Tcs sp8

Before choline treatment, choline chloride was dissolved in water to make a 100-mM choline solution. In the experiment, the choline solution was diluted to a concentration of 1 mM, and the seedlings were treated for the desired time.
For BFA treatment, 6-day-old Arabidopsis seedlings were immersed in 50 μM BFA for 1.5 hours, and then the BFA bodies were observed by confocal laser scanning microscopy (Leica TCS SP8). In the washout experiments, the BFA-treated seedlings were incubated in water for 2 hours, after which the BFA bodies were examined by confocal laser scanning microscopy (Leica TCS SP8).
For CHX and BFA treatment, 6-day-old Arabidopsis seedlings were pretreated with 50 μM CHX for 30 minutes, followed by being immersed in a solution with 50 μM CHX and 50 μM BFA for 1.5 hours, and then the BFA bodies were observed by confocal laser scanning microscopy (Leica TCS SP8). In the washout experiments, above seedlings were incubated in water for 2 hours, and the BFA bodies were then examined by confocal laser scanning microscopy (Leica TCS SP8).
For 1-butanol treatment, the Arabidopsis seeds were grown on 1/2 MS salt medium plate for six days, and the seedlings were then transplanted to the 1/2 MS salt medium plate containing 0.4% 1-butanol and grew for another five days. The root length was measured by Image J.

Samples were imaged using a Leica TCS SP8 laser scanning confocal microscope and Spinning disk confocal microscope. Images were obtained with ×25 objective for propidium iodide (PI) staining and ×40 and ×60 objective for microtubule labelling. For the microtubule labelling, maximum Z-projections were used and the Z-step size was 1 μm. Images were captured by a Leica TCS SP8 at 488 and 561 nm laser excitation and 500–550 nm and 590–630 nm emission for GFP and PI staining. Images captured by a spinning disk confocal microscope were obtained with ×40 and ×60 objective. For the microtubule labelling, maximum Z-projections were used and the Z-step size was 1 μm. Images were captured at 488 nm laser excitation and 500–550 nm emission.
PI (5 μg ml⁻¹)-stained cotyledon pavement cells from 7-day-old seedlings were imaged. ImageJ software was used to measure the lobe number, lobe length, neck width, perimeter, and area (http://rsb.info.nih.gov/ij). Circularity was analysed according to Zhang et al. (2011) (link).
Microtubule alignment was quantified according to Gomez et al. (2016) . At least 50 cells were used. The values were recorded and the significance was analysed using Student’s paired t-test.

The constructs of UBQ10-GGL-GFP or 35S-GGL-YFP were transformed into the Agrobacterium strain GV3101, and the strains were infiltrated into N. benthamiana leaf epidermis. Protoplasts of infiltrated tobacco leaves were prepared as described previously (Walter et al., 2004 (link)). Images were obtained by a confocal microscope (TCS-SP8; Leica, Weztlar, Germany) with a 40× water-immersion objective in the sequential scan, between frames mode. For localization in ER, an ER-marker HDEL-OFP (orange fluorescent protein; excitation at 561nm, emission range is 580nm to 630nm) was coexpressed for co-localization. Nile Red staining was performed for localization in lipid droplets, as described in our previous publication (Tang et al., 2020 (link)).
To confirm the subcellular localization of GGL13, GGL17, and GGL27 in Arabidopsis, GGL13-GFP, GGL17-GFP, or GGL27-GFP was transformed into Arabidopsis mesophyll protoplasts with HDEL-OFP (Yoo et al., 2007 (link)), respectively. The GFP and OFP signals of protoplasts were recorded 10–12h after transformation under a confocal microscope (TCS-SP8; Leica, Weztlar, Germany).