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1.  Recovery and characterization of a Citrus clementina Hort. ex Tan. 'Clemenules' haploid plant selected to establish the reference whole Citrus genome sequence 
BMC Plant Biology  2009;9:110.
In recent years, the development of structural genomics has generated a growing interest in obtaining haploid plants. The use of homozygous lines presents a significant advantage for the accomplishment of sequencing projects. Commercial citrus species are characterized by high heterozygosity, making it difficult to assemble large genome sequences. Thus, the International Citrus Genomic Consortium (ICGC) decided to establish a reference whole citrus genome sequence from a homozygous plant. Due to the existence of important molecular resources and previous success in obtaining haploid clementine plants, haploid clementine was selected as the target for the implementation of the reference whole genome citrus sequence.
To obtain haploid clementine lines we used the technique of in situ gynogenesis induced by irradiated pollen. Flow cytometry, chromosome counts and SSR marker (Simple Sequence Repeats) analysis facilitated the identification of six different haploid lines (2n = x = 9), one aneuploid line (2n = 2x+4 = 22) and one doubled haploid plant (2n = 2x = 18) of 'Clemenules' clementine. One of the haploids, obtained directly from an original haploid embryo, grew vigorously and produced flowers after four years. This is the first haploid plant of clementine that has bloomed and we have, for the first time, characterized the histology of haploid and diploid flowers of clementine. Additionally a double haploid plant was obtained spontaneously from this haploid line.
The first haploid plant of 'Clemenules' clementine produced directly by germination of a haploid embryo, which grew vigorously and produced flowers, has been obtained in this work. This haploid line has been selected and it is being used by the ICGC to establish the reference sequence of the nuclear genome of citrus.
PMCID: PMC2747335  PMID: 19698121
2.  Modification of tobacco plant development by sense and antisense expression of the tomato viroid-induced AGC VIIIa protein kinase PKV suggests involvement in gibberellin signaling 
BMC Plant Biology  2009;9:108.
The serine-threonine protein kinase gene, designated pkv (protein kinase- viroid induced) was previously found to be transcriptionally activated in tomato plants infected with the plant pathogen Potato spindle tuber viroid (PSTVd). These plants exhibited symptoms of stunting, and abnormal development of leaf, root, and vascular tissues. The encoded protein, PKV, is a novel member of the AGC VIIIa group of signal-transducing protein kinases; however, the role of PKV in plant development is unknown. In this communication, we report the phenotypic results of over expression and silencing of pkv in transgenic tobacco.
Over expression of pkv in Nicotiana tabacum cv. Xanthi (tobacco) resulted in stunting, reduced root formation, and delay in flowering, phenotypes similar to symptoms of PSTVd infection of tomato. In addition, homozygous T2 tobacco plants over expressing PKV were male sterile. Antisense expression of pkv, on the other hand, resulted in plants that were taller than non-transformed plants, produced an increased number of flowers, and were fertile. Exogenous application of GA3 stimulated stem elongation in the stunted, sense-expressing plants. PKV sense and antisense expression altered transcript levels of GA biosynthetic genes and genes involved in developmental and signaling pathways, but not genes involved in salicylic acid- or jasmonic acid-dependent pathways. Our data provide evidence suggesting that PKV plays an important role in a GA signaling pathway that controls plant height and fertility.
We have found that the over expression of the tomato protein kinase PKV resulted in stunting, modified vascular tissue development, reduced root formation, and male sterility in tobacco, and we propose that PKV regulates plant development by functioning in critical signaling pathways involved in gibberellic acid metabolism.
PMCID: PMC2735738  PMID: 19689802
3.  Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca) BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome 
BMC Plant Biology  2009;9:106.
Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs) and full-length (FL)cDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer.
We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR) and a cytochrome P450 (CYP720B4) from a non-arrayed genomic BAC library of white spruce (Picea glauca). Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR) and 94 kbp (CYP720B4) long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs), high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing.
We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene families can be isolated in a gene-specific fashion. The results of the present work provide important new information about the structure and content of conifer genomic DNA that will guide future efforts to sequence and assemble conifer genomes.
PMCID: PMC2729077  PMID: 19656416
5.  Virus infection elevates transcriptional activity of miR164a promoter in plants 
BMC Plant Biology  2009;9:152.
Micro RNAs (miRs) constitute a large group of endogenous small RNAs that have crucial roles in many important plant functions. Virus infection and transgenic expression of viral proteins alter accumulation and activity of miRs and so far, most of the published evidence involves post-transcriptional regulations.
Using transgenic plants expressing a reporter gene under the promoter region of a characterized miR (P-miR164a), we monitored the reporter gene expression in different tissues and during Arabidopsis development. Strong expression was detected in both vascular tissues and hydathodes. P-miR164a activity was developmentally regulated in plants with a maximum expression at stages 1.12 to 5.1 (according to Boyes, 2001) along the transition from vegetative to reproductive growth. Upon quantification of P-miR164a-derived GUS activity after Tobacco mosaic virus Cg or Oilseed rape mosaic virus (ORMV) infection and after hormone treatments, we demonstrated that ORMV and gibberellic acid elevated P-miR164a activity. Accordingly, total mature miR164, precursor of miR164a and CUC1 mRNA (a miR164 target) levels increased after virus infection and interestingly the most severe virus (ORMV) produced the strongest promoter induction.
