Biology subjects

Aro, E.-M.

Publications and source records attributed to Aro, E.-M..

16 recordsLinked to original sources

bioRxiv2026

Overexpression of flavodiiron protein Flv3 in engineered Synechocystis stimulates sucrose production and growth by altering cellular redox balance through enhanced sulfur metabolism

Biotechnological applications of oxygenic photosynthetic organisms depend on conversion of light energy into chemical energy through photosystems (PS). This energy can then be used to drive engineered metabolic pathways that are designed as strong electron sinks. For optimal performance, the engineered host metabolism must also be balanced with the native photoprotective electron transfer network. This includes the energy-consuming function of flavodiiron (Flv) proteins, which are universal to cyanobacteria and all other oxygenic photosynthetic organisms except angiosperms. In the cyanobacterium Synechocystis sp. PCC 6803, four different Flv proteins have been shown to function in a Mehler-like reaction within two heterodimeric forms (Flv1/Flv3 and Flv2/Flv4), donating electrons to O2 without generating oxidative stress. Previously, deleting Flv3 in the Synechocystis sucrose-producing (S02) strain was shown to cause drastic metabolic changes in S02{Delta}flv3, shifting it from photoautotrophic to mixotrophic growth (Muth-Pawlak, et al., 2024). In this study, we took an opposite approach by complementing S02 with Flv3 overexpression at different levels using RBS tuning. Interestingly, this resulted in S02oeFlv3 strains with significantly increased overall photosynthetic activity and sucrose production, enhanced cell growth, and storage compound accumulation. However, these outcomes are shown not to be due to conventional O2 photoreduction activity catalysed by Flv1/Flv3. Instead, we postulate that the observed changes are linked to the previously unidentified function of homomeric Flv3/Flv3 and the strongly increased sulphate redox metabolism. Based on extensive proteomic and metabolite analyses, we hypothesise that the Flv3 homooligomer uses sulfate metabolites directly or indirectly as the final electron acceptor instead of O2. This would also explain the upregulation of sulfate-related enzymes, as well as SQR, which passes the electrons back to the PQ pool in the Flv3 overexpression strain.

biochemistry↗

bioRxiv2026

Optimizing photoautotrophic production of soluble 3-hydroxybutyrate in Synechocystis sp. PCC 6803 through combinatorial translational tuning

While photosynthetic cyanobacteria are potential biotechnological hosts for light-driven production of sustainable chemicals from CO2, engineering more efficient strains is critical for the development of competitive industrial processes. This study demonstrates significantly enhanced production of the soluble bioplastic precursor (R)-3-hydroxybutyrate (3HB) that has been engineered based on the polyhydroxybutyrate (PHB) pathway in photoautotrophic cyanobacterium Synechocystis sp PCC 6803. As the key novelty, we generated a library of engineered 3HB pathway variants that express the three key heterologous pathway enzymes PhaA, PhaB and TesB at varying efficiencies, followed by the screening of most efficient 3HB producers. This was achieved by placing each of the pathway enzymes under the translational regulation of three alternative RBSs in different combinations, resulting in strains with wide dynamic range of 3HB productivities. The best strains accumulated over 5 gl-1 under 200 mol photons m-2s-1 and 3% CO2 in a 14-day flask batch culture, with the highest titer reaching 12 gl-1, corresponding to nearly 3 gl-1 d-1 during the peak production phase. These are the highest 3HB production levels reported so far in cyanobacteria, and comparable to those previously established in heterotrophic production systems. Proteomic comparison of selected strains revealed that the different RBS combinations result in varying expression patterns of the pathway proteins, and that the strain-specific enzyme levels remained relatively constant over the monitored six-day period. The results show that altering the levels of the target pathway enzymes can dramatically improve product yield in Synechocystis, while even very small quantitative differences in the strain-specific expression profiles can have marked effects on the production efficiency. This could be used as a general tool for optimizing engineered pathways in cyanobacteria, provided that the flux to the end-product is not critically restricted by substrate availability but rather determined by the balance between the consecutive pathway steps.

synthetic biology↗

bioRxiv2025

The EGY1-SGR1 module controls chloroplast development and senescence by modulating photosynthetic functions

