Confirmation of mesophyll signals controlling stomatal responses by a newly devised transplanting method

There are opposing views on whether the responses of stomata to environmental stimuli are all autonomous reactions of stomatal guard cells or whether mesophyll is involved in these responses. Transplanting isolated epidermis onto mesophyll is a potent methodology for examining the roles of mesophyll-derived signals in stomatal responses. Here we report on development of a new transplanting method. Leaf segments of Commelina communis L. were pretreated in the light or dark at 10, 39 or 70Pa ambient CO2 for 1h. Then the abaxial epidermises were removed and the epidermal strips prepared from the other leaves kept in the dark at 39Pa CO2, were transplanted onto the mesophyll. After illumination of the transplants for 1h at 39Pa CO2, stomatal apertures were measured. We also examined the molecular sizes of the mesophyll signals by inserting the dialysis membrane permeable to molecules smaller than 100-500Da or 500-1000Da between the epidermis and mesophyll. Mesophyll pretreatments in the light at low CO2 partial pressures accelerated stomatal opening in the transplanted epidermal strips, whereas pretreatments at 70Pa CO2 suppressed stomatal opening. Insertion of these dialysis membranes did not suppress stomatal opening significantly at 10Pa CO2 in the light, whereas insertion of the 100-500Da membrane decelerated stomatal closure at high CO2. It is probable that the mesophyll signals inducing stomatal opening at low CO2 in the light would permeate both membranes, and that those inducing stomatal closure at high CO2 would not permeate the 100-500Da membrane. Possible signal compounds are discussed.