tasiRNA-ARF pathway moderates floral architecture in Arabidopsis plants subjected to drought stress.

In plants, miRNAs and siRNAs, such as transacting siRNAs (ta-siRNAs), affect their targets through distinct regulatory mechanisms. In this study, the expression profiles of small RNAs (smRNAs) in Arabidopsis plants subjected to drought, cold, and high-salinity stress were analyzed using 454 DNA sequencing technology. Expression of three groups of ta-siRNAs (TAS1, TAS2, and TAS3) and their precursors was downregulated in Arabidopsis plants subjected to drought and high-salinity stress. Analysis of ta-siRNA synthesis mutants and mutated ARF3-overexpressing plants that escape the tasiRNA-ARF target indicated that self-pollination was hampered by short stamens in plants under drought and high-salinity stress. Microarray analysis of flower buds of rdr6 and wild-type plants under drought stress and nonstressed conditions revealed that expression of floral development- and auxin response-related genes was affected by drought stress and by the RDR6 mutation. The overall results of the present study indicated that tasiRNA-ARF is involved in maintaining the normal morphogenesis of flowers in plants under stress conditions through fine-tuning expression changes of floral development-related and auxin response-related genes.

Jun activation domain-binding protein 1 (JAB1) is required for the optimal response to interferons.

Degradation of IFN receptor (IFNR) protein is one of the mechanisms to limit the extent of cellular responses to interferons. Tyrosine kinase 2 (TYK2), a JAK family kinase, has been reported to bind to and stabilize IFNR, indicating that TYK2 is a fundamental component of IFNR complex. Herein, we identified Jun activation domain-binding protein 1 (JAB1) as a new TYK2 binding partner and investigated its role in the regulation of IFN responses. siRNA knockdown of JAB1 resulted in suppression of IFN-induced phosphorylation of STAT proteins and their transcriptional activation. Importantly, JAB1 knockdown induced the activation of SCF ubiquitin ligase complex containing Cullin 1 (CUL1), as judged by the enhancement of covalent modification of CUL1 with the ubiquitin-like protein NEDD8, and markedly reduced the basal protein level of IFNR. In contrast, NEDD8 knockdown or inhibition of NEDD8 modification by NEDD8-activating enzyme inhibitor resulted in increased IFNR protein concomitantly with a reduction of NEDD8-modified CUL1. Furthermore, NEDD8-activating enzyme inhibitor treatment enhanced the susceptibility to IFN-α in HeLa cells. These data suggest that the NEDD8 modification pathway is involved in the proteolysis of IFNR and that JAB1 acts as a positive regulator of IFN responses by stabilizing IFNR through antagonizing the NEDD8 pathway.

Fine definition of the pedigree haplotypes of closely related rice cultivars by means of genome-wide discovery of single-nucleotide polymorphisms.

BACKGROUND: To create useful gene combinations in crop breeding, it is necessary to clarify the dynamics of the genome composition created by breeding practices. A large quantity of single-nucleotide polymorphism (SNP) data is required to permit discrimination of chromosome segments among modern cultivars, which are genetically related. Here, we used a high-throughput sequencer to conduct whole-genome sequencing of an elite Japanese rice cultivar, Koshihikari, which is closely related to Nipponbare, whose genome sequencing has been completed. Then we designed a high-throughput typing array based on the SNP information by comparison of the two sequences. Finally, we applied this array to analyze historical representative rice cultivars to understand the dynamics of their genome composition. RESULTS: The total 5.89-Gb sequence for Koshihikari, equivalent to 15.7 x the entire rice genome, was mapped using the Pseudomolecules 4.0 database for Nipponbare. The resultant Koshihikari genome sequence corresponded to 80.1% of the Nipponbare sequence and led to the identification of 67,051 SNPs. A high-throughput typing array consisting of 1917 SNP sites distributed throughout the genome was designed to genotype 151 representative Japanese cultivars that have been grown during the past 150 years. We could identify the ancestral origin of the pedigree haplotypes in 60.9% of the Koshihikari genome and 18 consensus haplotype blocks which are inherited from traditional landraces to current improved varieties. Moreover, it was predicted that modern breeding practices have generally decreased genetic diversity CONCLUSIONS: Detection of genome-wide SNPs by both high-throughput sequencer and typing array made it possible to evaluate genomic composition of genetically related rice varieties. With the aid of their pedigree information, we clarified the dynamics of chromosome recombination during the historical rice breeding process. We also found several genomic regions decreasing genetic diversity which might be caused by a recent human selection in rice breeding. The definition of pedigree haplotypes by means of genome-wide SNPs will facilitate next-generation breeding of rice and other crops.

Arabidopsis transcriptome analysis under drought, cold, high-salinity and ABA treatment conditions using a tiling array.

