Improving tolerance to fluctuating light through adaptive laboratory evolution in the cyanobacterium Synechocystis

Improving tolerance to fluctuating light through adaptive laboratory evolution in the cyanobacterium Synechocystis | Research Square Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article Improving tolerance to fluctuating light through adaptive laboratory evolution in the cyanobacterium Synechocystis Dario Leister, Theo Figueroa-Gonzalez, Eslam Abel-Salam, Weiyang Chen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6305715/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 May, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Fluctuating light (FL) poses a challenge to cyanobacteria by disrupting photosynthesis and damaging photosystems. Although key FL tolerance components are known, their genetic enhancement remains unexplored. We evolved Synechocystis PCC 6803 under two FL regimes (one lethal to the starter strain, LT) in order to identify adaptive alleles. Our analysis revealed 44 novel mutations, 28 of which impact proteins/RNAs. Mutations in Pam68 (PSII assembly) and Sll0518, present in all strains, enhanced non-lethal FL tolerance in LT. Mutated Pam68 increased PSII abundance and activity. A gain-of-function mutation in RpaB (regulator of phycobilisome association B) significantly increased tolerance to both lethal FL and high-light conditions. This was associated with an increased PSI/PSII ratio and downregulation of light harvesting. In summary, our results suggest that adaptive laboratory evolution can simultaneously identify new FL tolerance factors and their advantageous alleles. The identified point mutations rewire multiple protective responses by as yet unknown molecular mechanisms. Biological sciences/Biophysics/Bioenergetics Biological sciences/Biological techniques/Genetic engineering Biological sciences/Evolution/Experimental evolution adaptation adaptive laboratory evolution fluctuating light Pam68 photosynthesis RpaB Synechocystis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Cyanobacteria uniquely perform oxygenic photosynthesis using photosystems I and II (PSI, PSII), capturing light to drive water-splitting and create a proton gradient for ATP synthesis and CO 2 fixation 1,2 . While light is essential, high (HL) or fluctuating light (FL) induces photoinhibition 3,4 . Cyanobacteria often encounter HL and FL 5,6,7 , yet FL tolerance is less understood than HL tolerance, which involves diverse adaptive mechanisms 6,8,9,10,11,12,13,14,15,16,17,18,19,20,21 . Studies on Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803 (" Synechocystis ") reveal the importance of inorganic carbon and alternative electron pathways for FL tolerance 22,23,24 . Flavodiiron proteins 23,25,26 , nitrogen assimilation 27,28 , and thylakoid respiratory activity 29 enhance FL tolerance, and Fluctuating-light acclimation protein 1 (FLAP1) 30 also plays a role. Although more FL tolerance components likely exist, no genetic enhancement of cyanobacterial FL tolerance has been reported, hindering the development of suitable production strains for FL-prone photobioreactors 31 . Similarly, improving flowering plant FL tolerance through genetic engineering has seen limited success, with a few exceptions in tobacco and soybean 32 . Further increases in acclimation potential may require an evolutionary approach entailing the identification of new FL tolerance factors and the evolution of advantageous alleles 33 . Previous adaptive laboratory evolution (ALE) studies on Synechocystis for increased HL tolerance have demonstrated the general accessibility of photosynthetic robustness to evolutionary improvement 34,35,36 . Therefore, we applied ALE to evolve novel alleles conferring FL tolerance in Synechocystis , resulting in the identification of distinct candidate mechanisms for tolerance to different types of light fluctuations. Mutations in Sll0518 and Pam68 conferred tolerance to moderate FL, while a RpaB mutation increased tolerance to both FL and HL. RESULTS Generation of FL-tolerant batch cultures and isolation and characterization of monoclonal strains We adapted Synechocystis to tolerate FL using ALE, adapting previous methods for generating HL tolerance 35 and relying on the natural mutation rate of Synechocystis 33 . Two experimental protocols were designed to progressively increase FL intensity, modifying an existing FL regimen for Arabidopsis thaliana 37 , which alternated between high (HL) and low light (LL) phases. The "FL0" protocol maintained the original 1-min HL / 5-min LL rhythm but increased light intensity: starting with 700 μmol photons m -2 s -1 for HL (HL 700 ) and 50 μmol photons m -2 s -1 for LL (LL 50 ), the intensities were gradually altered to HL 1200 and LL 12 (FL0 final ), respectively ( Table 1, Fig. 1a ). While continuous HL 1200 is lethal to the non-adapted starter strain (LT) 35 , the FL0 final conditions allowed for recovery and growth during the LL phase, as demonstrated by the productive growth of the non-evolved LT strain under both FL0 and FL0 final conditions ( Fig. 1b ). The "FL+" protocol began with the same initial conditions as FL0 but progressively shortened the LL phase to 1 min. The final cycles alternated between 1 min at LL 12 and 1 min at HL 1200 . These conditions (FL+ final ) proved lethal for the LT strain ( Fig. 1b ). Both FL0 and FL+ protocols involved 20 selective cultivation cycles on triplicate batch cultures over 20 months. After ALE, cultures showed phenotypic variations within and between triplicates ( Fig. 1c-f, Source Data 1 ). Under their respective final FL conditions, FL+ strains exhibited higher growth rates and increased cell density compared to FL0 cultures, although with lower chlorophyll content. This suggests that the FL+ final adapted strains not only tolerated the lethal light regime, but also made productive use of the greater light energy available in the FL+ condition compared to the FL0 strains, which received fewer total photons. The time-integrated FL+ final photon flux (286%) corresponded to approximately three times that of FL0 final (100%). The batch cultures were diluted, plated on solid media, and incubated under constant LL 20 ( Fig. 1g ), before individual clones were isolated, imaged and analysed with respect to their 'apparent' quantum yield of PSII ( Fig. 1h ). Note that analysing the quantum yield (Fv/Fm) in cyanobacteria can be problematic due to phycobilisome contribution to basal fluorescence and interference from respiration, and therefore this parameter is designated as apparent quantum yield or Fv - /Fm - 38,39 . The FL+ clones exhibited greater heterogeneity in Fv - /Fm - values compared to FL0 clones ( Fig. 1h, i ). Four clones from each batch were selected to represent the quantiles of Fv - /Fm - , sampling a wide range of phenotypic diversity. FL0 isolates showed a homogeneous phenotype with dark green colour and Fv - /Fm - of 0.39 ± 0.02. In contrast, FL+ clones displayed clear heterogeneity, with colours ranging from cyan to ochre and Fv - /Fm - values between 0.19 and 0.53 (0.40 ± 0.10) ( Fig. 1h, i; Source Data 1 ). Most FL0 isolates grew denser than LT under FL0 final condition, while all FL+ clones survived and accumulated high cell densities under FL+ final condition, which was lethal to the non-adapted LT ( Fig. 1j; Source Data 1 ). Mutations in FL-tolerant monoclonal strains Whole-genome analysis of the 12 FL0 and 12 FL+ monoclonal strains, using the LT strain from which these adapted strains were derived and the original motile Synechocystis PCC 6803 strain (designated "WT") as controls, yielded a mutation matrix revealing 412 mutations (234 in FL0 and 269 in FL+) absent in both LT and WT strains ( Supplementary Fig. 1a, Source Data 2 ). The majority of these mutations (349 total, 201 in FL0 and 223 in FL+) were located within coding regions. Almost all (342 total, 198 in FL0 and 218 in FL+) were single nucleotide polymorphisms (SNPs), while seven (3 in FL0 and 5 in FL+) were insertions or deletions (InDels). Among the coding region SNPs, 277 (157 in FL0 and 182 in FL+) resulted in non-synonymous exchanges, modifying the amino acid sequence of 89 proteins (53 and 56 in FL0 and FL+, respectively) with known function and