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At least 91 records · Page 5

Dynamic in vivo monitoring of granum structural changes of Ctenanthe setosa (Roscoe) Eichler during drought stress and subsequent recovery

Investigating the effects of drought stress and subsequent recovery on the structure and function of chloroplasts is essential to understanding how plants adapt to environmental stressors. We investigated Ctenanthe setosa (Roscoe) Eichler, an ornamental plant that can tolerate prolonged drought periods (40 and 49 days of water withdrawal). Conventional biochemical, biophysical, physiological and (ultra)structural methods combined for the first time in a higher plant with in vivo small-angle neutron scattering (SANS) were used to characterize the alterations induced by drought stress and subsequent recovery. Upon drought stress, no significant changes occurred in the chloroplast ultrastructure, chlorophyll content, 77K fluorescence emission spectra and maximal quantum efficiency of PSII (Qy dark), but the actual quantum efficiency of PSII (Qy light) decreased, the amounts of PSI-LHCII complexes and PSII monomers declined, and that of PSII supercomplexes increased. Thickness of the leaf and of the adaxial hypodermis, chloroplast length and granum repeat distance (RD) values decreased upon drought stress, as shown by light microscopy and SANS, respectively. Because of the very slight (nm-range) changes in RD values, the large biological variability (significant differences in RD values among the leaves and studied leaf regions) and the invasive sampling required for this method, transmission electron microscopy (TEM) hardly showed significant differences. On the other side, in situ SANS analyses provided a unique insight in vivo into the fast structural recovery of the granum structure of drought-stressed leaves, which happened already 18 h after re-watering, while functional and biochemical recovery took place on a longer time scale.

59 BASIC BIOLOGICAL SCIENCES↗

Proteins associated with the Arabidopsis thaliana plastid rhomboid-like protein RBL10

Rhomboid-like proteins are intramembrane proteases with a variety of regulatory roles in cells. Though many rhomboid-like proteins are predicted in plants, their detailed molecular mechanisms or cellular functions are not yet known. Of the 13 predicted rhomboids in Arabidopsis thaliana, one, RBL10, affects lipid metabolism in the chloroplast, because in the respective rbl10 mutant the transfer of phosphatidic acid through the inner envelope membrane is disrupted. Here we show that RBL10 is part of a high-molecular-weight complex of 250 kDa or greater in size. Nine likely components of this complex are identified by two independent methods and include Acyl Carrier Protein 4 (ACP4) and Carboxyltransferase Interactor1 (CTI1), which have known roles in chloroplast lipid metabolism. The acp4 mutant has decreased C16:3 fatty acid content of monogalactosyldiacylglycerol, similar to the rbl10 mutant, prompting us to offer a mechanistic model of how an interaction between ACP4 and RBL10 might affect chloroplast lipid assembly. We also demonstrate the presence of a seventh transmembrane domain in RBL10, refining the currently accepted topology of this protein. Taken together, the identity of possible RBL10 complex components as well as insights into RBL10 topology and distribution in the membrane provide a stepping-stone towards a deeper understanding of RBL10 function in Arabidopsis lipid metabolism.

59 BASIC BIOLOGICAL SCIENCES↗

Assembly and Repair of Photosystem II in Chlamydomonas reinhardtii

Oxygenic photosynthetic organisms use Photosystem II (PSII) to oxidize water and reduce plastoquinone. Here, we review the mechanisms by which PSII is assembled and turned over in the model green alga Chlamydomonas reinhardtii. This species has been used to make key discoveries in PSII research due to its metabolic flexibility and amenability to genetic approaches. PSII subunits originate from both nuclear and chloroplastic gene products in Chlamydomonas. Nuclear-encoded PSII subunits are transported into the chloroplast and chloroplast-encoded PSII subunits are translated by a coordinated mechanism. Active PSII dimers are built from discrete reaction center complexes in a process facilitated by assembly factors. The phosphorylation of core subunits affects supercomplex formation and localization within the thylakoid network. Proteolysis primarily targets the D1 subunit, which when replaced, allows PSII to be reactivated and completes a repair cycle. While PSII has been extensively studied using Chlamydomonas as a model species, important questions remain about its assembly and repair which are presented here.

