Near-complete de novo assembly of Tricholoma bakamatsutake chromosomes revealed the structural divergence and differentiation of Tricholoma genomes.

Tricholoma bakamatsutake, which is an edible ectomycorrhizal fungus associated with Fagaceae trees, may have diverged before the other species in Tricholoma section Caligata. We generated a highly contiguous whole-genome sequence for T. bakamatsutake SF-Tf05 isolated in an Oak (Quercus salicina) forest in Japan. The assembly of high-fidelity long reads, with a median read length of 12.3 kb, resulted in 13 chromosome-sized contigs comprising 142,068,211 bases with an average guanine and cytosine (GC) content of 43.94%. The 13 chromosomes were predicted to encode 11,060 genes. A contig (122,566 bases) presumably containing the whole circular mitochondrial genome was also recovered. The chromosome-wide comparison of T. bakamatsutake and Tricholoma matsutake (TMA_r1.0) indicated that the basic number of chromosomes (13) was conserved, but the structures of the corresponding chromosomes diverged, with multiple inversions and translocations. Gene conservation and cluster analyses revealed at least 3 phylogenetic clades in Tricholoma section Caligata. Specifically, all T. bakamatsutake strains belonged to the "bakamatsutake" clade, which is most proximal to the "caligatum" clade consisting of Tricholoma caligatum and Tricholoma fulvocastaneum. The constructed highly contiguous nearly telomere-to-telomere genome sequence of a T. bakamatsutake isolate will serve as a fundamental resource for future research on the evolution and differentiation of Tricholoma species.

In Vitro Evaluation of the Reductase Activities of Human AKR1C3 Allelic Variants.

Aldo-keto reductase 1C3 (AKR1C3) plays a role in the detoxification and activation of clinical drugs by catalyzing reduction reactions. There are approximately 400 single-nucleotide polymorphisms (SNPs) in the AKR1C3 gene, but their impact on the enzyme activity is still unclear. This study aimed to clarify the effects of SNPs of AKR1C3 with more than 0.5% global minor allele frequency on the reductase activities for its typical substrates. Recombinant AKR1C3 wild-type and R66Q, E77G, C145Y, P180S, or R258C variants were constructed using insect Sf21 cells, and reductase activities for acetohexamide, doxorubicin, and loxoprofen by recombinant AKR1C3s were measured by liquid chromatography-tandem mass spectrometry. Among the variants tested, the C145Y variant showed remarkably low (6%-14% of wild type) intrinsic clearances of reductase activities for all three drugs. Reductase activities of these three drugs were measured using 34 individual Japanese liver cytosols, revealing that heterozygotes of the SNP g.55101G>A tended to show lower reductase activities for three drugs than homozygotes of the wild type. Furthermore, genotyping of the SNP g.55101G>A causing C145Y in 96 Caucasians, 166 African Americans, 192 Koreans, and 183 Japanese individuals was performed by polymerase chain reaction-restriction fragment length polymorphism. This allelic variant was specifically detected in Asians, with allele frequencies of 6.8% and 3.6% in Koreans and Japanese, respectively. To conclude, an AKR1C3 allele with the SNP g.55101G>A causing C145Y would be one of the causal factors for interindividual variabilities in the efficacy and toxicity of drugs reduced by AKR1C3. SIGNIFICANCE STATEMENT: This is the first study to clarify that the AKR1C3 allele with the SNP g.55101G>A causing C145Y results in a decrease in reductase activity. Since the allele was specifically observed in Asians, the allele would be a factor causing an interindividual variability in sensitivity of drug efficacy or toxicity of drugs reduced by AKR1C3 in Asians.

Development and Validation of a Proteomic Correlation Profiling Technique to Detect and Identify Enzymes Involved in Metabolism of Drugs of Concern.