This work shows for the first time that the alteration of miR pathways produced by viral infections possesses a transcriptional component. In addition, the degree of miR alteration correlates with virus severity since a more severe virus produces a stronger P-miR164a induction.
PMCID: PMC2809068  PMID: 20042107
6.  Glycosylation-mediated phenylpropanoid partitioning in Populus tremuloides cell cultures 
BMC Plant Biology  2009;9:151.
Phenylpropanoid-derived phenolic glycosides (PGs) and condensed tannins (CTs) comprise large, multi-purpose non-structural carbon sinks in Populus. A negative correlation between PG and CT concentrations has been observed in several studies. However, the molecular mechanism underlying the relationship is not known.
Populus cell cultures produce CTs but not PGs under normal conditions. Feeding salicyl alcohol resulted in accumulation of salicins, the simplest PG, in the cells, but not higher-order PGs. Salicin accrual reflected the stimulation of a glycosylation response which altered a number of metabolic activities. We utilized this suspension cell feeding system as a model for analyzing the possible role of glycosylation in regulating the metabolic competition between PG formation, CT synthesis and growth. Cells accumulated salicins in a dose-dependent manner following salicyl alcohol feeding. Higher feeding levels led to a decrease in cellular CT concentrations (at 5 or 10 mM), and a negative effect on cell growth (at 10 mM). The competition between salicin and CT formation was reciprocal, and depended on the metabolic status of the cells. We analyzed gene expression changes between controls and cells fed with 5 mM salicyl alcohol for 48 hr, a time point when salicin accumulation was near maximum and CT synthesis was reduced, with no effect on growth. Several stress-responsive genes were up-regulated, suggestive of a general stress response in the fed cells. Salicyl alcohol feeding also induced expression of genes associated with sucrose catabolism, glycolysis and the Krebs cycle. Transcript levels of phenylalanine ammonia lyase and most of the flavonoid pathway genes were reduced, consistent with down-regulated CT synthesis.
Exogenous salicyl alcohol was readily glycosylated in Populus cell cultures, a process that altered sugar utilization and phenolic partitioning in the cells. Using this system, we identified candidate genes for glycosyltransferases that may mediate the glycosylation, and for transporters that mediate the subcellular compartmentalization of sugars and phenolic glycosides. The suspension cells appear to represent a facile system for dissecting the regulation of phenolic carbon partitioning, and in turn, its effects on growth in Populus.
PMCID: PMC2808312  PMID: 20040108
7.  Copia and Gypsy retrotransposons activity in sunflower (Helianthus annuus L.) 
BMC Plant Biology  2009;9:150.
Retrotransposons are heterogeneous sequences, widespread in eukaryotic genomes, which refer to the so-called mobile DNA. They resemble retroviruses, both in their structure and for their ability to transpose within the host genome, of which they make up a considerable portion. Copia- and Gypsy-like retrotransposons are the two main classes of retroelements shown to be ubiquitous in plant genomes. Ideally, the retrotransposons life cycle results in the synthesis of a messenger RNA and then self-encoded proteins to process retrotransposon mRNA in double stranded extra-chromosomal cDNA copies which may integrate in new chromosomal locations.
The RT-PCR and IRAP protocol were applied to detect the presence of Copia and Gypsy retrotransposon transcripts and of new events of integration in unstressed plants of a sunflower (Helianthus annuus L.) selfed line. Results show that in sunflower retrotransposons transcription occurs in all analyzed organs (embryos, leaves, roots, and flowers). In one out of sixty-four individuals analyzed, retrotransposons transcription resulted in the integration of a new element into the genome.
These results indicate that the retrotransposon life cycle is firmly controlled at a post transcriptional level. A possible silencing mechanism is discussed.
PMCID: PMC2805666  PMID: 20030800
8.  Over-expression of miR172 causes loss of spikelet determinacy and floral organ abnormalities in rice (Oryza sativa) 
BMC Plant Biology  2009;9:149.
Regulation of gene expression by microRNAs (miRNAs) plays a crucial role in many developmental and physiological processes in plants. miRNAs act to repress expression of their target genes via mRNA cleavage or translational repression. Dozens of miRNA families have been identified in rice, 21 of which are conserved between rice and Arabidopsis. miR172 is a conserved miRNA family which has been shown to regulate expression of APETALA2 (AP2)-like transcription factors in Arabidopsis and maize. The rice genome encodes five AP2-like genes predicted to be targets of miR172. To determine whether these rice AP2-like genes are regulated by miR172 and investigate the function of the target genes, we studied the effect of over-expressing two members of the miR172 family on rice plant development.
Analysis of miR172 expression showed that it is most highly expressed in late vegetative stages and developing panicles. Analyses of expression of three miR172 targets showed that SUPERNUMERARY BRACT (SNB) and Os03g60430 have high expression in developing panicles. Expression of miR172 was not inversely correlated with expression of its targets although miR172-mediated cleavage of SNB was detected by 5' rapid amplification of cDNA ends (RACE). Over-expression of miR172b in rice delayed the transition from spikelet meristem to floral meristem, and resulted in floral and seed developmental defects, including changes to the number and identity of floral organs, lower fertility and reduced seed weight. Plants over-expressing miR172b not only phenocopied the T-DNA insertion mutant of SNB but showed additional defects in floret development not seen in the snb mutant. However SNB expression was not reduced in the miR172b over-expression plants.
