Short-term effects of combined light and chilling stress on non-photochemical quenching in bryophytes

Overexpression of violaxanthin de-epoxidase gene alleviates photoinhibition of PSII and PSI in tomato during high light and chilling stress

Diatoms dominate phytoplankton communities in turbulent waters, where light fluctuations can be frequent and intense. Due to this complex environment, these heterokont microalgae display remarkable photoprotection strategies, including a fast Non-Photochemical Quenching (NPQ). However, in nature, several abiotic parameters (such as temperature) can influence the response of photosynthetic organisms to light stress in a synergistic or antagonistic manner. Yet, the combined effects of light and these other drivers on the photosynthetic and photoprotective capacity of diatoms are still poorly understood. In this work, we investigated the impact of short-term temperature and light stress on the model diatom Phaeodactylum tricornutum, combining NPQ induction-recovery assays or light curves with a broad gradient of superimposed temperature treatments (5 to 35°C). We employed mutant lines deficient in NPQ generation (vde KO) or recovery (zep3 KO) and wild type. We found that temperature and light have a synergistic effect: lower temperatures limited both the photosynthetic capacity and NPQ, while the general photophysiological performance was enhanced with warming, up to a heat-stress limit (above 30°C). We discuss the temperature effects on NPQ induction and recovery and propose that these are independent from the energy requirements of the cells and result from altered xanthophyll cycle dynamics. Namely, we found that de-epoxidation activity strongly increases with temperature, outweighing epoxidation and resulting in a positive increase of NPQ with temperature. Finally, we propose that in a short-term time frame, temperature and light have a synergistic and not antagonistic effect, with a positive relationship between increasing temperature and NPQ.

Different responses of tobacco antioxidant enzymes to light and chilling stress

Behavioral and physiological responses to light are the two major mechanisms by which natural microphytobenthic assemblages adapt to the intertidal environment and protect themselves from light stress. The present study investigated these photoresponses with different light intensities over 8 h of illumination, and used a specific inhibitor (Latrunculin A, Lat A) for migration to compare migratory and non-migratory microphytobenthos (MPB). Photosynthetic activity was detected using rapid light curves and induction curves by chlorophyll fluorescence. It showed distinct variation in migratory responses to different light intensities; high light induced downward migration to avoid photoinhibition, and low and medium light (50–250 μmol/(m2·s)) promoted upward migration followed by downward migration after certain period of light exposure. No significant difference in non-photochemical quenching (NPQ) or PSII maximal quantum yield (Fv/Fm) was detected between low and medium light irradiance, possibly indicating that only high light influences the photosynthetic capability of MPB. Decreased photosynthetic activity, indicated by three parameters, the maximum relative electron transport rate (rETR max), minimum saturating irradiance (Ek) and light utilization coefficient (α), was observed in MPB after exposure to prolonged illumination under low and medium light. Lat A effectively inhibited the migration of MPB in all light treatments and induced lower Fv/Fm under high light (500 and 100 μmol/(m2·s)) and prolonged illumination at 250 μmol/(m2·s), but did not significantly influence Fv/Fm under low light (0–100 μmol/(m2·s)) or NPQ. The increase of NPQ in Lat A treatments with time implied that the MPB assemblages can recover their physiological photoprotection capacity to adapt to light stress. Non-migratory MPB exhibited lower light use efficiency (lower α) and lower maximum photosynthetic capacity (lower rETR max) than migratory MPB under light intensities above 250 μmol/(m2·s) after 4.0 h illumination.

