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. Author manuscript; available in PMC: 2018 Feb 22.
Published in final edited form as: Nature. 2017 May 10;545(7654):311–316. doi: 10.1038/nature22077

Extended Data Figure 1. Nitrate promotes plant development and induces Ca2+ signatures in both leaves and roots.

Extended Data Figure 1

a, Nitrate promotes both shoot and root development. Plants were germinated without exogenous nitrogen source for 4 days and then transferred to the plates supplemented with different concentrations of KNO3, NH4Cl or glutamine (Gln) for 7 days. Scale bar, 1 cm. The experiments were repeated twice with 10 seedlings for each treatment with consistent results. b, c, Distinct Ca2+ signatures induced by nitrate and flg22 in aequorin transgenic plants. Arabidopsis transgenic seedlings constitutively expressing the Ca2+ reporter protein apoaequorin were grown in liquid medium containing 2.5 mM ammonium succinate as the sole nitrogen source for 6 days. Aequorin was reconstituted with 10 μM coelenterazine for overnight in the dark. The results are presented as relative light units (RLU) in response to 10 mM KCl or KNO3 (b) or in response to 100 nM flg22 or water (c) at intervals of 1 sec. Error bars, ±s.e.m, n=10 seedlings. The experiments were repeated three times with similar results. The RLU value is cut off at 3,500. d, NRT1.1/CHL1/NPF6.3/NRG1 is highly expressed in shoots and mesophyll protoplasts. The signal counts of the genes in roots and shoots were derived from previously published microarray data1. The signal counts of the genes for mesophyll protoplasts were derived from previously published microarray data61. LHCB2.2 (PHOTOSYSTEM II LIGHT HARVESTING COMPLEX GENE 2.2) serves as a leaf-specific expression control. The control gene UBQ10 is constitutively and highly expressed in roots, shoots and mesophyll protoplasts. Error bars, s.d., n=3 biological replicates from mesophyll protoplasts. e, Nitrate induction of the endogenous NIR gene as a primary nitrate responsive marker gene in seedlings and mesophyll protoplasts. NIR expression was quantified by RT-qPCR analysis. Arabidopsis seedlings or mesophyll protoplasts were treated with 10 mM KCl or KNO3 for 2 h. Error bars, s.d., n=3 biological replicates. f, g, Time-lapse images of nitrate stimulated Ca2+ signalling in roots of intact transgenic GCaMP6 plants. The entire time-lapse recording of Ca2+ signals stimulated by 10 mM KCl or KNO3 in the root tip (f) or the elongated region (g) was presented in Supplementary Video 3 and 4. Seedlings were grown on basal medium without nitrogen for 4 days and then stimulated by KCl or KNO3. Scale bar, 10 μm. The experiments were repeated three times with 10 seedlings for each treatment with consistent results. Source data for d, e can be found in Supplementary Information.