Molecular characterization and genetic mapping of two clusters of genes encoding chlorophyll a/b-binding proteins in Lycopersicon esculentum (tomato)

We have constructed a tomato genomic library in the λ Charon 4 phage vector. The library was screened with a pea cDNA probe encoding a chlorophyll a/b-binding protein (CAB), and several recombinant phages containing tomato CAB genes were isolated and characterized by restriction mapping, heteroduplex analysis and nucleotide sequencing. Two phages with overlapping segments of the tomato genome contain a total of four CAB genes, all arranged in tandem. A third phage contains three CAB genes, two arranged in tandem and one in opposite orientation, and an additional, truncated CAB gene. Genetic mapping experiments showed that the four CAB genes on the first two phages belong to a locus, previously designated Cab-1, on chromosome 2. The CAB genes from the third phage belong to the Cab-3 locus on chromosome 3. Complete sequence determination of two CAB genes, one from each locus, and additional sequence determination of about 50% of each of the other five CAB genes showed that each gene within a CAB locus is more similar to other CAB genes in the same locus than it is to the CAB genes from the second locus. Furthermore, the polypeptides encoded by Cab-1 genes diverge significantly from those encoded by Cab-3 genes in the domains of transit peptide and the N terminus of the mature polypeptide but are essentially identical in the rest of the sequence.     

Determination of Residues Responsible for Substrate and Product Specificity of Solanum habrochaites Short-Chain cis-Prenyltransferases

Prenyl residues confer divergent biological activities such as antipathogenic and antiherbivorous activities on phenolic compounds, including flavonoids, coumarins, and xanthones. To date, about 1,000 prenylated phenolics have been isolated, with these compounds containing various prenyl residues. However, all currently described plant prenyltransferases (PTs) have been shown specific for dimethylallyl diphosphate as the prenyl donor, while most of the complementary DNAs encoding these genes have been isolated from the Leguminosae. In this study, we describe the identification of a novel PT gene from lemon (Citrus limon), ClPT1, belonging to the homogentisate PT family. This gene encodes a PT that differs from other known PTs, including flavonoid-specific PTs, in polypeptide sequence. This membrane-bound enzyme was specific for geranyl diphosphate as the prenyl donor and coumarin as the prenyl acceptor. Moreover, the gene product was targeted to plastid in plant cells. To our knowledge, this is the novel aromatic PT specific to geranyl diphosphate from citrus species.Prenylation is an important derivatization of plant aromatics, contributing to the chemical diversification of phenolic secondary metabolites in plants due to differences in prenylation positions, prenyl chain lengths, and further modifications of prenyl chains. To date, about 1,000 prenylated aromatic compounds have been isolated as biologically active substances from various plant species, including many medicinal plants.Coumarins (α-benzopyrones) are a large group of plant secondary metabolites. Many biologically active coumarins are prenylated, with the prenyl residue enhancing the biological activities of the aromatic core compound. For example, imperatorin (dimethylallylated xanthotoxol), a strong inhibitor of a Manduca sexta midgut cytochrome P450, has 100-fold greater activity than the nonprenylated coumarin compound, suggesting that prenylation is involved in chemoprevention against biotic stress in plants (Neal and Wu, 1994). Prenylated compounds are also