Aging can be experimentally induced in Hydra oligactis, making it an exception among the "immortal" cnidarian genus Hydra. In response to cold temperatures, H. oligactis polyps switch from asexual to "emergency" sexual reproduction and eventually age and die. We used GC-MS-based metabolite profiling to characterize cold-induced (CI) metabolic reprogramming during concurrent sexual differentiation and aging in H. oligactis. Metabolites in four clusters either decreased or increased progressively until week 8, when animals were severely aged, whereas others peaked at sexual maturity after 4-6 weeks. At week 4, signatures of failing cytoprotection and neurotransmitter function appeared in both sexes, including a drastic reduction of taurine, whose deficiency is a known driver of aging in other organisms. Taurine supplementation partly reversed interstitial stem cell loss during the first 2 weeks of cold induction and subsequent sexual differentiation. Metabolites in the NAD+ synthesis and salvage pathways, such as picolinate and niacin, also declined. Changes in pyruvate and TCA cycle intermediates indicated increased energy demands associated with both gametogenesis and aging. Urate, the final oxidation product of purine metabolism, showed the highest fold-change, peaking at sexual maturity in both sexes and remaining elevated. The abundance of most purine and pyrimidine bases also increased, in line with nucleotide cleavage by apoptosis, as part of gametogenesis and aging. Sexual maturity was clearly reflected by high levels of polyamines, such as putrescine, cadaverine, and 3-hydroxybutyrate, required for spermatogenesis. Our study reveals metabolic processes related to gametogenesis and aging and highlights differences between female and male metabolism.

Salicylic acid (SA) is an important plant hormone but is also produced by microorganisms. Contrary to the well-described roles and biosynthetic pathways of SA in plants, its role in fungal physiology and its biosynthesis within fungi remains largely unclear. Here, we sought to investigate the role of SA in the physiology of Trichoderma spp. and to identify fungal genes responsible for SA biosynthesis in Trichoderma virens, while applying and optimizing a transformation approach recently adapted for Trichoderma atroviride. Significant strain- and species-dependent differences in both SA biosynthesis and growth in the presence of exogenous SA were observed. Furthermore, in certain Trichoderma species SA biosynthesis turned out to be induced by the presence of plant volatile organic compounds (VOCs). Based on plant SA biosynthesis pathways, candidate fungal SA biosynthesis genes were screened and respective T. virens gene deletion mutants generated through application and optimization of an enhanced transformation approach. Gene deletion did not result in a decrease in SA biosynthesis, providing evidence that SA biosynthesis in T. virens is distinct from the canonical plant pathways. Although we were not able to identify genes responsible for SA biosynthesis in T. virens, we uncovered how certain Trichoderma and fungal phytopathogen species are affected by SA in their environment and how SA release by Trichoderma spp. can be affected by the presence of a plant host. Furthermore, we were able to optimize an approach to measuring phytohormones produced by Trichoderma spp. in plate culture and proved the applicability of an optimized transformation approach in T. virens.

Aeroterrestrial algae often encounter a semi-hydrated state between full hydration and dehydration, in which the cytoplasm has fluidity but metabolism is impaired, potentially inducing stress. We hypothesised that not all desiccation tolerant (DT) algae tolerate semi-hydration equally, but that longevity in a semi-hydrated state aligns with species habitat. To test this, we monitored photochemical activity between 75-99% relative humidity (RH), as well as resistance to being held at ∼90% RH, 20°C, under a diurnal light cycle, in six DT algae from a range of habitats. Compositions of photosynthetic pigments, tocopherols (lipid antioxidant) and lipid peroxidation-derived reactive carbonyl species were assessed as stress markers after 0 and 3 weeks of semi-hydration. Initial dehydration activated the photoprotective xanthophyll cycle in all species and on average elevated carbonyls 14-fold more in species less tolerant of semi-hydration, while tocopherols accumulated ∼4-fold in more tolerant species. The least DT species, Zygnema circumcarinatum, with primarily an aquatic habit, had barely detectable photochemistry at 95% RH and pigments broke down during desiccation. In contrast, Asterochloris glomerata (aeroterrestrial lichen photobiont) was photochemically active at 87.5% RH and least stressed by semi-hydration. However, even in aeroterrestrial species, cells aged at ∼90% RH, showing that cellular repair was impaired in a semi-hydrated state. Overall, semi-hydration is a vulnerable state, whereby certain algal species have adapted to tolerate it more than others.

