FIGURE 4. CAS is required for guard-cell Ca²⁺_o signalling and plant development.

a, Northern blot analysis of CAS mRNA levels in leaves from wild-type and CAS antisense transgenic plants (AS1 to AS3). b, Western blot analysis of CAS protein levels using total proteins from leaves. An 81-kDa band stained non-specifically by anti-CAS antibodies. c, Defect in guard-cell CICI in AS1 plants. AS1 was transformed with a cameleon construct carrying a basta resistance marker. 2 mM Ca²⁺ was added as described. Other conditions were the same as in Supplementary Fig. S-1. d, Increase in guard-cell [Ca²⁺]_i (F_535 nm/F_480 nm) in response to Ca²⁺ treatment in the wild type, AS1 and AS2 from experiments as in c (n = 48 to 56 guard cells). [Ca²⁺]_i increases were normalized to that of the wild type. r.u., relative unit. e, Defects in Ca²⁺_o-induced stomatal closure in CAS antisense lines. Stomatal apertures were normalized to those without 2 mM Ca²⁺ treatment (n = 120 stomata). f, CAS is required for bolting in response to reduced Ca²⁺ supplies. Both wild-type and AS1 plants were grown in media containing 1 mM (left) or 0.1 mM Ca²⁺ (right), and plant photos were taken after 23 days of growth. g, Quantitative analysis of the late-bolting phenotype of AS1 grown at 0.1 mM Ca²⁺. Data from four separate experiments and total of 60 seedlings are shown.