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4.  Pscroph, a parasitic plant EST database enriched for parasite associated transcripts 
BMC Plant Biology  2005;5:24.
Parasitic plants in the Orobanchaceae develop invasive root haustoria upon contact with host roots or root factors. The development of haustoria can be visually monitored and is rapid, highly synchronous, and strongly dependent on host factor exposure; therefore it provides a tractable system for studying chemical communications between roots of different plants.
Triphysaria is a facultative parasitic plant that initiates haustorium development within minutes after contact with host plant roots, root exudates, or purified haustorium-inducing phenolics. In order to identify genes associated with host root identification and early haustorium development, we sequenced suppression subtractive libraries (SSH) enriched for transcripts regulated in Triphysaria roots within five hours of exposure to Arabidopsis roots or the purified haustorium-inducing factor 2,6 dimethoxybenzoquinone. The sequences of over nine thousand ESTs from three SSH libraries and their subsequent assemblies are available at the Pscroph database . The web site also provides BLAST functions and allows keyword searches of functional annotations.
Libraries prepared from Triphysaria roots treated with host roots or haustorium inducing factors were enriched for transcripts predicted to function in stress responses, electron transport or protein metabolism. In addition to parasitic plant investigations, the Pscroph database provides a useful resource for investigations in rhizosphere interactions, chemical signaling between organisms, and plant development and evolution.
PMCID: PMC1325228  PMID: 16288663
5.  A fully automatable enzymatic method for DNA extraction from plant tissues 
BMC Plant Biology  2005;5:23.
DNA extraction from plant tissues, unlike DNA isolation from mammalian tissues, remains difficult due to the presence of a rigid cell wall around the plant cells. Currently used methods inevitably require a laborious mechanical grinding step, necessary to disrupt the cell wall for the release of DNA.
Using a cocktail of different carbohydrases, a method was developed that enables a complete digestion of the plant cell walls and subsequent DNA release. Optimized conditions for the digestion reaction minimize DNA shearing and digestion, and maximize DNA release from the plant cell. The method gave good results in 125 of the 156 tested species.
In combination with conventional DNA isolation techniques, the new enzymatic method allows to obtain high-yield, high-molecular weight DNA, which can be used for many applications, including genome characterization by AFLP, RAPD and SSR. Automation of the protocol (from leaf disks to DNA) is possible with existing workstations.
PMCID: PMC1298311  PMID: 16269076
6.  Callose (β-1,3 glucan) is essential for Arabidopsis pollen wall patterning, but not tube growth 
BMC Plant Biology  2005;5:22.
Callose (β-1,3 glucan) separates developing pollen grains, preventing their underlying walls (exine) from fusing. The pollen tubes that transport sperm to female gametes also contain callose, both in their walls as well as in the plugs that segment growing tubes. Mutations in CalS5, one of several Arabidopsis β-1,3 glucan synthases, were previously shown to disrupt callose formation around developing microspores, causing aberrations in exine patterning, degeneration of developing microspores, and pollen sterility.
Here, we describe three additional cals5 alleles that similarly alter exine patterns, but instead produce fertile pollen. Moreover, one of these alleles (cals5-3) resulted in the formation of pollen tubes that lacked callose walls and plugs. In self-pollinated plants, these tubes led to successful fertilization, but they were at a slight disadvantage when competing with wild type.
Contrary to a previous report, these results demonstrate that a structured exine layer is not required for pollen development, viability or fertility. In addition, despite the presence of callose-enriched walls and callose plugs in pollen tubes, the results presented here indicate that callose is not required for pollen tube functions.
PMCID: PMC1274334  PMID: 16212660
7.  PineappleDB: An online pineapple bioinformatics resource 
BMC Plant Biology  2005;5:21.
A world first pineapple EST sequencing program has been undertaken to investigate genes expressed during non-climacteric fruit ripening and the nematode-plant interaction during root infection. Very little is known of how non-climacteric fruit ripening is controlled or of the molecular basis of the nematode-plant interaction. PineappleDB was developed to provide the research community with access to a curated bioinformatics resource housing the fruit, root and nematode infected gall expressed sequences.