Plants respond and adapt to drought, cold and high-salinity stresses in order to survive. In this study, we applied Arabidopsis Affymetrix tiling arrays to study the whole genome transcriptome under drought, cold, high-salinity and ABA treatment conditions. The bioinformatic analysis using the tiling array data showed that 7,719 non-AGI transcriptional units (TUs) exist in the unannotated "intergenic" regions of Arabidopsis genome. These include 1,275 and 181 TUs that are induced and downregulated, respectively, by the stress or ABA treatments. Most of the non-AGI TUs are hypothetical non-protein-coding RNAs. About 80% of the non-AGI TUs belong to pairs of the fully overlapping sense-antisense transcripts (fSATs). Significant linear correlation between the expression ratios (treated/untreated) of the sense TUs and the ratios of the antisense TUs was observed in the SATs of AGI code/non-AGI TU. We studied the biogenesis mechanisms of the stress- or ABA-inducible antisense RNAs and found that the expression of sense TUs is necessary for the stress- or ABA-inducible expression of the antisense TUs in the fSATs (AGI code/non-AGI TU).

IDENTIFICATION OF GENES PREFERENTIALLY EXPRESSED DURING ASEXUAL SPORULATION IN PORPHYRA YEZOENSIS GAMETOPHYTES (BANGIALES, RHODOPHYTA)(1).

Asexual reproduction via archeospores in Porphyra yezoensis Ueda gametophytes is a very valuable character to nori farming; however, there is little information available on the molecular basis of the developmental process. To identify genes involved in the Porphyra asexual sporulation, we compared the gene expression profiles derived from four developmental stages of the life cycle (three from gametophytes; one from sporophytes) using cDNA macroarray, which includes 4,896 nonredundant expressed sequence tag (EST) groups. Candidate genes were screened by two different macroarray data analyses combined with reverse transcription-PCR (RT-PCR) analysis or Northern analysis. RT-PCR analysis revealed that nine genes (one: similarity to 5'-3' exoribonuclease; the other eight: no sequence similarity to known proteins) were expressed with a gametophyte (G)-specific manner, and two genes (named ASPO2608, ASPO1527) were expressed only in gametophytes that formed archeospores. The deduced amino acid sequences for the latter two genes are predicted to contain signal peptides for secretion at their N-termini. Northern analysis revealed that expression levels of Calvin cycle genes in the gametophytic stage that formed archeospores (G-A stage) were higher than those of the gametophyte blade with no archeospores (G-NA stage). In the macroarray analysis based on the rank data of G-preferentially expressed genes, which were detected in the previous P. yezoensis EST analysis, one gene encoding the cyclase associated protein (CAP) exhibited a change upwardly in the G-A stage >1,000 ranks to the G-NA stage. We propose that ASPO2608 and CAP may function in a signaling pathway of asexual sporulation.

Crosstalk in the responses to abiotic and biotic stresses in Arabidopsis: analysis of gene expression in cytochrome P450 gene superfamily by cDNA microarray.

From Arabidopsis full-length cDNA libraries, we collected ca. 7000 (7K) independent full-length cDNAs to prepare a cDNA microarray. The 7K cDNA collection contains 49 cytochrome P450 genes. In this study, expression patterns of these cytochrome P450 genes were analyzed by a full-length cDNA microarray under various treatments, such as hormones (salicylic acid, jasmonic acid, ethylene, abscisic acid), pathogen-inoculation ( Alternaria brassicicola , Alternaria alternata ), paraquat, rose bengal, UV stress (UV-C), heavy metal stress (CuSO4), mechanical wounding, drought, high salinity and low temperature. Expression of 29 cytochrome P450 genes among them was induced by various treatments. Inoculation with A. brassicicola and A. alternata as biotic stresses increased transcript levels of 12 and 5 genes in Arabidopsis plants, respectively. In addition, some of the genes were also expressed by abiotic stresses. This suggests crosstalk between abiotic and biotic stresses. The promoter sequences and cis -acting elements of each gene were studied on the basis of full-length cDNA sequences. Most cytochrome P450 genes induced by both abiotic and biotic stresses contained the recognition sites of MYB and MYC, ACGT-core sequence, TGA-box and W-box for WRKY transcription factors in their promoters. These cis -acting elements are known to participate in the regulation of plant defense. The response of each gene to multiple stresses is strictly regulated.

Monitoring the expression profiles of genes induced by hyperosmotic, high salinity, and oxidative stress and abscisic acid treatment in Arabidopsis cell culture using a full-length cDNA microarray.