To predict the variation of pharmacological or toxicological effect caused by pharmacokinetic variance, it is important to be able to detect previously unknown and unsuspected enzymes involved in drug metabolism. We investigated the use of proteomic correlation profiling (PCP) as a technique to identify the enzymes involved in metabolism of drugs of concern. By evaluating the metabolic activities of each enzyme (including isoforms of cytochrome P450, uridine 5' diphospho-glucuronosyltransferase, and hydrolases, plus aldehyde oxidase and carbonyl reductase) on their typical substrates using a panel of human liver samples, we were able to show the validity of PCP for this purpose. R or Rs and P values were calculated for the association between the protein abundance profile of each protein and the metabolic rate profile of each typical substrate. For the 18 enzymatic activities examined, 13 of the enzymes reported to be responsible for the reactions had correlation coefficients higher than 0.7 and were ranked first to third. For the remaining five activities, the responsible enzymes had correlation coefficients lower than 0.7 and lower rankings. The reasons for this were diverse, including confounding resulting from low protein abundance ratios, artificially high correlations of other enzymes due to limited sample numbers, the presence of inactive enzyme forms, and genetic polymorphisms. Overall, PCP was able to identify the majority of responsible drug-metabolizing enzymes across several enzyme classes (oxidoreductase, transferase, hydrolase); use of this methodology could allow more timely and accurate identification of unknown drug-metabolizing enzymes. SIGNIFICANCE STATEMENT: Proteomic correlation profiling using samples from individual human donors was proven to be a useful methodology for the identification of enzymes responsible for drug-metabolism. This methodology could accelerate the identification of unknown drug-metabolizing enzymes in the future.

Identification of HSD17B12 as an enzyme catalyzing drug reduction reactions through investigation of nabumetone metabolism.

Nabumetone, a nonsteroidal anti-inflammatory prodrug, is converted to a pharmacologically active metabolite, 6-methoxy-2-naphthylacetic acid (6-MNA); however, it is 11-fold more efficiently converted to 4-(6-methoxy-2-naphthyl)butan-2-ol (MNBO) via a reduction reaction in human hepatocytes. The goal of this study was to identify the enzyme(s) responsible for MNBO formation from nabumetone in the human liver. MNBO formation by human liver microsomes (HLM) was 5.7-fold higher than in the liver cytosol. In a panel of 24 individual HLM samples with quantitative proteomics data, the 17ß-hydroxysteroid dehydrogenase 12 (HSD17B12) protein level had the high correlation coefficient (r = 0.80, P < 0.001) among 4457 proteins quantified in microsomal fractions during MNBO formation. Recombinant HSD17B12 expressed in HEK293T cells exhibited prominent nabumetone reductase activity, and the contribution of HSD17B12 to the activity in the HLM was calculated as almost 100%. MNBO formation in HepG2 and Huh7 cells was significantly decreased by the knockdown of HSD17B12. We also examined the role of HSD17B12 in drug metabolism and found that recombinant HSD17B12 catalyzed the reduction reactions of pentoxifylline and S-warfarin, suggesting that HSD17B12 prefers compounds containing a methyl ketone group on the alkyl chain. In conclusion, our study demonstrated that HSD17B12 is responsible for the formation of MNBO from nabumetone. Together with the evidence for pentoxifylline and S-warfarin reduction, this is the first study to report that HSD17B12, which is known to metabolize endogenous compounds, such as estrone and 3-ketoacyl-CoA, plays a role as a drug-metabolizing enzyme.

Quantitative Evaluation of the Contribution of Each Aldo-Keto Reductase and Short-Chain Dehydrogenase/Reductase Isoform to Reduction Reactions of Compounds Containing a Ketone Group in the Human Liver.

Enzymes of the aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase superfamilies are involved in the reduction of compounds containing a ketone group. In most cases, multiple isoforms appear to be involved in the reduction of a compound, and the enzyme(s) that are responsible for the reaction in the human liver have not been elucidated. The purpose of this study was to quantitatively evaluate the contribution of each isoform to reduction reactions in the human liver. Recombinant cytosolic isoforms were constructed, i.e., AKR1C1, AKR1C2, AKR1C3, AKR1C4, and carbonyl reductase 1 (CBR1), and a microsomal isoform, 11ß-hydroxysteroid dehydrogenase type 1 (HSD11B1), and their contributions to the reduction of 10 compounds were examined by extrapolating the relative expression of each reductase protein in human liver preparations to recombinant systems quantified by liquid chromatography-mass spectrometry. The reductase activities for acetohexamide, doxorubicin, haloperidol, loxoprofen, naloxone, oxcarbazepine, and pentoxifylline were predominantly catalyzed by cytosolic isoforms, and the sum of the contributions of individual cytosolic reductases was almost 100%. Interestingly, AKR1C3 showed the highest contribution to acetohexamide and loxoprofen reduction, although previous studies have revealed that CBR1 mainly metabolizes them. The reductase activities of bupropion, ketoprofen, and tolperisone were catalyzed by microsomal isoform(s), and the contributions of HSD11B1 were calculated to be 41%, 32%, and 104%, respectively. To our knowledge, this is the first study to quantitatively evaluate the contribution of each reductase to the reduction of drugs in the human liver. SIGNIFICANCE STATEMENT: To our knowledge, this is the first study to determine the contribution of aldo-keto reductase (AKR)-1C1, AKR1C2, AKR1C3, AKR1C4, carbonyl reductase 1, and 11ß-hydroxysteroid dehydrogenase type 1 to drug reductions in the human liver by utilizing the relative expression factor approach. This study found that AKR1C3 contributes to the reduction of compounds at higher-than-expected rates.

