Elucidation of the core betalain biosynthesis pathway in Amaranthus tricolor.

Amaranthus tricolor L., a vegetable Amaranthus species, is an economically important crop containing large amounts of betalains. Betalains are natural antioxidants and can be classified into betacyanins and betaxanthins, with red and yellow colors, respectively. A. tricolor cultivars with varying betalain contents, leading to striking red to green coloration, have been commercially produced. However, the molecular differences underlying betalain biosynthesis in various cultivars of A. tricolor remain largely unknown. In this study, A. tricolor cultivars with different colors were chosen for comparative transcriptome analysis. The elevated expression of AmCYP76AD1 in a red-leaf cultivar of A. tricolor was proposed to play a key role in producing red betalain pigments. The functions of AmCYP76AD1, AmDODAα1, AmDODAα2, and AmcDOPA5GT were also characterized through the heterologous engineering of betalain pigments in Nicotiana benthamiana. Moreover, high and low L-DOPA 4,5-dioxygenase activities of AmDODAα1 and AmDODAα2, respectively, were confirmed through in vitro enzymatic assays. Thus, comparative transcriptome analysis combined with functional and enzymatic studies allowed the construction of a core betalain biosynthesis pathway of A. tricolor. These results not only provide novel insights into betalain biosynthesis and evolution in A. tricolor but also provide a basal framework for examining genes related to betalain biosynthesis among different species of Amaranthaceae.

miRgo: integrating various off-the-shelf tools for identification of microRNA-target interactions by heterogeneous features and a novel evaluation indicator.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression and biological processes through binding to messenger RNAs. Predicting the relationship between miRNAs and their targets is crucial for research and clinical applications. Many tools have been developed to predict miRNA-target interactions, but variable results among the different prediction tools have caused confusion for users. To solve this problem, we developed miRgo, an application that integrates many of these tools. To train the prediction model, extreme values and median values from four different data combinations, which were obtained via an energy distribution function, were used to find the most representative dataset. Support vector machines were used to integrate 11 prediction tools, and numerous feature types used in these tools were classified into six categories-binding energy, scoring function, evolution evidence, binding type, sequence property, and structure-to simplify feature selection. In addition, a novel evaluation indicator, the Chu-Hsieh-Liang (CHL) index, was developed to improve the prediction power in positive data for feature selection. miRgo achieved better results than all other prediction tools in evaluation by an independent testing set and by its subset of functionally important genes. The tool is available at http://predictor.nchu.edu.tw/miRgo.

CoII(Chromomycin)2 Complex Induces a Conformational Change of CCG Repeats from i-Motif to Base-Extruded DNA Duplex.

We have reported the propensity of a DNA sequence containing CCG repeats to form a stable i-motif tetraplex structure in the absence of ligands. Here we show that an i-motif DNA sequence may transition to a base-extruded duplex structure with a GGCC tetranucleotide tract when bound to the (CoII)-mediated dimer of chromomycin A3, CoII(Chro)2. Biophysical experiments reveal that CCG trinucleotide repeats provide favorable binding sites for CoII(Chro)2. In addition, water hydration and divalent metal ion (CoII) interactions also play a crucial role in the stabilization of CCG trinucleotide repeats (TNRs). Our data furnish useful structural information for the design of novel therapeutic strategies to treat neurological diseases caused by repeat expansions.

Evolution of microRNA827 targeting in the plant kingdom.

Unlike most ancient microRNAs, which conservatively target homologous genes across species, microRNA827 (miR827) targets two different types of SPX (SYG1/PHO81/XPR1)-domain-containing genes, NITROGEN LIMITATION ADAPTATION (NLA) and PHOSPHATE TRANSPORTER 5 (PHT5), in Arabidopsis thaliana and Oryza sativa to regulate phosphate (Pi) transport and storage, respectively. However, how miR827 shifted its target preference and its evolutionary history are unknown. Based on target prediction analysis, we found that in most angiosperms, miR827 conservatively targets PHT5 homologs, but in Brassicaceae and Cleomaceae it preferentially targets NLA homologs, and we provide evidence for the transition of target preference during Brassicales evolution. Intriguingly, we found a lineage-specific loss of the miR827-regulatory module in legumes. Analysis of miR827-mediated cleavage efficiency and the expression of PHT5 in A. thaliana indicated that accumulation of mutations in the target site and the exclusion of the target site by alternative transcriptional initiation eliminated PHT5 targeting by miR827. Here, we identified a transition of miR827 target preference during plant evolution and revealed the uniqueness of miR827-mediated regulation among conserved plant miRNAs. Despite the change in its target preference, upregulation of miR827 by Pi starvation and its role in regulating cellular Pi homeostasis were retained.