The phenotypes resulting from over-expression of miR172b suggests it represses SNB and at least one of the other miR172 targets, most likely Os03g60430, indicating roles for other AP2-like genes in rice floret development. miR172 and the AP2-like genes had overlapping expression patterns in rice and their expression did not show an obvious negative correlation. There was not a uniform decrease in the expression of the AP2-like miR172 target mRNAs in the miR172b over-expression plants. These observations are consistent with miR172 functioning via translational repression or with expression of the AP2-like genes being regulated by a negative feedback loop.
PMCID: PMC2803185  PMID: 20017947
9.  'Who's who' in two different flower types of Calluna vulgaris (Ericaceae): morphological and molecular analyses of flower organ identity 
BMC Plant Biology  2009;9:148.
The ornamental crop Calluna vulgaris is of increasing importance to the horticultural industry in the northern hemisphere due to a flower organ mutation: the flowers of the 'bud-flowering' phenotype remain closed i.e. as buds throughout the total flowering period and thereby maintain more colorful flowers for a longer period of time than the wild-type. This feature is accompanied and presumably caused by the complete lack of stamens. Descriptions of this botanical particularity are inconsistent and partially conflicting. In order to clarify basic questions of flower organ identity in general and stamen loss in detail, a study of the wild-type and the 'bud-flowering' flower type of C. vulgaris was initiated.
Flowers were examined by macro- and microscopic techniques. Organ development was investigated comparatively in both the wild-type and the 'bud-flowering' type by histological analyses. Analysis of epidermal cell surface structure of vegetative tissues and perianth organs using scanning electron microscopy revealed that in wild-type flowers the outer whorls of colored organs may be identified as sepals, while the inner ones may be identified as petals. In the 'bud-flowering' type, two whorls of sepals are directly followed by the gynoecium. Both, petals and stamens, are completely missing in this flower type. The uppermost whorl of green leaves represents bracts in both flower types.
In addition, two MADS-box genes (homologs of AP3/DEF and SEP1/2) were identified in C. vulgaris using RACE-PCR. Expression analysis by qRT-PCR was conducted for both genes in leaves, bracts, sepals and petals. These experiments revealed an expression pattern supporting the organ classification based on morphological characteristics.
Organ identity in both wild-type and 'bud-flowering' C. vulgaris was clarified using a combination of microscopic and molecular methods. Our results for bract, sepal and petal organ identity are supported by the 'ABCDE model'. However, loss of stamens in the 'bud-flowering' phenotype is an exceptional flower organ modification that cannot be explained by modified spatial expression of known organ identity genes.
PMCID: PMC2803492  PMID: 20003430
10.  A high-density collection of EMS-induced mutations for TILLING in Landsberg erecta genetic background of Arabidopsis 
BMC Plant Biology  2009;9:147.
Arabidopsis thaliana is the main model species for plant molecular genetics studies and world-wide efforts are devoted to identify the function of all its genes. To this end, reverse genetics by TILLING (Targeting Induced Local Lesions IN Genomes) in a permanent collection of chemically induced mutants is providing a unique resource in Columbia genetic background. In this work, we aim to extend TILLING resources available in A. thaliana by developing a new population of ethyl methanesulphonate (EMS) induced mutants in the second commonest reference strain. In addition, we pursue to saturate the number of EMS induced mutations that can be tolerated by viable and fertile plants.
By mutagenizing with different EMS concentrations we have developed a permanent collection of 3712 M2/M3 independent mutant lines in the reference strain Landsberg erecta (Ler) of A. thaliana. This population has been named as the Arabidopsis TILLer collection. The frequency of mutations per line was maximized by using M1 plants with low but sufficient seed fertility. Application of TILLING to search for mutants in 14 genes identified 21 to 46 mutations per gene, which correspond to a total of 450 mutations. Missense mutations were found for all genes while truncations were selected for all except one. We estimated that, on average, these lines carry one mutation every 89 kb, Ler population providing a total of more than five million induced mutations. It is estimated that TILLer collection shows a two to three fold higher EMS mutation density per individual than previously reported A. thaliana population.
Analysis of TILLer collection demonstrates its usefulness for large scale TILLING reverse genetics in another reference genetic background of A. thaliana. Comparisons with TILLING populations in other organisms indicate that this new A. thaliana collection carries the highest chemically induced mutation density per individual known in diploid species.
PMCID: PMC2803491  PMID: 20003424
11.  A recent duplication revisited: phylogenetic analysis reveals an ancestral duplication highly-conserved throughout the Oryza genus and beyond 
BMC Plant Biology  2009;9:146.
The role of gene duplication in the structural and functional evolution of genomes has been well documented. Analysis of complete rice (Oryza sativa) genome sequences suggested an ancient whole genome duplication, common to all the grasses, some 50-70 million years ago and a more conserved segmental duplication between the distal regions of the short arms of chromosomes 11 and 12, whose evolutionary history is controversial.
