Singlet oxygen-induced signalling depends on the metabolic status of the Chlamydomonas reinhardtii cell

Youssef, Waeil Al; Feil, Regina; Saint-Sorny, Maureen; Johnson, Xenie; Lunn, John E.; Grimm, Bernhard; Brzezowski, Pawel

doi:10.1038/s42003-023-04872-5

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Published: 16 May 2023

Singlet oxygen-induced signalling depends on the metabolic status of the Chlamydomonas reinhardtii cell

Communications Biology volume 6, Article number: 529 (2023) Cite this article

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Abstract

Using a mutant screen, we identified trehalose 6-phosphate phosphatase 1 (TSPP1) as a functional enzyme dephosphorylating trehalose 6-phosphate (Tre6P) to trehalose in Chlamydomonas reinhardtii. The tspp1 knock-out results in reprogramming of the cell metabolism via altered transcriptome. As a secondary effect, tspp1 also shows impairment in ¹O₂-induced chloroplast retrograde signalling. From transcriptomic analysis and metabolite profiling, we conclude that accumulation or deficiency of certain metabolites directly affect ¹O₂-signalling. ¹O₂-inducible GLUTATHIONE PEROXIDASE 5 (GPX5) gene expression is suppressed by increased content of fumarate and 2-oxoglutarate, intermediates in the tricarboxylic acid cycle (TCA cycle) in mitochondria and dicarboxylate metabolism in the cytosol, but also myo-inositol, involved in inositol phosphate metabolism and phosphatidylinositol signalling system. Application of another TCA cycle intermediate, aconitate, recovers ¹O₂-signalling and GPX5 expression in otherwise aconitate-deficient tspp1. Genes encoding known essential components of chloroplast-to-nucleus ¹O₂-signalling, PSBP2, MBS, and SAK1, show decreased transcript levels in tspp1, which also can be rescued by exogenous application of aconitate. We demonstrate that chloroplast retrograde signalling involving ¹O₂ depends on mitochondrial and cytosolic processes and that the metabolic status of the cell determines the response to ¹O₂.

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Introduction

Nearly 40 years ago, tetrapyrrole biosynthesis (TBS) intermediates were proposed to be involved in chloroplast retrograde (organelle-to-nucleus) signalling. This conclusion was based on the observation that accumulation of chlorophyll precursors negatively affects transcript level of light-harvesting chlorophyll a/b-binding (LHCB) protein¹. Later studies on chloroplast retrograde signalling involved mutants², treatment with a carotenoid biosynthesis inhibitor, norflurazon³, or TBS inhibitors, such as α,α-dipyridyl¹ or thujaplicin⁴. Experiments with norflurazon led to the discovery of the genomes uncoupled (gun) mutants with a common phenotype of chloroplast status-independent expression of photosynthesis-associated nuclear genes³.

It is noteworthy that treatment with inhibitors causing carotenoid deficiencies results in generation of reactive oxygen species (ROS) and photooxidative damage to chloroplasts. Furthermore, the end-products of TBS, heme and chlorophyll, as well as many of their intermediates (Supplementary Fig. 1) are light-absorbing and redox-reactive molecules, capable to generate ROS. Singlet oxygen (¹O₂) can be produced through interaction of ground (triplet)-state oxygen (³O₂) with triplet-state chlorophyll or TBS intermediates, e.g. protoporphyrin IX (Proto), which are excited by light⁵. ROS are also metabolic products of other cellular processes in plants, such as photosynthesis and respiration.

Although ¹O₂ is not considered to be the most reactive oxygen species, it is thought to be the major ROS involved in photooxidative damage⁶. However, it was shown that production of ¹O₂ in the chloroplast induces stress responses that do not result exclusively from physicochemical damage, but also rely on signal transduction triggered by ROS⁷. ¹O₂ has a short half-life (about 200 ns) in the cell⁸ and as a result, the distance that it may move was calculated to be ~10 nm, based on predicted diffusion rates^9,10. Its diffusion range is also limited due to its high reactivity with membrane lipids¹¹. Therefore, ¹O₂ could play a specific role as an activator of a stress response only if it is detected close to its source, which strongly suggests that other components mediating the ¹O₂ signals should exist. Alternatively, altered metabolite contents triggered by ¹O₂ may also mediate ¹O₂-retrograde signalling, or certain metabolic signature may be required to trigger changes in nuclear gene expression^12,13.

Despite the profound effect of ¹O₂ on the chloroplast redox state and its apparent involvement in altering nuclear gene expression, little is known about the components involved in ¹O₂-dependent retrograde signalling. The protein factors identified so far include executer 1 (EX1) and EX2 in the fluorescence (flu) mutant of Arabidopsis thaliana¹⁴, the P-subunit of photosystem II family protein (PSBP2)¹⁵ and singlet oxygen acclimation knockedout 1 (SAK1) in Chlamydomonas reinhardtii¹⁶, or methylene blue sensitivity (MBS1) shown to be involved in ¹O₂-signalling in both A. thaliana and C. reinhardtii¹⁷.

We hypothesized that ¹O₂ generated via the photosensitizing activity of Proto triggers signalling cascades that alter nuclear gene expression. Therefore, we used a C. reinhardtii mutant chlD-1¹⁸ that does not produce chlorophyll and accumulates Proto due to a dysfunctional Mg-chelatase (MgCh, Supplementary Fig. 1), but with introduced over-expression of the gene encoding the genomes uncoupled 4 (GUN4) protein, chlD-1/GUN4¹⁹. Endogenous accumulation of Proto in mutants is advantageous for studying ¹O₂-signalling, because it eliminates the need for exogenous application of TBS or carotenoid biosynthesis inhibitors, as well as photosensitizers, such as rose bengal or neutral red to induce ¹O₂ generation^15,20,21, which are not natural products of the cell, do not localise specifically to any subcellular compartment, and as a consequence may result in artefactual responses. In these terms, the system used in our study is similar to the research conducted on the conditional flu mutant of A. thaliana. However, instead of Proto, etiolated seedlings of flu accumulate another TBS intermediate, protochlorophyllide (Pchlide; Supplementary Fig. 1), which generates ¹O₂ in light²². Nonetheless, it should be noted that neither accumulation of Pchlide in A. thaliana nor accumulation of Proto in C. reinhardtii can be observed in their respective wild types (WTs) in any conditions, and both systems were artificially generated to facilitate study of the ¹O₂-signalling. In contrast to flu, which shows WT level of chlorophyll in continuous light²³, C. reinhardtii mutant used in our study is devoid of chlorophyll and accumulates Proto both in dark and light. The lack of chlorophyll in the mutants used in our study provides another advantage, because such mutants do not have functional photosynthetic electron transport (PET), so that ¹O₂ production and signalling originating in photosynthesis is avoided. The accumulating Proto is thus the dominant source of generated ¹O₂ in the chloroplast and we hypothesized that this approach should allow us to isolate novel components or mechanisms governing ¹O₂-signalling, which otherwise might be difficult to detect due to the dominant signal(s) originating in PET. The GUN4 protein involved in MgCh function (Supplementary Fig. 1) and signalling degrades upon Proto accumulation¹⁹. However, chlD-1 overexpressing GUN4 showed higher GUN4 content than chlD-1, while it retained the chlorophyll-free phenotype¹⁹. Additionally, chlD-1/GUN4 demonstrated higher expression of GLUTATHIONE PEROXIDASE 5 (GPX5) and higher tolerance to ¹O₂ than chlD-1¹⁹. Thus, to minimize the GUN4-deficient phenotype and to maintain high and stable ¹O₂-inducibility of GPX5 expression, chlD-1/GUN4 instead of chlD-1 was used as the receiver strain for the gene construct that allowed us to monitor ¹O₂-signalling. The gene construct introduced into the chlD-1/GUN4 genome consisted of the promoter region of the GPX5 gene fused to the promoterless ARYLSULFATASE 2 gene (ARS2). GPX5 was shown to be specifically induced by ¹O₂ in C. reinhardtii^20,24, while the ARS2 activity can be assessed by the enzymatic assay²⁵. The resulting GPX5-ARS2 gene construct was shown previously to be an effective reporter to study ¹O₂-signalling^15,24,26. Following introduction of GPX5-ARS2, transformant strain showing high inducibility of GPX5-ARS2 in response to ¹O₂ was named signalling Reporter (sigRep) and was subjected to further studies.