beneficial for human health. For example, geranylation of umbelliferone at the OH position to form auraptene results in a 25-fold enhancement of the inhibition of Epstein Barr virus activity, a test used to screen antitumor compounds (Murakami et al., 1997). Moreover, in tuberculosis, 8-geranyloxypsoralen was reported to decrease the growth rate of Mycobacterium smegmatis (Adams et al., 2006).There are many reports on the detection of prenyltransferase (PT) activities for coumarins in various plant species. For example, umbelliferone-dimethylallyltransferase activities were reported in cultured parsley (Petroselinum crispum) cells, Ruta graveolens, and Ammi majus, and plastidial localization of the enzyme activity is also reported (Ellis and Brown, 1974; Dhillon and Brown, 1976; Tietjen and Matern, 1983; Hamerski and Matern, 1988; Hamerski et al., 1990). In addition, bergaptol 5-O-geranyltransferase activity, which yields bergamottin, a major coumarin derivative, was characterized using the microsomal fraction of lemon (Citrus limon) peel flavedo, the outer part of the lemon fruit (Frérot and Decorzant, 2004; Munakata et al., 2012). In the lemon flavedo, 8-geranyltransferase activity for umbelliferone was also detected (Munakata et al., 2012). To date, only one gene encoding these enzymes has been described; this gene, which encodes a parsley PT (PcPT), was very recently isolated (Karamat et al., 2014).The first flavonoid-specific PT identified was naringenin 8-dimethylallyltransferase (SfN8DT1) from a leguminous medicinal plant, Sophora flavescens (Sasaki et al., 2008). Since then, genes encoding various flavonoid PTs have been identified in Leguminosae (Akashi et al., 2009; Sasaki et al., 2011; Shen et al., 2012). Although other prenylated aromatic compounds, including coumarins, xanthons, phenylpropanoids, and phloroglucinols, have been isolated from many plant species, no gene encoding a PT for those aromatics has been isolated, except for the gene encoding a phloroglucinol-specific enzyme (HlPT1) from hops (Humulus lupulus) and a the recently isolated coumarin dimethylallyltransferase from parsley (Tsurumaru et al., 2010, 2012; Karamat et al., 2014). These isolated plant aromatic PTs show strong preference for dimethylallyl diphosphate (DMAPP) as the prenyl donor substrate, although in nature, many geranylated phenolics and less farnesylated phenolics have been described. This raises questions about the enzymes and reaction mechanisms involved in the synthesis of these phenolic compounds, such as substrate specificity and prenylation sites. Better understanding of these reactions requires the identification of PTs with other enzymatic activities. It is also necessary to identify PTs producing prenylated phenolics in nonleguminosaeous plants. Four different tracks should be explored to identify enzymes that (1) recognize nonflavonoid substrates, e.g. coumarins, phenylpropanoids, and xanthons, (2) are specific for longer chain prenyl diphosphates such as geranyl diphosphate (GPP) and farnesyl diphosphate (FPP), (3) are from nonlegume origins, and (4) catalyze O-prenylation.Citrus species, including lemons, contain large quantities of geranylated coumarins. We therefore isolated a complementary DNA (cDNA) encoding a PT from lemon peel, identifying the novel PT-encoding gene ClPT1. Phylogenetic analysis showed that this enzyme shares homologies with homogentisate PTs involved in vitamin E and plastoquinone biosynthesis but is located in a new clade. We provide evidence showing that this unique enzyme is highly specific for GPP as a prenyl donor and coumarin as a prenyl acceptor. We also show that the gene product is targeted to plastid in plant cells.     

