stigmas of self-incompatible plants (Hatakeyama et al. 2001; Mable et al. 2003; Prigoda et al. 2005). Such structure–function studies require an experimental system that allows efficient in vivo functional analysis of large numbers of SRK and SCR sequence variants generated in vitro by site-directed mutagenesis or domain swapping between proteins that determine different SI specificities. The recent transfer of the SI trait into Arabidopsis thaliana has established this species as a model organism for mechanistic and evolutionary studies of mating systems in crucifers (Nasrallah et al. 2002, 2004). However, to date, only one SI specificity, that which is determined by the Sb haplotype of A. lyrata, has been successfully introduced into A. thaliana and shown to alter the plant''s mating reaction from strict autogamy to full SI. To exploit fully the A. thaliana transgenic SI model, additional S haplotypes must be introduced into this species. In addition to facilitating mechanistic studies of the SRK–SCR interaction and dominance relationships, the expression of multiple SI specificities in A. thaliana promises to shed light on processes underlying the diversification of SRK and SCR genes. For example, expression in A. thaliana of SI specificities derived from different crucifer species will allow direct assays of the functional equivalence or nonequivalence of the corresponding S haplotypes, an issue that is difficult to resolve on the basis of sequence information alone.Although conceptually simple, expressing different SI specificities by transformation with different SRK/SCR gene pairs is not a straightforward proposition. Difficulties stem largely from the availability of appropriate cloned SRK/SCR variants for use in transformation experiments. A large number of SRK/SCR gene pairs are available from Brassica species as a result of extensive and long-standing studies of SI. However, attempts to restore SI in transgenic A. thaliana using Brassica S-locus genes had met with failure (Bi et al. 2000; J. B. Nasrallah, unpublished data), possibly because of the inability of Brassica SRKs to interact productively with A. thaliana components of the SI signal transduction pathway. In the past few years, studies of SI were initiated in self-incompatible species more closely related to A. thaliana, such as A. lyrata, A. halleri, and Capsella grandiflora. However, with a few exceptions, these studies produced only partial SRK and SCR sequences amplified from genomic DNA (Schierup et al. 2001; Prigoda et al. 2005; Bechsgaard et al. 2006; Paetsch et al. 2006). The challenging task of cloning the very highly polymorphic SCR sequences and complete SRK and SCR genes, which requires genomic library construction and in many cases chromosome walking, has only been accomplished for two S haplotypes of A. lyrata, Sb (hereafter AlSb, which was used in previous transformation studies (Nasrallah et al. 2002, 2004), and Sa (AlSa; Kusaba et al. 2001), and for the S7 haplotype of C. grandiflora (CgS7; Nasrallah et al. 2007).In this article, we report the isolation of two new SRK/SCR gene pairs from genomic libraries of A. lyrata and expression of the corresponding SI specificities in A. thaliana. We also describe a novel strategy for rapid and efficient transfer of several distinct SI specificities into A. thaliana, which only requires knowledge of the eSRK sequence and SCR second-exon sequences that encode the mature SCR protein.     

Genome-wide crossover distribution in Arabidopsis thaliana meiosis reveals sex-specific patterns along chromosomes

In most species, crossovers (COs) are essential for the accurate segregation of homologous chromosomes at the first meiotic division. Their number and location are tightly regulated. Here, we report a detailed, genome-wide characterization of the rate and localization of COs in Arabidopsis thaliana, in male and female meiosis. We observed dramatic differences between male and female meiosis which included: (i) genetic map length; 575 cM versus 332 cM respectively; (ii) CO distribution patterns: male CO rates were very high at both ends of each chromosome, whereas female CO rates were very low; (iii) correlations between CO rates and various chromosome features: female CO rates correlated strongly and negatively with GC content and gene density but positively with transposable elements (TEs) density, whereas male CO rates correlated positively with the CpG ratio. However, except for CpG, the correlations could be explained by the unequal repartition of these sequences along the Arabidopsis chromosome. For both male and female meiosis, the number of COs per chromosome correlates with chromosome size expressed either in base pairs or as synaptonemal complex length. Finally, we show that interference modulates the CO distribution both in male and female meiosis.     

