Michael P. Timko

Four genes in two diverged subfamilies encode the ribulose-1,5-bisphosphate carboxylase small subunit polypeptides of Arabidopsis thaliana.

The multigene family encoding the small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase/oxygenase in the crucifer Arabidopsis thaliana has been isolated and the organization and structure of the individual members determined. The family consists of four genes which have been divided into two subfamilies on the basis of linkage and DNA and amino acid sequence similarities. Three of the genes, designated ats1B, ats2B, and ats3B, reside in tandem on an 8 kb stretch of the chromosome. These genes share greater than 95% similarity in DNA sequence and encode polypeptides identical in length and 96.7% similar in amino acid sequence. The fourth gene, ats1A, is at least 10 kb removed from, or completely unlinked to the B subfamily. The B subfamily genes are more similar to each other than to ats1A in nucleotide and amino acid sequence. All four genes are interupted by two introns whose placement within the coding region of the genes is conserved. The introns of the B subfamily genes are similar in length and nucleotide sequence, but show no similarity to the introns of ats1A. Comparison of the DNA sequences within the immediate 5′ and 3′ flanking sequences among the genes revealed only limited regions of homology. S1 analysis shows that all four genes are expressed.

Light-inducible and tissue-specific expression of a chimaeric gene under control of the 5'-flanking sequence of a pea chlorophyll a/b-binding protein gene

We have investigated the regulatory functions of the 5′‐flanking sequences of a chlorophyll a/b‐binding protein gene from Pisum sativum, using the neomycin phosphotransferase (II) activity from Tn5 as an enzymatic reporter. We show that 0.4 kb of the upstream flanking sequences of this gene are sufficient for both organ‐specific and light‐regulated expression of our chimaeric constructs in transformed tobacco plants. In addition, we show that sequences farther upstream have a significant influence on the level of transcription of these constructions.

Tobacco Transcription Factors: Novel Insights into Transcriptional Regulation in the Solanaceae

Tobacco (Nicotiana tabacum) is a member of the Solanaceae, one of the agronomically most important groups of flowering plants. We have performed an in silico analysis of 1.15 million gene-space sequence reads from the tobacco nuclear genome and report the detailed analysis of more than 2,500 tobacco transcription factors (TFs). The tobacco genome contains at least one member of each of the 64 well-characterized TF families identified in sequenced vascular plant genomes, indicating that evolution of the Solanaceae was not associated with the gain or loss of TF families. However, we found notable differences between tobacco and non-Solanaceae species in TF family size and evidence for both tobacco- and Solanaceae-specific subfamily expansions. Compared with TF families from sequenced plant genomes, tobacco has a higher proportion of ERF/AP2, C2H2 zinc finger, homeodomain, GRF, TCP, zinc finger homeodomain, BES, and STERILE APETALA (SAP) genes and novel subfamilies of BES, C2H2 zinc finger, SAP, and NAC genes. The novel NAC subfamily, termed TNACS, appears restricted to the Solanaceae, as they are absent from currently sequenced plant genomes but present in tomato (Solanum lycopersicum), pepper (Capsicum annuum), and potato (Solanum tuberosum). They constitute approximately 25% of NAC genes in tobacco. Based on our phylogenetic studies, we predict that many of the more than 50 tobacco group IX ERF genes are involved in jasmonate responses. Consistent with this, over two-thirds of group IX ERF genes tested showed increased mRNA levels following jasmonate treatment. Our data are a major resource for the Solanaceae and fill a void in studies of TF families across the plant kingdom.

Molecular cloning, nuclear gene structure, and developmental expression of NADPH: protochlorophyllide oxidoreductase in pea (Pisum sativum L.)

