桃和苹果库源关系中源叶光合作用，光抑制及其光保护机制的研究

以‘早久保’（Prunus persica (L.) Batch.）为试材，在果实最后迅速生长期，通过去果处理降低库力，同时设留果对照，并通过环剥和保留相同数量叶片严格控制库源关系，进行了源叶净光合速率（Pn）、叶绿素荧光、叶黄素循环、抗氧化酶及抗氧化同化物日变化的研究。结果表明，和留果对照相比，去果处理显著降低了源叶Pn、气孔导度（gs）和蒸腾速率（E），但显著增加了胞间二氧化碳浓度（Ci）、叶面饱和蒸汽压亏缺（VPDl）和叶片温度（Tl）。光系统II光化学效率（ΦPSII）以及羧化速率（CE）与Pn平行降低。中午去果降低Pn主要归因于非气孔限制。在低库需条件下，开放的PSII反应中心捕获能量的降低以及关闭的PSII反应中心的增加导致了ΦPSII的降低。去果处理叶片中依赖于叶黄素循环的热耗散以及抗氧化系统的上调保护叶片免受光氧化破坏。和留果对照相比，去果处理的叶片有更大的叶黄素循环库，更高的脱环氧化状态以及更高的抗氧化酶活性，包括超氧化物歧化酶（SOD）、抗坏血酸过氧化物酶（APX）、单脱氢抗坏血酸还原酶（MDAR）和脱氢抗坏血酸还原酶（DHAR）的活性以及更高的还原型抗坏血酸（AsA）和还原型谷胱甘肽（GSH）的含量。但与此同时，去果显著增加了过氧化氢（H2O2）以及丙二醛（MDA）的含量，这意味着在去果处理的叶片中可能会发生光氧化破坏。 以一年生‘皇家嘎拉’苹果（Malus domestica Borkh.）组培苗为试材，通过环剥降低库力，进行了源叶Pn、叶绿素荧光、核酮糖-1,5-二磷酸羧化酶/氧化酶（Rubisco）以及光系统II（PSII）复合体关键蛋白PsbA和PsbO含量日变化的研究。和对照相比，环剥显著降低了源叶Pn、gs和E，但是却显著增加了Ci、Tl和淀粉的含量。在低库需下，开放的PSII反应中心捕获能量的降低以及关闭的PSII反应中心的增加导致了ΦPSII的降低。另一方面，环剥降低了光合作用关键酶Rubisco以及PSII复合体PsbA和放氧复合体PsbO的含量。以上结果表明，环剥降低Pn主要归因于非气孔限制。

Localization of the mechanism of pH-dependent non- photochemical fluorescence quenching in thylakoid membranes

Higher plants have evolved a well-conserved set of photoprotective mechanisms, collectively designated Non-Photochemical Quenching of chlorophyll fluorescence (qN), to deal with the inhibitory absorption of excess light energy by the photosystems. Their main contribution originates from safe thermal deactivation of excited states promoted by a highly-energized thylakoid membrane, detected via lumen acidification. The precise origins of this energy- or LlpH-dependent quenching (qE), arising from either decreased energy transfer efficiency in PSII antennae (~ Young & Frank, 1996; Gilmore & Yamamoto, 1992; Ruban et aI., 1992), from alternative electron transfer pathways in PSII reaction centres (~ Schreiber & Neubauer, 1990; Thompson &Brudvig, 1988; Klimov et aI., 1977), or from both (Wagner et aI., 1996; Walters & Horton, 1993), are a source of considerable controversy. In this study, the origins of qE were investigated in spinach thylakoids using a combination of fluorescence spectroscopic techniques: Pulse Amplitude Modulated (PAM) fluorimetry, pump-probe fluorimetry for the measurement of PSII absorption crosssections, and picosecond fluorescence decay curves fit to a kinetic model for PSII. Quenching by qE (,..,600/0 of maximal fluorescence, Fm) was light-induced in circulating samples and the resulting pH gradient maintained during a dark delay by the lumenacidifying capabilities of thylakoid membrane H+ ATPases. Results for qE were compared to those for the addition of a known antenna quencher, 5-hydroxy-1,4naphthoquinone (5-0H-NQ), titrated to achieve the same degree of Fm quenching as for qE. Quenching of the minimal fluorescence yield, F0' was clear (8 to 130/0) during formation of qE, indicative of classical antenna quenching (Butler, 1984), although the degree was significantly less than that achieved by addition of 5-0H-NQ. Although qE induction resulted in an overall increase in absorption cross-section, unlike the decrease expected for antenna quenchers like the quinone, a larger increase in crosssection was observed when qE induction was attempted in thylakoids with collapsed pH gradients (uncoupled by nigericin), in the absence of xanthophyll cycle operation (inhibited by DTT), or in the absence of quenching (LlpH not maintained in the dark due to omission of ATP). Fluorescence decay curves exhibited a similar disparity between qE-quenched and 5-0H-NQ-quenched thylakoids, although both sets showed accelerated kinetics in the fastest decay components at both F0 and Fm. In addition, the kinetics of dark-adapted thylakoids were nearly identical to those in qEquenched samples at F0' both accelerated in comparison with thylakoids in which the redox poise of the Oxygen-Evolving Complex was randomized by exposure to low levels of background light (which allowed appropriate comparison with F0 yields from quenched samples). When modelled with the Reversible Radical Pair model for PSII (Schatz et aI., 1988), quinone quenching could be sufficiently described by increasing only the rate constant for decay in the antenna (as in Vasil'ev et aI., 1998), whereas modelling of data from qE-quenched thylakoids required changes in both the antenna rate constant and in rate constants for the reaction centre. The clear differences between qE and 5-0H-NQ quenching demonstrated that qE could not have its origins in the antenna alone, but is rather accompanied by reaction centre quenching. Defined mechanisms of reaction centre quenching are discussed, also in relation to the observed post-quenching depression in Fm associated with photoinhibition.

