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BackgroundThe DNA sequences encoding ribosomal RNA genes (rRNAs) are commonly used as markers to identify species, including in metagenomics samples that may combine many organismal communities. The 16S small subunit ribosomal RNA (SSU rRNA) gene is typically used to identify bacterial and archaeal species. The nuclear 18S SSU rRNA gene, and 28S large subunit (LSU) rRNA gene have been used as DNA barcodes and for phylogenetic studies in different eukaryote taxonomic groups. Because of their popularity, the National Center for Biotechnology Information (NCBI) receives a disproportionate number of rRNA sequence submissions and BLAST queries. These sequences vary in quality, length, origin (nuclear, mitochondria, plastid), and organism source and can represent any region of the ribosomal cistron.ResultsTo improve the timely verification of quality, origin and loci boundaries, we developed Ribovore, a software package for sequence analysis of rRNA sequences. The ribotyper and ribosensor programs are used to validate incoming sequences of bacterial and archaeal SSU rRNA. The ribodbmaker program is used to create high-quality datasets of rRNAs from different taxonomic groups. Key algorithmic steps include comparing candidate sequences against rRNA sequence profile hidden Markov models (HMMs) and covariance models of rRNA sequence and secondary-structure conservation, as well as other tests. Nine freely available blastn rRNA databases created and maintained with Ribovore are used for checking incoming GenBank submissions and used by the blastn browser interface at NCBI. Since 2018, Ribovore has been used to analyze more than 50 million prokaryotic SSU rRNA sequences submitted to GenBank, and to select at least 10,435 fungal rRNA RefSeq records from type material of 8,350 taxa.ConclusionRibovore combines single-sequence and profile-based methods to improve GenBank processing and analysis of rRNA sequences. It is a standalone, portable, and extensible software package for the alignment, classification and validation of rRNA sequences. Researchers planning on submitting SSU rRNA sequences to GenBank are encouraged to download and use Ribovore to analyze their sequences prior to submission to determine which sequences are likely to be automatically accepted into GenBank.

Petunia (Petunia x hybrida cv 'Mitchell Diploid' [MD]) flowers emit high levels of multiple floral volatile benzenoid/phenylpropanoid (FVBP) compounds from anthesis to senescence in a concerted manner. Here we show seven genes responsible for the production of emitted FVBPs share similar transcript accumulation profiles through an analysis of four expression criteria. As a group, the FVBP gene transcripts accumulate to high levels in petal limb tissue of MD flowers from anthesis to senescence. Two to four hours of exogenous ethylene exposure reduces transcript levels of all FVBP genes examined, but 2h of treatment will not accelerate senescence or reduce volatile emissions in MD flowers. The FVBP genes show two obvious rhythmic patterns of transcript accumulation; however, corresponding enzyme activities of a subset of FVBP gene products do not. Together, these results depict floral volatile benzenoid/phenylpropanoid biosynthesis as a specific system with multiple regulatory features. One such feature is the highly regulated transcript accumulation of the FVBP genes. Additionally, ethylene may have a regulatory role in the FVBP system prior to a floral senescence program.

Ethylene is a gaseous plant hormone involved in many physiological processes including senescence, fruit ripening, and defence. Here the effects of pollination and wound-induced ethylene signals on transcript accumulation of benzoyl CoA:benzyl alcohol/phenylethanol benzoyltransferase (PhBPBT) are shown in Petuniaxhybrida cv. Mitchell 'Diploid' (MD). In petunia, PhBPBT is responsible for the biosynthesis of both benzyl benzoate and phenylethyl benzoate from benzyl alcohol and phenylethanol, respectively. RNAi-silenced lines, with reduced PhBPBT transcript, displayed reduced benzyl benzoate emission, and increased benzyl alcohol levels. Detailed expression analysis showed that PhBPBT is regulated by both light and an endogenous circadian rhythm, while it is also differentially regulated in response to ethylene in a tissue-specific manner. Twenty-four hours following pollination of MD flowers, expression of PhBPBT decreases in the corolla, while it increases in the ovary after 48 h. This is caused by ethylene that is emitted from the flower coinciding with fertilization as this is not observed in transgenic ethylene-insensitive plants (CaMV35S::etr1-1; 44568). Ethylene is also emitted from vegetative tissue of petunia following mechanical wounding, resulting in an increase in PhBPBT expression in the leaves where expression is normally below detection levels. Indicative of this pattern of expression, we hypothesize that PhBPBT and subsequent benzyl benzoate production is involved in defence-related processes in the corolla prior to pollination, in the ovary immediately following fertilization, and in vegetative tissue in response to wounding.