Kelp forests are essential marine ecosystems providing high-density habitats, nurseries, and food for countless species, including fish, invertebrates, and mammals. An increasing number of rafting thalli have been observed recently. This phenomenon may represent a natural dispersal strategy of kelp populations, or alternatively, it may be associated with rising ocean temperatures and the increased frequency and intensity of storm events. In the latter case, one can assume that thalli would be degrading during rafting and be associated with declining physiology. With this rationale in mind, we tested the physiological condition and ultrastructure of floating thalli of giant kelp (Macrocystis pyrifera) and featherboa kelp (Egregia menziesii), collected in July 2023 in La Jolla, California, USA. Both species were photosynthetically active with Fv/Fm values > 0.6, maximum relative electron transport rate of PSII (rETRIImax) values between 11 and 16, and total chlorophyll (Chl) contents of 3.0 µmol g− 1 DW. The ultrastructure was typical for Phaeophyceae with a central medulla region and peripheral meristoderm containing chloroplasts, mitochondria and many Golgi bodies indicative of intact and active cells. However, it is not known for how long the kelps were in raft. After exposure to a strong short term experimental UV stress treatment (high UV/PAR ratio), the Chl a and Chl c contents as well as the Chl a/Chl c ratio did not change. However, rETRIImax values decreased by 75

Water loss is a major challenge for photosynthetic organisms. Most are prone to drought stress and only few can tolerate full desiccation. Here, we investigated regulation of photosynthetic electron flow during dehydration and rehydration in Haematococcus lacustris, a desiccation tolerant green alga. During dehydration, non-photochemical quenching (NPQ) increased for dissipating excess light energy, while light-use efficiency of photosystems II (PSII) and I (PSI) decreased. The reaction centre of PSI (P700) became electron-limited at its donor side, helping form photoprotective P700+. Inhibiting alternative oxidases with octyl gallate delayed chlorophyll fluorescence quenching, indicating that plastid terminal oxidases (PTOX) supported formation of NPQ during desiccation. Reduction rates of P700+ during a saturating pulse were slower if cells dehydrated slower, showing that photoprotection was upregulated during desiccation acclimation. During rehydration, octyl gallate and diphenyleneiodonium (DPI), a flavoenzyme inhibitor, slowed oxidation of P700 under actinic light, indicating PTOX and flavodiiron proteins (FLV) were involved in maintaining P700+. A similar response occurred with the protonophore nigericin. We conclude that beyond preventing over-reduction of the electron transport chain, PTOX and FLV facilitated thylakoid luminal acidification under low water stress, protecting photosystems via NPQ, photosynthetic control and P700+ formation.

Improper seed storage conditions, such as elevated temperature and moisture, accelerate ageing and compromise seed quality. However, the impacts of ageing on the seed microbiome and the resulting consequences for germination performance remain poorly understood. Here, we characterised how ageing soybean (Glycine max) seeds at 45 °C and 75% RH affects seed fungal communities, metabolism relevant to microbial growth, and sensitivity of germination to fungal challenge. Additionally, we assessed a role for endophytic bacteria in controlling pathogenic fungi. Amplicon sequencing revealed that ageing decreased fungal richness and reshaped community structure and composition of dominant taxa in seeds. Fusarium and Rhizopus isolates inhibited germination of non-aged seeds, whereas Sarocladium, Plectosphaerella, and Cladosporium impaired germination of aged seeds only. During imbibition, ageing increased seed metabolite leakage, including pinitol, glucose, and fructose, which promoted fungal growth in vitro. Among 39 endophytic bacteria previously isolated from soybean seeds, Bacillus toyonensis C55 and B. pumilus AM26 antagonised fungal growth, consistent with genomic regions associated with antifungal activity. Ageing oxidised the seed cellular redox state, and fungi tolerated oxidative growth conditions better than bacteria. In two cultivars, seed inoculation with B. toyonensis C55 increased germination, supporting a role in regulating fungal infections, whereas B. pumilus AM26 impaired germination. Notably, neither Bacillus strain affected germination of high-vigour non-aged seeds. Fluorescence in situ hybridisation microscopy revealed that both strains recolonised the seed endosphere following ageing. We conclude that oxidation during seed ageing contributes to increased sensitivity to fungal pathogens, which can be modulated by certain bacteria.