Deleterious amino acid polymorphisms in Arabidopsis thaliana and rice

Plant genetic diversity has been mainly investigated with neutral markers, but large-scale DNA sequencing projects now enable the identification and analysis of different classes of genetic polymorphisms, such as non-synonymous single nucleotide polymorphisms (nsSNPs) in protein coding sequences. Using the SIFT and MAPP programs to predict whether nsSNPs are tolerated (i.e., effectively neutral) or deleterious for protein function, genome-wide nsSNP data from Arabidopsis thaliana and rice were analyzed. In both species, about 20% of polymorphic sites with nsSNPs were classified as deleterious; they segregate at lower allele frequencies than tolerated nsSNPs due to purifying selection. Furthermore, A. thaliana accessions from marginal populations show a higher relative proportion of deleterious nsSNPs, which likely reflects differential selection or demographic effects in subpopulations. To evaluate the sensitivity of predictions, genes from model and crop plants with known functional effects of nsSNPs were inferred with the algorithms. The programs predicted about 70% of nsSNPs correctly as tolerated or deleterious, i.e., as having a functional effect. Forward-in-time simulations of bottleneck and domestication models indicated a high power to detect demographic effects on nsSNP frequencies in sufficiently large datasets. The results indicate that nsSNPs are useful markers for analyzing genetic diversity in plant genetic resources and breeding populations to infer natural/artificial selection and genetic drift.     

Subcellular distribution of glutathione precursors in Arabidopsis thaliana

Glutathione is an important antioxidant and has many important functions in plant development, growth and defense. Glutathione synthesis and degradation is highly compartment-specific and relies on the subcellular availability of its precursors, cysteine, glutamate, glycine and γ-glutamylcysteine especially in plastids and the cytosol which are considered as the main centers for glutathione synthesis. The availability of glutathione precursors within these cell compartments is therefore of great importance for successful plant development and defense. The aim of this study was to investigate the compartment-specific importance of glutathione precursors in Arabidopsis thaliana. The subcellular distribution was compared between wild type plants (Col-0), plants with impaired glutathione synthesis (glutathione deficient pad2-1 mutant, wild type plants treated with buthionine sulfoximine), and one complemented line (OE3) with restored glutathione synthesis. Immunocytohistochemistry revealed that the inhibition of glutathione synthesis induced the accumulation of the glutathione precursors cysteine, glutamate and glycine in most cell compartments including plastids and the cytosol. A strong decrease could be observed in γ-glutamylcysteine (γ-EC) contents in these cell compartments. These experiments demonstrated that the inhibition of γ-glutamylcysteine synthetase (GSH1) - the first enzyme of glutathione synthesis - causes a reduction of γ-EC levels and an accumulation of all other glutathione precursors within the cells.     

Characterization of the rice circadian clock-associated pseudo-response regulators in Arabidopsis thaliana

Members of the small family of Arabidopsis PSEUDO-RESPONSE REGULATORS (PRR1/TOC1, PRR3, PRR5, PRR7, and PRR9) play roles close to the circadian clock in Arabidopsis thaliana. We have reported that the rice (Oryza sativa) genome also encodes a set of PRR counterparts (designated OsPRR1, OsPRR37, OsPRR59, OsPRR73, and OsPRR95 respectively). To gain new insight into the molecular functions of OsPRRs, we carried out genetic complementation analyses by introducing two representative rice genes, OsPRR1 and OsPRR37, into the corresponding Arabidopsis loss-of-function mutants (toc1 and prr7 respectively). The results showed that these OsPRR and AtPRR genes are genetically interchangeable at least in part, suggesting the conserved clock-associated function of these OsPRRs.     