The chloroplast plays essential roles not only in production of biochemical energy and metabolism, but also in plant development, including senescence. We describe a mutant with an unusual progression of senescence and identify it as an allele of ethylene-dependent gravitropism-deficient and yellow-green 1 (egy1-5). EGY1 encodes a chloroplast-localized metalloprotease that regulates chlorophyll metabolism. The single egy1-5 mutant exhibits defects in various aspects of chloroplast function, including abnormal accumulation of photosynthetic complexes, defects in photosynthetic electron transfer chain and altered responses to the chemicals methyl viologen and lincomycin. Additionally, egy1 lacks guard cell chloroplasts. We performed a suppressor mutant screen in the egy1 background to identify new regulators of senescence and chloroplast development. We found two suppressors that restored green leaves and mapping revealed that both suppressors had mutations in STAY-GREEN 1 (SGR1). SGR1 encodes the Mg2+ dechelatase enzyme, which catalyses the first step in chlorophyll breakdown. Several defects in the egy1 mutant were either fully or partially suppressed in egy1 sgr1. This enabled the function of EGY1 to be isolated, independent of the pleiotropic effects of chlorophyll degradation. Our findings suggest that chlorophyll homeostasis regulates the overall development and function of the chloroplast as well as plant senescence. HighlightIdentifying new mutants with defects in chlorophyll metabolism and chloroplast development shows that proper chlorophyll homeostasis is necessary for maintaining functional chloroplasts.

plant biology↗

bioRxiv2024

Sustained non-photochemical quenching and regulation of PSII repair cycle during combined low temperature and high light stress in lettuce

In nature, light and other environmental conditions are constantly changing, requiring plants to have several overlapping regulatory mechanisms to keep light reactions and metabolism in balance. Here, we show that high light (HL) induces a much stronger down-regulation of light reactions when lettuce plants are exposed to 1500 {micro}mol photons m-2 s-1 for 4 h at 13{degrees}C (low temperature, LT) compared to 23{degrees}C (growth temperature, GT). GT/HL treatment induced non-photochemical quenching (NPQ), which relaxed during 1 h recovery in darkness. In contrast, LT/HL treatment induced an exceptionally high NPQ that only partially relaxed during 1 h in darkness at GT. Such a high sustained NPQ (sNPQ) cannot be explained by canonical NPQ mechanism(s). Instead, sNPQ was associated with partial disassembly of PSII-LHCII complexes and a transient increase in phosphorylation of the minor antenna proteins LHCB4.1/LHCB4.2. This coincided with increased expression of the light-harvesting-like proteins SEP2 and ELIP1.2, the PSII assembly proteins HCF173 and LPA3, and accumulation of the pre-D1 protein. These results lead us to propose that LHCB4.1/LHCB4.2 phosphorylation- dependent disassembly of PSII-LHCII supercomplexes allows SEP2 to bind to CP47 and hypothetically quenches the inner PSII core antenna, while free CP43 released during PSII repair is proposed to be protected by LPA3.

plant biology↗

bioRxiv2024

Jasmonic acid signaling and glutathione coordinate plant recovery from high light stress

High light-induced chloroplast retrograde signaling originates from the photosynthetic apparatus and regulates nuclear gene expression to enhance photoprotection and coordination of cell metabolism. Here, we analyzed the transcript profiles and accumulation of ROS, stress hormones and small molecule antioxidants to investigate the signaling mechanisms operating under HL stress, and in particular during plant recovery under growth conditions. Exposure to high light for 15 min induced a number of 1O2- and H2O2-responsive genes and accumulation of an oxidative form of glutathione and ascorbate, the hallmarks of oxidative stress in cells. Prolonged exposure to high light resulted in accumulation of transcripts encoding oxylipin biosynthesis enzymes, leading to accumulation of 12-oxo-phytodienoic acid and jasmonic acid. However, the expression of several jasmonic acid -responsive genes, already induced by HL, peaked during the recovery together with accumulation of jasmonic acid, reduced glutathione and ascorbate, highlighting the critical role of jasmonic acid signaling in restoring chloroplast redox balance following high light stress. The involvement of jasmonic acid signaling in recovery-sustained gene expression was further confirmed by conducting experiments with jasmonic acid receptor mutants. High light exposure of only two min was sufficient to induce some recovery-sustained genes, indicating rapid response of plants to changing light conditions. We propose that ROS production at high light induces the signaling cascade for early oxylipin biosynthesis and 12-oxo-phytodienoic acid accumulation, while increased accumulation of jasmonic acid in recovery-phase activates the genes that fully restore the glutathione metabolism, and ultimately allow recovery from short-term high light stress.

