Guard Cell Starch Concentration Quantitatively Related to Stomatal Aperture

Abstract

Gas exchange between the environment and leaf interior is regulated by stomatal aperture. Stomatal opening results from guard cell swelling which is caused by increased osmotica in the cell. Most of the change in osmotic potential can be accounted for by K+ influx (2, 6). Potassium uptake is balanced by 18,19) and organic anion synthesis (1,14 The principle used for starch determination has been described by Lust et al. (9). Details of the initial portions of the assay are given below.Step 1. Guard cell contents were extracted in 20 nl of 0.2 N KOH containing 100 mm ethanol using the "oil well technique" (8). Incubation was for 20 min at 80 C. Starch standards were carried through all steps of the assay.Step 2. One hundred nl of specific step reagent was added to the oil well droplet. The reagent was 100 mm sodium acetate (70 mm acid, 30 mm base) and 200 Ag/ml amyloglucosidase (from Rhizopus, EC 3.2.1.3). Incubation was for 60 min at 30 C. This step was terminated by heating to 80 C for 20 min. Glucose standards were incorporated into some aliquots of this step and were carried through the remaining steps of the assay.Step 3. Fifty nl of glucose reagent was added to the reaction droplet. The reagent was 360 mm Tris-HCl (50 mm acid, 310 mM base), 3.6 mm MgCl2, 1 mM ATP, 0.1 mm NADP+, 3.6 mm DTT, 0.2 ,ug/ml glucose-6-P dehydrogenase (from yeast, EC 1.1.1.49) and 3.6 ,ug/ml hexokinase (from yeast, EC 2.7.1.1). Incubation was for 30 min at 24 C.Step 4. One pl of 0.1 N NaOH was added. The droplets were heated to 80 C for 20 min.Step 5. The NADP in a 0.5-,lI aliquot was amplified by an enzymic cycling technique (8).Heating in 0.2 N ethanolic KOH was necessary to make amylaceous polymers susceptible to enzymic degradation. When wheat starch, corn starch, "soluble" potato starch, shellfish glycogen, and rabbit liver glycogen were heated in only 0.02 N NaOH, the end point of glucose release by Aspergillus amyloglucosidase was 34, 53, 55, 88, and 97%, respectively, of

Sucrose: a solute that accumulates in the guard‐cell apoplast and guard‐cell symplast of open stomata

“…Although he found a strong relationship between time of day and 'guard cell' sucrose content, the relationship between aperture and sucrose content was weak (r ~ = 0.46 for Vicia and r 2 = 0.29 for Commelina). The experimental approach of Outlaw and Manchester [12] was different from Pearson's approach [23] and also provides results that complement those presented here. These workers [12] made a technically unambiguous measurement of guard-cell-sucrose content of stomata that were stimulated to open under non-photosynthetic conditions.…”

“…The experimental approach of Outlaw and Manchester [12] was different from Pearson's approach [23] and also provides results that complement those presented here. These workers [12] made a technically unambiguous measurement of guard-cell-sucrose content of stomata that were stimulated to open under non-photosynthetic conditions. The values thus obtained were, on average, only one-half of those reported here.…”

“…[14]) because peeling ruptures many epidermal cells and thus relieves the back-pressure they exert on guard cells. Also importantly, the rate and extent of stomatal opening were less in these experiments than in comparable ones [12] in which two stimuli (viz. low [CO2] + light) were applied simultaneously.…”

“…Initially, the whole-leaf guard-cell sucrose content was 356 + 27 (S.E.) fmol.guard-cell-pair -1, which is similar to the control ('closed stomata') values (77 mmol-kg<~rly)= 460 fmol' guard-cell-pair -1) of Outlaw and Manchester [12], who made their measurements on detached leaflets in midmorning. Initially, the rinsed-peel guard-cell sucrose content was 140 fmol-guard-cell-pair ~, indicating that 39% of the sucrose of guard cells in closed stomata was …”

“…Subsequent investigations [9][10][11] also failed to show the obligatory relationship between aperture size and potassium concentration. Corroboratively, elevation of guard-cell sugars [12][13][14] and a unique pattern of sucrose-metabolizing enzymes in guard cells [15] have been the additional impetuses for our reinvestigation of the role of sucrose and potassium in stomatal movements. In the experiments reported here, both greenhouse-and growth-chamber-grown plants were investigated over a daily time course of normal stomatal movements in planta.…”