BackgroundPhytochromes are plant photoreceptors that have long been associated with photomorphogenesis in plants; however, more recently, their crucial role in the regulation of variety of abiotic stresses has been explored. Chilling stress is one of the abiotic factors that severely affect growth, development, and productivity of crops. In the present work, we have analyzed and compared physiological, biochemical, and molecular responses in two contrasting phytochrome mutants of tomato, namely aurea (aur) and high pigment1 (hp1), along with wild-type cultivar Micro-Tom (MT) under chilling stress. In tomato, aur is phytochrome-deficient mutant while hp1 is a phytochrome-sensitive mutant. The genotype-specific physiological, biochemical, and molecular responses under chilling stress in tomato mutants strongly validated phytochrome-mediated regulation of abiotic stress. ResultsHere, we demonstrate that phytochrome-sensitive mutant hp1 show improved performance compared to phytochrome-deficient mutant aur and wild-type MT plants under chilling stress. Interestingly, we noticed significant increase in several photosynthetic-related parameters in hp1 under chilling stress that include photosynthetic rate, stomatal conductance, stomatal aperture, transpiration rate, chlorophyll a and carotenoids. Whereas most parameters were negatively affected in aur and MT except a slight increase in carotenoids in MT plants under chilling stress. Further, we found that PSII quantum efficiency (Fv/Fm), PSII operating efficiency (Fq′/Fm′), and non-photochemical quenching (NPQ) were all positively regulated in hp1, which demonstrate enhanced photosynthetic performance of hp1 under stress. On the other hand, Fv/Fm and Fq′/Fm′ were decreased significantly in aur and wild-type plants. In addition, NPQ was not affected in MT but declined in aur mutant after chilling stress. Noticeably, the transcript analysis show that PHY genes which were previously reported to act as molecular switches in response to several abiotic stresses were mainly induced in hp1 and repressed in aur and MT in response to stress. As expected, we also found reduced levels of malondialdehyde (MDA), enhanced activities of antioxidant enzymes, and higher accumulation of protecting osmolytes (soluble sugars, proline, glycine betaine) which further elaborate the underlying tolerance mechanism of hp1 genotype under chilling stress. ConclusionOur findings clearly demonstrate that phytochrome-sensitive and phytochrome-deficient tomato mutants respond differently under chilling stress thereby regulating physiological, biochemical, and molecular responses and thus establish a strong link between phytochromes and their role in stress tolerance.

ISHS International Symposium on Vegetable Safety and Human Health EFFECTS OF 5-AMINOLEVULINIC ACID (ALA) ON PHOTOSYNTHESIS AND CHLOROPHYLL FLUORESCENCE OF WATERMELON SEEDLINGS GROWN UNDER LOW LIGHT AND LOW TEMPERATURE CONDITIONS

The xanthophyll cycle and the water-water cycle had different functional significance in chilling-sensitive sweet pepper upon exposure to chilling temperature (4 °C) under low irradiance (100 µmol m−2 s−1) for 6 h. During chilling stress, effects of non-photochemical quenching (NPQ) on photosystem 2 (PS2) in dithiothreitol (DTT) fed leaves remained distinguishable from that of the water-water cycle in diethyldithiocarbamate (DDTC) fed leaves. In DTT-fed leaves, NPQ decreased greatly accompanied by visible inhibition of the de-epoxidized ratio of the xanthophyll cycle, and maximum photochemical efficiency of PS2 (Fv/Fm) decreased markedly. Thus the xanthophyll cycle-dependent NPQ could protect PS2 through energy dissipation under chilling stress. However, NPQ had a slighter effect on photosystem 1 (PS1) in DTT-fed leaves than in DDTC-fed leaves, whereas effects of the water-water cycle on PS1 remained distinguishable from that of NPQ. Inhibiting superoxide dismutase (SOD) activity increased the accumulation of $$\operatorname{O} \overline {^-- } _2$$ , the oxidation level of P700 (P700+) decreased markedly relative to the control and DTT-fed leaves. Both Fv/Fm and NPQ changed little in DDTC-fed leaves accompanied by little change of (A+Z)/(V+A+Z). This is the active oxygen species inducing PS1 photoinhibition in sweet pepper. The water-water cycle can be interrupted easily at chilling temperature. We propose that during chilling stress under low irradiance, the xanthophyll cycle-dependent NPQ has the main function to protect PS2, whereas the water-water cycle is not only the pathway to dissipate energy but also the dominant factor causing PS1 chilling-sensitivity in sweet pepper.