affecting an additional 188 proteins (104 and 126 in FL0 and FL+, respectively) with unknown functions ( Source Data 2 ). Moreover, the 24 strains showed considerable variability in the ratios of non-synonymous to synonymous mutations (Supplementary Fig. 1b, Source Data 2 ). Almost two-thirds of the mutations were classified as 'low-frequency' (≤ 10% of the reads), while about one-quarter of the alleles were fully segregated (100% frequency) ( Supplementary Fig. 1c , Source Data 3 ). In our previous ALE for HL tolerance, we also adapted a batch culture to HL without exposing it to external mutagens in order to increase the mutation rate 35 . This provides a useful point of comparison with our FL-ALE experiment. Comparing the segregation patterns of the mutations obtained during this HL-ALE experiment reveals that the high proportion of low-frequency alleles is likely to be a characteristic of the FL-ALE rather than being due to the absence of external mutagens. FL adaptive haplotype 101 mutations were fully segregated in the evolved strains, LT, or WT, but absent from the published Synechocystis reference genome ( Source Data 2 ). Their phylogeny exhibited shorter genetic distances among themselves in the FL0 strains compared to FL+ clones ( Fig. 2, Source Data 2 ). Of the 101 fully segregated mutations, 44 were novel (not present in LT or WT), including 16 mutations in non-coding regions, 24 protein-altering mutations, and four mutations in structural RNAs. Of these 28 fully segregated mutations, affecting proteins or structural RNAs, five were common to both FL0 and FL+ strains, three were specific to FL0 strains, and 20 were specific to FL+ strains ( Table 2, Fig. 2 ). Three mutations (in rpoDI , sll0518 , pam68 ) occurred in all 24 strains, and five loci harboured multiple mutations. Functionally, the mutated genes were involved in various processes ( Table 2 ). Three mutations were selected for further analysis: sll0518 A133V , affecting a cyanobacteria-specific protein of unknown function, pam68 S113G , a missense mutation in pam68 / sll0933 , encoding a factor involved in the early assembly of PSII 40 , and rpaB T183P , affecting the regulator of phycobilisome association B, the inactivation of which reduces the efficiency of energy transfer from phycobilisomes to PSII 41 . The sll0518 A133V and pam68 S113G mutations were common to all 24 strains, suggesting that they arose early in the adaptive process. RpaB T183P was exclusive to three FL+ strains and another mutation of RpaB (RpaB D194G ) independently occurred in three other FL+ strains ( Table 2, Fig. 2 ). Recapitulating FL tolerance in LT cells and interplay of FL and HL tolerance To assess their individual contributions to FL tolerance, the sll0518 A133V , pam68S 113G , and rpaB T183P mutations were introduced into LT at the corresponding wild-type gene loci via marker-less homologous recombination. This generated three sets of strains that differed from LT only in terms of these single SNPs. According to Alphafold3 42 , all three amino-acid substitutions were predicted to cause no folding differences in their corresponding proteins ( Supplementary Fig. 2a ). The sll0518 A133V and pam68 S113G mutants showed significantly enhanced FL0 final tolerance compared to LT ( Fig. 3, Source Data 4 ). However, these strains failed to grow productively under FL+ final . In contrast, rpaB T183P displayed growth comparable to LT under FL0 final but outperformed all other strains under FL+ final . Under constant LL 12 , sll0518 A133V exhibited slightly increased growth, rpaB T183P showed slightly decreased growth compared to LT, while pam68 S113G grew similarly to LT ( Supplementary Fig. 2 , Source Data 4 ). Under constant HL 1200 , lethal for non-adapted LT cells, only rpaB T183P demonstrated productive growth. To test whether mutations conferring HL tolerance could also impart resistance to FL+ final conditions, we evaluated the growth of three previously characterized HL-tolerant strains 35 . However, none of the HL-tolerant strains exhibited growth under FL+ final ( Supplementary Fig. 3 , Source Data 4 ), implying that the mechanisms underlying tolerance to constant HL and fluctuating HL may be distinct, and that adaptations to one condition do not necessarily confer tolerance to the other. In summary, sll0518 A133V and pam68 S113G , identified in all strains adapted to FL0 final or FL+ final , confer enhanced tolerance to FL0 final but not to FL+ final . Conversely, rpaB T183P , specific to FL+ final adaptation, imparts tolerance to both FL+ final and constant HL, even in the absence of the four common mutations found in the FL-adapted haplotypes. This suggests that the FL-adapted haplotype mutations are not prerequisite for FL+ final or HL tolerance and that mutations conferring HL tolerance do not necessarily provide tolerance to FL+ final . Pam68 S113G enhances the accumulation and activity of PSII under FL To understand the effects of the pam68 S113G mutation, we created a Pam68 overexpression strain ( pam68oe ) and compared its growth to a pam68 knockout mutant ( ins0933 ) 40 under both LL 12 and FL0final ( Supplementary Fig. 4a-c , Source Data 5 ). Under FL0 final , ins0933 exhibited slower growth than LT for up to 120 h. This was followed by a sudden acceleration, resulting in an OD 730nm seven days after inoculation that was 32% higher than that of LT. The final OD 730nm of pam68 S113G , in contrast, was 92% higher. The pam68oe strain grew 67% better than LT, slightly less than pam68 S113G , but not significantly different from ins0933 and pam68 S113G . No significant growth differences were seen among the strains under LL 12 . Since Pam68 is involved in PSII assembly 40,43 , we examined thylakoid protein levels. To this end, we grew LT and two independent pam68 S113G mutant strains under FL0 final conditions. We also tested our standard LL control condition (LL 50 ) and found that pam68 S113G cultures had a significantly higher OD 730nm than LT under this condition ( Fig. 4a-c , Source Data 5 ). Therefore, we used the LL 50 and FL0 final conditions to study thylakoid protein accumulation in the pam68 S113G and LT strains. Immunoblot analysis was used to quantify the levels of Pam68 and representative thylakoid proteins (D1, PsaA, AtpB), while allophycocyanin (APC) and phycocyanin (PC) were quantified via in-gel fluorescence ( Fig. 5a, b; Source Data 5 ). Under LL 50 conditions, the levels of Pam68 (+1%), D1 (+7%), PsaA (-9%), and AtpB (+8%) were found to be non-significantly altered in pam68 S113G compared to LT, while APC+PC levels were found to be significantly increased (+14%; p=1.94x10 -3 ). Under FL0 final conditions, pam68 S113G cells showed reduced levels of Pam68 (-17%; p=4.90x10 -2 ) and APC+PC (-9%; p=3.88x10 -4 ) compared to LT. Meanwhile, the levels of D1 (+35%; p=3.45x10 -5 ) and PsaA (+16%; p=1.14x10 -3 ) were increased, while AtpB levels remained unchanged. These results suggest an increase in photosynthetic complex accumulation in pam68 S113G mutants under FL0 final conditions, but not under constant LL 50 conditions. This also suggests that pam68 S113G is likely a gain-of-function mutation, as its beneficial effect on growth under FL0 final exceeds that of Pam68 overexpression, despite lowered Pam68 protein levels. The reasons for the slight decrease in mutant Pam68 levels compared to WT Pam68 under FL conditions remain unclear. The serine that mutated to glycine at position 113 in Synechocystis Pam68 is conserved from cyanobacteria to flowering plants, corresponding to serine at position 174 of Arabidopsis thaliana PAM68 (AtPAM68, At4g19100) 40 . To evaluate the adaptive potential of this S→G mutation across species, WT AtPAM68 ( AtPAM68 WT ) and AtPAM68 S174G were expressed in Synechocystis LT and ins0933 , and growth was observed under LL 50 , HL 700 and FL0 final conditions ( Fig. 4a-c ). No increase in growth was observed under LL 50 and FL0 final conditions compared to the corresponding controls (LT or ins0933 ). However, the overexpression of AtPAM68 WT in the ins0933 mutant background significantly increased growth at HL 700 . Conversely, the mutated AtPAM68 S174G did not exhibit this effect in the ins0933 background. This demonstrates that, despite the evolutionary distance