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. 2016 May 26;6:26685. doi: 10.1038/srep26685

Figure 3. Generation of a new allele of the ost2 mutant using a tru-gRNA and Cas9 driven by a tissue-specific promoter, and the altered stomatal responses in Arabidopsis.

Figure 3

(a) Schematic structure of the CRISPR/Cas9 vectors harboring a tissue-specific promoter for Cas9 expression using Arabidopsis elongation factor α-1 (AtEF1) (pEgP126_Paef1-2A-GFPSD2) and histoneH4 (AtH4) (pEgP126_Phis4-2A-GFPSD2) promoters. (b) GFP fluorescence in the T1 transgenic plants of the tissue-specific CRISPR/Cas9 vectors. Bar = 100 μm. (c) Generation of a new allele of ost2 mutant (ost2_crispr-1) using the tissue-specific CRISPR/Cas9. A base substitution (1492C to T, red character and asterisk in the sequence data) was detected in genomic DNA in the mutants. This mutation creates a stop codon (underlined) just after the mutation site. (d) A new mutant for OST2 gene (ost2_crispr-1) generated by the tissue-specific CRISPR/Cas9. Bar = 1 cm. (e) Thermal infrared imaging of the wild-type and ost2_crispr-1 showing the temporal variability in stomatal conductance. (f ) Stomatal conductance of rossette leaves in the wild-type and ost2_crispr-1 measured by leaf polometer. Values are means and SD (n = 20). Asterisks indicate statistically significant differences between the wild-type and mutant plants, determined by Student’s t tests. *P < 0.0001 (g) Effects of various concentrations of ABA on stomatal closure in ost2_crispr-1 and wild-type. Epidermal peels were treated with or without ABA for 1 h after stomatal preopening under light for 2 h, and the stomatal aperture was measured under a microscope. Values are means and SD (n = 30). Asterisks indicate statistically significant differences between the wild-type and mutant plants, determined by Student’s t tests. *P < 0.0001. L1, L2 are T2 individuals harboring mutations in OST2 locus. (h) Transpirational water loss in ost2_crispr-1 and wild-type at the indicated time points. Water loss is expressed as a percentage of the initial fresh weight. Values are means and SD of five samples of two shoots of each ost2_crispr-1 and wild-type.