59 BASIC BIOLOGICAL SCIENCES↗

Episomal DNA vectors for plant genetic engineering

This disclosure pertains to a novel platform for genetic engineering of chloroplasts. The disclosure provides episomal DNA vectors containing a chloroplast origin of replication. These vectors remain extra-plastomic and sustainably and autonomously replicate in chloroplasts of the plant cells transformed with the vectors and in the plants regenerated from the transformed plant cells. The episomal DNA vectors do not contain any sequence that shares sequence homology with the plastome DNA and, thus, do not get integrated into the plastome DNA. The vectors can also comprise one or more genes of interest that confer desirable characteristics to the transformed plant cells. The disclosure also provides methods of transforming plant cells with the episomal DNA vectors and regenerating from the transformed plant cells plants having desirable characteristics. The vectors and methods disclosed herein provide a significant advancement in speed, flexibility, and prospects of introducing genes into plant cells for effective metabolic engineering.

Lenaghan, Scott C.↗

Stromules, functional extensions of plastids within the plant cell

We report stromules are thin tubular extensions of the plastid compartment surrounded by the envelope membrane. A myriad of functions have been proposed for them, and they likely have multiple roles. Recent work has illuminated aspects of their formation, especially the important of microtubules in their movement and microfilaments in anchoring. A variety of biotic and abiotic stresses result in induction of stromule formation, and in recent years, stromule formation has been strongly implicated as part of the innate immune response. Both stromules and chloroplasts relocate to surround the nucleus when pathogens are sensed, possibly to supply signaling molecules such as reactive oxygen species. In addition to the nucleus, stromules have been observed in close proximity to other compartments such as mitochondria, endoplasmic reticulum, and the plasma membrane, potentially facilitating exchange of substrates and products to carry out important biosynthetic pathways. Much remains to be learned about the identity of proteins and other molecules released from chloroplasts and stromules and how they function in plant development and defense.

60 APPLIED LIFE SCIENCES↗

Acclimation to high and low diurnal light is flexible in Chlamydomonas reinhardtii

Chlamydomonas acclimates to repeated low (LL) or high light (HL) days by changing the abundance of photosynthetic complexes and the ultrastructure of its thylakoid membranes. These phenotypes persist through the night phases, suggesting a readiness for the daylight environment that is routinely experienced despite the intervening dark periods [S. Dupuis et al., Plant Cell 37, koaf086 (2025), 10.1093/plcell/koaf086]. Here, we investigate how prior acclimation impacts algal fitness upon a change in daylight intensity and how quickly Chlamydomonas can reprogram its photoprotective strategy in a diurnal context. We performed a systems analysis of synchronized populations acclimated to diurnal LL when subjected to HL days and of populations acclimated to diurnal HL when subjected to LL days. In the latter case, diurnal photoacclimation decreased fitness during the first day at a new light intensity: HL-acclimated cells barely increased in size over the first LL period, and they failed to complete a cell cycle. However, although LL-acclimated cells showed severe photodamage after 6 h of HL, they recovered chloroplast form and function later that afternoon and successfully divided at nightfall. These cells rapidly altered their thylakoid membrane ultrastructure, increased their photoprotective quenching capacity, and decreased their inventory of photosystem and antenna proteins by the end of the first HL day. Transcriptomic and proteomic analyses revealed rapid induction of thousands of genes, including those encoding proteases, chaperones, and other proteins involved in the chloroplast unfolded protein response. These results show that the alga is highly flexible and competent to rapidly acclimate to changes in diurnal light intensity.

