Non-photochemical quenching (NPQ) is an important photoprotective process in plants, but all molecular details of the process(es) involved are yet not understood. We have used advanced spectroscopic techniques (including simultaneous time- and spectrally-resolved room-temperature chlorophyll fluorescence analysis and spectro-kinetic deconvolution) to analyse the processes in Arabidopsis, hybrid aspen and Scots pine plants. We used four well-characterized Arabidopsis lines (npq1, npq2, npq4 and L17) affected in NPQ, together with hybrid aspen lines with corresponding modifications that we generated. The data are described best by a model for NPQ induction with up to five fluorescence components representing distinct biochemical entities. A dominant fluorescing species at the end of NPQ induction was identified as functionally detached and quenched LHCII but most importantly we believe that one represents a "PSII-PSI (Photosystem II-Photosystem I) complex" where direct energy transfer between PSII and PSI (spillover) take place. This provides strong quenching in all three plant species. We suggest a new integrated model for NPQ in higher plants where spillover is a major element and suggest roles for PsbS and zeaxanthin. Moreover, we discuss the link between NPQ and thylakoid rearrangements as thylakoid destacking facilitates direct contact between PSII and PSI; a prerequisite for spillover. ### Competing Interest Statement Alfred R. Holzwarth is a cofounder of ChloroSpec B.V.

Chlorophyll fluorescence is a ubiquitous tool in basic and applied plant science research. Various standard commercial instruments are available for characterization of photosynthetic material like leaves or microalgae, most of which integrate the overall fluorescence signals above a certain cut-off wavelength. However, wavelength-resolved (fluorescence signals appearing at different wavelengths having different time dependent decay) signals contain vast information required to decompose complex signals and processes into their underlying components that can untangle the photo-physiological process of photosynthesis. Hence, to address this we describe an advanced chlorophyll fluorescence spectrometer - ChloroSpec - allowing three-dimensional simultaneous detection of fluorescence intensities at different wavelengths in a time-resolved manner. We demonstrate for a variety of typical examples that most of the generally used fluorescence parameters are strongly wavelength dependent. This indicates a pronounced heterogeneity and a highly dynamic nature of the thylakoid and the photosynthetic apparatus under actinic illumination. Furthermore, we provide examples of advanced global analysis procedures integrating this three-dimensional signal and relevant information extracted from them that relate to the physiological properties of the organism. This conveniently obtained broad range of data can make ChloroSpec a new standard tool in photosynthesis research.

Boreal conifers – the ‘Christmas trees’ – maintain their green needles over the winter by retaining their chlorophyll. These conifers face the toughest challenge in February and March, when subzero temperatures coincide with high solar radiation. To balance the light energy they harvest with the light energy they utilise, conifers deploy various mechanisms in parallel. These include, thylakoid destacking, which facilitates direct energy transfer from Photosystem II (PSII) to Photosystem I (PSI), and excess energy dissipation through sustained nonphotochemical quenching (NPQ). Additionally, they upregulate alternative electron transport pathways to safely reroute excess electrons while maintaining ATP production. From an evolutionary and ecological perspective, we consider these mechanisms as part of a comprehensive photosynthetic alteration, which enhances our understanding of winter acclimation in conifers and their dominance in the boreal forests.

Plants have a protective mechanism called non-photochemical quenching to prevent damage caused by excessive sunlight. A critical component of this mechanism is energy-dependent quenching (qE). In Chlamydomonas reinhardtii, the protein expression called light-harvesting complex stress-related protein 3 (LHCSR3) is crucial for the qE mechanism. LHCSR3 expression is observed in various conditions that result in photooxidation, such as exposure to high light or nutrient deprivation, where the amount of captured light surpasses the maximum photosynthetic capacity. Although the role of LHCSR3 has been extensively studied under high light (HL) conditions, its function during nutrient starvation remains unclear. In this study, we demonstrate that LHCSR3 expression can occur under light intensities below saturation without triggering qE, particularly when nutrients are limited. To investigate this, we cultivated C. reinhardtii cells under osmotic stress, which replicates conditions of nutrient scarcity. Furthermore, we examined the photosynthetic membrane complexes of wild-type (WT) and npq4 mutant strains grown under osmotic stress. Our analysis revealed that LHCSR3 expression might modify the interaction between the photosystem II core and its peripheral light-harvesting complex II antennae. This alteration could potentially impede the transfer of excitation energy from the antenna to the reaction center.