The Fast Regulation of Photosynthesis in Diatoms: an inquiry into the physiological and physical origins of non-photochemical chlorophyll fluorescence quenching.

Diatoms are renowned for their robust ability to perform NPQ (Non-Photochemical Quenching of chlorophyll fluorescence) as a dissipative response to heightened light stress on photosystem II, plausibly explaining their dominance over other algal groups in turbulent light environs. Their NPQ mechanism has been principally attributed to a xanthophyll cycle involving the lumenal pH regulated reversible de-epoxidation of diadinoxanthin. The principal goal of this dissertation is to reveal the physiological and physical origins and consequences of the NPQ response in diatoms during short-term transitions to excessive irradiation. The investigation involves diatom species from different originating light environs to highlight the diversity of diatom NPQ and to facilitate the detection of core mechanisms common among the diatoms as a group. A chiefly spectroscopic approach was used to investigate NPQ in diatom cells. Prime methodologies include: the real time monitoring of PSII excitation and de-excitation pathways via PAM fluorometry and pigment interconversion via transient absorbance measurements, the collection of cryogenic absorbance spectra to measure pigment energy levels, and the collection of cryogenic fluorescence spectra and room temperature picosecond time resolved fluorescence decay spectra to study excitation energy transfer and dissipation. Chemical inhibitors that target the trans-thylakoid pH gradient, the enzyme responsible for diadinoxanthin de-epoxidation, and photosynthetic electron flow were additionally used to experimentally manipulate the NPQ response. Multifaceted analyses of the NPQ responses from two previously un-photosynthetically characterised species, Nitzschia curvilineata and Navicula sp., were used to identify an excitation pressure relief ‘strategy’ for each species. Three key areas of NPQ were examined: (i) the NPQ activation/deactivation processes, (ii) how NPQ affects the collection, dissipation, and usage of absorbed light energy, and (iii) the interdependence of NPQ and photosynthetic electron flow. It was found that Nitzschia cells regulate excitation pressure via performing a high amplitude, reversible antenna based quenching which is dependent on the de-epoxidation of diadinoxanthin. In Navicula cells excitation pressure could be effectively regulated solely within the PSII reaction centre, whilst antenna based, diadinoxanthin de-epoxidation dependent quenching was implicated to be used as a supplemental, long-lasting source of excitation energy dissipation. These strategies for excitation balance were discussed in the context of resource partitioning under these species’ originating light climates. A more detailed investigation of the NPQ response in Nitzschia was used to develop a comprehensive model describing the mechanism for antenna centred non-photochemical quenching in this species. The experimental evidence was strongly supportive of a mechanism whereby: an acidic lumen triggers the diadinoxanthin de-epoxidation and protonation mediated aggregation of light harvesting complexes leading to the formation of quencher chlorophyll a-chlorophyll a dimers with short-lived excited states; quenching relaxes when a rise in lumen pH triggers the dispersal of light harvesting complex aggregates via deprotonation events and the input of diadinoxanthin. This model may also be applicable for describing antenna based NPQ in other diatom species.

Can we measure terrestrial photosynthesis from space directly, using spectral reflectance and fluorescence?