between land plants and cyanobacteria, the plant PAM68 can still functionally replace its cyanobacterial counterpart. Moreover, the importance of this amino acid position is also conserved in the two Pam68 proteins, with a serine-to-glycine exchange resulting in pronounced phenotypic changes under specific light conditions. However, it seems that the rest of the protein sequence determines whether an increased tolerance to FL0 final or HL 700 results from a serine or a glycine at this conserved position (glycine in Synechocystis Pam68 for tolerance to both FL0 final and HL 700 ; serine in AtPAM68 for HL 700 tolerance). Therefore, it can be concluded that the role of PAM68 in FL tolerance is most likely not conserved in flowering plants. To investigate the role of Pam68 S113G in the increase of PSII levels (see Fig. 5a,b ), we analysed PSII complex assembly using two-dimensional clear-native (CN)/SDS-PAGE, followed by immunoblot analysis ( Fig. 5c, Source Data 5 ). In-gel Chl a fluorescence indicated an increase in PSII dimer abundance under both LL 50 and FL0 final conditions ( Fig. 5c ), thus corroborating the results of the SDS-PAGE immunoblots. Analysis of the second dimension by immunoblotting showed a similar distribution of Pam68 and Pam68 S113G signals between the low- and high-molecular-weight fractions, indicating no overall change in the interaction patterns of mutant Pam68 and corroborating the SDS-PAGE immunoblot results (see Fig. 5a, b ). Furthermore, pulse labelling experiments showed that the pam68 S113G mutant exhibited a pronounced reduction in de-novo biosynthesis of membrane proteins, with a clear decline in unassembled D1 and CP43 protein in RCIIa and CP43m assembly intermediates as compared to LT under LL 50 ( Supplementary Fig. 5 ). This reduction was confirmed by immunodetection of D1 and CP43 on the 2D blot ( Fig. 5c ), which may indicate enhanced stability of mature PSII complexes, as these showed no decrease in steady-state levels ( Fig. 5a, b ) 44 . To further assess the physiological effects of the S113G mutation of Pam68, PSII activity, respiration rates, apparent PSII quantum yield (Fv - /Fm - ) and P700 oxidation kinetics were determined ( Supplementary Fig. 6 ). Under LL 50 , PSII activity, as measured as O 2 evolution (see Methods ), was slightly lower in pam68 S113G than in LT when normalized to OD 730nm , but slightly higher when normalized to Chl a ( Supplementary Fig. 6a ). This suggests that the pam68 S113G mutant has a lower Chl a /OD 730nm ratio. Under FL0 final , however, PSII activity significantly increased per unit OD 730 (+227%; p=1.22x10 -23 ) and per mg Chl a (+212%; p=5.11x10 -22 ). Respiration per unit OD 730 was moderately decreased (-16%; p=2.70x10 -2 ) under LL 50 and not significantly decreased (-5%; p=7.21x10 -1 ) under FL0 final ( Supplementary Fig. 6b ). In-vivo fluorimetry also indicated an increase in Fv - /Fm - of LL 20 - (+21%; p=4.55x10 -16 ) and FL0 final - (+118%; p=2.75x10 -21 ) adapted pam68 S113G mutants ( Supplementary Fig. 6c ), further suggesting that pam68 S113G enhances PSII performance. In addition, pam68 S113G was also observed to display delayed P700 oxidation when exposed to far-red (FR) light following a prolonged period of incubation in the dark, and to undergo accelerated P700 re-reduction when FR was switched off ( Supplementary Fig. 6d,e ). This corresponds to an increase in the time required for half-maximum P700 oxidation (t 0.5 P700 ox ) under LL 50 (+50%; p=6.3x10 -10 ) and FL0 final (+15%; p=5.20x10 -4 ) conditions, and a decrease in the half-time of re-reduction (t 0.5 P700 red ) (LL 50 : -66%; p=2.33x10 -12 ). FL0 final : -43%; p=2.46x10 -7 ) ( Supplementary Fig. 6d,e ). High t 0.5 P700 ox and low t 0.5 P700 red values can be taken as an indirect measure of high CEF, as the oxidation of P700 upon exposure to FR light following the depletion of respiratory donors by prolonged dark incubation is primarily mediated by CEF 45,46 . The increased CEF could contribute to enhanced FL tolerance by alleviating PSI acceptor site limitation. The rpaB T183P mutation is associated with downregulation of light harvesting RpaB/Slr0947 plays a crucial role in regulating energy transfer from phycobilisomes to photosystems 47,48,49 . This suggests that reduced light harvesting capacity may contribute to the enhanced tolerance of rpaB T183P cultures to HL and FL+ final that are lethal to the starter strain. To investigate the functional implications of this mutation, an RpaB overexpression strain ( rpaBoe ) was generated and compared with a previously characterized RpaB knock-down mutant ( rpaBkd ) 49 under constant HL 1200 and FL+ final . Notably, only the rpaB T183P mutant demonstrated productive growth under both conditions, while LT, rpaBkd , and rpaBoe strains failed to grow ( Supplementary Fig. 7, Source Data 6 ). Furthermore, LT and rpaB T183P had similar endpoint OD 730nm values under LL 50 ( Fig. 6a , Source Data 6 ). However, under HL 700 , rpaB T183P showed markedly higher values, and under FL+ final , only rpaB T183P grew while LT failed. Immunoblot analysis and in-gel fluorescence quantification showed slightly increased RpaB levels in rpaB T183P under LL 50 (+37%; p=1.10x10 -3 ) and HL 700 (+22%; p=1.94x10 -3 ) conditions, compared to LT. At LL 50 , no significant changes were observed in D1 (+11%; p=2.30x10 -1 ), PsaA (+6%; p=2.74x10 -1 ), or AtpB levels (-3%; p=5.31x10 -1 ), while APC+PC levels were significantly reduced (-22%; 1.03x10 -8 ) in rpaB T183P ( Fig. 6b, c; Source Data 6) . Under HL 700 , D1 levels were significantly reduced (-21%; p=2.32x10 -3 ), while non-significant changes in PsaA (+16%), AtpB (+2%) and APC+PC levels (-15%) were observed ( Fig. 6b, c ). This indicates a general downregulation of peripheral antennas in rpaB T183P , with differential effects on PSI and PSII under LL and HL conditions. Consistently, the ratio between PC and Chl a absorption maxima was significantly reduced in rpaB T183P under both LL 50 and HL 700 ( Supplementary Fig. 8 a, Source Data 6 ), indicating downregulation of light harvesting. A comparison of the 77K emission spectra of blue-light-treated and dark-treated cells grown in LL 50 revealed a decrease in the S2-to-S1 state transition in rpaB T183P compared to LT (-9% at an excitation wavelength of 600 nm; p=6.85x10 -5 ) ( Fig. 6d ). The same trend was observed in cells grown at HL 700 (-9%, p=3.81x10 -2 ), thus supporting previous suggestions that RpaB regulates state transitions 50 . Importantly, HL 700 -acclimated cells showed strong evidence of blue-light-induced phycobilisome decoupling in both LT and rpaB T183P , as indicated by a strong increase in emission around 650 nm. No such effect was observed in LL 50 -acclimated cells ( Fig. 6d ). Fluorescence emission spectra were collected at low temperature (77K) with an excitation wavelength of 435 nm. This revealed a significantly lower PSI:PSII ratio in LL 50 -acclimated rpaB T183P cells compared to LT (-19%; p=1.25x10 -8 ), but a significantly higher ratio in HL 700 -acclimated cells (+30%; p=1.12x10 -5 ) ( Supplementary Fig. 8b, c ). These results are consistent with the quantification of D1 and PsaA immunoblots ( Fig. 6b,c ). Moreover, 77K analyses performed at an excitation wavelength of 600 nm ( Source Data 6 ) showed a statistically significant increase in the PC:APC ratio in rpaB T183P under LL 50 (+27%; p=3.96x10 -14 ) and HL 700 (+43%; p=7.99x10 -16 ) conditions ( Supplementary Fig. 8c ), which suggests an increase in average rod length under both light conditions. Meanwhile, PSII:APC levels in rpaB T183P significantly increased under both LL 50 (+16%; p=2.56x10 -7 ) and HL 700 (+17%; p=5.13x10 -5 ) conditions ( Supplementary Fig. 8c ). These observations suggest that rpaB T183P has a differential effect on photosystem stoichiometry and peripheral antenna accumulation: PSII accumulation increases under continuous LL, while phycobilisome accumulation decreases under LL and moderate HL. In vivo fluorescence measurements under orange-red actinic light also revealed that non-photochemical quenching (qN) 51 in rpaB T183P cells increased in LL 50 -acclimated cells and decreased in HL 700 -acclimated cells compared to LT ( Supplementary Fig. 8 d,e). These results suggest