一、Kochian 水培铝处理
Wild-type (Col-0 and Ler-0) and mutant seeds were surface-sterilized and cold stratified in dark at 4℃ for 3 days before sown on 250 μm polypropylene meshes floating on hydroponic growth solutions supplemented without or with Al or other metal ions in Magenta boxes.
The hydroponic solution consisted of the following macronutrients in mM: MgCl2, 3.0; (NH4)2SO4, 0.25; Ca(NO3)2, 1.0; KCl, 2.0; CaCl2, 2.75; KH2PO4, 0.18; and the following micronutrients in μM: H3BO3, 5.0; MnSO4, 1.0; CuSO4, 0.05; ZnSO4, 0.2; Na2MoO4, 0.02; CoCl2, 0.001. Two milimolar (mM) of Homo-PIPES were included in the solutions to maintain the pH at 4.2. Plants were grown in a plant growth chamber with a setting of 16/8 h day/night at 22℃.
二、Larsen Al-soaked gel 处理
Experiments using Al-soaked gel and Al-containing hydroponics were conducted
on common plates as previously described (Larsen et al., 1996, 2005). For all seedling growth experiments, Col-0 wild type and mutant Arabidopsis thaliana seeds were surface-sterilized and cold stratified at 4°C for 4 d in the dark to synchronize germination. Seeds were then sown on either soaked gel plates or hydroponic plates. For soaked gel plates, nutrient medium (pH 4.2)
consisted of 80mL of 1mMKNO3, 0.2mMKH2PO4, 2 mMMgSO4, 0.25mM
(NH4)2SO4, 1mMCa(NO3)2, 1mMCaSO4, 1 mMK2SO4, 1 mMMnSO4, 5 mM
H3BO3, 0.05 mM CuSO4, 0.2 mM ZnSO4, 0.02 mM NaMoO4, 0.1 mM CaCl2,
0.001 mM CoCl2, 1% sucrose, and 0.125% Gellan gum (Gell-8Gro; ICN Biomedicals).
For Alexperiments,thesolidifiednutrientmediumwassoakedwith
20 mL of nutrient medium 6 AlCl3 (pH 4.2) for 2 d, after which the soaking
solution was removed and seeds were planted and allowed to grow for 7 d
unless otherwise specified. For dose-response analyses in a hydroponic
environment, seedlingsweregrown on250-mmmeshpolypropylene screen in
100 3 15-mm X-plate dishes with 40 mL of liquid nutrient medium (pH 4.2)
describedabovewithvaryingconcentrations of AlCl3.Allgrowthanalyseswere
performed in a Percival 136LLVL plant growth chamber (Percival Scientific)
with light intensity at 40mmol of photons/s/m2 under a 24-h light cycle at 20°C.
参考文献
[1] Caroline A. Sjogren, Stephen C. Bolaris, and Paul B. Larsen.(2015). Aluminum-Dependent Terminal Differentiation of theArabidopsis Root Tip Is Mediated through an ATR-, ALT2-, andSOG1-Regulated Transcriptional Response. Plant Cell 27: 2501–2515.
[2] Yuqi Wang, Ruihong Li, Demou Li, Xiaomin Jia, Dangwei Zhou, Jianyong Li, Sangbom M. Lyi, Siyu Hou, Yulan Huang, Leon V. Kochian, and Jiping Liu.(2017). NIP1;2 is a plasma membrane-localized transporter mediating aluminum uptake, translocation, and tolerance in Arabidopsis. Proc. Natl. Acad. Sci. USA 114:5047-5052.
