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1.  Arabidopsis Gene Family Profiler (aGFP) – user-oriented transcriptomic database with easy-to-use graphic interface 
BMC Plant Biology  2007;7:39.
Microarray technologies now belong to the standard functional genomics toolbox and have undergone massive development leading to increased genome coverage, accuracy and reliability. The number of experiments exploiting microarray technology has markedly increased in recent years. In parallel with the rapid accumulation of transcriptomic data, on-line analysis tools are being introduced to simplify their use. Global statistical data analysis methods contribute to the development of overall concepts about gene expression patterns and to query and compose working hypotheses. More recently, these applications are being supplemented with more specialized products offering visualization and specific data mining tools. We present a curated gene family-oriented gene expression database, Arabidopsis Gene Family Profiler (aGFP; ), which gives the user access to a large collection of normalised Affymetrix ATH1 microarray datasets. The database currently contains NASC Array and AtGenExpress transcriptomic datasets for various tissues at different developmental stages of wild type plants gathered from nearly 350 gene chips.
The Arabidopsis GFP database has been designed as an easy-to-use tool for users needing an easily accessible resource for expression data of single genes, pre-defined gene families or custom gene sets, with the further possibility of keyword search. Arabidopsis Gene Family Profiler presents a user-friendly web interface using both graphic and text output. Data are stored at the MySQL server and individual queries are created in PHP script. The most distinguishable features of Arabidopsis Gene Family Profiler database are: 1) the presentation of normalized datasets (Affymetrix MAS algorithm and calculation of model-based gene-expression values based on the Perfect Match-only model); 2) the choice between two different normalization algorithms (Affymetrix MAS4 or MAS5 algorithms); 3) an intuitive interface; 4) an interactive "virtual plant" visualizing the spatial and developmental expression profiles of both gene families and individual genes.
Arabidopsis GFP gives users the possibility to analyze current Arabidopsis developmental transcriptomic data starting with simple global queries that can be expanded and further refined to visualize comparative and highly selective gene expression profiles.
PMCID: PMC1963329  PMID: 17645793
2.  Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers 
BMC Plant Biology  2007;7:11.
Beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein (in the beta-epsilon branch) and violaxanthin (in the beta-beta branch). None of these carotenoids have provitamin A activity. We have previously shown that tuber-specific silencing of the first step in the epsilon-beta branch, LCY-e, redirects metabolic flux towards beta-beta carotenoids, increases total carotenoids up to 2.5-fold and beta-carotene up to 14-fold.
In this work, we silenced the non-heme beta-carotene hydroxylases CHY1 and CHY2 in the tuber. Real Time RT-PCR measurements confirmed the tuber-specific silencing of both genes . CHY silenced tubers showed more dramatic changes in carotenoid content than LCY-e silenced tubers, with beta-carotene increasing up to 38-fold and total carotenoids up to 4.5-fold. These changes were accompanied by a decrease in the immediate product of beta-carotene hydroxylation, zeaxanthin, but not of the downstream xanthophylls, viola- and neoxanthin. Changes in endogenous gene expression were extensive and partially overlapping with those of LCY-e silenced tubers: CrtISO, LCY-b and ZEP were induced in both cases, indicating that they may respond to the balance between individual carotenoid species.
Together with epsilon-cyclization of lycopene, beta-carotene hydroxylation is another regulatory step in potato tuber carotenogenesis. The data are consistent with a prevalent role of CHY2, which is highly expressed in tubers, in the control of this step. Combination of different engineering strategies holds good promise for the manipulation of tuber carotenoid content.
PMCID: PMC1828156  PMID: 17335571
3.  Discovery of chemically induced mutations in rice by TILLING 
BMC Plant Biology  2007;7:19.
Rice is both a food source for a majority of the world's population and an important model system. Available functional genomics resources include targeted insertion mutagenesis and transgenic tools. While these can be powerful, a non-transgenic, unbiased targeted mutagenesis method that can generate a range of allele types would add considerably to the analysis of the rice genome. TILLING (Targeting Induced Local Lesions in Genomes), a general reverse genetic technique that combines traditional mutagenesis with high throughput methods for mutation discovery, is such a method.
To apply TILLING to rice, we developed two mutagenized rice populations. One population was developed by treatment with the chemical mutagen ethyl methanesulphonate (EMS), and the other with a combination of sodium azide plus methyl-nitrosourea (Az-MNU). To find induced mutations, target regions of 0.7–1.5 kilobases were PCR amplified using gene specific primers labeled with fluorescent dyes. Heteroduplexes were formed through denaturation and annealing of PCR products, mismatches digested with a crude preparation of CEL I nuclease and cleaved fragments visualized using denaturing polyacrylamide gel electrophoresis. In 10 target genes screened, we identified 27 nucleotide changes in the EMS-treated population and 30 in the Az-MNU population.
We estimate that the density of induced mutations is two- to threefold higher than previously reported rice populations (about 1/300 kb). By comparison to other plants used in public TILLING services, we conclude that the populations described here would be suitable for use in a large scale TILLING project.
PMCID: PMC1858691  PMID: 17428339
4.  Characterization and transferability of microsatellite markers of the cultivated peanut (Arachis hypogaea) 
BMC Plant Biology  2007;7:9.
