Protoplast Isolation and Transfection.

Mesophyll protoplasts were isolated from 4-wk-old leaves of Arabidopsis plants expressing C-AT1.03-nD/nA or TKTP-AT1.03-nD/nA by the tape–Arabidopsis sandwich method (47). Leaves were fixed on tape with the lower epidermal surface facing upward and peeled with a strip of tape. The exposed mesophyll cells were digested in digestion buffer containing 20 mM 2-(N-morpholino)ethanesulfonic acid (Mes) buffer (pH 5.7) containing 1% (wt/vol) cellulose (Yakult), 0.25% (wt/vol) macerozyme (Yakult), 10 mM CaCl₂, 20 mM KCl, 0.1% (wt/vol) BSA, and 0.4 M mannitol with gentle agitation (50 rpm) for 1 h. After washing the released protoplasts with W5 solution (2 mM Mes, pH 5.7, 154 mM NaCl, 125 mM CaCl₂, and 5 mM KCl) twice, protoplasts were incubated on ice for 30 min, centrifuged, and resuspended into MMg solution (4 mM Mes, pH 5.7, 15 mM MgCl₂, and 0.4 M mannitol). Protoplasts were transfected with plasmids by incubating for 10 min at room temperature in the presence of 20% (wt/vol) PEG4000 with 0.1 M CaCl₂ and 0.2 M mannitol. After washing with W5 solution twice, transfected protoplasts were incubated in 1 mL of WI solution [0.5 M mannitol, 4 mM Mes (pH 5.7), and 20 mM KCl] in the dark at 25 °C overnight.