Parity-dependent hairpin configurations of repetitive DNA sequence promote slippage associated with DNA expansion.

Repetitive DNA sequences are ubiquitous in life, and changes in the number of repeats often have various physiological and pathological implications. DNA repeats are capable of interchanging between different noncanonical and canonical conformations in a dynamic fashion, causing configurational slippage that often leads to repeat expansion associated with neurological diseases. In this report, we used single-molecule spectroscopy together with biophysical analyses to demonstrate the parity-dependent hairpin structural polymorphism of TGGAA repeat DNA. We found that the DNA adopted two configurations depending on the repeat number parity (even or odd). Transitions between these two configurations were also observed for longer repeats. In addition, the ability to modulate this transition was found to be enhanced by divalent ions. Based on the atomic structure, we propose a local seeding model where the kinked GGA motifs in the stem region of TGGAA repeat DNA act as hot spots to facilitate the transition between the two configurations, which may give rise to disease-associated repeat expansion.

Phytoplasma SAP11 alters 3-isobutyl-2-methoxypyrazine biosynthesis in Nicotiana benthamiana by suppressing NbOMT1.

Phytoplasmas are bacterial phytopathogens that release virulence effectors into sieve cells and act systemically to affect the physiological and morphological state of host plants to promote successful pathogenesis. We show here that transgenic Nicotiana benthamiana lines expressing the secreted effector SAP11 from Candidatus Phytoplasma mali exhibit an altered aroma phenotype. This phenomenon is correlated with defects in the development of glandular trichomes and the biosynthesis of 3-isobutyl-2-methoxypyrazine (IBMP). IBMP is a volatile organic compound (VOC) synthesized by an O-methyltransferase, via a methylation step, from a non-volatile precursor, 3-isobutyl-2-hydroxypyrazine (IBHP). Based on comparative and functional genomics analyses, NbOMT1, which encodes an O-methyltransferase, was found to be highly suppressed in SAP11-transgenic plants. We further silenced NbOMT1 through virus-induced gene silencing and demonstrated that this enzyme influenced the accumulation of IBMP in N. benthamiana In vitro biochemical analyses also showed that NbOMT1 can catalyse IBHP O-methylation in the presence of S-adenosyl-L-methionine. Our study suggests that the phytoplasma effector SAP11 has the ability to modulate host VOC emissions. In addition, we also demonstrated that SAP11 destabilized TCP transcription factors and suppressed jasmonic acid responses in N. benthamiana These findings provide valuable insights into understanding how phytoplasma effectors influence plant volatiles.

Inheritance of gene expression level and selective constraints on trans- and cis-regulatory changes in yeast.

Gene expression evolution can be caused by changes in cis- or trans-regulatory elements or both. As cis and trans regulation operate through different molecular mechanisms, cis and trans mutations may show different inheritance patterns and may be subjected to different selective constraints. To investigate these issues, we obtained and analyzed gene expression data from two Saccharomyces cerevisiae strains and their hybrid, using high-throughput sequencing. Our data indicate that compared with other types of genes, those with antagonistic cis-trans interactions are more likely to exhibit over- or underdominant inheritance of expression level. Moreover, in accordance with previous studies, genes with trans variants tend to have a dominant inheritance pattern, whereas cis variants are enriched for additive inheritance. In addition, cis regulatory differences contribute more to expression differences between species than within species, whereas trans regulatory differences show a stronger association between divergence and polymorphism. Our data indicate that in the trans component of gene expression differences genes subjected to weaker selective constraints tend to have an excess of polymorphism over divergence compared with those subjected to stronger selective constraints. In contrast, in the cis component, this difference between genes under stronger and weaker selective constraint is mostly absent. To explain these observations, we propose that purifying selection more strongly shapes trans changes than cis changes and that positive selection may have significantly contributed to cis regulatory divergence.

