Photosynthetic characteristics of Pinaceae: From evolution to environmental acclimation

Boreal evergreen conifers are important for Northern countries and have functioned for centuries as main drivers of the economy. Nowadays, the attention has shifted to the sustainability of the boreal forests, their protection against ever increasing biodiversity loss and highlighting their irreplaceable role as carbon sinks in climate change mitigation. Climate change drastically changes the growth environment of boreal forests, which exerts a serious threat for the acclimation capacity to new environmental condition. As photosynthesis is the key process to sustain plant growth and biomass production, but also the most vulnerable process upon environmental changes, I decided to focus my PhD thesis on boreal evergreen conifer species, in particular the members of the Pinaceae family including pine and spruce. In current genome and molecular level photosynthesis research, the members of Pinaceae are drastically underrepresented, as the research has almost exclusively been based on few and only distantly related model species. Therefore, I aimed to further characterize the photosynthetic machinery of members of Pinaceae and investigate their seasonal regulation mechanisms necessary for successful overwintering and subsequent spring restoration of photosynthesis. Thereby also paving the way for a better understanding of photosynthesis across different scales, which become more and more accessible with new advances in remote-sensing technologies. The key findings of my thesis work include the elucidation of the unique subunit composition of the photosynthetic apparatus of Pinaceae across the land plants with the help of a custom build mass spectrometry database. Furthermore, the identification of specific and highly dynamic thylakoid protein phosphorylations, particularly the triple phosphorylation of the LHCB1 and multiple phosphorylations PSBS proteins as prerequisites for the sustained thermal energy dissipation, allowed building a new model conferring photoprotection for spruce during winter and spring. And at last, the derivation of new in vivo parameters to disentangle the seasonal dynamics of PSI and PSII, suggesting a re-evaluation of the role of cyclic electron flow in conifer photoprotection--- Boreaaliset ikivihreät havupuumetsät ovat elintärkeitä pohjoiselle luonnolle ja ne ovat myös toimineet vuosisatojen ajan talouden vetureina pohjoismaissa. Nykyään huomio on siirtynyt yhä enemmän metsien kestävyyteen eli biologisen monimuotoisuuden sälyttämiseen ja metsien korvaamattomaan roolin ilmastonmuutoksen hillinnässä hiilinieluina. Ilmastonmuutos vaikuttaa voimakkaasti boreaalisten metsien kasvuympäristöön, mikä uhkaa vakavasti niiden sopeutumiskykyä. Fotosynteesi on keskeisin prosessi kasvien kasvun ja biomassan tuotannon ylläpitämisessä. Fotosynteesi on kuitenkin myös hyvin haavoittuva prosessi ympäristöolosuhteiden muuttuessa epäsuotuisimmiksi. Tästä syystä päätin keskittyä väitöskirjassani boreaalisten ikivihreiden havupuulajien, erityisesti Pinaceae-suvun männyn ja kuusen fotosynteesikoneiston kykyyn sopeutua ympäristöolosuhteiden muutoksiin. Nykyisessä genomi- ja molekyylitason fotosynteesitutkimuksessa Pinaceae-suvun jäsenet ovat aliedustettuina. Tästä syystä olen selvittänyt tarkemmin kuusen ja männyn fotosynteesikoneiston rakennetta ja fotosynteesin toiminnan vuodenaikaisia säätelymekanismeja, jotka ovat välttämättömiä onnistuneelle talvehtimiselle ja sitä seuraavalle fotosynteesin palautumiselle keväällä. Säätelymekanismien molekyylitason ymmärtämistä tarvitaan myös nopeasti kehittyvien kaukokartoitusteknologioiden tulosten fysiologisen merkityksen tulkinnassa. Väitöskirjatyöni tärkeimpiin tuloksiin kuuluu Pinaceae-heimon fotosynteesikoneiston ainutlaatuisen alayksikkökoostumuksen selvittäminen näille lajeille räätälöimäni massaspektrometriatietokannan avulla. Lisäksi ympäristöolosuhteiden muutokseen dynaamisesti reagoivien tylakoidiproteiinien fosforylaatioin, erityisesti LHCB1:n kolmoisfosforylaation sekä PSBS-proteiinin monipaikkaisen fosforylaation tunnistaminen sekä vuosirytmi olivat tärkeitä uusia havaintoja. Biofysikaalisiin mittaustuloksiini pohjautuen kehitin myös uusia in vivo -parametreja, joiden avulla PSI:n ja PSII:n kausittainen dynamiikka ja dynamiikan takana toimivat molekyylimekanismit voidaan erottaa toisistaan. Tutkimustyöni avulla on mahdollista kehittää malli siitä, miten havupuiden fotosynteesikoneiston sopeutumiskykyä ilmastonmuutoksen aiheuttamiin haasteisiin voitaisiin parantaa