PineappleDB is an online, curated database providing integrated access to annotated expressed sequence tag (EST) data for cDNA clones isolated from pineapple fruit, root, and nematode infected root gall vascular cylinder tissues. The database currently houses over 5600 EST sequences, 3383 contig consensus sequences, and associated bioinformatic data including splice variants, Arabidopsis homologues, both MIPS based and Gene Ontology functional classifications, and clone distributions. The online resource can be searched by text or by BLAST sequence homology. The data outputs provide comprehensive sequence, bioinformatic and functional classification information.
The online pineapple bioinformatic resource provides the research community with access to pineapple fruit and root/gall sequence and bioinformatic data in a user-friendly format. The search tools enable efficient data mining and present a wide spectrum of bioinformatic and functional classification information. PineappleDB will be of broad appeal to researchers investigating pineapple genetics, non-climacteric fruit ripening, root-knot nematode infection, crassulacean acid metabolism and alternative RNA splicing in plants.
PMCID: PMC1260026  PMID: 16202174
8.  Genetic chimerism of Vitis vinifera cv. Chardonnay 96 is maintained through organogenesis but not somatic embryogenesis 
BMC Plant Biology  2005;5:20.
Grapevine can be a periclinal chimera plant which is composed at least of two distinct cell layers (L1, L2). When the cell layers of this plant are separated by passage through somatic embryogenesis, regenerated plants could show distinct DNA profiles and a novel phenotype which proved different from that of the parent plant.
Genetically Chardonnay clone 96 is a periclinal chimera plant in which is L1 and L2 cell layers are distinct. Plants obtained via organogenesis through meristematic bulks are shown to be composed of both cell layers. However, plants regenerated through somatic embryogenesis starting from anthers or nodal explants are composed only of L1 cells. These somaclones do not show phenotypic differences to the parental clone up to three years after regeneration. Interestingly, the only somaclone showing an atypical phenotype (asymmetric leave) shows a genotypic modification.
These results suggest that the phenotype of Chardonnay 96 does not result from an interaction between the two distinct cell layers L1 and L2. If phenotype conformity is further confirmed, somatic embryogenesis will result in true-to-type somaclones of Chardonnay 96 and would be well suitable for gene transfer.
PMCID: PMC1285365  PMID: 16194273
9.  Purification and kinetic studies of recombinant gibberellin dioxygenases 
BMC Plant Biology  2005;5:19.
The 2-oxoglutarate-dependent dioxygenases (2ODDs) of gibberellin (GA) biosynthesis have a key role in the metabolism of a major plant hormone. The activity of recombinant GA 2ODDs from many species has been characterised in detail, however little information relates to enzyme purification. Native GA 2ODDs displayed lability during purification.
Two GA 2ODDs were expressed in Escherichia coli and purified to homogeneity. The GA 2-oxidase from Pisum sativum L., PsGA2OX1, was expressed as a glutathione s-transferase (GST) fusion. It was purified in the three steps of affinity chromatography, GST removal and gel filtration. Highly pure PsGA2OX1 was obtained at a yield of 0.3 mg/g of cells. It displayed a Km of 0.024 μM and a Vmax of 4.4 pkat/mg toward [1β,2β,3β-3H3]GA20. The GA 3-oxidase from Arabidopsis thaliana, AtGA3OX4, was expressed as a poly(His)-tagged thioredoxin fusion. It was purified by Immobilised Metal Affinity Chromatography followed by gel filtration. Cleavage of the fusion took place between the two purification steps. Highly pure AtGA3OX4 was obtained at a yield of 0.01 mg/g of cells. It displayed a Km of 0.82 μM and Vmax of 52,500 pkat/mg toward [1β,2β,3β-3H3]GA20.
Fusion tags were required to stabilise and solubilise PsGA2OX1 and AtGA3OX4 during E. coli expression. The successful purification of milligram quantities of PsGA2OX1 enables mechanistic and structural studies not previously possible on GA 2ODDs. A moderate yield of pure AtGA3OX4 requires the further optimisation of the latter stages of the enzyme purification schedule. PsGA2OX1's action in planta as deduced from the effect of the null mutation sln on GA levels in seeds is in agreement with the kinetic parameters of the recombinant enzyme.
PMCID: PMC1262730  PMID: 16181493
10.  Generation and analysis of 9792 EST sequences from cold acclimated oat, Avena sativa 
BMC Plant Biology  2005;5:18.