Light-harvesting complex stress-related protein 3 (LHCSR3) expression is observed in various protoxidizing conditions like high light and nutrient starvation. LHCSR3 expression is essential for energy-dependent quenching (qE), whereas its role under nutrient starvation is elusive. It is also unclear how nutrient starvation can induce LHCSR3 expression under subsaturating light intensities. To study the role of LHCSR3 under nutrient starvation, the C. reinhardtii cells are grown under osmotic stress that would prevent water uptake; therefore same holds true for soluble nutrients in the medium. In this work, we have shown that LHCSR3 expression can occur under osmotic stress and subsaturating light intensities, whereas it does not elicit qE. Further examination of thylakoid membrane architecture from wild-type and npq4 mutant grown under nutrient starvation revealed that LHCSR3 expression affects the interaction between the PSII core with its peripheral LHCII antenna and possibly can prevent excitation energy transfer. Thylakoid lumen acidification is essential for the expression and function of LHCSR3. Under saturating light intensities, this is achieved by the increased rate of photosynthetic electron flow coupled with proton translocation into the thylakoid lumen. Whereas, under nutrient starvation, the reports of LHCSR3 expression also showed reduced photosynthetic electron flow. Therefore, an alternative mechanism should exist for developing the proton gradient. We observed the downregulation of chloroplast (cp) ATP synthase activity and its abundance under osmotic stress, suggesting the role of (cp) ATP synthase in thylakoid lumen acidification under reduced photosynthetic electron flow. This observation is supported by the expression of LHCSR3 in (cp) ATP-synthase mutant atpF upon exposure to moderate light intensity. This study proposes that the mechanism of LHCSR3 expression and its functionality can vary with the type of photooxidizing stress.

Deciduous trees exhibit a spectacular phenomenon of autumn senescence driven by the seasonality of their growth environment, yet there is no consensus which external or internal cues trigger it. Senescence starts at different times in European aspen (Populus tremula L.) genotypes grown in same location. By integrating omics studies, we demonstrate that aspen genotypes utilize similar transcriptional cascades and metabolic cues to initiate senescence, but at different times during autumn. The timing of autumn senescence initiation appeared to be controlled by two consecutive "switches"; 1) first the environmental variation induced the rewiring of the transcriptional network, stress signalling pathways and metabolic perturbations and 2) the start of senescence process was defined by the ability of the genotype to activate and sustain stress tolerance mechanisms mediated by salicylic acid. We propose that salicylic acid represses the onset of leaf senescence in stressful natural conditions, rather than promoting it as often observed in annual plants.

Green organisms evolve oxygen (O2) via photosynthesis and consume it by respiration. Generally, net O2 consumption only becomes dominant when photosynthesis is suppressed at night. Here, we show that green thylakoid membranes of Scots pine (Pinus sylvestris L) and Norway spruce (Picea abies) needles display strong O2 consumption even in the presence of light when extremely low temperatures coincide with high solar irradiation during early spring (ES). By employing different electron transport chain inhibitors, we show that this unusual light-induced O2 consumption occurs around photosystem (PS) I and correlates with higher abundance of flavodiiron (Flv) A protein in ES thylakoids. With P700 absorption changes, we demonstrate that electron scavenging from the acceptor-side of PSI via O2 photoreduction is a major alternative pathway in ES. This photoprotection mechanism in vascular plants indicates that conifers have developed an adaptative evolution trajectory for growing in harsh environments.