Diel↗

Mechanism of Protein Transport on the Twin Arginine Translocation Pathway

This project was awarded in September 2018 and ran for one year. The overarching goals of this multi-award project was to understand the mechanism and energetics of the Tat protein transport system responsible to the transport of proteins into the chloroplast thylakoid lumen. This pathway is responsible for the delivery of a number of proteins required for chloroplast biogenesis and homeostasis, including subunits of the photosynthetic oxygen-evolving complex and of the three large protein complexes of the photosynthetic electron transport chain. The manner through which proteins are transported on this pathway is highly unusual, and through the research supported by his set of DOE grants are leading to a mechanism of action that is previously unknown for protein trafficking. During the course of this short project we have continued to uncover evidence that the Tat pathway allows proteins to cross membranes through lipid-lined toroidal pores. The experiments undertaken during this project have been continued during the subsequent on-going DOE award period.

59 BASIC BIOLOGICAL SCIENCES↗

Thylakoid Assembly and Folded Protein Transport by the Tat Pathway

Assembly of functional photosystems complete with necessary intrinsic and extrinsic proteins requires the function of at least three protein transport pathways in thylakoid membranes. Our research focuses on one of those pathways, a unique and essential protein transport pathway found in the chloroplasts of plants, bacteria, and some archaebacteria, the Twin arginine translocation (Tat) system. The chloroplast Tat (cpTat) system is thought to be responsible for the proper localization of ~50% of thylakoid lumen proteins, several of which are necessary for proper photosystem assembly, maintenance, and function. For example, two components of the extrinsic oxygen evolving complex, PsbP and PsbQ, are transported to the thylakoid lumen via the cpTat system. PsbP and PsbQ have been shown to be important in photosystem II function and maintenance (Yi et al. 2006; Yi et al. 2007). The current project addresses the issues of what forms the translocation conduit, how the translocon complex is arranged and what role Hcf106 and cpTatC may have in the process of forming the conduit.

59 BASIC BIOLOGICAL SCIENCES↗

Engineering Nannochloropsis oceanica for the production of diterpenoid compounds

Abstract Photosynthetic microalgae like Nannochloropsis hold enormous potential as sustainable, light‐driven biofactories for the production of high‐value natural products such as terpenoids. Nannochloropsis oceanica is distinguished as a particularly robust host with extensive genomic and transgenic resources available. Its capacity to grow in wastewater, brackish, and sea waters, coupled with advances in microalgal metabolic engineering, genome editing, and synthetic biology, provides an excellent opportunity. In the present work, we demonstrate how N. oceanica can be engineered to produce the diterpene casbene—an important intermediate in the biosynthesis of pharmacologically relevant macrocyclic diterpenoids. Casbene accumulated after stably expressing and targeting the casbene synthase from Daphne genkwa (DgTPS1) to the algal chloroplast. The engineered strains yielded production titers of up to 0.12 mg g −1 total dry cell weight (DCW) casbene. Heterologous overexpression and chloroplast targeting of two upstream rate‐limiting enzymes in the 2‐C‐methyl‐ d ‐erythritol 4‐phosphate pathway, Coleus forskohlii 1‐deoxy‐ d ‐xylulose‐5‐phosphate synthase and geranylgeranyl diphosphate synthase genes, further enhanced the yield of casbene to a titer up to 1.80 mg g −1 DCW. The results presented here form a basis for further development and production of complex plant diterpenoids in microalgae.

Du, Zhi‐Yan↗

In vivo localization of iron starvation induced proteins under variable iron supplementation regimes in Phaeodactylum tricornutum

