What happens when a cell is subjected to a solution where the water concentration is lower than that inside a cell?

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glycol tertdodecyl thioet her), ß amylase and 0.1 percent Nonic 218, distilled water, or 0.1 percent Nonic. Prior to placement, the stomata were checked to make sure they were closed and an estimate of guard cell starch was made for later comparative purposes.

The condition of the stomata was observed 21/2, 14, and 26 hours after treatment.

More stomata opened, and wider openings were observed, in either 1,000 p.p.m. a or ß amylase. With decreasing amylase concentration, stomatal opening resembled that found in the 0.1 percent Nonic 218. More stomata were open in Nonic than in distilled water. Epidermal strips containing guard cells floated on water have been reported to open, possibly a reflection of passive movements discussed in a preceding subsection (p. 18). Fourteen hours later, all stomata were found to have closed to some extent. At this time starch had disappeared in all but those guard cells subjected to the lowest concentration of amylase or in distilled water.

It was unknown why guard cell starch disappeared in 0.1 percent Nonic 218. It had originally been included in this experiment to increase wetting. The experimental results with amylase could not be evaluated until further information on Nonic action was available.

Experiment 2.—This experiment utilized the same solutions as experiment 1, but leaf disks were taken from the mature leaves of Zea mays. The condition of stomata was observed at 2 and 4 hours after treatment, and starch content in guard cells was determined after 24 hours.

None of the treatments unequivocally opened the stomata of corn. A few were opened in a amylase at 1,000 or 10 p.p.m. when checked at the 4-hour period, but the area of opening was correlated with a translucent appearance of the leaf at that point.

The 24-hour starch check showed a normal starch accumulation in the distilled water control and 0.1 p.p.m. a amylase. Guard cells treated with 1,000 p.p.m. and 10 p.p.m. a amylase and with Yonic 218 lacked starch.

Experiment 3.-Several wetting agents were included in this experiment to test their effect on guard cell activity. Leaf disks of bean leaves were submerged in three wetting agents; namely, 0.1 and 0.01 percent Tween 20 (polyoxyethylene glycol sorbiton monolaurate), Nonic 218, or Vatsol OT (sodium dioctylsulfosuccinate). The stomatal condition of these disks were checked 4, 7, and 27 hours later. Four hours after treatment, stomata in all treatments opened, but the opening occurred between the time the epidermis was stripped from disks and observed through the microscope. Seven hours after treatment, the condition of the stomata was observed without peeling. That the stomata opened after peeling was reconfirmed; open stomata in leaf disks were found only at the

edge of disks or in thoroughly wetted translucent areas. One-tenth percent Vatsol OT and Xonic 218 were evidently toxic, as coagulation of protoplasm was noted in some treated guard cells. The disappearance of starch in Nonic-treated guard cells in the previous experiment probably was caused by Nonic toxicity.

Experiment 4.-Since Hagen (8) reported he had to evacuate the tissue to facilitate opening of the stomata by diastase, it was surmised that penetration of the enzyme molecule was limiting amylase action. The lack of starch in the guard cells at the end of experiments 1 and 2 indicated a degradation of the starch. However, the degradation, if limited by enzyme concentration, could have been too slow to have had an observable effect on guard cell activity, or possibly the degradation products could have diffused out of the cell without an effect on stomatal condition. An attempt was made to increase the penetration of amylase into the guard cells.

It has been reported that ethyl alcohol as well as acetone increases the cellular penetration of some substances. Various mixtures of alcohol and acetone were tried. Whether alcohol or acetone had any effect on a or ß amylase activity was determined by following cornstarch digestion at various concentrations of enzyme in vitro. High concentrations of alcohol or acetone mixed with distilled water (50 percent by volume) interfered with the color reaction necessary for evaluation of amylase enzyme activity. A 25-percent or less alcohol or acetone solution had no apparent effect on the activity of the enzymes.

Concentrations of a and/or ß amylase at 500 and 5 p.p.m. were used in solutions of 25 and 12.5 percent acetone or ethyl alcohol, or in solutions of 1,000 p.p.m. Tween 20, Nonic 218, or Vatsol OT. With the same proportions maintained, all combinations of amylases, acetone, alcohol, and wetting agents were used also. The solutions were checked for stomatal effect by submerging leaf disks in well slides containing a solution or by applying a drop of solution directly on a mature leaf. The stomata were observed 60 minutes after application. Zea mays, Zebrina pendula, Lycopersicon esculentum var. commune, Brassica rapa, and Nicotiana tabacum were the species tested. Prior to use, all plants were placed in the dark to close stomata, after which the guard cells were stained with iodine-potassium iodide to insure that starch was present.

No reproducible effect by the amylase solutions was found in any of the species tested.

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8 PALLAS, J. E., JR. EFFECTS OF TEMPERATURE AND HUMIDITY ON THE ABSORPTION AND TRANSLOCATION OF 2,4-DICHLOROPHENOXYACETIC ACID AND BENZOIC ACID. 1958. (Unpublished dissertation; on file at University of California, Davis.)