The genus Arachis includes Arachis hypogaea (cultivated peanut) and wild species that are used in peanut breeding or as forage. Molecular markers have been employed in several studies of this genus, but microsatellite markers have only been used in few investigations. Microsatellites are very informative and are useful to assess genetic variability, analyze mating systems and in genetic mapping. The objectives of this study were to develop A. hypogaea microsatellite loci and to evaluate the transferability of these markers to other Arachis species.
Thirteen loci were isolated and characterized using 16 accessions of A. hypogaea. The level of variation found in A. hypogaea using microsatellites was higher than with other markers. Cross-transferability of the markers was also high. Sequencing of the fragments amplified using the primer pair Ah11 from 17 wild Arachis species showed that almost all wild species had similar repeated sequence to the one observed in A. hypogaea. Sequence data suggested that there is no correlation between taxonomic relationship of a wild species to A. hypogaea and the number of repeats found in its microsatellite loci.
These results show that microsatellite primer pairs from A. hypogaea have multiple uses. A higher level of variation among A. hypogaea accessions can be detected using microsatellite markers in comparison to other markers, such as RFLP, RAPD and AFLP. The microsatellite primers of A. hypogaea showed a very high rate of transferability to other species of the genus. These primer pairs provide important tools to evaluate the genetic variability and to assess the mating system in Arachis species.
PMCID: PMC1829157  PMID: 17326826
5.  Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach 
BMC Plant Biology  2007;7:1.
The large amount of available sequence information for the plant acyl-ACP thioesterases (TEs) made it possible to use a bioinformatics-guided approach to identify amino acid residues involved in substrate specificity. The Conserved Property Difference Locator (CPDL) program allowed the identification of putative specificity-determining residues that differ between the FatA and FatB TE classes. Six of the FatA residue differences identified by CPDL were incorporated into the FatB-like parent via site-directed mutagenesis and the effect of each on TE activity was determined. Variants were expressed in E. coli strain K27 that allows determination of enzyme activity by GCMS analysis of fatty acids released into the medium.
Substitutions at four of the positions (74, 86, 141, and 174) changed substrate specificity to varying degrees while changes at the remaining two positions, 110 and 221, essentially inactivated the thioesterase. The effects of substitutions at positions 74, 141, and 174 (3-MUT) or 74, 86, 141, 174 (4-MUT) were not additive with respect to specificity.
Four of six putative specificity determining positions in plant TEs, identified with the use of CPDL, were validated experimentally; a novel colorimetric screen that discriminates between active and inactive TEs is also presented.
PMCID: PMC1770913  PMID: 17201914
6.  TaMSH7: A cereal mismatch repair gene that affects fertility in transgenic barley (Hordeum vulgare L.) 
BMC Plant Biology  2007;7:67.
Chromosome pairing, recombination and DNA repair are essential processes during meiosis in sexually reproducing organisms. Investigating the bread wheat (Triticum aestivum L.) Ph2 (Pairing homoeologous) locus has identified numerous candidate genes that may have a role in controlling such processes, including TaMSH7, a plant specific member of the DNA mismatch repair family.
Sequencing of the three MSH7 genes, located on the short arms of wheat chromosomes 3A, 3B and 3D, has revealed no significant sequence divergence at the amino acid level suggesting conservation of function across the homoeogroups. Functional analysis of MSH7 through the use of RNAi loss-of-function transgenics was undertaken in diploid barley (Hordeum vulgare L.). Quantitative real-time PCR revealed several T0 lines with reduced MSH7 expression. Positive segregants from two T1 lines studied in detail showed reduced MSH7 expression when compared to transformed controls and null segregants. Expression of MSH6, another member of the mismatch repair family which is most closely related to the MSH7 gene, was not significantly reduced in these lines. In both T1 lines, reduced seed set in positive segregants was observed.
Results presented here indicate, for the first time, a distinct functional role for MSH7 in vivo and show that expression of this gene is necessary for wild-type levels of fertility. These observations suggest that MSH7 has an important function during meiosis and as such remains a candidate for Ph2.
PMCID: PMC2234410  PMID: 18096080
7.  Sequencing analysis of 20,000 full-length cDNA clones from cassava reveals lineage specific expansions in gene families related to stress response 
BMC Plant Biology  2007;7:66.
Cassava, an allotetraploid known for its remarkable tolerance to abiotic stresses is an important source of energy for humans and animals and a raw material for many industrial processes. A full-length cDNA library of cassava plants under normal, heat, drought, aluminum and post harvest physiological deterioration conditions was built; 19968 clones were sequence-characterized using expressed sequence tags (ESTs).
The ESTs were assembled into 6355 contigs and 9026 singletons that were further grouped into 10577 scaffolds; we found 4621 new cassava sequences and 1521 sequences with no significant similarity to plant protein databases. Transcripts of 7796 distinct genes were captured and we were able to assign a functional classification to 78% of them while finding more than half of the enzymes annotated in metabolic pathways in Arabidopsis. The annotation of sequences that were not paired to transcripts of other species included many stress-related functional categories showing that our library is enriched with stress-induced genes. Finally, we detected 230 putative gene duplications that include key enzymes in reactive oxygen species signaling pathways and could play a role in cassava stress response features.
The cassava full-length cDNA library here presented contains transcripts of genes involved in stress response as well as genes important for different areas of cassava research. This library will be an important resource for gene discovery, characterization and cloning; in the near future it will aid the annotation of the cassava genome.
PMCID: PMC2245942  PMID: 18096061
8.  Evolutionary conservation of plant gibberellin signalling pathway components 
BMC Plant Biology  2007;7:65.