Abstract The model pennate diatom Phaeodactylum tricornutum is able to assimilate a range of iron sources. It therefore provides a platform to study different mechanisms of iron processing concomitantly in the same cell. In this study, we follow the localization of three iron starvation induced proteins (ISIPs) in vivo, driven by their native promoters and tagged by fluorophores in an engineered line of P. tricornutum . We find that the localization patterns of ISIPs are dynamic and variable depending on the overall iron status of the cell and the source of iron it is exposed to. Notwithstanding, a shared destination of the three ISIPs both under ferric iron and siderophore‐bound iron supplementation is a globular compartment in the vicinity of the chloroplast. In a proteomic analysis, we identify that the cell engages endocytosis machinery involved in the vesicular trafficking as a response to siderophore molecules, even when these are not bound to iron. Our results suggest that there may be a direct vesicle traffic connection between the diatom cell membrane and the periplastidial compartment (PPC) that co‐opts clathrin‐mediated endocytosis and the “cytoplasm to vacuole” (Cvt) pathway, for proteins involved in iron assimilation. Proteomics data are available via ProteomeXchange with identifier PXD021172. Highlight The marine diatom P. tricornutum engages a vesicular network to traffic siderophores and phytotransferrin from the cell membrane directly to a putative iron processing site in the vicinity of the chloroplast.

59 BASIC BIOLOGICAL SCIENCES↗

Localization of proteins involved in the biogenesis and repair of the photosynthetic apparatus to thylakoid subdomains in Arabidopsis

Abstract Thylakoid membranes in chloroplasts and cyanobacteria harbor the multisubunit protein complexes that catalyze the light reactions of photosynthesis. In plant chloroplasts, the thylakoid membrane system comprises a highly organized network with several subcompartments that differ in composition and morphology: grana stacks, unstacked stromal lamellae, and grana margins at the interface between stacked and unstacked regions. The localization of components of the photosynthetic apparatus among these subcompartments has been well characterized. However, less is known about the localization of proteins involved in the biogenesis and repair of the photosynthetic apparatus, the partitioning of proteins between two recently resolved components of the traditional margin fraction (refined margins and curvature), and the effects of light on these features. In this study, we analyzed the partitioning of numerous thylakoid biogenesis and repair factors among grana, curvature, refined margin, and stromal lamellae fractions of Arabidopsis thylakoid membranes, comparing the results from illuminated and dark‐adapted plants. Several proteins previously shown to localize to a margin fraction partitioned in varying ways among the resolved curvature and refined margin fractions. For example, the ALB3 insertase and FtsH protease involved in photosystem II (PSII) repair were concentrated in the refined margin fraction, whereas TAT translocon subunits and proteins involved in early steps in photosystem assembly were concentrated in the curvature fraction. By contrast, two photosystem assembly factors that facilitate late assembly steps were depleted from the curvature fraction. The enrichment of the PSII subunit OE23/PsbP in the curvature fraction set it apart from other PSII subunits, supporting the previous conjecture that OE23/PsbP assists in PSII biogenesis and/or repair. The PSII assembly factor PAM68 partitioned differently among thylakoid fractions from dark‐adapted plants and illuminated plants and was the only analyzed protein to convincingly do so. These results demonstrate an unanticipated spatial heterogeneity of photosystem biogenesis and repair functions in thylakoid membranes and reveal the curvature fraction to be a focal point of early photosystem biogenesis.

Chotewutmontri, Prakitchai↗

Delayed leaf greening involves a major shift in the expression of cytosolic and mitochondrial ribosomes to plastid ribosomes in the highly phosphorus-use-efficient Hakea prostrata (Proteaceae)