The dynamics of non-photochemical quenching (NPQ) of chlorophyll fluorescence and the dynamics of xanthophyll conversion under different actinic light conditions were studied in intact leaves of Arabidopsis thaliana. NPQ induction was investigated during up to 180 min illumination at 450, 900, and 1,800 μmol photons m−2 s−1 (μE) and NPQ relaxation after 5, 30, 90, or 180 min of pre-illumination at the same light intensities. The comparison of wild-type plants with mutants affected either in xanthophyll conversion (npq1 and npq2) or PsbS expression (npq4 and L17) or lumen acidification (pgr1) indicated that NPQ states with similar, but not identical characteristics are induced at longer time range (15–60 min) in wild-type and mutant plants. In genotypes with an active xanthophyll conversion, the dynamics of two slowly (10–60 min) inducible and relaxing NPQ components were found to be kinetically correlated with zeaxanthin formation and epoxidation, respectively. However, the extent of NPQ was independent of the amount of zeaxanthin, since higher NPQ values were inducible with increasing actinic light intensities without pronounced changes in the zeaxanthin amount. These data support an indirect role of zeaxanthin in pH-independent NPQ states rather than a specific direct function of zeaxanthin as quencher in long-lasting NPQ processes. Such an indirect function might be related to an allosteric regulation of NPQ processes by zeaxanthin (e.g., through interaction of zeaxanthin at the surface of proteins) or a general photoprotective function of zeaxanthin in the lipid phase of the membrane (e.g., by modulation of the membrane fluidity or by acting as antioxidant). The found concomitant down-regulation of zeaxanthin epoxidation and recovery of photosystem II activity ensures that zeaxanthin is retained in the thylakoid membrane as long as photosystem II activity is inhibited or down-regulated. This regulation supports the view that zeaxanthin can be considered as a kind of light stress memory in chloroplasts, allowing a rapid reactivation of photoprotective NPQ processes in case of recurrent light stress periods.

Plants respond daily to ever-changing light conditions from morning to night, and from season to season, when they may at times be irradiated excessively. Short-term light stress is thereby frequently imposed on chloroplasts and leads to photoinhibition. However, under such conditions, chloroplasts adopt unique and effective strategies to overcome stress caused by high light. For the past several decades, studies have mostly focused on revealing the mechanism(s) of photoinhibition and subsequent repair of PSII, the key multisubunit protein complex required for initiating oxygenic photosynthesis. However, research in this field is now shifting to a new direction: thylakoid membrane dynamics and changes in the distribution and molecular arrangement of PSII/light-harvesting Chl protein complexes (LHCIIs) in response to light stress. One way to avoid light stress is through dissipation of excessive light energy as heat, which can be monitored by qE (energy-dependent quenching) of non-photochemical quenching (NPQ) of Chl fluorescence (Briantais et al. 1980). This dissipative process occurs by changes in the distribution and molecular orientation of Chl proteins in the thylakoid membrane (Goral et al. 2010, Johnson et al. 2011, Herbstova et al. 2012), and results in the formation of reversible aggregates of LHCIIs, which are necessary to quickly dissipate excessive light energy as heat (Horton et al. 1991, Ruban et al. 2012). However, PSII cannot deal effectively with excess high light intensity, and consequently the irreversible aggregation of its proteins, including the reaction center-binding proteins D1 and D2, and the core antenna Chl-binding protein CP43, takes place, which results in PSII dysfunction (Yamamoto et al. 2008). This Special Focus Issue is dedicated to reflecting our current understanding of ‘Photo-oxidative stress in chloroplasts and molecular dynamics of thylakoids’ and presents the latest exciting work produced from several leading laboratories in this field. In the first of three reviews presented here, Yamamoto et al. (2014) give an overview of the relationship between PSII protein aggregations and molecular dynamics of thylakoids under high light. They describe how two types of protein aggregation, reversible and non-reversible, were identified in spinach thylakoids under light stress by Chl fluorescence spectra measurements at 77K and Western blot analysis. In addition, they discuss how the membrane fluidity of the thylakoids changes in response to the light stress, and how this most probably forms the basis for the regulation of thylakoid membrane dynamics. The primary cause for photodamage to the D1 protein and aggregation of proteins described above is ascribed to reactive oxygen species (ROS) produced by irradiation of PSII with excessive light. In particular, singlet oxygen (O2) produced in PSII is suggested to be the primary cause for damage to the D1 protein. The mechanism of formation and action of singlet oxygen (O2) in PSII has been studied extensively. Telfer (2014) presents a historical overview of O2 research in PSII, where she explains the mechanism of O2 production in PSII under light stress and the methods used to measure O2. In addition, carotenoids serve as efficient scavengers of O2, and Telfer provides details of this scavenging process. Also discussed are the current views on the role of O2 as a signaling molecule. Pospisil (2014) provides an extensive and updated review on the roles of native metal cofactors (Mn, non-heme iron and heme iron) in the generation and elimination (by metalcontaining enzymes) of ROS in PSII, and their roles in the PSII photodamage process. In parallel to the production of ROS, lipid peroxidation takes place under various environmental stresses. Lipid peroxidederived aldehydes and ketones (reactive carbonyl species; RCS) as well as ROS can react with proteins to form protein carbonyls that are capable of causing damage to various proteins in plant cells. Mano et al. (2014) studied the effects of salt stress on Arabidopsis thaliana leaves and identified some common RCS and ROS target proteins. Although salt stress is not directly related to light stress, these data highlight an overlap of plant responses to abiotic stress, which may lead to the inactivation of proteins, dysfunction of chloroplasts and, eventually, cell death. Protein phosphorylation is crucial for disorganization of damaged PSII and its mobilization from the grana to the grana margins and stroma thylakoids (Goral et al. 2010, Fristedt and Vener 2011). After the lateral migration of the PSII complexes on the thylakoids, the phosphorylated D1