that state transitions, the main contributor to qN in cyanobacteria under low light 38,52 , may have been increased in LL 50 -acclimated and decreased in HL 700 -acclimated rpaB T183P mutants compared to the LT control. Notably, the increase in qN in LL 20 -acclimated rpaB T183P mutants, despite a decrease in the S2-to-S1 ratio ( Fig 5d ), suggests a disproportionate change in other qN components. Next, we determined the physiological effects of the T183P mutation of RpaB on PSII activity, respiration rate, Fv - /Fm - , and P700 oxidation kinetics. In addition to LL 50 conditions, only the effect of HL 700 could be investigated, as experiments with FL+ final conditions were impossible (it was lethal to the LT control), and FL0 final conditions were not informative ( rpaB T183P mutants performed LT-like under this condition; see Fig. 3 ). Moreover, P700 oxidation could not be monitored under HL 700 conditions due to P700 overreduction in both genotypes. Under LL 50 conditions, PSII activity increased significantly in the presence of DCBQ in rpaB T183P mutants, both per unit OD 730 (+35%; p=1.54x10 -5 ) and per mg Chl a (+35%; p=3.68x10 -5 ), compared to LT ( Supplementary Fig. 9a ). The respiration of rpaB T183P did not change significantly under LL 50 compared to LT (-3%; p=7.76x10 -1 ), but increased significantly under HL 700 per unit OD 730 (+107%; p=6.31x10 -5 ) ( Supplementary Fig. 9b ). Consistently, Fv - /Fm - of rpaB T183P cells incubated at LL 20 was significantly higher than that of LT cells (+72%; p =2.45x10 -38 ) ( Supplementary Fig. 9c ). At the same time, rpaB T183P showed a P700 oxidation and re-reduction behaviour indicative of a substantial rise in CEF activity. This is evidenced by a notably extended P700 oxidation half-time (+54% compared to LT; p =2.33x10 -13 ) and a significantly reduced re-reduction half-time (-76% compared to LT; p =4.4x10 -5 )( Supplementary Fig. 9d,e ). Taken together, these results suggest that the photosynthetic electron transport chain in rpaB T183P undergoes profound changes, reducing PSII abundance and phycobilisome-mediated light harvesting under moderate HL, while maintaining PSII efficiency under LL conditions. Consistent with the immunoblot and 77K analyses, an mRNA sequencing experiment on cells grown under LL 50 or HL 700 conditions showed that the RpaB T183P exchange had only a minor effect on the expression of most photosynthetic genes. Under LL 50 conditions, a uniform downregulation of PSI structural subunit and phycobilisome-related genes was observed ( Fig. 7 ). Conversely, under HL 700 , most PSII structural subunit genes were found to be upregulated, while many NDH genes, some PSI genes and most phycobilisome-related genes were downregulated in rpaB T183P ( Fig. 7 ). This contrasts with the results of immunoblot and 77K analyses, which indicated lowered PSII and increased PSI levels under HL 700 . These findings suggest a complex response involving altered transcript accumulation as a consequence of the T183P substitution. This altered transcript accumulation could be due to an altered binding affinity of the mutated RpaB for its target genes, an altered target spectrum, or secondary changes to the transcriptome in response to the physiological effects of the mutation. Nevertheless, the mutation does not result in corresponding changes in protein levels with respect to the two photosystems, but it does with respect to phycobilisome-related genes. DISCUSSION Fluctuations in light intensity represent one of the most rapid and severe environmental stressors that photosynthetic organisms must cope with 25,53 . Consequently, the molecular mechanisms underlying FL tolerance and their application in crop improvement are being intensively investigated. ALE using cyanobacteria as chloroplast proxies has been previously employed to address HL-related stress 34,35,36 . In this study, evolutionary screening under two complex FL regimes identified new FL tolerance factors and adaptive alleles. Among 412 candidate mutations, three non-synonymous SNPs in genes encoding the protein Sll0518 with unknown function, the PSII assembly factor Pam68, and the Response Regulator RpaB, were reconstituted in the parental LT background and confirmed to confer varying yet specific FL adaptation, thus demonstrating that FL tolerance can be improved through ALE. The sll0518 A133V mutation promotes growth under both non-lethal FL0 final and LL 12 , but not HL 1200 . Furthermore, it does not facilitate tolerance to FL+ final (see Fig. 3 and Supplementary Fig. 2 ), indicating a specific role in FL acclimation. This mutation was observed in all monoclonal strains (see Table 2 , Fig. 2 ), further supporting its adaptive nature. Sll0518 has recently been found to co-immunoprecipitate with the RNA recognition motif protein Rbp3, which interacts with ribosomes and the 3’-ends of mRNAs encoding photosynthesis proteins and the absence of which lowers the PSI:PSII ratio 54 . The precise function of Sll0518 remains unclear, but it can be speculated that the protein may play an indirect role (via Rpb3) in the accumulation of PSI, which is a key target of FL-induced photodamage 25 . The pam68 S113G mutation, present in all 24 monoclonal strains, enhanced FL0 final tolerance. However, it did not improve growth under LL 12 or HL 1200 conditions, nor could it mediate FL+ final tolerance (see Fig. 3 and Supplementary Fig. 2 ). This aligns with previous studies that reported a lethal phenotype in Pam68 deletion mutants exposed to alternating darkness and HL conditions 55 . Overexpression of WT Pam68 also enhanced FL0 final tolerance, albeit to a lesser extent than in pam68 S113G (see Supplementary Fig. 4 ). Total Pam68 S113G protein levels decreased under FL0 final conditions compared to LT Pam68 levels ( see Fig. 4 ). Additionally, pam68 S113G slightly stimulated the accumulation of PSII core and peripheral phycobililisome antenna proteins under LL 50 (see Fig. 4 ). However, as no growth improvement was observed under LL 12 conditions (see Supplementary Fig. 2 ), carbon assimilation, rather than light energy harvesting and conversion, may be the limiting factor for growth under LL conditions, as previously proposed 16,56 . Pam68 promotes the accumulation of PSII assembly intermediates RCa and RCb 40 , as well as CP47 biosynthesis and chlorophyll ligand insertion 55 . Surprisingly, the pam68 S113G mutant showed reduced de-novo biosynthesis of membrane proteins and decreased accumulation of PSII assembly intermediates, while dimeric PSII was found increased (see Fig. 4d ). This effect aligns with the increased abundance of dimeric PSII observed in Pam68-depleted strains 40 . However both the apparent PSII quantum yield and PSII activity were significantly increased in pam68 S113G cells (see Supplementary Fig. 6a,c ), indicating increased PSII stability in pam68 S113G . The apparently increase in CEF activity ( Supplementary Fig. 6d ) is difficult to explain in this context. A PAM68 orthologue (PAM68-LIKE) acts as an NDH-1 complex assembly factor in Arabidopsis 57 . As there is no second pam68 homologue in Synechocystis , it could be speculated that the Synechocystis Pam68 protein fullfils both functions, with the Pam68 S113G mutation increasing NDH-dependent CEF activity. However, inactivation of Pam68 had no effect on NDH-1 assembly in Synechocystis 57 , which suggests that this is not the case. In sum, our findings suggest that pam68 S113G is likely a gain-of-function mutation. Meanwhile, the precise molecular mechanism causing the depletion of mutant Pam68 S113G protein levels under FL conditions remains to be elucidated in future studies. The described pam68 gain-of-function mutation appears to be organism-specific, as a serine-to-glycine substitution at the homologous position of AtPAM68 did not increase FL tolerance when expressed in Synechocystis (see Fig. 4 ). Nevertheless, expressing the WT AtPAM68 protein, but not the mutant AtPAM68 S174G protein, in the pam68 knock-out background significantly increased Synechocysti s growth under HL 700 conditions. This suggests that, together with the glycine residue at position 174, substitutions in other positions of the Arabidopsis PAM68 protein confer HL 700 tolerance, likely due to evolutionary