Multicellular organisms produce small cysteine-rich antimicrobial peptides as an innate defense against pathogens. While defensins, a well-known class of such peptides, are common among eukaryotes, there are other classes restricted to the plant kingdom. These include thionins, lipid transfer proteins and snakins. In earlier work, we identified several divergent classes of small putatively secreted cysteine-rich peptides (CRPs) in legumes [Graham et al. (2004)Plant Physiol. 135, 1179-97]. Here, we built sequence motif models for each of these classes of peptides, and iteratively searched for related sequences within the comprehensive UniProt protein dataset, the Institute for Genomic Research's 33 plant gene indices, and the entire genomes of the model dicot, Arabidopsis thaliana, and the model monocot and crop species, Oryza sativa (rice). Using this search strategy, we identified approximately 13,000 plant genes encoding peptides with common features: (i) an N-terminal signal peptide, (ii) a small divergent charged or polar mature peptide with conserved cysteines, (iii) a similar intron/exon structure, (iv) spatial clustering in the genomes studied, and (v) overrepresentation in expressed sequences from reproductive structures of specific taxa. The identified genes include classes of defensins, thionins, lipid transfer proteins, and snakins, plus other protease inhibitors, pollen allergens, and uncharacterized gene families. We estimate that these classes of genes account for approximately 2-3% of the gene repertoire of each model species. Although 24% of the genes identified were not annotated in the latest Arabidopsis genome releases (TIGR5, TAIR6), we confirmed expression via RT-PCR for 59% of the sequences attempted. These findings highlight limitations in current annotation procedures for small divergent peptide classes.

In many flowering plants, such as petunia (Petunia × hybrida), ethylene produced in floral organs after pollination elicits a series of physiological and biochemical events, ultimately leading to senescence of petals and successful fertilization. Here, we demonstrate, using transgenic ethylene insensitive (44568) and Mitchell Diploid petunias, that multiple components of emission of volatile organic compounds (VOCs) are regulated by ethylene. Expression of benzoic acid/salicylic acid carboxyl methyltransferase (PhBSMT1 and 2) mRNA is temporally and spatially down-regulated in floral organs in a manner consistent with current models for postpollination ethylene synthesis in petunia corollas. Emission of methylbenzoate and other VOCs after pollination and exogenous ethylene treatment parallels a reduction in PhBSMT1 and 2 mRNA levels. Under cyclic light conditions (day/night), PhBSMT mRNA levels are rhythmic and precede emission of methylbenzoate by approximately 6 h. When shifted into constant dark or light conditions, PhBSMT mRNA levels and subsequent methylbenzoate emission correspondingly decrease or increase to minimum or maximum levels observed during normal conditions, thus suggesting that light may be a more critical influence on cyclic emission of methylbenzoate than a circadian clock. Transgenic PhBSMT RNAi flowers with reduced PhBSMT mRNA levels show a 75% to 99% decrease in methylbenzoate emission, with minimal changes in other petunia VOCs. These results implicate PhBSMT1 and 2 as genes responsible for synthesis of methylbenzoate in petunia.

The plant hormone ethylene regulates many aspects of growth and development. Loss-of-function mutations in ETHYLENE INSENSITIVE2 (EIN2) result in ethylene insensitivity in Arabidopsis, indicating an essential role of EIN2 in ethylene signaling. However, little is known about the role of EIN2 in species other than Arabidopsis. To gain a better understanding of EIN2, a petunia (Petunia × hybrida cv Mitchell Diploid [MD]) homolog of the Arabidopsis EIN2 gene (PhEIN2) was isolated, and the role of PhEIN2 was analyzed in a wide range of plant responses to ethylene, many that do not occur in Arabidopsis. PhEIN2 mRNA was present at varying levels in tissues examined, and the PhEIN2 expression decreased after ethylene treatment in petals. These results indicate that expression of PhEIN2 mRNA is spatially and temporally regulated in petunia during plant development. Transgenic petunia plants with reduced PhEIN2 expression were compared to wild-type MD and ethylene-insensitive petunia plants expressing the Arabidopsis etr1-1 gene for several physiological processes. Both PhEIN2 and etr1-1 transgenic plants exhibited significant delays in flower senescence and fruit ripening, inhibited adventitious root and seedling root hair formation, premature death, and increased hypocotyl length in seedling ethylene response assays compared to MD. Moderate or strong levels of reduction in ethylene sensitivity were achieved with expression of both etr1-1 and PhEIN2 transgenes, as measured by downstream expression of PhEIL1. These results demonstrate that PhEIN2 mediates ethylene signals in a wide range of physiological processes and also indicate the central role of EIN2 in ethylene signal transduction.