Natural selection on cis and trans regulation in yeasts.

Gene expression is regulated both by cis elements, which are DNA segments closely linked to the genes they regulate, and by trans factors, which are usually proteins capable of diffusing to unlinked genes. Understanding the patterns and sources of regulatory variation is crucial for understanding phenotypic and genome evolution. Here, we measure genome-wide allele-specific expression by deep sequencing to investigate the patterns of cis and trans expression variation between two strains of Saccharomyces cerevisiae. We propose a statistical modeling framework based on the binomial distribution that simultaneously addresses normalization of read counts derived from different parents and estimating the cis and trans expression variation parameters. We find that expression polymorphism in yeast is common for both cis and trans, though trans variation is more common. Constraint in expression evolution is correlated with other hallmarks of constraint, including gene essentiality, number of protein interaction partners, and constraint in amino acid substitution, indicating that both cis and trans polymorphism are clearly under purifying selection, though trans variation appears to be more sensitive to selective constraint. Comparing interspecific expression divergence between S. cerevisiae and S. paradoxus to our intraspecific variation suggests a significant departure from a neutral model of molecular evolution. A further examination of correlation between polymorphism and divergence within each category suggests that cis divergence is more frequently mediated by positive Darwinian selection than is trans divergence.

Abundance of tRNA-derived small RNAs in phosphate-starved Arabidopsis roots.

Several research advances have indicated an important role of transfer RNA (tRNA)-derived small RNAs in modulating developmental processes or stress responses. Recently, from the deep sequencing of small RNAs in Arabidopsis (Arabidopsis thaliana), we identified a new class of 19-nucleotide (nt) small RNAs corresponding to the 5' end of tRNA accumulated at high levels in phosphate-starved roots. In two very recent studies, 19-nt tRNA fragments were also observed in human cells, suggesting their widespread nature. In our study, tRNA halves cleaved at the anticodon loop, the most common tRNA fragments found, were predominant in roots. These results showed a spatial and temporal expression pattern of small RNAs derived from specific cleavage of tRNA molecules. Although the function of these tRNA-derived small RNAs under phosphate deficiency remains unknown, their diversity, biogenesis and potential function are henceforth summarized and discussed. Certainly, they will emerge as a novel class of regulatory small RNAs.

Uncovering small RNA-mediated responses to phosphate deficiency in Arabidopsis by deep sequencing.

Recent studies have demonstrated the important role of plant microRNAs (miRNAs) under nutrient deficiencies. In this study, deep sequencing of Arabidopsis (Arabidopsis thaliana) small RNAs was conducted to reveal miRNAs and other small RNAs that were differentially expressed in response to phosphate (Pi) deficiency. About 3.5 million sequence reads corresponding to 0.6 to 1.2 million unique sequence tags from each Pi-sufficient or Pi-deficient root or shoot sample were mapped to the Arabidopsis genome. We showed that upon Pi deprivation, the expression of miR156, miR399, miR778, miR827, and miR2111 was induced, whereas the expression of miR169, miR395, and miR398 was repressed. We found cross talk coordinated by these miRNAs under different nutrient deficiencies. In addition to miRNAs, we identified one Pi starvation-induced DICER-LIKE1-dependent small RNA derived from the long terminal repeat of a retrotransposon and a group of 19-nucleotide small RNAs corresponding to the 5' end of tRNA and expressed at a high level in Pi-starved roots. Importantly, we observed an increased abundance of TAS4-derived trans-acting small interfering RNAs (ta-siRNAs) in Pi-deficient shoots and uncovered an autoregulatory mechanism of PAP1/MYB75 via miR828 and TAS4-siR81(-) that regulates the biosynthesis of anthocyanin. This finding sheds light on the regulatory network between miRNA/ta-siRNA and its target gene. Of note, a substantial amount of miR399* accumulated under Pi deficiency. Like miR399, miR399* can move across the graft junction, implying a potential biological role for miR399*. This study represents a comprehensive expression profiling of Pi-responsive small RNAs and advances our understanding of the regulation of Pi homeostasis mediated by small RNAs.

