Membrane association of active genes organizes the chloroplast nucleoid structure.

DNA is organized into chromatin-like structures that support the maintenance and regulation of genomes. A unique and poorly understood form of DNA organization exists in chloroplasts, which are organelles of endosymbiotic origin responsible for photosynthesis. Chloroplast genomes, together with associated proteins, form membrane-less structures known as nucleoids. The internal arrangement of the nucleoid, molecular mechanisms of DNA organization, and connections between nucleoid structure and gene expression remain mostly unknown. We show that Arabidopsis thaliana chloroplast nucleoids have a unique sequence-specific organization driven by DNA binding to the thylakoid membranes. DNA associated with the membranes has high protein occupancy, has reduced DNA accessibility, and is highly transcribed. In contrast, genes with low levels of transcription are further away from the membranes, have lower protein occupancy, and have higher DNA accessibility. Membrane association of active genes relies on the pattern of transcription and proper chloroplast development. We propose a speculative model that transcription organizes the chloroplast nucleoid into a transcriptionally active membrane-associated core and a less active periphery.

Hydrogen isotope fractionation is controlled by CO2 in coccolithophore lipids.

Hydrogen isotope ratios (δ2H) represent an important natural tracer of metabolic processes, but quantitative models of processes controlling H-fractionation in aquatic photosynthetic organisms are lacking. Here, we elucidate the underlying physiological controls of 2H/1H fractionation in algal lipids by systematically manipulating temperature, light, and CO2(aq) in continuous cultures of the haptophyte Gephyrocapsa oceanica. We analyze the hydrogen isotope fractionation in alkenones (αalkenone), a class of acyl lipids specific to this species and other haptophyte algae. We find a strong decrease in the αalkenone with increasing CO2(aq) and confirm αalkenone correlates with temperature and light. Based on the known biosynthesis pathways, we develop a cellular model of the δ2H of algal acyl lipids to evaluate processes contributing to these controls on fractionation. Simulations show that longer residence times of NADPH in the chloroplast favor a greater exchange of NADPH with 2H-richer intracellular water, increasing αalkenone. Higher chloroplast CO2(aq) and temperature shorten NADPH residence time by enhancing the carbon fixation and lipid synthesis rates. The inverse correlation of αalkenone to CO2(aq) in our cultures suggests that carbon concentrating mechanisms (CCM) do not achieve a constant saturation of CO2 at the Rubisco site, but rather that chloroplast CO2 varies with external CO2(aq). The pervasive inverse correlation of αalkenone with CO2(aq) in the modern and preindustrial ocean also suggests that natural populations may not attain a constant saturation of Rubisco with the CCM. Rather than reconstructing growth water, αalkenone may be a powerful tool to elucidate the carbon limitation of photosynthesis.

Integrated multilevel investigation of photosynthesis revealed the algal response distinction to differentially charged nanoplastics.

Nanoplastics (NPs), especially those with different charges, as one of emerging contaminants pose a threat to aquatic ecosystems. Although differentially charged NPs could induce distinct biological effects, mechanistic understanding of the critical physiological processes of aquatic organisms from an integrated multilevel perspective on aquatic organisms is still uncertain. Herein, multi-effects of differentially charged nanosized polystyrene (nPS) including neutral nPS, nPS-COOH, and nPS-NH2 on the photosynthesis-related physiological processes of algae were explored at the population, individual, subcellular, protein, and transcriptional levels. Results demonstrated that both nPS and nPS-COOH exhibited hormesis to algal photosynthesis but nPS-NH2 triggered severe inhibition. As for nPS-NH2, the integrity of algal subcellular structure, chlorophyll biosynthesis, and expression of photosynthesis-related proteins and genes were interfered. Intracellular NPs' content in nPS treatment was 25.64 % higher than in nPS-COOH treatment, and the content of chloroplasts in PS and nPS-COOH treatment were 3.09 % and 4.56 % higher than control, respectively. Furthermore, at the molecular levels, more photosynthesis-related proteins and genes were regulated under nPS-COOH exposure than those exposed to nPS. Light-harvesting complex II could be recognized as an underlying explanation for different effects between nPS and nPS-COOH. This study first provides a novel approach to assess the ecological risks of NPs at an integrated multilevel.

Temporal dynamics of chloroplast biogenesis revealed initiation of photosynthesis-related gene expression and protein complexes during alfalfa seed germination.