The Arabidopsis thylakoid chloride channel ClCe regulates ATP availability for light-harvesting complex II protein phosphorylation

Coping with changes in light intensity is challenging for plants, but well-designed mechanisms allow them to acclimate to most unpredicted situations. The thylakoid K+/H+ antiporter KEA3 and the voltage-dependent Cl- channel VCCN1 play important roles in light acclimation by fine-tuning electron transport and photoprotection. Good evidence exists that the thylakoid Cl- channel ClCe is involved in the regulation of photosynthesis and state transitions in conditions of low light. However, a detailed mechanistic understanding of this effect is lacking. Here we report that the ClCe loss-of-function in Arabidopsis thaliana results in lower levels of phosphorylated light-harvesting complex II (LHCII) proteins as well as lower levels of the photosystem I-LHCII complexes relative to wild type (WT) in low light conditions. The phosphorylation of the photosystem II core D1/D2 proteins was less affected either in low or high light conditions. In low light conditions, the steady-state levels of ATP synthase conductivity and of the total proton flux available for ATP synthesis were lower in ClCe loss-of-function mutants, but comparable to WT at standard and high light intensity. As a long-term acclimation strategy, expression of the ClCe gene was upregulated in WT plants grown in light-limiting conditions, but not in WT plants grown in standard light even when exposed for up to 8 h to low light. Taken together, these results suggest a role of ClCe in the regulation of the ATP synthase activity which under low light conditions impacts LHCII protein phosphorylation and state transitions.</p

Specific thylakoid protein phosphorylations are prerequisites for overwintering of Norway spruce (Picea abies) photosynthesis

Coping of evergreen conifers in boreal forests with freezing temperatures on bright winter days puts the photosynthetic machinery in great risk of oxidative damage. To survive harsh winter conditions, conifers have evolved a unique but poorly characterized photoprotection mechanism, a sustained form of nonphotochemical quenching (sustained NPQ). Here we focused on functional properties and underlying molecular mechanisms related to the development of sustained NPQ in Norway spruce (Picea abies). Data were collected during 4 consecutive years (2016 to 2019) from trees growing in sun and shade habitats. When day temperatures dropped below -4 degrees C, the specific N-terminally triply phosphorylated LHCB1 isoform (3p-LHCII) and phosphorylated PSBS (p-PSBS) could be detected in the thylakoid membrane. Development of sustained NPQ coincided with the highest level of 3p-LHCII and p-PSBS, occurring after prolonged coincidence of bright winter days and temperatures close to -10 degrees C. Artificial induction of both the sustained NPQ and recovery from naturally induced sustained NPQ provided information on differential dynamics and light-dependence of 3p-LHCII and p-PSBS accumulation as prerequisites for sustained NPQ. Data obtained collectively suggest three components related to sustained NPQ in spruce: 1) Freezing temperatures induce 3p-LHCII accumulation independently of light, which is suggested to initiate destacking of appressed thylakoid membranes due to increased electrostatic repulsion of adjacent membranes; 2) p-PSBS accumulation is both light -and temperature-dependent and closely linked to the initiation of sustained NPQ, which 3) in concert with PSII photoinhibition, is suggested to trigger sustained NPQ in spruce