Human TRIM71 and its nematode homologue are targets of let-7 microRNA and its zebrafish orthologue is essential for development.

Animal microRNAs (miRNAs) are short RNAs that function as posttranscriptional regulators of gene expression by binding to the target mRNAs. Noting that some miRNAs are highly conserved in evolution, we explored the possibility of evolutionary conservation of their targets. We identified human orthologues of experimentally verified let-7 miRNA target genes in Caenorhabditis elegans and used the luciferase reporter system to examine whether these human genes are still the targets of let-7 miRNA. We found that in some cases, the miRNA-target relationship has indeed been conserved in human. Interestingly, human TRIM71, an orthologue of C. elegans let-7-target lin-41 gene, can be repressed by hsa-let-7a and hsa-let-7c. This repression was abolished when both predicted let-7 target sites of TRIM71 were mutated. Moreover, the zebrafish lin-41 orthologue was also repressed by let-7 to a similar degree as was TRIM71. When the expression of zebrafish lin-41 orthologue was silenced by microinjection of RNA interference or morpholino into zebrafish zygotes, retarded embryonic development was observed, providing direct evidence for an essential role of lin-41 in zebrafish development. Taken together, our results suggest that the regulation of TRIM71 expression by let-7 has been evolutionarily conserved and that TRIM71 likely plays an important role in development.

Shannon information in complete genomes.

Shannon information in the genomes of all completely sequenced prokaryotes and eukaryotes are measured in word lengths of two to ten letters. It is found that in a scale-dependent way, the Shannon information in complete genomes are much greater than that in matching random sequences--thousands of times greater in the case of short words. Furthermore, with the exception of the 14 chromosomes of Plasmodium falciparum, the Shannon information in all available complete genomes belong to a universality class given by an extremely simple formula. The data are consistent with a model for genome growth composed of two main ingredients: random segmental duplications that increase the Shannon information in a scale-independent way, and random point mutations that preferentially reduces the larger-scale Shannon information. The inference drawn from the present study is that the large-scale and coarse-grained growth of genomes was selectively neutral and this suggests an independent corroboration of Kimura's neutral theory of evolution.

Divergence and Shannon information in genomes.

Shannon information (SI) and its special case, divergence, are defined for a DNA sequence in terms of probabilities of chemical words in the sequence and are computed for a set of complete genomes highly diverse in length and composition. We find the following: SI (but not divergence) is inversely proportional to sequence length for a random sequence but is length independent for genomes; the genomic SI is always greater and, for shorter words and longer sequences, hundreds to thousands times greater than the SI in a random sequence whose length and composition match those of the genome; genomic SIs appear to have word-length dependent universal values. The universality is inferred to be an evolution footprint of a universal mode for genome growth.

Shannon information in complete genomes.

Shannon information in the genomes of all completely sequenced prokaryotes and eukaryotes are measured in word lengths of two to ten letters. It is found that in a scale-dependent way, the Shannon information in complete genomes are much greater than that in matching random sequences - thousands of times greater in the case of short words. Furthermore, with the exception of the 14 chromosomes of Plasmodium falciparum, the Shannon information in all available complete genomes belong to a universality class given by an extremely simple formula. The data are consistent with a model for genome growth composed of two main ingredients: random segmental duplications that increase the Shannon information in a scale-independent way, and random point mutations that preferentially reduces the larger-scale Shannon information. The inference drawn from the present study is that the large-scale and coarse-grained growth of genomes was selectively neutral and this suggests an independent corroboration of Kimura's neutral theory of evolution.

Minimal model for genome evolution and growth.

Textual analysis of typical microbial genomes reveals that they have the statistical characteristics of a DNA sequence of a much shorter length. This peculiar property supports an evolutionary model in which a genome evolves by random mutation but primarily grows by random segmental duplication. That genomes grew mostly by duplication is consistent with the observation that repeat sequences in all genomes are widespread and intragenomic and intergenomic homologous genes are preponderant across all life forms.