adaptation to the increased light intensities associated with a terrestrial light style. Therefore, while the Ser113/174 position is crucial and conserved for Pam68/PAM68 activity, the requirements for a gain of function under stressful light conditions have changed over evolutionary time. RpaB is a redox-responsive OmpR-type transcription factor. It was first identified in Synechocystis due to its ability to alter the energy distribution from phycobilisomes to PSI compared to PSII 41 . RpaB regulates at least 137 promoters of protein-coding genes or operons, as well as 22 non-coding RNAs, including many genes involved in photosynthesis 50 . The rpaB gene is essential 41,58 , and orthologues have been identified in the plastid genomes of algae that also possess genes encoding phycobiliproteins 47 . RpaB binds to the high-light regulatory 1 (HLR1) sequence in PSI gene promoters 49 , and under LL, it can function as either an activator or a repressor, depending on the position of the HLR1 sequence. Under HL, however, it loses binding activity. Consequently, RpaB can repress certain HL-inducible genes under LL and activate other genes, such as those encoding PSI subunits 48,59,60 . In Synechococcus , this involves the reversible phosphorylation of RpaB 58,61 . RpaB is also redox-regulated via a thiol switch; active dimers dissociate into less active monomers upon reduction by thioredoxin 62,63 . This links its function to the redox state of the photosynthetic transport chain. We identified two non-synonymous mutations in the rpaB gene ( Table 2 ). Notably, neither mutation affected the thiol switch residue (Cys59) or the likely phosphorylation site (Ser198 64 ). The rpaB T183P strains remained uniquely viable under FL+ final and HL 1200 , grew comparably to LT under FL0 final , and had a lower growth rate at LL 12 compared to LT (see Fig. 3, Supplementary Fig. 2 ). This suggests a trade-off: enhanced tolerance to HL in both continuous and fluctuating application at the expense of LL performance. RpaB T183P levels were significantly increased under both LL 50 and HL 700 . Previous studies have shown that rpaB knock-down impairs growth under LL but promotes growth under HL conditions 49,65 . However, in our study, neither knock-down nor overexpression of rpaB had positive effects on growth under HL 1200 and FL+ final (see Supplementary Fig. 7 ), demonstrating that rpaB T183P confers additional functionality to RpaB T183P , possibly by downregulation of PSII core and peripheral antenna proteins under elevated light intensities (see Fig. 6 and Supplementary Fig. 8 ). This aligns with observations from suppressor screens in Arabidopsis thaliana pgr5 mutants, where increased FL sensitivity was overcome through mutational disruption of the photosynthetic electron transport chain to prevent PSI damage 66 . The rpaB T183P mutant strains exhibited a notable decrease in PC:Chl a ratios, particularly under HL 700 (see Supplementary Fig. 8 ), suggesting that increased FL tolerance is linked to reduced light energy harvesting capacity through peripheral antennas. This was accompanied by a significant reduction in phycobilisome and PSII fluorescence emission, consistent with previous findings in rpaB knockdown strains 41 . Under HL 700 , the mutant cells showed a marked decrease in qN 67 (see Supplementary Fig. 8 ), likely due to reduced PSII and phycobilisome levels. In contrast, under LL 50 conditions, the mutant displayed a reduced abundance of peripheral antenna proteins, decreased S2-to-S1 state transition, as well as an increased qN (see Fig. 6 and Supplementary Fig. 8 ). The latter is consistent with reports of increased OCP-dependent quenching of PSII in state 2 cells 68 . Fostered state 2 persistence could also explain the increased CEF activity around PSI 69 (see Supplementary Fig. 9 ). In rpaB T183P , PsaA accumulation was found to be slightly, yet non-significantly, increased under LL 50 and HL 700 conditions (see Fig. 6 ). At the same time, mRNA-seq data indicate the repression of most phycobilisome-related and PSI genes in rpaB T183P under both LL 50 and HL 700 conditions (see Fig. 7 ). This suggests that RpaB T183P may have lost some of its PSI-gene activating activity under LL and some of its PSI-gene repressing activity under HL. At the same time, PsaA protein levels no longer directly reflect mRNA levels. Alternatively, the observed changes to the transcriptome may be independent of altered binding of the mutated RpaB to the corresponding genes and may instead represent compensatory effects due to the physiological changes triggered by the RpaB T183P mutation. Moreover, reduced PSII levels under HL 700 appear to be functionally uncoupled from both transcription regulation and PSI levels. This is evident from the slight increase in PSII gene transcripts and the fact that PSII levels do not closely track changes in PSI levels. In contrast, PSI levels tend to follow alterations in PSII levels more closely 70,71 . Combined with the improved viability of the mutant under HL and FL conditions, this suggests that rpaB T183P is a gain-of-function mutation. This shifts the regulatory activity of RpaB from PSI to PSII and its peripheral antennas (see Supplementary Fig. 9 ). At the same time, it suppresses phycobilisome accumulation and S2-to-S1 transition (see Fig. 6 and Supplementary Fig. 8 ). This, together with the apparently increased CEF, protects PSI from acceptor-site limitation. Given that RpaB-like proteins are conserved across cyanobacterial and some plastid phylogenies 47 , their homologues in eukaryotic algae could potentially be targeted to enhance FL tolerance. However, because land plants lack phycobilisomes, RpaB-based tolerance is not feasible. Nevertheless, the rationale behind RpaB photoprotection could be mimicked by increasing the PSI/PSII ratio and downregulating antenna size. Taken together, single amino acid exchanges in various cyanobacterial proteins can increase tolerance to FL. Two of these mutations, pam68 S113G and rpaB T183P , have been examined in greater detail in this study. This provides the first physiological insights into how these mutations modulate FL tolerance. Cross-species experiments with the Pam68 protein indicate that the FL tolerance function of the mutation found in Synechocystis is not conserved in land plant. Therefore, given that green algae are much closer relatives of flowering plants, it seems more practical to use ALE with green algae to identify adaptive mutations that might also function in flowering plants. METHODS Synechocystis strains: generation and culture conditions Synechocystis sp. PCC 6803 glucose-tolerant cells, referred to as “laboratory type” (LT) were kindly provided by Himadri Pakrasi (Washington University, St. Louis, USA). Previously described knock-out and knock-down mutants of pam68 and rpaB were utilized 40,49 . Reconstruction of sll0518 A133V , pam68 S113G , rpaB T183P in the LT background employed established methodology 35 with plasmid vectors constructed using a pUC57-mini vector backbone derived from IMBB2.4‐pUC57‐mini kindly provided by Professor Neil Hunter (University of Sheffield). The fragment of codon-optimized pam68 from Arabidopsis thaliana (AT4G19100.1) without cTP ( Atpam68 ) was synthesized by Invitrogen GeneArt Strings (Thermo Fisher Scientific, MA, USA). Overexpression mutants of pam68, rpaB, and Atpam68 were generated through homologous recombination using non-replicative vectors derived from IMBB2.4-pUC57-mini and pICH69822 (obtained from E. Weber, Icon Genetics GmbH, Halle, Germany), respectively. These constructs were assembled via Gibson assembly and targeted to the genomic neutral site slr0168 , with pam68 and rpaB coding sequences expressed under the control of the strong psbA2 and rbcL promoters, respectively. Cultures were typically grown under continuous illumination at 30 μmol photons m⁻² s⁻¹ of white fluorescent light (OSRAM HE28W/830 Lumilux warm white Hg fluorescent lamps) at 23 °C. This temperature aligns well with the average maximum temperature recorded in Oakland, California, over the year (https://weatherspark.com/y/541/Average-Weather-in-Oakland-California-United-States-Year-Round), where the original strain was isolated 72 . Liquid cultures were