Subsequent random insertional mutagenesis of sigRep, followed by a screening for decreased GPX5-ARS2 expression identified mutants with impaired ¹O₂-signalling. To reflect the impairment in ¹O₂-dependent signalling, these mutants were named genomes uncoupled Singlet Oxygen Signalling (gunSOS). The gunSOS1 mutant was selected for further analysis. Although employment of the Proto-accumulating mutant as the background strain provides advantage in studying ¹O₂-signalling, it also results in certain limitations, e.g. lack of chlorophyll enforces heterotrophic growth, alters nuclear gene expression and metabolism. Thus, due to the character of the background strain, phenotype of gunSOS1 was primarily compared with the parental sigRep, instead of the chlorophyll-synthesising WT.

The lack of the retrograde response to ¹O₂ in gunSOS1 was verified by decreased expression of GPX5, as well as SAK1, MBS, and PSBP2 compared with sigRep. The causal mutation in gunSOS1 was found in a gene annotated as TREHALOSE 6-PHOSPHATE PHOSPHATASE (hereafter TSPP1). Besides clear impairment in ¹O₂-signalling, mutation in TSPP1 also resulted in accumulation of trehalose 6-phosphate (Tre6P) in gunSOS1 compared with sigRep and WT. Therefore, TSPP1 is the first confirmed phosphatase acting on Tre6P in C. reinhardtii and in fact the first enzyme with a confirmed function in trehalose metabolism in this organism. Our data indicate that accumulation of Tre6P, alternatively the lack of the TSPP1 protein, causes changes to the expression of genes involved in several metabolic pathways. However, Tre6P or TSPP1 are rather intermediates than the primary cause of the impaired ¹O₂-signalling. Here, using a combination of genetics, gene expression and metabolic phenotypes, we reveal a complex interaction between chloroplast, mitochondria and cytosol in ¹O₂ signal transmission to the nucleus.

Results

Forward genetics screen to identify components of the ¹O₂-signalling

To generate a mutant impaired in ¹O₂-dependent signalling, we first created a reporter strain in a known ¹O₂-generating mutant, which was subsequently subjected to mutagenesis. The GPX5-ARS2 reporter construct (Supplementary Fig. 2) was introduced into the genome of chlD-1/GUN4 to express the ARS2 protein in a ¹O₂-dependent manner, and the transformants were tested for the enzymatic activity of ARS2. The reporter strain, which showed the lowest ARS2 activity in darkness and the highest activity in light was named signalling Reporter (sigRep; Fig. 1a) and was used in further applications. Expression kinetics of the cytosolic (GPX5_cyt) and chloroplastic (GPX5_cp) version of GPX5²⁷; Fig. 1b), as well as the GPX5-ARS2 construct (Fig. 1c) were examined by quantitative Real-Time PCR (qRT-PCR) upon transfer from dark to light. As expected, higher induction of GPX5_cp and GPX5_cyt were observed upon light illumination in sigRep compared with WT (Fig. 1b). Induced GPX5-ARS2 expression in sigRep in the light (Fig. 1c) confirmed the results obtained in the ARS activity assay (Fig. 1a) and a suitability of the sigRep reporter strain for further applications.

**Fig. 1: Generation and initial characterisation of the mutant impaired in ¹O₂-signalling.**

Subsequently, a random mutagenesis was performed on sigRep using bleomycin resistance cassette (ble^R) as an insert, followed by screening to isolate strains with lower or undetectable ARS2 activity in the light compared with sigRep, and thus, possibly disrupted ¹O₂-dependent signalling. Screen of 804 transformants allowed us to isolate nine gunSOS mutants. The relatively high number of the obtained mutants with a desired ¹O₂-signalling phenotype might be indicative of the multiple components involved or affecting ¹O₂-signalling and the response to photooxidative stress. These components may be key elements of the signalling pathways alternative to the dominant ROS-signalling originating in PET, or may constitute a part of it in an orchestrated complex network of the signalling pathways, which function becomes more apparent only in artificially generated chlorophyll- and PET-deficient conditions.

The gunSOS1 mutant (Fig. 1d) was selected for further analysis. Mutant impaired in ¹O₂-signalling showed similar Proto accumulation in light compared with sigRep and chlD-1/GUN4 (Supplementary Fig. 3), which eliminated the possibility that spurious mutations in gunSOS1, which may have been introduced during mutagenesis, resulted in reduced content of this photosensitizer. Because in our system Proto is considered as the main source of ¹O₂, a lower levels of this ROS in gunSOS1 compared with sigRep would not be expected.

The ble^R insertion in gunSOS1 was located in the first exon of TSPP1 (locus Cre12.g497750) in the JGI portal (Department of Energy Joint Genome Institute, https://phytozome-next.jgi.doe.gov, v13, genome v5.6; Fig. 1e). The TSPP1 transcript abundance was determined in gunSOS1 and compared with sigRep by qRT-PCR using primers annealing to the coding sequence upstream of the insertion site. At 2 h after transfer from dark to light, TSPP1 mRNA content increased 2.5-fold in sigRep compared with WT, while it was nearly absent in gunSOS1 (Fig. 1f). Expression of GPX5_cyt and GPX5_cp was about 11 and 9 times lower in gunSOS1 compared with sigRep, respectively (Fig. 1g). The transcript abundance for GPX5-ARS2 was 7 times lower in gunSOS1 than in sigRep (Fig. 1h), and explains undetectable ARS2 activity in the ¹O₂-signalling mutant (Fig. 1d).

C. reinhardtii TSPP1 is a functional Tre6P phosphatase

While gunSOS1 is clearly impaired in ¹O₂-signalling, it was necessary to determine the primary phenotype caused by the mutation in TSPP1. The content of Tre6P in sigRep and gunSOS1 was determined by anion-exchange high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Tre6P accumulation increased in gunSOS1 upon exposure to light in a time-dependent manner, while it decreased in sigRep (Fig. 2a). In photosynthetic WT, Tre6P content remained low throughout the entire course of the experiment (Fig. 2a). Accumulation of Tre6P in gunSOS1 indicates that C. reinhardtii TSPP1 is a functional phosphatase dephosphorylating Tre6P to trehalose. Subsequently, TSPP1 expression was found to be induced in the light in sigRep, but not in the WT, indicating its inducibility by photooxidative stress rather than light (Fig. 2b). Mutation of the TSPP1 gene would be expected to decrease dephosphorylation of Tre6P and lower trehalose content, but we also observed accumulation of trehalose in gunSOS1 compared with sigRep and WT (Fig. 2c). This apparent discrepancy could be explained by the existence of another enzyme in C. reinhardtii with possible Tre6P phosphatase activity, in combination with higher levels of Tre6P. However, this hypothesis was not verified (see the Discussion section).

**Fig. 2: *C. reinhardtii* TSPP1 is a functional phosphatase induced during photooxidative stress.**

Rescue of the TSPP1 deficiency in gunSOS1 was performed with the isolated genomic DNA fragment carrying the WT TSPP1 gene. Several independent transformants showed rescued ¹O₂-signalling, which was indicated by the GPX5-ARS2 expression determined in the ARS assay, and data from three representative strains are shown in Supplementary Fig. 4a. The analysed rescued strains also showed increased GPX5_cp transcript content compared with gunSOS1 (Supplementary Fig. 4b). A peptide-specific antibody for TSPP1 was produced to compare the protein content in gunSOS1 to strains showing ¹O₂-dependent signalling. A faint immune signal of ~43 kDa was detected in gunSOS1 and chlD-1, but two narrow bands were seen in chlD-1/GUN4, sigRep and the TSPP1-rescued strains (Fig. 2d). WT and gunSOS1 showed an additional unspecific immunoreaction with protein of ~53 kDa. As expected, the GPX5 protein content was lower in gunSOS1 compared with sigRep (Fig. 2d), which correlated with qRT-PCR results on GPX5 transcript levels (Fig. 1g). While higher sensitivity to light was recorded in gunSOS1 compared with the sigRep background strain (Fig. 2e), in agreement with a higher sensitivity or impaired acclimation to ¹O₂. Strains with rescued ¹O₂-signalling, TSPP1-R1, -R2, and -R3, showed increased content of GPX5 (Fig. 2d) and subsequently higher tolerance to light compared with gunSOS1 (Fig. 2e).