Rapidly changing light intensity is a natural challenge that photosynthetic organisms can tolerate. Regulatory mechanisms of light harvesting and alternative electron pathways are critical in dissipating and distributing energy under fluctuating light intensities (FL), but less is known about downstream metabolic regulations. Here, we compared the cellular responses of Chlamydomonas reinhardtii grown under FL to cells acclimated to constant high (HL) or low light (LL), either under high (2 %) or low (0.04 %) CO2. Under low CO2, the physiology of FL cells resembled HL cells and proteomics revealed an induction of the ATP consuming carbon-concentrating mechanism, and photorespiration particularly under FL. High CO2 promoted growth under FL, albeit by a lesser extent than under HL and led to higher ATP contents than under low CO2. To fuel ATP production under low CO2, cells upregulated mitochondrial respiration under FL, while enhanced cyclic electron flow and redox shuttling between intracellular compartments was most evident under FL and LL. Chloroplastic carbon metabolism rapidly responded to light changes, independent of CO2 availability, whereas metabolites associated with mitochondrial bioenergetics responded slower, and remained abundant under high CO2. The accumulation of enzymes involved in starch synthesis and breakdown under FL, together with the transient accumulation of hexoses and hexose phosphates, indicated that cells relied on sugars as a transient carbon pool to meet changing metabolic demands under FL. We conclude that the interplay between light intensity and CO₂ availability drives critical energy trade-offs, balancing photoprotection, repair, and carbon allocation, that regulate growth under FL. ### Competing Interest Statement The authors have declared no competing interest. Austrian Research Promotion Agency, https://ror.org/028jc0449 Oroboros Instruments (Austria), https://ror.org/02d84sx83

Light and inorganic carbon (Ci) drive photosynthesis, which fuels cellular maintenance, energy storage, and growth in photosynthetic organisms. Despite its pivotal role, how primary metabolism adjusts to contrasting light and Ci availability in algae remains elusive. Here, we characterized bioenergetics and profiled primary metabolites of photoautotrophic Chlamydomonas reinhardtii cultures grown under constant low/sub-saturating (LL) or high/saturating (HL) light with 2% (CO2) or ambient 0.04% (Amb) CO2. HL-Amb cells suffered photoinhibition and limitation of photosystem I electron flow at the donor side, but not the acceptor side, indicating use of alternative electron pathways to fuel ATP synthesis. Further, more glycolate was excreted under HL-Amb, indicative of photorespiration. In contrast, HL-CO2 cells upregulated the cytochrome b6f complex, ascorbate metabolism, and PTOX2 for maintaining plastid redox homeostasis. Enhanced glycerol excretion under HL enabled dissipation of excess reducing equivalents to adjust the cellular energy balance. CO2-enhanced photosynthesis promoted respiration and primary metabolite accumulation, driving faster growth while promoting nitrogen (N) metabolism. Hence, Ci-dependent photoacclimation influenced the interplay between the TCA cycle and N assimilation, as supported by proteomic data. Overall, abundant Ci supported growth by promoting electron flow for Ci assimilation, which supplied C skeletons for N assimilation while mitigating photorespiration and photoinhibition.

The trace gas carbonyl sulfide (COS) is used for estimating gross primary productivity at ecosystem level (GPP), as the net CO₂ flux is confounded by ecosystem respiration. Laboratory measurements studying the ratio of the deposition velocities of COS and CO2 at leaf level, i.e. leaf relative uptake rate (LRU), are required for calculating GPP. Under optimal conditions, the LRU has been suggested to be relatively constant. However, stress factors may affect the LRU and even lead to COS emission, which contradicts the prevailing scientific consensus. This study investigated the effect of drought on LRU in three C3 species, rapeseed, soybean and tobacco, and the C4 plant, amaranth.Our results revealed species-specific responses, with the LRU decreasing in C3 plants and increasing primarily in the C4 species under drought. We observed net COS emissions in soybean and rapeseed during drought and for the latter also under unstressed conditions. These emissions suggest bidirectional COS exchange, likely interfering with the unidirectional COS uptake concept underlying LRU even during net COS uptake.In all C3 species, drought induced an increase in leaf cysteine, supporting a cysteine-related COS emission pathway. However, in amaranth cysteine levels decreased in contrast to the COS flux, and were not the highest in rapeseed despite elevated COS emission, altogether showing that factors involved in COS flux require further investigation.Overall, our findings challenge the use of COS as a universal tracer for GPP and underscore the need for further research into COS emissions and LRU variability across species, particularly under environmental stress.

