Since the expression of AtINH3 was observed preferentially in guard cells, we initially attempted to characterize the physiological function of Inh3 in guard cells using Arabidopsis T-DNA insertion lines. We obtained two T-DNA insertion lines from the SALK and SAIL collections and designated the corresponding alleles inh3-1 (SALK_044593) and inh3-2 (SAIL_806_C02), respectively. The insertions were located in the second exon of AtINH3, 146 and 1 bp downstream of the ATG initiation codon in inh3-1 and inh3-2, respectively (Fig. 6A   ). A PCR-based screen using gene-specific primers and the T-DNA left border primer revealed that all the plants from the T3 or T4 generation were the heterozygous or wild-type strains, and there were no homozygous plants (Fig. 6, B and C). All the heterozygous plants were morphologically similar to the wild-type plants and did not show any impairment in stomatal responses (data not shown). Segregation analyses of >100 self-pollinated heterozygous (parent) plants revealed that there was a 2:1 ratio of T-DNA heterozygous plants to wild-type plants (Table I   ), suggesting that a homozygous insertion confers the lethality and/or reduced gametophytic transmission. To test whether the insertion was transmitted normally through both male and female gametophytes, we reciprocally crossed inh3-1 with the wild type and screened F1 plants for the T-DNA insertion by PCR. Transmission of the T-DNA insertion occurred through both gametophytes (Table I).