Blue Light Treatment but Not Green Light Treatment After Pre-exposure to UV-B Stabilizes Flavonoid Glycoside Changes and Corresponding Biological Effects in Three Different Brassicaceae Sprouts.

Ultraviolet-B (UV-B; 280-315 nm) radiation induces the biosynthesis of secondary plant metabolites such as flavonoids. Flavonoids could also be enhanced by blue (420-490 nm) or green (490-585 nm) light. Flavonoids act as antioxidants and shielding components in the plant's response to UV-B exposure. They are shown to quench singlet oxygen and to be reactive to hydroxyl radical. The aim was to determine whether treatment with blue or green light can alter flavonoid profiles after pre-exposure to UV-B and whether they cause corresponding biological effects in Brassicaceae sprouts. Based on their different flavonoid profiles, three vegetables from the Brassicaceae were selected. Sprouts were treated with five subsequent doses (equals 5 days) of moderate UV-B (0.23 kJ m-2 day-1 UV-BBE), which was followed with two subsequent (equals 2 days) doses of either blue (99 µmol m-2 s-1) or green (119 µmol m-2 s-1) light. In sprouts of kale, kohlrabi, and rocket salad, flavonoid glycosides were identified by HPLC-DAD-ESI-MSn. Both Brassica oleracea species, kale and kohlrabi, showed mainly acylated quercetin and kaempferol glycosides. In contrast, in rocket salad, the main flavonol glycosides were quercetin glycosides. Blue light treatment after the UV-B treatment showed that quercetin and kaempferol glycosides were increased in the B. oleracea species kale and kohlrabi while-contrary to this-in rocket salad, there were only quercetin glycosides increased. Blue light treatment in general stabilized the enhanced concentrations of flavonoid glycosides while green treatment did not have this effect. Blue light treatment following the UV-B exposure resulted in a trend of increased singlet oxygen scavenging for kale and rocket. The hydroxyl radical scavenging capacity was independent from the light quality except for kale where an exposure with UV-B followed by a blue light treatment led to a higher hydroxyl radical scavenging capacity. These results underline the importance of different light qualities for the biosynthesis of reactive oxygen species that intercept secondary plant metabolites, but also show a pronounced species-dependent reaction, which is of special interest for growers.

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.

Antioxidant defence in UV-irradiated tobacco leaves is centred on hydrogen-peroxide neutralization.

Greenhouse grown tobacco (Nicotiana tabacum L. cv. Petit Havana) plants were exposed to supplemental UV centred at 318 nm and corresponding to 13.6 kJ m(-2) d(-1) biologically effective UV-B (280-315 nm) radiation. After 6 days this treatment decreased photosynthesis by 30%. Leaves responded by a large increase in UV-absorbing pigment content and antioxidant capacities. UV-stimulated defence against ROS was strongest in chloroplasts, since activities of plastid enzymes FeSOD and APX had larger relative increases than other, non-plastid specific SODs or peroxidases. In addition, non-enzymatic defence against hydroxyl radicals was doubled in UV treated leaves as compared to controls. In UV treated leaves, the extent of activation of ROS neutralizing capacities followed a peroxidases > hydroxyl-radical neutralization > SOD order. These results suggest that highly effective hydrogen peroxide neutralization is the focal point of surviving UV-inducible oxidative stress and argue against a direct signalling role of hydrogen peroxide in maintaining adaptation to UV, at least in laboratory experiments.

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.

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.

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.

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.

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.