Complementary DNA clones and a corresponding nuclear gene (lpcr) encoding the NADPH-dependent protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) have been characterized from pea (Pisum sativum L.). The pea lpcr gene encodes a 43 118 Da precursor polypeptide comprised of a transit peptide of 64 amino acids and a mature protein of 336 amino acids. The coding portion of the gene is interrupted by four introns, two of which are located within the transit peptide coding portion of the gene. The deduced primary structure for the pea protein is similar to those reported for Arabidopsis and two monocot species. Northern blot analysis revealed little to no decrease in steady-state levels of mRNA encoding the enzyme in etiolated leaves illuminated with continuous white light for up to 48 h. In contrast, western blot analysis showed that the major immunoreactive species present in whole leaf extracts decreased to nearly undetectable levels during this same 48 h period. These results suggest that pchlide reductase activity in pea is primarily regulated post-transcriptionally, most likely at the level of translation initiation/elongation or protein turnover.

Sequencing and analysis of the gene-rich space of cowpea

BackgroundCowpea, Vigna unguiculata (L.) Walp., is one of the most important food and forage legumes in the semi-arid tropics because of its drought tolerance and ability to grow on poor quality soils. Approximately 80% of cowpea production takes place in the dry savannahs of tropical West and Central Africa, mostly by poor subsistence farmers. Despite its economic and social importance in the developing world, cowpea remains to a large extent an underexploited crop. Among the major goals of cowpea breeding and improvement programs is the stacking of desirable agronomic traits, such as disease and pest resistance and response to abiotic stresses. Implementation of marker-assisted selection and breeding programs is severely limited by a paucity of trait-linked markers and a general lack of information on gene structure and organization. With a nuclear genome size estimated at ~620 Mb, the cowpea genome is an ideal target for reduced representation sequencing.ResultsWe report here the sequencing and analysis of the gene-rich, hypomethylated portion of the cowpea genome selectively cloned by methylation filtration (MF) technology. Over 250,000 gene-space sequence reads (GSRs) with an average length of 610 bp were generated, yielding ~160 Mb of sequence information. The GSRs were assembled, annotated by BLAST homology searches of four public protein annotation databases and four plant proteomes (A. thaliana, M. truncatula, O. sativa, and P. trichocarpa), and analyzed using various domain and gene modeling tools. A total of 41,260 GSR assemblies and singletons were annotated, of which 19,786 have unique GenBank accession numbers. Within the GSR dataset, 29% of the sequences were annotated using the Arabidopsis Gene Ontology (GO) with the largest categories of assigned function being catalytic activity and metabolic processes, groups that include the majority of cellular enzymes and components of amino acid, carbohydrate and lipid metabolism. A total of 5,888 GSRs had homology to genes encoding transcription factors (TFs) and transcription associated factors (TAFs) representing about 5% of the total annotated sequences in the dataset. Sixty-two (62) of the 64 well-characterized plant transcription factor (TF) gene families are represented in the cowpea GSRs, and these families are of similar size and phylogenetic organization to those characterized in other plants. The cowpea GSRs also provides a rich source of genes involved in photoperiodic control, symbiosis, and defense-related responses. Comparisons to available databases revealed that about 74% of cowpea ESTs and 70% of all legume ESTs were represented in the GSR dataset. As approximately 12% of all GSRs contain an identifiable simple-sequence repeat, the dataset is a powerful resource for the design of microsatellite markers.ConclusionThe availability of extensive publicly available genomic data for cowpea, a non-model legume with significant importance in the developing world, represents a significant step forward in legume research. Not only does the gene space sequence enable the detailed analysis of gene structure, gene family organization and phylogenetic relationships within cowpea, but it also facilitates the characterization of syntenic relationships with other cultivated and model legumes, and will contribute to determining patterns of chromosomal evolution in the Leguminosae. The micro and macrosyntenic relationships detected between cowpea and other cultivated and model legumes should simplify the identification of informative markers for marker-assisted trait selection and map-based gene isolation necessary for cowpea improvement.

Transgenic expression of two marker genes under the control of an Arabidopsis rbcS promoter: sequences encoding the Rubisco transit peptide increase expression levels

SummaryChimeric gene constructs were made in which two reporter genes, the neo and bar genes, encoding neomycin phosphotransferase II and phosphinothricin acetyl transferase, respectively, were placed under the control of the promoter of ats1A, one of four genes encoding the ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit (SSU) in Arabidopsis thaliana. In one set of constructs the fusions were made at the initiation codons, while in the second set the sequences encoding the ats1 A transit peptide were included. Significantly higher steady-state levels of RNA and protein were observed in leaves of transgenic plants varrying the latter constructions. Individual transgenic plants varied in their degree of tissue specific expression of the chimeric genes as well as in absolute levels of expression. Preliminary results suggest that the ats1 A promoter may be only weakly responsive to phytochrome.