Abstract Background and aims Hakea prostrata (Proteaceae) is a highly phosphorus-use-efficient plant native to southwest Australia. It maintains a high photosynthetic rate at low leaf phosphorus (P) and exhibits delayed leaf greening, a convergent adaptation that increases nutrient-use efficiency. This study aimed to provide broad physiological and gene expression profiles across leaf development, uncovering pathways leading from young leaves as nutrient sinks to mature leaves as low-nutrient, energy-transducing sources. Methods To explore gene expression underlying delayed greening, we analysed a de novo transcriptome for H. prostrata across five stages of leaf development. Photosynthesis and respiration rates, and foliar pigment, P and nitrogen (N) concentrations were determined, including the division of P into five biochemical fractions. Key results Transcripts encoding functions associated with leaf structure generally decreased in abundance across leaf development, concomitant with decreases in foliar concentrations of 85% for anthocyanins, 90% for P and 70% for N. The expression of genes associated with photosynthetic function increased during or after leaf expansion, in parallel with increases in photosynthetic pigments and activity, much later in leaf development than in species that do not have delayed greening. As leaves developed, transcript abundance for cytosolic and mitochondrial ribosomal proteins generally declined, whilst transcripts for chloroplast ribosomal proteins increased. Conclusions There was a much longer temporal separation of leaf cell growth from chloroplast development in H. prostrata than is found in species that lack delayed greening. Transcriptome-guided analysis of leaf development in H. prostrata provided insight into delayed greening as a nutrient-saving strategy in severely phosphorus-impoverished landscapes.

59 BASIC BIOLOGICAL SCIENCES↗

Marine algae growth and carbon capture augmentation by antioxidant nanozymes

Large scale commercial cultivation of microalgae year-round is limited by seasonal stress conditions. The rapid growth and high CO 2 capture of the marine microalga Picochlorum celeri is largely inhibited under winter stress conditions of low temperature and high light. Herein, we demonstrated a nanotechnology approach to enhance the biomass productivity and CO 2 capture of P. celeri under abiotic stress by interfacing with antioxidant cerium oxide nanozymes (nanoceria). Antioxidant nanoceria catalytically scavenged reactive oxygen species (ROS) generated under stress conditions, reducing damage to the microalgae photosynthetic machinery in chloroplasts. Negatively charged poly-acrylic acid-coated nanoceria (PNC, 10 μM) were biocompatible in microalgae cells and colocalized with chloroplasts. In contrast, positively charged aminated nanoceria (ANC) resulted in microalgae aggregation (>50 μM) and were toxic at all concentrations tested (≥10 μM). PNC reduction of ROS levels in microalgae (78%) and superoxide levels (26%), enhanced microalgae growth (65%), photosynthetic performance (130%), and CO 2 uptake rate (380%) under low-temperature stress (15 °C) and high light (500 μmol/m 2 /s PAR) stress relative to controls without nanoceria. Nanoceria augmentation of microalgae provides a rapid and facile technology to increase algae CO 2 capture and biomass under stress conditions.

09 BIOMASS FUELS↗

Ocean current patterns drive the worldwide colonization of eelgrass (Zostera marina)

Currents are unique drivers of oceanic phylogeography and thus determine the distribution of marine coastal species, along with past glaciations and sea-level changes. Here we reconstruct the worldwide colonization history of eelgrass (Zostera marina L.), the most widely distributed marine flowering plant or seagrass from its origin in the Northwest Pacific, based on nuclear and chloroplast genomes. We identified two divergent Pacific clades with evidence for admixture along the East Pacific coast. Two west-to-east (trans-Pacific) colonization events support the key role of the North Pacific Current. Time-calibrated nuclear and chloroplast phylogenies yielded concordant estimates of the arrival of Z. marina in the Atlantic through the Canadian Arctic, suggesting that eelgrass-based ecosystems, hotspots of biodiversity and carbon sequestration, have only been present there for ~243 ky (thousand years). Mediterranean populations were founded ~44 kya, while extant distributions along western and eastern Atlantic shores were founded at the end of the Last Glacial Maximum (~19 kya), with at least one major refuge being the North Carolina region. The recent colonization and five- to sevenfold lower genomic diversity of the Atlantic compared to the Pacific populations raises concern and opportunity about how Atlantic eelgrass might respond to rapidly warming coastal oceans.

