Congo red

For evaluation of growth on agar plates under stress conditions, YAMM was used (47 (link)). For osmotic stress, 1 M NaCl, 1 M KCl, or 1.2 M sorbitol was added. For cell wall stress, calcofluor white (fluorescent brightener 28) (MP Biomedicals, Irvine, CA) or Congo red (Fujifilm Wako Pure Chemical Corp., Osaka, Japan) were added from 100-fold concentrated stock solutions. The response to temperature was evaluated at 30, 37, 42, and 45°C. Acid or alkaline stress was evaluated at pH 4 or 8. The medium pH was adjusted with HCl or NaOH.

Branches from adult corals of seven species (A. digitifera, G. fascicularis, G. aspera, M. digitata, P. divaricata, P. damicornis, and P. australiensis) were crushed using an iron mortar and pestle on ice and homogenized using stainless steel beads in PBS buffer (pH 7.4) at 3,000 rpm for 5 s. The crushed coral samples were centrifuged at 10,000 g for 10 min at 4 °C and the supernatants were concentrated using a disposable ultrafiltration unit (type USY-1; Advantec, Dublin, CA, USA). The protein concentration in the supernatants was adjusted to 2 mg/mL, and then, the supernatants were used as enzyme solutions. Agarose plates containing 1.5% agarose, acetate buffer (pH 5.5), and 0.1% carboxymethylcellulose (CMC) (Sigma) were prepared for assessing cellulase activity in the corals. To assess chitinase activity in the corals, agarose plates containing 0.1% glycol chitin were prepared according to the method of Yamada & Imoto (1981) (link). In brief, 5 µL of enzyme solution was deposited on the centre of each plate and the plate was incubated at 25 °C for 48 h. Then, the plates were stained with 0.1% Congo red (Wako) for 1 h and de-stained with 1 M NaCl solution.

For evaluation of fluorescent staining of pollen tubes on the medium and in the micropyle, aniline blue (415049; Merck, Germany), fluorescent brightener 28 (used as calcofluor white; F3543; Merck, Germany), Congo red (032-03922; Wako, Japan), PI (P4864; Merck, Germany), FM4-64 (F34653; Thermo Fisher Scientific, Waltham, MA, USA), and FDA (F7378; Merck, Germany) were prepared in the pollen germination medium without agarose. The final concentration of each dye is shown in Figure 1. At 5.5 h after pollination, 20 µL of each dye was dropped on the medium. After 5 min, staining of pollen tubes was observed with an Axio Imager A2 upright microscope (Zeiss, Jena, Germany) equipped with a cooled charge-coupled device (CCD) camera (Axiocam 506 color; Zeiss, Germany). Filter set 47 HE was used for aniline blue and calcofluor white. Filter set 31 was used for Congo red, PI, and FM4-64. Filter sets 09 and 38 were used for FDA. To quantify pollen tube attraction into ovules, pollen tubes were stained with 5 µM FDA dissolved in hydrated silicone oil (KF-96–100CS; Shin-Etsu Chemical, Japan). After 5 min of staining, pollen tubes were observed.