The chloroplast biogenesis occurs in cotyledon during alfalfa seed germination before true leaf formation, and is extremely important for the followed plant development and growth. In this study, we conducted a simulation of alfalfa seed germination in the soil by using tin foil and focused on 10 pivotal time points of chloroplast biogenesis in cotyledons before and after light exposure, which showed significant differences in multispectral images, and covered the whole process of chloroplast biogenesis from proplastid, etioplast to mature chloroplast. We revealed three phases that referred to the programmed involvements of photosynthesis promotion, ultrastructure maturity, transcriptomic expression, and protein complex construction, and observed distinct transcriptional expressions of genes from nuclear and chloroplast genomes. In phase I at dark germination before light exposure, chloroplast-encoded genes showed up-regulated expressions together with the importation of chloroplast proteins. In phase II for the first day after light exposure, nuclear-encoded genes' expressions were initiated at 2 h after light exposure (E2h), followed by swift assembly of chloroplast thylakoid membrane protein complexes, and roaring Fv/Fm and contents of chlorophyll a, chlorophyll b and carotenoid. The initiation at E2h was pronounced by the observation of gradual accumulation of single lamella, and facilitated the formation of granum stacks (thylakoid) at E8h in phase II. In phase III from the second day after light exposure, chloroplast became gradually complete with the fully established photosynthetic capacity. Altogether, our results layed a theoretical foundation for enhancing potential photosynthetic efficiency in alfalfa and related species.

Loss-of-function of PGL10 impairs photosynthesis and tolerance to high-temperature stress in rice.

High temperature (HT) affects the production of chlorophyll (Chl) pigment and inhibits cellular processes that impair photosynthesis, and growth and development in plants. However, the molecular mechanisms underlying heat stress in rice are not fully understood yet. In this study, we identified two mutants varying in leaf color from the ethylmethanesulfonate mutant library of indica rice cv. Zhongjiazao-17, which showed pale-green leaf color and variegated leaf phenotype under HT conditions. Mut-map revealed that both mutants were allelic, and their phenotype was controlled by a single recessive gene PALE GREEN LEAF 10 (PGL10) that encodes NADPH:protochlorophyllide oxidoreductase B, which is required for the reduction of protochlorophyllide into chlorophyllide in light-dependent tetrapyrrole biosynthetic pathway-based Chl synthesis. Overexpression-based complementation and CRISPR/Cas9-based knockout analyses confirmed the results of Mut-map. Moreover, qRT-PCR-based expression analysis of PGL10 showed that it expresses in almost all plant parts with the lowest expression in root, followed by seed, third leaf, and then other green tissues in both mutants, pgl10a and pgl10b. Its protein localizes in chloroplasts, and the first 17 amino acids from N-terminus are responsible for signals in chloroplasts. Moreover, transcriptome analysis performed under HT conditions revealed that the genes involved in the Chl biosynthesis and degradation, photosynthesis, and reactive oxygen species detoxification were differentially expressed in mutants compared to WT. Thus, these results indicate that PGL10 is required for maintaining chloroplast function and plays an important role in rice adaptation to HT stress conditions by controlling photosynthetic activity.

Strategies of Molecular Signal Integration for Optimized Plant Acclimation to Stress Combinations.

Plant growth and survival in their natural environment require versatile mitigation of diverse threats. The task is especially challenging due to the largely unpredictable interaction of countless abiotic and biotic factors. To resist an unfavorable environment, plants have evolved diverse sensing, signaling, and adaptive molecular mechanisms. Recent stress studies have identified molecular elements like secondary messengers (ROS, Ca2+, etc.), hormones (ABA, JA, etc.), and signaling proteins (SnRK, MAPK, etc.). However, major gaps remain in understanding the interaction between these pathways, and in particular under conditions of stress combinations. Here, we highlight the challenge of defining "stress" in such complex natural scenarios. Therefore, defining stress hallmarks for different combinations is crucial. We discuss three examples of robust and dynamic plant acclimation systems, outlining specific plant responses to complex stress overlaps. (a) The high plasticity of root system architecture is a decisive feature in sustainable crop development in times of global climate change. (b) Similarly, broad sensory abilities and apparent control of cellular metabolism under adverse conditions through retrograde signaling make chloroplasts an ideal hub. Functional specificity of the chloroplast-associated molecular patterns (ChAMPs) under combined stresses needs further focus. (c) The molecular integration of several hormonal signaling pathways, which bring together all cellular information to initiate the adaptive changes, needs resolving.

RIP5 Interacts with REL1 and Negatively Regulates Drought Tolerance in Rice.

