Thaliana Ecotype Columbia Research Articles

Mutagenic effects of carbon-ion irradiation on dry Arabidopsis thaliana seeds

To investigate the mutagenic effects of carbon ions on Arabidopsis thaliana (ecotype Columbia) and to isolate useful genes in plant development, dry seeds were exposed to 43MeV/u carbon ions at doses of 0, 100, 200, 300, 400, 500 and 600Gy. The survival rate, primary root length, and hypocotyl length of M1 plants were analyzed, and 200Gy was selected as the dose for the large-scale experiment. A total of 1363 lines of plants from 28,062 M2 populations displayed alterations in the leaf, stem, flower, or life cycle, with abnormal leaves and a premature life cycle as the main phenotypic variations. The mutated gene loci of five stable and inheritable mutations were roughly mapped on chromosomes. Novel mutants were obtained, although some of the mutants were similar to mutants induced by ethylmethane sulfonate (EMS) according to previous studies. This study provides a large body of specific information describing A. thaliana mutation phenotypes that were induced by carbon-ion irradiation. These results suggest that carbon-ion beams are as useful and effective as other mutagens for mutant breeding in plants, and that they will allow mutant breeding that is more diversified.

Light-harvesting complex B7 shifts the irradiance response of photosynthetic light-harvesting regulation in leaves of Arabidopsis thaliana

The nuclear LHCB7 gene is common in higher plants, encodes a transcript that is well expressed in a subset of leaf mesophyll cells, and is associated with a protein product that is homologous to pigment-binding components of the photosystem (PS) II peripheral antenna complex. We compared the physiological properties of wild type and LHCB7-deficient leaves [DNA insertion, Arabidopsis thaliana (At) ecotype Columbia] in terms of pigment content, CO2 exchange, in vivo transmittance at 810nm, and chlorophyll fluorescence. The latter two techniques are functional indicators for PSI and PSII, respectively. Key features of the mutant phenotype were confirmed using antisense technology and a hemizygote of two independent AtLHCB7 DNA insertion lines. Growth, leaf pigment composition, white light absorptance, and levels of AtLHCB1-6 were not significantly different in the mutant compared to wild type. Likewise, neither intrinsic PSII light capture efficiency nor partitioning of absorbed radiation to PSII was affected by the mutation. The absence of AtLHCB7 is associated with lower rates of light-saturated photosynthesis and a diminished irradiance threshold for induction of photoprotective non-photochemical quenching. Overall, the pattern of change in light utilization parameters and plastoquinol level indicated that loss of AtLHCB7 expression led to slower Rubisco turnover characterized by pH-dependent balancing of electron transport to reduced carbon assimilation capacity (photosynthetic control). No effect of AtLHCB7 genotype on xanthophyll de-epoxidation state was detected suggesting that factors in addition to lumenal pH influence zeaxanthin accumulation.

Comparative transcriptome analysis of energy-rich Arabidopsis thaliana under dark and light conditions

