Biology subjects

Hipsch, M.

Publications and source records attributed to Hipsch, M..

8 recordsLinked to original sources

bioRxiv2026

ACHT2 deactivates carbon assimilation during light-dark transition

In chloroplasts, photosynthetic efficiency relies on a delicate balance between reductive and oxidative thiol-based signaling networks. Members of the high-midpoint-potential atypical thioredoxins (Trxs) were shown to oxidize photosynthetic enzymes by channeling reducing equivalents to H2O2 through 2-Cys-Prx activity. However, it remains unclear which atypical-Trx isoforms regulate Calvin-Benson cycle (CBC) inactivation, and whether they possess distinct functional specificities or operate redundantly in vivo. To resolve this, electron transport and carbon assimilation were continuously monitored during physiological dynamic light transitions in CRISPR-generated single, double and triple atypical Trx mutants. Notably, ACHT2 was identified as a primary determinant of CBC inactivation during dark-to-light transitions, as evidenced by the alleviation of redox-mediated bottlenecks in electron flow downstream of Fd and a lower CBC inactivation state in acht2 plants, resembling the phenotype observed in plants lacking 2-Cys Prxs A and B (2cpab). In contrast, plants mutated in ACHT1, ACHT4, or TrxL2 displayed CBC inactivation kinetics comparable to those of the wild type. Furthermore, mutation of ACHT2 did not compromise plant fitness. In contrast, growth retardation was observed in the acht1/acht4 double mutant, suggesting that the severe phenotype of 2cpab does not arise from impaired CBC inactivation, but rather from disruption of oxidative regulation of other metabolic pathways mediated by distinct atypical Trxs. Collectively, these findings reveal a high degree of regulatory specificity within the chloroplast oxidative network and provide a foundation for a deeper understanding of how activation-inactivation cycles contribute to plant adaptation to dynamic light environments.

plant biology↗

bioRxiv2026

Dark-induced inactivation of the carbon assimilation process requires a water water cycle driven oxidative burst

Chloroplast metabolism is constantly fine-tuned by light availability through the perception and transmission of reductive and oxidative signals that activate or deactivate distinct metabolic enzymes. The reducing power originating from the photosynthetic electron transport chain has been shown to fuel the redox regulatory network, linking electron transport to the reductive activation of photosynthetic enzymes. However, the source of the oxidizing equivalents required to reverse photosynthetic enzyme activation and drive them toward an oxidized, inactive state has not yet been experimentally demonstrated. Here, we resolve redox dynamics associated with carbon assimilation inactivation by combining time-resolved redox imaging during the light-to-dark transition (LDT) with gas-exchange-based measurements. Dark-induced inactivation of carbon assimilation proved oxygen-dependent and coincided with an oxygen-dependent oxidative burst triggered during the LDT. This oxidative burst was suppressed under conditions that blocked electron transport to PSI or in plants in which PSI was photoinactivated. Notably, pgr5 and pgrl1ab mutants exhibited attenuated oxidative bursts and suppressed LDT-associated carbon assimilation inactivation, demonstrating that PGR5/PGRL1-dependent activity is required to generate the oxidative burst that drives CBC inactivation during LDT. These results establish a direct mechanistic link between oxygen- and PSI-dependent oxidative bursts and the inhibition of photosynthesis and mark the water-water cycle (WWC) as the primary source of the transient accumulation of oxidative equivalents that drive inactivation of Calvin-Benson cycle enzymes in darkness.

plant biology↗

bioRxiv2024

Quantification of electron transport-related oxidative signals by time and wavelength-resolved redox biosensors and chlorophyll fluorescence