The molecular mechanisms responsible for postpollination changes in floral scent emission were investigated in snapdragon cv Maryland True Pink and petunia cv Mitchell flowers using a volatile ester, methylbenzoate, one of the major scent compounds emitted by these flowers, as an example. In both species, a 70 to 75% pollination-induced decrease in methylbenzoate emission begins only after pollen tubes reach the ovary, a process that takes between 35 and 40 h in snapdragon and approximately 32 h in petunia. This postpollination decrease in emission is not triggered by pollen deposition on the stigma. Petunia and snapdragon both synthesize methylbenzoate from benzoic acid and S-adenosyl-l-methionine (SAM); however, they use different mechanisms to downregulate its production after pollination. In petunia, expression of the gene responsible for methylbenzoate synthesis is suppressed by ethylene. In snapdragon, the decrease in methylbenzoate emission is the result of a decrease in both S-adenosyl-l-methionine:benzoic acid carboxyl methyltransferase (BAMT) activity and the ratio of SAM to S-adenosyl-l-homocysteine ("methylation index") after pollination, although the BAMT gene also is sensitive to ethylene.

Background Although the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in Arabidopsis genome cannot be detected by microarray experiments. In addition, there are many more novel genes that have been discovered by experiments or predicted by new gene prediction programs. Another way to understand the function of individual genes is to investigate their in vivo expression patterns by reporter constructs in transgenic plants which can provide basic information on the patterns of gene expression. Results A high throughput pipeline was developed to generate promoter-reporter (GFP) transgenic lines for Arabidopsis genes expressed at very low levels and to examine their expression patterns in vivo. The promoter region from a total of 627 non- or low-expressed genes in Arabidopsis based on Arabidopsis annotation release 5 were amplified and cloned into a Gateway vector. A total of 353 promoter-reporter (GFP) constructs were successfully transferred into Agrobacterium (GV3101) by triparental mating and subsequently used for Arabidopsis transformation. Kanamycin-resistant transgenic lines were obtained from 266 constructs and among them positive GFP expression was detected from 150 constructs. Of these 150 constructs, multiple transgenic lines exhibiting consistent expression patterns were obtained for 112 constructs. A total 81 different regions of expression were discovered during our screening of positive transgenic plants and assigned Plant Ontology (PO) codes. Conclusions Many of the genes tested for which expression data were lacking previously are indeed expressed in Arabidopsis during the developmental stages screened. More importantly, our study provides plant researchers with another resource of gene expression information in Arabidopsis. The results of this study are captured in a MySQL database and can be searched at http://www.jcvi.org/arabidopsis/qpcr/index.shtml. Transgenic seeds and constructs are also available for the research community.

Biotechnology has the potential to modify commercially important traits of crops, such as fruit size and stress tolerance. To date, the floricultural industry has not profited significantly from these possibilities to manipulate, for example, flower size. Cytokinins are known to be involved in many aspects of plant development, including cell division. Increasing the amount of cytokinins has the potential to increase the size of an organ, such as the flower or the fruit. The Agrobacterium tumefaciens cytokinin biosynthesis gene isopentenyltransferase (ipt) has been shown to increase cytokinin levels when introduced into plants. Moreover, it has a dramatic effect on the vegetative development of plants. The expression of the ipt gene under the control of the flower-specific Arabidopsis APETALA3 promoter in petunia (Petunia hybrida) increases the flower size dramatically, but with no effect on vegetative development. The resulting transgenic plants produced flowers with larger corolla diameter and greater total floral fresh weight. This strategy has the potential for use in the production of ornamental crops with large flowers and crop species with larger fruit.

