Large-scale analysis of full-length cDNAs from the tomato (Solanum lycopersicum) cultivar Micro-Tom, a reference system for the Solanaceae genomics

<p>Abstract</p> <p>Background</p> <p>The Solanaceae family includes several economically important vegetable crops. The tomato (<it>Solanum lycopersicum</it>) is regarded as a model plant of the Solanaceae family. Recently, a number of tomato resources have been developed in parallel with the ongoing tomato genome sequencing project. In particular, a miniature cultivar, Micro-Tom, is regarded as a model system in tomato genomics, and a number of genomics resources in the Micro-Tom-background, such as ESTs and mutagenized lines, have been established by an international alliance.</p> <p>Results</p> <p>To accelerate the progress in tomato genomics, we developed a collection of fully-sequenced 13,227 Micro-Tom full-length cDNAs. By checking redundant sequences, coding sequences, and chimeric sequences, a set of 11,502 non-redundant full-length cDNAs (nrFLcDNAs) was generated. Analysis of untranslated regions demonstrated that tomato has longer 5'- and 3'-untranslated regions than most other plants but rice. Classification of functions of proteins predicted from the coding sequences demonstrated that nrFLcDNAs covered a broad range of functions. A comparison of nrFLcDNAs with genes of sixteen plants facilitated the identification of tomato genes that are not found in other plants, most of which did not have known protein domains. Mapping of the nrFLcDNAs onto currently available tomato genome sequences facilitated prediction of exon-intron structure. Introns of tomato genes were longer than those of Arabidopsis and rice. According to a comparison of exon sequences between the nrFLcDNAs and the tomato genome sequences, the frequency of nucleotide mismatch in exons between Micro-Tom and the genome-sequencing cultivar (Heinz 1706) was estimated to be 0.061%.</p> <p>Conclusion</p> <p>The collection of Micro-Tom nrFLcDNAs generated in this study will serve as a valuable genomic tool for plant biologists to bridge the gap between basic and applied studies. The nrFLcDNA sequences will help annotation of the tomato whole-genome sequence and aid in tomato functional genomics and molecular breeding. Full-length cDNA sequences and their annotations are provided in the database KaFTom <url>http://www.pgb.kazusa.or.jp/kaftom/</url> via the website of the National Bioresource Project Tomato <url>http://tomato.nbrp.jp</url>.</p

FlavonoidSearch: A system for comprehensive flavonoid annotation by mass spectrometry.

ほぼすべてのフラボノイドを検出する技術を開発--植物の機能性成分の研究が加速. 京都大学プレスリリース. 2017-05-08.Currently, in mass spectrometry-based metabolomics, limited reference mass spectra are available for flavonoid identification. In the present study, a database of probable mass fragments for 6, 867 known flavonoids (FsDatabase) was manually constructed based on new structure- and fragmentation-related rules using new heuristics to overcome flavonoid complexity. We developed the FlavonoidSearch system for flavonoid annotation, which consists of the FsDatabase and a computational tool (FsTool) to automatically search the FsDatabase using the mass spectra of metabolite peaks as queries. This system showed the highest identification accuracy for the flavonoid aglycone when compared to existing tools and revealed accurate discrimination between the flavonoid aglycone and other compounds. Sixteen new flavonoids were found from parsley, and the diversity of the flavonoid aglycone among different fruits and vegetables was investigated

TAK1 (MAP3K7) signaling regulates hematopoietic stem cells through TNF-dependent and -independent mechanisms.

A cytokine/stress signaling kinase Tak1 (Map3k7) deficiency is known to impair hematopoietic progenitor cells. However, the role of TAK1 signaling in the stem cell function of the hematopoietic system is not yet well defined. Here we characterized hematopoietic stem cells (HSCs) harboring deletion of Tak1 and its activators, Tak1 binding proteins 1 and 2 (Tab1 and Tab2) using a competitive transplantation assay in a mouse model. Tak1 single or Tab1/Tab2 double deletions completely eliminated the reconstitution activity of HSCs, whereas Tab1 or Tab2 single deletion did not cause any abnormality. Tak1 single or Tab1/Tab2 double deficient lineage-negative, Sca-1(+), c-Kit(+) (LSK) cells did not proliferate and underwent cell death. We found that Tnfr1 deficiency restored the reconstitution activity of Tak1 deficient bone marrow cells for 6-18 weeks. However, the reconstitution activity of Tak1- and Tnfr1-double deficient bone marrow cells declined over the long term, and the number of phenotypically identified long-term hematopoietic stem cells were diminished. Our results indicate that TAB1- or TAB2-dependent activation of TAK1 is required for maintenance of the hematopoietic system through two mechanisms: one is prevention of TNF-dependent cell death and the other is TNF-independent maintenance of long-term HSC