Oat is an important crop in North America and northern Europe. In Scandinavia, yields are limited by the fact that oat cannot be used as a winter crop. In order to develop such a crop, more knowledge about mechanisms of cold tolerance in oat is required.
From an oat cDNA library 9792 single-pass EST sequences were obtained. The library was prepared from pooled RNA samples isolated from leaves of four-week old Avena sativa (oat) plants incubated at +4°C for 4, 8, 16 and 32 hours. Exclusion of sequences shorter than 100 bp resulted in 8508 high-quality ESTs with a mean length of 710.7 bp. Clustering and assembly identified a set of 2800 different transcripts denoted the Avena sativa cold induced UniGene set (AsCIUniGene set). Taking advantage of various tools and databases, putative functions were assigned to 1620 (58%) of these genes. Of the remaining 1180 unclassified sequences, 427 appeared to be oat-specific since they lacked any significant sequence similarity (Blast E values > 10-10) to any sequence available in the public databases. Of the 2800 UniGene sequences, 398 displayed significant homology (BlastX E values ≤ 10-10) to genes previously reported to be involved in cold stress related processes. 107 novel oat transcription factors were also identified, out of which 51 were similar to genes previously shown to be cold induced. The CBF transcription factors have a major role in regulating cold acclimation. Four oat CBF sequences were found, belonging to the monocot cluster of DREB family ERF/AP2 domain proteins. Finally in the total EST sequence data (5.3 Mbp) approximately 400 potential SSRs were found, a frequency similar to what has previously been identified in Arabidopsis ESTs.
The AsCIUniGene set will now be used to fabricate an oat biochip, to perform various expression studies with different oat cultivars incubated at varying temperatures, to generate molecular markers and provide tools for various genetic transformation experiments in oat. This will lead to a better understanding of the cellular biology of this important crop and will open up new ways to improve its agronomical properties.
PMCID: PMC1236939  PMID: 16137325
11.  Design and fabrication of adjustable red-green-blue LED light arrays for plant research 
BMC Plant Biology  2005;5:17.
Although specific light attributes, such as color and fluence rate, influence plant growth and development, researchers generally cannot control the fine spectral conditions of artificial plant-growth environments. Plant growth chambers are typically outfitted with fluorescent and/or incandescent fixtures that provide a general spectrum that is accommodating to the human eye and not necessarily supportive to plant development. Many studies over the last several decades, primarily in Arabidopsis thaliana, have clearly shown that variation in light quantity, quality and photoperiod can be manipulated to affect growth and control developmental transitions. Light emitting diodes (LEDs) has been used for decades to test plant responses to narrow-bandwidth light. LEDs are particularly well suited for plant growth chambers, as they have an extraordinary life (about 100,000 hours), require little maintenance, and use negligible energy. These factors render LED-based light strategies particularly appropriate for space-biology as well as terrestrial applications. However, there is a need for a versatile and inexpensive LED array platform where individual wavebands can be specifically tuned to produce a series of light combinations consisting of various quantities and qualities of individual wavelengths. Two plans are presented in this report.
In this technical report we describe the practical construction of tunable red-green-blue LED arrays to support research in plant growth and development. Two light fixture designs and corresponding circuitry are presented. The first is well suited for a laboratory environment for use in a finite area with small plants, such as Arabidopsis. The second is expandable and appropriate for growth chambers. The application of these arrays to early plant developmental studies has been validated with assays of hypocotyl growth inhibition/promotion and phototropic curvature in Arabidopsis seedlings.
The presentation of these proven plans for LED array construction allows the teacher, researcher or electronics aficionado a means to inexpensively build efficient, adjustable lighting modules for plant research. These simple and effective designs permit the construction of useful tools by programs short on electronics expertise. These arrays represent a means to modulate precise quality and quantity in experimental settings to test the effect of specific light combinations in regulating plant growth, development and plant-product yield.
PMCID: PMC1198233  PMID: 16117835
12.  Development of ESTs from chickpea roots and their use in diversity analysis of the Cicer genus 
BMC Plant Biology  2005;5:16.