The mitochondrial and plastid genomes of Oryza sativa L. cv. Taichung 65.

A highly contiguous mitochondrial and plastid genome sequences of a japonica rice cultivar, Taichung 65, were determined by a hybrid approach with long- and short-read sequences. The assembled mitochondrial genome was 465,453 bases in length with an overall GC content of 43.8%. It was predicted to harbor 62 protein-encoding genes, 16 kinds (33 copies) of transfer RNA, and three kinds (six copies) of ribosomal RNA genes. The mitochondrial genome structure in Taichung 65 is largely the same as that of Nipponbare, but the first ∼9.5 kb sequence in Nipponbare (DQ167400) is replaced with a ∼27 kb sequence duplicated from other parts of the mitochondrial genome. Phylogenetic and sequence polymorphism analysis indicated that Taichung 65 is classified as typical japonica. The assembled plastid genome sequence was 134,551 bases in length and completely identical to the previously reported Nipponbare sequence. These near-complete organelle genome sequences will serve as fundamental resources for investigating alloplasmic cytoplasmic male sterile lines and other organelle-controlled phenomena in rice.

Arylacetamide deacetylase as a determinant of the hydrolysis and activation of abiraterone acetate in mice and humans.

AIM: Abiraterone acetate for metastatic castration-resistant prostate cancer is an acetylated prodrug to be hydrolyzed to abiraterone. Abiraterone acetate is known to be hydrolyzed by pancreatic cholesterol esterase secreted into the intestinal lumen. This study aimed to investigate the possibility that arylacetamide deacetylase (AADAC) expressed in enterocytes contributes to the hydrolysis of abiraterone acetate based on its substrate preference. MATERIALS AND METHODS: Abiraterone acetate hydrolase activity was measured using human intestinal (HIM) and liver microsomes (HLM) as well as recombinant AADAC. Correlation analysis between activity and AADAC expression was performed in 14 individual HIMs. The in vivo pharmacokinetics of abiraterone acetate was examined using wild-type and Aadac knockout mice administered abiraterone acetate with or without orlistat, a pancreatic cholesterol esterase inhibitor. KEY FINDINGS: Recombinant AADAC showed abiraterone acetate hydrolase activity with similar Km value to HIM and HLM. The positive correlation between activity and AADAC levels in individual HIMs supported the responsibility of AADAC for abiraterone acetate hydrolysis. The area under the plasma concentration-time curve (AUC) of abiraterone after oral administration of abiraterone acetate in Aadac knockout mice was 38% lower than that in wild-type mice. The involvement of pancreatic cholesterol esterase in abiraterone formation was revealed by the decreased AUC of abiraterone by coadministration of orlistat. Orlistat potently inhibited AADAC, implying its potential as a perpetrator of drug-drug interactions. SIGNIFICANCE: AADAC is responsible for the hydrolysis of abiraterone acetate in the intestine and liver, suggesting that concomitant use of abiraterone acetate and drugs potently inhibiting AADAC should be avoided.

Highly Efficient and Comprehensive Identification of Ethyl Methanesulfonate-Induced Mutations in Nicotiana tabacum L. by Whole-Genome and Whole-Exome Sequencing.