Leaf Developmental Age Controls Expression of Genes Encoding Enzymes of Chlorophyll and Heme Biosynthesis in Pea (Pisum sativum L.)

The effects of leaf developmental age on the expression of three nuclear gene families in pea (Pisum sativum L.) coding for enzymes of chlorophyll and heme biosynthesis have been examined. The steady-state levels of mRNAs encoding aminolevulinic acid (ALA) dehydratase, porphobilinogen (PBG) deaminase, and NADPH:protochlorophyllide reductase were measured by RNA gel blot and quantitative slot-blot analyses in the foliar leaves of embryos that had imbibed for 12 to 18 h and leaves of developing seedlings grown either in total darkness or under continuous white light for up to 14 d after imbibition. Both ALA dehydratase and PBG deaminase mRNAs were detectable in embryonic leaves, whereas mRNA encoding the NADPH:protochlorophyllide reductase was not observed at this early developmental stage. All three gene products were found to increase to approximately the same extent in the primary leaves of pea seedlings during the first 6 to 8 d after imbibition (postgermination) regardless of whether the plants were grown in darkness or under continuous white-light illumination. In the leaves of dark-grown seedlings, the highest levels of message accumulation were observed at approximately 8 to 10 d postgermination, and, thereafter, a steady decline in mRNA levels was observed. In the leaves of light-grown seedlings, steady-state levels of mRNA encoding the three chlorophyll biosynthetic enzymes were inversely correlated with leaf age, with youngest, rapidly expanding leaves containing the highest message levels. A corresponding increase in the three enzyme protein levels was also found during the early stages of development in the light or darkness; however, maximal accumulation of protein was delayed relative to peak levels of mRNA accumulation. We also found that although protochlorophyllide was detectable in the leaves immediately after imbibition, the time course of accumulation of the phototransformable form of the molecule coincided with NADPH:protochlorophyllide reductase expression. In studies in which dark-grown seedlings of various ages were subsequently transferred to light for 24 and 48 h, the effect of light on changes in steady-state mRNA levels was found to be more pronounced at later developmental stages. These results suggest that the expression of these three genes and likely those genes encoding other chlorophyll biosynthetic pathway enzymes are under the control of a common regulatory mechanism. Furthermore, it appears that not light, but rather as yet unidentified endogenous factors, are the primary regulatory factors controlling gene expression early in leaf development.

TOBFAC: the database of tobacco transcription factors

BackgroundRegulation of gene expression at the level of transcription is a major control point in many biological processes. Transcription factors (TFs) can activate and/or repress the transcriptional rate of target genes and vascular plant genomes devote approximately 7% of their coding capacity to TFs. Global analysis of TFs has only been performed for three complete higher plant genomes – Arabidopsis (Arabidopsis thaliana), poplar (Populus trichocarpa) and rice (Oryza sativa). Presently, no large-scale analysis of TFs has been made from a member of the Solanaceae, one of the most important families of vascular plants. To fill this void, we have analysed tobacco (Nicotiana tabacum) TFs using a dataset of 1,159,022 gene-space sequence reads (GSRs) obtained by methylation filtering of the tobacco genome. An analytical pipeline was developed to isolate TF sequences from the GSR data set. This involved multiple (typically 10–15) independent searches with different versions of the TF family-defining domain(s) (normally the DNA-binding domain) followed by assembly into contigs and verification. Our analysis revealed that tobacco contains a minimum of 2,513 TFs representing all of the 64 well-characterised plant TF families. The number of TFs in tobacco is higher than previously reported for Arabidopsis and rice.ResultsTOBFAC: the database of tobacco transcription factors, is an integrative database that provides a portal to sequence and phylogeny data for the identified TFs, together with a large quantity of other data concerning TFs in tobacco. The database contains an individual page dedicated to each of the 64 TF families. These contain background information, domain architecture via Pfam links, a list of all sequences and an assessment of the minimum number of TFs in this family in tobacco. Downloadable phylogenetic trees of the major families are provided along with detailed information on the bioinformatic pipeline that was used to find all family members. TOBFAC also contains EST data, a list of published tobacco TFs and a list of papers concerning tobacco TFs. The sequences and annotation data are stored in relational tables using a PostgrelSQL relational database management system. The data processing and analysis pipelines used the Perl programming language. The web interface was implemented in JavaScript and Perl CGI running on an Apache web server. The computationally intensive data processing and analysis pipelines were run on an Apple XServe cluster with more than 20 nodes.ConclusionTOBFAC is an expandable knowledgebase of tobacco TFs with data currently available for over 2,513 TFs from 64 gene families. TOBFAC integrates available sequence information, phylogenetic analysis, and EST data with published reports on tobacco TF function. The database provides a major resource for the study of gene expression in tobacco and the Solanaceae and helps to fill a current gap in studies of TF families across the plant kingdom. TOBFAC is publicly accessible at http://compsysbio.achs.virginia.edu/tobfac/.

