SE50:/S01/M01/D01

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 original paper is available at The Plant Journal.

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. <Real-time PCR> 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). <Assay of radical scavenging activity> Radical scavenging activity was measured using DPPH as described previously (Nakajima et al., 2004; Tohge et al., 2005b). <Oxidative stress treatment using methyl viologen> 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. <Drought stress treatment> 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. <3,3′-Diaminobenzidine staining> 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). <Water loss experiment> 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

Analytical Method Information

ID	M01
Title	Untargeted profiling by LC‒QTOF‒MS
Method Details ID	MS01
Sample Amount	1μl
Comment

Analytical Method Details Information

ID	MS01
Title	LC‒PDA‒MS and LC‒QTOF‒MS
Instrument	Waters Acquity UPLC system and Waters Xevo G2 Q-Tof
Instrument Type	UPLC-QTOF-MS
Ionization	ESI
Ion Mode	Positive(LC‒QTOF‒MS)
Description	<Flavonoid-targeted profiling by LC‒PDA‒MS> This analysis was performed as described previously (Tohge et al., 2005a), except that the column was changed to Atlantis T3 (5µm, 4.6 mm×150 mm; Waters, http://www.waters.com/). Reference: Tohge, T., Nishiyama, Y., Hirai, M.Y. et al. (2005a) Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor. Plant J. 42, 218–235. <Untargeted profiling by LC‒QTOF‒MS> Frozen or freeze-dried samples were extracted with 5 or 50 μl of 80% methanol (MeOH) containing 2.5 μm lidocaine per milligram fresh or dry weight, respectively, which was an internal standard for positive mode analysis, using a mixer mill (MM300; Retsch, http://www.retsch.com/) together with one zirconia bead per tube for 10 min at 20 Hz. After centrifugation at 15 000 g for 10 min and filtration using an Oasis HLB 96-well μElution Plate (Waters), the extracts (1 μl) were analyzed by LC‒QTOF‒MS (LC, Waters Acquity UPLC system; MS, Waters Xevo G2 Q-Tof). The analytical conditions were as follows. For LC: column, Acquity bridged ethyl hybrid (BEH) C18 (1.7 μm, 2.1 mm × 100 mm, Waters); solvent system, solvent A (water including 0.1% formic acid) and solvent B (acetonitrile including 0.1% formic acid); gradient program, 99.5% A/0.5% B at 0 min, 99.5% A/0.5% B at 0.1 min, 20% A/80% B at 10 min, 0.5% A/99.5% B at 10.1 min, 0.5% A/99.5% B at 12.0 min, 99.5% A/0.5% B at 12.1 min, and 99.5% A/0.5% B at 15.0 min; flow rate, 0.3 ml min−1; column temperature, 40°C. For MS: capillary voltage, +3.0 keV, cone voltage, 25.0 V; source temperature, 120°C; desolvation temperature, 450°C; cone gas flow, 50 L h−1; desolvation gas flow, 800 L h−1; collision energy, 6 V; mass range, m/z 100–1500; scan duration, 0.1 sec; interscan delay, 0.014 sec; mode, centroid; polarity, positive; lockspray (leucine enkephalin); scan duration, 1.0 sec; interscan delay, 0.1 sec.
Comment_of_details

Data Analysis Information

ID	D01
Title	Untargeted profiling by LC‒QTOF‒MS
Data Analysis Details ID	DS01
Recommended decimal places of m/z	Default
Comment

Data Analysis Details Information

ID	DS01
Title	Untargeted profiling by LC‒QTOF‒MS
Description	The data matrix was aligned using MassLynx ver. 4.1 (Waters). After alignment, de-isotoping, and cutoff of the low-intensity peaks (<500 counts) and the intensity values of the remaining peaks were divided by those of lidocaine ([M + H]+, m/z 235.1804) for normalization. The processed data were used for PCA using SIMCA-P 11.5. The log2-transformed value was used for HCA using MeV 4.8 (http://mev.tm4.org/). All metabolome data acquired by LC‒QTOF‒MS were deposited at DROP Met in PRIMe (http://prime.psc.riken.jp/) and are freely available. These data were also deposited in MetaboLights (accession number MTBLS57) (Steinbeck et al., 2012; Salek et al., 2013) and MetabolomeExpress (Carroll et al., 2010).
Comment_of_details

Related data are deposited in MetaboLights.

The raw data files are available at DROP Met web site in PRIMe database of RIKEN.