We have carried out a comparative analysis of this duplication within the wild species of the genus Oryza, using a phylogenetic approach to specify its origin and evolutionary dynamics. Paralogous pairs were isolated for nine genes selected throughout the region in all Oryza genome types, as well as in two outgroup species, Leersia perrieri and Potamophila parviflora. All Oryza species display the same global evolutionary dynamics but some lineage-specific features appear towards the proximal end of the duplicated region. The same level of conservation is observed between the redundant copies of the tetraploid species Oryza minuta. The presence of orthologous duplicated blocks in the genome of the more distantly-related species, Brachypodium distachyon, strongly suggests that this duplication between chromosomes 11 and 12 was formed as part of the whole genome duplication common to all Poaceae.
Our observations suggest that recurrent but heterogeneous concerted evolution throughout the Oryza genus and in related species has led specifically to the extremely high sequence conservation occurring in this region of more than 2 Mbp.
PMCID: PMC2797015  PMID: 20003305
12.  Ascorbate metabolism and the developmental demand for tartaric and oxalic acids in ripening grape berries 
BMC Plant Biology  2009;9:145.
Fresh fruits are well accepted as a good source of the dietary antioxidant ascorbic acid (Asc, Vitamin C). However, fruits such as grapes do not accumulate exceptionally high quantities of Asc. Grapes, unlike most other cultivated fruits do however use Asc as a precursor for the synthesis of both oxalic (OA) and tartaric acids (TA). TA is a commercially important product in the wine industry and due to its acidifying effect on crushed juice it can influence the organoleptic properties of the wine. Despite the interest in Asc accumulation in fruits, little is known about the mechanisms whereby Asc concentration is regulated. The purpose of this study was to gain insights into Asc metabolism in wine grapes (Vitis vinifera c.v. Shiraz.) and thus ascertain whether the developmental demand for TA and OA synthesis influences Asc accumulation in the berry.
We provide evidence for developmentally differentiated up-regulation of Asc biosynthetic pathways and subsequent fluctuations in Asc, TA and OA accumulation. Rapid accumulation of Asc and a low Asc to dehydroascorbate (DHA) ratio in young berries was co-ordinated with up-regulation of three of the primary Asc biosynthetic (Smirnoff-Wheeler) pathway genes. Immature berries synthesised Asc in-situ from the primary pathway precursors D-mannose and L-galactose. Immature berries also accumulated TA in early berry development in co-ordination with up-regulation of a TA biosynthetic gene. In contrast, ripe berries have up-regulated expression of the alternative Asc biosynthetic pathway gene D-galacturonic acid reductase with only residual expression of Smirnoff-Wheeler Asc biosynthetic pathway genes and of the TA biosynthetic gene. The ripening phase was further associated with up-regulation of Asc recycling genes, a secondary phase of increased accumulation of Asc and an increase in the Asc to DHA ratio.
We demonstrate strong developmental regulation of Asc biosynthetic, recycling and catabolic genes in grape berries. Integration of the transcript, radiotracer and metabolite data demonstrates that Asc and TA metabolism are developmentally regulated in grapevines; resulting in low accumulated levels of the biosynthetic intermediate Asc, and high accumulated levels of the metabolic end-product TA.
PMCID: PMC2797797  PMID: 19995454
13.  Cytosolic N-terminal arginine-based signals together with a luminal signal target a type II membrane protein to the plant ER 
BMC Plant Biology  2009;9:144.
In eukaryotic cells, the membrane compartments that constitute the exocytic pathway are traversed by a constant flow of lipids and proteins. This is particularly true for the endoplasmic reticulum (ER), the main "gateway of the secretory pathway", where biosynthesis of sterols, lipids, membrane-bound and soluble proteins, and glycoproteins occurs. Maintenance of the resident proteins in this compartment implies they have to be distinguished from the secretory cargo. To this end, they must possess specific ER localization determinants to prevent their exit from the ER, and/or to interact with receptors responsible for their retrieval from the Golgi apparatus. Very few information is available about the signal(s) involved in the retention of membrane type II protein in the ER but it is generally accepted that sorting of ER type II cargo membrane proteins depends on motifs mainly located in their cytosolic tails.
Here, using Arabidopsis glucosidase I as a model, we have identified two types of signals sufficient for the location of a type II membrane protein in the ER. A first signal is located in the luminal domain, while a second signal corresponds to a short amino acid sequence located in the cytosolic tail of the membrane protein. The cytosolic tail contains at its N-terminal end four arginine residues constitutive of three di-arginine motifs (RR, RXR or RXXR) independently sufficient to confer ER localization. Interestingly, when only one di-arginine motif is present, fusion proteins are located both in the ER and in mobile punctate structures, distinct but close to Golgi bodies. Soluble and membrane ER protein markers are excluded from these punctate structures, which also do not colocalize with an ER-exit-site marker. It is hypothesized they correspond to sites involved in Golgi to ER retrotransport.
Altogether, these results clearly show that cytosolic and luminal signals responsible for ER retention could coexist in a same type II membrane protein. These data also suggest that both retrieval and retention mechanisms govern protein residency in the ER membrane. We hypothesized that mobile punctate structures not yet described at the ER/Golgi interface and tentatively named GERES, could be involved in retrieval mechanisms from the Golgi to the ER.
