Over-accumulation of chloroplast-nucleus located WHIRLY1 in barley leads to a decrease in growth and an enhanced stress resistance.

WHIRLY1 is a chloroplast-nucleus located DNA/RNA-binding protein with functions in development and stress tolerance. By overexpression of HvWHIRLY1 in barley, one line with a 10-fold and two lines with a 50-fold accumulation of the protein were obtained. In these lines, the relative abundance of the nuclear form exceeded that of the chloroplast form. Growth of the plants was shown to be compromised in a WHIRLY1 abundance-dependent manner. Over-accumulation of WHIRLY1 in chloroplasts had neither an evident impact on nucleoid morphology nor on the composition of the photosynthetic apparatus. Nevertheless, oeW1 plants were found to be compromised in the light reactions of photosynthesis as well as in carbon fixation. The reduction in growth and photosynthesis was shown to be accompanied by a decrease in the levels of cytokinins and an increase in the level of jasmonic acid. Gene expression analyses revealed that in nonstress conditions the oeW1 plants had enhanced levels of pathogen response (PR) gene expression indicating activation of constitutive defense. During growth in continuous light of high irradiance PR gene expression increased indicating that under stress conditions oeW1 are capable to further enhance defense.

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.

Mitochondrial alternative NADH dehydrogenases NDA1 and NDA2 promote survival of reoxygenation stress in Arabidopsis by safeguarding photosynthesis and limiting ROS generation.

Plant submergence stress is a growing problem for global agriculture. During desubmergence, rising O2 concentrations meet a highly reduced mitochondrial electron transport chain (mETC) in the cells. This combination favors the generation of reactive oxygen species (ROS) by the mitochondria, which at excess can cause damage. The cellular mechanisms underpinning the management of reoxygenation stress are not fully understood. We investigated the role of alternative NADH dehydrogenases (NDs), as components of the alternative mETC in Arabidopsis, in anoxia-reoxygenation stress management. Simultaneous loss of the matrix-facing NDs, NDA1 and NDA2, decreased seedling survival after reoxygenation, while overexpression increased survival. The absence of NDAs led to reduced maximum potential quantum efficiency of photosystem II linking the alternative mETC to photosynthetic function in the chloroplast. NDA1 and NDA2 were induced upon reoxygenation, and transcriptional activation of NDA1 was controlled by the transcription factors ANAC016 and ANAC017 that bind to the mitochondrial dysfunction motif (MDM) in the NDA1 promoter. The absence of NDA1 and NDA2 did not alter recovery of cytosolic ATP levels and NADH : NAD+ ratio at reoxygenation. Rather, the absence of NDAs led to elevated ROS production, while their overexpression limited ROS. Our observations indicate that the control of ROS formation by the alternative mETC is important for photosynthetic recovery and for seedling survival of anoxia-reoxygenation stress.

The plastid-nucleus localized DNA-binding protein WHIRLY1 is required for acclimation of barley leaves to high light.

MAIN CONCLUSION: In accordance with a key role of WHIRLY1 in light-acclimation mechanisms, typical features of acclimation to high light, including photosynthesis and leaf morphology, are compromised in WHIRLY1 deficient plants. Acclimation to the environment requires efficient communication between chloroplasts and the nucleus. Previous studies indicated that the plastid-nucleus located WHIRLY1 protein is required for the communication between plastids and the nucleus in situations of high light exposure. To investigate the consequences of WHIRLY1 deficiency on the light acclimation of photosynthesis and leaf anatomy, transgenic barley plants with an RNAi-mediated knockdown of HvWHIRLY1 were compared to wild-type plants when growing at low and high irradiance. While wild-type plants showed the typical light acclimation responses, i.e. higher photosynthetic capacity and thicker leaves, the WHIRLY1 deficient plants were not able to respond to differences in irradiance. The results revealed a systemic role of WHIRLY1 in light acclimation by coordinating responses at the level of the chloroplast and the level of leaf morphology.

In-vivo quantification of electron flow through photosystem I - Cyclic electron transport makes up about 35% in a cyanobacterium.

Photosynthetic electron flow, driven by photosystem I and II, provides chemical energy for carbon fixation. In addition to a linear mode a second cyclic route exists, which only involves photosystem I. The exact contributions of linear and cyclic transport are still a matter of debate. Here, we describe the development of a method that allows quantification of electron flow in absolute terms through photosystem I in a photosynthetic organism for the first time. Specific in-vivo protocols allowed to discern the redox states of plastocyanin, P700 and the FeS-clusters including ferredoxin at the acceptor site of PSI in the cyanobacterium Synechocystis sp. PCC 6803 with the near-infrared spectrometer Dual-KLAS/NIR. P700 absorbance changes determined with the Dual-KLAS/NIR correlated linearly with direct determinations of PSI concentrations using EPR. Dark-interval relaxation kinetics measurements (DIRKPSI) were applied to determine electron flow through PSI. Counting electrons from hydrogen oxidation as electron donor to photosystem I in parallel to DIRKPSI measurements confirmed the validity of the method. Electron flow determination by classical PSI yield measurements overestimates electron flow at low light intensities and saturates earlier compared to DIRKPSI. Combination of DIRKPSI with oxygen evolution measurements yielded a proportion of 35% of surplus electrons passing PSI compared to PSII. We attribute these electrons to cyclic electron transport, which is twice as high as assumed for plants. Counting electrons flowing through the photosystems allowed determination of the number of quanta required for photosynthesis to 11 per oxygen produced, which is close to published values.