Distinct functional patterns of gene promoter hypomethylation and hypermethylation in cancer genomes

Background

Aberrant DNA methylation plays important roles in carcinogenesis. However, the functional significance of genome-wide hypermethylation and hypomethylation of gene promoters in carcinogenesis currently remain unclear.

Principal Findings

Based on genome-wide methylation data for five cancer types, we showed that genes with promoter hypermethylation were highly consistent in function across different cancer types, and so were genes with promoter hypomethylation. Functions related to “developmental processes” and “regulation of biology processes” were significantly enriched with hypermethylated genes but were depleted of hypomethylated genes. In contrast, functions related to “cell killing” and “response to stimulus”, including immune and inflammatory response, were associated with an enrichment of hypomethylated genes and depletion of hypermethylated genes. We also observed that some families of cytokines secreted by immune cells, such as IL10 family cytokines and chemokines, tended to be hypomethylated in various cancer types. These results provide new hints for understanding the distinct functional roles of genome-wide hypermethylation and hypomethylation of gene promoters in carcinogenesis.

Conclusions

Genes with promoter hypermethylation and hypomethylation are highly consistent in function across different cancer types, respectively, but these two groups of genes tend to be enriched in different functions associated with cancer. Especially, we speculate that hypomethylation of gene promoters may play roles in inducing immunity and inflammation disorders in precancerous conditions, which may provide hints for improving epigenetic therapy and immunotherapy of cancer.     

Abundance of plastid DNA insertions in nuclear genomes of rice and Arabidopsis

Pairwise comparison of whole plastid and draft nuclear genomic sequences of Arabidopsis thaliana and Oryza sativa L. ssp. indica shows that rice nuclear genomic sequences contain homologs of plastid DNA covering about 94 kb (83%) of plastid genome and including one or more full-length intact (without mutations resulting in premature stop codons) homologues of 26 known protein-coding (KPC) plastid genes. By contrast, only about 20 kb (16%) of chloroplast DNA, including a single intact plastid-derived KPC gene, is presented in the nucleus of A. thaliana. Sixteen rice plastid genes have at least one nuclear copy without any mutation or with only synonymous substitutions. Nuclear copies for other ten plastid genes contain both synonymous and non-synonymous substitutions. Multiple ESTs for 25 out of 26 KPC genes were also found, as well as putative promoters for some of them. The study of substitutions pattern shows that some of nuclear homologues of plastid genes may be functional and/or are under the pressure of the positive natural selection. The similar comparative analysis performed on rice chromosome 1 revealed 27 contigs containing plastid-derived sequences, totalling about 84 kb and covering two thirds of chloroplast DNA, with the intact nuclear copies of 26 different KPC genes. One of these contigs, AP003280, includes almost 57 kb (45%) of chloroplast genome with the intact copies of 22 KPC genes. At the same time, we observed that relative locations of homologues in plastid DNA and the nuclear genome are significantly different.     

Distinctive core histone post-translational modification patterns in Arabidopsis thaliana

Post-translational modifications of histones play crucial roles in the genetic and epigenetic regulation of gene expression from chromatin. Studies in mammals and yeast have found conserved modifications at some residues of histones as well as non-conserved modifications at some other sites. Although plants have been excellent systems to study epigenetic regulation, and histone modifications are known to play critical roles, the histone modification sites and patterns in plants are poorly defined. In the present study we have used mass spectrometry in combination with high performance liquid chromatography (HPLC) separation and phospho-peptide enrichment to identify histone modification sites in the reference plant, Arabidopsis thaliana. We found not only modifications at many sites that are conserved in mammalian and yeast cells, but also modifications at many sites that are unique to plants. These unique modifications include H4 K20 acetylation (in contrast to H4 K20 methylation in non-plant systems), H2B K6, K11, K27 and K32 acetylation, S15 phosphorylation and K143 ubiquitination, and H2A K144 acetylation and S129, S141 and S145 phosphorylation, and H2A.X S138 phosphorylation. In addition, we found that lysine 79 of H3 which is highly conserved and modified by methylation and plays important roles in telomeric silencing in non-plant systems, is not modified in Arabidopsis. These results suggest distinctive histone modification patterns in plants and provide an invaluable foundation for future studies on histone modifications in plants.     