Reductive and oxidative signals transmitted from the photosynthetic electron chain to target proteins through the redox signaling network are key regulators of carbon assimilation and downstream metabolism. However, despite their crucial role in activating and inhibiting photosynthetic activity, their relation to photosynthetic efficiency is hardly quantified due to the methodological gap between traditional spectroscopic approaches for investigating photosynthesis and biochemical analyses used in the redox regulation field. Here, we simultaneously quantified redox signals and photosynthetic activity by exploring time and wavelength-resolved fluorescence spectra that capture biosensor and chlorophyll fluorescence signals. Using a set of potato plants expressing genetically encoded redox biosensors, we demonstrated how reductive and oxidative signals are amplified with elevated light intensities and revealed the tight connection between electron transport rate (ETR) and the generation of peroxiredoxin-related oxidative signals. These results demonstrate how full spectrum analysis can pave the way for the integration of genetically encoded biosensors in photosynthesis research and demonstrate light-dependent activation of inhibitory oxidative signals in major crop plants.

plant biology↗

bioRxiv2024

Whole-Plant Physiological Identification and Quantification of Disease Progression

Visual estimates of plant symptoms are traditionally used to quantify disease severity. Yet, the methodologies used to assess these phenotypes are often subjective and do not allow tracking of disease progression from very early stages. Here, we hypothesized that quantitative analysis of whole-plant physiological vital functions can be used to objectively determine plant health, providing a more sensitive way to detect disease. We studied the tomato wilt that is caused by Fusarium oxysporum f. sp. lycopersici. Physiological performance of infected and non-infected tomato plants was compared using a whole-plant pot-based lysimeter functional-phenotyping system in a semi-environmentally controlled greenhouse. Water-balance traits of the plants were measured continuously and simultaneously in a quantitative manner. Infected plants exhibited early reductions in transpiration and biomass gain, which preceded visual disease symptoms. These changes in transpiration proved to be effective quantitative indicators for assessing both plant susceptibility to infection and virulence of the fungus. Physiological changes linked to fungal outgrowth and toxin release contributed to reduced hydraulic conductance during initial infection stages. The functional-phenotyping method objectively captures early-stage disease progression, advancing plant disease research and management. This approach emphasizes the potential of quantitative whole-plant physiological analysis over traditional visual estimates for understanding and detecting plant diseases.

plant biology↗

bioRxiv2023

Nitrogen assimilation plays a role in balancing the chloroplastic glutathione redox state under high-light conditions

Nitrate reduction and subsequent ammonium assimilation require reducing equivalents directly produced by the photosynthetic electron transport chain. Therefore, it has been suggested that nitrate assimilation provides a valuable sink for excess electrons under high-light (HL) conditions, which protects the photosynthetic apparatus from excessive harmful reactive oxygen species. This work experimentally tested this hypothesis by monitoring photosynthetic efficiency and the chloroplastic glutathione redox state (chl-EGSH) of plant lines with mutated glutamine synthetase 2 (GS2) and ferredoxin-dependent glutamate synthase 1 (GOGAT1), two key enzymes of the nitrogen assimilation pathway. Unlike wild-type (WT) plants, mutant lines incorporated significantly less isotopically-labeled nitrate into amino acids, demonstrating impaired nitrogen assimilation. When nitrate assimilation was compromised, photosystem II (PSII) proved more vulnerable to photodamage, as shown by the low PSII quantum yields recorded in the mutant lines. High temporal resolution monitoring of the redox state of chloroplast-targeted reduction-oxidation sensitive green fluorescent protein 2 (chl-roGFP2), expressed in the background of the mutant lines, enabled assessment of the effect of the nitrate assimilation pathway on the chl-EGSH. Remarkably, while oxidation followed by reduction of chl-roGFP2 was detected in WT plants in response to HL, oxidation values were stable in the mutant lines, suggesting that the relaxation of chl-EGSH after HL-induced oxidation is achieved by diverting excess electrons to the nitrogen assimilation pathway. Together, these findings indicate that the nitrogen assimilation pathway serves as a sustainable energy dissipation route, ensuring efficient photosynthetic activity and fine-tuning redox metabolism under light-saturated conditions.