The 26S proteasome is a conserved multi-subunit machinery in eukaryotes. It selectively degrades ubiquitinated proteins, which in turn provides an efficient molecular mechanism to regulate numerous cellular functions and developmental processes. Here, we studied a new loss-of-function allele of RPN12a, a plant ortholog of the yeast and human structural component of the 19S proteasome RPN12. Combining a set of biochemical and molecular approaches, we confirmed that a rpn12a knock-out had exacerbated 20S and impaired 26S activities. The altered proteasomal activity led to a pleiotropic phenotype affecting both the vegetative growth and reproductive phase of the plant, including a striking repression of leaf senescence associate cell-death. Further investigation demonstrated that RPN12a is involved in the regulation of several conjugates associated with the auxin, cytokinin, ethylene and jasmonic acid homeostasis. Such enhanced aptitude of plant cells for survival in rpn12a contrasts with reports on animals, where 26S proteasome mutants generally show an accelerated cell death phenotype.

European aspen ( Populus tremula L.) undergoes a coordinated senescence program during autumn; however, it is not known what exactly triggers it. To identify the cellular program leading to senescence, we utilized natural variation among Swedish aspen genotypes in a common garden to study senescence timing and the underlying changes in leaf phytohormone and transcriptome profiles. Apart from the patterns of major transcriptional cascade that was similar between the genotypes and closely associated with cytokinin and auxin metabolite levels and gradually decreasing air temperature during autumn, we detected patterns that consistently preceded or coincided with senescence onset in individual genotypes. Another cascade seemed to respond to short-term changes in weather conditions that re-wired the transcriptional network; the up-regulation of genes related to ethylene and abiotic stress, programmed cell death and translation occurred first in the early-senescing genotypes and later in the late one. Network analyses displayed a connection between the two cascades, metabolic stress and immunity responses mediated by salicylic acid (SA)-signalling pathway that was repressed along with SA levels at senescence onset. We propose that autumn senescence in aspen trees is affected by environmental variation that evokes stress and the timing is fine-tuned by their stress tolerance mechanism.One sentence summary Salicylic acid signalling pathway fine-tunes the timing of senescence onset under challenging environmental conditions in autumn.

Fertilization with nitrogen (N)-rich compounds leads to increased growth but may compromise phenology and winter survival of trees in boreal regions. During autumn, N is remobilized from senescing leaves and stored in other parts of the tree to be used in the next growing season. However, the mechanism behind the N fertilization effect on winter survival is not well understood, and it is unclear how N levels or forms modulate autumn senescence. We performed fertilization experiments and showed that treating Populus saplings with inorganic nitrogen resulted in a delay in senescence. In addition, by using precise delivery of solutes into the xylem stream of Populus trees in their natural environment, we found that delay of autumn senescence was dependent on the form of N administered: inorganic N (NO3-) delayed senescence, but amino acids (Arg, Glu, Gln, and Leu) did not. Metabolite profiling of leaves showed that the levels of tricarboxylic acids, arginine catabolites (ammonium, ornithine), glycine, glycine-serine ratio and overall carbon-to-nitrogen (C/N) ratio were affected differently by the way of applying NO3- and Arg treatments. In addition, the onset of senescence did not coincide with soluble sugar accumulation in control trees or in any of the treatments. We propose that different regulation of C and N status through direct molecular signaling of NO3- and/or different allocation of N between tree parts depending on N forms could account for the contrasting effects of NO3- and tested here amino acids (Arg, Glu, Gln, and Leu) on autumn senescence.

Photosynthetic light-harvesting antennae are pigment-binding proteins that perform one of the most fundamental tasks on Earth, capturing light and transferring energy that enables life in our biosphere. Adaptation to different light environments led to the evolution of an astonishing diversity of light-harvesting systems. At the same time, several strategies have been developed to optimize the light energy input into photosynthetic membranes in response to fluctuating conditions. The basic feature of these prompt responses is the dynamic nature of antenna complexes, whose function readily adapts to the light available. High-resolution microscopy and spectroscopic studies on membrane dynamics demonstrate the crosstalk between antennae and other thylakoid membrane components. With the increased understanding of light-harvesting mechanisms and their regulation, efforts are focusing on the development of sustainable processes for effective conversion of sunlight into functional bio-products. The major challenge in this approach lies in the application of fundamental discoveries in light-harvesting systems for the improvement of plant or algal photosynthesis. Here, we underline some of the latest fundamental discoveries on the molecular mechanisms and regulation of light harvesting that can potentially be exploited for the optimization of photosynthesis.