Tobacco (Nicotiana tabacum L.) is a complex allotetraploid species with a large 4.5-Gb genome that carries duplicated gene copies. In this study, we describe the development of a whole-exome sequencing (WES) procedure in tobacco and its application to characterize a test population of ethyl methanesulfonate (EMS)-induced mutations. A probe set covering 50.3-Mb protein coding regions was designed from a reference tobacco genome. The EMS-induced mutations in 19 individual M2 lines were analyzed using our mutation analysis pipeline optimized to minimize false positives/negatives. In the target regions, the on-target rate of WES was approximately 75%, and 61,146 mutations were detected in the 19 M2 lines. Most of the mutations (98.8%) were single nucleotide variants, and 95.6% of them were C/G to T/A transitions. The number of mutations detected in the target coding sequences by WES was 93.5% of the mutations detected by whole-genome sequencing (WGS). The amount of sequencing data necessary for efficient mutation detection was significantly lower in WES (11.2 Gb), which is only 6.2% of the required amount in WGS (180 Gb). Thus, WES was almost comparable to WGS in performance but is more cost effective. Therefore, the developed target exome sequencing, which could become a fundamental tool in high-throughput mutation identification, renders the genome-wide analysis of tobacco highly efficient.

Application of heavy-ion-beam irradiation to breeding large rotifer.

In larviculture facilities, rotifers are generally used as an initial food source, while a proper size of live feeds to connect rotifer and Artemia associated with fish larval growth is needed. The improper management of feed size and density induces mass mortality and abnormal development of fish larvae. To improve the survival and growth of target larvae, this study applied carbon and argon heavy-ion-beam irradiation in mutation breeding to select rotifer mutants with larger lorica sizes. The optimal irradiation conditions of heavy-ion beam were determined with lethality, reproductivity, mutant frequency, and morphometric characteristics. Among 56 large mutants, TYC78, TYC176, and TYA41 also showed active population growth. In conclusion, (1) heavy-ion-beam irradiation was defined as an efficient tool for mutagenesis of rotifers and (2) the aforementioned 3 lines that have larger lorica length and active population growth may be used as a countermeasure of live feed size gap during fish larviculcure.

Quantitative analysis of mRNA expression levels of aldo-keto reductase and short-chain dehydrogenase/reductase isoforms in human livers.

The aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies are responsible for the reduction in compounds containing the aldehyde, ketone, and quinone groups. In humans, 12 AKR isoforms (AKR1A1, AKR1B1, AKR1B10, AKR1B15, AKR1C1, AKR1C2, AKR1C3, AKR1C4, AKR1D1, AKR1E2, AKR7A2, and AKR7A3) and 6 SDR isoforms (CBR1, CBR3, CBR4, HSD11B1, DHRS4, and DCXR) have been found to catalyze the reduction in xenobiotics, but their hepatic expression levels are unclear. The purpose of this study is to determine the absolute mRNA expression levels of these 18 isoforms in the human liver. In 22 human livers, all isoforms, except for AKR1B15, are expressed, and AKR1C2 (on average 1.6 × 106 copy/µg total RNA), AKR1C3 (1.3 × 106), AKR1C1 (1.3 × 106), CBR1 (9.7 × 105), and HSD11B1 (1.1 × 106) are abundant, representing 67% of the total expression of reductases in the liver. The expression levels of AKR1C2, AKR1C3, AKR1C1, CBR1, and HSD11B1 are significantly correlated with each other, except between AKR1C2 and CBR1, suggesting that they might be regulated by common factor(s). In conclusion, this study comprehensively determined the absolute expression of mRNA expression of each AKR and SDR isoform in the human liver.

FLOURY ENDOSPERM11-2 encodes plastid HSP70-2 involved with the temperature-dependent chalkiness of rice (Oryza sativa L.) grains.