Gibberellins (GA) are plant hormones that can regulate germination, elongation growth, and sex determination. They ubiquitously occur in seed plants. The discovery of gibberellin receptors, together with advances in understanding the function of key components of GA signalling in Arabidopsis and rice, reveal a fairly short GA signal transduction route. The pathway essentially consists of GID1 gibberellin receptors that interact with F-box proteins, which in turn regulate degradation of downstream DELLA proteins, suppressors of GA-controlled responses.
Arabidopsis sequences of the gibberellin signalling compounds were used to screen databases from a variety of plants, including protists, for homologues, providing indications for the degree of conservation of the pathway. The pathway as such appears completely absent in protists, the moss Physcomitrella patens shares only a limited homology with the Arabidopsis proteins, thus lacking essential characteristics of the classical GA signalling pathway, while the lycophyte Selaginella moellendorffii contains a possible ortholog for each component. The occurrence of classical GA responses can as yet not be linked with the presence of homologues of the signalling pathway. Alignments and display in neighbour joining trees of the GA signalling components confirm the close relationship of gymnosperms, monocotyledonous and dicotyledonous plants, as suggested from previous studies.
Homologues of the GA-signalling pathway were mainly found in vascular plants. The GA signalling system may have its evolutionary molecular onset in Physcomitrella patens, where GAs at higher concentrations affect gravitropism and elongation growth.
PMCID: PMC2234411  PMID: 18047669
9.  The metal tolerance profile of Thlaspi goesingense is mimicked in Arabidopsis thaliana heterologously expressing serine acetyl-transferase 
BMC Plant Biology  2007;7:63.
The Ni hyperaccumulator Thlaspi goesingense is tolerant to Ni ≅ Zn, ≅ Co and slightly resistant to > Cd. We previously observed that elevated glutathione, driven by constitutive activation of serine acetyltransferase (SAT), plays a role in the Ni tolerance of T. goesingense.
Here we show that the elevated shoot concentration of glutathione, previously shown to cause elevated Ni tolerance in Arabidopsis thaliana heterologously expressing T. goesingense mitochondrial serine acetyltransferase (SATm), also causes tolerance to Co and Zn while slightly enhancing resistance to Cd. The level of tolerance afforded to each metal is ranked Ni ≅ Co, > Zn > Cd. The Ni ≅ Co, > Zn tolerances are positively correlated with both the accumulation of glutathione (GSH) and the ability to resist the oxidative damage induced by these different metals. Based on the relative concentrations of each metal used a relatively low level of resistance to Cd was observed in both T. goesingense and TgSATm expressing lines and Cd resistance was least correlated to GSH accumulation.
Such data supports the conclusion that elevated glutathione levels, driven by constitutively enhanced SAT activity in the hyperaccumulator T. goesingense, plays an important role in the Ni, Co and Zn tolerance of this and other Thlaspi species. The hyper-activation of S assimilation through SAT is an excellent strategy for engineering enhanced metal tolerance in transgenic plants potentially used for phytoremediation.
PMCID: PMC2233625  PMID: 18045473
10.  LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum 
BMC Plant Biology  2007;7:64.
The phospholipids of the plant plasma membrane are synthesized in the endoplasmic reticulum (ER). The majority of these lipids reach the plasma membrane independently of the secretory vesicular pathway. Phospholipid delivery to the mitochondria and chloroplasts of plant cells also bypasses the secretory pathway and here it has been proposed that lysophospholipids are transported at contact sites between specific regions of the ER and the respective organelle, followed by lysophospholipid acylation in the target organelle. To test the hypothesis that a corresponding mechanism operates to transport phospholipids to the plasma membrane outside the secretory pathway, we investigated whether lysolipid acylation occurs also in the plant plasma membrane and whether this membrane, like the chloroplasts and mitochondria, is in close contact with the ER.
The plant plasma membrane readily incorporated the acyl chain of acyl-CoA into phospholipids. Oleic acid was preferred over palmitic acid as substrate and acyl incorporation occurred predominantly into phosphatidylcholine (PC). Phospholipase A2 stimulated the reaction, as did exogenous lysoPC when administered in above critical micellar concentrations. AgNO3 was inhibitory. The lysophospholipid acylation reaction was higher in a membrane fraction that could be washed off the isolated plasma membranes after repeated freezing and thawing cycles in a medium with lowered pH. This fraction exhibited several ER-like characteristics. When plasma membranes isolated from transgenic Arabidopsis expressing green fluorescent protein in the ER lumen were observed by confocal microscopy, membranes of ER origin were associated with the isolated plasma membranes.
We conclude that a lysoPC acylation activity is associated with plant plasma membranes and cannot exclude a PC transacylase activity. It is highly plausible that the enzyme(s) resides in a fraction of the ER, closely associated with the plasma membrane, or in both. We suggest that this fraction might be the equivalent of the mitochondria associated membrane of ER origin that delivers phospholipids to the mitochondria, and to the recently isolated ER-derived membrane fraction that is in close contact with chloroplasts. The in situ function of the lysoPC acylation/PC transacylase activity is unknown, but involvement in lipid delivery from the ER to the plasma membrane is suggested.
PMCID: PMC2241621  PMID: 18045483
11.  Analysis of cDNA libraries from developing seeds of guar (Cyamopsis tetragonoloba (L.) Taub) 
BMC Plant Biology  2007;7:62.
