SE190:/MS1
Sample Set Information
| ID | TSE1349 |
|---|---|
| Title | Novel insights into the function of Arabidopsis R2R3-MYB transcription factors regulating aliphatic glucosinolate biosynthesis. |
| Description | Arabidopsis transcription factors, MYB28, MYB29 and MYB76, positively regulate aliphatic glucosinolate (AGSL) biosynthesis. Mutual transcriptional regulation among these MYB genes makes it difficult to elucidate their individual function simply by analyzing knock-out mutants or ectopically overexpressing lines of these genes. In this study, we constructed transgenic lines expressing each MYB gene driven by its own promoter in the myb28myb29 background, where the expression of the endogenous MYB28, MYB29 and MYB76 was repressed with no AGSL accumulation. In leaves, transgenic MYB28 expression activated AGSL biosynthetic genes and restored accumulation of AGSLs with short side chains. Transgenic MYB29 expression activated the same biosynthetic pathway, but induction of the genes involved in side chain elongation was weaker than that by MYB28, resulting in a weaker recovery of AGSLs. Neither MYB28 nor MYB29 recovered long-chain AGSL accumulation. MYB76 was considered to require both MYB28 and MYB29 for its normal level of expression in leaves, and could not activate AGSL biosynthesis on its own. Interestingly, the accumulation in seeds of long- and short-chain AGSLs was restored by transgenic expression of MYB28 and MYB76, respectively. A sulfur stress experiment indicated that MYB28 expression was induced by sulfur deficiency, while the expression levels of MYB29 and MYB76 were positively correlated with sulfur concentration. This study illustrated how the individual MYBs work in regulating AGSL biosynthesis when expressed alone under normal transcriptional regulation. |
| Authors | Li, Y., Sawada, Y., Hirai, A., Sato, M., Kuwahara, A., Yan, X. and Hirai, M.Y. |
| Reference | Plant Cell Physiol. 2013 Aug;54(8):1335-44. doi: 10.1093/pcp/pct085 |
| Comment |
Analytical Method Details Information
| ID | MS1 |
|---|---|
| Title | Glucosinolate and O-acetyl-L-serine analyses (LC-MS) |
| Instrument | LC; Waters GI Pump solvent system (Waters, Milford, MA, USA) ; MS TQMS (TQD, Waters) |
| Instrument Type | UPLC-QTOF-MS |
| Ionization | ESI |
| Ion Mode | positive and negative |
| Description | The contents of GSLs and OAS were measured by LC-MS using ultra performance liquid chromatography–tandem quadrupole mass spectrometry (UPLC-TQMS) according to Sawada et al. (2009a, 2009b). Approximately 10–50 mg of rosette leaves was sampled from 3-week-old plants and transferred to a pre-frozen, 2 ml tube containing 5 mm zirconia beads. After chilling in liquid nitrogen, samples were stored at −80°C until use. Plant samples were lyophilized using a freeze dryer (FDU-2100, EYELA) in a vacuum, homogenized using a Shake Master (Bio Medical Science) at a speed of 1,000 r.p.m. and homogenized again following the addition of 800 µl of the extraction buffer (methanol : milliQ water = 4 : 1, 0.1% formic acid, 5 µM sinigrin as the AGSL internal standard, 1.68 µM 10-camphorsulfonic acid as the internal standard for negative ion mode analysis and 0.0336 µM lidocaine as the internal standard for positive ion mode analysis). A 200 µl homogenate was transferred to a new tube, concentrated to dryness using a Speedvac (Thermo Fisher Scientific) vacuum centrifuge, dissolved with 200 µl of ultrapure water (LC-MS grade) and filtered using Amicon Ultrafree-MC centrifugal filter devices (0.2 µm, 100 PK). For seed analysis, five seeds were extracted with 500 µl of extraction buffer without sinigrin, and diluted to 1/6 with ultrapure water. For UPLC-TQMS analysis, samples were diluted to 1/500 with ultrapure water (LC-MS grade) using an automated liquid handling system (Microlab STARplus, Hamilton). Compounds were separated using a HSS T3 1.8 µm column (1.0 × 50 mm, Waters), and the UPLC gradient program: 0–0.25 min, 99.9% of solvent A; 0.4 min, 91% of solvent A; 0.8 min, 83% of solvent A; 1.9 min, 0.1% of solvent A; 1.9–2.1 min, 0.1% of solvent A; 2.11–2.7 min, 99.9% of solvent A; and a flow rate of 0.24 ml min−1. Conditions for the TQMS (Waters) detection were capillary voltage of −0.80 kV (negative ion mode) or 0.50 kV (positive ion mode), cone voltage of 50 V (negative ion mode) or 30 V (positive ion mode), source temperature of 150°C, and desolvation gas of 1,000 l h−1 at 600°C. |
| Comment_of_details |