Cowpea, a Multifunctional Legume

Cowpea [Vigna unguiculata (L.) Walp.] is an important warm-season legume grown primarily in the semi-arid tropics. The majority of cowpea is grown by subsistence farmers in west and central sub-Saharan Africa, where its grain and stover are highly valued for food and forage. Despite its economic and social importance in developing parts of the world, cowpea has received relatively little attention from a research standpoint. To a large extent it is an underexploited crop where relatively large genetic gains can likely be made with only modest investments in both applied plant breeding and molecular genetics. A major goal of many cowpea breeding and improvement programs is combining resistance to numerous pests and diseases and other desirable traits, such as those governing maturity, photoperiod sensitivity, plant type, and seed quality. New opportunities for improving cowpea exist by leveraging the emerging genomic tools and knowledge gained through research on other major legume crops and model species. The use of marker-assisted selection and other molecular breeding systems for tracking single gene traits and quantitatively inherited characteristics will likely increase the overall efficiency and effectiveness of cowpea improvement programs in the foreseeable future and provide new opportunities for development of cowpea as a food staple and economic resource.

Identification of AFLP markers linked to resistance of cowpea (Vigna unguiculata L.) to parasitism by Striga gesnerioides

Abstract AFLP and bulked segregant analysis were used to identify molecular markers linked to resistance of cowpea [Vigna ungiculata (L.) Walp.] to parasitism by Striga gesnerioides (Willd.) Vatke. Segregation analysis of F2 progeny from a cross of Tvx3236, a Striga-susceptible line, with IT82D-849, a resistant cultivar, showed that resistance to S. gesnerioides race 1 from Burkina Faso was controlled by a single dominant gene, designated Rsg2–1. Three AFLP markers were identified that are tightly linked to Rsg2–1: E-AAC/M-CAA300 (2.6 cM), E-ACT/M-CAA524 (0.9 cM), and E-ACA/M-CAT140/150 (0.9 cM), which appears to be codominant. Segregation analysis of a different F2 population resulting from a cross of the Striga-susceptible line IT84S-2246–4 with Tvu 14676, a S. gesnerioides race 3 resistant line, showed that resistance to S. gesnerioides race 3 was also controlled by a single dominant gene, designated Rsg4–3. Six AFLP markers linked to Rsg4–3 were identified: E-ACA/M-CAG120 (10.1 cM), E-AGC/M-CAT80 (4.1 cM), E-ACA/M-CAT150 (2.7 cM), E-AGC/M-CAT150 (3.6 cM), E-AAC/M-CAA300 (3.6 cM), and E-AGC/M-CAT70 (5.1 cM). Segregation analysis of the E-AAC/M-CAA300 and E-ACA/M-CAG120 markers in recombinant inbred lines derived from IT84S-2049×524B determined that both are located within linkage group 1 of the cowpea genetic map. The identification of AFLP markers linked to Striga resistance provides a stepping stone for a marker-assisted selection program and the eventual cloning and characterization of the gene(s) encoding resistance to this noxious parasitic weed.