Chickpea is a major crop in many drier regions of the world where it is an important protein-rich food and an increasingly valuable traded commodity. The wild annual Cicer species are known to possess unique sources of resistance to pests and diseases, and tolerance to environmental stresses. However, there has been limited utilization of these wild species by chickpea breeding programs due to interspecific crossing barriers and deleterious linkage drag. Molecular genetic diversity analysis may help predict which accessions are most likely to produce fertile progeny when crossed with chickpea cultivars. While, trait-markers may provide an effective tool for breaking linkage drag. Although SSR markers are the assay of choice for marker-assisted selection of specific traits in conventional breeding populations, they may not provide reliable estimates of interspecific diversity, and may lose selective power in backcross programs based on interspecific introgressions. Thus, we have pursued the development of gene-based markers to resolve these problems and to provide candidate gene markers for QTL mapping of important agronomic traits.
An EST library was constructed after subtractive suppressive hybridization (SSH) of root tissue from two very closely related chickpea genotypes (Cicer arietinum). A total of 106 EST-based markers were designed from 477 sequences with functional annotations and these were tested on C. arietinum. Forty-four EST markers were polymorphic when screened across nine Cicer species (including the cultigen). Parsimony and PCoA analysis of the resultant EST-marker dataset indicated that most accessions cluster in accordance with the previously defined classification of primary (C. arietinum, C. echinospermum and C. reticulatum), secondary (C. pinnatifidum, C. bijugum and C. judaicum), and tertiary (C. yamashitae, C. chrossanicum and C. cuneatum) gene-pools. A large proportion of EST alleles (45%) were only present in one or two of the accessions tested whilst the others were represented in up to twelve of the accessions tested.
Gene-based markers have proven to be effective tools for diversity analysis in Cicer and EST diversity analysis may be useful in identifying promising candidates for interspecific hybridization programs. The EST markers generated in this study have detected high levels of polymorphism amongst both common and rare alleles. This suggests that they would be useful for allele-mining of germplasm collections for identification of candidate accessions in the search for new sources of resistance to pests / diseases, and tolerance to abiotic stresses.
PMCID: PMC1215491  PMID: 16107212
13.  Highly syntenic regions in the genomes of soybean, Medicago truncatula, and Arabidopsis thaliana 
BMC Plant Biology  2005;5:15.
Recent genome sequencing enables mega-base scale comparisons between related genomes. Comparisons between animals, plants, fungi, and bacteria demonstrate extensive synteny tempered by rearrangements. Within the legume plant family, glimpses of synteny have also been observed. Characterizing syntenic relationships in legumes is important in transferring knowledge from model legumes to crops that are important sources of protein, fixed nitrogen, and health-promoting compounds.
We have uncovered two large soybean regions exhibiting synteny with M. truncatula and with a network of segmentally duplicated regions in Arabidopsis. In all, syntenic regions comprise over 500 predicted genes spanning 3 Mb. Up to 75% of soybean genes are colinear with M. truncatula, including one region in which 33 of 35 soybean predicted genes with database support are colinear to M. truncatula. In some regions, 60% of soybean genes share colinearity with a network of A. thaliana duplications. One region is especially interesting because this 500 kbp segment of soybean is syntenic to two paralogous regions in M. truncatula on different chromosomes. Phylogenetic analysis of individual genes within these regions demonstrates that one is orthologous to the soybean region, with which it also shows substantially denser synteny and significantly lower levels of synonymous nucleotide substitutions. The other M. truncatula region is inferred to be paralogous, presumably resulting from a duplication event preceding speciation.
The presence of well-defined M. truncatula segments showing orthologous and paralogous relationships with soybean allows us to explore the evolution of contiguous genomic regions in the context of ancient genome duplication and speciation events.
PMCID: PMC1201151  PMID: 16102170
14.  Arabidopsis ETO1 specifically interacts with and negatively regulates type 2 1-aminocyclopropane-1-carboxylate synthases 
BMC Plant Biology  2005;5:14.
In Arabidopsis, ETO1 (ETHYLENE-OVERPRODUCER1) is a negative regulator of ethylene evolution by interacting with AtACS5, an isoform of the rate-limiting enzyme, 1-aminocyclopropane-1-carboxylate synthases (ACC synthase or ACS), in ethylene biosynthetic pathway. ETO1 directly inhibits the enzymatic activity of AtACS5. In addition, a specific interaction between ETO1 and AtCUL3, a constituent of a new type of E3 ubiquitin ligase complex, suggests the molecular mechanism in promoting AtACS5 degradation by the proteasome-dependent pathway. Because orthologous sequences to ETO1 are found in many plant species including tomato, we transformed tomato with Arabidopsis ETO1 to evaluate its ability to suppress ethylene production in tomato fruits.