Overexpression of Arabidopsis thaliana purple acid phosphatases AtPAP2 in Arabidopsis can promote plant growth. The overexpression (OE) lines flower earlier, grow faster, and contain more ATP, sucrose and glucose. The seed yields and silique numbers of OE lines are also more than the control lines (Sun et al., 2012a). In this study, we compared the leaf transcriptomes of 20-d-old transgenic and wild-type Arabidopsis grown under long day (16h/8h) condition. Total RNAs were collected at three time points: end of night (t=0 hr), one hour after light was turned on (t=1 hr), and eight hours after light was turned on (t=8 hr). AtPAP2 is dually targeted to chloroplasts and mitochondria. To study the RNA encoded by chloroplast and mitochondrial genomes, ribosomal RNAs were removed before sequencing. Approximate 65 million clean reads (~6Gbp) were obtained by Illumina HiSeqTM 2000 sequencing from each library. In total, after assembly 29,435 transcripts are detected from the six libraries. More genes are suppressed in OE leaves (vs. WT) at all the three time points, 1,623 (downregulated) versus 945 (up-regulated), 1,908 (down-regulated) versus 712 (up-regulated) and 1,642 (down-regulated) versus 824 (up-regulated) at t=0, 1 and 8 hours, respectively. Expression profiles of light-induced transcripts based on K-means clustering were analyzed. There are significant changes in the transcription levels of various components of photosystems, light-harvesting chlorophyll protein complexes (LHC), respiratory complexes, RNA polymerases and ribosomes. Our data provide systemic portraits of global changes in Arabidopsis transcriptome exerted by dark to light transition (external energy input) and high internal energy status. Comparative transcriptome analysis of energy-rich Arabidopsis thaliana under dark and light conditions Chao LIANG and Boon Leong LIM* vechao@hku.hk School of Biological Sciences, the University of Hong Kong, Hong Kong, China Fig. 1 Growth phenotypes of four-wk-old Arabidopsis under long day (16hr light/8hr dark)) conditions. WT, wild type; T-DNA, AtPAP2 mutant line; OE, AtPAP2 over-expression lines (Sun et al., 2012b). Arabidopsis thaliana ecotypes Columbia (Col-0) (wild type: WT), AtPAP2 overexpressors (OE) in Col-0 background were used in this study (Sun et al. 2012b) . Arabidopsis seeds of WT and OE7 lines were surface sterilized and plated on Murashige and Skoog medium supplemented with 2% (w/v) sucrose for 10 days before the seedling were transferred to soil under 16 hours light (22°C) / 8 hours dark (18°C) period in a growth chamber at a light intensity of 120–150 μmol m-2 s-1. Leaves of 20-day-old plants without bolting were immediately frozen in liquid nitrogen for RNA extraction. Leaves were harvested at three different time points: t=0 hr (end of night), t=1 hr (one hour after light turn on) and t=8 hr (eight hours after light turn on) respectively. Flowchart Total RNA Removal of Ribosome RNA Raw Data Allignment with reference gene and genome file (SOAP aligner/SOAP2) Clean Reads QC Expression level(RPKM) Cluster analysisc Gene ontology analysisc KEGG pathway analysis Background Methodology

Identification of MicroRNA 395a in 24-Epibrassinolide-Regulated Root Growth of Arabidopsis thaliana Using MicroRNA Arrays

Brassinosteroids (BRs) are endogenous plant hormones and are essential for normal plant growth and development. MicroRNAs (miRNAs) of Arabidopsis thaliana are involved in mediating cell proliferation in leaves, stress tolerance, and root development. The specifics of BR mechanisms involving miRNAs are unknown. Using customized miRNA array analysis, we identified miRNAs from A. thaliana ecotype Columbia (Col-0) regulated by 24-epibrassinolide (EBR, a highly active BR). We found that miR395a was significantly up-regulated by EBR treatment and validated its expression under these conditions. miR395a was over expressed in leaf veins and root tissues in EBR-treated miR395a promoter::GUS plants. We integrated bioinformatics methods and publicly available DNA microarray data to predict potential targets of miR395a. GUN5—a multifunctional protein involved in plant metabolic functions such as chlorophyll synthesis and the abscisic acid (ABA) pathway—was identified as a possible target. ABI4 and ABI5, both genes positively regulated by ABA, were down-regulated by EBR treatment. In summary, our results suggest that EBR regulates seedling development and root growth of A. thaliana through miR395a by suppressing GUN5 expression and its downstream signal transduction.