Improving the drought resistance of rice is of great significance for expanding the planting area and improving the stable yield of rice. In our previous work, we found that ROLLED AND ERECT LEAF1 (REL1) protein promoted enhanced tolerance to drought stress by eliminating reactive oxygen species (ROS) levels and triggering the abscisic acid (ABA) response. However, the mechanism through which REL1 regulates drought tolerance by removing ROS is unclear. In this study, we identified REL1 interacting protein 5 (RIP5) and found that it directly combines with REL1 in the chloroplast. We found that RIP5 was strongly expressed in ZH11 under drought-stress conditions, and that the rip5-ko mutants significantly improved the tolerance of rice plants to drought, whereas overexpression of RIP5 resulted in greater susceptibility to drought. Further investigation suggested that RIP5 negatively regulated drought tolerance in rice by decreasing the content of ascorbic acid (AsA), thereby reducing ROS clearance. RNA sequencing showed that the knockout of RIP5 caused differential gene expression that is chiefly associated with ascorbate and aldarate metabolism. Furthermore, multiple experimental results suggest that REL1 is involved in regulating drought tolerance by inhibiting RIP5. Collectively, our findings reveal the importance of the inhibition of RIP5 by REL1 in affecting the rice's response to drought stress. This work not only explains the drought tolerance mechanism of rice, but will also help to improve the drought tolerance of rice.

Chloroplast envelope K+/H+ antiporters are involved in cytosol pH regulation.

Variations in light intensity induce cytosol pH changes in photosynthetic tissues, providing a possible signal to adjust a variety of biochemical, physiological and developmental processes to the energy status of the cells. It was shown that these pH changes are partially due to the transport of protons in or out of the thylakoid lumen. However, the ion transporters in the chloroplast that transmit these pH changes to the cytosol are not known. KEA1 and KEA2 are K+/H+ antiporters in the chloroplast inner envelope that adjust stromal pH in light-to-dark transitions. We previously determined that stromal pH is higher in kea1kea2 mutant cells. In this study, we now show that KEA1 and KEA2 are required to attenuate cytosol pH variations upon sudden light intensity changes in leaf mesophyll cells, showing they are important components of the light-modulated pH signalling module. The kea1kea2 mutant mesophyll cells also have a considerably less negative membrane potential. Membrane potential is dependent on the activity of the plasma membrane proton ATPase and is regulated by secondary ion transporters, mainly potassium channels in the plasma membrane. We did not find significant differences in the activity of the plasma membrane proton pump but found a strongly increased membrane permeability to protons, especially potassium, of the double mutant plasma membranes. Our results indicate that chloroplast envelope K+/H+ antiporters not only affect chloroplast pH but also have a strong impact on cellular ion homeostasis and energization of the plasma membrane.

Immunofluorescence staining of chloroplast proteins with frozen sections of plant tissues.

KEY MESSAGE: Immunofluorescence staining with frozen sections of plant tissues and a nest tube is convenient and effective, and broadens the applicability of immunofluorescence staining. Immunofluorescence staining is an indispensable and extensively employed technique for determining the subcellular localization of chloroplast division proteins. At present, it is difficult to effectively observe the localization of target proteins in leaves that are hard, or very thin, or have epidermal hair or glands with the current immunofluorescence staining methods. Moreover, signals of target proteins were predominantly detected in mesophyll cells, not the cells of other types. Thus, the method of immunofluorescence staining was further explored for improvement in this study. The plant tissue was embedded with 50% PEG4000 at -60℃, which was then cut into sections by a cryomacrotome. The sections were immediately immersed in fixation solution. Then, the sample was transferred into a special nested plastic tube, which facilitated the fixation and immunofluorescence staining procedures. The use of frozen sections in this method enabled a short processing time and reduced material requirements. By optimizing the thickness of the sections, a large proportion of the cells could be well stained. With this method, we observed the localization of a chloroplast division protein FtsZ1 in the wild-type Arabidopsis and various chloroplast division mutants. Meanwhile, the localization of FtsZ1 was also observed not only in mesophyll cells, but also in guard cells and epidermal cells in a lot of other plant species, including many species with hard leaf tissues. This method is not only easy to use, but also expands the scope of applicability for immunofluorescence staining.

Posttranslational regulation of photosynthetic activity via the TOR kinase in plants.