59 BASIC BIOLOGICAL SCIENCES↗

From photosynthetic electron flow to gene regulation: redox signal transduction in cyanobacteria

In cyanobacteria, the free-living ancestors of chloroplasts, photosynthesis simultaneously sustains growth and generates reactive oxygen species (ROS) that damage proteins, lipids, and DNA when light capture outpaces carbon fixation. Maintaining redox balance, therefore, requires cells to read photosynthetic electron flow as a signal that continuously tunes gene expression and protein activity. This review traces how these redox signals are transduced to transcription machinery through three routes: membrane-localized sensors, cytoplasmic redox sensors downstream of photosystem I, and ROS generated when electron sinks are saturated. Membrane-bound histidine kinases (two-component systems) relay the redox state of the plastoquinone pool to control photosystem remodeling, pigment biosynthesis, and circadian timing. Cytoplasmic one-component regulators, by contrast, sense redox directly through thiol-disulfide switches, glutathionylation, iron-sulfur clusters, and metal-catalyzed oxidation to control photosystem-cofactor, electron-carrier, and transition-metal homeostasis. Because many of these regulators persist in algal and plant chloroplasts, cyanobacteria illuminate principles of redox control across photosynthetic eukaryotes. Post-transcriptional and translational control further shapes redox-dependent gene expression programs through transcript stability, ribosome assembly, and translation initiation, extending redox regulation beyond transcription to every step of protein synthesis and even activity modulation. Finally, we connect redox regulation to photosynthetic physiology, stress resilience, and the rational engineering of cyanobacteria for sustainable bioproduction.

59 BASIC BIOLOGICAL SCIENCES↗

Leaf microscopy applications in photosynthesis research: identifying the gaps

Abstract Leaf imaging via microscopy has provided critical insights into research on photosynthesis at multiple junctures, from the early understanding of the role of stomata, through elucidating C4 photosynthesis via Kranz anatomy and chloroplast arrangement in single cells, to detailed explorations of diffusion pathways and light utilization gradients within leaves. In recent decades, the original two-dimensional (2D) explorations have begun to be visualized in three-dimensional (3D) space, revising our understanding of structure–function relationships between internal leaf anatomy and photosynthesis. In particular, advancing new technologies and analyses are providing fresh insight into the relationship between leaf cellular components and improving the ability to model net carbon fixation, water use efficiency, and metabolite turnover rate in leaves. While ground-breaking developments in imaging tools and techniques have expanded our knowledge of leaf 3D structure via high-resolution 3D and time-series images, there is a growing need for more in vivo imaging as well as metabolite imaging. However, these advances necessitate further improvement in microscopy sciences to overcome the unique challenges a green leaf poses. In this review, we discuss the available tools, techniques, challenges, and gaps for efficient in vivo leaf 3D imaging, as well as innovations to overcome these difficulties.

Plant Sciences↗

The HIGH CHLOROPHYLL FLUORESCENCE 244 homolog CrHCF244 is required for psbA (D1) translation in Chlamydomonas reinhardtii

Translation of psbA, the chloroplast gene that encodes the D1 subunit of PSII, is important for both PSII biogenesis and repair. The translation of psbA transcripts in the chloroplast is under the control of nuclear gene products. Using a forward genetic screen and whole-genome sequencing of the alga Chlamydomonas reinhardtii , we found a mutant defective in PSII activity and mapped the causative gene to be the homolog of Arabidopsis HIGH CHLOROPHYLL FLUORESCENCE 244 (HCF244) , namely CrHCF244 . We then demonstrated that CrHCF244 is required for psbA translation in the alga, consistent with the function of HCF244 in Arabidopsis, and found that AtHCF244 also partially complemented the algal mutant. These results experimentally support the functional conservation of the homologs in green algae and land plants. Intriguingly, the CrHCF244 mutant also exhibited a relatively high rate of suppressor mutants, pointing to the presence of alternative factor(s)/pathway(s) for D1 translational control. The establishment of CrHCF244 as a psbA translation factor in C. reinhardti i shows the similarities in psbA translation regulation in algae and plants. The future identification of the alternative factor(s) in this alga will provide insights on psbA translation in plants.

Arabidopsis↗