Production of a High-Efficiency TILLING Population through Polyploidization

Targeting Induced Local Lesions in Genomes (TILLING) provides a nontransgenic method for reverse genetics that is widely applicable, even in species where other functional resources are missing or expensive to build. The efficiency of TILLING, however, is greatly facilitated by high mutation density. Species vary in the number of mutations induced by comparable mutagenic treatments, suggesting that genetic background may affect the response. Allopolyploid species have often yielded higher mutation density than diploids. To examine the effect of ploidy, we autotetraploidized the Arabidopsis (Arabidopsis thaliana) ecotype Columbia, whose diploid has been used for TILLING extensively, and mutagenized it with 50 mm ethylmethane sulfonate. While the same treatment sterilized diploid Columbia, the tetraploid M1 plants produced good seed. To determine the mutation density, we searched 528 individuals for induced mutations in 15 genes for which few or no knockout alleles were previously available. We constructed tridimensional pools from the genomic DNA of M2 plants, amplified target DNA, and subjected them to Illumina sequencing. The results were analyzed with an improved version of the mutation detection software CAMBa that accepts any pooling scheme. This small population provided a rich resource with approximately 25 mutations per queried 1.5-kb fragment, including on average four severe missense and 1.3 truncation mutations. The overall mutation density of 19.4 mutations Mb(-1) is 4 times that achieved in the corresponding diploid accession, indicating that genomic redundancy engenders tolerance to high mutation density. Polyploidization of diploids will allow the production of small populations, such as less than 2,000, that provide allelic series from knockout to mild loss of function for virtually all genes.

<i>At</i>L1 a Non-LTR Retrotrasposon Fragment in the Genome of <i>Arabidopsis thaliana </i> with Homology to Plants and Animals

We report the isolation of AtL1, a 249 bp non-LTR retrotransposon fragment from Arabidopsis thaliana by fingerprinting mRNAs extracted from A. thaliana plants, ecotype Columbia, in different heat stress conditions. Southern blot and PCR analysis suggested that AtL1 occurs as a single- or low-copy insert in the genome of A. thaliana ecotype Columbia. The presence of AtL1 in the genome of different Arabidopsis ecotypes was confirmed by PCR amplification and sequencing thus excluding all possible contamination. A preliminary scan of the AtL1 nucleotide sequence against the EMBL and NCBI databases revealed a high degree of similarity to a group of LINE type L1 retrotransposons of mammals and with a cDNA sequence of Artemisia annua. A phylogenetic analysis of LINE elements from animals and plants placed AtL1 and A. annua sequences in close proximity to some mammalian sequences but distant from the other plants LINE elements including those from Arabidopsis.

Chromosome variations in regenerants of Arabidopsis thaliana derived from 2- and 6-week-old callus detected using flow cytometry and FISH analyses

Shoot organogenesis was induced from 2- and 6-week-old callus derived from the leaves of Arabidopsis thaliana ecotype Columbia (2n = 10). Regenerated plants were evaluated for chromosomal variations by means of flow cytometry and fluorescent in situ hybridization (FISH). Flow cytometric measurements revealed the occurrence of diploid, tetraploid, and octoploid plants among the regenerants of 2-week-old calli, whereas only diploid and tetraploid plants were regenerated from the 6-week-old calli. Chromosome counting showed that plants developed from the 2-week-old calli exhibited mixoploidy and a high frequency of aneuploid cells. These plants were infertile and displayed altered morphology. FISH with 5S and 25S rDNA probes allowed to detect some structural chromosomal rearrangements in regenerated plants. Along with cells which exhibited correct localisation of rDNA loci, also cells bearing chromosomal translocations, deletions or duplications were found. The type of structural aberrations varied between diploid and tetraploid regenerants.

Antisense reductions in the PsbO protein of photosystem II leads to decreased quantum yield but similar maximal photosynthetic rates.

Photosystem (PS) II is the multisubunit complex which uses light energy to split water, providing the reducing equivalents needed for photosynthesis. The complex is susceptible to damage from environmental stresses such as excess excitation energy and high temperature. This research investigated the in vivo photosynthetic consequences of impairments to PSII in Arabidopsis thaliana (ecotype Columbia) expressing an antisense construct to the PsbO proteins of PSII. Transgenic lines were obtained with between 25 and 60% of wild-type (WT) total PsbO protein content, with the PsbO1 isoform being more strongly reduced than PsbO2. These changes coincided with a decrease in functional PSII content. Low PsbO (less than 50% WT) plants grew more slowly and had lower chlorophyll content per leaf area. There was no change in content per unit area of cytochrome b6f, ATP synthase, or Rubisco, whereas PSI decreased in proportion to the reduction in chlorophyll content. The irradiance response of photosynthetic oxygen evolution showed that low PsbO plants had a reduced quantum yield, but matched the oxygen evolution rates of WT plants at saturating irradiance. It is suggested that these plants had a smaller pool of PSII centres, which are inefficiently connected to antenna pigments resulting in reduced photochemical efficiency.