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.

Hydroxycinnamic acids in sunflower leaves serve as UV-A screening pigments.

Flavonoids and hydroxycinnamic acid derivatives, which are located in the upper epidermis of plants, are well known to screen ultraviolet radiation, thus protecting the underlying tissue from these harmful wavelengths. Both classes of secondary products complement each other over the UV spectral region according to their absorption spectra: flavonoids are most efficient as UV-A attenuators while hydroxycinnamates (HCAs) screen well within the UV-B region. Analysis of epidermal transmittance revealed a substantial UV-A screen in Helianthus annuus L. cv. Peredovick. Identifying responsible pigments by HPLC-MS, we found surprisingly low amounts of flavonoids but dominant abundance of the HCA derivatives chlorogenic and di-caffeoyl quinic acid. Both display low UV-A absorbance and thus, should contribute only a little to UV-A protection. However, growth at high light led to a decrease of epidermal transmittance at 366 nm of up to 90%. Underpinning the screening role, HCA autofluorescence microscopy revealed storage to occur predominantly in vacuoles of the upper epidermis. UV-A treatment in the absence of D1-repair resulted in photosystem II inactivation proportional to epidermal UV-A transmittance. Our findings suggest that UV-A protection can be achieved solely with HCAs, apparently through accumulation of high amounts of these compounds.

The nucleoid-associated protein WHIRLY1 is required for the coordinate assembly of plastid and nucleus-encoded proteins during chloroplast development.

MAIN CONCLUSION: Chloroplasts deficient in the major chloroplast nucleoid-associated protein WHIRLY1 have an enhanced ratio of LHCs to reaction centers, indicating that WHIRLY1 is required for a coordinate assembly of the photosynthetic apparatus during chloroplast development. Chloroplast development was found to be delayed in barley plants with an RNAi-mediated knockdown of WHIRLY1 encoding a major nucleoid-associated protein of chloroplasts. The plastids of WHIRLY1 deficient plants had a reduced ribosome content. Accordingly, plastid-encoded proteins of the photosynthetic apparatus showed delayed accumulation during chloroplast development coinciding with a delayed increase in photosystem II efficiency measured by chlorophyll fluorescence. In contrast, light harvesting complex proteins being encoded in the nucleus had a high abundance as in the wild type. The unbalanced assembly of the proteins of the photosynthetic apparatus in WHIRLY1-deficient plants coincided with the enhanced contents of chlorophyll b and xanthophylls. The lack of coordination was most obvious at the early stages of development. Overaccumulation of LHC proteins in comparison to reaction center proteins at the early stages of chloroplast development did not correlate with enhanced expression levels of the corresponding genes in the nucleus. This work revealed that WHIRLY1 does not influence LHC abundance at the transcriptional level. Rather, WHIRLY1 in association with nucleoids might play a structural role for both the assembly of ribosomes and the complexes of the photosynthetic apparatus.

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.

The plastid-nucleus located DNA/RNA binding protein WHIRLY1 regulates microRNA-levels during stress in barley (Hordeum vulgare L.).

In this article a novel mechanism of retrograde signaling by chloroplasts during stress is described. This mechanism involves the DNA/RNA binding protein WHIRLY1 as a regulator of microRNA levels. By virtue of its dual localization in chloroplasts and the nucleus of the same cell, WHIRLY1 was proposed as an excellent candidate coordinator of chloroplast function and nuclear gene expression. Comparison of wild-type and transgenic plants with an RNAi-mediated knockdown of WHIRLY1 showed, that the transgenic plants were unable to cope with continuous high light conditions. They were impaired in production of several microRNAs mediating post-transcriptional responses during stress. The results support a central role of WHIRLY1 in retrograde signaling and also underpin a so far underestimated role of microRNAs in this process.

Survey of the occurrence of desiccation-induced quenching of basal fluorescence in 28 species of green microalgae.

