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Enhancing photosynthesis at high light levels by adaptive laboratory evolution

Abstract

Photosynthesis is readily impaired by high light (HL) levels. Photosynthetic organisms have therefore evolved various mechanisms to cope with the problem. Here, we have dramatically enhanced the light tolerance of the cyanobacterium Synechocystis by adaptive laboratory evolution (ALE). By combining repeated mutagenesis and exposure to increasing light intensities, we generated strains that grow under extremely HL intensities. HL tolerance was associated with more than 100 mutations in proteins involved in various cellular functions, including gene expression, photosynthesis and metabolism. Co-evolved mutations were grouped into five haplotypes, and putative epistatic interactions were identified. Two representative mutations, introduced into non-adapted cells, each confer enhanced HL tolerance, but they affect photosynthesis and respiration in different ways. Mutations identified by ALE that allow photosynthetic microorganisms to cope with altered light conditions could be employed in assisted evolution approaches and could strengthen the robustness of photosynthesis in crop plants.

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Fig. 1: ALE scheme.
Fig. 2: Characterization of HL-tolerant batch cultures.
Fig. 3: Heterogeneity between and within adapted batch cultures.
Fig. 4: Haplotypes and mutational epistasis.
Fig. 5: HL tolerance of NdhF1-F124L, EF-G2-R461C and NdhF1-F124L EF-G2-R461C cells and control strains.
Fig. 6: PSI/PSII ratio and thylakoid marker protein accumulation in NdhF1-F124L, EF-G2-R461C and NdhF1-F124L EF-G2-R461C cells and control strains.
Fig. 7: Rates of photosynthesis and respiration for NdhF1-F124L, EF-G2-R461C and NdhF1-F124L EF-G2-R461C cells and control strains.

Data availability

All data supporting the findings of this study are available within the paper and its Supplementary Information files. The biological material is available upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank P. Hardy for his critical reading of the manuscript, and we thank the German Science Foundation (DFG, grant nos. TR175 Z1, GRK2062 and EXC2089/1 (e-conversion, funding ID: 390776260) to D.L.) and the European Research Council (ERC Synergy Grant ‘PhotoRedesign’) for financial support.

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Contributions

D.L., M.D. and A.G. designed the adaptive evolution experiments and, together with M.T., performed them. E.M.O. and H.S. designed the sequencing strategy and analysed the next-generation sequencing data. M.L. performed the carotenoid profiling. D.L. was responsible for the conceptualization and management of the entire study and wrote the paper with contributions from all authors.

Corresponding author

Correspondence to Dario Leister.

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The authors declare no competing interests.

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Peer review information Nature Plants thanks Diana Kirilovsky and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Synechocystis genomic allele-swapping strategy.

Candidate point mutations were introduced into the Synechocystis LT genome employing a modified version of our established single-vector-based, marker-less gene replacement strategy62. The complete ORF of interest (5’ CDS+ 3’ CDS) and a second copy of the 3’ CDS were cloned into a non-replicative plasmid on either side of the double-selection cassette (DSC)62. SNPs were introduced by Q5 site-directed mutagenesis. The endogenous genomic ORF was replaced with the mutated ORF by homologous recombination upon positive selection on kanamycin (KanR; nptI), and the original gene structure was re-established by removal of the DSC by intra-chromosomal homologous recombination upon negative selection on sucrose (SucrS; sacB).

Extended Data Fig. 2 Growth curves for mutant and control strains at 50, 700 and 1200 μmol photons m-2 s-1 and HL tolerance at 2000 μmol photons m-2 s-1.