Transgenic tomato lines that overexpress Arabidopsis ETO1 (ETO1-OE) did not show a significant delay of fruit ripening. So, we performed yeast two-hybrid assays to investigate potential heterologous interaction between ETO1 and three isozymes of ACC synthases from tomato. In the yeast two-hybrid system, ETO1 interacts with LE-ACS3 as well as AtACS5 but not with LE-ACS2 or LE-ACS4, two major isozymes whose gene expression is induced markedly in ripening fruits. According to the classification of ACC synthases, which is based on the C-terminal amino acid sequences, both LE-ACS3 and AtACS5 are categorized as type 2 isozymes and possess a consensus C-terminal sequence. In contrast, LE-ACS2 and LE-ACS4 are type 1 and type 3 isozymes, respectively, both of which do not possess this specific C-terminal sequence. Yeast two-hybrid analysis using chimeric constructs between LE-ACS2 and LE-ACS3 revealed that the type-2-ACS-specific C-terminal tail is required for interaction with ETO1. When treated with auxin to induce LE-ACS3, seedlings of ETO1-OE produced less ethylene than the wild type, despite comparable expression of the LE-ACS3 gene in the wild type.
These results suggest that ETO1 family proteins specifically interact with and negatively regulate type 2 ACC synthases. Our data also show that Arabidopsis ETO1 can regulate type 2 ACS in a heterologous plant, tomato.
PMCID: PMC1199607  PMID: 16091151
15.  New insights into the tonoplast architecture of plant vacuoles and vacuolar dynamics during osmotic stress 
BMC Plant Biology  2005;5:13.
The vegetative plant vacuole occupies >90% of the volume in mature plant cells. Vacuoles play fundamental roles in adjusting cellular homeostasis and allowing cell growth. The composition of the vacuole and the regulation of its volume depend on the coordinated activities of the transporters and channels localized in the membrane (named tonoplast) surrounding the vacuole. While the tonoplast protein complexes are well studied, the tonoplast itself is less well described. To extend our knowledge of how the vacuole folds inside the plant cell, we present three-dimensional reconstructions of vacuoles from tobacco suspension cells expressing the tonoplast aquaporin fusion gene BobTIP26-1::gfp.
3-D reconstruction of the cell vacuole made possible an accurate analysis of large spanning folds of the vacuolar membrane under both normal and stressed conditions, and suggested interactions between surrounding plastids. Dynamic, high resolution 3-D pictures of the vacuole in tobacco suspension cells monitored under different growth conditions provide additional details about vacuolar architecture. The GFP-decorated vacuole is a single continuous compartment transected by tubular-like transvacuolar strands and large membrane surfaces. Cell culture under osmotic stress led to a complex vacuolar network with an increased tonoplast surface area. In-depth 3-D realistic inspections showed that the unity of the vacuole is maintained during acclimation to osmotic stress. Vacuolar unity exhibited during stress adaptation, coupled with the intimate associations of vacuoles with other organelles, suggests a physiological role for the vacuole in metabolism, and communication between the vacuole and organelles, respectively, in plant cells. Desiccation stress ensuing from PEG treatment generates "double" membrane structures closely linked to the tonoplast within the vacuole. These membrane structures may serve as membrane reservoirs for membrane reversion when cells are reintroduced to normal growth conditions.
3-D processing of a GFP-labeled tonoplast provides compelling visual constructions of the plant cell vacuole and elaborates on the nature of tonoplast folding and architecture. Furthermore, these methods allow real-time determination of membrane rearrangements during stresses.
PMCID: PMC1190192  PMID: 16080795
16.  Expressed sequence tags (ESTs) and simple sequence repeat (SSR) markers from octoploid strawberry (Fragaria × ananassa) 
BMC Plant Biology  2005;5:12.