Distinct properties of the five UDP-D-glucose/UDP-D-galactose 4-epimerase isoforms of Arabidopsis thaliana

Plant genomes contain genetically encoded isoforms of most nucleotide sugar interconversion enzymes. Here we show that Arabidopsis thaliana has five genes encoding functional UDP-D-glucose/UDP-D-galactose 4-epimerase (named UGE1 to UGE5). All A. thaliana UDP-d-glucose 4-epimerase isoforms are dimeric in solution, maximally active in vitro at 30-40 degrees C, and show good activity between pH 7 and pH 9. In vitro, UGE1, -3, and -5 act independently of externally added NAD+, whereas cofactor addition stimulates the activity of UGE2 and is particularly important for UGE4 activity. UGE1 and UGE3 are most efficiently inhibited by UDP. The five isoforms display kcatUDP-Gal values between 23 and 128 s(-1) and KmUDP-Gal values between 0.1 and 0.3 mm. This results in enzymatic efficiencies ranging between 97 and 890 mm(-1) s(-1) for UGE4 = UGE1 < UGE3 < UGE5 < UGE2. The KmUDP-Glc values, derived from the Haldane relationship, were 0.76 mm for UGE1, 0.56 mm for UGE4, and between 0.13 and 0.23 mm for UGE2, -3, and -5. The expression of UGE isoforms is ubiquitous and displays developmental and cell type-dependent variations. UGE1 and -3 expression patterns globally resemble enzymes involved in carbohydrate catabolism, and UGE2, -4, and -5 expression is more related to carbohydrate biosynthesis. UGE1, -2, and -4 are present in the cytoplasm, whereasUGE4 is additionally enriched close to Golgi stacks. All UGE genes tested complement the UGE4rhd1 phenotype, confer increased galactose tolerance in planta, and complement the galactose metabolization deficiency in the Saccharomyces cerevisiae gal10 mutant. We suggest that plant UGE isoforms function in different metabolic situations and that enzymatic properties, gene expression pattern, and subcellular localization contribute to the differentiation of isoform function.     

Genes and genomes: Towards construction of an overlapping YAC library of the Arabidopsis thaliana genome

Arabidopsis thaliana (Thale cress, Arabidopsis) is an ideal model organism for the molecular genetic analysis of many plant processes. The availability of a complete physical map would greatly facilitate the gene cloning steps in these studies. The small genome size of Arabidopsis makes the construction of such a map a feasible goal. One of the approaches to construct an overlapping library of the Arabidopsis genome takes advantage of the many mapped markers and the availability of Arabidopsis yeast artificial chromosome (YAC) libraries. Mapped molecular markers are used to identify corresponding YAC clones and thereby place them on the genetic map. Subsequently, these YAC clones provide the framework for directed walking experiments aimed at closing the gaps between the YAC contigs. Adopting this strategy, YAC clones comprising about 10% of the genome have been assigned to the top halves of Arabidopsis chromosomes 4 and 5. Extensive walking experiments in a 10 cM interval of chromosome 4 have resulted in two contiguous regions in the megabase size range.     

Distinct short-range ovule signals attract or repel Arabidopsis thaliana pollen tubes in vitro

Background  

Pollen tubes deliver sperm after navigating through flower tissues in response to attractive and repulsive cues. Genetic analyses in maize and Arabidopsis thaliana and cell ablation studies in Torenia fournieri have shown that the female gametophyte (the 7-celled haploid embryo sac within an ovule) and surrounding diploid tissues are essential for guiding pollen tubes to ovules. The variety and inaccessibility of these cells and tissues has made it challenging to characterize the sources of guidance signals and the dynamic responses they elicit in the pollen tubes.