Induced Systemic Drought and Salt Tolerance by Pseudomonas chlororaphis O6 Root Colonization is Mediated by ABA-independent Stomatal Closure

Institute of Environmentally-Friendly Agriculture, Department of Plant Biotechnology, Chonnam National University, Gwangju500-757, Korea (Received on November 13, 2011; Revised on January 5, 2012; Accepted on January 11, 2012)Root colonization by the rhizobacterium Pseudomonaschlororaphis O6 in Arabidopsis thaliana Col-0 plantsresulted in induced tolerance to drought and salinitycaused by halide salt-generated ionic stress but not byosmotic stress caused by sorbitol. Stomatal aperturesdecreased following root colonization by P. chlororaphisO6 in both wild-type and ABA-insensitive Arabidopsismutant plants. These results suggest that an ABA-independent stomatal closure mechanism in the guardcells of P. chlororaphis O6-colonized plants could be akey phenotype for induced systemic tolerance todrought and salt stress. Keywords : abiotic stress, induced tolerance to environ-mental stress, priming, stomatal closurePlants possess various survival systems against abioticstresses, such as cold, drought, and salinity (Zhu, 2001).Under environmental stresses, the level of the planthormone, abscisic acid (ABA) increases triggering adaptiveresponses essential for survival (Zhu, 2001). Duringdrought and salt stresses, ABA induces stomatal closure tominimize water loss through transpiration (Leung andGiraudat, 1998). Consequently, ABA-biosynthesis mutants(aba mutants) and some of the ABA-response plantmutants (i.e. the ABA-insensitive abi mutants) aresusceptible to drought stress, due to problem of stomatalaperture regulation (Leung and Giraudat, 1998; Schroederet al., 2001). However, another plant growth regulator,jasmonate (Evans, 2003; Suhita et al., 2004), also stimu-lates stomatal closure under drought conditions (Creelmanand Mullet, 1997).Root colonization of certain plant-associated microbeselicits physiological and biochemical change to enhancesystemic resistance against various biotic and abioticstresses in plants (Kim et al., 2011; Yang et al., 2010),termed to as “induced systemic resistance (ISR)” or“induced systemic tolerance (IST)”. IST against drought orsalt stresses in plants can be induced with systemicapplication of certain rhizobacteria including Gram-positive Bacillus strains (Ryu et al., 2004; Timmusk andWagner 1999; Zhang et al., 2010), an endophytic fungalisolate, Trichoderma harziarum (Bae et al., 2010), andcertain Gram-negative bacterial isolates, such as ACC-deaminase producing bacteria (Mayak et al., 2004). Rootcolonization by Pseudomonas chlororaphis O6 inducedsystemic resistance against a broad spectrum of plantdiseases caused by viral, bacterial, and fungal pathogens invarious plants by jasmonic acid-ethylene related pathways(Cho et al., 2008; Kim et al., 2004; Ryu et al., 2007;Spencer et al., 2003). Additionally, root colonization by P.chlororaphis O6 induced systemic tol erance against drought,a process accompanied by stomatal closure. Applying2R,3R-butanediol, a volatile produced by P. chlororaphisO6, resulted in induced tolerance through a salicylic acid(SA), jasmonic acid (JA) and ethylene-dependent mech-anism (Cho et al., 2008). However, mechanisms involvedin microbe-mediated IST against drought have not beencharacterized or elucidated.In this study, we tested the hypothesis that P. chloro-raphis O6 induces tolerance to other abiotic stresses, suchas salinity, osmotic pressure, cold and heat. We used addi-tional Arabidopsis mutants altered in ABA signalingpathways to identify the role of ABA in induced abioticstress tolerance. Parental A. thaliana ecotypes Columbia(Col-0) or Landsberg erecta (Ler), and mutant and trans-genic lines were obtained from the Ohio State University