PMCID: PMC2799409  PMID: 19995436
14.  Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor® 
BMC Plant Biology  2009;9:143.
TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM) analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor® software, aimed at simultaneous detection of mutations in three homoeologous genes.
We demonstrate that High Resolution Melting (HRM) analysis can be used in mutation scans in mixed PCR amplicons containing three homoeologous gene fragments. Combining HRM scanning with sequence analysis using Mutation Surveyor® is sensitive enough to detect a single nucleotide mutation in the heterozygous state in a mixed PCR amplicon containing three homoeoloci. The method was tested and validated in an EMS (ethylmethane sulfonate)-treated wheat TILLING population, screening mutations in the carboxyl terminal domain of the Starch Synthase II (SSII) gene. Selected identified mutations of interest can be further analysed by cloning to confirm the mutation and determine the genomic origin of the mutation.
Polyploidy is common in plants. Conserved regions of a gene often represent functional domains and have high sequence similarity between homoeologous loci. The method described here is a useful alternative to locus-specific based methods for screening mutations in conserved functional domains of homoeologous genes. This method can also be used for SNP (single nucleotide polymorphism) marker development and eco-TILLING in polyploid species.
PMCID: PMC2794869  PMID: 19958559
15.  Analysis of a post-translational steroid induction system for GIGANTEA in Arabidopsis 
BMC Plant Biology  2009;9:141.
To investigate the link between the flowering time gene GIGANTEA (GI) and downstream genes, an inducible GI system was developed in Arabidopsis thaliana L. Heynh. Transgenic Arabidopsis plant lines were generated with a steroid-inducible post-translational control system for GI. The gene expression construct consisted of the coding region of the GI protein fused to that of the ligand binding domain of the rat glucocorticoid receptor (GR). This fusion gene was expressed from the constitutive cauliflower mosaic virus 35S promoter and was introduced into plants carrying the gi-2 mutation. Application of the steroid dexamethasone (DEX) was expected to result in activation of the GI-GR protein and its relocation from the cytoplasm to the nucleus.
Application of DEX to the transgenic plant lines rescued the late flowering phenotype conferred by the gi-2 mutation. However, despite their delayed flowering in the absence of steroid, the transgenic lines expressed predicted GI downstream genes such as CONSTANS (CO) to relatively high levels. Nevertheless, increased CO and FLOWERING LOCUS T (FT) transcript accumulation was observed in transgenic plants within 8 h of DEX treatment compared to controls which was consistent with promotion of flowering by DEX. Unlike CO and FT, there was no change in the abundance of transcript of two other putative GI downstream genes HEME ACTIVATOR PROTEIN 3A (HAP3A) or TIMING OF CHLOROPHYLL A/B BINDING PROTEIN 1 (TOC1) after DEX application.
The post-translational activation of GI and promotion of flowering by steroid application supports a nuclear role for GI in the floral transition. Known downstream flowering time genes CO and FT were elevated by DEX treatment, but not other proposed targets HAP3A and TOC1, indicating that the expression of these genes may be less directly regulated by GI.
PMCID: PMC2789081  PMID: 19943973
16.  SolEST database: a "one-stop shop" approach to the study of Solanaceae transcriptomes 
BMC Plant Biology  2009;9:142.
Since no genome sequences of solanaceous plants have yet been completed, expressed sequence tag (EST) collections represent a reliable tool for broad sampling of Solanaceae transcriptomes, an attractive route for understanding Solanaceae genome functionality and a powerful reference for the structural annotation of emerging Solanaceae genome sequences.
We describe the SolEST database which integrates different EST datasets from both cultivated and wild Solanaceae species and from two species of the genus Coffea. Background as well as processed data contained in the database, extensively linked to external related resources, represent an invaluable source of information for these plant families. Two novel features differentiate SolEST from other resources: i) the option of accessing and then visualizing Solanaceae EST/TC alignments along the emerging tomato and potato genome sequences; ii) the opportunity to compare different Solanaceae assemblies generated by diverse research groups in the attempt to address a common complaint in the SOL community.
Different databases have been established worldwide for collecting Solanaceae ESTs and are related in concept, content and utility to the one presented herein. However, the SolEST database has several distinguishing features that make it appealing for the research community and facilitates a "one-stop shop" for the study of Solanaceae transcriptomes.
PMCID: PMC2794286  PMID: 19948013
17.  Salt-dependent regulation of a CNG channel subfamily in Arabidopsis 
BMC Plant Biology  2009;9:140.
In Arabidopsis thaliana, the family of cyclic nucleotide-gated channels (CNGCs) is composed of 20 members. Previous studies indicate that plant CNGCs are involved in the control of growth processes and responses to abiotic and biotic stresses. According to their proposed function as cation entry pathways these channels contribute to cellular cation homeostasis, including calcium and sodium, as well as to stress-related signal transduction. Here, we studied the expression patterns and regulation of CNGC19 and CNGC20, which constitute one of the five CNGC subfamilies.