inoculated at an initial OD = 0.05 in BG11 photoautotrophic medium, with 5 mM glucose added for pre-transformation cultures. Growth was conducted in Multi-Cultivator MC 1000-OD devices, equipped with an AC-700 cooling unit and a warm-white LED panel (Photon System Instruments, Drasov, Czech Republic). For solid media growth, BG11 was supplemented with 0.75% (w/v) bacteriological agar. Adaptive evolution of Synechocystis under fluctuating light Two FL adaptive evolution experiments were conducted using Synechocystis , relying on its natural mutation rates 35 to identify new adaptive alleles. The experiments began with six separate batch cultures derived from a Synechocystis LT stock culture. Each culture was grown in a 100 mL glass tube within a temperature-controlled water bath of a multicultivator. The propagation cycles involved 70 mL of medium with an initial OD 730nm of 0.05, incubated under constant aeration at 23 °C with fluctuating warm-white LED illumination for 7-14 days. The light fluctuations were progressively intensified in both amplitude and frequency throughout the selection process. The FL0 regime alternated between 1 min of high light (HL) and 5 min of low light (LL) throughout the entire adaptive laboratory evolution (ALE) protocol. The selection process began with five cycles of 50 µmol photons m -2 s -1 (LL 50 ) and 700 µmol photons m -2 s -1 (HL 700 ), followed by three cycles with varying LL and HL intensities. The final 12 cycles used a regime of 5 min at LL 12 and 1 min at HL 1200 . The FL+ regime also started with 1 min of HL followed by LL, but the LL period was progressively shortened from 5 min to 1 min. The initial cycle matched that of the FL0 regime, with subsequent cycles gradually reducing LL intensity and increasing HL intensity. The final 12 cycles alternated between 1 min of LL 12 and 1 min of HL 1200 . Both FL0 and FL+ experiments consisted of 20 selective cycles in total, resulting in three evolved batch cultures for each condition, labeled FL0_a 20 , FL0_b 20, FL0_c 20 and FL+_a 20 , FL+_b 20 , FL+_c 20 , respectively ( Fig. 1a ). Further details of the selective cycle protocols can be found in Table 1 . Isolation of FL-adapted Synechocystis clones for genome re-sequencing Single clones were isolated by plating dilutions (10 -6 -10 -7 ) of FL0_a 20 , FL0_b 20 , FL0_c 20 and FL+_a 20 , FL+_b 20 , FL+_g 20 , onto solid BG11 media. Isolation plates were incubated at 30 µmol photons m -2 s -1 continuous illumination and 23 °C for seven days. Representatives of mutant subpopulations were sampled by selecting different clones based on colony color and size and isolated clones were grown on solid BG11 media for seven days. Subsequently, clones for genome re-sequencing were selected as previously described 35 based on room-temperature fluorescence parameters measured by FluorCam 800MF (Photon Systems Instruments, Drasov, Czech Republic). For FL0 and FL+, n = 66 and n = 72 isolated clones were assessed. To capture the genetic variability within each batch culture, clones best representing the quartiles of the observed the Fv - /Fm - distributions ( i.e. , two extreme and two intermediate values of PSII quenched quantum efficiency) were selected for whole-genome resequencing, totaling n = 12 for FL0 and FL+, respectively. Synechocystis genomic DNA extraction and sequencing Genomic DNA for whole‐genome sequencing was isolated from cell pellets of 10–30 mg fresh weight following the manufacturer’s protocol (EasyPure® Plant Genomic DNA Kit, TransGen Biotech Co., Ltd., Beijing, China). Cells were broken in EasyPure®lysis buffer using a 1:1 mixture of small glass beads (425–600 µm + 212–300 µm, Sigma Aldrich, St. Louis, MO, United States) and a TissueLyser II (QIAGEN, Hilden, Germany). DNA isolates were then subjected to agarose gel electrophoresis to assess structural integrity. The genomes of 24 monoclonal mutants (four per adapted batch culture) were then re‐sequenced on the Illumina HiSeq platform (2 × 150‐bp paired‐end reads) by NovoGene Ltd. (Cambridge, United Kingdom). Sequence data quality control and filtering The previously published Synechocystis LT t=0 genome assembly 35 served as the control for excluding background mutations; all mutations identified in FL-ALE were tracked relative to the LT t=0 assembly. Adapting previously described methodology 35 , the quality of the WGS raw data was assessed using FastQC v0.11.9 73 . Pre-processing began with Cutadapt v4.1 74 , which filtered out low-quality reads and removed sequences containing adapter contamination or more than 10% undetermined bases (‘N’ bases). Rcorrector 75 was then used to perform k -mer correction on the filtered datasets, applying the default k -mer length setting. The resulting dataset, consisting of filtered and corrected reads, was used for subsequent mutation detection. Genome resequencing of the 24 single clones yielded an average coverage of 339±44/638±137/300±65/116±42/577±193-fold for chromosome/pSYSM/pSYSA/pSYSG/pSYX, respectively (see Source Data 2 ). Variant analysis The Breseq pipeline 76 were applied for the identification of potential mutations. The clean reads were aligned to the Synechocystis sp. PCC 6803 reference genome (ASM972v1) obtained from the NCBI database using bowtie2 v2.5.1 77 . The generated SAM alignment files were then used for variant calling. Breseq analysis was conducted in two distinct modes. The ‘consensus’ mode defined a mutation as ‘fixed’ when its frequency was ≥0.80, while considering a site as ‘polymorphic’ when the variant frequency ranged between 0.20 and 0.80. On the other hand, in the ‘polymorphism’ mode, a mutation was designated as ‘fixed’ at frequencies ≥0.95 and as ‘polymorphic’ if its occurrence spanned frequencies between 0.05 and 0.95. Identified variants are listed in Source Data 2 . For an overview of fully segregated, protein-affecting mutations identified in FL-ALE strains, see Table 2 . Phylogenetic analysis The phylogenetic analysis was carried out considering 101 polymorphic sites representing all deviations from the reference genome (ASM972v1) with a 100% frequency ( i.e ., identified as fully segregated in at least one sample). IQ-TREE multicore version 2.2.6 78 was used to perform the subsequent phylogenetic analysis. Briefly, the model selection method was applied with default parameters to identify the most suitable model for the data set. Afterwards, the selected model (according to Bayesian Information Criterion “BIC” values), that is Kimura 2 Parameter (K2P) with equal frequencies, was applied to infer the maximum likelihood phylogenetic relationships between the samples. The bootstrapping method was applied to validate the generated tree with 500 replicates. The resulting phylogenetic tree was generated using CLC Main Workbench (QIAGEN, Venlo, Netherlands). Pigment extraction and quantification, determination of phycocyanin:chlorophyll ratios Chlorophyll a (Chl a ) and total carotenoids (Cars), were extracted and quantified as previously described 35 . Molar ratios of the peripheral antenna pigment phycocyanin (PC) to core antenna pigment Chl a in cultures seven days past inoculation were estimated as previously described 35 . Protein extraction, detection and quantification Cells were collected from a 3-mL suspension at OD 730nm = 10 by gentle centrifugation, and the pellets were snap-frozen in liquid N 2 and stored at −80 °C. The cell pellets were then homogenized and lysed in 600 µL of homogenization buffer (0.4 M sucrose, 10 mM NaCl, 5 mM MgCl 2 , 20 mM Tricine, adjusted to pH 7.9 with HCl), supplemented with protease inhibitors (cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail, Roche AG, Basel, Switzerland) and approximately 300 µL of a glass bead mixture. Lysis was performed using a mixer mill (MM 400, RETSCH, Haan, Germany) with five cold-lysis cycles (5 min at 30 Hz). After centrifugation at 4 °C, the supernatant was collected, and protein concentration was estimated using Bradford (ROTI-Quant, Carl Roth, Karlsruhe, Germany) and BCA (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, Waltham, MA, USA) protein assays.  