Dx402

Primary root tissue close to the seed (the first 0.5 cm) was harvested for light microscopy and scanning electron microscopy (SEM) studies. For light microscopy, tissues were fixed in 96% ethyl alcohol and stored at 4° C. Tissues were then placed in a histocassette and gradually dehydrated in ethyl alcohol. Subsequently, they were placed in a 1:1 solution of ethyl alcohol and 100% xylol (Spintissue Processor STP120 and Histo Star Embedding Centre, Thermo Scientific). The dehydrated samples were embedded in Histoplast (cat. no. 22900700, Fisherbrand), and 5 μm-thick cross-sections were cut with a microtome (Microm HM 340E, Thermo Scientific) and incubated in a drying oven (DX402, Yamato) at 60° C for 30 min, then placed in xylene for 2 min to remove paraffin. Tissue samples were then rehydrated in 1× PBS and visualized for endogenous fluorescence by epifluorescence microscopy (DIM6000, Leica, Germany). For SEM, the samples were treated as previously described by Olivares-Garcia et al. (2020) (link).

To fabricate PDMS rotators, the replica molding method was used. The mold was made of an acrylic plate manufactured by a cutting machine (MODELA MDX-40A, Roland). The mold was then adhered to Pyrex glass. PDMS was made by mixing the base (SILPOT184 BASE) and the curing agent (SILPOT184 CAT) at a 1:1 ratio (wt/wt). Next, the prepolymer of the PDMS was fully mixed in a rotary mixer (AR-100, THINKY) and degassed in an aspirator (AS-01, AS ONE) for 15 min. The prepolymer PDMS was poured into the mold, covered with another piece of Pyrex glass, and clamped with a clip. The whole device was placed in a 60 °C oven for 24 h (DX 402, Yamato Scientific). After drying, the PDMS rotators were carefully released from the acrylic plates by a metal ruler.

We used the GF/F glass filter, which had been burned and dried before the weight was measured by electric balance (Sartorius CPA2252), prior to the SS analysis. The filtrate was dried in the 60 °C automatic oven (Yamato DX402, Yamato, Japan) over 12 h before the weight was measured. After that, we burned the dried filter at 450 °C for 2 h using an electric muffle furnace (Yamato FO410), then measured the weight again in the same way. The SS content was calculated as follows; (450 °C burned weight)–(60 °C dried weight).