The gunSOS1 mutant shows altered metabolism compared with sigRep

In A. thaliana and other angiosperms, Tre6P functions as a sucrose signal and homoeostatic regulator of sucrose metabolism^28,29, which links plant growth and development to the availability of sucrose³⁰, reviewed in³¹. To determine the effect of accumulated Tre6P on metabolism in C. reinhardtii, we performed comparative metabolite profiling of gunSOS1 and sigRep (Supplementary Table 1), which revealed a relatively wide range of variation. In our metabolite analyses we focused on the differences between gunSOS1 and sigRep. However, the content of the same metabolites was also determined in WT, which was used only as a reference for non-photosynthetic gunSOS1 and sigRep. Aside from Tre6P and trehalose, 11 out of 27 analysed metabolites showed significantly increased content in gunSOS1 compared with sigRep in the light (Supplementary Fig. 5a). By means of the MetaboAnalyst 5.0 portal (https://www.metaboanalyst.ca) these metabolites were assigned to the metabolic processes in the cell. A hypergeometric test indicated the most affected metabolic pathways, i.e. the TCA cycle, pyruvate metabolism, and starch and sucrose metabolism (Supplementary Fig. 5b and Supplementary Table 2). Eight metabolites showed significantly decreased content in gunSOS1 compared with sigRep (Supplementary Fig. 6a), with the highest impact (MetaboAnalyst 5.0) on fructose and mannose metabolism, amino sugar and nucleotide sugar metabolism, and starch and sucrose metabolism (Supplementary Fig. 6b and Supplementary Table 3). Metabolites, which did not show a significant change in gunSOS1 compared with sigRep are presented in Supplementary Fig. 7. The map-overview of the selected metabolic pathways with depicted metabolites that showed different content, or were not changed, in gunSOS1 relative to sigRep is presented in Fig. 3.

**Fig. 3: The overview of the selected metabolic pathways in *gunSOS1* compared with *sigRep*.**

Starch content was determined in gunSOS1 and compared with sigRep, because of changes in several intermediates of starch and sucrose metabolism (Supplementary Figs. 5b and 6b). Indeed, gunSOS1 accumulated only 60% of the starch content of sigRep when the strains were grown in acetate-supplemented media (TAP, Supplementary Fig. 6c). Both sigRep and gunSOS1 are obligate heterotrophs. SigRep contained less starch after transfer from TAP and cultivation in TP for 48 h, whereas the starch content of gunSOS1 was essentially unchanged (Supplementary Fig. 6c), indicating that there was net degradation of starch in sigRep but not in gunSOS1. Starch formation is strongly induced during nitrogen (N) deprivation³². The sigRep cells did not increase starch reserves after 3 days in N-deficient medium, suggesting that starch accumulation had already reached its maximum under non-stress conditions. The starch content in gunSOS1 deprived of N did increase by 50% and was similar to the content in sigRep (Supplementary Fig. 6c). We conclude that sigRep accumulates high levels of starch as a consequence of the chlD-1/GUN4 genetic background and that the accumulation of Tre6P represses this response in gunSOS1 leading to a lower accumulation of starch. The gunSOS1 cells are still capable of responding to the N-deprivation stress and accumulating starch despite disturbed metabolism. Starch degradation on the other hand, is compromised in gunSOS1, which could indicate a general impairment of catabolic processes or a defect in sensing carbon limitation.

Increased Tre6P content alters metabolism in C. reinhardtii via transcriptional changes

To determine the extent to which chloroplast retrograde signalling is affected in gunSOS1, we performed comparative RNA-sequencing (RNA-seq) of the transcriptomes of gunSOS1 and sigRep upon transfer from dark to light. The analysis showed that 8120 genes were expressed in both strains, but 1377 and 1087 additional genes were uniquely expressed in gunSOS1 and sigRep, respectively (Fig. 4a, detailed listing can be found in Supplementary Data 1). Furthermore, differential gene expression analysis showed that the transcript contents of 1606 genes and 1706 genes were lower or higher, respectively, in gunSOS1 compared with sigRep (Supplementary Data 2).

**Fig. 4: Transcriptome analysis based on RNA-seq.**

Based on the KEGG ontology (http://www.kegg.jp/) several genes associated with metabolic pathways were down- or up-regulated (Fig. 4b) in gunSOS1 compared with sigRep (Supplementary Data 3). Given the strong overlap between differentially expressed genes and corresponding pathway intermediates, we hypothesized that the altered metabolism can be directly explained by altered gene expression. To decipher a complex correlation between altered signalling, gene expression and metabolism, accumulation or deficiency in metabolites from given pathway(s) was correlated with altered gene expression in gunSOS1 compared with sigRep. Fumarate and aconitate will be described in more detail, as representative examples of the metabolites showing respectively increased or decreased content in gunSOS1 compared with sigRep.

KEGG enrichment of the RNA-seq data indicated increased expression of the gene encoding fumarate hydratase class II (FUM2, Enzyme Commission (EC) Number 4.2.1.2, Cre01.g020223) in gunSOS1 compared with sigRep. FUM is responsible for reversible stereospecific interconversion of malate to fumarate. However, specific isoforms of this protein act in defined pathways and may favour one direction over the other depending on the subcellular localisation and environment³³. The mitochondrial isoform catalyses hydration of fumarate to L-malate in the TCA cycle, while the cytosolic form catalyses dehydration of L-malate to fumarate^34,35. Two isoforms exist in C. reinhardtii, FUM1 encoded in locus Cre06.g254400 and FUM2. Based on the predicted subcellular localisation (PredAlgo; http://lobosphaera.ibpc.fr/cgi-bin/predalgodb2.perl?page=main), FUM1 localises to the mitochondria, while FUM2 is assigned to the “other” compartment and is likely to be a cytosolic protein. Pathway (KEGG) diagrams for the TCA cycle and pyruvate metabolism can be found in Supplementary Fig. 8, showing integration of metabolite, RNA-seq and qRT-PCR data. Comparison of the transcriptomes of gunSOS1 and sigRep also indicated increased expression of the FUMARYLACETOACETASE gene (locus Cre17.g732802, EC 3.7.1.2) involved in tyrosine catabolism. Fumarylacetoacetase catalyses hydrolysis of 4-fumarylacetoacetate to acetoacetate and fumarate (Supplementary Fig. 9). Increased expression of genes encoding FUM2 and fumarylacetoacetase could explain accumulation of fumarate in gunSOS1 compared with sigRep.

Aconitate deficiency in gunSOS1 compared with sigRep can be explained by transcriptional downregulation of ACONITATE HYDRATASE (ACH1, Cre01.g042750, EC 4.2.1.3). ACH1 interconverts citrate and isocitrate, via cis-aconitate, in the TCA cycle (Supplementary Fig. 10) and is also involved in glyoxylate and dicarboxylate metabolism. At equilibrium, the reactants of the ACH1 reaction are present in the following ratio: 91% citrate, 6% isocitrate and 3% aconitate. With the smallest pool of the three tricarboxylic acids, fluctuations in the level of aconitate are expected to be more pronounced compared with the other metabolites involved, especially citrate, whose content was similar in gunSOS1 and sigRep in the light (Supplementary Fig. 7). Taken together, our data indicate that altered fumarate and aconitate content in gunSOS1 compared with sigRep can be explained by altered expression of genes encoding enzymes involved in key metabolic processes affecting those metabolites.