Root system morphology and primary root anatomy in natural non-metallicolous and metallicolous populations of three Arabidopsis species differing in heavy metal tolerance

AbstractRoot system morphology was characterized in the seedlings of heavy-metal sensitive Arabidopsis thaliana, the non-metallicolous (NM) and metallicolous (M) populations of the tolerant A. arenosa and A. halleri, developed on the natural soils: the Zn-Pb-Cd-Cu-contaminated (C soils), the non-contaminated (NC soils), and on an identical nutrient-rich compost. Anatomy of primary roots grown on agar medium with control and elevated zinc concentrations was investigated also in the model A. thaliana ecotype Columbia.The three Arabidopsis species differed in morphological and/or quantitative responses to the varying soil qualities. Comparing to natural NC soil, the morphology of A. thaliana root system differed only on the compost with dominating lateral root lengths while the root lengths were reduced on the C soil. In NM and M populations of A. arenosa the lateral root elongation and density were reduced on the C soil and root growth but not lateral root density were stimulated on the compost. In NM and M populations of A. halleri the root system morphology remained unaltered in all three soils. The root elongation was reduced but lateral root initiation increased on the C soil while the roots were longer and lateral root density lower on the compost. The responses of A. arenosa or A. halleri populations differed only in absolute root lengths. The similarity in morphological responses to varying soil metal contents indicated plastic responses rather than heritable traits of the root systems.The root tissue organization three Arabidopsis species resembled the known A. thaliana ecotype Columbia. Quantitatively, the tolerant species and their M populations had thicker roots due to a greater number and size of cells in epidermis, cortex including a higher number of middle cortex cells, and endodermis. The rates of root growth and quantitative root anatomy may represent morphological traits contributing to heavy metal tolerance of the Arabidopsis species.

Expression analysis of MAP2K9 and MAPK6 during pathogenesis of Alternaria blight in Arabidopsis thaliana ecotype Columbia

Arabidopsis thaliana ecotype Columbia was used as a host in order to investigate the involvement of MAP kinase machinery in the pathogenesis of Alternaria blight. Semi-quantitative reverse transcriptase polymerase chain reaction and quantitative real time PCR based approaches were used to determine the change in transcript profile of MAP2K9 and MAPK6 in leaves of A. thaliana ecotpe Columbia at early, middle and late stages of Alternaria blight infection. It was observed that the expression of both MAP2K9 and MAPK6 simultaneously increased up to middle stage of disease progression. There was observed a positive correlation between the expression of MAPK6 and MAP2K9 as disease progressed from initial to middle stage of infection. Then, the expression of MAP2K9 decreased and that of MAPK6 increased as disease progressed towards late stage of infection. The increased levels of MAP2K9 and MAPK6, seem to be necessary for plant to defend the pathogen up to middle stage of infection. However, MAP2K9 may be down regulated at late stage of infection by pathogen to promote it's efficient colonization. Since MAPK6 expression remains unaltered till late stage, it suggests that it's expression is not only regulated by MAP2K9 but also by other MAP2K's. The above results are consistent with observations of earlier studies. In conclusion, the present study has suggested MAP2K9/MAPK6 module as possible target, which is influenced during pathogenesis of Alternaria blight in A. thaliana ecotype Columbia. Hence genetic modulation in expression levels of these components in Arabidopsis or Brassica could be a possible strategy for engineering defense against Alternaria blight disease.

Phytoplasma effector SAP54 induces indeterminate leaf-like flower development in Arabidopsis plants.

Phytoplasmas are insect-transmitted bacterial plant pathogens that cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches' Broom (AY-WB) readily infects the model plant Arabidopsis (Arabidopsis thaliana) ecotype Columbia, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches' broom). We found that the majority of genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein, SAP54, that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen and reports to our knowledge the first example of a microbial pathogen effector protein that targets flower development in a host.