Non-photochemical quenching (NPQ) is a fast acting photoprotective response to high light stress triggered by over excitation of photosystem II. The mechanism for NPQ in the globally important diatom algae has been principally attributed to a xanthophyll cycle, analogous to the well-described qE quenching of higher plants. This study compared the short-term NPQ responses in two pennate, benthic diatom species cultured under identical conditions but which originate from unique light climates. Variable chlorophyll fluorescence was used to monitor photochemical and non-photochemical excitation energy dissipation during high light transitions; whereas whole cell steady state 77 K absorption and emission were used to measure high light elicited changes in the excited state landscapes of the thylakoid. The marine shoreline species Nitzschia curvilineata was found to have an antenna system capable of entering a deeply quenched, yet reversible state in response to high light, with NPQ being highly sensitive to dithiothreitol (a known inhibitor of the xanthophyll cycle). Conversely, the salt flat species Navicula sp. 110-1 exhibited a less robust NPQ that remained largely locked-in after the light stress was removed; however, a lower amplitude, but now highly reversible NPQ persisted in cells treated with dithiothreitol. Furthermore, dithiothreitol inhibition of NPQ had no functional effect on the ability of Navicula cells to balance PSII excitation/de-excitation. These different approaches for non-photochemical excitation energy dissipation are discussed in the context of native light climate.

A tomato (Lycopersicon esculentum Mill.) zeaxanthin epoxidase gene (LeZE) was isolated and antisense transgenic tomato plants were produced. Northern, southern, and western blot analyses demonstrated that antisense LeZE was transferred into the tomato genome and the expression of LeZE was inhibited. The ratio of (A+Z)/(V+A+Z) in antisense transgenic plants was maintained at a higher level than in the wild type (WT) plants under high light and chilling stress with low irradiance. The value of non-photochemical quenching (NPQ) in WT and transgenic plants was not affected during the stresses. The oxidizable P700 and the maximal photochemical efficiency of PSII (Fv/Fm) in transgenic plants decreased more slowly at chilling temperature under low irradiance. These results suggested that suppression of LeZE caused zeaxanthin accumulation, which was helpful in alleviating photoinhibition of PSI and PSII in tomato plants under chilling stress.

Marine diatoms are prominent phytoplankton organisms that perform photosynthesis in extremely variable environments. Diatoms possess a strong ability to dissipate excess absorbed energy as heat via nonphotochemical quenching (NPQ). This process relies on changes in carotenoid pigment composition (xanthophyll cycle) and on specific members of the light-harvesting complex family specialized in photoprotection (LHCXs), which potentially act as NPQ effectors. However, the link between light stress, NPQ, and the existence of different LHCX isoforms is not understood in these organisms. Using picosecond fluorescence analysis, we observed two types of NPQ in the pennate diatom Phaeodactylum tricornutum that were dependent on light conditions. Short exposure of low-light-acclimated cells to high light triggers the onset of energy quenching close to the core of photosystem II, while prolonged light stress activates NPQ in the antenna. Biochemical analysis indicated a link between the changes in the NPQ site/mechanism and the induction of different LHCX isoforms, which accumulate either in the antenna complexes or in the core complex. By comparing the responses of wild-type cells and transgenic lines with a reduced expression of the major LHCX isoform, LHCX1, we conclude that core complex-associated NPQ is more effective in photoprotection than is the antenna complex. Overall, our data clarify the complex molecular scenario of light responses in diatoms and provide a rationale for the existence of a degenerate family of LHCX proteins in these algae.