Guar, Cyamopsis tetragonoloba (L.) Taub, is a member of the Leguminosae (Fabaceae) family and is economically the most important of the four species in the genus. The endosperm of guar seed is a rich source of mucilage or gum, which forms a viscous gel in cold water, and is used as an emulsifier, thickener and stabilizer in a wide range of foods and industrial applications. Guar gum is a galactomannan, consisting of a linear (1→4)-β-linked D-mannan backbone with single-unit, (1→6)-linked, α-D-galactopyranosyl side chains. To better understand regulation of guar seed development and galactomannan metabolism we created cDNA libraries and a resulting EST dataset from different developmental stages of guar seeds.
A database of 16,476 guar seed ESTs was constructed, with 8,163 and 8,313 ESTs derived from cDNA libraries I and II, respectively. Library I was constructed from seeds at an early developmental stage (15–25 days after flowering, DAF), and library II from seeds at 30–40 DAF. Quite different sets of genes were represented in these two libraries. Approximately 27% of the clones were not similar to known sequences, suggesting that these ESTs represent novel genes or may represent non-coding RNA. The high flux of energy into carbohydrate and storage protein synthesis in guar seeds was reflected by a high representation of genes annotated as involved in signal transduction, carbohydrate metabolism, chaperone and proteolytic processes, and translation and ribosome structure. Guar unigenes involved in galactomannan metabolism were identified. Among the seed storage proteins, the most abundant contig represented a conglutin accounting for 3.7% of the total ESTs from both libraries.
The present EST collection and its annotation provide a resource for understanding guar seed biology and galactomannan metabolism.
PMCID: PMC2241620  PMID: 18034910
12.  UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseus cell suspension cultures 
BMC Plant Biology  2007;7:61.
Elicitations are considered to be an important strategy towards improved in vitro production of secondary metabolites. In cell cultures, biotic and abiotic elicitors have effectively stimulated the production of plant secondary metabolites. However, molecular basis of elicitor-signaling cascades leading to increased production of secondary metabolites of plant cell is largely unknown. Exposure of Catharanthus roseus cell suspension culture to low dose of UV-B irradiation was found to increase the amount of catharanthine and transcription of genes encoding tryptophan decarboxylase (Tdc) and strictosidine synthase (Str). In the present study, the signaling pathway mediating UV-B-induced catharanthine accumulation in C. roseus suspension cultures were investigated.
Here, we investigate whether cell surface receptors, medium alkalinization, Ca2+ influx, H2O2, CDPK and MAPK play required roles in UV-B signaling leading to enhanced production of catharanthine in C. roseus cell suspension cultures. C. roseus cells were pretreated with various agonists and inhibitors of known signaling components and their effects on the accumulation of Tdc and Str transcripts as well as amount of catharanthine production were investigated by various molecular biology techniques. It has been found that the catharanthine accumulation and transcription of Tdc and Str were inhibited by 3–4 fold upon pretreatment of various inhibitors like suramin, N-acetyl cysteine, inhibitors of calcium fluxes, staurosporine etc.
Our results demonstrate that cell surface receptor(s), Ca2+ influx, medium alkalinization, CDPK, H2O2 and MAPK play significant roles in UV-B signaling leading to stimulation of Tdc and Str genes and the accumulation of catharanthine in C. roseus cell suspension cultures. Based on these findings, a model for signal transduction cascade has been proposed.
PMCID: PMC2213653  PMID: 17988378
13.  Genome-wide analysis of Aux/IAA and ARF gene families in Populus trichocarpa 
BMC Plant Biology  2007;7:59.
Auxin/Indole-3-Acetic Acid (Aux/IAA) and Auxin Response Factor (ARF) transcription factors are key regulators of auxin responses in plants. We identified the suites of genes in the two gene families in Populus and performed comparative genomic analysis with Arabidopsis and rice.
A total of 35 Aux/IAA and 39 ARF genes were identified in the Populus genome. Comparative phylogenetic analysis revealed that several Aux/IAA and ARF subgroups have differentially expanded or contracted between the two dicotyledonous plants. Activator ARF genes were found to be two fold-overrepresented in the Populus genome. PoptrIAA and PoptrARF gene families appear to have expanded due to high segmental and low tandem duplication events. Furthermore, expression studies showed that genes in the expanded PoptrIAA3 subgroup display differential expression.
The present study examines the extent of conservation and divergence in the structure and evolution of Populus Aux/IAA and ARF gene families with respect to Arabidopsis and rice. The gene-family analysis reported here will be useful in conducting future functional genomics studies to understand how the molecular roles of these large gene families translate into a diversity of biologically meaningful auxin effects.
PMCID: PMC2174922  PMID: 17986329
14.  DNA sequence diversity and the origin of cultivated safflower (Carthamus tinctorius L.; Asteraceae) 
BMC Plant Biology  2007;7:60.
Safflower (Carthamus tinctorius L.) is a diploid oilseed crop whose origin is largely unknown. Safflower is widely believed to have been domesticated over 4,000 years ago somewhere in the Fertile Crescent. Previous hypotheses regarding the origin of safflower have focused primarily on two other species from sect. Carthamus – C. oxyacanthus and C. palaestinus – as the most likely progenitors, although some attention has been paid to a third species (C. persicus) as a possible candidate. Here, we describe the results of a phylogenetic analysis of the entire section using data from seven nuclear genes.