Impaired ¹O₂-signalling in gunSOS1 correlates with decreased expression of PSBP2, MBS, and SAK1

The PSBP2¹⁵, MBS¹⁷, and SAK1¹⁶ proteins are required for ¹O₂-induced chloroplast retrograde signalling in C. reinhardtii. Therefore, we determined the transcript levels of PSBP2, MBS, and SAK1, which were 8-, 9-, and nearly 41-fold lower, respectively, in gunSOS1 compared with sigRep (Fig. 5a). Decreased expression of SAK1 in gunSOS1 prompted us to compare transcript levels of selected ¹O₂-responsive genes between gunSOS1 and the sak1 mutant, reported in Wakao et al.¹⁶. SAK1 is a key regulator of the gene expression response and its knockout abolishes acclimation response to ¹O₂¹⁶. The involvement of SAK1 in ¹O₂-signalling was determined following treatment with rose bengal¹⁶, which produces ¹O₂ in the light, while in gunSOS1 the main source of ¹O₂ is endogenously accumulating Proto (Supplementary Fig. 3). Nevertheless, based on our qRT-PCR analysis, all tested genes had the same reduced inducibility in gunSOS1 compared with sigRep (Fig. 5b), as it was observed in sak1 compared with its corresponding WT following ¹O₂-exposure¹⁶. Among the genes that were down-regulated in both gunSOS1 (Fig. 5b) and sak1¹⁶ were two CYCLOPROPANE FATTY ACID SYNTHASES, CFA1 and CFA2 (CPLD27), involved in lipid and sterol metabolism, as well as the gene encoding SOUL1 heme-binding protein (Fig. 5b). It is noteworthy that attenuated expression of both CFA1 and CFA2, as well as SOUL1, was also observed in studies on a gpx5 mutant³⁶. Furthermore, in agreement with the sak1 phenotype presented in ref. ¹⁶, gunSOS1 also showed decreases in transcript content of PEPTIDE METHIONINE SULFOXIDE REDUCTASE (MSRA3), ALCOHOL DEHYDROGENASE (ADH7), RETINALDEHYDE BINDING PROTEIN-RELATED (RABPR1), and LOW-CO₂-INDUCIBLE PROTEIN 7 (LCI7) (Fig. 5b). Thus, there is an overlap between the phenotypes of gunSOS1 and sak1¹⁶ with respect to their attenuated expression of genes induced during elevated ¹O₂ (Fig. 5b).

**Fig. 5: The qRT-PCR analysis of gene expression in *gunSOS1* compared to *sigRep*.**

Altered metabolite content affects ¹O₂-signalling

We hypothesized that at least some of the metabolites showing increased or decreased content in gunSOS1 compared with sigRep, attenuate or propagate ¹O₂-signalling, respectively. To test this hypothesis, we undertook the most straight-forward approach by application of selected metabolites individually, at pre-determined sub-lethal concentrations. Addition of metabolites was followed by incubation in light to induce ¹O₂-generation by Proto and subsequent examination of the GPX5 expression as readout for functional ¹O₂-signalling. Selection of the metabolites to be tested was mostly based on their involvement or effect on the intracellular signalling processes reported in the literature.

Concerning selected metabolites accumulating in gunSOS1 relative to sigRep exposed to ¹O₂-stress (Supplementary Fig. 5a), fumarate is a well-recognised oncometabolite in mammalian cells³⁷. 2-oxoglutarate lies at the intersection between the carbon and nitrogen metabolic pathways and it was shown to regulate (together with glutamine) expression of NITRATE REDUCTASE in Nicotiana tabacum³⁸. Other studies also suggested 2-oxoglutarate playing a role as a signal metabolite in plants^39,40. Myo-inositol is a building block for several molecules involved in signalling, such as myo-inositol (1,4,5)trisphosphate or phosphatidylinositol (4,5)bisphosphate (reviewed in⁴¹). Exogenously provided fumarate, 2-oxoglutarate, and myo-inositol, always significantly (detailed report from two-way ANOVA analyses is presented in Supplementary Tables 4-9) attenuated expression of GPX5 in a concentration-dependent manner in sigRep, with no change in gunSOS1 and WT, relative to their respective non-treated controls (Fig. 6a–c). Among the selected metabolites, which showed decreased content in gunSOS1 relative to sigRep (Supplementary Fig. 6a), were mannose 6-phosphate, glucose 6-phosphate, and aconitate. The effect on ¹O₂-signalling of exogenously applied sugar phosphates was tested because of their known inhibitory effect on sucrose-non fermenting (SNF)-related protein kinase 1 (SnRK1) in plants^42,43. Application of mannose 6-phosphate and glucose 6-phosphate had a similar effect on GPX5 expression in sigRep, with significant increase at 20 and 50 µM. However, no effect on GPX5 mRNA could be detected in gunSOS1 (Fig. 6d, e, Supplementary Tables 10–13). Based on the literature, aconitate was never implicated in the signalling and it was selected randomly. Nevertheless, application of 20 µM aconitate significantly (P < 0.0001, Supplementary Tables 14 and 15) increased expression of GPX5 in gunSOS1 and even further in sigRep compared with untreated cells, but 50 and 100 µM had a quenching effect on the GPX5 expression in sigRep (Fig. 6f). This indicates that aconitate up to a certain threshold concentration promotes ¹O₂ signalling in otherwise aconitate-deficient gunSOS1. This result encouraged us to study the effect of exogenously applied aconitate further.

**Fig. 6: Effect of selected metabolites applied exogenously on ¹O₂-signalling inducing *GPX5*_cyt or *GPX5*_cp expression.**

Subsequently, we determined the expression of SAK1 upon feeding gunSOS1 and sigRep with aconitate. Exogenous application of aconitate at 20 µM increased SAK1 transcript abundance in gunSOS1 >120 times compared with the untreated gunSOS1 control, and exceeded values observed in sigRep subjected to the same treatment by a factor of 5 (Fig. 6g). Due to the increased SAK1 expression in gunSOS1 upon feeding with aconitate, we also determined the expression of PSBP2 and MBS in the same conditions. The PSBP2 response to aconitate was similar to SAK1 in terms of the increased expression (Fig. 6g), but with less pronounced dependence on the metabolite concentration compared with SAK1. Although 20 µM aconitate increased PSBP2 expression in gunSOS1 by a factor of 48 compared to the untreated control, it was still 2.7 times lower compared with sigRep subjected to the same treatment (Fig. 6g). Similarly to SAK1 and PSBP2, 20 µM aconitate also increased expression of MBS in gunSOS1, although values did not exceed those observed in sigRep subjected to the same treatment (Fig. 6g). Statistical analyses indicated that the effect of 20 µM aconitate on SAK1, PSBP2 and MBS expression in sigRep and gunSOS1, interaction between mutants and aconitate, as well as the mutant-dependent expression of these genes were always significant (P < 0.001, Supplementary Tables 16–18).

The rescued expression of the genes encoding SAK1¹⁶, PSBP2¹⁵, and MBS¹⁷ (Fig. 6g), consequently rescued ¹O₂-signalling and the expression of GPX5 (Fig. 6f) upon exogenously applied aconitate is intriguing, but the underlying mechanism remains unknown. However, substantial amount of data indicated the key role of aconitase (EC 4.2.1.3) in responses triggered by ROS^44,45. The decreased expression of ACH1 in gunSOS1 relative to sigRep (Supplementary Data 2; Supplementary Fig. 10) does not affect the content of citrate and isocitrate (Supplementary Fig. 7), but only deficiency in aconitate was observed in gunSOS1 compared with sigRep (Supplementary Fig. 6a). Thus, we tested the possibility that exogenously provided aconitate affects expression of ACH1 in gunSOS1. As previously observed (Supplementary Fig. 10b), expression of ACH1 was lower in sigRep compared with WT and it did not change upon treatment with aconitate (Fig. 6h). However, upon application of 20 µM aconitate expression of ACH1 in gunSOS1 increased 2.5-fold compared with untreated gunSOS1 control and reached the levels observed in sigRep (Fig. 6h; P < 0.05, Supplementary Table 19). This result indicated that aconitate content can also affect expression of ACH1 and points to possible correlation between aconitase function and ¹O₂-signalling.

Specificity of the ¹O₂-signalling pathway(s) attenuated in gunSOS1

The profound effect of accumulating Tre6P on the transcriptome (Fig. 4a, b) and consequently the metabolism of gunSOS1 (Fig. 3 and Supplementary Figs. 5 and 6) may have been indicative of a general photooxidative stress response being impaired in gunSOS1. To determine the specificity of this response, we compared gunSOS1 and sigRep in terms of the expression of selected genes that are known to be induced by various reactive species or conditions causing photooxidative stress other than ¹O₂.

