Non-photochemical Chlorophyll Fluorescence Quenching Research Articles

Glyphosate hormesis stimulates tomato (Solanum lycopersicum L.) plant growth and enhances tolerance against environmental abiotic stress by triggering nonphotochemical quenching.

Glyphosate is the most widely applied herbicide in the world. Hormesis caused by low glyphosate doses has been widely documented in many plant species. However, the specific adaptative mechanism of plants responding to glyphosate hormesis stimulation remains unclear. This study focused on the biphasic relationship between glyphosate dose and tomato plant growth, and how glyphosate hormesis stimulates plant growth and enhances tolerance to environmental stress. We constructed a hormesis model to describe the biphasic relationship with a maximal stimulation (MAX) of 162% above control by glyphosate at 0.063 g ha-1 . Low-dose glyphosate increased photosynthetic pigment contents and improve photosynthetic efficiency, leading to plant growth stimulation. We also found that glyphosate hormesis enhanced plant tolerance to diuron (DCMU; a representative photosynthesis inhibitor) by triggering the nonphotochemical chlorophyll fluorescence quenching (NPQ) reaction to dissipate excess energy stress from photosystem II (PSII). Transcriptomic analysis and quantitative real-time polymerase chain reaction results revealed that the photosynthesis-antenna proteins pathway was the most sensitive to glyphosate hormesis, and PsbS (encoding photosystem II subunit S), ZEP (encoding zeaxanthin epoxidase) and VDE (encoding violaxanthin de-epoxidase) involved in NPQ played crucial roles in the plant response to glyphosate hormesis. These results provide novel insights into the mechanisms of plant hormesis and is meaningful to the application of glyphosate hormesis in agriculture. © 2024 Society of Chemical Industry.

Leaf color change and photosystem function evaluation under heat treatment revealed the stress resistance variation between Loropetalum chinense and L. chinense var. rubrum.

This research mainly focused on the leaf color change and photosystem function differentiation between Loropetalum chinense and its variety L. chinense var. rubrum under heat stress, which were tightly concerned about their ornamental traits and growth. L. chinense 'Xiangnong Xiangyun' (X) and L. chinense var. rubrum 'Xiangnong Fendai' (F) and L. chinense var. rubrum 'Hei Zhenzhu' (H) were chosen to be experimented on to investigate whether leaf color morphology and pigment composition could influence the adaptability of plants to high temperature in order to select foliage plants which posses stable leaf color and better adaptability for hot regions. The plants were cultured in hot environment (40 °C/33 °C, day/night) and normal environment (25 °C/18 °C, day/night). Phenotype and anatomic observation of three cultivars were made and leaf color indices and pigment contents were measured. During the experiment, H and F gradually turned green, total anthocyanins contents significantly decreased in them, however, chlorophyll b contents increased in all three cultivars. In addition, the initial fluorescence (Fo) decreased in X, while increased in H and F. For the maximum fluorescence (Fm) and maximum photochemical efficiency of PSII (Fv/Fm), they only increased in H and decreased in both F and X. The non-photochemical chlorophyll fluorescence quenching (NPQ) also increased in H and decreased in F. For X, it increased at first then gradually decreased. The coefficient of photochemical quenching all increased at first then gradually decreased. Correlation analysis between showed that there was relatively strong connection between anthocyanins, flavonoids and chlorophyll fluorescence parameters, especially NPQ, proved anthocyanins and flavonoids might not only involved in enriching leaf color, but also interfered with the protection of photosystem. Generally speaking, we found higher anthocyanin and flavonoids content level not only dramatically enriched the leaf color of L. chinense var. rubrum cultivars, but also offered more potential antioxidant to keep their normal growth when encountered heat stress.

Overexpression of a Gene Encoding Trigonelline Synthase from Areca catechu L. Promotes Drought Resilience in Transgenic Arabidopsis.