Attempts to estimate photosynthetic rate or gross primary productivity from remotely sensed absorbed solar radiation depend on knowledge of the light use efficiency (LUE). Early models assumed LUE to be constant, but now most researchers try to adjust it for variations in temperature and moisture stress. However, more exact methods are now required. Hyperspectral remote sensing offers the possibility of sensing the changes in the xanthophyll cycle, which is closely coupled to photosynthesis. Several studies have shown that an index (the photochemical reflectance index) based on the reflectance at 531 nm is strongly correlated with the LUE over hours, days and months. A second hyperspectral approach relies on the remote detection of fluorescence, which is a directly related to the efficiency of photosynthesis. We discuss the state of the art of the two approaches. Both have been demonstrated to be effective, but we specify seven conditions required before the methods can become operational.

Effects of N supply on the accumulation of photosynthetic pigments and photoprotectors in Gracilaria tenuistipitata (Rhodophyta) cultured under UV radiation

We have studied the effects of nitrate supply under photosynthetic active radiation (PAR) plus ultraviolet radiation (UVR) exposure on photosynthetic pigments (chlorophyll a and carotenoids), photoprotective UV screen mycosporine-like amino acids (MAAs), and photosynthetic parameters, including the maximum quantum yield (F(v)/F(m)) and electron transport rate (ETR) on the red agarophyte Gracilaria tenuistipitata. Apical tips of G. tenuistipitata were cultivated under ten different concentrations of NO(3)(-) for 7 days. It has been shown that G. tenuistipitata cultured under laboratory conditions has the ability to accumulate high amounts of MAAs following a nitrate concentration-dependent manner under PAR+UVR. Two MAAs were identified, shinorine and porphyra-334. The relative concentration of the first increased under high concentrations of nitrate, while the second one decreased. The presence of antheraxanthin is reported for the first time in this macro-algae, which also contains zeaxanthin, lutein, and beta-carotene. The accumulation of pigments, photoprotective compounds, and photosynthetic parameters of G. tenuistipitata is directly related to N availability. All variables decreased under low N supplies and reached constant maximum values with supplements higher than 0.5 mM NO(3)(-). Our results suggest a high potential to acclimation and photoprotection against stress factors (including high PAR and UVR) directly related to N availability for G. tenuistipitata.

Light energy management in peach: utilization, photoprotection , photodamage and recovery. Maximizing light absorption in orchard is not always the best solution