GIGANTEA (GI) genes have a central role in plant development and influence several processes such as light signaling, circadian rhythm and abiotic stress tolerance. Hybrid aspen T89 (Populus tremula x tremuloides) trees with low GI expression through RNAi have a severely compromised growth. In order to study the effect of reduced GI expression on leaf traits with special emphasis on leaf senescence, we grafted GI-RNAi scions onto wild type (WT) rootstocks and managed to restore scions’ growth. The RNAi line had distorted leaf shape and reduced photosynthesis, probably caused by modulating phloem or stomatal function, increased starch accumulation, higher carbon-to-nitrogen (C/N) ratio and a reduced capacity to withstand moderate light stress. GI-RNAi also induced senescence under long day (LD) and moderate light conditions. Furthermore, the GI-RNAi lines were affected in their capacity to respond to “autumn environmental cues” inducing senescence, a type of leaf senescence with characteristics different from senescence induced directly by stress under LD conditions. Whereas Overexpression of GI delayed senescence. The two different effects on leaf senescence were not affected by the expression of FT (Flowering locus T), were “local” – they followed the genotype of the branch independent on the position in the tree – and trees with modified gene expression grown in the field were affected in a similar way as under controlled conditions. Taken together, GI plays a central role to sense the environmental changes during autumn and determine the appropriate timing for leaf senescence in Populus. One sentence summary Leaf senescence is a complex process that is not well understood, but this paper shows that changing the expression of one gene could influence leaf senescence in Populus trees in two separate ways.

Boreal conifers possess a tremendous ability to survive and remain evergreen during harsh winter conditions and resume growth during summer. This is enabled by coordinated regulation of major cellular functions at the level of gene expression, metabolism, and physiology. Here we present a comprehensive characterization of the annual changes in the global transcriptome of Norway spruce (Picea abies) needles as a resource to understand needle development and acclimation processes throughout the year. In young, growing needles (May 15 until June 30), cell walls, organelles, etc., were formed, and this developmental program heavily influenced the transcriptome, explained by over-represented Gene Ontology (GO) categories. Later changes in gene expression were smaller but four phases were recognized: summer (July-August), autumn (September-October), winter (November-February), and spring (March-April), where over-represented GO categories demonstrated how the needles acclimated to the various seasons. Changes in the seasonal global transcriptome profile were accompanied by differential expression of members of the major transcription factor families. We present a tentative model of how cellular activities are regulated over the year in needles of Norway spruce, which demonstrates the value of mining this dataset, accessible in ConGenIE together with advanced visualization tools.

Photosynthesis is the main process by which sunlight is harvested and converted into chemical energy and has been a focal point of fundamental research in plant biology for decades. In higher plants, the process takes place in the thylakoid membranes where the two photosystems (PSI and PSII) are located. In the past few decades, the evolution of biophysical and biochemical techniques allowed detailed studies of the thylakoid organization and the interaction between protein complexes and cofactors. These studies have mainly focused on model plants, such as Arabidopsis, pea, spinach, and tobacco, which are grown in climate chambers even though significant differences between indoor and outdoor growth conditions are present. In this manuscript, we present a new mild-solubilization procedure for use with “fragile” samples such as thylakoids from conifers growing outdoors. Here, the solubilization protocol is optimized with two detergents in two species, namely Norway spruce (Picea abies) and Scots pine (Pinus sylvestris). We have optimized the isolation and characterization of PSI and PSII multimeric mega- and super-complexes in a close-to-native condition by Blue-Native gel electrophoresis. Eventually, our protocol will not only help in the characterization of photosynthetic complexes from conifers but also in understanding winter adaptation.