Areca catechu L. is a commercially important palm tree widely cultured in tropical and subtropical areas. Its growth and production are severely hindered by the increasing threat of drought. In the present study, we investigated the physiological responses of areca seedlings to drought stress. The results showed that prolonged drought-induced yellowing on the overall area of most leaves significantly altered the chlorophyll fluorescence parameters, including maximum chemical efficiency (Fv/Fm), photochemical efficiency of PSII (Y(II)), photochemical chlorophyll fluorescence quenching (qP) and non-photochemical chlorophyll fluorescence quenching (NPQ). On the 10th day of drought treatment, the contents of proline in the areca leaves and roots increased, respectively, by 12.2 times and 8.4 times compared to normal watering. The trigonelline levels in the leaves rose from 695.35 µg/g to 1125.21 µg/g under 10 days of water shortage, while no significant changes were detected in the content of trigonelline in the roots. We determined the gene encoding areca trigonelline synthase (AcTS) by conducting a bioinformatic search of the areca genome database. Sequence analysis revealed that AcTS is highly homologous to the trigonelline synthases in Coffea arabica (CaTS 1 and CaTS 2) and all possess a conserved S-adenosyl- L-methionine binding motif. The overexpression of AcTS in Arabidopsis thaliana demonstrated that AcTS is responsible for the generation of trigonelline in transgenic Arabidopsis, which in turn improves the drought resilience of transgenic Arabidopsis. This finding enriches our understanding of the molecular regulatory mechanism of the response of areca to water shortage and provides a foundation for improving the drought tolerance of areca seedlings.

Physiological and Biochemical Responses of Cucumis melo L. Chloroplasts to Low-Phosphate Stress.

Phosphorus (P) is a limiting plant soil nutrient. Long-term low inorganic phosphate (Pi) irreversibly damages plant cells and hinders plant growth. Plants have evolved several adaptive biochemical, physiological, and developmental responses to low-Pi stress. However, little is known about chloroplast responses to low-Pi stress. In this study, we used physiological and biochemical analyses to investigate melon chloroplast responses to low-Pi stress. The results indicated that low-Pi stress impeded melon seedling growth and reduced its dry matter content by inhibiting the photosynthesis. Low-Pi stress reduced the P content in shoots, which inhibited ATP synthase (ATP-ase) activity, and disturbed the proton and electron transport efficiency on chloroplast photosynthetic electron transport chain. In addition, low-Pi stress induced reactive oxygen species (ROS) production in the leaves, which caused membrane peroxidation. Therefore, redox homeostasis was not maintained, and the melon leaves presented with symptoms of photooxidative stress. To mitigate photoinhibition, the melon plants initiated non-photochemical chlorophyll fluorescence quenching (NPQ) initiated by acidification of the thylakoid lumen to dissipate excess excitation energy, significantly improved ROS-scavenging enzyme activity. Based on these experimental results, we concluded that low Pi inhibited photosystem activity and caused photooxidative stress and photoinhibition. To alleviate these negative effects, the plant activated its NPQ mechanism, alternative electron transport pathways, and antioxidant system to protect its chloroplasts.

Response of gas exchange and chlorophyll fluorescence of maize to alternate irrigation with fresh- and brackish water

ABSTRACTThe scarcity of good-quality water is forcing the use of brackish water for irrigation in many areas around the world. Alternate fresh- and brackish water irrigation is a feasible irrigation method (IM). A pot experiment was carried out with three brackish water IMs and at three levels (1, 3 and 5 g NaCl L−1). The various levels of brackish waters were irrigated at the seedling stage, the jointing and tasseling stage and the after tasseling stage, respectively. The responses of maize (Zea mays L.) to alternate irrigation were investigated through gas exchange and chlorophyll fluorescence. The results showed that the alternate use of fresh- and brackish water reduced the increasing soil salinity caused by brackish water irrigation. The changes in net photosynthesis rate (Pn), stomatal conduction (gs), intercellular CO2 concentration (Ci) and chlorophyll content (SPAD) values revealed that maize was more resistant to brackish water at the after tasseling stage. Moreover, significant reductions in maximum quantum yield (Φpo), effective quantum yield of photochemical energy conversion (Φ2), photochemical quenching (qp), non-photochemical quenching of variable chlorophyll fluorescence (qN) and non-photochemical chlorophyll fluorescence quenching (NPQ) of photosystem II, grain yield and biomass weight of maize subjected to high NaCl level brackish water at the jointing and tasseling stage were observed. This implied that maize plants were extremely sensitive to brackish water irrigation during the jointing and tasseling stage, and freshwater should be applied at this growth period. Our results would be helpful for sustainable maize production using alternate irrigation with fresh- and brackish water in arid and semi-arid areas.