The relation between the intercepted light and orchard productivity was considered linear, although this dependence seems to be more subordinate to planting system rather than light intensity. At whole plant level not always the increase of irradiance determines productivity improvement. One of the reasons can be the plant intrinsic un-efficiency in using energy. Generally in full light only the 5 – 10% of the total incoming energy is allocated to net photosynthesis. Therefore preserving or improving this efficiency becomes pivotal for scientist and fruit growers. Even tough a conspicuous energy amount is reflected or transmitted, plants can not avoid to absorb photons in excess. The chlorophyll over-excitation promotes the reactive species production increasing the photoinhibition risks. The dangerous consequences of photoinhibition forced plants to evolve a complex and multilevel machine able to dissipate the energy excess quenching heat (Non Photochemical Quenching), moving electrons (water-water cycle , cyclic transport around PSI, glutathione-ascorbate cycle and photorespiration) and scavenging the generated reactive species. The price plants must pay for this equipment is the use of CO2 and reducing power with a consequent decrease of the photosynthetic efficiency, both because some photons are not used for carboxylation and an effective CO2 and reducing power loss occurs. Net photosynthesis increases with light until the saturation point, additional PPFD doesn’t improve carboxylation but it rises the efficiency of the alternative pathways in energy dissipation but also ROS production and photoinhibition risks. The wide photo-protective apparatus, although is not able to cope with the excessive incoming energy, therefore photodamage occurs. Each event increasing the photon pressure and/or decreasing the efficiency of the described photo-protective mechanisms (i.e. thermal stress, water and nutritional deficiency) can emphasize the photoinhibition. Likely in nature a small amount of not damaged photosystems is found because of the effective, efficient and energy consuming recovery system. Since the damaged PSII is quickly repaired with energy expense, it would be interesting to investigate how much PSII recovery costs to plant productivity. This PhD. dissertation purposes to improve the knowledge about the several strategies accomplished for managing the incoming energy and the light excess implication on photo-damage in peach. The thesis is organized in three scientific units. In the first section a new rapid, non-intrusive, whole tissue and universal technique for functional PSII determination was implemented and validated on different kinds of plants as C3 and C4 species, woody and herbaceous plants, wild type and Chlorophyll b-less mutant and monocot and dicot plants. In the second unit, using a “singular” experimental orchard named “Asymmetric orchard”, the relation between light environment and photosynthetic performance, water use and photoinhibition was investigated in peach at whole plant level, furthermore the effect of photon pressure variation on energy management was considered on single leaf. In the third section the quenching analysis method suggested by Kornyeyev and Hendrickson (2007) was validate on peach. Afterwards it was applied in the field where the influence of moderate light and water reduction on peach photosynthetic performances, water requirements, energy management and photoinhibition was studied. Using solar energy as fuel for life plant is intrinsically suicidal since the high constant photodamage risk. This dissertation would try to highlight the complex relation existing between plant, in particular peach, and light analysing the principal strategies plants developed to manage the incoming light for deriving the maximal benefits as possible minimizing the risks. In the first instance the new method proposed for functional PSII determination based on P700 redox kinetics seems to be a valid, non intrusive, universal and field-applicable technique, even because it is able to measure in deep the whole leaf tissue rather than the first leaf layers as fluorescence. Fluorescence Fv/Fm parameter gives a good estimate of functional PSII but only when data obtained by ad-axial and ab-axial leaf surface are averaged. In addition to this method the energy quenching analysis proposed by Kornyeyev and Hendrickson (2007), combined with the photosynthesis model proposed by von Caemmerer (2000) is a forceful tool to analyse and study, even in the field, the relation between plant and environmental factors such as water, temperature but first of all light. “Asymmetric” training system is a good way to study light energy, photosynthetic performance and water use relations in the field. At whole plant level net carboxylation increases with PPFD reaching a saturating point. Light excess rather than improve photosynthesis may emphasize water and thermal stress leading to stomatal limitation. Furthermore too much light does not promote net carboxylation improvement but PSII damage, in fact in the most light exposed plants about 50-60% of the total PSII is inactivated. At single leaf level, net carboxylation increases till saturation point (1000 – 1200 μmolm-2s-1) and light excess is dissipated by non photochemical quenching and non net carboxylative transports. The latter follows a quite similar pattern of Pn/PPFD curve reaching the saturation point at almost the same photon flux density. At middle-low irradiance NPQ seems to be lumen pH limited because the incoming photon pressure is not enough to generate the optimum lumen pH for violaxanthin de-epoxidase (VDE) full activation. Peach leaves try to cope with the light excess increasing the non net carboxylative transports. While PPFD rises the xanthophyll cycle is more and more activated and the rate of non net carboxylative transports is reduced. Some of these alternative transports, such as the water-water cycle, the cyclic transport around the PSI and the glutathione-ascorbate cycle are able to generate additional H+ in lumen in order to support the VDE activation when light can be limiting. Moreover the alternative transports seems to be involved as an important dissipative way when high temperature and sub-optimal conductance emphasize the photoinhibition risks. In peach, a moderate water and light reduction does not determine net carboxylation decrease but, diminishing the incoming light and the environmental evapo-transpiration request, stomatal conductance decreases, improving water use efficiency. Therefore lowering light intensity till not limiting levels, water could be saved not compromising net photosynthesis. The quenching analysis is able to partition absorbed energy in the several utilization, photoprotection and photo-oxidation pathways. When recovery is permitted only few PSII remained un-repaired, although more net PSII damage is recorded in plants placed in full light. Even in this experiment, in over saturating light the main dissipation pathway is the non photochemical quenching; at middle-low irradiance it seems to be pH limited and other transports, such as photorespiration and alternative transports, are used to support photoprotection and to contribute for creating the optimal trans-thylakoidal ΔpH for violaxanthin de-epoxidase. These alternative pathways become the main quenching mechanisms at very low light environment. Another aspect pointed out by this study is the role of NPQ as dissipative pathway when conductance becomes severely limiting. The evidence that in nature a small amount of damaged PSII is seen indicates the presence of an effective and efficient recovery mechanism that masks the real photodamage occurring during the day. At single leaf level, when repair is not allowed leaves in full light are two fold more photoinhibited than the shaded ones. Therefore light in excess of the photosynthetic optima does not promote net carboxylation but increases water loss and PSII damage. The more is photoinhibition the more must be the photosystems to be repaired and consequently the energy and dry matter to allocate in this essential activity. Since above the saturation point net photosynthesis is constant while photoinhibition increases it would be interesting to investigate how photodamage costs in terms of tree productivity. An other aspect of pivotal importance to be further widened is the combined influence of light and other environmental parameters, like water status, temperature and nutrition on peach light, water and phtosyntate management.