Chl a concentration was estimated as described above. Samples were stored at −20 °C until further processing. For SDS-PAGE, protein extracts (equal Chl a content) were mixed with 5× SDS loading dye, denatured (37 °C, 60 min) and size-separated on 10% Tris-Tricine gels. Phycocyanin and allophycocyanin were quantified by recording fluorescence (λ emission ≥ 600 nm) directly from the gels 79 (Fusion FX imaging system, Vilber, Collégien, France; excitation wavelength of λ excitation = 530 nm). Image analysis and signal quantification were conducted using ImageJ 80 . Proteins were then transferred to PVDF membranes (Immobilon-PSQ, Millipore, Burlington, MA, USA) via electroblotting. Membranes were stained with Coomassie Brilliant Blue (CBB) for loading control, then de-stained before immunodetection. For specific protein detection, membranes were cut or used whole, blocked with 1.5% (w/v) BSA in TBST, and incubated with primary antibodies against PsaA, PsbA, and AtpB (Agrisera, Vännäs, Sweden), Pam68 (kindly provided by Prof. Dr. Jörg Nickelsen, LMU Munich, Germany), and RpaB (PhytoAB, San Jose, CA, USA). After overnight incubation with primary antibodies at 4 °C and 2-hour incubation at room temperature with horseradish-peroxidase coupled secondary antibodies, chemiluminescence was detected using SuperSignal™ West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA, USA) and imaging system (Fusion FX imaging system, Vilber, Collégien, France). Signal quantification was performed using ImageJ software 80 . Preparation of the membrane fraction and analysis of proteins by clear native PAGE To isolate the cellular membranes, the cells were disrupted using zirconia–silica beads in a Precellys Evolution tissue homogeniser (Bertin Instruments, France). The membrane and soluble fractions were then separated by centrifugation at 36,000×g for 20 min. The membranes were then resuspended in buffer A (25 mM MES/NaOH, pH6.5, 10 mMCaCl 2 , 10 mM MgCl 2 , 25% glycerol) and, after measuring the Chl concentration, solubilised with β-dodecyl-maltoside (DDM, final concentration 1% (w/v)) and analysed using two-dimensional PAGE consisting of clear native (CN) PAGE in a 4–14% gradient gel 81 and SDS-PAGE in a denaturing 16–20% gradient gel containing 7 M urea (2D-CN/SDS-PAGE). For autoradiography, the gels were stained with Coomassie Blue (CBB), destained, dried, and exposed to a phosphorimager plate for 144 hours. For protein detection, the gels were stained with SYPRO Orange and subsequently transferred to a polyvinylidene fluoride (PVDF) membrane. The primary antibodies used in the study were raised in rabbits against the following: (i) D1 (residues 58–86 of the spinach D1 polypeptide); (ii) CP47 (residues 380–394 of the barley polypeptide); (iii) CP43 (Agrisera catalogue no. AS11 1787); and (iv) PAM68 (residues 1–63 of the Synechocystis polypeptide 40 ). The antibodies were used sequentially in the following order: PAM, CP43, CP47 and D1. The blots were developed using an anti-rabbit secondary antibody conjugated with horseradish peroxidase (Merck, USA), alongside a chemiluminescence substrate (Immobilon Crescendo, Merck, USA). Radioactive labelling Radioactive pulse labelling of the cells was performed at an intensity of 500 μmol photons m −2 s −1 and a temperature of 30 °C, using a mixture of [ 35 S]Met and [ 35 S]Cys (Hartmann Analytic Gmbh, Braunschweig, Germany) as previously described 82 . Low-temperature fluorescence spectrometry Low-temperature fluorescence spectra of Chl a and phycobiliproteins were recorded using a HORIBA Fluoromax Plus FL-1013 spectrofluorometer (HORIBA Jobin Yvon GmbH, Oberursel, Germany). Cultures grown in MC photobioreactors under the specified light conditions were transferred into glass capillaries (Hilgenberg GmbH, Malsfeld, Germany) directly from the cultivation device at 7 days past inoculation and immediately snap-frozen in liquid nitrogen. Samples were stored in a light-occluding casing at −80 °C for 1–2 weeks and measured in a single batch. Measurements were conducted at 77K in a dewar filled with liquid nitrogen, using a signal integration time of 0.2 s nm⁻¹ and a detection bandwidth of 1 nm. Fluorescence signals were detected through a 500 nm long-pass filter upon excitation at 435 nm ( λexc = 435 nm) to stimulate Chl a absorption and at 600 nm ( λ exc = 600 nm) to selectively excite phycobiliproteins. Key fluorescence peaks were analyzed as ratios derived from relevant peaks. Relevant peaks corresponded to phycocyanin (PC; F645), allophycocyanin (APC; F662), Chl a in PSII core antenna CP43 (CP43; F685), Chl a in PSII core antenna CP47 (CP47; F695), and Chl a in PSI (PSI; F725) as described 35 . For the state transition analysis, the cells were cultivated for seven days at 23°C in a multi-cultivator. The cultures were transferred directly from multi-cultivator into NMR capillaries and exposed to blue light (150 μmol photons m −2 s −1 ) for State 1 and to darkness for State 2 for 15 min. Then the capillaries were then frozen in liquid nitrogen and stored at –80°C until testing. The measurements were performed as described above. Determination of PSII quantum yield parameters Apparent PSII quantum yield was measured as Fv - /Fm - 38 at room temperature as previously described 35 using a FluorCam 800MF (Photon Systems Instruments, Drásov, Czech Republic). Fv - /Fm - was measured in cells grown for 7 days at 23 °C and LL 20 on BG11 solid media after single-clone isolation, or cells collected from cultures grown at LL 50 in multi-cultivators and subsequently normalized to OD 730nm = 10 of which 10 µL droplets were placed on BG11-agar. After overnight acclimation at LL 20 and 23 °C, plates were dark-incubated for one hour before measurements. Instrument sensitivity was manually adjusted (10%–20%) before measuring. For samples under HL, suspensions were collected from the liquid cultures after 7 day cultivation and prepared as described previously 8 . P700 redox kinetics measurements The cells for the P700 measurements were cultivated for seven days at appropriate light conditions and 23 °C in a Multi-Cultivator. Cells were harvested by a 4-min centrifugation at 2800 g and 25 °C, washed twice in BG11, and adjusted to OD 730 nm = 5. Cell suspensions were aliquoted into 2-ml fractions and incubated in the dark overnight (≥16 h) at 25 °C and 120 rpm. Oxidation and re-reduction of P700 was analysed by measuring P700 absorbance at 820 nm relative to 870 nm using the DUAL-PAM 100 instrument (Walz, Effeltrich, Germany), as previously described 35,83 . The measurement routine involved 3 s in the dark, 60 s of FR light, and 30 s in the dark at measuring light intensity 4, FR light intensity 3 (38 μmol photons m -2 s -1 ), acquisition rate 200 s −1 , and high gain (5) and damping (1 ms). Curves were normalised by equating the absorbance baseline (average absorbance over 3 s of dark before onset of FR) to 0, and equating the absorbance maximum during 60 s FR illumination to 1. Rate constants (time required to reach 50% P700 oxidation or re-reduction) were acquired from normalised curves by extracting the time points at which Δabs exceeded 0.5. Oxygen evolution and respiration measurements Oxygen evolution was measured using an Oxytherm+ P Clark-type oxygen electrode (Hansatech Instruments, King’s Lynn, UK) in non-disturbed Synechocystis cultures cultivated for seven days at 23°C in a multi-cultivator. PSII activity was estimated as the steady-state O 2 evolution rate under saturating light at 23°C in the presence of 0.5 mM DCBQ (1,4-benzoquinone) and 1 mM K 3 Fe(CN) 6 . Respiration was estimated in terms of the steady-state O 2 consumption rate over 5 min in the dark. The oxygen evolution rate and respiration rates were normalized to the OD 730nm or Chl a content, respectively, for each sample. Determination of coefficient of non-photochemical quenching at room temperature To determine the coefficient of non-photochemical quenching at room temperature, fast fluorescence kinetics were measured using a FluorCam 800MF (Photon Systems Instruments, Drásov, Czech Republic). Cells collected from cultures grown at 50 µmol photons m ‐2 s ‐1 in MC were normalized to OD 730nm = 10, and 10 µL droplets were evenly distributed on BG11-agar. After overnight acclimation at 30 µmol photons m -2 s -1 and 23 °C, plates were dark-incubated for one hour before measurements. Instrument sensitivity was manually adjusted (20%–30%) before measuring fluorescence kinetics under incremental red-orange illumination (Fo 5 s > Fm pulse 0.8 s > AL 10 % 60 s > Fm pulse 0.8 s > AL 20 % 60 s > Fm pulse 0.8 s > AL 40 % 60 s > Fm pulse 0.8 s > AL 60 % 60 s > Fm pulse 0.8 s > AL 80 % 60 s > Fm pulse 0.8 s > AL 100 % 60 s > Fm pulse 0.8 s > END) and induction-relaxation regime (Fo 5 s > Fm pulse 0.8 s > dark 10 s > AL 100 % 60 s > dark relaxation 60 s; 9 Fm pulses during AL and dark relaxation, first Fm pulse 9 s after AL onset). Actinic light at 100% intensity approximated 215 µmol photons m⁻² s⁻¹ (λ max = 625 nm), while saturating pulses (Fm pulses; ~1200 µmol photons m⁻² s⁻¹) were provided by cold-white 6500 K LEDs. Quenching parameters were computed using FluorCam7 (Photon Systems Instruments). RNA extraction and transcriptome analysis For RNA extraction, 30 ml of cell culture (OD 730 ~1.0) was harvested from LT and rpaB T183P cells, which were grown under constant LL 50 and HL 700 conditions in multi-cultivators at 23°C. The cells were then centrifuged and resuspended in TRIzol (Invitrogen, Carlsbad, California, USA). After incubation of cells in the TRIzol at 65°C for 15 min, the RNA was extracted using phenol-chloroform and finally precipitated with isopropanol overnight at -20°C. The RNA was treated with the TURBO DNA-free TM kit (Invitrogen, MA, USA) to remove genomic DNA, and then purified using Direct-zol™ RNA MiniPrep Plus columns (Zymo Research, Irvine, California, USA). Ribosomal RNA depletion and RNA-seq library generation were performed by Novogene Biotech (Beijing, China) using standard Illumina protocols. The RNA-seq libraries were sequenced on an Illumina HiSeq 2500 system (Illumina, San Diego, Calif. USA) using a 150 bp paired-end sequencing strategy. Data quality was checked using FastQC (version 0.12.1) and cleaned using fastp (version 1.0.1). Paired-end reads were mapped to the Synechocystis sp. PCC6803 genome (GCF_000009725.1) using HISAT2 (version 2.2.1). Read counts were quantified using featureCounts (version 2.1.1). The rest of the analysis was performed in R (version 4.5.2.) via Rstudio (version 2025.09.2, build 418). DESeq2 (1.48.2) was then used to identify differentially expressed genes. Statistics and boxplot description Statistical analyses were conducted using two-tailed Student’s t -tests in Microsoft Excel. Post-hoc Bonferroni-Holm corrections for multiple comparisons were performed using astatsa when significant differences among groups were detected via one-way ANOVA at https://astatsa.com/. Box plots depicting data point distributions were created in Microsoft Excel. The horizontal middle lines represent inclusive medians, crosses indicate mean values, boxes denote the second and third quartiles, whiskers extend to the first and fourth quartiles, and points beyond the whisker ranges indicate outliers exceeding 1.5 times the interquartile range. Structural predictions AlphaFold3 (https://alphafoldserver.com/) was used to predict the tertiary structures of Sll0518, PAM68 and RpaB, as well as their variants with point mutations. The top-ranked predictions from AlphaFold3 were selected for further calculations and comparisons using UCSF Chimera X (University of California, San Francisco). Declarations ACKNOWLEDGEMENTS We thank Prof. Jörg Nickelsen (LMU Munich, Germany) for providing the Pam68 antibody and Prof. Yukako Hihara (Saitama University, Japan) for sharing the RpaB knock-down mutant ( rpaBkd ). AUTHOR CONTRIBUTIONS D.L., M.D. and T.F.-G. conceived the project. D.L. provided the funding. D.L., T.F.-G., W.C. and J.K. designed all experiments, with support from M.D.. T.F.-G., W.C., M.Z. and D.S. performed the experiments. W.C. and E.M.A.-S. performed the DNA and RNA sequence analyses and interpretation of the results. D.L. wrote the manuscript with the support of M.D., W.C. and T.F.-G. All authors read and approved the final manuscript. FUNDING We acknowledge support by the Deutsche Forschungsgemeinschaft (grant TRR175 to D.L.), the European Research Council (ERC Synergy Grant “PhotoRedesign”, to D.L. and J.K.), the Czech Ministry of Education (project PHOTOMACHINES, CZ.02.01.01/00/22_008/0004624 to D.S. and J.K.) and the Deutscher Akademischer Austauschdienst (DAAD, to T.F.-G.). COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material available at xxxx. Correspondence and requests for materials should be addressed to Dario Leister. Data availability Vector sequences are listed in Source Data 7 . 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Condition LL HL A t LL t HL Cycles FL0 initial 50 700 650 5 1 5 intermediate 50 1000 950 5 1 1 50 1200 1150 5 1 1 20 1200 1180 5 1 1 final 12 1200 1188 5 1 12 FL+ initial 50 700 650 5 1 1 intermediate 50 700 650 4 1 1 50 700 650 3 1 1 50 700 650 2 1 1 50 700 650 1 1 1 50 1000 950 1 1 1 50 1200 1150 1 1 1 20 1200 1180 1 1 1 final 12 1200 1188 1 1 12 Table 2. Fully segregated, protein-affecting mutations identified in FL-ALE strains. The table lists non-synonymous single nucleotide polymorphisms (SNPs) and deletions within coding regions that are absent in both the laboratory type (LT) and wild-type (WT) strains, and have achieved 100% allele frequency in at least one of the 24 monoclonal strains. Columns are organized as follows: "#": Corresponds to the mutation numbering in Fig. 2 . "Locus": Gene identifier; "–" denotes deletions within coding regions, with affected genes in brackets "[]". "Mutation": Specifies the nature and position of SNPs or indels, using the notation "x/y nt" where x is the affected position in a sequence of total length y. "No.": Indicates the number of monoclonal strains harboring the specific allele. "ALE": Specifies the ALE protocol(s) in which the allele was detected (0 for FL0, + for FL+, 0+ for both). "Function": Assigns Gene Ontology (GO) terms - M (metabolic processes), P (photosynthesis), Tc (transcription), Tl (translation), and ? (unknown function). Alleles associated with the FL-tolerant haplotype (present in ≥65% of monoclonal strains) are highlighted in orange. Alleles reconstituted and assessed for FL tolerance in this study are highlighted in bold font. # Locus Annotation Mutation No. ALE Function 1 sll0518 unknown protein A133V (GCC→GTC) 24 0+ ? 2 pam68 ( sll0933 ) PAM68 protein S113G (AGC→GGC) 24 0+ P 3 rpoDI ( slr0653 ) RNA polymerase s factor R96L (CGT→CTT) 24 0+ Tc 4 nadC ( slr0936 ) nicotinatenucleotide pyrophosphorylase Q183H (CAA→CAT) 16 0+ M 5 ssr7018 –[ ssl7019 ] hypothetical proteins Δ935 bp 6 0+ ? 6 rpoB ( sll1787 ) RNA polymerase beta subunit T791N (ACC→AAC) 3 + Tc 7 rpaB ( slr0947 ) OmpR subfamily T183P (ACC→CCC) 3 + Tc 8 D194G (GAC→GGC) 3 + 9 cpcB ( sll1577 ) phycocyanin b subunit S40P (TCT→CCT) 2 + P 10 topA ( slr2058 ) DNA topoisomerase I T504A (ACC→GCC) 3 + Tc 11 E149D (GAA→GAT) 2 + 12 slr0315 hypothetical protein V62L (GTT→CTT) 2 + ? 13 sll1647 hypothetical protein W97R (TGG→AGG) 2 + ? 14 dfr ( sll0698 ) drug sensory protein A T393A (ACC→GCC) 2 + M 15 F605L (TTC→TTG) 1 + 16 rrn16Sa 16S ribosomal RNA noncoding (254/1489 nt) 1 0 Tl 17 noncoding (247/1489 nt) 1 0 18 noncoding (243/1489 nt) 1 0 19 ycf45 ( slr0692 ) hypothetical protein Δ13 bp, coding (331-343/1770 nt) 1 + ? 20 E533G (GAA→GGA) 1 + 21 +T, coding (59/1770 nt) 1 + 22 rpoC1 ( slr1265 ) RNA polymerase g subunit R221L (CGG→CTG) 1 + Tc 23 slr1557 unknown protein Δ8 bp, coding (573-580/1110 nt) 1 + ? 24 rnb ( sll1290 ) ribonuclease II V500M (GTG→ATG) 1 + Tc 25 sll0209 hypothetical protein Q290E (CAA→GAA) 1 + ? 26 deaD ( slr0083 ) ATP dependent RNA helicase; DeaD R399L (CGG→CTG) 1 + Tc 27 trn D-GUC tRNA for aspartate A→G, noncoding (8/74 nt) 1 + Tl 28 slr0937 unknown protein G292S (GGT→AGT) 1 + ? Additional Declarations There is NO Competing Interest. Supplementary Files SupplementalData1.xlsx Source Data 1 SupplementalData2.xlsx Source Data 2 SupplementalData3.xlsx Source Data 3 SupplementalData4.xlsx Source Data 4 SupplementalData5new.xlsx Source Data 5 SupplementalData6new.xlsx Source Data 6 SupplementalData7.xlsx Source Data 7 260203FLsup.docx Supplementary Figures 260203FLrebuttal.docx Point-by-point reply Cite Share Download PDF Status: Published Journal Publication published 04 May, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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