Upon H₂O₂ or organic tert-butyl hydroperoxide (t-BOOH) treatment, Blaby, et al.⁴⁶ observed an increase in the expression of the MSD3 gene, encoding plastid-localised Mn superoxide dismutase 3. In our studies, we did not observe an increase in MSD3 transcript in sigRep compared with WT (Supplementary Fig. 11a), which shows that MSD3 expression is not inducible by ¹O₂ produced by Proto. However, based on qRT-PCR analysis, a 9-fold increase was observed in gunSOS1 compared with sigRep (Supplementary Fig. 11a, see also RNA-seq in Supplementary Data 2). Upon H₂O₂ or t-BOOH treatment, Urzica, et al.⁴⁷ also observed induced expression of genes involved in the glutathione-ascorbate system, GDP-L-GALACTOSE PHOSPHORYLASE (VTC2) and DEHYDROSASCORBATE REDUCTASE (DHAR1). Based on our qRT-PCR results, VTC2 transcript content was not changed in sigRep compared with WT, but an increase was observed in gunSOS1 (Supplementary Fig. 11b and Supplementary Data 2). DHAR1 expression was also stimulated in gunSOS1 compared with sigRep (Supplementary Fig. 11c and Supplementary Data 2).

Similarly to sak1 following treatment with rose bengal¹⁶, we observed increased expression of GLUTATHIONE S-TRANSFERASE (GSTS1) in gunSOS1 compared with sigRep (Supplementary Fig. 11d and Supplementary Data 2). However, in another study increased expression of GSTS1 was shown after treatment with acrolein, which suggests its transcriptional induction by reactive electrophile species (RES)⁴⁸. Acrolein was also shown to induce FSD1 encoding Fe superoxide dismutase (FeSOD)⁴⁸, which was expressed both in sigRep and gunSOS1 (Supplementary Data 1), and no significant difference could be observed in DEG analysis (Supplementary Data 2) or qRT-PCR (Supplementary Fig. 11e). It can be concluded that, despite impaired ¹O₂-signalling, gunSOS1 retained the ability to express selectively tested genes, which induction was previously associated with response to other ROS, such as H₂O₂, organic peroxides, or RES.

Discussion

ROS are formed as a by-product of biological redox reactions⁴⁹ mostly in the mitochondria or chloroplasts^50,51. Although excess ROS production can cause oxidative damage to cell components, ROS or the oxidation products play an important role in the signal transduction processes. Different retrograde signalling pathways have been proposed to involve also TBS-intermediates in plants and green algae (reviewed in ref. ^52,53). While involvement of Mg-porphyrins in chloroplast retrograde signalling is now excluded^54,55, in the present study we have demonstrated that ¹O₂ produced by the photosensitizing activity of Proto in the light triggers signalling cascades that alter nuclear gene expression in mutant that endogenously accumulate Proto.

The ¹O₂-induced signalling phenotype in the gunSOS1 mutant was due to a lesion in the TSPP1 gene. We demonstrated that TSPP1 is a functional phosphatase responsible for dephosphorylating Tre6P (Fig. 2a, d). TSPP1 is the only representative of the classical plant TPP gene family in C. reinhardtii, contrasting with the large TPP gene families in angiosperms⁵⁶. In addition, C. reinhardtii has one representative of the class I TREHALOSE-6-PHOSPHATE SYNTHASE (TPS) family (Cre16.g662350, hereafter TSPS1), and two members of the class II TPS family (Cre06.g278221, here TSSP1 and Cre16.g686200, here TSSP2). Both class I and class II TPS proteins have glucosyltransferase and TPP-like domains, but only the class I TPS proteins have demonstrated TPS activity ^57,58,59. TSPP1 belongs to the haloacid dehalogenase superfamily of proteins and contains the characteristic DXDX(T/V) active site motif – ₁₀₇DYDGT₁₁₂ – in which the initial Asp residue forms a phospho-acyl intermediate during catalysis⁶⁰. TSSP1 also contains the complete active site motif (₅₉₂DYDGT₅₉₇), so we cannot exclude the possibility that the TSSP1 protein in C. reinhardtii has a phosphatase activity, which could explain the increased content of trehalose in gunSOS1 compared with sigRep (Fig. 2c). Although the intracellular localisations of these proteins have not been determined experimentally in C. reinhardtii, PredAlgo analysis indicated possible localisation of TSPP1 and TSSP1 in mitochondria, while TSPS1 and TSSP2 are likely to be cytosolic proteins, because they were not assigned to any specific organelle.

Correlation between TSPP1 (Fig. 2b) and GPX5 (Fig. 1b, c) expression may be indicating that low Tre6P content is necessary for efficient ¹O₂-signalling. However, the apparent negative effect of accumulating Tre6P or the absence of TSPP1 on ¹O₂-signalling is not direct and involves a complex metabolic reprogramming (Fig. 4a, b) leading to altered metabolite content (Fig. 3 and Supplementary Figs. 5 and 6). Study on the contrasting phenotypes between A. thaliana overexpressing bacterial TPS, TPP, or trehalose phosphate hydrolase (TPH) pointed to Tre6P, rather than trehalose, playing a signalling function³⁰. Nevertheless, Tre6P was shown to be highly correlated with sucrose, leading to the proposal that it functions as a signal of sucrose status²⁸. Tre6P was also shown to inhibit starch degradation in A. thaliana^61,62, which is also true in Tre6P-acumulating gunSOS1 mutant of C. reinhardtii (Supplementary Fig. 6c). However, although our analyses revealed the sensitivity of ¹O₂-dependent retrograde signalling to metabolites, it is less clear if Tre6P or the TSPP1 protein itself, or both, play a direct role in metabolic reprogramming (Fig. 3 and Supplementary Figs. 5 and 6). TPP in Z. mays is encoded by RAMOSA3 (RA3) and the ra3 mutants showed reduced meristem determinacy⁶³, without altering the Tre6P content compared with WT plants⁶⁴. Additionally, a catalytically inactive version of RA3 complemented the ra3 phenotype, which revealed the “moonlighting” function of TPP, i.e. its function aside from the catalysis of Tre6P dephosphorylation⁶⁴. Subsequent study also demonstrated “moonlighting” function of RA3 in carpel suppression⁶⁵. Rather regulatory than a catalytic function was also proposed for TPP7 in Oryza sativa, due to its low activity in vitro and no apparent effect on Tre6P content in knockout mutants of TPP7⁶⁶. In either case, although in our study the lack of functional TSPP1 clearly results in accumulation of Tre6P, we cannot exclude that the TSPP1 protein itself is involved in inducing changes to the nuclear gene expression (Fig. 4) and metabolic reprogramming (Fig. 3 and Supplementary Figs. 5 and 6).

Nonetheless, it was demonstrated that Tre6P acts as an inhibitor of SnRK1 in developing tissues and that this is dependent on a so-far unidentified protein factor^43,67,68. Tre6P also inhibits the activation of SnRK1 by SnRK1-activating kinases/geminivirus Rep interacting kinases⁶⁹. SnRK1 belongs to the AMPK-SNF1-SnRK family of protein kinases, which is represented in all eukaryotes⁷⁰. In plants, SnRK1 plays a central role in energy and metabolic homeostasis, and is activated during energy deficient conditions caused by stresses like nutrient starvation, pathogen attack, or ROS⁷¹. Baena-González et al.⁷² established approximately 1000 genes as markers of SnRK1 in A. thaliana, which indicates an extensive SnRK1-dependent transcriptional reprogramming. In C. reinhardtii, involvement of various SnRKs in responses to stress was observed during sulphur^73,74 and nitrogen deprivation⁷⁵, or cold stress⁷⁶. Genome-wide analysis revealed the existence of 21 genes as potential orthologues of the plant SnRK α, β and γ/βγ subunits in C. reinhardtii. It was suggested that the proteins encoded by these genes play the same role in cell survival and stress response in C. reinhardtii as SnRKs in land plants⁷⁵.

In analogy to A. thaliana, altered SnRKs activity was shown to cause metabolic remodelling also in algae⁷⁶. In our study, if the majority of changes to gene expression observed in gunSOS1, relative to sigRep, originate from accumulation of Tre6P and not the absence of TSPP1, based on previous studies, this may lead to inhibition of one or more SnRKs (Fig. 7). Our analysis showed that altered gene expression in gunSOS1 relative to sigRep (Supplementary Figs. 8–10) is directly responsible for altered fumarate and aconitate content in gunSOS1 compared with sigRep. Thus, Tre6P accumulating in gunSOS1 is not directly involved in ¹O₂-signalling but controls other processes in the cell which affect ¹O₂-signalling more directly (Fig. 7).