Beziehungen zwischen den Xanthophyllzyklen und der Biosynthese von Lichtsammelxanthophyllen in Chlorophyll a/c-haltigen Algen

Zusammenfassung:In Chlorophyll(Chl) a/c-haltigen Algen leisten Xanthophylle einen wesentlichen Beitrag zur Lichtsammlung. Daneben finden sich weitere Xanthophylle, die an einem Schutzmechanismus bei überoptimalem Lichtangebot beteiligt sind, dem sog. Xanthophyllzyklus. Ein Teil der Chl a/c-haltigen Algen besitzt den auch bei Höheren Pflanzen anzutreffenden Violaxanthin/Antheraxanthin/Zeaxanthin-(Vx/Ax/Zx-)Zyklus. In anderen Gruppen wie den Dinophyta, Haptophyta und den Kieselalgen (Bacillariophyceae) ist statt dessen der Diadinoxanthin/Diatoxanthin-(Ddx/Dtx-)Zyklus zu finden. Die vorliegende Arbeit zeigt, daß schwachlichtadaptierte Turbidostatkulturen der Kieselalge Phaeodactylum tricornutum unter mehrstündiger Starklichtinkubation neben den Pigmenten des Ddx/Dtx-Zyklus auch die des Vx/Ax/Zx-Zyklus akkumulieren. Außerdem läßt sich ein dritter Xanthophyllzyklus zwischen beta-Cryptoxanthin (Cx) und beta-Cryptoxanthin-Epoxid (CxE) nachweisen, doch liegen diese beiden Pigmente nur in sehr geringen Konzentrationen vor. Für die Starklichtakkumulation von Zx ist eine hohe Deepoxidase-Aktivität und die de-novo-Synthese von Carotinoiden erforderlich. Aus Zx wird im anschließenden Schwachlicht über die Intermediate Vx und Ddx das Lichtsammelxanthophyll Fucoxanthin (Fx) synthetisiert. Dies bestätigt auch ein Vergleich der Kinetiken der einzelnen Umwandlungsschritte mit den anhand eines Modells der Xanthophyllbiosynthesewege ermittelten theoretischen Ratenkonstanten. Dieser Vergleich legt jedoch nahe, daß bei der Vx-Synthese aus beta-Carotin CxE anstelle von Zx involviert sein könnte. Eine Untersuchung weiterer Chl a/c-haltiger Algen mit Ddx/Dt-Zyklus ergab, daß sie unter Starklicht ebenfalls den Vx/Ax/Zx-Zyklus akkumulieren. Weiterhin sind, mit Einschränkungen bei den Dinophyten und Xanthophyceen, alle untersuchten Algen in der Lage, die unter Starklicht akkumulierten Xanthophyllzykluspigmente im nachfolgenden Schwachlicht zur Synthese des jeweiligen Lichtsammelxanthophylls zu nutzen. Unter energetischen Gesichtspunkten stellt dieses Pigment-Recycling insbesondere für die Fx-haltigen Algen einen Vorteil dar, da ihre Lichtsammelkomplexe im Vergleich zu denen der Höheren Pflanzen etwa die doppelte Anzahl an Xanthophyllen binden.

Untersuchung der Pigment- und Proteinzusammensetzung der peripheren Lichtsammelantenne des Photosystem I