The frequent occurrence of chalky rice (Oryza sativa L.) grains becomes a serious problem as a result of climate change. The molecular mechanism underlying chalkiness is largely unknown, however. In this study, the temperature-sensitive floury endosperm11-2 (flo11-2) mutant was isolated from ion beam-irradiated rice of 1116 lines. The flo11-2 mutant showed significantly higher chalkiness than the wild type grown under a mean temperature of 28°C, but similar levels of chalkiness to the wild type grown under a mean temperature of 24°C. Whole-exome sequencing of the flo11-2 mutant showed three causal gene candidates, including Os12g0244100, which encodes the plastid-localized 70-kDa heat shock protein 2 (cpHSP70-2). The cpHSP70-2 of the flo11-2 mutant has an amino acid substitution on the 259th aspartic acid with valine (D259V) in the conserved Motif 5 of the ATPase domain. Transgenic flo11-2 mutants that express the wild-type cpHSP70-2 showed significantly lower chalkiness than the flo11-2 mutant. Moreover, the accumulation level of cpHSP70-2 was negatively correlated with the chalky ratio, indicating that cpHSP70-2 is a causal gene for the chalkiness of the flo11-2 mutant. The intrinsic ATPase activity of recombinant cpHSP70-2 was lower by 23% at Vmax for the flo11-2 mutant than for the wild type. The growth of DnaK-defective Escherichia coli cells complemented with DnaK with the D201V mutation (equivalent to the D259V mutation) was severely reduced at 37°C, but not in the wild-type DnaK. The results indicate that the lowered cpHSP70-2 function is involved with the chalkiness of the flo11-2 mutant.

Genome sequencing of ion-beam-induced mutants facilitates detection of candidate genes responsible for phenotypes of mutants in rice.

Ion beams are physical mutagens used for plant and microbe breeding that cause mutations via a mechanism distinct from those of chemical mutagens or gamma rays. We utilized whole-exome sequencing of rice DNA in order to understand the properties of ion beam-induced mutations in a genome-wide manner. DNA libraries were constructed from selected carbon-ion-beam-induced rice mutants by capturing with a custom probes covering 66.3 M bases of nearly all exons and miRNAs predicted in the genome. A total of 56 mutations, including 24 single nucleotide variations, 23 deletions, and 5 insertions, were detected in five mutant rice lines (two dwarf and three early-heading-date mutants). The mutations were distributed among all 12 chromosomes, and the average mutation frequency in the M1 generation was estimated to be 2.7 × 10-7 per base. Many single base insertions and deletions were associated with homopolymeric repeats, whereas larger deletions up to seven base pairs were observed at polynucleotide repeats in the DNA sequences of the mutation sites. Of the 56 mutations, six were classified as high-impact mutations that caused a frame shift or loss of exons. A gene that was functionally related to the phenotype of the mutant was disrupted by a high-impact mutation in four of the five lines tested, suggesting that whole-exome sequencing of ion-beam-irradiated mutants could facilitate the detection of candidate genes responsible for the mutant phenotypes.

An improved and robust method to efficiently deplete repetitive elements from complex plant genomes.

Genome size and complexity often present major challenges to genome-based approaches in crop plants and other agricultural species. For instance, repetitive sequences comprise 80% to 90% of the genome of hexaploid wheat, which has a haploid genome size of approximately 17 Gb. In this study, we developed an improved design and procedure for short-read library preparation that uses a modified adaptor and duplex-specific nuclease (DSN) for the efficient elimination of highly repeated sequence elements within genomes. The improved adapter, which has a hairpin-like form for stability, was constructed from truncated sequences adjacent to the original Illumina TruSeq adapter and can be converted to a full-length adapter structure during PCR amplification. Using the hairpin-structured adaptor, we prepared randomly sheared genomic libraries from rice and diploid, tetraploid, and hexaploid wheat cultivars and evaluated the efficiency of DSN for the enzymatic depletion of repetitive elements. According to real-time quantitative PCR analysis, the relative abundances of 18S and 25S ribosomal DNA decreased respectively to 1.15% and 3.54% in rice and 1.70%-1.95% and 14.71%-20.01% in the three wheat cultivars. Whole-genome sequencing analysis of a diploid wheat cultivar, KU104-1, indicated that DSN treatment with the designed hairpin-structured adapter dramatically reduced highly repetitive elements, such as Ty1-Copia and Ty3-Gypsy retrotransposons and DNA transposons, within the genome, while sequencing reads derived from low-copy genes and protein coding sequences increased more than 50%. Our new procedure should be useful not only for wheat genomes but also for other agricultural plant species with relatively large and complex genomes.

Targeted exome sequencing of unselected heavy-ion beam-irradiated populations reveals less-biased mutation characteristics in the rice genome.