Robust Prediction of Expression Differences among Human Individuals Using Only Genotype Information

Many genetic variants that are significantly correlated to gene expression changes across human individuals have been identified, but the ability of these variants to predict expression of unseen individuals has rarely been evaluated. Here, we devise an algorithm that, given training expression and genotype data for a set of individuals, predicts the expression of genes of unseen test individuals given only their genotype in the local genomic vicinity of the predicted gene. Notably, the resulting predictions are remarkably robust in that they agree well between the training and test sets, even when the training and test sets consist of individuals from distinct populations. Thus, although the overall number of genes that can be predicted is relatively small, as expected from our choice to ignore effects such as environmental factors and trans sequence variation, the robust nature of the predictions means that the identity and quantitative degree to which genes can be predicted is known in advance. We also present an extension that incorporates heterogeneous types of genomic annotations to differentially weigh the importance of the various genetic variants, and we show that assigning higher weights to variants with particular annotations such as proximity to genes and high regional G/C content can further improve the predictions. Finally, genes that are successfully predicted have, on average, higher expression and more variability across individuals, providing insight into the characteristics of the types of genes that can be predicted from their cis genetic variation.     

Systematic Dissection of the Sequence Determinants of Gene 3’ End Mediated Expression Control

The 3’end genomic region encodes a wide range of regulatory process including mRNA stability, 3’ end processing and translation. Here, we systematically investigate the sequence determinants of 3’ end mediated expression control by measuring the effect of 13,000 designed 3’ end sequence variants on constitutive expression levels in yeast. By including a high resolution scanning mutagenesis of more than 200 native 3’ end sequences in this designed set, we found that most mutations had only a mild effect on expression, and that the vast majority (~90%) of strongly effecting mutations localized to a single positive TA-rich element, similar to a previously described 3’ end processing efficiency element, and resulted in up to ten-fold decrease in expression. Measurements of 3’ UTR lengths revealed that these mutations result in mRNAs with aberrantly long 3’UTRs, confirming the role for this element in 3’ end processing. Interestingly, we found that other sequence elements that were previously described in the literature to be part of the polyadenylation signal had a minor effect on expression. We further characterize the sequence specificities of the TA-rich element using additional synthetic 3’ end sequences and show that its activity is sensitive to single base pair mutations and strongly depends on the A/T content of the surrounding sequences. Finally, using a computational model, we show that the strength of this element in native 3’ end sequences can explain some of their measured expression variability (R = 0.41). Together, our results emphasize the importance of efficient 3’ end processing for endogenous protein levels and contribute to an improved understanding of the sequence elements involved in this process.     

Metabolomics, genomics, proteomics, and the identification of enzymes and their substrates and products

A large proportion of the genes in any plant genome encode enzymes of primary and specialized (secondary) metabolism. Not all plant primary metabolites, those that are found in all or most species, have been identified. Moreover, only a small portion of the estimated hundreds of thousand specialized metabolites, those found only in restricted lineages, have been studied in any species. The correlative analysis of extensive metabolic profiling and gene expression profiling has proven a powerful approach for the identification of candidate genes and enzymes, particularly those in secondary metabolism. The final characterization of substrates, enzymatic activities, and products requires biochemical analysis, which has been most successful when candidate proteins have homology to other enzymes of known function. The challenges are to identify new types of enzymes and to develop biochemical techniques that are suitable for large-scale analysis.     

Temporal mating isolation driven by a behavioral gene in Drosophila

Speciation is the evolutionary process in which new barriers to gene exchange are created. These barriers may be physical, leading to spatial separation of subpopulations and resulting in allopatric speciation, or they may be temporal, giving rise to allochronic speciation, and may include the time of day or the time of year when mating takes place. Drosophila melanogaster and D. pseudoobscura show different temporal patterns of circadian locomotor activity that are determined by the circadian clock gene period (per). Genes that control aspects of behavior that might be relevant to courtship and mating, such as locomotor patterns, become obvious candidates for involvement in the speciation process. However, evidence for the role of individual genes in the mechanism of mate choice has proved elusive. We have used transgenic flies carrying the natural per genes from these two Drosophila species to reveal that per has the potential to provide the permissive conditions for speciation, by affecting mate choice through a mechanism involving the species-specific timing of mating behavior.     

Nicotinamidase participates in the salvage pathway of NAD biosynthesis in Arabidopsis

Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), which is derived from NAD, have important roles as a redox carriers in metabolism. A combination of de novo and salvage pathways contribute to the biosynthesis of NAD in all organisms. The pathways and enzymes of the NAD salvage pathway in yeast and animals, which diverge at nicotinamide, have been extensively studied. Yeast cells convert nicotinamide to nicotinic acid, while mammals lack the enzyme nicotinamidase and instead convert nicotinamide to nicotinamide mononucleotide. Here we show that Arabidopsis thaliana gene At2g22570 encodes a nicotinamidase, which is expressed in all tissues, with the highest levels observed in roots and stems. The 244-residue protein, designated AtNIC1, converts nicotinamide to nicotinic acid and has a Km value of 118 +/- 17 microM and a Kcat value of 0.93 +/- 0.13 sec(-1). Plants homozygous for a null AtNIC1 allele, nic1-1, have lower levels of NAD and NADP under normal growth conditions, indicating that AtNIC1 participates in a yeast-type NAD salvage pathway. Mutant plants also exhibit hypersensitivity to treatments of abscisic acid and NaCl, which is correlated with their inability to increase the cellular levels of NAD(H) under these growth conditions, as occurs in wild-type plants. We also show that the growth of the roots of wild-type but not nic1-1 mutant plants is inhibited and distorted by nicotinamide.