plant biology↗

bioRxiv2022

Early detection of late blight in potato by whole-plant redox imaging

Late blight caused by the oomycete Phytophthora infestans is a most devastating disease of potatoes (Solanum tuberosum). Its early detection is crucial for suppressing disease spread. Necrotic lesions are normally seen in leaves at 4 dpi (days post inoculation) when colonized cells are dead, but early detection of the initial biotrophic growth stage, when the pathogen feeds on living cells, is challenging. Here, the biotrophic growth phase of P. infestans was detected by whole-plant redox imaging of potato plants expressing chloroplast-targeted reduction-oxidation sensitive green fluorescent protein (chl-roGFP2). Clear spots on potato leaves with a lower chl-roGFP2 oxidation state were detected as early as 2 dpi, before any visual symptoms were recorded. These spots were particularly evident during light-to-dark transitions and reflected mislocalization of chl-roGFP2 outside the chloroplasts, demonstrating perturbation of the chloroplast import system by the pathogen. Image analysis based on machine learning enabled systematic identification and quantification of spots and unbiased classification of infected and uninfected leaves in inoculated plants. Comparing redox to chlorophyll fluorescence imaging showed that infected leaf areas which exhibit mislocalized chl-roGFP2 also showed reduced non-photochemical quenching (NPQ) and enhanced quantum PSII yield ({Phi}PSII) compared to the surrounding leaf areas. The data suggest that mislocalization of chloroplast-targeted proteins is an efficient marker of late blight infection and demonstrate how it can be utilized for nondestructive monitoring of the disease biotrophic stage using whole-plant redox imaging.

plant biology↗

bioRxiv2021

Systematic monitoring of photosynthetically-derived oxidative signals using a 2-Cys peroxiredoxin-based redox probe

Transmission of reductive cues from the photosynthetic electron transport chain to redox-regulated proteins plays a crucial role in activating chloroplast metabolism. However, deciphering the role of their counterbalanced oxidative signals is challenging due to monitoring difficulties. Here, we demonstrate the light-depended redox modification of chloroplast-targeted 2-Cys peroxiredoxins and introduce peroxiredoxin-based biosensors to monitor photosynthetically-derived oxidative signals. By employing a set of genetically encoded biosensors, we show the induction of oxidative signals under habitual light intensities and their inverse relationship with NADPH levels, unraveling the combined activity of reducing and oxidizing signals in fine-tuning chloroplast metabolism. A faster increase in carbon assimilation rates during photosynthesis induction phase was measured in plants deficient in 2-Cys peroxiredoxins compared to wild-type, suggesting the involvement of oxidative signals in attenuating photosynthesis under variable light environments. We suggest that oxidative signals measured by peroxiredoxin-based biosensors reflect the limitation to photosynthesis imposed by the redox regulatory system. One-Sentence SummaryA genetically encoded biosensor unmasked the dominant role of photosynthetically-derived oxidative signals under habitual conditions.

plant biology↗

bioRxiv2020

Sensing stress responses in potato with whole-plant redox imaging

Environmental stresses are among the major factors that limit crop productivity and plant growth. Plant exposure to various abiotic stress, such as drought, cold temperatures, or high light, results in overproduction of reactive oxygen species (ROS). To avoid oxidative damage, critical mechanisms for their detoxification have evolved, consisting of ROS-scavenging enzymes and small antioxidant molecules, such as glutathione (GSH) and ascorbate. Thus, monitoring redox changes with high spatial and temporal resolution is critical for understanding oxidative stress signaling and has the potential to enable early detection of stress responses in crop plants. In this work, potato plants ( Solanum tuberosum) expressing a chloroplast-targeted reduction-oxidation-sensitive green fluorescent protein2 (roGFP2) were generated to report the redox potential of the glutathione (EGSH) in the chloroplast stroma. By applying whole-plant fluorescence imaging, we mapped alteration in the chloroplast EGSH under several stress conditions including, high-light, cold and drought. Extremely high increase in chloroplast EGSH was observed under the combination of high-light and low temperatures, conditions that specifically induce PSI photoinhibition. Intriguingly, whole-plant ratiometric imaging analysis noted a higher reduced state in newly developed as compared to mature leaves, suggesting a graded stress sensitivity as part of the plant strategies for coping with stress conditions. The presented observations suggest that whole-plant redox imaging can serve as a powerful tool for the basic understanding of plant stress responses as well as for applied agricultural research, such as improving phenotyping capabilities in breeding programs and early detection of stress responses in the field.

plant biology↗