Heavy-ion beams have been widely utilized as a novel and effective mutagen for mutation breeding in diverse plant species, but the induced mutation spectrum is not fully understood at the genome scale. We describe the development of a multiplexed and cost-efficient whole-exome sequencing procedure in rice, and its application to characterize an unselected population of heavy-ion beam-induced mutations. The bioinformatics pipeline identified single-nucleotide mutations as well as small and large (>63 kb) insertions and deletions, and showed good agreement with the results obtained with conventional polymerase chain reaction (PCR) and sequencing analyses. We applied the procedure to analyze the mutation spectrum induced by heavy-ion beams at the population level. In total, 165 individual M2 lines derived from six irradiation conditions as well as eight pools from non-irradiated 'Nipponbare' controls were sequenced using the newly established target exome sequencing procedure. The characteristics and distribution of carbon-ion beam-induced mutations were analyzed in the absence of bias introduced by visual mutant selections. The average (±SE) number of mutations within the target exon regions was 9.06 ± 0.37 induced by 150 Gy irradiation of dry seeds. The mutation frequency changed in parallel to the irradiation dose when dry seeds were irradiated. The total number of mutations detected by sequencing unselected M2 lines was correlated with the conventional mutation frequency determined by the occurrence of morphological mutants. Therefore, mutation frequency may be a good indicator for sequencing-based determination of the optimal irradiation condition for induction of mutations.

Heavy-ion beam mutagenesis of the ectomycorrhizal agaricomycete Tricholoma matsutake that produces the prized mushroom "matsutake" in conifer forests.

Tricholoma matsutake is an ectomycorrhizal agaricomycete that produces the prized mushroom "matsutake" in Pinaceae forests. Currently, there are no available cultivars or cultivation methods that produce fruiting bodies. Heavy-ion beams, which induce mutations through double-stranded DNA breaks, have been used widely for plant breeding. In the present study, we examined whether heavy-ion beams could be useful in isolating T. matsutake mutants. An argon-ion beam gave a suitable lethality curve in relation to irradiation doses, accelerating killing at 100-150 Gy. Argon-ion beam irradiation of the agar plate cultures yielded several transient mutants whose colony morphologies differed from that of the wild-type strain at the first screening, but which did not persist following culture transfer. It also generated a mutant whose phenotype remained stable after repeated culture transfers. The stable pleiotropic mutant not only exhibited a different colony morphology to the wild type, but also showed increased degradation of dye-linked water-insoluble amylose and cellulose substrates. Thus, heavy-ion beams may be useful for isolating mutants of T. matsutake, although precautions may be required to maintain the mutants, without phenotypic reversion, during repetitive culture of their mycelia.

Prediction of bipartite transcriptional regulatory elements using transcriptome data of Arabidopsis.

In our previous study, a methodology was established to predict transcriptional regulatory elements in promoter sequences using transcriptome data based on a frequency comparison of octamers. Some transcription factors, including the NAC family, cannot be covered by this method because their binding sequences have non-specific spacers in the middle of the two binding sites. In order to remove this blind spot in promoter prediction, we have extended our analysis by including bipartite octamers that are composed of '4 bases-a spacer with a flexible length-4 bases'. 8,044 pre-selected bipartite octamers, which had an overrepresentation of specific spacer lengths in promoter sequences and sequences related to core elements removed, were subjected to frequency comparison analysis. Prediction of ER stress-responsive elements in the BiP/BiPL promoter and an ANAC017 target sequence resulted in precise detection of true positives, judged by functional analyses of a reported article and our own in vitro protein-DNA binding assays. These results demonstrate that incorporation of bipartite octamers with continuous ones improves promoter prediction significantly.

Heavy-ion beam mutagenesis identified an essential gene for chloroplast development under cold stress conditions during both early growth and tillering stages in rice.

We isolated a cold sensitive virescent1 (csv1) mutant from a rice (Oryza sativa L.) population mutagenized by carbon ion irradiation. The mutant exhibited chlorotic leaves during the early growth stages, and produced normal green leaves as it grew. The growth of csv1 plants displayed sensitivity to low temperatures. In addition, the mutant plants that were transferred to low temperatures at the fifth leaf stage produced chlorotic leaves subsequently. Genetic and molecular analyses revealed translocation of a 13-kb genomic fragment that disrupted the causative gene (CSV1; LOC_Os05g34040). CSV1 encodes a plastid-targeted oxidoreductase-like protein conserved among land plants, green algae, and cyanobacteria. Furthermore, CSV1 transcripts were more abundant in immature than in mature leaves, and they did not markedly increase or decrease with temperature. Taken together, our results indicate that CSV1 supports chloroplast development under cold stress conditions, in both the early growth and tillering stages in rice.

