MYB30 transcription factor regulates oxidative and heat stress responses through ANNEXIN-mediated cytosolic calcium signaling in ArabidopsisIF：8.1

Materials and Methods
Plant material and growthArabidopsis thaliana L. Heynh. Columbia (Col-0) constitutivelyexpressing AEQUORIN (AQ) (Knight et al., 1991) driven by thecauliflower mosaic virus 35S promoter was used as the wild-type.Seeds were sterilized in a solution containing 20% (v/v) sodiumhypochlorite and 0.1% (v/v) Triton X-100 for 10 min, washedfive times with sterilized distilled water and sown on Murashigeand Skoog (MS) medium containing 2.5% (w/v) sucrose and0.3%(w/v) phytagar (Sigma-Aldrich), kept in the dark for 3 d at4°C, and then grown in a growth chamber at 23°C, 4000 luxunder continuous white light. High-throughput screening of [Ca2+]cytresponse mutants from a T-DNA insertion pool (pSKI015) inthe wild-type background was described previously (Pan et al.,2012). Mutants myb30-1 (SALK_122884), ann1 (SALK_015426)and ann4 (SALK_019725) were obtained from the ArabidopsisBiological Resource Center (ABRC). Laboratory consumables were obtained from Shanghai Yansheng industrial Co., Ltd. (Shanghai, China).

Cytosolic Ca2+ measurements
For AEQUORIN assays, 9-d-old seedlings growing on MSmedium were used for [Ca2+]cyt measurement. The [Ca2+]cytluminescence measurements were performed as described previously (Zhao et al., 2013), and the quantification of [Ca2+]cyt (inlmol l1) was performed as described by Rentel & Knight(2004). The plates were sprayed with 10 mM H2O2 and thephoton emissions were immediately collected for 3 min. For heattreatment, 30 ml of H2O at 45°C was poured into the plates andthe photon emissions were immediately collected for 8 min.For the Fluo-4 assay, the 7-d-old vertically cultured seedlingsof wild-type and myb30 were incubated with 10 lM Fluo-4-AMin the presence of 0.02% (v/v) pluronic F-127 for 2 h at 37°C.Fluorescence in the roots of stained seedlings was captured usingthe ANdor IQ confocal microscope (Andor, Belfast, Northern

Ireland) (excitation at 488 nm and emission at 520 nm) afterstress treatments.

感谢中国农业大学生物科学学院植物生理学与生物化学国家重点实验室引用文献