Cultivated strawberry (Fragaria × ananassa) represents one of the most valued fruit crops in the United States. Despite its economic importance, the octoploid genome presents a formidable barrier to efficient study of genome structure and molecular mechanisms that underlie agriculturally-relevant traits. Many potentially fruitful research avenues, especially large-scale gene expression surveys and development of molecular genetic markers have been limited by a lack of sequence information in public databases. As a first step to remedy this discrepancy a cDNA library has been developed from salicylate-treated, whole-plant tissues and over 1800 expressed sequence tags (EST's) have been sequenced and analyzed.
A putative unigene set of 1304 sequences – 133 contigs and 1171 singlets – has been developed, and the transcripts have been functionally annotated. Homology searches indicate that 89.5% of sequences share significant similarity to known/putative proteins or Rosaceae ESTs. The ESTs have been functionally characterized and genes relevant to specific physiological processes of economic importance have been identified. A set of tools useful for SSR development and mapping is presented.
Sequences derived from this effort may be used to speed gene discovery efforts in Fragaria and the Rosaceae in general and also open avenues of comparative mapping. This report represents a first step in expanding molecular-genetic analyses in strawberry and demonstrates how computational tools can be used to optimally mine a large body of useful information from a relatively small data set.
PMCID: PMC1182381  PMID: 15985176
17.  Analysis of xylem sap proteins from Brassica napus 
BMC Plant Biology  2005;5:11.
Substance transport in higher land plants is mediated by vascular bundles, consisting of phloem and xylem strands that interconnect all plant organs.
While the phloem mainly allocates photoassimilates, the role of the xylem is the transport of water and inorganic nutrients from roots to all aerial plant parts. Only recently it was noticed that in addition to mineral salts, xylem sap contains organic nutrients and even proteins. Although these proteins might have important impact on the performance of above-ground organs, only a few of them have been identified so far and their physiological functions are still unclear.
We used root-pressure xylem exudate, collected from cut Brassica napus stems, to extract total proteins. These protein preparations were then separated by high-resolution two-dimensional gel electrophoresis (2-DE). After individual tryptic digests of the most abundant coomassie-stained protein spots, partial peptide sequence information was deduced from tandem mass spectrometric (MS/MS) fragmentation spectra and subsequently used for protein identifications by database searches. This approach resulted in the identification of 69 proteins. These identifications include different proteins potentially involved in defence-related reactions and cell wall metabolism.
This study provides a comprehensive overview of the most abundant proteins present in xylem sap of Brassica napus. A number of 69 proteins could be identified from which many previously were not known to be localized to this compartment in any other plant species. Since Brassica napus, a close relative of the fully sequenced model plant Arabidopsis thaliana, was used as the experimental system, our results provide a large number of candidate proteins for directed molecular and biochemical analyses of the physiological functions of the xylem under different environmental and developmental conditions. This approach will allow exploiting many of the already established functional genomic resources, like i.e. the large mutant collections, that are available for Arabidopsis.
PMCID: PMC1182380  PMID: 15969751
18.  Construction of a bacterial artificial chromosome library from the spikemoss Selaginella moellendorffii: a new resource for plant comparative genomics 
BMC Plant Biology  2005;5:10.
The lycophytes are an ancient lineage of vascular plants that diverged from the seed plant lineage about 400 Myr ago. Although the lycophytes occupy an important phylogenetic position for understanding the evolution of plants and their genomes, no genomic resources exist for this group of plants.
Here we describe the construction of a large-insert bacterial artificial chromosome (BAC) library from the lycophyte Selaginella moellendorffii. Based on cell flow cytometry, this species has the smallest genome size among the different lycophytes tested, including Huperzia lucidula, Diphaiastrum digita, Isoetes engelmanii and S. kraussiana. The arrayed BAC library consists of 9126 clones; the average insert size is estimated to be 122 kb. Inserts of chloroplast origin account for 2.3% of the clones. The BAC library contains an estimated ten genome-equivalents based on DNA hybridizations using five single-copy and two duplicated S. moellendorffii genes as probes.
The S. moellenforffii BAC library, the first to be constructed from a lycophyte, will be useful to the scientific community as a resource for comparative plant genomics and evolution.
PMCID: PMC1177970  PMID: 15955246
19.  Development and evaluation of a Gal4-mediated LUC/GFP/GUS enhancer trap system in Arabidopsis 
BMC Plant Biology  2005;5:9.