Single gene phylogenetic analyses indicated some reticulation or incomplete lineage sorting. However, the analysis of the combined dataset revealed a close relationship between safflower and C. palaestinus. In contrast, C. oxyacanthus and C. persicus appear to be more distantly related to safflower.
Based on our results, we conclude that safflower is most likely derived from the wild species Carthamus palaestinus. As expected, safflower exhibits somewhat reduced nucleotide diversity as compared to its progenitor, consistent with the occurrence of a population genetic bottleneck during domestication. The results of this research set the stage for an investigation of the genetics of safflower domestication.
PMCID: PMC2241619  PMID: 17986334
15.  Subcellular localisation of Medicago truncatula 9/13-hydroperoxide lyase reveals a new localisation pattern and activation mechanism for CYP74C enzymes 
BMC Plant Biology  2007;7:58.
Hydroperoxide lyase (HPL) is a key enzyme in plant oxylipin metabolism that catalyses the cleavage of polyunsaturated fatty acid hydroperoxides produced by the action of lipoxygenase (LOX) to volatile aldehydes and oxo acids. The synthesis of these volatile aldehydes is rapidly induced in plant tissues upon mechanical wounding and insect or pathogen attack. Together with their direct defence role towards different pathogens, these compounds are believed to play an important role in signalling within and between plants, and in the molecular cross-talk between plants and other organisms surrounding them. We have recently described the targeting of a seed 9-HPL to microsomes and putative lipid bodies and were interested to compare the localisation patterns of both a 13-HPL and a 9/13-HPL from Medicago truncatula, which were known to be expressed in leaves and roots, respectively.
To study the subcellular localisation of plant 9/13-HPLs, a set of YFP-tagged chimeric constructs were prepared using two M. truncatula HPL cDNAs and the localisation of the corresponding chimeras were verified by confocal microscopy in tobacco protoplasts and leaves. Results reported here indicated a distribution of M.truncatula 9/13-HPL (HPLF) between cytosol and lipid droplets (LD) whereas, as expected, M.truncatula 13-HPL (HPLE) was targeted to plastids. Notably, such endocellular localisation has not yet been reported previously for any 9/13-HPL. To verify a possible physiological significance of such association, purified recombinant HPLF was used in activation experiments with purified seed lipid bodies. Our results showed that lipid bodies can fully activate HPLF.
We provide evidence for the first CYP74C enzyme, to be targeted to cytosol and LD. We also showed by sedimentation and kinetic analyses that the association with LD or lipid bodies can result in the protein conformational changes required for full activation of the enzyme. This activation mechanism, which supports previous in vitro work with synthetic detergent micelle, fits well with a mechanism for regulating the rate of release of volatile aldehydes that is observed soon after wounding or tissue disruption.
PMCID: PMC2180173  PMID: 17983471
16.  Complete plastid genome sequences suggest strong selection for retention of photosynthetic genes in the parasitic plant genus Cuscuta 
BMC Plant Biology  2007;7:57.
Plastid genome content and protein sequence are highly conserved across land plants and their closest algal relatives. Parasitic plants, which obtain some or all of their nutrition through an attachment to a host plant, are often a striking exception. Heterotrophy can lead to relaxed constraint on some plastid genes or even total gene loss. We sequenced plastid genomes of two species in the parasitic genus Cuscuta along with a non-parasitic relative, Ipomoea purpurea, to investigate changes in the plastid genome that may result from transition to the parasitic lifestyle.
Aside from loss of all ndh genes, Cuscuta exaltata retains photosynthetic and photorespiratory genes that evolve under strong selective constraint. Cuscuta obtusiflora has incurred substantially more change to its plastid genome, including loss of all genes for the plastid-encoded RNA polymerase. Despite extensive change in gene content and greatly increased rate of overall nucleotide substitution, C. obtusiflora also retains all photosynthetic and photorespiratory genes with only one minor exception.
Although Epifagus virginiana, the only other parasitic plant with its plastid genome sequenced to date, has lost a largely overlapping set of transfer-RNA and ribosomal genes as Cuscuta, it has lost all genes related to photosynthesis and maintains a set of genes which are among the most divergent in Cuscuta. Analyses demonstrate photosynthetic genes are under the highest constraint of any genes within the plastid genomes of Cuscuta, indicating a function involving RuBisCo and electron transport through photosystems is still the primary reason for retention of the plastid genome in these species.
PMCID: PMC2216012  PMID: 17956636
17.  Global expression analysis of nucleotide binding site-leucine rich repeat-encoding and related genes in Arabidopsis 
BMC Plant Biology  2007;7:56.
Nucleotide binding site-leucine rich repeat (NBS-LRR)-encoding genes comprise the largest class of plant disease resistance genes. The 149 NBS-LRR-encoding genes and the 58 related genes that do not encode LRRs represent approximately 0.8% of all ORFs so far annotated in Arabidopsis ecotype Col-0. Despite their prevalence in the genome and functional importance, there was little information regarding expression of these genes.
