On singlet oxygen, photoinhibition, plastoquinone, and their interconnections

In oxygenic photosynthesis light is captured in series by two protein complexes: photosystem II and photosystem I (PSI and PSII). Electron transfer from PSII to PSI is mediated by the plastoquinone (PQ) pool. Despite being the energy source, light also damages the photosynthetic machinery. Singlet oxygen (1O2), an excited state of O2, may be generated when a charge recombination reaction in PSII re-excites the reaction center chlorophylls (P680), producing a triplet state. In this thesis, massspectroscopy- based detection methods for 1O2 were developed further, to understand the role of this reactive oxygen species in photosynthesis. It was shown that even though both O2 and 1O2 are produced in pumpkin thylakoid membranes, most (if not all) of the 1O2 derives from the ambient dissolved O2, not from the nascent O2 produced due to the water splitting activity of PSII. The result shows that the O2 evolving ability of PSII as such does not render PSII vulnerable to oxidative damage. Nevertheless, light inactivates PSII, and no consensus about the mechanism(s) of the photoinhibitory damage exists. Therefore, the temperature dependence of the rate constant of PSII photoinhibition was measured under various conditions and compared with temperature dependencies of 1O2 production and recombinations. The results show that in plants and cyanobacteria the rate constant of photoinhibition and production of 1O2 increase similarly with temperature as the miss probability of the oxygen evolving complex. Photoinhibition proceeded under anaerobicity, where no 1O2 is produced, and was unaffected by quenchers of 1O2. We suggest that when a miss occurs, but a recombination does not re-reduce the PSII reaction center, P680+ lives long enough to oxidize a vital component of PSII, causing the photodamage. Plants have also ways to adjust to different light conditions. An initial fluorescence screen and subsequent high-performance liquid chromatography measurements revealed that 470 nm, 560 nm and 660 nm light favors PSII over PSI and reduces 80–90 % of the PQ pool, whereas 440 nm, 520 nm and 690 nm favors PSI and oxidizes 90–100 % of the pool in Arabidopsis thaliana, when moderate light was used. Light state curvilinearly followed the redox state of the PQ pool; state 2 was reached with 50 % reduction. All tested white lights, including light from the Sun, reduced less than 50 % of the PQ pool. This PSI light character of white light enables plants to respond to the intensity of light via the redox state of the PQ pool.Vaikka valo onkin yhteytyksen eli fotosynteesin energianlähde, se myös vahingoittaa yhteyttäviä eliöitä. Happea vapauttavassa yhteytyksessä kaksi proteiinikompleksia, valoreaktiot II ja I (PSII ja PSI), vastaanottavat valoa. Plastokinonivaranto välittää elektroninsiirtoa näiden välillä. Singlettihappi on reaktiivinen happiyhdiste, jota syntyy, kun PSII:n reaktiokeskusklorofylli (P680) siirtyy triplettitilaan elektronin palatessa rekombinaatioreaktioissa takaisin reaktiokeskukselle. Tässä työssä kehitettiin edelleen massaspektrometriamenetelmiä ja havaittiin, että vaikka eristetyt kasvin yhteytyskalvostot tuottavat sekä happea että singlettihappea, suurin osa singlettihapesta on peräisin ympäristöön liuenneesta hapesta eikä PSII:n veden hajotuksessa vastasyntyneestä hapesta. PSII:n kyky vapauttaa happea ei siis itsessään tee PSII:sta herkkää hapettavalle vahingolle. Valo kuitenkin vaurioittaa erityisesti PSII:ta, eikä valovahingon mekanismista ole yksimielisyyttä. Tästä syystä PSII:n fotoinhibition, singlettihapen tuoton ja rekombinaatioreaktioiden lämpötilavasteet mitattiin kasveista ja syanobakteereista. Sekä fotoinhibitio että singlettihappi lisääntyivät lämpötilan noustessa. Molemmat lämpötilavasteet selvästi muistuttavat PSII:n happea vapauttavan kompleksin “hutien” lämpötilariippuvuutta. Fotoinhibitio ei kuitenkaan vähentynyt, kun yhteytyskalvostoja valotettiin singlettihapen vaimentajien kanssa tai hapettomissa olosuhteissa, jolloin singlettihappea ei synny. Tulosten perusteella muotoiltiin hypoteesi: mikäli huti tapahtuu eli happea vapauttava kompleksi on tilapäisesti kykenemätön luovuttamaan elektronin, eikä rekombinaatiokaan uudelleen pelkistä reaktiokeskusta, P680+:lla on tarpeeksi aikaa hapettaa jokin PSII:n tärkeä osa. Kasvit kykenevät myös sopeutumaan valo-olosuhteiden muutoksiin. Plastokinonivarannon hapetus-pelkistystila mitattiin nestekromatokrafialla; PSII vastaanotti PSI:tä tehokkaammin 470, 560 ja 660 nm:n aallonpituuksien valoa ja nämä valot pelkistivät 80–90 % plastokinonivarannosta. 440, 520 ja 690 nm:n aallonpituudet sen sijaan suosivat PSI:tä ja hapettivat 90–100 % plastokinonivarannosta, kun kohtalaista valovoimakkuutta käytettiin. Kaikki testatut valkoiset valot, mukaan lukien auringonvalo, suosivat PSI:tä, joten kasvit voivat aistia valon voimakkuutta plastokinonivarannon pelkistyneisyyden avull