In order to estimate vegetation photosynthesis from remote sensing observations; some critical parameters need to be quantified. From all absorbed light; the plant needs to release any excess that is not used for photosynthesis; by non-photochemical quenching; by fluorescence emission and unregulated thermal dissipation. Non-photochemical quenching (NPQ) processes are controlled photoprotective mechanisms which; once activated; strongly control the dynamics of photochemical efficiency. With illumination conditions increasing and decreasing during a diurnal cycle; photoprotection mechanisms needs to change accordingly. The goal of this work is to quantify dynamic NPQ; measured from active fluorescence measurements; based on passive proximal sensing leaf measurements. During a 22-day controlled light and water stress experiment on a tobacco (Nicotiana tabacum L.) leaf we measured the diurnal dynamics of passive fluorescence (Chl F); the Photochemical Reflectance Index (PRI); the Absorbed Photosynthetically Active Radiation (APAR) and leaf temperature in combination with the actively retrieved non-photochemical quenching (NPQ) parameter. Based on a bi-linear combination of diurnal APAR and PRI (plane fit model) we succeeded to estimate NPQ with a RMSE of 0.08. The simple plane fit model estimation represents well the diurnal NPQ dynamics; except for the high light stress phase; when additional reversible photoinhibition processes took place. The present works presents a way of determining NPQ from passive remote sensing measurements; as a necessary step towards estimating photosynthetic rate.

We have isolated very high light resistant nuclear mutants (VHL (R)) in Chlamydomonas reinhardtii, that grow in 1500-2000 mumol photons m(-2) s(-1) (VHL) lethal to wildtype. Four nonallelic mutants have been characterized in terms of Photosystem II (PS II) function, nonphotochemical quenching (NPQ) and xanthophyll pigments in relation to acclimation and survival under light stress. In one class of VHL (R) mutants isolated from wild type (S4 and S9), VHL resistance was accompanied by slower PS II electron transfer, reduced connectivity between PS II centers and decreased PS II efficiency. These lesions in PS II function were already present in the herbicide resistant D1 mutant A251L (L (*)) from which another class of VHL (R) mutants (L4 and L30) were isolated, confirming that optimal PS II function was not critical for survival in very high light. Survival of all four VHL (R) mutants was independent of CO(2) availability, whereas photoprotective processes were not. The de-epoxidation state (DPS) of the xanthophyll cycle pigments in high light (HL, 600 mumol photons m(-2) s(-1)) was strongly depressed when all genotypes were grown in 5% CO(2). In S4 and S9 grown in air under HL and VHL, high DPS was well correlated with high NPQ. However when the same genotypes were grown in 5% CO(2), high DPS did not result in high NPQ, probably because high photosynthetic rates decreased thylakoid DeltapH. Although high NPQ lowered the reduction state of PS II in air compared to 5% CO(2) at HL in wildtype, S4 and S9, this did not occur during growth of S4 and S9 in VHL. L (*) and VHL (R) mutants L4 and L30, also showed high DPS with low NPQ when grown air or 5% CO(2), possibly because they were unable to maintain sufficiently high DeltapH due to constitutively impaired PS II electron transport. Although dissipation of excess photon energy through NPQ may contribute to VHL resistance, there is little evidence that the different genes conferring the VHL (R) phenotype affect this form of photoprotection. Rather, the decline of chlorophyll per biomass in all VHL (R) mutants grown under VHL suggests these genes may be involved in regulating antenna components and photosystem stoichiometries.

Plants respond to environmental changes by acclimation that activates defence mechanisms and enhances the plant's resistance against a subsequent more severe stress. Chloroplasts play an important role as a sensor of environmental stress factors that interfere with the photosynthetic electron transport and enhance the production of reactive oxygen species (ROS). One of these ROS, singlet oxygen ((1)O2), activates a signalling pathway within chloroplasts that depends on the two plastid-localized proteins EXECUTER 1 and 2. Moderate light stress induces acclimation protecting photosynthetic membranes against a subsequent more severe high light stress and at the same time activates (1)O2-mediated and EXECUTER-dependent signalling. Pre-treatment of Arabidopsis seedlings with moderate light stress confers cross-protection against a virulent Pseudomonas syringae strain. While non-pre-acclimated seedlings are highly susceptible to the pathogen regardless of whether (1)O2- and EXECUTER-dependent signalling is active or not, pre-stressed acclimated seedlings without this signalling pathway lose part of their pathogen resistance. These results implicate (1)O2- and EXECUTER-dependent signalling in inducing acclimation but suggest also a contribution by other yet unknown signalling pathways during this response of plants to light stress.