We analyzed the expression patterns of ~170 NBS-LRR-encoding and related genes in Arabidopsis Col-0 using multiple analytical approaches: expressed sequenced tag (EST) representation, massively parallel signature sequencing (MPSS), microarray analysis, rapid amplification of cDNA ends (RACE) PCR, and gene trap lines. Most of these genes were expressed at low levels with a variety of tissue specificities. Expression was detected by at least one approach for all but 10 of these genes. The expression of some but not the majority of NBS-LRR-encoding and related genes was affected by salicylic acid (SA) treatment; the response to SA varied among different accessions. An analysis of previously published microarray data indicated that ten NBS-LRR-encoding and related genes exhibited increased expression in wild-type Landsberg erecta (Ler) after flagellin treatment. Several of these ten genes also showed altered expression after SA treatment, consistent with the regulation of R gene expression during defense responses and overlap between the basal defense response and salicylic acid signaling pathways. Enhancer trap analysis indicated that neither jasmonic acid nor benzothiadiazole (BTH), a salicylic acid analog, induced detectable expression of the five NBS-LRR-encoding genes and one TIR-NBS-encoding gene tested; however, BTH did induce detectable expression of the other TIR-NBS-encoding gene analyzed. Evidence for alternative mRNA polyadenylation sites was observed for many of the tested genes. Evidence for alternative splicing was found for at least 12 genes, 11 of which encode TIR-NBS-LRR proteins. There was no obvious correlation between expression pattern, phylogenetic relationship or genomic location of the NBS-LRR-encoding and related genes studied.
Transcripts of many NBS-LRR-encoding and related genes were defined. Most were present at low levels and exhibited tissue-specific expression patterns. Expression data are consistent with most Arabidopsis NBS-LRR-encoding and related genes functioning in plant defense responses but do not preclude other biological roles.
PMCID: PMC2175511  PMID: 17956627
18.  Systematic analysis of alternative first exons in plant genomes 
BMC Plant Biology  2007;7:55.
Alternative splicing (AS) contributes significantly to protein diversity, by selectively using different combinations of exons of the same gene under certain circumstances. One particular type of AS is the use of alternative first exons (AFEs), which can have consequences far beyond the fine-tuning of protein functions. For example, AFEs may change the N-termini of proteins and thereby direct them to different cellular compartments. When alternative first exons are distant, they are usually associated with alternative promoters, thereby conferring an extra level of gene expression regulation. However, only few studies have examined the patterns of AFEs, and these analyses were mainly focused on mammalian genomes. Recent studies have shown that AFEs exist in the rice genome, and are regulated in a tissue-specific manner. Our current understanding of AFEs in plants is still limited, including important issues such as their regulation, contribution to protein diversity, and evolutionary conservation.
We systematically identified 1,378 and 645 AFE-containing clusters in rice and Arabidopsis, respectively. From our data sets, we identified two types of AFEs according to their genomic organisation. In genes with type I AFEs, the first exons are mutually exclusive, while most of the downstream exons are shared among alternative transcripts. Conversely, in genes with type II AFEs, the first exon of one gene structure is an internal exon of an alternative gene structure. The functionality analysis indicated about half and ~19% of the AFEs in Arabidopsis and rice could alter N-terminal protein sequences, and ~5% of the functional alteration in type II AFEs involved protein domain addition/deletion in both genomes. Expression analysis indicated that 20~66% of rice AFE clusters were tissue- and/or development- specifically transcribed, which is consistent with previous observations; however, a much smaller percentage of Arabidopsis AFEs was regulated in this manner, which suggests different regulation mechanisms of AFEs between rice and Arabidopsis. Statistical analysis of some features of AFE clusters, such as splice-site strength and secondary structure formation further revealed differences between these two species. Orthologous search of AFE-containing gene pairs detected only 19 gene pairs conserved between rice and Arabidopsis, accounting only for a few percent of AFE-containing clusters.
Our analysis of AFE-containing genes in rice and Arabidopsis indicates that AFEs have multiple functions, from regulating gene expression to generating protein diversity. Comparisons of AFE clusters revealed different features in the two plant species, which indicates that AFEs may have evolved independently after the separation of rice (a model monocot) and Arabidopsis (a model dicot).
PMCID: PMC2174465  PMID: 17941993
19.  Synthetic xylan-binding modules for mapping of pulp fibres and wood sections 
BMC Plant Biology  2007;7:54.
The complex carbohydrate composition of natural and refined plant material is not known in detail but a matter that is of both basic and applied importance. Qualitative assessment of complex samples like plant and wood tissues requires the availability of a range of specific probes. Monoclonal antibodies and naturally existing carbohydrate binding modules (CBMs) have been used in the past to assess the presence of certain carbohydrates in plant tissues. However, the number of natural CBMs is limited and development of carbohydrate-specific antibodies is not always straightforward. We envisage the use of sets of very similar proteins specific for defined targets, like those developed by molecular evolution of a single CBM scaffold, as a suitable strategy to assess carbohydrate composition. An advantage of using synthetic CBMs lies in the possibility to study fine details of carbohydrate composition within non-uniform substrates like plant cell walls as made possible through minor differences in CBM specificity of the variety of binders that can be developed by genetic engineering.
A panel of synthetic xylan-binding CBMs, previously selected from a molecular library based on the scaffold of CBM4-2 from xylanase Xyn10A of Rhodothermus marinus, was used in this study. The wild type CBM4-2 and evolved modules both showed binding to wood sections. However, differences were observed in the staining patterns suggesting that these modules have different xylan-binding properties. Also the staining stability varied between the CBMs, the most stable staining being obtained with one (X-2) of the synthetic modules. Treatment of wood materials resulted in altered signal intensities, thereby also demonstrating the potential application of engineered CBMs as analytical tools for quality assessment of diverse plant material processes.
In this study we have demonstrated the usefulness of synthetic xylan-binding modules as specific probes in analysis of hemicelluloses (xylan) in wood and fibre materials.
