Petra Majer

Developmental stage is an important factor that determines the antioxidant responses of young and old grapevine leaves under UV irradiation in a green-house.

The impact of UV irradiation was studied on photosynthesis, photosystem II photochemical yields and antioxidant responses using green-house grown grapevine (Vitis vinifera L. cv. Chardonnay) leaves. Supplemental UV irradiation (280-400 nm) was centred in the UV-B region, and corresponded to 8.95 kJ m⁻² d⁻¹ global (280-400 nm) or 8.04 kJ m⁻² d⁻¹ UV-B (280-315 nm) biologically effective dose. UV irradiation was applied daily and its effects were evaluated after 4-days. Younger (1-3 weeks-old) leaves (YL) and older (4-6 weeks-old) leaves (OL) were affected differently, UV irradiation decreased their photochemical yields to 78% and 56%, respectively. Unlike OL, YL responded by an increase in UV-B absorbing pigment, anthocyanin and total phenolics contents. UV irradiation increased total antioxidant capacities in YL but not in OL. YL were also different in their ability to increase specific hydroxyl radical and singlet oxygen neutralizing capacities in response to the supplemental UV irradiation, which is reported here for the first time. Our results suggest that the ability of maintaining photosynthesis under supplemental UV is not necessarily determined by base levels of antioxidants but rather by their inducibilities in response to the irradiation and emphasise the importance of comparing leaves of the same age in UV studies. Correlations between various antioxidant capacities, pigment contents and photosynthesis parameters were also examined. However, no single element of the defence system can be picked up as decisive factor of sensitivity to UV.

Leaf hue measurements offer a fast, high-throughput initial screening of photosynthesis in leaves

Experiments with tobacco and grapevine leaves having different color due to varying stages of senescence showed that leaf hue is significantly linearly correlated with chlorophyll content up to 80% loss of pigment. Samples from leaves with more pronounced loss of chlorophyll did not fit into this linear relationship, and the hue data set as a whole followed a saturating exponential dependence on chlorophyll content. In leaves with less than 80% chlorophyll loss, the hue parameter was also proportional to the photochemical yield of photosystem (PS) II measured in the light. These results suggest that leaf hue measurements offer a fast, high-throughput initial screening system to precede more specific but more time consuming photosynthesis measurements, with the possibility of applications not only for senescing plants, but also for stress conditions accompanied by chlorophyll loss.

Do leaf total antioxidant capacities (TAC) reflect specific antioxidant potentials? – A comparison of TAC and reactive oxygen scavenging in tobacco leaf extracts

Two traditional methods of total antioxidant capacity (TAC) assessment, Trolox equivalent antioxidant capacity (TEAC) and ferric reducing antioxidant power (FRAP) were applied to water extracts from tobacco leaves at various stages of senescence. Physiological status of the leaves was characterized by the effective photochemical quantum yield of photosynthesis (Y(II)). TAC values were compared to amounts of total phenolics, carotenoid contents and also to reactive oxygen scavenging capacities of the leaf extracts. To this end a new, simple fluorimetric assay was introduced for testing hydroxyl radical neutralizing capacity in leaf extracts. We found that while both TAC values increased with declining photosynthesis and decreasing pigment content, they were not characteristic to specific superoxide or hydroxyl radical scavenging and had limited connection to leaf antioxidant content. Good linear correlations were only found between the following pairs of parameters: Y(II) - total carotenoid, TEAC - total carotenoid, FRAP - total phenolics. Our data show that TEAC and FRAP are not interchangeable in leaf studies and do not represent antioxidant action on ROS.

Leaf hue measurements: A high-throughput screening of chlorophyll content

Computer analysis of digital photographic images provides fast, high-throughput screening of leaf pigmentation. Pixel-by-pixel conversion of red, green, blue (RGB) parameters to hue, saturation, value (HSV) showed that Hue values were proportional to total chlorophyll, offering an alternative to photometric analysis of leaf extracts. This is demonstrated using tobacco leaves with various chlorophyll contents due to senescence but shows the possibility of applications in studies of stress conditions accompanied by chlorophyll loss.

Stress tolerance of transgenic barley accumulating the alfalfa aldose reductase in the cytoplasm and the chloroplast

Barley represents one of the major crops grown worldwide; its genetic transformation provides an important tool for the improvement of crop quality and tolerance to environmental stress factors. Biotic and abiotic stresses produce reactive oxygen species in the plant cells that can directly oxidize the cellular components including lipid membranes; resulting in lipid peroxidation and subsequently the accumulation of reactive carbonyl compounds. In order to protect barley plants from the effects of stress-produced reactive carbonyls, an Agrobacterium-mediated transformation was carried out using the Medicago sativa aldose reductase (MsALR) gene. In certain transgenic lines the produced MsALR enzyme was targeted to the chloroplasts to evaluate its protective effect in these organelles. The dual fluorescent protein-based method was used for the evaluation of tolerance of young seedlings to diverse stresses; our results demonstrated that this technique could be reliably applied for the detection of cellular stress in a variety of conditions. The chlorophyll and carotenoid content measurements also supported the results of the fluorescent protein-based method and the stress-protective effect of the MsALR enzyme. Targeting of MsALR into the chloroplast has also resulted in increased stress tolerance, similarly to the observed effect of the cytosolic MsALR accumulation. The results of the DsRed/GFP fluorescent protein-based method indicated that both the cytosol and chloroplast accumulation of MsALR can increase the abiotic stress tolerance of transgenic barley lines.

Factors contributing to the high light tolerance of leaves in vivo - Involvement of photo-protective energy dissipation and singlet oxygen scavenging

Contributions of preventive and antioxidant (energy dissipating and singlet oxygen neutralizing) processes to tolerating high light stress (photoinhibition) were examined in green-house grown tobacco (Nicotiana tabacum) plants acclimated to high or low light conditions and also in sun and shade leaves collected from a natural grown linden tree (Tilia platyphyllos). Tobacco leaves survived a short (1 h) exposure to photoinhibition by activating non-regulated non-photochemical quenching [Y(NO)] rather than relying on photo-protective, regulated non-photochemical quenching [Y(NPQ)]. Low light acclimated leaves had lower singlet oxygen scavenging ability and activated Y(NO) to a larger extent than high light acclimated ones. Low light grown leaves also suffered singlet oxygen mediated photo-damage, while no singlet oxygen was detected in high light acclimated leaves during photoinhibition. Natural grown linden leaves, however, coped with prolonged daily exposures to high light mainly by activating regulated non-photochemical quenching Y(NPQ), although they also featured very efficient singlet oxygen neutralizing. Our results suggest that high light tolerance is achieved by preventing photoinhibition of photosystem II via efficient photo-protective energy dissipation rather than relying on quenching of stress-induced pro-oxidative agents.