Total nucleic acid was isolated from whole leaves and guard cell protoplasts, and the DNA was removed using RQ1 RNase free DNase I (Promega, Madison, WI). The absence of DNA in the samples was confirmed after saturating PCR (38 cycles) with actin-specific primers. One microgram of total RNA was used for cDNA synthesis using Omniscript RT (Qiagen, Valencia, CA) with oligo(dT)₂₀ as the primer. PCR reactions contained 1× buffer, 2 μL of the reverse transcription reaction, and 1.5 μg of forward and reverse primers in a total reaction volume of 25 μL using the following conditions: 94°C/5 min; 94°C/50 s, annealing temperature (see below)/50 s; 72°C/1 min, 72°C/5 min. PCR products were visualized on ethidium bromide–stained 1.2% agarose gels.

Primers used were as follows: actin (X63603) (annealing temperature, 52°C), forward, 5′-CGCGAAAAGATGACTCAAATC-3′ and reverse, 5′-AGATCCTTTCTGATATCCACG-3′; CAT (U93244) (annealing temperature, 55°C), forward, 5′-CGGATACCTGAGCGTGTTGTTCATG-3′ and reverse, 5′-GTGATTATTGTGATGAGCACAC-3′; MDHAR (BQ842867) (annealing temperature, 55°C), forward, 5′-ACTTCAAATAGCCGTTTTTAATCCA-3′ and reverse, 5′-AGTTGAACATGTTGATCATTCTC-3′; FeSOD (M55090) (annealing temperature, 53°C), forward, 5′-TGCTTTGGAGCCTCATATGAG-3′ and reverse, 5′-AAGTCCAGATAGTAAGCATGC-3′; tobacco DHAR (AY074787) (annealing temperature, 55°C), forward, 5′-AATTGGATCCCTGATTCTGATGT-3′ and reverse, 5′-GCGAAACAACGGGATTATAATTATG-3′; wheat DHAR (AY074784) (annealing temperature, 59°C), forward, 5′-AATTGGATCCCTGATTCTGATGT-3′ and reverse, 5′-GGATCCAGGGGCTTACGGGTTCACTTTC-3′; to detect wheat and tobacco DHAR (annealing temperature, 59°C), forward, 5′-AATTGGATCCCTGATTCTGATGT-3′ and reverse, 5′-AGATGGTA(G/C)AG(C/T)TTCGGAGCCA-3′.