Enhancement of DPPH-radical Scavenging Activity in Heat-processed Sugarcane Molasses

The effects of heating temperature and time on browning, DPPH-radical scavenging activity and polyphenollike activity of molasses from sugarcane were investigated. The browning, DPPH-radical scavenging activity and polyphenol-like activity of molasses heated to between 120℃ and 160℃ were increased in comparison with unheated molasses. The browning of molasses heated to 120℃ and 140℃ increased with heating time, and was nearly 9.5 times greater than unheated molasses after heating for 60 minutes. The browning of molasses heated to 160℃ exponentially increased after heating for 10 minutes, and was nearly 16.7 times greater than unheated molasses after heating for 20 minutes. The DPPH-radical scavenging activity of molasses heated to 120℃ for 50 minutes, 140℃ for 10 minutes, and 160℃ for 10 minutes was four times greater than that of unheated molasses. The alterations in DPPH-radical scavenging activity were similar to the polyphenol-like activity pattern with heat-processing. The heated molasses with the highest polyphenol-like activity, processed at 160℃ for 20 minutes, showed stronger antimutagenicity than unheated molasses. These results indicate that the heat-processing of sugarcane molasses is a viable method for the enhancement of food functions in sugarcane molasses

Lipid remodeling regulator 1 ( LRL

藻類のオイル生産を制御する因子を同定 --有用脂質生産の自在制御に向け大きな一歩--. 京都大学プレスリリース. 2019-08-05.Scientists identify protein factors increasing yield of a biofuel precursor in microscopic algae. 京都大学プレスリリース. 2019-08-02.The elucidation of lipid metabolism in microalgae has attracted broad interest, as their storage lipid, triacylglycerol (TAG), can be readily converted into biofuel via transesterification. TAG accumulates in the form of oil droplets, especially when cells undergo nutrient deprivation, such as for nitrogen (N), phosphorus (P), or sulfur (S). TAG biosynthesis under N‐deprivation has been comprehensively studied in the model microalga Chlamydomonas reinhardtii, during which TAG accumulates dramatically. However, the resulting rapid breakdown of chlorophyll restricts overall oil yield productivity and causes cessation of cell growth. In contrast, P‐deprivation results in oil accumulation without disrupting chloroplast integrity. We used a reverse genetics approach based on co‐expression analysis to identify a transcription factor (TF) that is upregulated under P‐depleted conditions. Transcriptomic analysis revealed that the mutants showed repression of genes typically associated with lipid remodeling under P‐depleted conditions, such as sulfoquinovosyl diacylglycerol 2 (SQD2), diacylglycerol acyltransferase (DGTT1), and major lipid droplet protein (MLDP). As accumulation of sulfoquinovosyl diacylglycerol and TAG were suppressed in P‐depleted mutants, we designated the protein as Lipid Remodeling reguLator 1 (LRL1). LRL1 mutants showed slower growth under P‐depletion. Moreover, cell size in the mutant was significantly reduced, and TAG and starch accumulation per cell were decreased. Transcriptomic analysis also suggested the repression of several genes typically upregulated in adaptation to P‐depletion that are associated with the cell cycle and P and lipid metabolism. Thus, our analysis of LRL1 provides insights into P‐allocation and lipid remodeling under P‐depleted conditions in C. reinhardtii

In vitro expansion of LSK population.