Arabidopsis Chloroplastic Glutaredoxin C5 as a Model to Explore Molecular Determinants for Iron-Sulfur Cluster Binding into Glutaredoxins

Unlike thioredoxins, glutaredoxins are involved in iron-sulfur cluster assembly and in reduction of specific disulfides (i.e. protein-glutathione adducts), and thus they are also important redox regulators of chloroplast metabolism. Using GFP fusion, AtGrxC5 isoform, present exclusively in Brassicaceae, was shown to be localized in chloroplasts. A comparison of the biochemical, structural, and spectroscopic properties of Arabidopsis GrxC5 (WCSYC active site) with poplar GrxS12 (WCSYS active site), a chloroplastic paralog, indicated that, contrary to the solely apomonomeric GrxS12 isoform, AtGrxC5 exists as two forms when expressed in Escherichia coli. The monomeric apoprotein possesses deglutathionylation activity mediating the recycling of plastidial methionine sulfoxide reductase B1 and peroxiredoxin IIE, whereas the dimeric holoprotein incorporates a [2Fe-2S] cluster. Site-directed mutagenesis experiments and resolution of the x-ray crystal structure of AtGrxC5 in its holoform revealed that, although not involved in its ligation, the presence of the second active site cysteine (Cys(32)) is required for cluster formation. In addition, thiol titrations, fluorescence measurements, and mass spectrometry analyses showed that, despite the presence of a dithiol active site, AtGrxC5 does not form any inter- or intramolecular disulfide bond and that its activity exclusively relies on a monothiol mechanism.

K252a-sensitive protein kinases but not okadaic acid-sensitive protein phosphatases regulate methyl jasmonate-induced cytosolic Ca 2+ oscillation in guard cells of Arabidopsis thaliana

Methyl jasmonate (MeJA) induces stomatal closure similar to abscisic acid (ABA), and MeJA signaling in guard cells shares some signal components with ABA signaling. As part of this process, MeJA as well as ABA induce the elevation and oscillation of cytosolic free-calcium concentrations ([Ca 2+] cyt) in guard cells. While abscisic acid-induced [Ca 2+] cyt oscillation has been extensively studied, MeJA-induced [Ca 2+] cyt oscillation is less well understood. In this study, we investigated the effects of K252a (a broad-range protein kinase inhibitor) and okadaic acid (OA, a protein phosphatase 1 and 2A inhibitor) on MeJA-induced [Ca 2+] cyt oscillation in guard cells of Arabidopsis thaliana ecotype Columbia expressing the Ca 2+ reporter yellow cameleon 3.6. The protein kinase inhibitor K252a abolished MeJA-induced stomatal closure and reduced MeJA-elicited [Ca 2+] cyt oscillation. The protein phosphatase inhibitor OA, on the other hand, did not inhibit these processes. These results suggest that MeJA signaling involves activation of K252a-sensitive protein kinases upstream of [Ca 2+] cyt oscillation but not activation of an OA-sensitive protein phosphatase in guard cells of A. thaliana ecotype Columbia.

A Novel Stress-Associated Protein ‘AtSAP10’ from Arabidopsis thaliana Confers Tolerance to Nickel, Manganese, Zinc, and High Temperature Stress

We describe here the functional characterization of a novel AtSAP10, a member of the Stress Associated Protein (SAP) gene family, from Arabidopsis thaliana ecotype Columbia. AtSAP10 contains an A20 and AN1 zinc-finger domain at the N- and C-terminal, respectively. Arabidopsis SAP10 showed differential regulation by various abiotic stresses such as heavy metals and metalloids (Ni, Cd, Mn, Zn, and As), high and low temperatures, cold, and ABA. Overexpression of AtSAP10 in Arabidopsis conferred strong tolerance to heavy metals such as Ni, Mn, and Zn and to high temperature stress. AtSAP10 transgenic plants under these stress conditions grew green and healthy, attained several-fold more biomass, and had longer roots as compared to wild type plants. Further, while these transgenic plants accumulated significantly greater amounts of Ni and Mn in both shoots and root tissues, there was no significant difference in the accumulation of Zn. AtSAP10 promoter-GUS fusion studies revealed a root and floral organ-specific expression of AtSAP10. Overexpression of AtSAP10-GFP fusion protein showed the localization in both nucleus and cytoplasm. Taken together, these results showed that AtSAP10 is a potentially useful candidate gene for engineering tolerance to heavy metals and to abiotic stress in cultivated plants.