**Fig. 7: Model of the chloroplast ¹O₂-induced retrograde signalling depending on the metabolites content.**

We demonstrated that accumulation of fumarate, 2-oxoglutarate, or myo-inositol (Fig. 3 and Supplementary Fig. 5a) is capable of attenuating ¹O₂-signalling (Fig. 6a-c, see also model in Fig. 7). On the other hand, application of mannose 6-phosphate and glucose 6-phosphate had a positive effect on GPX5 expression in sigRep, but not in gunSOS1 (Fig. 6d, e). The mechanism behind individual effect of any of these metabolites on ¹O₂-signalling remains unknown. However, considering the rescue of the ¹O₂-signallling upon treatment with aconitate (Fig. 6f), interplay between fumarate and aconitate content can be hypothesized to play a role in this signalling pathway. Fumarate is associated with development of tumours by competitive inhibition of 2-oxoglutarate-dependent oxygenases, including hypoxia inducible factor (HIF) hydroxylases, leading to stabilisation of HIF and activation of oncogenic HIF-dependent pathways⁷⁷. In fact, accumulation of fumarate in human cells was linked to an aggressive variant of hereditary kidney cancer⁷⁸. In mice, fumarate was also shown to directly modify some proteins by succination of cysteine residues to form 2-succinocysteine derivatives⁷⁹. Succination of three cysteines crucial for iron-sulphur cluster binding was identified in mitochondrial aconitase 2 (ACO2) in a fumarate hydratase 1 knockout (Fh1KO) mouse embryonic fibroblast (MEF) cell line⁷⁹. Analysis of tryptic peptides derived from ACO2 in Fh1KO MEFs, indicated succination of Cys385. Another tryptic peptide of ACO2 was identified as a mixture of two isomers in Fh1KO MEFs, which showed succination at Cys451 or Cys448⁷⁹. In vitro experiments indicated that succination of these cysteine residues in ACO2 leads to its inhibition⁷⁹. Thus, the diminished expression of ACH1 in gunSOS1 compared with sigRep (Supplementary Fig. 10a,b) and plausible inactivation of ACH1 by fumarate, together may constitute factors leading to aconitate depletion in gunSOS1 cells exposed to ¹O₂ stress.

Simultaneous increase in the ACH1 expression and the rescue of the ¹O₂-signalling upon exogenously applied aconitate might be pointing to rather aconitase function than the sole aconitate effect on these processes. In animal cells, aconitase redox-regulated moonlighting function modulates biosynthesis of proteins carrying Iron Response Element (IRP) in their mRNA^45,80,81,82. The dual function of aconitase is possible due to the reversible redox-dependent post-translational modifications (reviewed in⁸¹). Three cysteines crucial for binding the iron–sulphur cluster in mitochondrial ACO2 in mouse, Cys385, Cys448, and Cys451⁷⁹, are also present in ACH1 of C. reinhardtii, Cys426, Cys489, and Cys492, respectively.

Aconitase emerges as a factor involved in stress response also in plants. In A. thaliana, aconitase (ACO) is found in three isoforms. ACO3 in A. thaliana serves both as a target and mediator of mitochondrial dysfunction signalling, and it was shown to be critical for stress response in leaves⁸³. Phosphorylation of ACO3-Ser91 contributes to the UV-B and mitochondrial complex III inhibitor antimycin A-induced stress tolerance⁸³. ACH1 of C. reinhardtii contains four serine residues within a widely conserved eukaryotic phosphorylation motif R-x-x-S⁸⁴, Ser118, Ser211, Ser284, and Ser439, so their phosphorylation is likely. ACO3 was demonstrated to be part of the mitochondrial dysfunction response, which is dependent on the signalling involving transcription factor NAC domain containing protein 17 (ANAC017). ANAC017 is considered as a master regulator of the retrograde signalling and cellular stress response with mitochondria acting as central sensors, but also during repression of the chloroplast function⁸⁵. Based on the genome-wide association study concerning single nucleotide polymorphism, it was determined that promoter of ACO3 binds ANAC017⁸⁶.

ACO3 plays an important role in acclimation to submergence⁸⁶ and it was proposed to regulate the stability of chloroplast superoxide dismutase 2 (SOD2) mRNA⁸⁷. Both submergence-related hypoxia and reoxygenation during desubmergence are accompanied by increased ROS generation and oxidative stress^88,89. The aco3 knockout mutant and ACO3 overexpressing lines (ACO3OE) showed altered stress signalling correlated with decreased and increased stress tolerance, respectively. Furthermore, a decreased expression of fumarase 2 (FUM2) was noted in ACO3OE lines in the control conditions, and it decreased in aco3, ACO3OE, and in WT after submergence and desubmergence⁸⁶. The JGI portal blast-search analysis of A. thaliana FUM2 against C. reinhardtii proteome indicated, that the protein showing the highest similarity in amino acid sequence is the FUM2, which showed increased expression in gunSOS1 compared with sigRep (Supplementary Fig. 8c, see also Supplementary Data 2 and 3). These findings provide additional support for the hypothesis regarding a reciprocal interplay between fumarate and aconitate in response to stress.

Based on our results, including increased expression of ACH1 upon application of aconitate (Fig. 6h), and current knowledge, it can be hypothesized that not aconitate, but ACH1 is the crucial player in ¹O₂-signalling in C. reinhardtii, while accumulation of fumarate deactivates ACH1, thereby impairing oxidative stress sensing and ¹O₂-signalling. Chloroplasts and mitochondria are biochemically connected^90,91, while the function of the components localised in either compartment has been shown to be integrated into the functional signalling^{92,93,94,95,96}. Therefore, it seems plausible that the mitochondrial function participates, or is even required for functional chloroplast-emitted ¹O₂-signalling. The chloroplast ¹O₂-signal might be providing an input into the overall signalling network, which is subsequently integrated or dependent on the mitochondrial retrograde signalling and other internal or external stimuli.

Our results regarding increased content of fumarate and decreased aconitate in gunSOS1 compared with sigRep (Fig. 3 and Supplementary Figs. 5 and 6) as well as feeding experiments (Fig. 6) indicate, that there is a metabolic signature conditioning ¹O₂-signalling. However, it should be noted that the extent to which exogenously applied fumarate and aconitate are taken up into the cells was not determined. Nevertheless, treatment with fumarate and aconitate, both have a very clear impact on gene expression (Fig. 6), which would not take place, if these metabolites were not taken up or sensed by the cells. Although, there are no known receptors detecting the presence of these metabolites outside of the cell, their existence cannot be excluded at this point. In either case, it was shown that altered chloroplast redox conditions result in changes in the metabolome in A. thaliana, reflected also by the reallocation of energy resources⁹⁷. Importantly, although our study shows that the metabolic configuration of the cell is essential for ¹O₂-signalling, this does not exclude the involvement of protein components in this signalling network (Fig. 7). On the contrary, our findings support the direct involvement of proteins in ¹O₂-signalling, but we demonstrated that expression of PSBP2¹⁵, MBS¹⁷, and SAK1¹⁶ depends on the metabolic status (Fig. 5a and Fig. 6g). The overlap between the gunSOS1 and the SAK1-dependent transcriptomes (Fig. 5b) as well as other defined ROS or RES-dependent signalling pathways apparently being functional (Supplementary Fig. 11) indicate specificity of the impaired ¹O₂-signalling in gunSOS1. Our results indicate that fumarate and aconitate affect the expression of PSBP, MBS, or SAK1 upon ¹O₂-exposure in light, which subsequently convey information about the metabolic status of the cell to the nucleus and trigger specific responses (Fig. 7). It is not clear yet how the specificity of the signalling is achieved, because the mechanisms by which metabolites affect the expression of these proteins, as well as downstream components of these signalling pathways, remain unknown. Additionally, the protein components involved in ¹O₂-signalling may be subject to additional regulatory mechanisms, such as phosphorylation, as proposed for SAK1 by Wakao et al.¹⁶.

We have shown that retrograde signalling triggered by ¹O₂, which is mostly generated in the chloroplast, strictly depends on the mitochondrial metabolic status. Prolyl-tRNA synthetase (PRORS1) in A. thaliana, a component of the organellar gene expression machinery indicated in the previous study that mitochondria may contribute to chloroplast retrograde signalling⁹⁵. PRORS1 is targeted both to the plastid and to mitochondria, but downregulation of specific photosynthesis-associated nuclear genes was observed only in a plastidial prpl11 and mitochondrial mrpl11 double mutant, but not in prpl11 or mrpl11 single mutants⁹⁵. Another study showed that the ROS-dependent signals from chloroplast and mitochondria are integrated at the radical-induced cell death 1 (RCD1) protein located in the nucleus⁹⁶. RCD1 suppresses transcription factors ANAC013 and ANAC017, which mediate the ROS-signal from mitochondria, while chloroplast regulation of RCD1 takes place through 3’-phosphoadenosine 5’-phosphate. It was proposed that RCD1 may function at the intersection of mitochondrial and chloroplastic retrograde signalling pathways⁹⁶.