Chlorophyll a fluorescence illuminates a path connecting plant molecular biology to Earth-system science

Remote sensing methods enable detection of solar-induced chlorophyll a fluorescence. However, to unleash the full potential of this signal, intensive cross-disciplinary work is required to harmonize biophysical and ecophysiological studies. For decades, the dynamic nature of chlorophyll a fluorescence (ChlaF) has provided insight into the biophysics and ecophysiology of the light reactions of photosynthesis from the subcellular to leaf scales. Recent advances in remote sensing methods enable detection of ChlaF induced by sunlight across a range of larger scales, from using instruments mounted on towers above plant canopies to Earth-orbiting satellites. This signal is referred to as solar-induced fluorescence (SIF) and its application promises to overcome spatial constraints on studies of photosynthesis, opening new research directions and opportunities in ecology, ecophysiology, biogeochemistry, agriculture and forestry. However, to unleash the full potential of SIF, intensive cross-disciplinary work is required to harmonize these new advances with the rich history of biophysical and ecophysiological studies of ChlaF, fostering the development of next-generation plant physiological and Earth-system models. Here, we introduce the scale-dependent link between SIF and photosynthesis, with an emphasis on seven remaining scientific challenges, and present a roadmap to facilitate future collaborative research towards new applications of SIF.Peer reviewe

Chlorophyll a fluorescence illuminates a path connecting plant molecular biology to Earth-system science

Remote sensing methods enable detection of solar-induced chlorophyll a fluorescence. However, to unleash the full potential of this signal, intensive cross-disciplinary work is required to harmonize biophysical and ecophysiological studies.For decades, the dynamic nature of chlorophyll a fluorescence (ChlaF) has provided insight into the biophysics and ecophysiology of the light reactions of photosynthesis from the subcellular to leaf scales. Recent advances in remote sensing methods enable detection of ChlaF induced by sunlight across a range of larger scales, from using instruments mounted on towers above plant canopies to Earth-orbiting satellites. This signal is referred to as solar-induced fluorescence (SIF) and its application promises to overcome spatial constraints on studies of photosynthesis, opening new research directions and opportunities in ecology, ecophysiology, biogeochemistry, agriculture and forestry. However, to unleash the full potential of SIF, intensive cross-disciplinary work is required to harmonize these new advances with the rich history of biophysical and ecophysiological studies of ChlaF, fostering the development of next-generation plant physiological and Earth-system models. Here, we introduce the scale-dependent link between SIF and photosynthesis, with an emphasis on seven remaining scientific challenges, and present a roadmap to facilitate future collaborative research towards new applications of SIF

On the Generalization of Detecting Face Morphing Attacks as Anomalies: Novelty vs. Outlier Detection

Face morphing attacks are verifiable to multiple identities, leading to faulty identity links. Recent works have studied the face morphing attack detection performance over variations in morphing approaches, pointing out low generalization. This work studies detecting these attacks as anomalies and discusses the performance and generalization over different morphing types. We also analyze the accuracy and generalization effect of including different amounts of attack contamination in the anomaly training data (novelty vs. outlier). This is performed with two baseline 2-class classifiers, two approaches for anomaly detection, two image feature extractions, two morphing types, and variations in contamination levels and tolerated training errors. The results points out the relative lower performance, but higher generalization ability, of anomaly detection in comparison to 2-class classifiers, along with the benefits of contaminating the anomaly training data

Specific thylakoid protein phosphorylations are prerequisites for overwintering of Norway spruce (Picea abies) photosynthesis