Incomplete homogenization of 18 S ribosomal DNA coding regions in Arabidopsis thaliana

BackgroundAs a result of concerted evolution, coding regions of ribosomal DNA sequences are highly conserved within species and variation is generally thought to be limited to a few nucleotides. However, rDNA sequence variation has not been systematically examined in plant genomes, including that of the model plant Arabidopsis thaliana whose genome was the first to be sequenced.FindingsBoth genomic and transcribed 18 S sequences were sampled and revealed that most deviation from the consensus sequence was limited to single nucleotide substitutions except for a variant with a 270 bp deletion from position 456 to 725 in Arabidopsis numbering. The deletion maps to the functionally important and highly conserved 530 loop or helix18 in the structure of E. coli 16 S. The expression of the deletion variant is tightly controlled during developmental growth stages. Transcripts were not detectable in young seedlings but could be amplified from RNA extracts of mature leaves, stems, flowers and roots of Arabidopsis thaliana ecotype Columbia. We also show polymorphism for the deletion variant among four Arabidopsis ecotypes examined.ConclusionDespite a strong purifying selection that might be expected against functionally impaired rDNAs, the newly identified variant is maintained in the Arabidopsis genome. The expression of the variant and the polymorphism displayed by Arabidopsis ecotypes suggest a transition state in concerted evolution.

Role of aromatic aldehyde synthase in wounding/herbivory response and flower scent production in different Arabidopsis ecotypes

Aromatic L-amino acid decarboxylases (AADCs) are key enzymes operating at the interface between primary and secondary metabolism. The Arabidopsis thaliana genome contains two genes, At2g20340 and At4g28680, encoding pyridoxal 5'-phosphate-dependent AADCs with high homology to the recently identified Petunia hybrida phenylacetaldehyde synthase involved in floral scent production. The At4g28680 gene product was recently biochemically characterized as an L-tyrosine decarboxylase (AtTYDC), whereas the function of the other gene product remains unknown. The biochemical and functional characterization of the At2g20340 gene product revealed that it is an aromatic aldehyde synthase (AtAAS), which catalyzes the conversion of phenylalanine and 3,4-dihydroxy-L-phenylalanine to phenylacetaldehyde and dopaldehyde, respectively. AtAAS knock-down and transgenic AtAAS RNA interference (RNAi) lines show significant reduction in phenylacetaldehyde levels and an increase in phenylalanine, indicating that AtAAS is responsible for phenylacetaldehyde formation in planta. In A. thaliana ecotype Columbia (Col-0), AtAAS expression was highest in leaves, and was induced by methyl jasmonate treatment and wounding. Pieris rapae larvae feeding on Col-0 leaves resulted in increased phenylacetaldehyde emission, suggesting that the emitted aldehyde has a defensive activity against attacking herbivores. In the ecotypes Sei-0 and Di-G, which emit phenylacetaldehyde as a predominant flower volatile, the highest expression of AtAAS was found in flowers and RNAi AtAAS silencing led to a reduction of phenylacetaldehyde formation in this organ. In contrast to ecotype Col-0, no phenylacetaldehyde accumulation was observed in Sei-0 upon wounding, suggesting that AtAAS and subsequently phenylacetaldehyde contribute to pollinator attraction in this ecotype.

