Improvement of the Quality of Wild Rocket (Diplotaxis tenuifolia) with Respect to Health-Related Compounds by Enhanced Growth Irradiance.

For healthier human nutrition, it is desirable to provide food with a high content of nutraceuticals such as polyphenolics, vitamins, and carotenoids. We investigated to what extent high growth irradiance influences the content of phenolics, α-tocopherol and carotenoids, in wild rocket (Diplotaxis tenuifolia), which is increasingly used as a salad green. Potted plants were grown in a climate chamber with a 16 h day length at photosynthetic photon flux densities varying from 20 to 1250 µmol m-2 s-1. Measurements of the maximal quantum yield of photosystem II, FV/FM, and of the epoxidation state of the violaxanthin cycle (V-cycle) showed that the plants did not suffer from excessive light for photosynthesis. Contents of carotenoids belonging to the V-cycle, α-tocopherol and several quercetin derivatives, increased nearly linearly with irradiance. Nonintrusive measurements of chlorophyll fluorescence induced by UV-A and blue light relative to that induced by red light, indicating flavonoid and carotenoid content, allowed not only a semiquantitative measurement of both compounds but also allowed to follow their dynamic changes during reciprocal transfers between low and high growth irradiance. The results show that growth irradiance has a strong influence on the content of three different types of compounds with antioxidative properties and that it is possible to determine the contents of flavonoids and specific carotenoids in intact leaves using chlorophyll fluorescence. The results may be used for breeding to enhance healthy compounds in wild rocket leaves and to monitor their content for selection of appropriate genotypes.

WHIRLY1-deficient chloroplasts display enhanced formation of cyclobutane pyrimidine dimers during exposure to UV-B radiation.

The single-stranded DNA/RNA binding protein WHIRLY1 is a major chloroplast nucleoid-associated protein required for the compactness of nucleoids. Most nucleoids in chloroplasts of WHIRLY1-knockdown barley plants are less compact compared to nucleoids in wild-type plants. The reduced compaction leads to an enhanced optical cross-section, which may cause the plastid DNA to be a better target for damaging UV-B radiation. To investigate this hypothesis, primary foliage leaves, chloroplasts, and nuclei from wild-type and WHIRLY1-knockdown plants were exposed to experimental UV-B radiation. Thereafter, total, genomic and plastid DNA were isolated, respectively, and analyzed for the occurrence of cyclobutane pyrimidine dimers (CPDs), which is a parameter for genome stability. The results of this study revealed that WHIRLY1-deficient chloroplasts had strongly enhanced DNA damages, whereas isolated nuclei from the same plant line were not more sensitive than nuclei from the wild-type, indicating that WHIRLY1 has different functions in chloroplasts and nucleus. This supports the hypothesis that the compaction of nucleoids may provide protection against UV-B radiation.

How do barley plants with impaired photosynthetic light acclimation survive under high-light stress?

MAIN CONCLUSION: WHIRLY1 deficient barley plants surviving growth at high irradiance displayed increased non-radiative energy dissipation, enhanced contents of zeaxanthin and the flavonoid lutonarin, but no changes in α-tocopherol nor glutathione. Plants are able to acclimate to environmental conditions to optimize their functions. With the exception of obligate shade plants, they can adjust their photosynthetic apparatus and the morphology and anatomy of their leaves to irradiance. Barley (Hordeum vulgare L., cv. Golden Promise) plants with reduced abundance of the protein WHIRLY1 were recently shown to be unable to acclimatise important components of the photosynthetic apparatus to high light. Nevertheless, these plants did not show symptoms of photoinhibition. High-light (HL) grown WHIRLY1 knockdown plants showed clear signs of exposure to excessive irradiance such as a low epoxidation state of the violaxanthin cycle pigments and an early light saturation of electron transport. These responses were underlined by a very large xanthophyll cycle pool size and by an increased number of plastoglobules. Whereas zeaxanthin increased with HL stress, α-tocopherol, which is another lipophilic antioxidant, showed no response to excessive light. Also the content of the hydrophilic antioxidant glutathione showed no increase in W1 plants as compared to the wild type, whereas the flavone lutonarin was induced in W1 plants. HPLC analysis of removed epidermal tissue indicated that the largest part of lutonarin was presumably located in the mesophyll. Since lutonarin is a better antioxidant than saponarin, the major flavone present in barley leaves, it is concluded that lutonarin accumulated as a response to oxidative stress. It is also concluded that zeaxanthin and lutonarin may have served as antioxidants in the WHIRLY1 knockdown plants, contributing to their survival in HL despite their restricted HL acclimation.

Cysts of the Snow Alga Chloromonas krienitzii (Chlorophyceae) Show Increased Tolerance to Ultraviolet Radiation and Elevated Visible Light.