a, Growth of Synechocystis strains over the course of 7 days post inoculation was measured as apparent optical density at λ=720 nm (OD720nm). LT, our laboratory-adapted Synechocystis PCC6803 strain; WT, the original motile Synechocystis PCC6803 strain obtained from the Pasteur Collection; #1, NdhF1F124L; #2, EF-G2R461C; #12, NdhF1F124L EF-G2R461C. Note that cultures were inoculated to reach an OD730nm = 0.05, and that cultivation of strains in Multi-Cultivator OD-1000 devices and direct measurement with the in-built system leads to underestimation of the actual OD720nm if actual values exceed ~0.5. b, Duplication times during the exponential phase of growth curves shown in (a). Duplication time data was inferred from growth curve slopes over eight hours starting at OD = 0.1 (that is after the first full duplication of culture OD). c, HL tolerance at 2000 μmol photons m-2 s-1 of NdhF1F124L, EF-G2R461C, NdhF1F124L EF-G2R461C cells, LT and WT strains, analysed as in Fig. 5. Data are derived from six replicates (two biological replicates of three independent clones per genotype). Error bars indicated SDs from respective average values. Crosses in boxplots indicate average values, letters signify statistically significant differences with p ≤ 0.05 according to post-hoc Bonferroni-Holm simultaneous comparison of all measurements after significant among-group differences were detected by one-factorial ANOVA (two-sided).

Source data

Extended Data Fig. 3 Cell culture absorbance spectra.

Absorbance spectra shown are averages of six biological replicates of Synechocystis cultures 7 days post inoculation. Data have been baseline-corrected, such that absorbance at 750 nm equals 0, and were used to estimate phycocyanin-to-chlorophyll a (PC/Chl) molar ratios70.

Source data

Extended Data Fig. 4 Low temperature (77 K) fluorescence spectra.

a, 77 K fluorescence emission spectra upon excitation at 435 nm. b, 77 K fluorescence emission spectra upon excitation at 600 nm. With the exception of the data for LT at 700 μmol photons m-2 s-1 (for which n = 4), all spectra shown are averages of six biological replicates of Synechocystis cultures measured 7 days post inoculation. Data has been baseline corrected for emission at 600 nm (a) and 620 nm (b), and normalized to input culture OD730nm, respectively. Spectra were corrected for dark-signal (dark-offset ON) and the blank value (BG11 medium prior to inoculation) was subtracted.

Extended Data Fig. 5 HL tolerance and motility of the original Synechocystis sp. PCC6803 strain.

Time course of the growth of the original (motile) Synechocystis sp PCC6803 strain at 2000 µmol photons m-2 s-1 and 23 °C over 7 days, after inoculation at OD730nm = 0.05. The white arrowheads indicate the initial level of the liquid culture; the dashed outline indicates an area of reduced light exposure owing to the absence of LEDs in the cultivator design. Note that the motile cells migrate out of the liquid medium and only begin to return at 6 days post inoculation (dpi). This observation indicates that this strain requires an extended period of acclimation before it can cope with such high light intensities.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2.

Reporting Summary

Supplementary Data 1

Processed sequencing data output with identified potentially HL-adaptive mutations categorized by types, frequencies and occurrences in epistatic series and haplotypes.

Supplementary Data 2

Sequencing data SNP analysis output—all candidate SNPs identified in the HL ALE experiment.

Source data

Source Data Fig. 2

Dry mass raw data and statistical analyses for ALE final batch cultures.

Source Data Fig. 3

FluorCam raw data for ALE isolated clones.

Source Data Fig. 5

OD, dry mass, pigment raw data and statistical analyses for ALE reconstituted point mutants.

Source Data Fig. 6

77 K fluorescence and immunoblot raw data and statistical analyses for ALE reconstituted point mutants.

Source Data Fig. 7

Chlorophyll fluorescence, oxygen evolution, P700 redox kinetics raw data and statistical analyses for ALE reconstituted point mutants.

Source Data Extended Data Fig. 2

Growth curves and WT PCC6803 final OD and dry mass at 2,000 µmol photons per m2 per s for ALE reconstituted point mutants.

Source Data Extended Data Fig. 3

77 K fluorescence spectra at excitation wavelengths 435 and 600 nm for ALE reconstituted point mutants.

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Dann, M., Ortiz, E.M., Thomas, M. et al. Enhancing photosynthesis at high light levels by adaptive laboratory evolution. Nat. Plants 7, 681–695 (2021). https://doi.org/10.1038/s41477-021-00904-2

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