Light-induced damage to photosystem II at a very low temperature (195 K) depends on singlet oxygen

Photosynthetic organisms, like evergreen plants, may encounter strong light at low temperatures. Light, despite being the energy source of photosynthesis, irreversibly damages photosystem II (PSII). We illuminated plant thylakoid membranes and intact cyanobacterial cells at -78.5? and assayed PSII activity with oxygen evolution or chlorophyll fluorescence, after thawing the sample. Both UV radiation and visible light damaged PSII of pumpkin (Cucurbita maxima) thylakoids at -78.5?, but visible-light-induced photoinhibition at -78.5?, unlike at +20?, proceeded only in the presence of oxygen. A strong magnetic field that would decrease triplet chlorophyll formation by recombination of the primary radical pair slowed down photoinhibition at -78.5?, suggesting that singlet oxygen produced via recombination of the primary pair is a major contributor to photoinhibition at -78.5?. However, a magnetic field did not affect singlet oxygen production at +25?. Thylakoids of winter leaves of an evergreen plant, Bergenia, were less susceptible to photoinhibition both at -78.5? and +20?, contained high amounts of carotenoids and produced little singlet oxygen (measured at +20?), compared to thylakoids of summer leaves. In contrast, high carotenoid amount and low singlet oxygen yield did not protect a Synechocystis mutant from photoinhibition at -78.5?. Thylakoids isolated from Arabidopsis thaliana grown under high light, which reduces PSII antenna size, were more resistant than control plants against photoinhibition at -78.5? but not at +20?, although carotenoid amounts were similar. The results indicate that visible-light-induced photoinhibition at -78.5? depends on singlet oxygen, whereas photoinhibition at +20? is largely independent of oxygen

Singlet oxygen production by photosystem II is caused by misses of the oxygen evolving complex

Singlet oxygen (O-1(2)) is a harmful species that functions also as a signaling molecule. In chloroplasts, O-1(2) is produced via charge recombination reactions in photosystem II, but which recombination pathway(s) produce triplet Chl and O-1(2) remains open. Furthermore, the role of O-1(2) in photoinhibition is not clear. We compared temperature dependences of O-1(2) production, photoinhibition, and recombination pathways. O-1(2) production by pumpkin thylakoids increased from -2 to +35 degrees C, ruling out recombination of the primary charge pair as a main contributor. S(2)Q(A)(-) or S(2)Q(B)(-) recombination pathways, in turn, had too steep temperature dependences. Instead, the temperature dependence of O-1(2) production matched that of misses (failures of the oxygen (O-2) evolving complex to advance an S-state). Photoinhibition in vitro and in vivo (also in Synechocystis), and in the presence or absence of O-2, had the same temperature dependence, but ultraviolet (UV)-radiation-caused photoinhibition showed a weaker temperature response. We suggest that the miss-associated recombination of P(680)(+)Q(A)(-) is the main producer of O-1(2). Our results indicate three parallel photoinhibition mechanisms. The manganese mechanism dominates in UV radiation but also functions in white light. Mechanisms that depend on light absorption by Chls, having O-1(2) or long-lived P-680(+) as damaging agents, dominate in red light

Oxygen and ROS in photosynthesis

Oxygen is a natural acceptor of electrons in the respiratory pathway of aerobic organisms and in many other biochemical reactions. Aerobic metabolism is always associated with the formation of reactive oxygen species (ROS). ROS may damage biomolecules but are also involved in regulatory functions of photosynthetic organisms. This review presents the main properties of ROS, the formation of ROS in the photosynthetic electron transport chain and in the stroma of chloroplasts, and ROS scavenging systems of thylakoid membrane and stroma. Effects of ROS on the photosynthetic apparatus and their roles in redox signaling are discussed.</p

Evaluation of visible-light wavelengths that reduce or oxidize the plastoquinone pool in green algae with the activated F0 rise method

We recently developed a chlorophyll a fluorescence method (activated F0 rise) for estimating if a light wavelength preferably excites PSI or PSII in plants. Here, the method was tested in green microalgae: Scenedesmus quadricauda, Scenedesmus ecornis, Scenedesmus fuscus, Chlamydomonas reinhardtii, Chlorella sorokiniana, and Ettlia oleoabundans. The Scenedesmus species displayed a plant-like action spectra of F0 rise, suggesting that PSII/PSI absorption ratio is conserved from higher plants to green algae. F0 rise was weak in a strain of C. reinhardtii, C. sorokiniana, and E. oleoabundans. Interestingly, another C. reinhardtii strain exhibited a strong F0 rise. The result indicates that the same illumination can lead to different redox states of the plastoquinone pool in different algae. Flavodiiron activity enhanced the F0 rise, presumably by oxidizing the plastoquinone pool during pre-illumination. The activity of plastid terminal oxidase, in turn, diminished the F0 rise, but to a small degree </p