GUS, GFP and luciferase reporter assays were used to study the expression of CNGC19 and CNGC20 genes from Arabidopsis thaliana in response to developmental cues and salt stress. CNGC19 and CNGC20 were differentially expressed in roots and shoots. The CNGC19 gene was predominantly active in roots already at early growth stages. Major expression was observed in the phloem. CNGC20 showed highest promoter activity in mesophyll cells surrounding the veins. Its expression increased during development and was maximal in mature and senescent leaves. Both genes were upregulated in the shoot in response to elevated NaCl but not mannitol concentrations. While in the root, CNGC19 did not respond to changes in the salt concentration, in the shoot it was strongly upregulated in the observed time frame (6-72 hours). Salt-induction of CNGC20 was also observed in the shoot, starting already one hour after stress treatment. It occurred with similar kinetics, irrespective of whether NaCl was applied to roots of intact plants or to the petiole of detached leaves. No differences in K and Na contents of the shoots were measured in homozygous T-DNA insertion lines for CNGC19 and CNGC20, respectively, which developed a growth phenotype in the presence of up to 75 mM NaCl similar to that of the wild type.
Together, the results strongly suggest that both channels are involved in the salinity response of different cell types in the shoot. Upon salinity both genes are upregulated within hours. CNGC19 and CNGC20 could assist the plant to cope with toxic effects caused by salt stress, probably by contributing to a re-allocation of sodium within the plant.
PMCID: PMC2794285  PMID: 19943938
18.  Dominance induction of fruitlet shedding in Malus × domestica (L. Borkh): molecular changes associated with polar auxin transport 
BMC Plant Biology  2009;9:139.
Apple fruitlet abscission is induced by dominance, a process in which hormones such as auxin, cytokinins and strigolactone play a pivotal role. The response to these hormones is controlled by transcription regulators such as Aux/IAA and ARR, whereas auxin transport is controlled by influx and efflux carriers.
Seven partial clones encoding auxin efflux carriers (MdPIN1_A, MdPIN1_B, MdPIN10_A, MdPIN10_B, MdPIN4, MdPIN7_A and MdPIN7_B), three encoding auxin influx carriers (MdLAX1, MdLAX2 and MdLAX3) and three encoding type A ARR cytokinin response regulators (MdARR3, MdARR4 and MdARR6) were isolated by the use of degenerate primers. The organization of the PIN multigene family in apple is closer to Medicago truncatula than to Arabidopsis thaliana. The genes are differentially expressed in diverse plant organs and at different developmental stages. MdPIN1 and MdPIN7 are largely more expressed than MdPIN10 and MdPIN4. During abscission, the transcription of these genes increased in the cortex whereas in the seed a sharp fall was observed. The expression of these genes was found to be at least partially controlled by ethylene and auxin.
The ethylene burst preceding abscission of fruitlets may be responsible for the decrease in transcript level of MDPIN1, MDARR5 and MDIAA3 in seed. This situation modulates the status of the fruitlet and its fate by hampering the PAT from the seeds down through the abscission zone (AZ) and this brings about the shedding of the fruitlet.
PMCID: PMC2809502  PMID: 19941659
19.  AtKinesin-13A is located on Golgi-associated vesicle and involved in vesicle formation/budding in Arabidopsis root-cap peripheral cells 
BMC Plant Biology  2009;9:138.
AtKinesin-13A is an internal-motor kinesin from Arabidopsis (Arabidopsis thaliana). Previous immunofluorescent results showed that AtKinesin-13A localized to Golgi stacks in plant cells. However, its precise localization and biological function in Golgi apparatus is unclear.
In this paper, immunofluorescent labeling and confocal microscopic observation revealed that AtKinesin-13A was co-localized with Golgi stacks in Arabidopsis root tip cells. Immuno-electron microscopic observations indicated that AtKinesin-13A is primarily localized on Golgi-associated vesicles in Arabidopsis root-cap cells. By T-DNA insertion, the inactivation of the AtKinesin-13A gene (NM-112536) resulted in a sharp decrease of size and number of Golgi vesicles in root-cap peripheral cells. At the same time, these cells were vacuolated in comparison to the corresponding cells of the wild type.
These results suggest that AtKinesin-13A decorates Golgi-associated vesicles and may be involved in regulating the formation of Golgi vesicles in the root-cap peripheral cells in Arabidopsis.
PMCID: PMC2790454  PMID: 19939242
20.  Characterization of microsatellites and gene contents from genome shotgun sequences of mungbean (Vigna radiata (L.) Wilczek) 
BMC Plant Biology  2009;9:137.
Mungbean is an important economical crop in Asia. However, genomic research has lagged behind other crop species due to the lack of polymorphic DNA markers found in this crop. The objective of this work is to develop and characterize microsatellite or simple sequence repeat (SSR) markers from genome shotgun sequencing of mungbean.