plant biology↗

bioRxiv2021

Flv3A facilitates O2 photoreduction and affects H2 photoproduction independently of Flv1A in diazotrophic Anabaena filaments

The model heterocyst-forming filamentous cyanobacterium, Anabaena sp. PCC 7120 (Anabaena) represents multicellular organisms capable of simultaneously performing oxygenic photosynthesis in vegetative cells and the O2-sensitive N2-fixation inside the heterocysts. The flavodiiron proteins (FDPs) have been shown to participate in photoprotection of photosynthesis by driving excess electrons to O2 (Mehler-like reaction). Here, we addressed the physiological relevance of the vegetative cell-specific Flv1A and Flv3A on the bioenergetic processes occurring in diazotrophic Anabaena under variable CO2. We demonstrate that both Flv1A and Flv3A are required for proper induction of the Mehler-like reaction upon a sudden increase in light intensity, which is likely important for the activation of carbon-concentrating mechanisms (CCM) and CO2 fixation. Under ambient CO2 diazotrophic conditions, Flv3A is capable of mediating moderate O2 photoreduction, independently of Flv1A, but in coordination with Flv2 and Flv4. Strikingly, the lack of Flv3A resulted in strong downregulation of the heterocyst-specific uptake hydrogenase, which led to enhanced H2 photoproduction under both oxic and micro-oxic conditions. These results reveal a novel regulatory network between the Mehler-like reaction and the diazotrophic metabolism, which is of great interest for future biotechnological applications.

plant biology↗

bioRxiv2021

Recording true oxygen reduction capacity during photosynthetic electron transfer in Arabidopsis thylakoids and intact leaves

Reactive oxygen species (ROS) are generated in electron transport processes of living organisms in oxygenic environments. Chloroplasts are plant bioenergetics hubs where imbalances between photosynthetic inputs and outputs drive ROS generation upon changing environmental conditions. Plants have harnessed various site-specific thylakoid membrane ROS products into environmental sensory signals. Our current understanding of ROS production in thylakoids suggests that oxygen (O2) reduction takes place at numerous components of the photosynthetic electron transfer chain (PETC). To refine models of site- specific O2 reduction capacity of various PETC components in isolated thylakoids, the stoichiometry of oxygen production and consumption reactions, associated with H2O2 accumulation, was quantified using membrane inlet mass spectrometry and specific inhibitors. Combined with P700 spectroscopy and electron paramagnetic resonance spin trapping, we demonstrate that electron flow to PSI is essential for H2O2 accumulation during light-induced photosynthetic electron transport process. Further leaf disc measurements provided clues that H2O2 from PETC has a potential of increasing mitochondrial respiration and CO2 release. One sentence summaryPhotosynthetically derived H2O2 only accumulates at Photosystem I and may trigger cooperation with mitochondria during stress

plant biology↗

bioRxiv2019

A commonly used photosynthetic inhibitor fails to block electron flow to photosystem I in intact systems

In plant science, 2,4-dinitrophenylether of iodonitrothymol (DNP-INT) is frequently used as an alternative to 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone (DBMIB) to examine the capacity of plastoquinol and semiquinone to reduce O2. DNP-INT is considered an effective inhibitor of the photosynthetic electron transfer chain (PETC) through its binding at the Q0 site of Cyt-b6f. The binding and action of DNP-INT has been previously characterized spectroscopically in purified Cyt-b6f complex reconstituted with Plastocyanin, PSII membranes and plastoquinone, as well as in isolated thylakoids based on its property to block MV-mediated O2 consumption. Contrary to the conclusions made from these experiments, we observed clear reduction of P700+ in samples incubated with DNP-INT during our recent investigation into the sites of oxygen consumption in isolated thylakoids. Therefore, we carried out an extensive investigation of DNP-INTs chemical efficacy in isolated thylakoids and intact leaves. This included examination of its capacity to block the PETC before PSI, and therefore its inhibition of CO2 fixation. P700 redox kinetics were measured using Dual-PAM whilst Membrane Inlet Mass Spectrometry (MIMS) was used for simultaneous determination of the rates of O2 evolution and O2 consumption in isolated thylakoids and CO2 fixation in intact leaves, using two stable isotopes of oxygen (16O2,18O2) and CO2 (12C,13C), respectively. Based on these investigations we confirmed that DNP-INT is unable to completely block the PETC and CO2 fixation, therefore its use may produce artefacts if applied to isolated thylakoids or intact cells, especially when determining the locations of reactive oxygen species formation in the photosynthetic apparatus.