MAIN CONCLUSION: Desiccation-induced chlorophyll fluorescence quenching seems to be an indispensable part of desiccation resistance in the surveyed 28 green microalgal species. Lichens are desiccation tolerant meta-organisms. In the desiccated state photosynthesis is inhibited rendering the photobionts potentially sensitive to photoinhibition. As a photoprotective mechanism, strong non-radiative dissipation of absorbed light leading to quenching of chlorophyll fluorescence has been proposed. Desiccation-induced quenching affects not only variable fluorescence, but also the so-called basal fluorescence, F0. This phenomenon is well-known for intact lichens and some free living aero-terrestrial algae, but it was often absent in isolated lichen algae. Therefore, a thorough screening for the appearance of desiccation-induced quenching was undertaken with 13 different aero-terrestrial microalgal species and lichen photobionts. They were compared with 15 aquatic green microalgal species, among them also three marine species. We asked the following questions: Do isolated lichen algae show desiccation-induced quenching? Are aero-terrestrial algae different in this respect to aquatic algae and is the potential for desiccation-induced quenching coupled to desiccation tolerance? How variable is desiccation-induced quenching among species? Most of the aero-terrestrial algae, including all lichen photobionts, showed desiccation-induced quenching, although highly variable in extent, whereas most of the aquatic algae did not. All algae displaying quenching were also desiccation tolerant, whereas all algae unable to perform desiccation-induced quenching were desiccation intolerant. Desiccation-induced fluorescence quenching seems to be an indispensable part of desiccation resistance in the investigated species.

Quantification of light screening by anthocyanins in leaves of Berberis thunbergii.

MAIN CONCLUSION: Up to 40% of incident light was screened in red Berberis leaves in vivo by anthocyanins, resulting also in up to 40% reduction of light-limited photosynthesis. The biological function of anthocyanins in leaves has been strongly discussed, but the hypothesis of a screening function is favored by most authors. For an evaluation of the function as photoprotective pigments, a quantification of their screening of the mesophyll is important. Here, chlorophyll fluorescence excitation of leaves of a red and a green variety of Berberis thunbergii was used to estimate the extent of screening by anthocyanins at 545 nm and over the whole photosynthetically active wavelength range. Growth at high light (430 µmol m-2 s-1) resulted in 90% screening at 545 nm corresponding to 40-50% screening over the whole wavelength range, depending on the light source. The concomitant reduction of photosynthetic quantum yield was of the same size as the calculated reduction of light reaching the chloroplasts. The induction of anthocyanins in the red variety also enhanced the epoxidation state of the violaxanthin cycle under growth conditions, indicating that red leaves were suffering less from excessive irradiance. Pool sizes of violaxanthin cycle carotenoids indicated a shade acclimation of the light harvesting complexes in red leaves. The observed reduction of internal light in anthocyanic leaves has by necessity a photoprotective effect.

Acceleration of leaf senescence is slowed down in transgenic barley plants deficient in the DNA/RNA-binding protein WHIRLY1.

WHIRLY1 in barley was isolated as a potential regulator of the senescence-associated gene HvS40. In order to investigate whether the plastid-nucleus-located DNA/RNA-binding protein WHIRLY1 plays a role in regulation of leaf senescence, primary foliage leaves from transgenic barley plants with an RNAi-mediated knockdown of the WHIRLY1 gene were characterized by typical senescence parameters, namely pigment contents, function and composition of the photosynthetic apparatus, as well as expression of selected genes known to be either down- or up-regulated during leaf senescence. When the plants were grown at low light intensity, senescence progression was similar between wild-type and RNAi-W1 plants. Likewise, dark-induced senescence of detached leaves was not affected by reduction of WHIRLY1. When plants were grown at high light intensity, however, senescence was induced prematurely in wild-type plants but was delayed in RNAi-W1 plants. This result suggests that WHIRLY1 plays a role in light sensing and/or stress communication between chloroplasts and the nucleus.

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.

A simple indicator for non-destructive estimation of the violaxanthin cycle pigment content in leaves.

The photosynthetic apparatus of higher plants acclimates to irradiance. Among the features which are changing is the pool size of the pigments belonging to the violaxanthin cycle, in which zeaxanthin is formed. In high light grown leaves, the violaxanthin cycle pool size is up to five times larger than in low light. The changes are reversible on a time scale of several days. Since it has been published that violaxanthin cycle pigments do not transfer absorbed energy to chlorophyll, we hypothesized that excitation of chlorophyll fluorescence in the blue spectral region may be reduced in high light-acclimated leaves. Fluorescence excitation spectra of leaves of the Arabidopsis thaliana tt3 mutant showed strong differences between high and low light-acclimated plants from 430 to 520 nm. The resulting difference spectrum was similar to carotenoids but shifted by about 20 nm to higher wavelengths. A good correlation was observed between the fluorescence excitation ratio F 470/F 660 and the violaxanthin cycle pool size when leaves were acclimated to a range of irradiances. In parallel to the decline of F 470/F 660 with high light acclimation also the quantum yield of photosynthetic oxygen evolution in blue light decreased. The data confirm that violaxanthin cycle carotenoids do not transfer absorbed light to chlorophyll. It is proposed to use the ratio F 470/F 660 as an indicator for the light acclimation status of the chloroplasts in a leaf.