The xanthophyll cycle affects reversible interactions between PsbS and light-harvesting complex II to control non-photochemical quenching.

To maintain high photosynthetic rates, plants must adapt to their light environment on a timescale of seconds to minutes. Therefore, the light-harvesting antenna system of photosystem II in thylakoid membranes, light-harvesting complex II (LHCII), has a feedback mechanism, which determines the proportion of absorbed energy dissipated as heat: non-photochemical chlorophyll fluorescence quenching (NPQ). This is crucial to prevent photo-oxidative damage to photosystem II (PSII) and is controlled by the transmembrane pH differences (ΔpH). High ΔpH activates NPQ by protonation of the protein PsbS and the enzymatic de-epoxidation of LHCII-bound violaxanthin to zeaxanthin. But the precise role of PsbS and its interactions with different LHCII complexes remain uncertain. We have investigated PsbS-LHCII interactions in native thylakoid membranes using magnetic-bead-linked antibody pull-downs. The interaction of PsbS with the antenna system is affected by both ΔpH and the level of zeaxanthin. In the presence of ΔpH alone, PsbS is found to be mainly associated with the trimeric LHCII protein polypeptides, Lhcb1, Lhcb2 and Lhcb3. However, a combination of ΔpH and zeaxanthin increases the proportion of PsbS bound to the minor LHCII antenna complex proteins Lhcb4, Lhcb5 and Lhcb6. This pattern of interaction is not influenced by the presence of PSII reactions centres. Similar to LHCII particles in the photosynthetic membrane, PsbS protein forms clusters in the NPQ state. NPQ recovery in the dark requires uncoupling of PsbS. We suggest that PsbS acts as a 'seeding' centre for the LHCII antenna rearrangement that is involved in NPQ.

Nonphotochemical Chlorophyll Fluorescence Quenching: Mechanism and Effectiveness in Protecting Plants from Photodamage.

We review the mechanism underlying nonphotochemical chlorophyll fluorescence quenching (NPQ) and its role in protecting plants against photoinhibition. This review includes an introduction to this phenomenon, a brief history of major milestones in our understanding of NPQ, definitions, and a discussion of quantitative measurements of NPQ We discuss the current knowledge and unknown aspects in the NPQ scenario, including the following: ΔpH, the proton gradient (trigger); light-harvesting complex II (LHCII), PSII light harvesting antenna (site); and changes in the antenna induced by ΔpH (change), which lead to the creation of the quencher We conclude that the minimum requirements for NPQ in vivo are ΔpH, LHCII complexes, and the PsbS protein. We highlight the most important unknown in the NPQ scenario, the mechanism by which PsbS acts upon the LHCII antenna. Finally, we describe a novel, emerging technology for assessing the photoprotective "power" of NPQ and the important findings obtained through this technology.

Spectral Radiation Dependent Photoprotective Mechanism in the Diatom Pseudo-nitzschia multistriata

Phytoplankton, such as diatoms, experience great variations of photon flux density (PFD) and light spectrum along the marine water column. Diatoms have developed some rapidly-regulated photoprotective mechanisms, such as the xanthophyll cycle activation (XC) and the non-photochemical chlorophyll fluorescence quenching (NPQ), to protect themselves from photooxidative damages caused by excess PFD. In this study, we investigate the role of blue fluence rate in combination with red radiation in shaping photoacclimative and protective responses in the coastal diatom Pseudo-nitzschia multistriata. This diatom was acclimated to four spectral light conditions (blue, red, blue-red, blue-red-green), each of them provided with low and high PFD. Our results reveal that the increase in the XC pool size and the amplitude of NPQ is determined by the blue fluence rate experienced by cells, while cells require sensing red radiation to allow the development of these processes. Variations in the light spectrum and in the blue versus red radiation modulate either the photoprotective capacity, such as the activation of the diadinoxanthin-diatoxanthin xanthophyll cycle, the diadinoxanthin de-epoxidation rate and the capacity of non-photochemical quenching, or the pigment composition of this diatom. We propose that spectral composition of light has a key role on the ability of diatoms to finely balance light harvesting and photoprotective capacity.