plant biology↗

bioRxiv2019

Growth under high light and elevated temperature affects metabolic responses and accumulation of health-promoting metabolites in kale varieties

Plants are highly sensitive to changes in the light environment and respond to alternating light conditions by coordinated adjustments in foliar gene expression and metabolism. Here we assessed how long-term growth under high irradiance and elevated temperature, a scenario increasingly associated with the climate change, affects foliar chemical composition of Brassicaceous plants. Transcript profiling of Arabidopsis suggested up-regulation of phenylpropanoid metabolism and down-regulation of processes related to biotic stress resistance and indole glucosinolates (GSL). These observations prompted metabolite profiling of purple (Black Magic) and pale green (Half Tall) varieties of kale, an economically important crop species. Long-term acclimation to high light and elevated temperature resulted in reduced levels of 4-methoxy-indol-3-yl-methyl GSL in both kale varieties. The total levels of aliphatic GSLs increased under these conditions, although the profiles of individual GSL structures showed cultivar-dependent differences. Black Magic became rich in 4-methylsulfinylbutyl GSL and 2-phenylethyl GSL, which have health-promoting effects in human diet. Additionally, the purple pigmentation of Black Magic became intensified due to increased accumulation anthocyanins, especially derivatives of cyanidin. These findings demonstrate that the potentially stressful combination of high light and elevated temperature can have beneficial effects on the accumulation of health-promoting metabolites in leafy vegetables.

plant biology↗

bioRxiv2019

GUN1 influences the accumulation of NEP-dependent transcripts and chloroplast protein import in Arabidopsis cotyledons upon perturbation of chloroplast protein homeostasis

Correct chloroplast development and function require coordinated expression of chloroplast and nuclear genes. This is achieved through chloroplast signals that modulate nuclear gene expression in accordance with the chloroplasts needs. Genetic evidence indicates that GUN1, a chloroplast-localized pentatricopeptide-repeat (PPR) protein with a C-terminal Small MutS-Related (SMR) domain, is involved in integrating multiple developmental and stress-related signals in both young seedlings and adult leaves. Recently, GUN1 was found to interact physically with factors involved in chloroplast protein homeostasis, and with enzymes of tetrapyrrole biosynthesis in adult leaves that function in various retrograde signaling pathways. Here we show that, following perturbation of chloroplast protein homeostasis i) by growth in lincomycin-containing medium, or ii) in mutants defective in either the FtsH protease complex (ftsh), plastid ribosome activity (prps21-1 and prpl11-1) or plastid protein import and folding (cphsp70-1), GUN1 influences NEP-dependent transcript accumulation during cotyledon greening and also intervenes in chloroplast protein import.

plant biology↗

bioRxiv2019

Increased expression of mitochondrial dysfunction stimulon genes affects chloroplast redox status and photosynthetic electron transfer in Arabidopsis

Mitochondrial retrograde signals control expression of nuclear mitochondrial dysfunction stimulon (MDS) genes. Although MDS gene products mostly affect mitochondrial functions, they also influence production of reactive oxygen species (ROS) and redox status of chloroplasts. To study this inter-organellar interaction, we analysed the response of the Arabidopsis MDS-overexpressor mutant rcd1 to methyl viologen (MV), which catalyses electron transfer from Photosystem I (PSI) to molecular oxygen, generating ROS in Mehlers reaction. The response of plants to MV was investigated by imaging chlorophyll fluorescence in aerobic and hypoxic environments, and by membrane inlet mass spectrometry. Hypoxic treatment abolished the effect of MV on photosynthetic electron transfer in rcd1, but not in wild type. A similar reaction to hypoxia was observed in other MDS-activating lines and treatments. This suggests that MDS gene products contribute to oxygen depletion at the PSI electron-acceptor side. In unstressed growth conditions this MDS-related effect is likely masked by endogenous oxygen evolution and gas exchange with the atmosphere. In rcd1, altered Mehlers reaction coincided with more reduced state of the chloroplast NADPH-thioredoxin oxidoreductase C (NTRC) and its targets, suggesting that NTRC performs feedback control of photosynthesis. This regulation may represent a novel mechanism whereby mitochondrial retrograde signalling affects chloroplast functions.