Several aquatic top predators suffer from deficiency in vitamin B1 (thiamine), sometimes combined with low levels of carotenoid pigments, e.g., astaxanthin. The mechanisms leading to correlations between carotenoid pigmentation and thiamine status are not known. These substances and their precursors are produced by single-celled organisms and transferred to higher trophic levels via zooplankton. However, little is known about the factors regulating this transfer process and how it is affected by environmental stressors and zooplankton diet. We therefore exposed a common copepod, Temora longicornis, to ultraviolet radiation (UVR), which is an important environmental stressor, and to food items of different quality in terms of carotenoid profile. Astaxanthin was the most abundant carotenoid found in copepods. Its concentrations were negatively affected by UVR regardless of diet type, and the availability of an astaxanthin precursor (β-carotene) in the diet did not affect the response. Thiamine, on the other hand, showed a varying response, with elevated levels in copepods exposed to UVR at low β-carotene diet and lower levels in copepods exposed to UVR and high β-carotene diet. Altogether, this indicates that astaxanthin was consumed for photoprotection in the zooplankton and that thiamine dynamics might be modulated by UVR under certain dietary conditions. Hence, the concentrations of astaxanthin and thiamine in copepods are dynamic and to some extent regulated by exposure to UVR. Thus, the ability of zooplankton to transfer these substances to higher trophic levels depends, to some extent, on the exposure to environmental stressors.

Regulation of photosynthetic electron transfer is fundamental for energetic efficiency and coping with changing environmental factors. All plant groups, except angiosperms, use flavodiiron proteins (FDPs) on the acceptor side of photosystem I (PSI), putatively protecting from PSI photoinhibition under fluctuating light. An alternative electron flow during photosynthesis is the direct reduction of O2 2 with consequential production of reactive oxygen species (ROS; the Mehler reaction), but to what extent FDPs prevent this is unknown. Here, we quantified O2 2-dependent electron flow, photosystem activity and the Mehler reaction in Chlamydomonas reinhardtii. . Near- infra red absorbance measurement of PSI reaction centre (P700) showed that FDPs remain active long after a dark-to-light transition and their activity increased under hyperoxia. Light-induced hydrogen peroxide (H2O2) 2 O 2 ) production, as a marker of the Mehler reaction, was influenced by O2 2 concentration, and was up to 67 % higher in an FDP-deficient mutant ( flvb ) than in the wild-type under saturating constant light. In cultures kept under sub-saturating constant light, flvb produced 315 % more H2O2 2 O 2 and had lower PSII efficiency than wild-type. Inhibiting electron transfer out of photosystem II (PSII) with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) only partially blocked H2O2 2 O 2 production, particularly under hyperoxia, indicating that PSII was an additional ROS source. P700+ + reduction in the dark in the presence of DCMU was faster in flvb than wild-type, revealing enhanced cyclic electron flow, which may also have led to PSI mediated Mehler reaction. We conclude that FDPs remain active in constant light and can prevent PSI mediated Mehler reaction, with relevance to PSII photoinhibition.

During photosynthesis, reactive oxygen species (ROS) are formed, including hydrogen peroxide (H2O2) and singlet oxygen (1O2), which have putative roles in signalling, but their involvement in photosynthetic acclimation is unclear. Due to extreme reactivity and a short lifetime, 1O2 signalling occurs via its reaction products, such as oxidised poly-unsaturated fatty acids in thylakoid membranes. The resulting lipid peroxides decay to various aldehydes and reactive electrophile species (RES). Here, we investigated the role of ROS in the signal transduction of high light (HL), focusing on GreenCut2 genes unique to photosynthetic organisms. Using RNA seq. data, the transcriptional responses of Chlamydomonas reinhardtii to 2 h HL were compared with responses under low light to exogenous RES (acrolein; 4-hydroxynonenal), β-cyclocitral, a β-carotene oxidation product, as well as Rose Bengal, a 1O2-producing photosensitiser, and H2O2. HL induced significant (p < 0.05) up- and down-regulation of 108 and 23 GreenCut2 genes, respectively. Of all HL up-regulated genes, over half were also up-regulated by RES, including RBCS1 (ribulose bisphosphate carboxylase small subunit), NPQ-related PSBS1 and LHCSR1. Furthermore, 96% of the genes down-regulated by HL were also down-regulated by 1O2 or RES, including CAO1 (chlorophyllide-a oxygnease), MDH2 (NADP-malate dehydrogenase) and PGM4 (phosphoglycerate mutase) for glycolysis. In comparison, only 0–4% of HL-affected GreenCut2 genes were similarly affected by H2O2 or β-cyclocitral. Overall, 1O2 plays a significant role in signalling during the initial acclimation of C. reinhardtii to HL by up-regulating photo-protection and carbon assimilation and down-regulating specific primary metabolic pathways. Our data support that this pathway involves RES.