<p>(A) The mice with indicated genotypes were i.p. injected with tamoxifen (160 mg/kg) for three consecutive days and sacrificed at day 4. The total LSK, MP and CLP cell numbers in the femurs and tibias from each mouse were determined. Data are presented as mean ± S.D. (n.s. not significant, n = 3) (B) Whole BMN cells isolated at day 4 described in (A) were cultured in STIFA medium. At the time of plating, 2.5×10⁶ cells/well, 2×10⁶ cells/well and 1×10⁶ cells/well were plated for 3-, 6- and 9-day culture, respectively. Cells were harvested and analyzed by flow cytometry for lineage, Sca-1 and c-Kit surface markers. The absolute number of the LSK population in the harvested cells is shown as per 1×10⁶ initial BMN cells. Data are presented as mean ± S.D. (*p<0.05, n = 3) (C, D) Annexin V-binding assay of LSK (C) and lineage positive cells (D) after nine days of cell expansion in STIFA medium. Data are presented as mean ± S.D. (*p<0.05, n = 3).</p

TAK1, TAB1 and TAB2 in bone marrow cells.

<p>(A) Expression of TAK1, TAB1 and TAB2 proteins. Whole cell extracts of the BMN cells (BM), splenocytes (Spleen) and thymocytes (Thymus) were analyzed by SDS-PAGE and Western blotted (WB) using the indicated antibodies. The amount of β-actin is shown as a loading control. (B) Expression of <i>Tak1</i>, <i>Tab1</i> and <i>Tab2</i> mRNA. Total RNA was isolated from BMN cells (BM), splenocytes (Spleen) and thymocytes (Thymus), and analyzed by qPCR. Expression level of each gene was normalized to that of <i>Actb</i> and shown as the relative value to BM. Data are presented as mean ± S.D. of three independent experiments. (C) Expression of <i>Tak1</i>, <i>Tab1</i> and <i>Tab2</i> mRNA. The cells in the LT-HSC (CD34⁻ Flt3⁻ LSK), ST-HSC (CD34⁺ Flt3⁻ LSK), MPP (CD34⁺ Flt3⁺ LSK), MP (Lineage⁻ c-Kit⁺ Sca-1⁻) or Lineage+ fractions from wild type mouse bone marrow were sorted by FACS, and total RNA was prepared. The relative amounts of <i>Tak1</i>, <i>Tab1</i>, <i>Tab2</i> and <i>Actb</i> mRNA were determined by qPCR. Expression level of each gene was normalized to that of <i>Actb</i> and shown as the relative value to LT-HSC. Data are presented as mean ± S.D. of four independent experiments.</p

Ablation of TNF signaling partially restores the reconstitution potential of <i>Tak1</i>-deficient HSCs.

<p>(A) Competitive reconstitution assay. 2×10⁵ BMN cells from control <i>Tak1^Het Tnfr1^−/−</i> (n = 3) or <i>Tak1^iKO Tnfr1^−/−</i> mice (n = 4) (CD45.2⁺) were transplanted into lethally irradiated recipients (CD45.1⁺) together with 2×10⁵ competitor wild type BMN cells (CD45.1⁺). At six weeks post transplantation (designated as Week 0), the chimerism of myeloid, T and B cells in the recipients’ PB was analyzed, and then the recipients were i.p. injected with tamoxifen at 160 mg/kg body weight for three consecutive days. The chimerism of PB cells was monitored every three weeks, and is shown as the mean ± S.D. (*p<0.05) (B) Competitive reconstitution assay of Cre-alone (n = 3) and <i>Tak1^iKO</i> (n = 3) was also performed as described above, and compared with <i>Tak1^Het Tnfr1^−/−</i> (n = 3) and <i>Tak1^iKO Tnfr1^−/−</i> (n = 4) at 18 weeks post-tamoxifen injection. The table shown below the graphs indicates statistical significance for the indicated comparisons. <i>P</i> values of less than 0.05 are highlighted. (C) Expression of TAK1 and TAB1 proteins in the donor-derived splenocytes. Whole spleen cells from control <i>Tak1^Het</i> and two independent <i>Tak1^iKO Tnfr1^−/−</i> transplanted mice (#1 and #2) at 15 weeks post-tamoxifen injection were sorted into the CD45.1+ or CD45.2+ population. Total cell lysates from the sorted splenocytes from control <i>Tak1^Het</i>, and <i>Tak1^iKO Tnfr1^−/−</i> #1 and #2 were analyzed by SDS-PAGE and Western blotted with anti-TAK1, TAB1 or anti-β-actin antibodies. The positions of molecular weight markers are shown on the right. The arrows indicate the bands corresponding to endogenous TAK1 and TAB1, and truncated TAK1 (TAK1Δ) resulting from Cre-mediated recombination.</p