PMCID: PMC2099432  PMID: 17935619
20.  An EST database from saffron stigmas 
BMC Plant Biology  2007;7:53.
Saffron (Crocus sativus L., Iridaceae) flowers have been used as a spice and medicinal plant ever since the Greek-Minoan civilization. The edible part – the stigmas – are commonly considered the most expensive spice in the world and are the site of a peculiar secondary metabolism, responsible for the characteristic color and flavor of saffron.
We produced 6,603 high quality Expressed Sequence Tags (ESTs) from a saffron stigma cDNA library. This collection is accessible and searchable through the Saffron Genes database The ESTs have been grouped into 1,893 Clusters, each corresponding to a different expressed gene, and annotated. The complete set of raw EST sequences, as well as of their electopherograms, are maintained in the database, allowing users to investigate sequence qualities and EST structural features (vector contamination, repeat regions). The saffron stigma transcriptome contains a series of interesting sequences (putative sex determination genes, lipid and carotenoid metabolism enzymes, transcription factors).
The Saffron Genes database represents the first reference collection for the genomics of Iridaceae, for the molecular biology of stigma biogenesis, as well as for the metabolic pathways underlying saffron secondary metabolism.
PMCID: PMC2221943  PMID: 17925031
21.  Erwinia carotovora elicitors and Botrytis cinerea activate defense responses in Physcomitrella patens 
BMC Plant Biology  2007;7:52.
Vascular plants respond to pathogens by activating a diverse array of defense mechanisms. Studies with these plants have provided a wealth of information on pathogen recognition, signal transduction and the activation of defense responses. However, very little is known about the infection and defense responses of the bryophyte, Physcomitrella patens, to well-studied phytopathogens. The purpose of this study was to determine: i) whether two representative broad host range pathogens, Erwinia carotovora ssp. carotovora (E.c. carotovora) and Botrytis cinerea (B. cinerea), could infect Physcomitrella, and ii) whether B. cinerea, elicitors of a harpin (HrpN) producing E.c. carotovora strain (SCC1) or a HrpN-negative strain (SCC3193), could cause disease symptoms and induce defense responses in Physcomitrella.
B. cinerea and E.c. carotovora were found to readily infect Physcomitrella gametophytic tissues and cause disease symptoms. Treatments with B. cinerea spores or cell-free culture filtrates from E.c. carotovoraSCC1 (CF(SCC1)), resulted in disease development with severe maceration of Physcomitrella tissues, while CF(SCC3193) produced only mild maceration. Although increased cell death was observed with either the CFs or B. cinerea, the occurrence of cytoplasmic shrinkage was only visible in Evans blue stained protonemal cells treated with CF(SCC1) or inoculated with B. cinerea. Most cells showing cytoplasmic shrinkage accumulated autofluorescent compounds and brown chloroplasts were evident in a high proportion of these cells. CF treatments and B. cinerea inoculation induced the expression of the defense-related genes: PR-1, PAL, CHS and LOX.
B. cinerea and E.c. carotovora elicitors induce a defense response in Physcomitrella, as evidenced by enhanced expression of conserved plant defense-related genes. Since cytoplasmic shrinkage is the most common morphological change observed in plant PCD, and that harpins and B. cinerea induce this type of cell death in vascular plants, our results suggest that E.c. carotovora CFSCC1 containing HrpN and B. cinerea could also induce this type of cell death in Physcomitrella. Our studies thus establish Physcomitrella as an experimental host for investigation of plant-pathogen interactions and B. cinerea and elicitors of E.c. carotovora as promising tools for understanding the mechanisms involved in defense responses and in pathogen-mediated cell death in this simple land plant.
PMCID: PMC2174466  PMID: 17922917
22.  Inorganic polyphosphate occurs in the cell wall of Chlamydomonas reinhardtii and accumulates during cytokinesis 
BMC Plant Biology  2007;7:51.
Inorganic polyphosphate (poly P), linear chains of phosphate residues linked by energy rich phosphoanhydride bonds, is found in every cell and organelle and is abundant in algae. Depending on its localization and concentration, poly P is involved in various biological functions. It serves, for example, as a phosphate store and buffer against alkali, is involved in energy metabolism and regulates the activity of enzymes. Bacteria defective in poly P synthesis are impaired in biofilm development, motility and pathogenicity. PolyP has also been found in fungal cell walls and bacterial envelopes, but has so far not been measured directly or stained specifically in the cell wall of any plant or alga.
Here, we demonstrate the presence of poly P in the cell wall of Chlamydomonas reinhardtii by staining with specific poly P binding proteins. The specificity of the poly P signal was verified by various competition experiments, by staining with different poly P binding proteins and by correlation with biochemical quantification. Microscopical investigation at different time-points during growth revealed fluctuations of the poly P signal synchronous with the cell cycle: The poly P staining peaked during late cytokinesis and was independent of the high intracellular poly P content, which fluctuated only slightly during the cell cycle.
The presented staining method provides a specific and sensitive tool for the study of poly P in the extracellular matrices of algae and could be used to describe the dynamic behaviour of cell wall poly P during the cell cycle. We assume that cell wall poly P and intracellular poly P are regulated by distinct mechanisms and it is suggested that cell wall bound poly P might have important protective functions against toxic compounds or pathogens during cytokinesis, when cells are more vulnerable.