LDSS-P: an advanced algorithm to extract functional short motifs associated with coordinated gene expression.

Identifying functional elements in promoter sequences is a major goal in computational and experimental genome biology. Here, we describe an algorithm, Local Distribution of Short Sequences for Prokaryotes (LDSS-P), to identify conserved short motifs located at specific positions in the promoters of co-expressed prokaryotic genes. As a test case, we applied this algorithm to a symbiotic nitrogen-fixing bacterium, Sinorhizobium meliloti The LDSS-P profiles that overlap with the 5' section of the extracytoplasmic function RNA polymerase sigma factor RpoE2 consensus sequences displayed a sharp peak between -34 and -32 from TSS positions. The corresponding genes overlap significantly with RpoE2 targets identified from previous experiments. We further identified several groups of genes that are co-regulated with characterized marker genes. Our data indicate that in S. meliloti, and possibly in other Rhizobiaceae species, the master cell cycle regulator CtrA may recognize an expanded motif (AACCAT), which is positionally shifted from the previously reported CtrA consensus sequence in Caulobacter crescentus Bacterial one-hybrid experiments showed that base substitution in the expanded motif either increase or decrease the binding by CtrA. These results show the effectiveness of LDSS-P as a method to delineate functional promoter elements.

Construction and characterization of a copy number-inducible fosmid library of Xanthomonas oryzae pathovar oryzae MAFF311018.

A fosmid library of Xanthomonas oryzae pathovar oryzae MAFF311018 (T7174), the causative agent of bacterial blight on rice, was constructed and characterized. The average fosmid library insert size was >34kb, and 967 clones were uniquely positioned on its sequenced genome. The entire Xoo MAFF311018 genome was covered by end-sequenced clones with at least 5kb of overlap. The fosmid vector contains both the single-copy Escherichia coli fertility factor origin, which enhances fosmid stability, and the multi-copy IncPα origin, allowing amplification of copy number upon induction with l-arabinose. Real-time quantitative PCR on 12 randomly picked fosmid library clones determined that fosmid copy number increased 8- to 58-fold after 5hour induction. This library provides a new resource for complementation experiments and systematic functional studies in Xoo and related species.

Characterization of a heavy-ion induced white flower mutant of allotetraploid Nicotiana tabacum.

KEY MESSAGE : We characterized a white flower mutant of allotetraploid N. tabacum as a DFR-deficient mutant; one copy of DFR has a cultivar-specific frameshift, while the other was deleted by heavy-ion irradiation. In most plants, white-flowered mutants have some kind of deficiency or defect in their anthocyanin biosynthetic pathway. Nicotiana tabacum normally has pink petals, in which cyanidin is the main colored anthocyanidin. When a relevant gene in the cyanidin biosynthetic pathway is mutated, the petals show a white color. Previously, we generated white-flowered mutants of N. tabacum by heavy-ion irradiation, which is accepted as an effective mutagen. In this study, we determined which gene was responsible for the white-flowered phenotype of one of these mutants, cv. Xanthi white flower 1 (xwf1). Southern blot analysis using a DNA fragment of the dihydroflavonol 4-reductase (DFR) gene as a probe showed that the xwf1 mutant lacked signals that were present in wild-type genomic DNAs. Sequence analysis demonstrated that one copy of the DFR gene (NtDFR2) was absent from the genome of the xwf1 mutant. The other copy of the DFR gene (NtDFR1) contained a single-base deletion resulting in a frameshift mutation, which is a spontaneous mutation in cv. Xanthi. Introduction of NtDFR2 cDNA into the petal limbs of xwf1 by particle bombardment resulted in production of the pink-colored cells, whereas introduction of NtDFR1 cDNA did not. These results indicate that xwf1 is a DFR-deficient mutant. One copy of NtDFR1 harbors a spontaneous frameshift mutation, while the other copy of NtDFR2 was deleted by heavy-ion beam irradiation.