plant biology↗

bioRxiv2019

A novel Ca2+-binding protein influences photosynthetic electron transport in Anabaena sp. PCC 7120

Ca2+ is a potent signalling molecule that regulates many cellular processes. In cyanobacteria, Ca2+ has been linked to cell growth, stress response and photosynthesis, and to the development of specialist heterocyst cells in certain nitrogen-fixing species. Despite this, the pathways of calcium signal transduction in cyanobacteria are poorly understood, and only a few protein components are known. The current study describes a previously unreported calcium-binding protein which was called the Calcium Sensor EF-hand (CSE), which is conserved in filamentous, nitrogen-fixing cyanobacteria. CSE is shown to bind calcium, which induces a conformational change in the protein structure. Poor growth of a strain of Anabaena sp. PCC 7120 overexpressing CSE was attributed to diminished photosynthetic performance. Transcriptomics, biophysics and proteomics analyses revealed modifications in the light-harvesting phycobilisome and photosynthetic reaction center-protein complexes, and downregulated respiration.

plant biology↗

bioRxiv2019

Flv1-4 proteins function in versatile combinations in O2 photoreduction in cyanobacteria

Flavodiiron proteins (FDPs) constitute a group of modular enzymes widespread in all life Domains. Synechocystis sp. PCC 6803 has four FDPs (Flv1-4) essential for photoprotection of photosynthesis. A direct comparison of the Mehler-like reaction (O2 photoreduction) in high Ci (3% CO2, HC) and low Ci (air level CO2, LC) acclimated cells demonstrated that the Flv1/Flv3 heterodimer is responsible for an efficient steady-state O2 photoreduction under HC, with flv2 and flv4 expression strongly down-regulated. Conversely, under LC conditions Flv1/Flv3 acts only as a transient electron sink due to competing withdrawal of electrons by the highly induced NDH-1 complex. Further, in vivo evidence is provided indicating that Flv2/Flv4 contributes to the Mehler-like reaction when naturally expressed under LC conditions, or when artificially overexpressed under HC. The O2 photoreduction driven by Flv2/Flv4 occurs down-stream of PSI in a coordinated manner with Flv1/Flv3 and supports slow and steady-state O2 photoreduction.

biochemistry↗

bioRxiv2019

A Genome-Wide Association Study of Non-Photochemical Quenching in response to local seasonal climates in Arabidopsis thaliana

Field-grown plants have variable exposure to sunlight as a result of shifting cloud-cover, seasonal changes, canopy shading, and other environmental factors. As a result, they need to have developed a method for dissipating excess energy obtained from periodic excessive sunlight exposure. Non-photochemical quenching (NPQ) dissipates excess energy as heat, however the physical and molecular genetic mechanics of NPQ variation are not understood. In this study, we investigated the genetic loci involved in NPQ by first growing different Arabidopsis thaliana accessions in local and seasonal climate conditions, then measured their NPQ kinetics through development by chlorophyll fluorescence. We used genome-wide association studies (GWAS) to identify 15 significant quantitative trait loci (QTL) for a range of photosynthetic traits, including a QTL co-located with known NPQ gene PSBS (AT1G44575). We found there were large alternative regulatory segments between the PSBS promoter regions of the functional haplotypes and a significant difference in PsbS protein concentration. These findings parallel studies in rice showing recurrent regulatory evolution of this gene. The variation in the PSBS promoter and the changes underlying other QTLs could give insight to allow manipulations of NPQ in crops to improve their photosynthetic efficiency and yield. B.P. & J.B. conceived the project; B.P., J.B., P.W. and T.R. designed the research plan and analysis; P.W. supervised the experiments; T.R. performed most of the experiments and analysis; P.G., T.S., A.A. & E.A. designed and undertook experimental design, experiments and analysis for Figure 4; R.C. did the GWAS analysis; P.W., T.R. & A.A. wrote the article with contributions of all the authors. O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/539379v1_fig4.gif" ALT="Figure 4"> View larger version (57K): org.highwire.dtl.DTLVardef@12ba2f9org.highwire.dtl.DTLVardef@1d88c15org.highwire.dtl.DTLVardef@1d4f21eorg.highwire.dtl.DTLVardef@16396aa_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 4:C_FLOATNO (A-D) Coverage tracks of the PSBS genomic regions from five low NPQ (A and B) and five high NPQ (C and D) Arabidopsis accessions aligned with the TAIR 10 Col-0 reference genome (low NPQ accession; A and C) and KBS-Mac-74 genome (high NPQ accession; B and D). Values along x-axes indicate the base pair distance relative to the PSBS transcription start site. Genes along the track are coloured green and the intergenic region is coloured pink. (E) Graphical view of the alignment of the TAIR 10 Col-0 and the KBS-Mac-74 PSBS genomic regions. Axis values refer to base pair positions within the respective tracks. (F) Comparison of the average relative PsbS protein abundance between low and high NPQ accessions. Error bars represent standard deviations. N=30; *** P < 0.001 with paired Students T test. C_FIG