Higher Plant Photosystem II Light-Harvesting Antenna, Not the Reaction Center, Determines the Excited-State Lifetime—Both the Maximum and the Nonphotochemically Quenched

The maximum chlorophyll fluorescence lifetime in isolated photosystem II (PSII) light-harvesting complex (LHCII) antenna is 4 ns; however, it is quenched to 2 ns in intact thylakoid membranes when PSII reaction centers (RCIIs) are closed (Fm). It has been proposed that the closed state of RCIIs is responsible for the quenching. We investigated this proposal using a new, to our knowledge, model system in which the concentration of RCIIs was highly reduced within the thylakoid membrane. The system was developed in Arabidopsis thaliana plants under long-term treatment with lincomycin, a chloroplast protein synthesis inhibitor. The treatment led to 1), a decreased concentration of RCIIs to 10% of the control level and, interestingly, an increased antenna component; 2), an average reduction in the yield of photochemistry to 0.2; and 3), an increased nonphotochemical chlorophyll fluorescence quenching (NPQ). Despite these changes, the average fluorescence lifetimes measured in Fm and Fm′ (with NPQ) states were nearly identical to those obtained from the control. A 77 K fluorescence spectrum analysis of treated PSII membranes showed the typical features of preaggregation of LHCII, indicating that the state of LHCII antenna in the dark-adapted photosynthetic membrane is sufficient to determine the 2 ns Fm lifetime. Therefore, we conclude that the closed RCs do not cause quenching of excitation in the PSII antenna, and play no role in the formation of NPQ.

Silencing of the Violaxanthin De-Epoxidase Gene in the Diatom Phaeodactylum tricornutum Reduces Diatoxanthin Synthesis and Non-Photochemical Quenching

Diatoms are a major group of primary producers ubiquitous in all aquatic ecosystems. To protect themselves from photooxidative damage in a fluctuating light climate potentially punctuated with regular excess light exposures, diatoms have developed several photoprotective mechanisms. The xanthophyll cycle (XC) dependent non-photochemical chlorophyll fluorescence quenching (NPQ) is one of the most important photoprotective processes that rapidly regulate photosynthesis in diatoms. NPQ depends on the conversion of diadinoxanthin (DD) into diatoxanthin (DT) by the violaxanthin de-epoxidase (VDE), also called DD de-epoxidase (DDE). To study the role of DDE in controlling NPQ, we generated transformants of P. tricornutum in which the gene (Vde/Dde) encoding for DDE was silenced. RNA interference was induced by genetic transformation of the cells with plasmids containing either short (198 bp) or long (523 bp) antisense (AS) fragments or, alternatively, with a plasmid mediating the expression of a self-complementary hairpin-like construct (inverted repeat, IR). The silencing approaches generated diatom transformants with a phenotype clearly distinguishable from wildtype (WT) cells, i.e. a lower degree as well as slower kinetics of both DD de-epoxidation and NPQ induction. Real-time PCR based quantification of Dde transcripts revealed differences in transcript levels between AS transformants and WT cells but also between AS and IR transformants, suggesting the possible presence of two different gene silencing mediating mechanisms. This was confirmed by the differential effect of the light intensity on the respective silencing efficiency of both types of transformants. The characterization of the transformants strengthened some of the specific features of the XC and NPQ and confirmed the most recent mechanistic model of the DT/NPQ relationship in diatoms.

