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 chambers 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-concentrated (0.05%; HF) fertilizer solution. Discs between second-order veins in the central portion of the leaves were excised and immediately cryofixed in liquid nitrogen for low-temperature scanning electron-microscopy (LTSEM) at 1000 hours and 1400 hours. Stomatal width, area and density were measured by digital image-analysis. X-ray counts of potassium (K) and calcium (Ca) ions were determined by means of energy-dispersive 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 with the brightness of the guard cells and the cuticular ledges. Regression analysis showed a close relationship between the stomatal width and the pore area (r = 0.938, P < 0.01). At all harvest times, the stomatal 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 had 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 effects, 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 fertilization. Stomatal widening was accompanied by increased K count rates in the guard cells, in contrast to constant K values in the subsidiary cells, irrespective of the fumigation or fertilization regimes. Calcium counts in the guard cells were similar to those in the subsidiary cells, and were independent of the aperture width. In samples with established ozone injury, the K/Ca ratio in collapsed guard cells increased compared with turgid guard cells irrespective of the pore aperture. Collapsed subsidiary cells only differed from turgid subsidiary cells when the guard cells had also collapsed and thus closed the pore.