Advanced imaging of root chemicals offers new insights on plantgrowth ucsandiego NatureComms
was detected in roots using the 2',7'-dichlorodihydrofluorescein diacetate staining method. The roots from 7-day-old seedlings were incubated in HDCFDA for 30 min under dark conditions. Then, the roots were rinsed with sterilized water three times. The fluorescent signals were observed with an Echo Revolution microscope . The excitation and emission wavelengths used for detection of the signals were 488 nm and 520 nm, respectively.
To detect superoxide, seedlings were stained for 2 min in a solution of 2.5 mM Nitroblue tetrazolium in 50 mM phosphate buffer in the dark and then rinsed three times with distilled waterConfocal images, most of the roots were stained by PI and observed under a Zeiss LSM880 confocal microscope . Some of the roots were stained by PI and observed under a Leica SP8 confocal microscope , thanks to the UCSD Microscopy Core—NS047101.
The parameters of the HPLC-MS/MS method are as follows: Analysis was performed on a Thermo Scientific Vanquish UHPLC system coupled with an Orbitrap-MS with a heated electrospray ionization source. Chromatographic separation was carried out on a Kinetex® C18 column with a Kinetex® C18 guard column maintained at 35 °C. The mobile phases were A , 1000/1, v/v) and B /FA, 1000/1, v/v) with the gradient program: 0–6 min, 1% B; 6–7 min, 1% B to 90% B; 7–9 min, 90% B; 9–9.
Two days after germination, the primary root tip was cut, flash-frozen in liquid nitrogen, and mounted in optimal cutting temperature compound. 20 μm-thick tissue sections were cut with a cryostat and thaw-mounted onto a glass slide. While use of O.C.T. has been reported to cause ion suppression due to polyethylene glycol and benzalkonium salts in the positive ion mode
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