Comparative study on energy partitioning in photosystem II of two Arabidopsis thaliana mutants with reduced non-photochemical quenching capacity

Lhcb1-2 and PsbS proteins of photosystem II (PSII) have important roles in photoprotective thermal energy dissipation of the absorbed excess light energy. The light responses of chlorophyll fluorescence parameters were analyzed to examine how the absence of Lhcb1-2 or PsbS proteins can modify the energy allocation patterns of absorbed light energy in PSII using an antisense construct of lhcb2 and a psbS deletion (npq4-1) mutant of Arabidopsis thaliana. Both mutants exhibit reduced Stern–Volmer non-photochemical chlorophyll fluorescence quenching (NPQ). Here, we have adopted an approach, presented by Hendrickson et al. (Photosynth Res 82:73–81, 2004), to gain a better insight into the mechanism of the NPQ in these mutants. We have found no significant differences in the quantum yields of photochemical energy conversion (ΦPSII) between the mutants and the wild type. Nevertheless, as it was expected, the fraction of the energy, which is dissipated as heat via regulated pathways in PSII (ΦNPQ) for both mutants, were reduced as compared to the wild type. In a complementary way, the extent of non-regulated non-photochemical energy loss in PSII (ΦNO) for both mutants was significantly higher than that in the wild type. This reflects, together with the lower ΦNPQ (or NPQ) values, suboptimal capacity of photoprotective reactions at higher light intensities.

Non-photochemical quenching of chlorophyll fluorescence and xanthophyll cycle responses after excess PAR and UVR in Chaetoceros brevis, Phaeocystis antarctica and coastal Antarctic phytoplankton

Sensitivity to photoinhibition from near-surface irradiance was determined in coastal phytoplankton from Potter Cove (King George Island, Antarctica) in relation to hydrographic conditions shaped by summertime meltwater influx. Slow (indicative for PSII damage) and fast (indicative for xanthophyll cycle activity) relaxing non-photochemical chlorophyll fluorescence quenching (NPQ) was measured during recovery from excess photosynthetically active radiation (PAR) and PAR + ultraviolet radiation (UVR) for 3 stations differentially affected by sediment influx. Additionally, assemblages were incubated outside to study xanthophyll cycle activity during PAR and PAR+UVR exposure. For comparison, NPQ and xanthophyll cycle activity were determined for high (125 mu mol photons m(-2) s(-1)) and low (20 mu mol photons m(-2) s(-1)) light acclimated Chaetoceros brevis (Bacillariophyceae) and Phaeocystis antarctica (Haptophyceae) cultures. Pigment analysis (CHEMTAX) revealed diatom dominance (average 74%) at all locations, with haptophytes being of minor importance (average 13%). All stations were stratified by a vertical density gradient, whereas sediment influx was significantly higher at the innermost station. Taxonomic composition, NPQ characteristics, and photoprotective pigment ratios were not different among stations. At the chlorophyll maximum, phytoplankton showed high light acclimation at all locations, and fast relaxing NPQ dominated after excess PAR and PAR+UVR. Samples from below the chlorophyll maximum were enriched in haptophytes, showed lower photoprotective pigment ratios, reduced capacity for NPQ, and increased contribution of slow relaxing NPQ. The photoprotective capacity of high and low light acclimated C. brevis and P. antarctica differed significantly. P. antarctica induced less NPQ and there was a stronger slow relaxing NPQ component than in the diatom, particularly after low light acclimation. Although total NPQ decreased and slow relaxing NPQ increased after UVR in field samples, NPQ and the xanthophyll cycle de-epoxidation state of cultures were not affected by UVR. A higher photoprotective capacity of diatoms over haptophytes may explain their dominance in stratified meltwater-affected coastal Antarctic waters.

Lutein from deepoxidation of lutein epoxide replaces zeaxanthin to sustain an enhanced capacity for nonphotochemical chlorophyll fluorescence quenching in avocado shade leaves in the dark.