Die Lichtsammelantenne des PSI (LHCI) ist hinsichtlich ihrer Protein- und Pigmentzusammensetzung weniger gut untersucht als die des PSII. Im Rahmen dieser Arbeit wurde deshalb zunächst die Isolation von nativen LHCI-Subkomplexen optimiert und deren Pigmentzusammensetzung untersucht. Zusätzlich wurde die Pigmentbindung analysiert sowie das Pigment/Protein-Verhältnis bestimmt. Die Analyse der Proteinzusammensetzung des LHCI erfolgte mittels einer Kombination aus ein- oder zweidimensionaler Gelelektrophorese mit Westernblotanalysen mit Lhca-Protein-spezifischen Antikörpern und massenspektrometrischen Untersuchungen. Dabei stellte sich heraus, dass der LHCI mehr Proteine bzw. Proteinisoformen enthält als bisher vermutet. So gelang durch die massenspektrometrischen Untersuchungen die Identifizierung zweier bisher noch nicht nachgewiesener Lhca-Proteine. Bei diesen handelt es sich um eine Isoform des Lhca4 und ein zusätzliches Lhca-Protein, das Tomaten-Homolog des Lhca5 von Arabidopsis thaliana. Außerdem wurden in 1D-Gelen Isoformen von Lhca-Proteinen mit unterschiedlichem elektrophoretischen Verhalten beobachtet. In 2D-Gelen trat zusätzlich eine große Anzahl an Isoformen mit unterschiedlichen isoelektrischen Punkten auf. Es ist zu vermuten, dass zumindest ein Teil dieser Isoformen physiologischen Ursprungs ist, und z.B. durch differentielle Prozessierung oder posttranslationale Modifikationen verursacht wird, wenn auch die Spotvielfalt in 2D-Gelen wohl eher auf die Probenaufbereitung zurückzuführen ist. Mittels in vitro-Rekonstitution mit anschließenden biochemischen Untersuchungen und Fluoreszenzmessungen wurde nachgewiesen, dass Lhca5 ein funktioneller LHC mit spezifischen Pigmentbindungseigenschaften ist. Außerdem zeigten in vitro-Dimerisierungsexperimente eine Interaktion zwischen Lhca1 und Lhca5, wodurch dessen Zugehörigkeit zur Antenne des PSI gestützt wird. In vitro-Dimerisierungsexperimente mit Lhca2 und Lhca3 führten dagegen nicht zur Bildung von Dimeren. Dies zeigt, dass die Interaktion in potentiellen Homo- oder Heterodimeren aus Lhca2 und/oder Lhca3 schwächer ist als die zwischen Lhca1 und Lhca4 oder Lhca5. Die beobachtete Proteinheterogenität deutet daraufhin, dass die Antenne des PSI eine komplexere Zusammensetzung hat als bisher angenommen. Für die Integration „neuer“ LHC in den PSI-LHCI-Holokomplex werden zwei Modelle vorgeschlagen: geht man von einer festen Anzahl von LHCI-Monomeren aus, so kann sie durch den Austausch einzelner LHC-Monomere erreicht werden. Als zweites Szenario ist die Bindung zusätzlicher LHC vorstellbar, die entweder indirekt über bereits vorhandene LHC oder direkt über PSI-Kernuntereinheiten mit dem PSI interagieren. In Hinblick auf die Pigmentbindung der nativen LHCI-Subfraktionen konnte gezeigt werden, dass sie Pigmente in einer spezifischen Stöchiometrie und Anzahl binden, und sich vom LHCIIb vor allem durch eine verstärkte Bindung von Chlorophyll a, eine geringere Anzahl von Carotinoiden und die Bindung von ß-Carotin an Stelle von Neoxanthin unterscheiden. Der Vergleich von nativem LHCI mit rekonstituierten Lhca-Proteinen ergab, dass Lhca-Proteine Pigmente in einer spezifischen Stöchiometrie binden, und dass sie Carotinoidbindungsstellen mit flexiblen Bindungseigenschaften besitzen. Auch über die Umwandlung des an die einzelnen Lhca-Proteine gebundenen Violaxanthins (Vio) im Xanthophyllzyklus war nur wenig bekannt. Deshalb wurden mit Hilfe eines in vitro-Deepoxidationssystems sowohl native als auch rekonstituierte LHCI hinsichtlich ihrer Deepoxidationseigenschaften untersucht und der Deepoxidationsgrad von in vivo deepoxidierten Pigment-Protein-Komplexen bestimmt. Aus den Deepoxidationsexperimenten konnte abgeleitet werden, dass in den verschiedenen Lhca-Proteinen unterschiedliche Carotinoidbindungsstellen besetzt sind. Außerdem bestätigten diese Experimente, dass der Xanthophyllzyklus auch im LHCI auftritt, wobei jedoch ein niedrigerer Deepoxidationsgrad erreicht wird als bei LHCII. Dies konnte durch in vitro-Deepoxidationsversuchen auf eine geringere Deepoxidierbarkeit des von Lhca1 und Lhca2 gebundenen Vio zurückgeführt werden. Damit scheint Vio in diesen Lhca-Proteinen eher eine strukturelle Rolle zu übernehmen. Eine photoprotektive Funktion von Zeaxanthin im PSI wäre folglich auf Lhca3 und Lhca4 beschränkt. Damit enthält jede LHCI-Subfraktion ein LHC-Monomer mit langwelliger Fluoreszenz, das möglicherweise am Lichtschutz beteiligt ist. Insgesamt zeigten die Untersuchungen der Pigmentbindung, der Deepoxidierung und der Fluoreszenzeigenschaften, dass sich die verschiedenen Lhca-Proteine in einem oder mehreren dieser Parameter unterscheiden. Dies lässt vermuten, dass schon durch leichte Veränderungen in der Proteinzusammensetzung des LHCI eine Anpassung an unterschiedliche Licht-verhältnisse erreicht werden kann.

Evaluation of the photosynthetic efficiency of sweet sorghum under drought and cold conditions

Sweet sorghum, a C4 crop of tropical origin, is gaining momentum as a multipurpose feedstock to tackle the growing environmental, food and energy security demands. Under temperate climates sweet sorghum is considered as a potential bioethanol feedstock, however, being a relatively new crop in such areas its physiological and metabolic adaptability has to be evaluated; especially to the more frequent and severe drought spells occurring throughout the growing season and to the cold temperatures during the establishment period of the crop. The objective of this thesis was to evaluate some adaptive photosynthetic traits of sweet sorghum to drought and cold stress, both under field and controlled conditions. To meet such goal, a series of experiments were carried out. A new cold-tolerant sweet sorghum genotype was sown in rhizotrons of 1 m3 in order to evaluate its tolerance to progressive drought until plant death at young and mature stages. Young plants were able to retain high photosynthetic rate for 10 days longer than mature plants. Such response was associated to the efficient PSII down-regulation capacity mediated by light energy dissipation, closure of reaction centers (JIP-test parameters), and accumulation of glucose and sucrose. On the other hand, when sweet sorghum plants went into blooming stage, neither energy dissipation nor sugar accumulation counteracted the negative effect of drought. Two hybrids with contrastable cold tolerance, selected from an early sowing field trial were subjected to chilling temperatures under controlled growth conditions to evaluate in deep their physiological and metabolic cold adaptation mechanisms. The hybrid which poorly performed under field conditions (ICSSH31), showed earlier metabolic changes (Chl a + b, xanthophyll cycle) and greater inhibition of enzymatic activity (Rubisco and PEPcase activity) than the cold tolerant hybrid (Bulldozer). Important insights on the potential adaptability of sweet sorghum to temperate climates are given.