Gal4 enhancer trap systems driving expression of LacZ and GFP reporters have been characterized and widely used in Drosophila. However, a Gal4 enhancer trap system in Arabidopsis has not been described in the primary literature. In Drosophila, the reporters possess a Gal4 upstream activation sequence (UAS) as five repeats (5XUAS) and lines that express Gal4 from tissue specific enhancers have also been used for the ectopic expression of any transgene (driven by a 5XUAS). While Gal4 transactivation has been demonstrated in Arabidopsis, wide use of a trap has not emerged in part because of the lack of detailed analysis, which is the purpose of the present study.
A key feature of this study is the use of luciferase (LUC) as the primary reporter and rsGFP-GUS as secondary reporters. Reporters driven by a 5XUAS are better suited in Arabidopsis than those containing a 1X or 2X UAS. A 5XUAS-LUC reporter is expressed at high levels in Arabidopsis lines transformed with Gal4 driven by the full, enhanced 35S promoter. In contrast, a minimum 35S (containing the TATA region) upstream of Gal4 acts as an enhancer trap system. Luciferase expression in trap lines of the T1, T2, and T3 generations are generally stable but by the T4 generation approximately 25% of the lines are significantly silenced. This silencing is reversed by growing plants on media containing 5-aza-2'-deoxycytidine. Quantitative multiplex RT-PCR on the Gal4 and LUC mRNA indicate that this silencing can occur at the level of Gal4 or LUC transcription. Production of a 10,000 event library and observations on screening, along with the potential for a Gal4 driver system in other plant species are discussed.
The Gal4 trap system described here uses the 5XUAS-LUC and 5XUAS rsGFP-GUS as reporters and allows for in planta quantitative screening, including the rapid monitoring for silencing. We conclude that in about 75% of the cases silencing is at the level of transcription of the Gal4 transgene and is at an acceptable frequency to make the Gal4 trap system in Arabidopsis of value. This system will be useful for the isolation and comprehensive characterization of specific reporter and driver lines.
PMCID: PMC1164422  PMID: 15941484
20.  Biomarker metabolites capturing the metabolite variance present in a rice plant developmental period 
BMC Plant Biology  2005;5:8.
This study analyzes metabolomic data from a rice tillering (branching) developmental profile to define a set of biomarker metabolites that reliably captures the metabolite variance of this plant developmental event, and which has potential as a basis for rapid comparative screening of metabolite profiles in relation to change in development, environment, or genotype. Changes in metabolism, and in metabolite profile, occur as a part of, and in response to, developmental events. These changes are influenced by the developmental program, as well as external factors impinging on it. Many samples are needed, however, to characterize quantitative aspects of developmental variation. A biomarker metabolite set could benefit screening of quantitative plant developmental variation by providing some of the advantages of both comprehensive metabolomic studies and focused studies of particular metabolites or pathways.
An appropriate set of biomarker metabolites to represent the plant developmental period including the initiation and early growth of rice tillering (branching) was obtained by: (1) determining principal components of the comprehensive metabolomic profile, then (2) identifying clusters of metabolites representing variation in loading on the first three principal components, and finally (3) selecting individual metabolites from these clusters that were known to be common among diverse organisms. The resultant set of 21 biomarker metabolites was reliable (P = 0.001) in capturing 83% of the metabolite variation in development. Furthermore, a subset of the biomarker metabolites was successful (P = 0.05) in correctly predicting metabolite change in response to environment as determined in another rice metabolomics study.
The ability to define a set of biomarker metabolites that reliably captures the metabolite variance of a plant developmental event was established. The biomarker metabolites are all commonly present in diverse organisms, so studies of their quantitative relationships can provide comparative information concerning metabolite profiles in relation to change in plant development, environment, or genotype.
PMCID: PMC1175851  PMID: 15927065
22.  Maintenance of the plant genome under natural light 
BMC Plant Biology  2005;5(Suppl 1):S7.
PMCID: PMC1810291
23.  Regulation of programmed cell death in plant embryogenesis 
BMC Plant Biology  2005;5(Suppl 1):S6.
PMCID: PMC1810290
24.  Ambient levels of UV radiation 
BMC Plant Biology  2005;5(Suppl 1):S5.
PMCID: PMC1810289
25.  Effect of UV-B radiation on some cereals 
BMC Plant Biology  2005;5(Suppl 1):S4.
PMCID: PMC1810288

Results 1-25 (63)