Leaves of avocado (Persea americana) that develop and persist in deep shade canopies have very low rates of photosynthesis but contain high concentrations of lutein epoxide (Lx) that are partially deepoxidized to lutein (L) after 1 h of exposure to 120 to 350 μmol photons m(-2) s(-1), increasing the total L pool by 5% to 10% (ΔL). Deepoxidation of Lx to L was near stoichiometric and similar in kinetics to deepoxidation of violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z). Although the V pool was restored by epoxidation of A and Z overnight, the Lx pool was not. Depending on leaf age and pretreatment, the pool of ΔL persisted for up to 72 h in the dark. Metabolism of ΔL did not involve epoxidation to Lx. These contrasting kinetics enabled us to differentiate three states of the capacity for nonphotochemical chlorophyll fluorescence quenching (NPQ) in attached and detached leaves: ΔpH dependent (NPQ(ΔpH)) before deepoxidation; after deepoxidation in the presence of ΔL, A, and Z (NPQ(ΔLAZ)); and after epoxidation of A+Z but with residual ΔL (NPQ(ΔL)). The capacity of both NPQ(ΔLAZ) and NPQ(ΔL) was similar and 45% larger than NPQ(ΔpH), but dark relaxation of NPQ(ΔLAZ) was slower. The enhanced capacity for NPQ was lost after metabolism of ΔL. The near equivalence of NPQ(ΔLAZ) and NPQ(ΔL) provides compelling evidence that the small dynamic pool ΔL replaces A+Z in avocado to "lock in" enhanced NPQ. The results are discussed in relation to data obtained with other Lx-rich species and in mutants of Arabidopsis (Arabidopsis thaliana) with increased L pools.

Photosynthetic responses of lettuce to downy mildew infection and cytokinin treatment.

Changes in primary metabolism of lettuce, Lactuca sativa L. (cv. Cobham Green), induced by compatible interaction with the biotrophic oomycete pathogen Bremia lactucae Regel (race BL 16), under two intensities of illumination in the presence and absence of exogenous cytokinins were studied by chlorophyll fluorescence imaging. Thirteen days post-inoculation leaf discs infected by B. lactucae exhibited impairments of photosynthesis associated with biotrophic infections, including: reductions in photosynthetic pigment contents and the maximum quantum yield of photosystem II photochemistry (F(V)/F(M)), inhibition of electron transport (Phi(PSII)) and increased non-photochemical chlorophyll fluorescence quenching (NPQ). Detected changes in photosynthetic parameters correlated with the leaf area colonized by the pathogen's intercellular hyphae. Applications of two cytokinins, benzylaminopurine and meta-topolin, previously shown to suppress B. lactucae sporulation if applied 24 h prior to inoculation at a concentration of 200 microM, retarded the pathogen's asexual reproduction with no apparent negative effects on the host's photosynthetic apparatus. However, long-lasting treatment of healthy tissues with this high concentration of exogenous cytokinin led to effects parallel to pathogenesis: reductions in photosynthetic pigment contents accompanied by inhibition of photosystem II photochemistry and electron transport. These effects of both prolonged exposure to cytokinins and the pathogenesis were weaker in discs exposed to the lower photosynthetic photon flux density. The role of cytokinins in plant-biotrophic pathogen interactions and their potential as disease control agents are discussed.

Imaging of NPQ and ROS Formation in Tobacco Leaves: Heat Inactivation of the Water-Water Cycle Prevents Down-Regulation of PSII

Non-photochemical chlorophyll fluorescence quenching (NPQ) plays a major role in the protection of the photosynthetic apparatus against damage by excess light, which is closely linked to the production of reactive oxygen species (ROS). The effect of a short heat treatment on NPQ and ROS production was studied with detached tobacco leaves by fluorescence imaging of chlorophyll and of the ROS sensor dye HO-1889NH. NPQ was stimulated >3-fold by 3 min pre-treatment at 44 degrees C, in parallel with suppression of CO(2) uptake, while no ROS formation could be detected. In contrast, after 3 min pre-treatment at 46 degrees C, NPQ was suppressed and ROS formation was indicated by quenching of HO-1889NH fluorescence. After 3 min pre-treatment at 46 degrees C and above, partial inactivation of ascorbate peroxidase and light-driven accumulation of H(2)O(2) was also observed. These data are discussed as evidence for a decisive role of the Mehler ascorbate peroxidase or water-water cycle in the formation of the NPQ that reflects down-regulation of PSII.