The retrograde signalling pathways identified so far assume the existence of separate signalling routes. However, chloroplast retrograde signalling involves a network of different but inter-connected mechanisms, and observation of specific signalling pathways within the network depends on the experimental conditions. Pharmacological or genetic interventions that disrupt specific pathways can be used to characterise alternative pathways. In the manuscript presented here, we report that the metabolic status of the cell, reflecting levels of Tre6P, and components of the mitochondrial TCA cycle, are integral factors in the ¹O₂-dependent retrograde signalling network in C. reinhardtii. It remains a matter of debate whether Tre6P or TSPP1 protein itself plays a direct role in the signalling and metabolic reprogramming. Additionally, further study should elucidate whether aconitate or ACH1 is the key player in the ¹O₂-signalling. Nevertheless, our results shed an important light on the signalling processes triggered by ¹O₂, by revealing their sensitivity to metabolites and thereby their potential modulation by cytosolic and mitochondrial metabolism.

Methods

Chlamydomonas reinhardtii cultures and genetic manipulations

All strains were cultivated heterotrophically in Tris-acetate-phosphate (TAP) medium in the dark until reaching the mid-log phase of 3–5 × 10⁶ cells mL⁻¹. Additionally, starch measurements were performed in TAP, Tris-phosphate (TP) medium without acetate, or nitrogen-depleted (TAP-N) medium. Experiments were performed in TAP medium in the dark or upon a shift to 20 µmol photons m⁻² s⁻¹ light. Sampling of three separate cultures grown in parallel in the same conditions was considered as biological triplicates. Three strains used in this study were described elsewhere, chlD-1 in von Gromoff, et al.¹⁸, chlD-1/GUN4 in ref. ¹⁹, and WT (4A+) in ref. ⁹⁸.

To generate the GPX5-ARS2 construct (Supplementary Fig. 2), the GPX5 regulatory region (GPX5 5’ RR) was amplified by PCR using forward and reverse primers carrying XhoI and EcoRV restrictions sites, respectively (Supplementary Table 20). The obtained fragment was subcloned and ligated between the XhoI/EcoRV sites of the pSL18 vector⁹⁹, replacing the existing promoter region of the gene encoding the PSAD and positioning GPX5 5’RR in reverse orientation with respect to the paromomycin resistance cassette (paroR¹⁰⁰). The DNA fragment carrying paroR and GPX5 5’ RR was excised by restriction digestion with KpnI and EcoRV and ligated into the corresponding restriction sites of pJD54¹⁰¹, which carries a copy of the promoterless version of the gene encoding arylsulfatase 2 (ARS2), which allowed ARS2 expression to be controlled by the GPX5 5’RR (Supplementary Fig. 2). The GPX5-ARS2 construct was verified by sequencing.

The sigRep strain was produced in the chlD-1/GUN4 background by transformation with a GPX5-ARS2 construct. Transformant selection was performed on TAP agar plates with 10 µg mL⁻¹ paromomycin in dark, followed by a screen for GPX5-ARS2 inducibility by ¹O₂. The sigRep strain showed low ARS2 activity in the dark and high activity in the light (Fig. 1a) and was selected for further applications. The gunSOS1 mutant was generated by random insertional mutagenesis performed on sigRep using a bleomycin resistance cassette (ble^R) isolated from the pMS188 vector¹⁰² using NotI and KpnI. Following mutagenesis, selection was performed on TAP agar plates containing 15 µg mL⁻¹ zeocin in the dark. More than 800 obtained colonies were screened for decreased or not detectable ARS2 activity in the light and nine mutants that showed impaired inducibility of GPX5-ARS expression in light were selected for further analysis. Restriction enzyme site-directed amplification PCR¹⁰³ allowed us to identify the genomic DNA flanking the ble^R insertion sites in five out of the nine mutants. Only analysis of the gunSOS1 mutant is presented here. Rescue of gunSOS1 (tspp1) was conducted with 5242 bp fragment of genomic DNA carrying 3241 bp TSPP1 amplified by PCR (see Supplementary Table 20 for primers) using bacterial artificial chromosome PTQ5987 (Clemson University Genomics Institute, Clemson, SC, USA) as a template. The amplified DNA fragment included 1500 bp upstream and 501 bp downstream of the annotated TSPP1. WT TSPP1 was introduced into gunSOS1 by co-transformation with a spectinomycin resistance cassette isolated from the pALM32 vector¹⁰⁴ with AleI and KpnI endonucleases. Transformant selection was performed on TAP agar plates supplemented with 100 µg mL^-1 spectinomycin in the dark. All genetic transformations were performed by electroporation.

Arylsulfatase activity assay

To assess the level of GPX5-ARS2 expression in transformed cells, enzymatic assays for arylsulfatase activity²⁵ were performed essentially as described before ref. ¹⁵. Cells were spotted onto agar-solidified TAP medium plates and cultured in either light or dark conditions. Arylsulfatase is expected to be secreted into the medium if GPX5-ARS2 is expressed. After removal of the cells, plates were flooded with detection solution containing 0.1 mg mL⁻¹ 2-naphthyl sulphate (potassium salt; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) as a chromogenic substrate coupled with 1 mg mL⁻¹ tetrazotized-o-dianisidine chloride (Fast Blue B salt, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Following 1 h incubation, purple spots appearing on the agar plates identified expressed ARS2.

Analysis of protoporphyrin IX content

The Proto content was analysed by High Pressure Liquid Chromatography (HPLC) essentially as described in ref. ¹⁰⁵, with modified sample preparation for C. reinhardtii¹⁹. In short, cultures were grown in the dark and transferred to 20 μmol photons m⁻² s⁻¹ light for 2 h. Samples containing 1.2 × 10⁸ cells were centrifuged at 3000 × g for 5 min at 4 °C and the pellets were snap-frozen in liquid N₂. Proto was extracted in cold (−20 °C) acetone/0.1 M NH₄OH (9/1, v/v) in a three-step cycle of resuspension and centrifugation. Proto analysis was performed using a Nova-Pak C18 column (Waters, 3.9 × 150 mm, 4 μm, at 20 °C). The results were normalised to pmol/10⁶ cells.

RNA isolation, qRT-PCR, and RNA-seq

The total RNA was isolated, after a shift from dark to 20 µmol photons m⁻² s⁻¹ light for 2 h, using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s protocol. RNA quality was assessed by electrophoresis on a 1% (w/v) agarose gel, while quantity was determined using a Nanodrop 2000 (Thermo Fischer Scientific, Waltham, MA). Aliquots of 2 µg RNA were treated with DNase and RiboLock RNase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA) and used to synthesise cDNA with RevertAid Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) and oligo(dT)₁₈ primer. Transcript analysis by qRT-PCR were performed using 2× ChamQ Universal SYBR qPCR Master Mix (Viazyme Biotech Co., Ltd., Nanjing, China) and a CFX96-C1000 96-well plate thermocycler (Bio-Rad, Hercules, CA, USA). In the initial experiments, determining expression kinetics of GPX5 (Fig. 1b) and GPX5-ARS2 (Fig. 1c), we observed change in the 18 S rRNA quantification cycle (Cq) by ≤ 3.3 in dark compared with subsequent exposure to 2 h light (Supplementary Table 21), both in sigRep and WT. Nevertheless, the difference in averaged 18 S Cq between sigRep and WT from the same conditions was always ≤ 0.9, which indicated sufficient consistency in 18 S expression regardless of the cell line and photooxidative stress. These results indicated suitability of 18 S to be used as a reference gene for qRT-PCR experiments. Most primers for transcript analyses were designed using Primer3Plus (http://primer3plus.com). Wherever applicable, the same primers were used as in the previous gene expression studies. All primers and their references are listed in Supplementary Table 20.