Transcriptional regulation in response to hyperosmotic, high-salinity and oxidative stress, and abscisic acid (ABA) treatment in Arabidopsis suspension-cultured cell line T87 was investigated with a cDNA microarray containing 7000 independent full-length Arabidopsis cDNAs. The transcripts of 102, 11, 84 and 73 genes were increased more than 5-fold within 5h after treatment with 0.5M mannitol, 0.1M NaCl, 50 microM ABA and 10mM H2O2, respectively. On the other hand, the transcripts of 44, 57, 25 and 34 genes were down-regulated to less than one-third within 5h after treatment with 0.5M mannitol, 0.1M NaCl, 50 microM ABA and 10mM H2O2, respectively. Venn diagram analysis revealed 11 genes were induced significantly by mannitol, NaCl, and ABA, indicating crosstalk among these signaling pathways. Comparison of the genes induced by each stress revealed that 32%, 17% and 33% of mannitol-, NaCl- and ABA-inducible genes were also induced by H2O2, indicating the crosstalk between the signaling pathways for osmotic stress and oxidative stress. Although the expression profiles revealed that the T87 cells had most of the regulatory systems seen in Arabidopsis seedlings, the T87 cells did not have one of ABA-dependent signaling pathways.

RIKEN Arabidopsis full-length (RAFL) cDNA and its applications for expression profiling under abiotic stress conditions.

Full-length cDNAs are essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. 155,144 RIKEN Arabidopsis full-length (RAFL) cDNA clones were isolated. The 3'-end expressed sequence tags (ESTs) of all 155,144 RAFL cDNAs were clustered into 14,668 non-redundant cDNA groups, about 60% of predicted genes. The sequence database of the RAFL cDNAs is useful for promoter analysis and the correct annotation of predicted transcription units and gene products. Recently, cDNA microarray analysis has been developed for quantitative analysis of global and simultaneous analysis of expression profiles. RAFL cDNA microarrays were prepared, containing independent full-length cDNA groups for analysing the expression profiles of genes under various stress- and hormone-treatment conditions and in various mutants and transgenic plants. In this review, recent progress on transcriptome analysis using the RAFL cDNA microarray is highlighted.

Monitoring the expression pattern of around 7,000 Arabidopsis genes under ABA treatments using a full-length cDNA microarray.

Full-length cDNAs are essential for functional analysis of plant genes. Recently, cDNA microarray analysis has been developed for quantitative analysis of global and simultaneous analysis of expression profiles. Microarray technology is a powerful tool for identifying genes induced by environmental stimuli or stress and for analyzing their expression profiles in response to environmental signals. We prepared an Arabidopsis full-length cDNA microarray containing around 7,000 independent full-length cDNA groups and analyzed the expression profiles of genes. The transcripts of 245, 299, 54 and 213 genes increased after abscisic acid (ABA), drought-, cold-, and salt-stress treatments, respectively, with inducibilities more than fivefold compared with those of control genes [corrected]. The cDNA microarray analysis showed that many ABA-inducible genes were induced after drought- and high-salinity-stress treatments, and that there is more crosstalk between drought and ABA responses than between ABA and cold responses. Among the ABA-inducible genes identified, we identified 22 transcription factor genes, suggesting that many transcriptional regulatory mechanisms exist in the ABA signal transduction pathways.

Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray.

Full-length cDNAs are essential for functional analysis of plant genes in the post-sequencing era of the Arabidopsis genome. Recently, cDNA microarray analysis has been developed for quantitative analysis of global and simultaneous analysis of expression profiles. We have prepared a full-length cDNA microarray containing approximately 7000 independent, full-length cDNA groups to analyse the expression profiles of genes under drought, cold (low temperature) and high-salinity stress conditions over time. The transcripts of 53, 277 and 194 genes increased after cold, drought and high-salinity treatments, respectively, more than fivefold compared with the control genes. We also identified many highly drought-, cold- or high-salinity- stress-inducible genes. However, we observed strong relationships in the expression of these stress-responsive genes based on Venn diagram analysis, and found 22 stress-inducible genes that responded to all three stresses. Several gene groups showing different expression profiles were identified by analysis of their expression patterns during stress-responsive gene induction. The cold-inducible genes were classified into at least two gene groups from their expression profiles. DREB1A was included in a group whose expression peaked at 2 h after cold treatment. Among the drought, cold or high-salinity stress-inducible genes identified, we found 40 transcription factor genes (corresponding to approximately 11% of all stress-inducible genes identified), suggesting that various transcriptional regulatory mechanisms function in the drought, cold or high-salinity stress signal transduction pathways.

Functional annotation of a full-length Arabidopsis cDNA collection.

Full-length complementary DNAs (cDNAs) are essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. We isolated 155,144 RIKEN Arabidopsis full-length (RAFL) cDNA clones. The 3'-end expressed sequence tags (ESTs) of 155,144 RAFL cDNAs were clustered into 14,668 nonredundant cDNA groups, about 60% of predicted genes. We also obtained 5' ESTs from 14,034 nonredundant cDNA groups and constructed a promoter database. The sequence database of the RAFL cDNAs is useful for promoter analysis and correct annotation of predicted transcription units and gene products. Furthermore, the full-length cDNAs are useful resources for analyses of the expression profiles, functions, and structures of plant proteins.