PMCID: PMC2096623  PMID: 17892566
23.  Evaluation of reference genes for real-time RT-PCR expression studies in the plant pathogen Pectobacterium atrosepticum 
BMC Plant Biology  2007;7:50.
Real-time RT-PCR has become a powerful technique to monitor low-abundance mRNA expression and is a useful tool when examining bacterial gene expression inside infected host tissues. However, correct evaluation of data requires accurate and reliable normalisation against internal standards. Thus, the identification of reference genes whose expression does not change during the course of the experiment is of paramount importance. Here, we present a study where manipulation of cultural growth conditions and in planta experiments have been used to validate the expression stability of reference gene candidates for the plant pathogen Pectobacterium atrosepticum, belonging to the family Enterobacteriaceae.
Of twelve reference gene candidates tested, four proved to be stably expressed both in six different cultural growth conditions and in planta. Two of these genes (recA and ffh), encoding recombinase A and signal recognition particle protein, respectively, proved to be the most stable set of reference genes under the experimental conditions used. In addition, genes proC and gyrA, encoding pyrroline-5-carboxylate reductase and DNA gyrase, respectively, also displayed relatively stable mRNA expression levels.
Based on these results, we suggest recA and ffh as suitable candidates for accurate normalisation of real-time RT-PCR data for experiments investigating the plant pathogen P. atrosepticum and potentially other related pathogens.
PMCID: PMC2151947  PMID: 17888160
24.  Differential gene expression in an elite hybrid rice cultivar (Oryza sativa, L) and its parental lines based on SAGE data 
BMC Plant Biology  2007;7:49.
It was proposed that differentially-expressed genes, aside from genetic variations affecting protein processing and functioning, between hybrid and its parents provide essential candidates for studying heterosis or hybrid vigor. Based our serial analysis of gene expression (SAGE) data from an elite Chinese super-hybrid rice (LYP9) and its parental cultivars (93-11 and PA64s) in three major tissue types (leaves, roots and panicles) at different developmental stages, we analyzed the transcriptome and looked for candidate genes related to rice heterosis.
By using an improved strategy of tag-to-gene mapping and two recently annotated genome assemblies (93-11 and PA64s), we identified 10,268 additional high-quality tags, reaching a grand total of 20,595 together with our previous result. We further detected 8.5% and 5.9% physically-mapped genes that are differentially-expressed among the triad (in at least one of the three stages) with P-values less than 0.05 and 0.01, respectively. These genes distributed in 12 major gene expression patterns; among them, 406 up-regulated and 469 down-regulated genes (P < 0.05) were observed. Functional annotations on the identified genes highlighted the conclusion that up-regulated genes (some of them are known enzymes) in hybrid are mostly related to enhancing carbon assimilation in leaves and roots. In addition, we detected a group of up-regulated genes related to male sterility and 442 down-regulated genes related to signal transduction and protein processing, which may be responsible for rice heterosis.
We improved tag-to-gene mapping strategy by combining information from transcript sequences and rice genome annotation, and obtained a more comprehensive view on genes that related to rice heterosis. The candidates for heterosis-related genes among different genotypes provided new avenue for exploring the molecular mechanism underlying heterosis.
PMCID: PMC2077334  PMID: 17877838
25.  The cyclic nucleotide gated cation channel AtCNGC10 traffics from the ER via Golgi vesicles to the plasma membrane of Arabidopsis root and leaf cells 
BMC Plant Biology  2007;7:48.
The cyclic nucleotide-gated ion channels (CNGCs) maintain cation homeostasis essential for a wide range of physiological processes in plant cells. However, the precise subcellular locations and trafficking of these membrane proteins are poorly understood. This is further complicated by a general deficiency of information about targeting pathways of membrane proteins in plants. To investigate CNGC trafficking and localization, we have measured Atcngc5 and Atcngc10 expression in roots and leaves, analyzed AtCNGC10-GFP fusions transiently expressed in protoplasts, and conducted immunofluorescence labeling of protoplasts and immunoelectron microscopic analysis of high pressure frozen leaves and roots.
AtCNGC10 mRNA and protein levels were 2.5-fold higher in roots than leaves, while AtCNGC5 mRNA and protein levels were nearly equal in these tissues. The AtCNGC10-EGFP fusion was targeted to the plasma membrane in leaf protoplasts, and lightly labeled several intracellular structures. Immunofluorescence microscopy with affinity purified CNGC-specific antisera indicated that AtCNGC5 and AtCNGC10 are present in the plasma membrane of protoplasts. Immunoelectron microscopy demonstrated that AtCNGC10 was associated with the plasma membrane of mesophyll, palisade parenchyma and epidermal cells of leaves, and the meristem, columella and cap cells of roots. AtCNCG10 was also observed in the endoplasmic reticulum and Golgi cisternae and vesicles of 50–150 nm in size. Patch clamp assays of an AtCNGC10-GFP fusion expressed in HEK293 cells measured significant cation currents.
AtCNGC5 and AtCNGC10 are plasma membrane proteins. We postulate that AtCNGC10 traffics from the endoplasmic reticulum via the Golgi apparatus and associated vesicles to the plasma membrane. The presence of the cation channel, AtCNGC10, in root cap meristem cells, cell plate, and gravity-sensing columella cells, combined with the previously reported antisense phenotypes of decreased gravitropic and cell enlargement responses, suggest roles of AtCNGC10 in modulating cation balance required for root gravitropism, cell division and growth.
PMCID: PMC2031891  PMID: 17877833

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