Coping of evergreen conifers in boreal forests with freezing temperatures on bright winter days puts the photosynthetic machinery in great risk of oxidative damage. To survive harsh winter conditions, conifers have evolved a unique but poorly characterized photoprotection mechanism, a sustained form of nonphotochemical quenching (sustained NPQ). Here we focused on functional properties and underlying molecular mechanisms related to the development of sustained NPQ in Norway spruce (Picea abies). Data were collected during 4 consecutive years (2016 to 2019) from trees growing in sun and shade habitats. When day temperatures dropped below -4 degrees C, the specific N-terminally triply phosphorylated LHCB1 isoform (3p-LHCII) and phosphorylated PSBS (p-PSBS) could be detected in the thylakoid membrane. Development of sustained NPQ coincided with the highest level of 3p-LHCII and p-PSBS, occurring after prolonged coincidence of bright winter days and temperatures close to -10 degrees C. Artificial induction of both the sustained NPQ and recovery from naturally induced sustained NPQ provided information on differential dynamics and light-dependence of 3p-LHCII and p-PSBS accumulation as prerequisites for sustained NPQ. Data obtained collectively suggest three components related to sustained NPQ in spruce: 1) Freezing temperatures induce 3p-LHCII accumulation independently of light, which is suggested to initiate destacking of appressed thylakoid membranes due to increased electrostatic repulsion of adjacent membranes; 2) p-PSBS accumulation is both light -and temperature-dependent and closely linked to the initiation of sustained NPQ, which 3) in concert with PSII photoinhibition, is suggested to trigger sustained NPQ in spruce

The unique photosynthetic apparatus of Pinaceae : analysis of photosynthetic complexes in Picea abies

Pinaceae are the predominant photosynthetic species in boreal forests, but so far no detailed description of the protein components of the photosynthetic apparatus of these gymnosperms has been available. In this study we report a detailed characterization of the thylakoid photosynthetic machinery of Norway spruce (Picea abies (L.) Karst). We first customized a spruce thylakoid protein database from translated transcript sequences combined with existing protein sequences derived from gene models, which enabled reliable tandem mass spectrometry identification of P. abies thylakoid proteins from two-dimensional large pore blue-native/SDS-PAGE. This allowed a direct comparison of the two-dimensional protein map of thylakoid protein complexes from P. abies with the model angiosperm Arabidopsis thaliana. Although the subunit composition of P. abies core PSI and PSII complexes is largely similar to that of Arabidopsis, there was a high abundance of a smaller PSI subcomplex, closely resembling the assembly intermediate PSI* complex. In addition, the evolutionary distribution of light-harvesting complex (LHC) family members of Pinaceae was compared in silico with other land plants, revealing that P. abies and other Pinaceae (also Gnetaceae and Welwitschiaceae) have lost LHCB4, but retained LHCB8 (formerly called LHCB4.3). The findings reported here show the composition of the photosynthetic apparatus of P. abies and other Pinaceae members to be unique among land plants

DataSheet_1_The Arabidopsis thylakoid chloride channel ClCe regulates ATP availability for light-harvesting complex II protein phosphorylation.pdf

Coping with changes in light intensity is challenging for plants, but well-designed mechanisms allow them to acclimate to most unpredicted situations. The thylakoid K+/H+ antiporter KEA3 and the voltage-dependent Cl− channel VCCN1 play important roles in light acclimation by fine-tuning electron transport and photoprotection. Good evidence exists that the thylakoid Cl− channel ClCe is involved in the regulation of photosynthesis and state transitions in conditions of low light. However, a detailed mechanistic understanding of this effect is lacking. Here we report that the ClCe loss-of-function in Arabidopsis thaliana results in lower levels of phosphorylated light-harvesting complex II (LHCII) proteins as well as lower levels of the photosystem I-LHCII complexes relative to wild type (WT) in low light conditions. The phosphorylation of the photosystem II core D1/D2 proteins was less affected either in low or high light conditions. In low light conditions, the steady-state levels of ATP synthase conductivity and of the total proton flux available for ATP synthesis were lower in ClCe loss-of-function mutants, but comparable to WT at standard and high light intensity. As a long-term acclimation strategy, expression of the ClCe gene was upregulated in WT plants grown in light-limiting conditions, but not in WT plants grown in standard light even when exposed for up to 8 h to low light. Taken together, these results suggest a role of ClCe in the regulation of the ATP synthase activity which under low light conditions impacts LHCII protein phosphorylation and state transitions.</p