We have generated and characterized a total of 470,024 genome shotgun sequences covering 100.5 Mb of the mungbean (Vigna radiata (L.) Wilczek) genome using 454 sequencing technology. We identified 1,493 SSR motifs that could be used as potential molecular markers. Among 192 tested primer pairs in 17 mungbean accessions, 60 loci revealed polymorphism with polymorphic information content (PIC) values ranging from 0.0555 to 0.6907 with an average of 0.2594. Majority of microsatellite markers were transferable in Vigna species, whereas transferability rates were only 22.90% and 24.43% in Phaseolus vulgaris and Glycine max, respectively. We also used 16 SSR loci to evaluate phylogenetic relationship of 35 genotypes of the Asian Vigna group. The genome survey sequences were further analyzed to search for gene content. The evidence suggested 1,542 gene fragments have been sequence tagged, that fell within intersected existing gene models and shared sequence homology with other proteins in the database. Furthermore, potential microRNAs that could regulate developmental stages and environmental responses were discovered from this dataset.
In this report, we provided evidence of generating remarkable levels of diverse microsatellite markers and gene content from high throughput genome shotgun sequencing of the mungbean genomic DNA. The markers could be used in germplasm analysis, accessing genetic diversity and linkage mapping of mungbean.
PMCID: PMC2788553  PMID: 19930676
21.  Proteins with an Euonymus lectin-like domain are ubiquitous in Embryophyta 
BMC Plant Biology  2009;9:136.
Cloning of the Euonymus lectin led to the discovery of a novel domain that also occurs in some stress-induced plant proteins. The distribution and the diversity of proteins with an Euonymus lectin (EUL) domain were investigated using detailed analysis of sequences in publicly accessible genome and transcriptome databases.
Comprehensive in silico analyses indicate that the recently identified Euonymus europaeus lectin domain represents a conserved structural unit of a novel family of putative carbohydrate-binding proteins, which will further be referred to as the Euonymus lectin (EUL) family. The EUL domain is widespread among plants. Analysis of retrieved sequences revealed that some sequences consist of a single EUL domain linked to an unrelated N-terminal domain whereas others comprise two in tandem arrayed EUL domains. A new classification system for these lectins is proposed based on the overall domain architecture. Evolutionary relationships among the sequences with EUL domains are discussed.
The identification of the EUL family provides the first evidence for the occurrence in terrestrial plants of a highly conserved plant specific domain. The widespread distribution of the EUL domain strikingly contrasts the more limited or even narrow distribution of most other lectin domains found in plants. The apparent omnipresence of the EUL domain is indicative for a universal role of this lectin domain in plants. Although there is unambiguous evidence that several EUL domains possess carbohydrate-binding activity further research is required to corroborate the carbohydrate-binding properties of different members of the EUL family.
PMCID: PMC2788552  PMID: 19930663
22.  Association mapping and marker-assisted selection of the lettuce dieback resistance gene Tvr1 
BMC Plant Biology  2009;9:135.
Lettuce (Lactuca saliva L.) is susceptible to dieback, a soilborne disease caused by two viruses from the family Tombusviridae. Susceptibility to dieback is widespread in romaine and leaf-type lettuce, while modern iceberg cultivars are resistant to this disease. Resistance in iceberg cultivars is conferred by Tvr1 - a single, dominant gene that provides durable resistance. This study describes fine mapping of the resistance gene, analysis of nucleotide polymorphism and linkage disequilibrium in the Tvr1 region, and development of molecular markers for marker-assisted selection.
A combination of classical linkage mapping and association mapping allowed us to pinpoint the location of the Tvr1 resistance gene on chromosomal linkage group 2. Nine molecular markers, based on expressed sequence tags (EST), were closely linked to Tvr1 in the mapping population, developed from crosses between resistant (Salinas and Salinas 88) and susceptible (Valmaine) cultivars. Sequencing of these markers from a set of 68 cultivars revealed a relatively high level of nucleotide polymorphism (θ = 6.7 × 10-3) and extensive linkage disequilibrium (r2 = 0.124 at 8 cM) in this region. However, the extent of linkage disequilibrium was affected by population structure and the values were substantially larger when the analysis was performed only for romaine (r2 = 0.247) and crisphead (r2 = 0.345) accessions. The association mapping approach revealed that one of the nine markers (Cntg10192) in the Tvr1 region matched exactly with resistant and susceptible phenotypes when tested on a set of 200 L. sativa accessions from all horticultural types of lettuce. The marker-trait association was also confirmed on two accessions of Lactuca serriola - a wild relative of cultivated lettuce. The combination of three single-nucleotide polymorphisms (SNPs) at the Cntg10192 marker identified four haplotypes. Three of the haplotypes were associated with resistance and one of them was always associated with susceptibility to the disease.
We have successfully applied high-resolution DNA melting (HRM) analysis to distinguish all four haplotypes of the Cntg10192 marker in a single analysis. Marker-assisted selection for dieback resistance with HRM is now an integral part of our breeding program that is focused on the development of improved lettuce cultivars.
PMCID: PMC2789080  PMID: 19930659
23.  Genome-wide analysis of major intrinsic proteins in the tree plant Populus trichocarpa: Characterization of XIP subfamily of aquaporins from evolutionary perspective 
BMC Plant Biology  2009;9:134.
Members of major intrinsic proteins (MIPs) include water-conducting aquaporins and glycerol-transporting aquaglyceroporins. MIPs play important role in plant-water relations. The model plants Arabidopsis thaliana, rice and maize contain more than 30 MIPs and based on phylogenetic analysis they can be divided into at least four subfamilies. Populus trichocarpa is a model tree species and provides an opportunity to investigate several tree-specific traits. In this study, we have investigated Populus MIPs (PtMIPs) and compared them with their counterparts in Arabidopsis, rice and maize.