Zygnematophyceae, a class of freshwater green algae, exhibit distinctive seasonal dynamics. The increasing frequency of cold snaps during the growing season might challenge the persistence of some populations. The present study explored the frost hardiness of two Mougeotia species isolated from different elevations and habitats. Additionally, a phylogenetic (rbcL sequence), ultrastructural and physiological characterization was performed. Both species, grown under standard culture conditions and cold acclimated cultures (+4 degrees C), were exposed to freezing temperatures down to -9 degrees C. Furthermore, ultrastructural-, hydrogen peroxide (H2O2)- and photosynthetic pigment analysis were performed on cells exposed to -2 degrees C, with and without induced ice nucleation. The alpine M. disjuncta showed a higher frost hardiness (LT50 = -5.8 degrees C), whereas the lowland M. scalaris was susceptible to ice. However, frost hardiness did not improve after cold acclimation in either species but rather decreased significantly in M. disjuncta (LT50 = -4.7 degrees C). Despite darkness, prolonged sub-zero temperatures or freezing induced the activation of the xanthophyll (VAZ) cycle in M. scalaris. Our results demonstrate that frost hardiness varies within the genus Mougeotia and that the VAZ cycle can be activated in the dark under subzero temperature- and freezing stress but does not necessarily increase frost hardiness. As highly frost hardy cell types are usually formed at the end of the growing season, the ability of young cells to survive ice formation in the upper subzero temperature range represents a crucial survival strategy in populations exposed to late spring frosts.

Antimicrobial photodynamic therapy (aPDT) is an evolving treatment strategy against human pathogenic microbes such as the Candida species, including the emerging pathogen C. auris . Using a modified EUCAST protocol, the light-enhanced antifungal activity of the natural compound parietin was explored. The photoactivity was evaluated against three separate strains of five yeasts, and its molecular mode of action was analysed via several techniques, i.e., cellular uptake, reactive electrophilic species (RES), and singlet oxygen yield. Under experimental conditions ( lambda =428nm, H=30J/cm (2) , PI=30min), microbial growth was inhibited by more than 90% at parietin concentrations as low as c=0.156mg/L (0.55 mu M) for C. tropicalis and Cryptococcus neoformans , c=0.313mg/L (1.10 mu M) for C. auris , c=0.625mg/L (2.20 mu M) for C. glabrata , and c=1.250mg/L (4.40 mu M) for C. albicans . Mode-of-action analysis demonstrated fungicidal activity. Parietin targets the cell membrane and induces cell death via ROS-mediated lipid peroxidation after light irradiation. In summary, parietin exhibits light-enhanced fungicidal activity against all Candida species tested (including C. auris ) and Cryptococcus neoformans , covering three of the four critical threats on the WHOs most recent fungal priority list.

Identifying traits that exhibit improved drought resistance is highly important to cope with the challenges of predicted climate change. We investigated the response of state transition mutants to drought. Compared with the wild type, state transition mutants were less affected by drought. Photosynthetic parameters in leaves probed by chlorophyll fluorescence confirmed that mutants possess a more reduced plastoquinone (PQ) pool, as expected due to the absence of state transitions. Seedlings of the mutants showed an enhanced growth of the primary root and more lateral root formation. The photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, leading to an oxidised PQ pool, inhibited primary root growth in wild type and mutants, while the cytochrome b6 f complex inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone, leading to a reduced PQ pool, stimulated root growth. A more reduced state of the PQ pool was associated with a slight but significant increase in singlet oxygen production. Singlet oxygen may trigger a, yet unknown, signalling cascade promoting root growth. We propose that photosynthetic mutants with a deregulated ratio of photosystem II to photosystem I activity can provide a novel path for improving crop drought resistance.

Heat and drought stresses are increasingly relevant topics in the context of climate change, particularly in the Alps, which are warming faster than the global average. Previously, we have shown that alpine plants, including Primula minima, can be gradually heat hardened under field conditions in situ to achieve maximum tolerance within a week. Here, we investigated the antioxidant mechanisms of P. minima leaves that had been heat hardened (H) without or with (H+D) additional drought stress. Lower free-radical scavenging and ascorbate concentrations were found in H and H+D leaves, while concentrations of glutathione disulphide (GSSG) were higher under both treatments without any change in glutathione (GSH) and little change in glutathione reductase activity. In contrast, ascorbate peroxidase activity in H leaves was increased, and H+D leaves had >two-fold higher catalase, ascorbate peroxidase and glucose-6-phosphate dehydrogenase activities compared with the control. In addition, the glutathione reductase activity was higher in H+D compared with H leaves. Our results highlight that the stress load from heat acclimation to maximum tolerance is associated with a weakened low-molecular-weight antioxidant defence, which may be compensated for by an increased activity of antioxidant enzymes, particularly under drought conditions.