Chloroplasts are the powerhouse of the plant cell, and their activity must be matched to plant growth to avoid photooxidative damage. We have identified a posttranslational mechanism linking the eukaryotic target of rapamycin (TOR) kinase that promotes growth and the guanosine tetraphosphate (ppGpp) signaling pathway of prokaryotic origins that regulates chloroplast activity and photosynthesis in particular. We find that RelA SpoT homolog 3 (RSH3), a nuclear-encoded enzyme responsible for ppGpp biosynthesis, interacts directly with the TOR complex via a plant-specific amino-terminal region which is phosphorylated in a TOR-dependent manner. Down-regulating TOR activity causes a rapid increase in ppGpp synthesis in RSH3 overexpressors and reduces photosynthetic capacity in an RSH-dependent manner in wild-type plants. The TOR-RSH3 signaling axis therefore regulates the equilibrium between chloroplast activity and plant growth, setting a precedent for the regulation of organellar function by TOR.

Reviving resilience: MEcPP-mediated ASK1-IMPα-9-TRP2 stress-responsive module.

Chloroplasts are not only critical photosynthesis sites in plants, but they also participate in plastidial retrograde signaling in response to developmental and environmental signals. MEcPP (2-C-Methyl-D-erythritol-2,4-cyclopyrophosphate) is an intermediary in the methylerythritol phosphate (MEP) pathway in chloroplasts. It is a critical precursor for the synthesis of isoprenoids and terpenoid derivatives, which play crucial roles in plant growth and development, photosynthesis, reproduction, and defense against environmental constraints. Accumulation of MEcPP under stressful conditions triggers the expression of IMPα-9 and TPR2, contributing to the activation of abiotic stress-responsive genes. In this correspondence, we discuss plastidial retrograde signaling in support of a recently published paper in Molecular Plant (Zeng et al. 2024). We hope that it can shed more insight on the retrograde signaling cascade.

Measurement of Photorespiratory Cycle Enzyme Activities in Leaves Exposed to Abiotic Stress.

Photorespiration, an essential metabolic component, is a classic example of interactions between the intracellular compartments of a plant cell: the chloroplast, peroxisome, mitochondria, and cytoplasm. The photorespiratory pathway is often modulated by abiotic stress and is considered an adaptive response. Monitoring the patterns of key enzymes located in different subcellular components would be an ideal approach to assessing the modulation of the photorespiratory metabolism under abiotic stress. This chapter describes the procedures for assaying several individual enzyme activities of key photorespiratory enzymes and evaluating their response to oxidative/photooxidative stress. It is essential to ascertain the presence of stress in the experimental material. Therefore, procedures for typical abiotic stress induction in leaves by highlighting without or with menadione (an oxidant that targets mitochondria) are also included.

Characterization of the DNA accessibility of chloroplast genomes in grasses.

Although the chloroplast genome (cpDNA) of higher plants is known to exist as a large protein-DNA complex called 'plastid nucleoid', researches on its DNA state and regulatory elements are limited. In this study, we performed the assay for transposase-accessible chromatin sequencing (ATAC-seq) on five common tissues across five grasses, and found that the accessibility of different regions in cpDNA varied widely, with the transcribed regions being highly accessible and accessibility patterns around gene start and end sites varying depending on the level of gene expression. Further analysis identified a total of 3970 putative protein binding footprints on cpDNAs of five grasses. These footprints were enriched in intergenic regions and co-localized with known functional elements. Footprints and their flanking accessibility varied dynamically among tissues. Cross-species analysis showed that footprints in coding regions tended to overlap non-degenerate sites and contain a high proportion of highly conserved sites, indicating that they are subject to evolutionary constraints. Taken together, our results suggest that the accessibility of cpDNA has biological implications and provide new insights into the transcriptional regulation of chloroplasts.

Alternative electron sinks in chloroplasts and mitochondria of halophytes as a safety valve for controlling ROS production during salinity.

Electron flow through the electron transport chain (ETC) is essential for oxidative phosphorylation in mitochondria and photosynthesis in chloroplasts. Electron fluxes depend on environmental parameters, e.g., ionic and osmotic conditions and endogenous factors, and this may cause severe imbalances. Plants have evolved alternative sinks to balance the reductive load on the electron transport chains in order to avoid overreduction, generation of reactive oxygen species (ROS), and to cope with environmental stresses. These sinks act primarily as valves for electron drainage and secondarily as regulators of tolerance-related metabolism, utilizing the excess reductive energy. High salinity is an environmental stressor that stimulates the generation of ROS and oxidative stress, which affects growth and development by disrupting the redox homeostasis of plants. While glycophytic plants are sensitive to high salinity, halophytic plants tolerate, grow, and reproduce at high salinity. Various studies have examined the ETC systems of glycophytic plants, however, information about the state and regulation of ETCs in halophytes under non-saline and saline conditions is scarce. This review focuses on alternative electron sinks in chloroplasts and mitochondria of halophytic plants. In cases where information on halophytes is lacking, we examined the available knowledge on the relationship between alternative sinks and gradual salinity resilience of glycophytes. To this end, transcriptional responses of involved components of photosynthetic and respiratory ETCs were compared between the glycophyte Arabidopsis thaliana and the halophyte Schrenkiella parvula, and the time-courses of these transcripts were examined in A. thaliana. The observed regulatory patterns are discussed in the context of reactive molecular species formation in halophytes and glycophytes.

