THE EFFECTS OF OZONE AND NUTRIENT SUPPLY ON STOMATAL RESPONSE IN BIRCH (BETULA-PENDULA) LEAVES AS DETERMINED BY DIGITAL AS IMAGE-ANALYSIS AND X-RAY-MICROANALYSIS

Cuttings of Betula pendula Roth were grown in field fumigation chamber s throughout one growing season in filtered air with < 3 nl l(-1) O-3 (control; C) or day/night = 90/40 nl l(-1) O-3 (ozone fumigation; O-3) . Plants were watered with either low (0.005%; LF) or high-concentrate d (0.05%; HF) fertilizer solution. Discs between second-order veins in the central portion of the leaves were excised and immediately cryofi xed in liquid nitrogen for low-temperature scanning electron-microscop y (LTSEM) at 1000 hours and 1400 hours. Stomatal width, area and densi ty were measured by digital image-analysis. X-ray counts of potassium (K) and calcium (Ca) ions were determined by means of energy-dispersiv e X-ray microanalysis in guard and subsidiary cells. Accurate and fast measurements of stomatal apertures by image analysis were possible in birch leaves, because the darkness of the stomatal pore contrasts wit h the brightness of the guard cells and the cuticular ledges. Regressi on analysis showed a close relationship between the stomatal width and the pore area (r = 0.938, P < 0.01). At all harvest times, the stomat al pores were significantly narrowed in the high fertilization control treatment (C/HF vs. C/LF), and in the ozone treatment at 1400 hours ( O-3/HF vs. O-3/LF). In addition to this fertilization effect, ozone ha d also narrowed the stomatal pores (O-3/HF vs. C/HF at 1400 hours, O-3 /LF vs. C/LF at all harvest times). In addition to these functional ef fects, morphological effects (individual leaf area, stomatal density) were determined. Single-leaf area was increased by high fertilization, with a tendency to decrease with O-3 fumigation. The stomatal density in intercostal fields was increased by O-3 but decreased by high fert ilization. Stomatal widening was accompanied by increased K count rate s in the guard cells, in contrast to constant K values in the subsidia ry cells, irrespective of the fumigation or fertilization regimes. Cal cium counts in the guard cells were similar to those in the subsidiary cells, and were independent of the aperture width. In samples with es tablished ozone injury, the K/Ca ratio in collapsed guard cells increa sed compared with turgid guard cells irrespective of the pore aperture . Collapsed subsidiary cells only differed from turgid subsidiary cell s when the guard cells had also collapsed and thus closed the pore.