RNA-sequencing was performed on total RNA samples isolated from biological triplicates of sigRep and gunSOS1 after the shift from dark to 20 µmol photons m⁻² s⁻¹ light for 2 h. Library preparation, sequencing, and analysis services were commercially provided by Novogene Europe (Novogene (UK) Company Ltd., Cambridge, UK) and performed using an Illumina NovaSeq 6000 platform operated in 150 bp pair-end sequencing mode, with a sequencing depth of 20 million reads per sample. Six gigabases of sequencing data per library was filtered for high-quality reads, which were mapped to the C. reinhardtii v5.5 (Department of Energy JGI). Estimated expression was obtained in fragments per kilobase of transcript sequence per millions base pairs sequenced (FPKM). Biological replicates were averaged to obtain sample expression estimate. Differential expression analysis between two mutants (three biological replicates per mutant) was performed using DESeq2.v1.20.0, which provides statistical routines for determining differential expression using a model based on the negative binomial distribution. The resulting p values were adjusted using the Benjamini and Hochberg’s approach for controlling the False Discovery Rate (FDR). Genes with a log₂FoldChange ≥ 2 and adjusted p value (padj) < 0.05 found by DESeq2.v1.20.0 were defined as differentially expressed (DEGs). Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG; www.genome.jp/kegg/pathway.html) pathway analysis were conducted to identify DEGs at the biologically functional level using clusterProfiler R package. GO terms and KEGG pathways with a padj < 0.05 were considered to be significantly enriched.

Metabolite analysis

Cultures were grown in TAP in the dark until they reached mid-log phase of 3 × 10⁶ cells mL⁻¹. Samples were always normalised to contain 6 × 10⁷ cells, all centrifugation steps were carried at 3000 × g at 4 °C for 5 min, followed by snap-freezing in liquid N₂. Samples from dark conditions were collected immediately, while the remainder of each culture was transferred to 20 μmol photons m−² s⁻¹ light. Subsequent sampling took place at 15-min intervals, up to 1 h. Metabolites were extracted using chloroform-methanol as described by Lunn et al.²⁸. Tre6P, other phosphorylated intermediates and organic acids were measured by anion-exchange HPLC coupled to tandem mass spectrometry (LC-MS/MS) as described in ref. ²⁸ with modifications as described in ref. ¹⁰⁶. Sugars and sugar alcohols were measured by LC-MS/MS as described in ref. ¹⁰⁷. Obtained results were normalised to pmol/10⁶ cells.

For starch analysis, cultures were grown in TAP in the dark until they reached mid-log phase of 3 × 10⁶ cells mL⁻¹. For starch determination cultures were either transferred in TAP to the light (20 μmol photons m⁻² s⁻¹) for 2 h before harvesting, or centrifuged cells were resuspended in Tris-phosphate (TP) medium (without acetate), keeping the same cell concentration, and transferred back to the dark for 24 h. Subsequently, cultures were transferred to 20 μmol photons m⁻² s⁻¹ for 2 h before harvesting. A similar protocol was applied for starch analysis in TAP devoid of N (TAP-N), except that cells were cultivated in TAP-N for 3 days in the dark, followed by exposure to 20 μmol photons m⁻² s⁻¹ for 2 h. For each sampling, 5 × 10⁶ cells were centrifuged and lyophilized. Starch content was determined by enzymatic degradation and glucose quantification following the protocol of ref. ¹⁰⁸.

Protein extraction and immunoblot analysis

Cultures were grown in the dark until reaching the mid-log phase of 3–5 × 10⁶ cells mL⁻¹, followed by a transfer to 20 μmol photons m⁻² s⁻¹ for 3 h. Cells were pelleted by centrifugation and total proteins were extracted in 400 μL buffer containing: 56 mM Na₂CO₃, 56 mM DTT, 2% (w/v) SDS, 12% (w/v) sucrose and 2 mM EDTA, pH 8.0. Proteins were quantified using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and separated by SDS-PAGE on a 12% polyacrylamide gel, followed by transfer by electroblotting to nitrocellulose membrane (GE Healthcare, Chicago, IL, USA). TSPP1 was detected using a rabbit crude antiserum (dilution 1:500) raised by inoculation with a TSPP1-specific peptide (VEWSKSDSNGWRAKPC) against C. reinhardtii TSPP1 (calculated MW 42 kDa). GPX5 was detected with a commercially available antibody (AS15 2882, dilution 1:1000) obtained from Agrisera (Vännäs, Sweden). CHLI1 antibody (PHY5510S, dilution 1:1000) was purchased from PhytoAB (San Jose, CA, USA). For application, all antibodies were diluted in CrossDown buffer (AppliChem, AppliChem GmbH, Darmstadt, Germany). The secondary antibody (AS09 602, goat anti-rabbit IgG, dilution 1:10,000) conjugated to horseradish peroxidase was obtained from Agrisera. The immunoblotting signals were detected using a CCD camera (Intas Biopharmaceuticals, Ahmedabad, India) after application of enhanced chemiluminescence detection kit (Clarity™ Western ECL Substrate; Bio-Rad, Hercules, CA, USA).

Chemical treatments of C. reinhardtii cells

Fumarate (sodium fumarate dibasic), 2-oxoglutarate (α-ketoglutaric acid), myo-inositol (D-myo-inositol 1,4,5-tris-phosphate trisodium salt), mannose 6-phosphate (D-mannose-6-phosphate, disodium salt), glucose 6-phosphate (D-glucose 6-phosphate sodium salt), and aconitate (cis-aconitic acid) were obtained from Sigma-Aldrich (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), dissolved in H₂O to a stock solutions of 100 mM and added individually to the mid-log phase (3 ×10⁶ cells mL^-1) cultures to final concentrations of 20, 50, and 100 μM, followed by exposure to light for 2 h.

Statistics and reproducibility

Statistical analyses were conducted using the GraphPad Prism 9 for Windows, version 9.5.0 (GraphPad Software, San Diego, California USA, www.graphpad.com). All the details concerning particular analysis are included in the main text, in the Figure legend, or in the Supplementary Table, wherever applicable.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data relevant for interpretation of this study are presented in the article and supplementary material, including uncropped and unedited blot images for Fig. 2d (Supplementary Fig. 12). The numerical source data for all the graphs presented in the main Figures are provided in Supplementary Data 4. The raw RNA-seq data were deposited in the National Center for Biotechnology Information Sequence Read Archive, accession number PRJNA954977. Any further information is available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (DFG-GR 936/20-2 to B.G. and P.B.), by the Agence Nationale de la Recherche (ChloroPaths: ANR-14-CE05-0041-01 to X.J.), and by the Max Planck Society (R.F. and J.E.L.). We acknowledge support by the Open Access Publication Fund of Humboldt-Universität zu Berlin.

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Pflanzenphysiologie, Institut für Biologie, Humboldt-Universität zu Berlin, 10115, Berlin, Germany
Waeil Al Youssef, Bernhard Grimm & Pawel Brzezowski
Max Planck Institute of Molecular Plant Physiology, 14476, Potsdam-Golm, Germany
Regina Feil & John E. Lunn
Photosynthesis and Environment Team, Commissariat à l’Energie Atomique et aux Energies Alternatives (CEA), CNRS, Institut de Biosciences et Biotechnologies d’Aix-Marseille, Aix-Marseille Université, UMR 7265, CEA Cadarache, F-13108, Saint-Paul-lez-Durance, France
Maureen Saint-Sorny & Xenie Johnson

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Waeil Al Youssef
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Contributions

P.B. designed the research. P.B. and W.A.Y. performed most of the experiments. R.F. and J.E.L. performed the LC-MS/MS measurements. M.S.-S. and X.J. performed the starch quantification. P.B., B.G., and J.E.L. analysed the data. P.B. wrote the manuscript. P.B., B.G., J.E.L., and X.J. revised the manuscript. All authors discussed the results and commented upon the manuscript.

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Correspondence to Pawel Brzezowski.

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Communications Biology thanks Krishna Niyogi and the other, anonymous, reviewer for their contribution to the peer review of this work. Primary Handling Editors: Joao Valente.

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Youssef, W.A., Feil, R., Saint-Sorny, M. et al. Singlet oxygen-induced signalling depends on the metabolic status of the Chlamydomonas reinhardtii cell. Commun Biol 6, 529 (2023). https://doi.org/10.1038/s42003-023-04872-5

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Received: 15 June 2022
Accepted: 24 April 2023
Published: 16 May 2023
DOI: https://doi.org/10.1038/s42003-023-04872-5

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