The effects of ozone and nutrient supply on stomatal response in birch (Betula pendula) leaves as determined by digital image‐analysis and X‐ray microanalysis

Abstract

SUMMARY Cuttings of Betula pendula Roth were grown in field fumigation chambers throughout one growing season in filtered air with < 3 nl l−1 O3 (control; C) or day/night = 90/40 nl 1−1 O3 (ozone fumigation: O3). 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 (O3/HF vs. O3/LF), and in the ozone treatment at 1400 hours (O3/HF vs. C/LF). In addition to this fertilization effect, ozone had also narrowed the stomatal pores (O3/HF vs. C/HF at 1400 hours, O3/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 O3 fumigation. The stomatal density in intercostal fields was increased by O3 but decreased by high fertilisation. 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.

Stomatal density and stomatal index as indicators of paleoatmospheric CO2 concentration

“…Elevated levels of ozone increase SD in Betula pendula (Frey et al, 1996;Pa Èa Èkko Ènen et al, 1998), Fraxinus excelsior (Wiltshire et al, 1996) and Olea europaea (Minnocci et al, 1999). Effects on SI were not reported.…”

Effects of elevated [CO2] and O3 on stomatal and surface wax characteristics in leaves of pubescent birch grown under field conditions

“…However, collapsed guard cells have been observed in birch leaves with established O 3 injury (Frey et al 1996). Apparently the O 3 doses here were not high enough to cause alterations in the wax formation or visible cell injury.…”

“…Previously, O 3 exposure has been seen to increase the stomatal density of birch (Maurer et al 1997), but not to affect the guard cell length (Frey et al 1996). The increase found in the guard cell length both in elevated CO 2 and CO 2 +O 3 levels may point to an effect of CO 2 on the growth and development of guard cells.…”

“…Ozone may interfere with control of the stomatal aperture and may have the potential to disturb the water balance of the leaf, or even the whole plant (Pearson and Mansfield 1993). Elevated O 3 levels have been shown to influence leaf differentiation during its formation (Frey et al 1996;Bäck et al 1999). Leaf differentiation changed towards xeromorphism, somewhat resembling sun leaves or leaf development under mild drought conditions (Matyssek et al 1991).…”

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“…The application of this method for coffee needs further information for the interpretation. Therefore, the localisation of mercury in intruded beans was studied in fractures of the frozen beans with cryo-scanning x-ray microanalysis and cryo-SEM (Frey et al 1996;reviewed by Echlin 1992).…”