(Table 1) Chlorophyll concentration and pigment composition of carotenoids in three heathland bryophytes, northern Sweden

Background and Aims: Anthropogenic depletion of stratospheric ozone in Arctic latitudes has resulted in an increase of ultraviolet-B radiation (UV-B) reaching the biosphere. UV-B exposure is known to reduce aboveground biomass and plant height, to increase DNA damage and cause accumulation of UV-absorbing compounds in polar plants. However, many studies on Arctic mosses tended to be inconclusive. The importance of different water availability in influencing UV-B impacts on lower plants in the Arctic has been poorly explored and might partially explain the observed wide variation of responses, given the importance of water in controlling bryophyte physiology. This study aimed to assess the long-term responses of three common sub-Arctic bryophytes to enhanced UV-B radiation (+UV-B) and to elucidate the influence of water supply on those responses. Results: Responses were species specific: H. splendens responded most to +UV-B, with reduction in both annual growth (-22%) and sporophyte production (-44%), together with increased b-carotene, violaxanthin, total chlorophyll and NPQ, and decreased zeaxanthin and de-epoxidation of the xanthophyll cycle pool (DES). Barbilophozia lycopodioides responded less to +UV-B, showing increased b-carotene and sclerophylly and decreased UV-absorbing compounds. Polytrichum commune only showed small morphogenetic changes. No effect of UV-B on bryophyte cover was observed. Water availability had profound effects on bryophyte ecophysiology, and plants showed, in general, lower growth and ETR, together with a higher photoprotection in the drier site. Water availability also influenced bryophyte responses to +UV-B and, in particular, responses were less detectable in the drier site. Conclusions: Impacts of UV-B exposure on Arctic bryophytes were significant, in contrast to modest or absent UV-B effects measured in previous studies. The impacts were more easily detectable in species with high plasticity such as H. splendens and less obvious, or more subtle, under drier conditions. Species biology and water supply greatly influences the impact of UV-B on at least some Arctic bryophytes and could contribute to the wide variation of responses observed previously.

Sample characteristics and pigment concentration of arctic snow and permafrost algal strains

Ten algal strains from snow and permafrost substrates were tested for their ability to produce secondary carotenoids and ?-tocopherol in response to high light and decreased nitrogen levels. The Culture Collection of Cryophilic Algae at Fraunhofer IBMT in Potsdam served as the bioresource for this study. Eight of the strains belong to the Chlorophyceae and two strains are affiliated to the Trebouxiophyceae. While under low light, all 10 strains produced the normal spectrum of primary pigments known to be present in Chlorophyta, only the eight chlorophyceaen strains were able to synthesize secondary carotenoids under stress conditions, namely canthaxanthin, echinenone and astaxanthin; seven of them were also able to synthesize minor amounts of adonixanthin and an unidentified hydroxyechinenone. The two trebouxiophyceaen species of Raphidonema exhibited an unusually high pool of primary xanthophyll cycle pigments, possibly serving as a buffering reservoir against excessive irradiation. They also proved to be good alpha-tocopherol producers, which might also support the deactivation of reactive oxygen species. This study showed that some strains might be interesting novel candidates for biotechnological applications. Cold-adapted, snow and permafrost algae might serve as valuable production strains still exhibiting acceptable growth rates during the cold season in temperate regions.

The roles of specific xanthophylls in photoprotection

Xanthophyll pigments have critical structural and functional roles in the photosynthetic light-harvesting complexes of algae and vascular plants. Genetic dissection of xanthophyll metabolism in the green alga Chlamydomonas reinhardtii revealed functions for specific xanthophylls in the nonradiative dissipation of excess absorbed light energy, measured as nonphotochemical quenching of chlorophyll fluorescence. Mutants with a defect in either the α- or β-branch of carotenoid biosynthesis exhibited less nonphotochemical quenching but were still able to tolerate high light. In contrast, a double mutant that was defective in the synthesis of lutein, loroxanthin (α-carotene branch), zeaxanthin, and antheraxanthin (β-carotene branch) had almost no nonphotochemical quenching and was extremely sensitive to high light. These results strongly suggest that in addition to the xanthophyll cycle pigments (zeaxanthin and antheraxanthin), α-carotene-derived xanthophylls such as lutein, which are structural components of the subunits of the light-harvesting complexes, contribute to the dissipation of excess absorbed light energy and the protection of plants from photo-oxidative damage.