Multicellular organisms produce small cysteine-rich antimicrobial peptides as an innate defense against pathogens. While defensins, a well-known class of such peptides, are common among eukaryotes, there are other classes restricted to the plant kingdom. These include thionins, lipid transfer proteins and snakins. In earlier work, we identified several divergent classes of small putatively secreted cysteine-rich peptides (CRPs) in legumes [Graham et al. (2004)Plant Physiol. 135, 1179-97]. Here, we built sequence motif models for each of these classes of peptides, and iteratively searched for related sequences within the comprehensive UniProt protein dataset, the Institute for Genomic Research's 33 plant gene indices, and the entire genomes of the model dicot, Arabidopsis thaliana, and the model monocot and crop species, Oryza sativa (rice). Using this search strategy, we identified approximately 13,000 plant genes encoding peptides with common features: (i) an N-terminal signal peptide, (ii) a small divergent charged or polar mature peptide with conserved cysteines, (iii) a similar intron/exon structure, (iv) spatial clustering in the genomes studied, and (v) overrepresentation in expressed sequences from reproductive structures of specific taxa. The identified genes include classes of defensins, thionins, lipid transfer proteins, and snakins, plus other protease inhibitors, pollen allergens, and uncharacterized gene families. We estimate that these classes of genes account for approximately 2-3% of the gene repertoire of each model species. Although 24% of the genes identified were not annotated in the latest Arabidopsis genome releases (TIGR5, TAIR6), we confirmed expression via RT-PCR for 59% of the sequences attempted. These findings highlight limitations in current annotation procedures for small divergent peptide classes.

Summary Comprehensive open reading frame (ORF) clone collections, ORFeomes, are key components of functional genomics projects. When recombinational cloning systems are used to capture ORFs in master clones, these DNA sequences can be easily transferred into a variety of expression plasmids, each designed for a specific assay. Depending on downstream applications, an ORF is cloned either with or without a stop codon at its original position, referred to as closed or open configuration, respectively. The former is preferred when the encoded protein is produced in its native form or with an amino-terminal tag; the latter is obligatory when the protein is produced as a fusion with a carboxyl-terminal tag. We developed a streamlined protocol for high-throughput, simultaneous cloning of both open and closed ORF entry clones with the Gateway™ recombinational cloning system. The protocol is straightforward to set up in large-scale ORF cloning projects, and is cost-effective, because the initial ORF amplification and the cloning in a pDONR vector are performed only once to obtain the two ORF configurations. We illustrated its implementation for the isolation and validation of 346 Arabidopsis ORF entry clones.

BackgroundSeveral lines of evidence support the existence of novel genes and other transcribed units which have not yet been annotated in the Arabidopsis genome. Two gene prediction programs which make use of comparative genomic analysis, Twinscan and EuGene, have recently been deployed on the Arabidopsis genome. The ability of these programs to make use of sequence data from other species has allowed both Twinscan and EuGene to predict over 1000 genes that are intergenic with respect to the most recent annotation release. A high throughput RACE pipeline was utilized in an attempt to verify the structure and expression of these novel genes.Results1,071 un-annotated loci were targeted by RACE, and full length sequence coverage was obtained for 35% of the targeted genes. We have verified the structure and expression of 378 genes that were not present within the most recent release of the Arabidopsis genome annotation. These 378 genes represent a structurally diverse set of transcripts and encode a functionally diverse set of proteins.ConclusionWe have investigated the accuracy of the Twinscan and EuGene gene prediction programs and found them to be reliable predictors of gene structure in Arabidopsis. Several hundred previously un-annotated genes were validated by this work. Based upon this information derived from these efforts it is likely that the Arabidopsis genome annotation continues to overlook several hundred protein coding genes.

The floriculture crop industry is technically diverse and is characterized by the use of hundreds of different plant species. Due to the large amount of genetic diversity used by this industry, there are often very complex issues that arise during crop production and during postharvest handling throughout the wholesale and retail markets. Crop production practices are often very complex, and may be complicated by the demands for precise crop timing, and the different cultural requirements of different cultivars. After the crops is produced, optimal conditions for postharvest shipping and handling are difficult to maintain, and this can lead to subsequent poor quality once the crop has arrived at the retail market. In addition to the demands of complex production and marketing systems, the demand from consumers for a constant supply of new and interesting flowering plants with unique characteristics continues to increase. Needs for product quality and availability and subsequent garden performance have driven the availability of new flowering crops to unprecedented levels over the last 5–10 years. With such a vast array of issues in floriculture crops, there is a great deal of potential for using some of these crops as model systems to study the potential for use of transgenic plants with improved horticultural characteristics. In many cases, biotechnology applications are proving to be very difficult, but there have been several advances made with engineering a wide variety of genetic traits in floriculture crops. There have also been significant gains made in cloning important genes that are proving to be involved with biological processes that scientists hope to manipulate in ornamental crops in the future.