Autumn senescence in aspen (Populus tremula) is precisely timed every year to relocate nutrients from leaves to storage organs before winter. Here we demonstrate how stem girdling, which leads to the accumulation of photosynthates in the crown, influences senescence. Girdling resulted in an early onset of senescence, but the chlorophyll degradation was slower and nitrogen more efficiently resorbed than during normal autumn senescence. Girdled stems accumulated or retained anthocyanins potentially providing photoprotection in senescing leaves. Girdling of one stem in a clonal stand sharing the same root stock did not affect senescence in the others, showing that the stems were autonomous in this respect. One girdled stem with unusually high chlorophyll and nitrogen contents maintained low carbon-to-nitrogen (C/N) ratio and did not show early senescence or depleted chlorophyll level unlike the other girdled stems suggesting that the responses depended on the genotype or its carbon and nitrogen status. Metabolite analysis highlighted that the tricarboxylic acid (TCA) cycle, salicylic acid pathway, and redox homeostasis are involved in the regulation of girdling-induced senescence. We propose that disrupted sink-source relation and C/N status can provide cues through the TCA cycle and phytohormone signaling to override the phenological control of autumn senescence in the girdled stems.

GIGANTEA (GI) genes have a central role in plant development and influence several processes. Hybrid aspen T89 (Populus tremula x tremuloides) trees with low GI expression engineered through RNAi show severely compromised growth. To study the effect of reduced GI expression on leaf traits with special emphasis on leaf senescence, we grafted GI-RNAi scions onto wild-type rootstocks and successfully restored growth of the scions. The RNAi line had a distorted leaf shape and reduced photosynthesis, probably caused by modulation of phloem or stomatal function, increased starch accumulation, a higher carbon-to-nitrogen ratio, and reduced capacity to withstand moderate light stress. GI-RNAi also induced senescence under long day (LD) and moderate light conditions. Furthermore, the GI-RNAi lines were affected in their capacity to respond to "autumn environmental cues" inducing senescence, a type of leaf senescence that has physiological and biochemical characteristics that differ from those of senescence induced directly by stress under LD conditions. Overexpression of GI delayed senescence under simulated autumn conditions. The two different effects on leaf senescence under LD or simulated autumn conditions were not affected by the expression of FLOWERING LOCUS T. GI expression regulated leaf senescence locally-the phenotype followed the genotype of the branch, independent of its position on the tree-and trees with modified gene expression were affected in a similar way when grown in the field as under controlled conditions. Taken together, GI plays a central role in sensing environmental changes during autumn and determining the appropriate timing for leaf senescence in Populus.

Photosynthesis is the major natural process that can harvest and harness solar energy into chemical energy. Photosynthesis is performed by a vast number of organisms from single cellular bacteria to higher plants and to make the process efficient, all photosynthetic organisms possess a special type of pigment protein complex(es) that is (are) capable of trapping light energy, known as photosynthetic light-harvesting antennae. From an evolutionary point of view, simpler (unicellular) organisms typically have a simple antenna, whereas higher plants possess complex antenna systems. The higher complexity of the antenna systems provides efficient fine tuning of photosynthesis. This relationship between the complexity of the antenna and the increasing complexity of the organism is mainly related to the remarkable acclimation capability of complex organisms under fluctuating environmental conditions. These antenna complexes not only harvest light, but also provide photoprotection under fluctuating light conditions. In this review, the evolution, structure, and function of different antenna complexes, from single cellular organisms to higher plants, are discussed in the context of the ability to acclimate and adapt to cope under fluctuating environmental conditions.

Significance During overwintering, evergreen conifers of boreal forests must cope with freezing temperatures and high light intensities that often occur simultaneously and expose the photosynthetic apparatus to oxidative damage. To mitigate damage, conifers have developed a specific photoprotection mechanism, called sustained nonphotochemical quenching (sustained NPQ). Here we provide evidence for involvement of unique posttranslational phosphorylations of thylakoid proteins in sustained NPQ of spruce. The triply phosphorylated LHCB1 isoform and phospho-PSBS protein appear as prerequisites for development of sustained NPQ that safely dissipate absorbed light energy as heat. While the induction of Lhcb1 triple phosphorylation requires only freezing temperatures, the triggering of sustained NPQ is additionally dependent on light-induced PSBS phosphorylation, likely in concert with limited photoinhibition of photosystem II.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-22013-6