Melting mountainous snowfields are populated by extremophilic microorganisms. An alga causing orange snow above timberline in the High Tatra Mountains (Poland) was characterised using multiple methods examining its ultrastructure, genetics, life cycle, photosynthesis and ecophysiology. Based on light and electron microscopy and ITS2 rDNA, the species was identified as Chloromonas krienitzii (Chlorophyceae). Recently, the taxon was described from Japan. However, cellular adaptations to its harsh environment and details about the life cycle were so far unknown. In this study, the snow surface population consisted of egg-shaped cysts containing large numbers of lipid bodies filled presumably with the secondary carotenoid astaxanthin. The outer, spiked cell wall was shed during cell maturation. Before this developmental step, the cysts resembled a different snow alga, Chloromonas brevispina. The remaining, long-lasting smooth cell wall showed a striking UV-induced blue autofluorescence, indicating the presence of short wavelengths absorbing, protective compounds, potentially sporopollenin containing polyphenolic components. Applying a chlorophyll fluorescence assay on intact cells, a significant UV-A and UV-B screening capability of about 30 and 50%, respectively, was measured. Moreover, intracellular secondary carotenoids were responsible for a reduction of blue-green light absorbed by chloroplasts by about 50%. These results revealed the high capacity of cysts to reduce the impact of harmful UV and high visible irradiation to the chloroplast and nucleus when exposed at alpine snow surfaces during melting. Consistently, the observed photosynthetic performance of photosystem II (evaluated by fluorometry) showed no decline up to 2100 µmol photons m-2 s-1. Cysts accumulated high contents of polyunsaturated fatty acids (about 60% of fatty acids), which are advantageous at low temperatures. In the course of this study, C. krienitzii was found also in Slovakia, Italy, Greece and the United States, indicating a widespread distribution in the Northern Hemisphere.

High impact of seasonal temperature changes on acclimation of photoprotection and radiation-induced damage in field grown Arabidopsis thaliana.

At temperate latitudes environmental factors such as irradiance, including ultraviolet-B radiation (UV-B, 280-315 nm), temperature and day length vary widely over the course of a year in a concerted way. In the present study physiological acclimation of photoprotection, growth and development of the model organism Arabidopsis thaliana were correlated to these strongly but gradually changing conditions in a one year field study. Plants were sown in the field avoiding any manipulation (and abrupt change) during their life. Developmental rate was strongly dependent on prevailing temperature. Moderate signs of light stress in form of photoinhibition at photosystem II were significantly related to solar irradiances while amount of DNA damage was low and not correlated to UV-B irradiance. Although all the markers were hypothesized to primarily react to radiation, multiple regression analysis showed at least a similarly strong influence of temperature as that of light. Especially for the classical UV screening compounds a positive correlation to UV-B radiation during the course of the year was absent, whereas there was a significant negative correlation between temperature and quercetin content. The sum of violaxanthin cycle pigments was correlated to both, irradiance and temperature, but with opposite sign. Epidermal UV-B transmittance was also much better related to air temperature than to UV-B irradiance. The data show that under natural conditions temperature has at least a similar importance for photoprotective acclimation and partially also for photosensitivity as solar irradiance.

Acclimation of Arabidopsis thaliana to low temperature protects against damage of photosystem II caused by exposure to UV-B radiation at 9 °C.

Various environmental variables interact with UV-B radiation (280-315 nm), among them temperature. In many plants epidermal UV screening is induced by low temperature even in the absence of UV irradiation. On the other hand, low temperature can aggravate damage caused by UV-B radiation. We investigated the interaction of UV-B radiation and low temperature in Arabidopsis thaliana (L.) Heynh. Exposure of plants grown at moderate temperature (21 °C) to UV-B radiation at 9 °C resulted in significantly higher damage of photosystem II (PS II) as compared to exposure at 21 °C. The higher damage at low temperature was related to slower recovery of maximal PS II quantum efficiency at this temperature. Epidermal UV-B transmittance was measured using a method based on chlorophyll fluorescence measurements. Acclimation to low temperature enhanced epidermal UV-B screening and improved the UV-B resistance considerably. Differences in the apparent UV-B sensitivity of PS II between plants grown in moderate or acclimated to cool temperatures were strongly diminished when damage was related to the UV-B radiation reaching the mesophyll (UV-Bint) as calculated from incident UV-B irradiance and epidermal UV-B transmittance. Evidence is presented that the remaining differences in sensitivity are caused by an increased rate of repair in plants acclimated to 9 °C. The data suggest that enhanced epidermal UV-B screening at low temperature functions to compensate for slower repair of UV-B damage at these temperatures. It is proposed that the UV-B irradiance reaching the mesophyll should be considered as an important parameter in experiments on UV-B resistance of plants.

Arabidopsis thaliana G2-LIKE FLAVONOID REGULATOR and BRASSINOSTEROID ENHANCED EXPRESSION1 are low-temperature regulators of flavonoid accumulation.