Changes in Chlorophyll Fluorescence in Maize Plants with Imposed Rapid Dehydration at Different Leaf Ages

A comparison of the effects of a rapidly imposed water deficit with different leaf ages on chlorophyll a fluorescence and gas exchange was performed in maize (Zea mays L.) plants. The relationships between photosynthesis and leaf relative turgidity (RT) and ion leakage were further investigated. Leaf dehydration substantially decreased net photosynthetic rate (A) and stomatal conductance (G s), particularly for older leaves. With dehydration time, F v /F m maintained a relatively stable level for youngest leaves but significantly decreased for the older leaves. The electron transport rate (ETR) sharply decreased with intensifying dehydration and remained at lower levels during continuous dehydration. The photochemical quenching of variable chlorophyll fluorescence (q P) gradually decreased with dehydration intensity for the older leaves but increased for the youngest leaves, whereas dehydration did not affect the nonphotochemical chlorophyll fluorescence quenching (NPQ) for the youngest leaves but remarkably decreased it for the older leaves. The leaf RT was significantly and positively correlated with its F v /F m, ETR, and q P, and the leaf ion leakage was significantly and negatively correlated with F v /F m and NPQ. Our results suggest that the photosynthetic systems of young and old leaves decline at different rates when exposed to rapid dehydration.

Light response curve methodology and possible implications in the application of chlorophyll fluorescence to benthic diatoms

Chlorophyll a fluorescence has been increasingly applied to benthic microalgae, especially diatoms, for measurements of electron transport rate (ETR) and construction of rapid light response curves (RLCs) for the determination of photophysiological parameters [mainly the maximum relative ETR (rETRmax), the light saturation coefficient (E k) and the maximum light use coefficient (α)]. Various problems with the estimation of ETR from the microphytobenthos have been identified, especially in situ. This study further examined the effects of light history of the cells and light dose accumulation during RLCs on the fluorescence measurements of ETR using the benthic diatom Navicula phyllepta. RLCs failed to saturate when using incremental increases in irradiance, however, curves with decreasing irradiance did saturate. Patterns indicating photoacclimation in response to light histories were observed, with higher rETRmax and E k, and lower α, at high light compared to low light. However, these differences could be negated by increasing the RLC irradiance duration from 30 to 60 s. It is suggested that problems arose as a result of rapid fluorescence variations due to ubiquinone (QA) oxidation and non-photochemical chlorophyll fluorescence quenching (NPQ) which depended upon the light history of the cells and the RLCs accumulated light dose. Also, RLCs with irradiance duration of 10 s were shown to have a high level of error possibly specific to the fluorimeter programming. It is suggested that RLCs, using a Diving-PAM fluorimeter on benthic diatoms, should be run using decreasing irradiance steps of 30 s duration.

The super-excess energy dissipation in diatom algae: comparative analysis with higher plants

When grown at intermittent light regime, diatom alga Phaeodactylum tricornutum is able to form photoprotective non-photochemical chlorophyll fluorescence quenching (NPQ) three to five times larger than that observed in the higher plants. This quenching is sustained in the dark for 5 to 10 min, reverses completely within approximately 1 h and seems to be very tightly related to the presence of the zeaxanthin analogue, diatoxanthin. Addition of the uncoupler NH4Cl before illumination can completely abolish formation of NPQ, revealing the DeltapH-dependency of the xanthophyll cycle activity. Once established, NPQ can also be almost completely reversed by the uncoupler. However, the higher NPQ is formed the more time is required for its reversal. At the point when the fluorescence was approximately 90% recovered the level of illumination-induced diatoxanthin was found to be only partially reduced. This indicates that the proton gradient is a key triggering factor of NPQ. It was also noticed that NPQ in Phaeodactylum cells was absent even when majority of reaction centers were closed and the plastoquinone pool was significantly reduced. The absence of NPQ at these conditions could be due to very low levels of DeltapH. It is likely that in diatoms alternative sources of protons such as the PS I cyclic electron transfer and/or chlororespiration are important in generating the proton gradient sufficient to trigger NPQ. Absorption changes associated with the xanthophyll cycle activity were found to be larger than those for higher plants. The position of the positive maximum in the difference spectrum illuminated-minus-dark was 512-514 nm in comparison to the 505-508 nm for leaves. The 535 nm band associated with NPQ in plants is absent in Phaeodactylum. An uncoupler-sensitive absorption change at 522 nm was discovered. Kinetics of NPQ showed linear correlation with the 522 nm absorption change.