Sequence-based physical mapping of complex genomes by whole genome profiling

We present whole genome profiling (WGP), a novel next-generation sequencing-based physical mapping technology for construction of bacterial artificial chromosome (BAC) contigs of complex genomes, using Arabidopsis thaliana as an example. WGP leverages short read sequences derived from restriction fragments of two-dimensionally pooled BAC clones to generate sequence tags. These sequence tags are assigned to individual BAC clones, followed by assembly of BAC contigs based on shared regions containing identical sequence tags. Following in silico analysis of WGP sequence tags and simulation of a map of Arabidopsis chromosome 4 and maize, a WGP map of Arabidopsis thaliana ecotype Columbia was constructed de novo using a six-genome equivalent BAC library. Validation of the WGP map using the Columbia reference sequence confirmed that 350 BAC contigs (98%) were assembled correctly, spanning 97% of the 102-Mb calculated genome coverage. We demonstrate that WGP maps can also be generated for more complex plant genomes and will serve as excellent scaffolds to anchor genetic linkage maps and integrate whole genome sequence data.

Ethylene suppression of sugar-induced anthocyanin pigmentation in Arabidopsis.

Anthocyanin accumulation is regulated negatively by ethylene signaling and positively by sugar and light signaling. However, the antagonistic interactions underlying these signalings remain to be elucidated fully. We show that ethylene inhibits anthocyanin accumulation induced by sucrose (Suc) and light by suppressing the expression of transcription factors that positively regulate anthocyanin biosynthesis, including GLABRA3, TRANSPARENT TESTA8, and PRODUCTION OF ANTHOCYANIN PIGMENT1, while stimulating the concomitant expression of the negative R3-MYB regulator MYBL2. Genetic analyses show that the ethylene-mediated suppression of anthocyanin accumulation is dependent upon ethylene signaling components responsible for the triple response. Furthermore, these positive and negative signaling pathways appear to be under photosynthetic control. Suc and light induction of anthocyanin accumulation was almost fully inhibited in wild-type Arabidopsis (Arabidopsis thaliana) ecotype Columbia and ethylene (ethylene response1 [etr1-1]) and light (long hypocotyl1 [hy1], cryptochrome1/2, and hy5) signaling mutants treated with the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The transcript level of the sugar transporter gene SUC1 was enhanced in ecotype Columbia treated with the ethylene-binding inhibitor silver and in etr1-1, ethylene insensitive2 (ein2-1), and ein3 ein3-like1 mutants. In contrast, 3-(3,4-dichlorophenyl)-1,1-dimethylurea treatment reduced SUC1 expression, which indicates strongly that SUC1 represents an integrator for signals provided by sugar, light, and ethylene. SUC1 mutations lowered accumulations of anthocyanin pigment, soluble sugar content, and ethylene production in response to Suc and light signals. These data demonstrate that the suppression of SUC1 expression by ethylene inhibits Suc-induced anthocyanin accumulation in the presence of light and, hence, fine-tunes anthocyanin homeostasis.

The subtelomeric region of the Arabidopsis thaliana chromosome IIIR contains potential genes and duplicated fragments from other chromosomes

The subtelomere and a portion of the associated telomeric region (together named 3RTAS) of chromosome IIIR from the Arabidopsis thaliana ecotypes Columbia (Col) and Wassilewskija (Ws) were specifically amplified by polymerase chain reaction and subsequently cloned and sequenced. The centromere-proximal portion of 3RTAS from both ecotypes contained two newly identified potential genes, one encoding the chloroplast luminal 19-kDa protein precursor and the other encoding three potential alternatively spliced CCCH-type zinc finger proteins. The telomere-proximal portion of 3RTAS from the Col ecotype contained short duplicated fragments derived from chromosomes I, II, and III, and that from the Ws ecotype contained a duplicated fragment derived from chromosome V. Each duplicated fragment has diverged somewhat in sequence from that of the ectopic template. Small patches of homologous nucleotides were found within the flanking sequences of both the duplicated fragments and the corresponding ectopic template sequences. The structural characteristics of these duplicated fragments suggest that they are filler DNAs captured by non-homologous end joining during double-strand break repair. Our characterization of 3RTAS not only filled up a gap in the chromosome IIIR sequence of A. thaliana but also identified new genes with unknown functions.