Flavonoid synthesis is predominantly regulated at the transcriptional level through the MYB-basic helix-loop-helix (bHLH)-WD40 (MBW) (MYB: transcription factor of the myeloblastosis protein family, WD40: tanscription factor with a short structural motif of 40 amino acids which terminates in an aspartic acid-tryptophan dipeptide) complex, and responds to both environmental and developmental stimuli. Although the developmental regulation of flavonoid accumulation in Arabidopsis thaliana has been examined in great detail, the response of the flavonoid synthesis pathway to abiotic stress (particularly low temperature) remains unclear. A screen of a Dissociation element (Ds) transposon-induced mutation collection identified two lines which exhibited an altered profile of phenylpropanoid accumulation following exposure to low-temperature stress. One of the mutated genes (BRASSINOSTEROID ENHANCED EXPRESSION1 (BEE1)) encoded a brassinosteroid enhanced expression transcription factor, while the other (G2-LIKE FLAVONOID REGULATOR (GFR)) encoded a G2-like flavonoid regulator. Phenylpropanoid-targeted analysis was performed using high-performance LC-MS, and gene expression analysis using quantitative reverse transcription-PCR. In both mutants, the accumulation of quercetins and scopolin was reduced under low-temperature growing conditions, whereas that of anthocyanin was increased. BEE1 and GFR were both shown to negatively regulate anthocyanin accumulation by inhibiting anthocyanin synthesis genes via the suppression of the bHLH (TRANSPARENT TESTA8 (TT8) and GLABROUS3 (GL3)) and/or the MYB (PRODUCTION OF ANTHOCYANIN PIGMENTS2 (PAP2)) components of the MBW complex. Our results provide new insight into the regulatory control of phenylpropanoid metabolism at low temperatures, and reveal that BEE1 and GFR act as important components of the signal transduction chain.

UV screening in higher plants induced by low temperature in the absence of UV-B radiation.

Epidermally located UV-B absorbing hydroxycinnamic acid derivatives and flavonoids serve as a screen against potentially damaging UV-B (280-315 nm) radiation in higher plants. We investigated the effect of low temperature on epidermal screening as assessed by a chlorophyll fluorescence technique. The epidermal UV-transmittance of greenhouse-grown Vicia faba plants was strongly dependent on growth temperatures between 21 and 9 degrees C, with significant differences already between 21 and 18 degrees C. There was a good correlation between epidermal UV-A and UV-B absorbance and the absorbance of whole leaf extracts at the respective wavelengths. Whereas in Oxyria digyna and Rumex longifolius no temperature dependence of epidermal transmittance could be detected, it was confirmed for seven other crop plant species, including summer and winter varieties, and for Arabidopsis thaliana. Dicotyledoneous plants showed a stronger response than monocotyledoneous ones. In all investigated species, the response in the UV-A spectral region was similar to that in the UV-B, suggesting that flavonoids were the responsible compounds. In V. faba, mature leaves did not respond with a change in epidermal transmittance upon transfer from warm to cool conditions or vice versa, whereas developing leaves did acclimate to the new conditions. We conclude that temperature is an important determinant of the acclimation of epidermal UV transmittance to environmental conditions in many plant species. The potential adaptive value of this response is discussed.

The lichens Xanthoria elegans and Cetraria islandica maintain a high protection against UV-B radiation in Arctic habitats.

This study reports UV screening pigments in the upper cortices of two widespread lichens collected in three sun-exposed locations along a latitudinal gradient from the Arctic lowland to alpine sites of the Central European Alps. Populations from the Alps receive 3-5 times higher UV-B irradiance than their Arctic counterparts from Svalbard because of latitudinal and altitudinal gradients in UV-B irradiance. In Cetraria islandica, the screening capacity of melanin in the upper cortices was assessed by direct measurements of cortical transmittance (250-1,000 nm). A comparison of cortical transmittances in brown sun-exposed and pale shade-adapted forest C. islandica thalli showed that fungal melanins strongly absorb both UV-B and photosynthetically active radiation (PAR). For Xanthoria elegans cortical UV-B absorbing pigments, mainly the orange parietin, were extracted and quantified. Field experiments with extracted, parietin-deficient X. elegans thalli cultivated under various filters showed that UV-B was essential for the induction of parietin synthesis. The parietin resynthesis in these parietin-deficient samples increased with decreasing latitude of their location in which they had been sampled, which may imply that the synthesis of pigments is habitat specific. However, no latitudinal gradient in cortical screening capacity was detected for any of the two species investigated in the field. This implies that Arctic populations maintain a high level of screening pigments in spite of low ambient UV-B, and that the studied lichen species presumably may tolerate an increase in UV-B radiation due to the predicted thinning of the ozone layer over polar areas.