SE50:/S01
Sample Set Information
| ID | SE50 |
|---|---|
| Title | Enhancement of oxidative and drought tolerance in arabidopsis by overaccumulation of antioxidant flavonoids |
| Description | We report that flavonoids with radical scavenging activity mitigate against oxidative and drought stress in Arabidopsis thaliana. Metabolome and transcriptome profiling and experiments with oxidative and drought stress in wild-type, single overexpressors of MYB12/PFG1 (PRODUCTION OF FLAVONOL GLYCOSIDES1) or MYB75/PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT1), double overexpressors of MYB12 and PAP1, transparent testa4 (tt4) as a flavonoid-deficient mutant, and flavonoid-deficient MYB12 or PAP1 overexpressing lines (obtained by crossing tt4 and the individual MYB overexpressor) demonstrated that flavonoid overaccumulation was key to enhanced tolerance to such stresses. Antioxidative activity assays using 2,2 diphenyl-1-picrylhydrazyl, methyl viologen, and 3,3′-diaminobenzidine clearly showed that anthocyanin overaccumulation with strong in vitro antioxidative activity mitigated the accumulation of reactive oxygen species in vivo under oxidative and drought stress. These data confirm the usefulness of flavonoids for enhancing both biotic and abiotic stress tolerance in crops. |
| Authors | Ryo Nakabayashi, Keiko Yonekura-Sakakibara, Kaoru Urano, Makoto Suzuki, Yutaka Yamada, Tomoko Nishizawa, Fumio Matsuda, Mikiko Kojima, Hitoshi Sakakibara, Kazuo Shinozaki, Anthony J. Michael, Takayuki Tohge, Mami Yamazak, Kazuki Saito |
| Reference | Nakabayashi R et al. (2014) The Plant Journal 77: 367-379 |
| Comment |
The raw data files are available at DROP Met web site in PRIMe database of RIKEN.
Sample Information
| ID | S01 |
|---|---|
| Title | Arabidopsis |
| Organism - Scientific Name | Arabidopsis thaliana |
| Organism - ID | NCBI taxonomy:3702 |
| Compound - ID | |
| Compound - Source | |
| Preparation | Arabidopsis thaliana (Col-0) plants were used as the wild type in this study. MYB12OX and pap1-D were described previously (Borevitz et al., 2000; Mehrtens et al., 2005). Homozygous WOX1-1 and WOX1-2 were prepared by crossing MYB12OX and pap1-D, followed by self-fertilization of the F1 generation. Two progenies, exhibiting both Basta and kanamycin tolerance in the F2 and F3 generations, were obtained and named WOX1-1 and WOX1-2. The homozygous MYB12OX/tt4 and pap1-D/tt4 lines were prepared by crossing MYB12OX or pap1-D with tt4, respectively. Progenies that exhibited both Basta and kanamycin tolerance (F2 and F3 generations) were obtained. Sterilized seeds were kept for 2 days at 4°C in the dark and were sown on plates of growth medium (GM) agar containing 1% sucrose (Valvekens et al., 1988). Black paper was used for wrapping the plates used for the light-shielding condition. All plates were set in a growth chamber at 22°C and a relative humidity of approximately 60% under a 16-h light (approximately 40 μmol sec-1 m-2)/8-h dark cycle. The drought stress experiment was conducted at 21°C and 40 ± 5% relative humidity under 16 h/8 h light/dark conditions in a plant growth room. Harvested plant samples were immediately frozen in liquid nitrogen and stored at -80°C until use. |
| Sample Preparation Details ID | SS01 |
| Comment |
Sample Preparation Details Information
| ID | SS01 |
|---|---|
| Title | DNA microarray, Real-time PCR, Assay of radical scavenging activity, and stress treatment |
| Description | <Transcriptome profiling by DNA microarray>
Total RNA samples were extracted using an RNeasy Plant Mini Kit (Qiagen, http://www.qiagen.com/). Transcriptome profiling using Affymetrix ATH1 GeneChips (24K) was performed according to the manufacturer’s instructions. The R package from Bioconductor (http://www.bioconductor.org/) was used for the normalization (MAS5) of the CELL files. After filtering using Student’s t-test (P < 0.05) between the wild type and other plants, all genes were then filtered with a fold change of ≥2.0 in the expression level between the wild type and each mutant. Only 907 genes in the other lines were considered. The fold change value was calculated as log2 (Dataset S1). Gene Ontology analysis (TAIR10; http://www.arabidopsis.org/) was performed to annotate 245 biotic and abiotic stress-related genes. The log2-transformed value was used for HCA using MeV 4.8. The data discussed here were also deposited in NCBI’s Gene Expression Omnibus (Edgar et al., 2002) and are accessible through the GEO Series accession number GSE51215.
Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) and cDNA was synthesized using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, http://www.invitrogen. com/) according to the manufacturer's instructions. The primers for MYB12 and PAP1 were used as described previously (Stracke et al., 2007; Gonzalez et al., 2008). The PCR programme for amplification was as follows: 95°C for 20 sec, 95°C for 3 sec, and 60°C for 30 sec (40 cycles).
Radical scavenging activity was measured using DPPH as described previously (Nakajima et al., 2004; Tohge et al., 2005b).
Three-week-old plants grown on GM plates were transferred to GM plates containing 10 lM methyl viologen (Wako Pure Chemical Industries) and were incubated for 21 days. After the stress treatment, frozen aerial parts were extracted with 20 ll MeOH per milligram fresh weight (FW). The UV absorption of the remaining chlorophylls (663 nm) was measured.
This experiment was conducted using Arasystem (Beta-Tech BVBA, http://www.arasystem.com/). Seeds of each line were sown in soil [Pro-Mix BX (Premier Tech Horticulture Inc., http://www.pthorticulture.com/): vermiculite = 2:1, supplemented with fertilizer] and were grown in the plant growth room. After germination, three plants per pot were used for the drought stress experiments. The plants were last watered on day 20 after germination. Three-week-old plants were exposed to drought stress by stopping watering. Trays with pots were turned 90° every day. The plants were rewatered after 16 days. The number of surviving plants was counted 3 days after watering.
Three-week-old plants were incubated in 2-ml tubes with 2 ml of water for 3 days in the growth chamber to assess drought stress. The detached leaves of the incubated plants were used for DAB staining (Thordal-Christensen et al., 1997).
Two-week-old plants were transferred from agar plates to soil conditions (five plants per pot). After a 1-week conditioning period, the plants were subjected to drought stress by withholding water.The weight of the pots was calculated at each time point. Another experiment was performed using detached leaves. The weight of 10 leaves from 3-week-old plants grown on GM medium was calculated at each time point. |
| Comment_of_details |