Mechanism of Non-Photochemical Chlorophyll Fluorescence Quenching. II. Resolution of Rapidly Reversible Absorbance Changes at 530 Nm and Fluorescence Quenching by the Effects of Antimycin, Dibucaine and Cation Exchanger, A23187

The putative relationship between the light-induced absorbance increase at 530 nm (ΔA530), the so-called light-scattering change, and non-photochemical chlorophyll fluorescence quenching (NPQ) was examined by the effect of inhibitors. Antimycin at a low concentration (350 nM) completely inhibited fluorescence quenching while only partially inhibiting A530. This effect was independent of the mode of thylakoid energisation and preinduction of violaxanthin de-epoxidation. Dibucaine at 20 FM abolished NPQ but had little effect on ΔA530. Moreover, the light-induced ΔA530 signal was present even in the absence of de-epoxidised xanthophylls. The cation exchanger A23187 blocked the development of NPQ as well as relaxed fluorescence quenching at steady state without involving a major portion of ΔA530. Thus, the relationship between energy-dependent A530 changes and fluorescence quenching was non-linear under all conditions tested. The light-induced absorbance increase at 530 nm, therefore, is insufficient for NPQ. The differential effects of inhibitors are explained schematically, depicting three phases for NPQ: (a) formation of zeaxanthin and antheraxanthin by the xanthophyll cycle; (b) formation of a state reflected by A530 that is induced by the transthylakoid ApH, possibly involving aggregation of LHCII; and (c) fluorescence quenching by the combined effect of both steps and by the H+-cation exchange properties of thylakoid membranes.

Mechanism of Non-Photochemical Chlorophyll Fluorescence Quenching. I. The Role of De-Epoxidised Xanthophylls and Sequestered Thylakoid Membrane Protons as Probed by Dibucaine

Dibucaine reportedly inhibits the light-induced transthylakoid proton gradient of chloroplasts without inhibiting energy-dependent non-photochemical chlorophyll fluorescence quenching (Laasch, H. and Weis, E. (1989). Photosynthesis Research 22, 137-146). We show that dibucaine can inhibit fluorescence quenching, depending on the de-epoxidation state of the xanthophyll cycle. Whereas dibucaine (20-40 μM) had little effect on fluorescence quenching in pre-illuminated-type thylakoids (loaded with zeaxanthin and antheraxanthin), it strongly inhibited quenching in dark-adapted-type thylakoids (no preinduction of de-epoxidation). Dibucaine inhibited lumen acidification similarly in both types of thylakoids and also the induction of violaxanthin de-epoxidation in dark-adapted thylakoids. Thus dark-adapted and pre-illuminated thylakoids differed in de-epoxidation states and their suspectibility to dibucaine inhibition of fluorescence quenching corresponded to this difference. The mechanism of inhibition of de-epoxidation by dibucaine is unclear. It could be due to the inhibition of lumen acidification but an inhibition of the violaxanthin available for de-epoxidation is not excluded. High dibucaine concentrations inhibited de-epoxidase activity directly. Dibucaine inhibition of fluorescence quenching, however, is not limited to the inhibition of de-epoxidation. Small but clear effects on fluorescence quenching were present in thylakoids even with de-epoxidation preinduced. Moreover, thylakoids with preinduced de-epoxidation were more resistant to dibucaine inhibition of fluorescene quenching when poised by salt treatments for proton partitioning into membrane-sequestered domains than when poised for proton partitioning into delocalised domains. We conclude that non-photochemical quenching of chlorophyll fluorescence depends on both de-epoxidised xanthophylls and sequestered proton domains in the thylakoid membranes