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Science 315 (5817): 1423-1426

Copyright © 2007 by the American Association for the Advancement of Science

Coupling Diurnal Cytosolic Ca2+ Oscillations to the CAS-IP3 Pathway in Arabidopsis

Ru-Hang Tang1*, Shengcheng Han1*, Hailei Zheng2,3,1*, Charles W. Cook1, Christopher S. Choi1, Todd E. Woerner4, Robert B. Jackson1, and Zhen-Ming Pei1{dagger}

1 Department of Biology, Duke University, Durham, NC 27708, USA.
2 Department of Biology, Xiamen University, Xiamen, Fujian 361005, China.
3 State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, Fujian 361005, China.
4 Department of Chemistry, Duke University, Durham, NC 27708, USA.


Figure 1 Fig. 1.. Media Ca2+ and CAS control diurnal [Ca2+]i oscillations. Blue, WT; red, CASas. (A) Imaging the resting [Ca2+]i in Arabidopsis wild-type (WT) and CAS antisense (CASas). The bright light-field and aequorin luminescence images were taken at 8 hours after dawn from plants grown on 1/2 MS media under long-day conditions. (B)[Ca2+]i oscillations in leaves. The aequorin luminescence images were taken from plants as in (A) every 3 hours starting from dawn. The luminescence was normalized to that discharged with excessive Ca2+, and the maximum value was arbitrarily set to 1. The white and black bars represent objective light on and off, respectively. r.u., relative unit. Data from five separate experiments are shown [mean ± SD; n = 150 plants; two-way analysis of variance (ANOVA), P< 0.001]. (C) The effect of media Ca2+ on [Ca2+]i. Plants were grown on agar media containing varied [Ca2+] under long-day conditions for 2 to 3 weeks. Aequorin luminescence images were taken as in (B). Data from three experiments are shown (mean ± SD; n = 120 plants; two-way ANOVA, P < 0.001). (D) [Ca2+]i oscillations in plants grown in 1 mM (solid lines) or 30 mM Ca2+ (triangles) (mean ± SD; n = 120 plants; two-way ANOVA, P< 0.001). [View Larger Version of this Image (57K GIF file)]
 

Figure 2 Fig. 2.. Media Ca2+ and stomatal conductance control diurnal [Ca2+]o oscillations. Blue, WT; red, CASas. (A) [Ca2+]o oscillations in leaves. The luminescence images were taken every 3 hours, starting at dawn from plants expressing apoplastic aequorin grown on 1/2 MS. The luminescence was normalized to that discharged with excessive Ca2+, and the maximum value was set to 1. Data from three separate experiments are shown (mean ± SD; n = 75 plants; two-way ANOVA, P >0.5). (B) The effect of media Ca2+ on the resting [Ca2+]o. Luminescence images were taken as in Fig. 1C. The other conditions were the same as in (A). Data are shown as the mean ± SD (n = 75 plants; two-way ANOVA, P >0.5). (C) WT plants were grown in soil for 3 weeks under long-day conditions, and stomatal conductance was monitored (mean ± SEM; n = 3). (D) 45Ca uptake measured over a long day. 45Ca was added to the growth solution at dawn. Shoots were collected every 2 hours for 2 days, and 45Ca radioactivity was measured (17). The data for the second day are shown. cpm/cm2, counts per minute/leaf area (cm2). (E) The effect of media Ca2+ on the shoot Ca content in plants grown under conditions as in Fig. 1C. mg/g DW, milligrams Ca per gram dry weight (mean ± SD; n = 15 to 25 plants; two-way ANOVA, P >0.5). (F) The analysis of CAS promoter::GUS reporter (17). [View Larger Version of this Image (32K GIF file)]
 

Figure 3 Fig. 3.. CAS is a cell-surface receptor in the IP3 pathway. (A) Plasma membrane localization of CAS in HEK293 cells expressing a CAS-GFP construct (upper) with GFP as a control (lower). Scale bar, 5 µm. (B) The effect of Ca2+o on IP3 generation in HEK293 cells expressing CAS (red). The control (blue) was cells transfected with empty vector. Cells were bathed in 0.1 mM Ca2+ before addition of 2.5 mM Ca2+ and harvested at the times indicated. The IP3 content was determined by [3H]IP3 radioreceptor assay (17) (mean ± SEM; n = 6). (C) Inhibition of CICI by PLC blockers in HEK293 cells expressing CAS. Cells were incubated with 0.1 mM Ca2+ without (white circles) or with 100 µMneomycin (black circles) or U-73122 (black squares) before Ca2+ stimulation. Ca2+ at 2.5 mM was added at the time indicated (arrow), and [Ca2+]i was monitored by Fura-2 imaging (17). (D) Inhibition of CICI by neomycin in guard cells. Epidermal peels carrying the Ca2+-indicator cameleon were incubated in solution containing 50 µMCa2+ for 2 hours, and then treated with 2.5 mM Ca2+ (10). Neomycin (100 µM) was added 15 min before addition of Ca2+.[Ca2+]i is shown as changes in emission fluorescence ratios ({Delta}[Ca2+]i; n = 15 cells; P< 0.001). (E) Ca2+o induces IP3 generation in Arabidopsis leaves. Leaves were incubated in the solution containing 50 µMCa2+ for 2 hours, then transferred to the same solution (white circles) or the solution containing 10 mM Ca2+ (black circles), and harvested at the times indicated. IP3 was determined by [3H]IP3 assay (mean ± SEM; n = 6). (F) Neomycin inhibits Ca2+o-induced IP3 generation in leaves. The samples were collected before addition of Ca2+ (white bars) or 60 s after addition of 10 mM Ca2+ (black bars). Neomycin (100 µM) was added 15 min before Ca2+ stimulation (mean ± SEM; n = 6). pmol/gFW, picomole per gram fresh weight. [View Larger Version of this Image (25K GIF file)]
 

Figure 4 Fig. 4.. CAS is required for the Ca2+o-evoked IP3 pathway. (A and B) Ca2+o-induced IP3 production in single WT guard cells. The leaf epidermis was incubated with 50 µMCa2+ and treated with 10 mM Ca2+. Ca2+o triggered PHPLCd-GFP translocation from the plasma membrane (blue) to the cytosol (red). The images in (B) correspond to before ({circ}) and after (bullet) Ca2+ stimulationin (A). (C) IP3 content in WT and CASas leaves with (black bars) and without (white bars) addition of 10 mM Ca2+ (mean ± SEM; n = 6; P < 0.02 for WT; P > 0.5 for CASas1 and CASas2). (D and E) Defect in Ca2+o-induced IP3 generation in CASas guard cells. The CASas line expressing PHPLCd-GFP was generated from a cross between CASas and the wild-type PHPLCd-GFP line, as in (A). (F) Increases in PHPLC{delta}-GFP fluorescence in the cytosol with addition of Ca2+ were analyzed from experiments as in (A) and (D). Thirty-eight out of 133 guard cells (28.6%) for the wild type (A) and 4 out of 76 (5.3%) for CASas (E) showed increases. Data are shown as the mean ± SEM (P< 0.001). Scale bars, 5 µm. [View Larger Version of this Image (37K GIF file)]
 


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