plant biology↗

bioRxiv2018

RCD1 Coordinates Chloroplastic and Mitochondrial Electron Transfer through Interaction with ANAC Transcription Factors

Signaling from chloroplasts and mitochondria, both dependent on reactive oxygen species (ROS), merge at the nuclear protein RADICAL-INDUCED CELL DEATH1 (RCD1). ROS produced in the chloroplasts affect the abundance, thiol redox state and oligomerization of RCD1. RCD1 directly interacts in vivo with ANAC013 and ANAC017 transcription factors, which are the mediators of the ROS-related mitochondrial complex III retrograde signa and suppresses activity of ANAC013 and ANAC017. Inactivation of RCD1 leads to increased expression of ANAC013 and ANAC017-regulated genes belonging to the mitochondrial dysfunction stimulon (MDS), including genes for mitochondrial alternative oxidases (AOXs). Accumulating AOXs and other MDS gene products alter electron transfer pathways in the chloroplasts, leading to diminished production of chloroplastic ROS and increased protection of photosynthetic apparatus from ROS damage. RCD1-dependent regulation affects chloroplastic and mitochondrial retrograde signaling including chloroplast signaling by 3-phosphoadenosine 5-phosphate (PAP). Sensitivity of RCD1 to organellar ROS provides feedback control of nuclear gene expression.

plant biology↗

bioRxiv2018

Regulation of chloroplast NADH dehydrogenase-like complex by NADPH-dependent thioredoxin system

Linear electron transport in the thylakoid membrane drives both photosynthetic NADPH and ATP production, while cyclic electron flow (CEF) around photosystem I only promotes the translocation of protons from stroma to thylakoid lumen. The chloroplast NADH-dehydrogenase-like complex (NDH) participates in one CEF route transferring electrons from ferredoxin back to the plastoquinone pool with concomitant proton pumping to the lumen. CEF has been proposed to balance the ratio of ATP/NADPH production and to control the redox poise particularly in fluctuating light conditions, but the mechanisms regulating the NDH complex remain unknown. We have investigated potential regulation of the CEF pathways by the chloroplast NADPH-thioredoxin reductase (NTRC) in vivo by using an Arabidopsis knockout line of NTRC as well as lines overexpressing NTRC. Here we present biochemical and biophysical evidence showing that NTRC activates the NDH-dependent CEF and regulates the generation of proton motive force, thylakoid conductivity to protons and redox balance between the thylakoid electron transfer chain and the stroma during changes in light conditions. Further, protein-protein interaction assays suggest a putative thioredoxin-target site in close proximity to the ferredoxin binding domain of NDH, thus providing a plausible mechanism for regulation of the NDH ferredoxin:plastoquinone oxidoreductase activity by NTRC.\n\nOne sentence summaryChloroplast thioredoxins regulate photosynthetic cyclic electron flow that balances the activities of light and carbon fixation reactions and improves plant fitness under fluctuating light conditions.

plant biology↗