Ultraviolet screening by slug tissue and tight packing of plastids protect photosynthetic sea slugs from photoinhibition

One of the main mysteries regarding photosynthetic sea slugs is how the slug plastids handle photoinhibition, the constant light-induced damage to Photosystem II of photosynthesis. Recovery from photoinhibition involves proteins encoded by both the nuclear and plastid genomes, and slugs with plastids isolated from the algal nucleus are therefore expected to be incapable of constantly repairing the damage as the plastids inside the slugs grow old. We studied photoinhibition-related properties of the sea slug Elysia timida that ingests its plastids from the green alga Acetabularia acetabulum. Spectral analysis of both the slugs and the algae revealed that there are two ways the slugs use to avoid major photoinhibition of their plastids. Firstly, highly photoinhibitory UV radiation is screened by the slug tissue or mucus before it reaches the plastids. Secondly, the slugs pack the plastids tightly in their thick bodies, and therefore plastids in the outer layers protect the inner ones from photoinhibition. Both properties are expected to greatly improve the longevity of the plastids inside the slugs, as the plastids do not need to repair excessive amounts of damage.</p

Automatic detection of cereal rows by means of pattern recognition techniques

Automatic locating of weeds from fields is an active research topic in precision agriculture. A reliable and practical plant identification technique would enable the reduction of herbicide amounts and lowering of production costs, along with reducing the damage to the ecosystem. When the seeds have been sown row-wise, most weeds may be located between the sowing rows. The present work describes a clustering-based method for recognition of plantlet rows from a set of aerial photographs, taken by a drone flying at approximately ten meters. The algorithm includes three phases: segmentation of green objects in the view, feature extraction, and clustering of plants into individual rows. Segmentation separates the plants from the background. The main feature to be extracted is the center of gravity of each plant segment. A tentative clustering is obtained piecewise by applying the 2D Fourier transform to image blocks to get information about the direction and the distance between the rows. The precise sowing line position is finally derived by principal component analysis. The method was able to find the rows from a set of photographs of size 1452 x 969 pixels approximately in 0.11 s, with the accuracy of 94 per cent

Singlet oxygen, flavonols and photoinhibition in green and senescing silver birch leaves

During autumn senescence, deciduous trees degrade chlorophyll and may synthesize flavonols. We measured photosynthetic parameters, epidermal flavonols, singlet oxygen production in vivo and photoinhibition of the photosystems (PSII and PSI) from green and senescing silver birch (Betula pendula) leaves. Chlorophyll a fluorescence and P700 absorbance measurements showed that the amounts of both photosystems decreased throughout autumn senescence, but the remaining PSII units stayed functional until ~ 90% of leaf chlorophyll was degraded. An increase in the chlorophyll a to b ratio, a decrease in > 700 nm absorbance and a blue shift of the PSI fluorescence peak at 77 K suggest that light-harvesting complex I was first degraded during senescence, followed by light-harvesting complex II and finally the photosystems. Senescing leaves produced more singlet oxygen than green leaves, possibly because low light absorption by senescing leaves allows high flux of incident light per photosystem. Senescing leaves also induced less non-photochemical quenching, which may contribute to increased singlet oxygen production. Faster photoinhibition of both photosystems in senescing than in green leaves, under high light, was most probably caused by low absorption of light and rapid singlet oxygen production. However, senescing leaves maintained the capacity to recover from photoinhibition of PSII. Amounts of epidermal flavonols and singlet oxygen correlated neither in green nor in senescing leaves of silver birch. Moreover, Arabidopsis thaliana mutants, incapable of synthesizing flavonols, were not more susceptible to photoinhibition of PSII or PSI than wild type plants; screening of chlorophyll absorption by flavonols was, however, small in A. thaliana. These results suggest that flavonols do not protect against photoinhibition or singlet oxygen production in chloroplasts

Differences in susceptibility to photoinhibition do not determine growth rate under moderate light in batch or turbidostat-a study with five green algae

To understand growth limitations of photosynthetic microorganisms, and to investigate whether batch growth or certain photosynthesis-related parameters predict a turbidostat (continuous growth at constant biomass concentration) growth rate, five green algal species were grown in a photobioreactor in batch and turbidostat conditions and their susceptibilities to photoinhibition of photosystem II as well as several photosynthetic parameters were measured. Growth rates during batch and turbidostat modes varied independently of each other; thus, a growth rate measured in a batch cannot be used to determine the continuous growth rate. Greatly different photoinhibition susceptibilities in tested algae suggest that different amounts of energy were invested in repair. However, photoinhibition tolerance did not necessarily lead to a fast growth rate at a moderate light intensity. Nevertheless, we report an inverse relationship between photoinhibition tolerance and minimum saturating irradiance, suggesting that fast electron transfer capacity of PSII comes with the price of reduced photoinhibition tolerance