Fifty five full-length MIPs have been identified in Populus genome. Phylogenetic analysis reveals that Populus has a fifth uncharacterized subfamily (XIPs). Three-dimensional models of all 55 PtMIPs were constructed using homology modeling technique. Aromatic/arginine (ar/R) selectivity filters, characteristics of loops responsible for solute selectivity (loop C) and gating (loop D) and group conservation of small and weakly polar interfacial residues have been analyzed. Majority of the non-XIP PtMIPs are similar to those in Arabidopsis, rice and maize. Additional XIPs were identified from database search and 35 XIP sequences from dicots, fungi, moss and protozoa were analyzed. Ar/R selectivity filters of dicots XIPs are more hydrophobic compared to fungi and moss XIPs and hence they are likely to transport hydrophobic solutes. Loop C is longer in one of the subgroups of dicot XIPs and most probably has a significant role in solute selectivity. Loop D in dicot XIPs has higher number of basic residues. Intron loss is observed on two occasions: once between two subfamilies of eudicots and monocot and in the second instance, when dicot and moss XIPs diverged from fungi. Expression analysis of Populus MIPs indicates that Populus XIPs don't show any tissue-specific transcript abundance.
Due to whole genome duplication, Populus has the largest number of MIPs identified in any single species. Non-XIP MIPs are similar in all four plant species considered in this study. Small and weakly polar residues at the helix-helix interface are group conserved presumably to maintain the hourglass fold of MIP channels. Substitutions in ar/R selectivity filter, insertion/deletion in loop C, increasing basic nature of loop D and loss of introns are some of the events occurred during the evolution of dicot XIPs.
PMCID: PMC2789079  PMID: 19930558
24.  In vivo imaging of the tonoplast intrinsic protein family in Arabidopsis roots 
BMC Plant Biology  2009;9:133.
Tonoplast intrinsic proteins (TIPs) are widely used as markers for vacuolar compartments in higher plants. Ten TIP isoforms are encoded by the Arabidopsis genome. For several isoforms, the tissue and cell specific pattern of expression are not known.
We generated fluorescent protein fusions to the genomic sequences of all members of the Arabidopsis TIP family whose expression is predicted to occur in root tissues (TIP1;1 and 1;2; TIP2;1, 2;2 and 2;3; TIP4;1) and expressed these fusions, both individually and in selected pairwise combinations, in transgenic Arabidopsis. Analysis by confocal microscopy revealed that TIP distribution varied between different cell layers within the root axis, with extensive co-expression of some TIPs and more restricted expression patterns for other isoforms. TIP isoforms whose expression overlapped appeared to localise to the tonoplast of the central vacuole, vacuolar bulbs and smaller, uncharacterised structures.
We have produced a comprehensive atlas of TIP expression in Arabidopsis roots, which reveals novel expression patterns for not previously studied TIPs.
PMCID: PMC2784467  PMID: 19922653
25.  Genome scale transcriptome analysis of shoot organogenesis in Populus 
BMC Plant Biology  2009;9:132.
Our aim is to improve knowledge of gene regulatory circuits important to dedifferentiation, redifferentiation, and adventitious meristem organization during in vitro regeneration of plants. Regeneration of transgenic cells remains a major obstacle to research and commercial deployment of most taxa of transgenic plants, and woody species are particularly recalcitrant. The model woody species Populus, due to its genome sequence and amenability to in vitro manipulation, is an excellent species for study in this area. The genes recognized may help to guide the development of new tools for improving the efficiency of plant regeneration and transformation.
We analyzed gene expression during poplar in vitro dedifferentiation and shoot regeneration using an Affymetrix array representing over 56,000 poplar transcripts. We focused on callus induction and shoot formation, thus we sampled RNAs from tissues: prior to callus induction, 3 days and 15 days after callus induction, and 3 days and 8 days after the start of shoot induction. We used a female hybrid white poplar clone (INRA 717-1 B4, Populus tremula × P. alba) that is used widely as a model transgenic genotype. Approximately 15% of the monitored genes were significantly up-or down-regulated when controlling the false discovery rate (FDR) at 0.01; over 3,000 genes had a 5-fold or greater change in expression. We found a large initial change in expression after the beginning of hormone treatment (at the earliest stage of callus induction), and then a much smaller number of additional differentially expressed genes at subsequent regeneration stages. A total of 588 transcription factors that were distributed in 45 gene families were differentially regulated. Genes that showed strong differential expression included components of auxin and cytokinin signaling, selected cell division genes, and genes related to plastid development and photosynthesis. When compared with data on in vitro callogenesis in Arabidopsis, 25% (1,260) of up-regulated and 22% (748) of down-regulated genes were in common with the genes regulated in poplar during callus induction.
The major regulatory events during plant cell organogenesis occur at early stages of dedifferentiation. The regulatory circuits reflect the combinational effects of transcriptional control and hormone signaling, and associated changes in light environment imposed during dedifferentiation.
PMCID: PMC2784466  PMID: 19919717

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