Production of biologically active human basic fibroblast growth factor (hFGFb) using Nicotiana tabacum transplastomic plants.

MAIN CONCLUSION: We generated transplastomic tobacco lines that stably express a human Basic Fibroblast Growth Factor (hFGFb) in their chloroplasts stroma and purified a biologically active recombinant hFGFb. MAIN: The use of plants as biofactories presents as an attractive technology with the potential to efficiently produce high-value human recombinant proteins in a cost-effective manner. Plastid genome transformation stands out for its possibility to accumulate recombinant proteins at elevated levels. Of particular interest are recombinant growth factors, given their applications in animal cell culture and regenerative medicine. In this study, we produced recombinant human Fibroblast Growth Factor (rhFGFb), a crucial protein required for animal cell culture, in tobacco chloroplasts. We successfully generated two independent transplastomic lines that are homoplasmic and accumulate rhFGFb in their leaves. Furthermore, the produced rhFGFb demonstrated its biological activity by inducing proliferation in HEK293T cell lines. These results collectively underscore plastid genome transformation as a promising plant-based bioreactor for rhFGFb production.

Transcriptomic study of the role of MeFtsZ2-1 in pigment accumulation in cassava leaves.

MeFtsZ2-1 is a key gene for plant plastid division, but the mechanism by which MeFtsZ2-1 affects pigment accumulation in cassava (Manihot esculenta Crantz) through plastids remains unclear. We found that MeFtsZ2-1 overexpression in cassava (OE) exhibited darker colors of leaves, with increased levels of anthocyanins and carotenoids. Further observation via Transmission Electron Microscopy (TEM) revealed no apparent defects in chloroplast structure but an increase in the number of plastoglobule in OE leaves. RNA-seq results showed 1582 differentially expressed genes (DEGs) in leaves of OE. KEGG pathway analysis indicated that these DEGs were enriched in pathways related to flavonoid, anthocyanin, and carotenoid biosynthesis. This study reveals the role of MeFtsZ2-1 in cassava pigment accumulation from a physiological and transcriptomic perspective, providing a theoretical basis for improving cassava quality.

Cutinsomes of Malus Mill. (Rosaceae) leaf and pericarp: genesis, localization, and transport.

New data were obtained on specific bionanostructures, cutinsomes, which are involved in the formation of cuticles on the surface of leaf blades and pericarp of Malus domestica Borkh (Malus Mill., Rosaceae)introduced to the mountains at the altitudes of 1200 and 1700 m above sea level. Cutinsomes, which are electron-dense structures of spherical shape, have been identified by transmission electron microscopy. It was demonstrated that plastids can be involved in the synthesis of their constituent nanocomponents. The greatest number of nanoparticles was observed in the granal thylakoid lumen of the chloroplasts in palisade mesophyll cells and pericarp hypodermal cells. The transmembrane transport of cutinsomes into the cell wall cuticle proper by exocytosis has been visualized for the first time. The plasma membrane is directly involved in the excretion of nanostructures from the cell. Nanoparticles of cutinsomes in the form of necklace-like formations line up in a chain near cell walls, merge into larger conglomerates and are loaded into plasmalemma invaginations, and then, in membrane packing, they move into the cuticle, which covers both outer and inner cell walls of external tissues. The original materials obtained by us supplement the ideas about the non-enzymatic synthesis of cuticle components available in the literature and expand the cell compartment geography involved in this process.

A novel PLS-DYW type PPR protein OsASL is essential for chloroplast development in rice.