Antioxidative Defense System, Pigment Composition, and Photosynthetic Efficiency in Two Wheat Cultivars Subjected to Drought1

We analyzed antioxidative defenses, photosynthesis, and pigments (especially xanthophyll-cycle components) in two wheat (Triticum durum Desf.) cultivars, Adamello and Ofanto, during dehydration and rehydration to determine the difference in their sensitivities to drought and to elucidate the role of different protective mechanisms against oxidative stress. Drought caused a more pronounced inhibition in growth and photosynthetic rates in the more sensitive cv Adamello compared with the relatively tolerant cv Ofanto. During dehydration the glutathione content decreased in both wheat cultivars, but only cv Adamello showed a significant increase in glutathione reductase and hydrogen peroxide-glutathione peroxidase activities. The activation states of two sulfhydryl-containing chloroplast enzymes, NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase and fructose-1,6-bisphosphatase, were maintained at control levels during dehydration and rehydration in both cultivars. This indicates that the defense systems involved are efficient in the protection of sulfhydryl groups against oxidation. Drought did not cause significant effects on lipid peroxidation. Upon dehydration, a decline in chlorophyll a, lutein, neoxanthin, and β-carotene contents, and an increase in the pool of de-epoxidized xanthophyll-cycle components (i.e. zeaxanthin and antheraxanthin), were evident only in cv Adamello. Accordingly, after exposure to drought, cv Adamello showed a larger reduction in the actual photosystem II photochemical efficiency and a higher increase in nonradiative energy dissipation than cv Ofanto. Although differences in zeaxanthin content were not sufficient to explain the difference in drought tolerance between the two cultivars, zeaxanthin formation may be relevant in avoiding irreversible damage to photosystem II in the more sensitive cultivar.

Interactions between Senescence and Leaf Orientation Determine in Situ Patterns of Photosynthesis and Photoinhibition in Field-Grown Rice1

Photosynthesis and photoinhibition in field-grown rice (Oryza sativa L.) were examined in relation to leaf age and orientation. Two varieties (IR72 and IR65598-112-2 [BSI206]) were grown in the field in the Philippines during the dry season under highly irrigated, well-fertilized conditions. Flag leaves were examined 60 and 100 d after transplanting. Because of the upright nature of 60-d-old rice leaves, patterns of photosynthesis were determined by solar movements: light falling on the exposed surface in the morning, a low incident angle of irradiance at midday, and light striking the opposite side of the leaf blade in the afternoon. There was an early morning burst of CO2 assimilation and high levels of saturation of photosystem II electron transfer as incident irradiance reached a maximum level. However, by midday the photochemical efficiency increased again almost to maximum. Leaves that were 100 d old possessed a more horizontal orientation and were found to suffer greater levels of photoinhibition than younger leaves, and this was accompanied by increases in the de-epoxidation state of the xanthophyll cycle. Older leaves had significantly lower chlorophyll content but only slightly diminished photosynthesis capacity.

Expression Studies of the Zeaxanthin Epoxidase Gene in Nicotiana plumbaginifolia1

Abscisic acid (ABA) is a plant hormone involved in the control of a wide range of physiological processes, including adaptation to environmental stress and seed development. In higher plants ABA is a breakdown product of xanthophyll carotenoids (C40) via the C15 intermediate xanthoxin. The ABA2 gene of Nicotiana plumbaginifolia encodes zeaxanthin epoxidase, which catalyzes the conversion of zeaxanthin to violaxanthin. In this study we analyzed steady-state levels of ABA2 mRNA in N. plumbaginifolia. The ABA2 mRNA accumulated in all plant organs, but transcript levels were found to be higher in aerial parts (stems and leaves) than in roots and seeds. In leaves ABA2 mRNA accumulation displayed a day/night cycle; however, the ABA2 protein level remained constant. In roots no diurnal fluctuation in mRNA levels was observed. In seeds the ABA2 mRNA level peaked around the middle of development, when ABA content has been shown to increase in many species. In conditions of drought stress, ABA levels increased in both leaves and roots. A concomitant accumulation of ABA2 mRNA was observed in roots but not in leaves. These results are discussed in relation to the role of zeaxanthin epoxidase both in the xanthophyll cycle and in the synthesis of ABA precursors.