Oryza longistaminata (OL), an AA-genome African wild rice which can propagate clonally via rhizome, is an important germplasm for improvement of Asian cultivated rice, however recessive lethal alleles can hitchhike clonal propagation in heterozygous state. Selfing of OL is difficult due to its self-incompatibility, but simple selfing of hybrid progeny between OL and O. sativa is effective to disclose and eliminate recessive lethal alleles. Here, we identified an exhibited albino-lethal phenotype mutant, from an F2 population between OL and O. sativa, named it albino seedling-lethal (asl). The leaves of asl mutant showed abnormal chloroplast development. The albino characteristics of asl were determined to be governed by a set of recessive nuclear genes through genetic analysis. Map-based cloning experiments found that a single nucleotide variation (G to A) was detected in the exon of OsASL in OL, which causes a premature stop codon. OsASL encodes a PLS-type PPR protein with 12 pentratricopeptide repeat domains, and is translocalized to chloroplasts. Complementation and knockout transgenic experiments further confirmed that OsASL is responsible for the albino-lethal phenotype. Loss-of-function OsASL (i.e. osasl) resulted in devoid of intron splicing of chloroplast RNA atpF, ndhA, rpl2 and rps12, and also RNA editing of ndhB, but facilitates the RNA editing of rpl2 in the plastid. Transcriptome sequencing showed that OsASL was mainly involved in chlorophyll synthesis pathway. The expression of Chlorophyll-associated genes were significantly decreased in asl plants, especially PEP (plastid-encoded RNA polymerase)-mediated genes. Our results suggest that OsASL is crucial for RNA editing, RNA splicing of chloroplast RNA group II genes, and plays an essential role in chloroplast development during early leaf development in rice.

The role of the Golden2-like (GLK) transcription factor in regulating terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

KEY MESSAGE: A GLK homologue was identified and functionally characterized in Catharanthus roseus. Silencing CrGLK with VIGS or the chloroplast retrograde signaling inducer lincomycin increased terpenoid indole alkaloid biosynthesis. Catharanthus roseus is the sole source of the chemotherapeutic terpenoid indole alkaloids (TIAs) vinblastine and vincristine. TIA pathway genes, particularly genes in the vindoline pathway, are expressed at higher levels in immature versus mature leaves, but the molecular mechanisms responsible for this developmental regulation are unknown. We investigated the role of GOLDEN2-LIKE (GLK) transcription factors in contributing to this ontogenetic regulation since GLKs are active in seedlings upon light exposure and in the leaf's early development, but their activity is repressed as leaves age and senesce. We identified a GLK homologue in C. roseus and functionally characterized its role in regulating TIA biosynthesis, with a focus on the vindoline pathway, by transiently reducing its expression through two separate methods: virus-induced gene silencing (VIGS) and application of chloroplast retrograde signaling inducers, norflurazon and lincomycin. Reducing CrGLK levels with each method reduced chlorophyll accumulation and the expression of the light harvesting complex subunit (LHCB2.2), confirming its functional homology with GLKs in other plant species. In contrast, reducing CrGLK via VIGS or lincomycin increased TIA accumulation and TIA pathway gene expression, suggesting that CrGLK may repress TIA biosynthesis. However, norflurazon had no effect on TIA gene expression, indicating that reducing CrGLK alone is not sufficient to induce TIA biosynthesis. Future work is needed to clarify the specific molecular mechanisms leading to increased TIA biosynthesis with CrGLK silencing. This is the first identification and characterization of GLK in C. roseus and the first investigation of how chloroplast retrograde signaling might regulate TIA biosynthesis.

Net O2 exchange rates under dark and light conditions across different stem compartments.

Woody plants display some photosynthetic activity in stems, but the biological role of stem photosynthesis and the specific contributions of bark and wood to carbon uptake and oxygen evolution remain poorly understood. We aimed to elucidate the functional characteristics of chloroplasts in stems of different ages in Fraxinus ornus. Our investigation employed diverse experimental approaches, including microsensor technology to assess oxygen production rates in whole stem, bark, and wood separately. Additionally, we utilized fluorescence lifetime imaging microscopy (FLIM) to characterize the relative abundance of photosystems I and II (PSI : PSII chlorophyll ratio) in bark and wood. Our findings revealed light-induced increases in O2 production in whole stem, bark, and wood. We present the radial profile of O2 production in F. ornus stems, demonstrating the capability of stem chloroplasts to perform light-dependent electron transport. Younger stems exhibited higher light-induced O2 production and dark respiration rates than older ones. While bark emerged as the primary contributor to net O2 production under light conditions, our data underscored that wood chloroplasts are also photosynthetically active. The FLIM analysis unveiled a lower PSI abundance in wood than in bark, suggesting stem chloroplasts are not only active but also acclimate to the spectral composition of light reaching inner compartments.